M.Tech. / Irritable bowel syndrome (IBS) is a multifactoral disorder of the gastrointestinal tract causing disturbances in gastric motility, resulting in abdominal pain, bloating and abnormal bowel movements. It is defined as a 'disorder of gut function in the absence of structural pathology' (Palmer et a!, 2002). It is the most commonly encountered functional gastrointestinal disorder in the primary and secondary health care system with a prevalence in the general population of five to twenty percent (Bellini et a!, 2005). No definite aetiological factor has been isolated, but factors such as psychological stress, anxiety and depression, certain dietary intolerances, increase in abnormal sensitivity to visceral distension, and hormonal changes in women have been implicated in compounding the symptoms of IBS (Ohman & Simren, 2007). Current treatment regimes include dietary changes and symptomatic relief using allopathic medications, which come with the risk of side-effects and may lead to dependency (University of Maryland Medical Center, 2007). Homoeopathic studies which have addressed the physical symptoms as well as the psychological contributing factors associated with IBS, have recorded favourable results when treating this syndrome (Mathie & Robinson, 2006).The aim of this research was to determine the efficacy of Homoeopathic Similimum treatment in IBS. In order to recruit volunteers, this study was advertised in local newspapers, pharmacies and at the University of Johannesburg's Doornfontein Campus. Volunteers completed the Rome III Criteria evaluation to determine their suitability for this study, with the likelihood of any other bowel pathology having been excluded. Ten suitable female volunteers, ranging in age from twenty to thirty five, were selected having met the inclusion criteria. Over a period of three months, each participant partook in four homoeopathic consultations. The initial consultation involved the completion of the information and consent form, an explanation of the research procedures, general well-being and general symptom rating questionnaires were completed, a full homoeopathic case history was taken, and a physical examination was performed. A baseline of four weeks without treatment followed. Participants were requested to complete daily symptom rating scales and keep a daily food diary in the four weeks between consultations. This was continued throughout the study period. The subsequent follow-ups, of which there were three, consisted of a follow-up on the initial consultation, completion of general well-being and symptom rating questionnaire, and a physical examination. A homoeopathic similimum remedy was chosen based on each participant's unique symptoms. Using physical, mental, and emotional symptoms in accordance with classical homoeopathic principles, a single remedy that most suited the individual was chosen and prescribed. It was predicted that the study would provide an alternative and safe treatment option to relieve the symptoms ofiBS. The results of the study showed that the homoeopathic similimum remedy does not provide a statistically significant improvement in the symptoms of IBS. Clinically, however, most participants experienced a general trend of improvement in physical symptoms and general well-being .

Ces dernières années, l'implication du microbiote intestinal dans la physiopathogénie des maladies inflammatoires chroniques de l'intestin (MICI) a été mise en évidence. Le but de nos travaux est de déterminer l'impact de la dysbiose sur l'écosystème intestinal au cours des MICI. Ces trois études nous ont permis de confirmer le rôle central de la dysbiose associée aux MICI : d'une part comme outil potentiellement prédictif de rechute, précédant une inflammation locale ou systémique, d'autre part comme acteur dans l'apparition d'un déséquilibre de l'écosystème intestinal. Ce déséquilibre est marqué par l'altération de l'activité enzymatique du microbiote modifiant le pool d'acides biliaires dans la lumière intestinale et pouvant affecter les effets anti-inflammatoires de certains acides biliaires sur les cellules épithéliales intestinales participant ainsi à une inflammation chronique au cours des MICI. Par ailleurs, cette dysbiose est possiblement entretenue par un déficit en défensine hBD1 et HD5, perpétuant une inflammation chronique intestinale.Ces résultats renforcent le rôle proéminent du microbiote dans l'évolution des MICI et suggèrent que la restauration de la normobiose au cours de la maladie devrait être un nouveau but dans la prise en charge de ces patients. / In recent years, the involvement of intestinal microbiota in the pathogenesis of inflammatory bowel disease (IBD) has been established. The aim of our study was to determine the impact of dysbiosis in intestinal ecosystem of IBD patients.These three studies allowed us to confirm the fundamental role of IBD-associated dysbiosis. First, IBD-associated dysbiosis has been identified as a potential predictive tool of relapse, before local or systemic inflammation. Second, IBD-associated dysbiosis has been involved as an actor in the emergence of an imbalance of intestinal ecosystem. This imbalance was characterised by an alteration of microbiota enzymatic activity leading to modifications in the luminal bile acid pool composition and may affect the anti-inflammatory effects of some bile acids on gut epithelial cells and could participate in the chronic inflammation loop of IBD. Moreover, a deficiency in the antimicrobial defense systems of defensins may be an explanation for the break of the antibacterial barrier function in inflammatory bowel diseases maintaining dysbiosis.These results reinforce the prominent role of the microbiota in the development of IBD and suggest that restoring normobiosis could be a new goal for optimal IBD management.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Background: A subtype of inflammatory bowel disease, Crohn’s disease is thought to represent a complex interaction between environmental factors, a defective immune system, the gastrointestinal microbiome and genetic susceptibility. Aim: The focus of this study was to investigate the association between environmental exposures during childhood and the subsequent development of Crohn’s disease, thus the two primary aims were to: 1) conduct a systematic review of the literature evaluating environmental risk factors during childhood, defined by studies either as, age intervals (e.g., 0-5, 6-10 and 11-18 years), or more 'broadly' as 0-18 years; and 2) investigate the association between childhood environmental exposures during three age intervals (0-5, 6-10 and 11-18 years), as well as frequency of childhood infections and the future development of Crohn's disease based on a score analysis, using a subset of previously collected data from a completed doctoral thesis involving a case control study design in study population, in the Western Cape, South Africa. The aim included a primary analysis of the latter dataset for childhood infections. Design: For the first aim of the study, a systematic search was conducted during March 2015 in electronic databases, such as EMBASE, EBSCOhost (Medline), Ovid, Scopus and World Cat, PubMed and Biomed Central, to identify epidemiological studies that examined the association between childhood environmental exposures and the subsequent development of Crohn's disease. Studies evaluating childhood exposure either by age intervals, or more broadly, from birth until 18 years were included. The environmental exposures evaluated in the review were; farm animal contact, place of upbringing, sibship size, household pets, primary water source and hot water availability. Of the 181 identified articles, 16 were included in the final systematic review. The second aim of the study involved a post hoc analysis of a subset of findings from the completed doctoral research by Abigail Basson with regard to the multiple logistic regression analysis evaluating environmental risk factor exposure during three age intervals; 0-5 years, 6-10 years and 11-18 years. In the present research, two different methodological approaches were undertaken. Briefly, exposure variables, of similar nature, were combined into subgroups and assigned weighting scores. The two 'subgroup models' were designated as: Group A and Group B. Based on these premises, a score analysis was performed, and the difference in scores, between case and control groups, was compared. In addition, multiple logistic regression models were conducted on a subset of original data from the aforementioned completed doctoral study to assess the association between the frequency of childhood infections between 0-20 years and risk of Crohn’s disease development. Following this, a score analysis was again performed. Results: Sixteen studies were included in the systematic review. Of the five studies that investigated the association between place of upbringing during the age interval 0-5 years and the subsequent development of Crohn's disease, three found no significant association; however of the three studies evaluating place of upbringing during the age intervals 6-10 and 11-18 years, only one study identified a significant association. Three studies investigated exposure to farm animals during the age interval 0-5 years, of which, two identified a significant association. Of the latter three studies, two investigated farm animal contact during the age intervals 6-10 and 11-18 years, but only one reported a significant association during these age intervals. Notably, this was the study which had failed to identify an association during the 0-5 year age interval. Both studies which broadly evaluated farm animal exposure during 'childhood' reported that not having contact with animals significantly increased the risk of developing Crohn's disease. Of the five studies that investigated exposure to pets during the age interval 0-5 years, only one identified a significant risk association, namely with exposure to cats. Of the three which investigated pet exposure during the age intervals 6-10 years and 11-18 years, one identified a significant association, for both age intervals. Five studies investigated pet exposure during 'childhood'; one found that having a pet significantly increased the risk of developing Crohn's disease, two reported that not having a pet significantly increased risk in developing Crohn's disease, whereas the remaining studies found no significant association. Only one study evaluated primary water source during the three age intervals; during the age interval 0-5 years and 11-18 years, having piped tap or bottled water was significantly associated with CD development. Of the four studies investigating primary water source during ‘childhood’, only one reported a significant association between primary water source and the development of Crohn's disease. The availability of hot water during the age interval 0-5 years was significantly associated with Crohn's disease development in one of the three relevant studies. Two studies investigated hot water availability during 6-10 and 11-18 years, however both failed to identify a significant association. When broadly evaluated, hot water availability during 'childhood' was significantly associated with Crohn's disease risk, in two of the three relevant studies. None of the studies which investigated sibship size and the risk of future Crohn's disease development during defined age intervals reported a significant association. Only two of the seven studies that evaluated sibship size during childhood reported a significant association. Results of the score analysis revealed a significant difference during all three age intervals between the case and control groups with Group A and Group B, with cases having significantly lower exposure scores (approximately 30% and 40% lower, respectively), when compared with that of controls. On multiple logistic regression analysis, subjects who never had tooth decay/cavity (OR = 1.78; 95% CI, 1.05-3.04), periodontitis (OR = 1.95; 95% CI, 1.10, 3.48), diarrhoea (OR = 2.71; 95% CI, 1.62-4.62), gastritis (OR = 2.13; 95% CI, 1.30-3.35), or mouth ulcers (OR = 2.02; 95% CI, 1.12-3.70), at least once per year or more, were at an increased risk for later development of Crohn's disease, when compared to those who were exposed to these infections at least once per year or more. There was a significant difference in exposure scores between the case and control groups (OR = 0.88; 95% CI, 0.82-0.94), thus indicating that cases had 12% less exposure to childhood infections from birth until the age of 20 years, when compared to the controls. Conclusion: The systematic review of the literature provides evidence in support of the hygiene hypothesis, in that delayed exposure to immunostimulatory microbes through the environmental exposures increases the risk for future CD development, in genetically susceptible individuals. In addition, the literature supports that the childhood environment plays an important role in the aetiology of Crohn's disease. However, the lack of consistent findings between studies, particularly those which have broadly defined ‘childhood’ implies that timing of exposure plays a crucial role in this ever evolving paradigm. Results from the score analysis provide insight into the 'compound' effects from multiple environmental exposures in the aetiology of Crohn's disease. While the present research was unable to provide any explanation for the underlying mechanism of disease pathogenesis, overall, the findings have important implications for future IBD-related studies as they demonstrate the importance of accounting for environment as a 'whole' when conducting epidemiological studies, as opposed to focusing on individual environmental factors, as well as that it is imperative to investigate environmental exposures within the context of defined age intervals.

Introduction: Inflammatory bowel disease (IBD), Crohn’s Disease and ulcerative colitis, are long term conditions which follow a relapsing and remitting pattern. The rising incidence of IBD in adults and children has implications for the lifelong burden of disease and the provision of specialist services. Patients are predominantly managed by secondary care and follow a traditional, scheduled follow-up cycle, which is unsustainable and unsatisfactory. Patients with IBD should have access to specialist care which is delivered according to their values and needs. However few studies have examined patients’ views of follow-up care. There is also concern in the UK that services for patients with long term conditions are not orgnised to promote independence with silo working in primary and secondary care.These may be brought together formally through the development of models of care. Utilization of current out-patient spaces to regularly review stable patients is inappropriate and is challenged by commissioners. The question remains as to what models of follow-up are we able to offer patients which are acceptable and what is the role of the general practitioner (GP) and primary care within this. The aim of this study was to develop an integrated, acceptable, model of follow-up care for patients with IBD.Methods The study follows the development phase of the MRC Framework for complex interventions. A best evidence synthesis was undertaken to identify the follow-up care models in IBD. A meta-synthesis of the health and social care needs of patients with IBD was conducted to explore the impact of living with IBD. Qualitative interviews with 24 IBD patients (18 patients had CD, and 6 UC, age range 27-72 years, disease duration range 2 – 40yr) and 20 GPs purposively selected from across NW England were carried out. Patients were asked about their experience, values and preference of follow-up care. The GPs were questioned about their current and potential role in IBD. Analysis was undertaken using Framework Analysis. The best evidence synthesis, meta-synthesis and interviews were synthesised by an expert panel, Consultant Gastroenterologist, patient, GP, IBD Nurse, to develop the model of follow-up care.Results There were similarities and commonalities between the patient and general practitioner interviews. Patients did not want to be seen when well, GPs wanted more involvement in care and there is scope for an IBD outreach nurse at the interface of primary/secondary care. Discharging quiescent patients into enhanced GP care, to ensure equitable treatment, was acceptable to all, as was the concept of ‘virtual’ clinics. Patients would initiate self referral within the ‘virtual’ arm whilst patients under GP care would be referred back into secondary care as a rapid referral < 7days and not using a new patient tariff. Complex IBD patients would remain under secondary care. A stratified model of follow-up care was developed.Conclusion This study provides an acceptable integrated model of follow-up for patients with IBD. It takes into account the growing incidence of IBD and UK policy to reduce inappropriate follow-up. It emphasises role of self management, the integration of primary and secondary care, placing the patient closer to home whilst allowing secondary care to concentrate on complex patient management.

The human GI tract has evolved to simultaneously absorb nutrients and be the frontline in host defence. These seemingly mutually exclusive goals are achieved by a single cell thick epithelial barrier, and a complex resident immune system which lives in symbiosis with the intestinal microflora and is also able to rapidly respond to invading pathogens. An immunological balance is therefore required to permit tolerance to the normal intestinal microflora, but also prevent the dissemination of pathogenic micro-organisms to the rest of the host. Inappropriate immune responses in genetically susceptible individuals are the hallmark of human inflammatory bowel disease (IBD) and are thus targeting effector immune cells and their cytokines remains the mainstay of treatment. However despite vigorous efforts to delineate the genetic contribution to IBD disease susceptibility using large multinational cohorts, the majority of disease heritability remains unknown. Epigenetics describes heritable changes in chromatin that are not conferred by DNA sequence. These incorporate changes to histones, chromatin structure and DNA methylation, which confer changes to gene transcription and thus gene expression and cellular function. Methylbinding proteins (MBD) have the ability to bind to methylated DNA and recruit large chromatin remodeling complexes that underpin a variety of epigenetic modifications. Methyl- CpG-binding domain protein 2 (MBD2) is one such MBD that is required for appropriate innate (dendritic cell) and adaptive (T cell) immune function, though its role has not been investigated in the GI tract. We hypothesized that alterations in chromatin are central to the reprogramming of normal gene expression that occurs in disease states. By defining the phenotype of immune cells in the absence of MBDs we hope to understand the mechanisms of chromatin-dysregulation that lead to immune-mediated diseases such as IBD. We therefore aimed to assess the role of MBD2 in colon immune cells in the steady state and in murine models of GI tract inflammation, thereafter identifying the culprit cell types and genes responsible for any observed changes. We envisaged that investigating heritable, epigenetic changes in gene expression that are inherently more amenable to environmental manipulation than our DNA code, may provide novel insight to a poorly understood mechanism of disease predisposition. In addition identifying the cellular and gene targets of Mbd2 mediated changes to immune homeostasis that may provide exciting and novel approaches to therapeutic modulation of pathological inflammatory responses. In chapter 3 we assessed the expression of Mbd2/MBD2 in the murine/human GI tract. Consistent with existing mouse data, levels of Mbd2 mRNA increased between anatomical divisions of small (duodenum, ileum, terminal ileum) and large intestine (caecum, colon, rectum). In addition MBD2 mRNA was greater in the rectum versus ileum, with active IBD associated with lower rectal MBD2 mRNA compared to quiescent IBD controls. Thus we sought to understand the role of Mbd2 in the colon, where mRNA levels were the highest in the GI tract and where appropriate immune function is central to prevent damaging inflammation. To address these aims required the development of existing methods of cell surface marker expression analysis using flow cytometry techniques to simultaneously identify multiple innate and adaptive immune populations. Using naïve Mbd2 deficient mice (Mbd2-/-) we observed CD11b+ CD103+ DCs were significantly reduced in number in Mbd2 deficiency. To understand the role of Mbd2 in colonic inflammation we employed a mouse model of chemical (DSS) and infectious (T. gondii) colitis comparing Mbd2-/- and littermate controls (WT). Mbd2-/- were extremely sensitive to DSS and T. gondii mediated colonic inflammation, characterized by increased symptom score, weight loss and histological score of tissue inflammation (DSS) and increased antibody specific cytokine responses (T. gondii) in Mbd2 deficient animals. Flow cytometry analysis of colon LP cells in both infectious and chemical colitis revealed significant accumulation of monocytes and neutrophils in Mbd2-/-. Indeed monocytes and neutrophils were the principal myeloid sources of IL-1b and TNF in DSS colitis and the number of IL-1b/TNF+ monocytes/neutrophils was significantly greater in Mbd2-/-. Lastly we employed our colon LP isolation techniques to analyse immune populations in active and quiescent IBD and healthy controls, using endoscopically acquired biopsy samples. Analysis revealed that as in murine colitis, active human IBD is characterized by the accumulation of CD14High monocyte-like cells, with an associated increased ratio of macrophage:monocyte-like cells. In Chapter 4 we sought to understand the cellular sources of Mbd2 that may explain the predisposition of Mbd2-/- to colitis. Firstly we restricted Mbd2 deficiency to haematopoietic cells using grafting Mbd2-/- bone marrow (BM) into lethally irradiated WT mice. These animals treated with DSS displayed increased weight loss, symptom score, neutrophil accumulation and histopathology score compared to mice irradiated and grafted with WT BM. Given the accumulation of monocytes in Mbd2-/- DSS treated mice, and existing literature supporting a pathogenic role in this model, we then investigated the role of Mbd2 in monocyte function. Colon monocytes sorted from Mbd2-/- and WT DSS treated mice displayed similar expression for many pro-inflammatory genes (Il6, Il1a, Il1b, Tnf), but demonstrated significantly dysregulated expression for some others (Regb, Lyz1, Ido1, C4a). To investigate this in a more refined model, we lethally irradiated WT mice and repopulated them with a WT:Mbd2-/- BM mix. This enabled the analysis of WT and Mbd2-/- haematopoietic cells in the same animal. Colon WT and Mbd2-/- monocyte recruitment and cytokine production in DSS treated mixed BM chimeras was equivalent between genotypes suggesting that Mbd2 deficiency in monocytes alone did not explain the increased susceptibility of Mbd2-/- to DSS colitis. We then restricted Mbd2 deficiency to CD11c expressing cells, given the known role for Mbd2 in their function, and for CD11c+ cells in DSS, using a CD11cCreMbd2Fl/Fl system. DSS treated mice with Mbd2 deficient CD11c+ cells demonstrated increased weight loss, symptoms score, histolopathology score, monocyte and neutrophil colon accumulation compared to controls. To further explore the role of Mbd2 in colon CD11c+ cells, macrophage and DCs from DSS treated WT and Mbd2-/- mice were purified and their gene expression analysed. Mbd2-/- versus WT macrophages demonstrated significantly altered expression of both pro- (Il1a, C6, Ido1, Trem2) and antiinflammatory (Tgfbi, Retnla) pathways that we hypothesized was a method for attempted host control of excessive colon damage in Mbd2-/- mice. DC gene expression analysis was hampered by small sample size, but demonstrated a large number of small expression changes, including IL-12/IL-23 (Jak2) and autophagy (Lrrk2) pathways. Lastly levels of costimualtory molecules (CD40/CD80) were increased in Mbd2-/- but not CD11cΔMbd2 colon LP DCs/macrophages suggesting that non-CD11c+ cellular sources of Mbd2 were required to produce increased activation phenotype in these cells. Finally in Chapter 5 we explored the role for Mbd2 in non-haematopoietic cells, namely the colonic epithelium. Here we first developed a novel method for identifying and purifying these cells using flow cytometry. Mbd2 deficient colonic epithelium demonstrated increased expression of activation markers MHC II and LY6A/E in the steady state and in DSS / T. muris mediated colonic inflammation. Indeed FACS purified colon epithelial cells from naive and DSS treated, Mbd2-/- and WT mice revealed conserved dysregulated gene expression independent of inflammation: Both naïve and inflamed Mbd2 deficient epithelium displayed significantly increased expression of genes responsible for antigen processing/presentation (MHC I, MHC II, immunoproteasome) and decreased expression of genes involved in cell-cell adhesion (Cldn1, Cldn4). Lastly we investigated whether the observed differences in Mbd2-/- cell types conferred alterations in the makeup of the intestinal microflora. Interestingly independent of co-housing of Mbd2-/- and WT animals, Mbd2 deficiency consistently predicted the microbial composition, with increased levels of Clostridales and decreased levels of Parabacteroides bacteria. Collectively we have identified CD11c+ cells, monocytes and colon epithelial cells as key cell types for Mbd2 mediated changes in gene expression that affect mucosal immune responses. These data thus identify Mbd2 gene targets within these cell types as exciting new areas for investigation and therapeutic modulation to limit damaging GI tract inflammation.

Introduction: Inflammatory bowel disease is a common, complex relapsing disorder characterised by immune dysregulation, altered intestinal permeability and microbial insult. Limited knowledge is available regarding the metabolic changes observed during progression of the disease, and limited biomarkers of disease available that have been validated and shown to be of sound clinical value. Aim of Study: A two stage metabolomics approach was adopted to determine if metabolic signature profiles, could distinguish inflammatory bowel disease Crohn’s disease (CD) patients from ulcerative colitis (UC) patients and from healthy controls. Methods: A serum metabolomics approach was undertaken to define metabolic changes associated with UC and CD. Serum samples from a discovery study of 30 UC, 30 CD and 29 ethnically, age and gender matched controls were analysed by ultra-performance liquid chromatography mass spectrometry. A subsequent validation study was preformed using 28UC, 31CD, and 29 gender matched controls were also analysed using UPLC-MS.ResultsClasses of metabolites, identified as biologically interesting and at significantly different levels (p<0.05) in comparisons of control and CD and UC cohorts included: steroids and steroid derivatives, phosphocholine, Vitamin D metabolites, fatty acids and conjugates, glycerolipids, isoprenoids, amino acids, and phosphosphingolipids. There were fewer discriminatory metabolites differentiating the CD and UC cohorts. Conclusion: Serum Metabolomic profiling may represent a novel technology which could be used to distinguish individuals with CD from those with UC and healthy controls.

Orientador: Maria Palmira Daflon Gremião / Coorientador: Flávia Chiva Carvalho / Banca: Beatriz Stringuetti Ferreira Cury / Banca: Marco Vinícius Chaud / Resumo: O metotrexato (MTX) é um fármaco com solubilidade pH dependente, utilizado no tratamento de doenças do trato gastrointestinal, como a doença de Crohn, retocolite ulcerativa e carcinomas colorretal. O desenvolvimento de micro/nanossuspensões podem ser uma estratégia promissora para incorporação de fármacos que possuem solubilidade pH dependente, oferecendo vantagens como a redução do tamanho da partícula do fármaco que pode levar ao aumento da taxa de dissolução e da permeabilidade. Adicionalmente, polímeros estímulo-responsivos podem sofrer transição de fase sol-gel in situ, promovendo a mucoadesão e uma maior retenção do sistema no local de ação. Dessa forma, o objetivo deste trabalho foi desenvolver micro/nanossuspensões contendo polímeros mucoadesivos para liberação cólon-específica do MTX. As partículas foram obtidas pelo método Bottom-up, utilizando reações de ácido-base e estabilizadas com hidroxipropilmetilcelulose (HPMC) e poloxâmero 407 (P407), através de agitação em Ultra-turrax ou Sonicador. Foram incorporados polímeros derivados do ácido poliacrílico como, Carbopol® ETD 2020 e Ultrez 10NF, poloxâmero 407 e goma gelana, pois estes polímeros atuam por diferentes mecanismos na mucoadesão como pH, temperatura e concentração iônica. A caracterização fíquico-química das micro/nanossuspensões foi realizada por espalhamento de luz, medida do potencial zeta, microscopia eletrônica de varredura (MEV), análise de textura (TPA) e ensaios de mucoadesão ex vivo. No método Bottom-up foram otimizadas condições para alcançar a solubilização e principalmente a precipitação do fármaco. Essa variação nas condições de precipitação, demonstrou que o aumento da proporção de ácido, permite modular o tamanho da partícula para a escala micro/nanométrica, na ausência... / Abstract: The methotrexate (MTX) is pH-dependent solubility drug used in the treatment of gastrintestinal diseases, such as Crohn's disease, ulcerative colitis and colorectal carcinomas. The micro/nanosuspensions development is a promising strategy to incorporate this type of drug, since it offers important advantages, such as particle size reducing of drug, which may lead to the increase of the dissolution and permeability rates of the drug. Additionaly, responsive-polymers can undergo in situ sol-gel phase transition, promoting mucoadhesion process followed by a greater retention of system in the action site. In this sense, the aim of this work was to develop micro and nanosuspensions associated to mucoadhesive polymers for colon specific release of MTX. The particles were obtained from Bottom-up method by acid-base reactions, employing the Ultra- Turrax or Sonicator and hydroxypropylmethylcellulose (HPMC) and poloxamer 407 (P407) as stabilizing. Polyacrylic acid (PAA), poloxamer 407 and gellan gum, which have different mucoadhesion mechanisms (pH, temperature and ionic concentration) were incorporated. The physic-chemical characterization of micro/nanosuspensions was realized by light scattering, measurement of zeta potential, scanning electron microscopy (SEM), texture profile analysis (TPA) and ex vivo mucoadhesion test. In the bottom-up method, the conditions were optimized to reach the solubilization and, mainly, the precipitation of drug. This variation in precipitation conditions showed that increasing the acid proportion is possible to modulate the particle size to the micro/nano-range, in the absence of stabilizer. The nanosuspensions exhibited pH above 5.0, while the microsuspensions below 4.0. For microsuspensions, analysis of the results showed that depending on the stirring process and type of stabilizer used it was possible ... / Mestre

Orientador: Alba Regina Monteiro Souza Brito / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T17:16:46Z (GMT). No. of bitstreams: 1 Almeida_AnaCristinaAlvesde_D.pdf: 2447485 bytes, checksum: 029d5569d7c4d1eaaffa8f4a530937dd (MD5) Previous issue date: 2015 / Resumo: A Doença Inflamatória Intestinal (DII), que compreende a Doença de Crohn (DC) e a Retocolite Ulcerativa (RCU), é marcada por resposta inflamatória exacerbada a componentes da microbiota, com danos à mucosa do cólon. O tratamento de DII envolve drogas ineficazes para a remissão de todos os parâmetros da doença, com vários efeitos colaterais e custo elevado, o que motiva a busca de novos agentes terapêuticos. Sendo os produtos naturais uma importante fonte para desenvolvimento de medicamentos, buscou-se, nesse trabalho, avaliar o efeito do alcaloide índigo em modelos experimentais de colite. Inicialmente, foi avaliado o tratamento oral com índigo em colite aguda induzida por ácido trinitrobenzeno sulfônico (TNBS) em ratos HanUnib: WH (Wistar), um modelo experimental de DC. O tratamento com índigo resultou em redução da lesão macroscópica, medida através de escore, na dose de 3 mg/kg, e da área de lesão ulcerativa (doses 0,1; 3; 10 e 30 mg/kg). Apenas a administração da maior dose (30 mg/kg) evitou o aumento da razão peso/comprimento do cólon e não houve diminuição da diarreia e aderência do cólon, após tratamento com o alcaloide. Danos histológicos foram minimizados no cólon de animais tratados com índigo (3, 6 e 12 mg/kg). Como a lesão macroscópica por TNBS é bastante severa, a dose de 3 mg/kg, que reduziu tanto o índice (escore) quanto a área de lesão, foi selecionada para as análises posteriores. Nos ratos tratados com índigo (3 mg/kg), houve aumento na atividade da superóxido dismutase (SOD) e redução da atividade da glutationa peroxidase (GPx), redução dos níveis de peroxidação lipídica (LPO) e, parcialmente, de glutationa (GSH), sem alteração significativa na atividade da glutationa redutase (GR) e catalase (CAT). O tratamento com índigo (3 mg/kg) evitou aumento da expressão de ciclooxigenase 2 (COX-2), e não mostrou efeito significativo na expressão de fator nuclear de transcrição ?B (NF-?B) e na concentração de citocina anti-inflamatória interleucina 10 (IL-10). O segundo modelo experimental empregado neste trabalho foi a indução de colite aguda por dextrana sal sódico (DSS), em camundongos Unib: SW (Swiss), que apresenta similaridades com a RCU. A administração de índigo (3 mg/kg) não levou à redução significativa do índice de atividade da doença (DAI), o qual engloba alteração de peso corporal, presença de diarreia e sangue nas fezes; entretanto, foi eficaz em evitar o aumento da razão peso/comprimento do cólon. Em análise histológica, notou-se menor gravidade dos danos causados pelo DSS (aumento da parede do cólon, com infiltração celular na mucosa e submucosa, desorganização do epitélio). No cólon de animais tratados com índigo, observou-se aumento na atividade da SOD, não acompanhada de mudanças nos níveis de GSH e atividade da GPx, GR e CAT. O alcaloide inibiu aumento na concentração cólica da citocina IL-6, mas não da interleucina IL-1?. Em modelo experimental de colite crônica, com recidiva, associada a câncer de cólon por azoximetano e DSS (AOM/DSS) em camundongos Unib: SW (Swiss) machos, a administração de índigo reduziu a mortalidade, minimizou a perda de massa corporal dos animais e evitou o aumento da razão peso/comprimento do cólon. A substância teste, entretanto, não foi capaz de reduzir o DAI, nesse experimento, por não minimizar a perda de consistência e o aparecimento de sangue nas fezes. No modelo de colite crônica por AOM/DSS (9 semanas), animais sadios tratados com salina fisiológica (veículo) ou índigo (3 mg/kg) foram monitorados para análise de sinais de toxicidade do alcaloide, a partir dos parâmetros: mortalidade, evolução de peso corporal, consumo de ração, peso e avaliação macroscópica dos órgãos coração, pulmões, rins e fígado. Não foram encontrados indícios de toxicidade nos parâmetros avaliados, mas devido à mortalidade de 17 % dos camundongos tratados com índigo, foi realizado teste de toxicidade aguda de dose única. Após 14 dias da administração oral e intraperitoneal de índigo (1000 mg/kg), em camundongos Unib:SW (Swiss) machos e fêmeas, não foram observadas alterações na evolução de peso corporal, consumo de água e ração, peso de órgãos vitais, comportamento e sobrevivência dos animais. Em suma, o alcaloide índigo apresentou efeito anti-inflamatório em modelos de colite por TNBS, DSS e AOM/DSS. A redução do estresse oxidativo deve ter papel central na redução das lesões causadas pelo TNBS, enquanto que na colite por DSS, a regulação da IL-6 parece ser determinante para redução dos danos. Apesar de não minimizar todos os parâmetros de danos causados pela colite, o tratamento com índigo possibilitou que os animais ficassem, em geral, menos debilitados (evidenciado pelo maior peso e consumo de ração) que os animais não tratados / Abstract: Inflammatory Bowel diseases (IBD) are known as na exacerbated imune response within the intestinal tract, mainly the mucosa of the colon. The IBD treatment is rather ineffective, including various side effects and high costs. Thus, the research with active compounds may bring therapeutic alternatives for IBD. Since natural products have been a vast source for pharmacology, we decided to investigate the effect of Indigo alkaloid in experimental models of IBD. The oral administration of Indigo (3 mg/Kg) in trinitrobenzenesulphonic acid (TNBS) -induced colitis showed beneficial results in the macroscopic and microscopic lesions, without significant results in the other evaluated parameters (diarrhea and intestine adhesion). We observed a reduction in the sulfhydryl groups (GSH) and in the activity of Glutathione peroxidase (GPx), an increase in the activity of Superoxide Dismutase (SOD) and Lipid Peroxidation (LPO). The treatment with Indigo (3 mg/Kg) prevented an increase in the LPO levels, and partially, the reduction of GSH levels. Furthermore, Indigo inhibited the increase of Cycloxigenase 2 (COX-2) expression. In the Indigo-treated animals, the expression of the Nuclear Factor kB (NF-kB) and the concentration of interleukin 10 (IL-10) were kept at intermediary levels between the healthy group and the non-treated colitic group (Veículo + TNBS). In the Dextran Sodium Salt (DSS), Indigo showed no effect on the disease associated index (DAI), which includes body weight reduction, consistence and blood in feces. However, the 7-day oral treatment with Indigo was capable of avoiding the weight/length ratio of the colon, which is usually augmented in the intestinal inflammation. In the histological, we observed a thickening of the intestinal wall, with mucosal and submucosal cell infiltration, necrotic areas as well as epithelium disorganization in the DSS-induced inflammation. The treatment with Indigo showed less severe morphological lesions. In the Dss-induced colitis, IL-6 and IL-1? levels were higher in the negative control group (DSS), which was prevented with the treatment with Indigo. In the azoximethane/DSS-cancer associated recidive model of colitis, the administration of Indigo lowered the death rate, minimized the body weight loss and also prevented the increase in the wejght/length ratio of the colon. The test substance, however, was not capable of of reducing DAI in this model, since it didn't minimize the loss of consistence and neither the blood in feces. Animals treated with saline or Indigo for 9 weeks were used for the analyses of Indigo toxicity through the following parameters: body weight evolution, chaw consumption, organ weight and macroscopic evaluation (heart, kidneys, lung and liver). In this analysis, no signs of toxicity were found for this dose of Indigo. Although it did not enhance all the parameters studied in this model of colitis, we observed that the Indigo-treated animals were, in general, less debilitated than the non-treated ones. Other studies and parameters have to be performed for a better understanding of the alkaloid effects in the intestinal inflammation / Doutorado / Fármacos, Medicamentos e Insumos para Saúde / Doutora em Ciências

As Doenças inflamatórias intestinais (DII) são multifatoriais e sua etiologia envolve susceptibilidade genética, fatores ambientais, disbiose e ativação exacerbada do sistema imunológico no intestino. Essas doenças também tem sido relacionadas a baixos níveis de dehidroepiandrosterona (DHEA), um hormônio precursor de diversos esteroides e relacionado à modulação das respostas imunes. Porém, os mecanismos precisos que relacionam as ações deste hormônio com a proteção ou susceptibilidade à doença de Crohn ou colite ulcerativa ainda não são totalmente conhecidos. Sendo assim, este projeto buscou entender o papel imunomodulador do DHEA exógeno in vitro e in vivo durante a inflamação intestinal experimental induzida por dextran sulfato de sódio (DSS) em camundongos C57BL/6. Inicialmente, in vitro, DHEA inibiu a proliferação de células do baço de forma dose dependente nas concentrações de 5?M, 50?M ou 100?M, com diminuição da produção de IFN-?. Este hormônio não foi tóxico para células de linhagem mieloide, embora tenha causado necrose em leucócitos nas doses mais elevada (50 ?M e 100?M), o que pode ter influenciado a diminuição das citocinas in vitro. Nos ensaios in vivo, os camundongos tratados com DHEA (40 mg/Kg) foram avaliados na fase de indução da doença (dia 6) e durante o reparo tecidual, quando os animais expostos ao DSS e ao DHEA por 9 dias foram mantidos na ausência destas drogas até o dia 15. Houve diminuição do escore pós-morte, melhora no peso e nos sinais clínicos da inflamação intestinal, com redução de monócitos no sangue periférico com 6 dias e aumento de neutrófilos circulantes na fase de reparo tecidual (15 dias). Ainda, a suplementação com DHEA levou à redução da celularidade da lâmina própria (LP) e ao restabelecimento do comprimento normal do intestino. O uso deste hormônio também diminuiu a expressão do RNAm de IL-6 e TGF-?, enquanto aumentou a expressão de IL-13 no colón dos animais durante a fase de indução da doença, o que provavelmente ajudou na atenuação da inflamação intestinal. Além disso, houve acúmulo de linfócitos CD4+ e CD8+ no baço e diminuição apenas de linfócitos CD4+ nos linfonodos mesentéricos (LNM), indicando retenção das células CD4+ no baço após uso do DHEA. O tratamento foi também capaz de aumentar a frequência de células CD4 produtoras de IL-4 e diminuir CD4+IFN-?+ no baço, além de reduzir a frequência de CD4+IL-17+ nos LNM, sugerindo efeito do DHEA no balanço das respostas Th1/Th2/Th17 relacionadas à colite. Em adição, as células de baço dos animais tratados com DHEA e expostos ao DSS se tornaram hiporresponsivas, como visto pela diminuição da proliferação após re-estímulos in vitro. Finalmente, DHEA foi capaz de atuar no metabolismo dos camundongos tratados, levando à diminuição de colesterol total e da fração LDL no soro durante a fase de indução da doença, sem gerar quaisquer disfunções hepáticas. Com isso, podemos concluir que o DHEA atua por meio do balanço das respostas imunes exacerbadas, minimizando os danos locais e sistêmicos causados pela inflamação intestinal induzida por DSS. / Inflammatory bowel diseases (IBD) are multifactorial diseases whose etiology involves genetic susceptibility, environmental factors, dysbiosis and exacerbated activation of the immune system in the gut. These diseases have also been associated to lower levels of dehydroepiandrosterone (DHEA), a precursor of various steroid hormones, related to modulation of immune responses. However, the precise mechanisms that link the actions of this hormone with protection or susceptibility to Crohn\'s disease or ulcerative colitis are still not fully understood. Thus, this project aimed to understand the immunomodulatory role of exogenous DHEA in vitro and in vivo in experimental intestinal inflammation induced by dextran sodium sulfate (DSS) in C57BL/6 mice. Initially, in vitro, DHEA inhibited the proliferation of spleen cells in a dose dependent way on the concentrations of 5?M, 50?M and 100?M, with decreased production of IFN-?. This hormone was not toxic to myeloid lineage cells, although it caused necrosis of leukocytes at the highest doses (50?M and 100?M), which may have influenced the decrease of the cytokines in vitro. Mice treated with DHEA (40 mg / kg) were evaluated at the induction phase of the disease (day 6) and during tissue repair, when animals exposed to DSS and DHEA for 9 days were maintained in the absence these drugs until the day 15. There was decrease of postmortem score, improved weight and clinical signs of intestinal inflammation, besides reduced peripheral blood monocytes on day 6, together with an increase in circulating neutrophils in tissue repair phase (15 days). Supplementation with DHEA also led to a reduction in cellularity of the lamina propria (LP) and to the restoration of normal length of the gut. The use of this hormone also decreased the expression of of IL-6 and TGF-? mRNA, while IL-13 was augmented in the colon of mice during the induction phase of the disease, a fact probably related to attenuation of intestinal inflammation. Furthermore, there was accumulation of CD4+ and CD8+ cells in the spleen along with decreased CD4+ leukocytes in mesenteric lymph nodes (MLN), indicating retention of CD4+ cells in the spleen after use of DHEA. The treatment was also able to increase the frequency of CD4+ cells producing IL-4 and decrease CD4+IFN-?+ in spleen, with reduced frequency of CD4+IL-17+ in the MLN, suggesting a role for DHEA on the balance of Th1/Th2/Th17 responses related colitis. In addition, splenocytes of mice treated with DHEA and exposed to DSS became hiporresponsives as seen by decreased proliferation after re-stimulation in vitro. Finally, DHEA was able to act on the metabolism of treated mice, leading to decreased total cholesterol and LDL cholesterol in serum during the induction phase of the disease, without generating any liver dysfunction. Thus, we concluded that DHEA acts by balancing the exacerbated immune responses, minimizing local and systemic damages caused by intestinal inflammation induced by DSS.

Bakgrund: Inflammatorisk tarmsjukdom räknas som en folksjukdom i Sverige. Tillstånden drabbar oftast personer i ett tidigt skede av livet och symtomen kommer i skov. Patientens vardagliga liv kan påverkas markant efter insjuknande och behandling går ut på att minska risken för nya skov och förhindra en försämring av patientens livskvalitet. Stödjande faktorer är viktigt för patienterna samt att skapa strategier för att hantera sjukdomens påverkan på dagligt liv. Syfte: Syftet med litteraturstudien var att beskriva kvinnors upplevelser av att leva med IBD. Metod: En litteraturstudie med en kvalitativ design baserad på 12 vetenskapliga artiklar utfördes. Litteratursökningen gjordes i databaserna CINAHL, PubMed och Psycinfo. De utvalda studierna kvalitetsgranskades och analyserades med innehållsanalys. Resultat: Smärta och fatigue var de två fysiologiska konsekvenser av inflammatorisk tarmsjukdom som framförallt beskrevs påverka dagligt liv. Kvinnorna kände sig ofta begränsade av sina symtom och i sin kost vilket påverkade både aktivitetsnivån, arbetslivet och socialt samliv. Begränsningarna orsakade emotionella konsekvenser och support och stöd var viktigt. Kvinnorna beskrev att de var i större behov av information gällande sin sjukdom men att det behovet inte alltid kunde tillgodoses av vårdpersonal. Konklusion: Med den ökad förståelse för sjukdomens yttrande i vardagligt liv kan sjuksköterskan genom ett helhetsperspektiv tillämpa en personcentrerad vård utifrån patientens behov och vara uppmärksam på outtalade behov som kan behövas lyftas fram i vården av patienten. / Background: Inflammatory bowel disease is considered an endemic disease in Sweden. The conditions usually affect people at an early stage of life and the symptoms come inflare-ups. The patient's daily life can be significantly affected by their illness and treatment aims to reduce the risk of new relapses and prevent a deterioration in the patient's quality of life. Supporting factors are important for patients and to create strategies for managing the impact the disease has on daily life. Aim: The aim of the literature study was to describe women's experiences of living with IBD. Method: A literature study with a qualitative design based on 12 scientific articles was conducted. The literature search was performed in the databases CINAHL, PubMed and Psycinfo. The chosen studies were quality reviewed and a content analysis was conducted. Results: Pain and fatigue were the two physiological consequences of inflammatory bowel disease that were primarily described to affect daily life. The women often felt limited by their symptoms and in their diet, which affected both the level of activity, working life and social cohabitation. The limitations caused emotional consequences and support was important. The women emphasized that they were in greater need of information regarding their illness, but that this need could not always be met by healthcare staff. Conclusion: With the increased understanding of the disease's manifestation in everyday life, the nurse can, through a holistic perspective, apply a person-centered care based on the patient's needs and be aware of unspoken needs that may need to be highlighted inthe care of the patient.