Publications by authors named "Daniel J MacPhee"

Article Synopsis
  • This study focuses on decellularized uterine extracellular matrix as a key biomaterial for uterine tissue regeneration and transplantation, investigating different decellularization protocols for porcine uterine tissues.
  • Six distinct protocols involving various chemicals, including sodium dodecyl sulfate and DNase I, were tested for their effectiveness in DNA removal and preservation of bioactive molecules.
  • Results showed that one specific combination (0.1% SDS + 1% Triton X-100 with agitation) was most effective for DNA removal, with DNase I significantly improving the process by reducing cell debris, which could enhance future uterine transplantation efforts.
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Background: Porcine reproductive and respiratory syndrome virus 2 (PRRSV-2) infection during late gestation substantially lowers fetal viability and survival. In a previous genome-wide association study, a single nucleotide polymorphism on chromosome 7 was significantly associated with probability of fetuses being viable in response to maternal PRRSV-2 infection at 21 days post maternal inoculation. The iodothyronine deiodinase 2 (DIO2) gene, located ~ 14 Kilobase downstream of this SNP, was selected as a priority candidate related to fetal susceptibility following maternal PRRSV-2 infection.

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The porcine epitheliochorial placenta creates a barrier for the transplacental transfer of some nutrients from the dam to the fetus, as well as feto-lethal viruses such as porcine reproductive and respiratory syndrome virus 2 (PRRSV-2). Areolae are specialized structures within the porcine placenta with a high absorptive and substance transport capacity that facilitate embryonic development. The overarching aim of this study was to characterize the localization of PRRSV-2 in and adjacent to areolae to provide insight into whether transplacental transmission might occur through placental areolae.

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Angiogenesis and cell proliferation in reproductive tissues are essential events for the maintenance of pregnancy, and alterations can lead to compromised fetal development and survival. Porcine reproductive and respiratory syndrome virus 2 (PRRSV-2) induces reproductive disease with negative financial and production impact on the swine industry. PRRSV-2 infection alters placental physiology through inflammatory and apoptotic pathways, yet fetal susceptibility varies.

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Understanding why intrauterine growth restricted (IUGR) fetuses are more resilient to transplacental porcine reproductive and respiratory syndrome virus-2 (PRRSV-2) infection compared to normal fetuses may lead to alternative approaches to control PRRS. Our objective was to compare gene expression of a subset of tight junction proteins in the endometrium (END) and placenta (PLC) of i) IUGR vs N-IUGR fetuses, and ii) across disease progression phenotypes following PRRSV-2 infection. In experiment 1, snap frozen END and PLC from fetuses of non-infected control dams (CTRL) and from high viral load viable (HVL-VIA) fetuses, with both groups further classified as either IUGR or non(N)-IUGR based on brain: liver weight ratio were strategically selected from a large challenge trial.

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Limb wounds are common in horses and often develop complications. Intravenous multipotent mesenchymal stromal cell (MSC) therapy is promising but has risks associated with intravenous administration and unknown potential to improve cutaneous wound healing. The objectives were to determine the clinical safety of administering large numbers of allogeneic cord blood-derived MSCs intravenously, and if therapy causes clinically adverse reactions, accelerates wound closure, improves histologic healing, and alters mRNA expression of common wound cytokines.

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Article Synopsis
  • Thyroid hormones, like T3 and T4, play crucial roles in growth, development, and metabolism in pigs, but their levels can drop significantly when pigs are exposed to stressors like disease.
  • In a study with nursery pigs that faced a natural disease challenge, T3 levels rebounded somewhat after 48 days, while T4 levels remained low; both hormones were linked to the pigs' growth rates.
  • Additionally, in a separate analysis involving PRRSV-infected pigs, both thyroid hormones decreased at first but returned to near baseline levels over time, with higher hormone levels associated with better growth, highlighting the impact of disease on thyroid function and growth performance in pigs.
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Introduction: Existing strategies to control porcine reproductive and respiratory syndrome (PRRS) are not completely effective and require alternative approaches. Although intrauterine growth restricted (IUGR) fetuses are more resilient to transplacental PRRS virus-2 (PRRSV2) infection compared to normal fetuses, the exact mechanisms are unknown. The objective of this research was to assess abundance and localization of a subset of tight junction (TJ) proteins in the maternal-fetal interface and any alterations that may affect the movement of nutrients or PRRSV2 across the epitheliochorial placenta.

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Background: Mechanisms of fetal death following maternal PRRSV2 infection remain uncharacterized, although hypoxia from umbilical cord lesions and/or placental detachment due to apoptosis are hypothesized. We performed two experiments examining hypoxia and apoptosis in PRRSV-infected and non-infected, third-trimester fetuses to elucidate possible associations with fetal death. Fetuses were selected based on four phenotypic infection groups: fetuses from non-challenged control gilts (CTRL); low viral load fetuses (LVL; Exp 1) or uninfected fetuses (UNINF; Exp 2) from inoculated gilts; viable high viral load fetuses (HVL-VIA); and HVL meconium-stained fetuses (HVL-MEC).

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Porcine reproductive and respiratory syndrome virus (PRRSV) is transmitted vertically, causing fetal death in late gestation. Spatiotemporal distribution of virus at the maternal-fetal interface (MFI) is variable, and accurate assessment of viral concentration and lesions is thus subject to sampling error. Our objectives were: 1) to assess whether viral load and lesion severity in a single sample of endometrium (END) and placenta (PLC), collected near the base of the umbilical cord (the current standard), are representative of the entire organ; and 2) to compare sampling strategies and evaluate if spatial variation in viral load can be overcome by pooling of like-tissues.

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PRRSV infection in third-trimester pregnant sows can lead to fetal death and abortions, although the mechanisms triggering these effects are not well understood. Since resistant and susceptible fetuses can coexist in the same litter, we propose that there may be differential mechanisms used by some fetuses to evade infection and/or disease progression. Our objectives were to investigate possible differences in the metabolome of PRRSV-infected and non-infected fetuses, as well as the interaction of altered intrauterine growth development and PRRSV infection to elucidate possible causes of fetal death following PRRSV infection.

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Background: A pregnant gilt infected with porcine reproductive and respiratory syndrome virus (PRRSV) can transmit the virus to her fetuses across the maternal-fetal-interface resulting in varying disease outcomes. However, the mechanisms leading to variation in fetal outcome in response to PRRSV infection are not fully understood. Our objective was to assess targeted immune-related gene expression patterns and pathways in the placenta and fetal thymus to elucidate the molecular mechanisms involved in the resistance/tolerance and susceptibility of fetuses to PRRSV2 infection.

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Limb wounds on horses are often slow to heal and are prone to developing exuberant granulation tissue (EGT) and close primarily through epithelialization, which results in a cosmetically inferior and non-durable repair. In contrast, wounds on the body heal rapidly and primarily through contraction and rarely develop EGT. Intravenous (IV) multipotent mesenchymal stromal cells (MSCs) are promising.

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Gonocytes in the neonatal testis have male germline stem cell potential. The objective of the present study was to examine the behavior and ultrastructure of gonocytes in culture. Neonatal porcine testis cells were cultured for 4 weeks and underwent live-cell imaging to explore real-time interactions among cultured cells.

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To better understand the host response to porcine reproductive and respiratory virus-2 (PRRSV2) we evaluated circulating thyroid hormone and associated gene expression in a late gestation challenge model. Pregnant gilts were inoculated at gestation day 85 and fetal samples collected at either 12 or 21 days post-infection (dpi). A subset of fetuses was selected for analysis based on viability and viral load categorized as either uninfected-viable (UNIF), high viral load viable (HV-VIA) or high viral load meconium stained (HV-MEC) and were compared with gestational age matched controls (CON).

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To understand the fetal immune response to porcine reproductive and respiratory virus-2 (PRRSV) and to evaluate the association with fetal viability, pregnant gilts were challenged on gestation day 85 and euthanized 21 days post infection. Based on preservation status and viral load in serum and thymus, fetuses were classified as either uninfected-viable (UNIF), high viral load viable (HV-VIA), or high viral load meconium stained (HV-MEC) and were compared with age matched control (CON) fetuses derived from mock infected gilts. Gene expression of IFNB, IFNG, CCL2, CCL5, CXCL10 and IL10, were all found to be significantly upregulated in the thymus and spleen of both high viral load groups.

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During pregnancy and labour the myometrium undergoes structural and physiological adaptations as part of a program of development. Heat shock factor 1 (HSF1) is a master regulator of both stress and developmental processes. A noted HSF1-induced gene is the 90 kDa heat shock protein (HSP90), which acts as a chaperone and regulator of cellular processes.

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Horses often sustain cutaneous wounds and healing can be prolonged and difficult to treat. Compared to body wounds, limb wounds heal slower and are more likely to develop exuberant granulation tissue. Differences in healing rates and exuberant granulation tissue formation is attributed to abnormal cytokine profiles.

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The cellular marker CD163 is a type 1 transmembrane scavenger protein found either on the surface of antigen-presenting cells or in a soluble form (sCD163), released in response to inflammation. Despite an obligatory role in porcine reproductive and respiratory virus (PRRSV) infection, information on sCD163 as a biomarker of disease outcome in swine remains limited. In the present study, we developed a sandwich ELISA using an anti-bovine CD163 antibody, LND68A, in conjunction with the porcine specific 2A10/11 antibody.

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Although porcine reproductive and respiratory syndrome virus (PRRSV) readily crosses the maternal fetal interface (MFI) in third trimester, fetal resilience varies within litters. The aim of this study was to characterize PRRSV-2 concentration in MFI and fetuses at five time points after experimental inoculation of late gestation gilts and use this information to classify potentially resistant, resilient and susceptible fetuses. The secondary objective was to verify the relationship between PRRS viral load and intrauterine growth retardation (IUGR).

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The efficient extraction of proteins of interest from cells and tissues can be challenging. Here we demonstrate the differences in extraction of the focal adhesion protein Kindlin-2 and the transcriptional repressor Snail from choriocarcinoma cells using NP-40 and RIPA lysis buffer. We also show the use of a more denaturing urea/thiourea lysis buffer for solubilization, by comparing its effectiveness with the often utilized RIPA lysis buffer for solubilization of heat shock proteins (HSP) B1 and B5 and the cytoplasmic adapter protein integrin-linked kinase (ILK) from smooth muscle.

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Background: Integrins are transmembrane receptors that mediate cell-extracellular matrix (ECM) and cell-cell adhesion and trophoblast cells undergo changes in integrin expression as they differentiate. However, the mechanism(s) of integrin activation leading to integrin-mediated signaling in trophoblast cell differentiation is unknown. The Fermitin family proteins are integrin activators that help mediate integrin-mediated signaling, but have never been studied in detail within the human placenta.

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The placenta is a vital organ providing the developing fetus with nutrient and gas exchange, thermoregulation, and waste elimination necessary for fetal development, as well as producing hormones to maintain pregnancy. It is hypothesized that fetal pig death in porcine reproductive and respiratory syndrome may be attributed to pathology of the maternal-fetal interface leading to premature placental separation. This study was designed to evaluate the chronologic progression of porcine reproductive and respiratory syndrome virus (PRRSV)-induced lesions at the maternal-fetal interface, with particular focus on placental separation in experimentally challenged third-trimester gilts.

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The placenta is the physiological bridge between mother and fetus and has life-sustaining functions during pregnancy, including metabolic regulation, fetal protection and hormone secretion. Nucleobindin-2 (NUCB2) is a calcium- and DNA-binding protein and precursor of nesfatin-1, a signalling peptide with multiple functions, including regulation of energy homeostasis and glucose transport. These are also key functions of the placenta, yet NUCB2/nesfatin-1 expression has never been comprehensively studied in this organ.

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The small heat shock protein B (HSPB) family is comprised of eleven members with many being induced by physiological stressors. In addition to being molecular chaperones, it is clear these proteins also play important roles in cell death regulation, cytoskeletal rearrangements, and immune system activation. These processes are important for the uterine smooth muscle or myometrium during pregnancy as it changes from a quiescent tissue, during the majority of pregnancy, to a powerful and contractile tissue at labor.

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