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Keywords:

  • horse;
  • equine physiology;
  • intensive exercise;
  • elastase;
  • myeloperoxidase;
  • inflammation

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. Manufacturers' addresses
  10. References

Reasons for performing study: Intensive exercise induces a systemic inflammatory response characterised by an increase of blood neutrophil count and myeloperoxidase (MPO) release. Neutrophil elastase (NE) could also contribute to tissues lesions by its proteinase activities.

Objective: To compare plasmatic NE concentrations before and after different forms of intensive exercise.

Materials and methods: EDTA blood samples were taken from 51 eventing horses (EvH) and 32 endurance horses (EndH) were sampled before the race (T0). Blood sampling was performed 2 h (T1) after completing either phase D of a one or 2 star eventing competition (n = 51), or a 120 or 160 km endurance race (n = 32). Plasmatic NE and MPO were measured by a specific equine ELISA. Neutrophil counts and creatine kinase (CK) levels were also measured. A Wilcox on test for paired samples was used to compare mean values of neutrophils, CK, MPO and NE at T0 and T1 in EvH and in EndH. Correlations were calculated between all the 4 parameters in EvH and EndH.

Results: At T0, mean NE levels were 14.43 ± 3.63 ng/ml for EvH and 11.7 ± 2.11 ng/ml for EndH. The competition induced a significant increase of NE levels in (58.57 ± 24.06 ng/ml) EvH and (95.74 ± 22.70 ng/ml) EndH (P<0.05). NE was significantly (P<0.0001) correlated to MPO in EvH (r = 0.293) and EndH (r = 0.594) and to CK (r = 0.297) in EndH (P<0.0001). Neutrophils, CK and MPO were significantly increased between T0 and T1 in both types of horses.

Conclusions: Significant increase of NE (EndH) was observed after intense exercise with a significant correlation between NE and MPO. The huge variability in MPO and NE indicates that not all horses show the same intensity of systemic inflammatory response.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. Manufacturers' addresses
  10. References

Ultra-endurance exercise, such as an Ironman triathlon, induces muscle damage and a systemic inflammatory response (Neubauer et al. 2008). Prolonged strenuous physical exercise also attenuates many components of immunity in man (Pedersen and Hoffman-Goetz 2000; Malm 2004; Gleeson 2007). It was shown that intense physical exercise can induce the degranulation of neutrophils that leads to an increase in plasma concentration of neutrophil marker proteins including myeloperoxidase (MPO) and elastase (NE) (Kokot et al. 1988; Camus et al. 1998; Gleeson et al. 1998; Walsh et al. 2000; Morozov et al. 2001,2006). The extent of degranulation tends to increase with increasing intensity of exercise (Peake et al. 2004).

The knowledge of exercise-induced inflammatory reaction in horses is sparse even if horses are particularly sensitive and exposed to excessive inflammatory responses (De la Rebière de Pouyade et al. 2009a). Neutrophil contribution to the acute inflammatory processes may lead to an excessive generation of reactive oxygen metabolites species (ROS) and secretion of granule enzymes (Franck et al. 2009). Consequently, the activation of neutrophils is one of the key events of the inflammatory process. MPO is considered as a valuable marker of this activation (Franck et al. 2005).

Some studies have demonstrated the effect of strenuous exercise on the innate immune system in horses (Robson et al. 2003; Donovan et al. 2007). These studies demonstrated an impairment of the neutrophil function that could persist for several days after intense exercise. Further, increased levels of circulating MPO have been shown to occur in the post exercise period in eventing and endurance horses (Art et al. 2006). In this last study, Art and collaborators found a positive correlation between MPO and neutrophil counts and between MPO and creatine kinase (CK) levels in endurance horses.

Elastase is the second neutrophil enzyme of the inflammatory reaction studied in man. NE released by activated neutrophils is recognised as an inflammatory and prognostic marker in various conditions (Gross et al. 1993; Zorn et al. 2003; Braga et al. 2006; Langhorst et al. 2008) and is implicated in inflammatory tissue damage (Jochum et al. 1994; Ginsburg 1999). This enzyme also plays a role in ischaemia-reperfusion injuries (Bzeizi et al. 1996; Okajima et al. 2004; Aoki et al. 2005; De la Rebière de Pouyade et al. 2009b).

Elastase was regularly pointed out in lung pathology in man and horses for its elastinolytic properties (Dubin et al. 1994; Dagleish et al. 1999). Despite its wellknown proteinolyticproperties, the role of NE is probably underestimated (De la Rebière de Pouyade et al. 2009b). Most studies concerning NE in animals were performed using NE activity assays and, therefore, cannot avoid the influence of its interaction with inhibitors. The purification of this enzyme was only recently made possible in horses from small samples of blood (De la Rebière de Pouyade et al. 2009a). An ELISA assay specific for equine NE was developed (De la Rebière de Pouyade et al. 2009b).

A better description of the exercise-induced inflammatory reaction and in particular of the proteolytic activity of neutrophil enzymes, could help identify a pathogenic process potentially leading to injuries. The aim of the study was to compare plasma NE concentration before and after 2 different forms of intensive exercise, either an endurance race or phase D of a one or 2 star eventing competition and to evaluate its correlation with MPO.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. Manufacturers' addresses
  10. References

Horses and sampling

Venous blood samples were obtained by jugular puncture and collected into 2 tubes containing EDTA (1 mg/ml) and one dry tube. The first EDTA tube was used to determine the neutrophil counts. The blood of the second EDTA tube was centrifuged at 1000 g for 10 min, then the plasma was aliquoted and frozen at -80°C until MPO and NE were assayed. The serum collected in the dry tube was used to determine creatine kinase (CK) level. Blood samples were taken from 51 eventing horses (EvH) and 32 endurance horses (EndH) were sampled before the race (T0). Blood sampling was performed 2 h (T1) after completing either phase D of an eventing competition or endurance race of 120 or 160 km. Horses were regularly trained sport horses aged ≥7 years.

Neutrophil counts and creatine kinase

Haematology testing was limited to neutrophil counts. Serum activity of CK was measured using a colorimetric method (Cobas-Roche)1.

MPO and NE assays

Sandwich ELISA for neutrophil myeloperoxydase: Concentration of MPO was determined by a commercial ELISA2 developed by Franck et al. (2005). The primary antibody, rabbit IgG against MPO, was coated onto microplate wells (Cliniplate EB)3. Equine MPO standards ranging from 0.78–50 ng/ml and EDTA plasma diluted 40 times were added (100 µl) into the wells, and microplates were incubated overnight at 4°C. After the plates were washed in 0.9% NaCl solution containing 0.1% Tween 204, the immobilised antibody–antigen complexes were incubated for 2 h at 37°C with the secondary antibody, guinea pig IgG against equine MPO labelled with alkaline phosphatase. After another washing, phosphatase activity was determined by incubation for 30 min at 37°C in the dark with paranitrophenyl phosphate-stabilised solution. The reaction was stopped with 2.5 mol/l NaOH and the absorbance at 405 nm was read with the Multiscan Ascent plate reader3. The absorbance was directly proportional to the MPO captured by the primary antibody and therefore to the concentration of MPO in the sample. Each sample was assayed twice and the mean value calculated.

Sandwich ELISA for neutrophil elastase: A specific ELISA for equine neutrophil elastase was recently developed by De la Rebière de Pouyade et al. (2009b) and commercialised by BiopTis2. Briefly, the microplate wells (Nunc MaxiSorp)5 were coated (overnight, at 4°C) with 150 ml of the rabbit anti-NE IgG solution (primary antibody). Several concentrations (0.5, 1, 2, 3, 4 and 5 mg/ml) of the primary antibody dissolved in the coating buffer (10 mmol/l phosphate, 137 mmol/l NaCl and 2.7 mmol/l KCl, pH 7.4) were tested to select the optimal (saturating) concentration for further use in the ELISA. After the primary antibody coating, the plates were washed 4 times with 300 ml of the first washing buffer (154 mmol/l NaCl solution with 0.1% Tween 20). Blocking buffer (200 µl coating buffer with 5 g/l BSA) was then added and the plates incubated for 150 min at room temperature (20°C). After 4 washes with the first washing buffer, equine elastase standards ranging from 1.875–60 ng/ml and EDTA plasma diluted 8× were added (100 µl) into the wells, and microplates were incubated overnight at 4°C. Control (blank) and dilutions of the samples were made with the dilution buffer (blocking buffer added with 0.1% Tween 20). After 4 washes with 300 ml of the second washing buffer (150 mmol/l NaCl, 50 mmol/l Tris–HCl, 0.1% Tween 20, pH 7.5), the plates were incubated (2 h, 37°C) with 100 ml (3 mg/ml) of the secondary antibody conjugated to AP and diluted with the second washing buffer. After washing (second washing buffer), phosphatase activity was detected by incubation (30 min, 37°C, in the dark) with the substrate paranitrophenyl phosphate. The reaction was stopped with 2.5 mol/l NaOH and the absorbance read at 405 nm. NE concentrations in samples were determined according to the standard curve made with equine elastase.

Intra- and interassay coefficients of variation (CVs) were previously determined with EDTA plasma. For the elastase assay, intra- and interassay CVs did not exceed 10 and 12%, respectively (De la Rebière de Pouyade et al. 2009b) and for the MPO assay, did not exceed 8 and 9%, respectively (Franck et al. 2005).

Statistics

The mean ± standard errors (s.e.) were calculated. The parametric distribution of the data was tested by a Levene's test and a Wilcoxon test for paired samples used to evaluate the effect of the time of sampling on the parameters: neutrophils, CK, MPO and NE. The level of significance was set at P<0.05.

Kendall Tau rank correlation coefficients were calculated for all four parameters in EndH and EvH as well as their significance levels. For all the correlation tests, the level of significance was set at P<0.0001.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. Manufacturers' addresses
  10. References

Data followed a nonparametric distribution except for the neutrophil counts. Therefore, a nonparametric analysis was performed.

At T0, mean NE levels were 14.43 ± 3.63 ng/ml for EvH and 11.7 ± 2.11 ng/ml for EndH. The competition induced a significant (P<0.05) increase of NE levels in EvH (58.57 ± 24.06 ng/ml) and EndH (95.74 ± 22.7) (Fig 1).

image

Figure 1. Plasma elastase (NE) concentrations in 51 eventing horses (EvH) and 32 endurance horses (EndH) before (T0) and 2 h after (T1) either phase D of an eventing competition or an endurance race. NE levels at T0 are significantly different from NE levels at T1 in EvH (*) and in EndH (**) (P<0.05).

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Neutrophil elastase was significantly (P<0.0001) correlated to MPO in EvH (r = 0.293) and EndH (0.594) and to CK (r = 0.297) in EndH (P<0.0001). Additional results concerned neutrophils, CK and MPO. All these parameters were significantly increased between T0 and T1 in both types of horses. The means of all the measured parameters are presented in Table 1 and the correlation coefficients are presented in Table 2.

Table 1. Neutrophil counts, creatine kinase serum concentration myeloperoxidase and elastase plasma concentrations in 51 eventing horses and 32 endurance horses before (T0) and 2 h after (T1) either phase D of an eventing competition or an endurance race
ParametersT0T1
Eventing horsesEndurance horsesEventing horsesEndurance horses
  1. Data are given as mean ± s.e.

Neutrophil counts (109/l)4.63 ± 0.245.53 ± 0,425.59 ± 0.3012.60 ± 0,46
Creatine Kinase (iu/l)179.42 ± 11.63382.17 ± 159.48269.04 ± 34.587185.09 ± 1253.91
Myeloperoxydase (µg/l)144.64 ± 13.33146.98 ± 16.54365.75 ± 51.211041.82 ± 225.48
Elastase (µg/l)14.43 ± 3.6311.70 ± 2.1158.57 ± 24.0695.74 ± 22.70
Table 2. Correlation coefficients and their significance between neutrophil counts, creatine kinase (CK) serum concentration and myeloperoxidase (MPO) and elastase (NE) plasma concentrations in 51 eventing horses (a) and 32 endurance horses (b)
HorsesNeutrophilsCKMPONE
  • *

    Significant correlations are indicated by a (P<0.0001).

a) Eventing    
 Neutrophils 0.338*0.3939*0.103
 CK0.338* 0.426*0.111
 MPO0.3939*0.426* 0.293*
 NE0.1030.1110.293* 
b) Endurance    
 Neutrophils 0.61*0.395*0.293*
 CK0.61* 0.448*0.297*
 MPO0.395*0.448* 0.594*
 NE0.293*0.297*0.594* 

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. Manufacturers' addresses
  10. References

It is well known that exercise causes an increase in CK plasmatic concentration (Castejón et al. 2006). In equine sports medicine, it is generally assumed that the increase in muscular enzymatic level after exercise is associated with changes in muscular membrane permeability, while in human sports medicine, increases in CK level are associated with muscular damages (Suzuki et al. 1999; Art et al. 2006). The level and type of training of the horse could also influence the intensity and duration of increase of CK concentration (Lindner et al. 2006) as observed in human medicine (Machado et al. 2009).

To the authors' knowledge, this is the first report regarding changes in concentrations of NE in blood relative to intensive exercise in horses. A significant increase in plasma NE was found in horses consecutive to a 120 km or a 160 km race or to phase D of a one or 2 star eventing competition.

Elastase, a serine protease, is widely recognised as a component and marker of inflammatory disorders (Gross et al. 1993; Jochum et al. 1994; Zorn et al. 2003; Langhorst et al. 2008). NE plays a role in the oxygen-independent microbiocidal pathway, and contributes to the tissue remodelling that occurs after injuries (Shapiro 2002; Chua and Laurent 2006). NE is able to digest elastin and other extracellular compounds (Shapiro 2002) and may also promote the extravasation of the PMNs through the endothelial barrier (Scholz et al. 2003). NE is known to have substrates other than elastin (Dagleish et al. 1999). It may also take part in the activation process of MMP-9 (Ferry et al. 1997). Higher levels of NE were found in the bronchoalveolar lavage fluid of horses with recurrent airway obstruction (Brazil et al. 2005), in the plasma of horses suffering from intestinal pathologies (De la Rebière de Pouyade et al. 2009b) and in ischaemia-reperfusion injuries (Okajima et al. 2004; Aoki et al. 2005). Consequently, elastase seems to take part in the pathological response of neutrophils during the inflammatory process.

The effect of intensive exercise in horses seems to follow the same profile as in man. Eventing and endurance races produce a significant increase of NE concentration.

Significant increase of NE was observed after intense exercise with a significant correlation between NE and MPO. The huge variability in NE indicates that not all horses show the same intensity of systemic inflammatory response. This could partly be explained by the fact that not all horses produce the same effort during the same competition, because of their innate qualities or their level of training. It could be interesting to perform further studies with experimental horses in order to monitor objectively their training and effort during the race. The inflammatory reaction is, however, evident. In this study, 2 different types of exercise are observed. Phase D of the cross country was short in duration and high in intensity, while the endurance race was long in duration and moderate in intensity.

For the observations relative to neutrophil counts, CK and MPO, our study confirms the results of Art et al. (2006). Significant increases (P<0.05) of those 3 parameters were observed in EvH and EndH. Even if the horses performing both type of exercise are not the same, the fact that neutrophils, CK, MPO and NE levels increase more in EndH could suggest that the endurance effort is more relevant concerning the intensity of the inflammatory reaction.

In endurance horses, Art et al. (2006) found a correlation between MPO and CK levels. In our study, we found a significant positive correlation between NE and MPO both in EvH and EndH as well as a significant positive correlation between NE and CK in EndH.

Because MPO release occurs prior to that of NE in stimulated neutrophils, an interaction between both enzymes could be suggested. MPO may yield an environment that favours the activity of serine proteases like NE (De la Rebière de Pouyade et al. 2009c).

The significant increase of MPO and NE plasma concentrations confirms the fact that intense exercise induces a neutrophil activation with degranulation and release of oxidative and proteolytic enzymes.

Further studies need to investigate the potential deleterious effects of elevated levels of MPO and NE in equine plasma after an intense exercise. The positive correlation between these enzymes concentrations and CK levels observed in this study could lead us to pay a particular attention in a potential localisation and action of these oxidative and proteolytic enzymes in muscles. As in human medicine (Neubauer et al. 2008), the possible use of these biomarkers has to be explored in order to allow the monitoring of the recovery period and finally predict the ability of the horses to undergo a new phase of training and competition.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. Manufacturers' addresses
  10. References

The authors wish to express their sincere gratitude to the riders and owners of horses. This work was supported by ‘Ministère de l'Agriculture et de la Ruralité de la Région Wallonne’ of Belgium.

Manufacturers' addresses

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. Manufacturers' addresses
  10. References

1 Roche Diagnostics, Vilvoorde, Belgium.

2 BiopTis SA, Liège, Belgium.

3 Thermo Labsystems, Helsinki, Finland.

4 Sigma Chemical Company, St Louis, Missouri, USA.

5 Thermo Scientific, Roskilde, Denmark.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. Manufacturers' addresses
  10. References
  • Aoki, T., Tsuchida, M., Takekubo, M., Saito, M., Sato, K. and Hayashi, J. (2005) Neutrophil elastase inhibitor ameliorates reperfusion injury in a canine model of lung transplantation. Eur. Surg. Res. 37, 274-280.
  • Art, T., Franck, T., Gangl, M., Votion, D., Kohnen, S., Deby-Dupont, G. and Serteyn, D. (2006) Plasma concentrations of myeloperoxidase in endurance and 3-day event horses after a competition. Equine vet. J., Suppl. 36, 298-302.
  • Braga, P.C., Dal Sasso, M., Culici, M., Bianchi, T., Bordoni, L. and Marabini, L. (2006) Anti-inflammatory activity of thymol: inhibitory effect on the release of human neutrophil elastase. Pharmacol. 77, 130-136.
  • Brazil, T.J., Dagleish, M.P., McGorum, B.C., Dixon, P.M., Haslett, C. and Chilvers, E.R. (2005) Kinetics of pulmonary neutrophil recruitment and clearance in a natural and spontaneously resolving model of airway inflammation. Clin. exp. Allergy 35, 854-865.
  • Bzeizi, K.I., Jalan, R., MacGregor, I., Drummond, O., Lee, A. and Hayes, P.C. (1996) Neutrophil elastase: A determinant of endothelial damage and reperfusion injury after liver transplantation? Transplantation 62, 916-920.
  • Camus, G., Nys, M., Poortmans, J., Venneman, I., MonWls, T., Deby-Dupont, G., Juchmes-Ferir, A., Deby, C., Lamy, M. and Duchateau, J. (1998) Possible in vivo tolerance of human polymorphonuclear neutrophil to low-grade exercise-induced endotoxaemia. Mediators Inflamm. 7, 413-415.
  • Castejón, F., Trigo, P., Muñoz, A. and Riber, C. (2006) Uric acid responses to endurance racing and relationships with performance, plasma biochemistry and metabolic alterations. Equine vet. J., Suppl. 36, 70-73.
  • Chua, F. and Laurent, G.J. (2006) Neutrophil elastase: mediator of extracellular matrix destruction and accumulation. Proc. Am. Thorac. Soc. 3, 424-427.
  • Dagleish, M., Pemberton, A., Brazil, T., McAleese, S., Miller, H. and Scudamor, C. (1999) Kinetics of equine neutrophil elastase release and superoxide anion generation following secretagogue activation: a potential mechanism for antiproteinase inactivation. Vet. Immunol. Immunopathol. 72, 257-275.
  • De la Rebière de Pouyade, G., Franck, T., Salciccia, A., Deby-Dupont, G., Grulke, S., Vander Heyden, L., Sandersen, C. and Serteyn, D. (2009b) Development of an enzyme-linked immunosorbent assay for equine neutrophil elastase measurement in blood: Preliminary application to colic cases. Vet. Immunol. Immunopathol. 135, 282-288.
  • De la Rebière de Pouyade, G., Riggs, L.M., Moore, J.N., Franck, T., Deby-Dupont, G., Hurley, D.J. and Serteyn, D. (2009c) Equine neutrophil elastase in plasma, laminar tissue, and skin of horses administered black walnut heartwood extract. Vet. Immunol. Immunopathol. 135, 181-187.
  • De la Rebière de Pouyade, P.G., Serteyn, D., Deby-Dupont, G. and Franck, T. (2009a) Method for co-purification of equine neutrophil elastase and myeloperoxidase from a limited blood volume. Res. vet. Sci. 87, 358-363.
  • Donovan, D.C., Jackson, C.A., Colahan, P.T., Norton, N.N., Clapper, J.L., Moore, J.N. and Hurley, D.J. (2007) Assessment of exercise-induced alterations in neutrophil function in horses. Am. J. vet. Res. 68, 1198-1204.
  • Dubin, A., Potempa, J. and Travis, J. (1994) Structural and functional characterization of elastases from horse neutrophils. Biochem. J. 300, 401-406.
  • Ferry, G., Lonchampt, M., Pennel, L., De Nanteuil, G., Canet, E. and Tucker, G.C. (1997) Activation of MMP-9 by neutrophil elastase in an in vivo model of acute lung injury. FEBS Lett. 402, 111-115.
  • Franck, T., Grulke, S., Deby-Dupont, G., Deby, C., Duvivier, H., Peters, F. and Serteyn, D. (2005) Development of an enzyme-linked immunosorbent assay for specific equine neutrophil myeloperoxidase measurement in blood. J. Vet. Diagn. Invest. 17, 412-419.
  • Franck, T., Kohnen, S., Rebière, G., Deby-Dupont, G., Deby, C., Niesten, A. and Serteyn, D. (2009) Activation of equine neutrophils by phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine induces a different response in reactive oxygen species production and release of active myeloperoxidase. Vet. Immunol. Immunopathol. 130, 243-250.
  • Ginsburg, I. (1999) Multi-drug strategies are necessary to inhibit the synergistic mechanism causing tissue damage and organ failure in post infectious sequelae. Inflammopharmacol. 7, 207-217.
  • Gleeson, M. (2007) Immune function in sport and exercise. J. appl. Physiol. 103, 693-699.
  • Gleeson, M., Walsh, N., Blannin, A., Robson, P., Cook, L., Donnelly, A. and Day, S. (1998) The effect of severe eccentric exercise-induced muscle damage on plasma elastase, glutamine, and zinc concentrations. Eur. J. appl. Physiol. Occup. Physiol. 77, 543-546.
  • Gross, V., Leser, H.G., Heinisch, A. and Scholmerich, J. (1993) Inflammatory mediators and cytokines-new aspects of the pathophysiology and assessment of severity of acute pancreatitis? Hepatogastroenterol. 40, 522-530.
  • Jochum, M., Gippner-Steppert, C., Machleidt, W. and Fritz, H. (1994) The role of phagocyte proteinases and proteinase inhibitors in multiple organ failure. Am. J. Respir. crit. care Med. 150, S123-S130.
  • Kokot, K., Schaefer, R.M., Teschner, M., Gilge, U., Plass, R. and Heidland, A. (1988) Activation of leukocytes during prolonged physical exercise. Adv. exp. Med. Biol. 240, 57-63.
  • Langhorst, J., Elsenbruch, S., Koelzer, J., Rueffer, A., Michalsen, A. and Dobos, G.J. (2008) Noninvasive markers in the assessment of intestinal inflammation in inflammatory bowel diseases: Performance of fecal lactoferrin, calprotectin, and PMN-elastase, CRP, and clinical indices. Am. J. Gastroenterol. 103, 162-169.
  • Lindner, A., Signorini, R., Brero, L., Arn, E., Mancini, R. and Enrique, A. (2006) Effect of conditioning horses with short intervals at high speed on biochemical variables in blood. Equine vet. J., Suppl. 36, 88-92.
  • Machado, M. and Willardson, J. (2009) Short recovery augments the magnitude of muscle damage in high responders. Med. Sci. Sports Exerc. (Abstract) [Epub ahead of print.
  • Malm, C. (2004) Exercise immunology: The current state of man and mouse. Sports Med. 34, 555-566.
  • Morozov, V.I., Usenko, T.N. and Rogozkin, V.A. (2001) Neutrophil antiserum response to decrease in proteolytic activity in loaded rat muscle. Eur. J. appl. Physiol. 84, 195-200.
  • Morozov, V.I., Tsyplenkov, P.V., Golberg, N.D. and Kalinski, M.I. (2006) The effects of high-intensity exercise on skeletal muscle neutrophil myeloperoxidase in untrained and trained rats. Eur. J. appl. Physiol. 97, 716-722.
  • Neubauer, O., König, D. and Wagner, K. (2008) Recovery after an Ironman triathlon: sustained inflammatory responses and muscular stress. Eur. J. appl. Physiol. 104, 417-426.
  • Okajima, K., Harada, N., Uchiba, M. and Mori, M. (2004) Neutrophil elastase contributes to the development of ischemia-reperfusion-induced liver injury by decreasing endothelial production of prostacyclin in rats. Am. J. Physiol. Gastrointest. Liver Physiol. 287, G1116-G1123.
  • Peake, J., Wilson, G., Hordern, M., Suzuki, K., Yamaya, K., Nosaka, K., Mackinnon, L. and Coombs, J.S. (2004) Changes in neutrophil surface receptor expression, degranulation, and respiratory burst activity after moderate- and high-intensity exercise. J. appl. Physiol. 97, 612-618.
  • Pedersen, B.K. and Hoffman-Goetz, L. (2000) Exercise and the immune system: Regulation, integration, and adaptation. Physiol. Rev. 80, 1055-1081.
  • Robson, P.J., Alston, T.D. and Myburgh, K.H. (2003) Prolonged suppression of the innate immune system in the horse following an 80 km endurance race. Equine vet. J. 35, 133-137.
  • Scholz, M., Wimmer-Greinecker, G., Simon, A., Dzemali, O., Chang, H.Y., Kleine, P., Matheis, G. and Moritz, A. (2003) Perioperative elastase activity in cardiac surgery and its role in endothelial leakage. Inflamm. Res. 52, 433-438.
  • Shapiro, S.D. (2002) Neutrophil elastase: Path clearer, pathogen killer, or just pathologic? Am. J. Respir. cell mol. Biol. 26, 266-268.
  • Suzuki K., Totsuka M., Nakaji S., Yamada M., Kudoh S., Lui Q., Sugawara K., Yamaya K., Sato K. (1999) Endurance exercise causes interaction among stress hormones, cytokines, neutrophil dynamics, and muscle damage. J. appl. Physiol. 87, 1360-1367.
  • Walsh, N., Blannin, A., Bishop, N., Robson, P. and Gleeson, M. (2000) Effect of oral glutamine supplementation on human neutrophil lipopolysaccharide- stimulated degranulation following prolonged exercise. Int. J. Sport Nutr. Exerc. Metab. 10, 39-50.
  • Zorn, B., Sesek-Briski, A., Osredkar, J. and Meden-Vrtovec, H. (2003) Semen polymorphonuclear neutrophil leukocyte elastase as a diagnostic and prognostic marker of genital tract inflammation–a review. Clin. Chem. Lab. Med. 41, 2-12.