Introdução: O desequilibrio protease-antiprotease é fundamental para a fisiopatologia da doença pulmonar obstrutiva crónica (DPOC). No entanto, poucos estudos para a inibição da elastase têm sido investigados. Objetivo: O nosso estudo avaliou a capacidade do inibidor proteinase derivada da planta Bauhinia bauhinioides (BbKI) na modulação da inflamação pulmonar induzida pela elastase. Métodos: Camundongos C57BL receberam instilação intratraqueal de elastase (0,025 mg, ELA n=6) ou solução salina (SAL n=6) e foram tratados por via intraperitoneal com BbKI (2 mg/kg, de ELA-BbKI n=6, SAL-BbKI n=6) nos dias 1, 14 e 21. No dia 28 foram realizadas as seguintes análises: (I) avaliação da mecânica pulmonar (II) medida do óxido nítrico exalado (ENO), (III) a determinação do número de céluas no lavado broncoalveolar (FLBA), e ( IV) coloração imunohistoquímica do fluído pulmonar, (V) intercepto linear médio (Lm), Resultados: Além de diminuir alterações mecânicas e a lesão do septo alveolar (Lm), BbKI reduziu o número de células no fluido de FLBA e diminuiu a expressão celular de TNF-alfa, MMP-9, MMP-12, TIMP-1, eNOS e iNOS em vias aéreas e nas paredes alveolares em comparação com o grupo de ELA (p < 0,05). BbKI diminuiu a proporção de volume de 8-iso-PGF2, as fibras colagenas e as elásticas nas vias aéreas e paredes alveolares em comparação com o grupo de ELA (p < 0,05). Houve redução do número de células para positivas MUC-5 nas paredes das vias aéreas (p < 0,05). Houve redução do número de neutrófilos em vias aereas e parenquima e de macrófagosnas paredes alveolares. Conclusão: BbKI foi eficaz na redução da inflamação pulmonar, mecânica pulmonar e do remodelamento da matriz extracelular induzida por elastase. BbKI pode ser uma ferramenta farmacológica potencial para o tratamento da DPOC; no entanto, são necessárias análises adicionais / Introduction: The protease-antiprotease imbalance is essential to the pathophysiology of chronic obstructive pulmonary disease (COPD). However, few studies for inhibition of elastase have been investigated. Objective: Our study evaluated the ability of proteinase inhibitor derived Bauhinia bauhinioides plant (BbKI) in modulating lung inflammation induced by elastase. Methods: Mice C57BL received intratracheal elastase instillation (0.025 mg, ELA n = 6) or saline (SAL n = 6) and were treated intraperitoneally with BbKI (2 mg/kg of ELA-BbKI n = 6, SAL-BbKI n = 6) on days 1, 14 and 21. On the 28th the following analyzes were performed: (i) assessment of pulmonary mechanics (II) measurement of exhaled nitric oxide (ENO), (III) determining the number of cells in bronchoalveolar lavage fluid (BALF), and (IV) immunohistochemical staining of lung fluid, (V) mean linear intercept (Lm) Results: In addition to reducing mechanical changes and Lm, BbKI reduced the number of cells in BALF fluid and decreased cellular expression of TNF-alfa, MMP-9, MMP-12, TIMP-1, eNOS and iNOS in the airway and alveolar walls compared with ELA group (p < 0.05). BbKI decreased volume proportion of 8-iso-PGF2, collagen fibers and elastic airway and alveolar walls compared with ELA group (p < 0.05). There was a reduction from MUC-5 positive cells in the airway walls (p < 0.05). There was a reduction in the number of neutrophils in airway and alveolar walls (p < 0.005) and a reduction in macrophages in alveolar walls (p < 0.005). Conclusion: BbKI was effective in reducing inflammation, pulmonary mechanics and remodeling of the extracellular matrix induced by elastase. BbKI may be a potential pharmacological agent for the treatment of COPD; however, additional tests are required

O objetivo deste trabalho foi avaliar se a proteína bifuncional de planta, CrataBL, que tem lectina e as propriedades inibidoras de enzima, modula alterações de mecânica pulmonar, inflamatórias e remodelamento induzidas por elastase intratraqueal em camundongos. Métodos: 36 camundongos C57BL6 receberam elastase (0,025 mg) por instilação intratraqueal (grupo ELA e ELA-CrataBL). Os grupos controles receberam salina (grupo SAL e SAL-CrataBL). Os camundongos foram tratados com instilação intraperitoneal de CrataBL (2mg/kg) nos dias 1, 14 e 21 após a instilação intratraqueal de elastase (grupo SAL-CrataBL e ELA-CrataBL) os animais controle receberam salina no mesmo volume. No dia 28, os camundongos foram anestesiados, ventilados mecanicamente e foram analisados a resistência e elastância do sistema respiratório (Ers e Rrs), elastância e resistência tecidual (Htis e Gtis), resistência das vias aéreas (Raw) e óxido nítrico exalado (NOex). Após, o lavado broncoalveolar (LBA) foi realizado, os pulmões foram retirados e por morfometria, e foram quantificados o intercepto linear médio (Lm), a quantidade de neutrófilos, células positivas para TNF-alfa, fibras colágenas, elásticas, células positivas para MMP-9, MMP-12, TIMP-1, eNOS e iNOS e isoprostano no parênquima pulmonar e vias aéreas. No parênquima foram avaliados os macrófagos nos septos alveolares e nas vias aéreas, foram também avaliadas as células para MUC-5. Resultados: No grupo ELA houve um aumento na Ers, Raw, Gtis, Htis, Lm, NOex, nas células totais, macrófagos, neutrófilos, eosinófilos e linfócitos no LBA em relação aos controles (p < 0,05), sendo que Raw, diminuiu também nos grupos SAL-CrataBL e ELA-CrataBL. Nos grupos tratados com CrataBL houve uma diminuição de Ers (37,0±2,2 cmH2O/L), Htis (37,9±3,5 cmH2O/ml/s), ENO (14,7±0,7 ppb), comparativamente ao grupo ELA (p < 0,05). No LBA houve atenuação de neutrófilos (0,003±0,001 104células/ml), linfócitos (0,003±0,001 104células/ml) e de Lm (54,6±6,0 mm). Complementando a avaliação, no grupo que recebeu elastase houve um aumento no número de macrófagos (22,88 +- 2,24 células/104um2), neutrófilos (1,18 +- 0,15 células/10 4um2), células positivas para TNF-ala (12,52 +- 0,42 células/104um2) no parênquima pulmonar. Nas alterações de remodelamento no parênquima pulmonar, houve um aumento da proporção de volume de fibras colágenas (11,5 +- 0,11%), elásticas (0,5 +- 0,03%), na quantidade de células positivas para MMP-9 (18,59 +- 1,87 células/104?m2), MMP-12 (20,17 +- 1,92 células/104?m2), TIMP-1 (14,42 +- 2,05 células/104um2) em comparação com os controlos (p < 0,001). No estresse oxidativo, houve um aumento de eNOS (13,15 +- 0,40 células/104um2), iNOS (10,49 +- 0,65 células/104um2) e isoprostano (18,11 =- 5,38%). O tratamento CrataBL (grupo ELA-CrataBL) reduziu no parênquima pulmonar a quantidade de macrófagos (9,58 +- 1,36 células/104um2), neutrófilos (0,75 +- 0,1 células/104um2), células positivas para TNF-alfa (10.4±0,49 células/104?m2), fibras colágenas (10,8 +- 0,13%), elásticas (0,3 +- 0,02%), a quantidade de células positivas para a MMP-9 (10,35±0,65 células/104um2), MMP-12 (14,15±0,59 células/104um2), TIMP-1 (9,89 +- 2,79 células/104um2), MUC-5 (3,56 +- 0,54 células/104um2), eNOS (6.98 +- 0.32 células/104um2) e iNOS (6,21 +- 0,42 células/104um2) e isoprostano (8,96 +- 3,08 %) em relação ao grupo ELA (p < 0,001). Nas vias aéreas também ocorreu um aumento significativo de neutrófilos (5,97 +- 1,03 células/104um2), células positivas para TNF-alfa (15,82 +- 1,03 células/104um2). Nas alterações de remodelamento pulmonar nas vias aéreas também ocorreu um aumento da proporção de volume de fibras colágenas (8,73 +- 2,59%), elásticas (2,56 +- 0,18%), na quantidade de células positivas para MMP-9 (14,86 +- 1,77 células/104um2), MMP-12 (18,56 +- 1,79 células/104um2), TIMP-1 (1,31 +- 0,12 células/104um2) e MUC-5 (7,09 +- 1,71 células/104um2) em comparação com os controlos (p < 0,001). No estresse oxidativo, houve um aumento de células positivas para eNOS (3,09 +- 0,08 células/104um2), iNOS (5,4 +- 0,3 células/104um2) e isoprostano (18,11 +- 5,38%) em comparação com os controlos (p < 0,001). O tratamento CrataBL (grupo ELA-CrataBL) reduziu nas vias aéreas a quantidade de neutrófilos (4,62 +- 0,61 células/104um2), TNF- alfa (14,30 +- 1,28 células/104um2), fibras colágenas (7,80 +- 1,37%), elásticas (1,4 +- 0,13%), a quantidade de células positivas para a MMP-9 (9,93 +- 1,39 células/104um2), MMP-12 (12,06 +- 1,15 células/104um2), TIMP-1 (0,73 +- 0,05 células/104?m2), MUC-5 (3,56 +- 0,54 células/104um2), eNOS (1,89 +- 0,16 células/104um2) e iNOS (4,3 +- 0,31 células/104um2), isoprostano (7,34 +- 2,31%) em relação ao grupo ELA (p < 0,001). Conclusão: CrataBL atenua as alterações de mecânica pulmonar, lavado bronco alveolar, responsividade inflamatória, controle do remodelamento e estresse oxidativo induzidas pela elastase. Embora mais estudos devam ser realizados, esta proteína bifuncional pode contribuir como potencial ferramenta terapêutica para o tratamento da DPOC / The aim of this study was to evaluate whether the bifunctional protein plant, CrataBL, which has lectin and enzyme inhibitory properties, modulates changes in lung mechanics, inflammatory and remodeling induced by intratracheal elastase in mice.Methods : 36 C57/Bl6 mice received elastase (0.025 mg) by intratracheal (group ELA and ELA-CrataBL). Control groups received saline (group SAL and SAL-CrataBL).The mice were treated with intraperitoneal instillation of CrataBL (2mg/kg) on days 1, 14 and 21 after intratracheal instillation of elastase (group SAL-CrataBL and ELA-CrataBL), control animals received saline in the same volume. On day 28, the mice were anesthetized and mechanically ventilated were analyzed resistance and respiratory system elastance (Ers and Rrs), elastance and tissue resistance (Htis and Gtis), airway resistance (Raw) and exhaled nitric oxide (ENO). After the bronchoalveolar lavage (BAL) was performed, the lungs were removed and morphometry were quantified and the linear intercept mean (Lm), the number of neutrophils, positive cells for TNF-alfa, collagen fibers, positive cells for MMP-9, MMP-12, TIMP-1, eNOS, iNOS and isoprostane in lung parenchyma and airways. Parenchyma was also evaluated macrophages in the alveolar septa. Airway was also evaluated MUC-5 cells. Results: In group ELA was an increase in Ers, Raw, Gtis, Htis, Lm, ENO, in total cells, macrophages, neutrophils, eosinophils and lymphocytes in BAL compared to controls (p < 0.05), and Raw, decreased in both groups SAL-CrataBL and ELA-CrataBL. In the groups treated with CrataBL there was a decrease in Ers (37.0±2.2 cmH2O/L) Htis (37 9±3.5 cmH2O/ml/s) and ENO (14.7±0.7 ppb) compared to the ELA group (p < 0.05). In BAL there was attenuation of neutrophils (0.003±0.001 104cells/ml), lymphocytes (0.003±0.001 104cells/ml) and Lm (54.6±6.0 mm). Complementing the assessment, the group that received elastase was an increase in the number of macrophages (22.88±2.24 cells/104um2), neutrophils (1.18±0.15 cells/104um2), positive TNF-alfa cells (12.52±0.42 cells/104um2) in the lung parenchyma. In remodeling changes in lung parenchyma, there was an increase in the volume ratio of collagen fibers (11.5 ± 0.11%), elastic (0.5±0.03%), the number of positive MMP-9 cells (18.59±1.87 cells/104um2), MMP-12 (20.17 ± 1.92 cells/104um2) TIMP-1 (14.42±2.05 cells/104um2) compared to controls (p < 0.001). Oxidative stress, was an increased of eNOS (13.15±0.40 cells/104um2), iNOS (10.49 ± 0.65 cells/104um2) and isoprostane (18.11±5.38%). Treatment CrataBL (ELA-CrataBL group) reduced the amount of parenchymal lung macrophages (9.58±1.36 cells/104um2), neutrophils (0.75±0.1 cells/104um2), positive TNF-alfa cells (10.4±0.49 cells/104um2), collagen (10.8±0.13%), elastic (0.3±0.02%), the number of positive MMP-9 cells (10.35±0.65 cells/104?m2), MMP-12 (14.15±0.59 cells/104um2), TIMP-1 (9.89±2.79 cells/104um2) MUC-5 (3.56±0.54 cells/104um2), eNOS (6.98±0:32 cells/104um2) and iNOS (6.21±0.42 cells/104um2) and isoprostane (8.96 ± 3.08%) compared to group ELA (p < 0.001). Airway was also a significant increase in neutrophils (5.97±1.03 cells/104um2), positive TNF-alfa cells (15.82±1.03 cells/104um2). Changes in lung airway remodeling also occurred an increase in the volume ratio of collagen fibers (8.73±2.59%), elastic (2.56±0.18%), the number of positive MMP-9 cells (14.86±1.77 cells/104um2), MMP-12 (18.56±1.79 cells/104um2) TIMP-1 (1.31±0.12 cells/104um2) and MUC-5 (7.09±1.71 cells/104um2) compared to controls (p < 0.001). Oxidative stress, an increase of eNOS (3.09 ± 0.08 cells/104um2), iNOS (5.4±0.3 cells/104um2) and isoprostane (18.11±5.38%) compared to controls (p < 0.001). Treatment CrataBL (ELA-CrataBL group) reduced the amount airway neutrophils (4.62±0.61 cells/104um2), TNF-alfa (14.30 ± 1.28 cells/104um2), collagen fibers (7 80±1.37%), elastic (1.4±0.13%), the number of positive MMP-9 cells (9.93±1.39 cells/104um2), MMP-12 (12.06±1.15), TIMP-1 (0.73±0.05 cells/104um2), MUC-5 (3.56±0.54 cells/104um2), eNOS (1.89±0,16 cells/104um2) and iNOS (4.3±0.31 cells/104um2), isoprostane (7.34±2.31%) compared to group ELA (p < 0.001). Conclusion: CrataBL attenuates changes in lung mechanics, broncho alveolar inflammatory responsiveness, control remodeling and oxidative stress induced by elastase. Although more studies should be conducted, this bifunctional protein may contribute as a potential therapeutic tool for the treatment of COPD

Os modelos experimentais têm sido utilizados para o estudo dos mecanismos fisiopatológicos envolvidos no desenvolvimento da Doença Pulmonar Obstrutiva Crônica (DPOC). O modelo que melhor mimetiza a doença em humanos é o que utiliza a exposição à fumaça de cigarro. No entanto, a utilização deste modelo experimental requer um longo tempo de exposição (6 meses) e a lesão do parênquima obtida é considerada leve. O desequilíbrio protease/anti-protease é considerado um importante mecanismo fisiopatológico envolvido no desenvolvimento da DPOC. Desta forma, neste estudo propomos o desenvolvimento de um modelo experimental no qual associamos a instilação de elastase previamente ao início da exposição ao fumo na tentativa de obter um maior grau de lesão tecidual em um menor espaço de tempo. Para tanto, camundongos C57Bl/6 foram divididos em quatro grupos: Controle, Elastase, Fumo, Fumo/Elastase 1 dose e Fumo/Elastase 2 doses e analisados após dois meses de exposição. Os animais do grupo Fumo/Elastase 1 dose e 2 doses foram submetidos à instilação intranasal de elastase pancreática de porco (0,33UI) e expostos a fumaça de cigarro por dois meses. O grupo controle recebeu o mesmo tratamento com solução fisiológica (NaCl 0.9%). A exposição ao fumo foi feita por 30min, 2 vezes/dia, 5 dias da semana. Após dois meses, os animais foram sacrificados e observamos aumento de LM no grupo Fumo/Elastase 1 dose e 2 doses comparado aos grupos Controle e Fumo; aumento de células positivas para MAC-2 no parênquima (Fumo/Elastase 2 doses) e vias aéreas (Fumo/Elastase 1 dose e 2 doses), MMP-12 no parênquima pulmonar (Fumo/Elastase 2 doses), GP91 no parênquima (Fumo/Elastase 1 dose e 2 doses) e vias aéreas (Fumo e Fumo/Elastase 1 dose) e aumento de proporção de fibras elásticas no parênquima pulmonar do grupo Fumo/Elastase 1 dose e do grupo Fumo, caracterizando presença de enfisema pulmonar. A instilação de elastase pancreática de porco juntamente com a exposição à fumaça de cigarro aumentou a susceptibilidade ao desenvolvimento do enfisema / Experimental models have been used to study the pathophysiological mechanisms involved in the development of COPD. Cigarette Smoke exposure (CS) is considered the best model to mimetize the disease in humans. However, the CS requires a long exposure time (6 months) and the parenchymal destruction obtained is considered mild. The protease / anti - protease imbalance is considered an important pathophysiological mechanism involved in the development of COPD. Thus, in this study we propose the development of an experimental model in which we associate instillation of elastase before the start of exposure to smoke, trying to increase the parenchymal destruction degree in a shorter time. For that, C57BL / 6 mice were divided into four groups: Control, Elastase, Smoke and Smoke/Elastase 1 dose and Smoke/Elastase 2 doses and analyzed in two months after the CS exposition. The Smoke/Elastase 1 dose and 2 doses animals group received an intranasal instillation of porcine pancreatic elastase (0.33 IU) and exposed to cigarette smoke for two months. The control group received the same treatment with saline (NaCl 0.9 %). Animals were exposed to CS for 30min, 2 times / day, 5 days a week. After two months, we observed increased mean linear intercept (LM) and positive cells for MAC-2, MMP-12 and GP91 in the airways and lung parenchyma and increase of elastic fibers in the lung parenchyma characterizing the presence of pulmonary emphysema. The instillation of porcine pancreatic elastase along the exposure to cigarette smoke increased susceptibility to the development of emphysema

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É possível que as alterações inflamatórias e estruturais nos pulmões com enfisema alterem a resposta dos mesmos à sepse influenciando no desenvolvimento da síndrome do desconforto respiratório agudo. Objetivo: Comparar a lesão pulmonar secundária à sepse, induzida por lipopolissacarídeo (LPS) intraperitoneal, em ratos com e sem enfisema induzido pela administração intratraqueal de elastase. Métodos: Vinte e quatro ratos Wistar adultos foram randomizados para quatro grupos de seis animais: controle (C-C), enfisema (E-C), controle com sepse (C-LPS) e enfisema com sepse (E-LPS). O enfisema foi induzido pela injeção intratraqueal de elastase pancreática de porco (12 UI/ animal). Após três semanas deste procedimento, a sepse foi induzida pela injeção intraperitoneal de LPS da Escherichia coli (10 mg/Kg). Vinte e quatro horas após a indução da sepse, os animais foram submetidos à ventilação mecânica por 10 minutos para posterior coleta da gasometria arterial. A seguir, foram eutanasiados e as seguintes análises foram realizadas: lavado broncoalveolar (LBA), permeabilidade pulmonar e histologia. Os resultados foram expressos em média ± desvio padrão ou mediana (intervalo interquartil), quando apropriado, e comparados por ANOVA seguida do teste de Tukey, ou por Kruskal-Wallis seguido do teste de Mann-Whitney. Resultados: O escore de lesão pulmonar foi significativamente maior nos grupos C-LPS [0,62 (0,19)] e E-LPS [0,59 (0,13)] em comparação com os grupos C-C [0,11 (0,09)] e E-C [0,15 (0,05)] (p < 0,05). A contagem total de células (C-LPS=2,37 ± 0,74; E-LPS=5,37 ± 0,13; C-C=0,73 ± 0,36; E-C=3,09 ± 7,53 x 105) e de neutrófilos [C-LPS=1,39 (1,48); E-LPS=4,39 (1,95); C-C=0,07 (0,11); E-C=0,68 (0,61) x 105] no LBA foi significativamente maior nos grupos C-LPS e E-LPS comparado aos grupos C-C e E-C (p < 0,05). Animais do grupo E-LPS apresentaram maior contagem de células totais e de neutrófilos no LBA em comparação com o grupo C-LPS (p < 0,05). Na avaliação da razão albumina LBA/soro, o grupo E-LPS apresentou aumento significativo quando comparado ao C-LPS [0,069 (1,243) vs. 0,007 (0,002), respectivamente, p < 0,05]. Não foram observadas diferenças significativas nas trocas gasosas entre os grupos. Conclusões: A presença de enfisema foi acompanhada de maior inflamação pulmonar em resposta à agressão sistêmica induzida pela injeção intraperitoneal de LPS, levando a uma maior lesão da barreira alvéolo-capilar. Palavras-chave: enfisema pulmonar, sepse, lesão pulmonar aguda, síndrome do desconforto respiratório agudo, elastase pancreática, lipopolissacarídeos. / Introduction: The response of lungs with emphysema to a systemic insult such as sepsis is not known. Structural and inflammatory abnormalities in lungs caused by emphysema might alter their response to sepsis and thus influence the incidence and severity of acute respiratory distress syndrome. We therefore aimed to compare the severity and extension of acute lung injury in response to intraperitoneal lipopolysaccharide (LPS) injection in rats with and without emphysema induced by elastase. Methods: Twenty four adult Wistar rats were randomized into 4 groups, each of them with 6 animals: control (C-C), emphysema without sepsis (E-C), control with sepsis (C-LPS) and emphysema with sepsis (E-LPS). Emphysema was induced by intratracheal instillation of pancreatic porcine elastase (12 IU/animal). Three weeks later, sepsis was induced by intraperitoneal Escherichia coli LPS injection (10 mg/kg). Twenty four hours after sepsis induction, animals underwent mechanical ventilation for 10 minutes and then blood was sampled for gasometric analysis. Thereafter, euthanasia and the following analysis were performed: BAL, lung permeability and histology. Results were expressed as mean ± standard deviation or median (interquartile range), when appropriate, and compared using ANOVA followed by Tukey test or Kruskal-Wallis followed by Mann-Whitney test. Results: Significant increase in lung injury score was observed in the C-LPS [0.62 (0.19)] and E-LPS [0.59 (0.13)] compared to the groups C-C [0.11 (0.09)] and E-C [0.15 (0.05)] (p < 0.05). Total cell (C-LPS=2.37 ± 0.74; E-LPS=5.37 ± 0.13; C-C=0.73 ± 0.36; E-C=3.09 ± 7.53 x 105) and neutrophil counts [C-LPS=1.39 (1.48); E-LPS=4.39 (1.95); C-C=0.065 (0.11); E-C=0.68 (0.61) x 105] in the BAL were significantly higher in the groups that received LPS (p < 0.05). Significantly higher total cell and neutrophil counts in the BAL were also observed in the E-LPS group compared to C-LPS (p < 0.05). The group E-LPS showed a significant increase in the BAL/serum albumin ratio compared to C-LPS [0.069 (1.243) vs. 0.007 (0.002), respectively] (p < 0.05). There were no significant differences in the gas exchange levels among the groups. Conclusions: The presence of emphysema increases the inflammatory response in the lungs to a systemic stimulus, represented in this model by the intraperitoneal injection of LPS.

A Doenca Pulmonar Obstrutiva Cronica (DPOC) e uma patologia que apresenta obstrucao ao fluxo aereo. No enfisema ocorre destruicao da matriz extracelular que resulta no alargamento do espaco aereo. A hiper-responsividade bronquica pode ocorrer em pacientes com DPOC. Nesse contexto o objetivo desse estudo foi averiguar a responsividade bronquica apos desafio com metacolina (MCh) em um modelo de enfisema induzido por elastase pancreatica de porco (PPE). Para tal utilizamos camundongos machos Swiss (25-30g), divididos aleatoriamente em tres grupos: o grupo controle foi instilado via intratraqueal com solucao salina e o grupo enfisema subdividido em dois grupos instilados com PPE (0,3 e 0,6U). Apos 20 dias, os animais foram anestesiados, traqueostomizados, acoplados em ventilador para pequenos animais e submetidos a inalacao de salina e MCh (50 e 100mg/mL). A avaliacao da mecanica respiratoria utilizou o modelo de fase constante e os parametros analisados foram resistencia das vias aereas (Raw), resistencia tecidual (G), elastancia tecidual (H) e histeresividade (ƒÅ). Apos o teste, os pulmoes foram removidos para analises histologicas e quantificacao de proteinas. Os resultados da mecanica respiratoria mostraram que os valores de Raw, G e ƒÅ, tanto basais quanto apos nebulizacao com salina, nao apresentaram diferencas entre os grupos quando comparado com o grupo controle, porem a H no grupo enfisema 0,6U foi menor quando comparado com o grupo controle. Apos o desafio com MCh 100mg/mL, a Raw foi atenuada nos animais com enfisema 0,3U. A G aumentou nos animais com enfisema 0,3U. A H nos animais com enfisema 0,6U diminuiu. A ƒÅ apos nebulizacao de MCh 50 e 100mg/ml elevou-se nos animais instilados com PPE 0,3 e 0,6U. Na analise morfologica, o grupo tratado com PPE 0,6U aumentou as areas de colapso alveolar, o numero de alveolos hiperinsuflados foi maior nos grupos enfisema 0,3 e 0,6U, o intercepto linear medio elevou-se em ambos os grupos tratados com PPE e o numero de alveolos normais reduziu em ambos os grupos enfisema quando comparado com o grupo controle. A quantificacao de proteinas mostrou diminuicao de elastina no grupo 0,6U e aumento de colageno tipo III nos grupos 0,3 e 0,6U. Os resultados mostraram que animais com enfisema induzido por PPE 0,3U apresentam atenuacao da responsividade bronquica durante a inalacao de MCh (100mg/mL), sugerindo que a perda da integridade do epitelio pulmonar e o remodelamento tecidual aumentam a heterogeneidade dos pulmoes. / Chronic Obstructive Pulmonary Disease (COPD) is a disease that presents airflow obstruction. In emphysema, there is destruction of the extracellular matrix resulting in enlargement of the airspace. The bronchial hyperresponsiveness can occur in patients with COPD. In this context, the objective of this study was to evaluate the responsiveness after bronchial challenge with methacholine (MCh) in a model of emphysema induced by porcine pancreatic elastase (PPE). Swiss male mice (25 - 30g) were randomly divided into three groups: the control group was intratracheally instilled with saline and emphysema group was subdivided into two other groups that were instilled with PPE (0.3 and 0.6 U). After 20 days the animals were anesthetized , tracheotomized , connected to ventilator for small animals and subjected to inhalation of salina and MCh (50 and 100mg/mL) . The evaluation of respiratory mechanics used the model of constant phase and the parameters analyzed were airway resistance (Raw), tissue resistance (G), tissue elastance (H) and hysteresivity (ç). The lungs were removed for histological analysis and quantification of proteins after the test. The results of respiratory mechanics showed that the baseline and after nebulization with saline the parameters Raw, G and ç showed no differences between groups when compared with the control group, but the H in group emphysema 0.6U was lower . After challenge with MCh 100mg/mL Raw was attenuated in animals with emphysema 0.3U. The G increased in animals with emphysema 0.3U. The H in animals with emphysema 0.6 U decreased. The ç after misting of MCh 50 and 100mg/mL increased in animals instilled with PPE 0.3 and 0.6U. In the morphological analysis the group treated with PPE 0.6U increased areas of alveolar collapse, alveolar number of hyperinflation was higher in groups emphysema 0.3 and 0.6U, linear mean intercept increased in both groups treated with PPE and reduced number of normal alveoli in both groups emphysema when compared with the control group. The protein quantification showed decrease of elastin in the group 0.6 U and increased collagen type III in the groups treated with PPE. Our results show that animals with elastase-induced emphysema 0.3U have attenuation of bronchial hyperresponsiveness during inhalation of MCh (100mg/mL), suggesting that loss of integrity of the epithelial lung tissue and tissue remodeling increased the heterogeneity of lungs. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES

Glycosaminoglycans (GAGs) are linear polysaccharides whose disaccharide building blocks consist of an amino sugar and either uronic acid or galactose. They are expressed on virtually all mammalian cells, usually covalently attached to proteins, forming proteoglycans. GAGs are highly negatively charged due to an abundance of sulfate and carboxylic acid groups, and are structurally very diverse, with differences arising from chain length, the type of monomeric units, the linkages between each monomeric unit, the position of sulfate groups, and the degree of sulfation. GAGs are known to interact with a multitude of proteins, impacting diverse physiological and pathological processes. In addition, most of the biological interactions mediated by proteoglycans are believed to be primarily because of the GAG chains present on their surface. Considering the involvement of GAGs in multiple diseases, their use in the development of drugs has been of significant interest in the pharmaceutical field. Heparin, the first GAG-based drug developed in 1935, is still the most widely used anticoagulant in the world. The therapeutic potential of GAGs for the treatment of many other disease states, including cancer, inflammation, infection, wound healing, lung diseases, and Alzheimer’s disease, is being actively studied with many GAGs currently in clinical trials. However, challenges associated with the heterogeneous and complex structure of GAGs, limit their successful development. To combat such issues, our lab has focused on developing Non- Saccharide GAG Mimetics (NSGMs) as structural mimics of GAGs. NSGMs, being synthetic molecules, offer multiple advantages over GAGs. The studies mentioned here describe our efforts in the development of NSGMs as potential therapeutics for cancer, and cystic fibrosis.

Ziel dieser Studie war es, Auswirkungen von subgingival applizierter Hyaluronsäure (Gengigel®) nach scaling and root planing (SRP) auf mikrobiologische Variablen bei Parodontitispatienten nachzuweisen. Dabei wurden die möglichen Zusammenhänge zwischen Ergebnissen der Bakterienspezies und zuvor ermittelten Ergebnissen klinischer Variablen untersucht. An der Untersuchung nahmen 20 Männer und 29 Frauen teil. Es erfolgte eine Randomisierung in zwei Gruppen, bestehend aus einer Testgruppe mit 23 und einer Kontrollgruppe mit 26 Probanden. Bei den Versuchteilnehmern wurde in zwei Sitzungen in einem 24-stündigen Abstand ein SRP mittels Hand- und Ultraschallinstrumenten durchgeführt. Am Ende des SRP wurde in der Testgruppe Gengigel prof® (mit 0,8% Hyaluronsäure) in die parodontalen Taschen eingebracht. Zusätzlich trugen die Probanden der Testgruppe während der folgenden 14 Tage zweimal täglich morgens und abends Gengigel® (0,2%) auf den Gingivarand auf. In der Kontrollgruppe erfolgte das übliche SRP ohne Verwendung von Gengigel®. Alle Probanden wurden zu Beginn der Untersuchung, nach drei und sechs Monaten aus parodontologischer Sicht untersucht. Des Weiteren wurden Proben der Sulkusflüssigkeit für eine spätere Analyse von zehn parodontopathogenen Keimen sowie der Peroxidase- und Granulozytenaktivität gewonnen. Im Gegensatz zur kontinuierlichen Zunahme in der Kontrollgruppe (p=0,035) konnte beim Verlauf der Gesamtbakterienzahl für die Testgruppe keine Veränderung der Keimzahlen (p=0,737) beobachtet werden. In der Testgruppe wurde nach sechs Monaten für Campylobacter rectus, Treponema denticola und Aggregatibacter actinomycetemcomitans eine Reduktion der Keimbelastung festgestellt (p=0,05; p=0,043; p=0,066). Am Ende der Untersuchung waren in der Testgruppe keine Unterschiede in der bakteriellen Besiedlung unterschiedlich tiefer Taschen mehr nachweisbar (p=1). In der Testgruppe bestand eine stark signifikante Korrelation der Granulozytenaktivität mit der Zeit (r=0,443; p<0,0001) und mit der Gesamtbakterienzahl (r=0,336; p=0,009). Die Ergebnisse der Studie weisen auf einen wachstumshemmenden Effekt der Hyaluronsäure auf parodontopathogene Bakterien hin, welcher womöglich auf einer indirekten Interaktion mit dem Immunsystem basiert.

L’adiponectine, une adipokine aux niveaux plasmatiques inversement associés aux composantes du syndrome métabolique, protège contre l’athérosclérose et réduit les risques d’infarctus du myocarde. Les cellules progénitrices endothéliales (EPCs) jouent un rôle dans la réparation vasculaire et leur nombre est réduit chez les patients atteints de maladies cardiovasculaires. Nous croyons que les effets de l’adiponectine peuvent s’expliquer entre autres via ses interactions avec les EPCs. Trois sous-population d’EPCs, isolées du sang de donneurs sains, ont été caractérisées par immunophénotypage par cytométrie en flux. L’expression des récepteurs de l’adiponectine, AdipoR1, AdipoR2 et H-cadherin par les EPCs et les cellules endothéliales a été évaluée par qPCR. Les effets de l’adiponectine sur la migration et l’apoptose des EPCs et sur l’apoptose des HUVECs ont été étudiés. L’expression de l’élastase des neutrophiles par les EPCs et son activité ont été testées. Les résultats de qPCR montrent que l’AdipoR1 est plus fortement exprimé que l’AdipoR2 alors qu’H-cadhérine n’est pas détectable dans les EPCs. Les EPCs précoces expriment aussi l’élastase. L’expression d’AdipoR1 a été confirmée par immunobuvardage. L’adiponectine augmente de façon significative la survie de deux sous-populations d’EPCs, mais pas celle des HUVECs, en condition de privation de sérum. L’activité de l’élastase a été confirmée dans le milieu conditionné par les EPCs. Les EPCs expriment les récepteurs de l’adiponectine et l’élastase. L’adiponectine protège les EPCs contre l’apoptose et pourrait augmenter leur capacité de réparation vasculaire. L’activité élastase des EPCs pourrait moduler localement l’activité de l’adiponectine par la génération de sa forme globulaire. / Adiponectin, an adipokine whose plasma levels are inversely correlated to metabolic syndrome components, protects against atherosclerosis and reduces myocardium infarction-associated risks. Endothelial progenitor cells (EPCs) are involved in vascular repair and their number is reduced in patients with cardiovascular disease. We hypothesized that positive effects of adiponectin against atherosclerosis are explained in part by its interaction with EPCs. EPCs were obtained from healthy volunteers’ venous blood by mononuclear cell isolation and plating on collagen-coated dishes. Three subpopulations of EPCs were characterized by flow cytometry immunophenotyping. Expression of adiponectin receptors, AdipoR1, AdipoR2 and H-cadherin was evaluated by qPCR in EPCs and endothelial cells. Effects of recombinant adiponectin on apoptosis of EPCs and HUVECs were assessed. Expression of neutrophil elastase by EPCs and enzymatic activity on adiponectin processing were assessed. Quantitative PCR of EPCs and HUVECs mRNA showed a higher expression of AdipoR1 compared to AdipoR2 and no expression of H-cadherin in EPCs. Expression of AdipoR1 in EPCs was confirmed by Western Blot. We demonstrated that early EPCs express neutrophil elastase. Adiponectin significantly increased survival of two subpopulations of EPCs in condition of serum deprivation but had no effects on HUVECs. Neutrophil elastase activity was confirmed in EPCs conditioned media. Adiponectin protects some EPCs subpopulations against apoptosis and therefore could modulate their involvement in vascular repair. Neutrophil elastase activity of EPCs could locally modify adiponectin activity by its ability to generate the globular form of adiponectin.

Les mécanismes à l'origine de la Bronchopneumopathie Chronique Obstructive (BPCO) et de ses complications cardiovasculaires restent partiellement connus. Le NO est produit par les NO synthases en quantité importante dans le poumon des sujets emphysémateux, mais son rôle dans la maladie n'est pas connu. Une dysfonction endothéliale précoce liée à une diminution de la disponibilité en NO au niveau vasculaire a aussi été observée chez les patients BPCO. Dans un premier travail, nous avons montré que iNOS et eNOS étaient induites de manière diffuse dans le poumon de souris développant un emphysème après une instillation d'élastase. Le recours à des souris iNOS-/- et eNOS -/-, ainsi qu'à un inhibiteur pharmacologique d'iNOS (1400W) ont permis de montrer que l'induction d'iNOS dans le poumon était responsable d'une accumulation de protéines nitratées dans les pneumocytes de type 2 et d'une diminution de l'oxydation protéique. Cependant ni iNOS ni eNOS n'étaient nécessaires au développement de l'emphysème induit par l'élastase. Dans un deuxième travail, nous avons exploré l'effet de la polyglobulie, une complication de la BPCO hypoxique, sur la fonction endothéliale chez 15 patients polyglobuliques et 13 normoglobuliques atteints de BPCO de sévérité égale. La polyglobulie était associée de base à une viscosité sanguine plus élevée et un diamètre artérielbrachial plus important mais avec des forces de cisaillement calculées similaires. L'étude de la vasodilatation en réponse à l'hyperhémie et celle du flux sanguin de l'avant bras mesuré par plethysmographie d'occlusion veineuse en réponse à une perfusion d'acétylcholine (ACh), et de N-monomethyl-L-arginine (L-NMMA) ont permis de montrer que les artères systémiques despatients polyglobuliques ajustent leur diamètre aux forces de cisaillement aigues et chroniques de manière adaptée grâce à une libération adaptée de NO. De plus, nos résultats suggèrent que la polyglobulie modérée n'a pas d'effet délétère sur la fonctionvasculaire chez les patients BPCO.

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