This work deals with effectiveness of coeliac disease management. It is a model case of chronic disease management. The analytical framework of this work is the multilevel Innovative Care for Chronic Conditions Framework. Crucial is the interaction between health care team that represents health care system, community partners and patients with their families. In the case of coeliac disease it is necessary to include also the role of representatives of market with gluten free products. The interaction of these participants takes place in wider political context, whose state influences this interaction fundamentaly and reflects so in final results - state of patients and quality of their live. Using the evaluation of the quality of life tool SF-36, it has been ascertained, that the quality of life of coealiac patients is lower in comparison with European standard of healthy population and that the causes of the problem have to be searched and systematically removed at all levels of the system of chronic health care. The findings validate the theoretic presumption, that the chronic health care is still considerably underestimated and that it does not reply to the needs of chronic ill patients, which reflects just on general quality of life.

The topic of this thesis is the production of gluten-free bread from naturally gluten-free flour, which will be enriched with suitable additives that will increase antioxidant activity and naturally effect on the structure of the pastry, so it will meet the sensory requirements of final consumers. The literary part define, which proteins can be found in cereals, what does the gluten means, its function in the pastry and which diseases can cause. In connection with these diseases, a gluten-free diet for improving the patient's health and suitable foodstuff are briefly described. The literary part is concluded with the theory of sensory evaluation. The practical part deals with the production of naturally gluten-free breads enriched with ingredients from kitchen onions (Allium cepa L.) and subsequent sensory evaluation of this bread. Selected statistical procedures were used to obtain, processed and evaluated data. Antioxidant activity was also monitored and evaluated. By using texture-meter was find out, what effect has additives on aging of gluten-free bread.

Dans les conditions physiologiques des robustes mécanismes immunologiques empêchent le développement des réponses exagérées aux antigènes alimentaires. En revanche, dans le cas de maladie céliaque, qui affecte environ 1% de la population occidentale, l’exposition au gluten alimentaire d’individus génétiquement prédisposés HLA-DQ2.5/DQ8 provoque l’entéropathie chronique de l’intestin grêle. Les études précédentes chez l’homme ont établi le rôle crucial de la réponse cellulaire T CD4+ restreinte par HLA-DQ2.5/DQ8 et spécifique du gluten. La réponse T CD4+ est nécessaire mais cependant insuffisante pour induire des lésions tissulaires. D’autres études ont suggéré le rôle de l’interleukine 15 (IL-15). Ainsi, l’IL-15 surexprimée dans la muqueuse des patients céliaques peut interférer avec les mécanismes d’immunorégulation et stimuler l’activation des lymphocytes intraépithéliaux T CD8+ cytotoxiques probablement induisant des lésions épithéliales. Comment les cellules T CD4+ spécifiques du gluten et l’IL-15 interagissent pour activer les lymphocytes intraépithéliaux T CD8+ et induisent des lésions n’a pas été toutefois établi. Pour répondre à cette question, nous avons créé un modèle murin basé en croisant des souris OTII possédant des cellules T CD4+ spécifiques de l’antigène modèle, ovalbumine, avec les souris transgéniques hétérozygotes surexprimant une forme secrétée de l’IL-15 humaine dans l’épithélium intestinale (souris hIL-15Tge). Les souris obtenues OTII+/- B6 and OTII+/- hIL-15Tge+/- ont été mises au régime riche en ovalbumine depuis la période prénatale jusqu’à l’âge de 3 mois. Les souris OTII+/- hIL-15Tge+/-, contrairement aux souris OTII+/- B6, exposées de façon chronique à l’ovalbumine ont développé un retard de croissance et une atrophie villositaire associée à l’expansion des cellules intestinales T CD8+ cytotoxiques, comme dans la maladie céliaque. En outre, nous avons démontré que l’IL-15 altérait l’immunorégulation par les cellules T FoxpP3+ et coopérait avec l’IL-2, produite par les cellules T CD4+ activées par l’OVA, pour l’expansion des cellules T CD8+ non-spécifiques de l’OVA. Nous suggérons que le scénario similaire pourrait opérer dans la maladie céliaque. Au cours de cette étude, j’ai observé que la surexpression chronique de l’IL-15 était associée avec l’expansion de cellules dendritiques CD103+CD11c+CD11b-. Dans la partie de résultats supplémentaires, j’ai démontré que cet effet dépend de la production de la cytokine GM-CSF secrétée par les cellules Natural Killer (NK) activées par l’IL-15 et que ces cellules dendritiques étaient enrichies en cellules CD103+ ayant une capacité accrue de cross-présentation in vitro. Ces derniers résultats illustrent comment l’IL-15 peut moduler les réponses immunes adaptatives en orchestrant la coopération entre les cellules NK et les phagocytes mononucléaires. / In physiological conditions, robust immunological mechanisms avoid adverse responses to food antigens. In contrast, in celiac disease that affects about 1% of Western populations, exposure to dietary gluten of genetically predisposed HLA-DQ2.5/ DQ8 individuals triggers a chronic small intestinal enteropathy. Previous studies in humans have established the crucial role of HLA-DQ2/DQ8 restricted gluten-specific intestinal CD4 T cell response. This CD4 T cell response is necessary but is however not sufficient to induce tissue damage. Other studies have pointed to the role of interleukin 15 (IL-15). Thus, IL-15 over-expressed in the mucosa of celiac patients can interfere with immunoregulatory mechanisms and stimulate the activation of cytotoxic CD8 T intraepithelial lymphocytes, thought to induce epithelial lesions. Whether and how gluten-specific CD4 T cells and IL-15 interact to activate CD8 T intraepithelial lymphocytes and to drive intestinal tissue damage has not been however established. To address this question, we have set up a mouse model based on the breeding of OTII mice possessing CD4 T cells specific of a model antigen, ovalbumin, with heterozygous transgenic mice overexpressing a secreted form of human IL-15 in intestinal epithelium (hIL-15Tge mice). Resulting OTII+/- B6 and OTII+/- hIL-15Tge+/- mice were exposed to dietary ovalbumin from the prenatal period until 3 months of age. Upon chronic exposure to ovalbumin, OTII+/- hIL-15Tge+ mice, contrary to their OTII+/- B6 littermates, developed growth retardation, and villous atrophy associated with expansion of intestinal cytotoxic CD8 T cells, as in celiac disease. Moreover, we showed that IL-15 impaired immunoregulation by FoxP3+ T cells and cooperated with IL-2 produced by OVA-activated CD4 T cells to stimulate the expansion of non-cognate cytotoxic CD8 T cells. We suggest that a comparable scenario can operate in celiac disease. During this study, I observed that chronic overexpression of IL-15 was associated with an expansion of CD103+CD11c+CD11b- mononuclear cells. In the Supplementary results, I have shown that this effect depends on the production of GM-CSF secreted by IL-15-activated NK cells and that CD11c+ DCs differentiated in mice overexpressing IL-15 were enriched in CD103+ cells and displayed enhanced cross-presentation abilities in vitro. The latter results illustrate how IL-15, by orchestrating a crosstalk between NK cells and mononuclear phagocytes, can modulate adaptive immune responses.

Abstract The function of the transmembrane protein FasL is to complex with the Fas receptor in a target cell and induce target cell apoptosis. Fas/FasL-mediated apoptosis plays important role in immunoregulation. FasL expression is mostly seen in activated lymphocytes. We have characterized endothelial FasL expression in different functional compartments of lymph nodes and gut-associated lymphoid tissue. Furthermore, we have explored the functional role of endothelial FasL expression by analyzing correlation with apoptosis of lymphocyte subpopulations in lymph nodes and by assessing endothelial expression under different conditions by activation of immune functions in gastrointestinal mucosa. Immunohistochemical stainings (Fas, FasL, CD3, CD20, CD19, CD23, CD56, FVIII) were performed on 20 reactive lymph node tissues (I and II), 60 pediatric endoscopy biopsy samples (III) or 60 samples from gut resections (IV). A double-staining method combining apoptosis detection with the TUNEL-method and lymphocyte classification with FasL, Fas and cell lineage markers was optimized. Patient groups included non-pathological lymph nodes, pediatric cow’s milk-sensitive enteropathy, pediatric celiac disease, appendicitis, ulcerative colitis and Crohn’s disease. Control groups included normal biopsy samples from pediatric patients and non-pathological resecate samples from the appendix, colon or ileum to correspond to patient groups. Quantitative analysis (positive vessels or cells per mm2) was performed thoroughly for each anatomical region. In a subset of patients, soluble FasL in the serum was quantified with standard enzyme-linked immunosorbent assay. In reactive lymph nodes FasL expression was predominantly present in high endothelial venules located in the paracortical area, where apoptotic T and B lymphocytes, some expressing Fas, were subsequently found. In the gut wall vascular FasL expression was seen in high endothelial vessels near lymphoid follicles. Serum FasL was elevated in children with an abundance of mucosal lymphoid follicles. In IBD, vascular FasL was upregulated in ulcers and in the submucosa of colons affected by Crohn’s disease. The results indicate that endothelial FasL is characteristically present in high endothelial venules of lymphoid tissues. Detection of apoptotic Fas expressing lymphocytes adjacent to such vessels supports the idea that endothelial FasL functions as a selective gatekeeper by inducing apoptosis of Fas+ lymphocytes entering from the blood stream. / Tiivistelmä Solukalvon läpäisevän proteiinin, FasL:n, tehtävä on sitoutua kohdesolun Fas-reseptoriin ja indusoida kohdesolun apoptoosi. Fas/FasL-välitteinen apoptoosi on merkittävä tekijä immunologisessa säätelyssä. FasL ilmentyy pääsääntöisesti aktivoituneissa lymfosyyteissä. Olemme kuvanneet tutkimuksessamme FasL:n endoteelistä ilmentymistä imukudoksen eri toiminnallisissa alueissa ja suoliston lymfaattisessa kudoksessa. Lisäksi kartoitimme endoteelin FasL:n toiminnallista merkitystä analysoimalla sen yhteyttä lymfosyyttien alaryhmien apoptoosiin imusolmukkeissa ja arvioimalla FasL:n endoteelistä ilmentymistä suoliston limakalvon immunologisesti erilaisissa sairauksissa. Teimme immunohistokemiallisia värjäyksiä (Fas, FasL, CD3, CD20, CD19, CD23, CD56 ja FVIII) 20 reaktiiviselle imusolmukkeelle (I ja II), 60 lapsen endoskooppiselle biopsianäytteelle (III) sekä 60 suoliresekaattinäytteelle (IV). Optimoimme kaksoisvärjäysmenetelmän, missä yhdistettiin apoptoosin havainnointimenetelmä TUNEL ja FasL-, Fas- tai solulinjamarkkeri. Potilasryhmiin kuului potilaita, joilla oli normaalit imusolmukkeet, sekä potilaita, jotka sairastivat lasten viivästynyttä lehmänmaitoallergiaa, lasten keliakiaa, umpilisäketulehdusta, haavaista paksusuolitulehdusta tai Crohnin tautia. Verrokkiryhmiin kuului normaaleja biopsianäytteitä lapsipotilailta sekä terveitä resekaattinäytteitä umpilisäkkeestä sekä paksu- tai sykkyräsuolesta potilasryhmien mukaisesti. Jokaiselle anatomiselle alueelle suoritimme perusteellisen määrällisen analyysin (positiivista suonta tai solua per mm2). Osalle ryhmistä suoritimme seerumin liukoisen FasL:n määrityksen entsyymivälitteisellä immunosorbenttimäärityksellä. Reaktiivisissa imusolmukkeissa FasL:n ilmentyminen näkyi pääsääntöisesti parakortikaalialueen korkeaendoteelisissä venuleissa, missä myös apoptoottiset T- ja B-lymfosyytit (joista osa ilmensi Fasia) sittemmin näkyivät. Suoliston seinämässä havaitsimme verisuoniperäistä FasL:n ilmentymistä korkeaendoteelisissä suonissa lymfaattisten itukeskusten lähettyvillä. Niillä lapsipotilailla, joilla havaitsimme limakalvon lymfaattisten itukeskuksien lisääntymistä, oli myös seerumin FasL-pitoisuus koholla. Tulehduksellisissa suolistosairauksissa verisuoniperäinen FasL oli lisääntynyt limakalvon haavaumissa sekä Crohnin tautia sairastavien potilaiden submukoosassa. Tulokset osoittavat verisuoniperäisen FasL:n tyypillisesti ilmentyvän imukudoksen korkeaendoteelisissa suonissa. Apoptoosin havaitseminen Fasia ilmentävissä lymfosyyteissä näiden suonien läheisyydessä tukee ajatusta siitä, kuinka verisuoniperäinen FasL toimii valikoivana portinvartijana ja aiheuttaa Fas-positiivisten lymfosyyttien apoptoosin estämällä niiden pääsyn verenkierrosta.