Validation of masks for determination of V̇O2 max in horses exercising at high intensity.

BACKGROUND: The need for a horse to be ridden while wearing a measurement device that allows unrestricted ventilation and gas exchange has hampered accurate measurement of its maximal oxygen consumption (V̇O2 max) under field conditions. OBJECTIVES: Design and validate a facemask with the potential to measure V̇O2 max accurately in the field. STUDY DESIGN: Experiment with 6 × 6 Latin square design. METHODS: Two variations of a mask and associated electronic control module (ECM) were designed to enable breath-by-breath measurement of airflows through two 7.8 cm diameter pneumotachometers located 7.5 cm in front of each narus. The ECM was comprised of an analogue-to-digital converter and a lithium-ion battery that provided power and signal filtering to the pneumotachometers and an oxygen sensing cell, and powered a pump connected to gas sampling ports between the nares and pneumotachometers. Airflow and oxygen content of inspired and expired gases were recorded through the ECM and electronically transferred to a notebook. V̇O2 was determined from these recordings using a customised software program. Mask B encased the lower jaw. Mask R left the jaw free so the horse could wear a bit if ridden. V̇O2 max and arterial blood gases were measured in 6 horses during multiple treadmill tests. Each mask was worn twice and results compared to those from an established open flow-through system (O) by ANOVA-RM (P<0.05). System utility was evaluated using the intraclass correlation coefficient of 4 independent raters. RESULTS: Blood gases and V̇O2 max (151.9±7.0 [mean±s.d.; O], 151.5±9.6 [B], 149.5±7.5 [R] ml/[kg.min]) were not different between masks. V̇O2 max measures were reproducible for each mask. Intraclass correlation coefficient between raters = 0.99. MAIN LIMITATIONS: Some rebreathing of expired air from mask dead space. CONCLUSION: Masks capable of measuring V̇O2 max during treadmill exercise were developed, tested and found to be accurate. Mask R has potential application to measurement of V̇O2 max under field conditions.

Reliability of breath by breath spirometry and relative flow-time indices for pulmonary function testing in horses.

BACKGROUND: Respiratory problems are common in horses, and are often diagnosed as a cause of poor athletic performance. Reliable, accurate and sensitive spirometric tests of airway function in resting horses would assist with the diagnosis of limitations to breathing and facilitate investigations of the effects of various treatments on breathing capacity. The evaluation of respiratory function in horses is challenging and suitable procedures are not widely available to equine practitioners. The determination of relative flow or flow-time measures is used in paediatric patients where compliance may limit conventional pulmonary function techniques. The aim of the current study was to characterise absolute and relative indices of respiratory function in healthy horses during eupnoea (tidal breathing) and carbon dioxide (CO2)-induced hyperpnoea (rebreathing) using a modified mask pneumotrachographic technique well suited to equine practice, and to evaluate the reliability of this technique over three consecutive days. Coefficients of variation, intra-class correlations, mean differences and 95% confidence intervals across all days of testing were established for each parameter. RESULTS: The technique provided absolute measures of respiratory function (respiratory rate, tidal volume, peak inspiratory and expiratory flows, time to peak flow) consistent with previous studies and there was no significant effect of day on any measure of respiratory function. Variability of measurements was decreased during hyperpnea caused by rebreathing CO2, but a number of relative flow-time variables demonstrated good agreement during eupnoeic respiration. CONCLUSIONS: The technique was well tolerated by horses and study findings suggest the technique is suitable for evaluation of respiratory function in horses. The use of relative flow-time variables provided reproducible (consistent) results, suggesting the technique may be of use for repeated measures studies in horses during tidal breathing or rebreathing.

The effect of prone positioning with surgical bolsters on liver blood flow in healthy volunteers.

This study sought to identify changes in hepatic flood flow and cardiac output during prone positioning on surgical bolsters in awake volunteers, and was prompted by a local incident of significant hepatic dysfunction following surgery in the prone position. Cardiac output was determined using the non-invasive Peñáz technique, and plasma disappearance rate of indocyanine green (ICG-PDR) was measured as a surrogate maker for hepatic blood flow along with serum hepatic enzyme assays. Measurements were made after one hour in supine, prone and returned supine positions. Ten volunteers completed the study. There were significant changes in the disappearance rate of indocyanine green, which decreased this from mean (SD) 31.1 (9.70) supine to 19.6 (4.37)%.min prone, respectively (p = 0.02), increasing on return to the supine position to 24.6 (5.54)%.min (p = 0.019). Cardiac output was also significantly reduced when changing from the supine to the prone position, from mean (SD) 4.7 (1.0 to 3.5 (1.1) (l.min(-1) ), respectively (p = 0.002). We demonstrated an acute and reversible change in both hepatocellular function and cardiac output associated with the prone position.

Number, causes and destinations of horses leaving the Australian Thoroughbred and Standardbred racing industries.

OBJECTIVE: Significant proportions of horses leave the Australian Thoroughbred and Standardbred racing industries, which has ramifications for both the economic sustainability and the public perception of racing. The aim of this study was to quantify potential horse wastage, describe the destinations of exiting horses and identify risk factors for horses going to these destinations. METHODS: Questionnaires were sent to 1258 selected Thoroughbred and 981 Standardbred trainers, with response rates of 30% and 32%, respectively. The survey investigated the role of various risk factors for wastage, including horse age, sex and number of years in training. The destination of departing horses was also examined in relation to these risk factors. RESULTS: Total horse exit rates for the 2002-03 official race year were 39.7% and 38.7% for the Thoroughbred and Standardbred racing industries, respectively. Reasons for leaving included 'poor performance/slow' (36.5% Thoroughbreds, 35.2% Standardbreds), 'illness/injury' (31.0%, 27.1%), 'to breed' (9.4%, 10.1%), 'unsuitable temperament/behaviour' (6.4%, 6.4%) and 'other' (16.8%, 21.2%). Statistically significant (P < 0.001) risk factors influencing the destinations of both Thoroughbred and Standardbred racing horses included whether the trainer owned the horses, sex, age and reasons for leaving. In addition, some factors were specific to one breed or the other. CONCLUSIONS: Improved behaviour training and early identification of the causes of poor performance could assist in reducing wastage.

Pulmonary function measurements immediately after exercise are correlated with neutrophil percentage in tracheal aspirates in horses with poor racing performance.

Inflammatory airway disease (IAD) is common in racehorses, and is a cause of wastage in the industry. IAD has been diagnosed by measurement of percent neutrophils (N%) in tracheal aspirates (TA). The aim of this study was to investigate whether spirometric indices of pulmonary function were correlated with N% in TAs. Limits to breathing were measured by analyses of relationships between relative times and relative respiratory gas flows during inspiration and expiration in individual breaths recorded after exercise. Horses with higher N% had significantly lower relative gas flows at the same relative times during inspiration and expiration, suggesting a limit to breathing. These findings confirm a physiological basis for the measurement of N% in TA after exercise for diagnosis of IAD. Spirometric pulmonary function testing using analyses of individual breaths after exercise has application for assessment of pulmonary function and poor exercise performance.

Incidence rate of musculoskeletal injuries and determinants of time to recovery in young Australian Thoroughbred racehorses.

OBJECTIVE: To describe the incidence rate of musculoskeletal (MS) injuries and investigate the factors that influence recovery from MS injury in young Thoroughbred horses in Australia. METHODS: Training and injury data were collected at fortnightly intervals from 14 trainers in New South Wales. Incidence rates were calculated for initial injuries stratified by age-class and sex. Survival analysis methods were used to investigate the time to recovery after the first MS injury (measured as a start in a race or barrier trial). RESULTS: For the 248 racehorses enrolled, there were 428 injuries recorded; 2-year-olds were 2.99-fold more likely than 3-year-olds to sustain an MS injury. The cumulative percentage of horses that had recovered within 6 months was 55%. The time to recovery was longest in horses whose fast gallop speed prior to injury was < 890 m/min. CONCLUSION: This study supports the finding that shin soreness is the most common MS injury in young Australian Thoroughbreds. There was an increased rate of MS injury in 2-year-olds compared with 3-year-olds. However, it was impossible to determine if the increased risk in 2-year-olds was related to age, the start of training or a combination of both. The study also found that horses performing at higher exercise speeds prior to injury were more likely to recover. There was a significant effect of trainer, suggesting that other factors related to trainer may influence the time to the recovery. Additional research is required to understand the determinants of time to recovery.

Profiling training preparation in young Australian Thoroughbred racehorses.

OBJECTIVE: To describe the impact of age, sex, and musculoskeletal (MS) injury on three preparation-level measures of performance: (i) the duration of preparation, (ii) time from the beginning of the preparation until the first start in a race or barrier trial, and (iii) time from the first start until the end of the preparation. METHODS: Daily training and injury data for 2- and 3-year old Thoroughbred racehorses were collected fortnightly from 14 trainers in NSW, Australia. Training data were aggregated into units referred to as 'preparations'. Univariate and multivariate analytical methods were used to examine the associations between the explanatory variables and each of the measures of performance. RESULTS: There were 1274 preparations recorded in 451 horses. Younger horses had a shorter interval from the first start in a race or barrier trial to the end of the preparation. MS injury was not conditionally associated with any of the outcomes. CONCLUSION: MS injuries in young racehorses have limited impact on long-term performance, although care must be taken in interpreting the results because they could be confounded by variables not considered in this analysis. This study also begins to unravel the complicated relationship between age and previous exposure to training, with results supporting the hypothesis that the intensity of training increases with increasing age.

Rat survival to anthrax lethal toxin is likely controlled by a single gene.

We examined whether survival of different rat strains administered anthrax lethal toxin is genetically determined. A reproducible test population of first filial generation hybrid rats was bred based on the susceptibility of progenitors to anthrax lethal toxin and to maximize genetic diversity across the strains. These rats were then tested with varying doses of anthrax lethal toxin. We found that all 'sensitive' strains died within 2 h following systemic administration of 240 mug/kg lethal toxin, while one strain survived following a five times higher dose (1.4 mg/kg). The ability of lethal toxin to lyse macrophage cultures derived from the bone marrow of these strains corresponded with in vivo results. We conclude that a rat test population can detect strain differences in response to anthrax lethal toxin. Survival is influenced by the host genome background and is likely due to a single gene with a recessive mode of inheritance.

Physiology of equine performance and associated tests of function.

This review addresses the physiology of performance horses in the context of functional tests aimed at assisting clinicians in the search for explanations of the individual's limitations, actual or perceived as below expectation. The intention is to assist clinicians who are faced frequently by owners or trainers seeking explanations for poor performance; and for research workers as a basis on which to proceed in their search for new methodologies, particularly those that can be applied in the field as for treadmill exercise. The review sets out fundamental physiological pathways, the entailment of which may represent a limiting factor to performance, while taking into account the individual's status, including anamnesis and the possibility of actual disease being present. Tests that assess the possible roles of several body systems, as causes of limited performance, are discussed. These systems include pulmonary, cardiovascular and neuromuscular systems. The evaluation of anaerobic and aerobic energy pathways is also discussed. Emphasis is given to the assessment of pulmonary ventilation by techniques such as spirometry and ergospirometry. These techniques are considered to be of particular potential for further development.

Paroxetine for prevention of depressive symptoms induced by interferon-alpha and ribavirin for hepatitis C.

BACKGROUND: Whether antidepressants prevent depression during interferon-alpha/ribavirin treatment for hepatitis C virus infection has yet to be established. AIM: To investigate the use of paroxetine in a prospective, double-blind, placebo-controlled study for this indication. METHODS: Sixty-one hepatitis C virus-infected patients were randomly assigned to the antidepressant, paroxetine (n = 28), or placebo (n = 33), begun 2 weeks before and continued for 24 weeks during interferon-alpha/ribavirin treatment. Primary endpoints included development of major depression and severity of depressive symptoms measured by the Montgomery Asberg Depression Rating Scale (MADRS). RESULTS: Rates of major depression during the study were low (17%) and did not differ between groups. Nevertheless, using published MADRS cut-off scores, the percent of subjects who met criteria for mild, moderate or severe depression during interferon-alpha/ribavirin therapy was significantly lower in paroxetine- vs. placebo-treated subjects (P = 0.02, Fisher's exact test). Assignment to paroxetine was also associated with significantly reduced depressive symptom severity. This effect was largely accounted for by participants with depression scores above the median (MADRS > 3) at baseline in whom paroxetine was associated with a maximal reduction in MADRS scores of 10.3 (95% CI: 2.1-18.5) compared with placebo at 20 weeks (P < 0.01). Study limitations included a small sample size and high drop-out rate. CONCLUSION: This double-blind, placebo-controlled trial provides preliminary data in support of antidepressant pre-treatment in hepatitis C virus patients with elevated depressive symptoms at baseline.

Risk factors for musculoskeletal injuries in 2-year-old Thoroughbred racehorses.

Musculoskeletal injuries (MSI) in racehorses have been identified as the most common cause of lost training days and weeks spent resting at pasture. Several training-, track- and horse-related risk factors have been identified. However, there is no clear understanding of the inter-relationship between these factors. The aim of this longitudinal cohort study was to investigate risk factors for MSI in 2-year-old Thoroughbred racehorses. Australian Thoroughbred trainers were convenience sampled and enrolled in a 27-month longitudinal cohort study. The study population consisted of all 2-year-old Thoroughbred racehorses trained by a participating trainer. Horses were followed from the time of enrollment until the completion of the study or until they were lost to follow-up. Trainers were visited at approximately 14-day intervals to collect training and injury data. Training days were categorised as fast days if the maximum speed during training exceeded 800 m/min. For each horse, the first training period that included one or more fast days was analysed. A multiple logistic regression model was used to determine those horse- and training-related variables that were associated with MSI. Data from 274 horses trained by 14 trainers at five racetracks were analysed. Forty percent of the horses sustained a MSI during their first fast preparation. The average distance trained at speeds >or=800 m/min and the percentages of fast days from the first fast day until the end of the preparation were the only variables associated with MSI. After accounting for these variables there were still significant differences between trainers. This suggests that there are other training-related variables such as the rate of increase in distance trained at speeds >or=800 m/min that may be risk factors for MSI. In addition, other factors such as the level of veterinary involvement and the training centre/racetrack may be risk factors for MSI. More analysis using multi-level modelling is required to determine the relationship between trainer- and track-related risk factors.

Measurements of fitness in thoroughbred racehorses using field studies of heart rate and velocity with a global positioning system.

REASONS FOR PERFORMING STUDY: Field based studies of changes in fitness are difficult to conduct in galloping Thoroughbreds. Measurements of heart rate (HR) and real time velocity with a global positioning system (GPS) could provide a method for routine field studies of fitness. OBJECTIVE: To investigate measurements of fitness in the field without using multiple, standardised steps of increasing velocity in the exercise test. METHODS: Twelve healthy 3- to 5-year-old Thoroughbreds were used in the study of reliability, and twelve 2-year-old Thoroughbreds in the investigation of the effect of training. Five second averages of velocity and HR were recorded during a typical fast exercise training session, using a GPS and Polar HR monitor. A standardised exercise test protocol was not used. Regression analyses using trot and gallop data were used to calculate velocities at HR of 200 beats/min (V200), and at maximal HR (VHRmax). Data were collected on consecutive 'fast' day training sessions to assess the reliability of measurements. The effect of training was investigated with fitness tests in weeks 2 and 6 of 'fast' training. Absolute and relative differences were calculated to evaluate reliability, and paired t tests were used to detect an effect of training. RESULTS: VHRmax, V200 and HRmax were reliable measurements of fitness, with mean differences of 2% or less. Reliability of VHRmax was not dependent on VHRmax. VHRmax and V200 both increased significantly with training (P<0.01), but there was no effect of training on HRmax. There were no significant changes in the slope or intercept of the regression equations after training. CONCLUSIONS: Velocity and HR measurements during field gallop exercise provided reliable measures of fitness which enabled a measurement of the response to training. POTENTIAL RELEVANCE: This approach offers a simple, noninvasive method for monitoring adaptations to training in the field.

Correlation of race earnings with velocity at maximal heart rate during a field exercise test in thoroughbred racehorses.

REASONS FOR PERFORMING STUDY: Running ability of Thoroughbred racehorses is correlated with maximal oxygen uptake, and the velocity at maximal oxygen uptake is highly correlated with the velocity at maximal heart rate (VHRmax). OBJECTIVE: To investigate the relationship between VHRmax and racing performance, expressed as 'peak dollars earned per race start'. METHODS: Heart rate (HR) and velocity were recorded in 25 Thoroughbred racehorses during trotting and subsequent fast gallops in the field at velocities of 15-16 m/sec. Velocity was recorded by a global positioning system (GPS). Maximal HR (HRmax) and maximal velocity (Vmax) were identified, and a linear regression of HR on velocity for trotting and galloping data was constructed to derive VHRmax. Horses followed the training programme designed by one trainer, had at least 6 race starts and were clinically sound at the time of testing. Race earnings were expressed as the peak dollars per start in the horse's race career. Data were normalised using the results for the square root of 'peak dollars earned per race start' and the significance of associations between variables was determined by correlation coefficient and least square analyses. RESULTS: Horses with higher VHRmax earned significantly more dollars per race start (r = 0.41, P<0.05), and horses with VHRmax less than 14.5 m/sec had mean earnings of less than A$2500 per race. There were no correlations between race earnings and either HRmax or Vmax. CONCLUSION: Field studies of the relationship between HR and velocity with a GPS enable identification of horses with limited earnings. POTENTIAL RELEVANCE: This study demonstrates that a field test of fitness of Thoroughbred racehorses that correlates with retrospective racing ability is feasible. The technique has potential application in commercial training environments assisting with decisions concerning racing careers of individual racehorses.

Effect of corticosteroid and bronchodilator therapy on bronchoalveolar lavage cytology following intrapulmonary blood inoculation.

REASON FOR PERFORMING STUDY: Intrapulmonary blood causes an inflammatory response. Modifying this inflammatory response may be useful in the treatment of exercise induced pulmonary haemorrhage (EIPH). OBJECTIVES: To evaluate the efficacy of 3 treatments, an oral bronchodilator, inhaled steroid and oral steroid in modifying the cellular response to intrapulmonary blood inoculation. MATERIALS AND METHODS: Experimental design was a randomised block divided into 4 experimental segments of 2 weeks. Eight normal horses were randomly assigned to pairs and treatments. Autologous blood was inoculated into the lungs on 4 occasions over an 8 week period. For each treatment, a bronchoalveolar lavage (BAL) sample was collected on Day 0 and then 2 randomly selected segmental bronchi (one in each lung) were inoculated with blood (50 ml). BAL fluid was collected from one inoculated bronchus on Day 3 and from the other on Day 10. From BAL samples, total RBC and total and differential WBC were determined. Results for percentages and absolute cell counts were expressed as mean +/- s.e. and analysed using one, 2 and 3 way analysis of variance. RESULTS: All treatments significantly reduced the number of erythrophages at Day 3 (P = 0.02) Treatment with oral prednisolone resulted in a significantly higher percentage of macrophages (P = 0.03) and also reduced the relative and absolute number of haemosiderophages in BAL compared to the other 2 treatments at Day 10 (P = 0.07). CONCLUSION: We concluded that treatments altered the dynamics of cell populations in BAL following blood inoculation. Whether this alters the evolution of lung lesions following episodes of EIPH and indeed whether there are benefits for equine lungs following episodes of EIPH, remain to be determined. POTENTIAL RELEVANCE: Future studies into treatments for EIPH may benefit from incorporating a variety of dose rates for oral prednisolone and a longer period of evaluation. Such studies may also benefit from the use of cell activation markers. The concomitant use of pulmonary function testing and cytology in the field may assist in ascertaining whether treatments influence the performance and welfare of athletic horses subsequent to episodes of EIPH.

Observations on respiratory flow strategies during and after intense treadmill exercise to fatigue in thoroughbred racehorses.

REASONS FOR PERFORMING STUDY: Locomotor-respiratory coupled (LRC) breath types are a feature of galloping exercise in horses. Differences in breath type have been demonstrated during exercise in particular the 'big respiratory cycle' (BRC) and 'flow hesitation'. To investigate breath types during recovery and quantitatively investigate BRCs during exercise to understanding the mechanism driving BRCs. OBJECTIVES: To investigate the occurrence of different breath types during and after intense treadmill exercise, and test the hypothesis that large breaths (BRCs) were a function of respiratory frequency. METHODS: Six trained and clinically normal Thoroughbred horses were exercise tested on a treadmill (slope 10%). Breath-by-breath pulmonary ventilation was measured continuously during exercise and recovery using a Quadflow mask. RESULTS: Five different breath types were identified, and classified as normal monophasic, normal biphasic, deglutition, effort pause, or large breaths. Exercising at 10 m/sec, the number of large breaths was significantly related to Rf (r = -0.86, P = 0.03). During 120 sec after exercise there were 2 distinct populations of breaths, large and normal monophasic. CONCLUSIONS: BRC type breaths are a normal feature of ventilation during and after intense exercise. In recovery there are two distinct breath populations. During exercise BRC frequency is inversely associated with respiratory frequency and highly dependant on the individual horse. From intense exercise to recovery, high flow rates and LRC limited tidal volumes are replaced by high tidal volumes and progressively decreasing flow rates. There is a temporal association between BRC occurrence and PECO2. POTENTIAL RELEVANCE: Breath types and the physiological mechanism for driving each type is important in the clinical interpretation of respiratory disease or dysfunction. The demonstration of BRC association with PECO2 may help understand the driving mechanism for the BRC. In pulmonary function testing, breath type is important in quantitative results. The demonstration that high tidal flows with limited tidal volumes during intense exercise being replaced by high tidal volumes and progressively decreasing flows in recovery has potential clinical relevance.

Activation of natural killer-like YT-INDY cells by oligodeoxynucleotides and binding by homologous pattern recognition proteins.

The present study was designed to examine the binding and signalling effects of single base and CpG dinucleotide phosphodiester (Po) oligodeoxynucleotides (ODN) on the human natural killer (NK)-like cell line (YT-INDY). Single base Po ODN composed of 20-mers of guanosine (dG20), adenosine (dA20), cytosine (dC20) or thymidine (dT20) as well as 'conventional' Po CpG ODN were examined for their ability to bind and activate YT-INDY cells. Binding by dG20 and CpG ODN to YT-INDY cells was saturable and specific. dG20 binding was competitively inhibited by homologous dG20 and heterologous CpG ODN but not by dC20 and dA20. Two different YT-INDY membrane proteins (18 and 29 kDa) were identified by ligand (Southwestern) blotting with biotinylated dG20 and CpG. The specificity of the ODN-binding protein(s) was further confirmed by ODN depletion experiments using a teleost recombinant protein orthologue [nonspecific cytotoxic cells (NCC) cationic antimicrobial protein-1 (ncamp-1)] known to bind CpG and dG20. Cell proliferation and activation studies showed that dG20 and CpG treatment of YT-INDY cells induced cellular DNA synthesis (i.e. G1 to S-phase conversion). This signalling function was accompanied in dG20-treated cells by proliferation 10 h posttreatment. Both dG20 and CpG ODN binding induced a calcium flux in YT-INDY cells within seconds of treatment. These experiments demonstrated that Po single base dG20 and CpG ODN bind to a (potential) new class of cell-surface proteins that mediate the activation of YT-INDY cells.

Thoracolaryngeal reflex latencies in Thoroughbred horses with recurrent laryngeal neuropathy.

Electrolaryngeography was used to study the latencies of the thoracolaryngeal adductor reflex in Thoroughbred horses with and without recurrent laryngeal neuropathy (RLN). Latencies were compared in horses with grades 1 and 2 RLN, diagnosed by endoscopy in resting horses. The reliability of the measurements, effect of sedation and correlations of latencies with age of the horse were also studied. There was no effect of sedation on reflex latency periods. The latency of the reflex period measured to a convolved peak of the electromyographic response was significantly different in horses with grades 1 and 2 disease; medians and quartile ranges were 0.067 (0.065-0.073) and 0.072 (0.068-0.074) s, respectively (P<0.05). Significant associations were found between reflex latencies and both horse age and the grade of RLN. Reflex latency measurements are reliable and sensitive, and may assist with the clinical appraisal of Thoroughbred horses with RLN.

Reliability of cardiorespiratory measurements with a new ergospirometer during intense treadmill exercise in Thoroughbred horses.

This study investigated the reliability of measurements with a new equine ergospirometer (Quadflow). Heart rate and blood lactate responses during exercise in horses wearing the Quadflow and an open flow mask were also compared. The mean percentage error of the oxygen uptake measurements was 8.2% (range 2.1-12.5%). Percent error for peak expiratory flow rates ranged from 6.1% to 9.4 %, and for minute ventilation from 2.5% to 7.4%. The coefficients of variation of the means of four measurements in two horses exercising continuously at 9.0 m/s were <5% for variables related to pulmonary ventilation, and was 7.7% for oxygen uptake. The Quadflow mask resulted in small increases in blood lactate concentration and relative heart rate during submaximal exercise. It was concluded that between- and within-test reliability statistics for important measurements in equine clinical exercise testing were acceptable for routine use in a veterinary practice or research laboratory.

Single-base oligodeoxyguanosine-binding proteins on nonspecific cytotoxic cells: identification of a new class of pattern-recognition receptors.

The present study was designed to identify a possible new class of pathogen-recognition proteins that bind single-base oligodeoxynucleotide (ODN) ligands. Binding by the teleost natural killer cell equivalent [referred to as nonspecific cytotoxic cells (NCC)] was compared with mammalian cells (mouse RAW264.7 cells and human THP-1 cells). The ODN analysed were composed of 20-mers of guanosine (dG20), adenosine (dA20), thymidine (dT20) or cytosine (dC20). Binding studies first determined the 50% saturation levels for NCC (1.25 microg/ml), RAW264.7 (0.2 microg/ml) and THP-1 (0.8 microg/ml). Binding by dG20 to all the three cell types was saturable. Ligand blots of NCC membrane lysates with biotinylated dG20 revealed two different major molecular weight species (16-18 and 29 kDa) of binding proteins. The 29-kDa protein was identified with the help of Western blot analysis using a polyclonal antibody specific to an NCC antimicrobial protein (ncamp-1). The membrane expression of the 29-kDa ncamp-1 was determined by the binding of surface-biotinylated NCC membrane proteins with digoxigenin dG20 followed by immunoprecipitation using anti-digoxigenin agarose beads. The 29 and 14-18 kDa NCC membrane proteins were cross-reactive using Western blot examination with a polyclonal anti-histone 1 antibody. Function studies revealed that dG20 activated a twofold upregulation of membrane binding by homologous dG20-biotin. dG20 also stimulated NCC-increased membrane expression of NCC receptor protein 1. Additional experiments were performed to determine the DNase sensitivity of the different ODN. dG20 appeared to be more resistant to DNase treatment, compared to dC20, dA20 and dT20. The single-base ODN-binding proteins may represent a new class of pattern-recognition receptors that are involved in innate anti-bacterial resistance mediated by NCC.