Celiac disease is an autoimmune enteropathy driven by the ingestion of gluten in genetically predisposed individuals carrying HLA-DQ2 and/or -DQ8 genes. Currently, the only available treatment is a strict, life-long, gluten-free diet (GFD), which is very restrictive and not always effective, highlighting the need for alternative therapies. Celiac disease requires activation of both the innate (intraepithelial lymphocytes or IELs) and adaptive (lamina propria CD4+ T cells) arms of the immune system. Activation of these two pathways leads to the destruction of IEC and villous atrophy. Thus, IEC damage is a hallmark of CeD. However, IECs are not only the target of tissue damage; they also actively participate in CeD pathogenesis by translocating gluten peptides, expressing stress-induced markers, and releasing TG2 into the gut lumen to generate TG2-gluten complexes. Although IECs are known to express MHC, their role in gluten-dependent T cell activation has never been proven, partly because of the lack of an appropriate in vitro epithelial model expressing human MHC class II. This thesis aims to address this gap by developing a humanized organoid monolayer expressing the CeD risk gene HLA-DQ2.5, to investigate the interaction between IEC-gluten-T cells. The expression of epithelial MHC class II was evaluated in active and treated CeD patients, as well as in gluten-immunized and control (non-immunized; NI) DR3-DQ2.5 transgenic mice that express only CeD-associated MHC class II (HLA-DQ2.5). Active CeD patients and gluten-immunized DR3-DQ2.5 mice demonstrated higher expression of epithelial MHC class II compared with their treated and NI counterparts. Organoid monolayers developed from these mice and were treated with or without IFN-. Organoid monolayers derived from gluten-immunized DR3-DQ2.5 mice showed higher expression of MHC class II compared with NI mice, and this expression was upregulated by IFN- treatment. The functional consequences of MHC class II expression were determined by co-culturing organoid monolayers with CD4+ T cells in the presence of gluten and zein (a non-gluten protein). In the co-culture, gluten, but not zein, enhanced CD4+ T cell proliferation, activation, and release of cytokines, including IL-2, IFN- and IL-15, in the co-culture supernatants. Bacteria have recently emerged as modulators of inflammation in patients with CeD. It has been shown that opportunistic pathogens, including Pseudomonas aeruginosa, partially metabolize gluten into more immunogenic peptides. As such, the role of bacterially modified gluten in modulating the T cell response was assessed using the in vitro co-culture system I described. For this, monolayers were treated with the gluten pre-digested, or not, by elastase-producing P. aeruginosa or its lasB mutant. Gluten metabolized by P. aeruginosa, but not by the lasB mutant, significantly increased CD4+ T cell responses. In conclusion, MHC class II-expressing organoid monolayers are a functional model that can promote T cell responses under certain conditions. The model described in this thesis reveals a new immunomodulatory role for IECs in activating CD4+ T cells through MHC class II. This mechanism may serve to localize and further increase injury to the epithelium caused by gluten-specific CD4+ T cells in CeD. Therefore, therapeutics directed at IECs may offer a novel approach for modulating both adaptive and innate immunity in CeD, providing an alternative or adjuvant therapy to the current GFD treatment. / Thesis / Doctor of Philosophy (PhD) / Celiac disease is one of the most common food sensitivities, affecting approximately 1 in 100 people worldwide, including Canada. It occurs in people with specific genes (DQ2 and/or DQ8) when they eat gluten-containing foods such as wheat, barley, and rye. In people with celiac disease the immune system overreacts to gluten, damaging the lining of the upper gut, which we call “epithelium. This lining of cells constitutes the first barrier between the external world and our body, allowing in healthy conditions for nutrients to be absorbed but blocking the passage of gut microbes, some of which can cause disease or worsen gut inflammation. In patients with celiac disease, gluten crosses the epithelium into the gut tissue, where it activates specific cells of the immune system called “T cells”. Recently, there has been growing interest in whether the gut lining itself plays a role in triggering this immune response in celiac disease, though this has not yet been proven. If proven, this would suggest that the gut lining is responsible for directing the harmful immune response to gluten and should be considered a target site when developing therapies to prevent or treat celiac disease. This concept has been difficult to prove because we do not have a model to investigate this question. Such a model would require a gut lining that carries the genes linked to celiac disease. My thesis describes the development of such a model, made of a gut lining from a mouse genetically modified to carry human celiac disease genes. Using this model, I found that when the gut lining was exposed to certain molecules present in celiac patients (cytokines), it switched on other molecules that ultimately activated T cells. Additionally, I demonstrated that certain microbes, such as Pseudomonas aeruginosa, which are present in higher numbers in the upper gut of patients with celiac disease, can break down gluten into fragments that further activate T cells. The results validated the use of this model to understand what other co-factors can tip the balance in a person with celiac genes to remain healthy or develop inflammation. In summary, I demonstrated that the gut lining expressing celiac genes actively participates in the activation of immune cells that drive intestinal damage in celiac disease. This new model is a novel tool to continue to identify additional co-factors that predispose patients to celiac disease, as well as to screen for novel therapies for celiac disease. This is important, as the only currently available treatment is a strict lifelong gluten-free diet, which has many limitations, including frequent contamination and celiac disease reactivation.

The epithelial cells lining the intestinal mucosa separate the underlying tissue from components of the intestinal lumen. Innate immunity mediated by intestinal epithelial cells (IECs) provides rapid protective functions against microorganisms. Innate immunity also participates in orchestrating adaptive immunity. Key components in innate defence are defensins. To study the production of defensins and how it is affected by intestinal inflammation IECs were isolated from the small and large intestines of patients suffering from ulcerative colitis (UC), Crohn´s disease (MbC), celiac disease (CD), and from controls, and analyzed by quantitative RT-PCR (qRT-PCR) and immunoflow cytometry. Defensin expressing cells were also studied by in situ hybridization and immunohistochemistry. Normally, only small intestinal Paneth cells express human α-defensin 5 (HD-5) and HD-6. In UC colon IECs, HD-5, HD-6, and lysozyme mRNAs were expressed at high levels. In Crohn´s colitis colon the levels of HD-5 and lysozyme mRNAs were also increased although not to the same extent as in UC. No increase was detected in MbC with ileal localization. Metaplastic Paneth cell differentiation in UC colon was primarily responsible for the expression of the antimicrobial components. Human β-defensin 1 (hBD-1) mRNA was more abundant in large than in small intestine of controls, and remained unchanged in UC and MbC. hBD-2 mRNA was barely detectable in normal intestine and was induced in UC IECs but not in MbC IECs. mRNAs for the recently discovered hBD-3 and hBD-4, were detected in IECs from both small and large intestine. Both hBD-3 and hBD-4 mRNA were significantly increased in IECs of UC patients but not of MbC patients. Bacteria and IL-1β induced hBD-2 but not hBD-1 mRNA in colon carcinoma cell lines. IFN-γ, but not TNF-α or IL-1β, augmented hBD-3 expression in these cells, while none of the agents induced hBD-4. High antimicrobial activity of IECs in UC may be a consequence of changes in the epithelial lining, which permit the adherence of microorganisms. Unexpectedly, in situ hybridization revealed expression of hBD-3 and hBD-4 mRNAs by numerous lamina propria cells in colonic tissue from UC patients. These cells were identified as plasma cells (CD138+). hBD-3 and hBD-4 mRNAs were also demonstrated in the plasmacytoma cell line U266. This is the first demonstration of defensins in plasma cells. The four prominent constituents of the intestinal glycocalyx, carcinoembryonic antigen (CEA), CEA cell adhesion molecule 1 (CEACAM1), CEACAM6 and CEACAM7 all seem to play a critical role in innate defence of the intestinal mucosa by trapping and expelling microorganisms at the epithelial surface. The inducibility of these molecules in colonic epithelial cell lines was analyzed by qRT-PCR, immunoflow cytometry, and immunoelectron microscopy. IFN-g but not bacteria, LPS, TNF-α, or IL-1β modified the expression of CEA, CEACAM1 and CEACAM6. None of these agents modified CEACAM7 expression. IFN-γ was shown to have two effects: a direct effect on CEACAM1 transcription, and promotion of cell differentiation resulting in increased CEA and CEACAM6 and decreased CEACAM7 expression. Scanning electron microscopy of jejunal biopsies from children with CD revealed the presence of rod shaped bacteria in ~40% of patients with active CD, but only in 2% of controls. 19% of treated CD patients still had adhering bacteria. Presence of bacteria is not due to lack of antimicrobial factors. In fact, HD-5, HD-6, and lysozyme mRNA levels were significantly increased in IECs of patients with active CD. hBD-1 and hBD-2 were unchanged. Lack of induction of hBD-2 may reflect disturbed signalling in IECs of CD patients. Analysis of CEA and CEACAM1 mRNA/protein expression showed no differences between CD patients and controls. Analysis of the mucins MUC2 and MUC3 revealed significantly increased MUC2 levels in active disease and unchanged MUC3. Immunohistochemistry demonstrated goblet cell metaplasia as well as staining of the apical portion of absorptive cells. Glycosylation status of proteins was studied by lectin histochemistry. Goblet cells in the mucosa of CD patients were stained by the lectin UEAI. This was not seen in controls. The lectin PNA stained the glycocalyx of controls but not that of CD patients. Thus, unique carbohydrate structures of the glycocalyx/mucous layer are likely discriminating features of CD patients and may allow bacterial binding. We conclude that the intestinal epithelium is heavily involved in the innate defence of the mucosa and that its reactive pattern is affected by intestinal inflammation. Keywords: human intestinal mucosa; epithelial cells; innate immunity; defensin; ulcerative colitis; Crohn´s disease; celiac disease; glycoαcalyx; mucin

A doença celíaca (DC) é uma doença inflamatória induzida pela ingestão de glúten em indivíduos geneticamente predispostos e seu tratamento é baseado em uma dieta sem glúten por toda a vida. A doença celíaca refratária é um problema comum que afeta de 10% a 19% dos pacientes célicos tratados. Provavelmente, a contaminação da dieta por glúten é uma das razões principais para a persistência de sintomas em pacientes celíacos tratados, assim como a ingestão inadvertida de glúten, devido a rotulagem incorreta. Assim, o objetivo deste estudo foi avaliar a confiabilidade dos rótulos dos alimentos brasileiros processados, através de testes de contaminação de glúten nos seguintes grupos (a) produtos \"livres de glúten\" - preparados especificamente para a população celíaca; (b) produtos \"naturalmente sem glúten\" feitos com arroz, milho, soja e mandioca, utilizados por toda a população e (c) produtos rotulados com \"contém glúten\", mas que não apresentam glúten em sua composição no rótulo. Foram analisados 213 produtos alimentícios agrupados em: 115 produtos do grupo \"sem glúten\"; 86 produtos do grupo \"naturalmente sem glúten\" e 12 produtos do grupo rotulados com \"contém glúten\". O teor de glúten foi detectado e quantificado por ELISA-R5 (Ridascreen®gliadin) e os resultados foram expressos em ppm e mg/100 g de alimento. A linha de corte foi estabelecida em 20 ppm para a contaminação de glúten. Todas as contaminações por glúten foram confirmadas por Western-blotting. Resultados: (a) alimentos livres de glúten 15 das 115 (13%) apresentaram contaminação por glúten (20 ppm), (b) grupo de alimentos naturalmente sem glúten - 8 de 86 (9,3%) apresentaram contaminação por glúten (20 ppm); (c) grupo de alimentos rotulados com contem glúten - somente 2 de 12 (16,7%) apresentaram contaminação por glúten (20 ppm). A análise de Western-blotting confirmou 36 das 38 (95%) contaminações encontradas no ELISA-R5. CONCLUSÕES: Ambos os grupos de alimentos \"sem glúten\" e \"naturalmente sem glúten\" comercializados no Brasil apresentaram razoável porcentagem de contaminação por glúten, o que dificulta a realização de uma dieta adequada ao paciente celíaco. O grupo de alimentos rotulado \"com glúten\" não apresentou 100% de contaminação, o que revela que a rotulagem desses produtos deve ser feita como uma medida preventiva. Uma maior chance de contaminação pelo glúten foi observada para os produtos a base de arroz (13,6x), soja (13,3x) e milho (9,3x), mas não naqueles à base de mandioca. Em média, encontramos 10,8% (23 de 213) de contaminação de glúten para os alimentos analisados, um panorama positivo para a população brasileira celíaca, principalmente devido ao uso da mandioca, uma alternativa para a farinha de trigo. No entanto, a contaminação de glúten encontrada mostra a importância da quantificação de glúten em todos os alimentos industrializados. / Celiac disease (CD) is an inflammatory disorder induced by ingestion of gluten in genetically predisposed individuals and its treatment is based on a life-time gluten-free diet. Nonresponsive celiac disease is a common problem affecting from 10% to 19% of treated celiac patients. Probably a gluten contamination in diet is one of the major reasons for symptoms persistence in celiac patients as well as an inadvertent gluten intake due to a misleading nutritional label. The aim of this study was to evaluate the reliability of Brazilian processed food labels by testing gluten contamination in (a) gluten-free products - prepared specifically for the celiac population; (b) in naturally gluten-free products made with rice, corn, soy bean and cassava and used by all population and (c) in not gluten-free products labeled to contain gluten but not having it in their composition. We analyzed 213 food samples grouped accordingly to its type: 115 samples of \"gluten-free food, 86 samples of \"naturally gluten-free food and 12 samples of not-gluten free labeled products. The gluten content was detected and quantified by ELISA-R5 (Ridascreen® Gliadin) and the results were expressed in ppm and mg/100 g of food. A cut-off line was established in 20 ppm for gluten contamination. All gluten contaminations were confirmed by Western-blotting. Results: (a) Gluten-free foods - we found 100 of 115 samples (87%) with no contamination (< 20 ppm) and 15 of 115 (13%) showed gluten contamination 20 ppm; (b) Naturally Gluten-free foods - we found 78 of 86 samples (90,7%) showing no contamination (< 20 ppm) and 8 of 86 (9,3%) with gluten levels 20 ppm; (c) Not gluten-free foods - we found 10 of 12 samples (83,3%) showing no contamination (< 20 ppm) and 2 of 12 (16,7%) with gluten contamination 20 ppm. The Western-blotting analysis confirmed 36 of 38 (95%) contaminations found in the ELISA-R5. CONCLUSIONS: Both \"gluten-free and \"naturally gluten-free foods commercialized in Brazil have presented some gluten contamination making a restricted gluten-free diet hard to be achieved by the celiac population. Unexpectedly the not gluten-free group was not entirely contaminated showing a preventive measure in labeling by food companies. A higher odds ratio for gluten contamination was observed for products made with rice (13.6), soy bean (13.3) and corn (9.3) but not to cassava products (not significant). In general, we found a 10.8% (23 of 213) of gluten contamination for all food products analyzed, a positive panorama for the Brazilian celiac population mainly due to cassava products, an alternative for wheat starch. Nevertheless the gluten contamination found here leads us to the importance for a gluten quantification in all industrialized food to guarantee an appropriated diet to the Brazilian celiac group

A doença celíaca é uma desordem autoimune caracterizada pela intolerância ao glúten, provavelmente subdiagnosticada devido ao amplo espectro de apresentação clínica, e que se não tratada pode ocasionar graves complicações. Considerando o fato de que certas manifestações bucais podem ser indicativas da doença celíaca, os cirurgiões-dentistas podem desempenhar um importante papel na diagnose desta condição. Neste estudo, 52 celíacos entre 2 e 23 anos, além de um grupo controle composto por 52 indivíduos pareados por sexo e idade, foram sistematicamente avaliados quanto à presença de estomatite aftosa recorrente, defeitos do esmalte dental e ocorrência de cárie dental tanto na dentição decídua quanto na permanente, tendo também sido submetidos a análise salivar (pH, fluxo e capacidade tampão). Paralelamente, 10 molares decíduos de pacientes celíacos e 10 provenientes de um grupo controle sem a doença tiveram a composição química de seu esmalte analisada através da Espectroscopia no Infravermelho por Transformada de Fourier e Espectroscopia por Energia Dispersiva de Raios-X. E, finalmente, 27 crianças com defeitos de formação no esmalte dental foram submetidas ao diagnóstico sorológico da doença celíaca. Foi observado que os defeitos específicos do esmalte dental são mais comuns entre os celíacos que em controles (57,7% vs 13,46%), bem como a estomatite aftosa recorrente (40,38% vs 17,31%). Contrariamente, uma menor frequência de lesões de cáries dentais foi observada no grupo de doentes celíacos (2,11 vs 3,9). A análise salivar mostrou maior ocorrência de baixo fluxo no grupo celíaco (36% vs 12%), enquanto o pH e a capacidade tampão foram estatisticamente semelhantes em ambos os grupos. A composição química, apesar de não apresentarem diferenças nas concentrações de cálcio e fósforo entre os grupos, revelou uma menor proporção Ca/P nos dentes decíduos dos celíacos (1,35 vs 1,58). E por fim, os testes sorológicos (anti-tTG IgA) não evidenciaram doença celíaca nas crianças portadoras de defeitos específicos do esmalte dental. / Celiac disease is an autoimmune disorder characterized by gluten intolerance, believed to be underdiagnosed owing to its wide spectrum of clinical presentation, and if untreated it can lead to severe complications. Considering that particular oral manifestations might be indicative of celiac disease, dentists could play a pivotal role in helping the diagnosis of this condition. In this study, a group of 52 Brazilian celiac patients aged 2 to 23 years, along with an age and sex-matched control group of 52 individuals, were systematically evaluated for the presence of recurrent aphthous stomatitis, dental enamel defects and caries in both deciduous and permanent dentition, and were also subjected to salivary analysis pH, stimulated saliva flow and buffering capacity. Concurrently, 10 deciduous molar teeth from celiac patients and 10 from a non-celiac control group had their enamel chemically analyzed using Fourier Transform Infrared Spectroscopy and Energy Dispersive X-ray analysis. Finally, 27 healthy children with specific dental enamel defects underwent serologic screening for celiac disease. Specific dental enamel defects were found to be more common among celiac patients than in controls (57,7% vs 13,46%), as well as recurrent aphthous stomatitis (40,38% vs 17,31%). Conversely, a smaller frequency of dental caries was observed in the disease group (2,11 vs 3,9). Salivary analysis showed a higher frequency of reduced flow in the celiac group (36% vs 12%), while saliva consistency, pH and buffering capacity were statistically similar in both groups. Chemical composition, although presenting no statistical differences in calcium and phosphorus concentrations between the groups, revealed a lower proportion Ca/P in the deciduous teeth of celiac individuals (1,35 vs 1,58). Lastly, after serological testing (anti-tTG IgA), no celiac disease was found among the children with specific dental enamel defects.

A doença celíaca (DC) é uma doença autoimune causada pela intolerância ao glúten e o único tratamento existente é sua exclusão permanente na dieta. O glúten apresenta propriedades viscoelásticas que proporcionam características físicas e sensoriais desejáveis nos produtos de panificação e sua remoção, muitas vezes pode dificultar o desenvolvimento desses produtos. Dessa maneira, o objetivo desta pesquisa foi estudar o efeito da farinha de chia sobre as características reológicas das massas sem glúten e avaliar a influência dessas características sobre os atributos físicos dos biscoitos. A farinha de chia foi usada para substituir (10, 20 e 30) % de fécula de mandioca e também foi formulado um biscoito sem farinha de chia, como controle. A adição de farinha de chia alterou as propriedades de hidratação da massa, seu alto conteúdo de fibras, proteínas e mucilagem elevaram significativamente (p &lt; 0,05) os teores de capacidade de absorção de água (CAA), capacidade de retenção de água (CRA) e volume de inchamento (VI). Os resultados da varredura de frequência mostraram que o módulo de armazenamento (G\') foi superior ao módulo de perda (G\'\') para todas as massas, indicando um comportamento sólido-elástico. À medida que aumentou a porcentagem de farinha de chia nas formulações dos biscoitos foram obtidas massas com menor viscosidade extensional biaxial e maior recuperação elástica. Os modelos matemáticos da abordagem da derivada fracionária e de Burgers foram ajustados aos pontos experimentais do teste de fluência e recuperação com bons coeficientes de determinação (R2 ~ 0,97). Os parâmetros reológicos desses modelos obtidos para as massas foram correlacionados às propriedades físicas do biscoito e podem ser usados como indicadores de qualidade na elaboração de biscoito sem glúten. A substituição da fécula de mandioca por 30% de farinha de chia resultou em biscoitos com maior densidade aparente, umidade e firmeza, menor valor para o fator diâmetro/espessura e coloração na superfície mais escura. Correlações significativas entre as propriedades físicas dos biscoitos e o parâmetro de recuperação elástica (ensaio de fluência e recuperação) e de viscosidade extensional biaxial da massa foram obtidas. Os resultados encontrados sugerem que os ensaios reológicos aplicados à massa poderiam ser correlacionados com algumas propriedades finais (densidade, fator de espalhabilidade e textura) dos biscoitos, e que a substituição parcial de farinha de chia poderia ser utilizada para melhorar as características físicas e nutricionais na produção de biscoitos sem glúten. / Celiac disease (CD) is an autoimmune disease caused by gluten intolerance, and the only existing treatment is its permanent exclusion from the diet. Gluten has viscoelastic properties that provide desirable physical and sensory characteristics in bakery products and its removal can often hamper the development of these products. Thus, the aim of this study was to investigate the effect of chia flour on rheological characteristics of gluten-free doughs and to evaluate the influence of these characteristics on the physical attributes of the biscuits. Chia flour was used to replace (10, 20 and 30) % of cassava starch and also as control biscuit without chia flour was formulated. The addition of chia flour altered the hydration properties of the dough, its high content of fibers, proteins and mucilage significantly increased (p&lt;0.05) water absorption capacity (WCA), water retention capacity (WCR) and swelling volume (VS). The frequency sweep results showed that the storage modulus (G \') was higher than loss modulus (G\') for all doughs, indicating a solid-elastic behavior. As the percentage of chia flour increased in the biscuit formulations the doughs with lower apparent biaxial extensional viscosity and greater elastic recovery. The mathematical models of the fractional derivative modelling approach and Burgers were adjusted to the experimental points obtained in the fluency and recovery test with a good coefficient of determination (R2 ~ 0.97) and the rheological parameters of these models were correlated to the physical properties of the biscuit and can be used as indicators of quality in the preparation of gluten-free biscuits. The replacement of cassava starch with 30% of chia flour resulted in biscuits with higher bulk apparent density, moisture and firmness, lower diameter/thickness factor and darker surface coloration. Significant correlations between the physical properties of the biscuit doughs and the rheological parameters (creep and recovery test) and biaxial extensional viscosity were obtained. The results suggest that rheological tests applied to the doughs could be correlated with some final properties (density, spreading factor, texture) of the biscuits, and that the partial substitution of cassava starch with chia flour could be used to improve the physical and nutritional characteristics in the manufacture of gluten-free biscuits.

A doença celíaca é uma desordem autoimune caracterizada pela intolerância ao glúten, provavelmente subdiagnosticada devido ao amplo espectro de apresentação clínica, e que se não tratada pode ocasionar graves complicações. Considerando o fato de que certas manifestações bucais podem ser indicativas da doença celíaca, os cirurgiões-dentistas podem desempenhar um importante papel na diagnose desta condição. Neste estudo, 52 celíacos entre 2 e 23 anos, além de um grupo controle composto por 52 indivíduos pareados por sexo e idade, foram sistematicamente avaliados quanto à presença de estomatite aftosa recorrente, defeitos do esmalte dental e ocorrência de cárie dental tanto na dentição decídua quanto na permanente, tendo também sido submetidos a análise salivar (pH, fluxo e capacidade tampão). Paralelamente, 10 molares decíduos de pacientes celíacos e 10 provenientes de um grupo controle sem a doença tiveram a composição química de seu esmalte analisada através da Espectroscopia no Infravermelho por Transformada de Fourier e Espectroscopia por Energia Dispersiva de Raios-X. E, finalmente, 27 crianças com defeitos de formação no esmalte dental foram submetidas ao diagnóstico sorológico da doença celíaca. Foi observado que os defeitos específicos do esmalte dental são mais comuns entre os celíacos que em controles (57,7% vs 13,46%), bem como a estomatite aftosa recorrente (40,38% vs 17,31%). Contrariamente, uma menor frequência de lesões de cáries dentais foi observada no grupo de doentes celíacos (2,11 vs 3,9). A análise salivar mostrou maior ocorrência de baixo fluxo no grupo celíaco (36% vs 12%), enquanto o pH e a capacidade tampão foram estatisticamente semelhantes em ambos os grupos. A composição química, apesar de não apresentarem diferenças nas concentrações de cálcio e fósforo entre os grupos, revelou uma menor proporção Ca/P nos dentes decíduos dos celíacos (1,35 vs 1,58). E por fim, os testes sorológicos (anti-tTG IgA) não evidenciaram doença celíaca nas crianças portadoras de defeitos específicos do esmalte dental. / Celiac disease is an autoimmune disorder characterized by gluten intolerance, believed to be underdiagnosed owing to its wide spectrum of clinical presentation, and if untreated it can lead to severe complications. Considering that particular oral manifestations might be indicative of celiac disease, dentists could play a pivotal role in helping the diagnosis of this condition. In this study, a group of 52 Brazilian celiac patients aged 2 to 23 years, along with an age and sex-matched control group of 52 individuals, were systematically evaluated for the presence of recurrent aphthous stomatitis, dental enamel defects and caries in both deciduous and permanent dentition, and were also subjected to salivary analysis pH, stimulated saliva flow and buffering capacity. Concurrently, 10 deciduous molar teeth from celiac patients and 10 from a non-celiac control group had their enamel chemically analyzed using Fourier Transform Infrared Spectroscopy and Energy Dispersive X-ray analysis. Finally, 27 healthy children with specific dental enamel defects underwent serologic screening for celiac disease. Specific dental enamel defects were found to be more common among celiac patients than in controls (57,7% vs 13,46%), as well as recurrent aphthous stomatitis (40,38% vs 17,31%). Conversely, a smaller frequency of dental caries was observed in the disease group (2,11 vs 3,9). Salivary analysis showed a higher frequency of reduced flow in the celiac group (36% vs 12%), while saliva consistency, pH and buffering capacity were statistically similar in both groups. Chemical composition, although presenting no statistical differences in calcium and phosphorus concentrations between the groups, revealed a lower proportion Ca/P in the deciduous teeth of celiac individuals (1,35 vs 1,58). Lastly, after serological testing (anti-tTG IgA), no celiac disease was found among the children with specific dental enamel defects.

A doença celíaca (DC) é uma doença autoimune causada pela intolerância ao glúten e o único tratamento existente é sua exclusão permanente na dieta. O glúten apresenta propriedades viscoelásticas que proporcionam características físicas e sensoriais desejáveis nos produtos de panificação e sua remoção, muitas vezes pode dificultar o desenvolvimento desses produtos. Dessa maneira, o objetivo desta pesquisa foi estudar o efeito da farinha de chia sobre as características reológicas das massas sem glúten e avaliar a influência dessas características sobre os atributos físicos dos biscoitos. A farinha de chia foi usada para substituir (10, 20 e 30) % de fécula de mandioca e também foi formulado um biscoito sem farinha de chia, como controle. A adição de farinha de chia alterou as propriedades de hidratação da massa, seu alto conteúdo de fibras, proteínas e mucilagem elevaram significativamente (p &lt; 0,05) os teores de capacidade de absorção de água (CAA), capacidade de retenção de água (CRA) e volume de inchamento (VI). Os resultados da varredura de frequência mostraram que o módulo de armazenamento (G\') foi superior ao módulo de perda (G\'\') para todas as massas, indicando um comportamento sólido-elástico. À medida que aumentou a porcentagem de farinha de chia nas formulações dos biscoitos foram obtidas massas com menor viscosidade extensional biaxial e maior recuperação elástica. Os modelos matemáticos da abordagem da derivada fracionária e de Burgers foram ajustados aos pontos experimentais do teste de fluência e recuperação com bons coeficientes de determinação (R2 ~ 0,97). Os parâmetros reológicos desses modelos obtidos para as massas foram correlacionados às propriedades físicas do biscoito e podem ser usados como indicadores de qualidade na elaboração de biscoito sem glúten. A substituição da fécula de mandioca por 30% de farinha de chia resultou em biscoitos com maior densidade aparente, umidade e firmeza, menor valor para o fator diâmetro/espessura e coloração na superfície mais escura. Correlações significativas entre as propriedades físicas dos biscoitos e o parâmetro de recuperação elástica (ensaio de fluência e recuperação) e de viscosidade extensional biaxial da massa foram obtidas. Os resultados encontrados sugerem que os ensaios reológicos aplicados à massa poderiam ser correlacionados com algumas propriedades finais (densidade, fator de espalhabilidade e textura) dos biscoitos, e que a substituição parcial de farinha de chia poderia ser utilizada para melhorar as características físicas e nutricionais na produção de biscoitos sem glúten. / Celiac disease (CD) is an autoimmune disease caused by gluten intolerance, and the only existing treatment is its permanent exclusion from the diet. Gluten has viscoelastic properties that provide desirable physical and sensory characteristics in bakery products and its removal can often hamper the development of these products. Thus, the aim of this study was to investigate the effect of chia flour on rheological characteristics of gluten-free doughs and to evaluate the influence of these characteristics on the physical attributes of the biscuits. Chia flour was used to replace (10, 20 and 30) % of cassava starch and also as control biscuit without chia flour was formulated. The addition of chia flour altered the hydration properties of the dough, its high content of fibers, proteins and mucilage significantly increased (p&lt;0.05) water absorption capacity (WCA), water retention capacity (WCR) and swelling volume (VS). The frequency sweep results showed that the storage modulus (G \') was higher than loss modulus (G\') for all doughs, indicating a solid-elastic behavior. As the percentage of chia flour increased in the biscuit formulations the doughs with lower apparent biaxial extensional viscosity and greater elastic recovery. The mathematical models of the fractional derivative modelling approach and Burgers were adjusted to the experimental points obtained in the fluency and recovery test with a good coefficient of determination (R2 ~ 0.97) and the rheological parameters of these models were correlated to the physical properties of the biscuit and can be used as indicators of quality in the preparation of gluten-free biscuits. The replacement of cassava starch with 30% of chia flour resulted in biscuits with higher bulk apparent density, moisture and firmness, lower diameter/thickness factor and darker surface coloration. Significant correlations between the physical properties of the biscuit doughs and the rheological parameters (creep and recovery test) and biaxial extensional viscosity were obtained. The results suggest that rheological tests applied to the doughs could be correlated with some final properties (density, spreading factor, texture) of the biscuits, and that the partial substitution of cassava starch with chia flour could be used to improve the physical and nutritional characteristics in the manufacture of gluten-free biscuits.

Zöliakie ist eine immunologisch vermittelte Erkrankung bei der die Dünndarm-schleimhaut durch das in zahlreichen Getreidesorten vorkommende Klebereiweiß Gliadin geschädigt wird. Dabei wird die typische Architektur der Mukosa zerstört und imponiert histologisch als Zottenatrophie. In Folge dessen zeigen Betroffene Mangelerscheinungen und Verdauungsbeschwerden sowie zahlreiche extra-intestinale, atypische Symptome. Bei Kindern können zusätzlich gravierende Wachstums- und Entwicklungsstörungen auftreten. Die Therapie besteht in einer lebenslangen glutenfreien Diät. Die Diagnostik der Erkrankung basiert auf vier Säulen: Neben der Beurteilung der klinischen Symptomatik werden zöliakie-typische Antikörper nachgewiesen, welche bei hoher Konzentration die Indikation zur Biopsie darstellen. Die bioptische Untersuchung mit anschließendem histolo-gischem Nachweis der Zottenatrophie stellt den Goldstandard der Diagnostik dar und wird durch die Besserung der klinischen Symptomatik unter glutenfreier Diät gestützt. Bei der serologischen Untersuchung haben Antikörper gegen natives Gliadin auf Grund niedriger diagnostischer Genauigkeit an Bedeutung verloren. Sie wurden durch die Bestimmung von Autoantikörpern gegen die Gewebstransglutaminase abgelöst, die eine höhere Sensitivität und Spezifität aufweisen. Im Jahr 2000 konn-te jedoch gezeigt werden, dass sich Antikörper von Zöliakiepatienten an Gliadin-peptide besser nach selektiver Deamidierung (Austausch der Aminosäure Glutamin durch Glutaminsäure) binden. Darauf aufbauend entwickelte die Firma INOVA Diagnostics im Jahr 2006 erst-mals einen ELISA zur Zöliakiediagnostik mit synthetisch hergestellten, deamidier-ten Gliadinpeptiden (DGP) als Antigen. Zu Beginn unserer Arbeit existierten keine Veröffentlichungen zur diagnostischen Genauigkeit dieser Tests bei Kindern und nur eine Veröffentlichung, die zwei der ELISA an Seren von erwachsenen Patienten untersuchte. Bei Kindern ist es jedoch besonders wichtig, durch die Antikörperbestimmung eine hohe Diagnosesicher-heit zu erlangen, weil für sie die Biopsie eine große Belastung darstellt und eine nicht erkannte Zöliakieerkrankung zu schwerwiegenden Entwicklungsstörungen führen kann. Aus diesem Grund soll die vorliegende Studie den Nutzen dieser neuen ELISA in der Diagnostik der Zöliakie im Kindesalter evaluieren. Es wurden dazu insgesamt 340 Seren von bioptisch bestätigten Zöliakiepatienten und Kontrollen, bei denen die Erkrankung histologisch ausgeschlossen wurde, gesammelt, verblindet und retrospektiv analysiert. Dabei wurden vier verschiede-ne ELISA der Firma INOVA Diagnostics eingesetzt: drei ELISA mit DGP als An-tigen sowie ein weiterer ELISA, in dem DGP mit Gewebstransglutaminase kombiniert war. Es wurden folgende Erkenntnisse gewonnen: 1. Alle vier ELISA eignen sich zur Diagnostik von Zöliakie bei Kindern und weisen eine hohe Trennschärfe auf. Die Fläche unter der Receiver Operating Characteristic (ROC)-Kurve ist für alle vier Tests größer als 0,96. Mit den Tests kann somit die Indikation zur bioptischen Untersuchung mit großer Sicherheit gestellt werden. 2. Die Bestimmung der Antikörper gegen DGP ist der Antikörperbestimmung gegen natives Gliadin überlegen. Die DGP-Tests weisen eine signifikant größe-re Fläche unter der ROC-Kurve als die Tests auf Antikörper gegen natives Gli-adin auf. Die Bestimmung der Antikörper gegen DGP ist der Bestimmung von Antikörpern gegen Gewebstransglutaminase nicht unterlegen. Die Flächen un-ter den ROC-Kurven unterscheiden sich nicht signifikant voneinander. 3. Überraschenderweise zeigt die IgG-Klasse der DGP eine signifikant höhere diagnostische Genauigkeit als die IgA-Klasse. Somit ist auch bei Patienten mit IgA-Mangel eine sichere Diagnosestellung gegeben und es kann auf die gene-relle Bestimmung des Gesamt-IgA verzichtet werden. 4. Der DGP-IgG-Test weist von den vier validierten Antikörpertests bei einer Sensitivität von 100 % die höchste Spezifität (90 %) und bei einer Spezifität von 100 % die höchste Sensitivität (64 %) auf. 5. Durch den kombinierten Test mit zwei Antigenen und den gleichzeitigen Nachweis von IgA und IgG in einem ELISA (tTG/DGP-Screen-Test) lassen sich Kosten und Zeit sparen. Dieser kombinierte Test weist die höchste diagnosti-sche Genauigkeit der untersuchten DGP-Tests auf. 6. Durch Angabe von Likelihood Ratios und mittels grafischer Darstellung der Posttest-Wahrscheinlichkeit in Abhängigkeit der Antikörperkonzentration und Prävalenz können den behandelnden Ärzten wertvolle Informationen zur Di-agnosesicherheit eines einzelnen Testergebnisses vermittelt werden. Mit den evaluierten DGP-Tests stehen neue und zuverlässige ELISA zur Diagnos-tik der Zöliakie im Kindesalter zur Verfügung. Sie weisen eine hohe diagnostische Sicherheit auf und unterscheiden sicher zwischen Gesunden und Zöliakiepatien-ten. Die Indikation für eine Dünndarmbiopsie kann mit Hilfe der DGP-Tests sehr sicher gestellt werden. Ob darüber hinaus auf der Basis sehr hoher Antikörper-konzentrationen ohne histologische Untersuchung die Diagnose Zöliakie ausrei-chend sicher gestellt werden kann oder bei sehr niedrigen Konzentrationen auf einen bioptischen Ausschluss verzichtet werden kann, soll in einer weiteren be-reits in Planung befindlichen prospektiven Studie geklärt werden. Zusätzlich sollte die Diagnosestellung durch DGP-Tests bei besonders jungen Kindern in prospektiven Studien untersucht werden. Die vorliegende Arbeit legt gemeinsam mit anderen Untersuchungen den Grundstein dafür.

Inflammatory bowel disease (including Crohn’s disease and ulcerative colitis) and coeliac disease are debilitating gastrointestinal diseases that seriously affect the quality of life of those affected. Under normal circumstances, the intestinal immune system is maintained in a state of controlled inflammation, whereby balance exists between protective immunity, mediated by effector cells, and tolerance mediated by cells with regulatory function. However, an aberrant immune response is believed to contribute to the intestinal inflammation present in individuals afflicted by these diseases. This thesis investigated the involvement of CD4⁺ CD25[superscript]high Foxp3⁺ Regulatory T cells (Treg) and Th17 Effector cells in both inflammatory bowel disease (IBD) and coeliac disease. The reciprocal relationship between Treg and Th17 cells under certain cytokine conditions, has prompted the exploration of these two cell types in IBD and coeliac disease. Previous studies have examined these factors individually in a range of diseases, however, to our knowledge the study of both Treg and Th17 in IBD and coeliac disease subjects represents a novel area of research. Crohn’s disease (CD), ulcerative colitis (UC) and coeliac disease subjects were recruited through the Department of Gastroenterology and Hepatology at The Queen Elizabeth Hospital (QEH) in Adelaide, South Australia. In total, one-hundred and seventeen subjects were enlisted in this study to donate blood samples. In addtion, intestinal biopsy samples were collected from fifty-six subjects undergoing colonoscopy at the QEH Department of Gastroenterology and Hepatology. All subjects participated, with informed consent and ethics approval. Treg and Th17 cell numbers were investigated in the peripheral blood of Crohn’s disease, ulcerative colitis, coeliac disease and control subjects using multi-colour, intracellular flow cytometry. A decrease in Treg cell numbers and an increase in Th17 cell numbers was observed in IBD, but not in coeliac disease. Closer investigation into the ratio of Treg and Th17 cells within patients identified a near 1:1 Treg/Th17 ratio in control subjects, but a lower Treg/Th17 ratio in IBD patients. This suggested a disturbance in regulatory and effector cell equilibrium. Furthermore, the excess of Th17 cells and deficiency of Tregs could contribute to the pathologies observed in IBD. The discovery of an imbalance in Treg and Th17 cell numbers in IBD prompted further investigation of these cells in intestinal biopsies collected from IBD, coeliac and control subjects. Real time RT-PCR of intestinal biopsy samples demonstrated increased expression of the Th17 cytokine, IL-17a, in both IBD and coeliac disease. Elevated levels of the Treg transcription factor Foxp3 were also identified in intestinal biopsies from IBD subjects. It was therefore hypothesised that Treg cells may have been actively recruited from the periphery in an attempt to control inflammation in the gut; however, the intestinal cytokine microenvironment may have restricted the regulatory function of these cells. Cytokines known to promote human Th17 differentiation, namely IL-1β, IL-6, TGF-β, IL-21 and IL-23, were explored in intestinal biopsy samples from IBD, coeliac and control subjects. High levels of IL-1β and IL-6 were detected in IBD patient samples, however, no change in levels of IL-21 or IL-23 were observed in IBD or coeliac disease subjects. Elevated levels of TGF-β were only identified in UC. No changes in cytokine expression were observed between control and coeliac subjects, except a significant decrease in IL-6 levels was identified in coeliac disease sufferers. The pro-inflammatory microenvironment identified in intestinal biopsies from IBD subjects may have promoted the continual differentiation and development of Th17 cells, whilst restricting Treg activity. Moreover, the observed deficiency of Treg in IBD patients may have impaired the ability of the immune system to limit excessive pathogenic Th17 driven immune responses in the intestinal mucosa. Therefore, therapeutic approaches that aim to re-establish regulatory and effector cell homeostasis by increasing Treg numbers in IBD patients, and specifically targeting Th17 cells, may prove effective in the treatment of IBD. Approaches such as these could provide greater focus to treatment strategies for IBD management compared to current broad-spectrum immunosuppressive therapies that could increase susceptibility to cancer or infection in IBD patients. In addition, the imbalance of regulatory and effector cells demonstrated in the peripheral blood of IBD patients may potentially provide new options for a noninvasive diagnostic tool. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1457580 / Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 2009

Inflammatory bowel disease (including Crohn’s disease and ulcerative colitis) and coeliac disease are debilitating gastrointestinal diseases that seriously affect the quality of life of those affected. Under normal circumstances, the intestinal immune system is maintained in a state of controlled inflammation, whereby balance exists between protective immunity, mediated by effector cells, and tolerance mediated by cells with regulatory function. However, an aberrant immune response is believed to contribute to the intestinal inflammation present in individuals afflicted by these diseases. This thesis investigated the involvement of CD4⁺ CD25[superscript]high Foxp3⁺ Regulatory T cells (Treg) and Th17 Effector cells in both inflammatory bowel disease (IBD) and coeliac disease. The reciprocal relationship between Treg and Th17 cells under certain cytokine conditions, has prompted the exploration of these two cell types in IBD and coeliac disease. Previous studies have examined these factors individually in a range of diseases, however, to our knowledge the study of both Treg and Th17 in IBD and coeliac disease subjects represents a novel area of research. Crohn’s disease (CD), ulcerative colitis (UC) and coeliac disease subjects were recruited through the Department of Gastroenterology and Hepatology at The Queen Elizabeth Hospital (QEH) in Adelaide, South Australia. In total, one-hundred and seventeen subjects were enlisted in this study to donate blood samples. In addtion, intestinal biopsy samples were collected from fifty-six subjects undergoing colonoscopy at the QEH Department of Gastroenterology and Hepatology. All subjects participated, with informed consent and ethics approval. Treg and Th17 cell numbers were investigated in the peripheral blood of Crohn’s disease, ulcerative colitis, coeliac disease and control subjects using multi-colour, intracellular flow cytometry. A decrease in Treg cell numbers and an increase in Th17 cell numbers was observed in IBD, but not in coeliac disease. Closer investigation into the ratio of Treg and Th17 cells within patients identified a near 1:1 Treg/Th17 ratio in control subjects, but a lower Treg/Th17 ratio in IBD patients. This suggested a disturbance in regulatory and effector cell equilibrium. Furthermore, the excess of Th17 cells and deficiency of Tregs could contribute to the pathologies observed in IBD. The discovery of an imbalance in Treg and Th17 cell numbers in IBD prompted further investigation of these cells in intestinal biopsies collected from IBD, coeliac and control subjects. Real time RT-PCR of intestinal biopsy samples demonstrated increased expression of the Th17 cytokine, IL-17a, in both IBD and coeliac disease. Elevated levels of the Treg transcription factor Foxp3 were also identified in intestinal biopsies from IBD subjects. It was therefore hypothesised that Treg cells may have been actively recruited from the periphery in an attempt to control inflammation in the gut; however, the intestinal cytokine microenvironment may have restricted the regulatory function of these cells. Cytokines known to promote human Th17 differentiation, namely IL-1β, IL-6, TGF-β, IL-21 and IL-23, were explored in intestinal biopsy samples from IBD, coeliac and control subjects. High levels of IL-1β and IL-6 were detected in IBD patient samples, however, no change in levels of IL-21 or IL-23 were observed in IBD or coeliac disease subjects. Elevated levels of TGF-β were only identified in UC. No changes in cytokine expression were observed between control and coeliac subjects, except a significant decrease in IL-6 levels was identified in coeliac disease sufferers. The pro-inflammatory microenvironment identified in intestinal biopsies from IBD subjects may have promoted the continual differentiation and development of Th17 cells, whilst restricting Treg activity. Moreover, the observed deficiency of Treg in IBD patients may have impaired the ability of the immune system to limit excessive pathogenic Th17 driven immune responses in the intestinal mucosa. Therefore, therapeutic approaches that aim to re-establish regulatory and effector cell homeostasis by increasing Treg numbers in IBD patients, and specifically targeting Th17 cells, may prove effective in the treatment of IBD. Approaches such as these could provide greater focus to treatment strategies for IBD management compared to current broad-spectrum immunosuppressive therapies that could increase susceptibility to cancer or infection in IBD patients. In addition, the imbalance of regulatory and effector cells demonstrated in the peripheral blood of IBD patients may potentially provide new options for a noninvasive diagnostic tool. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1457580 / Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 2009