IL-33 is a pleiotropic cytokine that orchestrates both innate and adaptive immunity. It is commonly associated with type 2 immune responses but recently expression of the IL-33 receptor, ST2, was reported on Treg cells found preferentially in non-lymphoid tissues, such as the visceral adipose tissue, muscle or colon. A crucial role of Tregs in maintaining intestinal homeostasis has been well described. However, little is known about the functional relevance of the ST2-expressing Treg population in the colon. Phenotypic and functional characterisation of Tregs in the gut revealed the presence of two distinct populations: ST2⁺/Gata3⁺ and Rorγt⁺ Tregs. Thymic-derived ST2⁺/Gata3⁺ Tregs showed a more activated phenotype and produced IL-10 under homeostatic conditions. Upon microbial challenge and colitis, ST2+/Gata3+ Tregs were decreased, while Rorγt⁺ Tregs expanded. Furthermore, in vitro experiments demonstrated that IL-33 directly induced activation of the Gata3 pathway in Tregs, which enhanced expression of Foxp3 and ST2. Additionally, amphiregulin was also induced in Tregs upon stimulation with IL-33. However, in vivo, IL-33 was dispensable for both the maintenance of Treg cells under homeostatic conditions and Treg function in Helicobacter hepaticus-driven colitis. Investigation of the negative regulators of IL-33 showed that IL-23 inhibited IL-33-mediated effects on Tregs. We also observed increased production of soluble ST2 by stromal cells during intestinal inflammation, which likely contributed to the reduction of IL-33 bioavailability. Finally, a systematic analysis of the cellular source of IL-33 revealed that PDGRFα⁺ stromal cells located in the T cell zone of secondary and tertiary lymphoid tissues were a major IL-33-producing cell population in the gut. Collectively, our findings suggest that signals received by the stromal compartment upon cell injury may trigger a specific phenotype of Tregs with a repair capacity, and thus, promote intestinal homeostasis. These findings improve our understanding of tissue-resident Tregs and open an exciting avenue to explore heterocellular signalling between stromal cells and Tregs.

Inflammatory Bowel Disease (IBD) is a debilitating, life- long disease that affects about 1.4 millions Americans. Little is known about the pathogenesis of IBD and an effective cure still remains to be discovered. While there are numerous T cell targeting therapies for IBD, more research is still needed. Bispecific T Cell Engagers, BiTES, is a modified protein capable of engaging two antigens simultaneously; it is capable of activating T cells by circumventing the MHC protein molecule. This provides an alternative to the current molecular therapies for IBD. In addition to monoclonal therapy research, there has been a plethora of research on immunomodulatory molecules, such as β- glucan. The benefit of β-glucan has been shown with supplements and food sources alike in animal models. In this study, we used BiTES, CMPD-1, with an anti-CD3/ Dectin-1 epitopes capable of engaging T Cells and β-glucan in beads and fungi cell wall. CMPD-1 is capable of engaging Splenic and Lamina Propia T Cells from a C57BL/6 mice. Likewise, CMPD-1 engaged T cells to hyphae of C. albicans and A. fumigatus, which have a higher concentration of β-glucan than in the candida form. The data show a delayed in hyphae growth in yeast with CMPD-1 and a decrease in yeast growth for the first four hours when compared to non- BiTES molecules. Additionally, qualitative analysis of CMPD-1 shows a decrease A. fumigatus growth after a 72-hour incubation period. Splenic T cells from mice lacking Dectin-1 and Wild-type (WT) mouse strains where incubated with BiTES compound and yeast for 23 hours followed by a PrestoBlue killing assay to assess yeast cell viability. The PrestoBlue assay showed that CMPD-1 killed more A. fumigatus in both T cell subsets; although, the difference lacked statistical significance. The applications of this molecule as a therapeutic agent for IBD are promising, although, still in its infancy. An alternative use for this molecule is to train the immune system with the BiTES molecule in conjunction with β-glucan supplements to build immunity against opportunistic pathogens such as A. fumigatus and C. albicans that often cause havoc in IBD patients as a result of the changes in microbiota, and compromised integrity of the GI tract. / 2017-06-16T00:00:00Z

INTRODUCTION: The management of pediatric patients with ulcerative colitis (UC) is dependent upon the ability to detect meaningful changes in disease status. This is currently done using validated patient-reported clinical disease activity indices, including the Pediatric Ulcerative Colitis Activity Index (PUCAI). While useful for global assessments completed during ambulatory office visits, the sensitivity of this metric may be insufficient to reflect more subtle changes in disease activity or response to medical therapy in hospitalized patients. Intravenous steroids are typically employed in the management of patients admitted for acute exacerbations of UC symptoms. These are typically manifest by worsening bloody diarrhea, abdominal pain, and worsening anemia. There is presently no way of predicting whether a patient admitted for UC will respond to steroid therapy. Current paradigms dictate a five-day trial before considering a transition to more potent medical or definitive surgical approaches to the management of refractory colitis. The development of more sensitive and reliable biomarkers or disease activity metrics could enable clinicians to more expediently identify steroid non-responders. This would minimize patient morbidity, decrease risk of complication, and lower overall cost of care. Previous studies have demonstrated that changes in fecal lactoferrin (FLA) correlate with disease activity in patients with UC. OBJECTIVES: To analyze the predictive value of FLA in the response to steroid treatment of patients admitted for management of UC. METHODS: We recruited pediatric inpatients with UC in the Division of Gastroenterology, Hepatology and Nutrition at Boston Children’s Hospital who were hospitalized for treatment of a flare of their UC symptoms. After obtaining patient consent, we collected a stool sample on days 1 and 3 of their hospital stay. We sent samples to TECHLAB® Inc. (Blacksburg, VA) to be analyzed for levels of FLA. We compared Day 1, Day 3 and ∆FLA (Day 1 – Day 3) in two patient groups: those that responded to conventional steroid therapy and those that required rescue medical or surgical therapy. We reported statistical significance with the Wilcoxon signed-rank test. RESULTS: Of 67 patients consented for the study, 30 provided stool samples on both days 1 and 3 of their inpatient hospitalization. Of the 30 patients, 63.3% responded to steroids while 36.7% required rescue therapy with immunomodulators. ∆FLA for responders, 43.6μg/mL(-239.0, 331.6) (median(interquartile range)), did not differ significantly from non-responders, -74.1μg/mL(-296.7, 221.7), P = 0.3. CONCLUSIONS: Our findings do not demonstrate that measurement of changes in quantitative FLA over three days can be used to assess acute responses to steroid therapy. Increasing the sample size may allow us to better delineate subtle differences between responders and non-responders to steroid therapy.

Introduction Inflammatory bowel disease (IBD) is a chronic gastrointestinal disorder that encompasses two major subtypes; Crohn’s Disease (CD) and Ulcerative Colitis (UC). Our knowledge regarding disease pathogesis is rapidly increasing. However, these disease entities provide challenges in diagnosis, monitoring of disease activity and assessing individual response to treatment, because there is a lack of validated clinical biomarkers. Metabolomics involves the study of numerous analytes that have very diverse physical and chemical properties and occur in a wide concentration range. Early evidence suggests there is potential for metabolomic profiling to be used in the differentiation of CD and UC. However, knowledge is limited regarding the metabolic changes seen in relation to disease activity or to medical or surgical treatments. Aims A metabolomics approach was taken to determine whether metabolomic profiles could distinguish between patients with CD or UC and healthy controls. We also aimed to define the relationship between metabolomic profile and disease activity, and to determine the effect of medical (anti-TNFa agents) and surgical treatment on the metabolome. Methods A metabolomics approach was undertaken. Serum and urine sample sets were collected from a total of 41 patients with ulcerative colitis, 43 patients with Crohn’s disease, and 62 healthy controls (HC). In order to allow a comparison of metablomic profile and disease activity, 4 sample sets were taken from the same patient at 3 monthly intervals over the period of one year. Those patients undergoing either surgical or biological treatment had sample sets taken pre and post intervention. Metabolomic analysis using gas chromatography time of flight mass spectrometry (GC-ToF-MS) and ultra-high performance liquid chromatography Fourier Transform mass spectrometry (UHPLC-FTMS) was carried out on both serum and urine. Results Serum and urine GC-ToF-MS and UHPLC-FTMS metabolomic analyses show differentiation between UC, CD and healthy controls, most significantly in urine analyses. No significant differentiation was seen in pre- and post-surgical patients, or pre- and post-biological therapy patients. It was possible to differentiate surgical patients from healthy controls, especially in the urine analyses. Metabolite identification revealed consistently more dietary variation in the healthy controls than in the IBD patients. Significant differences (p < 0.05) were seen between healthy controls and IBD patients in classes of metabolites relating to the citric acid cycle and the uronic acid pathway, as well as amino acids, fatty acids and cholesterols. The behaviour or location of disease, or the disease activity score did not appear to influence the metabolome in either serum or urine analyses using GC-ToF-MS and UHPLC-FTMS. Conclusion Metabolomic profiling of urine and serum in IBD may provide a novel methodology aiding both clinical diagnosis through biomarker development, and advancing knowledge of disease pathogenesis.

Defensinas são peptídeos antimicrobianos produzidos na mucosa intestinal e fazem parte da imunidade inata, agindo sobre vários microrganismos luminais. Deficiência na expressão de defensinas tem sido relatada em doenças inflamatórias intestinais (DII), no entanto a contribuição de cada tipo de defensina, num cenário de polimorfismo genético, mantém alguma controversa. Βeta-defensinas humanas (HBDs) têm atividade antimicrobiana contra uma ampla variedade de fungos, bactérias e vírus e têm também, um papel na ligação entre a imunidade inata e adaptativa atuando como quimiotáticos. O gene DEFB1 (8p23), codificando a beta-defensina humana 1 (HBD-1), é expresso normalmente por células epiteliais de uma série de tecidos, mas sua expressão pode variar entre indivíduos e pode ser modificada durante processo inflamatório. Produção deficiente de defensinas parece contribuir para a patogênese de DII, e uma diminuição na expressão de HBD-1 tem sido relatada na mucosa de pacientes com doença de Crohn (DC) e retocolite ulcerativa (RCU). Nós avaliamos a possível associação de três polimorfismos do gene DEFB1 com a suscetibilidade a DII, RCU e DC, em 149 pacientes, 79 com DC e 70 com RCU; e 200 controles saudáveis do sul do Brasil. No nosso estudo não se observou diferença estatisticamente significativa entre a distribuição das frequências alélicas para DEFB1 SNPs -52G>A. -44C>G e -20G>A entre o total de pacientes com DII e controles. Porém, quando pacientes com DC foram estratificados de acordo com a localização anatômica, o alelo -20G>A foi mais frequente em pacientes com DC colônica do que em controles (65 % VS 44 %, p=0,048). De forma similar, o genótipo A/A foi mais frequente em pacientes com DC colônica do que em controles (36 % VS 16 %), mas neste caso, a diferença não foi estatisticamente significativa (p=0,07). Embora não se achou uma clara e forte associação entre os SNPs 5’-UTR DEFB1 e suscetibilidade/proteção à doença inflamatória intestinal, nossos resultados sugerem possível envolvimento do gene DEFB1 nestas enfermidades, especialmente com a localização colônica da doença de Crohn. Estudos com amostras maiores e populações diversas serão úteis para avaliar a tendência observada no nosso grupo. / Defensins are antimicrobial peptides produced by the intestinal mucosa and are part of the innate immune system, playing a protective role against various intestinal microorganisms. Deficiency in the expression of defensins has been reported in inflammatory bowel diseases (IBD), however there is some controversy over the contribution of each type of defensine, in a setting of great genetic polymorphism. Beta-defensins (HBDs) have an antimicrobial activity against a great variety of fungi, bacteria and viruses, and also have a role in connecting the innate and the adaptive immunity, acting as a chemostatic agent. Deficient production of defensins appears to contribute to the pathogenesis of IBD, and the lower expression of HBD-1 has been reported on the mucosa of Ulcerative colitis (UC) and Crohn’s disease (CD) patients. We evaluated a possible association of three polymorphisms of gene DEFB1 with susceptibility to develop IBD, UC and CD in 149 patients, 79 with CD and 70 with UC; and 200 healthy controls from the south of Brazil. The gene DEFB1 (8p23), which codifies human beta-defensin 1 (HBD-1), is constitutivelly expressed by epithelial cells of several tissues, but its expression may vary among different individuals and may be modified by inflammation. In our study we did not find a statistically significant difference between the distribution of the allelic frequencies for DEFB1 SNPs -52G>A, -44C.G and -20G>A between the total number of patients and controls. However, when patients were stratified according to the anatomic location, the allele -20G>A was more frequent in patients with colonic CD than in contros (65% VS 44%, p=0,048). Similarly, the genotype A/A was more frequent in patients with colonic CD than in controls (36% vs 16%), however, in this case, the difference wasn’t statistically significant (p=0,07). Although we did not find a clear and strong association between the 5’-UTR DEFB1 SNP and susceptibility to IBD, our results suggest a possible involvement of the DEFB1 gene and these diseases, particularlly colonic CD. Further studies with larger samples and diverse populations will be usefull to evaluate the trend observed by our group.

O objetivo dessa tese foi avaliar a expressão de citocinas Th1 (IL-12 e INF&#61543;), citocinas Th2 (IL-4, IL-6 e IL-10) e das citocinas pró-inflamatórias IL-18, IL-1&#61538; e TNF&#61537; no fluido gengival de pacientes com periodontite crônica portadores da doença de Crohn (DC), de retocolite ulcerativa idiopática (RCUI) e em indivíduos saudáveis (o grupo controle, GC). Como objetivo secundário, avaliamos a função dos neutrófilos no fluido gengival desses pacientes através da mensuração das metaloproteinases da matriz -8, -9 (MMP-8 e MMP-9) e da atividade da elastase. Quinze pacientes com DC (idade média 38.2 11.4 anos), 15 pacientes com RCUI (idade média 45.0 10.5 anos) e 15 pacientes saudáveis (idade média 42.1 7.8 anos) participaram desse estudo. Todos os dentes presentes, com exceção dos terceiros molares, foram examinados. Profundidade de bolsa (PB), nível de inserção clínica (NI), presença de placa e de sangramento a sondagem foram avaliados em seis sítios por dente. Em cada paciente, o fluido de 4 sítios com periodontite (PB &#61619; 5 mm e NI&#61619; 3mm) e de 4 sítios com gengivite (PB&#61603; 3 mm e NI &#61603; 1 mm) foram coletados através de pontas de papel absorvente pré-fabricadas. O sistema LUMINEX foi utilizado na mensuração das IL-1&#61538;, IL-4, IL-6, IL-10, IL-12p70, TNF&#61537;, INF&#61543;, MMP-8 e MMP-9. A IL-18 foi analisada através do ensaio ELISA e a atividade de elastase através de uma reação enzimática. O soro desses pacientes também foi analisado e o coeficiente de correlação de Pearson foi utilizado na análise da correlação entre as citocinas no soro e no fluido gengival. Nos sítios com gengivite, a quantidade total de IL-4 foi significativamente menor no grupo RCUI do que no grupo GC (p=0.016). Nos sítios com periodontite, a quantidade total de IL-4 foi significativamente menor no grupo DC do que no grupo GC (p=0.029). A quantidade total da IL-6 (p=0.028) assim como sua concentração (p=0.044) foram significativamente maiores no grupo RCUI do que no grupo GC. No soro, os níveis da IL-18 foram significativamente mais altos nos grupos DC (p=0.011) e RCUI (p=0.019) do que no grupo GC. No grupo RCUI, a IL-18 do soro se correlacionou positivamente com a IL-1&#61538; do fluido gengival (r=0. 667, p=0.01). Concluindo, nos pacientes com doença inflamatória intestinal, os níveis da IL-4 estavam mais baixos no fluido gengival e os níveis da IL-18 estavam aumentados no soro quando comparados aos controles. A função dos neutrófilos foi similar entre os pacientes com doença inflamatória intestinal e os controles. Com exceção da correlação positiva entre a IL-18 sérica e a IL-1&#61538; no fluido gengival dos pacientes com retocolite ulcerativa idiopática, não houve correlação entre as diversas citocinas mensuradas no soro e as citocinas mensuradas no fluido gengival nos controles nem nos pacientes com doença inflamatória intestinal. Não houve a caracterização de um padrão de resposta Th1 ou Th2 no fluido gengival dos pacientes com doença inflamatória intestinal. / The aim of this thesis was to evaluate the expression of Th1 cytokines (IL-12 and INF-&#947;), Th2 cytokines (IL-4, IL-6 and IL-10) and the pro-inflammatory cytokines IL-18, IL-1&#61538; and TNF-&#945; in the gingival crevicular fluid (GCF) from Crohns disease (CD) patients, ulcerative colitis (UC) patients and healthy individuals (control group, CG) who had chronic periodontitis. Besides, we measured elastase activity, matrix metalloproteinase -8 and -9 (MMP-8 and -9) to address the neutrophil function in the GCF. Fifteen CD patients (mean age 38.2 &#61617; 11.4 years), 15 UC patients (mean age 45.0 &#61617; 10.5 years) and 15 systemically healthy controls (mean age 42.1 &#61617; 7.8 years) were enrolled in this study. All the present teeth, except for the third molars were examined. Probing pocket depth (PPD), clinical attachment loss (CAL), presence of plaque and presence of bleeding on probing were assessed in six sites per tooth. In every subject, GCF from 4 gingivitis sites (PPD &#61603; 3mm and CAL &#61603; 1mm) and from 4 periodontitis sites (PPD &#61619; 5mm and CAL&#61619; 3mm) were collected with filter strips. The data were reported as total amount and concentration. IL-1&#61538;, IL-4, IL-6, IL-10, IL-12p70, TNF&#945;, INF&#947;, MMP-8 and MMP-9 were analyzed by the Luminex analyzer. IL-18 was analyzed using a commercially available ELISA assay and the elastase activity by an enzymatic reaction. The serum was also analysed and the correlations between the cytokines in the GCF and in the serum were calculated by Pearson correlation analysis. In gingivitis sites, the total amount of IL-4 was significantly lower in the UC group than in the CG group (p=0.016). In periodontitis sites, the total amount of IL-4 was significantly lower in CD group than in the CG group (p=0.029). The total amount of IL-4 was lower in UC group than in CD group (p=0.077). Similarly, IL-4 concentrations in both CD (p=0.096) and UC (p=0.064) groups were lower than in CG group. IL-6 total amount (p=0.028) and IL-6 concentration (p=0.044) were significantly higher in the UC group than in the CG group. In the serum, IL-18 levels were significantly higher in CD (p=0.011) and UC (p=0.019) groups than in the CG group. In UC group, there was a positive correlation between serum IL-18 levels and IL-1&#61538; in the GCF (r=0. 667, p=0.01). In conclusion, IBD patients had lower IL-4 levels in the GCF and higher IL-18 levels in the serum than healthy controls. The neutrophil function in IBD patients is similar to controls. Except for the positive correlation between IL-18 in the serum and IL-1&#61538; in the GCF, there was no correlation between the cytokines in serum and in the GCF either in IBD patients or in controls. There was not a Th1 or Th2 polarization in the GCF from IBD patients.

Estudos recentes têm avaliado a presença de polimorfismos do gene multidroga resistente 1 (MDR1), que codifica o transportador de membrana de efluxo chamado de P-glicoproteína, seu potencial papel na suscetibilidade das doenças inflamatórias intestinais (DII) e suas possíveis correlações com aspectos clínicos das DII. Dados conflitantes podem resultar da análise genética de populações distintas. Investigamos se os polimorfismos do gene MDR1 estão associados com as DII em população do sudeste do Brasil e suas possíveis correlações com fenótipos, atividade de doença, resposta ao tratamento e efeitos colaterais. Como métodos, a presente pesquisa trabalhou com 146 pacientes com Doença de Crohn (DC) e 90 com Retocolite Ulcerativa Idiopática (RCUI), que foram recrutados através de critérios diagnósticos estabelecidos. Os polimorfismos do MDR1 mais comumente descritos na literatura, C1236T, G2677T e C3435T, foram avaliados por PCR. As frequências genotípicas de pacientes com RCUI e DC foram analisadas na população de estudo. Associações de genótipo-fenótipo com características clínicas foram estabelecidas e riscos estimados para as mutações foram calculados. Nenhuma diferença significativa foi observada nas freqüências genotípicas para os polimorfismos G2677T/A e C3435T do MDR1 na DC ou na RCUI. O polimorfismo C1236T foi significativamente mais comum na DC do que na RCUI (p = 0,036). Na RCUI foram encontrados mais homens nos polimorfismos C1236T e G2677T no grupo de heterozigotos. Foram encontradas associações significativas entre o polimorfismo C3435T do gene MDR1 em pacientes com fenótipo estenosante na DC (OR: 3,16, p = 0,036), em oposição ao comportamento penetrante (OR: 0,31, p = 0,076). Na DC, associações positivas também foram encontradas entre o polimorfismo C3435T, à atividade moderada/severa da doença (OR: 3,54, p = 0,046), e à resistência / refratariedade ao corticosteróide (OR: 3,29, p = 0,043) nos homozigotos polimórficos. Nenhuma associação significativa foi encontrada entre os polimorfismos do MDR1 e categorias fenotípicas, atividade de doença ou resposta ao tratamento da RCUI. Em conclusão, os resultados do presente estudo sugerem que os polimorfismos do gene MDR1 poderiam estar implicados na susceptibilidade a DC e no seu fenótipo estenosante, como também estarem associados com uma resposta inadequada ao tratamento em um grupo de pacientes com DC. A forte relação com a DC suporta a existência de papéis adicionais para o MDR1 em mecanismos específicos subjacentes na patogênese da DC, como o controle da microbiota intestinal, mediação e regulação da fibrose. Além disso, compreender os efeitos de vários fármacos associados a estas variantes do MDR1 pode contribuir para a prescrição personalizada de regimes terapêuticos.

IgA is the dominant antibody isotype found at mucosal surfaces during homeostasis. However, genetic variation in Fcγ receptors (FcγRs), a family of receptors that mediate immune cell activation by IgG, influences susceptibility to inflammatory bowel disease (IBD), suggesting that IgG may be important during gut inflammation. IBD is a chronic relapsing condition with two major subtypes, Crohn’s disease (CD) and ulcerative colitis (UC), both driven by aberrant immune responses to commensals. In the first part of this thesis, we sought to investigate anti-commensal IgG responses in patients with UC and to determine the mechanism by which local IgG might contribute to intestinal inflammation. We found that UC and murine dextran sodium sulfate (DSS)-induced colitis are associated with a significant increase in anti-commensal IgG and local enrichment of FcγR signalling pathway genes. The genes most robustly correlated with FCGR2A, an activating FcγR associated with UC susceptibility, were IL1B andCXCL8. Ex vivo stimulation of human and murine lamina propria mononuclear cells with IgG immune complexes (IC) resulted in an increase in these cytokines/chemokines. In vivo manipulation of the macrophage FcγR A/I ratio in transgenic mice determined IL-1β and Th17 cell induction. Finally, IL-1β blockade in mice with a high FcγR A/I ratio reduced IL-17 and IL-22-producing T cells and the severity of colitis. Our data reveal that commensal-specific IgG contributes to intestinal inflammation via FcγR-dependent, IL-1β-mediated Th17 activation. In this thesis, we have also addressed the interplay between IgG and group 3 innate lymphoid cells (ILC3s). ILC3s are closely related to natural killer cells, which are known to express FcγRs, and are characterised by their production of Th17 cytokines. Here, we have shown that ILC3s express FcγRs, that ICs drive IL-22 production and MHC class II expression by ILC3s, and FcγR signalling induces a transcriptional programme that reinforces ILC3 maintenance and functionality. These results represent a new paradigm for ILC activation, with direct regulation by the adaptive immune response. Finally, we have begun to address the role played by ILC3-derived cytokines in the regulation of local tissue-resident immune cells. We have demonstrated that ILC depletion significantly alters the activation state of intestinal macrophages, resulting in detrimental bacterial outgrowth following C. rodentium infection but protection from overwhelming DSS-induced inflammation. We have shown that GM-CSF promotes macrophage IL-1β and IL-23 production, which in turn act to reinforce ILC3-derived GM-CSF and IL-22 secretion in vitro, respectively. Therefore, ILC3s are essential coordinators of the local inflammatory response within the gut through activation and possible recruitment of immune cells, and their modulation may be beneficial in the treatment of IBD.

Made available in DSpace on 2014-06-11T19:25:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-02-28Bitstream added on 2014-06-13T20:13:52Z : No. of bitstreams: 1 chagas_as_me_botib_parcial.pdf: 147304 bytes, checksum: f84c21b758e3dbb707fc30cee5ecea6e (MD5) Bitstreams deleted on 2015-02-04T11:39:28Z: chagas_as_me_botib_parcial.pdf,Bitstream added on 2015-02-04T11:40:13Z : No. of bitstreams: 1 000722900.pdf: 359844 bytes, checksum: 2318a21c5548347f5c7493251309fe9e (MD5) / A Doença Inflamatória Intestinal (DII) é uma doença com etiologia desconhecida e sem terapêutica curativa disponível, englobando, fundamentalmente, duas doenças distintas: a Doença de Crohn (DC) e a Retocolite Ulcerativa (RCU), ambas caracterizadas por uma inflamação crônica do intestino, com períodos de exacerbação seguidos de intervalos prolongados com remissão dos sintomas, cujo tratamento com os fármacos disponíveis apresentam sérios efeitos colaterais. Portanto, o desenvolvimento de novas estratégias de tratamento que combinem eficácia e segurança é uma importante meta na terapia da DII. A espécie Baccharis dracunculifolia DC (família Asteraceae) é uma planta medicinal brasileira usada popularmente contra úlceras, inflamação e problemas hepáticos e tem sido amplamente reconhecida como a principal fonte botânica da resina e dos constituintes químicos da própolis brasileira (própolis verde), a qual tem sido incorporada em alimentos e bebidas para a melhora da saúde. Estudos mostram que as inúmeras atividades biológicas da própolis verde decorrem exclusivamente dos constituintes presentes em Baccharis dracunculifolia, a qual possui várias atividades biológicas, especialmente agindo como imunomoduladora, anti-inflamatória e anti-inflamatória intestinal, sequestradora de radicais livres, antiúlcera e analgésica. A atividade anti-inflamatória intestinal está associada à presença de diferentes ácidos fenólicos presentes na espécie, tais como ácidos cafeico, p-cumárico, 3-prenil-p-cumárico (drupanina), 3,5-diprenil-p-cumárico (artepilina C), bacarina e metil-éster de aromadendrina. Estes compostos possuem íntima associação biossintética com os derivados cumarínicos também ativos na doença inflamatória intestinal, justamente por fazerem parte de sua rota biossintética. Com base nestas... / The Inflammatory Bowel Disease (IBD) is a disease with unknown etiology and no curative treatment available, encompassing essentially two distinct diseases: Crohn's disease (CD) and ulcerative colitis (UC), both characterized by chronic inflammation of the intestine, with periods of exacerbation followed by long intervals with symptom remission whose treatment with available drugs have serious side effects. Therefore, the development of new treatment strategies that combine effectiveness and safety is an important goal in the treatment of IBD. The species Baccharis dracunculifolia DC (Asteraceae) is a Brazilian medicinal plant popularly used against ulcers, inflammation and liver problems and has been widely recognized as the main botanical source of the resin and the chemical constituents of Brazilian propolis (green-propolis), which has been incorporated in foods and beverages to improve health. Studies show that the numerous biological activities of green propolis derived exclusively from the constituents present in Baccharis dracunculifolia, which has several biological activities, especially acting as immunomodulatory, anti-inflammatory and anti-inflammatory intestinal free radical scavenging, antiulcer and analgesic. The intestinal anti-inflammatory activity is associated with the presence of different phenolic acids in this specie, such as caffeic acid, p-coumaric acid, 3-prenyl-p-coumaric (drupanina), 3,5-diprenyl-p-coumaric acid (C artepilina ) bacarina and methyl ester aromadendrin. These compounds have a close association with the biosynthetic coumarin derivatives also active in inflammatory bowel disease, just by being part of their biosynthetic pathway. Based on this information, the aim of this study was to identify potential pharmacological targets of anti-inflammatory action of the extract... (Complete abstract click electronic access below)

Made available in DSpace on 2014-12-17T14:10:30Z (GMT). No. of bitstreams: 1 AlessandraMM_DISSERT.pdf: 2030240 bytes, checksum: 2080f624a4d75ae70190a46a38c96a10 (MD5) Previous issue date: 2014-03-21 / As doen?as inflamat?rias intestinais s?o enfermidades onde a toler?ncia e homeostase da resposta inflamat?ria est?o comprometidas, gerando les?es teciduais e favorecendo o surgimento de neoplasias. H? dois importantes exemplos de doen?as inflamat?rias intestinais, a colite ulcerativa, foco do modelo desse estudo, e a doen?a de Crohn. Os medicamentos utilizados no tratamento dessas doen?as desencadeiam diversos efeitos adversos; al?m disso, em alguns pacientes, eles n?o s?o eficazes. Os extratos de algas t?m demonstrado v?rias atividades biol?gicas, entre elas a atividade anti-inflamat?ria. Os extratos das algas do g?nero Caulerpa foram utilizados em v?rios estudos, onde modelos inflamat?rios foram analisados, entre eles o modelo de colite ulcerativa. A utiliza??o do extrato metan?lico da C. mexicana como terap?utica nesse modelo atenuou o quadro clinico desenvolvido pelos animais. Sendo assim, o presente estudo teve como objetivo analisar a a??o terap?utica da Caulerpina (CLP), extra?da da C. racemosa, no modelo murino de colite ulcerativa. Camundongos C57BL/6 machos foram expostos a uma solu??o de Dextrana Sulfato de S?dio (DSS) a 3% por sete dias. A partir do primeiro dia de exposi??o ao DSS os animais foram tratados em dias alternados com a CLP nas doses de 4 e 40 mg/kg e com a dexametasona (3 mg/kg) por via oral. O desenvolvimento da doen?a foi analisado atrav?s do ?ndice de atividade da doen?a (IAD), que leva em considera??o a perda de peso corporal, a consist?ncia e a presen?a de sangue nas fezes. Ap?s a eutan?sia, o c?lon foi removido e mensurado, e amostras do tecido col?nico foram destinadas a an?lise histol?gica e ? cultura para dosagem de citocinas. Os n?veis de citocinas no sobrenadante da cultura do c?lon foram mensurados por ELISA. O tratamento com a CLP (4 mg/kg) desencadeou significativa melhora quanto ? perda de peso corporal e ao IAD, e atenuou o encurtamento do c?lon em resposta ao DSS. Tal dose foi capaz de reduzir os n?veis de citocinas pr?-inflamat?rias analisadas (TNF-&#61537;, IFN-&#61543;, IL-6, IL-17), mas n?o teve efeito significativo nas citocinas anti-inflamat?rias IL-10 e TGF-&#61538;. O tratamento com a CLP (40 mg/kg) n?o foi eficaz quanto a perda de peso e ao IAD, al?m de n?o ter atenuado a redu??o do c?lon em resposta ao DSS. Essa dose conseguiu reduzir os n?veis das citocinas pr?-inflamat?rias, por?m n?o os n?veis de IL-6. O tratamento com a dexametasona obteve melhora discreta quanto ? perda de peso corporal e ao IAD, por?m n?o atenuou a redu??o do c?lon em resposta ao DSS. Esse tratamento tamb?m conseguiu reduzir todas as citocinas pr?-inflamat?rias testadas. Deste modo a CLP (4 mg/kg) demonstrou ser uma alternativa promissora no tratamento da colite ulcerativa, em raz?o dos seus efeitos ben?ficos sobre par?metros cl?nicos, morfol?gicos e moleculares do modelo em estudo