alpha-chymotrypsin and Sepsis

Inflammation and oxidative stress play a major role in the development of sepsis and its associated complications, leading to multiple organ failure and death. The lungs, liver, and kidneys are among the early affected organs correlated with mortality in sepsis. Alpha-chymotrypsin (α-ch) is a serine protease that exerts anti-inflammatory, anti-edematous, and anti-oxidant properties.. This study was undertaken to elucidate if the anti-inflammatory and anti-oxidant effects of α-ch observed in previous studies can alleviate lung, liver, and kidney injuries in a cecal ligation and puncture (CLP)-induced sepsis model, and thus decrease mortality.. Septic animals were given α-ch 2 h post CLP procedure. Sepsis outcomes were assessed in the lungs, liver, and kidneys. Separate animal groups were investigated for a survival study.. CLP resulted in 0% survival, while α-chymotrypsin post-treatment led to 50% survival at the end of the study. Administration of α-chymotrypsin resulted in a significant attenuation of sepsis-induced elevated malonaldehyde (MDA) and total nitrite/nitrate (NOx) levels. In addition, there was a significant increase in reduced glutathione (GSH) content and superoxide dismutase (SOD) activity in the lungs, liver, and kidneys. Administration of α-ch reduced elevated tissue expression of toll-like receptor-4 (TLR4), nuclear factor kappa-B (NF-κB), myeloperoxidase (MPO), and inducible nitric oxide synthase (iNOS). Alpha-chymotrypsin resulted in a significant reduction in serum levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6). Alpha-chymotrypsin attenuated the rise in serum creatinine, cystatin C, blood urea nitrogen (BUN), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels that was observed in the septic group. In addition, α-ch significantly reduced the lung wet/dry weight ratio, total protein content, and leukocytic counts in bronchoalveolar lavage fluid (BALF). Histopathological examination of the lungs, liver, and kidneys confirmed the protective effects of α-ch on those organs.. α-ch has protective potential against sepsis through lowering tissue expression of TLR4, NF-κB, MPO, and iNOS leading to decreased oxidative stress and inflammatory signals induced by sepsis. This effect appeared to alleviate the damage to the lungs, liver, and kidneys and increase survival in rats subjected to sepsis.

Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Chymotrypsin; Inflammation; Kidney; Liver; Lung; NF-kappa B; Pneumonia; Punctures; Rats; Sepsis; Toll-Like Receptor 4

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

Although the proteasome inhibitor known as bortezomib can modulate the inflammatory process through the nuclear factor-kappa B signaling pathway, the immunomodulatory effect of pre-incubated bortezomib has not been fully evaluated for inflammation by infectious agents. Therefore, we evaluated the effect of bortezomib on the expression of inflammatory cytokines and mediators in macrophage cell lines and on survival in a murine peritonitis sepsis model.. Bortezomib was applied 1 hr before lipopolysaccharide (LPS) stimulation in RAW 264.7 cells. The cecal ligation and puncture (CLP) experiments were performed in C57BL/6J mice.. Pre-incubation with bortezomib (25 nM or 50 nM) prior to LPS (50 ng/mL or 100 ng/mL) stimulation significantly recovered the number of viable RAW 264.7 cells compared to those samples without pre-incubation. Bortezomib decreased various inflammatory cytokines as well as nitric oxide production in LPS-stimulated cells. The 7-day survival rate in mice that had received bortezomib at 0.01 mg/kg concentration 1 hr prior to CLP was significantly higher than in the mice that had only received a normal saline solution of 1 mL 1 hr prior to CLP. In addition, the administration of bortezomib at 0.01 mg/kg concentration 1 hr before CLP resulted in a significant decrease in inflammation of the lung parenchyma. Collectively, pretreatment with bortezomib showed an increase in the survival rate and changes in the levels of inflammatory mediators.. These results support the possibility of pretreatment with bortezomib as a new therapeutic target for the treatment of overwhelming inflammation, which is a characteristic of severe sepsis.

Topics: Animals; Boronic Acids; Bortezomib; Cecum; Cell Adhesion Molecules; Cell Line; Cell Proliferation; Cell Survival; Chymotrypsin; Cytokines; Disease Models, Animal; Inflammation Mediators; Ligation; Lipopolysaccharides; Lung; Male; Mice, Inbred C57BL; Nitric Oxide; Proteasome Inhibitors; Punctures; Pyrazines; Sepsis

We studied the role of the ubiquitin-proteasome system in rat skeletal muscle during sepsis and subsequent recovery. Sepsis was induced with intraperitoneal zymosan injections. This model allows one to study a sustained and reversible catabolic phase and mimics the events that prevail in septic and subsequently recovering patients. In addition, the role of the ubiquitin-proteasome system during muscle recovery is poorly documented. There was a trend for increased ubiquitin-conjugate formation in the muscle wasting phase, which was abolished during the recovery phase. The trypsin- and chymotrypsin-like peptidase activities of the 20S proteasome peaked at day 6 following zymosan injection (i.e. when both muscle mass and muscle fiber cross-sectional area were reduced the most), but remained elevated when muscle mass and muscle fiber cross-sectional area were recovering (11 days). This clearly suggests a role for the ubiquitin-proteasome pathway in the muscle remodeling and/or recovery process. Protein levels of 19S complex and 20S proteasome subunits did not increase throughout the study, pointing to alternative mechanisms regulating proteasome activities. Overall these data support a role for ubiquitin-proteasome dependent proteolysis in the zymosan septic model, in both the catabolic and muscle recovery phases.

Topics: Animals; Body Weight; Chymotrypsin; Eating; Male; Muscle, Skeletal; Proteasome Endopeptidase Complex; Protein Subunits; Rats; Rats, Wistar; Sepsis; Ubiquitin; Zymosan

In previous studies, dantrolene prevented sepsis-induced muscle proteolysis, but the influence of dantrolene on the expression and activity of the ubiquitin-proteasome proteolytic pathway is not known. In addition, the role of glucocorticoids in the anabolic effect of dantrolene is not well understood.. Sepsis was induced in rats by cecal ligation and puncture (CLP). Other rats underwent sham-operation. Groups of rats were treated with dantrolene (10 mg/kg) administered 2 h before and 8 h after sham-operation or CLP. Sixteen hours after sham-operation or CLP, mRNA levels for ubiquitin, the ubiquitin ligase E3alpha, and the 20S proteasome subunit C3 were determined by dot-blot analysis. In additional experiments, the effect of dantrolene on protein degradation was tested in L6 myotubes cultured in the absence or presence of dexamethasone.. Treatment of rats with dantrolene prevented the sepsis-induced increase in mRNA levels for ubiquitin, the ubiquitin ligase E3alpha, and the 20S proteasome subunit C3. In additional experiments, treatment of cultured myotubes with dantrolene reduced protein degradation in both the absence and the presence of dexamethasone.. The results suggest that dantrolene can prevent sepsis-induced activation of the ubiquitin-proteasome proteolytic pathway in skeletal muscle and that dantrolene reduces muscle protein degradation independent of glucocorticoids.

Topics: Animals; Calcium; Cecum; Chymotrypsin; Corticosterone; Cysteine Endopeptidases; Dantrolene; Gene Expression Regulation; Glucocorticoids; Ligases; Ligation; Male; Multienzyme Complexes; Muscle Proteins; Muscle Relaxants, Central; Muscle, Skeletal; Proteasome Endopeptidase Complex; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sepsis; Ubiquitin; Ubiquitin-Protein Ligases

Kallistatin, a human serine proteinase inhibitor, is a newly identified tissue kallikrein inhibitor. It binds strongly to tissue kallikrein but weakly to other serine proteinases such as chymotrypsin and elastase. The tissue distribution and changes in kallistatin levels in human diseases were characterized by using specific monoclonal and polyclonal antibodies against kallistatin. Kallistatin antigen levels in blood cells, fluids, and tissues measured with a specific enzyme-linked immunosorbent assay showed displacement curves that were parallel with those in purified kallistatin, indicating their immunologic identity. Expression of kallistatin mRNA in platelets, neutrophils, lymphocytes, monocytes, endothelial cells, hepatocytes, and colon and prostate carcinoma cells was identified by reverse transcription-polymerase chain reaction followed by Southern blot analysis. Plasma kallistatin concentration was 22.1 +/- 3.5 micrograms/ml in 30 normal subjects and 21.1 +/- 3.8 micrograms/ml in 5 patients with C1 inhibitor deficiency. A significantly reduced kallistatin level (7.2 +/- 2.5 micrograms/ml, p < 0.001) was seen in plasma samples from 9 patients with liver disease and 10 patients with sepsis (7.7 +/- 3.5 micrograms/ml, p < 0 .001). Further, kallistatin levels in 10 women taking oral contraceptives (19.8 +/- 3.8 micrograms/ml) and 21 pregnant women (14.9 +/- 3.3 microg/ml) were significantly lower than those seen in healthy individuals. These data suggest that kallistatin is found in plasma, is produced mostly in the liver, and can be consumed during sepsis. Its consumption in sepsis may indicate a protective role to prevent blood pressure lowering.

Topics: Antibodies, Monoclonal; Base Sequence; Blood Cells; Blotting, Western; Body Fluids; Carrier Proteins; Chymotrypsin; Enzyme-Linked Immunosorbent Assay; Female; Humans; Kallikreins; Liver Diseases; Male; Molecular Sequence Data; Pancreatic Elastase; Pregnancy; Sepsis; Serpins; Tissue Distribution

Catheter-related infections pose a hazard to both humans and laboratory animals. The aim of this study was to develop a technique preventing bacterial colonization of intravascular catheters. In 27 dogs a total of 70 catheters were implanted. On an average catheters were used for 207 days. Three protocols were compared: (1) flushing the catheters with a heparinized solution; (2) filling only the catheter lumen with alpha-chymotrypsin solution (225 units/ml); (3) filling only the catheter lumen with a solution containing a mixture of the aminoglycoside antibiotic gentamicin (20 mg/ml) and chymotrypsin (225 units/ml). Catheter fillings were always withdrawn before catheter use. Catheter exit sites were all treated with povidone iodine ointment once a day. Body temperatures and weights were recorded, bacteriological and electron microscopical examinations of catheters performed. Without gentamicin filling all catheters were colonized after a few weeks. The dogs showed clinical signs of chronic bacteraemia. Gentamicin filling eradicated colonization. No further bacteraemia was observed. We conclude that filling only the catheter lumen with a concentrated solution of chymotrypsin and gentamicin, combined with measures to prevent infections via the subcutaneous catheter tunnel, is an effective and safe technique to prevent catheter-related infections.

Topics: Animals; Bacteria; Body Temperature; Catheters, Indwelling; Chymotrypsin; Dogs; Female; Gentamicins; Heparin; Sepsis

The levels of 12 serum proteins including 'acute-phase reactants', immunoglobulins and albumin were measured in 20 patients suffering from thermal burns. The acute-phase reactants: C-reactive protein, alpha-l antitrypsin, alpha-l antichymotrypsin, haptoglobin and orosomucoid, all increased in concentration. Highest levels, which showed significant correlations with injury severity, occurred at 6-8 days post-burn. The levels of albumin, alpha-l lipoprotein and transferrin were decreased. The immunoglobulins IgG, IgA and IgM showed an initial decrease followed by a steady return to normal levels. Four patients, of whom three died, developed serious sepsis. The levels of alpha-l antichymotrypsin and C-reactive protein were much higher in patients with sepsis than in those without sepsis. The highest levels occurred during and often before the episode of sepsis was clinically evident. The immunoglobulins especially IgG and IgA were lower in those patients who developed sepsis than in those who did not. The results suggest that the serum levels of either C-reactive protein or alpha-l antichymotrypsin could be used both as an aid to diagnosis of sepsis and also to monitor the effect of therapy.

Topics: Adult; Aged; alpha 1-Antichymotrypsin; alpha 1-Antitrypsin; Blood Proteins; Burns; C-Reactive Protein; Chymotrypsin; Female; Haptoglobins; Humans; Immunoglobulins; Lipoproteins, HDL; Male; Middle Aged; Orosomucoid; Sepsis; Serum Albumin; Transferrin

Serum lactoferrin concentrations were elevated in almost all children with meningococcal septicemia, in whom the disease had been clinically apparent for less than 18 hours, while the concentrations were normal or only moderately elevated in patients who had had the disease longer before being admitted. Concentrations of C-reactive protein (CRP) were markedly elevated, even with a time lapse of less than six hours, making this the most suitable parameter for the early diagnosis of severe meningococcal infection. Following an operative injury on children the lactoferrin concentrations changed very little. More than six hours after an operation, however, a marked increase in CRP-values was observed, possibly indicating differentiation of this response from that of bacterial infection. The concomitant study of serum alpha 1-antitrypsin, alpha 1-antichymotrypsin, orosomucoid and haptoglobin did not uncover results of great significance with regard to early changes.

Topics: Acute Disease; alpha 1-Antichymotrypsin; alpha 1-Antitrypsin; C-Reactive Protein; Chymotrypsin; Haptoglobins; Humans; Infant, Newborn; Lactoferrin; Lactoglobulins; Meningococcal Infections; Orosomucoid; Sepsis; Time Factors

To show whether direct proteolysis of coagulation factors may play a role in patients with so-called consumption coagulopathy, granulocytic neutral proteases in the plasma of patients with acute myelocytic leukemia and septicemia were assayed by one- and two-dimensional Laurell electrophoresis. Complexes between serum alpha1-antitrypsin and elastase-like granulocytic protease could be demonstrated in those patients with acute myelocytic leukemia and septicemia who also had moderate or severe coagulation defects. Despite the presence of a high antiprotease potential, addition of the elastase-like enzyme to normal plasma resulted in coagulation defects in vitro comparable to those seen in the patients. These results and the ability of the elastase-like protease to destroy isolated clotting factors suggested that in certain types of coagulation factor deficiencies direct proteolysis rather than consumption of clotting factors due to disseminated intravascular coagulation may be operational.

Topics: alpha 1-Antitrypsin; Blood Coagulation Disorders; Chymotrypsin; Electrophoresis, Agar Gel; Factor XIII; Fibrin Fibrinogen Degradation Products; Granulocytes; Humans; Immunoelectrophoresis, Two-Dimensional; Leukemia, Myeloid; Leukocytes; Pancreatic Elastase; Peptide Hydrolases; Sepsis

Topics: Acute Disease; Blood Coagulation Disorders; Chymotrypsin; Granulocytes; Humans; Leukemia; Leukocytes; Pancreatic Elastase; Peptide Hydrolases; Sepsis