Contains errata sheet in back pocket. / Bibliography: leaves 167-211. / xviii, 211, [8] leaves, [4] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Explores the hypothesis that growth of the small intestine at weaning is promoted by an activated mucosal immune system in the gut. Tests by observing rats, guinea pigs and human infants. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1995?

It is currently believed that the etiology of inflammatory bowel disease involves an aberrant immune response towards the gastrointestinal microbial flora. In addition, an increase in intestinal paracellular permeability may also be a contributing factor of disease, as it precedes disease in several animal models. However, it remains unclear whether increased intestinal permeability is an epiphenomenon of disease or if it can lead to it. The goal of this thesis is to elucidate this cause-effect relationship. The IL-10-/- mouse is a model of IBD that spontaneously develops colitis after 12 weeks of age. We measured intestinal permeability in this mouse from 4-17 weeks of age and observed that there was a significant increase in small intestinal permeability early in life and before the onset of colitis. When small intestinal permeability was selectively decreased with AT-1001 (a ZOT antagonist peptide) colitis was significantly ameliorated. In contrast, when it was increased with AT-1002 (a ZOT agonist peptide) colitis worsened, indicating that modifications in the paracellular traffic of the small intestine had a significant effect on the severity of colonic disease. In order to study the possible mechanisms by which small intestinal permeability modulated disease in the colon, we measured the effect of increasing small intestinal permeability on the colonic microbial flora of IL-10-/- mice. After AT-1002 treatment from 4-12 weeks of age, there was an evident shift in colonic adherent flora. This effect was not a consequence of inflammation as there was a similar effect in wild type mice. We also studied the effect of increasing small intestinal permeability in the development of oral tolerance to dietary antigens. When wild-type mice were fed OVA under conditions of increased small intestinal permeability there was a significant increase in the proliferation of B cells in the spleen and an increase in OVA-specific humoral response, compared to animals fed OVA alone. Moreover, the production of IL-10 in response to oral OVA was prevented when OVA was given with AT-1002, both in the small intestine and the colon. The studies presented in the doctoral thesis suggest that small intestinal permeability has a critical role in the development of colitis in IL-10-/-mice, and that increasing paracellular traffic in the small intestine may lead to changes in colonic bacterial flora and the abrogation of tolerance to oral antigens, two features of inflammatory bowel disease in humans. / Experimental Medicine

Immunomodulatory effects of lactic acid bacteria vary with strain and may vary with growth phase and medium. The ability of different lactobacilli strains (Lactobacillus helveticus R0052, L. rhamnosus R0011, L. rhamnosus GG) at different growth phases to modulate macrophage and intestinal epithelial cell cytokine production following a pro-inflammatory challenge was examined. Modulation of cytokine production by human macrophage cell lines (U-937) and intestinal epithelial cells (HT-29) induced by Tumor Necrosis Factor α was assayed by ELISA for interleukin-8 (IL-8). Granulocyte-macrophage colony stimulating factor (GM-CSF) production was assayed by ELISA in the HT-29 cell line. Strain-dependent differences were observed in the ability of viable bacteria and spent de Mann-Rogosa- Sharpe (MRS) broths from log versus stationary growth phase in HT-29 and U-937 cells. Overall, variation in the immunomodulatory activity of these lactic acid bacteria and spent broths reflects not only strain variation but potentially also differences in growth phase and substrate. / UOIT

Las paredes celulares de levadura (PCL) de S. cerevisiae vienen siendo utilizadas en avicultura por hace más de una década, algunas de las finalidades de esta práctica son las de mejorar la productividad y salud del ave. Los polisacáridos tipo beta-glucanos y mánanos presentes en la PCL, pueden ejercer efectos en el sistema inmune del pollo y exclusión de patógenos a escala digestiva. No obstante, debido a que la industria de la fermentación genera gran diversidad levaduras y PCL de diferente origen y características, el estudio acerca del mejor conocimiento y definición de estos nuevos aditivos puede ser clave para optimizar su utilización alimentación animal. En la presente tesis, se realizaron 6 experimentos (Exp.) con pollos de engorde machos (Ross 308) con el objetivo de evaluar las respuestas en la productividad y la salud de las aves al proporcionarles dietas elaboradas con maíz o trigo-cebada-centeno (TCC), con diferentes levaduras activas de S. cerevisiae (pecuario, panadería y "killer" vinícola) y sus componentes: 2 distintas paredes celulares de levadura (PCL), extractos, beta-glucanos (BG) y manano-proteínas (MP), en ninguno de los experimentos se emplearon aditivos en el alimento de tipo antibióticos, coccidiostatos o enzimas. En los Exp. 1 y 2, se observo que las características propias de las distintas levaduras de S. cerevisiae y de sus componentes (extractos y PCL), así como el tipo de dieta empleada (TCC o maíz), adquieren importantes implicaciones en las respuestas que pueden ejercer en el animal. Las PCL-2 derivada de la producción de extractos de levadura, y con mayor contenido de MP y BG, adicionadas (500 mg/kg) en dietas con TCC (Exp. 1) o maíz (Exp. 2), fueron capaces de mejorar el peso vivo y el índice de conversión del ave de forma similar al empleo de avilamicina (0.01 mg/kg alimento). Los distintos aditivos derivados de S. cerevisiae no afectaron claramente variables ileales como la viscosidad, absorción de nutrientes y recuentos bacterianos. En el Exp. 2, se encontró que el empleo de la PCL-2, favoreció el desarrollo de la mucosa digestiva del ave al incrementar la altura, el grosor de la mucina y el número de células caliciformes de las vellosidades del yeyuno (dietas con TCC o maíz). Además, la utilización en la dieta (TCC) de MP y BG purificados de PCL, resultó en mayor altura de las vellosidades de la mucosa del yeyuno (Exp. 3). Por otra parte, el empleo de MP+BG en concentraciones parecidas a las presentes en la PCL-2, representó efectos similares en el peso relativo del timo respecto a la PCL-2 (Exp. 4). La utilización de la PCL-2, MP, BG, o MP+BG en la dieta de pollos, no mostró diferencias claras en la productividad del ave en favor de alguno de los aditivos (Exp.4 y 5). En el Exp. 6, se observo que las PCL-2, incrementaron la reacción de hipersensibilidad cutánea tardía o determinación indirecta de la inmunidad celular en los pollos. Este efecto inmunomodulador de las PCL, pudo brindar beneficios en las pollos sometidos a estrés inflamatorio por la inoculación con lipopolisacárido (LPS) de E. coli, los cuales fueron traducidos en una mejor eficiencia alimenticia y % de peso relativo de la bolsa de Fabricio similares a los observados en pollos control sin estrés inmunitario (no desafiados con LPS). Los resultados de estos experimentos mostraron que las PCL adicionadas a dietas de pollos de engorde pudieron mejorar su eficiencia productiva, parte del mecanismo podría estar relacionado con favorecer el desarrollo de la mucosa digestiva y mantener un mejor estado de inmunocompentencia del ave, situación que puede tener beneficios en ambientes con mayor presencia desafíos microbianos. / The yeast cell walls (YCW) from S. cerevisiae have been used in poultry feeding for more than one decade; some of the purposes of this practice are to improve the chicken productivity and health. The polysaccharides such as beta-glucans and mannans presents in the YCW can exert effects in the immune system of chicken and also pathogen exclusion at digestive scale. However, the fermentation industry generates great diversity of active yeast and YCW from different origin and characteristics thus, the study about the correct definition and origin of these new additives can be necessary to optimize its use animal feeding. In the present thesis, there were carried out six experiments (Exp.) with male broiler chickens (Ross 308), with the objective of evaluating the effects of dietary supplementation of active yeast from S. cerevisiae (pecuary, bakery and "killer" winery) and their components: 2 YCW, extract, beta-glucans (BG) and manno-proteins (MP), to maize and wheat+barley+rye based diets on productivity and health of broiler chickens. In all experimental diets there were not used additives such as antibiotic, coccidiostates and feed enzymes. In the Exp. 1 and 2, It was observed that the own characteristics of different yeast (S. cerevisiae) and its components (extracts and YCW) as well as the type of diet used (TCC or maize) in these experiments, were important in the productive and digestive responses evaluated in the broiler chickens. The YCW-2 from the industrial production of yeast extracts, and with greater content of MP and BG, added (500 mg/kg) to WBR (Exp. 1) and maize (Exp. 2) based diets, improved the live weight and feed conversion ratio of broiler chickens in a similar magnitude to that obtained with dietary avilamycin (0.01 mg/kg feed). The different dietary additives derived from S. cerevisiae did not affect clearly variables at ileal level such as viscosity, bacterial counts and nutrients absorption. In the Exp. 2, the use of YCW-2 in WBR and maize based diets, improved the mucosa development of chickens by increasing the height, the mucin mucus thickness and the number of goblets cells of villous (jejune mucosa). In addition, in WBR based diet, dietary MP and BG purified from YCW, increased the villous height of jejune mucosa (Exp. 3). On the other hand, dietary MP+BG in a similar concentration respect to that present in YCW-2, represented similar effects in the percentage relative thymus weight respect to the dietary YCW-2 use (Exp. 4). The dietary use of YCW-2, MP, BG, or MP+BG in broiler diets did not show clearly differences in the productivity of chickens in favour of some of them (Exp.4 and 5). In the Exp. 6, dietary YCW-2 increased the delayed cutaneous hypersensitivity response or indirect determination of cellular immune response in chickens. This immunomoduladory effect of dietary YCW-2, could bring some benefits to chickens with immunitary stress (challenge with lipopolysaccharide or E. coli-LPS), because chickens fed YCW-2 and challenge with LPS showed similar feed efficiency and percentage relative bursa of Fabricius weighs than those not challenge chickens. The results of these experiments showed that the dietary supplementation with YCW-2 to broiler chickens diets can improve the animal productive performance; part of the mode of acting of dietary YCW could involve favouring intestinal health and maintaining a better state of immune-competence in the chickens. These positive effects can bring some benefits to chickens when raised under stress conditions or in environments causing great microbial challenges.

Bajo el nombre de Enfermedad Inflamatoria Intestinal (EII) se agrupan dos patologías inflamatorias crónicas y recidivantes de etiología desconocida: la enfermedad de Crohn y la colitis ulcerosa. La relativamente elevada incidencia de estas patologías hace que estudiar su etiopatogenia sea un objetivo importante, ya que actualmente no existe ningún tratamiento curativo y/o que prevenga la recidiva A pesar de que los pacientes afectados de EII presentan síntomas relacionados con alteraciones de la motilidad digestiva se desconoce la implicación que pueden tener estos desordenes en la patogénesis de la enfermedad. Uno de los principales objetivos de esta tesis doctoral ha sido la obtención y caracterización de un nuevo modelo experimental de Enfermedad Inflamatoria Intestinal crónica en la rata, con el objetivo de valorar los cambios que la inflamación provoca sobre los mecanismos fisiopatológicos que intervienen en la regulación tanto de la actividad motora como de la función de la barrera intestinal, así como la implicación que tienen estas alteraciones en la cronificación del proceso inflamatorio.Este nuevo modelo, que se ha obtenido a partir de la modificación realizada sobre el modelo clásico de enteritis inducida por indometacina en rata, ofrece una gran ventaja respecto al modelo clásico, ya que hemos reproduce las recidivas de inflamación intestinal que se producen de forma espontánea en los pacientes humanos. Así, en este modelo los animales presentan una alternancia de fases de inflamación activa, caracterizadas por leucocitosis, incremento de TNF en suero y aumento de la actividad MPO, con otras de aparente recuperación de la normalidad. Estudios realizados en este modelo demuestran que las diferentes fases van acompañadas de cambios en la flora intestinal y alteraciones de la motilidad. Mientras que durante la fase activa se observa una hipomotilidad generalizada acompañada de sobrecrecimiento de bacterias luminales, la fase de recuperación está asociada a un incremento en los parámetros motores y el restablecimiento de la carga microbiana La regulación de la síntesis de NO juega un papel clave en el curso de las alteraciones motoras asociadas al proceso inflamatorio. Mientras que el incremento de motilidad observado durante las fases de recuperación parece ser debido a la reducción del tono inhibitorio intestinal derivado de la inhibición de la expresión de nNOS, la disminución de motilidad y sobrecrecimiento bacteriano asociado a las fases activas parecen ser debidos a un incremento de la síntesis de NO producido por la sobre-expresión de iNOS. Recientemente también hemos demostrado que los animales con inflamación inducida presentan un aumento sostenido de la permeabilidad intestinal, sugiriendo que la vulnerabilidad de la función de la barrera epitelial es uno de los factores involucrados en la cronificación del proceso inflamatorio. Además, durante las fases de inflamación activa se observa una estrecha correlación entre el aumento de la permeabilidad transcelular y la translocación de bacterias luminales, sugiriendo que éste podría ser uno de los mecanismos involucrados en las recaídas cíclicas de inflamación intestinal.De manera conjunta, todos estos resultados dan pie a la siguiente hipótesis: mientras que la carga antigénica se mantiene dentro de unos límites, debido a una normal o incrementada motilidad, no hay respuesta inflamatoria a pesar del incremento de permeabilidad intestinal. Sin embargo, cuando la actividad motora decrece, el incremento de la interacción antígeno-mucosa facilita el paso de moléculas a través del epitelio, incluidas bacterias, resultando en la activación del sistema inmune y la liberación de mediadores inflamatorios. / Inflammatory Bowel Disease (IBD) includes two chronic inflammatory pathologies characterized by alternation of active and quiescent phases of inflammation with yet unknown aetiology: Crohn's disease and ulcerative colitis. The ultimate goal of gaining a better understanding of the pathological mechanisms involved in the development of IBD is to find an effective treatment. Although IBD patients present symptoms that suggest motor alterations, their implication in the pathogenesis of the disease has not been well established. The purpose of this Thesis have been to obtain and to characterize a new experimental model of Inflammatory Bowel Disease in the rat to evaluate the effects of inflammation in the mechanisms involved in the regulation of both intestinal motility and epithelial barrier function, as well as the implication of both disorders in the development of chronic inflammation.This new model has been obtained by the modification of the classic model of indomethacin-induced enteritis. Similarly to human IBD, this model is characterized by spontaneous alternation of active and quiescent phases of inflammation. Active phases are related to leukocytosis, increased TNF and increased tissular MPO. In contrast, quiescent phases are related to a reduction of inflammatory parameters. Studies made in this model have also demonstrated changes in both bacterial load and motor activity. Whereas active phases are characterized by hypomotility and bacterial overgrowth, quiescent phases are related to hypermotility and the reduction of bacterial load.The regulation of the synthesis of NO plays a key role in the motility disturbances associated to the inflammatory process. Whereas hypermotility observed during the quiescent phases seems to be consequence of the reduction of the intestinal inhibitory tone due to nNOS down-regulation, hypomotility and bacterial overgrowth are related to an over-production of NO by up-regulated iNOS. Recently we have demonstrated animals with induced inflammation present a sustained enhancement of paracellular permeability, suggesting epithelial barrier dysfunction is involved in the chronicity of inflammation. By contrast, trancellular permeability and bacterial translocation only increased during the active phases, when hypomotility and bacterial overgrowth are also present, suggesting these factors may play a critical role in the course of acute relapses in IBD. The results obtained are in agreement with the following hypothesis: while antigen load is maintained at lower level, due to normal or enhanced motility, there is no inflammatory response despite the increased intestinal permeability (mainly paracellular). However, when motor activity decreases, the increase in the interaction antigen-mucosa facilitate massive antigenic uptake and bacterial translocation, leading to the activation of the immune system and the release of inflammatory mediators.

El Síndrome del Intestino Irritable (IBS) se define como una alteración de la función gastrointestinal caracterizada por dismotilidad e hipersensibilidad visceral. En la mayoría de pacientes con este síndrome el número de mastocitos de mucosa intestinal (IMMC) y su actividad están aumentados. Este hecho podría jugar un papel central en las alteraciones funcionales, sensitivas y motoras del IBS ya que la activación de esta población celular provocaría la liberación de mediadores que podrían afectar a la excitabilidad de las neuronas entéricas y a la de las aferentes primarias, así como a la contractilidad del músculo liso intestinal. No obstante, este síndrome presenta un diagnóstico difícil y un tratamiento poco efectivo ya que los factores que lo causan aún no están claros. Recientemente los procesos alérgicos no diagnosticados hacia componentes de la dieta se han propuesto como posible factor desencadenante del IBS. Se requiere sin embargo disponer de modelos in vivo para estudiar los mecanismos implicados e identificar nuevas dianas terapéuticas. El modelo experimental inducido en rata Sprague Dawley (SD) a la que se expone a ovo-albúmina (OVA) por vía oral reproduce algunos de los hallazgos observados en pacientes con IBS: un aumento de la actividad mastocitaria y una alteración en la motilidad.El objetivo de esta tesis doctoral ha sido contribuir a caracterizar el modelo de exposición oral a OVA en rata SD; determinar el tipo de respuesta inmune inducida por el antígeno, evaluar la actividad mastocitaria y su implicación en la dismotilidad inducida por OVA y valorar la participacion del factor de crecimiento nervioso (NGF) en el proceso.La exposición oral a OVA, a diferencia de otras proteínas como la gelatina, produjo un aumento en la actividad mastocitaria en el intestino delgado y el colon, como reflejan el aumento del número de IMMC y el incremento de la concentración intestinal de RMCPII. Sin embargo, parámetros como la concentración de IgE o IgG específicas anti-OVA, IL4 o la presencia de eosinófilos y celulas IgE+ en tejido intestinal no se vieron afectados por la exposición a OVA. Estos resultados, junto con el aumento de liberación de histamina tras la estimulación directa de mastocitos con OVA in Vitro, sugirieron que el efecto de esta proteína podría deberse a una interacción directa, no mediada por anticuerpos, entre OVA y el mastocito. La exposición oral a OVA también produjo un incremento en parámetros relacionados con la respuesta motora principalmente en colon, así como en la expresión de NGF en tejido intestinal. Finalmente, el tratamiento con ketotifeno disminuyó significativamente el aumento inducido por OVA sobre la actividad mastocitaria y la respuesta motora y la expresión de NGF. Además se observó una correlación entre el efecto de OVA sobre la actividad mastocitaria y la respuesta motora especialmente en colon, que contribuye a proponer la hipótesis de que los mastocitos están implicados en la alteración de la motilidad intestinal.La exposición de ratas SD a OVA por vía oral induce una alteración de la motilidad intestinal que no es mediada por una reacción de naturaleza alérgica. A la disfunción motora provocada por la OVA contribuyen sin embargo los mastocitos, probablemente mediante la interacción directa entre esta población celular y la proteína que puede generar, entre otros mediadores, la liberación de NGF. Este mecanismo no alérgico que implica a los mastocitos puede ser relevante en ciertos tipos de pacientes con IBS, cuyo abordaje terapéutico quizás requiera de un replanteamiento. Este trabajo ha apuntado el camino para una caracterización más profunda del modelo con el objetivo de identificar nuevas dianas terapéuticas. / Irritable Bowel Syndrome (IBS) is an alteration of the gastrointestinal function characterised by dismotility and visceral hypersensitivity. In most patients with this syndrome the number of intestinal mucosal mast cells (IMMC) and its activity are increased. This could play a central role in any physiological, sensory and motor IBS because activation of this cell population would result in the release of mediators that could affect the excitability of enteric neurons and primary afferents, as well as the intestinal smooth muscle contractility. However, this syndrome presents a difficult diagnosis and ineffective treatment because the factors that cause it are still unclear. Recently undiagnosed allergic processes to dietary components have been proposed as a possible trigger of IBS. However is required to have in vivo models to study the mechanisms involved and identify new therapeutic targets. The rat experimental model induced in Sprague Dawley (SD) which is exposed to ovo-albumin (OVA) orally play some of the findings in patients with IBS: increased mast cell activity and altered motility. The aim of this PhD was to characterize the model of oral exposure to OVA in SD rat; determine the type of immune response induced, the mast cell activity and assess their involvement in the OVA-induced dysmotility and evaluate the participation of Nerve growth factor (NGF) in the process. Oral exposure to OVA, unlike other proteins such as gelatin, induced an increase in mast cell activity in the small intestine and colon, as reflected in the increased number of IMMC and intestinal RMCPII concentration. However, parameters such as concentration of specific IgE or IgG anti-OVA, IL4 or the presence of eosinophils and IgE + cells in intestinal tissue were not affected by exposure to OVA. These findings, coupled with increased histamine release after direct stimulation of mast cells with OVA in vitro, suggested that the effect of this protein could be due to direct interaction between OVA and the mast cell. Oral exposure to OVA also produced an increase in parameters related to motor response mainly in the colon, as well as in the expression of NGF in intestinal tissue. Finally, treatment with ketotifen significantly reduced the OVA-induced increase of mast cell activity and motor response and the expression of NGF. We observed a correlation between the effect of OVA on mast cell activity and motor response especially in the colon, which contributes to hypothesize that mast cells are involved in altering intestinal motility.Oral exposure to OVA in SD rats induces a disturbance in motor response that is not mediated by an allergic reaction. However, mast cells have a key role in the disturbance of intestinal motility, probably through direct interaction between this cell population and the protein, which can generate, among other mediators, the release of NGF. This nonallergic mechanism involving mast cells may be relevant in certain types of patients with IBS, whose therapeutic approach may require a restatement. This work has pointed the way for a deeper characterization of the model with the aim of identifying new therapeutic targets.

Le cadmium (Cd) est un métal lourd auquel la population en générale est exposée par l'alimentation. L'épithélium intestinal accumule beaucoup de Cd ingéré et représente un organe cible. Étant donné le rôle de cet épithélium dans la biotransformation de xénobiotiques ingérés, l'objectif de notre étude était d'évaluer dans quelle mesure une exposition chronique au Cd peut perturber l'expression des enzymes de biotransformation lors de la différenciation des entérocytes. Comme modèle in vitro, nous avons utilisé la lignée cellulaire humaine Caco-2 qui développe spontanément un phénotype entérocytaire. Le Cd étant connu pour troubler certaines voies de signalisation cellulaires, nous avons testé les hypothèses suivantes : 1) ce métal pourrait modifier l'expression (niveaux et/ou profil) des enzymes de biotransformation; 2) il serait susceptible d'altérer le processus de différenciation. Le profil d'expression d'enzymes de biotransformation (CYP1A1, CYP3A4 et GSTP1) a été caractérisé par RT-PCR en fonction du temps de culture : les niveaux d'ARNm de la GSTP1, de la P-gp et du CYP1A1 augmentent durant la différenciation. Parallèlement, nous avons estimé par mesure d'activité MTT (viabilité cellulaire), la LC5, soit la concentration d'exposition menant à 5% de mortalité. Lorsque les cellules sont exposées durant la phase de prolifération, une période de récupération augmente considérablement la viabilité (LC5 = 87 ± 4 vs. 15.2 ± 0.7 µM). Une LC5 de 36 ± 2 µM est obtenue lorsque les cellules sont traitées pendant la phase de différenciation montrant que la période de sensibilité maximale est pendant la prolifération. Étant donné les profils d'expression obtenus selon le temps de culture des quatre enzymes d'intérêt dans les cellules Caco-2 témoins, nous avons choisi la LC5 de 36 ± 2 µM comme concentration d'exposition pendant la phase de différenciation pour tester l'effet du Cd. Une exposition chronique à cette concentration de Cd perturbe le profil d'expression d'enzymes. De plus hauts niveaux d'ARNm de GSTP1 et de la P-gp sont alors observés mais cette induction par le Cd est diminuée en présence de vitamine D3 ou de NAC, toutes deux antioxydantes. Ainsi, l'effet du Cd sur la GSTP1 et la P-gp serait médié par un déséquilibre rédox. Des mesures d'activités enzymatiques de CYP1A1 et de GSTP1 ont été effectuées afin de corréler l'effet du Cd sur les niveaux d'ARNm aux activités. Une activité extrêmement faible d'environ 0,290 pmol/min/mg de protéines microsomales a été mesurée dans les cellules contrôles et traitées au Cd de 21 jours, soit presque 100 fois moins que ce qui est rapporté dans la littérature, mais plusieurs études ont révélé l'effet inhibiteur du Cd sur l'activité EROD. Deux types de réponses sont observées quant à l'effet du Cd sur l'activité de la GST: (1) une légère stimulation, (2) une faible inhibition. Nous avons également mesuré une diminution de la résistance électrique transépithéliale confirmant ainsi que le Cd affecte l'intégrité de l'épithélium intestinal en augmentant la perméabilité paracellulaire. Par ailleurs, nos récents résultats suggèrent que le Cd diminue l'activité de la phosphatase alcaline, un marqueur de différenciation. Néanmoins, les enzymes de biotransformation n'étaient pas toutes affectées de la même façon, l'effet du Cd sur ces enzymes n'est pas le résultat (indirect) d'une action "non spécifique" sur le processus global de différenciation. Nos résultats montrent que l'exposition intestinale au Cd pourrait avoir des répercussions sur le métabolisme de premier passage des médicaments et autres xénobiotiques absorbés oralement. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : cellules Caco-2, épithélium intestinal, cadmium, entérocyte, CYP1A1, CYP3A4, GSTP1

Cytomegalievirus (CMV) ist bei immunsupprimierten Patienten nach Organ- oder Stammzelltransplantation sowie bei Patienten mit chronisch entzündlichen Darmerkrankungen und nicht immunsupprimierten kritisch kranken Patienten ein ernstzunehmender Risikofaktor für eine manifeste CMV-Infektion. Diese ist durch eine hohe Morbidität und Letalität gekennzeichnet. Daher ist eine zeitnahe und zuverlässige Diagnosestellung für die Einleitung einer adäquaten Therapie und damit für das klinische Ergebnis der Patienten von entscheidender Bedeutung. Ziel dieser retrospektiven Arbeit war es, die diagnostischen Verfahren der endoskopischen, pathologischen und virologischen Untersuchung bezüglich ihrer Treffsicherheit bei der gastrointestinalen CMV-Infektion zu untersuchen. Dafür wurde eine quantitative molekularvirologische Nachweismethode für CMV aus formalinfixiertem, in Paraffin eingebettetem Gewebe etabliert. Des Weiteren wurde das Procedere der Entnahme und der Verteilung der Biopsien an die Institute für Virologie und Pathologie evaluiert und die Ergebnisse von endoskopischer und molekularvirologischer Untersuchung in Blut- und Gewebeproben miteinander verglichen. In einem weiteren Schritt konnte die Korrelation der molekularvirologischen mit den immunhistochemischen Ergebnissen untersucht werden. Für die genannten Fragestellungen wurde CMV spezifisches Genom aus Blutproben und Gewebeproben des Gastrointestinaltraktes von 164 Patienten im Zeitraum von Oktober 2008 bis September 2010 quantitativ ausgewertet. Insgesamt wurden 860 Gewebeproben und 2550 Plasma- und Serumproben untersucht. Basierend auf den Ergebnissen der Datenerhebung zeigt die vorliegende Arbeit, dass ohne eine Anpassung der Vorgehensweise einer von vier Fällen mit gastrointestinaler CMV-Infektion nicht diagnostiziert werden würde und es wird macht einen Vorschlag zur weiteren Optimierung des diagnostischen Procedere.

This study was conducted to determine if electroporation could deliver membrane-impermeant molecules intracellularly to intact, physiologically competent monolayers that mimic the intestinal epithelium. The long-term effects of electroporation on these monolayers were studied to determine the kinetics with which monolayers recover barrier function. The ability of electroporation to introduce biologically active molecules, e.g., plasmid DNA and siRNA, into these monolayers, to either express a protein of interest or modify cellular function, was also studied. Results showed that intracellular uptake of calcein, a small tracer molecule, and bovine serum albumin, a globular protein, occurred uniformly throughout the monolayers and increased as a function of voltage, pulse length, and pulse number. There was no significant difference in uptake resulting from single and multiple pulses of the same total exposure time. Barrier function recovery depended on the electroporation conditions applied, with some monolayers recovering normal physiologic function within a day. Electroporation also increased the permeability of the monolayers to calcein and BSA, possibly through a combination of increased paracellular and transmonolayer transport. When compared to cationic lipid transfection (lipofection), transfection of intestinal epithelial monolayers with reporter plasmids by electroporation was more efficient in situations where high concentrations of DNA, and as a result, higher levels of expression were needed. Although uptake of DNA was high after electroporation and increased with increasing amounts of DNA, overall expreseion efficiency was still low (~3%). Electroporation-mediated transfection of intestinal epithelial monolayers with a plasmid that expressed inflammation inhibitor protein, IκВα was not always successful, probably because of low levels of protein expression. Introduction of the much smaller siRNA molecules into the monolayers by electroporation, on the other hand, was very successful at inhibiting the production of the nuclear envelope proteins lamin A and lamin C. The results of these experiments demonstrated that electroporation can introduce a wide variety of molecules intracellularly into model intestinal epithelia. These results should be useful to identify optimal electroporation conditions for transporting drugs, proteins, and genes into intestinal and, possibly, other epithelia for local drug and gene therapy, as well as for development of improved models of intestinal epithelium.

A series of four experiments were conducted to evaluate whether a preconditioning period, in which adult leghorn cockerels are allowed 3 weeks to adapt to diets containing relatively high levels of guar meal (GM) resulted in changes in digestive tract morphology and ecology. It was expected that these changes would result in increased utilization of GM as birds became acclimated to consuming it at high levels. In the 1st experiment, 28 birds were fed diets containing 0, 6, 12, or 24% GM. Consuming the 24% GM diet resulted in decreased body weight, increased small intestine, liver, and pancreas weight, increased ceca and villus length, and increased severity of injury to the intestinal mucosa (P < 0.05). In the 2nd experiment, 28 cockerels were subjected to a partial cross-over experiment. Preconditioning to the 24% GM diet resulted in decreased TMEn of GM, decreased body weight, and decreased absorptive capacity of the GI tract as evidenced by increased energy lost via the excreta (P < 0.05). The 3rd experiment evaluated how differences in endogenous energy losses (EEL) from 30 cockerels affected the TMEn content of GM in birds consuming 0, 6, or 12% GM. Decreased EEL was observed in birds conditioned to 6 and 12% GM relative to the control group (P < 0.05). Further, decreased TMEn of GM was observed in birds consuming 6% GM relative to those consuming the control diet (P < 0.05). In the 4th experiment, changes in cecal microbiota were evaluated in 24 cockerels consuming 0, 6, or 12% dietary GM using denaturing gradient gel electrophoresis of amplified bacterial DNA sequences. Cecal microbiota of birds consuming 12% GM was considerably different from the control group (similarity coefficient = 84%) with an apparent decrease in the complexity of microbial communities. Results of these studies show that consuming 12% dietary GM results in changes in the cecal microbial community which may be responsible for modest increases in the TMEn of GM when compared to 6% dietary GM, which consistently resulted in decreased TMEn values of GM. Further, determining the TMEn value of GM appears to be highly dependent upon factors such as preconditioning diet and timeframe and innate physiological "thresholds" which seem to initiate or limit the maximum physiological response to preconditioning that the bird is capable of.