Porcine Cells Research Articles

Comparative transcriptomics of porcine liver-resident CD8αdim, liver CD8αhigh and circulating blood CD8αhigh NK cells reveals an intermediate phenotype of liver CD8αhigh NK cells.

Liver-resident NK (lrNK) cells have been studied in humans as well as in mice. Unfortunately, important differences have been observed between murine and human lrNK cells, complicating the extrapolation of data obtained in mice to man. We previously described two NK cell subsets in the porcine liver: A CD8αhigh subset, with a phenotype much like conventional CD8αhigh NK cells found in the peripheral blood, and a specific liver-resident CD8αdim subset which phenotypically strongly resembles human lrNK cells. These data suggest that the pig might be an attractive model for studying lrNK cell biology. In the current study, we used RNA-seq to compare the transcriptome of three porcine NK cell populations: Conventional CD8αhigh NK cells from peripheral blood (cNK cells), CD8αhigh NK cells isolated from the liver, and the liver-specific CD8αdim NK cells. We found that highly expressed transcripts in the CD8αdim lrNK cell population mainly include genes associated with the (adaptive) immune response, whereas transcripts associated with cell migration and extravasation are much less expressed in this subset compared to cNK cells. Overall, our data indicate that CD8αdim lrNK cells show an immature and anti-inflammatory phenotype. Interestingly, we also observed that the CD8αhigh NK cell population that is present in the liver appears to represent a population with an intermediate phenotype. Indeed, while the transcriptome of these cells largely overlaps with that of cNK cells, they also express transcripts associated with liver residency, in particular CXCR6. The current, in-depth characterization of the transcriptome of porcine liver NK cell populations provides a basis to use the pig model for research into liver-resident NK cells.

Co-expression of HLA-E and HLA-G on genetically modified porcine endothelial cells attenuates human NK cell-mediated degranulation.

Natural killer (NK) cells play an important role in immune rejection in solid organ transplantation. To mitigate human NK cell activation in xenotransplantation, introducing inhibitory ligands on xenografts via genetic engineering of pigs may protect the graft from human NK cell-mediated cytotoxicity and ultimately improve xenograft survival. In this study, non-classical HLA class I molecules HLA-E and HLA-G were introduced in an immortalized porcine liver endothelial cell line with disruption of five genes (GGTA1, CMAH, β4galNT2, SLA-I α chain, and β-2 microglobulin) encoding three major carbohydrate xenoantigens (αGal, Neu5Gc, and Sda) and swine leukocyte antigen class I (SLA-I) molecules. Expression of HLA-E and/or HLA-G on pig cells were confirmed by flow cytometry. Endogenous HLA-G molecules as well as exogenous HLA-G VL9 peptide could dramatically enhance HLA-E expression on transfected pig cells. We found that co-expression of HLA-E and HLA-G on porcine cells led to a significant reduction in human NK cell activation compared to the cells expressing HLA-E or HLA-G alone and the parental cell line. NK cell activation was assessed by analysis of CD107a expression in CD3-CD56+ population gated from human peripheral blood mononuclear cells. CD107a is a sensitive marker of NK cell activation and correlates with NK cell degranulation and cytotoxicity. HLA-E and/or HLA-G on pig cells did not show reactivity to human sera IgG and IgM antibodies. This in vitro study demonstrated that co-expression of HLA-E and HLA-G on genetically modified porcine endothelial cells provided a superior inhibition in human xenoreactive NK cells, which may guide further genetic engineering of pigs to prevent human NK cell mediated rejection.

Experimental infection of a porcine kidney cell line with hepatitis A virus

The hepatitis A virus (HAV) induces severe acute liver injury and is adapted to human and monkey cell lines but not other cells. In this study, the HAV was inoculated into porcine kidney (PK-15) cells to determine its infectivity in porcine cells. The growth pattern of the HAV in PK-15 cells was compared with its growth pattern in fetal rhesus kidney (FRhK-4) cells. The growth of HAV was less efficient in PK-15 cells. In conclusion, HAV replication was verified in PK-15 cells for the first time. Further investigations will be needed to identify the HAV-restrictive mechanisms in PK-15 cells.

A Porcine DNMT1 Variant: Molecular Cloning and Generation of Specific Polyclonal Antibody.

DNA methyltransferase 1 (DNMT1), the first-identified DNA methyltransferase in mammals, has been well studied in the control of embryo development and somatic homeostasis in mice and humans. Accumulating reports have demonstrated that DNMT1 plays an important role in the regulation of differentiation and the activation of immune cells. However, little is known about the effects of porcine DNMT1 on such functional regulation, especially the regulation of the biological functions of immune cells. In this study, we report the cloning of DNMT1 (4833 bp in length) from porcine alveolar macrophages (PAMs). According to the sequence of the cloned DNMT1 gene, the deduced protein sequence contains a total of 1611 amino acids with a 2 amino acid insertion, a 1 amino acid deletion, and 12 single amino acid mutations in comparison to the reported DNMT1 protein. A polyclonal antibody based on a synthetic peptide was generated to study the expression of the porcine DNMT1. The polyclonal antibody only recognized the cloned porcine DNMT1 and not the previously reported protein due to a single amino acid difference in the antigenic peptide region. However, the polyclonal antibody recognized the endogenous DNMT1 in several porcine cells (PAM, PK15, ST, and PIEC) and the cells of other species (HEK-293T, Marc-145, MDBK, and MDCK cells). Moreover, our results demonstrated that all the detected tissues of piglet express DNMT1, which is the same as that in porcine alveolar macrophages. In summary, we have identified a porcine DNMT1 variant with sequence and expression analyses.

Human and porcine aortic valve endothelial and interstitial cell isolation and characterization.

Calcific aortic valve stenosis (AVS) is defined by pathological changes in the aortic valve (AV) and their predominant cell types: valvular interstitial (VICs) and endothelial cells (VECs). Understanding the cellular and molecular mechanisms of this disease is a prerequisite to identify potential pharmacological treatment strategies. In this study, we present a unique aortic valve cell isolation technique to acquire specific human and porcine cell populations and compared VICs and VECs of these species with each other for the first time. AV cells were isolated from tissue obtained from human patients undergoing surgical aortic valve replacement (SAVR) or from porcine hearts. Functional analysis and in vitro experiments revealed that endothelial-to-mesenchymal transition (EndMT) can be induced in hVECs, leading to a significant increase in mesenchymal markers. In vitro calcification experiments of VICs demonstrated pronounced expression of calcification markers and visible calcific deposits in Alizarin Red staining in both species after incubation with pro-calcific media. Cells isolated from patient-derived AVs showed mesenchymal and endothelial-specific gene signatures (VIC and VEC, respectively). For instance, von Willebrand factor (vWF) and platelet endothelial adhesion molecule-1 (PECAM1) were upregulated in VECs, while the myofibroblastic markers alpha-smooth muscle actin (α-SMA) and vimentin (VIM) were downregulated in VECs compared to VICs. Analysis of cell function by migration revealed that VECs are more migratory than VICs. Induction of EndMT in vitro in VECs displayed increased expression of EndMT markers and decreased expression of endothelial markers, confirming their mesenchymal transdifferentiation ability. In vitro calcification of VICs revealed upregulation of alkaline phosphatase (ALPL), a hallmark of calcification. In addition, other calcification-related genes such as osteocalcin (BGLAP) and runt-related factor 2 (RUNX2) were upregulated. Alizarin red staining of calcified cells provided a further layer of confirmation that the isolated cells were VICs with osteoblastic differentiation capacity. This study aims to take a first step towards standardizing a reproducible isolation technique for specific human and porcine VEC and VIC populations. A comparison of human and porcine aortic valve cells demonstrated that porcine cells may serve as an alternative cellular model system in settings where human tissue is difficult to obtain.

Development of a chronic focal equine arteritis virus infection of a male reproductive tract cell line

Equine arteritis virus (EAV) is an Alphaarterivirus (family Arteriviridae, order Nidovirales) that frequently causes an influenza-like illness in adult horses, but can also cause the abortions in mares and death of newborn foals. Once primary infection has been established, EAV can persist in the reproductive tract of some stallions. However, the mechanisms enabling this persistence, which depends on testosterone, remain largely unknown. We aimed to establish an in vitro model of non-cytopathic EAV infection to study viral persistence. In this work, we infected several cell lines originating from the male reproductive tract of different species. EAV infection was fully cytopathic for 92BR (donkey cells) and DDT1 MF-2 (hamster cells) cells, and less cytopathic for PC-3 cells (human cells); ST cells (porcine cells) seemed to eliminate the virus; LNCaP (human cells) and GC-1 spg (murine cells) cells were not permissive to EAV infection; finally, TM3 cells (murine cells) were permissive to EAV infection without any overt cytopathic effects. Infected TM3 cells can be maintained at least 7 days in culture without any subculture. They can also be subcultured over 39 days (subculturing them at 1:2 the first time at 5 dpi and then every 2–3 days), but in this case, the percentage of infected cells remains low. Infected TM3 cells may therefore provide a new model to study the host-pathogen interactions and to help determine the mechanisms involved in EAV persistence in stallion reproductive tract.

Anti-inflammatory IL-8 Regulation via an Advanced Drug Delivery System at the Oral Mucosa.

Oral inflammatory diseases are highly prevalent in the worldwide population. Topical treatment of inflammation is challenging due to dilution effects of saliva and crevicular fluid. Thus, there is a great medical need to develop smart anti-inflammatory drug delivery systems for mucosa treatment. We compared two promising anti-inflammatory dendritic poly(glycerol-caprolactone) sulfate (dPGS-PCL) polymers for their applicability to the oral mucosa. Using an ex vivo porcine tissue model, cell monolayers, and full-thickness 3D oral mucosal organoids, the polymers were evaluated for muco-adhesion, penetration, and anti-inflammatory properties. The biodegradable dPGS-PCL97 polymers adhered to and penetrated the masticatory mucosa within seconds. No effects on metabolic activity and cell proliferation were found. dPGS-PCL97 revealed a significant downregulation of pro-inflammatory cytokines with a clear preference for IL-8 in cell monolayers and mucosal organoids. Thus, dPGS-PCL97 exhibits excellent properties for topical anti-inflammatory therapy, suggesting new therapeutic avenues in the treatment of oral inflammatory diseases.

Preventive effects of quercetin against foot-and-mouth disease virus in vitro and in vivo by inducing type I interferon.

Foot-and-mouth disease (FMD) is an acute contagious infectious disease that affects cloven-hoofed animals. Although current emergency FMD vaccines only take effect 7 days after vaccination, antiviral agents, such as quercetin, which is a common flavonoid, could reduce the spread of FMD virus (FMDV) during outbreaks. We investigated the in vitro and in vivo antiviral effects of quercetin against FMDV. Analysis of viral copy numbers showed that quercetin had a dose-dependent inhibitory effect on FMDV at concentrations between 19.5 and 1,250 μM in porcine cells. In addition, we observed a quercetin-induced interferon (IFN)-α protein and interferon-stimulated gene (ISG) upregulation in swine cells. Enzyme-linked immunosorbent assay of sera revealed that quercetin induces the production of IFN-α, IFN-β, IFN-γ, interleukin (IL)-12, and IL-15 in mice. Inoculation of mice with quercetin or a combination of quercetin with an inactivated FMD vaccine enhanced both the survival rate and neutralizing antibody titer. Therefore, we suggest the use of quercetin as a novel and effective antiviral agent for controlling FMDV infection; however, further investigation of its application in livestock is required.

Development of swine immune reagents for swine immunity and biomedical research

Abstract The USDA-NIFA Swine Immune Toolkit Initiative has been involved in generating priority immune reagents for swine immunity and biomedical research and pipelining them for marketing. With the help of commercial partners we expressed each target immune molecule as a soluble protein using yeast expression systems and then produced panels of monoclonal antibodies (mAbs) against each target. Most recently we generated panels of mAbs reactive with porcine IL-5, IL-6, IL-13, IL-21, IL-28B, CXCL10, and BAFF and screened their reactivity in multiple immune assays. We determined mAb reactivity with orthologous proteins for most panels of mAbs. A sensitive sandwich ELISA is now available for IL-13 and CXCL10; other targets are being screened for best mAb pairs. Reactivity tests of labeled α-CXCL10, α-IL-6 and α-IL-13 mAbs for intracellular staining of porcine immune cells using flow cytometry assays are underway using different cell stimulation conditions. Immunohistochemistry analyses for binding of α-CXCL10 and other mAbs on formalin fixed pig lymph nodes and spleen tissues are in progress. Further, analysis of a large panel of commercial α-human CD antigen mAbs for cross-reactivity with porcine cells is ongoing. For each target, our goal is to provide the veterinary community with new commercial reagents and standardized assay techniques for their research efforts. Finally, efforts are proceeding to develop SLA tetramers for antigen presentation studies. Tools and reagents generated by this project will undoubtedly advance our understanding of swine immune responses to disease and vaccine and use for biomedical research efforts. Supported by USDA-NIFA AFRI grant # 2019-67015-29815 and USDA ARS project 8042-32000-117.

Chia (Salvia hispanica L.) can directly suppress basic ovarian cell functions in two farm animal species and protect ovarian cells from the proliferation-stimulating influence of xylene.

The influence of the functional food plant chia (Salvia hispanica L.) on reproduction functions and its ability to prevent the negative effects of environmental contaminants has not yet been studied. Our study aimed to examine the effect of chia seed extract alone and in combination with xylene on the markers of proliferation, apoptosis and hormones release by cultured bovine and porcine ovarian granulosa cells. The extract of chia reduced all of the measured parameters in bovine and porcine ovarian cells but had no effect on the proliferation of porcine cells. Xylene, stimulated proliferation and IGF-I release and inhibited the release of progesterone and testosterone but not apoptosis of bovine granulosa cells. It promoted proliferation, apoptosis and progesterone output by porcine cells. Chia mitigated the stimulatory effect of xylene on proliferation but not on other parameters in both species. The present results are the first demonstration of a direct effect of chia on basic ovarian cell functions. They confirmed a direct influence of xylene on these functions and found a similar stimulatory action of xylene on bovine and porcine ovarian cell proliferation. The present observations demonstrated species-specific differences in the characteristics of xylene influences on ovarian cell apoptosis and secretory activity. Finally, the present results indicate that chia can be a natural protector against the proliferation-stimulating effects of xylene on ovarian cells in both species.

Highly Efficient A-to-G Editing in PFFs via Multiple ABEs.

Cytosine base editors (CBEs) and adenine base editors (ABEs) are recently developed CRISPR-mediated genome-editing tools that do not introduce double-strand breaks. In this study, five ABEs, ABE7.10, ABEmax, NG-ABEmax, ABE8e and NG-ABE8e, were used to generate A-to-G (T-to-C) conversions in five genome loci in porcine fetal fibroblasts (PFFs). Variable yet appreciable editing efficiencies and variable activity windows were observed in these targeting regions via these five editors. The strategy of two sgRNAs in one vector exhibited superior editing efficiency to that of using two separate sgRNA expression vectors. ABE-mediated start-codon mutation in APOE silenced its expression of protein and, unexpectedly, eliminated the vast majority of its mRNA. No off-target DNA site was detected for these editors. Substantial off-target RNA events were present in the ABE-edited cells, but no KEGG pathway was found to be significantly enriched. Our study supports that ABEs are powerful tools for A-to-G (T-to-C) point-mutation modification in porcine cells.

Production of MSTN knockout porcine cells using adenine base-editing-mediated exon skipping

Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9 and cytosine base editing (CBE) technologies, adenine base editing (ABE) shows better safety and accuracy in gene modification. However, because of the characteristics of gene sequences, the ABE system cannot be widely used in gene knockout. Alternative splicing of mRNA is an important biological mechanism in eukaryotes for the formation of proteins with different functional activities. The splicing apparatus recognizes conserved sequences of the 5' end splice donor and 3' end splice acceptor motifs of introns in pre-mRNA that can trigger exon skipping, leading to the production of new functional proteins, or causing gene inactivation through frameshift mutations. This study aimed to construct a MSTN knockout pig by inducing exon skipping with the aid of the ABE system to expand the application of the ABE system for the preparation of knockout pigs. In this study, first, we constructed ABEmaxAW and ABE8eV106W plasmid vectors and found that their editing efficiencies at the targets were at least sixfold and even 260-fold higher than that of ABEmaxAW by contrasting the editing efficiencies at the gene targets of endogenous CD163, IGF2, and MSTN in pigs. Subsequently, we used the ABE8eV106W system to realize adenine base (the base of the antisense strand is thymine) editing of the conserved splice donor sequence (5'-GT) of intron 2 of the porcine MSTN gene. A porcine single-cell clone carrying a homozygous mutation (5'-GC) in the conserved sequence (5'-GT) of the intron 2 splice donor of the MSTN gene was successfully generated after drug selection. Unfortunately, the MSTN gene was not expressed and, therefore, could not be characterized at this level. No detectable genomic off-target edits were identified by Sanger sequencing. In this study, we verified that the ABE8eV106W vector had higher editing efficiency and could expand the editing scope of ABE. Additionally, we successfully achieved the precise modification of the alternative splice acceptor of intron 2 of the porcine MSTN gene, which may provide a new strategy for gene knockout in pigs.

Role of heat shock protein 90 as an antiviral target for swine enteric coronaviruses

A variety of swine enteric coronaviruses (SECoVs) have emerged and are prevalent in pig populations, including porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome (SADS)-CoV, a newly identified bat-origin CoV with zoonotic potential. Unfortunately, available traditional, inactivated and attenuated SECoV vaccines are of limited efficacy against the variants currently circulating in most pig populations. In this study, we evaluated the role of host factor heat shock protein 90 (Hsp90) as an antiviral target against SECoVs, exemplified by SADS-CoV. Pharmacological inhibition of Hsp90 diminished SADS-CoV replication significantly in porcine and human cell lines, and also decreased replication of SADS-CoV in a porcine intestinal enteroid model. Further mechanistic experiments revealed that both porcine and human isoforms of Hsp90 interact with the SADS-CoV nucleocapsid (N) protein, and inhibition of Hsp90 resulted in autophagic degradation of N protein. Moreover, we linked Hsp90 to virus-induced cellular pyroptosis, as SADS-CoV was found to trigger caspase-1/gasdermin-d-mediated pyroptotic cell death, which was mitigated by inhibition of Hsp90. Finally, we demonstrated that Hsp90 also associated with N proteins and was involved in propagation of PEDV, PDCoV and TGEV. This study thus extends our understanding of immune responses to SADS-CoV infection and offers a new potential therapeutic option against four SECoVs.

Development and Characterization of Efficient Cell Culture Systems for Genotype 1 Hepatitis E Virus and Its Infectious cDNA Clone.

Hepatitis E virus (HEV) is a major cause of acute viral hepatitis globally. Genotype 1 HEV (HEV-1) is responsible for multiple outbreaks in developing countries, causing high mortality rates in pregnant women. However, studies on HEV-1 have been hindered by its poor replication in cultured cells. The JE04-1601S strain recovered from a Japanese patient with fulminant hepatitis E who contracted HEV-1 while traveling to India was serially passaged 12 times in human cell lines. The cell-culture-generated viruses (passage 12; p12) grew efficiently in human cell lines, but the replication was not fully supported in porcine cells. A full-length cDNA clone was constructed using JE04-1601S_p12 as a template. It was able to produce an infectious virus, and viral protein expression was detectable in the transfected PLC/PRF/5 cells and culture supernatants. Consistently, HEV-1 growth was also not fully supported in the cell culture of cDNA-derived JE04-1601S_p12 progenies, potentially recapitulating the narrow tropism of HEV-1 observed in vivo. The availability of an efficient cell culture system for HEV-1 and its infectious cDNA clone will be useful for studying HEV species tropism and mechanisms underlying severe hepatitis in HEV-1-infected pregnant women as well as for discovering and developing safer treatment options for this condition.

Multiplexed genome engineering for porcine fetal fibroblasts with gRNA–tRNA arrays based on CRISPR/Cas9

Multiplex gene modifications are highly required for various fields of porcine research. In many species, the CRISPR/Cas9 system has been widely applied for genomic editing and provides a potential tool for introducing multiplex genome mutations simultaneously. Here, we present a CRISPR–Cas9 gRNA–tRNA array (GTR–CRISPR) for multiplexed engineering of porcine fetal fibroblasts (PFFs). We successfully produced multiple sgRNAs using only one Pol III promoter by taking advantage of the endogenous tRNA processing mechanism in porcine cells. Using an all-in-one construct carrying GTR and Cas9, we disrupted the IGFBP3, MSTN, MC4R, and SOCS2 genes in multiple codon regions in one PFF cell simultaneously. This technique allows the simultaneous disruption of four genes with 5.5% efficiency. As a result, this approach may effectively target multiple genes at the same time, making it a powerful tool for establishing multiple genes mutant cells in pigs.

Identification AAVS1-like locus from the porcine genome and site-specific integration of recombinase-mediated cassette exchange using CRISPR/Cas9

Gene integration at site-specific loci is a critical approach for understanding the function of a gene in cells or animals. The AAVS1 locus is a well-known safe harbor for human and mouse studies. In this study, we found an AAVS1-like sequence (pAAVS1) in the porcine genome using the Genome Browser and designed TALEN and CRISPR/Cas9 to target the pAAVS1. The efficiency of CRISPR/Cas9 in porcine cells was superior to that of TALEN. We added a loxP-lox2272 sequences to the pAAVS1 targeting donor vector containing GFP for further exchange of various transgenes via recombinase-mediated cassette exchange (RMCE). The donor vector and CRISPR/Cas9 components were transfected into porcine fibroblasts. Targeted cells of CRISPR/Cas9-mediated homologous recombination were identified by antibiotic selection. Gene knock-in was confirmed by PCR. To induce RMCE, another donor vector containing the loxP-lox2272 and inducible Cre recombinase was cloned. The Cre-donor vector was transfected into the pAAVS1 targeted cell line, and RMCE was induced by adding doxycycline to the culture medium. RMCE in porcine fibroblasts was confirmed using PCR. In conclusion, gene targeting at the pAAVS1 and RMCE in porcine fibroblasts was successful. This technology will be useful for future porcine transgenesis studies and the generation of stable transgenic pigs.

Chicken or Porcine Aminopeptidase N Mediates Cellular Entry of Pseudoviruses Carrying Spike Glycoprotein from the Avian Deltacoronaviruses HKU11, HKU13, and HKU17.

Members of deltacoronavirus (DCoV) have mostly been identified in diverse avian species as natural reservoirs, though the porcine DCoV (PDCoV) is a major swine enteropathogenic virus with global spread. The important role of aminopeptidase N (APN) orthologues from various mammalian and avian species in PDCoV cellular entry and interspecies transmission has been revealed recently. In this study, comparative analysis indicated that three avian DCoVs, bulbul DCoV HKU11, munia DCoV HKU13, and sparrow DCoV HKU17 (Chinese strain), and PDCoV in the subgenera Buldecovirus are grouped together at whole-genome levels; however, the spike (S) glycoprotein and its S1 subunit of HKU17 are more closely related to night heron DCoV HKU19 in Herdecovirus. Nevertheless, the S1 protein of HKU11, HKU13, or HKU17 bound to or interacted with chicken APN (chAPN) or porcine APN (pAPN) by flow cytometry analysis of cell surface expression of APN and by coimmunoprecipitation in APN-overexpressing cells. Expression of chAPN or pAPN allowed entry of pseudotyped lentiviruses with the S proteins from HKU11, HKU13 and HKU17 into nonsusceptible cells and natural avian and porcine cells, which could be inhibited by the antibody against APN or anti-PDCoV-S1. APN knockdown by siRNA or knockout by CRISPR/Cas9 in chicken or swine cell lines significantly or almost completely blocked infection of these pseudoviruses. Hence, we demonstrate that HKU11, HKU13, and HKU17 with divergent S genes likely engage chAPN or pAPN to enter the cells, suggesting a potential interspecies transmission from wild birds to poultry and from birds to mammals by certain avian DCoVs. IMPORTANCE The receptor usage of avian deltacoronaviruses (DCoVs) has not been investigated thus far, though porcine deltacoronavirus (PDCoV) has been shown to utilize aminopeptidase N (APN) as a cell receptor. We report here that chicken or porcine APN also mediates cellular entry by three avian DCoV (HKU11, HKU13, and HKU17) spike pseudoviruses, and the S1 subunit of three avian DCoVs binds to APN in vitro and in the surface of avian and porcine cells. The results fill the gaps in knowledge about the avian DCoV receptor and elucidate important insights for the monitoring and prevention of potential interspecies transmission of certain avian DCoVs. In view of the diversity of DCoVs, whether this coronavirus genus will cause novel virus to emerge in other mammals from birds, are worthy of further surveillance and investigation.

Large-gap peripheral nerve repair using xenogeneic transplants in rhesus macaques.

Surgical intervention is required to successfully treat severe, large-gap (≥4cm) peripheral nerve injuries. However, all existing treatments have shortcomings and an alternative to the use of autologous nerves is needed. Human and porcine nerves are physiologically similar, with comparable dimensions and architecture, presence and distribution of Schwann cells, and conserved features of the extracellular matrix (ECM). We report the repair of fully transected radial nerves in 10 Rhesus Macaques using viable, whole sciatic nerve from genetically engineered (GalT-KO), designated pathogen free (DPF) porcine donors. This resulted in the regeneration of the transected nerve, and importantly, recovery of wrist extension function, distal muscle reinnervation, and recovery of nerve conduction velocities and compound muscle action potentials similar to autologous controls. We also demonstrate the absence of immune rejection, systemic porcine cell migration, and detectable residual porcine material. Our preliminary findings support the safety and efficacy of viable porcine nerve transplants, suggest the interchangeable therapeutic use of cross-species cells, and highlight the broader clinical potential of xenotransplantation.

The relationship between autophagy and apoptosis during pseudorabies virus infection.

Both autophagy and apoptosis are mechanisms that maintain homeostasis in cells and that play essential roles in viral infections. Previous studies have demonstrated that autophagy and apoptosis pathways occurred with complex relationships in virus-infected cells. However, the regulation between these two processes in Pseudorabies virus (PRV) infection remains unclear. In the present study, we demonstrated that activated autophagy was induced at the early stage of PRV infection and that apoptosis was induced at the late stage of infection. Autophagy induction inhibited apoptosis and decreased viral replication, and autophagy inhibition promoted apoptosis and increased viral replication. We also found that viral infection resulted in an increase in the production of reactive oxygen species (ROS) and activation of apoptosis in autophagy-impaired cells, suggesting that ROS may participate in the cross-talk between autophagy and apoptosis in PRV-infected cells. Our studies provide possible molecular mechanisms for the cross-talk between apoptosis and autophagy induced by PRV infection in porcine cells. This suggests that these two cell death processes should be considered as the same continuum rather than as completely separate processes.

Efficient and Safe Editing of Porcine Endogenous Retrovirus Genomes by Multiple-Site Base-Editing Editor.

Gene-modified miniature pigs serve as alternative tissue and organ donors for xenotransplantation to alleviate the shortage of human allogenic organs. However, the high copy number of porcine endogenous retrovirus (PERV) genomes integrates with the porcine genome, which has a potential risk of cross-species transmission and hinders the clinical practice of xenotransplantation. Recently, CRISPR/Cas9 has been used to inactivate PERVs. However, Cas9 also triggers severe DNA damage at multiple integrated PERV sites in the porcine genome, which induces senescence and apoptosis of porcine cells. In this study, the cytosine base editor (CBE), an efficient and safe editor that does not cause DNA double strand breaks (DSBs), was used for PERV editing to reduce cytotoxic effects. Seven sgRNAs were set to target gag and pol loci of PERVs to induce premature stop codons. We found that approximately 10% of cell clones were completely inactivated for PERVs in pig ST cells, and the plasmid that was used for editing the PERVs did not integrate into host genome and influence the karyotype of the modified cells. Our studies offer a powerful and safe strategy for further generating PERV-knockout pigs using base editors.