Neonatal Pigs Research Articles

Establishment of a porcine bronchial epithelial cell line and its application to study innate immunity in the respiratory epithelium.

In vitro culture models that precisely mirror the porcine respiratory epithelium are needed to gain insight into how pathogens and host interact. In this study, a new porcine bronchial epithelial cell line, designated as PBE cells, was established from the respiratory tract of a neonatal pig. PBE cells assumed a cobblestone-epithelial like morphology with close contacts between the cells when they reached confluence. The PBE cell line was characterized in terms of its expression of pattern recognition receptors (PRRs) and its ability to respond to the activation of the Toll-like receptor 3 (TLR3) and TLR4 signaling pathways, which are key PRRs involved in the defense of the respiratory epithelium against pathogens. PBE cells stimulated with poly(I:C) were able to up-regulate the expression of IFN-β, IFN-λ1 (IL-29), IFN-λ3 (IL-28B), the antiviral factors Mx1, OAS1, and PKR, as well as the viral PRRs RIG-1 and MDA5. The expression kinetics studies of immune factors in PBE cells allow us to speculate that this cell line can be a useful in vitro tool to investigate treatments that help to potentiate antiviral immunity in the respiratory epithelium of the porcine host. In addition, poly(I:C) and LPS treatments increased the expression of the inflammatory cytokines TNF-α, IL-6, IL-8, and MCP-1/CCL2 and differentially modulated the expression of negative regulators of the TLR signaling pathways. Then, PBE cells may also allow the evaluation of treatments that can regulate TLR3- and TLR4-mediated inflammatory injury in the porcine airway, thereby protecting the host against harmful overresponses.

Activation of renal vascular smooth muscle TRPV4 channels by 5-hydroxytryptamine impairs kidney function in neonatal pigs.

Control of microvascular reactivity by 5-hydroxytryptamine (5-HT; serotonin) is complex and may depend on vascular bed type and 5-HT receptors. 5-HT receptors consist of seven families (5-HT1-5-HT7), with 5-HT2 predominantly mediating renal vasoconstriction. Cyclooxygenase (COX) and smooth muscle intracellular Ca2+ levels ([Ca2+]i) have been implicated in 5-HT-induced vascular reactivity. Although 5-HT receptor expression and circulating 5-HT levels are known to be dependent on postnatal age, control of neonatal renal microvascular function by 5-HT is unclear. In the present study, we demonstrate that 5-HT stimulated human TRPV4 transiently expressed in Chinese hamster ovary cells. 5-HT2A is the predominant 5-HT2 receptor subtype in freshly isolated neonatal pig renal microvascular smooth muscle cells (SMCs). HC-067047 (HC), a selective TRPV4 blocker, attenuated cation currents induced by 5-HT in the SMCs. HC also inhibited the 5-HT-induced increase in renal microvascular [Ca2+]i and constriction. Intrarenal artery infusion of 5-HT had minimal effects on systemic hemodynamics but reduced renal blood flow (RBF) and increased renal vascular resistance (RVR) in the pigs. Transdermal measurement of glomerular filtration rate (GFR) indicated that kidney infusion of 5-HT reduced GFR. HC and 5-HT2 receptor antagonist ritanserin attenuated 5-HT effects on RBF, RVR, and GFR. Moreover, the serum and urinary COX-1 and COX-2 levels in 5-HT-treated piglets were unchanged compared with the control. These data suggest that activation of renal microvascular SMC TRPV4 channels by 5-HT impairs kidney function in neonatal pigs independently of COX production.

Review: Nutritional regulation of muscle growth in neonatal swine.

Despite advances in the nutritional support of low birth weight and early-weaned piglets, most experience reduced extrauterine growth performance. To further optimize nutritional support and develop targeted intervention strategies, the mechanisms that regulate the anabolic response to nutrition must be fully understood. Knowledge gained in these studies represents a valuable intersection of agriculture and biomedical research, as low birth weight and early-weaned piglets face many of the same morbidities as preterm and low birth weight infants, including extrauterine growth faltering and reduced lean growth. While the reasons for poor growth performance are multifaceted, recent studies have increased our understanding of the role of nutrition in the regulation of skeletal muscle growth in the piglet. The purpose of this review is to summarize the published literature surrounding advances in the current understanding of the anabolic signaling that occurs after a meal and how this response is developmentally regulated in the neonatal pig. It will focus on the regulation of protein synthesis, and especially the upstream and downstream effectors surrounding the master protein kinase, mechanistic target of rapamycin complex 1 (mTORC1) that controls translation initiation. It also will examine the regulatory pathways associated with the postprandial anabolic agents, insulin and specific amino acids, that are upstream of mTORC1 and lead to its activation. Lastly, the integration of upstream signaling cascades by mTORC1 leading to the activation of translation initiation factors that regulate protein synthesis will be discussed. This review concludes that anabolic signaling cascades are stimulated by both insulin and amino acids, especially leucine, through separate pathways upstream of mTORC1, and that these stimulatory pathways result in mTORC1 activation and subsequent activation of downstream effectors that regulate translation initiation Additionally, it is concluded that this anabolic response is unique to the skeletal muscle of the neonate, resulting from increased sensitivity to the rise in both insulin and amino acid after a meal. However, this response is dampened in skeletal muscle of the low birth weight pig, indicative of anabolic resistance. Elucidation of the pathways and regulatory mechanisms surrounding protein synthesis and lean growth allow for the development of potential targeted therapeutics and intervention strategies both in livestock production and neonatal care.

Synthesis of glycine from 4-hydroxyproline in tissues of neonatal pigs.

Glycine from sow's milk only meets 20% of the requirement of suckling piglets. However, how glycine is synthesized endogenously in neonates is not known. This study determined glycine synthesis from 4-hydroxyproline (an abundant amino acid in milk and neonatal blood) in tissues of sow-reared piglets with normal birth weights. Piglets were euthanized at 0, 7, 14 and 21 days of age, and their tissues were used to determine glycine synthesis from 0 to 5 mM 4-hydroxyproline, activities and mRNA expression of key glycine-synthetic enzymes, and their cell-specific localization. Activities of 4-hydroxyproline oxidase (OH-POX), proline oxidase (POX), serine hydroxymethyltransferase (SHMT), threonine dehydrogenase (TDH), alanine:glyoxylate transaminase (AGT), and 4-hydroxy-2-oxoglutarate aldolase (HOA) occurred in the kidneys and liver from all age groups of piglets, and in the pancreas of 7- to 21-day-old piglets. Activities of OH-POX and HOA were absent from the small intestine of newborn pigs but present in the small intestine of 7- to 21-day-old piglets and in the skeletal muscle of 14- to 21-day-old piglets. Between days 0 and 21 of age, the enzymatic activities of OH-POX, AGT, and HOA decreased in the liver and kidneys but increased in the pancreas and small intestine with age. The mRNA levels of these three enzymes changed in a manner similar to their enzymatic activities. In contrast to OH-POX, AGT, and HOA, the enzymatic activities of POX, SHMT, and TDH were present in the kidneys, liver, and intestine of all age groups of piglets. Glycine was synthesized from 0.1 to 5 mM 4-hydroxyproline in the liver and kidney from 0- to 21-day-old piglets, as well as the pancreas, small intestine, and skeletal muscle from 14- to 21-day-old piglets in a concentration-dependent manner. Collectively, our findings indicate that 4-hydroxyproline is used for the synthesis of glycine in tissues of piglets to compensate for the deficiency of glycine in milk.

Feeding medium-chain fatty acid-rich formula causes liver steatosis and alters hepatic metabolism in neonatal pigs.

Medium-chain fatty acids (MCFA) and long-chain fatty acids (LCFAs) are often added to enhance the caloric value of infant formulas. Evidence suggests that MCFAs promote growth and are preferred over LCFAs due to greater digestibility and ease of absorption. Our hypothesis was that MCFA supplementation would enhance neonatal pig growth to a greater extent than LCFAs. Neonatal pigs (n = 4) were fed a low-energy control (CONT) or two isocaloric high-energy formulas containing fat either from LCFAs, or MCFAs for 20 days. Pigs fed the LCFAs had greater body weight compared with CONT- and MCFA-fed pigs (P < 0.05). In addition, pigs fed the LCFAs and MCFAs had more body fat than those in the CONT group. Liver and kidney weights as a percentage of body weight were greater (P ≤ 0.05) for pigs fed the MCFAs than those fed the CONT formula, and in those fed LCFAs, liver and kidney weights as a percentage of body weight were intermediate (P ≤ 0.05). Pigs in the CONT and LCFA groups had less liver fat (12%) compared with those in the MCFA (26%) group (P ≤ 0.05). Isolated hepatocytes from these pigs were incubated in media containing [13C]tracers of alanine, glucose, glutamate, and propionate. Our data suggest alanine contribution to pyruvate is less in hepatocytes from LCFA and MCFA pigs than those in the CONT group (P < 0.05). These data suggest that a formula rich in MCFAs caused steatosis compared with an isocaloric LCFA formula. In addition, MCFA feeding can alter hepatocyte metabolism and increase total body fat without increasing lean deposition.NEW & NOTEWORTHY Our data suggest that feeding high-energy MCFA formula resulted in hepatic steatosis compared with isoenergetic LCFA or low-energy formulas. Steatosis coincided with greater laurate, myristate, and palmitate accumulation, suggesting elongation of dietary laurate. Data also suggest that hepatocytes metabolized alanine and glucose to pyruvate, but neither entered the tricarboxylic acid (TCA) cycle. In addition, the contribution of alanine and glucose was greater for the low-energy formulas compared with the high-energy formulas.

Sertoli Cells Express Accommodation, Survival, and Immunoregulatory Factors When Exposed to Normal Human Serum.

Transplantation is a clinical procedure that treats a variety of diseases yet is unattainable for many patients due to a nationwide organ shortage and the harsh side effects of chronic immune suppression. Xenografted pig organs are an attractive alternative to traditional allografts and would provide an endless supply of transplantable tissue, but transplants risk rejection by the recipient's immune system. An essential component of the rejection immune response is the complement system. Sertoli cells, an immunoregulatory testicular cell, survive complement as xenografts long term without any immune suppressants. We hypothesized that exposure to the xenogeneic complement influences Sertoli cell gene expression of other accommodation factors that contribute to their survival; thus, the purpose of this study was to describe these potential changes in gene expression. RNA sequencing of baseline neonatal pig Sertoli cells (NPSC) as compared to NPSC after exposure to normal human serum (NHS, containing complement) revealed 62 significantly differentially expressed genes (DEG) that affect over 30 pathways involved in immune regulation, cell survival, and transplant accommodation. Twelve genes of interest were selected for further study, and Sertoli cell protein expression of CCL2 and the accommodation factor A20 were confirmed for the first time. Functional pathway analyses were conducted in NPSC and three biological clusters were revealed as being considerably affected by NHS exposure: innate immune signaling, cytokine signaling, and T cell regulation. Better understanding of the interaction of Sertoli cells with complement in a xenograft environment may reveal the mechanisms behind immune-privileged systems to increase graft viability.

Mitochondrial transplantation preserves myocardial function and viability in pediatric and neonatal pig hearts donated after circulatory death

ObjectiveMitochondrial transplantation has been shown to preserve myocardial function and viability in adult porcine hearts donated after circulatory death (DCD) . Herein, we investigate the efficacy of mitochondrial transplantation for the preservation of myocardial function and viability in neonatal and pediatric porcine DCD heart donation. MethodsCirculatory death was induced in neonatal and pediatric Yorkshire pigs by cessation of mechanical ventilation. Hearts underwent 20 or 36 minutes of warm ischemia time (WIT), 10 minutes of cold cardioplegic arrest, and then were harvested for ex situ heart perfusion (ESHP). Following 15 minutes of ESHP, hearts received either vehicle (VEH) or vehicle containing isolated autologous mitochondria (MITO). A sham nonischemic group (SHAM) did not undergo WIT, mimicking donation after brain death heart procurement. Hearts underwent 2 hours each of unloaded and loaded ESHP perfusion. ResultsFollowing 4 hours of ESHP perfusion, left ventricle developed pressure, dP/dt max, and fractional shortening were significantly decreased (P < .001) in DCD hearts receiving VEH compared with SHAM hearts. In contrast, DCD hearts receiving MITO exhibited significantly preserved left ventricle developed pressure, dP/dt max, and fractional shortening (P < .001 each vs VEH, not significant vs SHAM). Infarct size was significantly decreased in DCD hearts receiving MITO as compared with VEH (P < .001). Pediatric DCD hearts subjected to extended WIT demonstrated significantly preserved fractional shortening and significantly decreased infarct size with MITO (P < .01 each vs VEH). ConclusionsMitochondrial transplantation in neonatal and pediatric pig DCD heart donation significantly enhances the preservation of myocardial function and viability and mitigates against damage secondary to extended WIT.

Neonatal Enteric Glia Enhance Intestinal Epithelial Restitution in vitro Following Exposure to Sterile Colonic Luminal Content of Mature but not Neonatal Pigs

Neonates exhibit significantly poorer outcomes from damage to the intestinal epithelium following ischemia for unknown reasons. In our pig model, we recently discovered an age-dependent epithelial restitution defect in ischemia-injured neonatal intestine that is rescued by application of homogenized juvenile mucosa, but the mechanism remains unknown. Enteric glial cells (EGC) have been shown to promote epithelial restitution following injury via paracrine signaling and are abundant in the intestinal mucosa of juvenile but not neonatal pigs. Co-culture studies have shown that pig EGC enhance restitution in epithelial monolayers following scratch wounding. In mice, postnatal maturation and maintenance of the EGC network is purportedly driven by colonization of gut microbiota. Furthermore, weaning of neonates leads to drastic shifts in the dynamics and populations of these gut microbiota. Therefore, we believe that changes to pig intestinal microbiota during weaning may play a key role in EGC maturation required for proper epithelial restitution. We hypothesized that treatment with mature (&gt;6 weeks of age), but not neonatal (2 weeks of age), luminal content would improve restitution of neonatal epithelial monolayers co-cultured with neonatal EGC. We compared the effect of sterile-filtered neonatal or mature luminal content addition to the apical chamber on scratch wound restitution in neonatal porcine IPEC-J2 monolayers in monoculture or co-culture with primary porcine neonatal submucosal EGC using a transwell system (n = 9). Our results support a significant effect of luminal content treatment in the presence of EGC (P=0.0085). Specifically, mature luminal content treatment enhanced IPEC-J2 wound closure in co-culture with EGC but not in IPEC-J2 monoculture (P=0.0156). EGC co-culture alone or treated with neonatal lumen content did not affect restitution. Both neonatal and mature luminal content treatment decreased restitution in monoculture, but this effect was not significant. These data suggest that EGC provide secretory signals to the intestinal epithelium that promote repair in response to a component of mature luminal content, which we believe to be microbial products. Future work aims to determine what is driving these differences in restitution by analyzing neonatal and mature luminal secretomes. NIH 5 T35 OD 11070-12; NIH K01 OD 028207; NIH P30 DK 034987; NIH-NICHD R01 HD095876; USDA-NIFA VMCG-0065. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Neonatal colon exhibits enhanced post-ischemic repair relative to the neonatal jejunum

Intestinal ischemia is the most common cause of gastrointestinal-related morbidity and mortality within the neonatal intensive care unit. Ischemia-induced loss of the intestinal epithelial barrier predisposes patients to life-threatening sepsis unless that barrier is rapidly restored. Early barrier restoration relies on epithelial restitution to achieve wound closure, followed by rearrangement of tight junction proteins to re-establish barrier resistance. To inform putative therapeutics that promote rapid barrier repair in neonates, we have developed a highly-translational model of intestinal ischemia in the neonatal pig. Previous studies have demonstrated a significant defect in post-ischemic repair of the neonatal pig (2-weeks-old) jejunum relative to the juvenile (6-weeks-old) pig. Therefore, we hypothesized that the neonatal colon will demonstrate a similar defect in epithelial barrier repair. Segments of distal jejunum and spiral colon were subjected to surgically-induced ischemia followed by ex vivo recovery on Ussing chambers. Barrier repair was calculated by percent mucosal epithelialization, a measure of restitution, as well as transepithelial electrical resistance, a measure of barrier resistance. Unexpectedly, the ischemically-injured, ex vivo-recovered neonatal colon demonstrated significantly increased epithelial wound healing by restitution (p&lt;0.005) as well as barrier resistance (p&lt;0.05) relative to the neonatal jejunum. Furthermore, scanning electron microscopy revealed that colonocytes demonstrate a restitution phenotype defined by loss of their columnar morphology, marked flattening of their cellular profile and frequent formation of lamellipodia-like processes. Under identical experimental conditions, enterocytes do not demonstrate this phenotypic change. These findings suggest the presence of a colon-specific mechanism of intestinal epithelial barrier repair that is not present in the jejunum. Ongoing experiments aim to better elucidate the inter- and intracellular pathways that define this colon-specific mechanism of repair. Defining these pathways may inform the formulation of future pharmaceuticals that directly upregulate these pathways along the entire length of the intestine and therefore mitigate the effects of ischemic injury in neonates. NIH K01 OD 028207, NIH-NICHD R01 HD095876, U01 TR002953, T32OD011130 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Activation of renal vascular smooth muscle TRPV4 channels by 5-hydroxytryptamine impairs kidney function in neonatal pigs

Control of microvascular reactivity by 5-hydroxytryptamine (5-HT; serotonin) is complex and may depend on vascular bed type and 5-HT receptors. 5-HT receptors consist of seven families (5-HT1-5-HT7), with 5-HT2 predominantly mediating renal vasoconstriction. Regulation of cyclooxygenase (COX) and smooth muscle cell (SMC) intracellular Ca2+ concentration ([Ca2+]i) levels have been implicated in 5-HT-induced vascular reactivity. Although 5-HT receptor expression and circulating 5-HT levels are known to be dependent on postnatal age, control of neonatal renal microvascular function by 5-HT is unclear. The objective of this study is to use the translational swine model to examine the effects of 5-HT on newborn renal microcirculation. We demonstrate that 5-HT activates human transient receptor potential cation channel subfamily V member 4 (TRPV4) channels that were transiently expressed in Chinese hamster ovary cells. 5-HT2A is the predominant 5-HT2 receptor subtype in freshly isolated neonatal pig renal microvascular SMC. HC-067047 (HC), a selective TRPV4 blocker, attenuated cation currents induced by 5-HT in neonatal pig renal vascular SMC. HC also inhibited the 5-HT-induced increase in renal microvascular [Ca2+]i and constriction. Intrarenal artery infusion of 5-HT had minimal effects on systemic hemodynamics but reduced renal blood flow (RBF) and increased renal vascular resistance (RVR) in the pigs. Transcutaneous determination of glomerular filtration rate (GFR) indicated that kidney infusion of 5-HT reduced GFR. HC diminished 5-HT-induced alteration of RBF, RVR, and GFR. Moreover, plasma and urinary COX-1 and COX-2 levels in 5-HT-treated pigs were unchanged. These data suggest that activation of renal microvascular SMC TRPV4 channels by 5-HT impairs kidney function in neonatal pigs independently of COX production. NIH R01DK120595 and R01DK127625 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Vaginal Birth Alters Gut Transcriptome of Neonatal Pigs

Background: Women at risk of preterm birth are routinely administered prenatal doses of glucocorticoids to facilitate lung maturation and improve pulmonary outcomes in preterm infants. Additionally, preterm infants are often administered low doses of glucocorticoids postnatally for prevention of bronchopulmonary dysplasia. However, there is limited understanding about the impact of perinatal glucocorticoid exposure on the gut and liver in preterm infants. In pigs, there is a spike in plasma cortisol in newborns during parturition. We previously showed in pigs that plasma cortisol levels positively correlate with plasma fibroblast growth factor 19 (FGF19), a gut hormone that negatively regulates hepatic bile acid synthesis. FGF19 is secreted by epithelial cells predominantly in the distal ileum in response to bile acids. However, in vaginally-born pigs, FGF19 mRNA levels were higher in the proximal jejunum. This led us to hypothesize that exposure to glucocorticoids in the perinatal period has a profound impact on the transcription of genes involved in the development of gut and liver. Objective: The objective of this study was to characterize the impact of spontaneous vaginal birth and the exposure to elevated cortisol on the transcriptome in gut and liver tissue of neonatal pigs Methods: Two sows underwent a cesarean section on gestation day 114 (“Term”; term=115 day), two sows underwent a cesarean section on gestation day 105 (“Preterm”) and two additional sows were allowed to farrow at term gestational age (“Vaginal”). Small intestine tissue and plasma was collected from newborn pigs (n= 10 Preterm; n=8 Term; n=6 Vaginal). RNA-seq analysis was performed on intestinal samples mapped to the latest Sus scrofa Sscrofa11.1 reference genome (NCBI). Results: Cortisol levels were statistically highest in the vaginally-born pigs (139 ng/mL), followed by the Term (101 ng/mL) and lowest in the Preterm (61 ng/mL) pigs. Gene Ontology (GO) analysis showed significant enrichment in pathways of upregulated genes involved in response to toxic substances, stress, detoxification and inorganic substances in the jejunum of Vaginal pigs compared to Preterm and Term pigs. KEGG analysis of the proximal jejunum indicated enrichment in pathways of upregulated genes in steroid hormone biosynthesis, bile secretion, TNF signaling and IL-17 Vaginal, compared to Term and Preterm pigs. The same pattern was not the same in the distal ileum where GO analysis showed pathway enrichment of upregulated genes in oxidation-reduction processes, and transition metal ion binding in Vaginal compared to Term and Preterm pigs. KEGG analysis of the distal ileum identified pathway enrichment of upregulated genes in complement and coagulation, bile acid synthesis and secretion, fatty acid metabolism, and amino acid metabolism in Vaginal pigs. Conclusion: Genes in metabolic and cellular signaling pathways associated with cortisol are upregulated in vaginally-born pigs. This work was supported in part by federal funds from the USDA, Agricultural Research Service under Cooperative Agreement Number 3092-51000-060-01, and grants from the National Institutes of Health Grant DK-094616 (D.G.B), and the Texas Medical Center Digestive Diseases Center (NIH Grant P30 DK-56338). Caitlin Vonderohe was supported by T32 DK007664 and Greg Guthrie was supported by K01 DK129408. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effect of phage vB_EcoM_FJ1 on the reduction of ETEC O9:H9 infection in a neonatal pig cell line

Enterotoxigenic Escherichia coli (ETEC) colonizes the intestine of young pigs causing severe diarrhoea and consequently bringing high production costs. The rise of antibiotic selective pressure together with ongoing limitations on their use, demands new strategies to tackle this pathology. The pertinence of using bacteriophages as an alternative is being explored, and in this work, the efficacy of phage vB_EcoM_FJ1 (FJ1) in reducing the load of ETEC EC43-Ph (serotype O9:H9 expressing the enterotoxin STa and two adhesins F5 and F41) was assessed. Foreseeing the oral application on piglets, FJ1 was encapsulated on calcium carbonate and alginate microparticles, thus preventing phage release under adverse conditions of the simulated gastric fluid (pH 3.0) and allowing phage availability in simulated intestinal fluid (pH 6.5). A single dose of encapsulated FJ1, provided to IPEC-1 cultured cells (from intestinal epithelium of piglets) previously infected by EC43, provided bacterial reductions of about 99.9% after 6 h. Although bacteriophage-insensitive mutants (BIMs) have emerged from treatment, the consequent fitness costs associated with this new phenotype were demonstrated, comparatively to the originating strain. The higher competence of the pig complement system to decrease BIMs’ viability, the lower level of colonization of IPEC-1 cells observed with these mutants, and the increased survival rates and health index recorded in infected Galleria mellonella larvae supported this observation. Most of all, FJ1 established a proof-of-concept of the efficiency of phages to fight against ETEC in piglet intestinal cells.

Prediction and Verification of Curcumin as a Potential Drug for Inhibition of PDCoV Replication in LLC-PK1 Cells.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus (CoV) that causes lethal watery diarrhea in neonatal pigs and poses economic and public health burdens. Currently, there are no effective antiviral agents against PDCoV. Curcumin is the active ingredient extracted from the rhizome of turmeric, which has a potential pharmacological value because it exhibits antiviral properties against several viruses. Here, we described the antiviral effect of curcumin against PDCoV. At first, the potential relationships between the active ingredients and the diarrhea-related targets were predicted through a network pharmacology analysis. Twenty-three nodes and 38 edges were obtained using a PPI analysis of eight compound-targets. The action target genes were closely related to the inflammatory and immune related signaling pathways, such as the TNF signaling pathway, Jak-STAT signaling pathway, and so on. Moreover, IL-6, NR3C2, BCHE and PTGS2 were identified as the most likely targets of curcumin by binding energy and 3D protein-ligand complex analysis. Furthermore, curcumin inhibited PDCoV replication in LLC-PK1 cells at the time of infection in a dose-dependent way. In poly (I:C) pretreated LLC-PK1 cells, PDCoV reduced IFN-β production via the RIG-I pathway to evade the host's antiviral innate immune response. Meanwhile, curcumin inhibited PDCoV-induced IFN-β secretion by inhibiting the RIG-I pathway and reduced inflammation by inhibiting IRF3 or NF-κB protein expression. Our study provides a potential strategy for the use of curcumin in preventing diarrhea caused by PDCoV in piglets.

Transplantation of fecal filtrate to neonatal pigs reduces post-weaning diarrhea: A pilot study.

Post-weaning diarrhea (PWD) remains a major source of mortality and morbidity in swine production. Transplantation of bacteria-free filtrate of feces (fecal filtrate transplant, FFT) has shown gut protective effects in neonatal pigs, and early postnatal establishment of the gut microbiome is suggested to determine later stability and robustness of the gut. We, therefore, hypothesized that early postnatal transplantation of bacteria-free feces would have a protective effect against PWD. Using fecal filtrates derived from healthy lactating sows, we compared oral administration of fecal filtrate transplantation (FFT, n = 20) and saline (CON, n = 18) in newborn piglets. We assessed growth, diarrhea prevalence, blood parameters, organ measurements, morphology, and gut brush border enzymes and analyzed luminal bacterial composition using 16S rRNA gene amplicon sequencing. The two groups showed similar average daily gain (ADG) during the suckling period, whereas in the post-weaning period, a negative ADG was observed in both groups. While diarrhea was largely absent in both groups before weaning, there was a lower diarrhea prevalence on days 27 (p = 2.07*10-9), 28 (p = 0.04), and 35 (p = 0.04) in the FFT group relative to CON. At weaning on day 27, the FFT group had higher numbers of red blood cells, monocytes, and lymphocytes, while on day 35, i.e., 1 week after weaning, the two groups were similar regarding hematology. The biochemical profile was largely similar between FFT and CON on days 27 and 35, except for a higher level of alanine aminotransferase and a lower level of Mg in the FFT group. Likewise, organ weights relative to body weight were largely similar on day 35, albeit with a lower stomach weight and more colon content in FFT relative to CON. Gut mucosal percentage and mucosal enzyme activity were similar between the two groups on days 27 and 35. Gut bacterial composition was slightly different on day 35 but not on day 27. In conclusion, early postnatal administration of FFT, showed positive clinical effects in post-weaning pigs, albeit with subtle effects on the gut mucosa and microbiome. Prophylactic treatment with FFT may offer a means to reduce morbidity, yet larger studies are required to document effect size.

Contribution of D-(-)-3-Hydroxybutyrate to the Energy Expenditure of Neonatal Pigs

In vivo oxidation rate of arterially infused D-(-)-3-hydroxybutyrate (3HB) was measured in 1-2-d-old-piglets. Twelve piglets (1.4 kg) were randomly assigned to a 12 h continuous infusion of 3HB at 19.5, 37.8, 55.8 or 74.5 µmol/min along with ∼31 kBq/h of [3-14C]3HB. Piglets were housed in respiration chambers allowing collection of total expired CO2 over 20-min intervals for the 12 h infusion and 6 h washout. Oxidation of 3HB was calculated from the quantity and specific radioactivity of expired CO2 for 20-min collection periods at 6, 9 and 12 h for each piglet and collectively plotted against plasma 3HB concentration measured in blood drawn during those 20-min periods. A Lineweaver-Burk plot of these data yielded a Km of 0.62 ± 0.07 mmol/L and Vmax of 0.74 ± 0.02 mmol ATP equivalents/(min·kg0.75) (parameter estimate ± SD), which could account for 32% of the piglet mean total ATP turnover of 2.3 mmol/(min·kg0.75). These data show that 3HB oxidation is a linear function of plasma concentration in the physiologic range measured in piglets (0.006 mmol/L to 0.1 mmol/L) and within this range would account for 0.3% to 4.5% of piglet energy requirement. Oxidation of 3HB can meet a maximum of 30 to 40% of piglet energy requirement at unphysiologically high 3HB concentrations (> mmol/L).

Carnitine Palmitoyltransferase Modulation of Hepatic Fatty Acid Metabolism and Radio-HPLC Evidence for Low Ketogenesis in Neonatal Pigs

A neonatal piglet model was used to study hepatic fatty acid metabolism during the early postnatal period. Hepatocytes were isolated from pigs at birth or after 24 h, in fed or unfed states (n = 4 pigs/group). Cells were incubated with 1 mmol/L [1-14C]-octanoate (C8) or -palmitate (C16) in the presence or absence of 1 mmol/L L-carnitine, carnitine plus tetra-decylglycidic acid (TDGA; 10 µmol/L) or carnitine plus glucagon (0.5 µg/L). Accumulation of radiolabel [nmol/(h · 106 cells)] in CO2 and acid-soluble products (ASP) was higher (3.5- and 4.5-fold, respectively) from C8 than from C16 (P < 0.0001). Glucagon, carnitine and TDGA had no effect on the oxidation of C8 (P > 0.1). Carnitine addition tended to increase C16 flux to ASP [from 5.3 to 7.6 nmol/(h · 106 cells); P < 0.1], whereas carnitine plus TDGA decreased flux (from 7.6 to 2.1; P < 0.001). Esterified products accounted for 70% of metabolized label in control C16 incubations; this was reduced to 62% by carnitine (P < 0.05) and increased to 80% by the addition of carnitine plus TDGA (P < 0.0001). The 1-14C flux to CO2 in cells from 24-h-old unfed piglets was 47% lower than from fed pigs (P < 0.01) but 28% higher than in pigs at birth. Radiolabel contained in ASP and total metabolized label were 48% lower from unfed pigs compared with the piglets at birth and 24-h-old fed pigs (P < 0.01) and were paralleled by changes in oxygen consumption. Radio-HPLC analysis of ASP revealed minimal radiolabel accumulation in ketone bodies. Up to 40% of radioactivity in ASP was presumptively identified as acetate. These data illustrate that hepatic C16 metabolism in piglets can be altered by changes in the activity of carnitine palmitoyltransferase I during the neonatal period and that ketone bodies do not represent a major route of hepatic fatty acid carbon flux.

Feeding Colostrum Rapidly Alters Enzymatic Activity and the Relative Isoform Abundance of Jejunal Lactase in Neonatal Pigs ,

Our objective was to determine the short-term effects of feeding colostrum and mature milk on the enzyme activity and relative isoform abundance of lactase in neonatal pigs. We studied newborn pigs that were fed colostrum, mature milk and water for 6 h (Study 1). We also studied unfed pigs shortly after birth, and newborn pigs fed either colostrum or mature milk for 24 h (Study 2). The specific activity (micromol x min(-1) x g protein(-1)) of lactase in ajejunal mucosal homogenate and a purified membrane fraction was lower in pigs fed colostrum than in unfed newborns or those fed either milk or water. However, after 24 h, total jejunal lactase activity (micromol x mol(-1)), jejunal mass and protein content were higher in the colostrum- and milk-fed pigs than in the unfed newborns. In colostrum-fed pigs, the reduction in lactase specific activity after 6 h was associated with 1) a marked increase in the relative abundance of a 180-kDa protein, which was shown to be one of three pro-lactase isoforms, and 2) a lower relative abundance of the 160-kDa isoform, considered to be the mature form of the enzyme. Our evidence suggests that feeding either colostrum or mature milk increases total jejunal lactase activity. The reduction in both the specific activity and abundance of the mature isoform in conjunction with an increased relative abundance of the 180-kDa pro-lactase isoform suggests that feeding colostrum alters the post-translational processing of intestinal lactase in neonatal pigs.

Directed self-assembly of a xenogeneic vascularized endocrine pancreas for type 1 diabetes

Intrahepatic islet transplantation is the standard cell therapy for β cell replacement. However, the shortage of organ donors and an unsatisfactory engraftment limit its application to a selected patients with type 1 diabetes. There is an urgent need to identify alternative strategies based on an unlimited source of insulin producing cells and innovative scaffolds to foster cell interaction and integration to orchestrate physiological endocrine function. We previously proposed the use of decellularized lung as a scaffold for β cell replacement with the final goal of engineering a vascularized endocrine organ. Here, we prototyped this technology with the integration of neonatal porcine islet and healthy subject-derived blood outgrowth endothelial cells to engineer a xenogeneic vascularized endocrine pancreas. We validated ex vivo cell integration and function, its engraftment and performance in a preclinical model of diabetes. Results showed that this technology not only is able to foster neonatal pig islet maturation in vitro, but also to perform in vivo immediately upon transplantation and for over 18 weeks, compared to normal performance within 8 weeks in various state of the art preclinical models. Given the recent progress in donor pig genetic engineering, this technology may enable the assembly of immune-protected functional endocrine organs.

Galacto-oligosaccharides fed during gestation increase Rotavirus A specific antibodies in sow colostrum, modulate the microbiome, and reduce infectivity in neonatal piglets in a commercial farm setting.

Rotavirus A is a major cause of acute dehydrating diarrhea in neonatal pigs resulting in significant mortality, morbidity, reduced performance and economic loss. Commercially available prebiotic galacto-oligosaccharides are similar to those of mammalian milk and stimulate the development of the microbiota and immune system in neonates. Little is known about the effects of supplementing sows' diets with galacto-oligosaccharides during gestation. This study aimed to determine if dietary galacto-oligosaccharide supplementation during gestation could improve immunity, reduce rotavirus infection and modulate the microbiota in sows and neonates in a commercial farm setting with confirmed natural endemic rotavirus challenge. In a randomized controlled trial, control sows received lactation diet with no galacto-oligosaccharide supplementation and test sows received lactation diet with 30 g/day galacto-oligosaccharide top-dressed into feed daily, seven days before farrowing. Colostrum was collected from sows 24 hours post-partum and tested for rotavirus specific antibodies. Fecal samples were collected from sows and piglets three days post-partum, tested for rotavirus A by qPCR and the microbiome composition assessed by 16s rRNA gene sequencing. Supplementation with galacto-oligosaccharides during gestation significantly increased rotavirus-specific IgG and IgA in sow colostrum and reduced the number of rotavirus positive piglet fecal samples. Abundance of potential pathogens Treponema and Clostridiales were higher in fecal samples from non-galacto-oligosaccharide fed sows, their piglets and rotavirus positive samples. This study demonstrates that galacto-oligosaccharide supplementation during gestation significantly increases rotavirus specific IgG and IgA in sow colostrum thereby reducing neonatal rotavirus infection and suppresses potential pathogenic bacteria in nursing sows and neonatal piglets.

The Sertoli Cell Complement Signature: A Suspected Mechanism in Xenograft Survival.

The complement system is an important component of transplant rejection. Sertoli cells, an immune regulatory testicular cell, survive long-term when transplanted across immunological barriers; thus, understanding the mechanisms behind this unique survival would be of great benefit to the transplantation field. This study focused on Sertoli cell inhibition of complement as relevant in xenotransplantation. Neonatal pig Sertoli cells (NPSCs) survived activated human complement in vitro while neonatal pig islet (NPI) aggregates and pig aortic endothelial cell (PAEC) survival were diminished to about 65% and 12%, respectively. PAECs cultured in NPSC-conditioned media and human complement demonstrated a 200% increase in survival suggesting that NPSCs secrete complement-inhibiting substances that confer protection. Bioinformatic and molecular analyses identified 21 complement inhibitors expressed by NPSCs with several significantly increased in NPSCs compared to NPIs or PAECs. Lastly, RNA sequencing revealed that NPSCs express 25 other complement factors including cascade components and receptors. Overall, this study identified the most comprehensive Sertoli cell complement signature to date and indicates that the expression of a variety of complement inhibitors ensures a proper regulation of complement through redundant inhibition points. Understanding the regulation of the complement system should be further investigated for extending xenograft viability.