A Doença de Crohn (CD) e a Colite Ulcerativa (UC) são as principais enfermidades componentes das Doenças Inflamatórias Intestinais (DII). Embora existam vários medicamentos atualmente empregados para atenuar a inflamação descontrolada no intestino, tratar as complicações ou prolongar os períodos de remissão clínica, não há, ainda, uma terapia que seja totalmente efetiva para estas doenças. Os glicocorticoides (GCs), anti-inflamatórios comumente usados nas DII, possuem eficácia limitada e mais da metade dos pacientes se tornam refratários ou dependentes da medicação. Por outro lado, as estatinas são conhecidas por possuírem propriedades pleiotrópicas e seu uso concomitante com os GCs tem gerado boas perspectivas em várias doenças autoimunes e inflamatórias, inclusive nas DII. Apesar de já existirem indicativos de melhora de pacientes com DII pela utilização combinada destas drogas, ainda há escassez de dados que mostrem as alterações causadas no sistema imunológico. Assim, o objetivo desse trabalho foi avaliar os efeitos imunomoduladores do uso concomitante de GCs e estatinas na colite experimental induzida por dextran sulfato de sódio (DSS) em camundongos C57BL/6. Os resultados mostraram que o uso contínuo de GCs (dexametasona - DX), associados ou não a estatinas (atorvastatina - ATO), não alterou o curso da doença e antecipou a morte dos camundongos, enquanto que o oposto foi observado com o uso isolado de ATO. Tratamentos em curto prazo (3 doses) contendo ATO (isolada ou associada à DX) causaram melhora clínica e histológica dos animais doentes, diminuíram o número de leucócitos circulantes (principalmente monócitos) e de células mononucleares na lâmina própria (LP), a frequência de células CD11b+ na LP, a frequência de células dendríticas (DCs) CD11b+CD11c+ e CD11b-CD11c+ no baço e a frequência de células CD4+ produtoras de IFN- nos linfonodos mesentéricos (LNM). Entretanto, ambos os esquemas terapêuticos aumentaram a frequência de linfócitos T CD8+ no baço e LNM. Ainda, as terapias inibiram a proliferação de esplenócitos tratados in vitro, diminuíram a síntese de IL-6 e, quando em baixas concentrações, aumentaram a produção de IL-10. Diferencialmente, o tratamento combinado pareceu exercer os efeitos acima descritos de modo mais pronunciado do que o uso isolado de estatina. Adicionalmente, diminuiu os níveis de expressão de RNAm das citocinas IL-1, IL-17 e IFN- no local da inflamação, reduziu o número de linfócitos circulantes, de leucócitos no baço e LNM e de linfócitos T CD4+ nos LNM, além de ter aumentado a frequência de DCs CD11b-CD11c+ na LP e a concentração de Fas-L no intestino grosso. Considerando o uso em curto prazo com ATO isolada, foi observado aumento da frequência de DCs CD11b-CD11c+ nos LNM e de células Natural killer (NK) no baço dos camundongos doentes e diminuição dos níveis de expressão de RNAm de PPAR- no intestino grosso. O uso isolado de DX em curto prazo melhorou os aspectos histológicos, diminuiu o número de macrófagos e os níveis de IFN- no cólon, diminuiu o número de leucócitos circulantes (principalmente linfócitos), aumentou a frequência de células CD11b+ no baço e a síntese de IL-10 por esplenócitos ex vivo. Apesar da frequência de células T reguladoras (Treg) e da susceptibilidade dos esplenócitos à sinais reguladores não terem sido modificados após os diferentes tratamentos, nossos resultados sugerem que as estatinas usadas isoladamente preservaram a resposta inflamatória do organismo de modo eficiente e controlado, enquanto que o uso associado das drogas causou a imunossupressão dos animais doentes, contribuindo para as complicações clínicas decorrentes da colite experimental induzida por DSS. / Crohn\'s disease (CD) and Ulcerative colitis (UC) are the main conditions that comprise the Inflammatory Bowel Diseases (IBD). The conventional drug therapies for IBD aim to attenuate the uncontrolled inflammation in the intestinal mucosa, to treat the complications and to extend clinical remission. However, all available drugs have unpredictable or limited effects. Glucocorticoids (GCs) are commonly anti-inflammatory drugs, which are associated to refractoriness and/or dependence in over half of IBD patients. On the other hand, statins have pleiotropic properties and the concomitant use with GCs has shown good prospects in several autoimmune and inflammatory diseases, including IBD. Despite the putative clinical improvement after combined use of GCs and statins in IBD, there is a lack of data indicating their additive effects on the immune system. Therefore, the purpose of this study was to evaluate the immune modulatory effects of the concomitant use of statins and GCs in experimental colitis induced by dextran sulfate sodium (DSS). The results showed that long-term use of GCs (dexamethasone - DX), alone or associated to statins (atorvastatin - ATO), did not improve the clinical signs and increased the death rates of C57BL/6 mice exposed to DSS, while the opposite was observed after treatment with statins alone. Short-term use of ATO (3 doses), alone or associated to DX, improved the clinical signs and histological parameters in DSS-exposed mice, decreased the number of white blood cells (mainly monocytes), the number of mononuclear cells in the lamina propria (LP), the frequency of CD11b+ cells in the LP, the frequency of CD11b+CD11c+ and CD11b-CD11c+ dendritic cells (DCs) in the spleen and the frequency of IFN--producing CD4+ T cells in the mesenteric lymph nodes (MLN). However, ATO alone or associated to DX lead to increased CD8+ T lymphocytes in the spleen and MLN. Moreover, both therapies containing ATO inhibited the proliferation of in vitro-treated splenocytes, besides decreasing IL-6 and increasing IL-10 synthesis. Differentially, the association of drugs led to a more pronounced effects over the changes mentioned above than the single use of statin and additionally decreased IL-1, IL-17 and IFN- mRNA expression levels at the intestinal tissue, the number of circulating lymphocytes, the number of leukocytes in spleen and MLN and the frequency of CD4+ T lymphocytes in the MLN. In addition, statins and GCs increased the frequency of CD11b-CD11c+ DCs in LP and the Fas-L concentrations in the large intestine. Considering the short-term use of ATO there was increased frequency of CD11b-CD11c+ DCs in MLN, increased frequency of natural killer (NK) cells in the spleen and decreased mRNA expression of PPAR- in the large intestine. The short-term use of DX improved the histology parameters, decreased the number of macrophages and IFN- levels in the colon, reduced the number of circulating leukocytes (mainly lymphocytes), and increased the frequency of CD11b+ cells in spleen and IL-10 synthesis by ex vivo splenocytes. Finally, since both regulatory T cells (Treg) frequency and the splenocytes susceptibility to regulatory signals have not been modified after the different treatments, our findings suggest that single use of statins preserved an efficient and controlled inflammatory response, while the combined use of GCs and statins led to immunosuppression, which probably contributed to long-term clinical complications of DSS-induced colitis.

Orientador: Daniela Cardoso Umbelini Cavallini / Orientador no exterior: Bruce A. Vallance / Coorientador: Caetano Martha Antunes / Banca: Bruce Vallance / Banca: Carla Fontana / Banca: Xiaonan Lu / Banca: Ligia Sassaki / Resumo: O trato gastrointestinal abriga a maior população de microrganismos no corpo humano, onde eles desempenham um papel importante na promoção da saúde do hospedeiro. A alteração na composição da microbiota pode levar à disbiose intestinal, que consequentemente desencadeia ou agrava doenças intestinais e extra-intestinais. Microrganismos benéficos também conhecidos como probióticos são constantemente investigados como uma terapia complementar nas doenças relacionadas à disbiose. No entanto, sua eficácia no tratamento de condições severas, como por exemplos as doenças inflamatórias intestinais (DII), é bastante variável e apresenta resultados controversos. Para abordar a importância de uma abordagem probiótica personalizada para tratar a inflamação intestinal, primeiro examinamos o efeito de bactérias personalizadas usando um modelo de colite induzida por produtos químicos. Os animais que receberam comensais isolados de suas próprias fezes foram mais protegidos contra a inflamação, pois mostraram sinais reduzidos de colite, menor dano histológico e menores níveis de marcadores inflamatórios, quando comparados aos ratos que receberam uma cepa probiótica comercial. Em seguida, o papel da mucina intestinal Muc2 e da enzima Core-1 que glicosilam foram explorados usando o modelo de colite infecciosa Citrobacter rodentium. A camada de muco intestinal é a primeira linha de defesa no intestino e é composta em grande parte pela mucina Muc2. Uma vez que quase todas as bactérias entéricas de... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The intestinal tract harbours the largest population of microbes in the human body where they play an important role in promoting the health of their host. If the composition of these microbes is altered, this may lead to dysbiosis that triggers or exacerbates intestinal and extra-intestinal diseases. Probiotics have been investigated as a complementary therapy in dysbiosis-related diseases. However, their effectiveness in treating severe conditions such as Inflammatory Bowel Disease (IBD) is quite variable and have shown controversial results. To address the importance of a personalized probiotic approach to treat intestinal inflammation, we first examined the effect of personalized bacteria using a model of chemical induced colitis. The animals that received commensals isolated from their own feces were more protected against inflammation as they showed reduced signs of colitis, less histological damage and lower levels of inflammatory markers as compared to mice given a commercial probiotic strain. Next, the role of the intestinal mucin Muc2 and the Core-1 enzyme that glycosylates it were explored using the Citrobacter rodentium model of infectious colitis. The intestinal mucus layer is the first line of defense in the intestine and is largely composed of the secreted mucin Muc2. Since almost all enteric bacteria must cross the overlying mucus layer to infect the host, the mucus-enteric bacterial interactions provide fundamental knowledge about infectious diseases as well as inflammatory conditions linked to dysbiosis... (Complete abstract click electronic access below) / Doutor

Submitted by CARINA DE FÁTIMA DE SIBIA null (carinasibia@gmail.com) on 2017-02-21T19:28:25Z No. of bitstreams: 1 tese final 21.2.pdf: 1461578 bytes, checksum: aaee8aaa84b0ad8485b183d6dc28a095 (MD5) / Rejected by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br), reason: O arquivo submetido está na orientação paisagem, o arquivo deve estar na orientação retrato on 2017-02-24T19:02:56Z (GMT) / Submitted by CARINA DE FÁTIMA DE SIBIA null (carinasibia@gmail.com) on 2017-02-24T22:08:47Z No. of bitstreams: 1 tese .pdf: 1294801 bytes, checksum: dd9a234277b605736e3567a3692c2d48 (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-03-06T16:57:54Z (GMT) No. of bitstreams: 1 sibia_cf_me_bot.pdf: 1294801 bytes, checksum: dd9a234277b605736e3567a3692c2d48 (MD5) / Made available in DSpace on 2017-03-06T16:57:54Z (GMT). No. of bitstreams: 1 sibia_cf_me_bot.pdf: 1294801 bytes, checksum: dd9a234277b605736e3567a3692c2d48 (MD5) Previous issue date: 2017-01-31 / Introdução: A doença de Crohn (DC) e a retocolite ulcerativa (RCU) são as duas principais doenças que compõem a doença inflamatória intestinal (DII). Estudos indicam que vários genes estão diferencialmente expressos em DC. vs. RCU. Entretanto, os mecanismos moleculares de desenvolvimento e progressão das diferentes formas da DII ainda não foram elucidados. Considerando que os microRNAs (miRNAs) são potentes reguladores da expressão gênica e têm papel importante em várias doenças humanas, estes podem constituir biomarcadores com potencial diagnóstico, prognóstico e terapêutico em DII. Objetivos: Identificar miRNAs desregulados em DII, distinguindo DC e RCU; identificar genes-alvo dos miRNAs alterados e redes de interação entre miRNAs e genes-alvo em DII. Materiais e Métodos: Foi utilizada estratégia de meta-análise para identificação de dados de expressão de miRNAs em DII. Após aplicação dos critérios de inclusão e exclusão, foram selecionados 10 estudos para extração dos dados. Desses estudos, foram identificados miRNAs significativamente desregulados (nível de alteração ou FC>=2 e p<0,05) e coletadas informações sobre o tipo e o número de amostras analisadas (soro, plasma ou tecido) de pacientes com DC ou RCU, plataformas utilizadas para análise de expressão de miRNAs e validação dos dados, entre outras. A seguir, foram aplicadas as ferramentas de bioinformática mirwalk 2.0 para predição de genes-alvo regulados pelos miRNAs e STRING e BiNGO para identificação de redes de interação entre miRNAs e genes-alvo e funções biológicas, respectivamente. Resultados: Entre os miRNAs com expressão aumentada, foram identificados 17 em DC e 62 em RCU. Entre os miRNAs com expressão diminuída, foram identificados 18 em DC e 31 em RCU. Os miRNAs que mostraram o maior número de interações com genes-alvo na DC foram: let-7a-5p, let-7b-5p, miR-199a-5p, miR-150-5p, miR-362-3p e miR-224-5p. Em RCU, os miRNAs desregulados e com maior número de interações foram miR-155-5p, miR-24-5p, miR-335-5p e miR-16-5p. Conclusões e Perspectivas Futuras: Foram identificadas redes de interação entre miRNAs e genes-alvo associados a processos biológicos de inflamação e resposta imune. Os miRNAs identificados modulam vias moleculares potencialmente envolvidas na patogênese da DII. Estudos como esse podem contribuir para a melhoria do diagnóstico e no desenvolvimento de tratamentos direcionados e mais precisos para pacientes com DC e RCU.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / IntroduÃÃo: A mucosite induzida por antineoplÃsicos à um fator limitante na terapia anticÃncer. Mucosite à um termo clÃnico que descreve uma sÃndrome caracterizada por reaÃÃo inflamatÃria da mucosa de todo o trato digestivo com ulceraÃÃes. A mucosite intestinal por antineoplÃsicos desencadeia alteraÃÃes da motilidade digestiva. A dexametasona à um glicocorticÃide cujo principal efeito farmacolÃgico decorre de sua aÃÃo antiinflamatÃria e imunossupressora Objetivos: Investigar a dismotilidade gastrintestinal associada à mucosite intestinal induzida por irinotecano em camundongos, bem como avaliar o efeito da modulaÃÃo farmacolÃgica da resposta inflamatÃria sobre estas alteraÃÃes motoras. MÃtodos: Camundongos Swiss machos (20â25g) foram tratados do D1-D4 com irinotecano (75 mg/Kg, i.p.) ou com salina (0,1ml, i.p.), o sacrificio foi realizado no sÃtimo dia experimental. Afim de estudar alteraÃÃes relacionadas à mucosite intestinal, amostras do duodeno, jejuno e Ãleo, foram removidas para avaliar a injÃria epitelial por morfometria, escores histolÃgicos, e atividade de MPO, tendo a diarrÃia sido avaliada antes do sacrifÃcio. Para avaliaÃÃo de citocinas amostras de duodeno foram retiradas e pelo mÃtodo de ELISA foi determinada a concentraÃÃo de TNF-&#61537;, IL-1&#61538;, KC e IL-10. Para avaliar a motilidade digestiva, os animais foram deixados em jejum de 18 horas do D6 para o D7. No D7, foram administrados 350ÂL da soluÃÃo glicosada (5%) contendo vermelho de fenol (VF) a 0,75 mg/ml em cada animal antes do sacrifÃcio. ApÃs os tempos de 10, 20 e 30 min, os animais foram sacrificados e submetidos a uma laparotomia mediana, sendo o estÃmago retirado completamente e o intestino delgado divido em 3 partes iguais: proximal, medial e distal. A seguir o esvaziamento gÃstrico, trÃnsito intestinal e transito gastrintestinal (centro geomÃtrico) foram determinados por espectofotometria. A seguir, em outro grupo experimental, foi avaliado se a modulaÃÃo farmacolÃgica do processo inflamatÃrio poderia reverter Ãs alteraÃÃes da motilidade digestiva associadas à mucosite por irinotecano. Para tanto, dexametasona (2,5mg/kgm s.c.) ou metoclopramida (10mg/kg i.p.) do D1-D7, foram administradas 30 minutos antes da administraÃÃo de irinotecano. A seguir foram avaliados todos os parÃmetros descritos acima. Resultados: O tratamento com irinotecano foi capaz de induzir uma lesÃo intestinal com um importante comprometimento da barreira epitelial funcional com a presenÃa das seguintes alteraÃÃes: diarrÃia, leucopenia, encurtamento acentuado das vilosidades intestinais, necrose parcial de criptas, aumento da atividade de MPO, aumento na concentraÃÃo de KC e IL-1&#61538; e retarde do esvaziamento gÃstrico e transito gastrontestinal e aceleraÃÃo do transito intestinal. O tratamento com dexametasona, mas nÃo metoclopramida, reduziu significativamente as lesÃes intestinais, com recuperaÃÃo da altura dos vilos, da profundidade das criptas, diminuiÃÃo da atividade de MPO, reduÃÃo da concentraÃÃo de IL-1&#61538;. Ademais, somente o tratamento com dexametasona, mas nÃo por metoclopramida, foi capaz de reverter retarde do esvaziamento gÃstrico, bem como a aceleraÃÃo do trÃnsito gastrintestinal. ConclusÃo: A mucosite intestinal induzida por irinotecano foi capaz de causar dismotilidade e resposta inflamatÃria gastrintestinal com participaÃÃo de KC e IL1&#61538; a qual se associa com o retarde do esvaziamento gÃstrico e trÃnsito gastrintestinal e aceleraÃÃo do trÃnsito intestinal. ConcluÃmos ainda que a dexametasona, mas nÃo metoclopramida, reduziu a resposta inflamatÃria e modulou parcialmente a dismotilidade gastrintestinal associada à mucosite por iriniotecano. / Introduction: Mucositis induced by antineoplasic drugs is a limiting factor in anticancer therapy. Mucositis is a clinical term which describes a syndrome characterized by mucosal ulceration of the entire digestive tract. Mucositis results from intestinal inflammatory events, which induces changes in gastrointestinal motility. Dexamethasone is a glucocorticoid whose main pharmacological effect is anti-inflammatory and immunosuppressive. Objectives: To investigate the gastrointestinal dismotility associated with irinotecan-induced intestinal mucositis in mice and to assess the effect of pharmacological modulation of the inflammatory response on these motor abnormalities. Methods: Swiss male mice (20-25g) were treated in the D1-D4 with irinotecan (75 mg / kg, ip) or saline (0.1 ml, ip), the sacrifice was performed on the seventh day trial in order to study changes related to intestinal mucositis. Samples of duodenum, jejunum and ileum were removed to assess the epithelial injury by morphometry, histological scores and MPO activity, and diarrhea were evaluated before sacrifice. For evaluation of cytokine samples of duodenum were removed and the ELISA was determined concentrations of TNF-&#61537;, IL-1 &#61538;, KC and IL-10. In order to evaluate gastrointestinal motility, the animals were kept fasting for 18 hours from D6 to D7. In D7, were administered 350&#956;L of glucose solution (5%) containing phenol red (VF) to 0.75 mg / ml in each animal prior to sacrifice. After the times of 10, 20 and 30min, animals were sacrifice. Stomach was totally removed, and small intestine was divided into 3 parts: proximal, medial and distal. The gastric emptying, intestinal transit and gastrointestinal transit were determinate by spectrofotometry. In other experimental group, we evaluated if pharmacological modulation of the inflammatory process could reverse the gastrointestinal dismotility associated with irinotecan- induced intestinal mucositis. Then, dexamethasone (2.5 mg / kg sc) or metoclopramide (10mg/kg ip) of D1-D7 were administrated 30 minutes before irinotecan administration and all of parameters described before were evaluated Results: Treatment with irinotecan was able to induce an intestinal lesion with significant impairment of epithelial barrier function in the presence of the following changes: diarrhea, leukopenia, marked shortening of the villus, crypts of partial necrosis, increase im MPO activity, increased concentrations of IL-1 &#61538; and changes in gastrointestinal motility. Treatment with dexamethasone, but not with metoclopramide, significantly reduced the intestinal lesions, with recovery of villous height, crypt depth, decreased MPO activity, reduced concentrations of IL-1&#61538;. In addition, dexamethasone, but not metoclopramide, was able to reverse the delay in gastric emptying and increase in intestinal transit. Conclusion: The intestinal mucositis induced by irinotecan was able to cause gastrointestinal dismotility and inflammatory response with the participation of KC and IL1&#61538; which is associated with delay gastric emptying and gastrointestinal transit and acceleration of intestinal transit. It is also concluded that dexamethasone, but not metoclopramide, reduced the inflammatory response and modulated in part the gastrointestinal dismotility associated with irinotecan-induced intestinal mucositis.

A microbiota intestinal é um ecossistema complexo que desempenha um importante papel na gênese da obesidade. A ocorrência e participação dos Mollicutes na microbiota intestinal é praticamente desconhecida. Deste modo, o objetivo do presente estudo foi analisar a participação dos Mollicutes e dos Filos Firmicutes e Bacteroidetes na microbiota intestinal de mulheres obesas e eutróficas. A casuística foi de 20 mulheres obesas e 20 mulheres em eutrofia. Foram obtidas amostras de fezes, sangue e aplicado questionário semiestruturado sobre fatores relacionados com obesidade, microbiota intestinal e ambiente, além de Bioimpedância e questionário de frequência alimentar. Constatou-se uma associação positiva estatisticamente significante entre a presença de Mollicutes e mulheres obesas. Foi observada maior proporção de Firmicutes/Bacteroidetes na microbiota intestinal das mulheres obesas. Os resultados obtidos permitiram obter evidências importantes da participação dos micro-organismos da classe Mollicutes. As alterações da microbiota intestinal também contribuíram na definição de subconjuntos de indivíduos com diferentes perfis de risco metabólico e a da heterogeneidade associada a fenótipos humanos relacionados com a adiposidade. / The gut microbiota is a complex ecosystem that plays an important role in the pathogenesis of obesity. The occurrence and participation of Mollicutes in the gut microbiota is pratically unknown. The aim of this study was to analyze the participation of Mollicutes and Firmicutes and Bacteroidetes phylos in the gut microbiota of obese and normal weight women. For the study, it was collected samples of 20 women with obesity and 20 women of normal weight. It was collected stool samples, blood, semi-structured questionnaire on factors associated with obesity, gut microbiota and the environment, and anthropometric measurements using bioelectrical impedance and food frequency questionnaire. It was detected a statistically significant positive association between the presence of Mollicutes and obese women, and there was a higher proportion of Firmicutes/Bacteroidetes in the gut microbiota of obese women. The results provide important evidence about the participation of Mollicutes class in the gut microbiota of the population studied and interactions in intestinal microbiota can define subsets of individuals with different metabolic risk profiles and thus contribute to investigation of the heterogeneity associated phenotypes related to adiposity.

Tryptases are trypsin-like enzymes found in mast cell granules. Although in vivo substrates have not been positively identified, tryptases cleave a limited number of potential physiological substrates in vitro, including high molecular weight kininogen (HMWK) and vasoactive intestinal peptide (VIP). Purified human lung mast cell tryptase (HLT) apparently exists as a tetramer with an M$\sb{\rm r}$ of 135-144 kDa by gel filtration, whereas SDS-PAGE yielded two bands of M$\sb{\rm r}$ 29 Kda and 33 Kda. Tryptases are resistant to inhibition by most natural trypsin inhibitors and display some affinity for heparin. The existence of tryptase isozymes has been implied from the cloning of two tryptase cDNAs from human lung tissue, but distinct isozymes have not been isolated and characterized. This knowledge gap has been filled by isolating and characterizing two electrophoretically different forms of human lung mast cell tryptase, designated high-HLT (high molecular weight HLT) and low-HLT (low molecular weight HLT). These two forms of HLT have been separated by chromatography on a cellulose phosphate column, with the high M$\sb{\rm r}$ form eluting with 10 $\mu$M heparin and the low M$\sb{\rm r}$ form subsequently eluting with 1 M NaCl. Using HMWK and VIP as substrates, these two forms of HLT were found to differ with regard to specificity and rate of cleavage. High-HLT initially cleaved HMWK at a single Arg residue, whereas low-HLT cleaved HMWK simultaneously at multiple sites. Both isozymes cleaved VIP at multiple sites, but differed with regard to the preferential site of cleavage. Low-HLT was, on an active site basis, 25 and 2 times more active than high-HLT on HMWK and VIP, respectively. In addition, gel filtration of the isozymes yielded M$\sb{\rm r}$s of 125 Kda for high-HLT and 28 kDa for low-HLT, indicating tetrameric and monomeric quaternary structures, respectively. Both isozymes were inhibited by human secretory leukocyte proteinase inhibitor (SLPI), but not by other trypsin inhibitors tested. This work provides the first evidence for the existence of distinct tryptase isozymes, with supposedly different in vivo functions, and identification of an inhibitor that may control tryptase activity in vivo.

Thesis (M.Pharm. (Pharmaceutics) -- University of Limpopo, 2018 / Griseofulvin is an antibiotic fungistatic drug used in the treatment of dermatophyte and ringworm infections. About 50% of a dose of griseofulvin passes the gastro- intestinal tract unabsorbed and is excreted in faeces. Since griseofulvin is highly soluble in acidic pH, a gastro-retentive floating matrix system was developed to control dissolution rate and thereby enhance solubility in an effort to develop an improved and convenient dosage form. Preformulation studies included selection of excipients and evaluation of their compatibility with griseofulvin. Using the chosen excipients, floating tablets of griseofulvin were formulated. Floating tablets containing 100 mg of griseofulvin were prepared by wet granulation technique with varying ratios of Methocel™, Accurel MP and Polyvinylpyrrolidone as determined by Design Expert software. Pre and post-compression studies, buoyancy capability and dissolution studies were carried out to assess the influence of the tablet components. Results obtained revealed that a density of less than 0.00091 g/cm3 was necessary for tablet floatation. Tablets that float immediately upon contact with dissolution medium and continue floating for over 12 hours were achieved with at least 28% Accurel MP by mass of the tablet. Dissolution studies revealed that an increase in tablet hardness reduced the rate of griseofulvin release only up to 120 minutes. From 120 minutes onwards, tablet hardness had no significant influence on griseofulvin release from tablets. Methocel™ had the most significant influence on griseofulvin release. The amount of Methocel™ included in the formulation was indirectly proportional to the rate of griseofulvin release. Using Design Expert software, optimized formulation was achieved with 1% Polyvinylpyrrolidone, 30% Methocel™, 60% Accurel MP and hardness ranging between 8 – 9 N. Pre and post-compression parameters of the optimized tablets were found to be within pharmacopoeial limits and thus compressed tablets were of acceptable quality. Tablets produced floated immediately upon contact with the medium and remained floating for at least 12 hours. Griseofulvin was released from the optimized tablets in a near zero order fashion, with a total of 80.8% griseofulvin released at the end of the 12 hour dissolution test period. Results of accelerated stability studies indicated potential stability of the manufactured tablets months.

Background: The traditional model of infection control has failed to stop the spread of emerging infectious diseases such as Clostridium difficile-associated diarrhea (CDAD) in the acute care environment. Ecological models, which rely upon identification of susceptible hosts, offer an alternative to the prevention of deadly outbreaks. Previous epidemiological research has identified a number of risk factors associated with CDAD. Utilization of this body of research by nurses is limited due to methodological issues that introduce bias and confounding, and use of variables that have limited meaning to the practicing clinical nurse. Aim: The aim of this study was to develop an ecological model useful for nurses in predicting the susceptibility of individuals to CDAD during an acute care hospital stay. Method: A case-control study compared 66 cases with CDAD to 66 controls matched for the temporal and spatial risk factors of hospital admission date and geographic nursing care unit within the institution. The two subject groups were compared on variables of age, antibiotic burden, laxative or bowel preparation exposure, nutritional status, gastric acid suppression therapy, enteral feeding exposure, and severity of illness as measured on the Horn Severity of Illness index. All subjects were hospitalized between January 1, 2000 and December 31, 2006. Results: On univariate analysis, age, severity of illness, serum albumin levels, length of exposure, and proton pump inhibitor drug burden were significantly associated with CDAD status. Following multivariate analysis, only severity of illness, length of exposure, and decreased antibiotic drug burden were significantly associated with the development of hospital-acquired CDAD. Conclusions: This study supports the use of an ecological perspective in identifying risk factors and interventions to prevent the future spread of this infectious disease.

Abstract not available

Throughout history a diversity of animal species have been used and studied extensively in the development of vaccines for the benefit of humans and animals alike. As mice are a relatively easy species to maintain, handle and manipulate, and have the advantage of being cost effective, they are commonly employed as animal models in the investigation of immunisation strategies against mucosal associated pathogens. Vaccine research against the human gastric pathogen Helicobacter pylori is extensively conducted in a mouse model and typically uses intra-gastric administration for the testing of potential vaccine candidates. An inherent complication with this route, however, is that the vaccine constituents may be inadequately delivered to sites of specific immunity and consequently may not be the optimal method for vaccine delivery. In the present study a mouse model of H. pylori infection was used to determine the efficacy of alternate mucosal routes of immunisation from examination of protective immunity, immune responses and the practical aspects of vaccine administration. Commencing with the optimisation of intra-intestinal immunisation, the direct injection of a H. pylori vaccine to initiator sites of the mucosal immune system established baseline data of dose rates for the comparative analysis of intra-gastric, intra-nasal and intra-rectal immunisation. Following the development of simple administration techniques whilst maintaining the welfare of the animals, intra-nasal immunisation was shown to elicit the highest level of prophylaxis against H. pylori challenge. Effective prophylaxis was also shown to be dependent upon a specific ratio of the vaccine constituents. When using whole cell lysate of H. pylori and the mucosal adjuvant cholera toxin, the ratio of antigen:adjuvant for optimal protective immunity was 10:1. The outcomes of this study have proved conclusively the necessity for optimisation of all aspects of immunisation in an animal model of infection. / Master of Science (Hons)