NOX1 Promotes Mesothelial-Mesenchymal Transition through Modulation of Reactive Oxygen Species-mediated Signaling.

Pleural organization may occur after empyema or complicated parapneumonic effusion and can result in restrictive lung disease with pleural fibrosis (PF). Pleural mesothelial cells (PMCs) may contribute to PF through acquisition of a profibrotic phenotype, mesothelial-mesenchymal transition (MesoMT), which is characterized by increased expression of α-SMA (α-smooth muscle actin) and other myofibroblast markers. Although MesoMT has been implicated in the pathogenesis of PF, the role of the reactive oxygen species and the NOX (nicotinamide adenine dinucleotide phosphate oxidase) family in pleural remodeling remains unclear. Here, we show that NOX1 expression is enhanced in nonspecific human pleuritis and is induced in PMCs by THB (thrombin). 4-Hydroxy-2-nonenal, an indicator of reactive oxygen species damage, was likewise increased in our mouse model of pleural injury. NOX1 downregulation blocked THB- and Xa (factor Xa)-mediated MesoMT, as did pharmacologic inhibition of NOX1 with ML-171. NOX1 inhibition also reduced phosphorylation of Akt, p65, and tyrosine 216-GSK-3ß, signaling molecules previously shown to be implicated in MesoMT. Conversely, ML-171 did not reverse established MesoMT. NOX4 downregulation attenuated TGF-ß- and THB-mediated MesoMT. However, NOX1 downregulation did not affect NOX4 expression. NOX1- and NOX4-deficient mice were also protected in our mouse model of Streptococcus pneumoniae-mediated PF. These data show that NOX1 and NOX4 are critical determinants of MesoMT.

Cinnamyl-3,4-dihydroxy-α-cyanocinnamate is a potent inhibitor of 5-lipoxygenase.

Lipoxygenases (LOs) are iron-containing enzymes that catalyze the conversion of arachidonic acid into hydroperoxyeicosatetraenoic acids (HPETEs) and other bioactive lipid mediators. In mammals, 5-LO, 15-LO, and 12-LO enzymes seem to have distinct roles in pathophysiological contexts, which have emphasized the need for selective inhibitors. Cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC) has been proposed as potent and selective inhibitor of platelet-type 12-LO (p12-LO). Here, we re-evaluated the selectivity profile of CDC on LOs, and we show that CDC is a potent and direct inhibitor of 5-LO. CDC reduced 5-LO activity in cell-free assays (purified human recombinant enzyme or leukocyte homogenates), with IC(50) values in the low nanomolar range (9-25 nM) and a selectivity index of approximately 35 and 15 over p12-LO and 15-LO1, respectively. Likewise, CDC inhibited 5-LO product formation in intact human polymorphonuclear leukocytes and monocytes (IC(50) = 0.45-0.8 µM). A lower potency was observed for 15-LO1, whereas p12-LO activity in platelets was hardly affected. In human whole blood, CDC efficiently reduced the formation of 5-LO products, and similar effects were observed for 12(S)-H(P)ETE and 15(S)-H(P)ETE. Finally, CDC (3.5 and 7 mg/kg i.p.) was effective in vivo in the platelet-activating factor-induced shock in mice and reduced formation of the 5-LO product leukotriene B(4) in the rat carrageenan-induced pleurisy after a single oral dose of 10 mg/kg. Together, our data demonstrate that CDC is a potent inhibitor of 5-LO with efficacy in vivo and encourage further development of CDC as the lead compound.

Cryptococcal pleuritis containing a high level of adenosine deaminase in a patient with AIDS: a case report.

Cryptococcal infection is the 4th most common opportunistic infection in patients with acquired immune deficiency syndrome (AIDS). Although pleural effusion alone is an unusual presentation, we present a case of cryptococcal pleuritis in an AIDS patient which was initially difficult to discriminate from tuberculous pleuritis because of the high level of pleural adenosine deaminase (ADA). Cryptococcus neoformans was detected in the culture of the pleural effusion after the initiation of antituberculous treatment. High levels of ADA in the pleural fluid can be observed in patients with cryptococcal pleuritis, and longer incubation of pleural fluid should be performed in all patients who present with pleuritis associated with a high ADA level as the only significant finding.

Contribution of the prostaglandin E2/E-prostanoid 2 receptor signaling pathway in abscess formation in rat zymosan-induced pleurisy.

Abscess formation is a classic host response to infection by many pathogenic microorganisms. Here, we studied the role of prostaglandins (PGs) and their signal transduction in abscess formation. Zymosan was injected into the pleural cavity of rats. Expression of enzymes involved in PG synthesis, their receptors, and cytokines in exudate leukocytes and abscesses were analyzed by polymerase chain reaction, Western blotting, and immunohistochemistry. Treatment with ketorolac, a cyclooxygenase (COX)-1 inhibitor, or N-[2-cyclohexyloxy-4-nitrophenyl] methanesulfonamide (NS-398), a COX-2 inhibitor, reduced the size of abscesses and the number of cells recovered from the abscess. COX-2 was detected in leukocytes of the exudate and a marginal area of abscesses. Among detected terminal PG synthases, the major one was cytosolic PGE synthase. Membrane-bound PGE synthase (mPGES)-1 was detected in cells that were similar to the COX-2-expressing cells in morphology and localization. A high level of the E-prostanoid (EP)(2) receptor and a low level of the EP(4) receptor were detected. The expression pattern of the EP(2) receptor paralleled that of COX-2 and mPGES-1. 11,15-O-Dimethyl PGE(2) (ONO-AE1-259), an EP(2) receptor agonist, and rolipram, a phosphodiesterase type-4 inhibitor, reversed the effects of COX inhibitors on abscess formation. In contrast, 16-(3-methoxymethyl) phenyl-omega-tetranor-3,7-dithia PGE(1) (ONO-AE1-329), an EP(4) receptor agonist, did not reverse the effects of NS-398. Moreover, NS-398 reduced the mRNA levels in exudate leukocytes of some proinflammatory and fibrogenic cytokines, which was reversed by ONO-AE1-259. These results suggest that PGE(2) generated via COX-1 and COX-2 may interact with the EP(2) receptor and may up-regulate in cAMP-dependent fashion the production of cytokines that promote abscess formation.

The diagnostic utility of adenosine deaminase isoenzymes in tuberculous pleural effusions.

SETTING: Pleural adenosine deaminase (ADA) levels have been found to be useful in diagnosing tuberculous pleuritis. Elevated ADA levels have been attributed to ADA2 isoenzyme, although no comprehensive studies have evaluated ADA2 as a diagnostic test. OBJECTIVE: To estimate the diagnostic accuracy of ADA and ADA2 in diagnosing tuberculous pleurisy. METHOD: A 3-year retrospective study was carried out. ADA and ADA2 were determined on patients diagnosed according to predetermined criteria. RESULTS: A total of 951 samples were received, including 387 patients with tuberculosis (TB). ADA values>or=52.4 U/l yielded a sensitivity, specificity and positive (PPV) and negative predictive value (NPV) respectively of 93.7% (95%CI 90.0-96.0), 88.7% (95%CI 85.7-91.3), 85.5% (95%CI 81.7-88.8) and 95.2% (95%CI 92.9-96.9). ADA2 values>or=40.6 U/l yielded a sensitivity, specificity and PPV and NPV of respectively 97.2% (95%CI 95.0-98.7), 94.2% (95%CI 91.8-96.0), 92.2% (95%CI 89.1-94.7) and 98.0% (95%CI 96.3-99.0). The chi2 and McNemar tests proved the superiority of ADA2 statistically. CONCLUSION: ADA2 is superior to ADA in the diagnosis of tuberculous pleuritis and should be used as a routine test in the diagnostic work-up of patients with pleural effusions in areas with high TB prevalence.

Levels of cathepsins S and H in pleural fluids of inflammatory and neoplastic origin.

Cathepsins S and H are present in immune cells and tissues and may play a role in the activation of an adoptive immune response. Our goal was to assess their protein levels in pleural fluids from 82 patients who underwent thoracentesis or thoracoscopy for therapeutic or diagnostic reasons and to relate them to an inflammatory, neoplastic or hemodynamic origin. Pleural effusions were also analyzed for a panel of 13 inflammatory or proliferative markers to test possible links to a nonspecific host reaction. Increased levels of cathepsin S were found in parainflammatory and cancer-related effusions compared to transudates. Cathepsin H levels were elevated only in parainflammatory effusions, whereas the levels in cancer-related effusions were comparable to transudates. Cathepsin S values significantly correlated with LDH, alpha-1-AT, VEGF, sICAM, sVCAM, MPO, uPA, MMP-9/TIMP-1, IL-8 and MCP-1, but not with CRP, IL-10 or cathepsin H. In contrast to cathepsin S, cathepsin H values did not correlate with markers of inflammation, indicating a specific role for cathepsin H in the pleural host response. In conclusion, the estimation of cathepsin S and cathepsin H may help to distinguish between effusions of different etiology.

Discovery of benzo[g]indol-3-carboxylates as potent inhibitors of microsomal prostaglandin E(2) synthase-1.

Selective inhibition of pro-inflammatory prostaglandin (PG)E(2) formation via microsomal PGE(2) synthase-1 (mPGES-1) might be superior over inhibition of all cyclooxygenase (COX)-derived products by non-steroidal anti-inflammatory drugs (NSAIDs) and coxibs. We recently showed that benzo[g]indol-3-carboxylates potently suppress leukotriene biosynthesis by inhibiting 5-lipoxygenase. Here, we describe the discovery of benzo[g]indol-3-carboxylates as a novel class of potent mPGES-1 inhibitors (IC(50)0.1 microM). Ethyl 2-(3-chlorobenzyl)-5-hydroxy-1H-benzo[g]indole-3-carboxylate (compound 7a) inhibits human mPGES-1 in a cell-free assay (IC(50)=0.6 microM) as well as in intact A549 cells (IC(50)=2 microM), and suppressed PGE(2) pleural levels in rat carrageenan-induced pleurisy. Inhibition of cellular COX-1/2 activity was significantly less pronounced. Compound 7a significantly reduced inflammatory reactions in the carrageenan-induced mouse paw edema and rat pleurisy. Together, based on the select and potent inhibition of mPGES-1 and 5-lipoxygenase, benzo[g]indol-3-carboxylates possess potential as novel anti-inflammatory drugs with a valuable pharmacological profile.

Effect of PD98059, a selective MAPK3/MAPK1 inhibitor, on acute lung injury in mice.

The aim of the present study is to evaluate the contribution of mitogen-activated protein kinase 1-3 MAPK3/MAPK1) in a model of acute lung inflammation in mice. Injection of carrageenan into the pleural cavity of mice elicited an acute inflammatory response characterized by: accumulation of fluid containing a large number of neutrophils (PMNs) in the pleural cavity, infiltration of PMNs in lung tissues and subsequent adhesion molecule expression (I-CAM and P-selectin), lipid peroxidation, and increased production of tumour necrosis factor-alpha, (TNF-alpha) and interleukin-1beta (IL-1beta). Furthermore, carrageenan induced lung apoptosis (Bax and Bcl-2 expression) as well as nitrotyrosine formation, NF-kB activation, and pJNK expression, as determined by immunohistochemical analysis of lung tissues and the degree of lung inflammation and tissue injury (histological score). Administration of PD98059, an inhibitor of MAPK3/MAPK1 (10 mg/kg) 1 h after carrageenan caused a reduction in all the parameters of inflammation measured. Thus, based on these findings we propose that inhibitors of the MAPK3/MAPK1 signaling pathways, such as PD98059, may be useful in the treatment of various inflammatory diseases.

[Serum activity of chitotriosidase, lysozyme and cathepsin H in patients with lung cancer and patients with inflammatory exudate (preliminary report)]. / Aktywnosc chitotriozydazy oraz lizozymu i katepsyny H w surowicy chorych na raka pluca i chorych z wysiekiem zapalnym (doniesienie wstepne).

UNLABELLED: Lung cancer remains the leading cause of cancer death over the world. Although new diagnostic methods have been discovered, new biomarkers of the cancer are still under studying. A human chitinolytic enzyme called chitotriosidase hydrolyzes chitin and chitotrioside substrates. It is specifically expressed by activating macrophages and seems to play a role in the defense against chitinous human pathogens. Recently it has been shown that chitotriosidase may also attend to the inflammatory process. The aim of the study was to determine chitotriosidase activity in serum of patients with lung cancer and patients with inflammatory exudate. We studied the usefulness of the above parameter determination in differentiation between lung cancer and inflammation. In addition, serum activity of lysozyme and cathepsin H was determined. MATERIALS AND METHODS: The study included 17 patients with inflammatory pleural exudate--group 1., 40 lung cancer patients with malignant pleural effusion--group 2. and 37 healthy subjects. All the patients of group 2. were divided into 2 subgroups: 2A without metastases (n = 23) and 2B with metastases (n = 17). Chitotriosidase and cathepsin H activity was determined in serum by a fluorometric methods. Serum lysozyme activity was measured by turbidimetric method with Micrococcus luteus as substrate. RESULTS: We observed an increase of the chitotriosidase activity in serum patients of both studied group in comparison with the control. The activity of the chitotriosidase in lung cancer patients was significantly higher than in the control (36.7 vs 68.1 nmol/ml/h; p < 0.01). There were no significant differences in serum lysozyme and cathepsin H activity in patients in comparison to healthy subjects. CONCLUSION: The results suggest that activity of the chitotriosidase can not be used to differentiation between inflammation and cancer in lung.

Interaction between cyclooxygenase (COX)-1- and COX-2-products modulates COX-2 expression in the late phase of acute inflammation.

Prostanoid production depends on the activity of two cyclooxygenase (COX) isoforms. It is appreciated that COX-1 plays a role in physiological processes, whereas COX-2 acts in pathological conditions. However their roles, particularly roles of COX-1, have not yet been fully established in inflammation. Here, we examined the effects of COX inhibitors, having differential isoform selectivity, on the late phase of rat carrageenin-induced pleurisy to elucidate the role of COX-2 expressed in the draining lymph nodes and found substantial contribution of COX-1-product(s). Protein and mRNA of COX-2 were detectable with Western blotting analysis and reverse-transcription polymerase chain reaction (RT-PCR) analysis in parathymic lymph nodes, peaking at 48 h after induction of pleurisy. Microsomal prostaglandin E synthase (mPGES)-1 was detectable by immunohistochemical analysis in cells with dendritic processes, a morphological characteristic similar to that of COX-2 expressing cells. Although aspirin, indomethacin and a COX-1 inhibitor, ketorolac, significantly decreased the volume of pleural exudate, they did not affect the levels of COX-2 and mPGES-1 in the lymph node 24 h after induction of pleurisy. In contrast, COX-2 inhibitors, nimesulide and NS-398, had no effect on the exudate volume, but they increased the number of COX-2- and mPGES-1-expressing cells and extension of their dendritic processes with significant increase in the COX-2 level, which were antagonised by ketorolac. These results suggest that COX-2-expressing cells may negatively self-regulate their functions by producing PGE2 via mPGES-1: migration into the draining lymph node and their differentiation. Moreover, COX-1- and COX-2-derived prostanoids may play differential or sometimes antagonistic roles in the late phase of acute inflammation.

Phosphoinositide-3 kinases critically regulate the recruitment and survival of eosinophils in vivo: importance for the resolution of allergic inflammation.

The phosphatidylinositol-3 kinase (PI3K) family of signaling enzymes plays a crucial role in leukocyte recruitment and activation and hence, likely regulates the induction and propagation phases of inflammation. However, little data have emerged showing a role for these processes in the resolution phase in models of in vivo inflammation. Here, we have evaluated the role of PI3K for the migration and survival of eosinophils in a model of allergic pleurisy in mice. Eosinophil accumulation in PI3Kgamma-deficient mice was inhibited at 48 h, as compared with wild-type mice but not at earlier time-points (6 and 24 h). Experiments with adoptive transfer of bone marrow showed that PI3Kgamma in eosinophils but not in non-bone marrow-derived cells was required for their accumulation. Systemic treatment with PI3K inhibitors before antigen challenge prevented the recruitment of eosinophils. This was associated with decreased Akt phosphorylation, interleukin-5 production, and eosinophil release from the bone marrow. Treatment with PI3K inhibitors 24 h after antigen challenge markedly cleared the accumulated eosinophils, an effect associated with inhibition of Akt phosphorylation and an increased number of apoptotic events. Altogether, our data demonstrate an important role of PI3Kgamma for the maintenance of eosinophilic inflammation in vivo, whereas other isoforms of PI3K may be relevant for the recruitment process.

Characterization of CGS 8515 as a selective 5-lipoxygenase inhibitor using in vitro and in vivo models.

CGS 8515 inhibited 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B4 synthesis in guinea pig leukocytes (IC50 = 0.1 microM). The compound did not appreciably affect cyclooxygenase (sheep seminal vesicles), 12-lipoxygenase (human platelets), 15-lipoxygenase (human leukocytes) and thromboxane synthetase (human platelets) at concentrations up to 100 microM. CGS 8515 inhibited A23187-induced formation of leukotriene products in whole blood (IC50 values of 0.8 and 4 microM, respectively, for human and rat) and in isolated rat lung (IC50 less than 1 microM) in vitro. The selectivity of the compound as a 5-lipoxygenase inhibitor was confirmed in rat whole blood by the 20-70-fold separation of inhibitory effects on the formation of leukotriene from prostaglandin products. Ex vivo and in vivo studies with rats showed that CGS 8515, at an oral dose of 2-50 mg/kg, significantly inhibited A23187-induced production of leukotrienes in whole blood and in the lung. The effect persisted for at least 6 h in the ex vivo whole blood model. CGS 8515, at oral doses as low as 5 mg/kg, significantly suppressed exudate volume and leukocyte migration in the carrageenan-induced pleurisy and sponge models in the rat. Inhibitory effects of the compound on inflammatory responses and leukotriene production in leukocytes and target organs are important parameters suggestive of its therapeutic potential in asthma, psoriasis and inflammatory conditions.

Inducible cyclooxygenase-derived 15-deoxy(Delta)12-14PGJ2 brings about acute inflammatory resolution in rat pleurisy by inducing neutrophil and macrophage apoptosis.

Failure of acute inflammation to resolve leads to persistence of the inflammatory response and may contribute to the development of chronic inflammation. Thus, an understanding of inflammatory resolution will provide insight into the etiology of chronic inflammation. In an acute pleurisy, polymorphonuclear leukocytes (PMNs) were found to predominate at the onset of the lesion but decreased in number by undergoing apoptosis, the principal mechanism by which PMNs died in this model. PMNs were progressively replaced by monocytes, which differentiated into macrophages. As with PMNs, macrophages also underwent programmed cell death leading to an abatement of the inflammatory response and eventual resolution. It was found that apoptosis of both these inflammatory cell types was mediated by pro-resolving cyclooxygenase 2-derived 15deoxyDelta12-14PGJ2, which is uniquely expressed during active resolution. Although PMN programmed cell death is well understood, the observation that macrophages apoptose during resolution of acute inflammation is less well described. These results provide insight into the mechanisms that switch off acute inflammation and prevent complications of wound healing and potentially the development of immune-mediated chronic inflammation.

A novel role for phospholipase A2 isoforms in the checkpoint control of acute inflammation.

Acute inflammation can be considered in terms of a series of checkpoints where each phase of cellular influx, persistence, and clearance is controlled by endogenous stop and go signals. It is becoming increasingly apparent that in addition to initiating the inflammatory response, eicosanoids may also mediate resolution. This suggests there are two phases of arachidonic acid release: one at onset for the generation of proinflammatory eicosanoids and one at resolution for the synthesis of proresolving eicosanoids. What is unclear is the identity of the phospholipase (PLA2) isoforms involved in this biphasic release of arachidonic acid. We show here that type VI iPLA2 drives the onset of acute pleurisy through the synthesis of PGE2, LTB4, PAF, and IL-1beta. However, during resolution there is a switch to a sequential induction of first sPLA2 (types IIa and V) that mediates the release of PAF and lipoxin A4, which, in turn, are responsible for the subsequent induction of type IV cPLA2 that mediates the release of arachidonic acid for the synthesis of proresolving prostaglandins. This study is the first of its kind to address the respective roles of PLA2 isoforms in acute resolving inflammation and to identify type VI iPLA2 as a potentially selective target for the treatment of inflammatory diseases.

Tuberculous pleurisy: a study of 254 patients.

OBJECTIVES: To determine the age at which tuberculous pleural effusions occur, the radiological and biochemical characteristics of the effusions, the sensitivities of the various diagnostic tests, and the utility of combining clinical, radiological, and analytic data in diagnosis. METHODS: We studied the case histories of 254 patients in whom tuberculous pleural effusions were diagnosed with certainty between January 1, 1989, and June 30, 1997, in a Spanish university hospital in a region with a high incidence of tuberculosis. RESULTS: The mean (+/-SD) age of the patients was 34.1+/-18.1 years, and 62.2% were younger than 35 years. The effusion was on the right side in 55.9% of patients, on the left side in 42.5% of patients, and on both sides in 1.6% of patients. In 81.5% of patients, less than two thirds of the hemithorax was affected. Associated pulmonary lesions were detected in 18.9% of patients, of whom 14.6% exhibited cavitation. In 93.3% of the effusions, more than 50% of leukocytes were lymphocytes, and almost all had the biologic characteristics of exudates (98.8% had high total protein contents, 94.9% had high cholesterol levels, and 82.3% had high lactate dehydrogenase levels). All but 1 effusion (99.6%) had an adenosine deaminase (ADA) concentration higher than 47 U/L, 96.8% (123/127) of the effusions had high ADA2 levels, and 89% (73/82) of the effusions had high interferon gamma levels. Adenosine deaminase 2 contributed 72.2%+/-12.5% (mean +/- SD) of total ADA activity. Total ADA activity was significantly correlated with ADA2 (r = 0.83) and with interferon gamma (r = 0.30) levels. Definitive diagnosis was based on the observation of caseous granulomas in pleural biopsy tissue samples in 79.8% of patients, on the results of biopsy cultures in 11.7% of patients, and on pleural effusion cultures in the remaining 8.5% of patients. Results of the tuberculin skin test were positive in only 66.5% of patients. CONCLUSIONS: In these patients, lymphocyte-rich exudative pleural effusions occurred, on average, at a young age, with no preference for either the right or the left side; normally affected no more than two thirds of the hemithorax; and were generally unaccompanied by pulmonary infiltrates. High ADA concentration was a highly sensitive diagnostic sign and was caused by a rise in ADA2 concentration. The most sensitive criterion based on pleural biopsy was the observation of caseous granulomas, and culture of biopsy material further increased overall sensitivity. Negative skin test results were no guarantee of the effusion being nontuberculous. This, together with the low mean age of the patients and the low frequency of associated pulmonary lesions, suggests that tuberculous pleural effusion is a primary form of tuberculosis in this region.

Role of type II phospholipase A2 in the inflammatory process of carrageenan-induced pleurisy in rats.

In order to investigate the role of type II phospholipase A2 (PLA2) in the inflammatory process, the effect of a monoclonal antibody specific to type II PLA2 on carrageenan-induced pleurisy was studied in rats. Intravenous injection of the antibody (MB5.2), which inhibits the catalytic activity of type II PLA2, significantly reduced both the pleural exudate volume and the intrapleural leukocyte count, while a control antibody did not have any appreciable effect. MB5.2 caused no change in the level of type II PLA2 in the pleural fluid. These results suggest that type II PLA2 generated at inflamed sites, at least in part, have a crucial role in the pathogenesis of acute inflammation.

Cyclo-oxygenase and nitric oxide synthase isoforms in rat carrageenin-induced pleurisy.

1. The profiles of cyclo-oxygenase (COX) and nitric oxide synthase (NOS) isoforms were determined in the rat carrageenin-induced pleurisy model of acute inflammation. 2. The enzymes were assessed in peripheral blood leucocyte (PBL) cell pellets taken from untreated animals and at 2, 6 and 24 h after injection of the irritant in pleural exudate cell pellets and lung homogenates. 3. COX activity was assessed by the generation of prostacyclin (PGI2, measured as the stable metabolite, 6-keto prostaglandin F1 alpha) and prostaglandin E2 (PGE2). Western blot analysis and immunohistochemistry were also carried out. 4. NOS activity was based on the conversion of [3H]-L-arginine to [3H]-L-citrulline in the presence (total NOS activity) or absence of Ca2+ (inducible NOS; iNOS). 5. Peripheral blood leucocyte samples contained low levels of COX activity. In pleural exudate cell pellets, COX activity peaked at 2 to 6 h after injection of the carrageenin. At 24 h, COX activity was significantly reduced. 6. Western blot analysis demonstrated that the inducible isoform of COX (COX-2), was the predominant enzyme at all time points. Low levels of COX-2 were seen in PBLs. In pleural exudate cell pellets maximal COX-2 protein levels were seen at 2 h. 7. Immunohistochemistry confirmed the findings of Western blot studies. Approximately 10% of polymorphonuclear neutrophils (PMNs) in PBLs from untreated animals were immunopositive for COX-2. In cell pellet smears from carrageenin-induced pleurisy taken 2 h after injection of the irritant, PMNs were also the major source of COX-2 immunoreactivity. A small proportion of macrophages and mesothelial cells were also immunolabelled for COX-2.8. Low levels of NOS activity were seen in PBLs. In pleural exudates NOS activity was maximum at 6 h and greatly reduced by 24 h. This activity was solely attributable to iNOS.9. The present results illustrated a similar profile of COX and NOS activity in the carrageenin-induced pleurisy model of acute inflammation. It was demonstrated that COX-2 and iNOS were the predominant isoforms of their respective enzymes.

Meloxicam: influence on arachidonic acid metabolism. Part II. In vivo findings.

Meloxicam is a new nonsteroidal anti-inflammatory drug (NSAID) derived from enolic acid. Preclinical studies have indicated that meloxicam has potent anti-inflammatory activity, together with a good gastrointestinal and renal tolerability profile. This report summarizes studies undertaken to compare meloxicam to other NSAIDs in the inhibition of the inducible cyclooxygenase (COX-2) in inflamed areas (pleurisy of the rat, peritonitis of mice) and their influence on the activity of the constitutive cyclooxygenase (COX-1) in stomach, kidney, brain, and blood. In pleurisy of the rat, meloxicam was twice as potent as tenoxicam, 3 times as potent as flurbiprofen, 8 times as potent as diclofenac, and 20 times as potent as tenidap at inhibiting prostaglandin E2 (PGE2) biosynthesis. In the peritonitis model in mice, meloxicam was approximately twice as active as piroxicam, and more than 10 times as active as diclofenac in the suppression of PGE biosynthesis. Doses of meloxicam sufficient to inhibit PGE2 biosynthesis in the pleural exudate and peritoneal exudate had no influence on leukotriene-B4 (LTB4) or leukotriene-C4 (LTC4) content. The effect of meloxicam on the PGE2 content of rat gastric juice and rat urine was weaker than that of piroxicam or diclofenac. Meloxicam was a weaker inhibitor of the increased PGE2 concentration in brain of rats and mice (induced by convulsant doses of pentetrazole) than piroxicam, diclofenac, or indomethacin. Meloxicam had a weaker effect on serum thromboxane-B2 (TXB2) concentration in rats than piroxicam or tenoxicam. The in vivo findings confirm the results of in vitro tests, conducted separately, showing that meloxicam preferentially inhibits COX-2 over COX-1. COX-2 is the inducible isoenzyme implicated in the inflammatory response, whereas COX-1 has cytoprotective effects in the gastric mucosa. Therefore, a preferential selectivity for one isoenzyme over another, as displayed by meloxicam, may have implications in the clinical setting in terms of a more favorable risk: benefit profile.

Etiology and pleural fluid characteristics of large and massive effusions.

STUDY OBJECTIVE: To report the etiology of large and massive pleural effusions, and to compare their biochemical fluid characteristics with those of smaller size, and between malignant and nonmalignant conditions. DESIGN: Retrospective chart review of all patients undergoing thoracentesis at an academic medical center in Lleida, Spain, during a 10-year period. PATIENTS: Posteroanterior chest radiographs were available in 766 patients during the study period. Large pleural effusions (ie, two thirds or more of the hemithorax without its complete obliteration) were identified in 70 patients (9%), and massive pleural effusions (ie, hemithorax was completely opacified) were identified in 93 patients (12%). RESULTS: A similar etiologic spectrum between large and massive pleural effusions was observed. The most frequent cause of these pleural effusions was malignancy (89 patients; 55%), followed by complicated parapneumonic or empyema (36 patients; 22%), and tuberculosis (19 patients; 12%). Compared with nonmalignant pleural effusions, patients with large or massive malignant pleural effusions were more likely to have pleural fluids with higher RBC counts (18.0 x 10(9) cells/L vs 2.7 x 10(9) cells/L, respectively; p < 0.001) and lower adenosine deaminase (ADA) activity (11.5 vs 31.5 U/L, respectively; p < 0.001), which were the two parameters that were selected by a stepwise logistic-regression model as independent predictors of malignancy. In addition, large/massive malignant pleural effusions showed higher median RBC counts (18.0 x 10(9) cells/L vs 4.3 x 10(9) cells/L, respectively; p < 0.001), higher lactate dehydrogenase levels (641 vs 409 U/L, respectively; p = 0.001), lower pH (7.39 vs 7.42, respectively; p = 0.006) content, but similar cytologic yield (63% vs 53%, respectively; p = 0.171) than smaller malignant pleural effusions. CONCLUSIONS: The presence of a large or massive pleural effusion enables the clinician to narrow the differential diagnosis of pleurisy, since most effusions are secondary to malignancy or infections (either bacterial or mycobacterial). Bloody pleural fluid with low ADA content favors a malignant condition.

Hydrolysis of platelet activating factor and its methylated analogs by acetylhydrolases.

We examined the substrate specificity of PAF-degrading enzymes from various sources using platelet activating factor (PAF) and its synthetic analogs. The results were as follows: 1) Tissue-originated acetylhydrolases, such as rat kidney soluble enzyme, deacetylated 1S-methyl-1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (1S-Me-PAF) slightly more rapidly than PAF, whereas plasma acetylhydrolase hydrolyzed PAF more effectively than 1S-Me-PAF. 2) Rat polymorphonuclear leukocytes, monocytes, and lymphocytes homogenates showed an appreciable acetylhydrolase activity, the substrate specificity of which resembled that of the plasma enzyme. 3) Pleural exudates in an experimental pleurisy induced in rats by carrageenan contained an acetylhydrolase activity, the properties of which were similar to those of the plasma enzyme. 4) An extracellular phospholipase A2 activity, which was also observed in the pleural exudate and required Ca2+ ion for maximum activity, seemed not to participate in the deacetylation of PAF, since addition of EDTA did not affect the PAF deacetylation catalyzed by the pleural exudate. These findings indicate that the inactivation reaction of PAF present in the extracellular space is mainly catalyzed by plasma acetylhydrolase, which yields lysoPAF.