The structure of a Michaelis serpin-protease complex.

Serine protease inhibitors (serpins) regulate the activities of circulating proteases. Serpins inhibit proteases by acylating the serine hydroxyl at their active sites. Before deacylation and complete proteolysis of the serpin can occur, massive conformational changes are triggered in the serpin while maintaining the covalent linkage between the protease and serpin. Here we report the structure of a serpin-trypsin Michaelis complex, which we visualized by using the S195A trypsin mutant to prevent covalent complex formation. This encounter complex reveals a more extensive interaction surface than that present in small inhibitor-protease complexes and is a template for modeling other serpin-protease pairs. Mutations of several serpin residues at the interface reduced the inhibitory activity of the serpin. The serine residue C-terminal to the scissile peptide bond is found in a closer than usual interaction with His 57 at the active site of trypsin.

Long-term enteral access in aspiration-prone patients.

Aspiration pneumonia is a serious complication of enteral feeding. Many critically ill patients are particularly at risk for aspiration. Few studies have rigorously compared various access devices. Risk factors for aspiration and studies examining aspiration associated with enteral feeding devices are reviewed. We recommend a surgical jejunostomy for all patients at high risk for aspiration who require more than 3 weeks of enteral nutrition support.

Glucocorticoid receptor blockade reverses postinjury macrophage suppression.

OBJECTIVES: To study the effects of the stress-induced surge of endogenous glucocorticoids on macrophage function and the role of inhibiting glucocorticoids with a receptor antagonist, mifepristone (RU 486). DESIGN: One hundred thirty female Swiss-Webster mice were randomly assigned to either injury by femur fracture or uninjured anesthesia control in this intervention study. SETTING: A university-based surgical laboratory and animal facility. INTERVENTION: Injured mice were randomized to receive either the glucocorticoid receptor antagonist mifepristone (10 mg/kg by oral gavage) or its vehicle. Mifepristone or its vehicle were given either 2 hours before or 2 hours after the injury. MAIN OUTCOME MEASURES: Peritoneal macrophages were harvested 24 hours after the injury. Macrophages were assayed for the stimulated (phorbol myristate acetate, 1 microgram/mL) production of superoxide anion, secretion of interleukin-6, tumor necrosis factor alpha, and prostaglandin E2 in response to endotoxin (lipopolysaccharide at 10 micrograms/mL) and killing of Candida albicans. RESULTS: Pretreatment with mifepristone significantly prevented or reduced suppression of several macrophage functions following injury, including superoxide production and C albicans killing. Treatment after the injury preserved only C albicans. Mifepristone failed to block the increased secretion of prostaglandin E2 after injury. CONCLUSION: Pretreatment with mifepristone before an injury prevented suppression of several macrophage functions. Further studies are required on the effects of glucocorticoid inhibition on other aspects of the immune and metabolic responses to injury to define the potential clinical applications of mifepristone trauma.

Inhibition of nitric oxide synthesis is detrimental during endotoxemia.

BACKGROUND: Increased production of nitric oxide has been implicated as a mediator during septic shock and sepsis syndrome. Inhibition of nitric oxide production could be beneficial during endotoxemia to improve the individual's hemodynamic status and possibly outcome. OBJECTIVE: To evaluate the effects of nitric oxide inhibition on macrophage function and survival in a murine sepsis model. DESIGN: Sixty-eight female Swiss-Webster (ND4) mice were injected with a sublethal dose of Escherichia coli lipopolysaccharide (25 mg/kg). INTERVENTION: The treated group (n = 34) received 10 mg/kg of NG-nitro-L-arginine methyl ester at the time of lipopolysaccharide injection. MAIN OUTCOME MEASURES: Blood samples and peritoneal macrophages were obtained at baseline and at 2, 4, and 8 hours after injection. Nitrite levels were measured in 36 mice from plasma and supernatant samples of cultured peritoneal macrophages stimulated with interferon gamma (100 micrograms/mL) for 48 hours. Thirty-two animals were observed for survival. RESULTS: Administration of N-nitro-L-arginine methyl ester after lipopolysaccharide injection caused significant reductions in macrophage mean nitrite production from 13 and 15 mumol/L to 7 and 11 mumol/L (P < .05) and reduced mean plasma nitrite concentrations from 100 and 118 mumol/L to 46 and 108 mumol/L (P < .05) at 2 and 4 hours, respectively. The rate of survival was significantly decreased to 0% in the group receiving N-nitro-L-arginine methyl ester after septic challenge compared with 87.5% in controls (P < .005). CONCLUSIONS: Inhibition of nitric oxide production is detrimental in this murine model of endotoxemia.

Role of bactericidal permeability-increasing protein in the treatment of gram-negative pneumonia.

BACKGROUND: Gram-negative infections are a major cause of morbidity and death. Bactericidal permeability-increasing protein (BPI) is an endotoxin-neutralizing protein that also exhibits potent bactericidal activity. This study compared the efficacy of a 23 kd recombinant N-terminal fragment of BPI (rBPI23) with that of antiendotoxin antibody E5 in a model of gram-negative sepsis. METHODS: Sixty Swiss-Webster mice (Carworth farm) received an intratracheal inoculation of Escherichia coli (7 x 10(6) colony-forming units) and were randomized to three groups (20 per group). Starting immediately after inoculation, the groups received either rBPI23 (4 mg/kg intravenously every 2 hours for four doses), E5 (11 mg/kg intravenously every 24 hours for two doses), or an isotype control antibody B55 (11 mg/kg intravenously every 24 hours for two doses) and were followed up for survival. In a second survival study, 40 mice received the same intratracheal inoculation of E. coli and were randomized to two groups. Starting 2 hours after inoculation, the groups received either rBPI23 (4 mg/kg intravenously every 2 hours for four doses) or E5 (8 mg/kg intravenously every 12 hours for four doses) and were followed up for survival. In a third study, mice received an intratracheal inoculation of 3 x 10(6) colony-forming units E. coli, a sublethal dose, and were killed to determine pulmonary and blood clearance of bacteria. RESULTS: rBPI23 conferred significantly greater protection from death than either E5 or B55 when started immediately (95% survival vs 20% and 10%, respectively; p < 0.001) or 2 hours after inoculation (65% survival vs 25% for E5; p < 0.05). Both pulmonary and vascular clearance of bacteria was enhanced significantly by treatment with rBPI23. CONCLUSIONS: rBPI23 may be a novel therapeutic agent in the management of gram-negative sepsis.

A broad-spectrum human lung fibroblast-derived mitogen is a variant of hepatocyte growth factor.

A heparin-binding mitogen was isolated from conditioned medium of human embryonic lung fibroblasts. It exhibited broad target-cell specificity whose pattern was distinct from that of any known growth factor. It rapidly stimulated tyrosine phosphorylation of a 145-kDa protein in responsive cells, suggesting that its signaling pathways involved activation of a tyrosine kinase. Purification identified a major polypeptide with an apparent molecular mass of 87 kDa under reducing conditions. Partial amino acid sequence analysis and cDNA cloning revealed that it was a variant of hepatocyte growth factor, a mitogen thought to be specific for hepatic cells and structurally related to plasminogen. Recombinant expression of the cDNA in COS-1 cells established that it encoded the purified growth factor. Its site of synthesis and spectrum of targets imply that this growth factor may play an important role as a paracrine mediator of the proliferation of melanocytes and endothelial cells, as well as cells of epithelial origin.