Neonatal necrotizing enterocolitis (NEC) is the most common gastrointestinal disease in premature babies. Despite significant morbidity and mortality, the cause of this disease remains unclear and there are no preventative treatments available. Prematurity and enteral feeding of infant formula are considered to be the primary risk factors for development of NEC. Interestingly, the incidence of NEC is six to ten times lower in breast-fed babies compared to those that were formula-fed. The factors responsible for the protective effect of breast milk against NEC have not been identified, but epidermal growth factor (EGF) is one of the most promising candidates. EGF is found at high concentrations in human milk, but is not present in any commercial formula. Mothers with extremely premature babies have 50-80% higher levels of EGF in their breast milk compared to mothers with full term infants. This suggests that EGF plays an important role in the development of premature infants. Our studies have shown that supplementation of EGF into formula significantly reduces the incidence of NEC in a neonatal rat model. However, the mechanisms underlying this EGF-mediated reduction of NEC are not understood. The overall hypothesis of this dissertation is that the protective effect of EGF in NEC pathogenesis is mediated via increased expression of pro-survival genes and strengthening of the mucosal barrier. The results of the studies within this dissertation demonstrate that treatment with EGF significantly decreases intestinal epithelial cell apoptosis at the site of NEC injury by up-regulating anti-apoptotic genes and down-regulating pro-apoptotic genes. Furthermore, supplementation of formula with EGF strengthens the mucosal barrier by inducing accelerated maturation of ileal goblet cells and mucin-2 production. In addition, EGF treatment normalizes expression of crucial tight junction proteins in the ileum. Consequently, EGF treatment results in a significant decrease in intestinal paracellular permeability and improved barrier function. Results from these studies will provide significant contributions to the understanding of EGF-mediated reduction of NEC, which may lead to development of therapeutic strategies for the treatment of human NEC.

No description available.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A deficiÃncia de vitamina A (DVA) à considerada um importante problema de saÃde pÃblica em todo o mundo e o papel da DVA na integridade epitelial intestinal ainda nÃo està bem compreendido. O objetivo do trabalho foi investigar a associaÃÃo do retinol (vitamina A) e carotenÃides com a barreira funcional intestinal em crianÃas de uma comunidade urbana em Fortaleza, no Nordeste do Brasil. Participaram do estudo 102 crianÃas de 2 meses a 9 anos de idade. O estado nutricional das crianÃas foi avaliado atravÃs do escore âzâ de peso por altura (WHZ), mostrando que 19,6% (20/102) das crianÃas apresentaram desnutriÃÃo leve (âzâ escore de -1 a -2). Todas as crianÃas tiveram as concentraÃÃes de retinol determinadas e nenhuma estava severamente deficiente (< 0,35 ÂM); 2,9% (3/102) das crianÃas apresentaram nÃveis moderados de deficiÃncia de vitamina A (0,36 - 0,70 ÂM); 20,6% (21/102) apresentaram leve deficiÃncia (0,71 - 1,05 ÂM) e 76,5% (78/102) apresentaram concentraÃÃes suficientes de retinol no sangue (> 1,05 ÂM). A razÃo de lactulose/manitol (L/M) estava anormal em 49% (47/97) das crianÃas quando comparadas com crianÃas saudÃveis de uma mesma regiÃo geogrÃfica. Os carotenÃides, luteÃna, &#946;-criptoxantina e beta-caroteno no soro das crianÃas do Parque UniversitÃrio, apresentaram correlaÃÃes inversas significativas com a razÃo de L/M. As proteÃnas de fase aguda (proteÃna C reativa, CRP e glicoproteÃna &#945;1-Ãcida, AGP) apresentaram correlaÃÃes inversas com retinol. O retinol correlacionou-se significativamente com a proteÃna ligadora de retinol (RBP) e com a transtiretina (TTR). Esses dados sugerem que a alteraÃÃo na barreira funcional intestinal provavelmente ocorreu atravÃs do caminho paracelular com baixas concentraÃÃes de carotenÃides no soro. Os carotenÃides, precursores do retinol, poderiam assim promover um melhor marcador para a barreira funcional intestinal alterada do que as medidas de RBP ou retinol. As concentraÃÃes sanguÃneas de retinol correlacionaram-se com a RBP e TTR e correlacionaram-se inversamente com a AGP e com a CPR / Vitamin A deficiency (VAD) is considered an important public health problem worldwide and the role of VAD on intestinal epithelial integrity is not well understood. To investigate the association of retinol (vitamin A) and carotenoids with the intestinal barrier function from children in an urban community in Fortaleza, Northeastern Brazil, one hundred and two children from 2 months to 9 years old participated in the study. The nutritional status of these children, measured by weight for height z-score (WHZ), showed that 19.6% (20/102) of them had mild malnutrition (-1 to â2 z-score). All children had their serum retinol concentration measured and none were severely deficient (<= 0.35 ÂM), 2.9% (3/102) were moderately (0.36 â 0.70 ÂM), 20.6% (21/102) were mildly (0.71 â 1.05 ÂM) deficiencies; 76.5% (78/102) were vitamin A sufficient (> 1.05 ÂM). The lactulose:mannitol (L/M) ratio was abnormal in 49% (47/97) of children when compared to healthy children in the same geographic area. Serum carotenoids, lutein, beta-cryptoxanthin and beta-carotene showed significant inverse correlation with the L/M ratio. Acute phase proteins (C-reactive protein; CRP; and &#945;-acid glycoprotein; AGP) were significantly inversely correlated with retinol. Retinol was significantly correlated with retinol-binding protein (RBP), and with transthyretin (TTR). These data suggest a disruption of intestinal barrier function due to paracellular pathway with low serum concentrations of carotenoids. The retinol precursors, carotenoids, may provide a better marker for disrupted intestinal barrier function than measurements of RBP or retinol. Serum retinol concentrations correlate with RBP and TTR and inversely correlate with AGP and CRP

Les maladies inflammatoires chroniques de l’intestin (MICI) représentent un problème de santé publique majeur touchant de jeunes patients. Aux récidives inflammatoires, à la mauvaise qualité de vie et à l’espérance de vie réduite des patients viennent s’ajouter la durée, l’efficacité parfois limitée et le coût des traitements actuellement proposés. La recherche de nouvelles stratégies permettant de prévenir les récidives inflammatoires est primordiale. Ainsi, le rôle du S-nitrosoglutathion (GSNO, donneur et forme de stockage naturelle de NO) dans le maintien de l’intégrité de la barrière intestinale est étudié dans cette thèse, en décrivant : i) l’effet concentration-dépendante du GSNO sur la perméabilité intestinale (modèle de chambre de Ussing, expression des protéines de jonctions cellulaires…), ii) des formulations innovantes de GSNO (nanoparticules composites) à base de nanoparticules d’Eudragit®RL, elles-mêmes encapsulées dans une matrice polymérique à base d’alginate. Différents procédés de formulation ont été testés. Une caractérisation physicochimique des objets développés et des études de perméabilité sur des modèles cellulaires (Caco-2) ont conduit à l’obtention de trois formulations, adaptées à la voie orale et permettant de délivrer de façon prolongée le GSNO. Cependant, la prise en charge du GSNO libre ou formulé, l’identification des cibles du NO au niveau intestinal, mais aussi les doses et durées du traitement in vivo restent encore à définir avant de proposer ce donneur de NO comme candidat pour un traitement préventif des MICI / Inflammatory bowel diseases (IBD) (Crohn’s disease, ulcerative colitis…), are disabling pathologies affecting young patients and presenting the particularity that most of the current agents act by down regulating chronic inflammation in the intestine mucosa and cannot cure the disease. As the pivotal role of S-nitrosoglutathione (GSNO) in preventing intestinal inflammation and gut barrier failure has been clearly pointed out, therapies based on pharmaceutical technology to limit chronic inflammation and prevent relapses of barrier failure by an optimized dosage form of GSNO appears as an interesting challenge. The objective of this project relies on i) studying GSNO concentration/response functions in intestine by using Ussing chamber ex vivo model and following protein cell junction expression, ii) developing polymer nanocomposite particles encapsulating GSNO and adapted to the oral route, with GSNO protection, controlled delivery and local effect in intestine as major requirements. GSNO loaded polymeric (Eudragit® RL) nanoparticles were included in polymer matrix based on alginate; different processes were tested and particles were characterized: high GSNO loading, GSNO sustained release and local retention for 4 h in cellulo study (Caco-2) were obtained. Thus, therapies based on GSNO administration should represent a novel strategy to limit chronic inflammation and prevent relapses of barrier failure in IBD patients

FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / IntroduÃÃo: Apesar do reconhecimento das alteraÃÃes intestinais associadas à desnutriÃÃo e dos efeitos trÃficos da alanil-glutamina (AG) na funÃÃo de barreira intestinal, medida pela taxa de excreÃÃo urinÃria de Lactulose: Manitol, ainda sÃo escassos os estudos para determinar o efeito de suplementaÃÃo oral da AG em crianÃas desnutridas. Objetivos: Examinar o efeito de suplementaÃÃo oral de AG ou placebo glicina (G) na funÃÃo de barreira intestinal e crescimento em crianÃas sob risco nutricional residentes na comunidade do Parque UniversitÃrio do Pici. MÃtodos: Ensaio clÃnico randomizado controlado em crianÃas maiores de 6 meses e menores de 8 anos de idade, com pelo menos um dos escores z para os indicadores antropomÃtricos (IAs) (peso-para-idade, estatura-para-idade e peso-para-estatura) < -1. Cento e sete crianÃas foram randomizadas, entre julho de 2003 a novembro de 2004, para receberem AG (24g/dia) ou G (25g/dia) em quantidades isonitrogÃnicas por 10 dias. A excreÃÃo urinÃria de Lactulose:Manitol foi utilizada como medida da permeabilidade intestinal e realizada nos 1 e 10 dias do protocolo de estudo. O peso e estatura das crianÃas foram coletados nos 1Â, 10Â, 30 e 120 dias do protocolo de estudo para cÃlculo dos IAs. Resultados: O percentual da excreÃÃo urinÃria de lactulose diminuiu significantemente no grupo que recebeu AG, mas nÃo no grupo que recebeu G ( p < 0,05 teste t de Student). A melhora cumulativa nos indicadores antropomÃtricos, peso-para-estatura e peso-para-idade, no 120 dia do protocolo de estudo foi significante quando realizado a anÃlise de covariÃncia no grupo que recebeu AG versus o grupo que recebeu glicina (respectivamente, p <0,04 e <0,029). Durante o perÃodo de estudo foram observados um total de vinte (19%) Eventos Adversos (EAs) na seguinte ordem de freqÃÃncia: vÃmitos (8,4%), diarrÃia (2,8%), nÃuseas (1,9%), infecÃÃo respiratÃria (1,9%), escabiose (1,9%), asma (0,9%), epistaxe (0,9%). Os EAs nÃo foram diferentes na anÃlise estatÃstica entre os grupos estudados e somente trÃs dos EAs foram considerados como SÃrios Eventos Adversos (SEAs ), dois no grupo da alanil-glutamina (1 asma e 1 diarrÃia) e um no grupo da glicina (1 diarrÃia). ConclusÃes: A suplementaÃÃo oral de AG por 10 dias melhorou a permeabilidade intestinal e o estado nutricional em crianÃas sob risco nutricional residentes em uma comunidade de Fortaleza / Introduction: In spite of the recognition of alterations in intestinal barrier function associated with malnutrition and the trophic effects of alanyl-glutamine (AG) on intestinal barrier function, measured by the rate of urinary excretion of Lactulose:Manitol, there are still few studies determining the effect of oral supplementation of AG in malnourished children. Objectives: Examine the effect of oral AG supplementation or the placebo Glycine (G) on intestinal barrier function and growth in children who are at risk for malnutrition and are residents of the University Park of Pici community. Methods: Double blind randomized study in children between 6 months and 8 years of age, with at least one of the z scores for the anthropometrical indicators (AIs) (weight-for-age, height-for-age, and weight-for-height) less than minus one. One hundred and seven children were randomized between July 2003 and November 2004, to receive AG (24 g/day) or G (25 g/day) in iso-nitrogenic quantities for 10 days. The urinary excretion of Lactulose:Mannitol was used as a measure of intestinal permeability, and was performed on the first and tenth days of the study protocol. The weight and height of the children were collected on the 1, 10, 30, and 120 days of the study protocol to calculate the AIs. Results: The percentage of urinary excretion of Lactulose decreased significantly in the group that received AG (p < 0.05 Student T test), but not in the group that received G. The cumulative improvement in the Anthropometrical Indicators, weight-for-height and weight-for-age on the 120 day of the study protocol were significant when covariant analysis was performed in the group which received AG versus the control group G (p < 0.04 and < 0.029, respectively). During the study period a total of 20 (19%) Adverse Events (AEs) were observed in the following order of frequency: vomiting (8.4%), diarrhea (2.8%), nausea (1.9%), respiratory infection (1.9%), scabies (1.9%), asthma (0.9%), epistaxis (0.9%). The AEs were not statistical different between the study groups, and only three of the AEs were considered Serious Adverse Events (SEAs), two in the AG group (1 asthma and 1 diarrhea) and one in the G group (1 diarrhea). Conclusions: The oral supplementation of AG for 10 days improved intestinal permeability and nutritional status in children at risk for malnutrition in a Fortaleza community

Les phycotoxines lipophiles sont des métabolites secondaires produits par certaines espèces phytoplanctoniques. Elles s'accumulent dans les mollusques filtreurs et peuvent provoquer une intoxication chez l'homme avec une grande variété de symptômes. Ce travail s’est attaché à mieux connaitre leur devenir chez l’homme et leurs effets après ingestion en apportant des données sur leur absorption intestinale et leur métabolisme hépatique puisque ces phénomènes affectent la quantité de toxine circulant dans l’organisme et donc la génération d’effets toxiques. En outre, plusieurs phycotoxines se retrouvent parfois simultanément dans les coquillages alors que les effets de ces mélanges sont encore méconnus. Quatre phycotoxines lipophiles, l'acide okadaïque (AO), la pecténotoxine-2 (PTX-2), la yessotoxine (YTX) et le spirolide (SPX-1), ont été sélectionnées. Le passage intestinal a été évalué à l’aide de cellules intestinales humaines Caco-2 mimant l’épithélium intestinal. Si des différences d’absorption ont été observées pour les 4 phycotoxines, nos résultats ont montré que l’épithélium intestinal était également capable de les renvoyer dans la lumière intestinale, limitant ainsi la quantité circulant dans l’organisme. De même, à l’aide d’extraits de foie, nous avons montré que la structure des 4 phycotoxines était modifiée, principalement par des réactions d'hydroxylation. Concernant les effets mélanges, l'ajout d'une autre toxine (PTX-2, YTX ou SPX-1) à l'AO entraîne des effets moins importants avec de faibles concentrations, et des effets additifs ou plus importants avec des concentrations plus élevées. Ces résultats apportent des données complémentaires pouvant servir à confirmer ou réviser les seuils réglementaires établis pour ces toxines. / Lipophilic phycotoxins are secondary metabolites produced by some phytoplankton species. They accumulate in filter-feeding molluscs and can cause intoxication in humans with a wide variety of symptoms. This work aimed at bringing better knowledge on their fate in humans and their effects after ingestion by providing data on their intestinal absorption and their hepatic metabolism since these phenomena affect the amount of toxin circulating in the body and therefore the generation of toxic effects. In addition, several phycotoxins are sometimes found simultaneously in shellfish while the effects of these mixtures are still unknown. Four lipophilic phycotoxins, okadaic acid (OA), pectenotoxin-2 (PTX-2), yessotoxin (YTX) and spirolide (SPX-1) were selected. The intestinal passage was evaluated using human intestinal Caco-2 cells mimicking the intestinal epithelium. If differences in absorption were observed for the 4 phycotoxins, our results showed that the intestinal epithelium was also able to send them back into the intestinal lumen, thus limiting the amount circulating in the body. Similarly, using liver extracts, we showed that the structure of the 4 phycotoxins was modified, mainly by hydroxylation reactions. For mixtures effects, the addition of another toxin (PTX-2, YTX or SPX-1) to OA results in lower effects at low concentrations, and additive or larger effects at higher concentrations. These results provide additional data that can be used to confirm or revise regulatory thresholds established for these toxins.

<p>Abstract Redacted.</p> / Master of Science (MSc)

The purpose of lactation is for nutrient provision and also importantly for protection from various environmental stressors. Milk polar lipids reduce cholesterol, protect against bacterial infection, reduce inflammation and help maintain gut integrity. Dynamic interactions within dietary fat, lipid metabolism, gut permeability and inflammatory cytokines remain unclear in the context of obesity and systemic inflammation. A rat model and three mouse models were developed to test the hypotheses that dietary milk polar lipids may affect lipid metabolism and intestinal integrity and may protect against systemic inflammation in the context of stressful diet, systemic inflammation, and obesity. The milk polar lipids isolates had complex effects on lipid metabolism and associated gene expression in the rat model. There were complex dynamics in lipid metabolism, gut permeability and systemic inflammation at different time points in all mouse models. The milk phospholipids increased gut permeability in genetic and diet-induced obesity and during the lipopolysaccharide (LPS) -induced inflammation. The phospholipids increased the plasma LPS level in genetic obesity and during the LPS stress. The phospholipids reduced liver mass and liver lipids in genetic obesity and during the LPS-induced inflammation. The phospholipids increased the body fat in the diet-induced obesity model. The milk gangliosides did not significantly affect gut permeability, systemic inflammation, and lipid metabolism in all three mouse models. Current estimate by the Centers for Disease Control is that about 1/3 Americans are obese (body mass index, BMI ≥ 30) and 1/3 Americans are overweight (25 ≤ BMI < 30). More than 25% of Americans today have a fatty liver which could lead to further health problems. The data from this dissertation shed light on the complicated interrelationships between gut permeability, systemic inflammation, and lipid metabolism in obesity. The results contribute to our understanding of the bioactivities of milk polar lipids and provide scientific evidence for the role of milk polar lipids rich materials in affecting biological functions. The study of the influence of milk polar lipids on gut barrier integrity adds new information on understanding the mechanisms of gut leakiness and recovery. The investigation of the impact of milk polar lipids on lipid metabolism reveals new perspectives for the development of diet-induced obesity.

The purpose of lactation is for nutrient provision and also importantly for protection from various environmental stressors. Milk polar lipids reduce cholesterol, protect against bacterial infection, reduce inflammation and help maintain gut integrity. Dynamic interactions within dietary fat, lipid metabolism, gut permeability and inflammatory cytokines remain unclear in the context of obesity and systemic inflammation. A rat model and three mouse models were developed to test the hypotheses that dietary milk polar lipids may affect lipid metabolism and intestinal integrity and may protect against systemic inflammation in the context of stressful diet, systemic inflammation, and obesity. The milk polar lipids isolates had complex effects on lipid metabolism and associated gene expression in the rat model. There were complex dynamics in lipid metabolism, gut permeability and systemic inflammation at different time points in all mouse models. The milk phospholipids increased gut permeability in genetic and diet-induced obesity and during the lipopolysaccharide (LPS) -induced inflammation. The phospholipids increased the plasma LPS level in genetic obesity and during the LPS stress. The phospholipids reduced liver mass and liver lipids in genetic obesity and during the LPS-induced inflammation. The phospholipids increased the body fat in the diet-induced obesity model. The milk gangliosides did not significantly affect gut permeability, systemic inflammation, and lipid metabolism in all three mouse models. Current estimate by the Centers for Disease Control is that about 1/3 Americans are obese (body mass index, BMI ≥ 30) and 1/3 Americans are overweight (25 ≤ BMI < 30). More than 25% of Americans today have a fatty liver which could lead to further health problems. The data from this dissertation shed light on the complicated interrelationships between gut permeability, systemic inflammation, and lipid metabolism in obesity. The results contribute to our understanding of the bioactivities of milk polar lipids and provide scientific evidence for the role of milk polar lipids rich materials in affecting biological functions. The study of the influence of milk polar lipids on gut barrier integrity adds new information on understanding the mechanisms of gut leakiness and recovery. The investigation of the impact of milk polar lipids on lipid metabolism reveals new perspectives for the development of diet-induced obesity.

Οι ασθενείς µε αποφρακτικό ίκτερο, ιδιαίτερα όταν εκτίθενται στο επιπρόσθετο stress ενός επεµβατικού διαγνωστικού ή θεραπευτικού χειρισµού, είναι επιρρεπείς στην ανάπτυξη σηπτικών επιπλοκών και νεφρικής δυσλειτουργίας που οδηγούν σε υψηλά ποσοστά νοσηρότητας και θνητότητας. Πειραµατικές και κλινικές µελέτες έχουν δείξει ότι ο αποφρακτικός ίκτερος προκαλεί δυσλειτουργία του βλεννογόνιου εντερικού φραγµού, οδηγώντας σε ενδοτοξιναιµία, η οποία φαίνεται να διαδραµατίζει κεντρικό ρόλο στην ανάπτυξη των επιπλοκών αυτών. Η απουσία χολής µεταβάλλει τη λειτουργία του εντερικού φραγµού χωρίς να διασπά τη συνέχεια του επιθηλίου. Οι µοριακοί µηχανισµοί που ενέχονται στο φαινόµενο αυτό δεν έχουν αποσαφηνιστεί. Η παρούσα διδακτορική διατριβή επιχειρεί να διερευνήσει την επίδραση του πειραµατικού αποφρακτικού ικτέρου στην έκφραση της αποφραξίνης, δοµικού συστατικού των αποφρακτικών ενώσεων, στον εντερικό βλεννογόνο, στην απόπτωση και στον πολλαπλασιασµό των επιθηλιακών κυττάρων στις κρύπτες και στα επίπεδα οξειδωτικού stress στο έντερο. Επιπλέον, σε µια προσπάθεια θεραπευτικής παρέµβασης, διερευνήθηκε ο πιθανός ρόλος των ρυθµιστικών εντερικών πεπτιδίων bombesin (BBS) και neurotensin (NT) στις ανωτέρω παραµέτρους, καθώς και στην ιστολογία και στα επίπεδα οξειδωτικού stress στο ήπαρ. Προηγούµενες µελέτες έχουν δείξει ότι τα πεπτίδια αυτά εξασκούν ένα ευρύ φάσµα δράσεων στον εντερο-ηπατικό άξονα και βελτιώνουν την ακεραιότητα του γαστρεντερικού βλεννογόνου έπειτα από την επίδραση διαφόρων βλαπτικών παραγόντων. Η απουσία χολής ενδοαυλικά, αποστερεί τον εντερικό βλεννογόνο από τις βακτηριοστατικές, αντι-ενδοτοξινικές και τροφικές της ιδιότητες, οδηγώντας σε αύξηση των βακτηριδίων και της ενδοτοξίνης ενδοαυλικά και σε εντερική ατροφία. Οι µεταβολές αυτές προάγουν τη µετακίνηση βακτηρίων και ενδοτοξινών στην πυλαία φλέβα και ακολούθως, µέσω µιας κατασταλµένης εκκαθαριστικής ικανότητας των κυττάρων Kupffer εξαιτίας της χολόστασης, στη συστηµατική κυκλοφορία. Η συστηµατική ενδοτοξιναιµία ενεργοποιεί τη συστηµατική φλεγµονώδη απάντηση, η οποία σχετίζεται µε τη δυσλειτουργία που αναπτύσσεται σε αποµακρυσµένα όργανα, ενώ συνεισφέρει και στην περαιτέρω επιδείνωση της λειτουργίας του εντερικού φραγµού και της ηπατικής βλάβης. Τα αποτελέσµατα της παρούσας µελέτης επιβεβαίωσαν την παρουσία πυλαίας και συστηµατικής ενδοτοξιναιµίας σε εξωηπατική απόφραξη των χοληφόρων. Η προκαλούµενη από τον αποφρακτικό ίκτερο ατροφία του εντερικού βλεννογόνου τεκµηριώθηκε µε µορφοµετρική ανάλυση και µε µετρήσεις του DNA και της πρωτεΐνης. Ένας πιθανός µηχανισµός προαγωγής της ατροφίας του εντερικού βλεννογόνου είναι η διαταραχή της ισορροπίας µεταξύ κυτταρικού πολλαπλασιασµού και κυτταρικού θανάτου στις κρύπτες, µε αύξηση της απόπτωσης και µείωση της µιτωτικής δραστηριότητας. Επίσης, ο αποφρακτικός ίκτερος οδήγησε σε αύξηση του οξειδωτικού stress στο έντερο, όπως τεκµηριώνεται από την αύξηση της υπεροξείδωσης των λιπιδίων, της οξείδωσης των πρωτεϊνών, της οξειδωµένης γλουταθειόνης (GSSG), των ολικών µη πρωτεϊνικών µεικτών δισουλφιδίων (NPSSR), και των πρωτεϊνικών δισουλφιδίων (PSSP), ενώ µειώθηκε το αντιοξειδωτικό µόριο της ανηγµένης γλουταθειόνης (GSH). Η ενδοτοξιναιµία και οι αυξηµένες συγκεντρώσεις χολικών αλάτων αποτελούν σηµαντικούς διεγέρτες της παραγωγής δραστικών µεταβολιτών οξυγόνου. Η παρουσία οξειδωτικού stress στο έντερο συνεισφέρει στην προαγωγή της αποπτωτικής διεργασίας και στην αναστολή του κυτταρικού πολλαπλασιασµού στις κρύπτες, οδηγώντας σε ατροφία του βλεννογόνου. Ένα άλλο πρωτότυπο εύρηµα αυτής της µελέτης είναι η διαταραχή του παρακυττάριου φραγµού του εντέρου στα πειραµατόζωα µε αποφρακτικό ίκτερο, η οποία τεκµηριώνεται µε την απώλεια της έκφρασης της αποφραξίνης περίπου στο 50% των επιθηλιακών κυττάρων στο κορυφαίο τµήµα της λάχνης. Συνεπώς, το άνοιγµα της παρακυττάριας οδού φαίνεται να είναι ένας σηµαντικός παράγων στη διαφυγή ενδοτοξίνης από τον εντερικό αυλό στην πυλαία κυκλοφορία. Η ενδοτοξιναιµία, η απελευθέρωση στη συστηµατική κυκλοφορία µεσολαβητών φλεγµονής και η παρουσία οξειδωτικού stress στο έντερο πιθανώς σχετίζονται µε τη µεταβολή της έκφρασης της αποφραξίνης στον εντερικό βλεννογόνο. Επιπλέον, στον αποφρακτικό ίκτερο ανεβρέθηκε µια διαβάθµιση της έκφρασης της αποφραξίνης κατά µήκος της λάχνης, µε µεγαλύτερη απώλεια της έκφρασής της στο άνω τριτηµόριο, µικρότερη στο µέσο και ακόµα µικρότερη στην κρύπτη. Μια πιθανή εξήγηση αυτής της διαβάθµισης της απώλειας της έκφρασης της αποφραξίνης είναι ότι, δεδοµένου ότι η ανανέωση του επιθηλίου γίνεται από την κρύπτη προς την κορυφή, τα κύτταρα της κορυφής της λάχνης έχουν εκτεθεί για περισσότερο χρονικό διάστηµα στις συνθήκες οξειδωτικού stress που επικρατούν στο έντερο. Τα ρυθµιστικά εντερικά πεπτίδια, BBS και ΝΤ, δρώντας είτε άµεσα, µέσω ειδικών υποδοχέων των επιθηλιακών κυττάρων του εντέρου, είτε έµµεσα, βελτιώνοντας τη µικροκυκλοφορία του εντέρου, αποκατέστησαν την έκφραση της αποφραξίνης στον εντερικό βλεννογόνο, µείωσαν σηµαντικά την απόπτωση και το οξειδωτικό stress και ανέστρεψαν την εντερική ατροφία. Η πρόληψη, από τα ρυθµιστικά πεπτίδια, των επαγόµενων από τον αποφρακτικό ίκτερο κυτταρικών και βιοχηµικών µεταβολών του εντερικού βλεννογόνου, οδήγησε σε σηµαντική µείωση της πυλαίας και συστηµατικής ενδοτοξιναιµίας. Επιπλέον, η BBS και η ΝΤ, εξασκώντας αντιοξειδωτική δράση και στο ήπαρ, προστατεύουν από δυο µείζονες παράγοντες ηπατικής βλάβης κατά τη χολόσταση, που είναι το οξειδωτικό stress και η ενδοτοξιναιµία, οδηγώντας σε βελτίωση των ιστολογικών αλλοιώσεων της αποφρακτικής χολαγγειοπάθειας. Συµπερασµατικά, τα αποτελέσµατα της παρούσας µελέτης δείχνουν ότι η δυσλειτουργία του εντερικού φραγµού στον αποφρακτικό ίκτερο σχετίζεται µε την επαγωγή κυτταρικών και βιοχηµικών µεταβολών στον εντερικό βλεννογόνο, οι οποίες χαρακτηρίζονται από κατά τόπους απώλεια της έκφρασης της αποφραξίνης, πρόκληση οξειδωτικού stress και επαγωγή της απόπτωσης. Τα ρυθµιστικά εντερικά πεπτίδια BBS και ΝΤ προλαµβάνοντας τις µεταβολές αυτές του εντερικού βλεννογόνου µειώνουν σηµαντικά την πυλαία και συστηµατική ενδοτοξιναιµία. Επίσης, εξασκούν προστατευτική δράση εναντίον του οξειδωτικού stress στο ήπαρ και διαφυλάσσουν την αρχιτεκτονική του πυλαίου διαστήµατος. Η συνδυασµένη ευεργετική επίδραση των ρυθµιστικών πεπτιδίων, τόσο στη δυσλειτουργία του εντερικού φραγµού και την ενδοτοξιναιµία, που ευθύνονται για την ανάπτυξη σηπτικών επιπλοκών και βλάβης αποµακρυσµένων οργάνων, όσο και στο οξειδωτικό stress και την ιστολογία του ήπατος, εισηγούνται µια νέα θεραπευτική προσέγγιση στον αποφρακτικό ίκτερο. Όπωσδήποτε απαιτούνται περαιτέρω µελέτες για τη διευκρίνιση των µηχανισµών δράσης και πιθανών παρενεργειών της BBS και της ΝΤ πριν την εφαρµογή τους στην κλινική πράξη. / Patients with obstructive jaundice, especially when exposed to the additional stress of an invasive diagnostic or therapeutic procedure, are prone to septic complications and renal dysfunction contributing to high morbidity and mortality rates. Experimental and clinical studies have shown that obstructive jaundice compromises intestinal barrier function resulting in endotoxemia, which appears to play a key role in the development of these complications. Lack of bile alters intestinal epithelial barrier function without disrupting epithelial continuity. The molecular mechanisms implicated in this phenomenon are poorly understood. This study was undertaken to investigate the influence of experimental obstructive jaundice on the expression of the key tight junction-associated protein occludin in the intestinal epithelium, epithelial cell apoptosis and proliferation in crypts and intestinal oxidative stress. In addition, in an attempt for therapeutic intervention in obstructive jaundice, we have explored the potential positive effect of gut regulatory peptides bombesin (BBS) and neurotensin (NT) on the above-described parameters and on liver histology and oxidative stress. These factors exert a wide spectrum of actions on the gut-liver axis and they improve gastrointestinal mucosa integrity after various injurious insults. The results of our study showed that experimental obstructive jaundice results in portal and aortic endotoxaemia. In jaundiced rats, there was total loss of occludin expression in numerous enterocytes and this effect was most profound at the upper third of the villi, while a gradient of positivity existed from crypt to tip. Intestinal cell apoptosis in crypts was significantly increased, while mitotic activity was reduced. This imbalance of cell proliferation and death in crypts was accompanied by induction of mucosal atrophy, as evidenced by morphometrical analysis and decreased DNA and protein content. Moreover, obstructive jaundice induced intestinal oxidative stress demonstrated by increased lipid peroxidation, protein oxidation, GSSG, NPSSR, PSSP and reduction of GSH. Administration of BBS or NT significantly reduced portal and systemic endotoxaemia. These agents restored occludin expression in the intestinal epithelium to the control state, significantly reduced apoptosis and oxidative stress and reversed mucosal atrophy. Moreover, both agents significantly ameliorated liver injury, as demonstrated by improvement of obstructive cholangiopathy, reduction of hepatic oxidative stress and prevention of neutrophilic accumulation in portal tracts. Absence of intraluminal bile deprives the gut from its bacteriostatic, endotoxin-neutralizing and mucosal-trophic effect leading to increased intestinal bacterial and endotoxin load and mucosal atrophy. These alterations promote bacterial and endotoxin translocation into portal circulation and subsequently, through a decreased clearance capacity of Kupffer cells because of cholestasis, into systemic circulation. Systemic endotoxemia activates a systemic inflammatory response, which is associated with dysfunction of remote organs, while it further aggravates intestinal barrier dysfunction and cholestatic liver injury. Endotoxin and bile acids represent important sources of reactive oxygen species formation. We have demonstrated increased intestinal oxidative stress in obstructive jaundice, which possibly contributes to induction of apoptosis and inhibition of cell proliferation in intestinal crypts, leading to mucosal atrophy. Another novel finding of this study is that the intestinal paracellular barrier in jaundiced rats is disrupted, as evidenced by loss of expression of the key tight junction-associated protein occludin in approximately 50% of enterocytes at the upper third of the villi. Therefore, the opened paracellular route may significantly contribute to the escape of endotoxin from the intestinal lumen into portal circulation. Endotoxemia, systemic release of inflammatory mediators and intestinal oxidative stress are possibly associated with decreased occludin expression. A possible explanation for the gradient of occludin expression from crypt to tip is that the cells at the tip of the villi have been exposed for a longer duration to the oxidative intestinal environment of jaundiced rats since epithelial renewal takes place from crypt to tip. Gut regulatory peptides, BBS and NT, acting either directly, through specific receptors located in intestinal epithelial cells, or indirectly, through improvement of intestinal microcirculation, prevent the above-described cellular and biochemical alterations of the intestinal mucosa, leading to lower portal and systemic endotoxin concentrations. Moreover, exerting an antioxidant effect on the liver as well, they prevent two major factors in the promotion of cholestatic liver injury, oxidative stress and endotoxemia, leading to significant amelioration of obstructive cholangiopathy. In conclusion, the results of the present study show that obstructive jaundice-induced gut barrier dysfunction is associated with regional loss of occludin expression in the intestinal epithelium, increased intestinal oxidative stress and induction of apoptosis / inhibition of proliferation in crypts leading to intestinal atrophy. Gut regulatory peptides BBS and NT exerting beneficial effects on these cellular and biochemical alterations of the intestinal mucosa prevent portal and systemic endotoxaemia. Moreover, these factors exert a protective action on portal tract architecture and hepatic oxidative stress. The combined beneficial effects of regulatory peptides on intestinal barrier dysfunction and endotoxemia, which account for septic complications and dysfunction of remote organs, and on liver oxidative status and histology, provide a novel therapeutic approach for obstructive jaundice. However, further investigation to elucidate the details of the functional mechanisms and possible side effects of BBS and NT are needed before any clinical application.