Elastase-like Activity Research Articles

Effect of Neurotropic and Immunotropic Drugs on Leukocyte Elastase Activity In Vitro.

Leukocyte elastase is a marker of inflammation. Previously, a relationship was found between the severity of mental disorders in patients and elastase-like activity of blood plasma. The effect of various neurotropic drugs on leukocyte elastase activity was analyzed in an in vitro experiment. We revealed an inhibitory effect of the benzodiazepine tranquilizers diazepam and bromodihydrochlorophenylbenzodiazepine and immunomodulators aminodihydrophthalazinedione and diclofenac on the plasma elastase-like activity of healthy donors and pure human neutrophil elastase. The antipsychotics chlorpromazine and alimemazine, as well as the nootropic vinpocetine increased elastase-like activity in a dose-dependent manner. The activating effect of chlorpromazine and vinpocetine, but not alimemazine, was reproduced in neutrophil elastase. We hypothesized that these drugs can affect the development of inflammatory reactions in the complex therapy of mental disorders.

A protease activity-based machine-learning approach as a complementary tool for conventional diagnosis of diarrhea-predominant irritable bowel syndrome.

Irritable bowel syndrome (IBS) has no clinically accepted biomarkers even though it affects a large number of individuals worldwide. To address this lack of understanding, we evaluated peptidase activity in fecal samples from 35 patients with diarrheal IBS without symptom exacerbation (IBS-n) and 35 healthy subjects using a library of 384 fluorescent enzymatic substrate probes. IBS-n patients had high trypsin-like peptidase activity for cleavage of C-terminal lysine and arginine residues and low elastase-like activity for cleavage of C-terminal serine and glycine residues. These fluorescent probe library data, together with diagnostic machine-learning techniques, were able to accurately predict IBS-n. This approach can be used to diagnose diseases where no clinically accepted biomarkers exist, in which fecal enzyme activity is altered and also suggests that the development of new therapies targeting enzyme activities is possible.

A7 OPPORTUNISTIC PATHOGEN MODULATION OF GLUTEN-REACTIVE CD4+ T CELL ACTIVATION BY DQ2-EXPRESSING ORGANOID MONOLAYERS

Abstract Background Bacteria have recently emerged as additional modulators of inflammation in CeD. We have shown that the elastase-like producing opportunistic pathogen, Pseudomonas (P) aeruginosa, partially metabolizes gluten into peptides that translocate the mucosal barrier and retain their immunogenicity. We previously demonstrated that organoid monolayers derived from DR3-DQ2 mice carrying the CeD risk gene HLA-DQ2 express MHC class II (HLA-DQ2) and co-stimulatory molecules under induced inflammatory conditions, priming the monolayers for gluten antigen presentation. Here we investigate the activation of human (h)CD4+ T cell co-cultured with DQ2 monolayers stimulated with gluten pre-digested, or not, by bacterial elastase. Purpose To investigate whether organoid monolayers expressing DQ2 activate T cell differentially in the presence of gluten metabolized by elastase-like producing Pseudomonas aeruginosa. Method Organoid monolayers were derived from the duodenum and proximal jejunum of gluten-sensitized DR3-DQ2 mice, following the gluten sensitization protocol previously described1. Monolayers were then stimulated with IFN-γ for 24h to induce MHC-II and co-stimulatory molecules expression. Splenic T-cell expressing hCD4 from gluten-sensitized DR3-DQ2-hCD4 mice were then co-cultured with monolayers in the presence of deamidated pepsin-trypsin-digested (DAPT)-gluten or Pseudomonas aeruginosa PA14 (WT)-digested DAPT-gluten. As a control, DAPT-gluten was incubated with a P. aeruginosa lasB mutant strain that lacks elastase-like activity (lasB△/△). Co-cultures stimulated with DAPT-gluten alone or WT-media were used as additional controls. Result(s) Increased hCD4+ T-cell proliferation was observed in co-cultures stimulated with WT-digested gluten compared with lasB△/△-digested gluten (p<0.0001), gluten alone (p=0.0002) or WT-media (p<0.0001). hCD4+ T cell co-cultured with organoid monolayers stimulated with WT-digested gluten, had an activated phenotype with increased expression of CD69, CD44 and CD25 versus those stimulated with gluten, lasB△/△-digested gluten, or WT-media. Increased levels of pro-inflammatory and T helper type 1 (Th1)-associated cytokines were detected in the supernatant of the co-cultures stimulated with WT-digested gluten, including IL-2, IFN-γ, IL-6, TNF-α, IL-1α, IL-β, and IL-15. Conclusion(s) Using a novel HLA-DQ2-expressing organoid monolayer, we demonstrate elastase-like producing P. aeruginosa, enhanced activation and proliferation of hCD4+ T cell through gluten metabolism. This in vitro model constitutes a relevant tool for studying microbial triggers and drivers of intestinal epithelial dysfunction in CeD. 1. Galipeau, H. J. et al. 1. Galipeau, H. J. et al. Sensitization to Gliadin Induces Moderate Enteropathy and Insulitis in Nonobese Diabetic-DQ8 Mice. J. Immunol.187, 4338–4346 (2011). Please acknowledge all funding agencies by checking the applicable boxes below CIHR, Other Please indicate your source of funding; Canadian Celiac Disease Association (CCA) Disclosure of Interest None Declared

The Effect of Serine Protease Inhibitors on Visceral Pain in Different Rodent Models With an Intestinal Insult.

Background: Serine proteases are believed to play a key role in the origin of abdominal pain in IBD and IBS. We previously demonstrated a reduction of visceral pain in a post-inflammatory IBS rat model after a single intraperitoneal or intracolonic administration of a serine protease inhibitor. The aim of this study was to investigate the efficacy of serine protease inhibition on visceral pain in two different animal models involving a colonic insult based either on acute inflammation or on neonatal irritation. Moreover, protease profiling was explored in the acute colitis model. Methods: An acute 2,4,6-trinitrobenzenesulphonic acid (TNBS) colitis rat model and a chronic neonatal acetic acid mouse model were used in this study. Visceral sensitivity was quantified by visceromotor responses (VMRs) to colorectal distension, 30 min after intraperitoneal administration of the serine protease inhibitors nafamostat, UAMC-00050 or their vehicles. Colonic samples from acute colitis rats were used to quantify the mRNA expression of a panel of serine proteases and mast cell tryptase by immunohistochemistry. Finally, proteolytic activities in colonic and fecal samples were characterized using fluorogenic substrates. Key Results: We showed a significant and pressure-dependent increase in visceral hypersensitivity in acute colitis and neonatal acetic acid models. UAMC-00050 and nafamostat significantly reduced VMRs in both animal models. In acute colitis rats, the administration of a serine protease inhibitor did not affect the inflammatory parameters. Protease profiling of these acute colitis animals revealed an increased tryptase immunoreactivity and a downregulation of matriptase at the mRNA level after inflammation. The administration of UAMC-00050 resulted in a decreased elastase-like activity in the colon associated with a significantly increased elastase-like activity in fecal samples of acute colitis animals. Conclusion: In conclusion, our results suggest that serine proteases play an important role in visceral hypersensitivity in an acute TNBS colitis model in rats and a neonatal acetic acid model in mice. Moreover, we hypothesize a potential mechanism of action of UAMC-00050 via the alteration of elastase-like proteolytic activity in acute inflammation. Taken together, we provided fundamental evidence for serine protease inhibitors as a promising new therapeutic strategy for abdominal pain in gastrointestinal diseases.

A versatile inhibitor of digestive enzymes in Aedes aegypti larvae selected from a pacifastin (TiPI) phage display library

In Brazil, the major vector of arboviruses is Aedes aegypti, which can transmit several alpha and flaviviruses. In this work, a pacifastin protease inhibitor library was constructed and used to select mutants for Ae. aegypti larvae digestive enzymes. The library contained a total of 3.25 × 105 cfu with random mutations in the reactive site (P2–P2’). The most successfully selected mutant, TiPI6, a versatile inhibitor, was able to inhibit all three Ae. aegypti larvae proteolytic activities, trypsin-like, chymotrypsin-like and elastase-like activities, with IC50 values of 0.212 nM, 0.107 nM and 0.109 nM, respectively. In conclusion, the TiPI mutated phage display library was shown to be a useful tool for the selection of an inhibitor of proteolytic activities combined in a mix. TiPI6 is capable of controlling all three digestive enzyme activities present in the larval midgut extract. To our knowledge, this is the first time that one inhibitor containing a Gln at the P1 position showed inhibitory activity against trypsin, chymotrypsin, and elastase-like activities. TiPI6 can be a candidate for further larvicidal studies.

Local Colonic Administration of a Serine Protease Inhibitor Improves Post-Inflammatory Visceral Hypersensitivity in Rats

Dysregulation of the protease–antiprotease balance in the gastrointestinal tract has been suggested as a mechanism underlying visceral hypersensitivity in conditions such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). We aimed to study the potential therapeutic role of an intracolonically administered serine protease inhibitor for the treatment of abdominal pain in a post-inflammatory rat model for IBS. An enema containing 2,4,6-trinitrobenzene sulfonic acid (TNBS) was used to induce colitis in male Sprague–Dawley rats, whereas controls received a saline solution. Colonoscopies were performed to confirm colitis and follow-up mucosal healing. In the post-inflammatory phase, the serine protease inhibitor UAMC-00050 (0.1–5 mg/kg) or its vehicle alone (5% DMSO in H2O) was administered in the colon. Thirty minutes later, visceral mechanosensitivity to colorectal distensions was quantified by visceromotor responses (VMRs) and local effects on colonic compliance and inflammatory parameters were assessed. Specific proteolytic activities in fecal and colonic samples were measured using fluorogenic substrates. Pharmacokinetic parameters were evaluated using bioanalytical measurements with liquid chromatography–tandem mass spectrometry. Post-inflammatory rats had increased trypsin-like activity in colonic tissue and elevated elastase-like activity in fecal samples compared to controls. Treatment with UAMC-00050 decreased trypsin-like activity in colonic tissue of post-colitis animals. Pharmacokinetic experiments revealed that UAMC-00050 acted locally, being taken up in the bloodstream only minimally after administration. Local administration of UAMC-00050 normalized visceral hypersensitivity. These results support the role of serine proteases in the pathophysiology of visceral pain and the potential of locally administered serine protease inhibitors as clinically relevant therapeutics for the treatment of IBS patients with abdominal pain.

The effect of dry extract of cabbage garden on the local non-specific proteinase-inhibitory potential of the mucous membrane of the stomach on the model of acute gastric ulcer

Peptic ulcer of the stomach and duodenum is a complex gastroenterological disease multifactorial in pathogenesis. One of the key components of non-specific inflammation in peptic ulcer disease is the activation of proteinases in the gastric mucosa and their interaction with proteinase inhibitors, which determines the degree of proteolytic aggression in terms of the development of destructive changes in the tissues that they damage.
 The aim of the work was to study the effect of dry extract of cabbage garden on the components of the proteinase-inhibitor system in experimental gastric ulcer.
 The effect of dry extract of cabbage garden on the local non-specific proteinase-inhibitory potential of the gastric mucosa was studied on the model of acute alcohol-prednisolone ulcers. Dry extract of cabbage garden at a dose of 50 mg/kg and comparison drugs – omeprazole and altan were administered intragastrically once a day for 7 days from the second day of the experiment. On the 8th day, elastase-like, trypsin-like activity and acid-stable inhibitors in the swabs from the stomach and intestines, biopsy samples of the gastric mucosa from the esophagus and fundus, and small intestine were determined.
 It has been established that with an experimental alcohol-prednisolone gastric ulcer, a local non-specific proteinase-inhibitory system is involved in the mechanism of mucosal damage. At the same time, the level of trypsin-like and elastase-like activity of gastric juice increases and the activity of acid-stable inhibitors decreases, which normally suppress the enzymatic activity of proteinases. According to the effect on the non-specific proteinase-inhibitor system, the dry cabbage extract was significantly superior to altan and not inferior to omeprazole. The most pronounced effect of normalizing the activity of nonspecific proteinases and their inhibitors was established using a combination of dry cabbage extract with omeprazole. One of the mechanisms of the antiulcer action of the dry cabbage extract is its effect on the local nonspecific proteinase-inhibitory potential of the gastric mucosa.
 In the model of acute alcohol-prednisolone gastric ulcer, dry extract of cabbage garden caused a decrease in the activity of local non-specific proteolytic enzymes and activation of proteinase inhibitors of the gastric mucosa and duodenum, exceeding the activity of altan. Under the combined use of the dry extract of cabbage and omeprazole, a complete normalization of the state of the local non-specific proteinase-inhibitory potential of the gastroduodenal zone was observed.

Chymotrypsin- and trypsin-like activities secreted by the multidrug-resistant yeasts forming the Candida haemulonii complex.

Candida haemulonii complex (C. haemulonii, C. haemulonii var. vulnera and C. duobushaemulonii) consists of emergent multidrug-resistant pathogens that cause bloodstream and deep-seated infections. However, little is known about their virulence factors. Herein, we evaluated the presence of extracellular serine peptidases in this fungal complex. Serine peptidase activity was measured by spectrophotometry using chromogenic peptide substrates to the S1 family. Chymotrypsin-, trypsin- and elastase-like activities were detected in all fungal isolates. Since higher chymotrypsin- and trypsin-like activities were observed from the cleavage of N-succinyl-Ala-Ala-Pro-Phe-pNa and N-benzoyl-Phe-Val-Arg-pNa, respectively, these substrates were selected for further experiments. Overall, pHs 7.0 and 9.0 were those in which higher chymotrypsin- and trypsin-like activities were observed, respectively, displaying higher hydrolytic activities at 37-45°C. Additionally, the serine peptidases produced by C. haemulonii complex were inhibited by PMSF and AEBSF in a typically concentration-dependent manner. Although the Michaelis constant (Km) values obtained for chymotrypsin-like peptidases were similar, greater differences were observed for trypsin-like enzymes secreted by the different fungal isolates. This is the first time that peptidases belonging to the S1 family are described in the C. haemulonii species complex. Thus, these data open the doors for more detailed studies into potential roles of these peptidases in fungal virulence.

MARKERS OF INFLAMMATION IN DIFFERENT FORMS OF CHRONIC ABACTERIAL PROSTATITIS

Introduction.Chronic prostatitis is the most common and difficult to diagnose androurologic disease. However, the etiology, pathogenesis and pathophysiology of chronic abacterial prostatitis are not well understood.Objective.Analysis of the role of the body’s proteolytic systems in inflammatory processes in the prostate, detection of markers of inflammation in the blood, prostate secretion in various forms of chronic abacterial prostatitis / chronic pelvic pain syndrome (CAP/CPPS).Materials and methods.The study included 52 patients with inflammatory (CAP/CPPS IIIA) and 46 patients with non - inflammatory (CAP/CPPS IIIB) forms of chronic abacterial prostatitis. We determined the activity of kallikrein, level of prekallikrein, inhibitory activity α1-proteinase inhibitor and α2- macroglobulin, total arginine-esterase activity, activity of leukocyte elastase and elastase-like activity in the blood serum and the prostate secret.Results.Disturbance of metabolic processes of inflammation development in chronic abacterial prostatitis occurs against the background of imbalance of the proteinase inhibitors system, uncontrolled enhancement of proteolytic processes in the prostate in conditions of weakening of natural resistance of the organism is the most significant factor in the development of CAP/CPPS.Conclusions.The analysis of the activity of proteolytic processes in the blood and prostate secretion can serve as an additional diagnostic criterion for chronic abacterial prostatitis/chronic pelvic pain syndrome.Disclosure: TInformation about sponsorship. The publication was prepared within the framework of the implementation of the State Task of the SSC RAS for 2018. The state registration of the project № 01201363192. The author declares no conflict of interest.

Preservation with α1-antitrypsin improves primary graft function of murine lung transplants.

BackgroundVascular damage and primary graft dysfunction increase with prolonged preservation times of transplanted donor lungs. Hence, storage and conservation of donated lungs in protein-free, dextran-containing electrolyte solutions, like Perfadex, is limited to about 6 hours. We hypothesized that transplanted lungs are protected against neutrophil-mediated proteolytic damage by adding α1-anti-trypsin (AAT), a highly abundant human plasma proteinase inhibitor, to Perfadex.MethodsA realistic clinically oriented murine model of lung transplantation was used to simulate the ischemia–reperfusion process. Lung grafts were stored at 4°C in Perfadex solution supplemented with AAT or an AAT mutant devoid of elastase-inhibiting activity for 18 hours. We examined wild-type and proteinase 3/neutrophil elastase (PR3/NE) double-deficient mice as graft recipients. Gas exchange function and infiltrating neutrophils of the transplanted lung, as well as protein content and neutrophil numbers in the bronchoalveolar lavage fluid, were determined.ResultsAAT as a supplement to Perfadex reduced the extent of primary graft dysfunction and early neutrophil responses after extended storage for 18 hours at 4°C and 4-hour reperfusion in the recipients. Double-knockout recipients that lack elastase-like activities in neutrophils were also protected from early reperfusion injury, but not lung grafts that were perfused with a reactive center mutant of AAT devoid of elastase-inhibiting activity.ConclusionsPR3 and NE, the principal targets of AAT, are major triggers of post-ischemic reperfusion damage. Their effective inhibition in the graft and recipient is a promising strategy for organ usage after storage for >6 hours.

The potentialities of enotherapy in the correction of experimental hypoxia’s effects

The aim of the work was to study the influence of sparkling wines on the main markers of hypoxia. Material and methods. The experiment was carried out on 25 white male rats weighing 180–200 grams (age 10–12 weeks.). Animals of group 1 were control animals and received drinking water. Animals of the second group received water and were subjected to bloodletting on the 1st week. Animals of the 3rd group were subjected to bloodletting on the 1st week and received diluted sparkling wine as drinking water. Animals of group 4 were not subjected to bloodletting and received diluted sparkling wine as drinking water throughout the experiment. Sparkling wine was given at a dose of 300 ml of wine per 70 kg of body weight. In the blood serum, markers of free radical oxidation and markers of inflammation formed in hypoxic damage were studied. Results: the use of sparkling wines for correction of biochemical disorders in hypoxia led to the strengthening of antioxidant protection systems: the level of superoxide dismutase showed an increase of 77 % (p < 0,05) comparing to the group with simulated hypoxia, the growth of catalase activity by 5 times, the values of trypsin-like activity and elastase-like activity were lower than the hypoxic level by 20 % (p < 0,05) and 59 % (p < 0,01), respectively. Conclusion: the use of sparkling red wine in the correction of free-radical and proteolytic damage, typical for hypoxia, revealed a complex positive effect on a number of important indicators of antioxidant and antiproteolytic protection of the body. This indicates the prospects for further experimental and clinical studies in this direction.

Изменения неспецифических протеиназ и провоспалительных цитокинов при остром инфаркте миокарда различной степени тяжести

to study changes of serum levels of nonspecific proteinases, their inhibitors, and proinflammatory cytokines during short term observation of patients with acute myocardial infarction (MI). We included in this prospective short-term study 82 patients (27 with uncomplicated non-Q wave MI, 30 with Q-MI complicated by Killip class I-II acute left ventricular failure [ALVF], 17 with Q-MI complicated by Killip class III-IV ALVF, and 8 non-survivors due to development of cardiogenic shock) and 12 healthy controls. Serum levels of interleukin (IL) - 1β, IL-6 and tumor necrosis factor (TNF)-α were measured by ELISA. Elastaselike (ELA) and trypsin-like (TLA) activities as well as characteristics of proteinase inhibitors (antitrypsin activity and acid-stable inhibitors) were also determined. Blood samples were taken at hospital admission within 24 hours after onset of symptoms. The GUSTO Score at admission was used for risk stratification. All cytokines levels were significantly elevated in MI patients in comparison to controls. Mean concentrations of IL-6 and TNF-α at baseline were higher among patients with MI complicated by ALVF than in the group with uncomplicated MI (27.45 vs 16.04 pikogram / mL, р.

Changes of nonspecific proteinases and their inhibitors in different clinical types of myocardial infarction

Purpose: to elucidate the relationships between serum levels of nonspecific proteinases and their inhibitors in patients with complications of myocardial infarction (MI). Material and methods. This prospective short-term study included 82 patients with uncomplicated non-Q wave MI (n=27), Killip I-II Q-MI complicated by acute left ventricular failure (ALVF) (n=30), Killip III-IV Q-MI, and Killip III-IV ALVF (n=17) as well as non-survivors due to development of cardiogenic shock (n=8) and healthy controls (n=12). Serum levels of elastase-like (ELA) and trypsine-like (TLA) activities and of proteinase inhibitors (antitrypsin activity and acid-stable inhibitors) was evaluated by enzymatic method within 24 hours, 3 and 14 days after the onset of symptoms. Risk stratification was done at admission according to the GUSTO Score. Results. The levels of all nonspecific proteinases were elevated in MI patients in comparison to controls. Mean baseline ELA (0.29 vs 0.37 nMol/mL min; р&lt;0.05) and TLA (0.33 vs 0.40 mcMol/mL min; р&lt;0.05) were higher in patients with MI complicated by ALVF than in those with uncomplicated MI. Non-survivors showed significantly higher ELA and TLA and lower levels of proteinase inhibitors than patients with uncomplicated MI. Discussion. Proteolytic enzyme activities varied parallel to the changes of proteinase inhibitor levels. Proteinase activities were much more elevated in non-survirvors than in patients with uncomplicated MI. Decreased proteinase inhibitor levels can be considered as a marker of imbalance of proteinase-inhibitor system. Conclusions. Increase of nonspecific proteinase serum levels within 24 hours after the onset of MI is associated with the development of ALVF and unfavorable prognosis; it suggests the involvement of the proteinase-inhibitor system in pathogenesis of MI.

Prolonged pharmacological inhibition of cathepsin C results in elimination of neutrophil serine proteases

Cathepsin C (CatC) is a tetrameric cysteine dipeptidyl aminopeptidase that plays a key role in activation of pro-inflammatory serine protease zymogens by removal of a N-terminal pro-dipeptide sequence. Loss of function mutations in the CatC gene is associated with lack of immune cell serine protease activities and cause Papillon-Lefèvre syndrome (PLS). Also, only very low levels of elastase-like protease zymogens are detected by proteome analysis of neutrophils from PLS patients. Thus, CatC inhibitors represent new alternatives for the treatment of neutrophil protease-driven inflammatory or autoimmune diseases. We aimed to experimentally inactivate and lower neutrophil elastase-like proteases by pharmacological blocking of CatC-dependent maturation in cell-based assays and in vivo. Isolated, immature bone marrow cells from healthy donors pulse-chased in the presence of a new cell permeable cyclopropyl nitrile CatC inhibitor almost totally lack elastase. We confirmed the elimination of neutrophil elastase-like proteases by prolonged inhibition of CatC in a non-human primate. We also showed that neutrophils lacking elastase-like protease activities were still recruited to inflammatory sites. These preclinical results demonstrate that the disappearance of neutrophil elastase-like proteases as observed in PLS patients can be achieved by pharmacological inhibition of bone marrow CatC. Such a transitory inhibition of CatC might thus help to rebalance the protease load during chronic inflammatory diseases, which opens new perspectives for therapeutic applications in humans.

Secretion of Proteases by an Opportunistic Fungal Pathogen Scedosporium aurantiacum.

Scedosporium aurantiacum is an opportunistic filamentous fungus increasingly isolated from the sputum of cystic fibrosis patients, and is especially prevalent in Australia. At the moment, very little is known about the infection mechanism of this fungus. Secreted proteases have been shown to contribute to fungal virulence in several studies with other fungi. Here we have compared the profiles of proteases secreted by a clinical isolate Scedosporium aurantiacum (WM 06.482) and an environmental strain (WM 10.136) grown on a synthetic cystic fibrosis sputum medium supplemented with casein or mucin. Protease activity was assessed using class-specific substrates and inhibitors. Subtilisin-like and trypsin-like serine protease activity was detected in all cultures. The greatest difference in the secretion of proteases between the two strains occurred in mucin-supplemented medium, where the activities of the elastase-like, trypsin-like and aspartic proteases were, overall, 2.5–75 fold higher in the clinical strain compared to the environmental strain. Proteases secreted by the two strains in the mucin-supplemented medium were further analyzed by mass spectrometry. Six homologs of fungal proteases were identified from the clinical strain and five from the environmental strain. Of these, three were common for both strains including a subtilisin peptidase, a putative leucine aminopeptidase and a PA-SaNapH-like protease. Trypsin-like protease was identified by mass spectrometry only in the clinical isolate even though trypsin-like activity was present in all cultures. In contrast, high elastase-like activity was measured in the culture supernatant of the clinical strain but could not be identified by mass spectrometry searching against other fungi in the NCBI database. Future availability of an annotated genome will help finalise identification of the S. aurantiacum proteases.

Changes in non-specific proteases and their inhibitors in the course of acute purulent rhinosinusitis treatment with mucoactive drugs

Objective of the study is to determine the influence of mucoactive drugs of various groups on the activity of non-specific proteases and their inhibitors in the blood serum and sinonasal discharge during treatment of patients with acute purulent rhinosinusitis. Materials and methods. 48 patients with acute purulent rhinosinusitis were treated; they were randomly assigned to four groups: N-acetylcysteine was used in Group 1 (n = 11), S-carboxymethylcysteine – in Group 2 (n = 14), phytoneering herbal drug (BNO-101) Sinupret – in Group 3 (n = 13). The control group consisted of patients (n = 10) who did not receive mucoactive agents during the treatment. Normal values were determined in 12 healthy volunteers. The material for the study included blood serum and sinonasal discharge of the patients. We studied elastase-like activity, trypsin-like activity, antitryptic activity and acid-stable inhibitors. Results. Conducted treatment course resulted in improved clinical symptoms and decreased proteolytic activity in the blood serum and sinonasal discharge of the patients with acute purulent rhinosinusitis as compared to the patients’ parameters before treatment. Against this background, the state of proteinase inhibitors did not significantly change. This is primarily caused by the fact that antitryptic activity in acute purulent rhinosinusitis remained practically unchanged as compared to the parameter at the beginning of treatment. However, there is a tendency towards an increase in the inhibitory potential during treatment with mucoactive drugs. The highest level of this increase was observed when patients were taking BNO-101. On Day 10 of the study it resulted in the significant increase in antitryptic activity(ATA) as compared to the group prior to the treatment. The level of acid-stable inhibitors (ASI) in patients with purulent rhinosinusitis significantly decreased in the settings of acute purulent inflammation, and the activity of ASI increased during the treatment. Conclusion. The study of non-specific proteases and antiprotease potential of sinonasal discharge demonstrates that the decrease in protease activity and normalization of their inhibitors level depends on the mucoactive agent chosen. The most effective treatment option reduces the aggressive effect of non-specific proteases on mucosa and connective tissue structures. The mucoactive drugs’ effect of improved drainage increases antiprotease potential by restoring the structure and functional activity of the mucosa. Restoration of mucosa increases synthesis and secretion of acid-stable protease inhibitors which are the key component of local antiprotease protection.

Fluorescent diphenylphosphonate-based probes for detection of serine protease activity during inflammation

Activity-based probes are small molecules that covalently bind to the active site of a protease in an activity-dependent manner. We synthesized and characterized two fluorescent activity-based probes that target serine proteases with trypsin-like or elastase-like activity. We assessed the selectivity and potency of these probes against recombinant enzymes and demonstrated that while they are efficacious at labeling active proteases in complex protein mixtures in vitro, they are less valuable for in vivo studies. We used these probes to evaluate serine protease activity in two mouse models of acute inflammation, including pancreatitis and colitis. As anticipated, the activity of trypsin-like proteases was increased during pancreatitis. Levels of elastase-like proteases were low in pancreatic lysates and colonic luminal fluids, whether healthy or inflamed. Exogenously added recombinant neutrophil elastase was inhibited upon incubation with these samples, an effect that was augmented in inflamed samples compared to controls. These data suggest that endogenous inhibitors and elastase-degrading proteases are upregulated during inflammation.

Conservative Mechanisms of Extracellular Trap Formation by Annelida Eisenia andrei: Serine Protease Activity Requirement.

Formation of extracellular traps (ETs) capturing and immobilizing pathogens is now a well-established defense mechanism added to the repertoire of vertebrate phagocytes. These ETs are composed of extracellular DNA (extDNA), histones and antimicrobial proteins. Formation of mouse and human ETs depends on enzymes (i) facilitating decondensation of chromatin by citrullination of histones, and (ii) serine proteases degrading histones. In invertebrates, initial reports revealed existence of ETs composed of extDNA and histones, and here we document for the first time that also coelomocytes, immunocompetent cells of an earthworm Eisenia andrei, cast ETs which successfully trap bacteria in a reactive oxygen species (ROS)-dependent and -independent manner. Importantly, the formation of ETs was observed not only when coelomocytes were studied ex vivo, but also in vivo, directly in the earthworm coelom. These ETs were composed of extDNA, heat shock proteins (HSP27) and H3 histones. Furthermore, the formation of E. andrei ETs depended on activity of serine proteases, including elastase-like activity. Moreover, ETs interconnected and hold together aggregating coelomocytes, a processes proceeding encapsulation. In conclusion, the study confirms ET formation by earthworms, and unravels mechanisms leading to ET formation and encapsulation in invertebrates.

Endometrial hyperplasia-related inflammation: its role in the development and progression of endometrial hyperplasia.

Endometrial hyperplasia (EH) is one of the most common gynecologic diseases in the world. Different statistical categories implicate an imbalance of estrogens and progestogens in the etiology of this disease. We propose that inflammation also plays a key role in the progression of endometrial hyperplasia. The aim of this study is to evaluate the role of inflammation in the transformation and progression of endometrial hyperplasia, using local inflammatory cytokines and nonspecific protease levels, CD 45(+) expression, and histological examination. The study included 107 patients (ages 29-49years) with different forms of endometrial hyperplasia. The enrolled patients were randomized into one of the four groups: normal endometrium (n=18) as the control group, simple hyperplasia (n=41), complex hyperplasia without atypia (n=36), complex atypical hyperplasia or endometrioid adenocarcinoma (n=12). The following were evaluated for patients with different forms of EH: steroid hormone levels in blood serum and uterine flushings, immunohistochemical estrogen and progesterone receptor expression patterns in the endometrial tissue, CD 45(+) (common leukocyte antigen) expression, the levels of the cytokines IL-1β, IL-6, and TNF-α, and nonspecific proteases and their inhibitors. The level of estradiol in blood serum and especially in uterine flushings was elevated dramatically in simple EH as compared to that of controls, but there was no significant difference between estradiol levels among the different forms of EH. The estimation of CD 45(+), the levels of the cytokines IL-1β, IL-6, and TNF-α, and the activity of proteases (elastase-like and trypsin-like activities) and their inhibitors showed that levels of nonspecific inflammatory markers increase with EH progression. We suggest that the initial responsibility for the development of simple endometrial hyperplasia belongs to systemic hyperestrogenemia and, in particular, local hyperestrogenia, but that the role of inflammatory processes increases in complex and atypical EH. Development of inflammatory changes in endometrial hyperplasia may be considered as a factor in the promotion and progression of pathology, as well as an attributed risk factor for malignancy in endometrial hyperplasia. In this study, we have established a role for CD 45(+) expression cells, non-specific proteases, and the inflammatory cytokines IL-1β, IL-6, and TNF-α in endometrial hyperplasia-related inflammation.

COMBINED USE OF HAEMOSTATIC SYSTEM INDICES FOR EVALUATION OF UPPER RESPIRATORY TRACT CANCER PROGRESSION

To analyze whether comprehensive assessment of haemostatic system components, in particular, indices of coagulation and fibrinolytic systems along with functionally related proteins, could be indicative of upper respiratory tract (URT) cancer progression. Indices of coagulation and fibrinolytic systems along with functionally related proteins, in particular, trypsin-like amidolytic activity, trypsin-like proteolytic activity, thrombin-like amidolytic activity, elastase-like amidolytic activity, fibrinolytic activity, potential amidolytic plasmin activity, content of fibrinogen, antithrombin III, α1-proteinase inhibitor, and α2-macroglobulin, and prothrombin time were evaluated in blood plasma of patients with URT cancer of II (n = 10) and III (n = 25) stages with the use of routine biochemical methods. For both groups of patients with URT cancer there have been shown notable differences for the majority of the studied indices, especially the indexes of proteolytic activities, from these of healthy donors, and in the case of URT cancer of III stage they reached statistical significance. In contrary, the changes in the content of antithrombin III, α1-proteinase inhibitor, and α2-macroglobulin were insignificant. In both groups of patients significant increase of fibrinogen content has been registered, while the content of soluble fibrinogen didn't change. Also, in both groups of patients there a significant increase of potential activity of plasminogen was documented, while clot lysis time was significantly increased only in patients with III stage URT cancer. Multifactorial analysis of haemostatic system indices evidenced for efficacy of their combined use for evaluation of URT cancer progression risk. Combined use of fibrinogen and α2-macroglobulin content and the level of amidolytic thrombin-like activity could serve as an indicator of URT cancer progression.