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The hippocampus participates in spatial navigation and behavioral processes, displays molecular plasticity in response to environmental challenges, and can play a role in neuropsychiatric diseases. The combined effects of inflammatory prenatal and postnatal challenges can disrupt the hippocampal gene networks and regulatory mechanisms. Using a proven pig model of viral maternal immune activation (MIA) matched to controls and an RNA-sequencing approach, the hippocampal transcriptome was profiled on two-month-old female and male offspring assigned to fasting, mimetic viral, or saline treatments. More than 2600 genes presented single or combined effects (FDR-adjusted p-value < 0.05) of MIA, postnatal stress, or sex. Biological processes and pathways encompassing messenger cyclic adenosine 3′,5′-monophosphate (cAMP) signaling were enriched with genes including gastric inhibitory polypeptide receptor (GIPR) predominantly over-expressed in the MIA-exposed fasting males relative to groups that differed in sex, prenatal or postnatal challenge. While this pattern was amplified in fasting offspring, the postnatal inflammatory challenge appeared to cancel out the effects of the prenatal challenge. The transcription factors C-terminal binding protein 2 (CTBP2), RE1 silencing transcription factor (REST), signal transducer and activator of transcription 1 (STAT1), and SUZ12 polycomb repressive complex 2 subunit were over-represented among the genes impacted by the prenatal and postnatal factors studied. Our results indicate that one environmental challenge can influence the effect of another challenge on the hippocampal transcriptome. These findings can assist in the identification of molecular targets to ameliorate the effects of pre-and post-natal stressors on hippocampal-associated physiology and behavior.

The influence of proinflammatory challenges, such as maternal immune activation (MIA) or postnatal exposure to drugs of abuse, on brain molecular pathways has been reported. On the other hand, the simultaneous effects of MIA and drugs of abuse have been less studied and sometimes offered inconsistent results. The effects of morphine exposure on a pig model of viral-elicited MIA were characterized in the prefrontal cortex of males and females using RNA-sequencing and gene network analysis. Interacting and main effects of morphine, MIA, and sex were detected in approximately 2000 genes (false discovery rate-adjusted p-value < 0.05). Among the enriched molecular categories (false discovery rate-adjusted p-value < 0.05 and −1.5 > normalized enrichment score > 1.5) were the cell adhesion molecule pathways associated with inflammation and neuronal development and the long-term depression pathway associated with synaptic strength. Gene networks that integrate gene connectivity and expression profiles displayed the impact of morphine-by-MIA interaction effects on the pathways. The cell adhesion molecules and long-term depression networks presented an antagonistic effect between morphine and MIA. The differential expression between the double-challenged group and the baseline saline-treated Controls was less extreme than the individual challenges. The previous findings advance the knowledge about the effects of prenatal MIA and postnatal morphine exposure on the prefrontal cortex pathways.

Magnetic Resonance Imaging is an important tool for characterizing volumetric changes of the piglet brain during development. These analyses have been aided by the development of piglet brain atlases which are based on averages drawn from multiple piglets. Because these atlases typically contain only brain tissue, their use is limited to "brain extracted" images from which the surrounding tissues have been removed. Brain extractions, or segmentations, are typically performed manually. This approach is time-intensive and can lead to variation between segmentations when multiple raters are used. Automated segmentations processes are important for reducing the time required for analyses and improving the uniformity of the segmentations. Here we demonstrate the use of region-based recurrent convolutional neural networks (RCNNs) on a dataset consisting of 32 piglet brains. The RCNNs are trained from manual segmentations of sets of 27 piglets and then applied to sets of the remaining 5 piglets. The volumes of the machine-generated brain masks are highly correlated with those of the manually generated masks, and visual inspection of the segmentations show acceptable accuracy. These results demonstrate that neural networks provide a viable tool for the segmentation of piglet brains.

The effects of the practice of weaning in pigs have been documented, and include changes in organ function and immune mechanisms. However, the effects of weaning following exposure to maternal infection during gestation are incompletely characterized. We hypothesize that the disruption of physiological processes triggered by weaning can be influenced by the exposure of the pig to maternal immune signals elicited by a challenge during gestation. To test our hypothesis, the concentrations of biochemical indicators and cytokines were measured in the blood from 72 female and male pigs distributed across two weaning groups (nursed or weaned), and two gestational challenge groups (gilts infected with porcine reproductive and respiratory virus or healthy controls). Weaning had a significant effect on the concentration of albumin among females born from healthy gilts (P < 0.01). The albumin concentrations were higher in weaned relative to nursed pigs from healthy gilts. This pattern may be associated with the transition to a higher protein diet, whereas no differences were observed among pigs from infected gilts. The interaction between gestation and weaning stresses had a significant effect on the concentration of bicarbonate. The highest levels of bicarbonate were detected among nursed pigs from infected gilts. The lowest levels of bicarbonate were detected among weaned pigs from health gilts, and this could indicate metabolic acidosis. An interaction effect (P < 0.02) was detected for pro-inflammatory interleukin 4. The highest concentrations of interleukin 4 were detected in weaned females from infected gilts, while the lowest levels were detected among weaned males from health gilts. The studied biomarker patterns indicate that the effect of weaning on blood indicators can be influenced by the exposure of the pigs to challenges during gestation. This study is supported by USDA NIFA AFRI, grant number 2018-67015-27413.

Simple Summary The present study of blood chemical, cytokine and hormone indicators in male and female pigs offered insights into the combined effects of maternal immune activation and weaning stress. Our results indicate that maternal immune activation may grant higher tolerance to anion gap and bilirubin disruptions triggered by weaning stress. Therefore, management practices aimed at minimizing the effects of weaning should consider the interacting effect of immune activation during development. Abstract Weaning stress can elicit changes in the metabolic, hormone and immune systems of pigs and interact with prolonged disruptions stemming from maternal immune activation (MIA) during gestation. The present study advances the characterization of the combined effects of weaning stress and MIA on blood chemistry, immune and hormone indicators that inform on the health of pigs. Three-week-old female and male offspring of control gilts or gilts infected with the porcine reproductive and respiratory syndrome virus were allocated to weaned or nursed groups. The anion gap and bilirubin profiles suggest that MIA enhances tolerance to the effects of weaning stress. Interleukin 1 beta and interleukin 2 were highest among weaned MIA females, and cortisol was higher among weaned relative to nursed pigs across sexes. Canonical discriminant analysis demonstrated that weaned and nursed pigs have distinct chemistry profiles, whereas MIA and control pigs have distinct cytokine profiles. The results from this study can guide management practices that recognize the effects of the interaction between MIA and weaning stress on the performance and health of pigs.

Changes at the molecular level capacitate the plasticity displayed by the brain in response to stress stimuli. Weaning stress can trigger molecular changes that influence the physiology of the offspring. Likewise, maternal immune activation (MIA) during gestation has been associated with behavior disorders and molecular changes in the amygdala of the offspring. This study advances the understanding of the effects of pre- and postnatal stressors in amygdala gene networks. The amygdala transcriptome was profiled on female and male pigs that were either exposed to viral-elicited MIA or not and were weaned or nursed. Overall, 111 genes presented interacting or independent effects of weaning, MIA, or sex (FDR-adjusted P-value

In this work, we show that deep learning and SLIM can be combined to quickly deliver superior results in tissue screening applications. This concept of combining QPI label-free data with AI with the purpose of extracting molecular specificity has been recently introduced by our laboratory as phase imaging with computational specificity (PICS) [Nat. Comm., in press]. Training on ten thousand SLIM images of piglet brain tissue with the 71-layer transfer learning model Xception, we created a two-parameter classification to differentiate the gestational size: either appropriate for gestational age (AGA) or small for gestational age (SGA), and diet: either an experimental regimen high in hydrolyzed fats or a control diet, with an accuracy of 80% and 81%, respectively, and a four-parameter classification (diet and size) with 62% accuracy. These results are significant, as it would otherwise be impossible for a trained histopathologist to distinguish such discrepancies.

Inflammatory signals elicited in response to stressors during gestation can affect fetal development and can have prolonged postnatal effects in the offspring. The objectives of the present study were to assess the effects of viral infection during gestation on nursed piglets, and to investigate possible modulating effects of fish oil. A total of 36 pigs were studied, distributed across three gilt treatment groups and sexes within treatment group. One-third of the piglets served as reference, being born from gilts that were not exposed to viral infection and receiving a non-supplemented diet. Another third of the piglets were born from gilts that were challenged with porcine reproductive and respiratory virus during the last third of gestation and receiving a non-supplemented diet. The remaining piglets were born from gilts that were challenged with the virus and were supplemented with fish oil (2.5 g of docosahexaenoic acid and eicosapentaeonic acid). All gilts received diets that met nutritional requirements during gestation and lactation (~90% corn and soybean meal). The piglets remained with the gilts and nursed until 22 d of age at which time blood was drawn and the levels of metabolic parameters were measured. The immune challenge during gestation had a significant effect on the level of globulin (P < 0.02). The globulin levels were higher in female piglets from gilts infected during gestation relative to control gilts. The blood level of triglycerides was similar between piglets from reference gilts and viral-infected gilts supplemented with fish oil, and lower than in piglets from non-supplemented infected gilts. Our results demonstrate that fish oil supplementation can modulate effects of infection during gestation on the piglet. This study is supported by USDA NIFA AFRI, grant number 2018-67015-27413.

The prolonged and sex-dependent impact of maternal immune activation (MIA) on the molecular pathways of the amygdala, a brain region that influences social, cognitive, and sexually dimorphic behaviors, is only partially understood. To address this gap, the effect of MIA elicited by porcine reproductive and respiratory syndrome virus (PRRSV) infection during gestation on the amygdala transcriptome of pigs was studied. Gene expression levels were measured using RNA-Seq on the amygdala for 3-week-old female and male offspring from MIA and control groups. Among the 328 genes that exhibited an MIA-by-sex effect, the majority annotated to functional categories relevant to behavioral abnormalities, including neuroactive ligand-receptor pathways, glutamatergic functions, and neuropeptide systems. Genes in these categories included corticotropin releasing hormone receptor 2, glutamate metabotropic receptor 4, glycoprotein hormones, alpha polypeptide, parathyroid hormone 1 receptor, vasointestinal peptide receptor 2, neurotensin, proenkephalin, and gastrin releasing peptide. These genes and functional categories have been associated with MIA-related schizophrenia and autism spectrum behavior disorders. The transcript and network dysregulation uncovered in this study advances the understanding necessary to develop treatments that ameliorate the effects of neurodevelopmental disorders caused by gestational MIA exposure. This study is supported by USDA NIFA AFRI, grant number 2018-67015-27413.

Although both neurons and oligodendrocytes have been well studied individually, very little is known about how they interact with each other. New methods are needed to further study the intricacies of this interplay in terms of cellular and molecular dynamics. Spatial Light Interference Microscopy (SLIM) is a quantitative phase imaging technique that generates phase maps related to the dry mass content of the sample. In this work, we study the ability of SLIM to quantify myelination at the axonal level. We imaged a series of cocultures comprising hippocampal neurons and oligodendrocytes, of varying densities, using SLIM, and evaluated dry mass formation and growth of myelin.

Deficient myelination of the brain is associated with neurodevelopmental delays, particularly in high-risk infants, such as those born small in relation to their gestational age (SGA). New methods are needed to further study this condition. Here, we employ Color Spatial Light Interference Microscopy (cSLIM), which uses a brightfield objective and RGB camera to generate pathlength-maps with nanoscale sensitivity in conjunction with a regular brightfield image. Using tissue sections stained with Luxol Fast Blue, the myelin structures were segmented from a brightfield image. Using a binary mask, those portions were quantitatively analyzed in the corresponding phase maps. We first used the CLARITY method to remove tissue lipids and validate the sensitivity of cSLIM to lipid content. We then applied cSLIM to brain histology slices. These specimens are from a previous MRI study, which demonstrated that appropriate for gestational age (AGA) piglets have increased internal capsule myelination (ICM) compared to small for gestational age (SGA) piglets and that a hydrolyzed fat diet improved ICM in both. The identity of samples was blinded until after statistical analyses.

Sarcomas are rare and heterogeneous cancer variants of mesenchymal origin. Their genetic heterogeneity coupled with uncertain histogenesis makes them difficult to treat and results in poor prognosis. In this work, we show that structure-based drug discovery involving computational modeling can be used to identify a new retinoid X receptor (RXR) agonist ligand with a bis(indolyl)methane scaffold. This agent co-self-assembles with an amphiphilic diblock copolymer resulting in nanoparticles (Nano-RXR) with excellent kinetic stability, which were evaluated for efficacy and safety in transformed sarcoma cells, 63-3 Cre and 141-10 Cre of pig origin, and in rodent xenograft models. Responses at gene and protein levels established the treatment approach as a highly effective RXR agonist across cell, rodent, and "Oncopig" models. Interestingly, Nano-RXR was not only able to modulate metabolic and transporter genes related to orphan nuclear receptors but also played a major role in modulating programmed cell death in sarcomas developed in Oncopigs.

The hippocampus is involved in learning and memory and undergoes significant growth and maturation during the neonatal period. Environmental insults during this developmental timeframe can have lasting effects on brain structure and function. This study assessed hippocampal DNA methylation and gene transcription from two independent studies reporting reduced cognitive development stemming from early life environmental insults (iron deficiency and porcine reproductive and respiratory syndrome virus (PRRSv) infection) using porcine biomedical models. In total, 420 differentially expressed genes (DEGs) were identified between the reduced cognition and control groups, including genes involved in neurodevelopment and function. Gene ontology (GO) terms enriched for DEGs were associated with immune responses, angiogenesis, and cellular development. In addition, 116 differentially methylated regions (DMRs) were identified, which overlapped 125 genes. While no GO terms were enriched for genes overlapping DMRs, many of these genes are known to be involved in neurodevelopment and function, angiogenesis, and immunity. The observed altered methylation and expression of genes involved in neurological function suggest reduced cognition in response to early life environmental insults is due to altered cholinergic signaling and calcium regulation. Finally, two DMRs overlapped with two DEGs, VWF and LRRC32, which are associated with blood brain barrier permeability and regulatory T-cell activation, respectively. These results support the role of altered hippocampal DNA methylation and gene expression in early life environmentally-induced reductions in cognitive development across independent studies.

Costs and productivity losses associated with porcine reproductive and respiratory syndrome virus (PRRSV) outbreaks impact the swine industry. The objective of this study was to evaluate the effect of maternal immune activation (MIA) from a PRRSV challenge during gestation on the piglet serum concentrations of albumin and globulin, indicators of inflammation or organ damage. Eighteen pregnant Camborough gilts were inoculated with either PRRSV or saline on gestational day 76. After farrowing 106 piglets remained with the gilts until postnatal day (PD) 21. One group of piglets was weaned and group-housed while another group remained with the gilts until PD22. At PD 60, a group of weaned piglets was fasted for one day while the rest was fed to meet nutritional requirements. Serum concentrations of globulin and albumin were measured on PD 22 and 61 and were analyzed using a linear mixed model including the fixed effects of gilt challenge, piglet treatment and sex and the random effect of gilt. On PD 22, albumin was higher in weaned relative to suckled piglets from saline-treated gilts (P < 0.0305). Globulin was higher in weaned relative to suckled piglets (P < 0.0014) and in piglets from PRRSV-treated relative to saline-treated gilts (P < 0.0201). The albumin/globulin ratio was higher in weaned piglets from saline relative to PRRSV-treated gilts (P < 0.0367). On PD 61, albumin was higher in fasted relative to fed piglets (P < 0.0072). Globulin was higher in males relative to females from PRRSV-treated gilts (P < 0.0326), and the albumin/globulin ratio was higher in fasted relative to fed piglets (P < 0.0043). Our results indicate a positive correlation between serum protein concentrations and MIA, weaning or fasting stress that may be indicative of inflammation, organ dysfunction or nutritional imbalance. This study is supported by USDA NIFA AFRI, grant number 2018-67015-27413.

Maternal immune activation (MIA) during gestation can affect the ability of the offspring to cope with stress such as weaning. Cortisol, a glucocorticoid released in response to stress, is an established indicator of stress. The purpose of this study was to evaluate the cortisol response to weaning stress of offspring exposed to MIA during gestation. Camborough gilts were inseminated at approximately 235 days of age. On gestational day 76, gilts (n = 11) were inoculated with porcine reproductive and respiratory syndrome virus (PRRSV) intranasally whereas saline was intranasally administered to Control gilts (n = 7). On postnatal day (PD) 21 approximately half of the male and female piglets remained with sows (suckled) while the rest were weaned. Blood was collected from the piglets prior to weaning on PD21 and at PD22. ELISA was used to measure cortisol in the serum. The analysis of cortisol concentration tested for the fixed effects of gilt treatment (PRRSV or Control), piglet sex (female or male), management (suckled or weaned), day of sampling (PD21 or PD22), interactions, and for Normality assumptions. At PD22 female weaned piglets had a higher level of cortisol when compared to female suckled piglets in both Control (P < 0.05) and PRRSV (P < 0.05). Males exhibited a similar trend that did not reach significance. Cortisol concentrations were lower in male piglets from Control relative to PRRSV-treated gilts and the differences were more pronounced among the suckled group. The results suggest that MIA elevates basal serum cortisol in unstressed males. This study is supported by USDA NIFA AFRI, 2018-67015-27413.

Scope Activation of microglia, the resident immune cells of the central nervous system, has been related to the etiology and progression of neurodegenerative diseases; thus, finding novel approaches to suppress the neuroinflammatory process is of utmost relevance. Methods and results The anti-inflammatory activity of whey Cu-, Fe-, and Zn-binding peptides and their possible underlying mechanism of action were evaluated in microglia. Whey metal-binding peptides decreased nitric oxide production and tumor necrosis factor α (TNF-α) at mRNA and protein levels by stimulated BV-2 microglia in comparison to the control with no peptide treatment. The hydrophobicity, specific sequences, and possible synergistic effects seem to play a role. Cu-binding peptides (Cu-bp) presented anti-inflammatory activity both in BV-2 and primary microglia cultures. These peptides exert their action by suppressing nuclear factor kappa B (NF-kB) pathway since nuclear translocation of NF-kB p65 is decreased by roughly 30% upon Cu-bp treatment. Specific sequences identified in Cu-bp showed high affinity to bind NF-kB p65 by molecular docking (up to -8.8 kcal mol-1 ), corroborating the immunofluorescence studies. Conclusion Cu-bp represent food-derived peptides that may be useful for neuroprotective purposes. Chelation of copper excess in the CNS and the bioavailability of such peptides, as well as their behavior in in vivo models, deserve further research for future applications.

Inadequate myelination in the central nervous system is associated with neurodevelopmental complications. Thus, quantitative, high spatial resolution measurements of myelin levels are highly desirable. We used spatial light interference microcopy (SLIM), a highly sensitive quantitative phase imaging (QPI) technique, to correlate the dry mass content of myelin in piglet brain tissue with dietary changes and gestational size. We combined SLIM micrographs with an AI classifying model that allows us to discern subtle disparities in myelin distributions with high accuracy. This concept of combining QPI label-free data with AI for the purpose of extracting molecular specificity has recently been introduced by our laboratory as phase imaging with computational specificity (PICS). Training on nine thousand SLIM images of piglet brain tissue with the 71-layer transfer learning model Xception, we created a two-parameter classification to differentiate gestational size and diet type with an accuracy of 82% and 80%, respectively. To our knowledge, this type of evaluation is impossible to perform by an expert pathologist or other techniques.

Human soft-tissue sarcomas (STS) are rare mesenchymal tumors with a 5-year survival rate of 50%, highlighting the need for further STS research. Research has been hampered by limited human sarcoma cell line availability and the large number of STS subtypes, making development of STS cell lines and animal models representative of the diverse human STS subtypes critical. Pigs represent ideal human disease models due to their similar size, anatomy, metabolism, and genetics compared to humans. The Oncopig encodes inducible KRAS G12D and TP53 R167H transgenes, allowing for STS modeling in a spatial and temporal manner. This study utilized Oncopig STS cell line (fibroblast) and tumor (leiomyosarcoma) RNA-seq data to compare Oncopig and human STS expression profiles. Altered expression of 3,360 and 7,652 genes was identified in Oncopig STS cell lines and leiomyosarcomas, respectively. Transcriptional hallmarks of human STS were observed in Oncopig STS, including altered TP53 signaling, Wnt signaling activation, and evidence of epigenetic reprogramming. Furthermore, master regulators of Oncopig STS expression were identified, including FOSL1, which was previously identified as a potential human STS therapeutic target. These results demonstrate the Oncopig STS model’s ability to mimic human STS transcriptional profiles, providing a valuable resource for sarcoma research and cell line development.

Background The domestic pig (Sus scrofa) is important both as a food source and as a biomedical model given its similarity in size, anatomy, physiology, metabolism, pathology, and pharmacology to humans. The draft reference genome (Sscrofa10.2) of a purebred Duroc female pig established using older clone-based sequencing methods was incomplete, and unresolved redundancies, short-range order and orientation errors, and associated misassembled genes limited its utility. Results We present 2 annotated highly contiguous chromosome-level genome assemblies created with more recent long-read technologies and a whole-genome shotgun strategy, 1 for the same Duroc female (Sscrofa11.1) and 1 for an outbred, composite-breed male (USMARCv1.0). Both assemblies are of substantially higher (>90-fold) continuity and accuracy than Sscrofa10.2. Conclusions These highly contiguous assemblies plus annotation of a further 11 short-read assemblies provide an unprecedented view of the genetic make-up of this important agricultural and biomedical model species. We propose that the improved Duroc assembly (Sscrofa11.1) become the reference genome for genomic research in pigs.

Deficient myelination in the internal capsule of the brain is associated with neurodevelopmental delays, particularly in high-risk infants such as those born small for gestational age (SGA). New methods are needed to further study this condition and assess how it relates to early life nourishment. MRI technology has been effective at measuring brain growth and composition but lacks myelin specificity and is low resolution. The development of new quantitative approaches that are rapid and precise may complement MRI results with insight into the pathology of deficient myelination and efficacy of nutritional interventions. Color Spatial Light Interference Microscopy (cSLIM) uses a brightfield objective and RGB camera to generate phase map images in conjunction with a regular brightfield image. Using paraffin embedded brain tissue sections, stained myelin was segmented from a brightfield image and, with a binary mask, those portions were quantitatively analyzed in the corresponding phase maps. This technique was therefore sensitive to subtle variations in myelin density. The results of this study indicate a positive correlation between an experimental diet, rich in critical nutrients such as iron, and dry mass levels of myelin in the internal capsules of both appropriate (AGA) and SGA piglets. In summary, neonatal dietary treatments affect the degree of myelination in certain regions of the brain, irrespective of gestational size, and may therefore impact cognitive health.

The pork industry faces financial losses associated with outbreaks in porcine reproductive and respiratory virus (PRRSV). One type of loss stems from the effect of PRRSV-elicited maternal immune activation during gestation on the response of pigs to a second immune challenge later in life. The objective of this study was to assess the effects of PRRSV infection during gestation, a second immune challenge, and sex on pig behavior. Camborough gilts were inoculated with PRRSV or saline (Controls) during the last third of gestation. The piglets from these gilts were weaned at day 21, and were injected with Poly(I:C) to elicit a second immune challenge, similarly to a viral infection, or with Saline on day 60. Post injection, behaviors were recorded in 5-minute intervals for one hour by a trained experimenter. Recorded behaviors included laying, standing, and panting, and totaled 624 observations per behavior on 51 pigs from 10 gilts. The logistic generalized mixed effect model used to describe behavior included the effects of gilt and pig treatments, sex, and interactions, and accounted for the random effect of gilt, and repeated measurements within pig. Panting showed a significant gilt challenge-by-sex-by-pig treatment interaction effect (P < 0.031). The Poly(I:C) challenge had a significant and positive effect on panting among male pigs from Control gilts (P < 0.001), while this effect was not observed in pigs from PRRSV-treated gilts. Among female pigs challenged with Poly(I:C), PRRSV treatment has a significant effect on lateral laying (P < 0.015) and standing (P < 0.042). Pigs from PRRSV-treated gilts were less active than from Control gilts. Our results highlight that the effect on behaviors of immune challenge later in life depend on the exposure of pigs to PRRSV during gestation. This study is supported by USDA NIFA AFRI, grant number 2018-67015-27413.

The synthetic polyinosinic-polycytidylic acid Poly(I:C) is commonly used to mimic viral infection in rodents. A Poly(I:C) injection activates inflammatory receptors that recognize viral patterns. This immune-stimulating agent can be used to understand the effects of viral infection on pork production. The objective of this study was to compare the effects of multiple Poly(I:C) doses on sickness behaviors across time. Female and male pigs from the University of Illinois Swine herd, averaging 24.5 kg, were injected with 1 mg/kg or 0.5 mg/kg of Poly(I:C), or comparable volume of Saline at approximately 60 days of age. Post injection, sickness behaviors and activity levels were measured in 15-minute intervals by a trained experimenter for three hours. Observed sickness behaviors included vomiting, diarrhea, or shivering. The activity levels were scored 1 through 8 with lower levels indicating pigs running, medium levels indicating walking, and higher levels indicating laying behaviors. The binary sickness behavior and discrete activity levels from 130 observations from 10 pigs evenly distributed across sexes were analyzed using generalized mixed effect models. The model included the effects of Poly(I:C) dose, hour, sex, and interactions, and a repeated structure within pig. The administration of Poly(I:C) had a significant effect on activity level within one-hour post-injection, and Poly(I:C)-treated pigs had lower activity levels than the Saline group. The Poly(I:C) effect had a lower significance in males (P < 0.001) than females (P < 0.004). The Poly(I:C) treatment also had a significant effect on the probability of sickness behavior (P < 0.005). The probability of sickness behavior was 0.03, 0.18, and 0.23 for pigs treated with Saline, 0.5, and 1.0 mg/kg of Poly(I:C), respectively. Our results suggest that Poly(I:C) could be an effective agent in studying sickness response to viral infections in pigs. This study is supported by USDA NIFA AFRI, grant number 2018-67015-27413.

Maternal immune activation (MIA) during gestation can alter immune responses of offspring to many stressors including weaning. Interleukin (IL)-10 and IL-6 are anti- and pro-inflammatory cytokines, respectively, released during the immune response. This study assessed the effects of MIA on piglets’ serum cytokine concentrations in response to weaning. Camborough gilts were inseminated at approximately 235 days of age and were inoculated intranasally on gestational day 76 with porcine reproductive and respiratory syndrome virus (PRRSV) (n = 11) or saline (n = 7). On postnatal day (PD) 21 approximately half of the piglets remained with sows (suckled) while the rest were weaned. Blood was collected from piglets prior to weaning on PD21 and at PD22. Serum concentrations of IL-6 and IL-10 were measured by ELISA. Cytokine concentrations were tested for fixed effects of gilt treatment (PRRSV or Control), piglet sex (female or male), management (suckled or weaned), sampling day (PD21 or PD22), interactions, and Normality assumptions. IL-10 presented a significant interaction between sex, gilt treatment, and management at PD22 (P < 0.05). Weaned males from PRRSV-treated gilts had higher IL-10 concentrations versus suckled males whereas females from PRRSV-treated gilts had comparable levels of IL-10 between management groups. Overall, IL-10 was higher in piglets from PRRSV-treated gilts relative to Control gilts and matching sex and management groups. Higher IL-6 concentrations were found in weaned males from PRRSV-treated gilts versus suckled males at PD22. This pattern between management groups was reversed in females from PRRSV-treated gilts. Among piglets from Control gilts, males possessed higher IL-6 concentrations versus females across management groups. These results suggest MIA can alter inflammatory cytokine concentrations differently between males and females in response to weaning. This altered response may reduce offspring resiliency to subsequent immune or management stressors. This study is supported by USDA NIFA AFRI, 2018-67015-27413.

Porcine reproductive and respiratory syndrome virus (PRRSV) costs the U.S. pork industry millions of dollars yearly due to production losses and management costs. The objective of this study was to evaluate the effects of maternal immune activation (MIA) elicited by PRRSV infection during gestation on the behavioral response of the offspring to subsequent immune challenge. On gestational day 76, PRRSV negative Camborough gilts (n = 6) were either inoculated with PRRSV or saline intranasally. Nineteen female and male piglets from these PRRSV positive and Control gilts were weaned at day 21 and subsequently housed in groups. At day 60, piglets were randomized by sex to receive either saline or an intraperitoneal injection of Poly(I:C) to represent immune challenge. Poly(I:C) is a synthetic polyinosinic-polycytidylic acid that mimics double-stranded RNA that activates host defense similar to viral infections by stimulating the release of cytotoxic cytokines and inducing interferon-gamma production. Post injection, binary laying, eating, vomiting, panting, and overall sickness behaviors were collected by instantaneous scan sampling at 1.5-minute intervals for 60 minutes by a trained experimenter blinded to the experimental design. Fisher’s exact test and Cochran-Mantel-Haenszel statistics were used to test the effects of gilt treatment, piglet challenge, and sex within and across factor levels. Male piglets from PRRSV-treated gilts exhibited significantly (P < 0.0455) fewer sickness behaviors than piglets from control gilts between 31 and 45 minutes post-challenge. Significant associations between gilt treatment and both panting (P < 0.0162) and overall sickness behavior (P < 0.0245), while controlling for sex and piglet challenge, were observed. This study characterized the MIA and sex-dependent nature of the effect Poly(I:C) immune challenge on behavior. Our results indicate that MIA is correlated to increased behavioral response to secondary viral infection in male offspring. This study is supported by USDA NIFA AFRI, grant number 2018-67015-27413.

Although survival has improved in recent years, the prognosis of patients with advanced pancreatic ductal adenocarcinoma (PDAC) remains poor. Despite substantial differences in anatomy, physiology, genetics, and metabolism, the overwhelming majority of preclinical testing relies on transgenic mice. Hence, while mice have allowed for tremendous advances in cancer biology, they have been a poor predictor of drug performance/toxicity in the clinic. Given the greater similarity of sus scrofa pigs to humans, we engineered transgenic sus scrofa expressing a LSL-KRASG12D-TP53R167H cassette. By applying Adeno-Cre to pancreatic duct cells in vitro, cells self-immortalized and established tumors in immunocompromised mice. When Adeno-Cre was administered to the main pancreatic duct in vivo, pigs developed extensive PDAC at the injection site hallmarked by excessive proliferation and desmoplastic stroma. This serves as the first large animal model of pancreatic carcinogenesis, and may allow for insight into new avenues of translational research not before possible in rodents.

OBJECTIVEMinimally invasive procedures may allow surgeons to avoid conventional open surgical procedures for certain neurological disorders. This paper describes the iterative process for development of a catheter-based ultrasound thermal therapy applicator.METHODSUsing an ultrasound applicator with an array of longitudinally stacked and angularly sectored tubular transducers within a catheter, the authors conducted experimental studies in porcine liver, in vivo and ex vivo, in order to characterize the device performance and lesion patterns. In addition, they applied the technique in a rodent model of Parkinson’s disease to investigate the feasibility of its application in brain.RESULTSThermal lesions with multiple shapes and sizes were readily achieved in porcine liver. The feasibility of catheter-based focused ultrasound in the treatment of brain conditions was demonstrated in a rodent model of Parkinson’s disease.CONCLUSIONSThe authors show proof of principle of a catheter-based ultrasound system that can create lesions with concurrent thermode-based measurements.

The pig was first used in biomedical research in ancient Greece and over the past few decades has quickly grown into an important biomedical research tool. Pigs have genetic and physiological traits similar to humans, which make them one of the most useful and versatile animal models. Owing to these similarities, data generated from porcine models are more likely to lead to viable human treatments than those from murine work. In addition, the similarity in size and physiology to humans allows pigs to be used for many experimental approaches not feasible in mice. Research areas that employ pigs range from neonatal development to translational models for cancer therapy. Increasing numbers of porcine models are being developed since the release of the swine genome sequence, and the development of additional porcine genomic and epigenetic resources will further their use in biomedical research.

Despite an improved understanding of cancer molecular biology, immune landscapes, and advancements in cytotoxic, biologic, and immunologic anti-cancer therapeutics, cancer remains a leading cause of death worldwide. More than 8.2 million deaths were attributed to cancer in 2012, and it is anticipated that cancer incidence will continue to rise, with 19.3 million cases expected by 2025. The development and investigation of new diagnostic modalities and innovative therapeutic tools is critical for reducing the global cancer burden. Toward this end, transitional animal models serve a crucial role in bridging the gap between fundamental diagnostic and therapeutic discoveries and human clinical trials. Such animal models offer insights into all aspects of the basic science-clinical translational cancer research continuum (screening, detection, oncogenesis, tumor biology, immunogenicity, therapeutics, and outcomes). To date, however, cancer research progress has been markedly hampered by lack of a genotypically, anatomically, and physiologically relevant large animal model. Without progressive cancer models, discoveries are hindered and cures are improbable. Herein, we describe a transgenic porcine model-the Oncopig Cancer Model (OCM)-as a next-generation large animal platform for the study of hematologic and solid tumor oncology. With mutations in key tumor suppressor and oncogenes, TP53R167H and KRASG12D , the OCM recapitulates transcriptional hallmarks of human disease while also exhibiting clinically relevant histologic and genotypic tumor phenotypes. Moreover, as obesity rates increase across the global population, cancer patients commonly present clinically with multiple comorbid conditions. Due to the effects of these comorbidities on patient management, therapeutic strategies, and clinical outcomes, an ideal animal model should develop cancer on the background of representative comorbid conditions (tumor macro- and microenvironments). As observed in clinical practice, liver cirrhosis frequently precedes development of primary liver cancer or hepatocellular carcinoma. The OCM has the capacity to develop tumors in combination with such relevant comorbidities. Furthermore, studies on the tumor microenvironment demonstrate similarities between OCM and human cancer genomic landscapes. This review highlights the potential of this and other large animal platforms as transitional models to bridge the gap between basic research and clinical practice.

Betaine-homocysteine S-methyltransferase (BHMT) activity is only detected in the liver of rodents, but in both the liver and kidney cortex of humans and pigs; therefore, the pig was chosen as a model to define the spatial and temporal expression of BHMT during development. During fetal development, a total of ten splice variants of bhmt were expressed at varying levels across a wide range of porcine tissues. Two variants contained an identical ORF that encoded a C-terminal truncated form of BHMT (tBHMT). The bhmt transcripts were expressed at significant levels in the liver and kidney from day 45 of gestation (G45) onward. The transcripts encoding tBHMT represented 5-13% of the total bhmt transcripts in G30 fetus, G45 liver, and adult liver and kidney cortex. The dominant structural feature of wild type BHMT is an (βα)8 barrel, however, a modeled structure of tBHMT suggests that this protein would assume a horseshoe fold and lack methyltransferase activity. Low BHMT activity was detected in the G30 fetus, and slightly increased levels of activity were observed in the liver from G45 and G90 fetuses. The bhmt promoter contained three key CpG sites, and methylation of these sites was significantly higher in adult lung compared to adult liver. The data reported herein suggest that genomic DNA methylation and variation of the 5' and 3' UTRs of bhmt transcripts are key regulators for the level of BHMT transcription and translation.

Background Iron deficiency is a common childhood micronutrient deficiency that results in altered hippocampal function and cognitive disorders. However, little is known about the mechanisms through which neonatal iron deficiency results in long lasting alterations in hippocampal gene expression and function. DNA methylation is an epigenetic mark involved in gene regulation and altered by environmental factors. In this study, hippocampal DNA methylation and gene expression were assessed via reduced representation bisulfite sequencing and RNA-seq on samples from a previous study reporting reduced hippocampal-based learning and memory in a porcine biomedical model of neonatal iron deficiency. Results In total 192 differentially expressed genes (DEGs) were identified between the iron deficient and control groups. GO term and pathway enrichment analysis identified DEGs associated with hypoxia, angiogenesis, increased blood brain barrier (BBB) permeability, and altered neurodevelopment and function. Of particular interest are genes previously implicated in cognitive deficits and behavioral disorders in humans and mice, including HTR2A, HTR2C, PAK3, PRSS12, and NETO1. Altered genome-wide DNA methylation was observed across 0.5 million CpG and 2.4 million non-CpG sites. In total 853 differentially methylated (DM) CpG and 99 DM non-CpG sites were identified between groups. Samples clustered by group when comparing DM non-CpG sites, suggesting high conservation of non-CpG methylation in response to neonatal environment. In total 12 DM sites were associated with 9 DEGs, including genes involved in angiogenesis, neurodevelopment, and neuronal function. Conclusions Neonatal iron deficiency leads to altered hippocampal DNA methylation and gene regulation involved in hypoxia, angiogenesis, increased BBB permeability, and altered neurodevelopment and function. Together, these results provide new insights into the mechanisms through which neonatal iron deficiency results in long lasting reductions in cognitive development in humans. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3216-y) contains supplementary material, which is available to authorized users.

Growth and carcass traits are of great economic importance in livestock production. A large number of quantitative trait loci (QTL) have been identified for growth and carcass traits on porcine chromosome one (SSC1). A key positional candidate for this chromosomal region is TGFBR1 (transforming growth factor beta type I receptor). This gene plays a key role in inherited disorders at cardiovascular, craniofacial, neurocognitive, and skeletal development in mammals.In this study, 27 polymorphic SNPs in the porcine TGFBR1 gene were identified on the University of Illinois Yorkshire × Meishan resource population. Three SNPs (SNP3, SNP43, SNP64) representing major polymorphic patterns of the 27 SNPs in F1 and F0 individuals of the Illinois population were selected for analyses of QTL association and genetic diversity. An association analysis for growth and carcass traits was completed using these three representative SNPs in the Illinois population with 298 F2 individuals and a large commercial population of 1008 animals. The results indicate that the TGFBR1 gene polymorphism (SNP64) is significantly associated (p0.05) with growth rates including average daily gains between birth and 56 kg (p = 0.049), between 5.5 and 56 kg (p = 0.024), between 35 and 56 kg (p = 0.021). Significant associations (p0.05) were also identified between TGFBR1 gene polymorphisms (SNP3/SNP43) and carcass traits including loin-eye-area (p = 0.022) in the Illinois population, and back-fat thickness (p = 0.0009), lean percentage (p = 0.0023) and muscle color (p = 0.021) in the commercial population. These three SNPs were also used to genotype a diverse panel of 130 animals representing 11 pig breeds. Alleles SNP3_T and SNP43_G were fixed in Pietrain and Sinclair pig breeds. SNP64_G allele was uniquely identified in Chinese Meishan pigs. Strong evidence of association (p0.01) between both SNP3 and SNP64 alleles and reproductive traits including gestation length and number of corpora lutea were also observed in the Illinois population.This study gives the first evidence of association between the porcine TGFBR1 gene and traits of economic importance and provides support for using TGFBR1 markers for pig breeding and selection programs. The genetic diversities in different pig breeds would be helpful to understand the genetic background and migration of the porcine TGFBR1 gene.

Despite the potential benefits of selective redox-modulating strategies for cancer therapy, an efficacious methodology for testing therapies remains elusive because of the difficulty in measuring intracellular redox potentials over time. In this report, we have incorporated a new FRET-based biosensor to follow in real time redox-sensitive processes in cells transformed to be tumorigenic and cultured in a microfluidic channel. A microfluidic network was used to control micro-scale flow near the cells and at the same time deliver drugs exogenously. Subsequently, the response of a redox homeostasis circuit was tested, namely reduced glutathione (GSH)/oxidized glutathione(GSSG), to diamide, a thiol oxidant, and two drugs used for cancer therapies: BSO (L-buthionine-[SR]-sulfoximine) and BCNU (carmustine). The main outcome from these experiments is a comparison of the temporal depletion and recovery of GSH in single living cells in real-time. These data demonstrate that mammalian cells are capable of restoring a reduced intracellular redox environment in minutes after an acute oxidative insult is removed. This recovery is significantly delayed by (i) the inhibition of GSH biosynthesis by BSO; (ii) the inactivation of glutathione reductase by BCNU; and (iii) in tumorigenic cells relative to an isogenic non-tumorigenic control cell line.

Soft-tissue sarcomas (STS) are aggressive, often lethal mesenchymal tumors that represent roughly 1% of adult cancers. The 5-year survival rate of STS has remained unchanged for decades at 50%, highlighting the need for more effective therapies. We are developing protocols and assessing the treatment efficacy of 3D spatially-registered real-time image-guided needle/catheter based ultrasound (CBUS) ablative thermal therapy in STS induced in the transgenic Oncopig cancer model (OCM). The transgenic Oncopig carries a Cre recombinase inducible transgene encoding KRASG12D and TP53R167H; both mutations commonly in human cancers. Cre recombinase induces expression of the transgenes and transformation of cells in culture and rapid and reproducible development of leiomyosarcomas when introduced into the muscle of oncopigs. The flexibility of the model allowed induction of sarcomas in two clinically relevant intramuscular (IM) sites in both the hind limb and retroperitoneal (RP) regions, to closely simulate human disease. We have previously shown that these Oncopig tumors present the same transcriptional hallmarks as human STS including altered TP53 signaling, Wnt signaling activation and evidence of epigenetic reprogramming. Initial acute studies revealed that the tumors could be successfully grown in the muscle reaching mean tumor volume of 19.76cm3 (RP, n= 8) and 25.12cm3 (IM, n=14) cm3 within two weeks following AdCre (5.0x108 to 1.0x109 PFU) injections. Tumors were then treated utilizing ultrasound image guidance combined with 3D EM tracking to place the applicator in the target region. Sectored tubular transducers were introduced through a small skin incision and used to precisely deliver thermal energy to the treatment region. Tumors were treated for 6-9 minutes at 7 watts acoustic power (10.2W/cm2). Thermocouples were inserted into the tumors monitored temperature throughout the treatment. Post-treatment analysis revealed that the mean induced lesion volume exceeded the actual tumor volume for both IM (43.41 cm3) and RP (22.87 cm3) tumors with mean peak temperatures reaching 64.56 ° C (IM) and 58.75 ° C (RP). The mean peak thermal dose (thermal equivalent minutes at 43 ° C - TEM43) accumulated for IM tumors was 3.70E+08, and 1.46E+06 for RP tumors. Histopathology analysis verified that treatment areas reached past tumor margins using single applicator configuration. This study clearly demonstrates that the Oncopig is a suitable model in size and clinically relevant tumor type for evaluation of ultrasound ablation instrumentation. In addition, these STS treatment protocols can reliably and accurately target and treat tumor volumes plus surrounding margins. Future work will include monitoring of treatment lesions for up to 3 months post ablation. Funded by NIH 5R21CA195433 Citation Format: Laurie A. Rund, Goutam Ghoshal, Emery Williams, Paul Neubauer, Lance Frith, R Tamas Heffter, Patrick Roady, Lawrence B. Schook, E Clif Burdette. Image-guided catheter-based ultrasound thermal ablation of intramuscular and retroperitoneal sarcomas in the transgenic Oncopig cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2148.

The Oncopig cancer model (OCM) was developed to support a broad range of solid and liquid tumor malignancies while providing the appropriate anatomical and physiological models for preclinical and co-clinical trials to expedite human clinical trials. The OCM develops tumors following Cre recombinase induced expression of KRASG12D and TP53R167H transgenes in a temporally and spatially controlled manner. To date, in vitro transformation and in vivo tumor formation of soft tissue sarcomas (STS), hepatocellular carcinoma (HCC), and pancreatic ductal adenocarcinoma (PDAC) have been demonstrated. However, the OCM was designed to generate cancers of all tissue origins. To demonstrate the potential importance of the OCM, we have successfully isolated and transformed multiple OCM cell types, each a progenitor of clinically relevant human disease. Cell type specific cell isolations were performed, and cells cultured under appropriate conditions. Within the first 48 hours, isolated cells were treated with adenoviral vector encoding Cre recombinase (AdCre) at 200-400 MOI for 5 hours in low serum (5%) medium. Both control and transformed (AdCre treated) cells underwent basic histopathologic screening (H&E staining, immunohistochemical staining for vimentin and cytokeratin), and verification of transgene expression (RT-PCR) and tumorigenicity (xenograft into SCID mice). Cell types isolated include fibroblasts, hepatocytes, pancreatic ductal cells, dermal epithelial cells, splenocytes, ovarian surface epithelial cells, fallopian tube secretory epithelial cells, renal proximal tubule epithelial cells, urinary bladder epithelial cells, bone marrow (without specific cell isolation), and testis (no specific cell isolation). Each cell type was successfully transformed, as shown by in vitro phenotypic changes and tumor formation in vivo. Additional gene expression analysis (RNA-Seq) of the STS and HCC cell lines verified the OCM transformed cells recapitulate transcriptional hallmarks of human cancers. Consistent with human STS, OCM STS cell lines display altered TP53 signaling, Wnt signaling activation, and evidence of epigenetic reprogramming. OCM HCC cell lines show transcriptional evidence of TERT reactivation, apoptosis evasion, angiogenesis activation, and Wnt signaling activation, all similar to human HCC. While not an exhaustive list of Oncopig cell types, we have not encountered cells resistant to malignant transformation through Cre recombinase exposure. These results support the unprecedented potential for modeling of additional cancer types, including colorectal, ovarian, fallopian tube, uterine, renal, bladder, skin, and hematological cancers to support and expand preclinical, translational, and co-clinical investigations. Citation Format: Laurie A. Rund, Kyle M. Schachtschneider, Ron C. Gaba, Daniel R. Principe, Paul Grippo, Regina M. Schwind, Howard Ozer, Lawrence B. Schook. Oncopig carcinoma cell lines: A foundation for co-clinical trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3105.

BRCA1 is a breast and ovarian tumor suppressor. Hereditary mutations in BRCA1 result in a predisposition to breast cancer, and BRCA1 expression is down-regulated in ~30% of sporadic cases. The function of BRCA1 remains poorly understood, but it appears to play an important role in DNA repair and the maintenance of genetic stability. Mouse models of BRCA1 deficiency have been developed in an attempt to understand the role of the gene in vivo. However, the subtle nature of BRCA1 function and the well-known discrepancies between human and murine breast cancer biology and genetics may limit the utility of mouse systems in defining the function of BRCA1 in cancer and validating the development of novel therapeutics for breast cancer. In contrast to mice, pig biological systems and cancer genetics appear to more closely resemble their human counterparts. To determine if BRCA1 inactivation in pig cells promotes their transformation and may serve as a model for the human disease, we developed an immortalized porcine breast cell line and stably inactivated BRCA1 using miRNA. The cell line developed characteristics of breast cancer stem cells and exhibited a transformed phenotype. These results validate the concept of using pigs as a model to study BRCA1 defects in breast cancer and establish the first porcine breast tumor cell line.