Synovial fluid and plasma nitric oxide and endothelin-1 concentrations in horses with and without joint disease.

Houston Equine Research Organization. $5,876. 

DJ Burba, J de la Calle,

RM Moore, J Williams,

J VanSteenhouse, G Hosgood

Joint disease is an extremely common clinical condition and a major cause of lameness and loss of use in race horses.  Joint disease can be initiated by numerous inciting causes, but ultimately it results in joint inflammation which leads to increased joint fluid, decreased range of motion, lameness, and subsequently a decrease in athletic function.  The joint inflammation is caused by the release of numerous injurious substances into the joint space from white blood cells and other cells with the synovial membrane and joint cartilage.  The inflammatory cascade ultimately leads to articular cartilage degeneration and the ensuing degenerative joint diseases (arthritis).  Although joint disease has been extensively studied in the horse, relatively little is known regarding its pathophysiology.  Nitric oxide (NO) and endothelin-1 (ET-1) are two relatively recently discovered molecules that have been shown to contribute to the inflammatory process of the various arthritides in people and laboratory animals.  The purpose of this study is to quantify and compare synovial fluid and plasma NO and ET-1 concentrations in horses with and without joint disease to determine if the levels of these substances increase in diseased joints and if they vary among various types of joint diseases.  These variables will be assessed in four groups of horses:  1) acute synovitis; 2) septic arthritis, 3) chronic degenerative joint disease; and 4) normal controls.  This clinical study will provide the necessary information to determine if it is worthwhile to pursue additional experimental studies to further evaluate the role of NO and ET-1 in equine joint disease.  Depending upon the results of this study,  it is possible that therapeutic interventions directed toward interruption of the pathophysiologic pathways of these two inflammatory mediators may be developed which could potentially aid in the prevention and treatment of joint disease in horses.

Does cytokine production correlate with reversible airway obstruction in SPAOPD horses? 

Grayson Jockey Club Research Foundation, Inc.  $41,824. 

DW Horohov, RE Beadle,

LRR Costa

The overall goal of this project is to determine the immunological mechanism involved in chronic obstructive pulmonary disease (COPD) and summer pasture associated obstructive pulmonary disease (SPAOPD), both now referred to as recurrent airway obstruction (RAO).  Though the underlying mechanism of RAO in the horse remains unknown, data from a number of sources support an immunological mechanism.  The main approach for this study is to characterize the cytokine response of lymphocytes collected from horses suffering from COPD and SPAOPD using a quantitative procedure of equine cytokine mRNA.  The production of certain cytokines (IL-4, IL-5 and IL-13) is characteristic of human asthma.  Preliminary data from my laboratory demonstrated that both peripheral blood mononuclear cells (PBMC) and bronchoalveolar lymphocytes (BAL) from horses suffering from SPAOPD expressed elevated levels of mRNA for IL-4 and IL-13, though not IL-5.  This latter observation is consistent with the histological feature of few or no eosinophils in equine RAO that differentiates it from human asthma where eosinophils and IL-5 are predominant.  To determine if cytokine gene expression is temporally associated with clinical signs, we have been collecting PBMC and BAL samples from a group of SPAOPD-affected horses and unaffected controls left on pasture during the past six months.  As the horses began to show clinical signs of disease, they were brought indoors and lung function was assessed.  BAL and PBMC samples were also collected.  As the horses recovered, another set of samples was collected. The samples have been fractionated into various lymphocyte subpopulations, and mRNA has been isolated and frozen at -70^o for subsequent analysis. We will also collect samples from all of the horses during the winter months when they are asymptomatic.  We have also begun to receive BAL and PBMC samples collected by Dr. Bruce McGorum (Edinburgh) from COPD horses challenged with moldy hay.  These samples, along with those from the SPAOPD horses, will be analyzed for mRNA levels of IL-4, IL-5, IL-13 and interferon-gamma.  It is our expectation that those horses exposed to the aeroallergens will exhibit elevated levels of IL-4 and IL-13, compared to unaffected controls.

The role of Type 2 cytokines in recurrent airway obstruction (RAO) in the horse. USDA-NRICGP. $200,000. 

DW Horohov, RE Beadle,

TR Klei, B McGorum

The overall goal of this project is to determine the relationship between gastrointestinal parasitism and susceptibility to recurrent airway obstruction (RAO) in horses.  Our primary hypothesis is that RAO is the result of the induction of a Type 2 cytokine response following exposure to environmental allergens.  We also propose that helminth parasites may predispose horses to RAO by providing a cytokine environment conducive to the development of a Type 2 cytokine response in the lung.  To test this hypothesis, we propose the following objectives: 1) to further characterize cytokine production in respiratory system of RAO horses; and 2) to determine the effect of parasitism on the immune response to aerosol challenge with a novel antigen.

Equine T-cell responses to nematode parasites.

United States Department of Agriculture NRICGP.  $238,000.

TR Klei, DW Horohov,

RM Moore, HW Taylor,

PH Elzer 

The goal of these studies is to continue to characterize the equine T-cell response to nematode parasites.  Using the Strongylus vulgaris helminth naive pony model, we have demonstrated that immune ponies respond to challenge infections with a dominate Type 2 cytokine response characterized by increased levels of mRNA for IL-4 and IL-5 and decreased levels of interferon-gamma.  CD4+ T cells were shown to express these cytokines following stimulation with parasite antigen.  This study will investigate the potential cross regulation of the induction of this Type 2 cytokine response by Type 1 cytokines.  In light of recent findings on the importance of IL-13 in protective immunity against gastrointestinal nematodes in other species, we will also clone the gene for this Type 2 cytokine and characterize its expression in this model.  The two specific objectives designed to address these points are:  1) to determine the role of Type 1 cytokines in the regulation of T-cell expression of Type 2 cytokine genes and the effect of this down regulation on the induction of protective immunity against S. vulgaris.  The hypothesis to be tested is that in a milieu of Type 1 cytokines vaccination with irradiated larvae of S. vulgaris will not induce a dominant Type 2 cytokine profile and ponies will be susceptible to challenge infection.  2)  to determine the gene expression of IL-13 in tissues and cells of ponies undergoing a protective immune response to S. vulgaris.  The hypothesis to be tested is that IL-13, a Type 2 cytokine with many but not all the activities of IL-4, will be expressed in S. vulgaris immune but not nonimmune ponies and that the regulation of IL-13 will mimic that of IL-4.  This further characterization of the T-cell response to nematode infection in equids will advance the feasibility of the future vaccine development against equine nematodes particularly as this relates to adjuvant design and will also advance the basic understanding of the immune response in this livestock species.

Molecular basis of stress-induced immune

modulation.  

USDA-NRICGP.  $180,000. 

DW Horohov

The contribution of stress towards economically important diseases of animals has been widely recognized for a number of decades.  Shipping disease in cattle and pneumonia in horses are two prime examples of the important role stress appears to play in veterinary medicine.  A number of other associations have likewise been made in human medicine and experimental models.  Over the past several decades numerous studies have been able to link specific stress-induced factors with changes in immune function.  Nevertheless, questions remain regarding the precise mechanism of stress-induced immune modulation.  The focus of the studies described in this proposal is characterizing the molecular basis of this immune deviation. An understanding of the molecular basis of stress-induced immune function will lead not only to a better understanding of immune regulation but may also identify potential targets for therapeutic intervention.  The long-term goal of this project is to define the mechanism of stress-induced changes in immune responses.  Our overall approach will be to subject a group of horses to a defined stressor (exercise) and to characterize the mechanism of stress-induced alterations in the lymphoproliferative response.  The use of exercise as a model for stress offers several advantages, and we have been able to demonstrate that horses subjected to exercise stress exhibit similar alterations in immune function as have been described in other model systems.  Thus the results generated from this work may be applicable to other stressors and species. We propose to focus on the critical events in lymphocyte activation and proliferation.  As such we propose to:  1) characterize the effect of exercise stress on the early events in lymphocyte activation; 2) determine the effect of exercise stress on signal transduction via the IL-2 receptor; and 3) determine whether exercise induces apoptosis in peripheral blood mononuclear cells.  The overall approach will be to collect lymphocytes from exercise stressed horses immediately prior to and after exercise and to stimulate their PBMC in vitro with phytohemagglutinin (PHA).  The lymphocyte cultures will then be analyzed for stress induced alterations in the signal transduction pathways involved in lymphocyte activation and the response to IL-2 binding to its receptor.  We are currently analyzing samples collected from a recent exercise challenge study for alterations in intracellular signaling pathways.

Role of endothelin and nitric oxide in equine laminitis.    

Grayson-Jockey Club

Research Foundation, Inc. $89,892.

RM Moore, SC Eades,

AS Holm, CS Venugopal,

JL Oliver

The global hypothesis for this study is that the initiating factor in the onset of acute laminitis in horses is a disruption in the balance between endothelium-derived vasodilators (nitric oxide, NO; decreased) and vasoconstrictors (endothelin-1, ET-1; increased), which leads to digital vasoconstriction and subsequent laminar ischemic necrosis.  The purposes of this study are to determine:  1) the digital hemodynamic effects of ET-1 infusion into the digital vasculature of horses; 2) the effectiveness of an ET antagonist on reversing these ET-1-induced hemodynamic alterations; 3) the effectiveness of a NO donor in reversing the hemodynamic effect of ET-1; 4) if NO and ET-1 concentrations in the palmar digital venous blood are altered subsequent to administration of black walnut extract, a reproducible model for inducing laminitis; 5) the effectiveness of the ET antagonist in reversing the hemodynamic alterations associated with black walnut extract-induced laminitis; 6) the effectiveness of a NO donor in reversing the hemodynamic alterations associated with black walnut extract-induced laminitis; 7) the distribution and intensity of immunohistochemical staining for the inducible isoform of NO synthase and ET-1 in the presence of an ET antagonist or NO synthesis inhibitor, and determine the effects of endothelium-dependent (acetylcholine) and -independent (nitroglycerin) vasodilators on the digital vessels preconstricted with ET-1 in normal and laminitic horses.  The information gained from these studies will contribute to the understanding of the initiating factors involved in the pathophysiology of acute laminitis in horses.  Discoveries made during these investigations may offer insights into potential therapeutic regimens for prevention or treatment of horses susceptible to or affected by this devastating disease.

Use of acupuncture to stimulate cyclicity in anestrus mares.    

American Association of Equine Practitioners Alternative Therapies Grant Program. $4,970.

AS Murton, LRR Costa,

B Eilts, D Paccamonti,

C Pinto, E Garcia

The objective of this study is to induce earlier cyclicity and ovulation in anestrus mares using acupuncture treatments at the Bai Hui, GV2, BL-31, BL-32, and BL-33 acupuncture points.  Acupuncture stimulation of these sites along the back of the animal are said to stimulate the hypothalamic-pituitary axis.  Progesterone concentration, ovarian follicular activity, and ovulation will be documented. Twelve mares aged 5-18 years, will be used in this study.  During the months of November through January, seasonal anestrus will be documented in the mares by weekly transrectal ultrasound of their ovaries and weekly blood samples to determine progesterone concentration.  On January 24, the mares will be divided into two groups:  Six mares will receive dry needle acupuncture stimulation at specified points along their caudal spine.  The treatments will be carried out every three days for the first four weeks, and weekly thereafter.  All 12 mares will be examined for ovarian activity by an investigator blinded to the treatment.  Transrectal ultrasound examinations will be performed weekly to detect any changes in the ovaries, including follicular growth and ovulation.  The size and number of follicles on each ovary will be recorded at each examination.  Blood samples will be collected for hormonal analysis.  It is anticipated that the mares treated with acupuncture will have follicular development, and ovulate earlier than the controls.  This should occur within four to six weeks of treatment,  late February to early March.  Untreated mares are not expected to cycle until the end of March.  These results are expected to demonstrate the viability of acupuncture in inducing cyclicity in anestrus mares.

The effects of ATP-MgCl₂ during low-dose endotoxin infusion in conscious horses.    

Morris Animal Foundation. $28,240.

J Tetens, RM Moore, SC Eades, G Hosgood, DW Horohov

The objective of the study is to determine whether intravenous infusion of ATP-MgCl₂ can lessen the severity of the signs associated with administration of endotoxin.  If positive results are obtained, ATP-MgCl₂ may have clinical application in the treatment of numerous diseases, such as diarrhea or severe colic.

Role of ATP in maintenance of equine colonic epithelial cell tight junction integrity.   Comparative Gastroenterology Society.  $5,000.

J Tetens, RM Moore, WG Henk, G Hosgood, SC Eades,

CS Venugopal

The objective of the study is to determine the effects of ATP depletion on permeability of the cells in the large intestine.  During disease, the cells become more permeable which allows toxins and bacteria to enter the blood stream.  If a decrease in ATP is involved in the permeability changes, we may be able to replace the cell’s ATP content to prevent or diminish the changes in permeability that can occur.