http://hdl.handle.net/1957/18827 2013-05-26T07:31:48Z http://hdl.handle.net/1957/38590 Role of IL-8 and TIMP-2 on term canine trophoblast migration, invasion, and proliferation Gullaba, Justine Marie Placental pathologies are not uncommon in dogs. For example, in primiparous bitches, the reported incidence of subinvolution of placental sites is 21% - 100%. Despite years of research in multiple species, the mechanisms regulating late gestation trophoblast behavior in the dog remain unexamined. Therefore, the objective of this study was to characterize normal in vitro term canine trophoblast (TCT) physiology, with respect to migration, invasion, and proliferation. In addition, the effects of interleukin-8 (IL-8) and tissue inhibitor of metalloproteinase-2 (TIMP-2) on trophoblast physiology were examined. Following isolation of primary TCT, the cell suspension was seeded into the migration (wound-healing) assay, invasion (Matrigel) assay, and proliferation (MTT) assay. Cells were cultured under the same conditions at 37ºC with 5% CO₂ for each assay. Recombinant human IL-8 (200-08M, Peprotech, Rocky Hill, NJ, USA) was used at a concentration of 10 ng/ml for each assay. Recombinant human TIMP-2 (410-02, Peprotech) was used at a concentration of 0.5 μg/ml for each assay, respectively. For the migration assay, cells were suspended in medium supplemented with 10% fetal bovine serum (10% FBS). Primary TCT (1000X10³ cells/well) were cultured in 12-well tissue culture plates until a confluent monolayer was formed. A wound was added with a sterile pipette tip, the monolayer was rinsed, and 10% FBS with no factor (control), with IL-8 or TIMP-2 was then added. Photomicrographs of the wound were taken with phase-contrast microscopy (100X) after 8 h in culture and the area of the wound was measured using ImageJ v.1.34 software. The experiment was performed in triplicate (n=5 dogs). The invasion assay was performed using Matrigel invasion chambers (#354480, BD Biosciences). Primary TCT (250X10³) were suspended in protein-free medium (PFM) with no factor (control), with IL-8 or TIMP-2 and seeded onto Matrigel-coated filter membranes in the upper well. Chemoattractant (10% FBS) was placed in the lower well and the 24-well chambers were cultured for 22 h. Non-invading cells were removed from the filters that were then fixed and stained. Cells were counted under light microscopy (400X). The experiment was performed in triplicate (n=5 dogs). For the proliferation assay, cells were suspended in PFM. For the proliferation assay (CGD-1, Sigma, St. Louis, MO, USA) primary TCT (100X10³ cells/well) were suspended in PFM with no factor (control) or with IL-8 and cultured in 96-well tissue culture plate for 28 h. Cells were then incubated with MTT for 4 h, which was later replaced with 1-propanol and the plates were vigorously shaken. Absorbances were measured with a microplate reader at 570 nm and 690 nm (to remove background). Measurements were analyzed with SoftMax Pro (5.2 program SoftMax® Pro Data Acquistion & Analysis Software, Sunnyvale, CA). The experiment was performed in quadruplicate (n=4 dogs). Statistical analysis of the data ws performed using Repeated Measures ANOVA (migration), one-way ANOVA (invasion), or two-way ANOVA (proliferation) in PROC MIXED using SAS (Version 9.2, SAS Institute Inc., Cary, NC). For the migration assay, the mean of the control wound area at 0 h was set to 0% wound closure and the data were expressed as the percent wound closure of the control for each dog. For the invasion assay and proliferation assay, the mean of the controls was set to 100% and the data was expressed as the percentage of the control for each dog. Significance was defined as P<0.05. Analysis showed that IL-8 increased cell migration by 35% compared to the control (P<0.01). TIMP-2 had no significant effect on cell migration (P=0.38). There was no significant effect of IL-8 on cell invasion compared to the control (P=0.42), whereas TIMP-2 decreased cell invasion by 57% compared to the control (P<0.05). Also, for the proliferation assay IL-8 had no significant effect on cell proliferation (P=0.18). This study was the first to investigate term canine trophoblast physiology. Future studies should compare differences between normal term trophoblasts and those from cases of subinvolution of placental sites. It would also be of interest to compare differences in trophoblast physiology at different stages of gestation. Graduation date: 2013 2013-05-06T00:00:00Z http://hdl.handle.net/1957/38542 Applications of GnRH Immunization in Domestic Dogs; Applications of GnRH immunization in domestic dogs Donovan, Caitlin Elizabeth Reproductive function in the dog is controlled by feedback mechanisms that involve the hypothalamus, the anterior pituitary gland, and the gonads. Surgical gonad removal, a common procedure performed in dogs for the purposes of sterilization, disrupts the hypothalamic-pituitary-gonadal (HPG) axis and results in permanently elevated concentrations of gonadotropins. Manipulation of the HPG axis via gonadotropin releasing hormone (GnRH) immunization results in the synthesis of GnRH neutralizing antibodies, which bind to (neutralize) GnRH and prevent it from binding to its receptors. The end result of GnRH immunization is the cessation of pituitary gonadotropin secretion, namely luteinizing hormone (LH). In 2004, a commercial GnRH vaccine was launched in the United States (Canine Gonadotropin Releasing Factor Immunotherapeutic®; Pfizer Animal Health USA), labeled for the treatment of benign prostatic hyperplasia in intact male dogs. This research investigated two novel clinical applications of this vaccine in dogs. In the first study, physiologic responses to GnRH immunization in intact male dogs were observed. Four intact males were vaccinated with the GnRH vaccine twice at four week intervals. Blood samples were collected prior to each injection (at weeks 0 and 4) and at weeks 12 and 20 following initial vaccination. Scrotal measurements were also made at the time of each blood sample collection to calculate testicular volume. All four dogs developed a GnRH antibody titer and experienced a significant decrease in testosterone concentrations. Testicular volume also significantly decreased, and this effect was reversed by the end of the study. LH concentrations remained at basal levels. These results are indicative of temporary humoral response to the GnRH vaccine, and future studies should investigate prolonging these effects to potentiate GnRH immunization as a method of population control in dogs. Beyond use as a temporary immunosterilant, GnRH immunization has other promising clinical applications. Elevated gonadotropin concentrations as a result of gonad removal in female dogs decreases urethral pressure, and in some bitches, this results in the development of urethral sphincter mechanism incompetence (USMI). Therefore, lowering gonadotropin concentrations in incontinent ovariectomized bitches through GnRH immunization may restore continence. In the second study, sixteen incontinent dogs that were using phenylpropanolamine (PPA) to control incontinence were recruited. Eleven dogs were immunized against GnRH at week 0, and nine dogs were vaccinated again four weeks later. Five control dogs were vaccinated with a placebo twice at four week intervals. Vaccinated dogs discontinued PPA two weeks after re-vaccination, and control dogs remained on PPA for the duration of the study. Blood samples were collected before each injection and at 6, 8, 10, 12, 16, 20, and 24 weeks, and owners recorded episodes of incontinence throughout the study. Of the nine dogs that completed the vaccination series, four dogs remained continent after PPA was discontinued. For these four dogs, there was no difference in the episodes of incontinence when using PPA versus treatment with the vaccine. All nine vaccinated dogs developed a GnRH antibody titer, and LH concentrations decreased significantly in vaccinated dogs compared to controls. These results indicate that decreasing LH concentrations through GnRH immunization restores continence to some, but not all incontinent ovariectomized bitches. Because the development of USMI results from decreased urethral pressure that happens after ovariectomy, future studies should focus on preventing this decrease in urethral pressure to prevent USMI from occurring. Graduation date: 2013 2013-05-03T00:00:00Z http://hdl.handle.net/1957/38541 α-tocopherol is a potential diagnostic indicator of metabolic diseases in early lactation dairy cows; Alpha-tocopherol is a potential diagnostic indicator of metabolic diseases in early lactation dairy cows Qu, Yang Milk fever (MF), retained placenta (RP), and left displaced abomasum (LDA) are three common and costly metabolic diseases in cows during the first days of lactation. Some studies suggest that circulating concentrations of α-tocopherol (ATOC) are decreased by these three diseases. It is, however, unknown if and how long lower circulating ATOC precede and/or remain after recovery from these three diseases. The hypothesis or the thesis is that lower serum ATOC concentrations precede and persist in cows after MF, RP and LDA. The objective of the thesis is to examine the association between MF, RP, and LDA and serum concentrations of ATOC, metabolites, refore, the hypothesis of this project was to compare with healthy cows, lower serum ATOC concentration precede and persist in cows after MF, RP and LDA. Using a nested case-control study design, the relationship between the incidence of those three diseases and serum concentrations of ATOC, metabolites, acute phase proteins, and minerals, measured at day -21, -14, -7, -3, -1, 0, 1, 3, 7, 14, 21, 28, 35, 42, and 49 postpartum, was evaluated in multiparous Holstein cows. In chapter 2, serum concentrations of ATOC, metabolites, acute phase proteins, and minerals of 7 multiparous Holstein cows diagnosed with LDA between day 6 and 32 postpartum were compared with 10 healthy Holstein cows. Besides indicators of negative energy balance and inflammation, lower serum ATOC concentrations preceded LDA and persisted after LDA correction. At the last blood sampling before LDA diagnosis, cows had 45% lower serum ATOC concentrations (5.0 ± 0.9 vs. 9.1 ± 0.9 μM; P = 0.004) and 39% lower ATOC to cholesterol molar ratios (1.90 ± 0.19 vs. 3.09 ± 0.26; P = 0.003) compared with healthy cows. Serum ATOC concentrations remained lower (<10 vs. ~15 μM) in cows that had LDA up to day 49 postpartum (all P < 0.03). In chapter 3, serum concentrations of ATOC, metabolites, acute phase proteins, and minerals of 32 multiparous Holstein cows with retained fetal membranes for more than 24 h were compared with those of 32 diseased cows and those of 32 visually healthy cows. Besides indicators of negative energy balance and inflammation, cows that developed RP had prepartum 30% lower prepartal serum ATOC concentrations (8.7 ± 0.6 vs. 12.5 ± 0.6 μM; P< 0.001) and 23% lower ATOC to cholesterol molar ratios (3.12 vs. 4.03 μM/mM; P< 0.001) compared with visually healthy cows. These group differences were already significant three weeks before calving for ATOC concentrations (8.3 ± 0.7 vs. 11.9± 0.7 μM; P< 0.001) and ATOC to cholesterol molar ratios (2.68 vs. 3.66 μM/mM; P = 0.001). Up to day 28 postpartum, serum ATOC concentrations remained lower in RP than in visually healthy cows (<10 vs. ~13 μM; all P < 0.001). Serum ATOC concentrations and ATOC to cholesterol molar ratios did not differ between diseased cows with RP than with other diseases. In chapter 4, serum concentrations of ATOC, metabolites, acute phase proteins, and minerals of 9 multiparous Holstein cows with serum calcium concentrations below 6 mg/dl and being treated for milk fever within the first 48 hpostpartum were compared with those of 10 healthy cows and with those of 31 diseased cows with serum calcium concentrations above 6 mg/dL in the first 48 hpostpartum Besides indicators of negative energy balance and inflammation, cows that later developed MF had 37% lower prepartal serum ATOC concentrations (9.0 ± 0.9 vs. 14.2 ± 0.8 μM; P< 0.001) and 35% lower ATOC to cholesterol molar ratios (3.08 vs. 4.78 μM/mM; P< 0.001) compared with healthy cows. These group differences were already significant three weeks before calving for ATOC concentrations (8.3 ± 0.9 vs. 13.8 ± 0.8 μM; P< 0.001) and ATOC to cholesterol molar ratios (2.86 vs. 4.18 μM/mM; P = 0.003). Up to day 28 postpartum, serum ATOC concentrations remained lower in MF than in healthy cows (<9 vs. ~13 μM; all P < 0.002). Serum ATOC concentrations and ATOC to cholesterol molar ratios did not differ between diseased cows with MF than with other diseases. In summary, depleted serum ATOC concentrations preceded the three investigated metabolic diseases (MF, RP, and LDA). Thus, lower serum ATOC concentrations maybe a potential diagnostic indicator for metabolic diseases in multiparous dairy cows during early lactation. Serum ATOC concentrations remained lower than in healthy cows for several wk after disease treatment. The focus of future studies will be if and how vitamin E alimentation may prevent or improve response to conventional treatments of metabolic diseases in multiparous cows. Graduation date: 2013 2013-04-12T00:00:00Z http://hdl.handle.net/1957/38037 Fructokinase, Fructans, Intestinal Permeability, and Metabolic Syndrome: An Equine Connection? Johnson, Richard J.; Rivard, Chris; Lanaspa, Miguel A.; Otabachian-Smith, Silvia; Ishimoto, Takuji; Cicerchi, Christina; Cheeke, Peter R.; McIntosh, Bridgett; Hess, Tanja Fructose is a simple sugar present in honey and fruit, but can also exist as a polymer (fructans) in pasture grasses. Mammals are unable to metabolize fructans, but certain gram positive bacteria contain fructanases and can convert fructans to fructose in the gut. Recent studies suggest that fructose generated from bacteria, or directly obtained from the diet, can induce both increased intestinal permeability and features of metabolic syndrome, especially the development of insulin resistance. The development of insulin resistance is driven in part by the metabolism of fructose by fructokinase C in the liver, which results in oxidative stress in the hepatocyte. Similarly, the metabolism of fructose in the small bowel by intestinal fructokinase may lead to increased intestinal permeability and endotoxemia. While speculative, these observations raise the possibility that the mechanism by which fructans induce laminitis could involve intestinal and hepatic fructokinase. Further studies are indicated to determine the role of fructanases, fructose and fructokinase in equine metabolic syndrome and laminitis. This is the author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by Elsevier and can be found at: http://www.journals.elsevier.com/journal-of-equine-veterinary-science/. 2013-02-01T00:00:00Z