Modelo experimental de doença pleural maligna induzida por células LLC (Lewis Lung Carcinoma)

Derrame pleural maligno (DPM) é um fator de mau prognóstico em pacientes com câncer de pulmão avançado. Sua patogênese ainda é pouco compreendida e as opções terapêuticas são limitadas. Modelos animais de DPM podem demonstrar novos aspectos fisiopatológicos desta doença. Stathopoulos et al. em 2006 (Am J Respir Cell Mol Biol. 2006;34:142-50) descreveram um modelo de DPM com 1,5 x105 células de LLC injetadas diretamente no espaço pleural de camundongos. Os autores relataram carcinomatose com derrame pleural, tumores pleurais e 100% de mortalidade após 17 dias. [...

Anti-EGFR reduces malignant pleural effusion and morbidity in experimental model of adenocarcinoma

Introduction: A lot patients with lung cancer develop pleural effusion in an advanced stage of the disease, with high morbidity and mortality. The pathogenesis of the malignant pleural effusion is not well known and the therapeutic options are limited. Currently target therapy, like the antibody anti-EGFR, is been used more often in the treatment of the lung cancer. The EGFR is a tyrosine-kinase receptor considered oncogenic and responsible for the growing, the survival, the proliferation and the differentiation of loads of cell types. The use of antibodies anti-EGFR may entail positive effects in the fight against the tumor and the effects of the malignant pleural effusion. Goals: To evaluate the effects of intrapleural therapy with anti-EGFR in experimental model of malignant pleural effusion. Material e Methods: This study received the approval of the Comitê de Ética no Uso de Animais (CEUA) from HCFMUSP. Sixty C57BL/6 mice received intrapleural injection of 0,5x105 Lewis Lung Carcinoma (LLC) cells. The animals were divided in two groups that received the intrapleural injection with anti-EGFR or PBS (No treatment/Control) after 3, 7, 10 and 14 days from the induction of the pleural neoplasia. Ten animals from each group were observed until their death to evaluate a survival graphic curve. Forty animals were submitted to euthanasia after 7, 10, 14 or 21 days. We evaluated the weight gain or loss in grams (g), the mobility (score 0-3), the volume of recovered pleural effusion in milliliters (mL), and the presence of tumor in in the pleura, the pericardium, the inflammatory cells of the lungs and the histological changes in the kidneys, liver and spleen. Tumor apoptosis (TUNEL) and proliferation (PCNA) were evaluated by scores (0-4). Statistical analysis: One Way ANOVA, Kaplan-Meier, p <0,05. Results: On the survival analysis we did not observe any statistical difference between the groups. The mice weight loss was observed in all the groups after 21 days. It was observed a better mobility in the groups that receives the anti-EGFR  treatment. The pleural effusion volume was higher in the control group during all the study. The presence of pleural tumor implants was higher in the control group in comparison to the group that received treatment after 14 days. Pulmonary inflammation was discreet in all groups. The histological evaluation of the pericardium and the cardiac muscle showed tumor implants in the control group after 21 days. Hepatic and renal steatosis were more evident after 14 days in the control group. White flesh hyperplasia of the spleen was observed in all the periods of the study, especially in the 21st day in the control group. Higher levels of apoptosis and lower levels of tumor proliferation were observed in the groups that received treatment with anti-EGFR. Conclusions: In this experimental model, although it did not showed any benefic change in the animals survival, the target therapy with anti-EGFR reduced the pleural effusion volume, the morbidity and showed good histological parameters

Comparação de dois modelos experimentais de hipertensão pulmonar

Objective: To compare two models of pulmonary hypertension (monocrotaline and monocrotaline+pneumonectomy) regarding hemodynamic severity, structure of pulmonary arteries, inflammatory markers (IL-1 and PDGF), and 45-day survival. Methods: We used 80 Sprague-Dawley rats in two study protocols: structural analysis; and survival analysis. The rats were divided into four groups: control; monocrotaline (M), pneumonectomy (P), and monocrotaline+pneumonectomy (M+P). In the structural analysis protocol, 40 rats (10/group) were catheterized for the determination of hemodynamic variables, followed by euthanasia for the removal of heart and lung tissue. The right ventricle (RV) was dissected from the interventricular septum (IS), and the ratio between RV weight and the weight of the left ventricle (LV) plus IS (RV/LV+IS) was taken as the index of RV hypertrophy. In lung tissues, we performed histological analyses, as well as using ELISA to determine IL-1 and PDGF levels. In the survival protocol, 40 animals (10/group) were followed for 45 days. Results: The M and M+P rats developed pulmonary hypertension, whereas the control and P rats did not. The RV/LV+IS ratio was significantly higher in M+P rats than in M rats, as well as being significantly higher in M and M+P rats than in control and P rats. There were no significant differences between the M and M+P rats regarding the area of the medial layer of the pulmonary arteries; IL-1 and PDGF levels; or survival. Conclusions: On the basis of our results, we cannot conclude that the monocrotaline+pneumonectomy model is superior to the monocrotaline model.Comparar dois modelos de hipertensão pulmonar (monocrotalina e monocrotalina+pneumonectomia) em relação à gravidade hemodinâmica, estrutura de artérias pulmonares, marcadores inflamatórios (IL-1 e PDGF) e sobrevida em 45 dias. métodos: Foram utilizados 80 ratos Sprague-Dawley em dois protocolos de estudo: análise estrutural e de sobrevida. Os animais foram divididos em quatro grupos: controle, monocrotalina (M), pneumonectomia (P) e monocrotalina+pneumonectomia (M+P). Para a análise estrutural, 40 animais (10/grupo) foram cateterizados após 28 dias para a medição dos valores hemodinâmicos e sacrificados, obtendo-se tecidos cardíaco e pulmonar. O ventrículo direito (VD) foi dissecado do septo interventricular (SI), e a relação do peso \ud do VD e do peso do ventrículo esquerdo (VE) com o SI foi obtida como índice de hipertrofia de VD. No tecido pulmonar, foram realizadas análises histológicas e dosados IL-1 e PDGF por ELISA. Para o estudo de sobrevida, 40 animais (10/grupo) foram observados por 45 dias. Resultados: Os grupos M e M+P apresentaram hipertensão pulmonar em relação aos demais. Houve um aumento significativo da relação VD/VE+S no grupo M+P em \ud relação aos demais. Não houve diferenças significativas entre os grupos M e M+P quanto à área da camada média das artérias pulmonares, dosagens de IL-1 e PDGF ou sobrevida. Conclusões: Baseados nos resultados, não podemos afirmar que o modelo de monocrotalina+pneumonectomia é superior ao modelo de monocrotalina

Comparison of in vivo pleural response and in vitro mesothelial response to different asbestos fibers

Os produtos derivados de asbesto são amplamente utilizados pelo setor industrial, sendo descritas diversas doenças relacionadas à sua exposição, entre elas, o tumor primário da pleura, ou mesotelioma. O mecanismo fisiopatológico da lesão pelas fibras de asbesto no espaço pleural ainda não está totalmente estabelecido. Entre os fatores possivelmente implicados estão os efeitos provocados por uma resposta inflamatória com migração celular e liberação de mediadores moleculares levando à necrose, apoptose e alterações na proliferação e fibrogênese. No entanto, existem dificuldades no estudo da resposta in vivo ao asbesto, principalmente em virtude da população multicelular da cavidade pleural. Neste sentido, tem sido preconizado na literatura o estudo envolvendo animais geneticamente modificados ou selecionados, a fim de melhor compreender o papel das diversas populações envolvidas neste processo. Neste trabalho, tivemos como objetivo estudar comparativamente a resposta inflamatória aguda no líquido pleural e em células mesoteliais em cultura expostas a diferentes fibras de asbesto. Para tanto, animais controle e geneticamente selecionados para alta (AIR max) e baixa (AIR min) resposta inflamatória, e células mesoteliais em cultura foram expostas às fibras de asbesto crocidolita ou crisotila. Após 4, 24 ou 48 horas foram avaliadas a produção das citocinas IL-1b, IL-6 e MIP-2. Adicionalmente, no modelo in vivo foi avaliado o perfil celular do líquido pleural e a expressão do Ra PDGF em RESUMO fragmentos de pleura, e no modelo in vitro a resposta celular de apoptose e necrose. Como resultados, as fibras de asbesto crocidolita e crisotila produziram, em animais AIR max, uma elevação significativa no líquido pleural de leucócitos, neutrófilos e da IL-1b em comparação aos controles e aos animais AIR min. Entretanto, não houve diferença no número de macrófagos, IL-6 e MIP-2. As células mesoteliais em cultura expostas tanto às fibras crocidolita quanto crisotila apresentaram elevados índices de apoptose e necrose e da produção das citocinas IL-1b, IL-6 e MIP-2 quando comparadas aos controles. Houve forte correlação das citocinas MIP-2 e IL-1b em cultura com os níveis no líquido pleural para ambas as fibras estudadas. Foi demonstrado, para ambas as fibras, uma forte expressão do Ra PDGF na superfície pleural tanto nos animais com alta quanto com baixa resposta inflamatória quando comparado aos controles. Como conclusão, caracterizamos o perfil da resposta inflamatória aguda a diferentes fibras de asbesto em modelos experimentais in vivo e in vitro, contribuindo para a melhor compreensão do mecanismo de agressão celular secundário a este material de uso comercial tão freqüente.Asbestos-derived products are used thoroughly by industry. Several diseases related to asbestos exposition have been described, among them the primary tumor of the pleura mesothelioma. The mechanisms by which asbestos fibers produce injury to the pleural space are not clear. Among the factors possibly implicated are the effects secondary to an inflammatory response characterized by cellular migration and the release of molecular mediators leading to necrosis, apoptosis, cellular proliferation and fibrogenesis. However, it is difficulty to characterize the cellular response in vivo, mainly by virtue of the multi-cellular population present into the pleural cavity. Therefore, studies involving animals genetically modified or genetically selected have been proposed in the literature, in order to better understand the role of the several cellular populations involved in this complex process. In this study, our objective was to determine the inflammatory response of the pleural fluid and compare to the response of cultured mesothelial cells exposed to different asbestos fibers. Controls and mice genetically selected for high (AIR max) or low (AIR min) inflammatory response as well as mice cultured mesothelial cells were treated to crocidolite or chrysotile asbestos fibers. After 4, 24 or 48 hours the production of the cytokines IL-1b, IL-6 and MIP-2 were analyzed. In addition, the in vivo cellular profile of the pleural fluid and the Ra PDGF expression in the pleura fragments was documented. In parallel, the in vitro mesothelial cellular response of apoptosis and necrosis was quantified. Both asbestos fibers produced in AIR max mice a significant elevation in the pleural fluid total leukocytes, neutrophils and IL-1b levels in comparison to the controls and AIR min animals. However, no difference was found in the macrophage number, IL-6 and MIP-2 levels. Cultured mesothelial cells had a high apoptosis, necrosis, IL-1b, IL-6 and MIP-2 levels in comparison to the controls when exposed to either crocidolite or chrysotile. MIP-2 and IL-1b levels in cultured mesothelial cells strongly correlated with the levels of the pleural fluid for both fibers. In addition, a pronounced expression of Ra PDGF in the pleural surface was demonstrated in both high and low inflammatory selected mice when compared to the controls. In conclusion, we characterized the acute inflammatory response to the asbestos fibers crocidolite and chrysotile in an in vivo and in vitro experimental model, aiming to contribute to better understand the mechanism of cellular aggression secondary to this particulate material of such frequent commercial use

Characteristics of ascitic fluid from patients with suspected spontaneous bacterial peritonitis in emergency units at a tertiary hospital

CONTEXT AND OBJECTIVE: Spontaneous bacterial peritonitis (SBP) is a complication of ascites, especially in cirrhosis. Ascitic fluid with 250 or more neutrophils/mm³ is an acceptable criterion for diagnosis, even when bacterial fluid cultures are negative. The aims here were to estimate SBP frequency among emergency room patients based on cellular criteria and evaluate the biochemical profile of these fluids. DESIGN AND SETTING: Retrospective study at a public tertiary hospital. METHODS: Laboratory records of patients with ascites attended in emergency rooms between November 2001 and November 2006, from whom ascitic fluid samples were sent to the laboratory due to suspected SBP, were evaluated. The 691 samples included were divided into group A (presumed SBP: > 250 neutrophils/mm³; n = 219; 31.7%) and group B (no presumed SBP: < 250 neutrophils/mm3; n = 472; 68.3%). Patients' sex and age; ascitic fluid characteristics (numbers of neutrophils, leukocytes and nucleated cells); bacteriological characteristics; and protein, lactate dehydrogenase, adenosine deaminase and glucose concentrations were evaluated. RESULTS: Among group A cultured samples, 63 (33.8%) had positive bacterial cultures with growth of pathogens commonly associated with SBP. In total, the group A samples showed higher lactate dehydrogenase levels than seen in the group B samples. The latter presented predominance of lymphocytes and macrophages. CONCLUSION: Among the ascitic fluid samples with clinically suspected SBP, 31.7% fulfilled the cellular diagnostic criteria. Positive bacterial isolation was found in 33.8% of the cultured samples from the presumed SBP grou

Cytokine levels in pleural fluid as markers of acute rejection after lung transplantation

Our objective was to determine the levels of lactate dehydrogenase, IL-6, IL-8, and VEGF, as well as the total and differential cell counts, in the pleural fluid of lung transplant recipients, correlating those levels with the occurrence and severity of rejection. We analyzed pleural fluid samples collected from 18 patients at various time points (up to postoperative day 4). The levels of IL-6, IL-8, and VEGF tended to elevate in parallel with increases in the severity of rejection. Our results suggest that these levels are markers of acute graft rejection in lung transplant recipients