Detection of PRRSV-2 alone and co-localized with CD163 positive macrophages in porcine placental areolae.

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. Control (CON) plus three phenotypic fetal groups were selected based on levels of virus in fetal placenta, sera and thymus, to determine if fetal resilience was related to differences in PRRSV-2 localization, alone or co-localized with CD163+ macrophages. These fetal groups represented a range of susceptibility: uninfected (UNINF) being resistant, infected in placenta only (PLCO) being resilient, and high viral load viable (HVL-VIA) being most susceptible. Finally, potential factors related to PRRSV-2 localization, including the severity of inflammation in endometrium and placenta, and intrauterine growth restriction, known resilience factors, were assessed. Thirty-one pregnant gilts were inoculated with PRRSV-2 at gestation day 86 ± 0.4. Seven pregnant gilts were sham-inoculated. Gilts were euthanized at 12 days post-infection. Presence of PRRSV and CD163+ macrophages were determined using immunofluorescence in cryosections of maternal-fetal interface (MFI) with and without areolae. In the maternal, fetal and cavity of areolar region PRRSV particles were found both independently and co-localized with CD163+ macrophages. Similarly, individual, and co-localized particles were observed in the maternal and fetal sides of the MFI region of infected fetuses. Weak positive correlations were observed between the counts of CD163+ macrophages and some inflammation scores in endometrial and placental tissues, but no correlations with PRRSV-2 localization. There were no differences across the four fetal groups evaluated. These results suggest that transplacental transmission of PRRSV may occur through the areolae, either as non-cell associated or in association with infected CD163 macrophages.

Effect of porcine reproductive and respiratory syndrome virus 2 on angiogenesis and cell proliferation at the maternal-fetal interface.

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. This study aimed to evaluate angiogenesis and cell proliferation in the porcine maternal-fetal interface (MFI) and determine if these physiological processes were altered by PRRSV-2 infection. Thirty-one pregnant gilts were inoculated with PRRSV-2 at gestation day 86 ± 0.4 (mean ± SD). Seven control gilts were sham-inoculated. All gilts were euthanized at 12 days postinoculation. Angiogenesis and cell proliferation were determined through the detection of vascular endothelial growth factor (VEGF) and Ki-67, respectively, using immunofluorescence of the MFI from 4 fetal resilience groups: uninfected (UNIF), high viral load-viable (HVL-VIA), and HVL-meconium-stained (MEC) from PRRSV-infected gilts, as well from sham-inoculated (CON) gilts. VEGF immunolabeling in the uterine submucosa was significantly lower in MEC compared with UNIF and HVL-VIA groups. Significantly greater Ki67 immunolabeling was detected in the trophoblasts of CON fetuses versus all other groups, and in uterine epithelium of CON and UNIF fetuses versus HVL-VIA and MEC. These results suggest that fetal resilience may be related to greater cell proliferation in uterine epithelium, and fetal compromise with reduced uterine submucosal angiogenesis, except fetuses with intrauterine growth restriction, in which inherently lower submucosal angiogenesis may be protective against PRRSV infection.

Effect of porcine reproductive and respiratory syndrome virus 2 on tight junction gene expression at the maternal-fetal interface.

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. In experiment 2, similar tissues were randomly selected from CTRL and from uninfected thymus (UNIF), (HVL-VIA) and HVL meconium-stained in the body (HVL-MEC-B) of PRRSV-infected dams. The expression of claudin (CLDN) 1, 3, 4, 5, 6, 7, 10, tight junction protein 1 (TJP1) and occludin (OCLN) genes were evaluated by PCR. There were no significant group differences between IUGR and N-IUGR groups, regardless of infection status, that explained the resilience of IUGR fetuses. Regarding disease progression, elevated CLDN3 was observed in END of UNIF, CLDN6 expression was lower in PLC when the fetus became infected (HVL-VIA), and CLDN10 elevated in PLC in fetuses showing evidence of compromise (HVL-MEC-B). Lastly, OCLN gene expression was higher in the END and PLC following maternal infection. In conclusion, differences in TJ integrity were mainly observed following PRRSV-2 infection with stepwise changes corresponding with disease progression.

Macroscopic, Histologic, and Immunomodulatory Response of Limb Wounds Following Intravenous Allogeneic Cord Blood-Derived Multipotent Mesenchymal Stromal Cell Therapy in Horses.

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. Wounds were created on the metacarpus of 12 horses. Treatment horses were administered 1.51-2.46 × 108 cells suspended in 50% HypoThermosol FRS, and control horses were administered 50% HypoThermosol FRS alone. Epithelialization, contraction, and wound closure rates were determined using planimetric analysis. Wounds were biopsied and evaluated for histologic healing characteristics and cytokine mRNA expression. Days until wound closure was also determined. The results indicate that 3/6 of treatment horses and 1/6 of control horses experienced minor transient reactions. Treatment did not accelerate wound closure or improve histologic healing. Treatment decreased wound size and decreased all measured cytokines except transforming growth factor-ß3. MSC intravenous therapy has the potential to decrease limb wound size; however, further work is needed to understand the clinical relevance of adverse reactions.

Thyroid hormone suppression in feeder pigs following polymicrobial or porcine reproductive and respiratory syndrome virus-2 challenge.

Thyroid hormones are powerful regulators of growth, development, and basal metabolic rate and can be dysregulated under conditions of severe stress or illness. To understand the role of these hormones in porcine disease response, serum samples were obtained from three batches of nursery-aged pigs (n = 208) exposed to a natural polymicrobial disease challenge with an array of bacterial and viral pathogens. Levels of total thyroxin (T4) and triiodothyronine (T3) assessed in sera by radioimmunoassay, decreased significantly by 14 days post-exposure (DPE). Levels of T3 partially rebounded by 48 DPE, while T4 levels remain depressed. Post-exposure T3 and T4 levels were positively correlated with acute and long-term average daily gain (ADG). Cross-sectional sampling of animals maintained at the high health source farms, showed no equivalent change in either hormone when managed under standard industrial conditions. To further elucidate the effect of porcine reproductive and respiratory syndrome virus (PRRSV)-infection on thyroid hormone levels, archived sera over 42 days post inoculation (DPI) from nursery pigs (N = 190) challenged with one of two PRRSV2 strains by the PRRS Host Genetics Consortium were similarly assessed, with animals selected in a two-by-two design, to investigate biological extremes in ADG and viral load (VL). All animals showed a similar decrease in both thyroid hormones reaching a minimum at 7 DPI and returning to near pre-challenge levels by 42 DPI. Post-challenge T3 and T4 levels were significantly greater in high ADG groups, with no significant association with VL or strain. The results of this study demonstrate porcine susceptibility to thyroid disruption in response to disease challenge and demonstrate a relationship between this response and growth performance.

Decreased tight junction protein intensity in the placenta of porcine reproductive and respiratory syndrome virus-2 infected fetuses.

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. METHODS: Paraffin-embedded samples of placenta from non-infected control (CTRL) and PRRSV2 infected fetuses (IUGR, non(N)-IUGR, meconium-stained (MEC) (n = 6 per group) were randomly selected from a large challenge trial and immunostained for claudins (CLDN) 1, 3, 4, 7 and tight junction protein 1 (TJP1). Immunostaining intensity was semi-subjectively scored by region. RESULTS: Intensity of CLDN1 was lower in placenta of IUGR, MEC, and N-IUGR fetuses compared to CTRL, mainly in fetal epithelium and maternal endothelial cells (MECL). CLDN4 intensity was lower in MECL of IUGR compared to CTRL and MEC fetuses. TJP1 intensity was lower in maternal and fetal epithelia of placenta within IUGR, MEC, and N-IUGR fetuses versus CTRL. DISCUSSION: Differences were mainly observed between PRRSV2 infected and non-infected groups indicating TJ integrity was affected by PRRSV2 infection. These results provide insights into the potential mechanisms of transplacental transmission of PRRSV2; however, since only CLDN4 differed amongst the infected groups, PRRSV2 induced changes in TJ integrity do not appear to explain variation in fetal outcomes after infection.

Fetal hypoxia and apoptosis following maternal porcine reproductive and respiratory syndrome virus (PRRSV) infection.

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). RESULTS: In experiment 1, paraffin embedded fetal tissues collected 21 days post maternal infection (DPI) were examined for DNA fragmentation associated with apoptosis. Positively stained foci were larger and more numerous (P < 0.05) in heart, liver, and thymus of HVL-VIA and HVL-MEC compared to CTRL and LVL fetuses. In experiment 2, group differences in gene expression within the hypoxia (HIF1a, IDO1, VEGFa, LDHA, NOS2, NOX1) and apoptosis (CASP3, CASP7, CASP8, CASP9, RIPK1, RIPK3) pathways were assessed by RT-qPCR in fetal tissues collected at 12 DPI. High viral load fetuses showed differential expression relative to the CTRL and UNINF (P < 0.05 for all). Brain tissue from HVL-VIA and HVL-MEC fetuses presented increased expression of CASP7, CASP8, RIPK3, HIF1a and IDO1. Fetal heart showed increased expression of CASP8, HIF1a, IDO and NOX1 and a decrease in NOS2 expression in infected groups. CASP7, CASP9, RIPK1 and RIPK3 were only increased in the heart of HVL-VIA while VEGFa was only increased for HVL-MEC fetuses. Thymus from HVL-MEC had decreased expression of CASP9 and there was increased IDO1 in all infected fetuses. CONCLUSIONS: There is strong evidence of apoptosis occurring in the heart, liver and thymus of highly viral load fetuses at 21 DPI. Furthermore, there was clear upregulation of apoptotic genes in the heart of high viral load infected fetuses and less prominent upregulation in the brain of PRRSV-infected fetuses, whereas thymus appears to be spared at 12 DPI. There was no strong evidence of hypoxia at 12 DPI in brain and thymus but some indication of hypoxia occurring in fetal heart.

Samples sizes required to accurately quantify viral load and histologic lesion severity at the maternal-fetal interface of PRRSV-inoculated pregnant gilts.

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. Spatially distinct pieces of END and PLC of 24 fetuses from PRRSV-2-infected dams were collected. PRRSV RNA quantified by RT-qPCR was compared in 5 individual pieces per fetus and in respective pools of tissue and extracted RNA. Three distinct pieces of MFI were assessed for histologic severity. Concordance correlation and kappa inter-rater agreement were used to characterize agreement among individual samples and pools. The viral load of individual samples and pools of END had greater concordance to a referent standard than did samples of PLC. Larger pool sizes had greater concordance than smaller pool sizes. Average viral load and lesion severity did not differ by location sampled, and no technical advantages of pooling tissues versus RNA extracts were found. We conclude that multiple pieces of MFI tissues must be evaluated to accurately assess lesion severity and viral load. Three pieces per fetus provided a reasonable balance of cost and logistic feasibility.

Fetal Metabolomic Alterations Following Porcine Reproductive and Respiratory Syndrome Virus Infection.

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. Near-term serum samples collected from fetuses on gestation day 106, 21 days post PRRSV-2 infection, were processed by direct flow injection mass spectrometry (DI-MS) and nuclear magnetic resonance (NMR) techniques. Experiment one investigated disease progression with 24 fetuses selected from each of four phenotypic groups: fetuses from non-inoculated gilts (CTRL); fetuses from inoculated gilts that escaped infection (UNINF); infected high viral load viable fetuses (INF); and infected high viral load meconium-stained fetuses (MEC). Experiment two investigated the interaction of intrauterine growth retardation (IUGR) and PRRSV infection by analyzing differences among: non-infected normal development (CON-N); CON-IUGR; PRRS infected normal development (PRRS-N); and PRRS-IUGR. Univariate and multivariate (PCA, PLS-DA) statistics determined group differences among various contrasts, and the most important metabolites associated with disease progression and fetal development. Significant differences in the metabolome were observed, especially between PRRSV-negative fetuses (CTRL and UNINF) and MEC fetuses, while INF fetuses appear to span both groups. The two metabolites with highest variable importance in projection (VIP) scores related to disease progression were alpha-aminoadipic acid (alpha-AAA) and kynurenine (KYN), having the highest concentration in MEC and INF fetuses, respectively, compared to CTRL and UNINF. In experiment two, non-IUGR fetuses were found to have increased levels of lysoPCs, PCs and amino acids compared to IUGR fetuses, while the near complete absence of lysoPCs and PCs in IUGR fetuses, even during infection, indicate a distinctive response to infection compared to non-growth retarded fetuses. Possible markers of PRRSV fetal susceptibility, such as alpha-AAA, kynurenine and lysoPCs, are presented and discussed.

Differential responses in placenta and fetal thymus at 12 days post infection elucidate mechanisms of viral level and fetal compromise following PRRSV2 infection.

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. Fetuses were grouped by preservation status and PRRS viral load (VL): mock infected control (CTRL), no virus detected (UNINF), virus detected in the placenta only with viable (PLCO-VIA) or meconium-stained fetus (PLCO-MEC), low VL with viable (LVL-VIA) or meconium-stained fetus (LVL-MEC), and high VL with viable (HVL-VIA) or meconium-stained fetus (HVL-MEC). RESULTS: The host immune response was initiated only in fetuses with detectable levels of PRRSV. No differentially expressed genes (DEG) in either the placenta or thymus were identified in UNINF, PLCO-VIA, and PLCO-MEC when compared to CTRL fetuses. Upon fetal infection, a set of core responsive IFN-inducible genes (CXCL10, IFIH1, IFIT1, IFIT3, ISG15, and MX1) were strongly upregulated in both tissues. Gene expression in the thymus is a better differentiator of fetal VL; the strong downregulation of several innate and adaptive immune pathways (e.g., B Cell Development) are indicative of HVL. Gene expression in the placenta may be a better differentiator of fetal demise than the thymus, based-on principle component analysis clustering, gene expression patterns, and dysregulation of the Apoptosis and Ubiquitination pathways. CONCLUSION: Our data supports the concept that fetal outcome in response to PRRSV2 infection is determined by fetal, and more significantly placental response, which is initiated only after fetal infection. This conceptual model represents a significant step forward in understanding the mechanisms underpinning fetal susceptibility to the virus.

Live-cell imaging and ultrastructural analysis reveal remarkable features of cultured porcine gonocytes.

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. This included imaging every 1 h from day 0 to day 3, every 2 h from day 4 to day 7, and every 1 h for 24 h at days 14, 21, and 28. Samples also underwent scanning electron microscopy, transmission electron microscopy, morphometric evaluations, immunofluorescence, and RT-PCR. Live-cell imaging revealed an active amoeboid-like movement of gonocytes, assisted by the formation of extensive cytoplasmic projections, which, using scanning electron microscopy, were categorized into spike-like filopodia, leaf-like lamellipodia, membrane ruffles, and cytoplasmic blebs. In the first week of culture, gonocytes formed loose attachments on top of a somatic cell monolayer and, in week 2, formed grape-like clusters, which, over time, grew in cell number. Starting at week 3 of culture, some of the gonocyte clusters transformed into large multinucleated embryoid body-like colonies (EBLCs) that expressed both gonocyte- and pluripotent-specific markers. The number and diameter of individual gonocytes, the number and density of organelles within gonocytes, as well as the number and diameter of the EBLCs increased over time (P < 0.05). In conclusion, cultured porcine gonocytes displayed extensive migratory behavior facilitated by their various cytoplasmic projections, propagated, and transformed into EBLCs that increased in size and complexity over time.

Homing and Engraftment of Intravenously Administered Equine Cord Blood-Derived Multipotent Mesenchymal Stromal Cells to Surgically Created Cutaneous Wound in Horses: A Pilot Project.

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. They home and engraft to cutaneous wounds and promote healing in laboratory animals, but this has not been demonstrated in horses. Furthermore, the clinical safety of administering >1.00 × 108 allogeneic MSCs IV to a horse has not been determined. A proof-of-principle pilot project was performed with two horses that were administered 1.02 × 108 fluorescently labeled allogeneic cord blood-derived MSCs (CB-MSCs) following wound creation on the forelimb and thorax. Wounds and contralateral non-wounded skin were sequentially biopsied on days 0, 1, 2, 7, 14, and 33 and evaluated with confocal microscopy to determine presence of homing and engraftment. Results confirmed preferential homing and engraftment to wounds with persistence of CB-MSCs at 33 days following wound creation, without clinically adverse reactions to the infusion. The absence of overt adverse reactions allows further studies to determine effects of IV CB-MSCs on equine wound healing.

Maternal and fetal thyroid dysfunction following porcine reproductive and respiratory syndrome virus2 infection.

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). In dams, circulating levels of total T3 and T4 decreased in the acute period following infection and rebounded by 21 dpi. A similar effect was observed in fetuses, but was largely restricted to HV-VIA and HV-MEC, with minimal decrease noted in UNIF relative to CON at 21 dpi. Gene expression in fetal heart at 12 dpi showed significant decompensatory transcription of thyroid hormone transporters (SLC16A2) and deiodinases (DIO2, DIO3), which was not observed in brain. Correspondingly, genes associated with cell cycle progression (CDK1,2,4) were downregulated in only the heart of highly infected fetuses, while expression of their inhibitor (CDKN1A) was upregulated in both tissues. Finally, expression of genes associated with cardiac stress including CAMKD and AGT were upregulated in the hearts of highly infected fetuses, and a shift in expression of MYH6 to MYH7 was observed in HV-MEC fetuses specifically. Collectively, the results suggest PRRSV2 infection causes a hypothyroid state that disproportionally impacts the fetal heart over the brain.

Fetal cytokine response to porcine reproductive and respiratory syndrome virus-2 infection.

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. UNIF fetuses remained largely unaffected, with only small upregulations in IFNA and IL10 in the thymus, and IFNA, CCL5 and CXCL10 in the spleen. Regarding fetal viability, expression of CCL5 was significantly elevated in the thymus and spleen of HV-MEC compared to HV-VIA fetuses. The concentrations of IFNα, IFNγ, TNFα and CCL2 were elevated in the sera of all infected fetuses, whereas IFNß was below the detection limit in all fetal sera. Additional gene expression analysis in the thymus showed significant downregulation of CDK1, CDK2 and CDK4, and upregulation of the inhibitor CDKN1A, suggesting altered regulation of cell cycle progression. Collectively, these results show near complete compartmentalization of the fetal immune response to infected fetuses and suggest this immune response is not a major contributor to fetal death.

Expression of heat shock factor 1, heat shock protein 90 and associated signaling proteins in pregnant rat myometrium: Implications for myometrial proliferation.

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. Immunoblot analysis demonstrated HSF1 expression levels in pregnant rat myometrium on gestational day (d) 6 were maintained at a significantly higher level compared with d12 to post-partum (PP) time points (P < 0.05), while expression on d12 was significantly higher compared to d15 and d19. The transcriptionally active form pSer230-HSF1 was detected at a significantly greater level at d6 compared with d21 and d23 time points and also at d12 compared with d21, d22 and 23 (labour). Similarly, phosphorylated (P)-HSP90AA1 protein detection was significantly greater on d6 compared to d19 to d23 time points and on d12 compared with d15 to PP time points. In contrast, P-HSP90AB1 showed significantly greater detection levels on d12 compared with d15 while levels on d22 were significantly higher compared to d15, d17 and d19. Immunofluorescence analysis demonstrated that total HSF1 and HSP90 were localized mainly in the cytoplasm of myometrial cells with some detection of HSF1 in nuclei. This work advances our scientific knowledge of the myometrium during pregnancy and the expression profiles of HSF1 and HSP90 within the proliferative phase of myometrial programming suggests a role for them in this period of hyperplasia and myometrial adaptation.

Development and application of a porcine specific ELISA for the quantification of soluble CD163.

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. The ELISA demonstrated that CD163 shedding from porcine alveolar macrophages increased following in vitro exposure to lipopolysaccharide or PRRSV-2 strain NVSL 97-7895. Evaluation of serum sCD163 in healthy feeder pigs identified a significant age effect with concentration rising after birth to a peak at day 19 (P < 0.05) followed by a sharp decline to a minimal level of detection at 9 weeks of age (P < 0.05). Healthy sows showed substantial variation but no significant change in average concentration between early and late lactation. The serum concentration of sCD163 from pigs with homozygous gene edits disrupting translation of the CD163 protein was below the threshold of detection. However, when reformatted as a competitive ELISA the assay identified an interfering substance consistent with the release of a truncated form of the CD163 protein in sera from gene edited animals. With sCD163 shown to be both dynamic and responsive, the described ELISA represents a novel tool for investigation of this molecule as a potential biomarker of disease response in the pig.

Classification of fetal resilience to porcine reproductive and respiratory syndrome (PRRS) based on temporal viral load in late gestation maternal tissues and fetuses.

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). Three PRRSV-inoculated pregnant gilts and 1 sham-inoculated control were euthanized at five time points in days post infection (DPI; 2, 5, 8, 12, 14). The preservation status of each fetus was determined and MFI samples adjacent to the umbilical stump of each fetus, as well as serum, thymus, umbilical cord and amniotic fluid were collected. Viral load was quantified using probe-based reverse-transcriptase quantitative PCR (RT-qPCR) targeting PRRSV NVSL 97-7895 ORF7. Our result show the MFI was largely PRRSV infected by 2 DPI and virus was first detected in fetal sera and umbilical cord by 5 DPI, and in fetal thymus and amniotic fluid by 8 DPI. This indicates that PRRSV-2 quickly crossed the placenta and traveled toward the fetus via umbilical circulation within one week of the dam's inoculation. Fetal compromise was first observed on 8 DPI and increased progressively through to 14 DPI. However, several factors were associated with fetal resilience. The random forest model identified that 'viral load in fetal thymus' and duration of infection ('DPI') as the most important factors predicting fetal resilience and resistance. Moreover, IUGR fetuses had lower viral load and were less frequently compromised or dead compared to non-IUGR and average cohorts. Understanding the mechanisms of fetal resilience to PRRSV will improve selection strategies for replacement gilts.

Lysis Buffer Choices Are Key Considerations to Ensure Effective Sample Solubilization for Protein Electrophoresis.

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. Overall, the results demonstrate the importance of optimizing lysis buffers for specific protein solubilization prior to finalizing the experimental workflow.

Fermitin family homolog-2 (FERMT2) is highly expressed in human placental villi and modulates trophoblast invasion.

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. Thus, we examined the spatiotemporal pattern of expression of Fermitin family homolog-2 (FERMT2) in human chorionic villi throughout gestation and its role in trophoblast-substrate adhesion and invasion. METHODS: Placental villous tissue was obtained from patients undergoing elective terminations by dilatation and curettage at weeks 8-12 (n = 10), weeks 13-14 (n = 8), as well as from term deliveries at weeks 37-40 (n = 6). Tissues were fixed, processed and sections utilized for immunofluorescence analysis of FERMT2 expression during gestation. Additionally, HTR8-SVneo human trophoblast cells were transfected by electroporation with FERMT2-specific siRNAs or non-targeting siRNAs (control) and used in cell-substrate adhesion as well as invasion assays. RESULTS: FERMT2 was more commonly expressed in the basal domain of villous cytotrophoblast cells and prominently localized around the periphery of individual extravillous trophoblast cells. siRNA-mediated knockdown of FERMT2 in HTR8-SVneo cells resulted in significantly decreased trophoblast-substrate attachment (p < 0.05) as well as significantly decreased trophoblast invasion (p < 0.05) relative to control cells. CONCLUSIONS: The detection of FERMT2 throughout extravillous trophoblast columns and the results of invasion assays demonstrated that this protein is likely an important regulator of integrin activation in extravillous cells to modulate migration and invasion.

Histologic Changes Associated With Placental Separation in Gilts Infected with Porcine Reproductive and Respiratory Syndrome Virus.

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. Fifteen gilts were inoculated with a virulent strain of PRRSV-2 on gestation day 86 ± 0.4. On multiple days postinoculation, 3 gilts along with 1 sham-inoculated control per time point were euthanized, and uterine and fetal placental tissues corresponding to each fetus were collected for histopathologic evaluation. The presence of any fetal lesion was 23 times more likely in compromised (meconium-stained and decomposed) compared with viable fetuses ( P < .001). In PRRSV-infected gilts, endometritis was more severe than placentitis, and the severity of endometrial inflammation and vasculitis increased progressively from 2 to 14 days postinoculation. Neither placental vasculitis nor a chronologic progression in the severity of placental detachment was observed. Severe placental detachment was more frequently present in PRRSV-infected compared with noninfected samples and was most significantly associated with placental inflammation, compared with other uterine lesions, viral load, or termination day. The results of this study suggest that placental separation by itself is not sufficient to significantly compromise fetal viability in reproductive porcine reproductive and respiratory syndrome.