Plasma Elastase Research Articles

Abstract 13841: The Impact of Age on Coronary Perfusion, Cardiac Function and Inflammation in a Rat Model of Ashpyxial Cardiac Arrest

Introduction: Age is an independent predictor of poor outcome after cardiac arrest (CA), and age reduces organ function and increase infarct size, oxidative stress and inflammation in studies of regional ischemia reperfusion injury. Meanwhile, experimental CA studies are mostly performed in young, healthy animals, and our understanding of the CA pathophysiology may therefore fail to address the age-effects inherent to human CA. Objective: To investigate whether high age affects the reduction in cardiac function, increased inflammation, and endothelial activation observed after CA. Method: Aged (26 months, n=12) and young (5 months, n=10) male Sprague Dawley rats were subjected to 8 min of asphyxia induced CA, resuscitated using adrenalin and mechanical CPR, and observed for 360 min. Coronary perfusion pressure (CPP) was measured during CPR, and left ventricular end diastolic pressure, dP/dtmax, and dP/dtmin were determined at baseline and at the end of follow up. Blood samples at baseline, 120min, and 360min after CA were analysed for IL-6, IL-10, elastase, sE-selectin, and sI-CAM1. Results: We found no difference in CPP (aged: 16 [95%CI 13;20] vs young: 23 [95%CI 12;34]) or rate of ROSC (aged: 9/12 vs. young 9/10) between groups. During the first hour after resuscitation, a hyperperfusion phase was seen in the young group but not in the aged group (MAP aged: 50mmHg [95%CI 38;62] vs MAP young 105 [95%CI 88;123]). Left ventricular end diastolic pressure and dP/dtmax was higher in aged animals at baseline (p<0.02). We observed a significant decrease in dP/dtmax and increase in dP/dtmin after CA, with no group difference. Compared to young rats, aged animals showed a significantly higher increase in plasma elastase and sE-selectin levels after CA. In contrast, plasma IL-6 and IL-10 increased from baseline to end of follow up, but with no difference between the groups. sI-CAM1 showed no difference over time or between groups. Conclusion: We found increased endothelial activation and a blunted hyperperfusion in aged compared to young rats after CA. Age did not affect cardiac function and cytokine levels.

A novel minimal invasive mouse model of extracorporeal circulation.

Extracorporeal circulation (ECC) is necessary for conventional cardiac surgery and life support, but it often triggers systemic inflammation that can significantly damage tissue. Studies of ECC have been limited to large animals because of the complexity of the surgical procedures involved, which has hampered detailed understanding of ECC-induced injury. Here we describe a minimally invasive mouse model of ECC that may allow more extensive mechanistic studies. The right carotid artery and external jugular vein of anesthetized adult male C57BL/6 mice were cannulated to allow blood flow through a 1/32-inch external tube. All animals (n = 20) survived 30 min ECC and subsequent 60 min observation. Blood analysis after ECC showed significant increases in levels of tumor necrosis factor α, interleukin-6, and neutrophil elastase in plasma, lung, and renal tissues, as well as increases in plasma creatinine and cystatin C and decreases in the oxygenation index. Histopathology showed that ECC induced the expected lung inflammation, which included alveolar congestion, hemorrhage, neutrophil infiltration, and alveolar wall thickening; in renal tissue, ECC induced intracytoplasmic vacuolization, acute tubular necrosis, and epithelial swelling. Our results suggest that this novel, minimally invasive mouse model can recapitulate many of the clinical features of ECC-induced systemic inflammatory response and organ injury.

NETosis is induced by high glucose and associated with type 2 diabetes.

The role of neutrophils in diabetes and its complications is unclear. Upon challenge with microbes and inflammatory triggers, neutrophils release enzymes and nuclear material, forming neutrophils extracellular traps (NETs) and thereby dying by NETosis. We herein tested NET formation and NETosis products in high glucose and in the setting of type 2 diabetes (T2D). NETosis was assessed in vitro in cells exposed to 0, 5, 25mM glucose and 25mM mannitol, DMSO and PMA using immunofluorescence staining for elastase, DNA and chromatin. Single-cell morphometric analysis was used to detect enter of elastase in the nucleus and extrusion of nuclear material. Release of NETs was quantified by staining with Hoechst 33342. In 38 T2D and 38 age- and sex-matched non-diabetic individuals, we determined plasma elastase, mono- and oligonucleosomes and double-strand (ds) DNA, as circulating NETosis products. NETosis was accurately reproduced in vitro: high (25mM) glucose increased NETosis rate and release of NETs compared with 5mM glucose and 25mM mannitol. T2D patients showed increased plasma elastase, mono- and oligonucleosomes and dsDNA compared with non-diabetic control individuals. A positive correlation was found between HbA1c and mono- and oligonucleosomes, whereas dsDNA was correlated with the presence of nephropathy and cardiovascular disease. Serum IL-6 concentrations were higher in T2D compared with CTRL and correlated with serum dsDNA levels. High glucose and hyperglycemia increase release of NETs and circulating markers of NETosis, respectively. This finding provides a link among neutrophils, inflammation and tissue damage in diabetes.

Identification and biochemical characterization of a Kunitz‐type serine protease inhibitor from an insect, Manduca sexta (772.1)

Hemolymph (blood) of insects contains serine proteases and serine protease inhibitors that function in innate immune responses. The objective for this research was to investigate low molecular weight protease inhibitors from hemolymph of a lepidopteran insect, Manduca sexta. We identified serine protease inhibitor activity against trypsin, chymotrypsin, and elastase in plasma from naïve larvae and in plasma from larvae that were previously injected with Micrococcus luteus. We separated hemolymph plasma proteins using size exclusion chromatography, and the fractions with protease inhibitor activity were analyzed by SDS‐PAGE and liquid chromatography‐electrospray ionization tandem mass spectrometry. A serine protease inhibitor belonging to the Kunitz family was identified, and using primers based on the sequence encoding this protein found in the M. sexta genome sequence, we cloned its cDNA from larval fat body RNA. The cDNA encodes a protein with a secretion signal sequence followed by the mature secreted inhibitor protein of 56 amino acid residues. This sequence is highly similar to a Kunitz inhibitor previously purified from M. sexta (Ramesh, N., Sugumaran, M., Mole, J.E. 1988. J Biol Chem. 263:11523‐7). Using a synthesized and codon‐optimized DNA encoding the protease inhibitor, we are currently producing the recombinant inhibitor in E. coli for further biochemical characterization. We will study its inhibitory activity as a potential regulator of proteases in hemolymph that function in the innate immune system of M. sexta. The genome of M. sexta contains nine genes for single‐domain Kunitz proteins closely spaced on a single scaffold. The ultimate goal of this project is to understand the biological functions of these Kunitz protease inhibitors in innate immunity.Grant Funding Source: Supported by NIH grant GM41247.

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.

Male urogenital infections: impact of infection and inflammation on ejaculate parameters

Urogenital infections and inflammation may contribute significantly to ejaculate parameters essential for male infertility. For this review, data were acquired by a systematic search of the medical literature of the last 5 years. We address the andrological relevance of male urogenital infections and inflammation on ejaculate parameters. The different classification systems of the WHO and NIH are illustrated. In most cases, a separation of the different areas of the urogenital tract, for example, of the prostate, epididymis and testicles, is not possible. The significance of bacteriospermia with common bacteria is discussed. Furthermore, HIV, ascending chlamydial, mycoplasmal and gonococcal infections are relevant. Especially, the relevance of sexually transmitted microorganisms seems to be underestimated. Leukocytospermia is not well defined in its biological significance. Seminal plasma elastase and the cytokine expression reveal better insights into the inflammatory response of the seminal pathways. Sperm antibodies and reactive oxygen species are not usable as indicators for infection and inflammation. Different aspects for an impairment of ejaculate quality have been demonstrated although a direct ascension of microorganisms to the prostate has not been confirmed. Probably, lesions of the epididymis may sustain an ongoing disturbance of sperm parameters. A potential negative influence of urogenital infections and inflammation on sperm function is under discussion. However, the severity of impairment differs according to the underlying infections and the involved compartments. Signs of infections and inflammation in the ejaculate of infertile men are common, and the relevance is often doubtful in spite of microbiological, spermatological and immunological facilities.

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.

Rheumatoid arthritis患者における関節液ミエロペルオキシダーゼ, 血漿エラスターゼに関する研究

Rheumatoid arthritis患者における関節液ミエロペルオキシダーゼ, 血漿エラスターゼに関する研究

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.

Clinical immunology Assessment of neutrophil elastase plasma levels in children with intermediate uveitis

ENWEndNote BIBJabRef, Mendeley RISPapers, Reference Manager, RefWorks, Zotero AMA Polańska B, Turno-Kręcicka A, Kaźmierowska-Niemczuk M. Clinical immunologyAssessment of neutrophil elastase plasma levels in children with intermediate uveitis. Central European Journal of Immunology. 2012;37(3):275-279. doi:10.5114/ceji.2012.30806. APA Polańska, B., Turno-Kręcicka, A., & Kaźmierowska-Niemczuk, M. (2012). Clinical immunologyAssessment of neutrophil elastase plasma levels in children with intermediate uveitis. Central European Journal of Immunology, 37(3), 275-279. https://doi.org/10.5114/ceji.2012.30806 Chicago Polańska, Bożena, Anna Turno-Kręcicka, and Maria Kaźmierowska-Niemczuk. 2012. "Clinical immunologyAssessment of neutrophil elastase plasma levels in children with intermediate uveitis". Central European Journal of Immunology 37 (3): 275-279. doi:10.5114/ceji.2012.30806. Harvard Polańska, B., Turno-Kręcicka, A., and Kaźmierowska-Niemczuk, M. (2012). Clinical immunologyAssessment of neutrophil elastase plasma levels in children with intermediate uveitis. Central European Journal of Immunology, 37(3), pp.275-279. https://doi.org/10.5114/ceji.2012.30806 MLA Polańska, Bożena et al. "Clinical immunologyAssessment of neutrophil elastase plasma levels in children with intermediate uveitis." Central European Journal of Immunology, vol. 37, no. 3, 2012, pp. 275-279. doi:10.5114/ceji.2012.30806. Vancouver Polańska B, Turno-Kręcicka A, Kaźmierowska-Niemczuk M. Clinical immunologyAssessment of neutrophil elastase plasma levels in children with intermediate uveitis. Central European Journal of Immunology. 2012;37(3):275-279. doi:10.5114/ceji.2012.30806.

Systemic upregulation of neutrophil -defensins and serine proteases in neutrophilic asthma

The well-characterised airway inflammatory phenotypes of asthma include eosinophilic, neutrophilic, mixed eosinophilic/neutrophilic and paucigranulocytic asthma, identified based on the proportion of sputum granulocytes. Systemic inflammation is now recognised as an important part of some airway diseases, but the involvement of systemic inflammation in the pathogenesis of airway inflammatory phenotypes of asthma remains unknown. Induced sputum samples and peripheral blood were collected from participants with asthma (n=36). Airway inflammatory cell counts were performed from induced sputum and inflammatory phenotype assigned based on the airway eosinophil and neutrophil cut-offs of 3% and 61%, respectively. RNA was extracted from whole blood and gene expression profiles were generated (Illumina Humanref-8 V3) and analysed using GeneSpring GX11. There were six genes classified as differentially expressed between the four asthma phenotypes, including the α-defensins (DEFA) 1, 1B, 3 and 4 and neutrophil proteases cathepsin G (CTSG) and elastase (ELA2). Systemic expression of DEFA1,1B,3,4,CTSG and ELA2 was significantly higher in the neutrophilic asthma (NA) phenotype. Microarray results of the α-defensins and neutrophil proteases were successfully validated using real-time PCR. Plasma elastase was significantly increased in people with neutrophilic airway inflammation. There is systemic upregulation of α-defensin and neutrophil protease expression in NA, which may represent proinflammatory effects on the bone marrow and the release of immature neutrophils into the circulation. This demonstrates a systemic inflammatory component in NA that further differentiates this from other asthma phenotypes and indicates different mechanisms in NA.

Alpha-1 Antitrypsin is Markedly Decreased Following Pulmonary F. tularensis Challenge

Neutrophils form the first line of defense during infection and are indispensable in this function. The neutrophil elastase is a key effector molecule of the innate immune system with potent antimicrobial activity against Gram-negative bacteria, spirochaetes, and fungi. However, the release of neutrophil elastase during bacterial infection must be checked otherwise its release in the extracellular milieu will result in damage to surrounding tissues. Alpha-1 antitrypsin is a small glycoprotein clade A serpine serine protease inhibitor and has been shown to increase in humans following bacterial and viral infection. Francisella tularensis is a Gram-negative facultative intracellular bacterium and the causative agent of tularemia. Type A strains are the most virulent with an infectious dose as low as 10 colony forming units and a mortality rate of 30–60% among untreated cases of pneumonic tularemia. We report here significant reduction of this major inhibitor of the neutrophil elastase in plasma of F. tularensis LVS and F. tularensis (type A) SCHU S4 infected animals following pulmonary challenge. Associated with an imbalance of protease–antiprotease function at the alveolar level in lungs of infected animals, increased elastase activity was observed in lung lavage fluids accompanied by decrease lung function, i.e., loss of lung elastance with concomitant increase of pulmonary hysteresivity. Consistent with a competent acute phase response following F. tularensis LVS and F. tularensis (type A) SCHU S4 pulmonary challenge and proposed up-regulation of plasma haptoglobin during the course of the acute phase reaction, haptoglobin was observed significantly increased. These data suggest that unchecked neutrophil serine protease activity may arise from F. tularensis targeted reduction of plasma α1-antitrysin promoting lung tissue damage facilitating increased dissemination of this bacterium in infected animals.

Macrophage activity in semen is significantly correlated with sperm quality in infertile men

The presence of leucocytes within semen has the potential to impair sperm function. Neutrophils and macrophages make up 95% of seminal leucocytes, with both having the ability to damage sperm via the generation of reactive oxygen species, proteases and the induction of apoptosis. Existing cytological techniques for quantifying leucocyte activity within semen (peroxidase, CD45) are less than ideal as they merely count the number of leucocytes, rather than assess their activity. Seminal plasma elastase effectively determines neutrophil activity, yet gives no insight into macrophage activity. Neopterin, a molecule released from activated macrophages, may be a useful marker for macrophage activity in the male reproductive tract. To examine this possibility a total of 63 asymptomatic subjects with male factor infertility and 11 fertile controls provided semen samples for measurement of various inflammatory markers. We were able to confirm for the first time that seminal plasma does indeed contain neopterin and that the levels of this macrophage activity marker are threefold higher in infertile than fertile men. Furthermore, seminal plasma neopterin concentration was significantly correlated with sperm oxidative stress, DNA fragmentation (TUNEL) and apoptosis (Annexin V), making it a useful marker of sperm quality. By contrast, seminal plasma elastase showed no correlation with any marker of sperm quality.

Blood hibernation: a novel strategy to inhibit systemic inflammation and coagulation induced by cardiopulmonary bypass

Background Inflammation and coagulation are two intimately cross-linked defense mechanisms of most, if not all organisms to injuries. During cardiopulmonary bypass (CPB), these two processes are activated and interact with each other through several common pathways, which may result in subsequent organ dysfunction. In the present study, we hypothesized that the addition of nitric oxide, prostaglandin E1 (PGE1), and aprotinin to the systemic circulation, hereby referred to as blood hibernation, would attenuate the inflammation and coagulation induced by CPB. Methods Thirty adult mongrel dogs were equally divided into five groups, anesthetized and placed on hypothermic CPB (32°C). Each group received respectively the following treatments: (1) inhalation of 40 ppm nitric oxide; (2) intravenous infusion of 20 ng·kg-1·min-1 of PGE1; (3) 80 000 kallikrein inhibitor units (KIU)/kg of aprotinin; (4) the combination of all three agents (blood hibernation group); and (5) no treatment (control group) during CPB. Activation of leukocyte, platelet, endothelial cell, and formation of thrombin were assessed after CPB. Results As compared with the other four groups, leukocyte counts were higher, while plasma elastase, interleukin-8, CD11b mRNA expression, myeloperoxidase activities and lung tissue leukocyte counts were lower in the blood hibernation group (P &lt;0.05 versus other four groups after CPB). Plasma prothrombin fragment (PTF)1+2, and platelet activation factors were lower, while platelet counts were higher in the blood hibernation group (P &lt;0.05 versus other four groups at 6 and 12 hours after CPB). Electron microscopy showed endothelial pseudopods protrusion, with cell adherence in all four groups except the blood hibernation group where endothelial cells remained intact. Conclusion Blood hibernation, effected by the addition of nitric oxide, PGE1 and aprotinin to the circulating blood during extra-corporeal circulation, was observed to attenuate the inflammation and coagulation induced by cardiopulmonary bypass, most likely by inhibiting the important common intermediates between the two cross-linked processes. Chin Med J 2010;123(13):1741–1747

Branched-Chain Amino Acid Supplementation Before Squat Exercise and Delayed-Onset Muscle Soreness

The authors examined the effect of branched-chain amino acid (BCAA) supplementation on squat-exercise-induced delayed-onset muscle soreness (DOMS) using 12 young, healthy, untrained female participants. The experiment was conducted with a crossover double-blind design. In the morning on the exercise-session day, the participants ingested either BCAA (isoleucine:leucine:valine = 1:2.3:1.2) or dextrin at 100 mg/kg body weight before the squat exercise, which consisted of 7 sets of 20 squats/set with 3-min intervals between sets. DOMS showed a peak on Days 2 and 3 in both trials, but the level of soreness was significantly lower in the BCAA trial than in the placebo. Leg-muscle force during maximal voluntary isometric contractions was measured 2 d after exercise (Day 3), and the BCAA supplementation suppressed the muscle-force decrease (to ~80% of the value recorded under the control conditions) observed in the placebo trial. Plasma BCAA concentrations, which decreased after exercise in the placebo trial, were markedly elevated during the 2 hr postexercise in the BCAA trial. Serum myoglobin concentration was increased by exercise in the placebo but not in the BCAA trial. The concentration of plasma elastase as an index of neutrophil activation appeared to increase after the squat exercise in both trials, but the change in the elastase level was significant only in the placebo trial. These results suggest that muscle damage may be suppressed by BCAA supplementation.

Aprotinin combined with nitric oxide and prostaglandin E1 protects the canine kidney from cardiopulmonary bypass-induced injury☆☆☆

Aprotinin is frequently used to reduce blood loss during cardiac surgery; however, it also causes renal injury. Since aprotinin reduces nitric oxide (NO) and prostaglandin I(2) (PGI(2)), and both cause vasodilation and inhibit activation of neutrophils and platelets, their reduction may be responsible for the injury. This study was to determine whether the combination of aprotinin with NO and prostaglandin E(1) (PGE(1), an analogue of PGI(2)) can attenuate renal injury associated with aprotinin during cardiopulmonary bypass (CPB). Thirty mongrel dogs were equally divided into five groups, with each group receiving CPB and aprotinin, NO, PGE(1), a combination of the three or no treatment (control). Serum creatinine and creatinine clearance were determined. To elucidate the mechanism, neutrophil, platelet and thrombin activations were also assessed. After CPB, serum creatinine increased and creatinine clearance decreased in all dogs. These changes were similar among the NO, PGE(1), aprotinin and control groups, but were significantly smaller in the combination group. Similarly, myeloperoxidase activities in tissues, CD11b expression, plasma elastase, prothrombin fragment (PTF) 1+2 and platelet activation factor were lower, whereas neutrophil and platelet counts were higher in the combination group than in the other groups (P<0.05). Aprotinin combined with NO and PGE(1) produced synergistic protective effects and improved renal function, due partly to inhibition of platelet and neutrophil activation and suppression of thrombin formation.

Equine Neutrophil Elastase in Plasma, Laminar Tissue, and Skin of Horses Administered Black Walnut Heartwood Extract (BWHE)

Equine Neutrophil Elastase in Plasma, Laminar Tissue, and Skin of Horses Administered Black Walnut Heartwood Extract (BWHE)

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

Equine neutrophil elastase in plasma, laminar tissue, and skin of horses administered black walnut heartwood extract

Laminitis is a local manifestation of a systemic inflammatory response that is characterized by neutrophil activation and movement of neutrophils into the laminar tissues. Given the evidence for the involvement of neutrophils in the development of laminitis, we measured concentrations of neutrophil elastase, a serine protease released from the azurophilic granules of neutrophils, in plasma, skin and laminar tissues obtained from control horses and horses given black walnut heartwood extract (BWHE) to induce laminitis. Healthy horses (5–15 years old) were randomly assigned to 4 groups: 3 experimental groups given BWHE via nasogastric tube, and a control group given an equal volume of water. The experimental groups consisted of horses euthanized 1.5 h ( n = 5), 3 h ( n = 6) or 12 h ( n = 10) after BWHE administration. Control horses ( n = 7) were euthanized 12 h after intragastric administration of water. Plasma samples were collected in all horses of the control and 12 h BWHE groups at 0, 1, 2, 3, 4, 6, 8, 10, and 12 h after treatment, and laminar tissue and skin from the middle region of the neck were harvested at the time of euthanasia in all 1.5 and 3 h BWHE horses, in 6 of the 12 h BWHE horses and in 5 of the control horses. Plasma and tissue concentrations of neutrophil elastase were determined using an equine specific ELISA, and statistical significance was set at p < 0.05. Plasma concentrations of neutrophil elastase in the BWHE group were significantly higher at 6 and 8 h compared to the control group and at 8 and 10 h compared to time 0. Concentrations of neutrophil elastase in skin and laminar tissue were significantly higher in the 3 and 12 h BWHE groups compared to the control group. Concentrations of neutrophil elastase were significantly higher in the skin than in the lamina in the 12 h BWHE horses. The administration of BWHE thus results in significant increases in the concentration of neutrophil elastase in the circulation, skin and laminar tissue. These results confirm a role for neutrophils in the developmental phase of laminitis, and the systemic nature of the inflammatory process. Furthermore, neutrophil elastase may play a key role in the disintegration of the hoof basal membrane and be a target for the development of new treatments for laminitis.