Horse Neutrophils Research Articles

Histidine-Rich Glycoprotein Functions as a Dual Regulator of Neutrophil Activity in Horses

Histidine-rich glycoprotein (HRG) is an abundant plasma protein that has been identified in most mammals. We first identified whole genome sequence of equine HRG (eHRG) and succeeded to purify eHRG from plasma of horses. Since HRG interacts with various ligands, this protein is thought to be involved in immune response, coagulation, and angiogenesis. Systemic inflammatory response syndrome (SIRS) is characterized as a non-specific, clinical, pro-inflammatory immune response that damage organs and tissues in the host. Recent reports revealed that blood HRG levels in human patients with SIRS are approximately 50% lower than those in healthy controls, indicating the use of HRG as a biomarker or treatment for SIRS. SIRS is also a serious issue in equine medicine. In this study, we investigated various effects of eHRG on neutrophil functions, including adhesion, migration, phagocytosis, reactive oxygen species (ROS) production, and lysosome maturation using neutrophils isolated from horses. Microscopic observation showed that the addition of eHRG to the culture diminished adhesion of neutrophils stimulated with LPS. Using the Boyden chamber technique, we showed that eHRG reduced neutrophil chemotaxis induced by recombinant human IL-8. Luminol-dependent chemiluminescence assay demonstrated that eHRG restrained the peak of LPS-promoted ROS production from neutrophils. In contrast, eHRG promoted phagocytic activity evaluated with uptake of fluorescent dye conjugated particles, as well as lysosomal maturation assessed using fluorescent staining for lysosomes of equine neutrophils. These results indicated that eHRG acts as a dual regulator of neutrophils in horses.

Phagocytosis, bacterial killing, and cytokine activation of circulating blood neutrophils in horses with severe equine asthma and control horses.

OBJECTIVE To evaluate in vitro phagocytosis and bactericidal activity of circulating blood neutrophils in horses with severe equine asthma and control horses and to determine whether circulating blood neutrophils in horses with severe equine asthma have an increase in expression of the proinflammatory cytokine tumor necrosis factor (TNF)-α and the chemokine interleukin (IL)-8 and a decrease in expression of the anti-inflammatory cytokine IL-10 in response to bacteria. ANIMALS 6 horses with severe equine asthma and 6 control horses. PROCEDURES Circulating blood neutrophils were isolated from horses with severe equine asthma and control horses. Phagocytosis was evaluated by use of flow cytometry. Bactericidal activity of circulating blood neutrophils was assessed by use of Streptococcus equi and Streptococcus zooepidemicus as targets, whereas the cytokine mRNA response was assessed by use of a quantitative PCR assay. RESULTS Circulating blood neutrophils from horses with severe equine asthma had significantly lower bactericidal activity toward S zooepidemicus but not toward S equi, compared with results for control horses. Phagocytosis and mRNA expression of TNF-α, IL-8, and IL-10 were not different between groups. CONCLUSIONS AND CLINCAL RELEVANCE Impairment of bactericidal activity of circulating blood neutrophils in horses with severe equine asthma could contribute to an increased susceptibility to infections.

Equine neutrophils and their role in ischemia reperfusion injury and lung inflammation.

Horses are susceptible to a multitude of inflammatory conditions that are characterized by a strong neutrophilic response. Here, we review basic equine neutrophil biology and explore the role of neutrophils in inflammatory conditions with emphasis on intestinal ischemia and reperfusion injury and lung inflammation. In addition, unique aspects of equine neutrophil biology have been highlighted. Neutrophils comprise the highest proportion of circulating white blood cells in equine blood. The concentration of circulating equine neutrophils is a primary indicator of systemic inflammation. Additionally, equine neutrophils exposed to various stimulants develop "toxic" changes characterized as cytoplasmic basophilia, presence of Döhle bodies, cytoplasmic vacuolation and toxic granulation. In contrast to human neutrophils, equine neutrophils fail to undergo chemotaxis in response to the peptide N-formyl-methionyl-leucyl-phenylalanine and are dependent on the addition of arachidonic acid due to reduced activity of phospholipase A2 to synthesize leukotrienes as part of the arachidonic acid pathway. Understanding the biologic function of neutrophils in horses is integral to developing methods to modulate inflammation associated with ischemia reperfusion injury and lung disease.

The effect of free and carrier-bound cortisol on equine neutrophil function

Cortisol is a key anti-inflammatory hormone that increases in bacterial sepsis and circulates predominantly bound to cortisol binding globulin (CBG). Only unbound cortisol was believed to be biologically active, but recent evidence suggests that CBG-bound cortisol also regulates inflammation. The objective of this study was to evaluate the effects of free and CBG-bound cortisol on equine neutrophil function ex vivo. We hypothesized that CBG would enhance cortisol-mediated suppression of neutrophil pro-inflammatory responses. Neutrophils isolated from 8 foals and 6 adult horses were exposed to Staphylococcus aureus antigen (SAA) alone and with hydrocortisone (HC), CBG, or both (CBG+HC). Inflammatory cytokine (TNF-α, IL-8) and reactive oxygen species (ROS) production were measured and compared among stimulants and between ages with linear mixed-effects models. CBG and CBG+HC inhibited ROS production induced by SAA in both foal and horse neutrophils, maintaining it at levels comparable to baseline production (P≤0.060–0.907). TNF-α production was not significantly different among stimulants (P=0.284). CBG+HC significantly (P≤0.016) increased IL-8 production by neutrophils in response to SAA in both foals and adults, although the response of foals was significantly greater than that of adults (P<0.001). These findings suggest that CBG directly modulates equine neutrophil responses, but the effects are cytokine- and age-specific.

115 CHARACTERIZING NEUTROPHIL PROFILES IN HORSES FOR RHINOCEROS CAPTURE

Conservation of several African species is becoming essential, and efforts to move threatened animals are causing physiological and reproductive problems. To save these species, a more comprehensive knowledge of their biology and response to stressors is required. Capture stress of rhinoceroses has been quantified (Kruger et al. 2011 Reprod. Fertil. Dev. 23, 181–182) by evaluating leucocyte coping capacity (LCC). LCC is the measurement of the fluorescence of circulating active neutrophils, then expressed as optical density (OD)/1000 neutrophils. The LCC then provides a standardized value between species as we used identical conditions and reagents. To quantify the role of LCC in rhinoceros conservation, it is essential to characterize normal LCC profiles of healthy unstressed rhinoceroses. Horse neutrophils are very similar to rhinoceros neutrophils in their biological activity. The objective of the study was to characterize normal LCC profiles in stallions, geldings, nonpregnant mares, pregnant mares, as well as fillies and colts of various ages as a benchmark for adult and juvenile rhinoceroses. The LCC profiles are shown in Figures 1 to 3. For the colts (days, weeks, and months old) there was little difference in their LCC profile over time. For the fillies (days, weeks, and months old) the LCC response for fillies only days old was significantly (P &lt; 0.05) greater when compared to fillies that were weeks and months old. For the adult horses, the stallions had the lowest overall LCC and were very similar to nonpregnant mares. The LCC of pregnant mares was of significantly (P &lt; 0.5) greater magnitude than that of stallions and nonpregnant mares. The LCC response of gelding was significantly (P &lt; 0.5) greater than that of stallions and nonpregnant mares, but significantly (P &lt; 0.5) lower than that of pregnant mares. The stallion and pregnant mare responses mirrored the quantitative responses of breeding rhinoceros bulls and pregnant rhinoceros cows. From the data (Figure 4) we suggest that breeding males (stallions, elephants, and rhinoceros bulls) have the lowest LCC activity curves, as their breeding activities result in higher daily stresses. We hypothesized that the stressed bulls have their neutrophil activity down regulated by circulating corticosteroids. The converse is true for pregnant females (mares and cows), as they may have more vigorous neutrophils and therefore the highest LCC curves. These basic studies support and validate the role of rapid LCC in stress evaluation of wild caught rhinoceroses and horses could be applicable for captured elephant.

Evaluation of neutrophil apoptosis in horses with acute abdominal disease

To quantify peripheral blood neutrophil apoptosis in equine patients with acute abdominal disease (ie, colic) caused by strangulating or nonstrangulating intestinal lesions and compare these values with values for horses undergoing elective arthroscopic surgery. 20 client-owned adult horses. Peripheral blood was collected from horses immediately prior to and 24 hours after surgery for treatment of colic (n = 10) or elective arthroscopic surgery (10), and neutrophils were counted. Following isolation by means of a bilayer colloidal silica particle gradient and culture for 24 hours, the proportion of neutrophils in apoptosis was detected by flow cytometric evaluation of cells stained with annexin V and 7-aminoactinomycin D. Values were compared between the colic and arthroscopy groups; among horses with colic, values were further compared between horses with and without strangulating intestinal lesions. Percentage recovery of neutrophils was significantly smaller in preoperative samples (median, 32.5%) and in all samples combined (35.5%) for the colic group, compared with the arthroscopy group (median, 66.5% and 58.0%, respectively). No significant differences in the percentages of apoptotic neutrophils were detected between these groups. Among horses with colic, those with strangulating intestinal lesions had a significantly lower proportion of circulating apoptotic neutrophils in postoperative samples (median, 18.0%) than did those with nonstrangulating lesions (66.3%). The smaller proportion of apoptotic neutrophils in horses with intestinal strangulation suggested that the inflammatory response could be greater or prolonged, compared with that of horses with nonstrangulating intestinal lesions. Further investigations are needed to better understand the relationship between neutrophil apoptosis and inflammation during intestinal injury.

Neutrophil and cytokine dysregulation in hyperinsulinemic obese horses

Equine metabolic syndrome is characterized by obesity and regional adiposity coupled with evidence of recurrent laminitis. Although inflammation has been well characterized in several experimental models of acute laminitis, the inflammatory events associated with endocrinopathic laminitis are not well documented. The aim of this study was to characterize selected markers of inflammation in horses with clinical evidence of equine metabolic syndrome (EMS). Neutrophil phagocytosis and oxidative burst, as well as endogenous and stimulated cytokine expression were evaluated. A marked increase in neutrophil reactive oxygen species production upon phagocytosis was observed in horses with EMS that was strongly correlated to the blood insulin concentration. Increased oxidative burst activity of neutrophils in hyperinsulinemic horses may predispose horses with metabolic syndrome to laminitis. In contrast, peripheral blood cells of obese hyperinsulinemic horses showed decreased endogenous proinflammatory cytokine gene expression (IL-1 and IL-6) and similar cytokine response following immune stimulation compared to that of control horses. This may suggest that, unlike in people, cytokine-mediated inflammation does not increase in direct response to obesity or insulin resistance in horses. This species-specific disparity may explain the difference in clinical outcomes observed in obese horses compared to obese people.

Flow cytometric detection of myeloperoxidase in horse neutrophils: a novel technique in equine diagnostic research

Myeloperoxidase (MPO) is a protein of interest due to its involvement in equine pathologies. Until now, results in equine diagnostic research were achieved through extracellular MPO detection. However, studying the cellular MPO content in neutrophils has revealed important insights in human diseases. This study aimed to develop a technique for the specific detection of MPO on the single cell level defining a flow cytometric protocol for the detection of both equine surface-bound and cellular MPO. Both indirect and direct labeling techniques are described which include the comparison of two secondary antibodies and two linking-fluorochromes, respectively.

Clara cell secretory protein increases phagocytic and decreases oxidative activity of neutrophils

Horses suffer from recurrent airway obstruction, an asthma-like condition induced by repeat inhalation of environmental substances present in barn air. Clara cell secretory protein (CCSP) is much reduced during active inflammation when neutrophils predominate in the airways, and in chronic asthmatics. We sought to investigate morphologic and functional interactions of CCSP with neutrophils. Bronchoalveolar and blood neutrophils from healthy control animals, and from animals with recurrent airway obstruction in remission and exacerbation, were evaluated by immuno-cytochemistry and immuno-electron microscopy for presence of CCSP. Blood neutrophil oxidative burst and phagocytic activities were determined in the presence of different concentrations of recombinant equine CCSP. Bronchoalveolar lavage neutrophils from horses with exacerbated lung inflammation, but not from control horses, and not blood neutrophils from either group of animal, contained abundant immunoreactive CCSP. On immuno-electron microscopy, CCSP localized to the cytoplasm and nucleus. Incubation of blood neutrophils with CCSP significantly reduced oxidative burst activity ( P < 0.0001) and increased phagocytosis ( P < 0.001) of neutrophils. These findings indicate that CCSP enters neutrophils in horses with active neutrophilic lung inflammation and alters the function of neutrophils in blood. Presence in the nucleus suggests a potential transcriptional role of CCSP in neutrophils.

Effects of Training on Phagocytic and Oxidative Metabolism of Peripheral Neutrophils in Horses Exercised in the Aerobic–Anaerobic Transition Area

Using simple techniques, the neutrophil function, in its phagocytosis and oxidative metabolism stages, was evaluated in horses. This was done before and after moderate exercise at the aerobic-anaerobic threshold (standardized heart rate 150 beats/min and lactate level of 3.07 +/- 0.21 mmol/L). The objective was to determine whether regular training and moderate exercise improved the neutrophil function. A group of 19 horses was used; 11 of these were untrained and the remainder trained for national jumping events. The exercise test consisted of a 5 min trot followed by a 3 min gallop on a long lunge. Blood samples were taken for analysis before, immediately after and 15 min after exercise. The results showed that (a) there is a difference in the internalization of particles (PI, PP and PE) by neutrophils from trained and untrained horses at a single time point during active recovery, and PP is higher in trained horses immediately after exercise; and (b) oxidative metabolism is significantly lower in untrained animals before and 1 min after exercise. The moderate exercise at the aerobic-anaerobic threshold did not have any influence on the peripheral blood neutrophil function of the phagocytosis and oxidative metabolism of particles.

First molecular characterization of a granulocytic Ehrlichia strain isolated from a dog in South Italy

The genus Ehrlichia includes obligate intracellular bacteria which are able to infect specific haematopoietic cell lineages in several animal species, including humans (Waner et al., 2001). E. canis and E. chaffeensis preferentially infect monocytes and macrophages and their host spectrum includes dogs, foxes and humans (Shaw et al., 2001). E. equi, E. phagocytophila and Human Granulocytic Ehrlichia (HGE) invade neutrophils in ruminants, horses and people, respectively (Bjoersdorff et al., 2002). E. platys infects platelets in dogs (Hancock et al., 2001). Currently Ehrlichia species are classified on the basis of their 16S rRNA gene sequence (Hildebrandt et al., 2002). Three phylogenetic groups have been identified (Goldman et al., 1998): E. equi, E. phagocytophila and HGE are closely related; E. canis, E. chaffeensis, E. muris and E. ewingii are distinct from each other but related; and a third group with a genetic resemblance is formed by E. risticii, E. platys and E. sennetsu (Chen et al., 1994; Kakoma et al., 1994). Serological cross-reactivity among the different Ehrichia species occurs to a variable extent (Dumler et al., 1995). Most Ehrlichia species are vectorborne, with ticks as the proven vectors for many species (Fishbein et al., 1994). Even though Ehrlichia species have shown a limited preference for certain mammalian species, experimental infections have shown that Ehr-

Antigen challenge increases adherence of circulating neutrophils in horses with chronic obstructive pulmonary disease.

Activation of circulating neutrophils has been observed following challenge of horses with chronic obstructive pulmonary disease (COPD) and may facilitate the accumulation of these cells in the airways. In this study, no significant difference was observed between adherence to protein coated plastic of blood neutrophils from asymptomatic COPD-susceptible and normal horses stimulated by the mediators PAF, human recombinant (hr)IL-8 and hrC5a. Twenty-four hours after the start of a 7 h antigen challenge, adherence of unstimulated neutrophils from COPD-susceptible horses increased from 2.5 (0.5-4.1)% and 3.4 (0.6-6.6)% to 19.6 (16.9-20.3)% and 21.8 (10.6-23.1)% adherence for cells in medium containing 0.1% or 0.2% BSA, respectively; (median [range]; n = 4). Adherence of cells from normal horses remained unchanged. Addition of an anti-CD18 monoclonal antibody, H20A, inhibited the increase in adherence at 24 h by 96 (45-100)%, n = 3. The percentage of neutrophils in bronchoalveolar lavage fluid at 24 h increased from 1 (0-2) to 80 (65-94), (median (range), n = 4). These results suggest that antigen challenge results in exposure of circulating equine neutrophils to one or more factors that prime, or activate, these cells, which may enhance their recruitment to the lungs. Inhibition of circulating neutrophil activation may therefore represent a therapeutic target.

A comparison of foal and adult horse neutrophil function using flow cytometric techniques

Flow cytometric assays were used to compare phagocytic and oxidative burst activity of neutrophils from healthy foals less than 7 days of age with the activity of cells from healthy adult horses. The phagocytosis of Staphylococcus aureus by foal neutrophils was less than that observed for adult neutrophils when autologous serum was used as the source of opsonins in the assay. The use of adult serum did not significantly improve the ability of foal neutrophils to attach bacteria. The oxidative burst activity of foal neutrophils was equivalent to that of adult cells. However, when serum or plasma was incorporated into the oxidative burst assay, foal neutrophils demonstrated greatly reduced autofluorescence and a suppressed response to phorbol myristate acetate (PMA), relative to that demonstrated by adult cells. These results suggest that peripheral blood neutrophils from foals have a reduced ability to phagocytose bacteria relative to that exhibited by adult horse neutrophils and that the oxidative burst activity of foal neutrophils is down-regulated in response to an unidentified serum factor(s). Such changes may contribute to the increased susceptibility of foals to septic disease.

Structural and functional analysis of horse cathelicidin peptides.

Cathelicidin-derived antimicrobial peptides are a component of the peptide-based host defense of neutrophils and epithelia, with a widespread distribution in mammals. We recently reported the cDNA sequences of three putative horse myeloid cathelicidins, named eCATH-1, -2, and -3. A Western analysis was performed to investigate their presence in neutrophils and processing to mature peptides. eCATH-2 and eCATH-3, but not eCATH-1, were found to be present in uncleaved forms in horse neutrophils. The corresponding mature peptides were detected in inflammatory sites, suggesting that processing of the propeptides takes place upon neutrophil activation. A functional characterization was then performed with synthetic eCATH peptides. Circular dichroism measurements indicated an amphipathic alpha-helical conformation of these peptides in an anisotropic environment, and in vitro assays revealed a potent activity and a broad spectrum of antimicrobial activity for eCATH-1 and a somewhat more restricted spectrum of activity for eCATH-2. Conversely, a strong dependence on salt concentration was observed when the activity of eCATH-3 was tested. This peptide efficiently killed bacteria and some fungal species, i.e., Cryptococcus neoformans and Rhodotorula rubra, in low-ionic-strength media, but the activity was inhibited in the presence of physiological salt medium. This behavior could be modified by modulating the amphipathicity of the molecule. In fact, the synthetic analogue LLK-eCATH-3, with a slightly modified sequence that increases the hydrophobic moment of the peptide, displayed a potent activity in physiological salt medium against the strains resistant to eCATH-3 under these conditions.

Novel cathelicidins in horse leukocytes

Cathelicidins are precursors of defense peptides of the innate immunity and are widespread in mammals. Their structure comprises a conserved prepropiece and an antimicrobial domain that is structurally varied both intra- and inter-species. We investigated the complexity of the cathelicidin family in horse by a reverse transcription-PCR-based cloning strategy of myeloid mRNA and by Southern and Western analyses. Three novel cathelicidin sequences were deduced from bone marrow mRNA and designated equine cathelicidins eCATH-1, eCATH-2 and eCATH-3. Putative antimicrobial domains of 26, 27 and 40 residues with no significant sequence homology to other peptides were inferred at the C-terminus of the sequences. Southern analysis of genomic DNA using a probe based on the cathelicidin-conserved propiece revealed a polymorphic DNA region with several hybridization-positive fragments and suggested the presence of additional genes. A null eCATH-1 allele was also demonstrated with a frequency of 0.71 in the horse population analyzed and low amounts of eCATH-1-specific mRNA were found in myeloid cells of gene-positive animals. A Western analysis using antibodies to synthetic eCATH peptides revealed the presence of eCATH-2 and eCATH-3 propeptides, but not of eCATH-1-related polypeptides, in horse neutrophil granules and in the secretions of phorbol myristate acetate-stimulated neutrophils. These results thus suggest that eCATH-2 and eCATH-3 are functional genes, whereas eCATH-1 is unable to encode a polypeptide.

An indirect immunofluorescence assay using a cell culture-derived antigen for detection of antibodies to the agent of human granulocytic ehrlichiosis.

An indirect immunofluorescence assay for the detection of human antibodies to the agent of human granulocytic ehrlichiosis (HGE) was developed and standardized. Antigen was prepared from a human promyelocytic leukemia cell line (HL-60) infected with a tick-derived isolate of the HGE agent (USG3). Suitable antigen presentation and preservation of cellular morphology were obtained when infected cells were applied and cultured on the slide, excess medium was removed, and cells were fixed with acetone. Use of a buffer containing bovine serum albumin and goat serum reduced background fluorescence, and use of an immunoglobulin G (gamma-specific) conjugate reduced nonspecific binding. The assay readily detected specific antibody from HGE patients and did not detect antibody from healthy individuals. No significant reactivity was noted in sera from patients with high titers of antibodies to other rickettsial species. We were able to identify antibodies reactive to USG3 antigen in samples from areas where HGE is endemic that had tested negative to other rickettsial agents. Animal sera reactive against Ehrlichia equi or Ehrlichia phagocytophila bound to the HGE antigen, indicating that the assay may be useful for veterinary use. Comparability between two different laboratories was assessed by using coded human sera exchanged between laboratories. Results from the two laboratories were similar, indicating that the assay can be easily integrated into use for routine testing for HGE. The assay was then compared to an assay using horse neutrophils infected with ehrlichiae. The two assays gave comparable results, indicating that the cell culture-derived antigen can be used for testing samples that have been previously tested with E. equi as an antigen. The new assay offers several advantages over other immunofluorescence methods that use animal-derived antigen and is suitable for use in testing for human antibodies to the HGE agent.

Comparative ultrastructural study of camel, cow, and horse neutrophil

The ultrastructure of Arabian camel, cow, and horse neutrophils in peripheral blood was studied in order to provide detailed description of its constituents and to determine the similarities and the differences between these three species. Peripheral blood was taken from the three animals, centrifuged and the pellets were fixed in glutaraldehyde, dehydrated and embedded in Spurr‘s resin. Silver sections were mounted on copper grids and viewed by Zeiss electron microscope and microphotographs were taken.Camel's neutrophils were found to have smoother cell surface with small number of short pseudopodia (Fig. 1). The horse's neutrophil had longer pseudopodia while the cow's neutrophil had a large number of small ridges instead. The size of the cell in camel was larger than that in cow and horse. The nucleus of camel's neutrophil appeared less irregular compared with that of cow's and horse's, with marginated non-patchy chromatin, and with abundant heterochromatin.

Comparative analysis of phagocytosis of fungal cells by insect hemocytes versus horse neutrophils

In this study, the phagocytic ability of Spodoptera exigua hemocytes was compared to horse neutrophils. In vitro assays showed that the insect granulocytes and horse neutrophils actively phagocytose FITC-labeled Paecilomyces farinosus blastopores opsonized with S. exigua hemolymph lectin or horse serum, respectively. Killing of fungal cells by the neutrophils and hemocytes was analyzed under in vitro conditions. Neutrophils reduced the growth of P. farinosus up to 65% whereas no fungicidal activity was observed with hemocyte monolayers. The production of oxygen metabolites by both phagocytic cells incubated with various elicitors (fungal cells, bacteria, phorbol myristate acetate) was examined using luminol-enhanced chemiluminescence. Phagocytosis of opsonized microbes by horse neutrophils resulted in marked increased of chemiluminescence activity whereas no chemiluminescence was detected in similary challenged phagocytic insect hemocytes. Electron microscopy was used to examine phagocytic events and confirmed that insect phagocytes were unable to kill tested microbes.

Structural and functional characterization of elastases from horse neutrophils.

In order better to understand the pathophysiology of the equine form of emphysema, two elastinolytic enzymes from horse neutrophils, referred to as proteinases 2A and 2B, have been extensively characterized and compared with the human neutrophil proteinases, proteinase-3 and elastase. Specificity studies using both the oxidized insulin B-chain and synthetic peptides revealed that cleavage of peptide bonds with P1 alanine or valine residues was preferred. Further characterization of the two horse elastases by N-terminal sequence and reactive-site analyses indicated that proteinases 2A and 2B have considerable sequence similarity to each other, to proteinase-3 from human neutrophils (proteinase 2A), to human neutrophil elastase (proteinase 2B) and to a lesser extent to pig pancreatic elastase. Horse and human elastases differed somewhat in their interaction with some natural protein proteinase inhibitors. For example, in contrast with its action on human neutrophil elastase, aprotinin did not inhibit either of the horse proteinases. However, the Val15, alpha-aminobutyric acid-15 (Abu15), alpha-aminovaleric acid-15 (Nva15) and Ala15 reactive-site variants of aprotinin were good inhibitors of proteinase 2B (Ki < 10(-9) M) but only weak inhibitors of proteinase 2A (Ki > 10(-7) M). In summary, despite these differences, the horse neutrophil elastases were found to resemble closely their human counterparts, thus implicating them in the pathological degradation of connective tissue in chronic lung diseases in the equine species.

A study of manatee leukocytes using peroxidase stain.

Leukocytes of the manatee, dog, cat, horse, rabbit, bird, and human were stained with modified Wright-Giemsa stain and myeloperoxidase stain. The predominant segmented leukocyte of the manatee stained positive for myeloperoxidase in a manner similar to the human, dog, cat, and horse neutrophil. Rabbit and bird heterophils stained unlike the manatee predominant leukocyte with myeloperoxidase stain. Eosinophils of all species examined also stained differently from the predominant manatee leukocyte. We conclude that the predominant segmented cell type of the manatee is not biochemically similar to the heterophil of the bird and rabbit or to the eosinophil of the human, dog, cat, horse, bird, and rabbit.