alpha-chymotrypsin and Shock--Septic

Uncontrolled proteolysis contributes to cell injury and organ dysfunction in animal models of circulatory shock. We investigated in humans the relationship between septic shock, proteolysis, and outcome.. Intensive care patients with septic shock (n=29) or sepsis (n=6) and non-hospitalised subjects (n=9) were recruited as part of the prospective observational trial 'ShockOmics' (ClinicalTrials.gov Identifier NCT02141607). A mass spectrometry-based approach was used to analyse the plasma peptidomes and the origin of circulating peptides from proteolysis in the enrolled subjects.. Evidence of systemic proteolysis was indicated by a larger number of circulating peptides in septic shock patients, compared with septic patients and non-hospitalised healthy subjects. The peptide count and abundance in the septic shock patients were greater in patients who died (n=6) than in survivors (n=23), suggesting an association between magnitude of proteolysis and outcome. In silico analysis of the peptide sequences and of the sites of cleavage on the proteins of origin indicated a predominant role for serine proteases, such as chymotrypsin, and matrix metalloproteases in causing the observed proteolytic degradation.. Systemic proteolysis is a novel fundamental pathological mechanism in septic shock. Plasma peptidomics is proposed as a new tool to monitor clinical trajectory in septic shock patients.. NCT02141607.

Topics: Adult; Aged; Aged, 80 and over; Chymotrypsin; Computer Simulation; Critical Care; Female; Hospital Mortality; Humans; Male; Matrix Metalloproteinases; Middle Aged; Peptides; Prospective Studies; Proteolysis; Sepsis; Shock, Septic; Survival Analysis; Treatment Outcome; Young Adult

Lipopolysaccharide (LPS) is a major structural component of all Gram-negative organisms and has been implicated in Gram-negative sepsis and septic shock. In the present study, Affymetrix microarray analysis of RNA derived from murine macrophages treated with LPS in the absence or presence of the proteasome inhibitor lactacystin revealed that the vast majority of genes regulated by LPS is under control of the proteasome. Analysis of the data has revealed that the products of these genes participate in 14 distinct signaling pathways. This represents a novel approach to the identification of signaling pathways that are both toll-like receptor 4- and proteasome-dependent and may lead to the development of new drug targets in Gram-negative sepsis and septic shock.

Topics: Acetylcysteine; Animals; Cell Survival; Chymotrypsin; Gene Expression Regulation, Enzymologic; Inflammation; Lipopolysaccharides; Macrophages; Mice; Oligonucleotide Array Sequence Analysis; Proteasome Endopeptidase Complex; Shock, Septic; Signal Transduction; Toll-Like Receptor 4

We have developed a novel LPS probe using a highly purified and homogenous preparation of [(3)H] Escherichia coli LPS from the deep rough mutant, which contains a covalently linked, photoactivable 4-p-(azidosalicylamido)-butylamine group. This cross-linker was used to identify the LPS-binding proteins in membranes of the murine-macrophage-like cell line RAW 264.7. The alpha-subunit (PSMA1 C2, 29.5 kDa) and the beta-subunit (PSMB4 N3, 24.36 kDa) of the 20S proteasome complex were identified as LPS-binding proteins. This is the first report demonstrating LPS binding to enzymes such as the proteasome subunits. Functionally, LPS enhanced the chymotrypsin-like activity of the proteasome to degrade synthetic peptides in vitro and, conversely, the proteasome inhibitor lactacystin completely blocked the LPS-induced proteasome's chymotrypsin activity as well as macrophage TNF-alpha secretion and the expression of multiple inflammatory mediator genes. Lactacystin also completely blocked the LPS-induced expression of Toll-like receptor 2 mRNA. In addition, lactacystin dysregulated mitogen-activated protein kinase phosphorylation in LPS-stimulated macrophages, but failed to inhibit IL-1 receptor-associated kinase-1 activity. Importantly, lactacystin also prevented LPS-induced shock in mice. These data strongly suggest that the proteasome complex regulates the LPS-induced signal transduction and that it may be an important therapeutic target in Gram-negative sepsis.

Topics: Acetylcysteine; Acute-Phase Proteins; Animals; Carrier Proteins; Cell Line; Chymotrypsin; Cross-Linking Reagents; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Down-Regulation; Enzyme Activation; Escherichia coli; Glutamate Synthase; Leupeptins; Lipopolysaccharides; Macrophages; Macrophages, Peritoneal; Membrane Glycoproteins; Membrane Proteins; Methanosarcina; Mice; Mice, Inbred C3H; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Multienzyme Complexes; Phosphorylation; Proteasome Endopeptidase Complex; Shock, Septic; Signal Transduction; Tritium

To determine the exocrine pancreatic function in critically ill patients.. Prospective cohort study.. Medical intensive care unit.. A total of 18 critically ill patients (11 patients with septic shock according to the criteria of the American College of Chest Physicians/Society of Critical Care Medicine, 7 nonseptic patients). The results obtained in these subjects were compared with the data obtained in seven healthy volunteers.. Examination of exocrine pancreatic function was done by means of a cholecystokinin-secretin test. Intravenous stimulation of the exocrine pancreas with cholecystokinin and secretin (1 unit/kg body weight/hr each) and aspiration of duodenal fluid by a gastroscopically inserted oroduodenal tube was done during a period of 1 hr.. The content of amylase, chymotrypsin, and trypsin in aspirated duodenal fluid was significantly reduced in patients with septic shock compared with nonseptic patients as well as healthy subjects (p < .01). The volume of aspirated fluid was significantly reduced in patients with septic shock compared with healthy controls (p = .03), but not in nonseptic patients. The content of bicarbonate was not statistically different in the three groups. No significant correlation was to find between variables of exocrine pancreatic function and Acute Physiology and Chronic Health Evaluation III score, sepsis-related organ failure assessment score, systolic arterial pressure and mean arterial pressure in septic shock patients. Positive end-expiratory pressure was significantly correlated with the content of trypsin (r2 = 0.52; p = .02). Postmortem examinations of five septic patients who died during the intensive care stay did not reveal gross morphologic alterations of pancreatic tissue.. The study shows two pancreatic enzyme systems, namely, amylase as a carbohydrate splitting enzyme and the proteolytic enzymes trypsin and chymotrypsin, strongly affected in critically ill patients with septic shock.

Topics: Aged; Amylases; Cholecystokinin; Chymotrypsin; Cohort Studies; Critical Care; Exocrine Pancreatic Insufficiency; Female; Humans; Male; Middle Aged; Pancreas; Pancreatic Function Tests; Prospective Studies; Secretin; Shock, Septic; Trypsin