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PURPOSE: To document and explain the beneficial effects of non-invasive ventilation in correcting hypoxemia and hypoventilation in severe chronic obstructive pulmonary disease, during spinal anesthesia in the lithotomy position. CLINICAL FEATURES: A morbidly obese patient with severe chronic obstructive pulmonary disease underwent prostate surgery in the lithotomy position under spinal anesthesia. Hypoxemia was encountered during surgery, and a profound decrease of forced vital capacity associated with alveolar hypoventilation and ventilation/perfusion mismatching were observed. In the operating room, an M-mode sonographic study of the right diaphragm was performed, which confirmed that after spinal anesthesia and assuming the lithotomy position, there was a large decrease (-30%) in diaphragmatic excursion. Hypoxemia and alveolar hypoventilation were successfully treated with non-invasive positive pressure ventilation. CONCLUSIONS: Intraoperative application of non-invasive positive pressure ventilation improved diaphragmatic excursion and overall respiratory function, and reduced clinical discomfort in this patient.

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Mature, active cysteine cathepsins (CPs) were identified in human inflammatory bronchoalveolar lavage fluid (BALF) supernatants from patients suffering from silicosis by both western blot and surface plasmon resonance analyses. BALFs are not a reservoir of activatable proforms, since no autocatalytic maturation at acidic pH occurs. Cathepsin H is the most profuse among studied CPs (median value: 36.5 nM), while cathepsins B and L are the two most abundant thiol-dependent endoproteases. The overall concentration of active cathepsins B, H, K, L, and S is approximately 10-fold lower than their concentration in BALF supernatants from patients suffering from inflammatory acute lung injuries (962+/-347 nM).The cathepsins (approximately 70 nM)/cystatin-like inhibitors (approximately 9 nM) ratio is unbalanced in favor of enzymes ( approximately 8-fold). This presence of uncontrolled CPs suggests that they may contribute, in addition to matrix metalloproteases, to the lung tissue breakdown/remodeling occurring during silicosis, although their exact contribution to interstitial inflammation remains to be evaluated.

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BACKGROUND INFORMATION: Chronic inflammation and tissue remodelling result from an imbalance between proteolytic enzymes and their inhibitors in the lungs in favour of proteolysis. While many studies have examined serine proteases (e.g. cathepsin G and neutrophil elastase) and matrix metalloproteases, little is known about the role of papain-like CPs (cysteine proteases). The present study focuses on the thiol-dependent cathepsins (CPs) and their specific cystatin-like inhibitors [CPIs (CP inhibitors)] in human inflammatory BALFs (BAL fluids, where BAL stands for broncho-alveolar lavage). RESULTS: Cathepsins B, K and S found were mostly zymogens, whereas cathepsins H and L were predominantly in their mature forms. Little immunoreactive cystatin C was found and the high- and low-molecular-mass ('weight') kininogens were extensively degraded. The BALF procathepsins B and L could be activated autocatalytically, indicating that alveolar fluid pro-CPs are reservoirs of mature enzymes. Hydrolysis patterns of 7-amino-4-methylcoumarin-derived peptide substrates showed that extracellular alveolar CPs remain proteolytically active, and that cathepsins B and L are the most abundant thiol-dependent endoproteases. The CP/CPI balance was significantly tipped in favour of cathepsins (3- or 5-fold), as confirmed by the extensive CP-dependent degradation of exogenous kininogens by BALFs. CONCLUSIONS: Although their importance for inflammation remains to be clarified, the presence of active cathepsins L, K and S suggests that they contribute to the extracellular breakdown of the extracellular matrix.

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The protease-antiprotease imbalance that is characteristic of most inflammatory lung disorders depends on the spatial-temporal regulation of active inhibitor and protease concentrations in lung secretions. We have studied the competition between the three main serine proteases from human neutrophil primary granules in their binding to alpha1-Pi, the main serine proteases inhibitor in lung secretions. Elastase was the only target of alpha1-Pi when identical molar amounts of purified inhibitor and the three proteases were tested together. The other two proteases were only inhibited once elastase was saturated. Elastase remained the preferred target of inhibitors when bronchoalveolar lavage fluids from patients with lung pneumonia and acute respiratory distress syndrome were used as the source of inhibitors, in spite of the presence of additional inhibitors in lung secretions. Since neutrophil proteases are expressed at the neutrophil surface, we also measured residual activities of membrane-bound proteases after purified neutrophils were incubated with bronchoalveolar fluids. Again, elastase was the preferred target of the inhibitors. We conclude that protease 3 and cathepsin G are not controlled as efficiently as elastase in lung secretions, a feature that must be taken into account when developing inhibitor-based anti-inflammatory therapies.

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Human neutrophil elastase (HNE) has long been linked to the pathology of a variety of inflammatory diseases and therefore is a potential target for therapeutic intervention. At least two other serine proteases, proteinase 3 (Pr3) and cathepsin G, are stored within the same neutrophil primary granules as HNE and are released from the cell at the same time at inflammatory sites. HNE and Pr3 are structurally and functionally very similar, and no substrate is currently available that is preferentially cleaved by Pr3 rather than HNE. Discrimination between these two proteases is the first step in elucidating their relative contributions to the development and spread of inflammatory diseases. Therefore, we have prepared new fluorescent peptidyl substrates derived from natural target proteins of the serpin family. This was done because serpins are rapidly cleaved within their reactive site loop whether they act as protease substrates or inhibitors. The hydrolysis of peptide substrates reflects the specificity of the parent serpin including those from alpha-1-protease inhibitor and monocyte neutrophil elastase inhibitor, two potent inhibitors of elastase and Pr3. More specific substrates for these proteases were derived from the reactive site loop of plasminogen activator inhibitor 1, proteinase inhibitors 6 and 9, and from the related viral cytokine response modifier A (CrmA). This improved specificity was obtained by using a cysteinyl residue at P1 for Pr3 and an Ile residue for HNE and because of occupation of protease S' subsites. These substrates enabled us to quantify nanomolar concentrations of HNE and Pr3 that were free in solution or bound at the neutrophil surface. As membrane-bound proteases resist inhibition by endogenous inhibitors, measuring their activity at the surface of neutrophils may be a great help in understanding their role during inflammation.

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Activated human polymorphonuclear neutrophils at inflammatory sites release the chymotrypsin-like protease cathepsin G, together with elastase and proteinase 3 (myeloblastin), from their azurophil granules. The low activity of cathepsin G on synthetic substrates seriously impairs studies designed to clarify its role in tissue inflammation. We have solved this problem by producing new peptide substrates with intramolecularly quenched fluorescence. These substrates were deduced from the sequence of putative protein targets of cathepsin G, including the reactive loop sequence of serpin inhibitors and the N-terminal domain of the protease-activated receptor of thrombin, PAR-1. Two substrates were selected, Abz-TPFSGQ-EDDnp and Abz-EPFWEDQ-EDDnp, that are cleaved very efficiently by cathepsin G but not by neutrophil elastase or proteinase 3, with specificity constants (k(cat)/K(m)) in the 10(5) M(-1).s(-1) range. They can be used to measure subnanomolar concentrations of free enzyme in vitro and at the surface of neutrophils purified from fresh human blood. Purified neutrophils express 0.02-0.7 pg of cathepsin G/cell (n=15) at their surface. This means that about 10(4) purified cells may be enough to record cathepsin G activity within minutes. This may be most important for investigating the role of cathepsin G as an inflammatory agent, especially in bronchoalveolar lavage fluids from patients with pulmonary inflammatory disorders.

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