Polymorphonuclear Granulocyte Elastase Research Articles

Exudate collection using wound sponges-An easy, non-invasive and reliable method to explore protease activities in ulcers.

Proteases are important for wound healing, but in excessive amounts or left uncontrolled, they may cause healing impairment or other severe wound complications. Point-of-care testing for protease activities in wounds may be useful for monitoring the effectiveness of treatment, and for early identification of wounds that potentially fail to heal. Here we describe an easy, noninvasive method to collect wound fluid for evaluating the protease milieu of wounds. Wound fluids were collected using sterile sponges applied between wound surface and normal wound dressing. Wound fluid could be easily squeezed or centrifuged out of the sponges and was tested for gelatinase (MMP-2 and MMP-9) activities by gel zymography. In addition, we measured polymorphonuclear granulocyte elastase levels by ELISA. Both gelatinases were remarkably stable in sponge derived fluids, as no significant loss was observed even when samples were stored for 3 days at room temperature. Protease levels were highly diverse amongst patients and, in some cases, showed substantial variations in the course of the treatment. The here described wound sponge approach represents a patient-friendly and reliable method to collect wound fluid for evaluating wound healing relevant biomarkers, such as matrix metalloproteinases.

Human semen hyperviscosity: prevalence and effects on physical and biochemical semen parameters in subfertile Egyptian men

Background Semen hyperviscosity (SHV) is a condition that can markedly impair the physical and chemical characteristics of seminal fluid, which leads to an adverse impact on sperm function. The aim of this study was to assess the prevalence of SHV in infertile Egyptian men and to evaluate its effects on physical and biochemical semen parameters, and also to identify any correlation between SHV and infections or inflammation of the genital tract. Participants and methods Semen samples were studied for 300 infertile men; of these, 225 men had normal semen viscosity and were excluded from the study. Seventy-five men with SHV were selected and included in this study. In addition, 25 fertile men with completely normal semen parameters who had recently fathered children were included as a control group. All participants underwent seminal analysis and measurement of seminal plasma fructose, ascorbic acid, calcium, and zinc, in addition to a peroxidase test and polymorphonuclear granulocyte elastase (PMN elastase). Results Our study showed that the prevalence of SHV was 25% in infertile Egyptian men. Sperm motility and sperm vitality, in addition to fructose, ascorbic acid, calcium, and zinc levels, were significantly reduced, whereas PMN elastase levels were significantly increased in samples with hyperviscosity (P Conclusion Our results showed that hyperviscosity seems to be the result of infection or inflammation in 75% in our cases and hyperviscosity seems not to be because of a single pathogenic factor, but rather because of several (biochemical, enzymatic, and genetic) factors that act in synergy. These factors should be studied further.

Urogenital inflammation: changes of leucocytes and ROS

The presence of excess leucocytes in the semen has been associated with male infertility. According to the WHO, concentrations of more than 106 leucocytes ml-1 are considered as leucocytospermia, indicating genital tract infections. Up to now, no consensus has been achieved on how leucocytes should be quantified in semen. Using the peroxidase staining and monoclonal antibodies to CD15, CD45 and CD68, we found significant differences between the detection methods. Only 47.4% of the semen samples that were assessed as leucocytospermic by CD45 were identified as such by peroxidase staining. The concentration of peroxidase-positive cells was significantly correlated with polymorphonuclear granulocyte (PMN) elastase (P < 0.0001). However, a negative correlation of peroxidase-positive cells with the sperm concentration was only found in oligozoospermic patients (P < 0.0001). Moreover, the slightly positive correlation with normal sperm morphology seems to be applicable only in cases of oligozoospermia. Significant negative correlation of the number of peroxidase-positive cells were found for both maximal inducible acrosome reaction (P = 0.0219) and the inducibility of acrosome reaction (P = 0.0370), indicating a rather deleterious effect of leucocytes on this important sperm function. Concerning the result in the in vitro fertilization programme, none of the examined parameters (PMN elastase, concentration of round cells and peroxidase-positive cells) showed a correlation with either fertilization or pregnancy. This result seems to be reasonable as severely damaged spermatozoa and leucocytes are eliminated from the ejaculate by different sperm separation methods. Interestingly, a significant negative correlation of the TUNEL assay as a measure of sperm DNA fragmentation was found only with pregnancy (P = 0.006) but not with fertilization. As DNA fragmentation can also be caused by ROS that are generated by leucocytes, this causality should not be neglected.

Urogenital inflammation: changes of leucocytes and ROS

Summary. The presence of excess leucocytes in the semen has been associated with male infertility. According to the WHO, concentrations of more than 106 leucocytes ml−1 are considered as leucocytospermia, indicating genital tract infections. Up to now, no consensus has been achieved on how leucocytes should be quantified in semen. Using the peroxidase staining and monoclonal antibodies to CD15, CD45 and CD68, we found significant differences between the detection methods. Only 47.4% of the semen samples that were assessed as leucocytospermic by CD45 were identified as such by peroxidase staining. The concentration of peroxidase-positive cells was significantly correlated with polymorphonuclear granulocyte (PMN) elastase (P < 0.0001). However, a negative correlation of peroxidase-positive cells with the sperm concentration was only found in oligozoospermic patients (P < 0.0001). Moreover, the slightly positive correlation with normal sperm morphology seems to be applicable only in cases of oligozoospermia. Significant negative correlation of the number of peroxidase-positive cells were found for both maximal inducible acrosome reaction (P = 0.0219) and the inducibility of acrosome reaction (P = 0.0370), indicating a rather deleterious effect of leucocytes on this important sperm function. Concerning the result in the in vitro fertilization programme, none of the examined parameters (PMN elastase, concentration of round cells and peroxidase-positive cells) showed a correlation with either fertilization or pregnancy. This result seems to be reasonable as severely damaged spermatozoa and leucocytes are eliminated from the ejaculate by different sperm separation methods. Interestingly, a significant negative correlation of the TUNEL assay as a measure of sperm DNA fragmentation was found only with pregnancy (P = 0.006) but not with fertilization. As DNA fragmentation can also be caused by ROS that are generated by leucocytes, this causality should not be neglected.

Relationship between semen quality and the seminal plasma components carnitine, alpha-glucosidase, fructose, citrate and granulocyte elastase in infertile men compared with a normal population.

The seminal plasma components neutral alpha-glucosidase, carnitine, fructose, citrate, and polymorphonuclear granulocyte (PMN) elastase in 253 men were determined. The seminal plasma of 221 infertile men, a control group with proved fertility and 13 patients after vasectomy were investigated. The concentrations of free carnitine (212 versus 521 micromol/l, n = 219, P < 0.001), total carnitine (437 versus 743 micromol/l, n = 219, P < 0.001), and the activity of neutral alpha-glucosidase (15.1 versus 23.4 IU/l, n = 236, P < 0.05) were significantly reduced in the infertile patient group as compared to the fertile control group, the concentration of PMN elastase (102 versus 48 microg/l, n = 234, P < 0.05) being significantly increased in the infertile patients. In the patients after vasectomy the activity of neutral alpha-glucosidase was the only epididymal marker that was significantly reduced (4.3 versus 9. 8 IU/l, n = 35, P = 0.002) in comparison with the patients with testicular azoospermia. At a limit of 6 IU/l the sensitivity of the method was 92% and the specificity was 72%. Altogether, the epididymal markers were reduced in subfertile patients compared with the control group. For the differential diagnosis of azoospermia only the determination of the neutral alpha-glucosidase activity is useful.

Application of argatroban, direct thrombin inhibitor, in heparin-intolerant patients requiring extracorporeal circulation.

This study describes the present knowledge regarding the clinical application of argatroban, a direct competitive thrombin inhibitor for heparin-intolerant patients, including those with congenital and acquired antithrombin III deficiencies, those with heparin-induced thrombocytopenia, and those with high levels of polymorphonuclear granulocyte elastase. These patients are often associated with intracircuit clot formation with heparin anticoagulation during extracorporeal circulation. Therefore, argatroban may be chosen as one of the alternate anticoagulants. Because the anticoagulant effect of argatroban is reflected in the prolongation of activated thromboplastin time, monitoring is easy, similar to that for heparin. Because argatroban has a fast acting anticoagulant effect without any cofactors such as antithrombin III, this drug is a favorable anticoagulant for heparin-intolerant patients with antithrombin III deficiencies requiring extracorporeal circulation. In adverse reactions to heparin, heparin acts as an antigen after complexing with platelet factor 4, which leads to life-threatening heparin-induced thrombocytopenia. As argatroban prevents heparin-induced platelet aggregation, it is effective for use as a therapeutic anticoagulant. In other clinical applications, heparin decreases antithrombin activity and causes intracircuit clot formation during extracorporeal circulation when the polymorphonuclear granulocyte elastase level is very high. The antithrombin activity shows less decrease when argatroban is substituted for heparin. These findings indicate that argatroban is a useful alternative anticoagulant in these heparin-intolerant patients.

PERIOPERATIVE PATTERN OF PERITONEAL INTERLEUKIN 8, TUMOUR NECROSIS FACTOR-α, AND GRANULOCYTE ELASTASE RELEASE IN HUMAN SECONDARY PERITONITIS

Cytokine levels during infection and sepsis have been extensively studied in the past. In contrast to the excellent data on tumour necrosis factor α (TNF-α), interleukin 8 (IL-8), and polymorphonuclear (PMN) granulocyte elastase (PMN-E) concentrations in blood, little is known about cytokine and PMN-E levels in tissue or local fluids like abdominal exudate in secondary, purulent peritonitis of man. Therefore, the authors studied perioperative intra-abdominal levels of TNF-α, IL-8 and PMN-E in 21 patients with severe purulent peritonitis. The average pre-operative levels of TNF-α were 694+239 pg/ml in exudate and 26±6 pg/ml in plasma, for IL-8 100±34 ng/ml and 0.7±0.5 ng/ml, and for PMN-E 68±14 μg/ml and 0.7±0.1 μg/ml, respectively. Standard surgical procedures reduced the intra-abdominal concentrations of cytokines and PMN-E to as low as one tenth of the pre-operative levels. Postoperatively, TNF-α and IL-8 levels recovered rapidly and pre-operative levels of IL-8 were reached again after 1 h and for TNF-α after 8 h. PMN-E concentration remained below the initial baseline within 8 h of observation. TNF-α concentration, but not IL-8 or PMN-E, depended on the microbiological load of the abdominal exudate (< or >103cfu/ml). There were no significant differences in the intra-abdominal or plasma levels of cytokines or PMN-E between survivors and non-survivors at any observation time.

The Effect of Ulinastatin, a Human Protease Inhibitor, on the Transfusion-Induced Increase of Plasma Polymorphonuclear Granulocyte Elastase

The Effect of Ulinastatin, a Human Protease Inhibitor, on the Transfusion-Induced Increase of Plasma Polymorphonuclear Granulocyte Elastase

Comparison of serum phospholipase A2, polymorphonuclear granulocyte elastase, C-reactive protein and serum amyloid A with the APACHE II score in the prognosis of multiple injured patients

This prospective study of 35 multitraumatized intensive care unit patients requiring mechanical ventilation examined the relative utility of four biochemical parameters with a physiological scoring system for predicting lethal outcome. Levels of serum phospholipase A2 (PLA2), serum amyloid A (SAA), polymorphonuclear granulocyte elastase (PMN elastase), and C-reactive protein (CRP) were determined at short intervals during the patient's hospitalization. The first specimen was obtained at the time of admission, and subsequent specimens were drawn at 8 h intervals for the first 48 h and then twice daily until death or convalescence. Calculations of the APACHE II score used the most deranged variables during the first 24 h of admission to assess patient outcome. Additional calculations of the APACHE II score at the time of each blood draw served as an indicator of patient status. The results indicate that during the first 24 h after admission none of the four examined biochemical parameters gives reliable information about the outcome. The APACHE II score provided the earliest indicator of patient outcome (83% sensitivity, 65% specificity). PMN elastase provided useful information first at 32 h (83% sensitivity, 45% specificity) and better at 132 h (86% sensitivity, 86% specificity). CRP was of intermediate use in predicting outcome initially at 72 h (83% sensitivity, 50% specificity) and later at 132 h (86% sensitivity, 93% specificity). PLA2 and SAA were not useful as early indicators of lethal outcome.

Inflammatory response in the early prediction of severity in human acute pancreatitis.

The role of the inflammatory response in acute pancreatitis and its relation with the clinical course was examined. This study examined if the serial measurement of polymorphonuclear granulocyte (PMN) elastase/A1PI complex, phospholipase A catalytic activity, C reactive protein, and other acute phase proteins, and the protease inhibitor alpha 2-macroglobulin, provides meaningful information for prognosis. Eighty non-consecutive patients with acute pancreatitis, classified according to their clinical outcome into mild (n = 40) and severe pancreatitis (n = 40), were followed up daily. Between 48 hours, median values of PMN-elastase, C reactive protein--and most of the acute phase proteins--and phospholipase A activity, were significantly higher in the severe pancreatitis group. PMN elastase shows a dynamic course and it reaches an early peak value at days 1-2, followed by C reactive protein (days 2-4) phospholipase A (day 3), and a negative peak for alpha 2-macroglobulin (days 4-5). PMN elastase (day 1) and C reactive protein (day 2) were selected by discriminant analysis as the most useful variables studied to allow the early accurate prediction of severity (sensitivity 100%, specificity 95%). Little or no predictive additional value was found for all other variables studied. These results strongly suggest a close relation between inflammatory parameters and clinical course in acute pancreatitis, and discriminant analysis of these variables provides a useful method to classify severity.

Operative injury in spinal surgery in the management of polytrauma patients

The release of indicators and mediators of the posttraumatic inflammatory response following spine surgery was measured in patients with multiple injuries. Eight of a group of 113 patients (mean injury severity score 36) who underwent delayed operative stabilization of vertebral fractures (> 24 h after trauma) were studied. The following significant postoperative changes of blood levels (median values, Wilcoxon signed-rank test) compared with the preoperative starting point were found: polymorphonuclear granulocyte elastase rose from 220 to 337 ng/ml, cathepsin B from 84.5 to 135.5 mU/l, C-reactive protein from 9.1 to 11.6 mg/dl, lactate from 9.6 to 15.2 mg/dl and neopterin from 6.9 to 15.2 nmol/l, while antithrombin III fell from 107.5% to 84%, platelet count from 102 to 88 x 10(9)/l and pO2/FiO2-ratio from 361 to 260. The alterations in the blood levels of these parameters following spine surgery showed a pattern similar, albeit of lesser magnitude, to that which can be observed after severe accidental trauma. We conclude that the additional activation of the inflammatory response following surgery for vertebral lesions should be taken into account when planning these operations in patients with multiple injuries.

Impact of clinically silent inflammation on male genital tract organs as reflected by biochemical markers in semen.

Three hundred eight-nine healthy, infertile patients were studied to determine the effects of inflammation on genital tract organs. Clinically silent inflammation was diagnosed by measuring polymorphonuclear granulocyte (PMN) elastase in semen. Seminal vesicle, prostate, and epididymis functions were assessed by measuring fructose, citric acid, and neutral alpha-glucosidase in semen. There was a significant relationship between high PMN elastase levels and low citric acid levels in semen; fructose and neutral alpha-glucosidase were not related to PMN elastase. Semen samples with increased PMN elastase levels (greater than 250 and greater than 1,000 ng/ml) showed a high incidence of pathologic citric acid levels (67% and 73%, respectively). These biochemical data indicate that the prostate is the main target in clinically silent male genital tract inflammation.

Effect of piroxicam therapy on granulocyte function and granulocyte elastase concentration in peripheral blood and synovial fluid of rheumatoid arthritis patients

The effect of piroxicam therapy (20 mg/day for 15 days) on various polymorphonuclear granulocyte (PMN) responses and on PMN elastase concentration was investigated in nine patients with active rheumatoid arthritis. Peripheral blood and synovial fluid samples were collected before starting therapy and 12 h after the last dose of the drug. All patients were evaluable for peripheral blood analysis and six for synovial fluid analysis. Piroxicam therapy had no effect on PMN random migration and phagocytosis, while it significantly reduced both FMLP-induced aggregation and FMLP-induced chemotaxis. This seems mainly due to an effect on FMLP binding, as no differences were observed after therapy in PMA- and PHA-induced aggregation as well as in serum-induced chemotaxis. In contrast, a marked impairment of NBT test and PMA- and FMLP-induced superoxide anion (O2-) production was found after piroxicam therapy. This effect was as evident in peripheral blood as in synovial fluid PMN. Also, a significant reduction in synovial fluid PMN number and synovial fluid PMN elastase concentration (elastase-alpha 1-proteinase complex) was found after treatment. It is concluded that piroxicam may act at different sites on various PMN responses. Its effect on O2- generation and PMN elastase concentration in synovial fluid may have an important role in reducing destruction of arthritic joint tissue.

An elastase-specific inhibitor from human bronchial mucus. Isolation and characterization.

It was shown previously that human bronchial mucus contains an acid-stable proteinase inhibitor directed against trypsin and chymotrypsin, polymorphonuclear granulocyte elastase and cathepsin G. In addition to this well-characterized inhibitor, designated here as BSI-ATE (identical with the inhibitor HUSI-I from human seminal plasma or antileucoprotease), another acid-stable inhibitor BSI-E is present in the mucus which exerts inhibitory activity towards porcine pancreatic and human granulocytic elastase, but not against trypsin, chymotrypsin, or granulocytic cathepsin G. This elastase-specific inhibitor was isolated by affinity chromatography. Its molecular mass and its amino acid composition are very similar to those of BSI-TE. An immunological cross-reactivity between both inhibitor species was not observed. In the mucus of patients suffering from obstructive airway disease the elastase-specific inhibitor is not present in the free form but can be liberated by acidification.