Benjamin Darien | University of Wisconsin-Madison (original) (raw)
Papers by Benjamin Darien
Journal of Veterinary Internal Medicine, 2008
Background: Although adequate colostrum intake and properly used antibiotics can provide much pro... more Background: Although adequate colostrum intake and properly used antibiotics can provide much protection for the bovine neonate, increased antibiotic scrutiny and consumer demand for organic products have prompted investigations of natural immunomodulators for enhancing calf health. One plant-based immunomodulator, Morinda citrifolia (noni) fruit, is a wellrecognized natural product that has a broad range of immunomodulatory effects.
Journal of Veterinary Internal Medicine, 1999
... J Vet Intern Med 1998;12:317324. 6. Darien BJ, Williams AM. Possible hypercoagulation in 3 f... more ... J Vet Intern Med 1998;12:317324. 6. Darien BJ, Williams AM. Possible hypercoagulation in 3 foals with septicaemia. Equine Vet Educ 1993;5:1922. ... Equine Internal Medicine. Philadelphia, PA: WB Saunders; 1998:513557. 9. Carr EA, Carlson GP, Wilson DW, Read DH. ...
Shock, 1997
The technique used most commonly to quantitate pulmonary edema in in vivo animal models is postmo... more The technique used most commonly to quantitate pulmonary edema in in vivo animal models is postmortem gravimetric analysis (wet:dry) ratio. To determine whether lung water can be quantitated morphometrically, as accurately as by the commonly used gravimetric analysis, perivascular edema (cuff) area to vessel area ratio was correlated to wet:dry ratio. Anesthetized pigs were given either oleic acid (20 mg/kg/h, intravenously) or physiologic saline. At 4 h, lungs were excised and cuff:vessel and wet:dry ratio analysis was performed. The intermediate lobe was clamped across its main stem bronchus to maintain peak inspiratory inflation, excised, frozen in liquid nitrogen, and stored at -70 degrees C until cryostat sectioning and quantification of perivascular interstitial edema (cuff) area. Gravimetric analysis (wet:dry ratio) was performed on the remaining lung. Mean cuff:vessel and wet:dry analyzes showed that lung water increased significantly (p < .01) in the oleic-acid treated group (4.9 +/- .22 and 6.78 +/- .47, respectively), compared with the saline group (.03 +/- .02 and 2.55 +/- .27, respectively). The correlation coefficient between mean cuff:vessel and wet:dry ratios was .86 (p = .0016). This study demonstrates that cuff:vessel ratio analysis can be used to identify the distribution of edema fluid versus vessel diameter, and seems to be as effective a technique as gravimetric analysis to quantitate lung water changes in acute lung injury models. Moreover cuff:vessel ratio analysis can differentiate modest changes in pulmonary edema by direct quantitation, an important end-point not provided by wet:dry analysis. Therefore, it may be a more sensitive technique when investigating therapeutic interventions in in vivo models of acute lung injury.
Veterinary Immunology and Immunopathology, 2008
P-selectin glycoprotein ligand (PSGL-1) is a widely distributed adhesion molecule that plays a cr... more P-selectin glycoprotein ligand (PSGL-1) is a widely distributed adhesion molecule that plays a critical role in regulating lymphocyte homing and leukocyte trafficking during inflammation. The lack of specific reagents for equine PSGL-1 (ePSGL-1) has prevented mechanistic studies regarding its function and regulation in the horse. We synthesized a ePSGL-1 peptide to generate a monoclonal antibody (mAb), ePL1. Using flow cytometry and Western blot, we showed that ePL1 binds specifically to ePSGL-1 in transfected mammalian cells. We also demonstrated that ePL1 binds to equine leukocytes and recognized a protein with molecular weight 165 and 280 kDa under reducing and non-reducing condition, respectively, likely corresponding to ePSGL-1.Seventy percent of equine monocytes bound by both ePL1 and HECA-452, an antibody defining sLex-like carbohydrate epitope. Both ePL1 and HECA-452 recognized ePSGL-1 protein precipitated by equine P-selectin-IgG chimera. Neuraminidase treatment increased ePL1 binding and the molecular weight of ePSGL-1, O-sialoglycoprotein endopeptidase digestion and tyrosine mutation abolished ePL1 staining and recognition. The ePL1 specific binding epitope appears to be the polypeptide backbone of ePSGL-1 in the presence of tyrosine but the process is independent of sialylation modification. In conclusion, we provide evidence that this antibody can be used for cell surface staining and immune-blot analyses. Published by Elsevier B.V.
Mammalian Genome, 2005
P-selectin glycoprotein ligand-1 (PSGL-1, CD162) is a dimeric, mucin-like, transmembrane glycopro... more P-selectin glycoprotein ligand-1 (PSGL-1, CD162) is a dimeric, mucin-like, transmembrane glycoprotein constitutively expressed on leukocytes. A high baseline level of P-selectin expression in circulating equine platelets suggests a primed state toward inflammation and thrombosis via P-selectin/PSGL-1 adhesion. To investigate the potential role of equine P-selectin in these events, we first identified the cDNA sequence of equine PSGL-1 (ePSGL-1) using degenerate PCR and RACE-PCR and then compared the predicted sequence with that of human PSGL-1 (hPSGL-1). ePSGL-1 protein subunit is predicted to be 43 kDa and composed of 420 amino acids with a predicted 18-amino-acid signal sequence showing 78% homology to hPSGL-1. Previously published work has shown that binding of P-selectin requires sulfation of at least one of three tyrosines and O-glycosylation of one threonine in the N-terminus of human PSGL-1. However, the corresponding domain in ePSGL-1, spanning residues 19–43, contains only one tyrosine in the vicinity of two threonines at positions 25 and 41. ePSGL-1 contains 14 threonine/serine-rich decameric repeats as compared to hPSGL-1 which contains 14–16 threonine-rich decameric repeats. The transmembrane and cytoplasmic domains display 91% and 74% homology to corresponding human PSGL-1 domains, respectively. In summary, there is 71% homology in comparing the open reading frame (ORF) of ePSGL-1 with that of hPSGL-1. The greatest homologies between species exist in the transmembrane domain and cytoplasmic tail while substantial differences exist in the extracellular domain.
Equine Veterinary Journal, 1989
Comparisons were made between transtracheal aspirate (TTA) and bronchoalveolar lavage (BAL) cytol... more Comparisons were made between transtracheal aspirate (TTA) and bronchoalveolar lavage (BAL) cytology obtained from 50 horses with chronic lung disease and from 10 control horses. There was no significant correlation between the TTA cytology and the BAL cytology, suggesting that the cell population in the trachea is not representative of the cell population in the lower airways. In control horses the range of differential cell counts obtained from TTA fluid was remarkably large, whereas the variability in cell populations observed in BAL fluid was smaller. In the principal horses the total and differential cell counts of the TTA and BAL fluids were within the 95 per cent confidence interval in 38 and 24 per cent of cases, respectively; and an increase in percentage neutrophils was most common. It was concluded that BAL may be a useful diagnostic aid when evaluating horses with chronic lung disease, but that the clinical usefulness of cytological evaluations of TTA fluid may be limited in these cases.
Veterinary Immunology and Immunopathology, 2006
Human P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric membrane mucin expressed on leukocyt... more Human P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric membrane mucin expressed on leukocytes that binds selectins. Here, we report that the open reading frame (ORF) of bovine PSGL-1 (bPSGL-1) cDNA is 1284 base pairs in length, predicting a protein of 427 amino acids including an 18-amino-acid signal peptide, an extracellular region with a mucin-like domain, and transmembrane and cytoplasmic domains. The amino acid sequence of bPSGL-1 demonstrated 52, 49 and 40% overall homology to equine, human and mouse, respectively. A single extracellular cysteine, at the transmembrane and extracellular domain junction, suggests a disulfide-bonding pattern. Alignment of bovine with equine, human and mouse PSGL-1 demonstrates high conservation of transmembrane and cytoplasmic domains, but diversity of the extracellular domain, especially in the anionic NH 2 -terminal of PSGL-1, the putative P-selectin binding domain. In the NH 2 -terminal of bPSGL-1, there are three potential tyrosine sulfation sites and three potential threonine O-glycosylation sites, all of which are required for P-selectin binding in human PSGL-1 (hPSGL-1). bPSGL-1 shares only 57% homology in amino acid sequence with the corresponding epitope region which binds the monoclonal antibody PL1 for hPSGL-1, and no cross-reactivity was found in bovine leukocytes. In summary, bPSGL-1 shares homology with hPSGl-1, but has differences in the putative extracellular Pselectin binding domain. #
Journal of Veterinary Internal Medicine, 2006
This prospective study compared survival rates of critically ill and septic foals receiving 1 of ... more This prospective study compared survival rates of critically ill and septic foals receiving 1 of 2 different types of commercial equine plasma and analyzed admission variables as possible predictors of survival. Standardized clinical, hematologic, biochemical, and hemostatic admission data were collected and foals received either conventional commercially available hyperimmune equine plasma or equine plasma specifically rich in antiendotoxin antibodies in a double-blinded, coded fashion. Sepsis was defined as true bacteremia or sepsis score >11. Overall survival rate to discharge was 72% (49/68). Foals that were nonbacteremic and demonstrated a sepsis score of ≤11 at admission had a 95% (18/19) survival rate. The survival rate to discharge for septic foals was 28/49 (57%), with truly bacteremic foals having a survival rate of 58% (14/24), whereas that for nonbacteremic, septic foals was 56% (14/25). Sensitivity and specificity for sepsis score 11 as a predictor of bacteremia were 74 and 52%, respectively. For the entire study population, a higher survival rate to discharge was documented for those foals receiving hyperimmune plasma rich in antiendotoxin antibodies (P= .012, odds ratio [OR] 6.763, 95% confidence interval [CI]: 1.311, 34.903). Administration of plasma rich in antiendotoxin antibodies also was associated with greater survival in septic foals (P= .019, OR 6.267, 95% CI: 1.186, 33.109). Statistical analyses demonstrated that, among 53 clinical and clinicopathologic admission variables, high sepsis score (P <.001), low measured IgG concentration (P= .01), high fibrinogen concentration (P= .018), low segmented neutrophil count (P= .028), and low total red blood cell numbers (P= .048) were the most significant predictors of overall mortality.
Veterinary Immunology and Immunopathology, 2003
Acute inflammatory diseases, such as colic, septicemia and endotoxemia are common in equines and ... more Acute inflammatory diseases, such as colic, septicemia and endotoxemia are common in equines and have been shown to be correlated to vascular injury and thrombosis. In humans with similar thrombotic conditions, P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1)-mediated platelet-leukocyte adhesion contributes to the pathogenesis of these disorders through the generation of inflammatory mediators and tissue factor. As such, we hypothesized that a P-selectin-PSGL-1 (platelet-leukocyte) interaction, similar to that in humans, may also exist in the horse. The objective of this study was to investigate phenotypic and morphological properties of equine platelet activation with a focus on CD62P (P-selectin) expression and CD62P mediated platelet-leukocyte interactions. To study high levels of platelet activation, we used 1 U/ml thrombin to induce secondary, irreversible aggregation in both human and equine platelets. Addition of glycyl-L-prolyl-L-arginyl-L-proline amide (GPRP) prior to thrombin activation blocked fibrin polymerization, allowing the use of flow cytometry to study a-granule expression as a measure of platelet activation. Thrombin activation resulted in high levels of activation, measured as P-selectin expression, in both humans and equines. Interestingly, our research illustrates that in healthy horses, P-selectin is also constitutively expressed on 20-25% of resting platelets. This finding is in direct contrast to humans, in which P-selectin expression is negligible (<5%) in the absence of agonist activation. The high baseline level of P-selectin expression among equine platelets may suggest that they are primed for leukocyte adhesion, possibly resulting in prothrombotic conditions. This phenomenon could be of significant clinical relevance, as it may be related to the rapid clinical decline often seen in equine patients with colic and endotoxemia, where vascular injury and thrombotic complications compromise patient survival. Based on these findings, further investigation into the mechanisms of platelet P-selectin-mediated inflammation and platelet-leukocyte mediated vascular injury in the horse appears warranted. #
Veterinary Immunology and Immunopathology, 2009
B.J. Darien).
Veterinary Immunology and Immunopathology, 2007
The recent molecular characterization and sequencing of equine P-selectin (ePsel), and its glycop... more The recent molecular characterization and sequencing of equine P-selectin (ePsel), and its glycoprotein ligand, P-selectin glycoprotein ligand-1 (PSGL-1), have provided the tools for further investigation into their role in leukocyte trafficking. Here, we report the generation of a genetically engineered chimeric protein (ePsel-IgG) in which the equine P-selectin lectin and epithelial growth factor (EGF) domains were covalently linked to the equine IgG1 heavy chain constant region. The soluble ePsel-IgG was observed to bind to equine monocytes by confocal microscopy and flow cytometry. Furthermore, equine monocytes bound to immobilized ePsel-IgG in a time course and dose dependent manner. Not only did ePsel-IgG act as an adhesion molecule, it was also found to activate ERK1/2 kinase and induce IL-8 mRNA expression in equine monocytes. That all of the aforementioned ePsel-IgG-induced cell binding and cell signaling were abolished by the addition of EDTA, suggested that ePsel-IgG chimera mediated events occurred via the Pselectin ligand, PSGL-1. We were able to demonstrate that 78% of equine monocytes cross-reacted with anti-human HECA-452 antibody, which recognizes the sialy-Lewis X (sLex) epitope, a well-known carbohydrate binding site on human PSGL-1. Preincubation of equine PBMC with neuraminidase or O-sialoglycoprotein endopeptidase (OSGP) reduced ePsel-IgG monocyte binding to 36% or 60%, respectively. Taken together, these data suggest that there might be two ligand recognition sites on P-selectin, one of which recognizes sLex and another which recognizes P-selectin ligand core protein. The ePsel-IgG chimera can be a useful as a reagent for further studies on the role of equine P-selectin and signal transduction in inflammatory events in horse. #
Shock, 1998
Because platelet-activating factor (PAF) is an important mediator of inflammation and heparin has... more Because platelet-activating factor (PAF) is an important mediator of inflammation and heparin has anti-inflammatory effects, we hypothesized that low molecular weight heparin (LMWH) would inhibit PAF-induced activation and chemotaxis in porcine neutrophils. Citrated blood was obtained from pentobarbital-anesthetized pigs, and neutrophils were isolated over a 55%/65% Percoll gradient. The effect of LMWH on basal phorbol myristate acetate (PMA)-induced superoxide (SO) release, as well as its effect on PAF priming for PMA-induced SO release, were investigated. Additionally, the effect of LMWH on PAF-induced chemotaxis of neutrophils across transwell membranes was evaluated. Baseline SO release in response to PMA was .351+/-.046 nmol/10(6) cells/min, and this was decreased to .289+/-.034 nmol/10(6) cells/min by pretreatment with 50 U/mL LMWH. PMA-induced SO production was increased by .240+/-.042 nmol/10(6) cells/min when cells were primed with 10 microM PAF. This priming effect of PAF was reduced significantly by pretreatment of neutrophils with LMWH at 10 and 50 U/mL. Chemotaxis of neutrophils in response to 100 microM PAF was significantly decreased to 70.02+/-6.4% (n = 8) of the control response by pretreatment of cells with 50 U/mL LMWH. We conclude that LMWH has anti-inflammatory effects on porcine neutrophils, which includes attenuation of cell activation and chemotaxis in response to the lipid-derived inflammatory mediator, PAF.
Journal of Veterinary Internal Medicine, 2005
The cardiac biomarkers cardiac troponin T (cTnT) and I (cTnI) and the cardiac isoenzyme of creati... more The cardiac biomarkers cardiac troponin T (cTnT) and I (cTnI) and the cardiac isoenzyme of creatine kinase (CKMB) are used extensively in human medicine to diagnose and provide valuable prognostic information in patients with ischemic, traumatic, and septic myocardial injury. We designed a study to establish normal values for these markers in healthy, neonatal foals and to compare them with values obtained from septic neonates in a referral hospital population. The 25th, 50th, 75th, and 95th percentiles for cTnI and CKMB in the healthy-foal population were 0.08, 0.14, 0.25, 0.49 ng/mL and 1.4, 2.3, 4.0, 7.4 ng/mL, respectively. The values obtained for cTnT were frequently (43/52 foals; 83%) below the lower limit of detection of the assay (0.009 ng/mL), but the median and range were 0.009 and 0.009-0.041 ng/mL, respectively. In the septic foal population, the 25th, 50th, 75th, and 95th percentile values for cTnI and CKMB were 0.05, 0.12, 0.22, and 1.10 ng/mL and 2.0, 4.4, 7.8, and 24 ng/mL, respectively. The values obtained for cTnT were less frequently below the lower limit of detection (23/38 foals; 60%) compared with the healthy foal population, and the median and range were 0.009 and 0.009–0.20 ng/mL, respectively. Significantly higher values were observed for cTnT and CKMB in septic foals compared with the healthy neonatal foal population, but there were no differences among septic foals in survivors compared with nonsurvivors. These findings suggest that myocardial injury occurs during septicemia in neonatal foals but that the injury is not associated with survival among septic foals.
Journal of Veterinary Internal Medicine, 2005
The cardiac biomarkers cardiac troponin T (cTnT) and I (cTnI) and the cardiac isoenzyme of creati... more The cardiac biomarkers cardiac troponin T (cTnT) and I (cTnI) and the cardiac isoenzyme of creatine kinase (CKMB) are used extensively in human medicine to diagnose and provide valuable prognostic information in patients with ischemic, traumatic, and septic myocardial injury. We designed a study to establish normal values for these markers in healthy, neonatal foals and to compare them with values obtained from septic neonates in a referral hospital population. The 25th, 50th, 75th, and 95th percentiles for cTnI and CKMB in the healthy-foal population were 0.08, 0.14, 0.25, 0.49 ng/mL and 1.4, 2.3, 4.0, 7.4 ng/mL, respectively. The values obtained for cTnT were frequently (43/52 foals; 83%) below the lower limit of detection of the assay (0.009 ng/mL), but the median and range were 0.009 and 0.009-0.041 ng/mL, respectively. In the septic foal population, the 25th, 50th, 75th, and 95th percentile values for cTnI and CKMB were 0.05, 0.12, 0.22, and 1.10 ng/mL and 2.0, 4.4, 7.8, and 24 ng/mL, respectively. The values obtained for cTnT were less frequently below the lower limit of detection (23/38 foals; 60%) compared with the healthy foal population, and the median and range were 0.009 and 0.009-0.20 ng/mL, respectively. Significantly higher values were observed for cTnT and CKMB in septic foals compared with the healthy neonatal foal population, but there were no differences among septic foals in survivors compared with nonsurvivors. These findings suggest that myocardial injury occurs during septicemia in neonatal foals but that the injury is not associated with survival among septic foals.
We have previously reported that Morinda citrifolia (noni) puree modulates neonatal calves develo... more We have previously reported that Morinda citrifolia (noni) puree modulates neonatal calves developmental maturation of the innate and adaptive immune system. In this study, the effect of noni puree on respiratory and gastrointestinal (GI), health in preweaned dairy calves on a farm with endemic salmonellosis was examined. Two clinical trials were conducted whereby each trial evaluated one processing technique of noni puree. Trials 1 and 2 tested noni versions A and B, respectively. Puree analysis and trial methods were identical to each other, with the calf as the experimental unit. Calves were designated to 1 of 3 treatment groups in each trial and received either: 0, 15 or 30 mL every 12 hr of noni supplement for the first 3 weeks of life. Health scores, weaning age, weight gain from admission to weaning, and weaned by 6 weeks, were used as clinical endpoints for statistical analysis. In trial 1, calves supplemented with 15 mL noni puree of version A every 12 hr had a higher probability of being weaned by 6 weeks of age than control calves (P = 0.04). In trial 2, calves receiving 30 mL of version B every 12 hr had a 54.5% reduction in total medical treatments by 42 days of age when compared to controls (P = 0.02). There was a trend in reduced respiratory (61%), and GI (52%) medical treatments per calf when compared to controls (P = 0.06 and 0.08, respectively). There were no differences in weight gain or mortality for any treatment group in either trial.
Journal of Veterinary Internal Medicine, 2008
Background: Although adequate colostrum intake and properly used antibiotics can provide much pro... more Background: Although adequate colostrum intake and properly used antibiotics can provide much protection for the bovine neonate, increased antibiotic scrutiny and consumer demand for organic products have prompted investigations of natural immunomodulators for enhancing calf health. One plant-based immunomodulator, Morinda citrifolia (noni) fruit, is a wellrecognized natural product that has a broad range of immunomodulatory effects.
Journal of Veterinary Internal Medicine, 1999
... J Vet Intern Med 1998;12:317324. 6. Darien BJ, Williams AM. Possible hypercoagulation in 3 f... more ... J Vet Intern Med 1998;12:317324. 6. Darien BJ, Williams AM. Possible hypercoagulation in 3 foals with septicaemia. Equine Vet Educ 1993;5:1922. ... Equine Internal Medicine. Philadelphia, PA: WB Saunders; 1998:513557. 9. Carr EA, Carlson GP, Wilson DW, Read DH. ...
Shock, 1997
The technique used most commonly to quantitate pulmonary edema in in vivo animal models is postmo... more The technique used most commonly to quantitate pulmonary edema in in vivo animal models is postmortem gravimetric analysis (wet:dry) ratio. To determine whether lung water can be quantitated morphometrically, as accurately as by the commonly used gravimetric analysis, perivascular edema (cuff) area to vessel area ratio was correlated to wet:dry ratio. Anesthetized pigs were given either oleic acid (20 mg/kg/h, intravenously) or physiologic saline. At 4 h, lungs were excised and cuff:vessel and wet:dry ratio analysis was performed. The intermediate lobe was clamped across its main stem bronchus to maintain peak inspiratory inflation, excised, frozen in liquid nitrogen, and stored at -70 degrees C until cryostat sectioning and quantification of perivascular interstitial edema (cuff) area. Gravimetric analysis (wet:dry ratio) was performed on the remaining lung. Mean cuff:vessel and wet:dry analyzes showed that lung water increased significantly (p < .01) in the oleic-acid treated group (4.9 +/- .22 and 6.78 +/- .47, respectively), compared with the saline group (.03 +/- .02 and 2.55 +/- .27, respectively). The correlation coefficient between mean cuff:vessel and wet:dry ratios was .86 (p = .0016). This study demonstrates that cuff:vessel ratio analysis can be used to identify the distribution of edema fluid versus vessel diameter, and seems to be as effective a technique as gravimetric analysis to quantitate lung water changes in acute lung injury models. Moreover cuff:vessel ratio analysis can differentiate modest changes in pulmonary edema by direct quantitation, an important end-point not provided by wet:dry analysis. Therefore, it may be a more sensitive technique when investigating therapeutic interventions in in vivo models of acute lung injury.
Veterinary Immunology and Immunopathology, 2008
P-selectin glycoprotein ligand (PSGL-1) is a widely distributed adhesion molecule that plays a cr... more P-selectin glycoprotein ligand (PSGL-1) is a widely distributed adhesion molecule that plays a critical role in regulating lymphocyte homing and leukocyte trafficking during inflammation. The lack of specific reagents for equine PSGL-1 (ePSGL-1) has prevented mechanistic studies regarding its function and regulation in the horse. We synthesized a ePSGL-1 peptide to generate a monoclonal antibody (mAb), ePL1. Using flow cytometry and Western blot, we showed that ePL1 binds specifically to ePSGL-1 in transfected mammalian cells. We also demonstrated that ePL1 binds to equine leukocytes and recognized a protein with molecular weight 165 and 280 kDa under reducing and non-reducing condition, respectively, likely corresponding to ePSGL-1.Seventy percent of equine monocytes bound by both ePL1 and HECA-452, an antibody defining sLex-like carbohydrate epitope. Both ePL1 and HECA-452 recognized ePSGL-1 protein precipitated by equine P-selectin-IgG chimera. Neuraminidase treatment increased ePL1 binding and the molecular weight of ePSGL-1, O-sialoglycoprotein endopeptidase digestion and tyrosine mutation abolished ePL1 staining and recognition. The ePL1 specific binding epitope appears to be the polypeptide backbone of ePSGL-1 in the presence of tyrosine but the process is independent of sialylation modification. In conclusion, we provide evidence that this antibody can be used for cell surface staining and immune-blot analyses. Published by Elsevier B.V.
Mammalian Genome, 2005
P-selectin glycoprotein ligand-1 (PSGL-1, CD162) is a dimeric, mucin-like, transmembrane glycopro... more P-selectin glycoprotein ligand-1 (PSGL-1, CD162) is a dimeric, mucin-like, transmembrane glycoprotein constitutively expressed on leukocytes. A high baseline level of P-selectin expression in circulating equine platelets suggests a primed state toward inflammation and thrombosis via P-selectin/PSGL-1 adhesion. To investigate the potential role of equine P-selectin in these events, we first identified the cDNA sequence of equine PSGL-1 (ePSGL-1) using degenerate PCR and RACE-PCR and then compared the predicted sequence with that of human PSGL-1 (hPSGL-1). ePSGL-1 protein subunit is predicted to be 43 kDa and composed of 420 amino acids with a predicted 18-amino-acid signal sequence showing 78% homology to hPSGL-1. Previously published work has shown that binding of P-selectin requires sulfation of at least one of three tyrosines and O-glycosylation of one threonine in the N-terminus of human PSGL-1. However, the corresponding domain in ePSGL-1, spanning residues 19–43, contains only one tyrosine in the vicinity of two threonines at positions 25 and 41. ePSGL-1 contains 14 threonine/serine-rich decameric repeats as compared to hPSGL-1 which contains 14–16 threonine-rich decameric repeats. The transmembrane and cytoplasmic domains display 91% and 74% homology to corresponding human PSGL-1 domains, respectively. In summary, there is 71% homology in comparing the open reading frame (ORF) of ePSGL-1 with that of hPSGL-1. The greatest homologies between species exist in the transmembrane domain and cytoplasmic tail while substantial differences exist in the extracellular domain.
Equine Veterinary Journal, 1989
Comparisons were made between transtracheal aspirate (TTA) and bronchoalveolar lavage (BAL) cytol... more Comparisons were made between transtracheal aspirate (TTA) and bronchoalveolar lavage (BAL) cytology obtained from 50 horses with chronic lung disease and from 10 control horses. There was no significant correlation between the TTA cytology and the BAL cytology, suggesting that the cell population in the trachea is not representative of the cell population in the lower airways. In control horses the range of differential cell counts obtained from TTA fluid was remarkably large, whereas the variability in cell populations observed in BAL fluid was smaller. In the principal horses the total and differential cell counts of the TTA and BAL fluids were within the 95 per cent confidence interval in 38 and 24 per cent of cases, respectively; and an increase in percentage neutrophils was most common. It was concluded that BAL may be a useful diagnostic aid when evaluating horses with chronic lung disease, but that the clinical usefulness of cytological evaluations of TTA fluid may be limited in these cases.
Veterinary Immunology and Immunopathology, 2006
Human P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric membrane mucin expressed on leukocyt... more Human P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric membrane mucin expressed on leukocytes that binds selectins. Here, we report that the open reading frame (ORF) of bovine PSGL-1 (bPSGL-1) cDNA is 1284 base pairs in length, predicting a protein of 427 amino acids including an 18-amino-acid signal peptide, an extracellular region with a mucin-like domain, and transmembrane and cytoplasmic domains. The amino acid sequence of bPSGL-1 demonstrated 52, 49 and 40% overall homology to equine, human and mouse, respectively. A single extracellular cysteine, at the transmembrane and extracellular domain junction, suggests a disulfide-bonding pattern. Alignment of bovine with equine, human and mouse PSGL-1 demonstrates high conservation of transmembrane and cytoplasmic domains, but diversity of the extracellular domain, especially in the anionic NH 2 -terminal of PSGL-1, the putative P-selectin binding domain. In the NH 2 -terminal of bPSGL-1, there are three potential tyrosine sulfation sites and three potential threonine O-glycosylation sites, all of which are required for P-selectin binding in human PSGL-1 (hPSGL-1). bPSGL-1 shares only 57% homology in amino acid sequence with the corresponding epitope region which binds the monoclonal antibody PL1 for hPSGL-1, and no cross-reactivity was found in bovine leukocytes. In summary, bPSGL-1 shares homology with hPSGl-1, but has differences in the putative extracellular Pselectin binding domain. #
Journal of Veterinary Internal Medicine, 2006
This prospective study compared survival rates of critically ill and septic foals receiving 1 of ... more This prospective study compared survival rates of critically ill and septic foals receiving 1 of 2 different types of commercial equine plasma and analyzed admission variables as possible predictors of survival. Standardized clinical, hematologic, biochemical, and hemostatic admission data were collected and foals received either conventional commercially available hyperimmune equine plasma or equine plasma specifically rich in antiendotoxin antibodies in a double-blinded, coded fashion. Sepsis was defined as true bacteremia or sepsis score >11. Overall survival rate to discharge was 72% (49/68). Foals that were nonbacteremic and demonstrated a sepsis score of ≤11 at admission had a 95% (18/19) survival rate. The survival rate to discharge for septic foals was 28/49 (57%), with truly bacteremic foals having a survival rate of 58% (14/24), whereas that for nonbacteremic, septic foals was 56% (14/25). Sensitivity and specificity for sepsis score 11 as a predictor of bacteremia were 74 and 52%, respectively. For the entire study population, a higher survival rate to discharge was documented for those foals receiving hyperimmune plasma rich in antiendotoxin antibodies (P= .012, odds ratio [OR] 6.763, 95% confidence interval [CI]: 1.311, 34.903). Administration of plasma rich in antiendotoxin antibodies also was associated with greater survival in septic foals (P= .019, OR 6.267, 95% CI: 1.186, 33.109). Statistical analyses demonstrated that, among 53 clinical and clinicopathologic admission variables, high sepsis score (P <.001), low measured IgG concentration (P= .01), high fibrinogen concentration (P= .018), low segmented neutrophil count (P= .028), and low total red blood cell numbers (P= .048) were the most significant predictors of overall mortality.
Veterinary Immunology and Immunopathology, 2003
Acute inflammatory diseases, such as colic, septicemia and endotoxemia are common in equines and ... more Acute inflammatory diseases, such as colic, septicemia and endotoxemia are common in equines and have been shown to be correlated to vascular injury and thrombosis. In humans with similar thrombotic conditions, P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1)-mediated platelet-leukocyte adhesion contributes to the pathogenesis of these disorders through the generation of inflammatory mediators and tissue factor. As such, we hypothesized that a P-selectin-PSGL-1 (platelet-leukocyte) interaction, similar to that in humans, may also exist in the horse. The objective of this study was to investigate phenotypic and morphological properties of equine platelet activation with a focus on CD62P (P-selectin) expression and CD62P mediated platelet-leukocyte interactions. To study high levels of platelet activation, we used 1 U/ml thrombin to induce secondary, irreversible aggregation in both human and equine platelets. Addition of glycyl-L-prolyl-L-arginyl-L-proline amide (GPRP) prior to thrombin activation blocked fibrin polymerization, allowing the use of flow cytometry to study a-granule expression as a measure of platelet activation. Thrombin activation resulted in high levels of activation, measured as P-selectin expression, in both humans and equines. Interestingly, our research illustrates that in healthy horses, P-selectin is also constitutively expressed on 20-25% of resting platelets. This finding is in direct contrast to humans, in which P-selectin expression is negligible (<5%) in the absence of agonist activation. The high baseline level of P-selectin expression among equine platelets may suggest that they are primed for leukocyte adhesion, possibly resulting in prothrombotic conditions. This phenomenon could be of significant clinical relevance, as it may be related to the rapid clinical decline often seen in equine patients with colic and endotoxemia, where vascular injury and thrombotic complications compromise patient survival. Based on these findings, further investigation into the mechanisms of platelet P-selectin-mediated inflammation and platelet-leukocyte mediated vascular injury in the horse appears warranted. #
Veterinary Immunology and Immunopathology, 2009
B.J. Darien).
Veterinary Immunology and Immunopathology, 2007
The recent molecular characterization and sequencing of equine P-selectin (ePsel), and its glycop... more The recent molecular characterization and sequencing of equine P-selectin (ePsel), and its glycoprotein ligand, P-selectin glycoprotein ligand-1 (PSGL-1), have provided the tools for further investigation into their role in leukocyte trafficking. Here, we report the generation of a genetically engineered chimeric protein (ePsel-IgG) in which the equine P-selectin lectin and epithelial growth factor (EGF) domains were covalently linked to the equine IgG1 heavy chain constant region. The soluble ePsel-IgG was observed to bind to equine monocytes by confocal microscopy and flow cytometry. Furthermore, equine monocytes bound to immobilized ePsel-IgG in a time course and dose dependent manner. Not only did ePsel-IgG act as an adhesion molecule, it was also found to activate ERK1/2 kinase and induce IL-8 mRNA expression in equine monocytes. That all of the aforementioned ePsel-IgG-induced cell binding and cell signaling were abolished by the addition of EDTA, suggested that ePsel-IgG chimera mediated events occurred via the Pselectin ligand, PSGL-1. We were able to demonstrate that 78% of equine monocytes cross-reacted with anti-human HECA-452 antibody, which recognizes the sialy-Lewis X (sLex) epitope, a well-known carbohydrate binding site on human PSGL-1. Preincubation of equine PBMC with neuraminidase or O-sialoglycoprotein endopeptidase (OSGP) reduced ePsel-IgG monocyte binding to 36% or 60%, respectively. Taken together, these data suggest that there might be two ligand recognition sites on P-selectin, one of which recognizes sLex and another which recognizes P-selectin ligand core protein. The ePsel-IgG chimera can be a useful as a reagent for further studies on the role of equine P-selectin and signal transduction in inflammatory events in horse. #
Shock, 1998
Because platelet-activating factor (PAF) is an important mediator of inflammation and heparin has... more Because platelet-activating factor (PAF) is an important mediator of inflammation and heparin has anti-inflammatory effects, we hypothesized that low molecular weight heparin (LMWH) would inhibit PAF-induced activation and chemotaxis in porcine neutrophils. Citrated blood was obtained from pentobarbital-anesthetized pigs, and neutrophils were isolated over a 55%/65% Percoll gradient. The effect of LMWH on basal phorbol myristate acetate (PMA)-induced superoxide (SO) release, as well as its effect on PAF priming for PMA-induced SO release, were investigated. Additionally, the effect of LMWH on PAF-induced chemotaxis of neutrophils across transwell membranes was evaluated. Baseline SO release in response to PMA was .351+/-.046 nmol/10(6) cells/min, and this was decreased to .289+/-.034 nmol/10(6) cells/min by pretreatment with 50 U/mL LMWH. PMA-induced SO production was increased by .240+/-.042 nmol/10(6) cells/min when cells were primed with 10 microM PAF. This priming effect of PAF was reduced significantly by pretreatment of neutrophils with LMWH at 10 and 50 U/mL. Chemotaxis of neutrophils in response to 100 microM PAF was significantly decreased to 70.02+/-6.4% (n = 8) of the control response by pretreatment of cells with 50 U/mL LMWH. We conclude that LMWH has anti-inflammatory effects on porcine neutrophils, which includes attenuation of cell activation and chemotaxis in response to the lipid-derived inflammatory mediator, PAF.
Journal of Veterinary Internal Medicine, 2005
The cardiac biomarkers cardiac troponin T (cTnT) and I (cTnI) and the cardiac isoenzyme of creati... more The cardiac biomarkers cardiac troponin T (cTnT) and I (cTnI) and the cardiac isoenzyme of creatine kinase (CKMB) are used extensively in human medicine to diagnose and provide valuable prognostic information in patients with ischemic, traumatic, and septic myocardial injury. We designed a study to establish normal values for these markers in healthy, neonatal foals and to compare them with values obtained from septic neonates in a referral hospital population. The 25th, 50th, 75th, and 95th percentiles for cTnI and CKMB in the healthy-foal population were 0.08, 0.14, 0.25, 0.49 ng/mL and 1.4, 2.3, 4.0, 7.4 ng/mL, respectively. The values obtained for cTnT were frequently (43/52 foals; 83%) below the lower limit of detection of the assay (0.009 ng/mL), but the median and range were 0.009 and 0.009-0.041 ng/mL, respectively. In the septic foal population, the 25th, 50th, 75th, and 95th percentile values for cTnI and CKMB were 0.05, 0.12, 0.22, and 1.10 ng/mL and 2.0, 4.4, 7.8, and 24 ng/mL, respectively. The values obtained for cTnT were less frequently below the lower limit of detection (23/38 foals; 60%) compared with the healthy foal population, and the median and range were 0.009 and 0.009–0.20 ng/mL, respectively. Significantly higher values were observed for cTnT and CKMB in septic foals compared with the healthy neonatal foal population, but there were no differences among septic foals in survivors compared with nonsurvivors. These findings suggest that myocardial injury occurs during septicemia in neonatal foals but that the injury is not associated with survival among septic foals.
Journal of Veterinary Internal Medicine, 2005
The cardiac biomarkers cardiac troponin T (cTnT) and I (cTnI) and the cardiac isoenzyme of creati... more The cardiac biomarkers cardiac troponin T (cTnT) and I (cTnI) and the cardiac isoenzyme of creatine kinase (CKMB) are used extensively in human medicine to diagnose and provide valuable prognostic information in patients with ischemic, traumatic, and septic myocardial injury. We designed a study to establish normal values for these markers in healthy, neonatal foals and to compare them with values obtained from septic neonates in a referral hospital population. The 25th, 50th, 75th, and 95th percentiles for cTnI and CKMB in the healthy-foal population were 0.08, 0.14, 0.25, 0.49 ng/mL and 1.4, 2.3, 4.0, 7.4 ng/mL, respectively. The values obtained for cTnT were frequently (43/52 foals; 83%) below the lower limit of detection of the assay (0.009 ng/mL), but the median and range were 0.009 and 0.009-0.041 ng/mL, respectively. In the septic foal population, the 25th, 50th, 75th, and 95th percentile values for cTnI and CKMB were 0.05, 0.12, 0.22, and 1.10 ng/mL and 2.0, 4.4, 7.8, and 24 ng/mL, respectively. The values obtained for cTnT were less frequently below the lower limit of detection (23/38 foals; 60%) compared with the healthy foal population, and the median and range were 0.009 and 0.009-0.20 ng/mL, respectively. Significantly higher values were observed for cTnT and CKMB in septic foals compared with the healthy neonatal foal population, but there were no differences among septic foals in survivors compared with nonsurvivors. These findings suggest that myocardial injury occurs during septicemia in neonatal foals but that the injury is not associated with survival among septic foals.
We have previously reported that Morinda citrifolia (noni) puree modulates neonatal calves develo... more We have previously reported that Morinda citrifolia (noni) puree modulates neonatal calves developmental maturation of the innate and adaptive immune system. In this study, the effect of noni puree on respiratory and gastrointestinal (GI), health in preweaned dairy calves on a farm with endemic salmonellosis was examined. Two clinical trials were conducted whereby each trial evaluated one processing technique of noni puree. Trials 1 and 2 tested noni versions A and B, respectively. Puree analysis and trial methods were identical to each other, with the calf as the experimental unit. Calves were designated to 1 of 3 treatment groups in each trial and received either: 0, 15 or 30 mL every 12 hr of noni supplement for the first 3 weeks of life. Health scores, weaning age, weight gain from admission to weaning, and weaned by 6 weeks, were used as clinical endpoints for statistical analysis. In trial 1, calves supplemented with 15 mL noni puree of version A every 12 hr had a higher probability of being weaned by 6 weeks of age than control calves (P = 0.04). In trial 2, calves receiving 30 mL of version B every 12 hr had a 54.5% reduction in total medical treatments by 42 days of age when compared to controls (P = 0.02). There was a trend in reduced respiratory (61%), and GI (52%) medical treatments per calf when compared to controls (P = 0.06 and 0.08, respectively). There were no differences in weight gain or mortality for any treatment group in either trial.