Peritoneal adhesions pose a considerable postoperative burden. The mechanisms underlying adhesion formation remain poorly understood but relate principally to inflammation and fibrinolysis. The peritoneum may coordinate the inflammatory response that leads to adhesion formation but human adhesion formation cannot be studied directly so most research has employed animal models and individual transformed cell lines. Furthermore, the specific roles of the visceral or parietal peritoneum in adhesion formation are unclear. The aims of the studies undertaken in this thesis are to investigate the regulation of a gut bacteria-driven inflammatory response in human visceral peritoneum. A novel ex vivo model was designed using human small bowel visceral peritoneum, cultured with E. coli derived LPS, for 18 to 48 hours. ELISA and rtPCR were used to analyse supernatant protein concentration and gene expression respectively for key inflammatory mediators associated with adhesion formation including cytokines (IL-6, IL-10, TNFalpha), transforming growth factor-β1 (TGF-β1) and fibrinolytic factors (tPA and PAI-1) as well as the signalling receptors thought to be involved in LPS-mediated inflammation (TLR2 and 4). LPS exposure resulted in stimulation of IL-6, IL-10 and TNFalpha production from human visceral peritoneum at 18 hours. These increases correlated with increased gene expression for IL-6 and TNFalpha but not IL-10. TGF-β1 production and gene expression were unchanged at 18 & 24 hours but increased at 48 hours when co-incubated with IL-1β. PAI-1 was increased but tPA was unchanged following LPS stimulation, suggesting a reduction in peritoneal fibrinolytic activity. Peritoneal tissue expressed the signalling receptors TLR 2 and 4. However, inhibition of TLR 2 or 4 failed to abrogate production of cytokines in cultured primary mesothelial cells or peritoneal tissue post-LPS exposure. TLR 4 inhibition did reduce mesothelial cell production of chemokines, CCL2 and CXCL1. These novel findings demonstrated that human visceral peritoneum actively participates in LPS-induced peritoneal inflammatory responses and reduced fibrinolysis which may account for the visceral peritoneum's affinity to form adhesions. Ex vivo human peritoneal culture is a novel, feasible and reproducible method of investigating the role of the peritoneum in inflammation.

No description available.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Introduction: Intestinal mucositis is a common side effect of anticancer regimens for first-line treatment of colorectal cancer. Among such drugs are irinotecan and 5-FU used in combination. Many studies have been conducted on the pathogenesis of MI. However, further investigations are necessary, because the course of MI may vary according to the drug regimen and employees. Then aimed to develop a new experimental model of MI induced by the combination of IRI and 5-FU in mice. Methods and Results: C57BL/6 mice (20-25g, n=6) were injected with saline (100ÂL, i.p), IRI (30 or 45 mg/kg, i.p), 5-FU (25, 37.5 or 50 mg/kg, i.p) or IRI+5-FU for 4 days. On day 7, diarrhea, weight loss, and blood leukocyte count were registered. Following animal euthanasia, ileum samples were collected for histopathological analysis, myeloperoxidase activity (MPO, neutrophil/mg protein), TNF-&#945; and IL-6 levels (pg/mg tissue). Kaplan-Mayer log rank test, Kruskal Wallis/Dunnâs or ANOVA/Bonferroniâs test was used for statistical analysis. p<0.05 was accepted. The best dose combination able to induce IM with no important mortality on day 7 was 5-FU (37.5 mg/kg) +IRI (45 mg/kg) (0% mortality), which was then used for subsequent studies. IRI+5-FU induced (p<0.001) diarrhea (2[0-3]), weight loss (86.7Â3.9g), and leukopenia (7.3 2.3 x103) versus saline group (0[0-1]; 101.1Â0.6; 215.5 54.1, respectively) or each drug given alone (5-FU: diarrhea (0[0-1]), weight loss [92.6Â2.7], and leukopenia [30.4 13.4]; IRI: diarrhea (0[0-1]), weight loss [94.8Â2.1], and leukopenia [49.2 5.5]). In addition, IRI+5-FU induced inflammatory cells infiltration, and loss of villi and crypt architecture (4[3-4]), increased in MPO activity (14641Â1598 neutrophil/mgproteÃn), TNF-a (3.2Â0.9 pg/mg tissue), IL-6 (1.4Â0.5 pg/mg tissue) tissue levels versus saline-injected group (0[0-1], 5747Â1155; 0.7Â0.2; 0.3Â0.1) or the drugs injected alone (5-FU: Intestinal damage (2.5[2-3]), MPO [3788Â1212], TNF-&#945; [0.7Â0.2], IL-6 [0.2Â2.3]; IRI: Intestinal damage (1[0-2]), MPO [3580Â1613], TNF-&#945; [0.4Â0,2], IL-6 [0.07Â0.05]) (p<0.05). Conclusion: We developed a new experimental model of IM induced by the combination of IRI+5-FU in mice, which opens perspective for a more appropriate knowledge concerning the pathogenesis of IM. / IntroduÃÃo: Mucosite intestinal à um efeito adverso comum dos regimes anticÃncer de primeira linha para o tratamento do cÃncer colorretal. Dentre esses fÃrmacos estÃo o irinotecano e 5-FU utilizados em associaÃÃo. Muitos estudos tÃm sido realizados sobre a patogÃnese da MI. No entanto, novas investigaÃÃes sÃo necessarias, pois o curso da MI pode variar de acordo com o fÃrmaco e regime empregados. Objetivou-se entÃo desenvolver um novo modelo experimental de MI induzida pela combinaÃÃo de IRI e 5-FU em camundogos. MÃtodos e Resultados: camundongos C57BL/6 (20-25g, n=6) foram injetados com salina (100ÂL, i.p), IRI (30 ou 45 mg/kg, i.p), 5-FU (25, 37,5 ou 50 mg/kg, i.p) ou IRI+5-FU em doses combinadas por 4 dias. No 7 dia, diarreia, perda de peso e contagem de leucÃcitos foram registradas. ApÃs a eutanÃsia dos animais, amostras do Ãleo foram coletadas para anÃlise histopatolÃgica, atividade de mieloperoxidase (neutrÃfilo/mg proteÃna), nÃveis de TNF-&#945; e IL-6 (pg/mg tecido). Teste Kaplan-Mayer log rank, Kruskal Wallis/Dunnâs ou teste ANOVA/Bonferroni foram usados para analise estatistica. p<0,05 foi aceito como significativo. A melhor combinaÃÃo de dose para induzir a MI sem mortalidade importante no 7 dia foi 5-FU (37,5 mg/kg) + IRI (45 mg/kg) (0% mortalidade), que foi entÃo usada para estudos subsequentes. IRI+5-FU induziu (p<0,001) diarreia (2[0-3]), perda de peso (86,7Â3,9g), e leucopenia (7,3 2,3x103) versus grupo salina (0[0-1]; 101,1Â0,6; 215,5 54,1, respectivamente) ou cada droga dada sozinha (5-FU: diarreia (0[0-1]), perda de peso [92,6Â2,7], e leucopenia [30,4 13,4]; IRI: diarreia (0[0-1]), perda de peso [94,8Â2,1], e leucopenia [49,2 5,5]). Adicionalmente, IRI+5-FU induziu infiltrado de cÃlulas inflamatÃrias, perda de vilos e arquitetura das criptas (4[4-4]), aumento na atividade de MPO (14641Â1598 neutrÃfilo/mgproteÃna), TNF-a (3,2Â0,9 pg/mg de tecido), IL-6 (1,4Â0,5 pg/mg de tecido) nÃveis teciduais versus grupo injetado com salina (0[0-1], 5747Â1155; 0,7Â0,2; 0,3Â0,1) ou com fÃrmacos injetados isoladamente (5-FU: dano intestinal (2.5[2-3]), MPO [3788Â1212], TNF-&#945; [0,7Â0,2], IL-6 [0,2Â2,3]; IRI: dano intestinal (1[0-2]), MPO [3580Â1613], TNF-&#945; [0,4Â0,2], IL-6 [0,07Â0,05]) (p<0,05). ConclusÃo: Desenvolvemos um novo modelo experimental de MI induzida pela combinaÃÃo de IRI+5-FU em camundongos, que abre perspectiva para um maior conhecimento sobre a patogÃnese da MI.

Orientadores: José Flávio Affonso de Almeida, Maria Cristina Volpato / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-17T16:39:58Z (GMT). No. of bitstreams: 1 Monteiro_MariaRachelFigueiredoPenalva_M.pdf: 1282276 bytes, checksum: b7401c79964f58abbced19c5c4e238eb (MD5) Previous issue date: 2011 / Resumo: Processos inflamatórios com pulpite irreversível podem promover alterações que levam à hiperalgesia e alodínia, tornando mais difícil a obtenção de anestesia local efetiva. Este estudo teve como objetivo avaliar a eficácia anestésica de duas soluções e técnicas anestésicas em molares inferiores com pulpite irreversível, comparando a técnica infiltrativa vestibular mandibular com a solução de articaína 4% (grupo 1) com a técnica de bloqueio dos nervos alveolar inferior e lingual com o uso da lidocaína 2% (grupo 2), ambas associadas à epinefrina 1:100.000. Também foi avaliada a eficácia da técnica inicial mais uma complementação (técnica infiltrativa- articaína com técnica intraligamentar- articaína e, bloqueio- lidocaína com técnica infiltrativa- articaína) entre os grupos e comparadas às eficácias anestésicas da técnica inicial e técnica inicial mais uma complementação no mesmo grupo. Além disso, foi avaliada a relação entre o desempenho da articaína e a espessura da cortical óssea vestibular e a distância das raízes mesial e distal à mesma cortical, por meio de tomografia computadorizada ¿Cone Beam¿. Para tal, foram distribuídos 43 voluntários em 2 grupos experimentais 43 pacientes (28 do grupo 1; 15 do grupo 2) com molares inferiores diagnosticados com pulpite irreversível. Todos foram orientados a preencher escalas analógicas visuais (EAV) para demonstrar a intensidade de dor anteriormente ao tratamento (calibração), durante o exame de diagnóstico clínico (endofrost) e após 5 e 10 minutos da aplicação da anestesia. Foram considerados sucessos os casos tratados sem dor com a técnica inicial e com a técnica inicial mais complementação anestésica. Não foi observada diferença estatisticamente significante (p=0,0955) para a técnica inicial (articaína 4% sucesso de 39,7% e lidocaína 2% sucesso de 13,3%) nem para a técnica inicial mais uma complementação quando comparados os grupos (p=0,7448). No entanto, quando analisado o mesmo grupo, foi encontrada diferença estatisticamente significante para a técnica inicial mais complementação anestésica no grupo da lidocaína (p=0,0025). Em relação à análise tomográfica, não houve influência da espessura da cortical óssea vestibular e da distância entre as raízes mesial e distal até a mesma cortical óssea no desempenho da articaína (p>0,05). Concluiu-se que, a infiltração com articaína obteve desempenho semelhante ao bloqueio com lidocaína pela técnica inicial ou após complementação anestésica e que a espessura da cortical óssea e a distâncias das raízes à cortical óssea não tem relação direta com o sucesso ou insucesso da eficácia anestésica da articaína. / Abstract: Inflammatory process due to irreversible pulpits can cause hyperalgesia and allodynia that can difficult proper profound local anesthesia. The main goal of this study was to evaluate the anesthetic efficacy of two initial techniques and solutions in mandibular molars, one in mandibular infiltrations with 4% articaine (group 1) an alveolar nerve block with 2% lidocaine (group 2), both with 1:100.000 epinephrine. Likewise, evaluated the efficacy of the initial technique and one supplemental injection between groups and inside the same group (mandibular infiltration and intraligamentary infiltration both with articaine and alveolar nerve block with lidocaine and mandibular infiltration with articaine) and the relationship of success and failure of articaine with buccal cortical thicknesses and mesial and distal root distance to the same cortical (cone beam tomography). Data (43 volunteers), admitted with a clinical diagnostic of irreversible pulpits, were randomized into two experimental groups (28 from group 1 and 15 from group 2). All patients were instructed to assess their pain in a visual analogue scale (VAS) before treatment, after cold testing and 5 and 10 minutes after the injection. Success criteria were painless treatment with only an initial anesthesia or adding one supplemental injection. There was no statistic difference between group 1 (39.7%) and group 2 (13.3%) (p=0.0955) with the initial technique and with one supplemental injection when groups were compared (p=0.7448). However, there was statistical difference when analyzed inside the same group for lidocaine (p=0.0025). Yet, no statistical difference was observed in tomography evaluation between articaine efficacy and buccal cortical thickness in mesial and distal root or between articaine efficacy and mesial and distal root distance to the buccal cortical (p>0.05). It was concluded that articaine was similar to lidocaine effectiveness with the initial technique or with one supplemental injection and that the buccal cortical thickness and the mesial and distal root distance to the same cortical had no relationship to the success or failure of articaine efficacy. / Mestrado / Endodontia / Mestre em Clínica Odontológica

Orientador: José Barreto Campello Carvalheira / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T19:07:24Z (GMT). No. of bitstreams: 1 Rocha_GuilhermeZweig_D.pdf: 7543896 bytes, checksum: 5459a8edf8e3ca3315e980f52bbbb876 (MD5) Previous issue date: 2014 / Resumo: Embora a obesidade seja reconhecida como importante causa de diabetes e doença cardiovascular, a associação entre obesidade e diferentes tipos de câncer tem recebido muito menos atenção. A associação entre obesidade e o desenvolvimento de câncer de cólon representa um dos principais avanços conceituais na patogênese do câncer de cólon da última década. Recentemente a atuação da inflamação subclínica da obesidade na carcinogênese ganhou destaque. Mecanisticamente acredita-se que a obesidade atue como promotor tumoral, e seus efeitos pró-tumorigênicos dependam principalmente da resposta inflamatória de baixo grau ocasionada pela obesidade que envolve a produção de citocinas inflamatórias e pró-tumorigênicas (TNF e IL-6). Uma das principais características da inflamação induzida por obesidade é a infiltração de macrófagos no tecido adiposo, produzindo citocinas inflamatórias e outros mediadores que interferem na sinalização insulínica. Inflamação e estresse de retículo que são conectadas em diversos níveis, são sistemas adaptativos de curto período de expressão necessárias para a função e sobrevivência do organismo, e ambas são prejudiciais quando ativadas cronicamente. Neste sentido, a ativação da PKR durante a inflamação e posterior ativação de JNK pela PKR, também interfere e prejudica a via de sinalização da insulina. A relação entre o câncer de cólon e obesidade pode ser devido a ação, em nível molecular, da inflamação subclínica de baixo grau e ao estresse celular causado por essa sinalização inflamatória. Sendo a PKR responsiva à sinalização inflamatória e também à via insulínica em outros tecidos, e relacionada à carcinogênese e à progressão em diversos tipos de câncer, a investigação de sua participação é relevante a medida que propicia o entendimento da fisiopatologia molecular de tumores de cólon. Assim, o objetivo principal do estudo foi avaliar o papel da PKR no desenvolvimento de tumores de cólon em camundongos submetido a dieta hiperlipídica. A ausência de PKR previne a formação de tumores. Além disso, aparentemente a ausência de PKR em células mielóides também confere proteção contra a resistência à insulina induzida por dieta hiperlipídica, reduzindo a inflamação induzida pela obesidade. Essas observações demostram que a PKR pode ser um ponto principal durante a carcinogênese associada à inflamação e pode representar um promissor alvo para a intervenção terapêutica / Abstract: Although obesity is recognized as a major cause of diabetes and cardiovascular disease, the association between obesity and different types of cancer has received much less attention. The association between obesity and the development of colon cancer is one of the major conceptual advances in the pathogenesis of colon cancer in the last decade. Recently the role of subclinical inflammation in obesity and in carcinogenesis gained prominence. Mechanistically it is believed that obesity acts as a tumor promoter, and their pro-tumorigenic effects depend mainly on low-grade inflammatory response caused by obesity, involving the production of inflammatory and pro-tumorigenic cytokines (TNF and IL-6). A key feature of obesity-induced inflammation is the infiltration of macrophages in adipose tissue, producing inflammatory cytokines and other mediators that interfere with insulin signaling. Reticulum stress and inflammation are connected on many levels and work as short period adaptive systems required for the function and survival of the organism, and both are detrimental when chronically activated. In this regard, the activation of PKR during inflammation and subsequent activation of JNK by PKR also interferes and impairs insulin signaling pathway. Thus, PKR can form a metabolically active inflammatory complex which then becomes part of the of insulin pathway and of the pathogens response pathway and control of translation sensible to nutrients. The relationship between colon cancer and obesity may be due to action at the molecular level, subclinical low-grade inflammation and cellular stress caused by this inflammatory signaling. PKR is responsive to inflammatory signaling and also to the insulin pathway in other tissues, and related to carcinogenesis and progression in several types of cancer. Thus, investigation of it's participation is relevant as it provides the understanding of the molecular pathophysiology of colon tumors. Thus, the main objective of the study was to evaluate the role of PKR in the development of colon tumors in mice subjected to a high-fat diet. The absence of PKR prevents the formation of tumors. Moreover, apparently the absence of PKR in myeloid cells also confers protection against resistance to insulin induced by a high-fat diet, reducing inflammation induced by obesity. These observations demonstrate that PKR can be a primary point during carcinogenesis associated with inflammation and may represent a promising target for therapeutic intervention / Doutorado / Fisiopatologia Médica / Doutor em Ciências

Orientadores: Maria Heloísa de Souza Lima Blotta, Rômulo Tadeu Dias de Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T11:06:38Z (GMT). No. of bitstreams: 1 Mineiro_MarcelaFranco_M.pdf: 3128314 bytes, checksum: 8579ae8081ba0070be37eca4970a0ac6 (MD5) Previous issue date: 2013 / Resumo: A presença de IL-1? e IL-18 em lesões ateroscleróticas e o importante papel dessas citocinas na ativação endotelial, recrutamento celular e na polarização para a resposta TH1 constitui indício indireto do envolvimento dos NLRs na patogênese da aterosclerose. Nesse trabalho investigamos a presença do RNAm e proteína de diversos NLRs, AIM2, caspase-1, proteína adaptadora ASC e IL-1? em placas ateroscleróticas humanas. Observamos que amostras de ateroma de pacientes assintomáticos apresentaram maior expressão relativa do RNAm de NLRP1, 2 e 3, NLRC2, 3, 4 e 5, NAIP e ASC. A presença de fatores de risco não promoveu diferenças na expressão gênica do RNAm para nenhum deles. A marcação por imuno-histoquímica revelou a presença de NLRP1, 3, AIM-2, NLRC2, 3 e 5 e caspase-1, especialmente em macrófagos, células gigantes e células espumosas. Em culturas de monócitos do sangue periférico, macrófagos humanos derivados de monócitos e células da linhagem monocítica THP-1 estimuladas com LPS, a presença de cristais de colesterol teve efeito aditivo na produção de IL-1?. Por outro lado, os inibidores farmacológicos inibiram a produção dessa citocina em células THP-1 após o estímulo com cristais de colesterol. A atividade da citocina IL-1? também foi avaliada pela capacidade de induzir a produção de mediadores inflamatórios por células musculares lisas e células endoteliais. Catepsina S, MMP3, CXCL10, CXCL16 e CX3CL1 foram produzidas apenas por células musculares após o estímulo, mas CCL2, CXCL8 e CCL5 foram produzidos tanto por células musculares quanto por células endoteliais estimuladas. Em conclusão, nosso trabalho mostrou a presença de RNAm e proteína de diversos NLRs em placas de ateroma humano. Além disso, mostramos o papel dos cristais de colesterol na ativação de monócitos e macrófagos produtores de IL-1?, provavelmente via NLRP3, expresso em grande quantidade nas placas analisadas. Finalmente, os experimentos de estimulação de células musculares lisas e endoteliais ressaltaram a importância da IL-1? como agente inflamatório e desestabilizador das lesões, confirmando seu potencial como alvo terapêutico visando a contenção do processo inflamatório que caracteriza a aterosclerose / Abstract: The presence of IL-1? and IL-18 in atherosclerotic lesions and their important role in endothelial activation, cell recruitment and switch to Th1 response, are indirect indications of the involvement of NLRs in disease pathogenesis. In this study we investigated the presence of mRNA and protein of various NLRs, AIM2, caspase-1, adaptor protein ASC and IL-1? in human atherosclerotic plaques. We observed that atheroma samples from asymptomatic patients had higher mRNA relative expression of NLRP1, NLRP2, NLRP3, NLRC2, NLRC3, NLRC4, NLRC5, NAIP and ASC. Risk factors have no influence in mRNA expression for any of them. Immunohistochemistry technique revealed the presence of NLRP1, NLRP3, AIM-2, NLRC2, NLRC3, NLRC5 and caspase-1, especially among macrophages, giant cells and foam cells. The presence of cholesterol crystals in monocytes and macrophages cultures stimulated with LPS, had an additive effect on IL-1? production. On the other hand, pharmacological inhibitors inhibited this cytokine production by THP-1 cells stimulated with cholesterol crystals. The ability of IL-1?to induce inflammatory mediators production by smooth muscle cells and endothelial cells was also evaluated. Cathepsin S, MMP3, CXCL10, CXCL16 and CX3CL1 were produced only by smooth muscle cells after stimulation, but CCL2, CXCL8 and CCL5 were produced by both smooth muscle cells and endothelial cells. In conclusion, our study revealed the presence of various NLRs mRNA and protein in human atherosclerotic plaques. Furthermore, we demonstrated the role of cholesterol crystals in the activation of IL-1? producers monocytes and macrophages, probably due to NLRP3 expressed in large amounts on the lesions analyzed. Finally, stimulation of smooth muscle cells and endothelial cells highlighted the importance of IL-1? as an inflammatory and destabilizing agent in lesions, confirming its potential as a therapeutic target that could restrain the prevailing inflammation in atherosclerosis / Mestrado / Ciencias Biomedicas / Mestra em Ciências Médicas

Activation of the cannabinoid receptor CB₂ has been shown to induce directed leukocyte migration and inhibit leukocyte chemotaxis towards CC chemokines. However, the role that CB₂ plays in regulating macrophage chemotaxis remains understudied. Using a real-time chemotaxis assay and a panel of chemically diverse CB₂ agonists, I set out to examine whether CB₂ modulates primary macrophage chemotaxis. Of 14 agonists tested, only a subset acted as bona fide macrophage chemoattractants. Surprisingly, despite being pertussis toxin-sensitive, neither pharmacological inhibition nor genetic ablation of CB₂ had any effect on CB₂ agonist-induced macrophage chemotaxis. Furthermore, the activation of CB2 had no effect on CCL2 or CCL5- induced macrophage chemotaxis. Therefore, the activation of CB2 does not inhibit CC chemokine-induced macrophage migration and a non-CB₁/CB₂, G_i/o-coupled GPCR must transduce CB2 agonist-induced macrophage chemotaxis. To identify the GPCR responsible, I examined primary murine macrophage GPCR expression and found that they express 124 non-sensory GPCRs. Functional screening of candidate receptors demonstrated that the putative cannabinoid receptors GPR18 and GPR55 and the lipid binding GPCRs LPAR1&amp;5, CYSLTR1&amp;2 and GPER1, were not responsible for CB₂ agonist-induced macrophage chemotaxis. Alongside, a ligand-directed virtual screen, combined with functional testing, uncovered a novel chemotaxis positive chemical scaffold. Importantly, compounds in this series containing a photoaffinity label retained activity and will aid in the identification of the target(s) responsible for CB₂ agonist-induced macrophage chemotaxis in future photocrosslinking experiments. Finally, I assessed whether CB2 controls innate immune cell recruitment in vivo using the zymosan-induced dorsal air pouch inflammation model and animals genetically deleted for CB₂. I found that CB₂^-/- mice had increased air pouch neutrophil and monocyte numbers, as well as pro-inflammatory mediators, during the acute inflammatory phase. Interestingly, mixed bone marrow chimera experiments demonstrated that lack of CB₂ specifically in the myeloid population is responsible for increased neutrophil trafficking. Therefore these data demonstrate that CB₂ acts to regulate neutrophil recruitment during the acute inflammatory response.

Topical glucocorticoids (GCs), such as hydrocortisone (HC), are the main drugs used to treat inflammatory skin conditions including eczema and psoriasis, but their longterm use is limited by the onset of side effects such as skin thinning, impairment of wound healing and systemic metabolic dysfunction. For this reason, there is a substantial need for new compounds with the same anti-inflammatory effects but fewer adverse effects. Previous studies have suggested 5α-tetrahydrocorticosterone (5α-THB) as a new, more selective anti-inflammatory compound; this steroid is a metabolite of the major endogenous GC in rodents, corticosterone (B). Preliminary data indicated that 5α-THB may be as effective as HC in reducing mouse irritant dermatitis, but without the local or systemic side effects of HC. The aim of this thesis is to investigate the mechanisms through which 5α-THB delivers more selective anti-inflammatory effects, with the hypothesis that 5α-THB influences distinct signalling pathways from those of B. A mouse model of irritant dermatitis induced by topical application of croton oil on the ear was developed, and the anti-inflammatory properties of 5α-THB were analysed, in comparison with those of B, after 6 and 24 hours of treatment. In inflamed tissue, B reduced tissue oedema and cell infiltration at both time points; in contrast, 5α-THB did so at 24 but not 6 hours, at a dose five-fold higher than B. Real-time analysis at 24 hours showed that B and 5α-THB similarly reduced the croton oil-induced increase of transcripts of genes encoding vascular and cellular adhesion molecules. Interestingly, while B did not affect the abundance of transcripts of the anti-inflammatory gene Dusp1, 5α-THB increased it in croton oil-treated ears, suggesting a different mechanism of action between 5α-THB and B. The experiment was repeated with the injection of the glucocorticoid receptor (GR) antagonist RU486; RU486 relieved the effect of B on swelling but did not attenuate the anti-inflammatory effects of 5α-THB, indicating a further important difference between the two steroids. Angiogenesis is fundamental for the healing process, and it is known that topical GCs impair wound healing in part by inhibiting angiogenesis; for this reason, the effects of 5α-THB on the formation of new vessels, in comparison with B, were tested in a mouse model of inflammatory angiogenesis induced by sub-cutaneous implantation of polyurethane sponges. 5α-THB, at equipotent doses to B for the reduction of macrophage infiltration, inhibited angiogenesis to a lesser extent than its precursor. In addition, B had systemic effects in that it lowered adrenal gland weights, whereas 5α-THB did not. Histological analysis suggested that while B inhibits formation and maturation of new vessels, 5α-THB may affect only the former process. Molecular analysis showed that B reduced the abundance of transcripts of the majority of the tested genes involved in inflammation, angiogenesis and tissue remodelling, but 5α-THB had more selective effects. Ex vivo studies in mouse bone marrow-derived macrophages stimulated with LPS showed that 5α-THB inhibited release of pro-inflammatory cytokines in a weaker manner compared with B. This inhibition was partially prevented by co-incubation of RU486 with B but not with 5α-THB. In in vitro studies, molecular pathways activated by B and associated with adverse side effects were only weakly activated by 5α-THB. In particular, 5α-THB only weakly induced phosphorylation of GR, and activation of expression of GC-responsive reporter plasmids and endogenous metabolic genes. Interestingly, 5α-THB reduced B-induced trans-activation of some of these genes. In summary, 5α-THB effectively reduces skin inflammation, but, unlike B, has only moderate anti-angiogenic properties, and weakly activates molecular mechanisms associated with adverse metabolic side effects. Most importantly, its action may not be due to activation of GR. This work opens the intriguing possibility that GCs work through mechanisms not yet investigated, and this may be of pivotal importance in the search for new safer anti-inflammatory compounds.

Magister Scientiae Dentium - MSc(Dent) / Includes Bibliographical references (leaves 83- 94).Forty-three male athymic nude mice were implanted with human vaginal mucosal cysts under general anaesthesia with Ketamine [25mg/kg] and Medetomidine [0.5mg/kg]. Cysts in 37 mice were recovered after 9 weeks of growth. twenty three cyst linings had retained the original structure of the vaginal epithelium. No marked deifference was present between the thickness of 9 week old linings and donor vaginal epithelium. The contaminants isolated from the skin of mice before implantation were mainly normal commercals of healthy experimental animals. There was no distinct difference in the number of cases with intact cyst formation between the terramycin/vitamin cocktaik group. The frequency of poor wound healing and/ or murine epidermis ingrowth was three times higher in animals stitched with silk sutures that in those cases where nylon sutures were used. / South Africa

Acute kidney injury (AKI), resulting from chemotherapeutic agents such as cisplatin, remains an obstacle in the treatment of cancer. Cisplatin-induced AKI involves apoptotic and necrotic cell death, pathways regulated by sphingolipids such as ceramide and glucosylceramide. Results from this study indicate that C57BL/6J mice treated with cisplatin had increased ceramide and hexosylceramide levels in the renal cortex 72 h following cisplatin treatment. Pretreatment of mice with inhibitors of acid sphingomyelinase and de novo ceramide synthesis (amitriptyline and myriocin, respectively) prevented accumulation of ceramides and hexosylceramide in the renal cortex and protected from cisplatin-induced AKI. To determine the role of ceramide metabolism to hexosylceramides in kidney injury, we treated mice with a potent and highly specific inhibitor of glucosylceramide synthase, the enzyme responsible for catalyzing the glycosylation of ceramides to form glucosylceramides. Inhibition of glucosylceramide synthase attenuated the accumulation of the hexosylceramides and exacerbated ceramide accumulation in the renal cortex following treatment of mice with cisplatin. Increasing ceramides and decreasing glucosylceramides in the renal cortex sensitized mice to cisplatin-induced AKI according to markers of kidney function, kidney injury, inflammation, cell stress, and apoptosis. Under conditions of high ceramide generation, data suggest that metabolism of ceramides to glucosylceramides buffers kidney ceramides and helps attenuate kidney injury.-Dupre, T. V., M. A. Doll, P. P. Shah, C. N. Sharp, D. Siow, J. Megyesi, J. Shayman, A.Bielawska, J. Bielawski, L. J. Beverly, M. Hernandez-Corbacho, C. J. Clarke, A. J. Snider, R. G. Schnellmann, L. M. Obeid, Y. A. Hannun, and L. J. Siskind. Inhibiting glucosylceramide synthase exacerbates cisplatin-induced acute kidney injury.