Tout médicament administré par la voie orale doit être absorbé sans être métabolisé par l’intestin et le foie pour atteindre la circulation systémique. Malgré son impact majeur sur l’effet de premier passage de plusieurs médicaments, le métabolisme intestinal est souvent négligé comparativement au métabolisme hépatique. L’objectif de ces travaux de maîtrise est donc d’utiliser, caractériser et développer différents outils in vitro et in vivo pour mieux comprendre et prédire l’impact du métabolisme intestinal sur l’effet de premier passage des médicaments comparé au métabolisme hépatique. Pour se faire, différents substrats d’enzymes du métabolisme ont été incubés dans des microsomes intestinaux et hépatiques et des différences entre la vitesse de métabolisme et les métabolites produits ont été démontrés. Afin de mieux comprendre l’impact de ces différences in vivo, des études mécanistiques chez des animaux canulés et traités avec des inhibiteurs enzymatiques ont été conduites avec le substrat métoprolol. Ces études ont démontré l’impact du métabolisme intestinal sur le premier passage du métoprolol. De plus, elles ont révélé l’effet sur la vidange gastrique du 1-aminobenzotriazole, un inhibiteur des cytochromes p450, évitant ainsi une mauvaise utilisation de cet outil dans le futur. Ces travaux de maîtrise ont permis d’améliorer les connaissances des différents outils in vitro et in vivo pour étudier le métabolisme intestinal tout en permettant de mieux comprendre les différences entre le rôle de l’intestin et du foie sur l’effet de premier passage. / To reach the systemic circulation, orally administered drugs have to be absorbed and not metabolized by the intestine and the liver. Even though it has a major impact on the first pass effect of many xenobiotics, the intestinal metabolism is often neglect compare to the hepatic metabolism. The objective of this work is to use, characterize and develop multiple in vitro and in vivo tools to better understand and predict the impact of intestinal metabolism on the first pass effect of xenobiotics compared to the liver. To do so, multiple substrates of metabolic enzymes were incubated in intestinal and hepatic microsomes and differences between the rate of metabolism and the production of metabolites were demonstrated. To better understand the impact of these differences in vivo, mechanistic studies were undergone in rats cannulated or treated with enzymatic inhibitors with the substrate metoprolol. These studies demonstrated the impact of intestinal metabolism on the first pass of metoprolol. Moreover, they exposed the effect on gastric emptying of 1-aminobenzotriazole, a cytochrome p450 inhibitor, avoiding its wrong utilisation in future studies. This work helped increase the knowledge about the different in vitro and in vivo tools to study intestinal metabolism and to better understand the differences between the role of the intestine and the liver on the first pass effect.

Les maladies inflammatoires de l’intestin (MIIs, [MIM 266600]) sont caractérisées par une inflammation chronique au niveau du tube gastro-intestinal. Les deux principales formes sont la maladie de Crohn (MC) et la colite ulcéreuse (CU). Les MIIs résulteraient d’un défaut du système immunitaire et de l’épithélium intestinal. Ce dernier forme une barrière physique et biochimique qui sépare notre système immunitaire des microorganismes commensaux et pathogènes de la microflore intestinale. Un défaut dans la barrière épithéliale intestinale pourrait donc mener à une réponse immunitaire soutenue contre notre microflore intestinale. Les études d’association pangénomiques (GWAS) ont permis d’identifier 201 régions de susceptibilité aux MIIs. Parmi celles-ci, la région 1q32 associée à la MC (p<2x10-11) et à la CU (p<6x10-7) contient 4 gènes, dont C1orf106, un gène codant pour une protéine de fonction inconnue. Le re-séquençage de la région 1q32 a permis d’identifier une variante génétique rare de C1orf106 (MAF˂1%) associée aux MIIs (p=0,009), Y333F. Nous avons démontré que la substitution de la tyr333 par une phénylalanine semble avoir un effet sur la stabilité protéique de C1orf106 tel que démontré lors de l’inhibition de la synthèse protéique induite par le cycloheximide. Nous avons déterminé que C1orf106 est exprimé dans le côlon et l’intestin grêle. De plus, son expression est augmentée lors de la différenciation des cellules épithéliales Caco-2 en épithélium intestinal polarisé. Son profil d’expression correspond aux types cellulaires et tissulaires affectés dans les MIIs. De plus, C1orf106 est partiellement co-localisée avec le marqueur des jonctions serrées, ZO-1. Toutefois, son marquage reproduit parfaitement celui du marqueur des jonctions adhérentes, E-cadhérine. Les jonctions serrées et adhérentes sont localisées du côté apical de la jonction intercellulaire et sont toutes deux impliquées dans l’établissement de la barrière épithéliale. Nous avons donc testé l’impact de C1orf106 sur la perméabilité de l’épithélium intestinal. Nous avons observé une augmentation de la perméabilité épithéliale chez un épithélium intestinal formé par des cellules Caco-2 sous-exprimant C1orf106. Nos résultats suggèrent que C1orf106 pourrait être le gène causal de la région 1q32. / The Inflammatory bowel diseases (IBD, [MIM 266600]) involve chronic inflammation of the digestive tract and include ulcerative colitis (UC) and Crohn’s disease (CD). IBD may result from defects in the homeostasis of immune system and intestinal epithelium. The latter forms a physical and biochemical barrier to commensal and pathogenic microorganisms. A dysfunction in the epithelial barrier may lead to a sustained immune response against the gut flora. Genome wide association studies (GWAS) have identified 200 susceptibility regions in IBD. Among these, the 1q32 region associated with risk of both CD (p<2x10-11) and UC (p<6x10-7), contains the gene C1orf106. Our targeted re-sequencing study has identified a low-frequency variant, Y333F (p=0.009) in C1orf106, a protein of unknown function and in which tyrosine333 is predicted to be phosphorylated. We demonstrated that its substitution by a phenylalanine may have an effect on C1orf106 protein stability as shown by cycloheximide treatment experiments. Our RNA expression analyses of human tissues and cell lines demonstrated that C1orf106 is mostly expressed in the small intestine and colon. It is also detectable in monocytic cell lines but more highly expressed in colonic epithelial cell lines. Furthermore, its expression is increased by 40% during differentiation of colonic epithelial Caco-2 cells into polarized epithelium. To provide further biological context, we generated colorectal LS174T cells that stably overexpress the Y333F alleles and demonstrated that it is partially localized with ZO-1, used as a tight junction (TJ) marker. We did observe tighter colocalization with E-cadherin, a canonical marker for adherens junctions (AJ), typically located below the TJ complex. AJ and TJ play an essential role in the establishment of epithelial barrier. The localization of C1orf106 at these regions suggests its possible implication in epithelial barrier homeostasis. Using trans-epithelial measurement of ions movement across epithelium, we demonstrated an increased in permeability of an epithelium formed by C1orf106 knock-down Caco-2 cells. Our results suggest that C1orf106 could be the causal gene of the 1q32 susceptibility region.

Le corps humain est un ensemble de microflores où cohabitent bactéries, archées, virus et eucaryotes. Ces écosystèmes complexes sont appelés microbiotes. Parmi ceux-ci figure le microbiote intestinal qui compte 1011 à 1014 bactéries/g de selle. Les modifications de la flore intestinale peuvent être à l'origine de pathologies comme les diarrhées infectieuses. Il s'agit d'un véritable problème de santé publique puisqu'environ 2.16 millions de décès sont liés à cette pathologie chaque année. Les virus intestinaux jouent un rôle prépondérant mais les infections bactériennes restent également importantes. Le diagnostic de ces infections bactériennes reste compliqué puisque le microbiote intestinal comporte 75% d'espèces non cultivables. De plus, on ne dispose pas réellement d'une liste exhaustive des bactéries pouvant être responsables de diarrhées infectieuses. Nous avons donc choisi d'étudier le microbiote intestinal dans des selles normales et pathologiques, par une approche polyphasique alliant une étape préliminaire de concentration des selles diarrhéiques par la lyophilisation, à des techniques de culture et des méthodes de biologie moléculaire. Pour cela nous avons mis au point une nouvelle technologie pour la détection des entéropathogènes par hybridation sur puce à ADN permettant la détection des bactéries et des virus ADN entéropathogènes, en présence d'un témoin archae. Notre outil permet le diagnostic multiplexe des diarrhées infectieuses puisque nous avons correctement identifié un adénovirus et la bactérie Campylobacter jejuni présents dans une même selle. / The human body is a collection of microflora where cohabit bacteria, archaea, viruses and eukaryotes. These complex ecosystems are called microbiota. Among these is the intestinal microbiota that counts 1011 to 1014 bacteria/g of stool. Changes in the intestinal flora can cause of pathologies such as infectious diarrhea. This is a real public health problem since about 2.16 million deaths are related to this disease each year. Enteric viruses play a preponderant role but bacterial infections are also important. The diagnosis of bacterial infections is complicated because the intestinal microbiota includes 75% non-cultivable species. In addition, there is not really a list of bacteria could be responsible for infectious diarrhea. We therefore decided to study the intestinal microbiota in normal stool and also pathological stools by a polyphasic approach combining a preliminary step of diarrheal stools concentration by lyophilization, with cultivation techniques and molecular biology methods. We developed a new technology for the detection of enteropathogens by hybridization on DNA microarray for the detection of bacteria and enteric viruses (DNA) in the presence of a control archaea. Our tool allows multiplexed diagnostic of infectious diarrhea since we correctly identified an adenovirus and Campylobacter jejuni present in a same sample. This is the first DNA microarray for multiplex detection of bacteria and viruses (DNA) enteropathogens. An improvement of our protocol for nucleic acid extraction is proposed to allow the detection of RNA viruses such as rotavirus and calicivirus which are currently dominant.

Le tube digestif héberge une communauté microbienne complexe, le microbiote intestinal, dont les capacités métaboliques sont plus riches et diversifiées que celles codées par le génome de l'hôte. L'implication du microbiote intestinal dans divers aspects de la physiologie de l'hôte, comme le métabolisme nutritionnel et l'immunité, est depuis longtemps étudiée. En revanche, l'action potentielle du microbiote sur le développement et le fonctionnement du cerveau constitue une nouvelle piste de recherche, encore peu explorée. Dans ce contexte, nous avons réalisé une première étude générale de l'action du microbiote intestinal sur le cerveau en comparant les fonctions sensori-motrices, le comportement de type anxieux, l'état d'activation de l'axe hypothalamo-hypophyso-surrénalien et le profil cérébral des monoamines de rats F344 axéniques et conventionnels. Les résultats révèlent que, chez cette lignée particulièrement sensible au stress, l'absence de microbiote intestinal exacerbe le comportement de type anxieux et la réponse hormonale au stress, et atténue le métabolisme dopaminergique cérébral. Afin d'étudier par quel moyen le microbiote peut agir sur le cerveau, une seconde étude a été menée, ciblant un métabolite bactérien spécifique, l’indole, dont certains dérivés oxydés par le foie sont connus pour avoir des propriétés neuroactives. L'indole est un métabolite naturel du microbiote intestinal, dont la surproduction pourrait survenir lors d'une dysbiose du microbiote. Deux cas de surproduction ont été modélisés : chronique et aiguë. Dans les deux cas, des modifications importantes du comportement de l'hôte ont été observées. En situation de surproduction chronique, l'indole favorise des comportements de type anxieux et dépressif, tandis qu'une surproduction aiguë a un effet sédatif marqué. D'un point de vue mécanistique, nous confirmons que l’indole peut agir sur le système nerveux central par la voie sanguine impliquant les dérivés oxydés et montrons pour la première fois qu'il peut aussi agir en activant les noyaux cérébraux du nerf vague. / The gastro-intestinal tract hosts a complex microbial community, the gut microbiota, whose collective genome coding capacity vastly exceeds that of the host genome. The involvement of the gut microbiota in various aspects of the host physiology, such as the nutritional metabolism and the immunity, has long been studied. In contrast, the possible action of the gut microbiota on brain development and functioning is a new line of research, still poorly explored. In this context, we performed a first general study of the effect of gut microbiota on the brain by comparing the sensory-motor functions, the anxiety-like behaviour, the activation of the hypothalamic-pituitary-adrenal axis and the brain monoamine profile in germ-free and conventional F344 rats. The results show that, in this particularly stress-sensitive strain, absence of gut microbiota exacerbates the anxiety-like behaviour and neuroendocrine response to stress, and reduces brain dopamine metabolism. To investigate the means by which the microbiota can affect the brain, a second study was conducted, targeting a specific bacterial metabolite, indole, whose oxidative derivatives, produced by the liver, are known to have neuroactive properties. Indole is a natural metabolite of the gut microbiota, whoseoverproduction could occur during a microbiota dysbiosis. Two conditions of overproduction, namely chronic and acute, were modelled. In both cases, significant changes in the behaviour of the host were observed. In chronic overproduction, indole promotes anxiety- and depressive-like behaviours, while acute overproduction has a marked sedative effect. From a mechanistic point of view, we confirm that indole can act on the central nervous system through its oxidized derivatives and show for the first time that it can also act by activating the brain nuclei of the vagus nerve.

La maladie du greffon contre l’hôte (GVHD) reste la complication majeure de l’allogreffe de cellules soucheshématopoïétiques (allo-CSH). La GVHD résulte de l’activation de la réponse immunitaire et de la reconnaissanced’alloantigènes par les lymphocytes T (LT) du donneur, entrainant ainsi des lésions tissulaires principalement auniveau de la peau, des intestins et du foie. L’interleukine-22 (IL-22) est une cytokine sécrétée par les LT Th1,Th17 et les cellules de l’immunité innée (ILC). Compte tenu des propriétés de l’IL-22 dans les tissus cibles de laGVHD, nous avons évalué sa contribution dans la physiopathologie de la maladie à l’aide de modèlesexpérimentaux murins. Il apparaît que les souris qui reçoivent des lymphocytes T invalidés pour l’IL-22développent une maladie moins sévère, et leur mortalité est diminuée. L’IL-22 issue du greffon participe donc à lasévérité de la GVHD en favorisant l’inflammation systémique, mais aussi locale au niveau des organes cibles. Deplus, dans les intestins, l’IL-22 agit en synergie avec les interférons de type I pour amplifier l’inflammation de typeTh1 au cours de la GVHD. Chez l’homme, la GVHD est associée à une modification du microbiote intestinal.Nous avons montré que l’absence d’IL-22 semble favoriser la colonisation de lactobacilles au détriment declostridiums, ce qui pourrait également participer à la diminution de la GVHD intestinale. Enfin, nous avonsmontré que l’effet anti-tumoral est préservé malgré l’absence d’IL-22. Ces résultats permettent donc d’envisagerde nouvelles perspectives thérapeutiques dans le traitement de la GVHD. / Graft-versus-host disease (GVHD) is still the major complication after allogeneic stem cell transplantation. GVHDresults from the activation of the immune response and the recognition by donor T cells of alloantigens leading totissue injury, especially in skin, gut and liver. Interleukin-22 (IL-22) is a cytokine secreted by CD4+ T cells Th1 andTh17 but also by innate lymphoid cells (ILC). Given that IL-22 functions in the GVHD target tissues, weinvestigated its contribution in GVHD physiopathology using mouse experimental models. We showed that IL-22deficiency in donor cells reduced the severity of GVHD by limiting systemic and local inflammation. Moreover, inthe large intestine, IL-22 acts in synergy with type I interferon to increase Th1-like inflammation. In humans,GVHD severity is associated with microbiotal modification in the intestine. We demonstrated that IL-22 deficiencyin donor cells seems to favor lactobacillus colonization instead of clostridium. These changes of microbiotacomposition may reduce the severity of intestinal GVHD. Finally, we showed that the antitumor effect is preservedeven in absence of IL-22 donor cells. Overall, our data support the design of new clinical approaches aiming totarget IL-22 pathways in GVHD patients.

A obesidade é considerada uma doença multifatorial e pode envolver, em sua gênese, aspectos genéticos, metabólicos, ambientais, psicológicos e socioeconômicos. O crescimento expressivo da incidência mundial da obesidade desencadeia o surgimento contínuo de novas pesquisas em relação a esse tema e, nesse contexto, estudos recentes sugerem que a microbiota intestinal é um fator que pode contribuir com o desenvolvimento desta doença. Assim, existe a premissa que a alteração da composição da microbiota intestinal pode ser influenciada pelo estado nutricional (obesidade x eutrofia) e pela qualidade da alimentação. O presente estudo teve como objetivos: i. comparar a proporção dos filos predominantes da microbiota intestinal Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, Verrucomicrobia e a razão Firmicutes/Bacteroidetes entre mulheres com obesidade grau III e mulheres eutróficas, e ii. investigar o impacto de uma dieta hipocalórica para perda de peso na composição da microbiota intestinal. Estudo prospectivo longitudinal, no qual foram selecionadas 20 mulheres com média de idade 33±3,1 anos, as quais foram divididas em dois grupos: Grupo Intervenção (GI): 10 mulheres com obesidade grau III (Índice de Massa Corporal (IMC) >40 kg/m2) que foram submetidas à intervenção nutricional (dieta hipocalórica) durante 8 semanas e Grupo Controle (GC):10 mulheres eutróficas (IMC entre 18,5 a 24,9 kg/m2). No GI, as coletas foram realizadas antes e após oito semanas da intervenção (GI) e, no GC em um único momento. Em cada momento, foram aferidos o peso e estatura; realizado o cálculo do IMC, análise composição corporal, taxa metabólica de repouso, consumo alimentar, glicemia e lipidograma, e coleta de amostra fecal. A análise da composição da microbiota intestinal em relação à abundância relativa dos Filos foi realizada por reação em cadeia da polimerase em tempo real (qPCR). Após oito semanas de dieta hipocalórica, houve redução do peso (119,5±10,3 para 114,9±10,2 kg), IMC (43,6±2,4 para 41,9±2,6 kg/m2), massa corporal gorda (MG) (62,4±7,5 para 58,9±7,7 kg), triglicérides (TG) (143,2±60,9 para 117,94±48,3 mg/dL). Evidenciou-se que mulheres com obesidade apresentam menor abundância dos filos pesquisados em relação às eutróficas. Ainda, a dieta hipocalórica promoveu diminuição da abundância relativa do filo Proteobacteria, o qual apresentou correlações positivas com a ingestão de lipídio total (%), ômega 6 (g). Conclui-se que a intervenção com dieta hipocalórica foi eficaz na redução de peso, IMC, MG e TG e na modulação da microbiota intestinal, com a diminuição do filo Proteobacteria / Obesity is considered a multifactorial disease and it may involve, in its genesis, genetic, metabolic, environmental, psychological and socioeconomic aspects. The expressive growth of a worldwide incidence of obesity triggers the continual emergence of new researches on this subject and, in this context, recent studies have suggested that the intestinal microbiota is a factor that may contribute to the development of this disease. Thus, there is the premise that the changes in the composition of the intestinal microbiota can be influenced by the nutritional status (obesity x eutrophy) and by the diet quality.The present study aimed at: i. comparing the proportion of the predominant phyla of the intestinal microbiota Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, Verrucomicrobia and ratio Firmicutes/Bacteroidetes among degree III obese women and eutrophic women, and ii. investigating the impact of a low-calorie diet for weight loss on the intestinal microbiota composition. This was a longitudinal prospective study in which 20 women at the average age of 33 ± 3.1 years old were selected and divided into two groups: Intervention Group (IG): 10 women with grade III obesity (Body Mass Index (BMI) > 40 kg / m2) who were submitted to a nutritional intervention (a low-calorie diet) for 8 weeks, and the Control Group (CG): 10 eutrophic women (BMI from 18.5 to 24.9 kg / m2) who were not submitted to any intervention. In the IG, sampling was carried out before and after the eight-week intervention (IG) and in the CG it was carried out only in a single moment. At each sampling, weight and height were checked; BMI was calculated, body composition was analyzed, resting metabolic rate, food intake, fasting blood glucose, and lipidogram tests were performed, and fecal sample collected. The analysis of the intestinal microbiota composition in relation to a relative abundance of every phila was performed by real-time polymerase chain reaction (qPCR). After eight weeks of a low-calorie diet, some of the observed results were weight reduction (119.5 ± 10.3 to 114.9 ± 10.2 kg), BMI (43.6 ± 2.4 to 41.9 ± 2.6 kg / m2), fat body mass (FM) (62.4 ± 7.5 to 58.9 ± 7.7 kg), triglycerides (TG) (143.2 ± 60.9 to 117.94 ± 48.3 mg / dL). It was evidenced that obese women present a lower abundance of the studied phyla in relation to the eutrophic ones. Moreover, the lowcalorie diet promoted a decrease in the relative abundance of Proteobacteria phylum, which presented positive correlations with the intake of total lipid (%) and omega 6 (g). It was concluded that the intervention with a low-calorie diet was effective in reducing BMI, FM and TG and in modulating the intestinal microbiota, by reducing Proteobacteria phylum

No description available.

O trato gastrointestinal (TGI) é a principal rota de exposição ao fluoreto (F) e o seu mais importante sítio de absorção. Acredita-se que a toxicidade do F comprometa a fisiologia do intestino, devido à relevante sintomatologia gastrointestinal relatada em consequência da exposição excessiva ao F. A função intestinal é controlada por uma complexa rede neuronal interligada e incorporada à parede deste órgão, denominada Sistema Nervoso Entérico (SNE). Embora os efeitos tóxicos do F sobre o Sistema Nervoso Central sejam descritos na literatura, não há estudos relacionados à sua toxicidade sobre o SNE. Neste estudo realizado em ratos, foi avaliado o efeito da exposição aguda ou crônica ao F, sobre a população geral de neurônios entéricos e sobre as subpopulações que expressam os principais neurotransmissores entéricos: Acetilcolina (ACh), Óxido Nítrico (NO), Peptídeo Vasoativo Intestinal (VIP), Peptídeo Relacionado ao Gene da Calcitonina (CGRP) e Substância P (SP). Os animais foram divididos em 5 grupos: 3 destinados à exposição crônica (0 ppm, 10 ppm ou 50 ppm de F na água de beber) e 2 à exposição aguda (0 ou 25 mgF/Kg por gavagem gástrica). Foram coletados os 3 segmentos do intestino delgado (duodeno, jejuno e íleo) e processados para a detecção da HuC/D, ChAT, nNOS, VIP, CGRP e SP, através de técnicas de imunofluorescência, no plexo mioentérico. Foram obtidas imagens para a realização da análise quantitativa dos neurônios da população geral (HuC/D) e nitrérgicos (imunorreativos à nNOS); e morfométrica dos neurônios imunorreativos à HuC/D ou nNOS; e das varicosidades imunorreativas à ChAT, VIP, CGRP ou SP. Amostras dos 3 segmentos intestinais foram preparadas e coradas em Hematoxilina e Eosina para análise histológica da morfologia básica. O segmento intestinal considerado mais afetado na análise morfométrica da população geral de neurônios, o duodeno, foi selecionado para a realização da análise proteômica, com o objetivo de oferecer o seu perfil proteico e determinar diferenças na expressão proteica em decorrência da exposição crônica ou aguda ao F. A análise da concentração de F no plasma sanguíneo foi realizada para a confirmação da exposição. Na análise quantitativa, o grupo de 50 ppm F, apresentou uma diminuição significativa na densidade da população geral de neurônios do jejuno e do íleo e na densidade dos neurônios imunorreativos à nNOS no duodeno e no jejuno. Quanto à análise morfométrica, a população geral e as subpopulações neuronais entéricas avaliadas apresentaram alterações morfológicas significativas, tanto após a exposição crônica quanto a aguda. Para a análise proteômica do duodeno, verificou-se que da associação de seus genes a um termo, e assim classificadas de acordo com diferentes processos biológicos. No caso do grupo da dose aguda, o processo biológico com a maior porcentagem de genes associados foi a geração de metabólitos precursores e energia (27% das proteínas); enquanto para os grupos de 10 e 50 ppm F foram o processo metabólico da piridina (41%) e a polimerização proteica (33%), respectivamente. / The gastrointestinal tract (GIT) is the main route of fluoride (F) exposure, and the most important site of its absorption. It is believed that F toxicity compromises the intestine physiology, due to the relevant gastrointestinal symptomatology reported in consequence to excessive exposure. The intestinal function is controlled by a complex neuronal net, which is interconnected and embedded in the wall of this organ, named Enteric Nervous System (ENS). Although the toxic effects of F on the Central Nervous system are described in the literature, there are no studies related to its toxicity on the ENS. Therefore, in this study performed in rats, the effects of chronic and acute F exposure were evaluated, on the general population of enteric neurons and on the subpopulations that express the main enteric neurotransmitters: Acetylcholine (Ach), Nitric Oxide (NO), Vasoactive Intestinal Peptide (VIP), Calcitonin gene related peptide (CGRP), and Substance P (SP). The animals were divided into 5 groups: 3 designed to the chronic exposure (0 ppm, 10 ppm ou 50 ppm de F in the drinking water) and 2 to the acute exposure (0 ou 25 mgF/Kg - gastric gavage). Three intestinal segments were collected (duodenum, jejunum, and ileum) and processed for the immunofluorescence techniques to detect HuC/D, ChAT, nNOS, VIP, CGRP and SP, on the myenteric plexus. Images were obtained for the quantitative analysis of the general population of neurons (HuC/D immunoreactive) and the nitrergic neurons (nNOS immunoreactive), for the morphometric analysis of the general population and nitrergic neurons and also for the immunoreactive varicosities to ChAT, VIP, CGRP or SP. Samples of the 3 intestinal segments were prepared and stained with hematoxylin and eosin for histological analysis of the basic morphology. Duodenum, the intestinal segment considered the most affected in the morphological analysis of the general population of neurons, was selected for the proteomic analysis, with the objective to offer the entire protein profile and to determine differences in the protein expression due to chronic or acute F exposure. Plasma F concentration was analyzed to confirm the exposure. In the quantitative analysis, the 50 ppm F group presented a significant decrease in the density of the general population of neurons in the jejunum and ileum, and in the density of the nitrergic neurons in the duodenum and jejunum. Regarding the morphometric analysis, the general population of neurons and all the neuronal subpopulations evaluated presented significant morphological alterations, for both exposures, chronic and acute. In the proteomic analysis of the duodenum, it was verified that both exposures caused alterations in the expression of intestinal proteins. These proteins identified with differential expression were distributed according the association of their genes to a specific term, and by this term classified in different biological process. In the group that received the acute dose the biological process with the highest percentage of associated genes was the generation of precursor metabolites and energy (27% of proteins), and for the 10 and 50 ppm F groups, they were pyridine nucleotide metabolic process (41%) and protein polymerization (33%), respectively.

INTRODUÇÃO: Estudos demonstram que a solução salina hipertônica melhora a hemodinâmica, a microcirculação e modula o sistema imune, atenuando a resposta inflamatória associada ao choque e trauma. Este estudo tem por objetivo avaliar e comparar os efeitos da solução salina hipertônica (NaCl, 7,5%) e do Ringer lactato seguido da ressecção de segmento do intestino necrosado no tratamento da obstrução intestinal e isquemia, através da análise da translocação bacteriana, da disfunção microcirculatória mesentérica, dos distúrbios hemodinâmicos e metabólicos e da disfunção orgânica. MÉTODOS: Ratos Wistar machos (250 300 g) anestesiados (pentobarbital sódico, 50 mg/kg, i.p.) foram submetidos à obstrução intestinal e isquemia (OI, ligadura ao nível do íleo terminal seguida de ligadura de ramos da artéria mesentérica correspondentes à irrigação de 7 10 cm do íleo). Duas horas após os animais foram randomizados em: OI sem tratamento (OI); OI tratado com Ringer lactato (RL, 4 mL/kg, i.v.); e OI tratado com solução salina hipertônica (SH 7,5%, 4 mL/kg, i.v.). Vinte e quatro horas após os procedimentos cirúrgicos iniciais, os ratos obstruídos (OI, RL e SH) foram submetidos à enterectomia. Ratos controles falso-operados (FO) foram submetidos à lapatotomia. Os seguintes parâmetros foram analisados: 1) cultura bacteriana (E. coli) em amostras de linfonodos mesentéricos, fígado, baço e sangue; 2) análise das interações leucócito-endotélio na microcirculação mesentérica por técnica de microscopia intravital; 3) expressão de moléculas de adesão endoteliais (P-selectina e ICAM-1) por imunohistoquímica; 4) quantificação das citocinas e quimiocinas CINC-1 e CINC-2 no soro por enzimaimunoensaio; 5) histologia intestinal; 6) bioquímica sérica; 7) gasometria, hematócrito, lactato, glicose e leucograma; 8) insulina e corticosterona e; 9) sobrevida. RESULTADOS: O tratamento com SH reduziu a bacteremia, a incidência de animais com amostras positivas para E. coli (57%) e a quantidade de colônias bacterianas (p<0,05) comparado ao tratamento com RL ou aos animais não tratados (OI). As interações leucócito-endotélio e a expressão das moléculas de adesão, P-selectina e ICAM-1, reduziram-se após tratamento com SH seguido de enterectomia a valores observados no grupo controle FO (p>0,05). Ambos os tratamentos, SH e RL, associados à enterectomia normalizaram as concentrações séricas de CINC-1 e CINC-2 (p>0,05). Alterações de PaCO2, pH, lactato e glicemia normalizaram-se após o tratamento dos animais com SH ou RL seguido de enterectomia. A hipoinsulinemia registrada nos ratos OI foi prevenida pelo tratamento dos animais com SH ou RL. As concentrações séricas de corticosterona normalizaram-se após tratamento dos animais com SH associado à enterectomia. A magnitude da lesão intestinal, evidenciada por dano da membrana basal, edema, congestão e presença de células inflamatórias, foi menor no grupo tratado com SH comparado ao tratamento com RL, ambos associados à enterectomia. As concentrações de uréia, creatinina e a atividade das enzimas hepáticas normalizaram-se após tratamento com SH e enterectomia. Houve um aumento significativo da sobrevida dos animais (86%, 15 dias) e no ganho de peso corpóreo (p>0,05 vs FO) no grupo tratado com SH seguido de enterectomia. CONCLUSÕES: A estabilização de ratos submetidos à OI com SH associada à enterectomia resultou em aumento significativo da sobrevida dos animais. A SH reduziu a resposta inflamatória local (interações leucócito-endotélio e expressão de moléculas de adesão na microcirculação mesentérica, e lesão intestinal) e sistêmica (concentrações séricas de CINC-1 e CINC-2, disfunção renal e hepática) / BACKGROUND: It has been shown that hypertonic saline solution improve hemodynamics, the microcirculation, and modulate the immune system, attenuating the inflammatory response associated with shock and trauma. The present study aims to investigate and compare the effects of hypertonic saline solution (NaCl, 7.5%) and lactated Ringer´s solution in the treatment of intestinal obstruction and ischemia, analysing the bacterial translocation phenomenon, mesenteric microcirculatory dysfunctions, hemodynamic/metabolic disturbances, and organ dysfunction in a rat model of intestinal obstruction and ischemia (IO) followed by resection of the necrotic small bowel segment. METHODS: Anesthetized (pentobarbital 50 mg/kg, i.p.) male Wistar rats (250-300 g) were submitted to IO (ligature at the level of the terminal ileum, followed by ligation of mesenteric vessels that supply 7 10 cm of the ileal loop). Two hours thereafter animals were randomized into: IO without treatment; IO treated with lactated Ringer´s (LR, 4 ml/kg, i.v.) solution; IO treated with hypertonic saline (HS, 7.5%, 4 mL/kg, i.v.) solution. Twenty-four hours thereafter, IO rats (IO, LR, HS) were submitted to enterectomy. Control Sham-operated rats were submitted to laparotomy only. The following parameters were analysed: 1) bacterial cultures (E. coli) from mesenteric lymph nodes, liver, spleen, and blood samples; 2) analyses of leukocyte-endothelial interactions at the mesenteric microcirculation by intravital microscopy; 3) expression of endothelial adhesion molecules (P-selectin, ICAM-1) by immunohistochemistry; 4) quantification of serum cytokines and chemokines (CINC-1, CINC-2) by enzyme-linked immunosorbent assay; 5) intestinal histology; 6) serum biochemistry; 7) blood gases, hematocrit, lactate, glycemia, white blood cell counts; 8) serum insulin and corticosterone; 9) survival rate. RESULTS: Treatment with HS reduced the number of animals with positive samples for the presence of E. coli (57%) and the number of CFU/g tissue (p<0.05) compared to LR treated rats or IO rats without treatment. Leukocyteendothelial interactions and expression of the adhesion molecules, Pselectin and ICAM-1, were reduced after treatment with HS solution followed by enterectomy. Values attained matched those observed in Sham group (p>0.05). Both treatments, HS and LR associated with enterectomy, reduced serum levels of CINC-1 and CINC-2 to normal values (p>0.05 vs Sham). PaCO2, pH, lactate, and glucose were normalized after treatment of the animals with HS or LR followed by enterectomy. The hypoinsulinemia observed in the IO rats was prevented by treatment of the animals with HS or LR. Corticosterone concentrations were normalized after treatment of the animals with HS associated with enterectomy. The magnitude of the intestinal lesion, characterized by basal membrane injury, edema, congestion, and inflammatory cells, was smaller in the HS group compared to LR group, both treatments associated with enterectomy. Serum urea and creatinine levels, and the activity of hepatic enzymes were normalized after treatment with HS and enterectomy. A significant increase in survival rate (86%, 15 days) and in the body weight gain (p>0.05 vs Sham) were observed after treatment of the animals with HS and enterectomy. CONCLUSIONS: Treatment of the animals with HS solution followed by enterectomy significantly increased the survival rate. HS solution reduced the magnitude of the local inflammatory response (leukocyte-endothelial interactions, and adhesion molecules expression in the mesenteric microcirculation, and the intestinal lesion) and the systemic inflammatory response (CINC-1 and CINC-2 serum levels, renal and hepatic dysfunctions)

De todas as influências exógenas que possam alterar a microbiota intestinal, os antimicrobianos são capazes de causar as mais rápidas e drásticas mudanças. O impacto da exposição aos antimicrobianos na microbiota intestinal causa diminuição no número de microrganismos ou mesmo supressão, dependendo do antimicrobiano utilizado, da dose e do tempo de exposição. Assim, o objetivo deste estudo foi analisar de forma comparativa alguns microrganismos que compõem a microbiota fecal de crianças com e sem antibioticoterapia em idade escolar; bem como avaliar a susceptibilidade aos antimicrobianos e os genes de resistência envolvidos. Foram coletadas amostras fecais não diarreicas de 30 crianças sem antibiótico (controle) e 31 de crianças com antibioticoterapia. Na análise quantitativa foi observada redução no número de cópias por g/fezes de: Bifidobacterium spp., B. fragilis, C. perfringens, E. coli, M. smithii e do filo Firmicutes nas amostras das crianças com antibióticos em relação ao grupo controle, exceto para Lactobacillus spp. e P. distasonis que apresentaram quantificação maior no grupo antibióticos quando comparados com o controle. E. coli foi isolada em 26 (86,7%) crianças controles e em 23 (74,2%) tratadas com antibióticos. A resistência foi verificada para diversas drogas no grupo controle exceto para ciprofloxacina, meropenem e tigeciclina; entretanto o grupo com antibioticoterapia apresentou elevada resistência para todas as drogas avaliadas, caracterizando os isolados desse estudo como MDR. Todos os isolados do grupo controle e antibióticos albergaram diversos genes de resistência, entretanto o gene blaKPC foi o único não detectado nos isolados do grupo controle. Desta forma, nossos dados demonstram que a antibioticoteria causa alterações qualitativas e quantitativas na microbiota intestinal; além disso, a elevada resistência as diversas classes de antimicrobianos das cepas de E. coli, bem como a presença de diversos genes de resistência ressalta a importância de cepas comensais serem MDR e albergarem esses genes. / Of all the exogenous influences that may alter the intestinal microbiota, antimicrobial agents are able to cause the more rapid and dramatic changes. The impact of exposure to antimicrobial agents on intestinal microbiota causes a decrease in the number of certain genera and species, depending on the antimicrobial agent used, dose and duration of exposure. Thus, the aim of this study was to analyze comparatively some microorganisms that composing the fecal microbiota of children with and without antibiotic therapy in school age; and evaluates the antimicrobial susceptibility and resistance genes involved. Stool samples (not diarrhea) were collected of 30 children without antibiotic (control) and 31 children with antibiotic therapy. In quantitative analysis was observed decrease in the number of copies per g/feces: Bifidobacterium spp., B. fragilis, C. perfringens, E. coli, M. smithii and the phylum Firmicutes in samples of children with antibiotic therapy in relation to control group, except Lactobacillus spp. and P. distasonis that showed a higher quantification in the antibiotics group when compared with control group. E. coli was isolated in 26 (86.7 %) children controls and in 23 (74.2 %) children treated with antibiotics. The resistance was verified for several drugs in the control group except for ciprofloxacin, meropenem and tigecycline; however the group with antibiotic therapy showed high resistance to all drugs evaluated, characterizing isolates of this study as MDR. All isolates from control group and antibiotics harbored several resistance genes, however blaKPC gene was the only one not detected in isolates from the control group. Thus, our data demonstrate that the antibiotic therapy cause qualitative or quantitative changes in intestinal microbiota leading to a decrease in the diversity and the elimination of microorganisms; in addition, the high resistance the various classes of antimicrobial of the strains of E. coli, as well as the presence of several genes of resistance highlights the importance of commensal strains are MDR and harboring these genes.