Hysteroscopic insemination in mares with delayed uterine clearance (DUC mares) is controversial. While some authors proposed that insemination with a reduced volume and number of spermatozoa may reduce post-mating endometritis, others proposed that the hysteroscopic procedure is inflammatory and should not be used in DUC mares. The overall objectives of this study were to evaluate the incidence and severity of post-mating endometritis in reproductively normal and DUC mares after hysteroscopic insemination at the uterotubal junction, and to determine if hysteroscopic insemination could be used in DUC mares to reduce post-mating endometritis. The mares were classified as normal or DUC based on the presence of intrauterine fluid 24 or 48 hours after a semen challenge. In Experiment 1, the acute endometritis was evaluated 24 hours after insemination. Each mare (n=5 normal, n=5 DUC) received three treatments in three consecutive estrous cycles: UB: uterine body insemination (one billion spermatozoa, 20 mL), HYST: hysteroscopic insemination (five million spermatozoa, 0.5 mL) and SHAM: sham hysteroscopic insemination (semen extender, 0.5 mL). Intrauterine fluid accumulation and uterine leukocyte numbers were not influenced by treatment. In the second experiment, residual endometritis was assessed 48 hours after insemination, and the effect of insemination method on fertility was evaluated. Each mare (n=4 normal, n=5 DUC) received four treatments in four consecutive estrous cycles: UB: uterine body insemination (one billion spermatozoa, 20 mL), HYST: hysteroscopic insemination (five million spermatozoa, 0.5 mL), SHAM: sham hysteroscopic insemination (semen extender, 0.5 mL) and SP: hysteroscopic infusion of seminal plasma (0.5 mL). There was no difference in intrauterine fluid accumulation between treatments in normal mares. HYST and SHAM treatments resulted in more fluid accumulation 24 hours after the procedures in DUC mares than UB and SP. However, leukocyte numbers were not different. It was concluded that the hysteroscopic procedure itself is inflammatory, so it should not be used with the intention of reducing post-mating endometritis. However, the inflammation was not greater than that induced by routine uterine body insemination and fertility was not affected. Therefore, there is no contraindication to its use in mares with delayed uterine clearance.

The murine pathogen Helicobacter hepaticus has important confounding effects on research. Neonatal fostering has been studied in our laboratory for elimination of infection in mice. The purpose of our study was to examine fostering of pups from experimentally infected dams in male-absent parturition, and to determine the significance of gender and time on quantity of bacterial colonization in the cecum and feces of C57BL/6 mice. Approximately 20 C57BL/6 mice were fostered per day from one to four days of age. None of the C57BL/6 pups tested positive by PCR in fecal or cecal samples through four days of age. This data showed that removal of the male C57BL/6 mouse prior to parturition is crucial for extending the fostering period to obtain Helicobacter-free mice. In a second experiment, H. hepaticus infected mice were housed under varying arrangements to determine the effects of gender and housing on fecal and cecal colonization. Neither time or housing group affected bacterial fecal shedding. However, there was a significant overall effect of gender and a significant difference between male and female mice in both fecal and cecal bacterial copy number. Male fecal and cecal samples contained more copies of H. hepaticus than did female samples. Additionally, significant correlations between fecal and cecal H. hepaticus values were found both overall and by gender. Novel predictive algorithms were formulated to predict cecal bacterial colonization levels in fecal pellets. These findings should prove useful in Helicobacter elimination efforts, and in future work to further elucidate the role of H. hepaticus in transmission and disease.

Intraoperative reductions in serum fentanyl levels in dogs with induced moderate hypothermia and transdermal fentanyl patches (TDF) in place has been documented. The impact of such reductions has not been evaluated nor has the anesthetic sparing effect of the TDF. Reductions in serum levels may be the result of either a decreased uptake of fentanyl from the dermal depot, or a biophase shift. The objective of this investigation was to determine whether the minimum alveolar concentration (MAC) of isoflurane was altered in the presence of TDF in normothermic and hypothermic dogs. Six mature, healthy, mixed breed dogs were anesthetized on four separate occasions, and received each of the 4 following treatments in random order: 1. Sham patch-normothermia (C-NORM), 2. Sham patch-hypothermia (C-HYPO), 3. Fentanyl patch-normothermia (F-NORM), 4. Fentanyl hypothermia (F-HYPO). The appropriate patch was applied twenty four hours prior to induction of anesthesia. Anesthesia was induced with isoflurane in oxygen; the dogs were intubated and mechanical ventilation was initiated. Target temperatures (34.5ºC- HYPO, or within 1ºC of baseline- NORM) were held constant for one hour prior to beginning the MAC determinations. Supramaximal stimulation was produced by an electrical stimulator which delivered a current to needle electrodes in the buccal mucosa of the lower jaw of the dog. The MAC (± SD) for C-NORM, C-HYPO, F-NORM and F-HYPO was 1.2 ± 0.17, 0.855 ± 0.183, 0.763 ± 0.097, and 0.830 ± 0.172 respectively. MAC for C-NORM was significantly higher than the other groups. There was no significant difference between C-HYPO, F-NORM, and F-HYPO. Transdermal fentanyl reduces the isoflurane requirement in normothermic dogs. The presence of hypothermia appears to negate the effects of TDF.

Gopher tortoise (Gopherus polyphemus) populations have experienced precipitous declines from habitat loss, and human and disease related mortality. The goal of this study was to characterize the health status of free-ranging Louisiana gopher tortoises. Gopher tortoises were collected during two distinct trapping seasons: fall (August-October 2002) and spring (April-June 2003). Captured tortoises were given a physical exam and the carapace and plastron length and width, weight, and body temperature were recorded. Blood was collected from the subcarapacial vein and submitted for the following testing procedures: complete blood count, plasma chemistry, infectious disease serology (Mycoplasma), and toxicologic screen (copper, mercury, zinc, and lead). Fifty-nine tortoises were captured during the study. Fifty-seven (97%) of the tortoises were adult animals and two (3%) were juveniles. Twenty (34%) of the tortoises were captured in the fall and thirty nine (66%) were captured in the spring. There were thirty male (53%) and twenty-seven female (47%) tortoises captured between the two trapping periods. The gender of the hatchling tortoises could not be determined. Complete blood counts, plasma biochemistry analyses and toxin screens were performed on fifty seven adult tortoises. There were several differences detected in the white blood cell count between trapping season. The white blood cell count and heterophil count were significantly higher in the spring compared to the fall. Significant gender differences were observed for potassium, calcium, and phosphorus. Marked seasonal variation was observed with glucose, uric acid, and GGT. Only creatinine kinase levels differed significantly between sites. There was a difference in the mercury and copper levels between gender. Only copper differed significantly between season. Overall, 26% of the tortoises were serologically suspect or positive for Mycoplasma agassizii. There was no difference in parasite shedding between gender, site of capture, or season. The parasites identified in these tortoises were consistent with findings in gopher tortoises throughout their range. In general, our findings suggest that these tortoises are in good health.

Osteoarthritis is arguably the most common ailment in both dogs and people in the developed World. Treatment for osteoarthritis is currently symptomatic. Development of therapies designed at stopping the progression of osteoarthritis, require a method of evaluating efficacy. Objective analysis by measuring joint metabolism via clinical trials of the cross-over design is currently not possible with the methods utilized. Extracellular matrix degradation is a hallmark of osteoarthritis. The matrix metalloproteinases are major degradative enzymes. There are currently no commercially available assays to measure canine matrix metalloproteinases. Human and canine matrix metalloproteinases are highly homologous. The use of antibodies and assays designed to detect human matrix metalloproteinases may also detect canine molecules. A commercially available human enzyme-linked immunosorbent assay (ELISA) was tested against canine samples without success. Casein zymography detected the presence of canine enzymes weighing the same as human matrix metalloproteinase 3 (MMP-3) and matrix metalloproteinase 13 (MMP-13). Inhibition of the casein degradation was achieved by the addition of ethylenediamine tetra-acetic acid (EDTA). The presence of a protein of the correct molecular weight with the ability to digest casein that is inhibited by EDTA is only circumstantial and does not definitively identify the enzymes. Enzyme-assisted immunoelectroblotting (Western blotting) utilizing human polyclonal antibodies was unsuccessful at positively identifying the canine molecule.

Endothelin-1 (ET-1) has been implicated in allergic type of respiratory inflammatory diseases in various species of animals including horses. This peptide elicits its actions by acting through endothelin-A (ET-A) and endothelin-B (ET-B) receptor sub-types. In this project, we have hypothesized that endothelin receptors (both ET-A and ET-B) are altered in terms of affinity and expression, in the lungs of summer pasture-associated obstructive pulmonary disease (SPAOPD)-affected horses. Objective of this dissertation was to determine the alterations in the affinity and expression of endothelin receptors in the lungs of healthy and SPAOPD-affected horses. To pursue our hypothesis, we have employed pharmacological, immunohistochemical and molecular studies. Totally 33 horses were used in this study. All the horses were examined and grouped in to 16 healthy and 17 SPAOPD-affected, based on clinical evaluation, clinical scoring, pulmonary function testing and broncho-alveolar lavage fluid (BALF) analysis. Horses were then euthanatized, and tissue specimens were immediately collected from all lung lobes. In pharmacological studies, cumulative concentration response curves and pA2 values were determined and compared in both groups of horses. The pA2 value of ET-B receptors was significantly greater in the SPAOPD-affected horses when compared with healthy horses. In immunohistochemical studies, expression of these receptors was determined in the bronchial smooth muscles and epithelium of both groups of horses. The percentage of immunostaining was significantly greater in the smooth muscles of SPAOPD-affected horses when compared with healthy horses. In molecular studies, by employing RT-PCR, western blotting and realtime PCR, expression of these receptors has been determined and compared. The molecular expression of ET-B receptors was siginifcantly greater in the peripheral lungs of SPAOPD-affected horses when compared with healthy horses. Specific antagonists, primers and polyclonal primary antibodies of ET-A or ET-B receptors were used for these three studies. On the whole, it can be concluded that ET-B receptors show a clear tendency of up-regulation in the lungs of SPAOPD-affected horses. These findings help us in the better understanding of the pathogenesis of this progressive, career-ending disease.

In the last twenty years, interferon-α (IFN-α) has gained success as an immunotherapy for treating such cancers as hairy cell leukemia, malignant melanoma, and renal cell cancer. Our goal was to improve the effectiveness of IFN-α therapy by genetically modified the IFN-α gene to encode a tumor-targeting peptide fused to a functional IFN-α protein. To ensure the targeting peptide worked, a genetically modified reporter gene encoding a secreted alkaline phosphatase (SEAP) gene and different mini-peptides were used to determine distribution and targeting ability. The DNA fragment encoding the most effective peptide was selected to modify the IFN-α gene construct for therapeutic trials. This fusion gene encoded the peptide with the amino acid sequence of C-D-G-R-C, and demonstrated a higher localization of the genetically modified gene product in the tumor local area. Tumor volume and animal survival was measured over several weeks to compare the anti-tumor effects of the IFN-α to CDGRC-IFN-α treatments. Results indicate an increase in therapeutic efficacy due to treatment with the CDGRC-IFN-α gene over the wild-type IFN-α gene. Flow cytometry was performed and it was determined that both of the tumor targeted gene products, CDGRC-SEAP and CNGRC-SEAP share a high affinity for the receptor, Aminopeptidase N (CD13). In order to determine the mechanism responsible for the enhanced anti-tumor effect by CDGRC-IFN-α gene therapy, the T cell infiltration, subsequent CTL activity, and tumor vessel density were confirmed through immunostaining. An increase in number of CD8⁺ T cells was seen, as well as an increase in activity of cytotoxic T cells. Decreased vessel density in CDGRC-IFN-α treated animals suggest that this therapy enhanced anti-angiogenisis. A high level of non-specific activity was detected in the CTL assay, suggesting involvement of other immune cells, such as NK cells. Overall, this study describes the first example of using a genetically modified immunostimulatory gene encoding tumor-targeted IFN-α for treating tumors. This novel concept may have the potential for increasing therapeutic efficacy of several current cancer treatments.

Cancer metastasis is a frequent manifestation of malignant melanoma progression. Successful invasion into distant organs by tumor cells must include attachment to microvessel endothelial cells, and degradation of extracellular matrix. Heparan sulfate proteoglycans are ubiquitous macromolecules associated with cell surface and extracellular matrix of a wide range of cells and tissues. Heparanase is an extracellular matrix degradative enzyme which degrades the heparan sulfate chains of heparan sulfate proteoglycans. To investigate effects of changes in heparanase gene expression in metastatic melanoma cells, we constructed adenoviral vectors containing the full-length human heparanase cDNA in both sense (Ad-S/hep) and anti-sense orientations (Ad-AS/hep). We demonstrated increased heparanase expression and activity in melanoma cell lines following Ad-S/hep infection by Western blot analyses and heparanase activity assay. Conversely, heparanase content was significantly inhibited following infection with Ad-AS/hep. Alteration of heparanase protein expression by these adenoviral constructs correlated with invasive cellular properties in vitro and in vivo. Unexpectedly, overexpression of heparanase inhibited brain tumor formation in vivo possibly by extensive remodeling of the extracellular matrix which in turn modifies growth factor signaling and activity. Finally, cell-surface heparan sulfate is also known to inhibit or promote tumorigenesis depending on size and composition. We proposed that heparanase generates bioactive heparan sulfate chains from the melanoma cell-surface that modify biological activities associated with vascular endothelial growth factor, a molecule essential for brain metastasis. Heparanase-degraded melanoma cell-surface heparan sulfate stimulated migration, but not proliferation of melanoma in vitro. It also enhanced angiogenesis in vivo, independent of vascular endothelial growth factor activity, an unexpected finding. Interestingly, melanoma cell-surface heparan sulfate did not have an observed effect on endothelioma migration in vitro. We also attempted to characterize the melanoma cell-surface heparan sulfate isolated by heparanase degradation by ion-pair high pressure liquid chromatography. This method proved to be not sensitive enough to detect nanogram quantities of HSGAG present in our samples.

Environmental contamination with airborne particles has been a human health concern for many years. Epidemiologic studies in urban communities have linked ambient particle exposure to various health effects, including chronic obstructive pulmonary disease, lung cancer, and several cardiovascular disease conditions. The pathogenesis of these conditions with respect to ambient particle exposure is complex because ambient particles are complex in composition. The particles vary greatly in origin, size, surface area, and elemental composition; and a given particle type, such as those generated by petrochemical (gasoline, diesel, industrial substrate) combustion, may be coated with many other compounds, including polynuclear aromatic hydrocarbons (PAHs). Our laboratory group had previously characterized the generation of PAHs from incomplete combustion of the high volume petrochemical 1,3-butadiene (BD) and briefly described the biological effects of BDs incomplete combustion product, butadiene soot (BDS), in vitro. The studies presented here represent a continuation of these initial studies, where we first characterize BDS with respect to particle size distribution and assembly, PAH composition, and elemental content of BDS ultrafine particles. We also describe in vitro assays demonstrating that BDS ultrafine particles can transport and transfer adsorbed organic constituents directly to target respiratory cells, without uptake of the particles by the cells. Next, we demonstrate that combustion-derived PAHs adsorbed onto BDS particles are concentrated in lipid droplets of respiratory system cells and that, in vitro, these PAHs activate xenobiotic metabolism pathways. We also present an in vivo analysis of bronchoalveolar lavage fluid (BALF) with inflammatory cell infiltrates, histopathological evidence of inflammation and particle retention, and gene expression analysis revealing upregulation of several cytokines and AhR-responsive biotransformation enzymes. Finally, we present ultrastructural evidence that BDS particles can be internalized by bronchoepithelial cells in vitro and phagocytosed by alveolar macrophages in vivo. These studies were designed to characterize and promote BDS as both a model mixture and a real-life example of a petrochemical product of incomplete combustion with the potential both for environmental contamination and for contributing to health problems.

The ABC transporter P-glycoprotein (Pgp) and membrane permeability are determinant factors for absorption and disposition of xenobiotics. These studies investigated the effects of potential modulators of Pgp and/or membrane function on the disposition, bioavailability and bioaccumulation of environmentally relevant pharmaceuticals, tetracycline and ivermectin, and the carcinogen benzo[a]pyrene in catfish. The pesticide dieldrin and surfactant linear alkylbenzene sulfonate (LAS) were selected as mixture components thought to have potential interactions with these determinants of disposition. Initial in situ experiments demonstrated dieldrin and tetracycline both significantly inhibited biliary excretion of Pgp prototypic substrate Rhodamine-123 in isolated perfused livers. Further, dieldrin (20µM) reduced movement of 3H-tetracycline (8.8nM) into bile (55%) to a greater extent than Pgp prototypic competitive inhibitor verapamil. In contrast to inhibitory effects in situ, a 4-week dietary dieldrin preexposure (0.1mg/day/kg body weight) increased plasma clearance (17%) and reduced tissue concentrations of 3H-tetracycline equivalents (parent and metabolites) for a single intra-aortic administration of 3H-tetracycline (8.31µg/kg body weight) in catfish. A 23% increase in the immunoreactive Pgp level in the hepatic membranes following chronic dietary dieldrin exposure was correlated with in vivo changes in disposition. Additional in situ studies demonstrated LAS treatments (1, 5, and 20µM) reduced movement of Rhodamine-123 (1µM) into bile in isolated perfused livers (18.6, 38.1 and 66.7%, respectively). Fluorescent anisotropy measurements of the corresponding hepatic membranes showed a 29.7% decrease (increase in membrane fluidity) at the 1µM LAS concentration, with little additional change evident at higher concentrations. In sequential in vivo experiments, following six daily diet administrations of 3H-ivermectin (10 µg/day/kg body weight) or 3H-benzo[a]pyrene (40 µg/day/kg body weight) starting on day 7 during a twelve-day waterborne LAS exposure (0, 100 and 300µg/l), 3H-ivermectin and 3H-benzo[a]pyrene equivalent remaining in catfish and their blood and tissue concentrations increased in a dose-dependent fashion with increasing LAS concentrations. The first study indicates dieldrin inhibited Pgp transport at the high concentration with the inductive effect upon Pgp expression predominating at the low concentration exposures in vivo. Findings of the second study suggest that LAS at environmental concentrations altered membrane permeability and/or transporter function so to increase bioaccumulation of other xenobiotics from the diet.