O objetivo deste estudo foi avaliar, através de ensaios de tração, os efeitos do desenvolvimento de miopatia metabólica secundária a administração de corticosteróide sobre propriedades biomecânicas dos músculos diafragma e gastrocnêmio medial de coelhos. Foram estudadas 30 coelhas albinas adultas da raça Nova Zelândia, divididas em dois grupos de 15: Grupo Experimental (GE), que recebeu injeções subcutâneas de succinato sódico de 21 metil-prednisolona (Solumedrol Ò; Pharmacia - Up John N.N. / S.A. – Puurs - Bélgica) nas doses de 2 mg/kg/dia, e Grupo Controle (GC) que recebeu soro fisiológico a 0,9% por via subcutânea em volumes proporcionais. Ambos os grupos foram tratados durante 21 dias consecutivos. Os ensaios de tração foram realizados utilizando uma Máquina Universal de Ensaios do Laboratório de Bioengenharia da Faculdade de Medicina de Ribeirão Preto - USP. Para o hemi-diafragma esquerdo foram feitos 24 ensaios, 12 para o GE e 12 para o GC e excluídos 3 animais de cada grupo devido a problemas técnicos. Para o gastrocnêmio medial esquerdo foram feitos 30 ensaios, sendo 15 no GE e 15 no GC. A análise histoenzimológica dos músculos hemi-diafragma e gastrocnêmio medial direitos foi feita em 3 coelhos do GE e 3 do GC. O peso médio final dos animais no GE foi 3,6 Kg e no GC 4,0 Kg. A variação média percentual de peso final no GE foi - 8,4% e no GC 3,1%. O valor médio de peso final do gastrocnêmio no GE foi 5,6g e no GC 7,0g. Os valores médios de área, largura e espessura do gastrocnêmio no GE foram 2,4 x 10-4 m 2, 21,7 mm e 5,4 mm, respectivamente e no GC 2,8 x 10-4 m 2, 24,1 mm e 6,7.mm Nos diafragmas os valores médios de tensão e deformação do limite máximo, tensão e deformação do limite de proporcionalidade e módulo de elasticidade no GE comparado ao GC utilizando o teste t de Student e teste de Mann Whitney, não mostraram diferenças estatísticamente significantes. Nos gastrocnêmios os valores médios de carga máxima, carga e deformação do limite de proporcionalidade e rigidez no GE comparado ao GC, pelo teste t de Student não mostraram diferença estatística significante. Porém, o valor médio de deformação máxima no GE foi 26,63 x10-3 m e no GC 32,33 x10-3 m, mostrando significância estatística entre os grupos através do teste t de Student. Quanto aos locais de ruptura, no GE, 9 foram no ventre muscular, 2 na porção miotendínea distal e 4 na origem e no GC 8 ocorreram no ventre muscular, 5 na porção miotendínea distal e 2 na origem. Do ponto de vista histopatológico observamos que: a miopatia metabólica apresentou-se mais evidente nos diafragmas do GE; houve alterações metabólicas leves nos gastrocnêmios do GE e ocorreu aumento da succinato de desidrogenase (SDH) e Tricrômicro de Gomori modificado nos diafragmas do GC. Diante disto, concluímos que: 1) Os diafragmas não mostraram alterações de suas propriedade biomecânicas, nas fases plástica e elástica, apresentando a mesma capacidade de alongamento em ambos os grupos, suportando cargas semelhantes; 2) O músculo gastrocnêmio medial manteve suas características e capacidade de alongamento na fase elástica. Entretanto, o tratamento com esteróide, na fase plástica, levou a uma significativa redução do seu limite máximo de deformação, apresentando uma menor capacidade de alongamento, embora com mesma carga máxima do controle. / The aim of this study was to assess, using traction assays, the effects of the metabolic myopathy secondary to corticosteroids on biomechanical features of the diaphragm and the medial gastrocnemius muscles of rabbits. The study was composed of 30 albino adult rabbits of the New Zealand breed, which were divided into 2 groups of 15: Experimental Group (EG), which received subcutaneous injections of sodium succinate of 21 methil-prednisolone with doses of 2mg/Kg/day, and the Control Group (CG) which received subcutaneous Saline in proportional volumes. Both groups were treated during 21 consecutive days. The traction assays were performed by applying the Universal Machine of Assays from the Bioengenering Laboratory at the Medical School of Ribeirão Preto - USP. For the left hemi-diaphragm, 24 assays were performed, 12 for EG and 12 CG. Three animals from each group were excluded due to technical problems. For the left medial gastrocnemius, 30 assays were done, 15 for the EG and 15 for CG. The histoenzymologic analysis of both the hemi-diaphragm muscles and the right mild gastrocnemius were performed in three rabbits of the EG, and 3 of the CG. The final average weight for the animals in EG was 3,6Kg, and in the CG was 4,0Kg. The average percentage variation from the initial to the final weight for the EG was -8,4% and for the CG 3,1%. The final average weight of gastrocnemius was 5,1g in the EG and 7,0g in the CG. The average value of the area width and thickness of the EG was 2,4 x 10 -4 m 2, 21,7 mm and 5,4 mm, respectively, and for the CG 2,8 x 10-4 m 2, 24,1 mm and 6,7mm. In the diaphragms, the average values of the tension and deformity in the maximum threshold, tension and deformity at the proportional threshold and elasticity module in the EG compared to the CG, through the Student t Test and the Mann Whitney test, presented no significant statistical differences. In the gastrocnemius, average values of the maximum load, load and deformity within the threshold of proportions and stiffness in the EG compared to the CG, according to the Student t test, did not present a significant statistical difference. However, the average values of maximum deformity were 26,63 x10 -3 m in the EG and 32,33 x10-3 m in the GC, showing statistical significant difference between groups. Concerning the locations of the rupture in the EG, 9 were in the muscle belly, 2 in the miotendinous distal junction, and 4 in the origin; in the CG the rupture ocurred 8 times in the muscle belly , 5 in miotendinous distal junction and 2 in the origin. From a histopathological view, we have observed that metabolic myophaty was presented clearly evident in the diaphragms of the EG; there were slight metabolic alterations in the gastrocnemius of the EG, and there was an increase of the succinic dehydrogenase (SDH) and modified Gomori Trichrome in the diaphragms of the CG. In this manner, we conclude that: 1) The diaphragms didn´t show alterations of their biomechanical properties, in elastic and plastic phases, showing the same elongation capacity in both groups, with equal loads; 2) The medium gastrocnemius muscles, kept their biomechanical features and elongation capacity in elastic phase. However, the steroid treatment lead to a significant decrease of the elongation capacity in the plastic phase, with maximal loads similar to the control group; 3) It was not found a relationship between histological evidences of metabolic miopathy and changes in the biomechanical properties of the studied muscles.

Objetivos: Comparar os efeitos dos tratamentos com montelucaste e dexametasona no recrutamento eosinofílico e na avaliação de células positivas para eotaxina, RANTES, fibronectina, IGF-I e NF-B tanto no parênquima pulmonar distal, quanto nas vias aéreas de cobaias com inflamação alérgica crônica. Métodos: As cobaias receberam inalação com ovoalbumina (grupo OVA- 2 vezes semanais, durante 4 semanas, totalizando 7 inalações). Após a quarta inalação, as cobaias foram tratadas com montelucaste (grupo OVA-M: 10mg/Kg/VO/dia) ou dexametasona ( grupo OVA-D: 5mg/Kg/IP/dia). Após 72 horas da sétima inalação, as cobaias foram anestesiadas e os pulmões foram removidos e submetidos a avaliação histopatológica. Resultados: Os tratamentos com montelucaste e dexametasona reduziram o número de eosinófilos tanto no parênquima pulmonar distal quanto nas vias aéreas, quando comparados ao grupo OVA (p<0.05). No parênquima pulmonary distal, ambos os tratamentos foram efetivos na redução de células positivas para RANTES, NF-B e fibronectina, quando comparados ao grupo OVA (p<0.001). O tratamento com montelucaste mostrou melhor eficácia na redução de células positivas para eotaxina, quando comparado ao tratamento com dexametasona (p<0.001), por outro lado, o tratamento com dexametasona mostrou-se mais significativo na redução de células positivas para IGF-I, quando comparado ao tratamento com montelucaste (p<0.001). Nas vias aéreas, ambos os tratamentos foram efetivos na redução de células positivas para IGF-I, RANTES e fibronectina, quando comparados ao grupo OVA (p<0.05). O tratamento com dexametasona foi mais efetivo na redução de células positivas para eotaxina e NF-B, quando comparado ao tratamento com montelucaste (p<0.05). Conclusões: Neste modelo animal, ambos os tratamentos foram efetivos no controle da resposta inflamatória, tanto no parênquima pulmonar distal, quanto nas vias aéreas / Aims: Compare the effects of montelukast or dexamethasone treatments on eosinophilic recruitment, eotaxin, RANTES, fibronectin, IGF-I and NF-B positive cells of distal lung parenchyma and also in airway walls of guinea pigs (GP) with chronic allergic inflammation. Methods: GP were inhaled with ovalbumin (OVA group-2x/week/4weeks). After 4th inhalation, GP were treated with montelukast (M group: 10mg/Kg/PO/day) or dexamethasone (D group: 5mg/Kg/IP/day). After 72 hrs of 7th inhalation, GP were anesthetised, lungs were removed and submitted to histopathological evaluation. Results: Montelukast and dexamethasone treatments reduced the number of eosinophils both in airway wall as well as in distal lung parenchyma compared to OVA group (p<0.05). On distal parenchyma both montelukast and dexamethasone were effective in reducing RANTES, NF-B and fibronectin positive cells compared to OVA group (p<0.001). Montelukast was more effective in reducing the eotaxin positive cells on distal parenchyma compared to dexamethasone treatment (p<0.001), while there was a more expressive reduction of IGF-I positive cells in OVA-D group (p<0.001). On airway walls, both montelukast and dexamethasone were effective in reducing IGF-I, RANTES and fibronectin positive cells compared to OVA group (p<0.05). Dexamethasone was more effective reducing the number of eotaxin and NF-kB positive cells than Montelukast (p<0.05). Conclusions: In this animal model, both treatments were effective in modulating the eosinophilic response in distal lung parenchyma and in airway wall, contributing to a better control of the inflammatory response in distal lung parenchyma as well as in airway walls. Dexamethasone treatment induced a greater reduction of NF-B expression in airway walls which suggests one of the mechanisms that explains the higher efficacy of this therapeutic approach

Os corticosteróides são substância largamente empregadas em dertatologia devido ao seu potente efeito anti-inflamatório na pela. Entretanto, associado a este efeito benéfico tem-se o risco da ocorrência de efeitos colaterais, decorrentes principalmente da absorção sistêmica dos corticosteróides pela via cutânea. A penetração transcutânea e retenção cutânea dos corticosteróides é influenciada pelo veículo no qual estes princípios ativos são incorporados. Assim sendo, os objetivos desta pesquisa foram investigar, in vitro, formulações que proporcionassem uma alta retenção cutânea dos corticosteróides na pele e também mínima penetração transcutânea. O estudo foi realizado com géis de Poloxamer 407 contendo diferentes concentrações dos promotores de absorção cutânea, uréia ou lecitina. Os parâmetros liberação, penetração transcutânea e retençâo cutânea dos acetatos de hidrocortisona e dexametasona, desonida e triamcinolona acetonida foram avaliados por metodologia in vitro, utilizando-se de célula de difusão e membranas. A quantificação dos corticosteróides nas diferentes fases do experimento foi realizada por cromatografia líquida de alta eficiência. Os esultados obtidos mostraram a influência do veículo nos parâAmetros avaliados. As formulações obedeceram cinética de 1ª. ordem para a liberação e penetração transcutânea. As preparações contendo lecitina promoveram uma maior retenção cutânea dos corticosteróides, sugerindo, assim, que géis de Poloxamer 407, associados com lecitina, formam veículos adequados para a incorporação de corticosteróides. / Corticoids are drugs often used in dermatology due to its anti-inflamatory effect in the skin. Meanwhile, together with this beneficial effect it can have side effects, due to the systemic absorption of the corticoids by cutaneous way. Transcutaneous penetration and cutaneous retention of the corticoids is influenciated by the vehicle in which this drogs are incorporated. In this way, the objetive of this research was to investigate, in vitro, formulations that would provide both, high cutaneous retention of the corticoids in the Skin and minimmn transcutaneous penetration. For this work was used Poloxamer 407 gels containing different concentrations of the absorptions enhancers, urea and lecithin. The release transcutaneous penetration and cutaneous retention of hydrocortisone and dexamethasone acetate, desonide and triamcinolone acetonide was evaluated by in vitro methodology using difiusion cells and membranes. The corticoids was analysed by HPLC. The results obtained showed the influence of the vehicle in the evaluated parameters. The corticoid relesse and transcutaneous penetration appeared to fit first order kinetic. The formulations containing lecithin promoved higher cutaneous retetion of the corticoids than the containing urea, sugesting, in this way, that Poloxamer 407 gels in the presence of lecithin are adequated preparations to the corticoids.

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-07-21T19:09:46Z No. of bitstreams: 1 CiceroAgostinho.pdf: 718364 bytes, checksum: 5b373c12916005349c74311789694b95 (MD5) / Made available in DSpace on 2017-07-21T19:09:46Z (GMT). No. of bitstreams: 1 CiceroAgostinho.pdf: 718364 bytes, checksum: 5b373c12916005349c74311789694b95 (MD5) Previous issue date: 2011-03-31 / The aim of this study was to evaluate the effect of two different concentrations (4 mg and 12 mg) of dexamethasone to control pain, swelling and trismus after third molar surgery inclusos. This clinical study was conducted with 16 adult patients of both sex, displaying mandibular third molar in the same position bilaterally, with an indication for surgical removal. Patients were treated with 4 mg or 12 mg of dexamethasone 1 hour before surgery on the first or second surgery. The choice of which side was operated first and which dose of dexamethasone would be taken first was performed randomly and double blind. The trismus was assessed by measuring the distance interincisal. Pain was assessed by the amount of tablets (paracetamol 750mg) taken after the surgery and the visual analogue pain scale (VAS). These data were obtained before surgery, 24 and 48 hours after surgery. Based on statistical analysis (Student's t test, Wilcoxon and Friedman), the results showed no differences in variables (swelling, pain and trismus) between the two doses of dexamethasone (4mg and 12mg). / O objetivo deste trabalho foi avaliar o efeito de duas concentrações diferentes (4mg e 12mg) de dexametasona no controle da dor, edema e trismo após a cirurgia de terceiros molares inferiores inclusos. Este estudo clínico foi realizado com 27 pacientes adultos, de ambos os sexos, que apresentavam terceiros molares inferiores inclusos na mesma posição bilateralmente, com indicação para remoção cirúrgica. Os pacientes foram medicados com 4mg ou 12mg de dexametasona uma hora antes do procedimento cirúrgico na primeira ou na segunda cirurgia. A escolha de qual lado foi operado primeiro e qual dose de dexametasona seria empregada inicialmente era realizada de forma randomizada e duplo-cega. O trismo foi avaliado pela medida da distância interincisal. A dor foi avaliada mediante a contagem da quantidade de comprimidos (paracetamol 750mg) ingerida após a cirurgia e da Escala Visual Analógica da dor (VAS). Esses dados foram obtidos antes da cirurgia, 24 horas e 48 horas depois do procedimento cirúrgico. Com base na análise estatística (teste t-student, Wilcoxon e Friedman), os resultados não mostraram diferença nas variáveis (edema, dor e trismo) entre as duas doses de dexametasona.

The stress response is an evolutionarily conserved mechanism critical for survival that requires orchestration of different systems in the body. Corticotropin-releasing hormone (CRH) neurons of the hypothalamic paraventricular nucleus (PVN) represent the final common pathway leading to HPA axis activation in response to stress. Noradrenergic inputs to CRH neurons in the PVN provide a powerful drive to activate the HPA axis. Previous anatomical studies have shown that noradrenergic afferents synapse directly on CRH neurons, but electrophysiological analyses indicate that the noradrenergic activation of CRH neurons is mediated primarily by the stimulation of presynaptic glutamatergic neurons. Here, using whole cell patch clamp recordings in identified CRH neurons, I demonstrate that norepinephrine (NE) stimulates excitatory synaptic inputs by activating postsynaptic α1 adrenergic receptors in CRH neurons and inducing the release of the retrograde messenger nitric oxide, which drives upstream glutamate neurons to elicit spike-dependent synaptic glutamate release onto the CRH neurons. Notably, the NE effect is dependent on ATP transmission and astrocytic function, suggesting that astrocytes serve as an intermediary in the retrograde activation of glutamateregic synaptic inputs to the CRH neurons. In addition, I also show that the NE-induced excitation of CRH neurons is stress-status sensitive and corticosterone dependent, in that stress-induced corticosterone causes internalization of membrane α1 adrenergic receptors to desensitize the CRH neurons to NE. Taken together, my findings provide evidence that NE excites CRH neurons in a stress state-dependent manner by a retrograde NO stimulation of local glutamate circuits that is dependent on glial activation. This retrograde trans-neuronal-glial regulation of excitatory synaptic inputs to CRH neurons by NE provides a mechanism for the NE activation of the HPA axis in the early stage of stress response. The stress-/corticosterone-induced desensitization of CRH neurons to NE modulation by the internalization of α1 adrenergic receptors confers a stress state-dependent resistance of the CRH neurons to repeated noradrenergic activation, which provides a mechanism for the negative feedback regulation of the CRH neurons and the HPA axis by stress and glucocorticoids, and a means to restore neuroendocrine homeostasis after stress exposure. / acase@tulane.edu

Human adenovirus (HAdV) mainly causes minor illnesses, but can lead to severe disease and death in both immunocompromised and immunocompetent patients. In such cases, the current standards of treatment often do not improve disease outcome and no approved antiviral therapy against HAdV exists. Since HAdV relies on cellular machinery to assist in the progression of the virus lifecycle, we hypothesized that small molecules targeting certain cellular proteins/pathways, without severely affecting cell health, may serve as effective anti-HAdV compounds. Thus, we aimed to identify novel inhibitors of HAdV, and investigate the molecular mechanism to determine new therapeutic targets for intervention in HAdV infection. We first examined the antiviral properties of pan-histone deacetylase (HDAC) inhibitor SAHA and found that the drug affects multiple stages of the HAdV lifecycle, resulting in significant reductions in virus yield. SAHA was effective in decreasing gene expression from clinically relevant HAdV serotypes. Subsequent investigations on the role of HDACs in HAdV infection led us to determine that class I HDAC activity, mainly HDAC2, is necessary for optimal viral gene expression. Using a wildtype-like HAdV reporter construct that allows us to monitor virus replication by fluorescence microscopy, we then designed an efficient system for screening small molecules to identify novel HAdV inhibitors. We screened over 1300 small molecules, and the screen was sensitive enough to detect compounds with both robust and modest antiviral activity. Several positive hits were validated to reduce HAdV gene expression and yield from infected cells. Further investigation on the efficacy of these compounds and the mechanism behind their inhibition of HAdV can lead to the discovery of new pharmacological targets and the development of more effective antivirals.

The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP activated chloride channel in the apical membrane of epithelial cells, is defective in patients with cystic fibrosis (CF). Research efforts are focused on chloride channel function in order to find a cure for the disease. Genistein increased chloride transport in normal and delF508-CFTR cultured airway epithelial cells without cAMP stimulation. Prior pretreatment with phenylbutyrate did not affect the rate of the genistein-stimulated chloride efflux in these cells. S-nitrosoglutathione is an endogenous bronchodilator, present in decreased amounts in the lungs of CF patients. We studied the effect of GSNO on chloride (Cl-) transport in primary nasal epithelial cells from CF patients homozygous for the delF508-CFTR mutation, as well as in two CF cell lines, using a fluorescent Cl- indicator and X-ray microanalysis. GSNO increased chloride efflux in the CF cell lines and in primary nasal epithelial cells from CF patients. This effect was partly mediated by CFTR. If the cells were exposed to GSNO in the presence of L-cysteine, Cl- transport was enhanced after 5 min, but not after 4 h. GSNO may be a candidate for pharmacological treatment of CF patients. Chloride transport properties of cultured NCL-SG3 sweat gland cells were investigated. The CFTR protein was neither functional nor expressed in these cells. Ca2+-activated chloride conductance was confirmed and the putative Ca2+-activated chloride channel (CaCC) was further characterized in term of its pharmacological sensitivity. Corticosteroids, the primary treatment for asthma, cause necrosis/apoptosis of airway epithelial cells. It was investigated whether a newer generation of drugs used in asthma, leukotriene receptor antagonists, had similar effects. Both montelukast and dexamethasone, but not beclomethasone or budesonide induced apoptosis/necrosis in superficial airway epithelial cells. Montelukast and corticosteroids also caused decreased expression of intercellular adhesion molecule -1 (ICAM-1) in epithelial but not endothelial cells.

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Acute quadriplegic myopathy (AQM), or critical illness myopathy, is a common debilitating acquired disorder in critically ill intensive care unit (ICU) patients characterized by generalized muscle wasting and weakness of limb and trunk muscles. A preferential loss of the thick filament protein myosin is considered pathognomonic of this disorder, but the myosin loss is observed relatively late during the disease progression. In attempt to explore the potential role of factors considered triggering AQM in sedated mechanically ventilated (MV) ICU patients, we have studied the early effects, prior to the myosin loss, of neuromuscular blockade (NMB), corticosteroids (CS) and sepsis separate or in combination in a porcine experimental ICU model. Specific interest has been focused on skeletal muscle gene/protein expression and regulation of muscle contraction at the muscle fiber level. This project aims at improving our understanding of the molecular mechanisms underlying muscle specific differences in response to the ICU intervention and the role played by the different triggering factors. The sparing of masticatory muscle fiber function was coupled to an up-regulation of heat shock protein genes and down-regulation of myostatin are suggested to be key factors in the relative sparing of masticatory muscles. Up-regulation of chemokine activity genes and down-regulation of heat shock protein genes play a significant role in the limb muscle dysfunction associated with sepsis. The effects of corticosteroids in the development of limb muscle weakness reveals up-regulation of kinase activity and transcriptional regulation genes and the down-regulation of heat shock protein, sarcomeric, cytoskeletal and oxidative stress responsive genes. In contrast to limb and craniofacial muscles, the respiratory diaphragm muscle responded differently to the different triggering factors. MV itself appears to play a major role for the diaphragm muscle dysfunction. By targeting these genes, future experiments can give an insight into the development of innovative treatments expected at protecting muscle mass and function in critically ill ICU patients.

[Truncated abstract] In obstetric practice, pregnant women at risk of pre-term delivery between 24 and 34 weeks of gestation are administered synthetic glucocorticoids (betamethasone or dexamethasone) to induce fetal organ maturation. During this gestational period, the fetal kidney is undergoing a phase of rapid organogenesis with an increase in renal growth and active nephrogenesis occurring. The studies comprising this thesis examine the effects of prenatal betamethasone exposure on the fetal and adult ovine kidney. The central hypothesis of these studies was that exposure of the fetal kidney to betamethasone in late gestation would change renal structure and induce long-term alterations in the expression of glucocorticoid-sensitive genes and proteins. In the fetal studies, pregnant Merino ewes bearing single fetuses received single or repeated-weekly intra-muscular (i.m.) injections of betamethasone (0.5 mg/kg body weight) or saline commencing on day 104 of gestation (term is 150 days). Kidneys were collected from fetuses at 109, 116, 121 and 146 days of gestation (d). Using gold standard unbiased stereological techniques, the physical disector/fractionator method, total glomerular (nephron) number and glomerular volume were determined in 146 d fetal kidneys exposed to repeated maternal saline or betamethasone administration. In the adult study, kidneys were collected from 3.5-year-old sheep that had been exposed to ... In this thesis I have demonstrated that renal growth restriction as a result of betamethasone exposure is associated with a reduction in fetal nephron endowment. Although betamethasone does not appear to consistently alter nephron number or glomerular size, it may indirectly affect total nephron endowment through effects on renal growth. I have also provided evidence which suggests that lategestation betamethasone exposure in sheep does not program permanent alterations in the renal expression of genes or proteins involved in glucocorticoid hormone action or components of the renin-angiotensin system. Therefore, exposure of the fetal kidney to betamethasone during nephrogenesis may alter renal structure if kidney growth is perturbed; however, there are no persistent alterations in the expression of glucocorticoid-sensitive genes. These findings are consistent with the preservation of normal basal blood pressure in the adult sheep I studied and with the limited results from human studies of late-gestation maternal glucocorticoid administration.