Browsing by Author "Akers, Robert Michael"

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    Ability of Klebsiella spp. mastitis isolates to produce virulence factors for enhanced evasion of bovine innate immune defenses
    Nedrow, Alicia (Virginia Tech, 2009-11-24)
    Klebsiella spp. are coliform bacteria that cause mastitis in dairy cattle and account for high mortality rates in infected cows leading to a significant financial loss. Recent outbreaks indicate that within farms a single strain can be responsible for clinical signs in multiple animals. Identification of the virulence of factors enabling Klebsiella spp. survival in the mammary glands of multiple animals may provide insight into host adaptation. In this study, Klebsiella spp. strains were evaluated for their ability to evade neutrophil killing, the primary immune defense in the bovine mammary gland. Our research focused on capsule and biofilm production by Klebsiella spp. when strains were grown in Luria Broth or skim milk to examine the effects on evasion of neutrophil killing. Biofilm production was not significantly related to the ability to resist neutrophil killing nor was capsule (P = 0.29). Farm (P < 0.001), media type (P < 0.005), and strain type by cow (P < 0.001) were found to play significant roles in neutrophil evasion. This suggests farm of origin, media type used, and cow all may play a role in evasion of neutrophils by Klebsiella spp. Further evaluation of virulence factor expression in different media types and the role of individual cow immune responses may provide insight into ability of Klebsiella spp. to cause outbreaks of mastitis in multiple animals.
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    Abundance and Localization of  (Yes-associated protein) YAP in Prepubertal Bovine Mammary Tissue
    Granger, Paulnisha Davida (Virginia Tech, 2018-07-09)
    Most mammary development is postnatal. Mammary growth that occurs before puberty is diminutive in amount but consequential for future milk production, especially in dairy heifers. With advanced knowledge on fundamental aspects that govern prepubertal mammary development, scientists and farmers alike can ensure that heifers perform their best once they become cows. The Hippo pathway has been identified as an evolutionarily conserved pathway that regulates organ size in many animal species; it might contribute to mammary growth in dairy heifers. This pathway is mediated by yes-associated protein (YAP) and through downstream gene transcription activation, results in cell proliferation. Because YAP has never been identified in bovine mammary tissue, questions examined in this body of work mainly focused on the abundance and localization of YAP in mammary tissue of prepubertal heifers. The first trial investigated effects of in vivo estradiol administration on YAP abundance and localization in prepubertal bovine mammary epithelial and myoepithelial cells. While YAP was present in nuclei and cytoplasm of both cell types, it was also discovered that estrogen did not influence YAP abundance or location. The second research trial focused on determining the effects of in vivo estradiol blockade on YAP abundance and localization in prepubertal bovine mammary epithelial and myoepithelial cells. Similar to the first experiment, results indicate that YAP abundance and localization was not influenced by estrogen blockade. Despite not being responsive to in vivo estradiol administration (experiment 1) or estradiol blockade (experiment 2) under the conditions of our experiments, YAP was present in nearly all mammary epithelial cells and myoepithelial cells of the 21 total prepubertal heifers examined. Its presence hints at an underlying biological function but that function was not ascertained here. It will be up to the next researcher to deduce what YAP contributes to mammary growth in prepubertal dairy heifers.
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    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.
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    Alterations in Lipid Metabolism in Mouse Tissues and Hepatic Cell Lines in Response to the Trans10,Cis12-18:2 Isomer of Conjugated Linoleic Acid
    Viswanadha, Srikant (Virginia Tech, 2003-07-15)
    Conjugated linoleic acid (CLA) reduces adipose mass in several species. Studies were conducted to determine: 1) the effect of dietary trans10,cis12-CLA on growth, tissue fatty acid profile, mRNA expression for stearoyl-CoA desaturase (SCD) in adipose and liver, and mRNA expression for fatty acid synthase (FAS) in adipose of mice, 2) the effect of a dietary combination of trans-vaccenic acid (TVA) and trans10,cis12-CLA on delta9- desaturation, and 3) the effect of cis9,trans11-CLA, trans10,cis12-CLA, and carnitine palmitoyltransferase-1 (CPT-1) inhibitors on expression of mRNA for CPT-1 and fatty acid profile in mouse hepatocytes (AML-12) and human hepatoma cells (HepG2). In the first study, male or female mice were fed diets containing 0, 0.15%, or 0.30% trans10,cis12-CLA for 6 wk. Epididymal adipose weights (males) and inguinal adipose weights (females) decreased by 81% and 52%, respectively, in response to 0.30% trans10,cis12-CLA. Dry carcass weights decreased from 4.75 g for the control to 3.62 g for mice fed 0.30% trans10,cis12-CLA and the decrease was due to a reduction in ether extract. Liver weights increased linearly from 0.55 g (control) to 0.65 g (0.30% trans10,cis12-CLA). Dietary trans10,cis12-CLA (0.30%) reduced FAS and SCD mRNA in adipose by 60 and 30 % respectively, compared with the control, suggesting reduced lipogenesis and desaturation might be primary factors responsible for reducing body fat. In the second study, adult male or female mice were fed diets containing 0.40% TVA in combination with 0, 0.15, or 0.30% trans10,cis12-CLA for 10 d. Both TVA and trans10,cis12-CLA were incorporated into plasma, liver, adipose, muscle, and bone lipids proportional to their concentrations in the diets. Desaturation ratios were not affected in adipose, liver, and bone. However, ratios of 16:0 to 16:1 and 18:0 to 18:1 increased from 0.81 to 0.86 and 0.15 to 0.19 respectively, in response to dietary trans10,cis12-CLA (0.30%), suggesting inhibition of delta9 desaturation in muscle. In the third study, AML-12 or HepG2 cells were incubated with control media or media containing 15 uM etomoxir (ETM), 30 uM ETM, 15 uM hemipalmitoylcarnitinium (HPC), 30 uM HPC, 100 uM cis9,trans11-CLA, or 100 uM trans10,cis12-CLA for 24 h. Half the cells were harvested for analysis of fatty acids, mRNA for CPT-1, and cholesterol after 24 h. The remaining cells were incubated for an additional 24 h in control medium. Incorporation (% of total fatty acids) of trans10,cis12-CLA was greater than cis9,trans11-CLA in AML-12 (34 vs 23.6) and HepG2 (28 vs 18) cells. Cells incubated with trans10,cis12-CLA had higher ratios of 16:0 to 16:1, 18:0 to 18:1, and 18:2n6 to 20:4n-6, suggesting inhibition of delta9, delta5 , and delta6 desaturation. Cis9,trans11-CLA also reduced ratio of 18:2n-6 to 20:4n-6 in both cell lines. Trans10,cis12-CLA increased mRNA for CPT-1 in both cell lines compared with the control, suggesting enhanced oxidation of fatty acids. In addition, trans10,cis12-CLA caused a 4-fold and 5-fold increase in free cholesterol content of AML-12 and HepG2 cells, respectively. Overall, results demonstrated that trans10,cis12-CLA modulated lipid metabolism in tissues in vivo and altered fatty acid metabolism, cholesterol synthesis, and CPT-1 mRNA in hepatic cell lines in vitro.
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    Artificial Induction of Lactation in Nonbreeder Dairy Cows
    Jewell, Tracy Michelle (Virginia Tech, 2002-08-09)
    Thirty-four cows (26 Holsteins and 8 Jerseys) were subjected to an estrous synchronization protocol administering 2 PGF2Æ Ã injections 11 d apart prior to beginning the lactation-induction protocol. Artificial induction of lactation yielded a 92% success rate for Holstein cows with success defined as achieving >9 kg milk/d, and a 88% success rate for Jersey cows with success defined as achieving > 5 kg milk/d. Mean accumulated milk yield for induced cows at 150 DIM was 65% of mean yield for nontreated cows. Mean peak milk yield for lactation- induced Holsteins and Jerseys was 32 kg/d and 20 kg/d, respectively. Mean serum and milk progesterone concentrations for samples collected during the first 6 d of lactation were not different between lactation-induced and nontreated cows. However, mean serum estradiol concentrations for induced cows were higher (P <0.05) in samples collected 3 and 5 DIM. Lactation-induced cows exhibited an increase in serum alpha-lactalbumin concentrations 2 d prior to initiation of milking, reaching values of ~260 ng/ml. Mean days-to-first service was greatly reduced in cows induced into lactation compared to nontreated cows, while mean services per conception was similar between induced and nontreated cows. Mean days to conception was lower for induced cows than for nontreated cows. By 150 DIM, pregnancy rate of induced cows was 70%, whereas nontreated cows averaged 56% pregnancy rate.
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    Aspects of lactation endocrinology: I. lactogenic receptors in bovine mammary tissue at different stages of lactation: II. growth hormone concentrations in Holstein cattle of differing genetic merit
    Kazmer, Gary Wayne (Virginia Polytechnic Institute and State University, 1985)
    Mammary tissue from nine Holstein cows was collected within one week of parturition, at 60 and 180 days postpartum. Blood samples were collected at 6-hr intervals from two days prior to . until two days after surgery. A membrane-enriched fraction of tissue homogenates was prepared by differential centrifugation. Displacement curve data was analyzed by a microcomputer program. Mean prolactin (Prl) during the periparturient period was greater than either postpartum period, but not prior to biopsy. Dissociation constants (Kd) estimated with NIH-bPRL-6 as competitor were not different among stages of lactation, and averaged 8.97 x 10⁻⁸M. Receptor concentrations were less during _the periparturient period than later lactation. The Kd was 100-fold greater when estimated with human growth hormone as competitor. It is concluded that lactogenic hormone receptor concentrations in bovine mammary tissue increase with the onset of lactation, following a pattern similar to that observed in non-ruminants. Three experiments were conducted to investigate endocrine metabolic hormone profiles in Holstein cattle of differing genetic merit at several ages. Control animals were randomly bred to non-AI sires originating in the Virginia Tech Dairy herd. Selected animals were offspring of commercially available AI sires. In one experiment, mean plasma Prl was greater in control animals after feeding and insulin injection, while growth hormone (GH) was greater in selected animals at all ages. Free fatty acids were greater in selected animals at 6 and 24 months of age, while glucose (Glc) and urea were unaffected by genetic merit. In a second experiment, Holstein bull calves were administered Glc and thyrotropin releasing hormone (TRH) on different days. Plasma GH was greater in selected animals. Plasma Prl was greater in control animals after TRH. In the third experiment, Holstein cows received TRH at 30, 90 and 200 days postpartum (DPP). Net energy balance was negative at 30, while positive at 90 and 200 DPP. Plasma GH before and after TRH was greater in selected animals, and greater during early than later lactation. Thus, the results of the three experiments indicate that increased plasma GH may be associated with selection for increased milk yield.
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    Assessment of Murine Embryo Development Following Electroporation and Microinjection of a Green Fluorescent Protein DNA Construct
    Schmotzer, Carolyn Anne (Virginia Tech, 2001-07-31)
    Transgenic techniques have rapidly evolved in recent years. However, the efficiency of these techniques to produce viable offspring is still disappointingly low. The purpose of this study was to assess in vitro development, transgene expression, and integration following pronuclear or cytoplasmic microinjection of condensed or linear green fluorescent protein DNA into murine embryos using electroporation. In experiment 1, the effect of embryo orientation (group or linear) within the electroporation chamber on development was evaluated using zygotes which received one pulse duration (10 msec), and one of two voltages (250 or 400 V). Zygotes that received 400 V had the lowest development score (Group, 2.06 ? 0.12; Linear, 1.97 ? 0.13), irrespective of orientation. Embryos that received 250 V had the highest development of the voltage treated groups (Group 3.42 ? 0.12; Linear 3.32 ? 0.12), irrespective of orientation, and development was lower than the control embryos (Control 4.28 ? 0.12; Mannitol control 4.36 ? 0.18). In experiment 2, the efficiency of utilization of the prepared enhanced green fluorescent protein (EGFP) construct as a visual marker of protein expression was evaluated using pronuclear microinjection. Embryo development and fluorescence were evaluated following pronuclear injection of EGFP at a concentration of 3 μg/ml and compared to an uninjected control. Embryos injected with the EGFP had lower development scores (3.85 ± 0.15) than uninjected control embryos (5.72 ± 0.2). Of the embryos injected, 32.4% fluoresced due to expression of EGFP. Experiment 3 evaluated the effect of combining cytoplasmic injection of EGFP (425 μg/ml) with electroporation at 250 V on EGFP expression. The non-manipulated control embryos had significantly higher (P < 0.01) 4 d development scores (5.57 ± 0.11) than manipulated control embryos (4.6 ± 0.18), where the injection needle was inserted into the cytoplasm and no DNA was injected. Combining cytoplasmic DNA injection and electroporation caused a significant (P < 0.01) decrease in development scores, irrespective of DNA construct, when compared to embryos injected with a DNA construct alone. The mechanical effects of needle insertion combined with electroporation were not significantly different (P > 0.05) from embryos injected with DNA alone, irrespective of construct injected. Cytoplasmic injection of condensed DNA (0.38%), linear DNA (0.38%), and condensed DNA combined with electroporation (0.36%) resulted in one fluorescent embryo respectively. Cytoplasmic injection of linear DNA when combined with electroporation (3.57%) resulted in 13 fluorescent embryos. Pronuclear injection of the prepared EGFP construct results in lower development than control embryos. Electrical stimulation of zygotes reduces early embryo development. However, low amounts of electrical stimulation may allow for enhancement of gene integration in transgenic embryos.
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    Autocrine mechanisms of action of insulin-like growth factor-I (IGF-I) and hormonal regulation of expression of IGF-finding proteins in mammary epithelial cells
    Romagnolo, Donato (Virginia Tech, 1993)
    Limited information is available concerning the molecular and cellular mechanisms that regulate expression of insulin-like growth factor-I (IGF-I) and IGF-binding proteins (IGFBPs) genes in mammary epithelial cells. To test the hypothesis that IGF-I affects growth of bovine mammary epithelial cells through an autocrine and/or paracrine pathway, several cell lines were developed expressing an ovine exon-2 containing IGF-I cDNA under the control of the mouse mammary tumor virus-long terminal repeat (pMMTV-IGF-I), early simian virus (pSV40-IGF-I), and herpes simplex thymidine kinase (pTK-IGF-I) promoters. Stably transfected clones were generated by cotransfection of clonal MAC-T cells with the IGF-I expression vectors and a plasmid conferring resistance to hygromycin-B (HYG-B), using a calcium phosphate precipitation procedure. Induction of the MMTV-LTR with the glucocorticoid dexamethasone (DEX) was required for enhanced expression of IGF-I in MD-IGF-I (MD=Mammary Derived) cells, whereas SV40-IGF-I cells constitutively expressed the highest levels of IGF-I, followed by TK-IGF-I cells. Activity of the MMTV promoter in MD-IGF-I cells was coordinately regulated by lactogenic hormones and extracellular matrix. Acute secretion of DEX-induced recombinant IGF-I by MD-IGF-I cells stimulated cell proliferation through an autocrine/paracrine pathway and triggered the expression of IGFBP-3. Neither acute nor constitutive expression of IGF-I affected expression of type 1 IGF receptor mRNAs, but down-regulated cell surface receptor levels, in the order SV40-> TK- > MD-IGF-I. Secretion of IGF-I-induced IGFBP-3 potentiated the mitogenic actions of IGF-I as evidenced by enhancement of [³H]thymidine uptake into DNA of parental MAC-T cells. This study provides evidence that local production of IGF-I can stimulate cell proliferation of bovine mammary epithelial cells through an autocrine/paracrine mode of action. We suggest that secretion of IGF-I-induced IGFBP-3 by bovine mammary epithelial cells enhances cell responsiveness to IGF-I, but does not prevent down-regulation of the IGF-I receptor in cells constitutively expressing IGF-I.
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    Calcium: some aspects of subcellular accumulation and distribution in milk
    Shappell, Nancy W. (Virginia Polytechnic Institute and State University, 1988)
    Distribution and bioavailability of ⁴⁷calcium in milk labeled by extrinsic and intrinsic methods was investigated. Milk from Sprague Dawley rats was labeled by both methods, and milk from a cow was labeled by the extrinsic method. Retention of ⁴⁷Ca from milks administered to young male Sprague Dawley rats was determined through whole body counting for 6 days after administration of milk. Percent of ⁴⁷Ca dose retained was 72% for extrinsically labeled cow milk, 62% for extrinsically labeled rat milk, and 55% for intrinsically labeled rat milk. Samples were fractionated by ultracentrifugation and by gel exclusion chromatography. ⁴⁷Calcium distributions in rat milk labeled intrinsically or extrinsically were similar. The majority of ⁴⁷Ca was found in a particulate, > 30,000 molecular weight fraction (about 60% for cow milk, about 90% rat milks). The amount of milk calcium retained by rats appeared to be related to the amount of noncasein micelle-associated calcium. When administered by intraperitoneal injection into rats, ⁴⁵Ca specific activity of milk peaked in 60 to 90 minutes. Specific activity was highest in cytosol, and lower in Golgi apparatus and rough endoplasmic reticulum. Specific activities in subcellular fractions changed in parallel with specific activities of milk. Rapid turnover of Ca was observed in endoplasmic reticulum and Golgi apparatus; this was expected since secretory proteins and associated Ca are transported through these organelles for secretion. In vitro ⁴⁵Ca accumulation was compared in Golgi apparatus and endoplasmic reticulum from liver and mammary gland of lactating Dunkin Hartley guinea pigs. In the presence of ATP, highest accumulation per unit total fraction protein was found in Golgi apparatus (mammary gland 28% of available ⁴⁵Ca, liver 11%) while 8% was accumulated by endoplasmic reticulum fractions. Calcium accumulation was not the result of binding, as preincubation of vesicles with calcium ionophore resulted in less than 10% of the accumulation found without ionophore. The ATPase inhibitor sodium orthovanadate, and the ATP analog AMP-PNP, reduced ⁴⁵Ca accumulation in all fractions. Protonophore caused a small reduction in ⁴⁵Ca accumulation in all cases. Citrate accumulation by fractions was not observed under conditions used for ⁴⁵Ca accumulation.
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    Career Values and Perceptions of Agricultural Careers of Gifted and Talented Students in the Virginia Governor's School for Agriculture
    Overbay, Andrew Edward (Virginia Tech, 2006-10-18)
    Career choice is governed by what individuals value and their perception of the realities that exist in a given field. Agriculture career education of gifted and talented students, therefore, must begin with an assessment of the values of the students, their assumptions regarding fields within the agriculture industry, and factors that influence their career decisions. This descriptive study summarized values and perceptions held by participants in the 2006 Virginia Governor's School for Agriculture (VGSA). Originally, the VGSA hosted 98 students; one student withdrew from the program. The results of the study confirmed that there is still much controversy and misunderstanding about agriculture and careers in the agriculture arena. The testing process included a survey of career values called the Values Scale. This instrument was developed by Dorothy Nevill and Donald Super and last updated in 1989. The 106-question survey measured 21 personal career values of participants. Follow-up data were collected gauging the students' thoughts on agriculture careers, agriculture companies, their individual career goals, and the influences that shaped their career decisions. The career values of the VGSA Class of 2006 were surprisingly similar to high school student data collected in 1989. There were slight decreases in the value placed on economic rewards and security, but many of the other values mirrored past national data. Most students (n=73) were able to name five agriculture careers with "farmer" garnering most of the responses; however, 29 students did not name a single agriculture company. A majority of the students (n=56) stated that they had made a career decision; however, most of these (n=32) also stated their career was not in the field of agriculture. Half of those having a career goal made their decision prior to their sophomore year in high school. Parents were named by the students as the greatest single influence on career decision among ten choices. School experiences, work experiences, and people who work in the field were also high among influences. Suggestions for further research include identifying effective methods of agricultural career exploration within VGSA and value comparisons between gifted students and the general student population.
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    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 CD8α. 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.
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    Characterization of estrogen and glucocorticoid receptors, skeletal muscle protein turnover and tissue growth in lambs treated with trenbolone acetate and estradiol
    Frey, Randall Scott (Virginia Tech, 1988-02-15)
    A study was conducted to determine the effects of trenbolone acetate (TBA) and estradiol-17B (E2) implantation on the characteristics of the glucocorticoid and E2 receptor, skeletal muscle protein turnover and tissue growth. Twenty-four lambs were utilized. Trenbolone acetate did not ,affect (P>.10) degradation rates in the semitendinosus (ST) and triceps brachii (TB) muscles, the production of cortisol, adrenal weights and cytosolic glucocorticoid binding capacity (Bmax). Trenbolone acetate decreased synthesis rate of muscle protein (P<.Ol), the percent of [3H] dexamethasone binding in the nuclear fraction, Bmax and the disociation constant (Kd) of the cytosolic E2 receptor, only in the TB muscle. Deoxyribonucleic acid (DNA) of the TB was increased (P<.05) with TBA. Pituitary weights were decreased (P<.005) with TBA and increased (P<.Ol) with E2. Estradiol decreased (P<.05) Bmax of the cytosolic E2 receptor in the ST and decreased (P<.05) Bmax of the nuclear E2 receptor in the TB muscle. The TB muscle had greater (P<.05) synthesis rates than the ST and the protein:RNA ratio was decreased (P<.05) in the TB. The TB muscle had greater (P<.005) Bmax for the cytosolic glucocorticoid receptor.
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    Characterization of Insulin-like Growth Factor Binding Protein-3 (IGFBP-3) interaction with the Bovine Aortic Endothelial (BAE) cell surface: Examination of the Role of Heparan Sulfate Proteoglycans (HSPG)
    Parghi, Nirav (Virginia Tech, 1998-07-15)
    Insulin-like growth factor binding proteins (IGFBPs) are known to be important modulators of the insulin-like growth factor (IGF-I). However, their precise role is as yet unclear. Further, recent studies have indicated that IGFBP-3 has a receptor mediated growth inhibitory response of its own. In the present study, we quantified the binding characteristics of IGFBP-3 to bovine aortic endothelial (BAE) cells. Binding studies at 4 oC were conducted and a specific binding curve for IGFBP-3 was obtained. IGFBP-3 was found to bind with an equilibrium dissociation constant (KD) value of 3.1 x 10-10 M. The role of heparan sulfate proteoglycans (HSPG) in the IGFBP-3 binding mechanism was also examined. It was seen that inactivation of the cell surface HSPGs with 75 mM sodium chlorate did not affect IGFBP-3 binding. Further, there have been reports of inhibition of IGFBP-3 binding by heparin in the media. Hence, the most probable interaction of HSPG with IGFBP-3 occurs in the extracellular region, with soluble HSPGs acting as receptors for IGFBP-3 and decreasing the net cell associated ligand receptor interaction. This is likely, since IGFBP-3 is known to possess a heparin binding domain. Simultaneous introduction of IGF-I and IGFBP-3 into the extracellular media decreased IGFBP-3 binding to the cell surface, which might imply that IGF-I and IGFBP-3 regulate each other's action.
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    Chondrocyte Regulation by IL-I and IGF-I: Interconnection Between Anabolic and Catabolic Factors
    Porter, Ryan Michael (Virginia Tech, 2005-10-14)
    Articular cartilage functions to reduce the mechanical stresses associated with diarthrodial joint movement, protecting these joints over a lifetime of use. Tissue function is maintained through the balance between synthesis and resorption (i.e., metabolism) of extracellular matrix (ECM) by articular chondrocytes (ACs). Two important hormonal regulators of cartilage metabolism are interleukin-1 (IL-1) and insulin-like growth factor-I (IGF-I). These factors have antagonistic effects on chondrocyte activity, and during the progression of osteoarthritis, IL-1 is thought to promote chondrocyte hyporesponsiveness to IGF-I. To better understand how the anabolic (IGF-I) and catabolic (IL-1) stimuli are linked within articular cartilage, we examined the mechanisms by which IL-1 regulates the IGF-I signaling system of ACs. Equine chondrocytes from non-arthritic stifle joints were multiplied over serial passages, re-differentiated in alginate beads, and stimulated with recombinant equine IL-1β. Chondrocytes were assayed for type I IGF receptor (IGF-IR), IGF binding proteins (IGFBPs), and endogenously-secreted IGF-I. Our experimental findings solidify the significance of IL-1 as a key regulator of IGF-I signaling within articular cartilage, demonstrating that regulation of the IGF-I system occurs through both direct (transcription) and indirect (proteolysis) mechanisms. These results have implications for molecular therapies (e.g., gene transfer) directed at reversing osteoarthritic cartilage deterioration. The presented research concerns not only cartilage biology but also tissue engineering strategies for cartilage repair. Alginate hydrogel culture has been reported to re-establish chondrocytic phenotype following monolayer expansion, but studies have not addressed effects on the signaling systems responsible for chondrocyte metabolism. We investigated whether chondrocyte culture history influences the IGF-I system and its regulation by IL-1. ACs expanded by serial passaging were either encapsulated in alginate beads or maintained on tissue culture plastic (TCP). Bead and TCP cells were plated at high-density, stimulated with IL-1β, and assayed for expression of IGF-I signaling mediators. Intermediate alginate culture yielded disparate basal levels of IGF-IR and IGFBP-2, which were attributed to differential transcription. The distinct mediator profiles coincided with varied effects of exogenous IL-1β and IGF-I on collagen Ia1 expression and cell growth rate. This study demonstrates that culture strategy impacts the IGF-I system of ACs, likely impacting their capability to mediate cartilage repair.
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    Chronic Shear Stress Effects on Endothelial Cell Response
    Elhadj, Selim (Virginia Tech, 2001-12-10)
    The overall focus of this dissertation is on how chronic shear stress alters the synthesis and secretion of important regulatory molecules by endothelial cells. Our hypothesis was that inclusion of chronic pulsatile shear stress in our model would lead to changes in endothelial cell release of regulatory molecules. We distinguished between high arterial shear stresses and low venous shear stresses and used static cell cultures as reference. The first part of this research thus entailed the complete characterization of the flow dynamics in our experimental biomechanical model. Cell stretching can have a physiological effect on endothelial cells; hence we implemented a laser based optical technique for real time strain measurement of the growth fibers used in our culture system, and found that no significant strains were occurring during shear treatment. After characterization of the mechanical environment of the cells, we focused the scope of our research on metabolism of proteoglycans and insulin-like growth factor-I (IGF-I) and related IGF binding proteins (IGFBPs) in bovine aortic endothelial cells cultured under chronic pulsatile shear. We found that shear stress increased the release of proteoglycans and significantly altered proteoglycans distribution. We also found that there was an inverse relationship between the shear level treatment used to obtain the purified proteoglycans from endothelial cells and their potency in inhibiting coagulation. IGF-I release and message (IGF-I mRNA) was decreased at high shear stress compared to low shear stress. Further, the levels found under shear were significantly greater than those observed in the static cell culture model. IGFBPs released were also significantly increased by shear. This research thus establishes a link between chronic pulsatile shear stress and the metabolism of both primary (IGF-I) and secondary (IGFBPs, proteoglycans) regulators of vascular cell activity. The improved realism of our experimental biomechanical model has proved to be a valuable tool in improving the relevance of this study to vascular research. Ultimately, this research calls for further investigation in the molecular mechanisms underlying the phenomenological effects documented, which may help in understanding fundamental aspects in cardiovascular disease and its link to hemodynamics but our work is an important first step.
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    Comparisons of Holstein, Brown Swiss, and Jersey cows for age at first calving, first calving interval, and true herd-life up to five years in seven regions of the United States
    Garcia-Peniche, Teresa Beatriz (Virginia Tech, 2004-12-10)
    The objectives of this research were to evaluate breed differences for heat-stress resistance using age at first calving and first calving interval, and to assess breed by region interactions for seven regions of the United States for survival-related traits up to five years of age in Brown Swiss, Holstein, and Jersey cows. Age at first calving and first calving interval were studied in farms with two breeds, with Holstein and Brown Swiss or Holstein and Jersey cows. The survival-related traits were analyzed in farms with one or two breeds. Seven regions within the United States were defined: Northeast, Northwest, Central north, Central, Central south, Southwest and Southeast. The fertility traits were also analyzed in seven individual states: Wisconsin, Ohio, Oregon, California, Arizona, Florida, and Texas. Brown Swiss were older than Holsteins at first calving (833 ± 2.4 d vs. 806 ± 2.0 d in regions, and 830 ± 3.1 d vs. 803 ± 2.4 d in states), but Holsteins and Brown Swiss did not differ for first calving interval. Jerseys were younger than Holsteins at first calving and had shorter first calving intervals (P < 0.01). In data from individual states, Holsteins housed with Brown Swiss were older at first calving than Holsteins housed with Jerseys (800 ± 2.7 d vs. 780 ± 2.5 d). Holsteins housed with Jerseys had slightly shorter first calving intervals than Holsteins housed with Brown Swiss, and the interaction of "type of Holstein: with season of the first calving was highly significant (P < 0.01). Region and season effects were smaller for Jerseys than for Holsteins, thus, Jerseys showed evidence of heat-stress resistance with respect to Holsteins. Management modified age at first calving in Holsteins, depending on the type of herd they were located in. Longer calving intervals might have been partly due to voluntary waiting period to breed the cows. The survival-related traits were evaluated up to five years of age. They consisted of stayability, number of completed lactations, days lived, herd-life, and total days in milk. For herds with one breed, the order for stayability to five years of age, from longer to shorter-lived breed was: Brown Swiss, Jersey and Holstein, but for the ratio of days in milk to herd-life the order was: Holstein, Jersey and Brown Swiss, and for the ratio of days in milk to days lived, it was: Jersey, and Holstein and Brown Swiss tied. This last ordering was the same for number of lactations completed by five years of age. The results for two-breed herds were similar since Brown Swiss and Jerseys had larger (Chi-square P < 0.01) probabilities of living past five years of age than Holsteins, and for days in milk and number of lactations completed, Jerseys had higher values than Holsteins (P < 0.01), but Holsteins and Brown Swiss tied in some analyses. Breed by region interaction was always significant. If all other conditions were assumed equal, Jerseys would give fastest returns by five years of age. The overall conclusion is that Jerseys performed better for the traits analyzed, all of them highly influenced by environmental conditions.
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    Dietary vitamin B6 supplementation promotes the growth of 7,12-dimethylbenz(a)anthracene-induced mammary carcinoma in Sprague Dawley rats
    Hobbs, Lisa M. (Virginia Tech, 2001-07-20)
    In vitro data from our laboratory demonstrate that vitamin B6 (B6) supplementation of estrogen receptor - positive and - negative breast cancer cells is growth inhibitory. Others have reported that dietary B6 supplementation resulted in increased fibrosarcoma pyridoxal phosphate (PLP) concentrations and a significant inverse relationship between tumor PLP concentration and tumor volume in mice. This suggests that, in contrast to data reported for normal cells, tumor cells are capable of accumulating supplemental B6. In the current study, we investigated the effects of dietary B6 supplementation on 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in rats. Specifically, we aimed to identify the effect of pyridoxine (PN) supplementation on tumor growth and vitamin uptake by tumor cells. To accomplish this, 50 d old female Sprague Dawley rats were gavaged with 15 mg DMBA and fed a diet containing either 7, 350, or 1050 mg PN-HCl/kg diet, which is the equivalent of 1, 50, or 150x the National Research Council's B6 requirement for rats, respectively. These levels of PN have previously been shown to produce no overt signs of toxicity in rats. Throughout the experiment, the percent of rats with tumors and the average number of tumors per rat remained similar between groups. Mammary tumor growth rates were significantly increased in response to dietary B6 supplementation (P < 0.05). Liver PLP and pyridoxal (PL) concentrations did not differ between dietary treatment groups. Plasma PL and PLP concentrations were significantly higher in the group fed the 150x diet compared with the 1x diet (P < 0.001, P < 0.05). Mammary tissue PL concentrations of the 150x group were significantly higher (P < 0.05) than the 1x group, but no differences were observed in mammary PLP concentrations. Similarly to mammary tissue, no differences between groups were observed in tumor PLP concentration. However, tumor PL concentrations in both the 50x and 150x dietary treatment groups were significantly higher than those from the rats fed the 1x diet (P < 0.002). These data demonstrate that previously reported inhibitory effects of supplemental B6 on breast cancer growth in vitro do not occur in response to dietary supplementation at 50 or 150 times the B6 requirement in vivo. In fact, dietary B6 at 150x the requirement may actually promote mammary tumor growth. In light of these results, investigation of the effects of supplemental B6 on cancer growth in humans is warranted. Supported by American Cancer Society Grant # IRG-99-225-01.
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    Differential effect of melengestrol acetate or progesterone-releasing intravaginal devices on follicular development, progesterone and estradiol-17β concentrations and patterns of luteinizing hormone release during the bovine estrous cycle
    Custer, Edward E. (Virginia Tech, 1992-09-05)
    Two studies were conducted to determine if 7-d MGA or PRID treatment initiated on d 17 of the estrous cycle altered: 1) follicular development, 2) estradiol-17β (E2) and progesterone (P4) concentrations, and 3) patterns of release of luteinizing hormone (LH). In both studies, Angus, Angus x Holstein or Holstein cows 2 to 6 yr of age were randomly assigned to receive either MGA (.5 mg⋅hd⁻¹⋅d⁻¹; n = 23) or PRID (n = 26) for 7 d or to serve as untreated controls (n = 14). Real time, B-mode ultrasound, equipped with a 7.5 mHz linear-array transrectal transducer, was used to conduct daily ovarian scans beginning 3 (Study 1) or 9 d (Study 2) after onset of estrus. Jugular venous blood samples (45 ml) were collected coincident with ovarian scans. In study 2, cows were fitted with indwelling jugular catheters 17 (Control, MGA and PRID), 20 and 23 d (MGA and PRID) after onset of estrus and blood samples were collected at 15-min intervals for 6 h for determination of LH. Interestrus interval was extended (P<.05) for 3 to 5 d in MGA-treated cows exhibiting two or three dominant follicles (classified as MGA-2 and MGA-3, respectively) or PRID-treated cows compared to controls exhibiting two or three dominant follicles during the estrous cycle (control-2 and control-3, respectively). Forty-four percent of MGA-treated cows ovulated the dominant follicle present at the beginning of MGA treatment. In both studies, days from detection of the ovulatory follicle until ovulation were greater (P<.01) in MGA-2 and control-2 cows than control-3, MGA-3 and PRID cows. Diameter of the ovulatory follicle was greater (P<.01) 9 d before estrus and growth rate of the ovulatory follicle was less (P<.02) in MGA-2 and control- 2 cows than control-3, MGA-3 and PRID cows. Serum P4 decreased 3 d earlier (P<.02) during the estrous cycle of MGA-2 and control-2 cows than control-3, MGA-3 and PRID cows. Serum E2 was greater (P<.01) 7 d before estrus in MGA-2 cows than all other treatment groups. Changes in mean and baseline LH concentrations and amplitude of LH pulses on d 17, 20 and 23 after onset of estrus did not differ (P>.10) among treatments. Luteinizing hormone pulse frequency was greater (P<.03) on d-20 after onset of estrus in MGA-2 cows than MGA-3 and PRID cows (4.3 ± .6 vs 2.6 ± .3 and 3.2 ± .4, respectively). In addition, LH pulse frequency did not differ (P>.10) 17 or 23 d after onset of estrus among treatments. In conclusion, MGA treatment extended the dominance phase of development of ovulatory follicles, which resulted in the premature increase in serum E2 and frequency of LH release, whereas the dominant follicle present at the beginning of PRID treatment underwent atresia and another preovulatory follicle developed.
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    Differential expression profiling of proteomes of pathogenic and commensal strains of Staphylococcus aureus using SILAC
    Manickam, Manisha (Virginia Tech, 2011-11-29)
    Staphylococcus aureus (S. aureus) is the etiological agent of food-borne diseases, skin infections in humans and mastitis in bovines. S. aureus is also known to exist as a commensal on skin, nose and other mucosal surfaces of the host. This symbiotic association is a result of immune dampening or tolerance induced in the host by this pathogen. We proposed the variation in protein expression by commensal and pathogenic strain as an important factor behind the difference in pathogenicity. The identification of differentially expressed proteins was carried out using a quantitative mass spectrometry (MS)-based proteomic approach, known as stable isotope labeling of amino acids in cell culture (SILAC). Four commensal and pathogenic strains each were grown in the SILAC minimal media (RPMI 1640), containing light (12C) and heavy (13C) form of lysine, respectively, until early stationary growth phase. Various protein fractions, including cell wall, membrane and secreted, were extracted from the bacterial cultures and mixed in a 1:1 ratio. The relative abundance of proteins present in light and heavy labeled samples was determined using MS analysis. From a total of 151 differentially expressed proteins, 58 were found to be upregulated in the pathogenic strains. These proteins are involved in a variety of cellular functions, including immune modulation, iron-binding, cellular transport, redox reactions, and metabolic enzymes. The differentially expressed proteins can serve as putative candidates to improve current approach towards development of a vaccine against S. aureus.
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    Effect of body condition and ration protein source on performance of high producing cows during early lactation
    Seymour, William Matthew (Virginia Tech, 1985-04-05)
    Forty-two high producing Holstein cows were paired by body condition and mature equivalent milk production and fed either a high or low energy complete ration ad libitum during the last 16-20 weeks of lactation. Cows fed the high energy ration ate more feed, produced more milk and gained more body condition than cows fed the low energy ration. Cows were fed to maintain condition during the dry period. During weeks 3-15 of the next lactation, half the cows in each condition group (fat or thin) were fed a mixed ration with soybean meal (SBM) as the major protein source. The remaining cows were fed a ration with dried brewers grains (DBG) as the main protein source.