Background: Perianal fistulae are common and cause significant symptoms and psychosocial morbidity. Current theory suggests two distinct types: idiopathic (no established cause) and secondary, which usually arise from Crohn's disease. Research to date has assumed that idiopathic and Crohn's perianal fistulae differ in their pathogenesis and pathophysiology. This fundamental hypothesis has not yet been adequately tested. We therefore aimed to test the broad hypothesis that idiopathic and Crohn's perianal fistulae differ in their pathogenesis and pathophysiology. Methods: We prospectively recruited a cohort of 61 participants (48 idiopathic, 13 Crohn's disease) and phenotyped them in detail using clinical assessment, Perineal Disease Activity Index, EQ-5D-5L, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), operative assessment, biopsy, and clinical follow-up. We interrogated the biopsy samples in the laboratory to characterize their profiles for 30 cytokines and 39 phosphoproteins. Over 12,000 clinical and 23,500 laboratory measurements were made. Results: We found no clear differences in the clinical characteristics or the overall cytokine or phosphoprotein profiles between idiopathic and Crohn's perianal fistulae. We also found no differences in the DCE-MRI measurements between the groups. Conclusions: We did not find good evidence to support our hypothesis. The alternative hypothesis offers a shift in the current research paradigm: idiopathic and Crohn's perianal fistulae are the same pathology on a spectrum across which the pathogenic and pathophysiological features are distributed in a skewed manner. This may be the key to unravelling our understanding of the disease's aetiology. It would also be a construct within which trials of biological therapy, such as infliximab, could be justified for idiopathic disease.

Orientador: Celso Antonio Rodrigues / Resumo: Na abordagem dos bovinos com enfermidades digestivas, durante o exame físico, exames complementares são necessários, de forma a retificar a suspeita clínica, contribuir para o estabelecimento do prognóstico e decisão clínica. Tais exames compreendem a avaliação do perfil hematológico, bioquímico, líquido peritoneal, ruminal, exame ultrassonográfico abdominal e laparotomia exploratória. Este último por se tratar de uma abordagem cirúrgica convencional, está associado as complicações e custos inerentes a técnica. Neste cenário a videolaparoscopia se apresenta com uma possibilidade para reduzir as complicações trans e pós-operatórias. O objetivo deste trabalho foi revisar e comparar a utilização da videolaparoscopia, ultrassonografia e demais métodos diagnósticos das enfermidades digestivas dos bovinos. Foram utilizados 7 bovinos mestiços, atendidos na Clínica de Bovinos de Garanhuns, unidade hospitalar da UFRPE. Realizou-se exame físico, ultrassonografia abdominal, videolaparoscopia exploratória, coleta de amostras de sangue, para a realização de hemograma, determinação plasmática da proteína total e fibrinogênio, e do soro foram realizadas análises dos indicadores bioquímicos: Proteína total, albumina, globulinas, relação albumina/globulina, glicose, L-lactato, uréia, creatinina, as atividades enzimáticas da aspartato aminotransferase (AST), gama glutamiltransferase (GGT), creatino quinase (CK) e lactato desidrogenase (LDH). Foi coletado líquido peritoneal e realizada análise ... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Submitted by JOSÉ RICARDO BARBOZA SILVA null (jose.ricardo_medvet@hotmail.com) on 2018-03-10T14:57:32Z No. of bitstreams: 1 Silva, J.R.B., 2018. Dissertação vs final arquivo.pdf: 2884438 bytes, checksum: 1aeb3fe55c567c7423c443a4d83a7ae2 (MD5) / Approved for entry into archive by Maria Lucia Martins Frederico null (mlucia@fca.unesp.br) on 2018-03-12T13:06:22Z (GMT) No. of bitstreams: 1 silva_ jrb_me_botfca.pdf: 2796813 bytes, checksum: 80083164ac1f3e931aed7c335545c884 (MD5) / Made available in DSpace on 2018-03-12T13:06:22Z (GMT). No. of bitstreams: 1 silva_ jrb_me_botfca.pdf: 2796813 bytes, checksum: 80083164ac1f3e931aed7c335545c884 (MD5) Previous issue date: 2018-03-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Na abordagem dos bovinos com enfermidades digestivas, durante o exame físico, exames complementares são necessários, de forma a retificar a suspeita clínica, contribuir para o estabelecimento do prognóstico e decisão clínica. Tais exames compreendem a avaliação do perfil hematológico, bioquímico, líquido peritoneal, ruminal, exame ultrassonográfico abdominal e laparotomia exploratória. Este último por se tratar de uma abordagem cirúrgica convencional, está associado as complicações e custos inerentes a técnica. Neste cenário a videolaparoscopia se apresenta com uma possibilidade para reduzir as complicações trans e pós-operatórias. O objetivo deste trabalho foi revisar e comparar a utilização da videolaparoscopia, ultrassonografia e demais métodos diagnósticos das enfermidades digestivas dos bovinos. Foram utilizados 7 bovinos mestiços, atendidos na Clínica de Bovinos de Garanhuns, unidade hospitalar da UFRPE. Realizou-se exame físico, ultrassonografia abdominal, videolaparoscopia exploratória, coleta de amostras de sangue, para a realização de hemograma, determinação plasmática da proteína total e fibrinogênio, e do soro foram realizadas análises dos indicadores bioquímicos: Proteína total, albumina, globulinas, relação albumina/globulina, glicose, L-lactato, uréia, creatinina, as atividades enzimáticas da aspartato aminotransferase (AST), gama glutamiltransferase (GGT), creatino quinase (CK) e lactato desidrogenase (LDH). Foi coletado líquido peritoneal e realizada análise físico-química e bioquímica. Diante dos resultados, observamos que a videolaparoscopia e ultrassonografia abdominal auxiliam na determinação do diagnóstico e prognóstico precisos das enfermidades digestivas. / 2016/2006-4

Cryptosporidiosis is a common infectious diarrhoeal disease of mammalian livestock and humans worldwide. The etiological organisms responsible are intestinal apicomplexans of the genus Cryptosporidium, including C. parvum, that infect intestinal epithelial cells. Immunocompromised or malnourished hosts develop severe life-threatening disease. Immunological elimination of Cryptosporidium requires CD4+ T cells and IFN-γ. Nevertheless, studies have shown innate immune responses have a significant protective role. Importantly, in T cell-deficient mice, IFN-γ is important for control of C. parvum infection. In innate immunity natural killer (NK) cells are major producers of IFN-γ and are activated by cytokines including type I IFNs but the roles of these components in immunity to Cryptosporidium infection have not been investigated. Therefore, the purpose of this project was to study the involvement of type I IFNs and NK cells in immunity to C. parvum employing in vitro and in vivo (murine) infection models. Enterocytes were shown capable of the production of type I IFNs in response to C. parvum infection. These cytokines directly inhibited parasite development in epithelial cells. Also, in neonatal SCID mice the level of infection increased after treatment with anti-type I IFN neutralising serum. A higher level of infection was observed in Rag2-/-γc-/- mice deficient in T, B and NK cells in comparison to Rag2-/- mice with a normal NK cell population and early mortality during chronic infection of adult animals was associated with the absence of NK cells. Using cultures of SCID mouse splenocytes, NK cells were the main source of IFN-γ in response to C. parvum antigen stimulation. However, IFN-γ was also found to have a protective role in Rag2-/-γc-/- mice, implying cells other than lymphocytes produce this cytokine. In conclusion, this is the first study to indicate important protective roles for type I IFNs and NK cells in innate immunity against C. parvum.

Background: Colorectal cancer is the result of dysregulation within classic regulatory pathways in epithelial stem-cell(s): the precursors giving rise to all other intestinal lineages. The resulting cancer stem-cell (CSC) generates tumours utilising its innate properties, e.g. self-renewal and lineage plasticity. CSCs appear to persist within a tumour as a distinct subtype responsible for local recurrence/metastasis. Therefore, therapies targeting colorectal CSCs may lead to improved cancer-specific outcome measures. The PROM-1/CD/AC133 cell surface marker has been associated with colorectal CSCs and its expression is reported as an independent negative prognostic marker. Therefore, this thesis sought to investigate the role of PROM-1/CD/AC133 in colorectal cancer. Methods: Tissue-culture, RT-qPCR, IHC, Western blotting, siRNA, PCR-array and FACS analyses were used to quantify and profile mRNA/protein expression patterns. Results: PROM-1/CD/AC133 was widely expressed in patient-matched colorectal tumour, adjacent normal epithelium, vascular invasion, lymph node metastases with significantly decreased expression in liver metastases. Furthermore, PROM- 1/CD/AC133 expression was not found to enrich colorectal cancer cell line populations for additional stem cell phenotypes (expression of ABCB1/ABCG2/BMIJ Murphy 1/CD44/LGR5/MSI-1). The data confirm the presence of alternative splice variants of PROM-1, and show that transcripts specifying PDZ binding predominate in colorectal cancer cell lines. Concomitantly, siPROM-1 was shown to modulate the expression of several key transcripts in colorectal tumourigenesis as well as regulate signal transduction pathways including the central cancer, colorectal cancer, NF-kB and p53 signalling cascades. Conclusions: PROM-1/CD/AC133 does not identify rare colorectal cancer cells responsible for tumourigenesis. However, it is associated with the regulation of signalling networks associated with cell growth, differentiation and apoptosis suggesting a potential role for this marker in colorectal tumourigenesis. The identification of specific target genes and signalling pathways in this thesis provides a springboard for further investigations into the functional role of this marker in colorectal cancer, with the potential for better treatments for this disease.

Objectives: Tolerance is a hyporesponsive state caused by repeated exposure to a stimulus. In the intestine, dysregulation of tolerance to luminal stimuli may lead to chronic and deleterious inflammation, such as characterizes Inflammatory Bowel Disease. The role of T-cells in immune tolerance is well known, but that of the epithelium requires investigation. Epithelial tolerance is gene-specific and differentially regulated, but the role of and involvement of epigenetics in tolerance regulation is unknown. We hypothesized that prior stimulation may cause epithelial cells to become hyporesponsive (tolerized) and that modification of histone methylation may alter the response to pro-inflammatory stimulation. The aim of this work was to examine if known inhibitors of histone methylation modifying enzymes affected the expression of CXCL8 in response to IL-1β. Methods: CXCL8 production of intestinal epithelial cells was measured by ELISA after stimulation with the pro-inflammatory stimuli P3CK and IL-1β and small molecule epigenetic inhibitors. The CXCL8 production of cells stimulated with a pro-inflammatory stimulus was compared to pre-stimulated cells after a second stimulus. CXCL8 production of IL-1β-pre-stimulated cells was also compared to CXCL8 production when these cells were incubated with epigenetic inhibitors. The effects of these inhibitors on histone methylation levels were examined by Western blotting for the global effect and by ChIP-qPCR for specific effects at the CXCL8 locus. Results: Intestinal epithelial cells stimulated with pro-inflammatory stimuli produced a large CXCL8 response. Pre-stimulation significantly decreased CXCL8 production after a second stimulus. The time-course of CXCL8 expression was measured to ensure that CXCL8 expression due to pre-stimulation was over before the second IL-1β-stimulation. In the presence of specific epigenetic inhibitors, pre-stimulation by IL-1β did not reduce CXCL8 production after a second IL-1β- stimulation. The specific effect of these inhibitors on the epigenetic signature at the CXCL8 locus was confirmed by ChIP. Thus, histone methylation modification disrupted tolerization of intestinal epithelial cells to a pro-inflammatory stimulus. Conclusion: The inflammatory response of the intestinal epithelium can be tolerized by prior stimulation with pro-inflammatory cytokines. Tolerization is lost after incubation with inhibitors known to modify histone methylation status, indicating for the first time, the involvement of histone methylation in this phenomenon.

Linear growth retardation affects up to 40% of children with Crohn’s disease (CD). It is caused by a combination of under\nutrition, and by a direct effect of cytokines on the axis linking human growth hormone (GH), insulin like growth factor\1 (IGF\1) and the growth plate of growing bones. In particular,the inflammation causes a functional insensitivity to GH, resulting in low circulating IGF\1. Current management is synonymous with the optimal management of childhood CD:to eliminate inflammation,and maintain remission. However, there is currently no consensus as to how to treat growth failure in patients whose inflammation remains intractable to treatment. This thesis examined the hypothesis that successful treatment of inflammation would improve linear growth in children with CD. We performed a series of retrospective studies examining a cohort of children aged ≤17 years with CD treated at Bart’s and The London Children Hospital. We determined the height standard deviation scores (SDS) of patients at diagnosis and investigated growth outcomes following treatments used to induce and maintain remission. These were:exclusive enteral nutrition (EEN), thiopurines and infliximab. Finally, as a potential new therapy, we considered the use of exogenously administered recombinant human IGF\1 (rhIGF\1) to restore plasma IGF\1 levels. We performed an open\labelled pharmacokinetic (PK) study of rhIGF\1 in 8 children with CD and growth failure. 9% of our patients had height SDS of \2 SDS at diagnosis; a four\fold increase compared to the normal age matched population. In addition, symptom duration negatively correlated with height SDS at diagnosis (r=\0.06; p=0.02). From 89 5 patients receiving EEN for primary induction of remission, 62.9% (56/89) of patients achieved complete remission. Over the course of 5 years, responders to EEN grew significantly better than non\responders (change in height SDS +0.18 [0.12] vs to \ 0.37 [0.13] respectively; p=0.005). This occurred despite no differences in duration of remission or treatment escalation between groups. 51% (26/51) patients treated with thiopurines were in remission at 12 months. These patients had improved growth (median change height SDS [IQR] 0.08 [\0.06 – 0.19] vs \0.24 [\0.61 \ \0.07] in non\responders; p=0.001). 39.2% (20/51) of these patients started anti\TNF therapy. However, over 12 months, we found that this conferred no improvement in growth (median [IQR] at 0 months\0.38 [\1.73 to \0.10] as compared to \0.61 [\ 1.72 to \0.09] at 12 months; p=0.43), irrespective of response to treatment. Subcutaneous treatment with 120 μg/kg twice daily rhIGF\1 restored plasma levels of IGF\1 in our patients, albeit it in some to high levels (median [range] concentration +2.09 [\1.27 to +5.21]). We were able to develop a PK mathematical model from these results to determine a more appropriate dosage. We found that in addition to patient’s age and weight, PCDAI also needs to be taken into consideration when determining dose. In summary, growth retardation is very common finding in paediatric CD. Response to EEN and thiopurines seems to result in beneficial effects upon growth. However, a number of children continued to have poor growth despite treatment. Our results show it is possible to increase circulating IGF\1 concentrations with exogenous injections, and develop a mathematical model to devise a dose to use in further trials.

Inflammatory diseases such as PCOS, autoimmune diseases, irritable bowel syndrome, etc. are all highly uncomfortable diseases with several negative side effects. By adding antioxidants and omega-3 fatty acids to patients with inflammatory diseases diets, studies show that symptoms of these diseases will lessen. The objective of this study is to create a banana bread with increased omega-3 fatty acids and increased antioxidants to be served on trays of patients with inflammatory diseases and for patients to make at home to decrease symptoms related to inflammation. The experimental food should be an equal substitute for the control flavor, aroma, and texture wise. The control banana bread was substituted for an anti-inflammatory banana bread with the addition of cinnamon, dark chocolate, extra eggs, and pecans. The banana bread was made without dairy and grain for celiac patients and lactose intolerant patients. Both variations were equally accepted according to the hedonic scale, completed by 9 participants. Research was continued to confirm the of increased omega-3 fatty acids within the anti-inflammatory bread. Furthermore, walnuts were switched for pecans to test the antioxidant and fatty acid composition of both variations. Overall, we found that the walnut variation had more fatty acids, but pecans had more antioxidants. Our research suggests that both variations can be used to accommodate patients with inflammatory diseases. Further research can be done for long-term research for inflammatory disease patients that swapped the control for the variations.

Pneumatosis intestinalis, air within the bowel wall, continues to have an elusive etiology due to its varied clinical presentation and associated disease processes. Pneumatosis may be an incidental finding on a routine CT Scan or it could present as peritonitis with intra-abdominal free air. The pathogenesis, therefore, is likely to be multifactorial rather than directly related to one particular, inciting pathology. Here we present a case of a 73-year-old male scheduled for a non-emergent incisional hernia repair who was found to have peritoneal free air without physical exam findings of peritonitis. This unusual case illustrates a rare presentation of small bowel, omental, and abdominal wall pneumatosis. The objective of this presentation is to broaden the clinician’s understanding of pneumatosis intestinalis, including a recommendation to discern the underlying illness as emergent or benign. Finally, we make the case for clinical intuition and the physical exam.

No / Methionine dependence is unique to cancer cells and defined as the inability to grow in a methionine-deprived environment even if supplemented with the metabolic precursor homocysteine. Cobalamin-dependent methionine synthase (MS) catalyses the formation of methionine and tetrahydrofolate from homocysteine and methyltetrahydrofolate, thus linking the methionine and folate pathways. The apparent altered methionine metabolism in methionine-dependent cancer cells suggests a role for MS, although results to date are conflicting. We have analysed key metabolites of the MS-associated transmethylation, transsulphuration and folate pathways of the methionine-dependent MAC15A tumour model as a function of tumour progression over a 10-day period. MS activity increased 2-fold from day I to day 10. Cysteine, homocysteine, S-adenosylmethionine and S-adenosylhomocysteine levels in tumour cytosolic fractions decreased as a function of tumour progression. Plasma cysteine levels also decreased, whilst the distribution of folates in erythrocytes was altered, with a maximum increase in methyltetrahydrofolate observed by day 5. The increasing MS activity and decreasing cysteine levels suggest an increasing methionine requirement by the tumour, whilst the induction of enzyme activity indicates that MS is not defective in the methionine-dependent MAC15A tumour. The decrease in tumour S-adenosylmethionine and S-adenosylhomocysteine levels suggests that methionine is required for some function other than cellular methylation, e.g., incorporation into protein. Overall, the results support a theory of methionine conservation in response to tumour growth, where the methionine-dependent MAC15A tumour has a higher than normal methionine requirement.