VTechWorks Repository :: Browsing by Author "Crisman, Mark V."

Browsing by Author "Crisman, Mark V."

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Adaptation of Three Different Apoptotic Methods in Equine Bronchoalveolar Cells and Comparison of Bronchoalveolar Lavage Cell Apoptosis in Normal and COPD Affected Horses Before and After Dexamethasone Administration
Leichner, Teri Lynn (Virginia Tech, 2001-06-21)
Recent studies suggest that lymphocyte apoptosis serves to regulate pulmonary inflammation. Equine COPD, an allergic disease of the lower airway, is likely due to dysregulation of the pulmonary immune response. In this study, the hypothesis tested was COPD affected horses would have less apoptotic airway lymphocytes than control horses during clinical disease. To achieve this, 3 methods of measuring apoptosis, Vindelov's propidium iodide with Triton-X (PI/Triton-X), 7-aminoactinomycin D (7-AAD), and Annexin V with propidium iodide (Annexin/PI) were evaluated in equine airway lymphocytes. A significant linear relationship was found for equine bronchoalveolar lavage (BAL) lymphocytes stained with 7-AAD and Annexin/PI . No relationship was identified with cells stained with PI/Triton-X and Annexin/PI, and 7-AAD and PI/Triton-X indicating that methods which preserve cell membrane characteristics are more comparable when measuring BAL lymphocytes apoptosis in a heterogeneous population of cells. Additionally, all stains appear to perform the same in COPD and normal horses in remission and disease. Comparison of predominately BAL lymphocyte apoptosis using the above methods were performed at baseline, after natural challenge, and after dexamethasone administration in nine horses, five of which were affected with COPD. No differences in bronchoalveolar lavage lymphocyte apoptosis between COPD and control horses were detected either before or after dexamethasone administration, although numerical trends in COPD horses identified less apoptosis after natural challenge indicating that defective apoptosis may play a role in equine COPD pathogenesis. Dexamethasone administration was associated with trends of improvement in the pulmonary gas exchange and increased apoptosis toward baseline in the COPD horses.
Apoptosis in the equine small intestine following experimental ischemia-reperfusion injury
Nagy, Amy Dae (Virginia Tech, 2008-05-23)
This study was aimed at characterizing the apoptotic response equine small intestine subjected to experimental ischemia-reperfusion injury and determining if use of an angiotensin converting enzyme inhibitor (enalaprilat) would ameliorate the apoptotic response. It was designed to determine if mucosal epithelial cells undergo apoptosis during the ischemia phase and also examined if apoptosis is significantly exacerbated by reperfusion. It also investigated whether administration of enalaprilat decreased reperfusion injury secondary to reduced enterocyte apoptosis. Injury was induced using a low flow model of I-R. During celiotomy a single loop of jejunum was isolated and arterial flow decreased to 20% of baseline for one hour and complete occlusion for a second hour. Reperfusion was monitored for 3 hours. A control group (n=6) were not treated while the treatment group (n=6) received 0.5 mg/kg enalaprilat in 0.9% NaCl immediately following ischemia. Jejunal samples were taken prior to the induction of ischemia, immediately post-ischemia and at 1, 2 and 3 hours of reperfusion. Samples were evaluated for gross tissue pathology with standard hematoxylin and eosin staining, the presence of apoptotic cells via TUNEL staining, and gene expression of three apoptosis related genes (bax, bcl-2, p53) using qPCR. Serum enalaprilat and ACE concentrations were determined from blood samples drawn concurrent with jejunal sampling using HPLC/MS and standard HPLC. Plasma enalaprilat concentrations were comparable to previous reports in awake horses. Enalaprilat appeared to have no effect on serum ACE concentrations, however a significant spike in ACE concentration occurred in the treatment group at 1 hour of reperfusion (P=0.0001). Grade of mucosal damage was not significantly different between control and treatment groups at any time point. Subjectively apoptotic index appeared to be higher in the treatment group at end ischemia and during reperfusion. There were no changes in expression of p53 or bcl-2 in either group. Bax expression was significantly decreased (P= 0.02) in the control group at 2 hours of reperfusion. Based on our data administration of an ACE inhibitor during anesthesia in horses with an ischemic segment of intestine confers no protective benefit and may be associated with increased intestinal injury and apoptosis. Lack of expression of p53, bax and bcl-2 suggests another apoptotic mechanism in equine ischemic intestine.
Biomarkers of oxidative stress and inflammation in biological samples collected from recurrent airway obstruction (RAO)-affected horses and their controls
Tan, Rachel Hsing Hsing (Virginia Tech, 2008-03-12)
Multiple biomarkers of oxidative stress have been measured and used in human medicine to diagnose and monitor airway disease. The purpose of the study was to determine if similar relationships existed between inflammatory and oxidative stress biomarkers in exhaled breath condensate (EBC), bronchoalveolar lavage fluid (BALF), red blood cells, white blood cells, and plasma; and cytokine expression in airway inflammatory cells and mucosal biopsies of RAO-affected horses and their controls. Sixteen horses in pairs were used: 8 non-RAO-affected (controls) and 8 RAO-affected horses. Samples from all horses were collected at remission (S1), during environmental challenge (S2) and at recovery (S3). RAO-affected horses had significant alterations in cellular glutathione peroxidase (cGPx) activity, ascorbic acid and pH in a number of biological samples. Concentrations of 8-isoprostanes, isofurans, amino acids and mRNA expression of interleukin 4 (IL4), gamma interferon (INFγ), inducible nitric oxide synthase (iNOS), extracellular glutathione peroxidase (GPx-3), and cytosolic superoxide dismutase (SOD-1) were not significantly different or were at the limits of detection. Conductivity was measured and assessed as a potential correctional factor for respiratory fluid dilution. The alterations in biomarker concentrations demonstrate that oxidative stress is an important component of airway inflammation in RAO-affected horses. Further research is warranted in the use of biomarkers and the effects of dietary interventions.
Can isoprostanes be used to predict survival in horses with colic?
Noschka, Erik (Virginia Tech, 2010-11-09)
Approximately 4% of horses suffer from one colic episode per year. The outcome is fatal in 11% of cases. F2-isoprostanes are the "gold standard" for assessment of oxidative stress in vivo and have been used extensively to quantify lipid peroxidation in association with risk factors in various diseases in humans. Because horses with colic may have intestinal ischemia and/or inflammation characterized by oxidative stress and increased production of isoprostanes, measurement of isoprostane concentrations in colicky horses may be of clinical value. The purpose of this study was to gather preliminary data on the feasibility of using urine isoprostane concentrations as an early screening tool for the severity of colic and to determine the need for surgery. The long term goal of this investigation is to reduce the number of deaths due to colic by developing a stall-side test capable of identifying horses needing surgery as early as possible and expediting their timely referral. We hypothesized that urine isoprostanes and isoprostane metabolites would be significantly higher in horses with colic compared to normal horses and that they can be used an indicator for the need for surgical intervention. Urine samples were collected from 42 normal horses and 38 horses with colic (21 medical and 22 surgical). Urine isoprostane and isoprostane metabolite concentrations were measured by mass spectrometry and normalized by urine creatinine (Cr) concentrations. Statistical analysis was performed using a one way ANOVA (Tukey's post-hoc comparison) and a 2 sample t-test. Significance was set at P<0.05. Mean (± SD) concentrations of isoprostanes and isoprostane metabolites were significantly higher in urine samples of horses with colic (2.94 ± 1.69 ng/mg Cr and 0.31 ± 0.22 ng/mg Cr, respectively) compared to healthy horses (1.89 ± 1.39 ng/mg Cr and 0.22 ± 0.08 ng/mg Cr, respectively). Urine isoprostane metabolite concentrations were significantly higher in horses undergoing surgery (0.38 ± 0.28 ng/mg Cr) compared to healthy control horses and medically treated colic horses (0.26 ± 0.11 ng/mg Cr). Non-survivors had significantly higher mean urine isoprostane metabolite concentrations (0.47 ± 0.39 ng/mg Cr) compared to healthy control horses and surviving colic horses (0.29 ± 0.24 ng/mg Cr). Since urinary concentrations of isoprostane metabolites are increased in horses suffering from colic and in non-survivor colic horses, the measurement of urine concentrations of isoprostane metabolites may be an important prognostic indicator in equine colic.
Comparison of Two Aerosolized Bronchodilators in the Treatment of Severe Equine Chronic Obstructive Pulmonary Disease
Friday, Philippa Anne (Virginia Tech, 1999-08-04)
Aerosolized bronchodilator drugs are commonly used for treatment of horses with chronic obstructive pulmonary disease. Relative efficacy of sympathomimetic and parasympatholytic bronchodilators for relief of acute airway obstruction and improvement of pulmonary gas exchange was compared in 6 horses with COPD. Physical examination, arterial and venous blood gas analysis and measurement of end-tidal CO₂ tension were performed at time zero, 30 minutes, 1, 2, 3 and 4 hours after administration of aerosol ipratropium (0.35 μg/kg), albuterol (1 μg/kg) or placebo via an equine Aeromask and metered dose inhaler. Physiologic shunt fraction (QS/QT), alveolar dead space fraction (VD/VT) and alveolar to arterial oxygen tension difference (p(A-a)O₂) were calculated using standard formulas. At time zero, horses demonstrated severe respiratory compromise and marked alterations in pulmonary gas exchange, indicative of alveolar hypoventilation, VA/Q mismatching and diffusion impairment. Ipratropium treatment significantly (p< 0.05) reduced arterial CO₂ tension and end-tidal CO₂ tension toward normal, but significantly increased p(A-a)O₂ from baseline. The change in paCO₂ after ipratropium treatment was significantly different from albuterol and placebo treatment groups. There were no significant changes in response variables after albuterol and there were no treatment by time interactions. These results indicate that, under the conditions of this study, ipratropium (0.35 μg/kg) improved alveolar ventilation and had superior bronchodilator efficacy than albuterol (1 μg/kg) in horses with severe COPD. Marked impairment of pulmonary gas exchange persisted after bronchodilators, emphasizing that anti-inflammatory therapy and environmental control are also necessary for effective treatment of severe equine COPD.
Effects of Long-Term Exposure of Normal C57BL/6J Inbred Mice to 17β-Estradiol on Gene Expression in Lymphocytes: mRNA Analysis of Lymphokines and bcl-2/fas
Yin, Zhi-Jun (Virginia Tech, 1997-08-05)
It is now clear that human and animal exposure to estrogenic compound occurs through several sources. This include: i) naturally occurring endogenous estrogens, ii) exogenous or intentional estrogens for prophylactic (e.g. oral contraceptive) and therapeutic (e.g. as replacement therapy for ovulation in nulliparous women and in menopausal women, and in some men suffering from prostate cancer) purposes, iii) accidental via estrogenic chemical exposure (e.g. pesticides, industrial byproducts) and phytoestrogens (e.g. soybeans). It has long been recognized that estrogen, a female sex hormone, functions not only on the reproductive system, but also on various other systems including the immune system. Estrogens are thought to be of both physiologic and pathologic importance. Female in general, have better immune capabilities than males, a phenomenon attributed to the action of sex hormones on the immune system. There is also a female-gender bias in susceptibility to autoimmune diseases. Estrogens have been linked either directly or indirectly to the etiology and pathogenesis of various female-predominant autoimmune diseases. Estrogens have also been linked to the onset of cancer, and conditions where the immune system often malfunctions. Estrogen affects the functions of both B and T cells, possibly by regulating such factors as lymphokine gene expression and/or cellular death by apoptosis. However, the functioning of both B and T cells under the influence of long-term exposure to estrogen has not been fully understood. The primary aim of this thesis was to investigate the effect of long-term exposure to 17β-estradiol on lymphokine and bcl-2/fas (proto-oncogenes) mRNA expression. We evaluated the effects of estrogen on the expression of genes for lymphokines, which are essential for the immune response. It is hypothesized that estrogen may regulate the immune system by modifying the expression of lymphokine genes and/or genes that regulate apoptosis. The results demonstrated that long-term 17β-estradiol exposure reduced the viability of lymphocytes when compared to lymphocytes from placebo-treated mice. IL-2 and IFN-g mRNA was consistently higher in ConA-stimulated lymphocytes from estrogen-treated mice (P < 0.05). The mRNA for TGF-β₁ lymphokine was also increased but was not consistent at all time points of incubation. The expression of IL-4 mRNA was not noticeably affected by estrogen treatment of mice. Long-term exposure to 17β-estradiol appear to have some influence on the mRNA expression of proto-oncogenes fas and bcl-2 in splenic and thymic T lymphocytes. There was a trend of increased bcl-2 mRNA expression in estrogen-treated mice compared to placebo-treated mice, whereas the mRNA expression of fas gene appeared to be lower compared to controls. Overall, these findings suggest that 17β-estradiol may selectively influence lymphokine and proto-oncogene mRNA expression. These results suggest that the one mode of modulation of the immune response by 17β-estradiol may be through alterations in the lymphokine and proto-oncogene expression. Since estrogen-treatment markedly induces atrophy of the thymus and diminishes the cellularity of the lymphoid organs (e.g. Spleen), it became necessary to perform multiple assays on the same cells, particularly lymphokine and apoptosis gene expression. A secondary objective of this thesis was to investigate whether lymphocytes, which have undergone proliferation in Lympho-Pro™ assay (Alamar Blue assay), could be utilized for further analysis. In this regard, we found that a non-radioactive assay that utilizes Alamar Blue had significant advantages over the conventional ³H-thymidine incorporation assay. By using cells from estrogen and placebo-treated mice in the Alamar Blue assay, we found that this assay not only allowed determination of lymphocyte proliferation, but also the assessment of mRNA expression, cytogenetics, apoptosis and immunophenotyping of the same lymphocytes.
Evaluation of Iohexol Clearance to Estimate Glomerular Filtration Rate in Normal Horse Foals
Gonda, Kathleen A. (Virginia Tech, 2002-04-11)
Estimation of the glomerular filtration rate (GFR), accepted as one of the earliest and most sensitive indicators of renal dysfunction, can be determined in horses using standard techniques In adult horses and foals, renal dysfunction can occur as a secondary complication to gastrointestinal disorders, dehydration, septicemia, endotoxemia and nephrotoxic drug administration. Measurement of renal function is an important feature not only in the diagnosis, but also in the prognosis and management of renal disease. Commonly used drugs such as phenylbutazone and gentamicin can be highly nephrotoxic under certain conditions. Of particular concern are those drugs, including the aminoglycoside antibiotics, that are eliminated almost exclusively by the kidney. Knowledge of a patients renal status prior to treatment would direct efforts at; 1) restoring kidney function prior to protracted therapy with potentially damaging drugs, 2) adjusting the dose of a life-saving drug based on the magnitude of dysfunction, or 3) selecting a drug that is not dependant on renal function for elimination. such as endogenous or exogenous renal creatinine clearance. Unfortunately, these techniques can be time consuming, dangerous to perform on fractious patients, require trained personnel and are subject to errors most often associated with improper or incomplete urine collection. Recently, tests using iohexol, a radiographic contrast agent, have been developed to estimate the GFR in human beings, dogs and cats with results that have been validated by traditional standards. Most testing protocols require a single bolus injection of iohexol, followed by 2 or 3 blood samples obtained over a few hours. Compared to traditional testing methods, samples are easily and rapidly obtained making the testing procedure less stressful for the patient. A simple method to measure GFR in horses that does not require urine collection, would allow veterinarians in a clinical setting the ability to determine a patient's renal status easily and safely. The objectives of this study were; 1) model the pharmacokinetic profile of iohexol in horse foals, 2) compare creatinine clearance, an accepted standard for GFR determination in patients, with iohexol clearance, and 3) develop sampling parameters and calculation methods for a practical test, based on iohexol clearance, that compares favorably with creatinine clearance in horse foals. Iohexol concentration time data were best described using a 3-compartment open model. Mean creatinine clearance (2.17 ml/min/kg) and mean iohexol clearance (2.15 ml/min/kg) showed good agreement. In addition, GFR values for all foals using either method were within published reference ranges for this species. The results of this study indicate that a single intravenous injection of iohexol at a dose of 150 mg/kg, followed by collection of 2 serum samples at 4 and 6 hours post injection can be used to estimate the GFR in healthy horse foals. Mean corrected GFR value (CLpredicted) for 10 foals in this study was 2.15 ml/min/kg.
Evaluation of iohexol clearance to estimate glomerular filtration rate in normal horses
Wilson, Katherine E. (Virginia Tech, 2006-04-18)
In adult horses and foals, renal dysfunction can occur as a secondary complication to gastrointestinal disorders, dehydration, septicemia, endotoxemia and nephrotoxic drug administration. Measurement of renal function is an important feature not only in the diagnosis, but also in the prognosis and management of renal disease. Commonly used drugs such as phenylbutazone and gentamicin can be highly nephrotoxic under certain conditions. Estimation of the glomerular filtration rate (GFR), accepted as one of the earliest and most sensitive assessments of renal function, can be determined in horses using standard techniques such as endogenous or exogenous renal creatinine clearance. These techniques can be time consuming, dangerous to perform on fractious patients, require trained personnel and are subject to errors most often associated with improper or incomplete urine collection. Recently, tests using iohexol, a radiographic contrast agent, have been developed to estimate the GFR in human beings, pigs, sheep, dogs, cats and horse foals with results that have been validated by traditional standards. Serum clearance of a substance that is freely filtered by the kidneys without tubular secretion or reabsorption, that is not protein bound, and that is not metabolized, is a measurement of glomerular filtration rate. Iohexol meets all of these requirements and thus its clearance from serum should accurately estimate GFR. Utilization of serum clearance studies for estimation of GFR provides a clinically feasible and reproducible method in order to measure GFR in horses. Other commonly used methods to assess renal function in horses are fraught with inherent and operator error. Serum clearance of iohexol does not necessitate collection of urine and has been shown to be a safe, reproducible method using collection of timed blood samples to assess renal function in humans and animals. The objectives of this project were 1) to determine a method of estimation of GFR based on serum clearance of a substance that meets the requirements of a marker for GFR, and 2) to make the method clinically applicable by developing a method using two blood samples to derive clearance and thus GFR in normal adult horses. Results of this study showed good agreement between GFR derived by exogenous creatinine clearance and serum clearance of iohexol. In addition, GFR values for all horses using either method were within published reference ranges for this species. The results of this study indicate that a single intravenous injection of iohexol at a dose of 150 mg/kg, followed by collection of 2 serum samples at 3 and 4 hours post injection can be used to estimate the GFR in healthy horses.
Gene Expression in Endometrial Tissues of Normal Mares and Mares With Delayed Uterine Clearance
Gray, Giles Anthony (Virginia Tech, 2006-04-04)
Delayed uterine clearance (DUC) is a significant problem contributing to subfertility and infertility in the mare, characterized by an accumulation of fluid and inflammatory debris in the uterine lumen following breeding events, venereal disease or an estral cycle. This syndrome is typically seen in older, multiparous mares and mares with poor reproductive tract conformation. The etiopathogenesis of DUC has not been fully elucidated but suggested causes include poor genital conformation, a cranioventrally tilted uterus, defective myometrial contractions, decreased intrauterine immune activity, inappropriate lymphatic drainage or mucus overproduction. The objective of this research was to evaluate gene expression of selected genes in endometrial tissue samples taken from three categories of mares (young fertile [YF], older clinically normal [ON] and older susceptible [OS]). The genes assayed in this research were oxytocin receptor, PGF2Ã¡ receptor and progesterone receptor. The expression of each of these genes was normalized using the expression of two housekeeping genes, beta actin and ribosomal 18S RNA. Quantitative real-time polymerase chain reaction (QPCR) was used to evaluate gene expression of the selected genes. Results indicated that there was no statistically significant difference in the expression of any of the three experimental genes among any of the three categories of mares. From this research, the direction of further research regarding the pathogenesis of DUC can be made: myometrial tissues can be assayed for similar genes, the expression of other genes regulating myometrial contraction can be assayed or the expression of uterorelaxants can be studied.
Hydration of Colonic Ingesta and Feces in Horses Fed Large Grain Meals or Treated with Enteral Fluid Therapy, Saline Cathartics and Intravenous Fluid Therapy
Lopes, Marco A. F. (Virginia Tech, 2002-09-30)
Systemic hydration, plasma electrolytes, ingesta and fecal hydration and gastrointestinal passage of cobalt (after CoEDTA administration via nasogastric tube) in horses fed large grain meals or treated with enteral fluid therapy, IV fluid therapy and enteral laxatives were investigated. In the first study, 0.9% NaCl (10 L/h/8h) was administered slowly via a small-bore nasogastric tube or as 10 L boluses via a large-bore nasogastric tube to four normal horses. In the other studies, horses with a right dorsal colon fistula were used. To create the right dorsal colon fistula, a cannula with 5 cm internal diameter was implanted 2 to 6 weeks after a right dorsal colopexy had been created. Six horses with the right dorsal colostomy were alternately used to test three feeding regimes for 48 h: 1- hay free choice; 2- hay free choice plus 4.5 kg of sweet feed twice daily after a period of 5 days of adaptation; 3- sudden change from hay to hay plus sweet feed. Seven horses with the right dorsal colostomy were alternately used to test 6 experimental conditions while fasted for 24 h: 1- control (no treatment), 2- enteral MgSO4 (1 g/kg), 3- enteral Na2SO4 (1 g/kg), 4- IV lactated Ringer's solution (5 L/h/12 h), 5- enteral water (5 L/h/12 h), 6- enteral electrolyte solution (5 L/h/12 h). In the last study, four horses with the right dorsal colostomy were alternately treated with enteral electrolyte solution (10 L/h/6h) and enteral MgSO4 (1 g/kg) plus IV fluid therapy (10 L/h/6h). Despite the administration regimen, enteral administration of 0.9% NaCl produced diarrhea, hypernatremia and hyperchloremia. Colostomy allowed serial collection of large ingesta samples. Grain ingestion did not change PCV or plasma protein, but affected plasma electrolytes and produced dehydration of ingesta and formation of frothy ingesta. Fasting delayed gastrointestinal transit. Enteral fluid therapy was the most effective treatment in promoting ingesta hydration. Enteral water, MgSO4, Na2SO4, IV fluid therapy and enteral MgSO4 plus IV fluid therapy were either ineffective in promoting ingesta hydration or produced marked plasma electrolyte imbalance. These findings support the use of enteral fluid therapy in horses with gastrointestinal impaction.
Immunogenicity of Potomac horse fever vaccine when simultaneously co-administered with rabies vaccine in a multivalent vaccine or as two monovalent vaccines at separate sites
McKenzie, H. C.; Funk, Rebecca A.; Trager, L.; Werre, Stephen R.; Crisman, Mark V. (Wiley, 2019-11-01)
Background: Potomac horse fever (PHF) is a potentially fatal enterocolitis of horses caused by Neorickettsia risticii. The disease was originally recognised almost 40 years ago in the state of Maryland in the US. It is now known to occur in many areas of North America, as well as having been described in South America and Europe. Monocomponent PHF vaccines are available, but clinical protection with vaccination has been reported to be inconsistent. Objectives: This study was designed to assess the immunogenicity of a commercially available Potomac Horse Fever (PHF) vaccine when administered as either a monovalent PHF vaccine simultaneously co-administered with a separate monovalent Rabies vaccine or as a multivalent PHF/Rabies vaccine in horses. Study design: Randomised parallel group trial. Methods: Ninety-one client or University owned horses participated in this open-label randomised study, with 45 horses receiving the monovalent vaccines at separate sites and 46 receiving the multivalent vaccine at a single site. Serum PHF IFA titres were determined twice prior to vaccination and at 1, 2 and 3 months after vaccination. Results: Both vaccination protocols exhibited poor immunogenicity, with only one-third of all the animals demonstrating seroconversion, defined as an increase in titre of greater than 400 over baseline, at any time point after vaccination. The monovalent PHF vaccine exhibited significantly greater immunogenicity in terms of the number of horses exhibiting seroconversion, as compared to the multivalent vaccine, at one (20 vs. 11, P = 0.03) and two (18 vs. 9, p = 0.02) months post vaccination. The monovalent PHF vaccine also exhibited significantly greater immunogenicity in terms of the median (interquartile range) IFA titres, as compared to the multivalent vaccine, at one (800 [200–1600] vs. 400 [200–800], P = 0.009) and 2 months (400 [200–1600] vs. 400 [100–800], P = 0.02) post vaccination. There was no significant difference between groups at 3 months in either seroconversion rate or median IFA titers. Main limitations: This study did not assess the actual protective effects of PHF vaccination but rather used the serologic response to vaccination as a surrogate biomarker of immunity. Conclusions: The multivalent PHF/Rabies vaccine exhibited lower immunogenicity as compared to the monovalent PHF vaccine co-administered with a separate Rabies vaccine.
Immunotoxic Effects of Mixtures of Endosulfan and Permethrin Via Caspase Dependent Thymocyte Apoptosis
Keenan, James John (Virginia Tech, 2003-03-21)
Altered immune responses have been observed following occupational, inadvertent, or therapeutic exposure to xenobiotics. Many pesticides are known to cause immunotoxicity. Exposure to mixtures of pesticides, either concurrently or sequentially, may result in potentiating this effect partly because one can effect the metabolism of the other. The objective of this study was to determine the effect of the insecticides endosulfan, permethrin and their mixtures on C57/BL6 male mice thymocytes in vitro and to ascertain the mechanism by which these effects take place. Permethrin, a broad-spectrum synthetic pyrethroid, is a widely used insecticide in agriculture and public health. Endosulfan is a highly toxic chlorinated hydrocarbon insecticide used worldwide. We examined the immunotoxic potential of these pesticides using a flow cytometric technique in combination with 7-Amino Actinomycin D (7AAD) to distinguish live, early apoptotic, and late apoptotic/necrotic cells. DNA ladder assay, a hallmark of apoptosis, was also used to determine the occurrence of apoptosis. Both endosulfan and permethrin were found to cause significant apoptotic death of thymocytes in a dose- and time- dependent manner. Thus, permethrin at 50, 100 or 300 µM was found to cause 5.5, 11.5 and 26.1% increases in early apoptotic cell death relative to control, respectively. Endosulfan at 25, 50 or 250 µM was found to cause 11.9, 15.7 and 68.0% early apoptotic cell death, respectively. For the mixture study, concentrations of 100 µM permethrin and 50 µM endosulfan were selected and found to cause 27.1% apoptosis. Thus, these pesticides in mixture have an additive immunotoxic effect. Increases in late-apoptotic/necrotic cells were found at these concentrations for either pesticide when exposed for 12 hours. DNA ladder assay confirmed the presence of DNA fragments and therefore the presence of significant apoptotic cell death. Apoptosis is a morphologically distinct form of cell death that can be mediated by a variety of pathological and physiological stimuli. Because permethrin and endosulfan were found to induce apoptosis in C57/BL6 mice thymocytes in vitro, the objective of the second half of this study was to elucidate the potential mechanism by which these pesticides regulate apoptosis in immune cells. Caspases are a family of cystine-dependent, aspartate-directed proteases that have an integral role in apoptotic cell death. Caspases, which are normally inactive in healthy cells, are activated during apoptosis and form an irreversible cascade. There are two subsets of caspases, initiator caspases (i.e. caspase 8 and 9) and effector caspases (i.e. caspases 3 and 6). Caspase 3, a downstream effector of apoptosis, is activated by many different pathways and is an apoptotic marker in cells. Caspase 8 is the apical caspase in the extrinsic pathway. Caspase 9 is the apical caspase in the intrinsic pathway, therefore we investigated mechanisms of pesticide induced apoptosis involving the thymocyte caspase system. Thymocytes from C57/BL6 mice were incubated with varying concentrations of pesticides for varying amounts of time. Active caspase 3 was then measured using EnzCheck Caspase 3 Assay Kit. Relative fluorescence for permethrin exposed cells after 12 hours incubation in the presence of pesticides at 150, 100, and 50 µM and 40 minutes in the presence of AFC-substrate was found to be 387, 386, and 297, respectively. Relative fluorescence for endosulfan exposed cells at 150, 100 and 50 µM was 188, 177, and 294. Caspase 3 activity increased as permethrin concentrations increased and decreased as endosulfan concentrations were increased. Then the extrinsic and intrinsic pathways of apoptosis were further investigated. Active caspase 8 was measured using the ApoAlert Caspase Fluorescent Assay Kit. Relative fluorescence for permethrin exposed cells after 7 hours incubation in the presence of pesticides at 100, 150, and 200 µM was found to be 35.5, 10.5, and 0, respectively. Relative fluorescence for endosulfan exposed cells after 7 hours incubation at 25, 50, 100 and 150 µM was found to be 32.8, 63.8, 69.5, and 55.5, respectively. A mixture study was then performed using endosulfan (50, 100, 150 µM) combined with permethrin (100 µM). All combinations were found to have more than an additive effect, therefore the extrinsic pathway seems to be involved. Caspase 9 activity was measured using Caspase 9/Mch6 Fluorometric Protease Assay Kit. Relative fluorescence for endosulfan exposed cells after 7 hours incubation at 25, 50, 100 and 150 µM was found to be 43, 73, 78.9, and 5.12, respectively. Relative Fluorescence for permethrin exposed cells at 100, 150 and 200 µM was found to be 34.5, 39, and 55.5, respectively. A mixture study was then performed using endosulfan (25, 50 µM) combined with permethrin (100 µM). Both combinations were found to have less than an additive effect. These results suggest that apoptosis caused by both endosulfan and permethrin exert their effects via the caspase pathway. The results also show that mixtures of pesticides have a less than additive effect on caspase 9 activation and more than an additive effect on caspase 8 activation, therefore the extrinsic pathway is predominantly involved in thymocyte apoptosis caused by mixtures of permethrin and endosulfan.
In vitro cytotoxic activity of equine lymphocytes on equine herpesvirus-1 infected allogenic fibroblasts
Edens, Lucy Marie (Virginia Tech, 1994-05-15)
The objectives of this study were to: 1) develop a technique to analyze the in vitro cytotoxic activity of lymphocytes from adult horses against equine herpes virus-1 (EHV-1) infected allogenic equine dermal fibroblasts (EDF); 2) evaluate the ability of a 72 hour in vitro incubation with interleukin-2 (I L-2) to enhance the lymphocytic cytolytic activity against EHV-1 infected EDF; 3) compare the cytotoxic activity among lymphocytes isolated from pregnant mares and non-pregnant mares against EHV-1 infected EDF; 4) ascertain if any correlations existed between the percent cytotoxicity and percentage of lymphocytes phenotypically identified by five different mouse-anti-equine monoclonal antibodies; and 5) determine if any correlation existed between virus-neutralizing antibody titers and the percent cytotoxicity. Results of the study indicate that in vitro cytotoxic activity of equine lymphocytes against EHV-1 infected allogenic fibroblasts can be measured with a standard 4 hour 51Cr release assay. This activity was enhanced by an in vitro incubation with IL-2. The cytolytic activity of freshly isolated lymphocytes was greater for non-pregnant than pregnant mares. However, after IL-2 stimulation the cytolytic activity was greater for lymphocytes from pregnant mares. A positive correlation was not detected between the percentage of phenotypically identified cells and the percent cytoxicity, although several negative correlations were present. This suggests that the cytotoxic activity was either not mediated by any of the phenotypically identified cell populations or that the activity was mediated by several different cell populations. No correlation was detected between virus neutralizing antibody titers and the percent cytotoxicity.
In vitro susceptibility of Borrelia burgdorferi isolates to three antibiotics commonly used for treating equine Lyme disease
Caol, Sanjie; Divers, Thomas; Crisman, Mark V.; Chang, Yung-Fu (2017-09-29)
Background Lyme disease in humans is predominantly treated with tetracycline, macrolides or beta lactam antibiotics that have low minimum inhibitory concentrations (MIC) against Borrelia burgdorferi. Horses with Lyme disease may require long-term treatment making frequent intravenous or intramuscular treatment difficult and when administered orally those drugs may have either a high incidence of side effects or have poor bioavailability. The aim of the present study was to determine the in vitro susceptibility of three B. burgdorferi isolates to three antibiotics of different classes that are commonly used in practice for treating Borrelia infections in horses. Results Broth microdilution assays were used to determine minimum inhibitory concentration of three antibiotics (ceftiofur sodium, minocycline and metronidazole), for three Borrelia burgdorferi isolates. Barbour-Stoner-Kelly (BSK K + R) medium with a final inoculum of 106 Borrelia cells/mL and incubation periods of 72 h were used in the determination of MICs. Observed MICs indicated that all isolates had similar susceptibility to each drug but susceptibility to the tested antimicrobial agents varied; ceftiofur sodium (MIC = 0.08 μg/ml), minocycline hydrochloride (MIC = 0.8 μg/ml) and metronidazole (MIC = 50 μg/ml). Conclusions The MIC against B. burgorferi varied among the three antibiotics with ceftiofur having the lowest MIC and metronidazole the highest MIC. The MIC values observed for ceftiofur in the study fall within the range of reported serum and tissue concentrations for the drug metabolite following ceftiofur sodium administration as crystalline-free acid. Minocycline and metronidazole treatments, as currently used in equine practice, could fall short of attaining MIC concentrations for B. burgdorferi.
Insulin-like growth factor-I in growing horses and RNA isolation from small articular cartilage samples
Cosden, Rebekah Stacey (Virginia Tech, 2007-08-10)
A longitudinal study was designed to characterize developmental patterns of plasma (PL) and synovial fluid (SF) total insulin-like growth factor-I (IGF-I) concentrations, as well as their association with measurements of skeletal growth in Thoroughbred horses. Horses were randomly assigned to one of two dietary treatment groups and fed diets with either a high or low starch content to examine the effects of dietary energy source on PL and SF IGF-I. At 3, 6, 9, 12 and 15 mo of age, PL and carpal SF samples were collected for analysis of total IGF-I. Body weight gain, wither height gain and forearm length gain were calculated for the 90 day periods between SF and PL sampling. No influence of diet on PL or SF IGF-I was detected (P > 0.05). Average SF IGF-I concentrations were 30.1 ± 1.8% of that found in PL, and PL and SF IGF-I were positively correlated (r = 0.48, P = 0.0003) There was an effect of month of age on both PL and SF IGF-I concentrations (P < 0.05). There was a positive correlation between all measures of gain except forearm length gain with PL and SF IGF-I (r = 0.41 to 0.55, P < 0.05). In our second study, we evaluated the use of a liquid-nitrogen cooled mortar and pestle, motorized freezer mill and rotor-stator homogenizer for homogenization of small (<50mg) articular cartilage samples. The rotor-stator homogenizer produced quanitfiable RNA yields, and was used to evaluate three different RNA isolation protocols. Two of the protocols were commercially available RNA extraction kits, with the third a modified guanidinium isothiocyanate/acid-phenol extraction procedure. The combined average yield for all protocols was 91.9 ng RNA/mg of cartilage. All protocols yielded a sufficient quantity of quality RNA suitable for gene expression analysis.
Pesticide Mixtures Induce Immunotoxicity: Potentiation of Apoptosis and Oxidative Stress
Rabideau, Christine L. (Virginia Tech, 2001-07-10)
The three insecticides of interest were lindane (an organochlorine), malathion (an organophosphate) and piperonyl butoxide (PBO; a synergist). Based on minimum cytotoxicity (> LC25), the following concentrations were chosen for the pesticide mixture studies: 70Î¼M lindane (Lind), 50Î¼M malathion (Mal) and 55Î¼M PBO. In the AlamarBlue cytotoxicity assay, individual pesticide and mixtures of malathion/PBO (MP) and malathion/lindane (ML) prompted cytotoxicity with varying intensities (Mal 18.8%, Lind 20.4%, PBO 23.5%, ML 53.6% and MP 64.9%). Cytopathological analysis revealed apoptotic features in treated cells and the DNA Ladder Assay confirmed the presence of DNA fragments. The specific mode of cell death was examined via the 7-aminoactinomycin D (7-AAD) Staining Assay. Apoptosis was detected in each treatment (Mal 6.5%, Lind 12.0%, PBO 13.2%, ML 19.3% and MP 23.4%). Furthermore, 7-AAD staining in combination with fluorescent-labeled monoclonal antibodies, PE-CD45RB/220 and FITC-CD90, was performed. B-cells were more susceptible to Mal and PBO treatments than were T-cells. The pro-oxidant activity of the pesticides was monitored via the Dichlorofluorescin Diacetate assay. Exposure to pesticides for 15 minutes increased H2O2 production above the controls, Mal 21.1%; Lind 10.8%; PBO 25.9%; ML 26.8%; MP 37.8%. The activities of antioxidant enzymes, glutathione peroxidase (GSH-Px) and glutathione reductase (GR) were altered by these treatments. GR was significantly reduced for the pesticide mixtures only (control: 51.7; Mal: 48.2; Lind: 50; PBO: 52.3; ML: 40.5; MP: 42 Units/mg). GSH-Px activity was severely reduced for all the pesticide treatments (control: 44.9; Mal: 30.2; Lind: 30.6; PBO: 32.4; ML: 21.1; MP: 21.1 Units/mg). These results indicate that exposure to these pesticide and pesticide mixtures induces apoptosis and oxidative stress.
A pharmacokinetic and pharmacodynamic study of pioglitazone in a model of induced insulin resistance in normal horses
Wearn, Jamie Macquarie (Virginia Tech, 2010-05-28)
Equine Metabolic Syndrome (EMS) is a unique condition of horses characterized by adiposity, insulin resistance, and an increased risk of laminitis. Reducing insulin resistance may decrease the incidence of laminitis in horses with EMS. Pioglitazone, a thiazolidinedione class of anti-diabetic drug, has proven efficacy in humans with type 2 diabetes, a syndrome of insulin resistance sharing some similarities with EMS. The ability of pioglitazone to influence insulin sensitivity in an endotoxin-infusion model of induced insulin resistance was investigated. Our hypothesis was that piogltiazone would preserve insulin sensitivity in a model of induced insulin resistance. The specific aims were to investigate the pharmacokinetics and pharmacodynamics of pioglitazone in an endotoxin infusion model of insulin resistance. 16 normal adult horses were enrolled. Pioglitazone was administered to 8 horses (1 mg/kg, PO, q24h) for 14 days, and 8 horses served as their controls. Liquid chromatography with tandem mass spectroscopy was used to quantitate plasma concentration of pioglitazone. A frequently sampled intravenous glucose tolerance test with minimum model analysis was used to compare indices of glucose and insulin dynamics prior to, and following, endotoxin infusion in horses treated with pioglitazone and their controls. Parameters of clinical examination and lipid metabolism were compared prior to, and following, endotoxin administration. Pioglitazone administered orally at 1 mg/kg q 24 h resulted in plasma concentrations lower, and more variable, compared to those considered therapeutic in humans. No significant effect of drug treatment was detected on clinical parameters or indices of insulin dynamics or lipid homeostasis following endotoxin challenge.
The Pharmacokinetics of Firocoxib after Multiple Oral Doses to Neonatal Foals
Hovanessian, Natasha (Virginia Tech, 2012-07-05)
The purpose of this study was to determine the safety and pharmacokinetic profile of firocoxib in healthy neonatal foals. Foals are more sensitive to the side effects of nonsteroidal anti-inflammatory drugs, (NSAIDs), particularly due to immature renal clearance mechanisms and ulcerogenic effects on gastric mucosa. Firocoxib, a novel second generation NSAID, is reported to have reduced side effects due to its COX-2 selectivity. The pharmacokinetic profile of firocoxib in neonates has not been established, making reliable dosing difficult. We hypothesized that firocoxib given per os at the labeled dose to neonatal foals would be absorbed and not be associated with clinically significant adverse events. Seven healthy American Quarter Horse foals of mixed gender were administered 0.1mg/kg firocoxib orally q24h for nine consecutive days, commencing at 36h of age. Blood samples were collected for firocoxib analysis using high pressure liquid chromatography with fluorescence detection at 0 (dose #1 only), 0.25, 0.5, 1, 2, 4, 8, 16 and 24 hours after doses #1, 5 and 9. For all other doses (2, 3, 4, 6, 7 and 8) blood was collected immediately prior to the next dose (24 hour trough). Elimination samples (36, 48, 72, 96, 120 and 144 hours) were collected after dose #9. Safety was assessed via physical examinations, changes in body weight, gastroscopy, complete blood count, serum biochemistry and urinalysis. Firocoxib was rapidly absorbed following oral administration with minimal accumulation after repeat dosing. After the initial dose, an average peak serum concentration (Cmax) of 89.50 ° 53.36 ng/mL (mean ° SD) was achieved (Tmax) in 0.54 ° 0.65 hours. Steady state was obtained after approximately 4 doses and the average maximum concentration (Cavg) in serum was 39.1 ° 8.4 ng/mL. After the final dose, the mean terminal half-life (TÂ½?») was 10.46 ° 4.97 hours. Firocoxib was not detected in plasma 72 hours after the final dose (<2ng/mL). Bioavailability could not be determined as currently, there is no accompanying intravenous dose of firocoxib for this age group to permit the calculation. No significant abnormalities were noted on blood work, urinalysis or gastroscopy. This study demonstrated that firocoxib is absorbed after oral administration in neonatal foals with no observable adverse effects after multiple doses.
Quantitative Pertechnetate Thyroid Scintigraphy and the Ultrasonographic Appearance of the Thyroid Gland in Clinically Normal Horses
Davies, Sarah Elizabeth (Virginia Tech, 2010-04-28)
The purpose of this study was to report the scintigraphic and sonographic appearance of the thyroid gland in clinically normal horses so these modalities could be used to assess the thyroid gland in this species. Horses were divided into two age groups. Group A consisted of 8 horses between 3 and 10 years of age and Group B of 7 horses between 11 and 20 years of age. Total T4 concentrations were within the laboratory reference interval. Thyroid to salivary (T/S) ratio, percent dose uptake of pertechnetate and thyroid lobe volume were calculated. Echogenicity of thyroid lobes and presence of nodules were documented. The two groups were compared using appropriate parametric and nonparametic tests. Total T4 concentrations were significantly lower in the older group. Sixty minute mean ± standard deviation (SD) T/S ratios for older versus younger horses were 5.8 ± 3.0 and 5.3 ± 2.2, respectively. Sixty minute median and interquartile ranges for percent dose uptake of pertechnetate for older versus younger horses were 3.64% (1.5 to 3.98%) and 2.55% (2.33 to 2.90%), respectively. Mean ± SD thyroid lobe volumes for older versus younger horses were 18.93 ± 5.16 cm3 and 13.55 ± 3.56 cm3, respectively. Most thyroid lobes were hyper or isoechoic to the sternocephalicus muscle. Prevalence of thyroid nodules did not differ between groups. Older horses had trends for greater T/S ratios, percent dose uptakes and thyroid lobe volumes but had lower total T4 concentrations. Further studies using scintigraphy and ultrasound in horses with thyroid disease are planned.
Recombinant Equine Interleukin-1 Induced Models of Equine Joint Disease
Takafuji, Vivian Ann (Virginia Tech, 2003-11-13)
Osteoarthritis (OA) is a debilitating disease of joints that afflicts horses of all ages and breeds and can result in lameness, suboptimal performance, and decreased quality of life. The pro-inflammatory cytokine interleukin-1 (IL-1) has been associated with the initiation and pathogenesis of joint disease. In part, this occurs by induction of proteases and oxidative pathways that contribute to the degradation of structural components of the articular cartilage extracellular matrix. Elucidating the complex macromolecular and molecular effects of IL-1 on articular tissues may further our understanding of the roles of IL-1 and inflammation in OA pathobiology. Full-length gene sequences encoding three recombinant equine interleukin-1 proteins (EqIL-1a, EqIL-1b, and EqIL-1 receptor antagonist), were previously cloned and expressed in-vitro. The objectives of this dissertation were to 1) establish EqIL-1 induced experimental models of equine OA, and 2) to investigate specific IL-1-induced immuno-inflammatory responses. Effects of EqIL-1 on articular cartilage explant proteoglycan metabolism and synthesis of a downstream inflammatory product, prostaglandin E2, established culturing conditions and furthered the rationale to use EqIL-1 in the in-vitro modeling of early joint disease. A customized cDNA array was used to profile changes in mRNA levels resulting from EqIL-1 treatments of cultured articular cartilage chondrocytes. EqIL-1a induced elevated mRNA levels corresponding to six genes after 1 hour relative to media control chondrocytes (p<0.05). EqIL-1b increased transcript levels of seven genes after 6 hours (p<0.0004); 102 additional transcripts were elevated > 2-fold over controls. A subset of the array-generated data was verified using optimized reverse transcriptase-PCR amplification. Results of principal component analysis indicate co-regulation of EqIL-1 induced transcript levels to relate to chondrocyte differentiation and cell-cycle processes. Subtractive hybridization-PCR identified 148 differentially expressed cDNAs in synovium resulting from a 6-hour intra-articular EqIL-1b injection. Combined results demonstrate the potent bioactivity of our equine IL-1 proteins and support the argument for crucial roles of IL-1 in pro-inflammatory processes and cytokine imbalances underlying early OA pathogenesis. These results add to the current knowledge of IL-1 modulated transcription that may precede ECM catabolic processes characteristic of OA. The culture systems, assays, and techniques for gene expression analysis may be useful for future studies attempting to elucidate macromolecular and transcriptional events underlying inflammatory-associated joint disease processes in horses. Reported information may further efforts toward improved diagnostic and preventive strategies and development of anti-IL-1 directed therapies.

Browsing by Author "Crisman, Mark V."

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