Funding opportunities for veterinary medicine with USDA competitive programs.

Dramatic changes are underway in how public higher education and professional training are funded. The shift from a public to a beneficiary funding model is driving increases in tuition for students, the collection of user fees, and fee-for-service activities and has focused increased attention on the need for funding support from federal grants and contracts. The US Department of Agriculture (USDA) was the first federal agency to support university-based research and education. While the funding provided by the USDA is now only a fraction of what is available through other federal sources, the USDA remains an important source of funding dedicated specifically to farm-animal-related research and the training of the next generation of veterinary scientists and educators.

Lepirudin prevents lethal effects of Shiga toxin in a canine model.

Microvascular thrombosis is a major cause of organ damage in Shiga toxin-mediated hemolytic uremic syndrome (Stx-HUS). In vitro and clinical studies implicate thrombin-mediated mechanisms in the pathogenesis of Stx microvascular thrombosis. In a greyhound model, administration of 0.03 microg/kg to 0.05 microg/kg Stx1 or Stx2 causes severe bloody diarrhea and HUS with microvascular thrombosis requiring humane euthanasia within 65 hours. Using a greyhound model of Stx-HUS we analyzed early hemostatic changes, and tested the hypothesis that thrombin blockade with lepirudin would prevent lethal Stx effects. Two Stx1-exposed greyhounds were analyzed for hemostatic changes prior to onset of clinical manifestations. Serial hemostasis studies after Stx1 challenge revealed trends of increased aPTT, fibrinogen levels, and prothrombin fragment 1+2, and appearance of abnormally large von Willebrand factor multimers. Three greyhounds were anticoagulated with lepirudin to maintain activated partial thromboplastin times (aPTT) >2.5-fold normal, followed by administration of Stx2 and observation of clinical responses. Among the 3 lepirudin-treated, Stx2-challenged greyhounds, one developed severe illness requiring euthanasia. Remarkably, 2 of the 3 greyhounds developed only hypersalivation and restlessness that resolved (P <.03 compared to 14 historical controls). These two greyhounds were clinically, hematologically and biochemically normal 74 hours after Stx administration, well beyond the time of euthanasia of any previous greyhound. This study suggests that greyhounds exposed to Stx develop procoagulant changes similar to humans, and that thrombin may be a critical factor in the pathogenesis and treatment of Stx-HUS.

Normalization of lens protein kinase Cgamma in galactosemic dogs by a novel aldose reductase inhibitor.

The purpose of this study was to determine the effects of a novel aldose reductase inhibitor on lens protein kinase Cgamma (PKCgamma) levels in galactosemic dogs. Six-month old Beagles (12 total; 6 male and 6 female) were made galactosemic by feeding a diet of 40% galactose for 6 weeks. Three dogs per group were fed either control, normal diet, 40% galactose diet, 40% galactose diet with aldose reductase inhibitor at 100 mg/kg body weight per day given orally, or a control diet with aldose reductase inhibitor alone (1-H,7-H-5alpha,6,8,9-tetrahydro-1-oxopyran[4,3-beta](1) benzopyran, referred to herein as HAR-1). Lenses were removed and analyzed for toxicity by pathological examination. Lens polyol concentrations were determined by GC/MS. PKCgamma levels were determined by Western blot and by reverse transcriptase-polymerase chain reaction (RT-PCR). No toxicity was observed from the aldose reductase inhibitor when given at 100 mg/kg body weight per day for 6 weeks. Galactosemic dogs showed deterioration of lens cells. Deterioration included vacuole formation in the lens, cell lysis, and loss of cell nuclei. Galactosemic dogs given the HAR-1 appeared identical to control dogs. Polyol concentrations in the lenses were reduced by 50% in dogs fed the 40% galactose diet with the aldose reductase inhibitor, HAR-1. PKCgamma protein levels were reduced in the galactosemic dog lenses, but synthesis of PKCgamma was not affected, as measured by RT-PCR. The PKCgamma protein levels were similar to controls in dogs given the aldose reductase inhibitor, HAR-1, even when polyol concentrations remained 50% elevated above control levels. HAR-1, when given to control dogs, caused a reduction in the synthesis of PKCgamma mRNA but not in total PKCgamma protein levels. This study demonstrates the use of a novel aldose reductase inhibitor to control changes in PKCgamma in dog lens, a PKC that is known to control gap junction activity.

Biochemical entities that influence membrane-associated TNF RII (80-kDa) and IL-1 RI (80-kDa) complex expression and receptor fragment production in adherent vascular endothelium.

The research aim of the present investigation was to identify leukocyte enzyme-proteases that have the capacity to biochemically recruit the passive participation of vascular endothelium in cytokine receptor 'shedding' phenomenon involving membrane-associated TNF RII (80-kDa) and IL-1 RI (80-kDa) complexes. Achieving this research objective involved the design of a laboratory approach that delineated to what extent enzyme-proteases released by activated macrophages directly interact with, and liberate soluble fragments of membrane-associated cytokine receptor complexes. Results from this segment of the investigation revealed that cathepsin-D, a leukocyte carboxyl/aspartate protease, altered the integrity and generated soluble fragments of TNF RII (80-kDa) and IL-1 RI (80-kDa) receptor complexes expressed by vascular endothelium. Furthermore, laboratory findings also suggested that cathepsin-D possessed the ability to variably deplete biologically functional membrane-associated TNF RII (80-kDa) and IL-1 RI (80-kDa) complexes. Complementary investigations isolated a carboxyl/aspartate protease from activated macrophages utilizing pepstatin-A affinity chromatography. Exposure of vascular endothelium to pepstatin-A binding proteins resulted in a detectable depletion of membrane-associated TNF RII (80-kDa) and IL-1 RI (80-kDa) in addition to the generation of soluble receptor fragments. Analysis of macrophage pepstatin-A binding proteins by SDS-PAGE identified a primary fraction with a molecular mass of 47-52-kDa that closely correlated with the known molecular mass of leukocyte cathepsin-D. Evaluation of macrophage pepstatin-A binding-protein fractions by non-denaturing Hb-PAGE detected a lucent proteolytic band at 47-52-kDa compatible with the known molecular mass of leukocyte cathepsin-D. Macrophage pepstatin-A binding proteins also hydrolyzed a synthetic enzyme-specific substrate that selectively recognizes cathepsin-D biochemical activity. In conclusion, the leukocyte carboxyl/aspartate protease, cathepsin-D can biochemically alter the integrity and generate soluble fragments of membrane-associated TNF RII (80-kDa) and IL-1 RI (80-kDa) receptor complexes expressed by vascular endothelium. The relevance of this concept is in part based on investigations that have discovered that genetic 'knock-out' mice incapable of expressing IL-1 RI (80-kDa) or TNF RI (55-kDa) receptor complexes are highly resistant to developing the pathophysiological alterations classically associated with conditions of endotoxic-shock.

Alterations in membrane-associated CD14 expression and the simultaneous liberation of soluble CD14 fragment in adherent macrophages mediated by a leukocyte carboxyl/aspartate protease.

Investigations sought to discover the biochemical mechanisms in macrophages that mediate the 'shedding' of soluble CD14 fragment. Stimulated macrophages display both increased liberation of soluble CD14 fragment and decreases in residual membrane-associated CD14 complexes following exposure to activating agents (fMLP/A23187). Application of 'class-specific' protease inhibitors revealed that a thiol/cysteine was involved in the biochemical production of soluble CD14 fractions and that a metalloprotease enzymatically degraded soluble CD14 fragment. Exposure of macrophages to individual proteases revealed that both cathepsin-D and elastase promoted variable depletion of membrane-associated CD14 complexes. Additionally, cathepsin-D, and to a lesser extent elastase, generated soluble CD14 fragment. Related studies isolated a carboxyl/aspartate protease from activated macrophages using pepstatin-A affinity chromatography. The physical and functional properties of macrophage pepstatin-A binding protein fractions closely corresponded with the known characteristics of cathepsin-D with respect to: (i) cellular origin; (ii) binding-avidity of carboxyl/aspartate proteases for pepstatin-A; (iii) non-specific proteolysis of haemoglobin detected by Hb-PAGE zymography; and (iv) hydrolysis of a synthetic cathepsin-D-specific peptide substrate. Interpretation of these findings collectively implies that activated leukocytes can biochemically alter membrane-associated CD14 complex expression and promote the liberation of soluble CD14 fragment in both activated and non-activated cell populations.