Converging TLR9 and PI3Kgamma signaling induces sterile inflammation and organ damage.

Toll-like receptor 9 (TLR9) and Phosphatidylinositol-3-kinase gamma (PI3Kγ) are very important effectors of the immune response, however, the importance of such crosstalk for disease development is still a matter of discussion. Here we show that PI3Kγ is required for immune responses in which TLR9 is a relevant trigger. We demonstrate the requirement of PI3Kγ for TLR9-induced inflammation in a model of CpG-induced pleurisy. Such requirement was further observed in inflammatory models where DNA sensing via TLR9 contributes to disease, such as silicosis and drug-induced liver injury. Using adoptive transfer, we demonstrate that PI3Kγ is important not only in leukocytes but also in parenchymal cells for the progression of inflammation. We demonstrate this crosstalk between TLR9 and PI3Kγ in vitro using human PBMCs. The inhibition of PI3Kγ in CpG-stimulated PBMCs resulted in reduction of both cytokine production and phosphorylated Akt. Therefore, drugs that target PI3Kγ have the potential to treat diseases mediated by excessive TLR9 signalling.

Relationship of chemical structure and anti-inflammatory activity of dihydrocorynantheol and its analogues.

BACKGROUND: Dihydrocorynantheol (DHC) is an alkaloid compound isolated from Esenbeckia leiocarpa Engl. that has demonstrated anti-inflammatory properties in experimental models. The aim of this study was to investigate whether the modification of the chemical structure of DHC could alter its anti-inflammatory effect in a mouse model of pleurisy induced by carrageenan. METHODS: DHC was isolated from Esenbeckia leiocarpa Engl. Capillary electrophoresis, physical characteristics, spectral data produced by infrared analysis and nuclearmagnetic resonance ((1)H and (13)C), and mass spectrometry analysis were used to identify and elucidate DHC structure. The DHC compound was subjected to chemical structural modifications by nucleophilic substitution reactions, yielding five analogous compounds: acetyl (1), p-methylbenzoyl (2), benzoyl (3), p-methoxybenzoyl (4) and p-chlorobenzoyl (5). Swiss mice were used throughout the experiments. Pro-inflammatory parameters leukocyte migration, exudate concentrations and myeloperoxidase (MPO) activity were quantified in the fluid leakage from the mouse pleural cavities at 4 h after pleurisy induction. RESULTS: DHC and its analogues acetyl, p-methylbenzoyl, benzoyl, p-methoxybenzoyl and p-chlorobenzoyl inhibited total and differential leukocyte migration and MPO activity (p < 0.05). Only DHC significantly decreased the exudate concentrations (p < 0.01). CONCLUSIONS: DHC was more effective than its analogues as an anti-inflammatory agent in the mouse model of pleurisy induced by carrageenan. We did not determine what physicochemical modifications altered the anti-inflammatory effect of DHC, but this effect may be due to the modifications on the hydroxyl group at carbon 17 of the DHC.

Monoclonal antibodies anti-TGFß1 and anti-VEGF inhibit the experimental pleurodesis induced by silver nitrate.

BACKGROUND: The mechanisms underlying pleural inflammation and pleurodesis are poorly understood. We hypothesized that the cytokines transforming growth factor ß (TGFß1) and vascular endothelial growth factor (VEGF) play a major role in pleurodesis after intrapleural silver nitrate (SN) injection. METHOD: Forty rabbits received intrapleurally 0.5% SN alone or 0.5% SN + anti-TGFß1, anti-IL-8, or anti-VEGF. After 28 days, the animals were euthanized and macroscopic pleural adhesions, microscopic pleural fibrosis, and collagen deposition were analyzed for characterization of the degree of pleurodesis (scores 0-4). RESULTS: Scores of pleural adhesions, pleural fibrosis, total collagen, and thin collagen fibers deposition after 28 days were significantly lower for 0.5% SN + anti-TGFß1 and 0.5% SN + anti-VEGF. Significant correlations were found between macroscopic adhesion and microscopic pleural fibrosis with total collagen and thin collagen fibers. CONCLUSIONS: We conclude that both TGFß1 and VEGF, but not IL-8, mediate the pleural inflammatory response and pleurodesis induced by SN.

Blockage of vascular endothelial growth factor (VEGF) reduces experimental pleurodesis.

BACKGROUND AND OBJECTIVE: Chemical pleurodesis controls recurrent malignant pleural effusion. The mechanism that determines pleural symphysis involves the action of vascular endothelial growth factor (VEGF). We assessed the influence of the anti-VEGF antibody (bevacizumab) on pleurodesis induced by talc or silver nitrate and analyzed the temporal development of pleural angiogenesis. METHODS: Sixty New Zealand rabbits received intrapleural injection (2mL) of talc (400mg/kg) or 0.5% silver nitrate. In each group, half of the animals received an intravenous injection of bevacizumab 30min before the sclerosing agent. Five animals from each group were euthanized 7, 14, or 28 days after the procedure. Adhesions and inflammation (scores: 0-4), thickness (µm), vascular density (vessels/field), and collagen fibers (µm(2)) were evaluated in the visceral pleura. RESULTS: Antibody anti-VEGF interferes in pleurodesis induced by talc or silver nitrate. Pleural inflammation was discreet with no difference between the groups, regardless the anti-VEGF treatment. Concerning the vascular density of the visceral pleura, a smaller number of neoformed vessels was noted in the animals that received bevacizumab. In the animals receiving silver nitrate, the decrement in adhesions and vascular density was associated with reduced thick and thin collagen fibers, resulting in less pleural thickness. CONCLUSION: The anti-VEGF antibody inhibits adhesions between pleural layers. Despite being an experimental study in animals with normal pleura, the results call attention to a likely lack of success in pleurodesis when VEGF blockers are used.

Pleural mesothelial cells mediate inflammatory and profibrotic responses in talc-induced pleurodesis.

Intrapleural talc is used to produce pleurodesis in malignant pleural effusions. Prior in vivo studies have documented an acute inflammatory response to talc in the pleural space but the cellular source of cytokines has not been identified. The aim of this study was to investigate the acute response of rabbit pleural mesothelial cells challenged with talc used for pleurodesis and compare it to prior studies of the response to talc in the rabbit pleural space. Cultured rabbit pleural mesothelial cells (PMC) were exposed to talc (25 mug/cm(2)) for 6, 24, or 48 h and assessed for viability, necrosis, and apoptosis by flow cytometry, Trypan Blue exclusion, and immunocytochemistry, and for the production of interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), and transforming growth factor-beta(1) (TGF-beta(1)) by ELISA. More than 50% of the PMC remained viable 48 h after talc stimulation. The PMC that were nonviable were identified as either apoptotic or necrotic, with roughly 20% in each category over the 48 h. At 6 h, the IL-8, VEGF, and TGF-beta(1) levels produced by talc-exposed PMC increased significantly and remained elevated for up to 48 h. These cytokine levels rose at similar times and at the same or higher levels than have been measured in the rabbit pleural space in prior studies. We report that viable, talc-exposed, pleural mesothelial cells may actively mediate the primary inflammatory pleural response in talc-induced pleurodesis.

Low concentration silver nitrate pleurodesis in rabbits: optimal concentration for rapid and complete sclerosing effect.

Pleurodesis is a useful therapeutic tool when local treatment of a recurrent malignant pleural effusion or pneumothorax is needed. We have previously demonstrated that the intrapleural injection of 0.5% silver nitrate (SN) produces a significant pleurodesis, while 0.25% SN has no sclerosing effect in a rabbit model. The objective of this study was to determine the minimum concentration of SN needed to induce pleurodesis in our experimental model. One hundred twenty male New Zealand white rabbits received 0.3, 0.4, or 0.5% SN (40 animals per group) in a total volume of 2 mL instilled intrapleurally. These animals were sacrificed 3, 7, 14 or 28 days after the intrapleural injection (n = 10 animals per group), and the pleural spaces were then assessed grossly for evidence of pleurodesis and microscopically for evidence of fibrosis and inflammation. By 28 days, all concentrations of SN had produced a pleurodesis. There was evidence of a gross pleurodesis 7 days post-injection in animals that received 0.5% SN (score of 2.8 +/- 0.2 on a scale of 0-4). After 14 days, significant pleural adhesions were evident in the groups that received 0.4 or 0.5% SN. We conclude that SN concentrations as low as 0.3% can effectively produce a pleurodesis within 28 days of intrapleural injection. However, the precocious pleurodesis development observed 7 days after the intrapleural injection of 0.5% SN suggests that this concentration may be optimal when a fast result is necessary.

Cholesterol in the separation of transudates and exudates.

The Light criteria represent the most acceptable method to separate transudates and exudates. However, approximately 10% of patients with transudates, especially those with congestive heart disease, are misdiagnosed with these criteria. To improve diagnostic accuracy, many biochemical markers have been proposed as alternatives to differentiate transudates and exudates. Cholesterol has raised particular interest because only pleural fluid is needed, which makes blood samples unnecessary and simplifies the procedure. In most clinical studies, cholesterol has been shown to be as sensitive as the Light criteria, although it is less specific. Other randomized studies are necessary to determine the real potential value of pleural-fluid cholesterol measurements. Studies of pleural-fluid cholesterol are aimed at better understanding the mechanisms by which cholesterol enters the pleural cavity and its role in diseases. The ideal cutoff point of cholesterol to differentiate transudates and exudates is still unknown. Recently, aspects of the cholesterol turnover in diseases have raised great interest. Cholesterol generated great interest after it was related to coronary artery diseases. The involvement of cholesterol in the atherosclerotic process is well known, although its importance in body cavities is still unclear.

Silver nitrate is superior to talc slurry in producing pleurodesis in rabbits.

STUDY OBJECTIVE: The ideal agent for producing pleurodesis has not been identified. Although talc is the agent most commonly used at the present time, there are concerns about its safety. Silver nitrate is a possible alternative agent. The purpose of the present study was to compare the effectiveness of intrapleural silver nitrate and talc slurry in producing pleurodesis in rabbits. Additionally, the total amount of pleural collagen and the distribution of thick and thin collagen fibers were studied. DESIGN: Two groups of 10 rabbits received either 0.50% silver nitrate or 400 mg/kg talc in a total volume of 2 mL intrapleurally. The animals were killed 28 days after injection, and the pleural spaces were assessed grossly for evidence of pleurodesis and microscopically for evidence of inflammation and fibrosis. Collagen was assessed with the van Gieson's and picrosirius stains. RESULTS: The macroscopic pleurodesis (scale, 0 to 4; mean +/- SEM) resulting from the intrapleural injection of silver nitrate (3.4+/-0.2) was significantly better (p<0.001) than that resulting from talc (1.6+/- 0.1). The mean degree of microscopic pleural fibrosis induced by silver nitrate (3.3+/-0.3) was significantly higher (p = 0.003) than that induced by talc (1.8+/-0.1). The mean amount of microscopic pleural collagen (van Gieson's) was significantly greater (p<0.001) in the rabbits that received silver nitrate (3.0+/-0.2) than in those that received talc (1.6+/-0.2). The distribution of thick and thin collagen fibers did not differ between the groups. CONCLUSIONS: We conclude that, in our rabbit model, intrapleural silver nitrate was more effective than talc in producing a pleurodesis.

Nitric oxide production by BCG-infected pleural macrophages from C57Bl/6 and DBA-2 mice.

We studied the kinetics of in vivo nitrite production in the inflammatory reaction induced by M. bovis BCG into the pleural space. Pleural macrophages harvested from C57Bl/6 mice after acute BCG infection produced high levels of nitric oxide (NO). Enhanced production was obtained upon stimulation with LPS plus IFN-gamma. In sharp contrast, macrophages from DBA-2 mice produced low levels of NO, as nitrite, at the same time interval (24 h after BCG infection), being completely refractory to further stimulation. After the third day, NO production was similar in both strains. There was a close relationship between nitrite levels in the pleural exudate in vivo and those produced by harvested macrophages in vitro. In this in vivo system, the pattern of NO production by pleural macrophages one day after BCG infection was discrepant and unexpected in the response of C57Bl/6 and DBA-2 mice. However, this early response did not affect the late progressive NO production in both mice strains, that may be responsible to the late control of the mycobacteria growth.

The use of histological and immunohistochemical markers to distinguish pleural malignant mesothelioma and in situ mesothelioma from reactive mesothelial hyperplasia and reactive pleural fibrosis.

Distinguishing malignant mesothelioma from reactive mesothelial hyperplasia and reactive fibrosis can be a diagnostic problem in small pleural biopsies, made more difficult by the recent recognition of mesothelioma-in-situ. Antibodies to epithelial membrane antigen (EMA), p53, and bcl-2 have all been advocated for differentiating reactive from neoplastic conditions, but reports are inconsistent. These antibodies have therefore been applied to 31 cases of malignant mesothelioma, 34 cases of reactive pleural disease (20 reactive mesothelial hyperplasia and 14 reactive pleural fibrosis) and four small biopsies that were initially coded as suspicious, from patients who later developed frank mesothelioma. Thirty out of 31 (97 per cent) cases of mesothelioma showed positive nuclear staining for p53, with a higher incidence of positivity in epithelioid than in sarcomatoid elements and 30/31 (97 per cent) showed diffuse linear membrane staining for EMA, again more intense in the epithelioid elements. No mesothelioma was positive for bcl-2. In seven cases that contained both in situ and invasive mesothelioma, the in situ elements showed similar staining patterns to the invasive epithelioid elements. Thirteen out of 20 (65 per cent) cases of reactive mesothelial hyperplasia showed occasional nuclear positivity for p53 and 5/20 (25 per cent) cases showed focal weak membrane staining for EMA. Three out of 14 (21 per cent) cases of reactive pleural fibrosis showed positive nuclear staining for p53 and 6/14 (43 per cent) cases showed focal membrane staining with EMA. No reactive cases stained for bcl-2. All four suspicious cases showed diffuse linear staining with EMA and three showed focal staining for p53. It is concluded that strong diffuse linear staining for EMA is a good marker of malignancy when differentiating epithelioid malignant mesothelioma and mesothelioma-in-situ from reactive mesothelial hyperplasia, although weak focal staining may occur in reactive conditions. Nuclear staining for p53 is also suggestive of epithelioid mesothelioma, but should be regarded as no more than suspicious. The antibodies used in this investigation are less helpful in differentiating sarcomatoid mesothelioma from reactive pleural fibrosis.

Opposite cellular accumulation and nitric oxide production in vivo after pleural immunization with M. leprae or M. bovis BCG.

Mycobacteria as intracellular pathogens have evolved mechanisms to survive within macrophages. Our previous data showed that M. leprae (ML), unlike M. bovis BCG, did not induce an inflammatory response in the mice subcutaneous tissue. Further, ML inhibited BCG-induced foot pad oedema and seemed to transform macrophages in epithelioid cells. Since these mycobacteria share common antigens, here we seeked to compare the acute and chronic cellular response evoked by ML and BCG in pleurisy of a mycobacteria-susceptible mice (BALB/c). The total leukocytes, the cell type that migrated to the pleural cavity and macrophage activation assayed by nitric oxide release were determined. Live or dead BCG Moreau recruited the same extent of cells, essentially monocytes and neutrophils, dose-dependently, in both acute and chronic pleurisy. BCG-induced eosinophilia was observed only in the acute response (after 24 h of injection). A significant nitric oxide release by pleural macrophages was triggered by BCG Moreau without previous activation. Nevertheless, ML failed to recruit leukocytes to the pleural space or to lead to nitric oxide production despite the number of bacilli used and the time studied (1, 7 or 14 days after injection). Although these mycobacteria have common antigens that cross-react, these data show a distinct ability of ML or BCG to recruit cells to the pleural space and to activate pleural macrophage for nitric oxide production in vivo.

Inhibition by the anti-mitotic drug doxorubicin of platelet-activating-factor-induced late eosinophil accumulation in rats.

Platelet-activating factor (PAF) has been shown, in the rat model of pleural inflammation, to induce the generation of an intermediate proteic factor able to cause eosinophil proliferation in vitro. This study was undertaken to investigate the effect of the anti-mitotic compound doxorubicin on PAF-induced eosinophilia in rats, in order to evaluate the contribution of local cell proliferation to this phenomenon. The late eosinophil infiltration caused by another chemoattractant leukotriene B4 was used for comparison. We observed that local treatment with doxorubicin (20 and 40 microg/cavity), given 6 h after PAF (1 microg/cavity), suppressed the eosinophil accumulation within 24 h, whilst only the higher dose was effective when the drug was given 12 h post-PAF. An effect on chemotaxis was ruled out, since local doxorubicin (40 microg/cavity) failed to modify the eosinophil migration noted 24 h after leukotriene B4 (0.5 microg/cavity) and the neutrophil/eosinophil infiltration noted at 6 h after PAF injection. Transfer of the pleural fluids collected 6 h after PAF from donors to recipient rats caused significant eosinophil accumulation in the recipient rats, an effect which was inhibited by the co-administration of doxorubicin (40 microg/cavity). No inhibitory effect was noted when the drug was given 6 h after the pleural fluids were transferred. We also found no change in the number of blood or bone marrow eosinophils after PAF stimulation. We conclude that doxorubicin selectively impaired the late eosinophil accumulation triggered by PAF in the pleural cavity of rats, clearly indicating that local cell proliferation seems to contribute to the development of this inflammatory response.

Eosinophil granulocyte proliferation induced by an intermediate factor generated in the pleural cavity of rats injected with platelet-activating factor-acether.

In previous research, we have observed that intrathoracic administration of platelet-activating factor-acether (PAF) promoted a delayed eosinophilia in the pleural cavity of rats that lasted for at least 96 h. We investigated the ability of pleural washings from rats previously injected with PAF (1 micrograms/cavity) to stimulate in vitro murine hematopoietic eosinophil proliferation. We observed that pleural fluid sustained eosinophil proliferation but not differentiation, under conditions in which PAF itself had no effect. The phenomenon lasted for 3 days and was maximal on the 1st day of culture. Treatment with neutralizing antibodies against interleukin (IL)-5, granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-3, alone or in combination, did not modify the eosinophil proliferation induced by PAF pleural fluid, suggesting that these cytokines may not be involved in the studied phenomenon. We conclude that the rat pleural fluid obtained 6 h after PAF administration induces eosinophil proliferation in vitro by a mechanism probably independent of IL-5, GM-CSF or IL-3.

Lavado pleural en hemotórax reciente: estudio experimental / Pleural lavage in fresh hemothorax: experimental study

Con el objeto de determinar si el lavado pleural precoz, en un hemotórax estabilizado, es capaz de mejorar el aseo pleural, se realizó un estudio experimental en perros a los que se les provocó un hemotórax izquierdo de 300 cc, se les dejó reposar 1 hora y luego se les instaló un tubo tygon » multiperforado por la línea media axilar, 6- espacio intercostal. En 5 perros (grupo A), el tubo se conectó a una trampa de agua y se mantuvo durante 6 horas. En los 5 perros restantes (grupo B), se aspiró con jeringa de 50 cc la sangre que fluyó fácil, y luego se realizó lavados con suero fisiológico. Contemplando el tratamiento se retiró el tubo, se suturó la herida y el perro se dejó en observación por 24 horas, después de las cuales se sacrificó y se midió la cantidad de sangre retenida, pesando los coágulos. Se tomó biopsia de la pleura parietal, sobre la curva posterior de la 7ª costilla y de pleura visceral, en el borde inferior del lóbulo inferior del pulmón. Se realizó análisis de variancia y t test. La duración promedio del tratamiento fue: a) 6 horas; b) 45 minutos: el volumen promedio de sangre extraído con el tratamiento; a) 127 cc; b) 208 cc (p<0,01). El volumen promedio de sangre retenida: a) 51,9 gr; b) 10,8 gr (p=0,0001). Biopsia: a) inflamación y depósito hemático sobre las pleuras; b) inflamación. Se concluye que el lavado pleural con suero fisiolólogico, produce una mejor evacuación del hemotórax

Aminoglucósidos en el tratamiento del empiema: concentraciones séricas en líquido pleural / Aminoglycosides in the therapy of empiema: serum concentrations in pleural liquid

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