Plasma Concentrations Of Elastase Research Articles

Prospective analysis over time of semen parameters in spinal cord-injured patients: Results of a pilot study.

Spinal cord injury often results in erectile dysfunction and an ejaculation along with impaired semen parameters. Fertility is a major concern in spinal cord injury adult males and some fear that the delay post-spinal cord injury may negatively affect sperm quality. We aimed to (i) assess semen parameters over time in SCI patients according to age at spinal cord injury, time post-spinal cord injury, and the spinal cord injury level and completeness and (ii) measure markers in semen for inflammation and marker of oxidative stress to investigate their impact on sperm parameters. The study is a prospective, longitudinal, pilot study over 18 months. Thirty-five men with spinal cord injury from 18 to 60 years of age were enrolled. Their mean age was 29.4 ± 6.4 years. Semen retrieval was scheduled every 6 months, allowing analysis of four ejaculates, in association with measurement of granulocyte and seminal plasma elastase concentrations to assess markers in semen for inflammation and spermatozoa DNA fragmentation to assess oxidative stress. Based on reference limits, a normal total sperm number, decreased motility and vitality of the spermatozoa, and increased morphological abnormalities were found. Mean round cell and granulocyte concentrations were elevated in the semen. Markers in semen for inflammation and marker of oxidative stress were elevated in several semen samples, compared to reference limits. However, neither the presence of markers in semen for inflammation or oxidative stress, the completeness or the level of the spinal cord lesion, the age or the time post-spinal cord injury had a negative impact on the semen quality over time. There was no significant decline in semen quality in spinal cord injury patients over time within the limitations of this pilot study. Moreover, a chronic genital inflammatory status was not associated with impairment of semen quality. The present findings are reassuring for men with spinal cord injury and could guide the management of their reproductive ability. According to these preliminary data, not all spinal cord injury patients who are able to ejaculate require systematic freezing of their spermatozoa.

The Impact of a Dairy Milk Recovery Beverage on Bacterially Stimulated Neutrophil Function and Gastrointestinal Tolerance in Response to Hypohydration Inducing Exercise Stress.

The study aimed to determine the impact of a dairy milk recovery beverage immediately after endurance exercise on leukocyte trafficking, neutrophil function, and gastrointestinal tolerance markers during recovery. Male runners (N = 11) completed two feeding trials in randomized order, after 2hr of running at 70% V˙O2max, fluid restricted, in temperate conditions (25°C, 43% relative humidity). Immediately postexercise, the participants received a chocolate-flavored dairy milk beverage equating to 1.2g/kg body mass carbohydrate and 0.4g/kg body mass protein in one trial, and water volume equivalent in another trial. Venous blood and breath samples were collected preexercise, postexercise, and during recovery to determine the leukocyte counts, plasma intestinal fatty acid binding protein, and cortisol concentrations, as well as breath H2. In addition, 1,000µl of whole blood was incubated with 1μg/ml Escherichia coli lipopolysaccharide for 1hr at 37°C to determine the stimulated plasma elastase concentration. Gastrointestinal symptoms and feeding tolerance markers were measured preexercise, every 15min during exercise, and hourly postexercise for 3hr. The postexercise leukocyte (mean [95% confidence interval]: 12.7 [11.6, 14.0] × 109/L [main effect of time, MEOT]; p < .001) and neutrophil (10.2 [9.1, 11.5] × 109/L; p < .001) counts, as well as the plasma intestinal fatty acid binding protein (470pg/ml; +120%; p = .012) and cortisol (236nMol/L; +71%; p = .006) concentrations, were similar throughout recovery for both trials. No significant difference in breath H2 and gastrointestinal symptoms was observed between trials. The total (Trial × Time, p = .025) and per cell (Trial × Time, p = .001) bacterially stimulated neutrophil elastase release was greater for the chocolate-flavored dairy milk recovery beverage (+360% and +28%, respectively) in recovery, compared with the water trial (+85% and -38%, respectively). Chocolate-flavored dairy milk recovery beverage consumption immediately after exercise prevents the decrease in neutrophil function during the recovery period, and it does not account for substantial malabsorption or gastrointestinal symptoms over a water volume equivalent.

Increased proteinase 3 and neutrophil elastase plasma concentrations are associated with non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes

IntroductionNon-alcoholic fatty liver disease (NAFLD) is becoming a major health problem worldwide. Inflammation plays an important role in disease pathogenesis and recent studies have shown a potential role for the neutrophil serine proteases (NSPs) proteinase-3 (PR3) and neutrophil elastase (NE) in NAFLD as well as an imbalance between NSPs and their natural inhibitor alpha-1 antitrypsin (AAT). The aim of this study was to investigate whether PR3 and NE plasma concentrations are associated with NAFLD and/or type 2 diabetes.MethodsTo explore this hypothesis we used several cohorts: a cohort of 271 obese individuals with liver steatosis, a cohort of 41 patients with biopsy-proven NAFLD, a cohort of 401 obese type 2 diabetes patients and a cohort of 205 lean healthy controls; and measured PR3 and NE plasma concentrations. In addition, we measured AAT plasma concentrations in order to investigate if the ratios between NSPs and their natural inhibitor were altered in NAFLD and type 2 diabetes when compared to healthy controls.ResultsOur data shows an increase in PR3 and NE concentrations and a decrease in AAT concentrations in obese patients when compared to controls. Moreover, PR3 plasma concentrations are increased in patients with liver steatosis. Furthermore, PR3 and NE concentrations in the liver are associated with the advanced stages of NAFLD characterized by NASH and/ or liver fibrosis. Additionally, PR3 and NE concentrations were up-regulated in patients with type 2 diabetes when compared to lean and obese controls.ConclusionWe conclude that circulating levels of NSPs associate with obesity-related metabolic disorders. Further research is needed to clearly establish the role of these proteases and investigate whether they could be used as non-invasive markers for NAFLD and/or type 2 diabetes.

Elastin-Specific Autoimmunity in Smokers With Thoracic Aortic Aneurysm and Dissection is Independent of Chronic Obstructive Pulmonary Disease.

BackgroundThoracic aortic aneurysm (TAA) and dissection (TAD) are characterized by progressive disorganization of the aortic wall matrix, including elastin, a highly immunogenic molecule. Whether acquired autoimmune responses can be detected in TAA/TAD patients who are smokers is unknown. The objectives of this study were to determine whether TAA/TAD smokers have increased T‐cell responses to human elastin fragments, and to determine whether autoimmune responses in TAA/TAD smokers are dependent on chronic obstructive pulmonary disease.Methods and ResultsIn a cross‐sectional study (N=86), we examined peripheral blood CD4+ T cell responses to elastin fragments in never‐, former‐, or current‐smokers with or without TAA/TAD. CD4+ T cells were co‐cultured with irradiated autologous peripheral blood CD1a+/CD14+ antigen presenting cells pulsed with or without elastin fragments to measure cytokine production. Baseline plasma concentration of anti‐elastin antibodies and elastin‐degrading enzymes (eg, matrix metalloproteinase‐9, and ‐12, and neutrophil elastase) were measured in the same cohort. elastin fragment‐specific CD4+ T cell expression of interferon‐γ, and anti‐elastin antibodies were dependent on history of smoking in TAA/TAD patients but were independent of chronic obstructive pulmonary disease. Matrix metalloproteinase‐9, and ‐12, and neutrophil elastase plasma concentrations were also significantly elevated in ever‐smokers with TAA/TAD.ConclusionsCigarette smoke is associated with loss of self‐tolerance and induction of elastin‐specific autoreactive T‐ and B‐cell responses in patients with TAA/TAD. Development of peripheral blood biomarkers to track immunity to self‐antigens could be used to identify and potentially prognosticate susceptibility to TAA/TAD in smokers.

TGF-β1 and granulocyte elastase in the evaluation of activity of inflammatory bowel disease. A pilot study

The aim was to assess the usefulness of TGF-β1 and elastase in the evaluation of activity of ulcerative colitis (UC) and Crohn's disease (CD). 32 patients diagnosed with UC, 31 with CD and 30 healthy volunteers were enrolled in this study. Diagnosis of the disease was confirmed by videocolonoscopy and histopathological evaluation of intestinal biopsies. Disease activity was assessed by use of the Mayo Scoring System for Assessment of Ulcerative Colitis Activity in UC patients and by CDAI in CD patients. hsCRP was determined by the immunonephelometric method, TGF-β1 and elastase plasma concentration by ELISA. The results of the study were analyzed using Statistica and R statistical language. In UC a positive correlation between disease activity and platelet level, hsCRP and TGF-β1 concentration was noted. Elastase concentration in UC patients was significantly higher than in CD, but there was no correlation with the activity of the disease. In CD patients we observed a positive correlation between disease activity and leukocytes, platelet levels and elastase concentration, and a very low correlation with hsCRP and TGF-β1. Determination of TGF-β1 can be used for evaluation of inflammatory activity in UC and it is connected with elevated concentrations of CRP and platelets. To a lower extent TGF-β1 can also be used for evaluation of inflammatory activity in CD. Examination of elastase concentration may be useful in the assessment of CD activity. Plasma elastase concentration may be helpful in UC and CD differentiation. The preliminary results of this investigation seem promising; nevertheless, more studies are necessary.

Systemic inflammation and immune response after laparotomyvslaparoscopy in patients with acute cholecystitis, complicated by peritonitis

To evaluate acute cholecystitis, complicated by peritonitis, acute phase response and immunological status in patients treated by laparoscopic or open approach. From January 2002 to May 2012, we conducted a prospective randomized study on 45 consecutive patients (27 women, 18 men; mean age 58 years). These subjects were taken from a total of 681 patients who were hospitalised presenting similar preoperative findings: acute upper abdominal pain with tenderness, involuntary guarding under the right hypochondrium and/or in the flank; fever higher than 38 °C, leukocytosis greater than 10 × 10(9)/L or both, and ultrasonographic evidence of calculous cholecystitis possibly complicated by peritonitis. These patients had undergone cholecystectomy for acute calculous cholecystitis, complicated by bile peritonitis. Randomly, 23 patients were assigned to laparoscopic cholecystectomy (LC), and 22 patients to open cholecystectomy (OC). Blood samples were collected from all patients before operation and at days 1, 3 and 6 after surgery. Serum bacteraemia, endotoxaemia, white blood cells (WBCs), WBC subpopulations, human leukocyte antigen-DR (HLA-DR), neutrophil elastase, interleukin-1 (IL-1) and IL-6, and C-reactive protein (CRP) were measured at 0, 30, 60, 90, 120 and 180 min, at 4, 6, 12, 24 h, and then daily (8 A.M.) until post-op day 6. The two groups were comparable in the severity of peritoneal contamination as indicated by the viable bacterial count (open group = 90% of positive cultures vs laparoscopic group = 87%) and endotoxin level (open group = 33.21 ± 6.32 pg/mL vs laparoscopic group = 35.02 ± 7.23 pg/mL). Four subjects in the OC group (18.1%) and 1 subject (4.3%) in the LC group (P < 0.05) developed intra-abdominal abscess. Severe leukocytosis (range 15.8-19.6/mL) was observed only after OC but not after LC, mostly due to an increase in neutrophils (days 1 and 3, P < 0.05). This value returned to the normal range within 3-4 d after LC and 5-7 d after OC. Other WBC types and lymphocyte subpopulations showed no significant variation. On the first day after surgery, a statistically significant difference was observed in HLA-DR expression between LC (13.0 ± 5.2) and OC (6.0 ± 4.2) (P < 0.05). A statistically significant change in plasma elastase concentration was recorded post-operatively at days 1, 3, and 6 in patients from the OC group when compared to the LC group (P < 0.05). In the OC group, the serum levels of IL-1 and IL-6 began to increase considerably from the first to the sixth hour after surgery. In the LC group, the increase of serum IL-1 and IL-6 levels was delayed and the peak values were notably lower than those in the OC group. Significant differences between the groups, for these two cytokines, were observed from the second to the twenty-fourth hour (P < 0.05) after surgery. The mean values of serum CRP in the LC group on post-operative days (1 and 3) were also lower than those in the OC group (P < 0.05). Systemic concentration of endotoxin was higher in the OC group at all intra-operative sampling times, but reached significance only when the gallbladder was removed (OC group = 36.81 ± 6.4 ρg/mL vs LC group = 16.74 ± 4.1 ρg/mL, P < 0.05). One hour after surgery, microbiological analysis of blood cultures detected 7 different bacterial species after laparotomy, and 4 species after laparoscopy (P < 0.05). OC increased the incidence of bacteraemia, endotoxaemia and systemic inflammation compared with LC and caused lower transient immunological defense, leading to enhanced sepsis in the patients examined.

Relationship between exercise-induced systemic inflammatory like reaction and racing performance in endurance horses

Endurance race induces a rise of serum creatine kinase (CK) activity and a systemic inflammatory like response characterised by an increase of neutrophil counts, plasma and muscle myeloperoxidase (MPO) and elastase (ELT) concentrations in horses. Horses performing the same standardised exercise test do not respond with the same magnitude of inflammatory reaction. The aim of the present study was to measure the total neutrophil count, the ratio neutrophil:lymphocyte, the MPO and ELT plasma concentrations and concomitant increases of serum CK activities in competing endurance horse and to relate these results to their race performance. Twenty one horses participating in a 120 km 4 star endurance race recruited on a voluntary basis finished the race with a mean speed ranging from 13.1 to 19.8 km/h. Blood was taken the day before the race and two hours after the race. Mean values of neutrophil counts, ratio neutrophil:lymphocyte, plasma MPO and ELT concentrations and serum CK activities after the race were significantly higher than the pre-race values. There was no correlation between neutrophil counts, MPO, ELT or CK and the mean speed of the horses during the race except for the ratio neutrophil:lymphocyte where a significant negative correlation was observed. These results showed that systemic responses induced by strenuous exercise such as an endurance race is not clearly related to performance but also to horse-related factors, such as intrinsic capacity or training.

Prostaglandins E 2 And F 2α as Inflammatory and Vasoactive Mediators in Black Walnut Heartwood Extract-Induced Equine Laminitis

s Vol 30, No 2 (2010) 95 TAKE HOME MESSAGE Elastase concentrations are increased in the integument of horses during the developmental phase of BWHE-induced laminitis. INTRODUCTION Current evidence suggests the involvement of neutrophils and myeloperoxidase (MPO), a strong oxidative enzyme in the development of laminitis. Elastase is a protease recognized as a component of inflammatory disorders. Neutrophil elastase concentrations were measured in plasma, skin and laminar tissues from control horses and horses given BWHE MATERIALS & METHODS (INCLUDING STATISTICAL ANALYSES) The study included 4 groups, a control group (water) and 3 experimental groups (BWHE). Plasma was obtained hourly until euthanasia of BWHE-treated horses at 1.5 (n 1⁄4 5), 3 (n 1⁄4 6), and 12 hours (n 1⁄4 10 plasma/n 1⁄4 6 tissue) and control horses at 12 hours (n 1⁄4 7 plasma/ n 1⁄4 5 tissue). Elastase concentrations were determined via ELISA. Log-transformed data were analyzed with 1-way ANOVA (tissues) and unpaired t-test (plasma); significance was set at P < 0.05. RESULTS Plasma elastase concentrations in the BWHE group were significantly higher at 6, 8 and 10h compared to the control group. Concentrations in skin and laminar tissue were significantly higher at 3 and 12h in the BWHE group. Elastase concentrations were significantly higher in skin than in lamina at 12h in BWHE group. DISCUSSION Increased elastase concentrations are consistent with dynamic changes in circulating leukocytes in horses given BWHE. Collectively, MPO and elastase have the potential to cause significant tissue damage during the development of laminitis. CLINICAL RELEVANCE Elastase may become an important target as new treatments for laminitis are developed. CONCLUSION Elastase concentrations increase in the integumentary system during the development of laminitis and may participate in the disintegration of the hoof basal membrane. Prostaglandins E2 And F2a as Inflammatory and Vasoactive Mediators in Black Walnut Heartwood Extract-Induced Equine Laminitis Erik Noschka, James N. Moore, John F. Peroni, Stephen J. Lewis, and Tom P. Robertson, Department of Large Animal Clinical Sciences (Noschka), Virginia Maryland Regional College of Veterinary Medicine Duck Pond Drive, Blacksburg, VA, 24071, Department of Physiology and Pharmacology, Institute of Comparative Medicine (Robertson, Moore, T Lewis, Peroni), and Department of Large Animal Medicine (Robertson, Moore, Peroni), College of Veterinary Medicine, University of Georgia, Athens, GA 30602-7389, and Department of Pediatrics (S Lewis) University of Virginia, College of Medicine, Charlottesville, VA 22908 TAKE HOME MESSAGE The increases in plasma levels of prostaglandins E2 (PGE2) and F2a at an early stage of laminitis are consistent with systemic inflammatory events and alterations in laminar microvascular function. INTRODUCTION Links between the inflammatory and vascular events during the prodromal stages of laminitis have yet to be identified. The aim of this study was to provide initial insights into the role that PGE2 and F2a may play in the development of laminitis induced by black walnut heartwood extract (BWHE). MATERIALS AND METHODS Blood samples were collected before and after administration of either water (control horses) or BWHE for white blood cell counts and plasma concentrations of PGE2 and F2a. Laminar tissue was collected for the isolation of laminar vessels, and responses of these vessels to PGE2 and F2 a were determined. The data were analyzed by repeated measures analysis of variance (ANOVA). Differences between individual means were identified by Student’s modified t-test using the Bonferroni correction for multiple comparisons between means using the error mean square term from the ANOVA. RESULTS Plasma levels of PGE2 and F2a increased transiently and coincided with the nadir in white blood cell counts in BWHE horses. PGE2 elicited small dilator responses in laminar veins from control horses, but elicited a small constrictor response in laminar veins from BWHE horses. PGF2a was 96 Abstracts Vol 30, No 2 (2010) a potent contractile agonist for laminar veins, whereas laminar arteries were unresponsive. DISCUSSION PGE2 elicited vascular responses in laminar vessels in control and BWHE horses may be explained by different receptor populations and alterations in laminar tissues in BWHE horses. PGF2a selectively constricted laminar veins, and may play a role in the vascular dysfunction that characterizes acute laminitis. CLINICAL RELEVANCE The use of anti-inflammatory drugs in acute laminitis is supported by the results of this study. The effects of these drugs on responses of laminar microvessels to other PGs need to be determined. CONCLUSION Specific prostanoids, such as PGE2 and F2a, may be viable targets for the development of more effective therapeutic regimens for the treatment of equine laminitis. Proliferative Effects of Insulin on Equine Lamellar Epithelial Cells Mediated By the IGF-1 Receptor S.R. Bailey and S. Chockalingham, Faculty of Veterinary Science, University of Melbourne, Victoria 3010,

Hind Laminar Proinflammatory Response Is Present After Carbohydrate Overload

s Vol 30, No 2 (2010) 95 TAKE HOME MESSAGE Elastase concentrations are increased in the integument of horses during the developmental phase of BWHE-induced laminitis. INTRODUCTION Current evidence suggests the involvement of neutrophils and myeloperoxidase (MPO), a strong oxidative enzyme in the development of laminitis. Elastase is a protease recognized as a component of inflammatory disorders. Neutrophil elastase concentrations were measured in plasma, skin and laminar tissues from control horses and horses given BWHE MATERIALS & METHODS (INCLUDING STATISTICAL ANALYSES) The study included 4 groups, a control group (water) and 3 experimental groups (BWHE). Plasma was obtained hourly until euthanasia of BWHE-treated horses at 1.5 (n 1⁄4 5), 3 (n 1⁄4 6), and 12 hours (n 1⁄4 10 plasma/n 1⁄4 6 tissue) and control horses at 12 hours (n 1⁄4 7 plasma/ n 1⁄4 5 tissue). Elastase concentrations were determined via ELISA. Log-transformed data were analyzed with 1-way ANOVA (tissues) and unpaired t-test (plasma); significance was set at P < 0.05. RESULTS Plasma elastase concentrations in the BWHE group were significantly higher at 6, 8 and 10h compared to the control group. Concentrations in skin and laminar tissue were significantly higher at 3 and 12h in the BWHE group. Elastase concentrations were significantly higher in skin than in lamina at 12h in BWHE group. DISCUSSION Increased elastase concentrations are consistent with dynamic changes in circulating leukocytes in horses given BWHE. Collectively, MPO and elastase have the potential to cause significant tissue damage during the development of laminitis. CLINICAL RELEVANCE Elastase may become an important target as new treatments for laminitis are developed. CONCLUSION Elastase concentrations increase in the integumentary system during the development of laminitis and may participate in the disintegration of the hoof basal membrane. Prostaglandins E2 And F2a as Inflammatory and Vasoactive Mediators in Black Walnut Heartwood Extract-Induced Equine Laminitis Erik Noschka, James N. Moore, John F. Peroni, Stephen J. Lewis, and Tom P. Robertson, Department of Large Animal Clinical Sciences (Noschka), Virginia Maryland Regional College of Veterinary Medicine Duck Pond Drive, Blacksburg, VA, 24071, Department of Physiology and Pharmacology, Institute of Comparative Medicine (Robertson, Moore, T Lewis, Peroni), and Department of Large Animal Medicine (Robertson, Moore, Peroni), College of Veterinary Medicine, University of Georgia, Athens, GA 30602-7389, and Department of Pediatrics (S Lewis) University of Virginia, College of Medicine, Charlottesville, VA 22908 TAKE HOME MESSAGE The increases in plasma levels of prostaglandins E2 (PGE2) and F2a at an early stage of laminitis are consistent with systemic inflammatory events and alterations in laminar microvascular function. INTRODUCTION Links between the inflammatory and vascular events during the prodromal stages of laminitis have yet to be identified. The aim of this study was to provide initial insights into the role that PGE2 and F2a may play in the development of laminitis induced by black walnut heartwood extract (BWHE). MATERIALS AND METHODS Blood samples were collected before and after administration of either water (control horses) or BWHE for white blood cell counts and plasma concentrations of PGE2 and F2a. Laminar tissue was collected for the isolation of laminar vessels, and responses of these vessels to PGE2 and F2 a were determined. The data were analyzed by repeated measures analysis of variance (ANOVA). Differences between individual means were identified by Student’s modified t-test using the Bonferroni correction for multiple comparisons between means using the error mean square term from the ANOVA. RESULTS Plasma levels of PGE2 and F2a increased transiently and coincided with the nadir in white blood cell counts in BWHE horses. PGE2 elicited small dilator responses in laminar veins from control horses, but elicited a small constrictor response in laminar veins from BWHE horses. PGF2a was

The effect of different oxygenators on polymorphonuclear neutrophil apoptosis of heart surgery undergoing cardiopul-monary bypass

Objective To evaluate the effect of two kind oxygenators on polymorphonuclear neutrophil (PMN) apoptosis, and to give a method of inhibting the systemic inflammatory response syndrome(SIRS) to cardiopulmonary bypass(CPB). Methods Sixty VSD patients undergoing open heart surgery with CPB were randomly divided into two groups(n = 30): group B(bubble oxygenator), group M(membrane oxygenator). Blood samples were drawn from the central venous line before starting CPB, at the end of CPB, 4, 8 and 24 h after CPB. The PMN counts were performed by blood cell counter. PMN apoptosis and the expression of CD11b were evaluated by flow eytometry. The plasma concentration of elastase was determined by enzyme linked immunosorbent assays. Results The rates of PMN apoptosis were significantly reduced (P<0.05) and the PMN counts, the expression of CD11b and the plasma con-centration of elastase were remarkably increased after CPB(P<0.05), but at the end of CPB, 4 h and 8 h after CPB the rate of PMN apoptosis was higher in group M than that in group B. PMN counts, the expression of CDI lb and the plasma concentration of elastase were higher in group B than in group M (P<0.05). The PMN counts were higher in group B than in group M 24 h after CPB (P< 0.05). Conclusion XiJian-Ⅱ membrane oxygenator can increase PMN apoptosis and depress systemic inflammatory response better than XIJing-90 bubble oxygenator. Key words: Cardiopulmonary bypass; Inflammatory response; Membrane oxygenators; Bubble oxygenators

Relationship between elastase and total antioxidant status in young subjects with recent myocardial infarction

In a group of young subjects with acute myocardial infarction (AMI) (68 men and 7 women; mean age 39.6+/-5.7 years) we examined the plasma concentration of elastase, the thiobarbituric acid-reactive substances (TBARS) and the total antioxidant status (TAS) at the initial stage of AMI. In this group we found an increase of elastase (p<0.001) and TBARS (p<0.001) and a decrease of TAS (p<0.001). A statistical correlation was observed in the whole group of AMI patients between plasma elastase and TAS (p<0.01) and this correlation was more statistically significant in patients with more risk factors and not in those with more involved vessels.

Role of leucocytes in damage to the vascular endothelium during ischaemia-reperfusion injury

During this investigation, a model of tourniquet-induced forearm ischaemia-reperfusion injury is employed to investigate the role of leucocytes in damage to the vascular endothelium during ischaemia-reperfusion injury. Leucocyte entrapment is investigated by measuring the concentration of leucocytes in venous blood leaving the arm. Neutrophil and monocyte leucocyte subpopulations are isolated by density gradient centrifugation techniques. Cell surface expression of CD11b and the intracellular production of hydrogen peroxide are measured via flow cytometry. Plasma concentrations of elastase and von Willebrand factor (vWF) are measured using enzyme-linked immunosorbemt assay (ELISA) techniques. During ischaemia-reperfusion, there was an increase in CD11b cell surface expression on neutrophils (P=0.040) and monocytes (P=0.049), and a decrease in peripheral blood leucocytes (P=0.019). There was an increase in the intracellular production of hydrogen peroxide by leucocyte subpopulations (P=0.027 [neutrophils], P=0.091 [monocytes]) and in the plasma elastase concentration (P=0.05). There was also a trend to increasing plasma concentration of vWF (P=0.0562), which was measured as a marker of endothelial damage. Ischaemia-reperfusion results in increased adhesiveness, entrapment and activation of leucocytes. Even following a mild ischaemic insult, this leucocyte response was followed immediately by evidence of endothelial damage. These results may have important implications for understanding the development of chronic diseases that involve mild ischaemic episodes.

Analysis of plasma elastase levels in early and late onset preeclampsia

Circulatory neutrophils have been reported to be activated in preeclampsia. It has been suggested that maternal plasma levels of elastase may serve as a possible cell-free marker to quantify such activation. Although plasma elastase levels have been found to be elevated in cases with manifest preeclampsia and eclampsia, this has not yet been examined in cases with early and late onset preeclampsia. We have now examined this aspect. In this retrospective study, maternal plasma samples were examined from eight cases with early onset preeclampsia (<34 weeks of gestation), eight cases with late onset preeclampsia (>34 weeks of gestation) and an equal number of gestational age matched normotensive term controls. Plasma concentrations of elastase were measured by ELISA using a commercially available assay. Plasma elastase concentrations were significantly elevated the preeclampsia study group when compared to the normotensive control group (median=139.2 ng/ml versus median=72.1 ng/ml; P=0.0025). These elevations remained significant when the preeclampsia study group was stratified into case with early onset preeclampsia (median=118.8 ng/ml versus median=62.2 ng/ml; P=0.03), but jailed failed to attain significance for those cases with late onset preeclampsia (median=181.3 ng/ml versus median=86.3 ng/ml; P=0.061). Our data indicate that elastase levels are elevated in both early and late onset forms of preeclampsia, and imply that the activation of neutrophils may be more acute in the former than in the latter (238 words).

Fluctuations in C-reactive protein concentration and neutrophil activation during normal human pregnancy

Objectives To clarify the changes in serum C-reactive protein (CRP) levels and in the neutrophil activation state during normal human pregnancy. Materials and methods A longitudinal study ( n = 23) was performed during the three trimesters of pregnancy; a group of non-pregnant women ( n = 24) was used as control. Total and differential leukocyte count, serum concentration of CRP and plasma levels of granulocyte-macrophage colony stimulating factor (GM-CSF) and of lactoferrin and elastase (two indirect markers of neutrophil activation) were measured. Results Pregnancy imposed an inflammatory response in the mother, observed by the significant increment in total white blood cell (WBC) and neutrophil counts and in the circulating levels of CRP, GM-CSF and lactoferrin, in all trimesters of gestation compared with non-pregnant controls. Plasma elastase concentration was also significantly higher in pregnant women, but only in the first trimester of gestation. Regarding the ratios of lactoferrin and elastase per neutrophil, they were significantly lower in pregnant women (all trimesters). During gestation, WBC and neutrophil count increased significantly from the first to the second trimester and remained high in the third period. In contrast, the ratios of lactoferrin and elastase per neutrophil decreased significantly from the first to the second trimester, remaining low in the last trimester. Concerning CRP levels, no consistent changes were observed throughout gestation; 12 cases (52.2%) presented fluctuations, whereas 7 (30.4%) showed progressive reductions and 4 (17.4%) progressive increments throughout pregnancy. Conclusions Changes in CRP levels vary in a wide manner between subjects along pregnancy, even though median values are consistently elevated throughout pregnancy. Moreover, circulating levels of neutrophil-activation products are higher in normal human gestation.

Aktivierung von Granulozyten und Antiproteasen bei herzchirurgischen Eingriffen mit extrakorporaler Zirkulation

Cardiovascular surgical procedures with extracorporeal circulation (ECC) lead to neutrophil activation followed by the release of proteases such as neutrophil elastase (NE) and oxidants. The mis-balance between proteases and their physiological inhibitors may contribute to morbidity in the postoperative period. In this study, the effects of cardiac surgery on neutrophil mediators were evaluated. Release of neutrophil elastase and plasma levels of the natural NE antagonists alpha (1)-proteinase inhibitor (API) and secretory leukocyte proteinase inhibitor (SLPI) were measured. The oxidative burst and the phagocytic activity were also evaluated. Tissue destruction was quantified by measuring the serum concentration of fibronectin. Blood samples were obtained from 30 patients undergoing elective coronary artery bypass grafting (n = 30). NE and SLPI concentrations were measured by ELISA, API and fibronectin plasma levels were determined by nephelometry. Neutrophil phagocytic activity and oxidative burst were evaluated by flow cytometry. Neutrophil elastase plasma concentrations rose during ECC (245 +/- 107 microg/ml versus 44 +/- 14 microg/ml after induction, p < 0.001), whereas SLPI and API were decreased after onset of ECC. 24 h after ECC SLPI (54 +/- 17 ng/ml versus 41 +/- 10 ng/ml, p < 0.05) and API (3 +/- 0.5 g/l versus 1.6 +/- 0.3 g/l, p < 0.05) increased significantly compared to baseline values. A minor increase in phagocytic activity was observed after the onset of ECC. There were no significant changes in the oxidative burst. Cardiac surgery with ECC leads to neutrophil activation and elastase release. The imbalance between NE and the NE inhibitors API and SLPI may increase the risk for tissue damage due to granulocyte activation after cardiac surgery.

Damage Control Orthopaedics

In some groups of polytrauma patients, particularly those with chest injuries, head injuries, and those with mangled extremities, early total care of major bone fractures may be potentially harmful. Delaying all orthopaedic surgery, however, is also not always the best approach. In these situations, damage control orthopaedics, which emphasizes the stabilization and control of the injury rather than repair will add little additional physiologic insult to the patient and is a treatment option that should be considered.

Effect of stress doses of hydrocortisone on S-100B vs. interleukin-8 and polymorphonuclear elastase levels in human septic shock

Stress doses of hydrocortisone are known to have immunomodulatory effects in patients with hyperdynamic septic shock. The prognosis correlates with the presence and severity of septic encephalopathy. However, neurological evaluation is influenced by the use of analgesia sedation during artificial ventilation. The objective of this study was to demonstrate the effect of stress doses of hydrocortisone during the initial phase of human septic shock on the serum values of the neurospecific protein S-100B in comparison to the inflammation markers interleukin (IL)-8 in serum and polymorphonuclear (PMN) elastase in plasma. A total of 24 consecutive patients, who met the American College of Chest Physicians/Society of Critical Care Medicine criteria for septic shock, were enrolled in this prospective, randomized, double-blind, single-center trial. The severity of illness at recruitment was graded using the Acute Physiology and Chronic Health Evaluation II and the Simplified Acute Physiology Score II scoring systems. Multi-organ dysfunction syndrome was described by the Sepsis-related Organ Failure Assessment (SOFA) score. All patients were prospectively randomized to receive either stress doses of hydrocortisone or placebo. Hydrocortisone was started in 12 patients with a loading dose of 100 mg and followed by a continuous infusion of 0.18 mg/kg/h for 6 days. Median S-100B serum levels of the hydrocortisone group decreased from 0.32 ng/mL at study entry to 0.07 ng/mL 6 days later without significant differences compared to the placebo group. Initial IL-8 serum levels were significantly higher in the hydrocortisone group up to 12 h after study entry, and significantly decreased from 715 to 17 pg/mL at the end of the observation period. Median PMN elastase plasma levels were not affected by hydrocortisone infusion. Patients with initial S-100B serum levels > 0.50 ng/mL revealed significantly higher SOFA scores up to 30 h, IL-8 serum levels up to 12 h, and PMN elastase plasma levels up to 36 h after study entry than those patients with < or = 0.50 ng/mL. These effects were independent of the amount of fluid correction for hemodilution. Starting S-100B, IL-8 and PMN elastase values of the hydrocortisone group were within the ranges already known in patients with out-of-hospital cardiac arrest or severe traumatic brain injury. Stress doses of hydrocortisone resulted in a significant reduction in IL-8 serum, but not in S-100B serum and PMN elastase plasma concentrations in patients with hyperdynamic septic shock. For the first time, a similar extent of S-100B increase in serum of septic patients at the time of diagnosis was shown as reported for cardiac arrest or severe traumatic brain injury.

Pathological Uterine Perfusion in the Second Trimester Is Not Associated with Neutrophil Activation

Objective: Preeclampsia and intrauterine growth retardation (IUGR) are associated with elevated concentrations of myeloperoxidase (MPO) and polymorphonuclear (PMN) elastase, which indicate maternal neutrophil activation. The aim of the study was to measure maternal MPO and PMN elastase plasma concentrations in second trimester pregnancies with pathological uterine perfusion that are a high risk group for preeclampsia and IUGR, and compare them to normal controls. Methods: The study includes 25 pregnancies with normal and 25 pregnancies with pathological uterine perfusion. In both groups, doppler‐sonographic measurement of uterine perfusion was performed in the twenty‐first week of gestation. Maternal plasma concentrations of MPO and PMN elastase were measured using a specific ELISA for both enzymes. Results: The plasma MPO concentration of pregnant women with normal perfusion did not differ significantly from that of the group with pathological perfusion (27.4 ± 3.3 vs. 23.7 ± 2.0 ng/mL). Likewise, the plasma PMN elastase‐concentration also did not show a significant difference between the groups (5.7 ± 0.5 ng/mL normal vs. 8.0 ± 1.0 ng/mL pathological). Patients with pathological perfusion that later developed preeclampsia or IUGR (9/25) showed unchanged MPO and PMN elastase values in the second trimenon compared to those with pathological perfusion and normal outcome. Conclusions: Pathological uterine perfusion in the second trimester was not associated with maternal neutrophil activation. The measurement of the MPO and PMN elastase concentration suggested that neutrophil activation in preeclampsia or IUGR is a secondary effect of the disease rather than a primary pathophysiological factor.

Microparticles from Patients with Multiple Organ Dysfunction Syndrome and Sepsis Support Coagulation through Multiple Mechanisms

We investigated the occurrence and thrombin generating mechanisms of circulating microparticles (MP) in patients with multiple organ dysfunction syndrome (MODS) and sepsis. MP, isolated from blood of patients (n = 9) and healthy controls (n = 14), were stained with cell-specific monoclonal antibodies (MoAbs) or anti-tissue factor (anti-TF) MoAb and annexin V, and analyzed by flow cytometry. To assess their thrombin-generating capacity, MP were reconstituted in normal plasma. The coagulation activation status in vivo was quantified by plasma prothrombin fragment F1+2- and thrombin-antithrombin (TAT) measurements. Annexin V-positive MP in the patients originated predominantly from platelets (PMP), and to a lesser extent from erythrocytes, endothelial cells (EMP) and granulocytes (GMP). Compared to healthy controls, the numbers of annexin V-positive PMP and TF-exposing MP were decreased (p = <0.001 for both), EMP were decreased (E-selectin, p = 0.003) or found equal (CD144, p = 0.063), erythrocyte-derived MP were equal (p = 0.726), and GMP were increased (p = 0.008). GMP numbers correlated with plasma concentrations of elastase (r = 0.70, p = 0.036), but not with C-reactive-protein or interleukin-6 concentrations. Patient samples also contained reduced numbers of annexin V-negative PMP, and increased numbers of erythrocyte-derived MP and GMP (p = 0.005, p = 0.021 and p <0.001, respectively). Patient MP triggered thrombin formation, which was reduced compared to the healthy controls (p = 0.008) and strongly inhibited by an anti-factor XII MoAb (two patients), by anti-factor XI MoAb (eight patients) or by anti-TF MoAb (four patients). Concentrations of F1+2 and TAT were elevated (p = 0.005 and p = 0.001, respectively) and correlated inversely with the number of circulating MP (and r = -0.51, p = 0.013, and r = -0.65, p = 0.001, respectively) and their thrombin generation capacity (F1+2: r= -0.62, p = 0.013). In patients with MODS and sepsis relatively low numbers of MP are present that differ from controls in their cellular origin, numbers and coagulation activation mechanisms.

Neutrophil oxidative activity is differentially affected by exercise intensity and type

The differential effects of exercise intensity and type on neutrophil activation were assessed in eight well-trained male runners. Each subject undertook, on different days, three separate 40 min interval (8 x 5 min) treadmill bouts: an intense uphill run (90% VO2 max), a moderate-intensity near-level run and an eccentrically-biased downhill run (both at 52% VO2 max). Blood granulocyte count increased (p< 0.05) after all three treadmill bouts (range 25-108%). Chemiluminescence activity of isolated neutrophils decreased (p< 0.05) immediately after (-58%) and 1-h after (-72%) uphill running, but became significantly elevated (p< 0.05) at 6-h after the near-level (+71%) and downhill (+84%) runs. The ability of neutrophils to release the superoxide anion radical was reduced (p< 0.05) immediately after near-level (-29%) and uphill (-21%) running in cells stimulated with opsonized zymosan. Epinephrine concentration increased by 430% (p=0.01) after uphill but not with near-level or downhill running. The plasma concentration of elastase increased (p< 0.05) immediately after uphill and near-level running, and one hour after uphill running. These results suggest that a population of neutrophils mobilised into the circulation became directly activated in response to exercise, and that neutrophil oxidative activity is affected differentially by both the intensity and type of exercise undertaken.