VTechWorks Repository :: Browsing by Author "Huckle, William R."

Browsing by Author "Huckle, William R."

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Abscisic acid ameliorates glucose tolerance and obesity-induced inflammation
Guri, Amir Joseph (Virginia Tech, 2007-10-19)
Obesity is a disease characterized by chronic inflammation and the progressive loss in systemic insulin sensitivity. One of the more effective medications in the treatment of insulin resistance have been the thiazolidinediones (TZDs), which act through the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma ). Due to the many side-effects of TZDs, our laboratory sought out a natural phytochemical, abscisic acid (ABA), with chemical similarities to TZDs. Our first study demonstrated that ABA activates PPARgamma in vitro and significantly ameliorates white adipose tissue (WAT) inflammation and glucose tolerance in db/db mice. We next further examined the effect of ABA on the phenotype of adipose tissue macrophages (ATMs). In doing so, we discovered two separate ATM populations which differed in their expression of the macrophage surface glycoprotein and maturation marker F4/80 (F4/80hi vs F4/80lo). Dietary ABA-supplementation significantly reduced F4/80hiCCR2+ ATMs and had no effect on the F4/80lo population. Utilizing a tissue-specific knockout generated through Cre-lox recombination, we were able to determine that this effect was dependent on PPARgamma in immune cells. To further characterize the differences between the ATM subsets that were affected by ABA, we performed a multi-organ assessment (i.e., WAT, skeletal muscle and liver) of the effect of diet-induced obesity on the phenotype of infiltrating macrophages and T cells into metabolic organs. Based on our new data, we formulated a model by which F4/80hiCCR2hi ATMs infiltrate WAT and ultimately induce a CD11c+ pro-inflammatory phenotype in the resident F4/80loCCR2lo subset. Ultimately, our findings provide evidence that ABA has potential as an alternative preventive intervention, expound the role of PPARgamma in immune cells and, in general, expand our knowledge concerning the immunopathogenesis of obesity-induced insulin resistance.
Adipose-Derived Adult Stem Cells as Trophic Mediators of Tendon Regeneration
Stewart, Shelley Leigh (Virginia Tech, 2012-06-26)
The adipose-derived stromal vascular fraction (SVF) is a promising new therapy for equine flexor tendonitis. This heterogeneous population of cells may improve tendon healing via the production of growth and chemotactic factors capable of recruiting endogenous stem cells and increasing extracellular matrix production by tendon fibroblasts (TFBL). The purpose of this study was to evaluate the ability of adipose-derived cells (ADC) culture expanded from the SVF to act as trophic mediators in vitro. We hypothesized that ADCs would produce growth and chemotactic factors important in tendon healing and capable of inducing cell migration and matrix protein gene expression. Superficial digital flexor tendons and adipose tissue were harvested from eight adult horses and processed to obtain SVF cells, ADCs and TFBLs. Adipose-derived cells and TFBLs were grown in monolayer culture for growth factor quantification, to produce conditioned media for microchemotaxis, and in co-culture for quantification of matrix protein gene expression by TFBLs. Growth factor gene expression by SVF cells was significantly greater than in ADCs or TFBLs. Co-culture of TFBLs and ADCs resulted in modest up-regulation of matrix protein expression (collagen types I and III, decorin, and cartilage oligomeric matrix protein) by TFBLs. Media conditioned by ADCs induced ADC migration in a dose dependent manner. These findings support the role of both SVF and ADCs as trophic mediators in tendon regeneration. The differences detected in gene expression between SVF cells and ADCs indicate that additional studies are needed to evaluate the changes that occur during culture of these cells.
Advanced Echocardiographic Imaging In Dogs With Myxomatous Mitral Valve Disease
Menciotti, Giulio (Virginia Tech, 2017-05-23)
Myxomatous mitral valve degeneration (MMVD) is the most common canine cardiac disease. In the studies presented in this dissertation, we used advanced echocardiographic techniques to elucidate several aspects of MMVD in dogs. Our hypothesis was that the mitral valve (MV) morphology could have a role in the development of MMVD. First, we tested whether we could use real time three-dimensional transthoracic echocardiography (RT-3DTTE), and an offline software for MV analysis to evaluate canine MV. We described that the technique was feasible and repeatable, we evaluated the morphology of the MV in healthy dogs, and we provided reference values for MV morphologic variables in this species. Then, we used the same technique to compare healthy dogs to dogs affected by MMVD. We found that dogs affected by MMVD have more circular and flatter valve. We then analyzed the MV of healthy Cavalier King Charles Spaniels (CKCSs), given the high predisposition of this breed for MMVD. Our findings indicate that compared to healthy dogs of other breeds, the MV of healthy CKCSs is flatter and has less leaflet tenting, corroborating our hypothesis that an altered MV morphology could represent a predisposing factor for disease development. We also used RT–3DTTE to characterize the area of the regurgitant MV orifice of dogs affected with MMVD, finding that the technique requires further standardization in order to become clinically useful. The elevation of pulmonary venous pressure caused by MMVD can, in some dogs, cause pulmonary arterial hypertension (PH), which is a risk factor associated with worse outcome in dogs with MMVD. Diagnosis of PH in dogs with MMVD is usually made by estimating pulmonary pressure using Doppler echocardiography. We are currently evaluating the accuracy of this technique, compared to invasive measurement of pulmonary pressure. Only preliminary data are presented regarding this study, as the disclosure of the blinding would have infringed the power of the study. Our preliminary results demonstrate that there is only moderate agreement between the two techniques, indicating that caution should be used when deriving the non-invasive estimation of systolic pulmonary pressure in order to make clinical decisions.
Advanced Studies in Veterinary Anatomy: Angiogenesis in Caprine Reproductive Organs of Non-Pregnant and Pregnant Normal and Swainsonine-Treated Does
Hafez, Shireen Abdelgawad (Virginia Tech, 2005-04-20)
The female reproductive organs are among the few adult tissues in which periodic angiogenesis normally occurs. Pathological angiogenesis can occur in various conditions, such as solid tumors. Vascular endothelial growth factor (VEGF) signaling often represents a critical rate-limiting step in physiological and pathological angiogenesis. This study utilizes development of utero-ovarian vasculature during pregnancy in goats as a model of physiological angiogenesis. Non-pregnant does and does at 4, 7, 10, 13, 16, and 18 weeks of gestation were used. Arteries of the reproductive tract were injected in situ with Microfil®. The tracts were fixed, dehydrated, and rendered transparent to reveal the paths of arteries. The ovarian artery was tortuous and lay in close apposition to the uterine tributary of the ovarian vein in all specimens studied. In non-pregnant does, this arrangement may serve as a local utero-ovarian pathway for the corpus luteum (CL) luteolysis at the end of non-fertile estrous cycle. During pregnancy, this arterio-venous arrangement may transfer luteotropic substances from uterus to ovary, which may serve in maternal recognition of pregnancy and fit the fact that the goat is CL dependent throughout gestation. In some cases of triplets, the size of the uterine branch of the ovarian artery was equal to or even larger than that of its parent artery and/or the ipsilateral uterine artery; and the vaginal artery contributed a connecting branch to the uterine artery. These physiological adaptations of the ovarian and/or vaginal arteries correlate well with the increasing nutrient demands of the growing multiple fetuses. In a second experiment, the vasculature of the uterus and ovaries was injected in situ with a mixture of Batson's No.17® and methyl methacrylate and then processed for observation by SEM. The microvasculature differed between non-pregnant and pregnant does, and with advancing gestation. We concluded that goats possess a multivillous type placenta. Capillary sinusoids and crypts on the fetal surface of the caruncle may compensate for the negative effect of the increased interhemal distance. Intussusceptive angiogenesis should be considered as equally possible and important mechanism as sprouting angiogenesis during placental development. Capillary diameters increased significantly during pregnancy especially after 4 weeks. Capillary density index was 66.8, 68.7, 55.5, 63.5, 70.1, 70.4, 64.5 percent in non-pregnant, 4, 7, 10, 13, 16, and 18 weeks of pregnancy, respectively. In the ovary, coiling of the ovarian branch of the ovarian artery around the ovarian tributary of the ovarian vein was observed. This may represent a local channel required for product transport from ovarian vein to ovarian artery and might have a role in regulating blood pressure to various ovarian structures. Immunolocalization of VEGF was performed as a third experiment. Immunostaining was observed in cyto- trophoblasts, maternal epithelial tissues, and vascular endothelium and smooth muscle, but not in binucleate giant cells or connective tissue. No apparent differences were observed in intensity and pattern of VEGF staining associated with advancing gestation. Luteal and follicular cells, and endothelium and smooth muscles of the ovarian vasculature positively stained. Patterns and intensity of staining of VEGF suggest that the fetus is directing its own survival by producing growth factors affecting fetal and maternal tissues. VEGF may have a role in growth and differentiation of cytotrophoblasts, as well as, development and maintenance of CL. In the fourth experiment, the sequential expression of VEGF and its receptors (fms-like tyrosine kinase, Flt-1 and kinase-insert domain-containing receptor, KDR) was measured using real-time quantitative PCR. Targets were detected in all studied tissues; however, levels of expression differed according to the stage of pregnancy. Expression of VEGF and its receptor mRNAs increased with advancing pregnancy, which correlates with the expansion of vasculature during pregnancy. Differences in the time-courses of the expression of Flt-1 and KDR mRNAs during pregnancy suggest that each receptor plays a different role in the angiogenic process. As an application of our model of angiogenesis, we tested the effect of swainsonine (active compound of locoweed and a potential anti-cancer drug) on the process. Does treated with swainsonine were euthanized at 7 and 18 weeks. No significant differences were found in sinusoidal diameters in treated does at 7 weeks, but a decrease in capillary density index was noted. In the ovary, focal avascular areas were observed in the corpus luteum of swainsonine-treated does at 7 weeks of pregnancy. Swainsonine caused great distortion in the uterine and ovarian vasculature at 18 weeks. A decrease in intensity of the immunoreactivity to VEGF antibody was observed in tissues from swainsonine-treated does at 7 and 18 weeks. There was no substantial effect of swainsonine on the expression of VEGF and its receptors' mRNAs in any of the studied tissues (except in the left ovary, where it had an inhibitory effect) at 7 weeks of pregnancy, but it had an inhibitory effect at 18 weeks. Demonstration of swainsonine's potential to negatively affect vascular development and suppress genes likely involved in angiogenesis at critical stages of blood vessel proliferation lends credibility to its potential as anti-cancer drug.
The anti-diabetic mechanisms by isoflavone genistein
Fu, Zhuo (Virginia Tech, 2011-05-10)
Diabetes is growing public health problem in the United States. Both in Type 1 and Type 2 diabetes, the deterioration of glycemic control over time is largely due to insulin secretory dysfunction and significant loss of functional β-cells. As such, the search for novel agents that promote β-cell survival and preserve functional β-cell mass are one of the essential strategies to prevent and treat the onset of diabetes. Genistein, a flavonoid in legumes and some herbal medicines, has various biological actions. It was recently shown that dietary intake of foods containing genistein improves diabetes in both experimental animals and humans. However, the potential anti-diabetic mechanisms of genistein are unclear. In the present study, we first investigated the effect of genistein on β-cell insulin secretion and proliferation and cellular signaling related to these effects in vitro and in vivo. We then determined its anti-diabetic potential in insulin-deficient and obese diabetic mouse models. The results in our study showed that exposure of clonal insulin secreting (INS1E) cells or isolated pancreatic islets to genistein at physiologically relevant concentrations (1-10 μM) enhanced glucose-stimulated insulin secretion (GSIS), whereas insulin content was not altered, suggesting that genistein-enhanced GSIS is not due to a modulation of insulin synthesis. This genistein's effect is protein tyrosine kinase- and KATP channel-independent. In addition, genistein had no effect on glucose transporter-2 expression or cellular ATP production, but similarly augmented pyruvate-stimulated insulin secretion in INS1E cells, indicating that genistein improvement of insulin secretion in β-cells is not related to an alternation in glucose uptake or the glycolytic pathway. Further, genistein (1-10 μM) induced both INS1 and human islet β-cell proliferation following 24 h of incubation, with 5 μM genistein inducing a maximal 27% increase. The effect of genistein on β-cell proliferation was neither dependent on estrogen receptors, nor shared by 17β-estradiol or a host of structurally related flavonoid compounds. Pharmacological or molecular intervention of PKA or ERK1/2 completely abolished genistein-stimulated β-cell proliferation, suggesting that both molecules are essential for genistein action. Consistent with its effect on cell proliferation, genistein induced cAMP/PKA signaling and subsequent phosphorylation of ERK1/2 in both INS1 cells and human islets. Furthermore, genistein induced protein expression of cyclin D1, a major cell-cycle regulator essential for β-cell growth. Dietary intake of genistein significantly improved hyperglycemia, glucose tolerance, and blood insulin levels in both insulin deficient type 1 and obese type 2 diabetic mice, concomitant with improved islet β-cell proliferation, survival, and mass. These changes were not due to alternations in animal body weight gain, food intake, fat deposit, plasma lipid profile, or peripheral insulin sensitivity. Collectively, these findings provide better understanding of the mechanism underlying the anti-diabetic effects of genistein. Loss of functional β-cell mass through apoptosis is central to the development of both T1D and T2D and islet β-cell preservation and regeneration are very important components of β-cell adaptation to increased apoptosis and insulin resistance and therefore holds promise as a treatment for this disease. In this context, these findings may potentially lead to the development of novel low-cost natural agents for prevention and treatment of diabetes.
Application of Fluid Flow for Functional Tissue Engineering of Bone Marrow Stromal Cells
Kreke, Michelle Renee (Virginia Tech, 2005-04-19)
In the United States, nearly half a million bone graft operations are performed annually to repair defects arising from birth defects, trauma, and disease, making bone the second most transplanted tissue. Autogenous bone is the current gold standard for bone grafts; however it is in limited supply and results in a second injury at the donor site. A promising alternative is a tissue engineered bone graft composed of a biomaterial scaffold, pharmaceutics, and osteoprogenitor cells. One source of osteoprogenitor cells is bone marrow stroma, which can be obtained from the patient - minimizing the risk of an immune response - directed in vitro to proliferate, and differentiate into a bone-like tissue. To date, tissue engineered bone grafts have not been clinically effective; thus, strategies must be developed to improve efficacy. I hypothesize that to facilitate tissue healing in a manner similar to autogenous bone tissue engineering bone must possess a mineralized collagen matrix to support tissue integration, and angiogenic factors to stimulate vascular infiltration, and osteogenic factors to direct normal bone remodeling. I propose that these factors can be synthesized by osteoprogenitor cells in vitro when cultured under the appropriate conditions. Previous work has demonstrated that perfusion culture of osteoprogenitor cells within 3D scaffolds stimulates phenotypic markers of osteoblastic differentiation, but those studies did not determine whether the effects were a consequence of shear stress or increased nutrient availability. Consequently, this work has involved studies in a planar geometry, where nutrient effects are negligible. Three studies that characterize the effect of fluid flow on osteoblastic differentiation of osteoprogenitor cells are presented here. The objective of the first study was to determine the effect of shear stress magnitude on cell density and osteocalcin deposition. In this study, radial flow chambers were used to generate a spatially dependent range of shear stresses (0.36 to 2.7 dynes/cm2) across single substrates, and immunofluorescent techniques were used to assay cell phenotype as a function of shear stress. The objective of the second study was to determine the effect of the duration of fluid flow on cell density and phenotypic markers of differentiation. Here, parallel plate flow chambers were used to generate a single shear stress at the cell surface, and entire cell layers were assayed for expression of osteoblastic genes. The objective of the third study was to compare continuous and intermittent fluid flow strategies. In this study, a microprocessor-controlled actuator was added to the flow loop to periodically halt flow, and markers of mechanosensation and osteoblastic differentiation were measured. These studies demonstrated that shear stresses of 0.36 to 2.7 dynes/cm2 stimulate late phenotypic markers of osteoblastic differentiation but not cell proliferation. In addition, this osteogenic effect is sensitive to duration of fluid flow but insensitive to the magnitude of shear stress. Further, intermittent fluid flow enhances cell retention, biochemical markers of mechanotransduction, and synthesis of the angiogenic factor vascular endothelial growth factor (VEGF). Thus, these studies suggest that intermittent fluid flow may be an attractive component of a biodynamic bioreactor for in vitro manufacture of clinically effective tissue engineered bone grafts. Future studies will further investigate intermittent fluid flow strategies and three-dimensional studies with scaffolds suitable for bone tissue engineering.
Approaches towards therapeutic development against chronic brucellosis in a mouse model
Jain, Neeta (Virginia Tech, 2012-01-19)
Brucellosis is the most common zoonotic disease worldwide. The intracellular localization of Brucella hinders the action of drugs that poorly cross cell membrane barriers. Additionally, when the immune response fails to clear the infection, chronic brucellosis ensues that becomes more challenging to treat with antibiotics. Therefore, two approaches, intracellular drug delivery and immunostimulation, have been explored in this dissertation, with an aim to develop a better therapeutic against Brucella infection in mice. First, to overcome the cell membrane barriers, drug loaded nanoparticles were tested to treat B. melitensis infection in mice. Gentamicin loaded block-ionomer complexes (BICs) and magnetite block-ionomer complexes (MBICs) were tested in vitro and along with clusters of MBICs (MBIClusters) were tested in vivo as tools to deliver gentamicin intracellularly. While these complexes showed very high efficacy compared to free gentamicin against Brucella in macrophage cell culture, they failed to show similar efficacies in mice. Histopathological examination of kidneys from mice treated with MBICs or MBIClusters showed deposition of brown pigment-laden macrophages in peri-renal adipose tissue and the pigment was confirmed as MBICs or MBIClusters based on special staining for iron. Additionally, it was found that doxycycline-gentamicin (DG) treatment results in better clearance of Brucella from infected mice compared to doxycycline alone. Secondly, two vaccine candidates, irradiated B. neotomae (IBN) and outer membrane vesicles (OMVs), were tested as immunostimulants to treat chronic B. melitensis infection in mice in combination with antibiotics. The non-ionic block co-polymer Pluronic P85, when mixed with OMVs as an adjuvant showed significantly higher protection against B. melitensis challenge in vaccinated mice compared to those vaccinated with OMVs alone. When tested as immunostimulants, there was no additive effect of vaccines and antibiotics on Brucella clearance from mice. However, IBN enhanced the production of IFN-γ while OMVs were associated with enhanced antibody production. This enhancement in the immune system resulted in the control of Brucella growth after the end of treatment. When given without antibiotics, vaccine alone failed to clear any Brucella from infected mice. The use of these vaccine candidates in combination with antibiotics shows a potential to prevent relapses in cases of brucellosis.
Aqueous humor concentration and prostaglandin E2 suppression efficacy of topically applied ophthalmic ketorolac 0.5% and diclofenac 0.1% solutions in dogs with cataract
Waler, Kayla A. (Virginia Tech, 2020-06-01)
Background: Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for their analgesic, anti-pyretic and anti-inflammatory properties in both human and veterinary patients. Topical ophthalmic NSAIDs are commonly employed in the management of intraocular inflammation (uveitis), corneoconjunctival inflammatory disease and pre-operatively to prevent intraoperative miosis during cataract surgery. Despite their routine application in these clinical scenarios, little is known regarding the corneal penetration and relative anti-inflammatory efficacy of the available topical ophthalmic NSAIDs in the dog. Decisions regarding which of these agents to employ are therefore based upon factors such as cost and ease of acquisition as opposed to established efficacy. Objectives: To investigate the relative intraocular penetration and anti-inflammatory efficacy of two commonly utilized topical ophthalmic NSAIDs in dogs, diclofenac 0.1% and ketorolac 0.5%. Animals: Twenty-two client owned dogs (22 operated eyes) presenting to the VTH ophthalmology service for routine cataract surgery for mature or hypermature cataract. Methods: Subjects were randomized to be treated with either topical ketorolac 0.5% or topical diclofenac 0.1% ophthalmic solutions at specified times in the 24-hour period pre-operatively. Aqueous humor samples were obtained intra-operatively and stored for subsequent evaluation of drug concentrations and prostaglandin E2 (PGE2) concentrations via ultra performance liquid chromatography-mass spectrometry (UPLC-MS/MS) and enzyme-linked immunoassay (ELISA) analysis, respectively. Results: Median aqueous humor drug concentrations were significantly higher in dogs treated with ketorolac 0.5% (1311.6 ng/mL) compared to those treated with diclofenac 0.1% (284.9 ng/mL). There was no significant difference in aqueous humor PGE2 concentrations between the two treatment groups. No significant association was determined between aqueous humor drug concentration and PGE2 concentration. There was no significant association between diabetic status and aqueous humor drug concentration or PGE2 concentration in either group. Conclusions and clinical importance: This study suggests that topical ketorolac 0.5% and diclofenac 0.1% are efficacious in decreasing aqueous humor PGE2 concentrations and are equally suitable for use based on their comparable anti-inflammatory profiles. The results of these assays provide clinically relevant information regarding intraocular penetration and anti-inflammatory efficacy of these medications in dogs with cataract.
Bioactive Poly(Lactic-co-Glycolic Acid)-Calcium Phosphate Scaffolds for Bone Tissue Regeneration
Popp, Jenni Rebecca (Virginia Tech, 2009-03-27)
Bone is currently the second most transplanted tissue, second only to blood. However, significant hurdles including graft supply and implant failure continue to plague researchers and clinicians. Currently, standard clinical procedures include autologous and allogeneic grafting. Autologous grafts may achieve functional repair; yet, they are available in limited supply and are associated with donor site morbidity. Allogeneic grafts are available in greater supply, but have a higher risk of infection. To overcome the disadvantages of current grafts, tissue engineering has become a major focus for the regeneration of bone. The goal of tissue engineering is to use a multidisciplinary approach to create biomimetic constructs that stimulate osteogenic regeneration to heal bone defects and restore tissue function. Biodegradable scaffolds are used in tissue engineering strategies as an interim template for tissue regeneration. The scaffold architecture provides mechanical support for cell attachment and tissue regeneration. Biocompatible poly(lactic-co-glycolic acid) (PLGA) has been processed through a number of techniques to create porous 3D architectures. Hydroxyapatite (HAP) and tricalcium phosphate have been used in conjunction with polymer scaffolds due to their osteoconductivity and biocompatibility, but they often lack osteoinductivity and are resistant to biodegradation. Conversely, amorphous calcium phosphate (ACP) is a mineral that solubilizes under aqueous conditions, releasing calcium and phosphate ions, which have been postulated to enhance osteoblast differentiation and mineralization. Controlled dissolution can be achieved by stabilizing ACP with divalent cations such as zinc or copper. Furthermore, incorporation of such osteogenic ACPs within a biodegradable PLGA scaffold could enhance the osteoconductivity of the scaffold while providing calcium and phosphate ions to differentiating osteoprogenitor cells, thereby stimulating osteogenesis when implanted in vivo. In this research, the effect of zinc on the differentiation of osteoprogenitor cells was investigated. Zinc supplementation of the culture media had no stimulatory effect on cell proliferation or differentiation. ACPs were synthesized using zirconium (ZrACP) and zinc (ZnACP) as stabilizers to achieve sustained ion release. Elevated concentrations suggested sustained ion release over the course of 96 hours and enhanced solubility of ZrACP and ZnACP. X-ray diffraction analysis showed a conversion of ZrACP to a semi-crystalline material after 96 hours, but ZnACP showed no conversion after 96 hours. Composite scaffolds were fabricated by incorporating HAP, zirconium-stabilized ACP (ZrACP), or zinc-stabilized ACP (ZnACP) into a sintered PLGA microsphere matrix and then characterized to determine the effect of the minerals on the in vitro differentiation of MC3T3-E1 cells. Scanning electron microscopy revealed a porous microsphere matrix with calcium phosphate powders distributed on the surface of the microspheres. Measurements of mechanical properties indicated that incorporation of 0.5 wt% calcium phosphates resulted in a 30% decrease in compressive modulus. When cells were cultured in the scaffolds, composite ACP scaffolds stimulated proliferation and ALP activity, while HAP scaffolds stimulated osteoblast gene expression. Overall, the results of this work indicate the addition of calcium phosphate minerals to PLGA scaffolds supported cell growth and stimulated osteogenic differentiation, making the scaffolds a promising alternative for bone tissue regeneration.
Biochemical and Immunocytochemical Characterization of Canine Corneal Cells Cultured in Two Different Media
Schorling, Jamie J. (Virginia Tech, 2007-04-26)
The study purpose was to determine whether canine corneal cultures demonstrate superior growth when cultured in a fully defined epithelial selective medium, Epilife®, compared to Dulbecco's modification of Eagle's medium (DMEM) with fetal bovine serum (FBS), and to characterize cultured canine corneal cells. Superficial keratectomies were performed on three dogs. Samples were trypsinized to separate cell layers. Post-trypsinization, immunohistochemistry confirmed that epithelial cells had been released from the stroma. Both cell populations (presumed epithelial cells and stromal tissues) were cultured in DMEM with FBS or Epilife®. First passage cells were fixed for immunocytochemistry and prepared for PCR. Immunocytochemical staining for pancytokeratin, vimentin, and E-cadherin was evaluated, and immunofluorescence for zonula occludens-1 was attempted. Amplification of cytokeratin 5 (CK5) mRNA was assessed by PCR. Primary presumed epithelial cells grew faster when cultured in DMEM with FBS compared to Epilife®. Stromal tissue segments in Epilife® medium failed to adhere to culture plates, indicating that this medium may inhibit attachment and growth of non-epithelial tissues. Staining of corneal tissue segments confirmed that epithelial layers were pancytokeratin and E-cadherin positive, while stromal cells were vimentin positive. Immunocytochemistry of cultured cells revealed that epithelial cells stained positively for pancytokeratin, vimentin, and E-cadherin, while stromal cells remained only vimentin positive. Greater amplification of CK5 mRNA occurred from epithelial cells grown in Epilife® compared to epithelial cells in DMEM with FBS or the stromal cells. Based on PCR results, Epilife® medium may support retention of the epithelial characteristic of CK5 mRNA expression better than DMEM with FBS.
Birth Defect Amelioration and Placental Cytokine Expression in Mnu-Exposed Dams Treated With Ifn-Gamma
Laudermilch, Chelsea Lee (Virginia Tech, 2007-01-16)
Each year, 7.9 million babies are born with birth defects. Seventy percent of those could be prevented, ameliorated, or repaired; yet 3.2 million children still die by the age of three (March of Dimes Global Report 2006). We have found that non-specific maternal immune stimulation with the cytokine interferon-gamma (IFN-gamma) can successfully ameliorate some of these defects in the C57BL/6N mouse model. We have observed a reduction in the distal limb malformations syndactyly, polydactyly, and webbing by 47%, 100%, and 63% respectively when IFN-gamma is given 2 days prior to MNU administration. We have also observed that IFN-gamma works at the placental level to protect against MNU-induced damage. Trophoblast loss and associated cytokine alterations occur in gestation day (GD) 14 placenta following GD9 MNU exposure, showing that fetal-maternal communication can be hindered due to MNU. In the labyrinthine layer of the placenta, we observed multifocal fibrinous necrosis of endothelial cells due to MNU, however IFN-gamma almost completely protected the trophoblast and endothelial cells when given to the dam as an immune stimulant. To determine the genes participating in these processes, gene microarray studies were conducted. Hepatocyte growth factor (HGF), interleukin 1 beta (IL1Β), and insulin-like growth factor 2 (IGF2) were elucidated as genes that were significantly expressed in GD12 placenta. These genes are similar in that they are all connected to the Jak-Stat signaling pathway. These findings provide a possible mechanism for birth defect reduction by maternal immune stimulation with IFN-gamma in MNU-challenged mice.
Bone Regeneration Potential of Mesenchymal Stromal Cells derived from a Clinically Relevant Rat Model of Osteoporosis
Saverot, Scott-Eugene (Virginia Tech, 2020-04-09)
Falls among the elderly are a major source of injury, often leading to serious fractures, hospitalization, and death. Osteoporosis (OP) is a global problem intimately related with these fractures, characterized by reduced bone mass, increased bone fragility. There exists a high failure rate in the translation of treatments to osteoporotic populations. Mesenchymal stromal cell (MSC) transplantation as a therapeutic strategy for OP has not yet been examined in clinical trials. This may be attributed to the mixed findings of pre-clinical studies aimed at determining the efficacy of MSC therapy towards bone regeneration in OP. The most common animal model of OP is ovariectomy (OVX) that simulates post-menopausal estrogen loss. A plethora of bone regeneration studies have used OVX models with 12-16 weeks post-OVX periods and have generally reported positive results from a variety of treatment modalities, including MSC therapy. However, the use of the minimum post-OVX period may not be appropriate to reflect the global changes in regenerative potential of OP patients. In our research group's previous study, MSC were isolated from a minimum 60 week post-OVX rat model, representing a severe case of OP. The MSC isolated from these animals are a unique cell population that we expect may better represent the outcomes of autologous cell therapies for the older patient population in the clinic. In the present study, adipose and bone marrow derived MSC from OVX and age-matched animals were evaluated for their osteogenic and adipogenic differentiation potentials in culture through passage 10. Results from this study suggest that bone marrow derived-MSC maintain their phenotype and functionality more effectively than adipose derived-MSC in OP. Further investigations used regenerative medicine approaches for cell expansion on keratin protein coated microcarriers in static culture. Hair-derived keratin biomaterials have demonstrated their utility as carriers of biologics and drugs for tissue engineering. An optimal microcarrier was selected that demonstrated superior retention of the protein coating through electrostatic interactions and high cell viability. Finally, the integration of cell-microcarriers into a perfusion bioreactor system was explored. Preliminary results demonstrated the feasibility of MSC growth and differentiation on microcarrier based packed beds. Moreover, AD-MSC from OP rats were unresponsive to both inductive media and shear stress related osteogenic cues. These results highlight the complexity and challenges associated with the MSC regenerative strategy.
Changes in Kinetochore Structure and Molecular Composition in Response to Mis-attachment
Shen, Muyao (Virginia Tech, 2011-05-27)
Each mitotic chromosome is constituted by two sister chromatids whose correct segregation to the daughter cells is ensured by amphitelic attachment, in which the two sister kinetochores (KTs) are attached to microtubules (MTs) from opposite mitotic spindle poles. KT mis-attachments can occur in early mitosis and cause chromosome mis-segregation and aneuploidy if not corrected. These mis-attachments include monotelic (one attached and one unattached sister KT), syntelic (both sister KTs attached to the same spindle pole), and merotelic (a single KT attached to MTs from opposite spindle poles) attachments. A biochemical pathway named the Spindle Assembly Checkpoint (SAC) is responsible for delaying anaphase onset to allow correction of KT mis-attachments. SAC activation is believed to occur due to KT localization of certain SAC proteins and/or lack of tension, but only monotelic attachment has been proven to activate the SAC. To determine if and how other KT mis-attachments may activate the SAC, we studied how molecular composition and structure of the KT changes in response to different types of attachments. Our data suggest that monotelic attachment is the only type of attachment that can induce a SAC response thanks to the accumulation of the SAC protein Mad2 at the KT. Our data also indicate that structural changes of the KT, measured as intra- or inter-KT stretching, do not directly induce a SAC response. Instead, our findings suggest decreased KT stretching, especially in inter-KT stretching of syntelic chromosomes, may play a key role in bringing MCAK and other KT substrates closer to Aurora B kinase for rapid and efficient correction of KT mis-attachments.
Characterization of Dendritic Cells in the Bovine Mammary Gland
Maxymiv, Nicolas George (Virginia Tech, 2009-12-03)
Bacterial mastitis is a significant problem for the dairy industry. A vaccine against mastitis pathogens could potentially target dendritic cells (DC). While there has been some research describing bovine DC populations in-vitro, little is known about DC in mammary tissue. In this study, immunohistofluorescence was used to identify and localize bovine mammary DC. DC were found in alveoli, in epithelia, and in interalveolar tissue. Fluorescence-activated cell sorting (FACS) was used to characterize mammary DC as expressing CD11c, MHC-II, CD205, CD11b, and CD8Î±. FACS allowed us to distinguish DC (CD14lo) from macrophages (CD14hi). Two DC subsets, CD11a-, CD11alo, were evident in the mammary gland while an additional CD11ahi population was identified in the supramammary lymph node. After phagocytosis of bacterial components such as lipopolysaccharide (LPS), DC undergo a maturation process, in which they upregulate homing receptors, such as CCR7, and antigen presentation markers, including MHCII and CD80. A primary cell culture model was used to evaluate changes in transcription of CD80 and CCR7 after LPS stimulation. Cell cultures contained digested and Ficoll separated mammary tissue or supramammary lymph node tissue. While the presence of CCR7 and CD80 was confirmed, CD80 and CCR7 transcripts were not upregulated after LPS stimulation. Further, CD11c, CD14, MHCII, CD11b, CD11a, and CD205 protein levels, as assessed by FACS, were similar in LPS stimulated cultures and unstimulated controls. Overall, these studies provide a better understanding of mammary gland immunology, while potentially aiding in the development of novel DC based vaccines.
Characterization of the Expression of Angiogenic Factors in Cutaneous Squamous Cell Carcinoma of Domestic Cats
Gudenschwager-Basso, Erwin Kristobal; Stevenson, Valentina; Sponenberg, D. Phillip; Cecere, Thomas E.; Huckle, William R. (MDPI, 2022-07-21)
Cutaneous squamous cell carcinoma (CSCC) is a common malignant skin cancer with a significant impact on health, and it is important to determine the degree of reliance of CSCC on angiogenesis for growth and metastasis. Major regulators of angiogenesis are the vascular endothelial growth factor (VEGF) family and their associated receptors. Alternative pre-mRNA splicing produces multiple isoforms of VEGF-A and PLGF with distinct biological properties. Several studies highlight the function of VEGF-A in CSCC, but there are no studies of the different isoforms of VEGF-A and PLGF for this neoplasm. We characterized the expression of three isoforms of VEGF-A, two isoforms of PLGF, and their receptors in cat CSCC biopsies compared to normal haired skin (NHS). Although our results revealed no significant changes in transcript levels of panVEGF-A or their isoforms, the mRNA levels of PLGF I and the receptors Flt-1 and KDR were downregulated in CSCC compared to NHS. Differences were observed in ligand:receptor mRNA expression ratio, with the expression of VEGF-A relative to its receptor KDR higher in CSCC, which is consistent with our hypothesis and prior human SCC studies. Immunolocalization in tissue showed increased expression of all measured factors and receptors in tumor cells compared to NHS and surrounding vasculature. We conclude that the factors measured may play a pivotal role in CSCC growth, although further studies are needed to clarify the role of angiogenic factors in feline CSCC.
Characterization of the expression of angiogenic factors in the feline placenta during development and in feline cutaneous squamous cell carcinoma
Gudenschwager Basso, Erwin Kristobal Felipe (Virginia Tech, 2018-11-13)
Throughout gestation, the blood vessel network of the placenta is formed sequentially by processes known as vasculogenesis and angiogenesis, which together meet the needs of the growing fetus. Normal placental angiogenesis is critical to support adequate fetal growth and assure the health of the offspring. Proper angiogenesis requires precise regulation of expression of agents that modulate this process; otherwise, pathologies of pregnancy such as preeclampsia may occur. The placenta is composed of different layers of tissue, including the lamellar (LZ), junctional, and glandular zones, each with a vascular morphology attuned to its function. We hypothesized that higher expression of pro-angiogenic factors is associated with increased morphological metrics in the LZ, the major vascularized zone. Thus, we aimed to characterize the major changes in morphology and vascular development in the placenta throughout pregnancy in cats, alongside a compressive analysis of the expression of major angiogenic factors and their receptors in the placenta, with an emphasis on the identification and interaction of different isoforms of the VEGF family. Microscopic analysis of tissue specimens from different stages of pregnancy revealed increased thickness of the LZ, especially during early to mid-gestation, at which time the tissue is composed of abundant materno-fetal interdigitations that appears rich in capillaries. VEGF proteins were detected in placental tissue in both fetal and maternal cells of the placenta, suggesting stimulatory interactions between different cell types to promote growth and angiogenesis. Gene expression analysis of placenta revealed upregulation of the pro-angiogenic factor VEGF-A in mid-pregnancy, followed by a steady decline toward term, consistent with morphologic changes in the LZ. In contrast, another pro-angiogenic factor, PlGF, showed a marked increase toward term; Flt-1, which acts as a receptor or reservoir for PLGF and VEGF A, was also upregulated at late pregnancy. Increased ratios of PLGF:VEGF-A may contribute to LZ proliferation in the last trimester. These findings are consistent with the creation of a proangiogenic placental state during gestation. Overall, we expect that this research will help elucidate mechanisms of placental vascularization, which can be applied to the design of improved strategies to treat vascular complications of pregnancy. Lastly, we applied the tools developed for placental studies to investigate pathologic angiogenesis in cutaneous squamous cell carcinoma (CSCC), a common skin cancer with major economic and medical impacts in humans and veterinary species. The creation of a new blood supply is essential for growth and metastasis of many tumor types. The goal of this study was to measure expression of variants of proteins that stimulate angiogenesis or transmit an angiogenic stimulus in feline CSCC. The results were mixed, with differences detected in expression of some regulatory agents and, for others, unexpectedly lower expression in CSSC compared to controls. Interestingly, the expression of VEGF-A relative to the protein that transmits its signal (KDR) was elevated in CSCC, suggestive of an altered signaling relationship. This finding supports our hypothesis and is consistent with human SCC studies. Our results encourage further studies on angiogenic factor variants in feline CSCC.
Chronic Hypoxia and Cardiovascular Dysfunction in Sleep Apnea Syndrome
Chittenden, Thomas William (Virginia Tech, 2002-07-24)
The purpose of the current study was to test the hypothesis that chronic hypoxia associated with sleep-disordered breathing relates to abnormal Nitric Oxide (NO) production and vascular endothelial growth factor (VEGF) expression patterns that contribute to aberrancy of specific determinates of cardiovascular and cardiopulmonary function before, during, and after graded exercise. These patterns may further reflect pathologic alteration of signaling within the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt-1) transduction network. To this end, 7 medically diagnosed OSA patients (3 male, 4 female), mean age 48 years and 7 apparently healthy control subjects (3 male, 4 female), mean age 42 years, underwent baseline venous blood draws and maximal bicycle ergometry. Mononuclear cells isolated from peripheral blood were utilized as reporter cells for measurement of VEGF, Akt-1, hypoxia inducible factor-1 alpha (HIF-1 alpha), and vascular endothelial growth factor receptor-2 (VEGFR2) gene expression by redundant oligonucleotide DNA microarray and real-time PCR technologies. Circulating angiogenic progenitor cells expressing VEGFR2 were profiled by flow cytometry. Plasma and serum concentrations of VEGF, nitrates/nitrites, catecholamines, and dopamine were measured by enzyme-linked immunosorbent assay (ELISA) and high performance liquid chromatography (HPLC). Arterial blood pressure, cardiac output, oxygen consumption and total peripheral resistance were determined at Baseline, 100W, and peak ergometric stress by standard techniques. There were no apparent differences (p < .05) observed in biochemical markers relating to vascular function and adaptation including, serum nitrates/nitrites, norepinephrine, dopamine, and plasma VEGF. No differences were found relative to cardiac output, stroke volume, cardiopulmonary or myocardial oxygen consumption, expired ventilation, heart rate, arteriovenous oxygen difference, total peripheral resistance, and mean arterial pressure. Due to methodological issues related to the redundant oligonucleotide DNA microarray and real-time PCR gene expression analyses, results of these experiments were uninterpretable. Thus, the research hypothesis was rejected. Conversely, significant (p < .05) differences were observed in waist: hip ratios, recovery: peak systolic blood pressure ratio at 1 minute post-exercise and %VEGFR2 expression. OSA was associated with elevations in both waist: hip ratios and recovery: peak systolic blood pressure ratio at 1 minute post-exercise as well as significant depression of %VEGFR2 profiles. Moreover, significant negative correlations were found regarding waist: hip ratios and %VEGFR2 expression (r = -.69;p =.005) and recovery: peak systolic blood pressure ratio at 1 minute post-exercise and %VEGFR2 expression (r = -.65;p =.01). These findings did not provide evidence that NO-dependent vasoactive mechanisms are suppressed nor did they support the supposition that angiogenic mechanisms are pathologically activated in sleep-disordered breathing.
Circadian Control of Cell Cycle Progression
Santos, Carlo Steven (Virginia Tech, 2009-03-31)
Tumorigenesis is the result of uncontrolled cell growth due to the deregulation of cell cycle checkpoints 1. Period 2 (Per2) is a tumor suppressor that oscillate in expression in a 24-hour cycle 2, 3. Here, we show that Per2 interacts with the tumor suppressor protein p53. Both G1 and G2 checkpoint pathways involve a p53 dependent pathway which can trigger the cell to go through cell arrest or programmed cell death4. Understanding all the mitigating factors involved in regulating cell cycle progression under DNA damage can offer a better idea in how cells become immortal. Initially discovered through screening of a human liver cDNA library, the novel interaction between p53-Per2 was further documented using co-precipitation. Interestingly, under genotoxic stress conditions, p53 and Per2 were not found to bind which leads us to suspect that Per2 does not affect active p53 which may possibly be due to post translational modifications of its active state. Furthermore we investigated p53's ability to act as a transcription factor in the presence of Per2, showing that the Per2-p53 complex prevents p53 from binding to DNA. This implies that the tetramerization of p53 may also be another factor in Per2's ability to bind to p53. A truncated p53 lacking the last 30 amino acids that theoretically increase p53's ability to form a tetramer showed a drastic reduction in binding to Per2 5, 6. On the other hand, p53 lacking the tetramerization domain showed binding similar to wildtype. Consequently we speculate that the ability of Per2 to modulate p53 and act as a tumor suppressor protein may be dependent on either the post translational modifications of p53 or its oligomeric state.
Circadian modulation of the estrogen receptor alpha transcription
Villa, Linda Monique (Virginia Tech, 2012-07-12)
The circadian clock is a molecular mechanism that synchronizes physiological changes with environmental variations. Disruption of the circadian clock has been linked to increased risk in diseases and a number of disorders (e.g. jet lag, insomnia, and cancer). Period 2 (Per2), a circadian protein, is at the center of the clock's function. The loss or deregulation of per2 has been shown to be common in several types of cancer including breast and ovarian [1, 2]. Epidemiological studies established a correlation between circadian disruption and the development of estrogen dependent tumors. The expression of estrogen receptor alpha (ERα) mRNA oscillates in a 24-hour period and, unlike Per2, ERα peaks during the light phase of the day. Because up regulation of ERα relates to tumor development, defining the mechanisms of ERα expression will contribute to our comprehension of cellular proliferation and regulation of normal developmental processes. The overall goal of this project is to investigate the molecular basis for circadian control of ERα transcription. Transcriptional activation of ERα was measured using a reporter system in Chinese hamster ovary (CHO) cell lines. Data show that Per2 influences ERα transcription through a non-canonical mechanism independent of its circadian counterparts. Breast cancer susceptibility protein 1 (BRCA1) was confirmed to be an interactor of Per2 via bacterial two-hybrid assays, in accordance with previous studies [2]. BRCA1 is a transcriptional activator of ERα promoter in the presence of octamer transcription factor-1 (OCT-1) [3]. Our results indicate that the DNA binding domain of OCT-1, POU, to directly interact with Per2 and BRCA1, in vitro. Pull-down assays were used to map direct interaction of various Per2 and BRCA1 recombinant proteins and POU. Chromatin immunoprecipitation assays confirmed the recruitment of PER2 and BRCA1 to the estrogen promoter by OCT-1 and the recruitment of Per2 to the ERα promoter decreases ERα mRNA expression levels in MCF-7 cells. Our work supports a circadian regulation of ERα through the repression of esr1 by Per2 in MCF-7 cells.
The Clinical Pharmacology of Acetaminophen in Adult Horses
Mercer, Melissa Ann (Virginia Tech, 2022-08-18)
Non-steroidal anti-inflammatory drugs (NSAIDs) are a mainstay of the management of pain and inflammation associated with musculoskeletal disorders and systemic inflammation in horses. The most utilized NSAIDs in equine practice are non-selective cyclooxygenase (COX) inhibitors, such as flunixin meglumine and phenylbutazone, which act through global inhibition of prostaglandin synthesis and release. While non-selective COX inhibitors are effective as anti-inflammatory agents, they are mired with complications with prolonged or high-dose use, particularly in critically ill patients. Therefore, non-selective COX-inhibitors have been displaced by selective COX-2 inhibitors for many practitioners due to the perceived reduced risk of gastrointestinal complications. It should be noted, however, that the use of COX-2 selective inhibitors in horses is not without risk. Due to the potential for significant adverse events in horses with critical illness treated with traditional NSAIDs, there is clinical need for safe, and effective anti-inflammatories and anti-pyretics for administration in these patients. The studies presented in this dissertation explore the pharmacokinetics, efficacy, and safety of acetaminophen in adult horses for use in musculoskeletal pain and pyrexia. In the first study, the pharmacokinetics and efficacy of oral acetaminophen at two different doses (20 mg/kg and 30 mg/kg) were examined in an experimentally induced lameness model and the analgesic efficacy of acetaminophen was compared to placebo and the non-selective COX inhibitor phenylbutazone. Acetaminophen when administered at 30 mg/kg produced a more rapid onset of greater improvement in subjective lameness scores and heart rate compared to other treatments in this model, and therefore would be more suitable as a monotherapy than acetaminophen dosed at 20 mg/kg. Acetaminophen dosed at 30 mg/kg resulted in a more rapid improvement in lameness scores than phenylbutazone at 2.2 mg/kg and was equivalent to phenylbutazone in lameness score reduction. However, results of this study necessitated further evaluation of the pharmacokinetics and safety of repeated oral dosing of acetaminophen at 30 mg/kg orally every 12 hours to determine clinical utility. In the second study, the pharmacokinetics, efficacy, and safety of oral acetaminophen (30 mg/kg) were examined in adult horses with naturally occurring chronic lameness. In that study, following 21 days of twice daily oral dosing at 30 mg/kg, acetaminophen was found to be safe with no evidence of gastric ulceration or hepatopathy in horses. Acetaminophen at 30 mg/kg twice daily for 21 days provided transient improvement in subjective and objective lameness evaluation when compared to baseline evaluation; however, the study concluded that acetaminophen may not be suitable as a monotherapy for management of moderate to severe orthopedic pain in horses In the third study, the pharmacokinetics and efficacy of oral acetaminophen (30 mg/kg) was examined in adult horses with experimentally induced endotoxemia when compared to placebo and the nonselective COX inhibitor flunixin meglumine. That study found that acetaminophen was superior to placebo and not statistically different from flunixin meglumine in reducing rectal temperature in adult horses with experimentally induced endotoxemia and may be an option for antipyresis in clinical cases, particularly when administration of traditional NSAIDs is contraindicated. Furthermore, acetaminophen administered at 30 mg/kg orally to adult horses with experimentally induced endotoxemia is an effective antipyretic but is unlikely to provide any alteration in systemic inflammatory response.

Browsing by Author "Huckle, William R."

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