Pig Hepatocytes Research Articles

Identification of amino acids restricting HBV receptor function in porcine NTCP

With 254 million chronically infected patients, hepatitis B virus (HBV) continues to be a severe health threat. While animal models play a crucial role in developing new therapies, the availability of preclinical HBV models is very limited. Therefore, novel in vivo infection models are urgently needed. The bona fide HBV receptor, sodium-taurocholate cotransporting polypeptide (NTCP), determines HBV’s species and cell-type specificity. Recent studies have indicated that the expression of human NTCP is the only limiting factor for HBV infection in selected species, such as macaques or pigs. Here, we confirm HBV infection of pig hepatocytes expressing human NTCP and show that porcine NTCP does not support HBV binding. By gradually humanizing porcine NTCP and site-directed mutagenesis, we identified amino acids 158 and 167 in porcine NTCP, limiting HBV interaction. In a proof-of-concept experiment, we showed that the expression of porcine NTCP with humanized amino acids 157-167 renders primary porcine hepatocytes fully susceptible to HBV. These results pave the way for generating transgenic pigs with humanized porcine chimeric NTCP as a novel, fully immunocompetent infection model for developing and validating new curative HBV therapies.

AAV Capsid Screening for Translational Pig Research Using a Mouse Xenograft Liver Model.

In gene therapy, delivery vectors are a key component for successful gene delivery and safety, based on which adeno-associated viruses (AAVs) gained popularity in particular for the liver, but also for other organs. Traditionally, rodents have been used as animal models to develop and optimize treatments, but species and organ specific tropism of AAV desire large animal models more closely related to humans for preclinical in-depth studies. Relevant AAV variants with the potential for clinical translation in liver gene therapy were previously evolved in vivo in a xenogeneic mouse model transplanted with human hepatocytes. Here, we selected and evaluated efficient AAV capsids using chimeric mice with a >90% xenografted pig hepatocytes. The pig is a valuable preclinical model for therapy studies due to its anatomic and immunological similarities to humans. Using a DNA-barcoded recombinant AAV library containing 47 different capsids and subsequent Illumina sequencing of barcodes in the AAV vector genome DNA and transcripts in the porcine hepatocytes, we found the AAVLK03 and AAVrh20 capsid to be the most efficient delivery vectors regarding transgene expression in porcine hepatocytes. In attempting to validate these findings with primary porcine hepatocytes, we observed capsid-specific differences in cell entry and transgene expression efficiency where the AAV2, AAVAnc80, and AAVDJ capsids showed superior efficiency to AAVLK03 and AAVrh20. This work highlights intricacies of in vitro testing with primary hepatocytes and the requirements for suitable pre-clinical animal models but suggests the chimeric mouse to be a valuable model to predict AAV capsids to transduce porcine hepatocytes efficiently.

From a Single Cell to a Whole Human Liver: Disease Modeling and Transplantation.

Although the underlying cause may vary across countries and demographic groups, liver disease is a major cause of morbidity and mortality globally. Orthotopic liver transplantation is the only definitive treatment for liver failure but is limited by the lack of donor livers. The development of drugs that prevent the progression of liver disease and the generation of alternative liver constructs for transplantation could help alleviate the burden of liver disease. Bioengineered livers containing human induced pluripotent stem cell (iPSC)-derived liver cells are being utilized to study liver disease and to identify and test potential therapeutics. Moreover, bioengineered livers containing pig hepatocytes and endothelial cells have been shown to function and survive after transplantation into pig models of liver failure, providing preclinical evidence toward future clinical applications. Finally, bioengineered livers containing human iPSC-derived liver cells have been shown to function and survive after transplantation in rodents but require considerable optimization and testing prior to clinical use. In conclusion, bioengineered livers have emerged as a suitable tool for modeling liver diseases and as a promising alternative graft for clinical transplantation. The integration of novel technologies and techniques for the assembly and analysis of bioengineered livers will undoubtedly expand future applications in basic research and clinical transplantation.

Maternal and Fetal Bile Acid Homeostasis Regulated by Sulfated Progesterone Metabolites through FXR Signaling Pathway in a Pregnant Sow Model.

Abnormally elevated circulating bile acids (BA) during pregnancy endanger fetal survival and offspring health; however, the pathology and underlying mechanisms are poorly understood. A total of nineteen pregnant sows were randomly assigned to day 60 of gestation, day 90 of gestation (G60, G90), and the farrowing day (L0), to investigate the intercorrelation of reproductive hormone, including estradiol, progesterone and sulfated progesterone metabolites (PMSs), and BA in the peripheral blood of mother and fetuses during pregnancy. All data were analyzed by Student’s t-test or one-way ANOVA of GraphPad Prism and further compared by using the Student–Newman–Keuls test. Correlation analysis was also carried out using the CORR procedure of SAS to study the relationship between PMSs and BA levels in both maternal and fetal serum at G60, G90, and L0. Allopregnanolone sulphate (PM4S) and epiallopregnanolone sulphate (PM5S) were firstly identified in the maternal and fetal peripheral blood of pregnant sows by using newly developed ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) methods. Correlation analysis showed that pregnancy-associated maternal BA homeostasis was correlated with maternal serum PM4S levels, whereas fetal BA homeostasis was correlated with fetal serum PM5S levels. The antagonist activity role of PM5S on farnesoid X receptor (FXR)-mediated BA homeostasis and fibroblast growth factor 19 (FGF19) were confirmed in the PM5S and FXR activator co-treated pig primary hepatocytes model, and the antagonist role of PM4S on FXR-mediated BA homeostasis and FGF19 were also identified in the PM4S-treated pig primary hepatocytes model. Together with the high relative expression of FGF19 in pig hepatocytes, the pregnant sow is a promising animal model to investigate the pathogenesis of cholestasis during pregnancy.

Liver and Hepatocyte Transplantation: What Can Pigs Contribute?

About one-fifth of the population suffers from liver diseases in China, meaning that liver disorders are prominent causative factors relating to the Chinese mortality rate. For patients with end-stage liver diseases such as hepatocellular carcinoma or acute liver diseases with life-threatening liver dysfunction, allogeneic liver transplantation is the only life-saving treatment. Hepatocyte transplantation is a promising alternative for patients with acute liver failure or those considered high risk for major surgery, particularly for the bridge-to-transplant period. However, the lack of donors has become a serious global problem. The clinical application of porcine xenogeneic livers and hepatocytes remains a potential solution to alleviate the donor shortage. Pig grafts of xenotransplantation play roles in providing liver support in recipients, together with the occurrence of rejection, thrombocytopenia, and blood coagulation dysfunction. In this review, we present an overview of the development, potential therapeutic impact, and remaining barriers in the clinical application of pig liver and hepatocyte xenotransplantation to humans and non-human primates. Donor pigs with optimized genetic modification combinations and highly effective immunosuppressive regimens should be further explored to improve the outcomes of xenogeneic liver and hepatocyte transplantation.

A New lncRNA, lnc-LLMA, Regulates Lipid Metabolism in Pig Hepatocytes.

A large variety of long noncoding RNAs (lncRNAs) have been discovered through high-throughput sequencing technology and some have been demonstrated to play important roles in lipid metabolism regulation. In our study, we found a highly expressed lncRNA (lnc-LLMA, liver lipid metabolism-associated lncRNA) in the liver of Duroc pigs, which was enriched in the nucleus. It displays potent tissue specificity among different pig breeds. Overexpression of lnc-LLMA can cause a decline in intracellular triglyceride (TG) levels and increases in ATP and mitochondrial DNA levels in pig primary hepatocytes and HepG2 cells. In addition, the expression levels of MTTP, APOB, CPT1α, and other genes were increased by overexpression of lnc-LLMA. It downregulated expression of G6Pase and SREBP1 genes. Chromatin isolation by RNA purification (ChRIP) experiments demonstrated that microsomal triglyceride transfer protein (MTTP) and glycogen synthase 2 (GYS2) were the potential interacting proteins of lnc-LLMA. The overexpression of the GYS2 gene rescued the decreasing intracellular TG levels caused by the increase of lnc-LLMA. Similarly, overexpression of MTTP was also able to save the lnc-LLMA-induced decrease in intracellular TG. Our study demonstrated that this novel lncRNA was closely related to lipid metabolism and affected lipid transport and mitochondrial function through MTTP and GYS2. Our results provided a new direction for further studying the effect of lncRNA on lipid metabolism regulation.

Apply a Physiologically Based Pharmacokinetic Model to Promote the Development of Enrofloxacin Granules: Predict Withdrawal Interval and Toxicity Dose.

Enrofloxacin (ENR) granules were developed to prevent and control the infections caused by foodborne zoonotic intestinal pathogens in our previous studies. To promote the further development of ENR granules and standardize their usage in pigs, a physiologically based pharmacokinetic (PBPK) model of the ENR granule in pigs was built to determine the withdrawal time (WT) and evaluate the toxicity to pigs. Meanwhile, the population WT was determined by a Monte Carlo analysis to guarantee pork safety. The fitting results of the model showed that the tissue residual concentrations of ENR, ciprofloxacin, and ENR plus ciprofloxacin were all well predicted by the built PBPK model (R2 > 0.82). When comparing with the EMA’s WT1.4 software method, the final WT (6 d) of the ENR granules in the population of pigs was well predicted. Moreover, by combining the cytotoxicity concentration (225.9 µg/mL) of ENR against pig hepatocytes, the orally safe dosage range (≤130 mg/kg b.w.) of the ENR granules to pigs was calculated based on the validated PBPK model. The well-predicted WTs and a few uses in animals proved that the PBPK model is a potential tool for promoting the judicious use of antimicrobial agents and evaluating the toxicity of the veterinary antimicrobial products.

S1076 Successful Delivery of Human Factor IX Into Pig Liver via Endoscopic Retrograde Cholangio-Pancreatography

INTRODUCTION: Gene therapy has the promise to provide curative therapies to many inherited monogenic liver diseases. Hemophilia B is an attractive target for gene therapy as a modest increase in the clotting factor activity can result in clinical benefits and attenuation of bleeding risk. Herein, we developed an alternative approach employing non-viral delivery of human factor IX (hFIX) DNA into the liver via hydrodynamic injection into the biliary tree. METHODS: We delivered hFIX DNA via biliary hydrodynamic injection into the pig liver, as accessed by endoscopic retrograde cholangio-pancreatography (ERCP) while using clinical equipment employed in treating human patients. The catheter was advanced into the common hepatic duct, and a balloon seal to prevent retrograde flow. The hydrodynamic injection consisted of 30 mL of saline solution containing 3–5.5 mg plasmid DNA injected at 2mL/sec. Four pigs in total were injected between 35 and 40 kg. RESULTS: The procedure was well tolerated in all four pigs. The safety profile was examined, including vital signs, liver function panel, and histology. hFIX DNA was integrated into the hepatocyte genome with co-injected piggyBac transposase plasmid and was found in all liver lobes by PCR, indicating global liver delivery. No human factor IX could be detected in the pig serum, but hFIX protein was detected in the liver tissue of all five lobes by western blot. Immunohistochemical staining revealed abundant hFIX-positive hepatocytes in all five lobes, with the most intense expression around the central vein. The transfection efficiency and pattern was reproduced in the three other pigs. CONCLUSION: We demonstrated non-viral endoscopic gene delivery successfully achieved hFIX expression in all liver lobes of the pig. No hFIX was detected in the pig serum, similar to reports in hFIX-transgenic pigs, suggesting pig hepatocytes may have a species-related secretory defect for hFIX protein. Future studies will continue to optimize gene delivery to develop this strategy as a therapy in human patients.Current clinical trials have focused on using adeno-associated viral (AAV) vectors to deliver hFIX into the liver to treat hemophilia B, but the resultant hFIX levels have been modest, and AAV vectors could not be re-dosed to increased hFIX expression.The advantages of a retrobiliary approach include the ability through one access point to reach the entire vast network of hepatocytes.Such a strategy would also be re-dosable in hemophilia B patients.Figure 1.: Immunohistochemistry staining showed hFIX-positive hepatocytes (orange-brown) with the most intense expression around the central vein.

JAK2-STAT5 signaling is insensitive to porcine growth hormone (pGH) in hepatocytes of neonatal pig

ABSTRACT Porcine growth hormone (pGH) is most important hormone which is involved in the growth and development of pig. However, a series of studies have indicated that neonatal pig is insensitive to pGH; the reason for this phenomenon is still not fully understood. In this work, we try to investigate this issue from the angle of intracellular signaling induced by pGH. In the present study, porcine hepatocytes from neonatal pig were used as a model, and confocal laser scanning microscopy (CLSM), Western blot, co-immunoprecipitation and colocalization assay were used to study pGH’s signaling properties in hepatocytes of neonatal pig and explore the possible mechanism(s) for why intracellular signaling is insensitive to pGH. The results indicated that Janus kinase 2 and signal transducers and activators of transcription 5/3/1 (JAK2-STATs) signaling are not activated. We further investigated the possible mechanism(s) by which JAK2-STATs’ signaling is not activated by pGH and growth hormone receptor (GHR) and found that the negative regulatory molecules of JAK2-STATs signaling may be associated with this phenomenon in the hepatocytes of neonatal pig. In addition, we also explored pGH’s biology in hepatocytes from neonatal pig, it can be found that pGH/GHR could translocate into the cell nucleus, which implies that pGH/GHR may exhibit physiological roles based on their nuclear localization. We found that pGH could not trigger intracellular signaling in the hepatocytes of neonatal pigs, but not young pigs, which provides an important explanation for why the growth of neonatal pig is GH independent.

Different intracellular signalling sensitivity and cell behaviour of porcine insulin with aging

Insulin has many important biological functions. Insulin interacts with the insulin receptor (IR) to play its physiological role and execute its functions. Here, we isolated porcine hepatocytes from young and aged pigs, which endogenously express the IR, as a model to study the intracellular signalling properties and cellular behaviour of insulin with aging. Firstly, we analysed the intracellular signal transduction that is triggered by insulin in porcine hepatocytes that were isolated from young and aged pigs and found that insulin can strongly activate insulin receptor subunit (IRS), protein kinase B (AKT), and GSK in a time- and dose-dependent manner in hepatocytes from young pigs. On the contrary, the signalling response to insulin in hepatocytes from aged pigs was significantly reduced compared to that of the young pig. Secondly, the different subcellular locations of insulin/insulin receptor (IR) may result in different biological activities, although nuclear-localized insulin/IR still could exhibit important functions and roles. We found that insulin can translocate into cell nuclei in the hepatocytes of the young pigs; however, insulin/insulin receptor fails to transports into the cell nucleus in hepatocytes from aged pigs, although insulin/insulin receptor could internalize into cell cytoplasm. In summary, in the current study, we explored and compared for the first time insulin’s behaviour and signalling properties in the cells of young pig hepatocytes and aged pig hepatocytes. Furthermore, we found that the insulin signalling response in hepatocytes was significantly reduced with age; more importantly, we found that the cell behaviour of insulin was changed significantly in the hepatocytes from aged pigs compared to young pigs, and it is noteworthy that insulin/IR cannot translocate into the cell nuclei in the hepatocytes from the aged pig. This may be a potential new reason contributing to insulin resistance with aging, suggesting that we need to study the reason for insulin resistance from a new point of view.

HNTCP‑expressing primary pig hepatocytes are a valuable tool for investigating hepatitis B virus infection and antiviral drugs

Primary human hepatocytes (PHHs) are the ‘gold standard’ for investigating hepatitis B virus (HBV) infection and antiviral drugs. However, poor availability, variation between batches and ethical issues regarding PHHs limit their applications. The discovery of human sodium taurocholate co-transporting polypeptide (hNTCP) as a functional HBV receptor has enabled the development of a surrogate model to supplement the use of PHHs. In the present study, the evolutionary distance of seven species was assessed based on single-copy homologous genes. Based on the evolutionary distance and availability, PHHs and primary rabbit hepatocytes (PRHs) were isolated and infected with hNTCP-recombinant lentivirus, and susceptibility to HBV infection in the two cell types was tested and compared. In addition, HBV infection efficiency of hNTCP-expressing PPHs with pooled HBV-positive serum and purified particles was determined. The potential use of HBV-infected hNTCP-expressing PPHs for drug screening was assessed. The results demonstrated that pigs and rabbits are closer to humans in the divergence tree compared with mice and rats, indicating that pigs and rabbits were more likely to facilitate the HBV post-entry lifecycle. Following hNTCP complementation and HBV infection, PPHs and Huh7D human hepatocellular carcinoma cells, but not PRHs, exhibited increased hepatitis B surface antigen and hepatitis B e-antigen secretion, covalently closed circular DNA formation and infectious particle secretion. hNTCP-expressing PPHs were susceptible to infection with HBV particles purified from pooled HBV-positive sera, but were poisoned by raw HBV-positive sera. The use of HBV-infected hNTCP-expressing PPHs for viral entry inhibitor screening was revealed to be applicable and reproducible. In conclusion, hNTCP-expressing PPHs may be valuable tool for investigating HBV infection and antiviral drugs.

Establishment of gene-edited pigs expressing human blood-coagulation factor VII and albumin for bioartificial liver use.

Bioartificial livers (BALs) are considered as a solution to bridge patients with acute liver failure to liver transplantation or to assist in spontaneous recovery for patients with end-stage liver disease. Pig is the best donor of hepatocytes for BALs in clinical trials, because metabolic and detoxification function of its liver are close to human. However, using pig hepatocytes for BALs remains controversial for safety concern owing to nonhuman proteins secretion. Herein, we attempt to establish modified pigs expressing humanized liver proteins, blood-coagulation factor VII (F7), and albumin (ALB). These pigs should also be porcine endogenous retrovirus subtype C (PERV-C) free so that their ability of transmitting PERV to human could be diminished seriously. We devised both homology-dependent and independent knock-in approaches to insert a fusion of hF7 and hALB gene downstream the site of pig endogenous F7 promoter in pig fetal fibroblasts negative for PERV-C. The modified pigs were then generated through somatic cell nuclear transfer. We obtained 14 and 10 cloned pigs by homology-dependent and independent approaches, respectively. Among them, 19 cloned pigs were with expected gene modification and 13 are alive to date. These modified pigs can successfully express hF7 and hALB in the liver and serum, and the expressed hF7 exhibits normal coagulation activity. The gene-edited pigs expressing hF7 and hALB in the liver were generated successfully. We anticipate that our pigs could provide an alternative cell source for BALs as a promising treatment for patients with acute liver failure.

Impact of Hepatic Stellate Cells in Scaffold-Free 3D-Bioprinting of the Liver Model

Background and Hypothesis: Hepatic stellate cells (HSC), which compromise ~15% of liver cells, are vital to hepatocellular function. Scaffold-free 3D-bioprinting (SF3DBP) offers an avenue for the creation of realistic organ models without the use of biomaterials. Therefore, we hypothesized that co-culturing primary hepatocytes with HSC in SF3DBP liver model would uphold hepatocyte function over time, providing us a better 3D-liver model for research. 
 Experimental Design: We used freshly thawed primary pig hepatocytes and immortalized pig HSC to generate spheroids with hepatocytes alone, HSC alone, or a combination of hepatocytes and HSC (2.5:1 ratio). Spheroids were formed using low adhesion plates, then characterized for distance from well center, diameter, roundness, and smoothness. A column of spheroids was printed using a Regenova 3D-bioprinter. Remaining loose spheroids are incubated over two weeks for albumin secretion, mRNA transcription, and histological analysis. 
 Results: Co-cultures of hepatocytes and HSC (2.5:1 ratio) formed spheroids within 48 hours, as did HSC only spheroids (Figure 1). Spheroids composed of only hepatocytes failed to form round spheroids. The combination spheroids increased in roundness and decreased in diameter between characterizations over 6 days. 
 Conclusion and Potential Impact: Spheroids proved too large to print at 48 hours but were successfully recognized and placed by the 3D-bioprinter. SF3DBP of combination spheroids would be viable by day 6. Optimization of spheroid composition using different cell ratios including HSC, hepatocytes, liver sinusoidal endothelial cells and fibroblasts, as well as optimization of spheroid incubation time will allow for production and printing of more advanced liver models.

Beneficial Effects of Human Mesenchymal Stromal Cells on Porcine Hepatocyte Viability and Albumin Secretion

Aim Porcine hepatocytes transplanted during acute liver failure might support metabolic functions until the diseased liver recovers its function. Here, we isolated high numbers of viable pig hepatocytes and evaluated hepatocyte functionality after encapsulation. We further investigated whether co-culture and co-encapsulation of hepatocytes with human multipotent mesenchymal stromal cells (MSC) is beneficial on hepatocyte function. Methods Livers from 10kg pigs (n=9) were harvested and hepatocytes were isolated from liver suspensions for microencapsulation using alginate and poly(ethylene-glycol)(PEG)-grafted alginate polymeric hydrogels, either alone or in combination with MSC. Results Viability, albumin secretion and diazepam catabolism of hepatocytes were measured for one week. 9.2±3.6x109 hepatocytes with 95.2±3.1% viability were obtained after isolation. At day 3, free hepatocytes displayed 99% viability, whereas microencapsulation in alginate and PEG-grafted alginate decreased viability to 62% and 48%, respectively. Albumin secretion and diazepam catabolism occurred in free and microencapsulated hepatocytes. Co-encapsulation of hepatocytes with MSC significantly improved viability and albumin secretion at days 4 and 8 (p<0.05). Co-culture with MSC significantly increased and prolonged albumin secretion. Conclusions In conclusion, we established a protocol for isolation and microencapsulation of high numbers of viable pig hepatocytes and demonstrated that the presence of MSC are beneficial for viability and function of porcine hepatocytes.

Hepatocyte spheroids as an alternative to single cells for transplantation after ex vivo gene therapy in mice and pig models

BackgroundAutologous hepatocyte transplantation after ex vivo gene therapy is an alternative to liver transplantation for metabolic liver disease. Here we evaluate ex vivo gene therapy followed by transplantation of single-cell or spheroid hepatocytes. MethodsPig and mouse hepatocytes were isolated, labeled with zirconium-89 and returned to the liver as single cells or spheroids. Biodistribution was evaluated through positron emission tomography–computed tomography. Fumarylacetoacetate hydrolase–deficient pig hepatocytes were isolated and transduced with a lentiviral vector containing the Fah gene. Animals received portal vein infusion of single-cell or spheroid autologous hepatocytes after ex vivo gene delivery. Portal pressures were measured and ultrasound was used to evaluate for thrombus. Differences in engraftment and expansion of ex vivo corrected single-cell or spheroid hepatocytes were followed through histologic analysis and animals’ ability to thrive off 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione. ResultsPositron emission tomography–computed tomography imaging showed spheroid hepatocytes with increased heterogeneity in biodistribution as compared with single cells, which spread more uniformly throughout the liver. Animals receiving spheroids experienced higher mean changes in portal pressure than animals receiving single cells (P < .01). Additionally, two animals from the spheroid group developed portal vein thrombi that required systemic anticoagulation. Immunohistochemical analysis of spheroid- and single-cell–transplanted animals showed similar engraftment and expansion rates of fumarylacetoacetate hydrolase–positive hepatocytes in the liver, correlating with similar weight stabilization curves. ConclusionEx vivo gene correction of autologous hepatocytes in fumarylacetoacetate hydrolase–deficient pigs can be performed using hepatocyte spheroids or single-cell hepatocytes, with spheroids showing a more heterogeneous distribution within the liver and higher risks for portal vein thrombosis and increased portal pressures.

Differentially expressed genes in response to cyadox in swine liver analyzed by DDRT-PCR

Cyadox is a good antimicrobial growth-promoter of quinoxalines. However, the molecular mechanism of action remains unclear. A growth performance study and mRNA differential display reverse transcription polymerase chain reaction (DDRT-PCR) in combination with Northern dot-blot and reverse Northern dot-blot analysis were conducted to determine the differentially expressed genes in liver tissues of piglets after treatment with cyadox. Transcription levels of the differentially expressed genes were quantificated by realtime RT-PCR in porcine primary hepotocytes. Cyadox could significantly promote body weight of piglets via feed with average daily gain (ADG) improved by 24.7% and 64.8% in 100 and 500mg/kg group, compared with control. A total of eight differentially expressed genes were found, of which the expression levels of five genes had positive correlation with cyadox dose. One gene expression had a negative correlation with cyadox dose and it was a new gene. The other two genes were up-regulated by cyadox, but the expression quantity was invariably when the cyadox doses were increased. Among the up-regulated genes, one was transcriptional regulating factor, two were growth-related factors, one was involved in immune defense and immune-regulation and three might be involved in the maintenance of normal development. In primary cultured pig hepatocytes, cyadox treatment evoked a significant time-dependent effect of eight genes expression. The results suggest, at the transcriptional level in vitro and in vivo, that growth factor and metabolism may be associated with cyadox growth-promoting activity, whereas immune defense and immune-regulation could play major roles in prophylaxis of cyadox in piglets.

Serum amyloid A as a marker of inflammation in xenotransplantation

Increasing evidence indicates that inflammation plays a role in pig-to-baboon organ xenotransplantation. We have evaluated serum amyloid A (SAA) as a marker of inflammation in baboons with various pig xenografts. We measured SAA levels in recipient baboons with pig artery patch (n = 5), life-supporting kidney (n = 5), heterotopic heart (n = 2), or hepatocyte (n = 1) grafts and using an OmniChek-SAA for Inflammation &amp; Infection kit. C-reactive protein (CRP), another marker of inflammation (e.g. D-dimer), was also measured.SAA indicated increased inflammation when baboons developed consumptive coagulopathy (CC; e.g. thrombocytopenia) or infection. SAA also indicated that treatment of the recipient with tocilizumab reduced inflammation. There was significant positive correlation between SAA with changes in CRP (r = 0.6, P &lt; 0.05) and with D-dimer (r = 0.8, P &lt; 0.01), but SAA appeared at times to more accurately reflect the clinical state of the baboon. In sum, measurement of SAA proved simple and quick, and indicated (1) significant inflammation when CC or infection was present, and (2) reduced inflammation when treatment with tocilizumab was administered.

Beneficial Effects of Human Mesenchymal Stromal Cells on Porcine Hepatocyte Viability and Albumin Secretion.

Porcine hepatocytes transplanted during acute liver failure might support metabolic functions until the diseased liver recovers its function. Here, we isolated high numbers of viable pig hepatocytes and evaluated hepatocyte functionality after encapsulation. We further investigated whether coculture and coencapsulation of hepatocytes with human multipotent mesenchymal stromal cells (MSC) are beneficial on hepatocyte function. Livers from 10 kg pigs (n = 9) were harvested, and hepatocytes were isolated from liver suspensions for microencapsulation using alginate and poly(ethylene-glycol)- (PEG-) grafted alginate hydrogels, either alone or in combination with MSC. Viability, albumin secretion, and diazepam catabolism of hepatocytes were measured for one week. 9.2 ± 3.6 × 109 hepatocytes with 95.2 ± 3.1% viability were obtained after isolation. At day 3, free hepatocytes displayed 99% viability, whereas microencapsulation in alginate and PEG-grafted alginate decreased viability to 62% and 48%, respectively. Albumin secretion and diazepam catabolism occurred in free and microencapsulated hepatocytes. Coencapsulation of hepatocytes with MSC significantly improved viability and albumin secretion at days 4 and 8 (p < 0.05). Coculture with MSC significantly increased and prolonged albumin secretion. In conclusion, we established a protocol for isolation and microencapsulation of high numbers of viable pig hepatocytes and demonstrated that the presence of MSC is beneficial for the viability and function of porcine hepatocytes.

RETRACTED: L-leucine stimulates glutamate dehydrogenase activity and glutamate synthesis by regulating mTORC1/SIRT4 pathway in pig liver

The liver is the most essential organ for the metabolism of ammonia, in where most of ammonia is removed by urea and glutamine synthesis. Regulated by leucine, glutamate dehydrogenase (GDH) catalyzes the reversible inter-conversion of glutamate to ammonia. To determine the mechanism of leucine regulating GDH, pigs weighing 20 ± 1 kg were infused for 80 min with ammonium chloride or alanine in the presence or absence of leucine. Primary pig hepatocytes were incubated with or without leucine. In the in vivo experiments with either ammonium or alanine as the nitrogen source, addition of leucine significantly inhibited ureagenesis and promoted the production of glutamate and glutamine in the perfused pig liver (P < 0.05). Similarly, leucine stimulated GDH activity and inhibited sirtuin4 (SIRT4) gene expression (P < 0.01). Leucine could also activate mammalian target of rapamycin complex 1 (mTORC1) signaling (P < 0.05), as evidenced by the increased phosphorylation levels of ribosomal protein S6 kinase 1 (S6K1) and ribosomal protein S6 (S6). Interestingly, the leucine-induced mTORC1 pathway activation suitably correlated with increased GDH activity and decreased expression of SIRT4. Similar results were observed in primary cultured hepatocytes. Notably, leucine exerted no significant change in GDH activity in SIRT4-deficient hepatocytes (P > 0.05), while mTORC1 signaling was activated. Leucine exerted no significant changes in both GDH activity and SIRT4 gene expression in rapamycin treated hepatocytes (P > 0.05). In conclusion, L-leucine increases GDH activity and stimulates glutamate synthesis from different nitrogen sources by regulating mTORC1/SIRT4 pathway in the liver of pigs.

Sodium taurocholate cotransporting polypeptide is the limiting host factor of hepatitis B virus infection in macaque and pig hepatocytes

Ntcp is the key host factor limiting HBV infection in cynomolgus and rhesus macaques and in pigs. In rodents (mouse, rat) and dogs, transfer of hNTCP supports viral entry but additional host factors are required for the establishment of HBV infection. This finding paves the way for the development of macaques and pigs as immunocompetent animal models to study HBV infection in vivo, immunological responses against the virus and viral pathogenesis. (Hepatology 2017;66:703-716).