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Home parenteral nutrition (HPN) is an established treatment for the management of patients with severe intestinal failure.  At the moment, it is the treatment of choice for those patients who are unable to eat or drink sufficient food or fluid to maintain nutrition or fluid status. There are no quality of life assessment tools that have been developed and validated specifically for this patient population, previous studies have used generic instruments or techniques not validated in this patient population. A method of objectively assessing the quality of life of patients treated with HPN has been developed – the HPN-QOL questionnaire.  This was designed using qualitative research methods to identify the issues particular to that patient population, thereby eliciting patients’ perspectives on their own quality of life.  The questionnaire was subjected to rigorous psychometric analysis to validate its use in the HPN population. The HPN-QOL has been translated into seven European languages.

Oligopeptide transport in rat small intestine has been studied in intact tissue, using the luminally and vascularly perfused isolated jejunum in situ technique, and a hydrolysis-resistant dipeptide (D-Phe-L-Gln). The data in this thesis can be divided into two main sections: (l) identification of the transporter proteins, and (2) short-term regulation of transport. The basolateral peptide transporter protein has not, to date, been identified. A candidate protein was identified from membrane vesicles by a photo affinity labelling technique using a dipeptide derivative ([4-azido-3,5-3H-D-Phe]-L-Ala), previously shown to be an efficient substrate for the basolateral transporter. The labelled candidate protein was successfully isolated by 2-DE, which revealed an apparent Mr of 112 ± 2 kDa and a pI of approximately 6.5. Initial sequence analysis, tryptic digestion followed by MALDI-TOF analysis and Q-TOF fragmentation of a tryptic peptide, produced a peptide fingerprint and a sequence tag of 9 amino acids, respectively, which, together, did not completely and conclusively match to any known protein sequence contained within databases, therefore suggesting that the 112 kDa protein may be novel. Short-term regulation of peptide transport was also investigated using the vascular perfusion method. An amino acid-sensing pathway was discovered, using L-Leucine as the regulator, involving protein kinase cacades leading to p70S6k activation and subsequent stimulation ofbasolateral membrane peptide transport. A major conclusion arising from the data was the distinction between PepTl and the basolateral transporter, i.e. the sequence data obtained from the candidate protein did not match to the PepTl sequence; in addition there appeared to be distinct mechanisms of regulatory control at the two membranes. Efficient delivery of peptidomimetic drugs when adminsitered by the oral route requires knowledge of short-term regulation of intestinal peptide transport, in addition to the sequence and structure of the basolateral transporter. This thesis provides essential information, which may eventually contribute to the unequivocal identification and sequencing of the intestinal basolateral peptide transporter, ultimately leading to the future development of compounds with high bioavailability.

Dendritic cells (DCs) are regulators of the immune response and are thought to be critical in maintaining tolerance towards the intestinal microbiota. Recent data have identified distinct subsets of DCs with specific functional properties. The objective of this thesis was to further define CD103⁺ and CX3CR1⁺ DCs in the intestine and to determine how DCs and regulatory T (Treg) cell responses are influenced by the microbiota. Using multicolour flow cytometry, we identified two CD103⁺ DC subsets with differential aldehyde dehydgrogenase (ALDH) activity and two populations of CX3CR1⁺ cells. In the mesenteric lymph node CD103⁺ALDH⁺ DCs were highly mature (CD86^hi, MHCII^hi), likely migratory (CCR7⁺) and enhanced Treg cell induction compared with ALDH⁻ DCs. CX3CR1^int cells accumulated during bacterially-induced colitis suggesting a pro-inflammatory role whereas CX3CR1^hi cells were associated with the production of the anti-inflammatory cytokine IL-10 during homeostasis. We also assessed the generation of CD103⁺ DCs from bone marrow progenitors. Although only small proportions of CD103⁺ DCs were detected in culture with FLT3L or GM-CSF alone, the combination of FLT3L and GM-CSF induced CD103⁺ DCs with a phenotype similar to those found in the small intestine. Using this system we showed that TLR ligands and retinoic acid induce ALDH enzyme activity in vitro. In order to assess how DCs and Treg cells respond to changes in the microbiota we employed broad-spectrum antibiotic treatment to deplete endogenous bacteria and also analyzed the impact of colonization with the model organism Helicobacter hepaticus. Interestingly, we did not detect alterations in the proportions of different DC subsets following antibiotic treatment or H. hepaticus infection. However, using a novel FoxP3^huCD2-IL-10^GFP reporter mouse, we found that IL-10 production by Treg cells was ablated following antibiotic treatment and significantly elevated following H. hepaticus infection. Preliminary investigation of the mechanism underlying this effect suggests a role for IL-27. In summary, this thesis provides further detail on the phenotype of intestinal DCs and shows that Treg cell IL-10 production is sensitive to the composition of the microbiota.

Cartas al editor / Revisión por pares / Revisón por pares

Métodos in vitro utilizando epitélio intestinal animal são importantes ferramentas para avaliar a permeabilidade de fármacos, propriedade que é um importante parâmetro de biodiosponibilidade. Considerando que o maior objetivo na indústria farmacêutica é desenvolver novos fármacos com boa biodisponibilidade oral, o projeto teve como objetivo padronizar o modelo de permeação com membrana de intestino de rã (Rana catesbeiana) em células de Franz, comparando seus resultados com ensaios de células Caco-2. Os fármacos modelo utilizados foram os antivirais zidovudina e aciclovir. A quantidade de fármaco permeado foi determinada por método de eletroforese capilar para o método com intestino de rã touro e por HPLC-UV para os ensaios com células Caco-2. O parâmetro de permeação foi o coeficiente de permeabilidade aparente (Papp) dos fármacos para ambos modelos experimentais. Para estabelecimento do protocolo experimental dos estudos de permeabilidade intestinal de rã, foi proposto um projeto fracionado 24-1 com 4 ensaios adicionais usando o software Minitab, e as variáveis foram: secção intestinal, pH da solução de Ringer e temperatura. A análise do planejamento experimental feita pela estimativa dos parâmetros da regressão obtidos através dos resultados do modelo fatorial possibilitou a determinação dos coeficientes da equação matemática que definiu a influência das variáveis sobre o coeficiente de permeabilidade aparente dos fármacos. Os efeitos das variáveis pH e temperatura interpretados conjuntamente apresentaram interferência leve, porém as variáveis fármaco e secção intestinal interpretados juntos tiveram interferência importante, mostrando maior permeação dos fármacos através da secção inicial do intestino da rã. Os resultados de Papp foram: para o metoprolol, pelo método das células Caco-2 foi de 28 x 10-6 cm/s, valor que está de acordo com demais dados de células Caco-2 na literatura e o valor obtido com células de Franz que mais se adequada a estes resultados e demais disponíveis provenientes de outras técnicas na literatura, foi de 28,1 x 10-6 cm/s, em uma das condições do planejamento estatístico utilizando segmento final da membrana epitelial da rã. No caso do aciclovir, o resultado de Papp de 0,48 x 10-6 cm/s também obtido em uma das condições envolvendo segmento intestinal final da rã foi exatamente igual a o valor 0,48 x 10 - 6 cm/s, encontrado com células Caco-2 no presente estudo e estão de acordo com outros valores disponíveis na literatura por Trapani e colaboradores, 2004 e também com células Caco-2. Para zidovudina o valor de Papp obtido em uma das condições utilizando segmento intestinal inicial da rã, de 13 x 10-6 cm/s, foi a que mais se assemelhou ao obtido pela técnica de células Caco-2, 13,6 x 10-6 cm/s, e também está de acordo com demais dados da literatura. / There are lots of different in vitro technics in the literature using animal intestinal epithelium to estimate permeability of drugs, property that is an important parameter of bioavailabity. Considering that the main objective of Pharmaceutical Companies is the development of new drugs with good oral bioavailabity, the aim of this work was to standardize the permeability of antivirals using in vitro/ex vivo method of intestinal epithelium of Rana catesbeiana in Franz cells and compare these results to those obtained from studies using Caco-2 cells. Zidovudine and Acyclovir were selected as model drugs. The amount of drug permeated will be determined by the method of capillary electrophoresis for assays using Rana Catesbeiana and HPLC-UV for studies with Caco-2 cells. The permeation parameter determined was the apparent permeability coefficient (Papp) of drugs for both experimental models. To establish the experimental protocol to studies of intestinal permeability of frog, it was proposed a fractional 24-1 design with 4 additional tests using Minitab software and the variables were: intestinal section, pH of Ringer solution and temperature. The analysis of the experimental design made by the estimate of the regression parameters obtained from the factorial model results allowed the determination of the coefficients of the mathematical equation that defines the influence of the variables on the apparent permeability coefficient of acyclovir and zidovudine. The effects of pH and temperature interpreted jointly presented a slight interference, but the variables drug and intes tinal section interpreted together had major interference, showing greater permeation of drugs through the initial section of the intestine of the frog . The results of Papp were: for metoprolol, with the method of Caco-2 cells was 28 x 10-6 cm/s, a value which is consistent with other data of Caco-2 cells provided in the literature and the condition obtained with Franz cells that are most suitable for these and other results obtained from other techniques available in the literature, was 28.1 x 10-6 cm/s, provided with the final intestinal segment using frog epithelial membrane. In the case of acyclovir, the result of Papp of 0.48 x 10-6 cm/s obtained in one condition with final frog intestinal segment was exactly equal to the value of 0.48 x 10-6 cm/s, found with Caco-2 cells in the present study and are in agreement with other values available in the literature for Trapani and colegues, 2004 and also with Caco-2 cells. The Papp value for zidovudine obtained with the initial gut segment of frog, 13 x 10-6 cm /s was which more resembled that obtained by the technique of Caco-2 cells, 13.6 x 10-6 cm/s and is also consistent with other literature data.

Foram utilizados 160 leitões recém-desmamados, com peso médio inicial de 6,43 ± 0,71 kg, com o objetivo de avaliar os efeitos dos nucleotídeos sobre o desempenho, a frequência de diarreia, a morfometria de órgãos, a histologia do epitélio intestinal e a microbiota intestinal. Foram testados cinco tratamentos em um experimento em blocos casualizados, com oito repetições (blocos) por tratamento e quatro animais por unidade experimental (dois machos castrados e duas fêmeas). Os tratamentos foram: dieta basal à base de milho, farelo de soja, derivados lácteos e plasma com inclusão de 120 ppm de clorohidroxiquinolina (antimicrobiano) e dieta basal contendo 0, 100, 150 e 200 ppm de nucleotídeos. Para a determinação das variáveis de desempenho, os animais foram pesados ao 1º, 14º e 34º dia de experimentação e foram quantificadas as rações fornecidas e desperdiçadas. Para avaliar a frequência de diarreia, foram observadas presença e ausência de diarreia na baia, diariamente, no período da manhã. Ao final do experimento, um animal de cada baia (unidade experimental) foi abatido para avaliação da morfometria de órgãos (estômago vazio, intestino delgado vazio, pâncreas, fígado e baço) e da histologia do epitélio intestinal (altura e largura de vilosidade, profundidade de cripta, relação altura de vilosidade: profundidade de cripta), além da coleta de amostras do conteúdo do duodeno e do jejuno para avaliação da microbiota intestinal dos leitões (mesófilos, Gram positivos totais, Gram negativos totais, Lactobacillus spp., Escherichia coli, Salmonella spp., Staphylococcus aureus e Clostridium perfringens). No período de 1 a 14 dias de experimentação, as variáveis de desempenho não foram influenciadas (P>0,05) pelos tratamentos. Para o período total (1 a 34 dias), houve efeito linear benéfico da inclusão dos nucleotídeos no peso final (P=0,005; P34 = 0,0033X + 23,657, R2 = 0,87) e no ganho diário de peso (P=0,008; GDP = 0,0002X + 0,4955, R2 = 0,83) dos animais. Os leitões alimentados com o antibiótico tiveram menor (P=0,049) frequência de diarreia comparados aos animais alimentados com nucleotídeos no período de 1 a 14 dias de experimentação. Por outro lado, no período de 1 a 34 dias de experimentação, os tratamentos não afetaram (P>0,05) a frequência de diarreia, a morfometria de órgãos, a histologia do epitélio intestinal e a microbiota intestinal. Assim, de maneira geral, a inclusão de até 200 ppm de nucleotídeos em dietas complexas para leitões recém-desmamados melhora o desempenho dos animais, sem afetar a frequência de diarreia, a morfometria de órgãos, a histologia do epitélio intestinal e a microbiota intestinal de leitões na fase de creche. / The purpose of this study was to evaluate the effects of dietary nucleotide levels on performance, occurrence of diarrhea, organ morphometry, intestinal histology and intestinal microbiota of weanling pigs fed complex diets containing corn, soybean meal, milk products, and spray-dried plasma. One hundred and sixty 21d-weaned pigs, averaging 6.43 ± 0.71 kg BW, were used in a randomized complete block design experiment with 5 treatments, 8 replications (blocks) per treatment and 4 animals per experimental unit (pen). The treatments were: basal diet with 120 ppm of chloro-hydroxyquinoline (antimicrobial treatment), and basal diet with 0, 100, 150, and 200 ppm of nucleotides. The ADG, ADFI, G:F and occurrence of diarrhea were calculated during 1 to 14 and 1 to 34 d of experimental period. A day after the end of the experimental period, an animal from each pen was slaughtered to evaluate of organ morphometry (empty stomach, empty small intestine, pancreas, liver and spleen), intestinal histology (villus height, villus width, crypt depth and villus height-to-crypt depth ratio), and intestinal microbiota (mesophiles, Gram-positive bacteria, Gram-negative bacteria, Lactobacillus spp., Escherichia coli, Salmonella spp., Staphylococcus spp., and Clostridium perfringens). During 1-14 d of experimental period, performance was not affected (P>0.05) by treatments. For the total experimental period (1-34 d), beneficial linear effects of dietary levels of nucleotides on final BW (P=0.005; BW = 0.0033X + 23.657, R2 = 0.87) and ADG (P = 0.008; ADG = 0.0002X + 0.4955, R2 = 0.83) were observed, but not (P>0.05) on ADFI and G:F. Pigs fed antimicrobial treatment had lower (P=0.049) occurrence of diarrhea from d 1 to 14 than those fed nucleotide treatments. However, for the total experimental period (1-34 d), treatments did not affect (P>0.05) occurrence of diarrhea, organ morphometry, intestinal histology and intestinal microbiota. Therefore, adding up to 200 ppm of dietary nucleotides to complex diets for weanling pigs showed beneficial effect on growth performance, without affecting organ morphometry, intestinal histology, intestinal microbiota and occurrence of diarrhea of nursery pigs.

O trato gastrointestinal é um sítio de alta exposição antigênica, por isso requer a presença de mecanismos de regulação imunológica mediada por linfócitos T reguladores e T auxiliares produtores de IL-17 (Th17) na mucosa intestinal. Se houvera falha na indução desses mecanismos, pode ocorrer o desequilíbrio das populações de bactérias comensais da microbiota intestinal, denominado de disbiose, geralmente associado à ruptura da barreira intestinal e translocação de bactérias ou LPS para o sangue. Neste sentido, alguns estudos têm evidenciado a importância dos linfócitos Th17 no intestino, já que estas células tem a capacidade de manter a integridade da barreira intestinal e, como conseqüência controlar a colonização e translocação bacteriana. Em adição, em pacientes e animais diabéticos têm sido observada a correlação de altos níveis de LPS circulantes e resistência à insulina. Baseado nessas evidências, nosso objetivo foi avaliar o papel da citocina IL-17 no controle das alterações inflamatórias e metabólicas no modelo de diabetes do tipo 2 (DM2). Para isso, foram utilizados camundongos C57BL/6 selvagens (WT) ou deficientes do receptor da citocina IL-17 (IL-17R-/-) submetidos à dieta controle (DN), composta por 10% de gorduras, 70% de carboidratos e 20% de proteínas ou à dieta hiperlipídica (DH), composta por 60% de gorduras, 20% de carboidratos e 20% de proteínas. Nossos dados demonstraram que a deficiência do receptor de IL-17 protegeu os animais contra a obesidade, mas os mesmos desenvolveram maior hiperglicemia e hiperinsulinemia decorrente da resistência à insulina. Além disso, foi verificada a hiperplasia das ilhotas pancreáticas, anormalidades na arquitetura e intenso infiltrado inflamatório no intestino (íleo) dos animais IL-17R-/- comparados aos WT após DH. Esse fato parece estar correlacionado a um defeito da migração de neutrófilos para a mucosa intestinal, uma vez que foi detectada reduzida expressão gênica da quimiocina CXCL-1 e do receptor CXCR-2 no íleo desses animais. De maneira interessante, as populações de neutrófilos (CD11b+Ly6G+) e de macrófagos anti-inflamatórios (CD11b+CX3CR1+) mostraram-se aumentadas nos linfonodos mesentéricos dos animais IL-17R-/- após DH. Em seguida, foi constatada maior translocação bacteriana no sangue tanto de animais IL-17R-/- submetidos à DN como DH. Entretanto, a análise metagenômica do gene 16S revelou a prevalência de bactérias Bacteroidetes e Proteobacterias, principais representantes de bactérias gram-negativas, somente nas fezes dos animais IL-17R-/- submetidos à DH. Em conjunto, estes dados indicam que o eixo IL-17/IL-17R é importante na manutenção da homeostase intestinal e na regulação das alterações inflamatórias e metabólicas associadas ao DM2 / The gastrointestinal tract is a high antigenic exposure site, so it requires the presence of immune regulation mechanisms mediated by regulatory T lymphocytes and IL-17- producing T helper lymphocytes (Th17) in the intestinal mucosa. If there is a failure in the induction of these mechanisms, may occur the imbalance in the populations of commensal bacteria of the intestinal microbiota, called dysbiosis, generally associated with the break of the intestinal barrier and translocation of bacteria or their products like LPS into the blood. In this regard, some studies have evidenced the importance of Th17 lymphocytes in the intestine, since these cells have the ability to maintain the integrity of the intestinal barrier, and consequently controlling the colonization and bacterial translocation. In addition, in patients and diabetic animals have been observed correlation between high circulating levels of LPS and insulin resistance. Based on this evidence, our objective was to evaluate the role of IL-17 cytokine in the control of inflammatory and metabolic changes in the type 2 diabetes (T2DM). For this reason, were used C57BL/6 wild-type mice (WT) or lacking of IL-17 cytokine receptor (IL-17R-/-) mice undergoing to the control diet (ND) comprising 10% fat, 70% carbohydrate and 20% protein or high fat diet (DH), comprising 60% fat, 20% carbohydrates and 20% protein. These data demonstrate that IL-17 receptor deficiency protected the animals against obesity, but these mice developed hyperglycemia and hyperinsulinemia due to insulin resistance. Furthermore, we verified a hyperplasia of the pancreatic islets, abnormalities in architecture and intense inflammation in the intestine (ileum) of IL-17R-/- animals undergoing DH compared to WT. This appears to be correlated to a defect in the neutrophil migration to the intestinal mucosa, since was detected reduced gene expression of the CXCL-1 chemokine and CXCR-2 receptor in the ileum of these animals. Interestingly, the populations of neutrophils (CD11b+Ly6G+) and antiinflammatory macrophages (CD11b+CX3CR1+) were shown to be increased in the mesenteric lymph nodes of IL-17R-/- animals after DH. Later, it was found more bacterial translocation in blood, both in IL-17R-/- mice with ND or DH. However, the metagenomic analyzes of the 16S gene revealed increased of Proteobacteria and Bacteroidetes phyla, the main representatives of gram-negative bacteria, only in the faeces of IL-17R-/- mice underwent DH. Together, these data indicate that IL-17/IL-17R axis is important in maintaining intestinal homeostasis and the regulation of inflammatory and metabolic alterations associated to T2DM