Browsing by Author "Peralta, Oscar A."

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    Bovine fetal mesenchymal stem cells exert antiproliferative effect against mastitis causing pathogen Staphylococcus aureus
    Cahuascanco, Berly; Bahamonde, Javiera; Huaman, Olger; Jervis, Miguel; Cortez, Jahaira; Palomino, Jaime; Escobar, Alejandro; Retamal, Patricio; Torres, Cristian G.; Peralta, Oscar A. (2019-04-11)
    Staphylococcus aureus is the most commonly isolated pathogen from clinical bovine mastitis samples and a difficult pathogen to combat. Mesenchymal stem cells (MSC) are multipotent progenitor cells equipped with a variety of factors that inhibit bacterial growth. The aim of the present study was to evaluate the in vitro antibacterial potential against S. aureus of conditioned medium (CM) from MSC derived from fetal bovine bone marrow (BM-MSC) and adipose tissue (AT-MSC). BM-MSC, AT-MSC and fetal fibroblasts (FB) cultures were activated by infection with S. aureus. Bacterial growth was evaluated in presence of CM, concentrated CM (CCM), activated CM (ACM) and concentrated ACM (CACM) from BM-MSC, AT-MSC and FB. Gene expression of β-defensin 4A (bBD-4A), NK-lysine 1 (NK1), cathelicidin 2 (CATHL2), hepcidin (HEP) and indoleamine 2,3 dioxygenase (IDO) and protein expression of bBD-4A were determined in activated and non-activated cells. The majority of BM-MSC and AT-MSC expressed CD73, Oct4 and Nanog, and were negative for CD34. Growth of S. aureus decreased when it was exposed to CM from BM-MSC, AT-MSC and FB. Moreover, growth of S. aureus in CCM, ACM and CACM was lower compared to controls of CM from BM-MSC and AT-MSC. Activated AT-MSC increased mRNA levels of bBD4A and NK1, and protein levels of bBD4A in CM. Thus, CM from fetal bovine BM-MSC and AT-MSC has the capacity to reduce in average ~30% of S. aureus relative growth under in vitro conditions. The in vitro antibacterial effect of fetal bovine MSC may be mediated by bBD4A and NK1 activity.
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    Comparison of three estrus detection systems during summer heat stress in a large commercial dairy herd
    Peralta, Oscar A. (Virginia Tech, 2003-06-12)
    The objective of the study was to compare three systems for detection of estrus and combinations of these systems on a large commercial dairy (1000 lactating cows) during stress of summer heat. At 37 to 45 days in milk (DIM), 266 cows were fitted with a HeatWatch (HW) device (HeatWatch; DDx Inc., Boulder, CO), an activity (A) sensor (ALPRO; DeLaval Inc., Kansas City, MO), and observed visually (V) twice daily. Pregnancy status was determined by uterine palpation 35 to 49 d following artificial insemination (AI). The effects of DIM, parity, physical activity, standing events, months, AI technician, and interval between onset of estrus and AI on conception rate were determined using linear contrasts and logistic regression. Efficiencies for detection of estrus, determined by comparing detected periods of estrus with a theoretical total of 707 periods, were 45.8% (V), 33.2% (A), 40.3% (HW), and 71.6% for all three systems simultaneously. Conception rates (LSM ± SE) by method of detection were 16.7 ± 4.9 for HW, 19.8 ± 5.5 for A, 7.9 ± 3.4 for V, 16.3 ± 6.0 for V + A, 27.6 ± 4.6 for V + HW, 21.1 ± 4.9 for A + HW, and 21.9 ± 4.5 for V + A + HW. Conception rate and number of mounts decreased for cows in first versus second and third parity (P < 0.05). For periods of estrus detected by A, the lowest conception rate (P < 0.05) occurred >18 h after the onset of estrus (16.7 ± 7.9). The highest conception rate occurred with the combination of V + HW, which confirms the premise that combination of multiple systems enhances both the efficiency and accuracy of detection.
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    Developmental Regulation of Prion Expression in Cattle and Mouse Embryonic Stem Cells
    Peralta, Oscar A. (Virginia Tech, 2008-07-25)
    The host encoded cellular prion protein (PrPC) is an N-linked glycoprotein tethered to the cell membrane by a glycophosphatidylinositol (GPI) anchor. Under certain conditions, PrPC can undergo conversion into a conformationally-altered isoform (PrPSc) widely believed to be the pathogenic agent of transmissible spongiform encephalopathies (TSEs). Thus, tissues expressing PrPC are potential sites for conversion of PrPSc during TSE pathogenesis. Although much is known about the role of PrPSc in prion diseases, the normal function of PrPC is poorly understood. Lines of mice and cattle in which PrPC has been ablated by gene knockout show no major phenotypical alterations other than resistance to TSE infection. However, recent reports using Prnp-null mouse models have suggested the participation of PrPC in neural stem/progenitor cell proliferation and differentiation. The first objective in our study was to map the expression of PrPC in twenty six somatic and reproductive tissues in ruminants. Our second objective was to characterize the ontogeny of PrPC expression during bovine embryonic and early fetal development. Finally, we used a mouse embryonic stem cell (mESC) model to study the potential role of PrPC during neurogenesis. In adult tissues, intense expression of PrPC was detected in the central nervous system (CNS), thymus and testes, whereas the liver, striated muscle and female reproductive tissues showed the lowest expression. We observed that PrPC was associated with tissues undergoing cellular differentiation including spermatogenesis, lymphocyte activation and hair follicle regeneration. Analyses in bovine embryos and fetuses indicated peaks in expression of PrPC at days 4 and 18 post-fertilization, stages associated with the maternal-zygote transition and the maternal recognition of pregnancy and initiation of placental attachment, respectively. Later in development, PrPC was expressed in the CNS where it was localized in mature neurons of the neuroepithelium and emerging neural trunks. Based on these observations, we hypothesized that PrPC was involved in neurogenesis. We tested this hypothesis in a murine embryonic stem cell model (mESC). mESC were induced to form embryoid bodies (EBs) by placing them in suspension culture under differentiating conditions and allowed to differentiate in vitro for 20 days. We detected increasing levels of PrPC starting on day 12 (8.21- fold higher vs. day 0; P < 0.05) and continuing until day 20 (20.77-fold higher vs. day 0; P < 0.05). PrPC expression was negatively correlated with pluripotency marker Oct-4 (r= -0.85) confirming that mESC had indeed differentiated. To provide a more robust system for assessing the role of PrPC in neural differentiation, mESC were cultured with or without retinoic acid (RA) to encourage differentiation into neural lineages. Induction of EBs with retinoic acid (RA) resulted in an earlier up-regulation of PrPC and nestin (day 12 vs. day 16; P < 0.05). In addition, immunofluorescence studies indicated co-expression of PrPC and nestin in the same cells. The results of these experiments suggested a temporal link between PrPC expression and expression of nestin, a marker of neural progenitor cells. We next tested whether PrPC was involved in RA-enhanced neural differentiation from mESC using a PrPC knockdown model. Plasmid vectors designed to express either a PrP-targeted shRNA or scrambled, control shRNA were transfected into mESC. Stable transfectants were selected under G418 and cloned. PrP-targeted and control shRNA clones, as well as wild-type mESC, were differentiated in presence of RA and sampled as above. PrPC expression was knocked down in PrP-targeted shRNA cultures between days 12 and 20 (62.2 % average reduction vs. scrambled shRNA controls). Nestin expression was reduced at days 16 and 20 in PrPC knockdown cells (61.3% and 70.7%, respectively vs. scrambled shRNA controls). These results provide evidence that PrPC plays a role in the neural differentiation at a point up-stream from the stages at which nestin is expressed. In conclusion, the widely distributed expression of PrPC in ruminant tissues suggests an important biological role for this protein. In the present work we have provided evidence for the participation of PrPC in the differentiation of mESC along the neurogenic pathway.