Thesis (M.D.) -- University of Adelaide, Dept. of Medicine, 1975.

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211 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (M.D.)--University of Adelaide, Dept. of Medicine, 1975

La maladie cœliaque est la principale pathologie liée à un déficit en cholécystokinine (CCK) occasionnant une diminution de la fonction pancréatique et une réduction de la contraction vésiculaire. La maladie cœliaque ou intolérance au gluten est une affection auto-immunitaire qui se caractérise par une malabsorption intestinale causée par une atteinte inflammatoire de l’intestin grêle, suite à l’ingestion de gluten présent dans la farine de blé, d’orge ou de seigle. Le but de ce travail est d’étudier la raison de ce déficit en CCK chez des patients adultes souffrant de maladie cœliaque. La diminution de sécrétion de CCK a d’abord été attribuée à une réduction du nombre de cellules à CCK intestinales et de leur contenu hormonal dans le cadre d’une muqueuse intestinale atrophique, de type destructif ou hypoplasique. Elle pourrait cependant être liée à d’autres mécanismes: une diminution de la stimulation des cellules endocrines suite à une moindre hydrolyse des nutriments dans la lumière intestinale, un trouble fonctionnel des cellules, une altération de l’interaction avec d’autres cellules intestinales (endocrines, épithéliales ou nerveuses), l’absence d’un facteur stimulant la sécrétion de CCK ou la présence de facteurs inhibiteurs associés au processus inflammatoire dans la muqueuse intestinale. Nous avons montré que la sécrétion de CCK n’est pas seulement diminuée chez les patients cœliaques présentant une muqueuse duodénale de type hypoplasique ou destructif mais également chez les patients dont la muqueuse ne présente qu’un infiltrat excessif de lymphocytes intraépithéliaux (IEL). Seuls les patients cœliaques traités, sans caractère desctructif ou hypoplasique de leur muqueuse, et sans infiltrat inflammatoire, présentent une sécrétion de CCK normale et semblable aux volontaires sains. En utilisant l’immunocytochimie et la PCR semi-quantitative, nous avons démontré que le déficit en sécrétion de CCK n’est pas lié à une diminution du nombre de cellules à CCK dans la muqueuse duodénale mais plutôt à une diminution du taux d’ARN messager, et de la synthèse de CCK, ceci tant chez les patients avec des lésions muqueuses de type destructif ou hypoplasique que chez les patients ne présentant qu’un infiltrat de lymphocytes intraépithéliaux. Ces données suggèrent que l’infiltrat inflammatoire affecte l’expression du gène de la CCK dans les cellules entéroendocrines intestinales. La partie suivante de notre travail a consisté à investiguer le rôle d’un déficit d’hydrolyse intraduodénale des nutriments sur la sécrétion de CCK chez des patients cœliaques adultes. Nous avons montré que la sécrétion postprandiale de CCK n’est pas améliorée par l’ingestion d’un repas prédigéré, chez des patients présentant des lésions destructives ou hypoplasiques comme ceux présentant des lésions muqueuses infiltratives. De plus l’hydrolyse alimentaire intraduodénale devrait être normale en cas de lésion muqueuse limitée à un infiltrat de IEL. Les taux plasmatiques réduits de CCK dans ce groupe de patients suggèrent clairement que le déficit de sécrétion de CCK pourrait être lié au processus inflammatoire et à des facteurs provenant de l’infiltrat de lymphocytes intraépithéliaux. Pour cette raison, nous avons étudié les effets de surnageants de cultures lymphocytaires et de cytokines produites par les lymphocytes de la muqueuse intestinale sur des biopsies duodénales maintenues en culture de courte durée et sur des cellules sécrétant de la CCK et provenant d’une lignée cellulaire murine neuroendocrine STC-1. Nous avons montré un effet inhibiteur significatif sur la sécrétion de CCK du surnageant de cultures de lymphocytes de la lamina propria stimulés par de la phytohémagglutinine A, ainsi que de l’interleukine-1 et du TNF-. En conclusion, nous avons montré que le déficit de sécrétion de CCK est présent dès le stade initial infiltratif de la lésion muqueuse cœliaque et que ce déficit pourrait être lié au processus inflammatoire présent dans la muqueuse intestinale et à certains facteurs suppresseurs associés à ces cellules inflammatoires. La modulation de la sécrétion de CCK doit être considérée comme un processus complexe impliquant une co-régulation par les nutriments, des peptides stimulants, des neurotransmetteurs et des facteurs solubles comme les cytokines. Les données décrites dans ce travail nous font entrevoir de nouveaux mécanismes de régulation et de nouvelles pistes pour améliorer la prise en charge de patients cœliaques continuant à présenter des troubles digestifs malgré un régime sans gluten bien suivi. / Celiac sprue is the principal disease associated with impaired cholecystokinin (CCK) release that accounts for the diminished gallbladder contraction and pancreatic function. Celiac sprue, also known as celiac disease or gluten-sensitive enteropathy, is an autoimmune disorder characterized by malabsorption resulting from inflammatory injury to the small intestine mucosa after the ingestion of wheat gluten or related rye and barley proteins. The aim of our work was to study the defective CCK release that is found in adult patients with celiac disease. The CCK decreased secretion was first thought to be caused by a reduction in the number of intestinal CCK cells in the presence of mucosal atrophy, but could also be related to other mechanisms: a lack of stimulation of the mucosal CCK cells due to impaired intra-duodenal hydrolysis of nutrients, a functional abnormality of the CCK-cell, an impaired interaction of the CCK-cell with other intestinal cells (endocrine, epithelial or nervous cells), the absence of a stimulating factor (luminal cholecystokinin releasing factor) or the presence of inhibitory factors associated with the inflammatory process taking place in the mucosa. We have shown that CCK release is not only impaired in untreated celiac patients with a destructive/hypoplastic type duodenal mucosa but also in patients whose mucosa only shows a significant intraepithelial lymphocytic (IEL) infiltrate. Treated celiac patients without hypoplastic or destructive mucosa and without any increase in IEL infiltrate exhibit a basal and postprandial CCK release similar to the control group. Using immunocytochemistry and semi-quantitative PCR we have demonstrated that the defective release of CCK observed in patients with coeliac disease is not related to a decrease in the number of CCK cells present in the proximal part of the small intestine but rather to a decrease in CCK synthesis related to a decrease in mRNA content, even in patients with an infiltrative type of celiac disease. These data suggest that the inflammatory infiltrate in the mucosa of celiac patients affects the expression of the CCK gene. The next part of our work included the investigation of the role of the intraluminal nutrient hydrolysis in adult celiac patients. Our data indicates that ingestion of a pre-digested meal does not correct the defective CCK release in patients with a destructive or an infiltrative type of celiac mucosal lesion. Moreover, intraduodenal food digestion should be considered normal in celiac patients with mucosal lesions limited to an intraepithelial lymphocytic infiltrate. The decreased level of plasma CCK in this group of patients, given a pre-digested meal, strongly supports that the defective CCK release could be related to suppressive factors released by the inflammatory infiltrate. We therefore studied the effects of supernatants obtained from IEL and lamina propria lymphocyte cultures and various cytokines produced by mucosal lymphocytes on short-term organ cultures of duodenal biopsy specimens obtained from normal controls and on the CCK secreting murine neuroendocrine tumour cell line STC-1. We demonstrated a significant inhibitory effect of supernatants obtained from phytohaemagglutinin A-stimulated cultured lamina propria lymphocytes and from phytohaemagglutinin A-stimulated co-cultured lamina propria and intraepithelial lymphocytes, and of interleukin-1 and tumour necrosing factor- on CCK release. All together these data show that the defective CCK release is already present at the initial infiltrative type of mucosal lesion seen in celiac disease and that this defective release may be related to inflammation and suppressive factors associated with the inflammatory cells. Modulation of CCK release in the gut should be considered as a complex process implying co-regulation of nutrients, releasing peptides, neurotransmitters and soluble factors including cytokines. These results may be of help in finding new directions to improve the treatment of patients with celiac disease who continue to present with abdominal complaints in spite of a strict gluten-free diet.

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Introduction: Wheat is one of the most cultivated cereal grains in the world and is used for the manufacture of a wide range of food products; however its consumption has been linked to several health issues. Food products containing wheat flour commonly elicit a high glycaemic response (GR) through rapid breakdown of starch and absorption of the resulting glucose. Regular over-consumption of such foods has been linked to obesity and development of type 2 diabetes. Dietary fibre may alter GR after meal consumption indirectly through modification of chyme viscosity. Wheat can also elicit immune-mediated adverse reactions, such as immunoglobulin E(IgE)-mediated wheat allergy and coeliac disease (CD), which are most often associated with gluten proteins consisting of gliadins and glutenins. Resistance to digestion may impact the allergenicity of such protein components. Digestion of gluten and its epitopes important for CD have been enhanced in vitro and in vivo using a prolyl endopeptidase from Aspergillus niger (AnPEP) however the impact on IgE-mediated allergy has yet to be considered. Additional information is needed about the digestion of wheat. Specifically the impact of food matrix, digestion conditions and effect of AnPEP require further investigation. Methods: First, the effect of food matrix on proteolysis was tested by in vitro batch oral-gastric digestion of a purified total gliadin fraction (TGF), flour and bread. As the most physiologically relevant material, bread was also processed through the duodenal/intestinal phase in varying conditions to assess the impact of enzyme inclusions on macronutrient breakdown. Second, results from the batch digestions were compared to bread digestion in dynamic models, where the effect of natural variations in soluble fibre was also tested. Increasing levels of AnPEP were used in two in vitro batch oral-gastric models. Protein breakdown in digestions was assessed using a combination of 1D PAGE, immunoblots with a variety of wheat-specific antibodies, kinetic analysis and inhibition ELISA. Immunoassays were performed with sera from 23 wheat-allergic patients and some digestions were analysed in terms of starch digestion. Finally, LC-MS/MS was used to obtain specific sequence information and relative intensity of peptides from in vitro batch model digestions. Thus, digestion of selected allergens and key epitopes was monitored. Results and Discussion: Wheat proteins were very resistant to in vitro batch gastric digestion in bread compared to the TGF, with flour proteins somewhat intermediate. Thus, studies digesting purified proteins are not always indicative of protein digestion in a processed food matrix. Digestion of bread protein was enhanced by starch digestion and vice versa. This has implications for patients with deficiency in pancreatic amylase, which is often observed in childhood, so may play a role in food allergy development by influencing polypeptides reaching the gut mucosa. Digestion model conditions also had a large impact on wheat protein digestibility with differences observed between batch and dynamic models, and the two batch models used. This may reflect biological variations observed in vivo. Unexpectedly, the wheat cultivar with higher soluble fibre digested slightly more quickly which may be due to alterations in other macronutrients present. In most cases patient sera were poly-sensitized to a number of wheat proteins and IgE-binding was mostly unaffected by baking. Gastric digestion reduced IgE-reactivity of bread but large polypeptides of high relative intensity remained. Addition of AnPEP further reduced IgE-reactivity of digestion samples by digesting gluten proteins into smaller peptides of lower relative intensity. This reduced the presence of epitopes important for IgE-mediated allergy and CD. Therefore, AnPEP may have an application for treatment of accidental wheat consumption for patients with IgE-mediated wheat allergy.