Qualidade de vida relacionada à saúde em pacientes acometidos por doença inflamatória intestinal tratados com terapia biológica

Pós-graduação em Bases Gerais da Cirurgia - FMB

Zeyheria montana Mart. (Bignoniaceae) as source of antioxidant and immunomodulatory compounds with beneficial effects on intestinal inflammation

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Desenvolvimento de nano e micropartículas de quitosana encapsulando 4-metilesculetina e avaliação no tratamento da doença inflamatória intestinal

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Uso do amido resistente na promoção da saúde intestinal de cães idosos: formação de produtos de fermentação e características histológicas da mucosa intestinal

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Testes preliminares de motilidade intestinal e toxicidade oral aguda com extrato de cascas pulverizadas de Endopleura uchi (Huber) Cuatrec: (Humiriaceae) em camundongos

The aim of this work was to analyze the acute oral toxicity and the effects on intestinal motility of the extract obtained through decoction 20% (m/v) of Endopleura uchi, popularly known as "uxi-amarelo", a native plant from the Brazilian Amazon. The plant is used indiscriminately against several diseases: arthritis, cholesterol, diabetes, ulcers, myomas, and intestinal illnesses in general, among others. The present results show that there were no significant alterations in intestinal motility and that the extract does not present signs of toxicity, showing its safety for consumption purposes.

Importância dos genes fliC e motB de Salmonella enterica subsp. enterica sorovar Enteritidis na colonização intestinal e invasão sistêmica em aves (Gallus gallus domesticus)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influência do suco de laranja na microbiota intestinal humana

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Increased IgE serum levels are unrelated to allergic and parasitic diseases in patients with juvenile systemic lupus erythematosus

OBJECTIVE: The aim of this study was to assess the IgE serum levels in juvenile systemic lupus erythematosus patients and to evaluate possible associations with clinical and laboratory features, disease activity and tissue damage. METHODS: The IgE serum concentrations in 69 consecutive juvenile systemic lupus erythematosus patients were determined by nephelometry. IgG, IgM and IgA concentrations were measured by immunoturbidimetry. All patients were negative for intestinal parasites. Statistical analysis methods included the Mann-Whitney, chi-square and Fisher's exact tests, as well as the Spearman rank correlation coefficient. RESULTS: Increased IgE concentrations above 100 IU/mL were observed in 31/69 (45%) juvenile systemic lupus erythematosus patients. The mean IgE concentration was 442.0 +/- 163.4 IU/ml (range 3.5- 9936.0 IU/ml). Fifteen of the 69 patients had atopic disease, nine patients had severe sepsis and 56 patients presented with nephritis. The mean IgE level in 54 juvenile systemic lupus erythematosus patients without atopic manifestations was 271.6 +/- 699.5 IU/ml, and only nine of the 31 (29%) patients with high IgE levels had atopic disease. The IgE levels did not statistically differ with respect to the presence of atopic disease, severe sepsis, nephritis, disease activity, or tissue damage. Interestingly, IgE concentrations were inversely correlated with C4 levels ( r = -0.25, p = 0.03) and with the SLICC/ACR-DI score (r = -0.34, p = 0.005). The IgE concentration was also found to be directly correlated with IgA levels (r = 0.52, p = 0.03). CONCLUSIONS: The present study demonstrated for the first time that juvenile systemic lupus erythematosus patients have increased IgE serum levels. This increase in IgE levels was not related to allergic or parasitic diseases. Our results are in line with the hypothesis that high IgE levels can be considered a marker of immune dysregulation.

B Lymphocyte-Induced Maturation Protein-1 Contributes to Intestinal Mucosa Homeostasis by Limiting the Number of IL-17-Producing CD4(+) T Cells

The transcription factor B lymphocyte induced maturation protein-1 (Blimp-1) plays important roles in embryonic development and immunity. Blimp-1 is required for the differentiation of plasma cells, and mice with T cell specific deletion of Blimp-1 (Blimp-1CKO mice) develop a fatal inflammatory response in the colon. Previous work demonstrated that lack of Blimp-1 in CD4(+) and CD8(+) T cells leads to intrinsic functional defects, but little is known about the functional role of Blimp-1 in regulating differentiation of Th cells in vivo and their contribution to the chronic intestinal inflammation observed in the Blimp1CKO mice. In this study, we show that Blimp-1 is required to restrain the production of the inflammatory cytokine IL-17 by Th cells in vivo. Blimp-1CKO mice have greater numbers of IL-17 producing TCR beta(+)CD4(+)cells in lymphoid organs and in the intestinal mucosa. The increase in IL-17 producing cells was not restored to normal levels in wild-type and Blimp-1CKO mixed bone marrow chimeric mice, suggesting an intrinsic role for Blimp-1 in constraining the production of IL-17 in vivo. The observation that Blimp-1 deficient CD4(+) T cells are more prone to differentiate into IL-17(+)/IFN-gamma(+) cells and cause severe colitis when transferred to Rag1-deficient mice provides further evidence that Blimp-1 represses IL-17 production. Analysis of Blimp-1 expression at the single cell level during Th differentiation reveals that Blimp-1 expression is induced in Th1 and Th2 but repressed by TGF-beta in Th17 cells. Collectively, the results described here establish a new role for Blimp-1 in regulating IL-17 production in vivo. The Journal of Immunology, 2012,189: 5682-5693.

Mass spectrometry-based protein profiling strategies for biomarker discovery in liver and inflammatory bowel diseases

The study of protein expression profiles for biomarker discovery in serum and in mammalian cell populations needs the continuous improvement and combination of proteins/peptides separation techniques, mass spectrometry, statistical and bioinformatic approaches. In this thesis work two different mass spectrometry-based protein profiling strategies have been developed and applied to liver and inflammatory bowel diseases (IBDs) for the discovery of new biomarkers. The first of them, based on bulk solid-phase extraction combined with matrix-assisted laser desorption/ionization - Time of Flight mass spectrometry (MALDI-TOF MS) and chemometric analysis of serum samples, was applied to the study of serum protein expression profiles both in IBDs (Crohn’s disease and ulcerative colitis) and in liver diseases (cirrhosis, hepatocellular carcinoma, viral hepatitis). The approach allowed the enrichment of serum proteins/peptides due to the high interaction surface between analytes and solid phase and the high recovery due to the elution step performed directly on the MALDI-target plate. Furthermore the use of chemometric algorithm for the selection of the variables with higher discriminant power permitted to evaluate patterns of 20-30 proteins involved in the differentiation and classification of serum samples from healthy donors and diseased patients. These proteins profiles permit to discriminate among the pathologies with an optimum classification and prediction abilities. In particular in the study of inflammatory bowel diseases, after the analysis using C18 of 129 serum samples from healthy donors and Crohn’s disease, ulcerative colitis and inflammatory controls patients, a 90.7% of classification ability and a 72.9% prediction ability were obtained. In the study of liver diseases (hepatocellular carcinoma, viral hepatitis and cirrhosis) a 80.6% of prediction ability was achieved using IDA-Cu(II) as extraction procedure. The identification of the selected proteins by MALDITOF/ TOF MS analysis or by their selective enrichment followed by enzymatic digestion and MS/MS analysis may give useful information in order to identify new biomarkers involved in the diseases. The second mass spectrometry-based protein profiling strategy developed was based on a label-free liquid chromatography electrospray ionization quadrupole - time of flight differential analysis approach (LC ESI-QTOF MS), combined with targeted MS/MS analysis of only identified differences. The strategy was used for biomarker discovery in IBDs, and in particular of Crohn’s disease. The enriched serum peptidome and the subcellular fractions of intestinal epithelial cells (IECs) from healthy donors and Crohn’s disease patients were analysed. The combining of the low molecular weight serum proteins enrichment step and the LCMS approach allowed to evaluate a pattern of peptides derived from specific exoprotease activity in the coagulation and complement activation pathways. Among these peptides, particularly interesting was the discovery of clusters of peptides from fibrinopeptide A, Apolipoprotein E and A4, and complement C3 and C4. Further studies need to be performed to evaluate the specificity of these clusters and validate the results, in order to develop a rapid serum diagnostic test. The analysis by label-free LC ESI-QTOF MS differential analysis of the subcellular fractions of IECs from Crohn’s disease patients and healthy donors permitted to find many proteins that could be involved in the inflammation process. Among them heat shock protein 70, tryptase alpha-1 precursor and proteins whose upregulation can be explained by the increased activity of IECs in Crohn’s disease were identified. Follow-up studies for the validation of the results and the in-depth investigation of the inflammation pathways involved in the disease will be performed. Both the developed mass spectrometry-based protein profiling strategies have been proved to be useful tools for the discovery of disease biomarkers that need to be validated in further studies.

Development of new therapeutic approaches in the treatments of Inflammatory Bowel Diseases: functional food and nutraceuticals vs synthetic peptides based vaccines

Inflammatory Bowel Diseases (IBD) are intestinal chronic relapsing diseases which ethiopathogenesis remains uncertain. Several group have attempted to study the role of factors involved such as genetic susceptibility, environmental factors such as smoke, diet, sex, immunological factors as well as the microbioma. None of the treatments available satisfy several criteria at the same time such as safety, long-term remission, histopatological healing, and specificity. We used two different approaches for the development of new therapeutic treatment for Inflammatory Bowel Disease. The first is focused on the understanding of the potential role of functional food and nutraceuticals nutrients in the treatment of IBD. To do so, we investigated the role of Curcuma longa in the treatment of chemical induced colitis in mice model. Since Curcma Longa has been investigated for its antinflammatory role related to the TNFα pathway as well investigators have reported few cases of patients with ulcerative colites treated with this herbs, we harbored the hypothesis of a role of Curcuma Longa in the treatment f IBD as well as we decided to assess its role in intestinal motility. The second part is based on an immunological approach to develop new drugs to induce suppression in Crohn’s disease or to induce mucosa immunity such as in colonrectal tumor. The main idea behind this approach is that we could manipulate relevant cell-cell interactions using synthetic peptides. We demonstrated the role of the unique interaction between molecules expressed on intestinal epithelial cells such as CD1d and CEACAM5 and on CD8+ T cells. In normal condition this interaction has a role for the expansion of the suppressor CD8+ T cells. Here, we characterized this interaction, we defined which are the epitope involved in the binding and we attempted to develop synthetic peptides from the N domain of CEACAM5 in order to manipulate it.

Semi-synthetic bile acids as novel drug candidate in liver diseases: physico-chemical characterization and HPLC-ES-MS/MS methods for their quali-quantitative analysis in different experimental animal models

The physico-chemical characterization, structure-pharmacokinetic and metabolism studies of new semi synthetic analogues of natural bile acids (BAs) drug candidates have been performed. Recent studies discovered a role of BAs as agonists of FXR and TGR5 receptor, thus opening new therapeutic target for the treatment of liver diseases or metabolic disorders. Up to twenty new semisynthetic analogues have been synthesized and studied in order to find promising novel drugs candidates. In order to define the BAs structure-activity relationship, their main physico-chemical properties (solubility, detergency, lipophilicity and affinity with serum albumin) have been measured with validated analytical methodologies. Their metabolism and biodistribution has been studied in “bile fistula rat”, model where each BA is acutely administered through duodenal and femoral infusion and bile collected at different time interval allowing to define the relationship between structure and intestinal absorption and hepatic uptake ,metabolism and systemic spill-over. One of the studied analogues, 6α-ethyl-3α7α-dihydroxy-5β-cholanic acid, analogue of CDCA (INT 747, Obeticholic Acid (OCA)), recently under approval for the treatment of cholestatic liver diseases, requires additional studies to ensure its safety and lack of toxicity when administered to patients with a strong liver impairment. For this purpose, CCl4 inhalation to rat causing hepatic decompensation (cirrhosis) animal model has been developed and used to define the difference of OCA biodistribution in respect to control animals trying to define whether peripheral tissues might be also exposed as a result of toxic plasma levels of OCA, evaluating also the endogenous BAs biodistribution. An accurate and sensitive HPLC-ES-MS/MS method is developed to identify and quantify all BAs in biological matrices (bile, plasma, urine, liver, kidney, intestinal content and tissue) for which a sample pretreatment have been optimized.

TNF suppresses acute intestinal inflammation by inducing local glucocorticoid synthesis

Although tumor necrosis factor (alpha) (TNF) exerts proinflammatory activities in a variety of diseases, including inflammatory bowel disease, there is increasing evidence for antiinflammatory actions of TNF. In contrast, glucocorticoids (GCs) are steroid hormones that suppress inflammation, at least in part by regulating the expression and action of TNF. We report that TNF induces extraadrenal production of immunoregulatory GCs in the intestinal mucosa during acute intestinal inflammation. The absence of TNF results in a lack of colonic GC synthesis and exacerbation of dextran sodium sulfate-induced colitis. TNF seems to promote local steroidogenesis by directly inducing steroidogenic enzymes in intestinal epithelial cells. Therapeutic administration of TNF induces GC synthesis in oxazolone-induced colitis and ameliorates intestinal inflammation, whereas inhibition of intestinal GC synthesis abrogates the therapeutic effect of TNF. These data show that TNF suppresses the pathogenesis of acute intestinal inflammation by promoting local steroidogenesis.

The continuum of intestinal CD4+ T cell adaptations in host-microbial mutualism

How a mutualistic relationship between the intestinal microbiota and intestinal T cell compartments is established is important, as a breakdown of intestinal T cell homeostasis may cause inflammatory bowel diseases. A number of studies have shown that different bacterial species modulate the intestinal CD4+ T cell compartment in different ways. We performed mechanistic in vivo studies that demonstrated the crucial requirement for regulatory T cells (Treg) and interleukin-10 (IL-10) in the induction of intestinal T cell homeostasis even following colonization with a completely benign microbiota. In the absence of a functional Treg response or IL-10 receptor signaling, the same bacteria that induced a Treg response in wild-type animals now induced T helper type 17 responses, without intestinal inflammation. Therefore, Treg, IL-10 and Th17 are crucial regulatory mechanisms in the intestine not only for controlling inflammation, but also to establish a continuum of CD4+ T cell homeostasis upon commensal colonization.

Depletion of murine intestinal microbiota: effects on gut mucosa and epithelial gene expression

Background Inappropriate cross talk between mammals and their gut microbiota may trigger intestinal inflammation and drive extra-intestinal immune-mediated diseases. Epithelial cells constitute the interface between gut microbiota and host tissue, and may regulate host responses to commensal enteric bacteria. Gnotobiotic animals represent a powerful approach to study bacterial-host interaction but are not readily accessible to the wide scientific community. We aimed at refining a protocol that in a robust manner would deplete the cultivable intestinal microbiota of conventionally raised mice and that would prove to have significant biologic validity. Methodology/Principal Findings Previously published protocols for depleting mice of their intestinal microbiota by administering broad-spectrum antibiotics in drinking water were difficult to reproduce. We show that twice daily delivery of antibiotics by gavage depleted mice of their cultivable fecal microbiota and reduced the fecal bacterial DNA load by 400 fold while ensuring the animals' health. Mice subjected to the protocol for 17 days displayed enlarged ceca, reduced Peyer's patches and small spleens. Antibiotic treatment significantly reduced the expression of antimicrobial factors to a level similar to that of germ-free mice and altered the expression of 517 genes in total in the colonic epithelium. Genes involved in cell cycle were significantly altered concomitant with reduced epithelial proliferative activity in situ assessed by Ki-67 expression, suggesting that commensal microbiota drives cellular proliferation in colonic epithelium. Conclusion We present a robust protocol for depleting conventionally raised mice of their cultivatable intestinal microbiota with antibiotics by gavage and show that the biological effect of this depletion phenocopies physiological characteristics of germ-free mice.