To compare insertion temperatures and torques of Hydroxyapatite (HA) coated and uncoated 5.5 mm AO cortical screws in equine third metatarsal bones (MTIII) in vitro, and to compare insertion and extraction torques of HA coated and uncoated screws after 4, 8, 12 and 16 weeks of healing in equine third metacarpal bones (MCIII) in vivo. No significant temperature differences were recorded in cadaveric bones for AO and HA coated screws. Insertion torques were significantly higher for HA coated implants compared to uncoated screws. In vivo, the AO screws lost 50% of their initial stability within 4 weeks of healing and failed to gain stability over the next 12 weeks. The HA screws maintained stability (at 4 weeks), and roughly doubled (at 8 weeks) and tripled (at 12 weeks) their insertional torques over time. HA coated screws can safely be inserted in equine cortical bone. AO cortical stainless steel screws fail to maintain stability in equine cortical bone. The addition of HA coating to the screws enables active osseointegration over 3 months of healing, as indicated by significantly higher extractional torques after 8, 12 and 16 weeks respectively. Screw failure can occur under acute load and cyclic fatigue indicating the need for improved stability in the equine patient. HA coating, leading to active osseointegration, is commonly used in human implants for this purpose. Varying results in equine models led to guarded acceptance of implant coatings amongst equine surgeons. Our results support the osseointegrative properties of HA coated screws in horses.

Methadone is a mu-opioid receptor agonist which is a very effective analgesic used to treat moderate to severe acute and chronic pain in humans. Due to methadones minimal undesirable side-effects in people, we believed it could be of use in horses as an analgesic agent. As found with the majority of lipophilic drugs, absorption of methadone occurs primarily in the small intestine via transcellular transport and its absorption is regulated by P-glycoprotein. P-glycoprotein is a transmembrane transporter protein encoded by the multidrug resistance gene, which is constitutively expressed in the apical membrane of enterocytes of various species. This protein may impair the therapeutic efficacy of oral opioids including methadone, by decreasing absorption through the small intestinal mucosa and altering drugs pharmacokinetics. The overall hypothesize was that the expression of P-glycoprotein in the equine small intestine affects absorption and bioavailability of methadone after oral administration to horses. In Vivo and in vitro studies presented here investigated the oral pharmacokinetics of methadone, expression of the multidrug resistance (MDR1) gene, and the role of intestinal P-glycoprotein on methadone flux or transport in equine jejunal mucosa. The contribution of this protein to in vivo absorption of this opioid drug after oral administration in horses is evaluated. Oral administration of methadone hydrochloride to healthy horses showed rapid absorption, reaching high serum concentrations without typical undesirable opioid-induced side effects. Drug absorption appears to be limited in the small intestine, supported by the observed low drug serum concentrations, low area under the drug serum concentration vs. time curve, and low drug bioavailability after intragastric administration. In addition, methadone was absorbed by oral mucosa and may be an important way that methadone gains entrance into equine plasma. Multidrug resistance (MDR1) gene expression was determined in several different tissues including those of the small intestine of horses. High MDR1 mRNA levels mainly in the duodenum and jejunum of horses may explain, at least in part, the limited intestinal absorption of methadone in vivo. P-glycoprotein, located in the apical membrane of epithelial intestinal cells of jejunum in horses impairs the flux of methadone across the intestinal mucosa and its drug efflux activity is minimized by verapamil HCl, a P-glycoprotein inhibitor. Therefore, these studies confirmed that the expression of P-glycoprotein in the equine small intestine affects absorption and bioavailability of methadone after oral administration to horses.

Objectives 1) To measure conductance and permeability of canine colonic mucosa exposed to increasing concentrations of carprofen. 2) To compare conductance and permeability of canine colonic mucosa exposed to carprofen or 2,4-dinitrophenol (DNP) and tempol blockade. Design In vitro randomized block design Animal 20 mixed breed dogs Methods Conductance, mannitol flux, and histology were evaluated in colonic mucosa mounted in Ussing chambers. Mucosa was first exposed to increasing concentrations of carprofen. Mucosa was then exposed to either carprofen (200 μg/ml) or DNP (0.25mM) +/- tempol (1mM) pretreatment. Conductance over time, mannitol fluxes, and frequency of histologic categories were analyzed for treatment effects. Histopathology and electron microscopy were evaluated post experiment. Results Mean +/- SEM conductance*time for 400 μg/ml carprofen treated colon was significantly greater than control. Mean +/- SEM conductance*time for carprofen treated colon at 200, 100 and 40 μg/ml were not significantly different from control. Mean +/- SEM conductance*time for 400 μg/ml and 200 μg/ml carprofen treated colon were not significantly different. Period 3 mannitol flux was greater than period 1 for 400 μg/ml and 200 μg/ml carprofen treated colon but not significantly different for 100 μg/ml, 40 μg/ml, and control. Period 3 flux for 400 μg/ml and 200 μg/ml carprofen treated colon were not different but were greater than control. Mean +/- SEM conductance*time for carprofen or DNP treated colon were not significantly different from control regardless of blockade. Period 3 flux for carprofen and DNP treated colon were not different but were greater than control. Period 3 flux for carprofen treated colon with tempol pretreatment was not significantly different than control. Period 3 flux for DNP treated colon with tempol pretreatment was not different than without tempol but was greater than control. Cell sloughing and erosions were observed with high carprofen concentrations. Mitochondrial damage was seen with carprofen treatment compared to DNP treatment or control. Tempol pretreatment effect on mitochondrial morphology was inconsistent. Conclusion Carprofen exhibits concentration dependent toxicity to canine colonic mucosa. Carprofen and DNP induce similar mucosal damage evident by changes in electrical conductance, mannitol flux, and histopathology. Carprofen damages enterocyte mitochondria.

Objective -To compare the compression produced in reduction of experimentally created medial condylar fractures using lag screw fixation with medial vs. lateral approach, and to determine the maximum torque at screw failure. Materials and Methods- Twelve (12) pairs (left and right) 3rd metacarpal bones (MC3) were collected from adult (2-7 years) Thoroughbreds euthanized for reasons unrelated to orthopedic disease. Complete parasagittal medial condylar osteotomies were created at a measurement of 9, 13, and 21 mm axial to the epicondylar fossa on four pairs each of cadaveric MC3 bones resulting in fracture fragments measuring 8, 12, and 20 mm in thickness. For each pair of cadaveric MC3, a lateral or medial approach was randomly selected to repair the condylar fracture using a single 4.5 mm AO cortical screw. Each repair was tested for fracture plane compression and screw torque to failure. Results-There was no significant difference in compression between the medial and lateral approaches for the 8 or 12-mm fragment groups. There was significantly more compression generated in the lateral approach when compared to the medial approach for the 20-mm fragment group. Failure occurred at significantly lower torque in the 8-mm group. There was no significant difference between medial and lateral approach in torque to failure for the 12 and 20-mm groups. Conclusion-Based on this data we have concluded that there was no significant difference in torque to failure between a medial vs. lateral approach for the 12 mm fragments but there was a significant difference for the 8 mm fragments and that a lateral approach may be acceptable for the repair of medial condylar fractures in 12-mm or thicker fragments. The compression achieved by a medial approach was not significantly greater for the 8, 12 or 20-mm groups. Clinical Relevance- Based on our results the 20 mm size fragments reaches a higher compression at a faster rate when compared to the 8 and 12 mm size fragments. We recommend using caution when repairing medial condylar fractures with a lateral approach for fragment sizes measuring 8-mm thick. The smaller fragment torque to failure was low and not much higher than the insertional torque. Failure resulted from the screws stripping in the bone fragment. The screws in the thicker fragments (12 and 20 mm) engage more bone and have a higher torque to failure as a result.

ABSTRACT Objectives To compare the osseointegration of roughened and electropolished 5.5 mm cortical screws used to secure a 4.5 mm broad dynamic compression plate (DCP) in equine third metacarpal (MC3) bones. Study Design In vivo study Animals 5 Adult thoroughbred horses (2-7 years old). Methods For each horse one MC3 was randomly assigned to secure a 4.5 mm broad DCP with 4aluminum oxide (Al2O3) roughened screws on the dorsal mid diaphysis. Four regular electropolished screws used to secure a similar plate on the contralateral limb served as control. They were removed at 12 weeks and the extraction torque was recorded. A paired t-test was used for comparison and statistical significance was set at p < 0.05. Results At 12 weeks, the mean extraction torque for roughened screws was significantly greater (p < 0.0002) when compared to regular AO screws. The roughened screws had a mean extraction torque of 3.24± 0.56 N-m, which was twice the mean extraction torque of the AO screws (1.65 ± 0.34 N-m). Discussion/Conclusions Bone tissue infiltrates the pores of the roughened screws increasing the contact surface and the mechanical anchorage. In our study the screw surface roughness is 2.14± 0.48 µm. This roughness has the lowest pore size that will interact with the surrounding bone without altering the screw dimensions. We found that Al2O3 roughened screws have a significantly greater removal torque when compared to the electropolished AO cortical screws when removed at 12 weeks post implantation. Clinical relevance - Information obtained from this study may help in improving the contact surface of implants and subsequent fixation and stability of bone-implant construct in equine fracture repair.

Lidocaine, an amide local anesthetic agent, is commonly used in mammals, including humans. There is a general assumption that birds are more sensitive to lidocaine than mammals. Relatively low doses of lidocaine have been suggested to cause toxic effects in birds. While this information appears to be anecdotal, it has been perpetuated in the literature. The overall objective of this thesis research was to determine the tolerance and safety of intravenous lidocaine in broiler chickens. To assess the cardiovascular effects of lidocaine, relative changes on heart rate and mean blood pressure were calculated. Clinically significant cardiovascular effects were defined as relative decrease of heart rate and/or mean blood pressure equal to or greater than 30%. On the first study, doses below the reported toxic dose were assessed. The effects of 2.5, 3.0, 3.5 mg/kg intravenous lidocaine were compared with a control (saline) group. Each dose was used in 2 randomly selected animals. No significant cardiovascular effects were detected; therefore, higher doses were investigated. On a 2nd study, using an up-and-down study design, a total of 11 subjects were evaluated. The up-and-down method is a sequential design with binary response variables within a certain population which allows the determination of an effective dose to 50% of the population (ED50). The ED50 was defined as the dose that would cause clinically insignificant cardiovascular depression to 50% of the population. Using two statistical methods, the ED50 of cardiovascular function was 6.3 mg/kg and 6.22 mg/kg (95% confidence interval, 5.3 7.13 mg/kg). The safety of this dose was then tested in a new group of broiler chickens. The dose of 6 mg/kg was administered to 6 animals. No clinically significant cardiovascular effects were detected in any animal. In conclusion, the 3 studies performed for this thesis indicates that the reported toxic dose of lidocaine appears to be erroneous. Furthermore, this thesis determined the highest tolerable dose and its safety in a specific group of broiler chickens. Further studies assessing analgesia and anesthetic effects of lidocaine are necessary, both in chickens and other avian species.

Thesis (M. S.)--Oklahoma State University, 2007. / Vita. Includes bibliographical references.

T regulatory cells are critical in the maintenance of self-tolerance, immune homeostasis and regulation of the immune system. Cytokine signalling is a dominant component of environmental signals which controls the function of Forkhead box P3 (Foxp3) regulatory T cells. This thesis addressed the hypothesis that interleukin-4 receptor alpha (IL-4Rα) signalling on T regulatory cells (T reg) play a role in the stability of T reg cells. Loss of IL-4Rα signalling on T reg cells may shift the immune balance from a Foxp3+ T reg to a Th1 effector function essential for Th1 disease outcome. Regulatory cells have a major function to dampen cytokine production; however, this role can be detrimental for host-protective immune responses in diseases such as tuberculosis. Here, we used two Th1 models of intracellular pathogens Listeria monocytogenes (Lm) and Mycobacterium tuberculosis (Mtb), to understand the role of IL-4Rα signalling on Foxp3+ T regulatory cells. Infection studies with L. monocytogenes demonstrated an impairment of T reg responses, with a decreased bacterial burden and diminished pathology both in the liver and spleen at 7 days post-infection, ultimately translated in better survival. Mechanistically, enhanced Th1 signature with the characteristic T-bet transcriptional factor and increased effector T cells producing IFN-γ, IL-2 following ex-vivo stimulation with PMA/Ionomycin, and heat-killed Lm (HKLM) were observed in Foxp3creIL-4Rα-/lox mice. Furthermore, CD8+ T cells of Foxp3creIL-4Rα-/lox mice showed increased cytotoxicity (Granzyme-B secretion) with higher proliferation capacity (Ki-67), better survival (Bcl-2) and decreased apoptosis (activated caspase3), suggesting contribution towards the observed protection against listeriosis. Subsequently, we investigated the role of IL-4Rα on Foxp3 T reg cells in Mycobacterium tuberculosis infection. To our surprise, in contrast to Lm infection, survival Survival of Mtb-infected Foxp3creIL-4Rα-/lox mice was similar to littermate control following infection with an intermediate dose of Mtb (H37Rv). We observed no differences in acute and chronic stages of infection in bacterial burden and histopathological scores in Foxp3creIL-4Rα-/lox mice when compared to littermate control animals in acute and chronic stages of infection. Importantly, Mtb infected FoxP3creIL-4Rα-/lox mice, exhibited significantly enhanced CD4+ T effector functions with increased pro-inflammatory cytokine secretion upon stimulation ex-vivo.

Background: Medical Male Circumcision (MMC) reduces the risk of HIV-1 acquisition by up to 60% as shown in a number of randomized controlled trials in Uganda, Kenya and South Africa. MMC has also been shown to reduce the prevalence of other sexually transmitted infections (STIs) like Herpes Simplex Virus (HSV) -2 and Human Papillomavirus (HPV) by 25% and 35% respectively. Asymptomatic STIs may elevate the risk of HIV-1 acquisition by recruiting HIV-1 target cells to the foreskin. The higher permeability of the inner foreskin may play a role in HIV-1 acquisition as well as the number of target cells present in the foreskin. The more inflamed inner foreskin may be increasing the risk of a productive HIV-1 infection. The aims of this dissertation was to a) examine the levels of keratinisation in the inner and outer foreskins after MMC; b) investigate the number of Langerhans, Ki67+ and CD4+ T cells in the inner and outer foreskin and c) identify the impact of asymptomatic STIs on the numbers and proliferative capacity of foreskin-resident Langerhans and CD4+ T cells.

Keratinocytes represent the major cell type in the skin. During cutaneous leishmaniasis (CL) and schistosomiasis, the skin is important during the parasite life cycle. While Th1 immunity is required to control CL, protection during schistosomiasis requires Th2 immunity. Paradoxically, Th2 characteristic IL-4 secreted early during L. major infection in mice, can drive a Th1 response by instructing dendritic cells to produce IL-12. Additionally, keratinocytes at the site of L. major infection in C57BL/6 mice, were postulated to be the source of the IL-4. We investigated if IL-4/IL-13 signalling via the IL-4Rα on keratinocytes contributed to early immunity during CL and schistosomiasis. Keratinocyte-specific IL-4Rα deficient (KRT14creIL-4Rα-/lox) BALB/c and C57BL/6 mice were generated by gene targeting and site-specific recombination (cre/loxP) under control of the KRT14 locus. In the L. major footpad model, KRT14creIL-4Rα-/lox BALB/c mice developed increased swelling, high parasite burdens, and cytokine and antibody secretion similar to littermate controls. L. major-infected KRT14creIL-4Rα-/lox C57BL/6 mice had decreased footpad swelling, low parasite burdens, a dominant Th1 cytokine response, and low type 1 and 2 antibody titres, similar to littermate control and resistant C57BL/6. In the L major ear model, KRT14creIL-4Rα-/lox BALB/c mice developed increased swelling, high parasite burdens, Th1 and Th2 cytokines, and high antibody titres, similar to littermate controls. L. major LV39-infected KRT14creIL-4Rα-/lox BALB/c mice showed significantly decreased parasite burdens in the ear, compared to littermate controls. L. major-infected KRT14creIL-4Rα-/lox C57BL/6 mice in the ear model, had decreased swelling, low parasite burdens, a dominant Th1 immune response, and low type 1 and 2 antibody titres, similar to littermate control and C57BL/6 mice. In the Schistosoma model, survival of S. mansoni-infected KRT14creIL-4Rα-/lox BALB/c mice was similar to littermate controls during mortality studies. During acute infection, S. mansoni-infected KRT14creIL-4Rα-/lox BALB/c mice showed gut pathology, hepatosplenomegaly, cytokine production, low type 1 and high type 2 antibodies, similar to littermate controls. In comparison to littermate controls, S. mansoni-infected KRT14creIL-4Rα-/lox BALB/c mice had smaller granulomas. Collectively, our results indicate that IL-4/IL-13 signalling through the IL-4Rα on keratinocytes is not required for control during CL or acute schistosomiasis, but does contribute to efficient granuloma formation during acute schistosomiasis.