Swine Peripheral Blood Mononuclear Cells Research Articles

Effect of agro-industrial by-products on inflammation and oxidative stress using an in vitro cell model

Abstract Agro-industrial by-products can play an important role in the animal feeding, being an alternative to conventional raw materials. These two wastes can be considered as important sources of bioactive compounds that can be used in animal feed with multiple benefits for animal health. This study has investigated the anti-inflammatory and anti-oxidant in vitro capacity of grape seed and sea buckthorn meals, as waste resulted from the oil industry in order to be used in swine nutrition. The in vitro study was realised on swine peripheral blood mononuclear cells challenged with LPS and treated or not with grape seed meal and sea buckthorn meal extracts. Our results show that both extracts have the capacity to attenuate the inflammation and oxidative stress induced by LPS. The effect induced by the grape seed meal extract was in majority of cases more important than of the sea buckthorn, this effect might be due to a different composition in bioactive compounds (polyphenols, PUFA etc) of the two extracts.

Co-Contamination of Food and Feed with Mycotoxin and Bacteria and Possible Implications for Health

Food and feed safety is an issue of great concern for both animal and human health, due to the frequent contamination of food and feed with pathogens, such as bacteria, viruses, yeasts, molds, and parasites. The present paper assumes the possibility that a mycotoxin contamination could occur at the same time as a bacterial infection and investigates the effects of such co-contamination, in comparison with the individual effects of bacterial challenge. For this purpose, we have investigated the effects of simultaneous contamination of swine peripheral blood mononuclear cells (PBMCs) with lipopolysaccharide (LPS) (as a model for bacterial contamination) and mycotoxins (ochratoxin or zearalenone) on cell viability, cell cycles, oxydative stress, and inflammation. Our results show important additive/synergistic effects of co-exposure to fungal and bacterial toxins, and that these effects are more important when the cells are co-exposed to LPS and ochratoxin (OTA). The exposure of PBMCs to both OTA and LPS induced an exacerbation of the increase in the inflammation of concentrations of pro-inflamatory cytokines, compared with that of LPS-challenged cells: IL-1β (4.1 times increase), TNF-α (3.2 times increase), IL-6 (3.1 times increase). There was also a decrease in antioxidant defence (i.e., a significant decrease in the total antioxidant capacity and catalase activity) and a significant increase in the percentage of cells undergoing necrosis (24.3% vs. 15.3% in LPS-treated cells). The exposure to zearalenone (ZEA) and LPS led to less important effects and concerned mainly the parameters related to oxidative stress (i.e., a decrease in total antioxidant capacity). The present study provides important data for risk assessment, as the concomitant contamination with bacteria and mycotoxins can lead to a higher toxicity than that which results after an individual infection with Gram-negative bacteria.

Programmed death-ligand 1 expression in swine chronic infections and enhancement of interleukin-2 production via programmed death-1/programmed death-ligand 1 blockade.

IntroductionChronic infections lead to the functional exhaustion of T cells. Exhausted T cells are phenotypically differentiated by the surface expression of the immunoinhibitory receptor, such as programmed death‐1 (PD‐1). The inhibitory signal is produced by the interaction between PD‐1 and its PD‐ligand 1 (PD‐L1) and impairs the effector functions of T cells. However, the expression dynamics of PD‐L1 and the immunological functions of the PD‐1/PD‐L1 pathway in chronic diseases of pigs are still poorly understood. In this study, we first analyzed the expression of PD‐L1 in various chronic infections in pigs, and then evaluated the immune activation by the blocking assay targeting the swine PD‐1/PD‐L1 pathway.MethodsIn the initial experiments, anti‐bovine PD‐L1 monoclonal antibodies (mAbs) were tested for cross‐reactivity with swine PD‐L1. Subsequently, immunohistochemical analysis was conducted using the anti‐PD‐L1 mAb. Finally, we assessed the immune activation of swine peripheral blood mononuclear cells (PBMCs) by the blockade with anti‐PD‐L1 mAb.ResultsSeveral anti‐PD‐L1 mAbs tested recognized swine PD‐L1‐expressing cells. The binding of swine PD‐L1 protein to swine PD‐1 was inhibited by some of these cross‐reactive mAbs. In addition, immunohistochemical analysis revealed that PD‐L1 was expressed at the site of infection in chronic infections of pigs. The PD‐L1 blockade increased the production of interleukin‐2 from swine PBMCs.ConclusionsThese findings suggest that the PD‐1/PD‐L1 pathway could be also involved in immunosuppression in chronic infections in pigs. This study provides a new perspective on therapeutic strategies for chronic diseases in pigs by targeting immunosuppressive pathways.

African Swine Fever Virus CD2v Protein Induces β-Interferon Expression and Apoptosis in Swine Peripheral Blood Mononuclear Cells.

African swine fever (ASF) is a hemorrhagic disease of swine characterized by massive lymphocyte depletion in lymphoid tissues due to the apoptosis of B and T cells, a process likely triggered by factors released or secreted by infected macrophages. ASFV CD2v (EP402R) has been implicated in viral virulence and immunomodulation in vitro; however, its actual function(s) remains unknown. We found that CD2v expression in swine PK15 cells induces NF-κB-dependent IFN-β and ISGs transcription and an antiviral state. Similar results were observed for CD2v protein treated swine PBMCs and macrophages, the major ASFV target cell. Notably, treatment of swine PBMCs and macrophages with CD2v protein induced apoptosis. Immunoprecipitation and colocalization studies revealed that CD2v interacts with CD58, the natural host CD2 ligand. Additionally, CD58 knockdown in cells or treatment of cells with an NF-κB inhibitor significantly reduced CD2v-mediated NF-κB activation and IFN-β induction. Further, antibodies directed against CD2v inhibited CD2v-induced NF-κB activation and IFN-β transcription in cells. Overall, results indicate that ASFV CD2v activates NF-κB, which induces IFN signaling and apoptosis in swine lymphocytes/macrophages. We propose that CD2v released from infected macrophages may be a significant factor in lymphocyte apoptosis observed in lymphoid tissue during ASFV infection in pigs.

Production of a Locus- and Allele-Specific Monoclonal Antibody for the Characterization of SLA-1*0401 mRNA and Protein Expression Levels in MHC-Defined Microminipigs.

The class I major histocompatibility complex (MHC) presents self-developed peptides to specific T cells to induce cytotoxity against infection. The MHC proteins are encoded by multiple loci that express numerous alleles to preserve the variability of the antigen-presenting ability in each species. The mechanism regulating MHC mRNA and protein expression at each locus is difficult to analyze because of the structural and sequence similarities between alleles. In this study, we examined the correlation between the mRNA and surface protein expression of swine leukocyte antigen (SLA)-1*0401 after the stimulation of peripheral blood mononuclear cells (PBMCs) by Staphylococcus aureus superantigen toxic shock syndrome toxin-1 (TSST-1). We prepared a monoclonal antibody (mAb) against a domain composed of Y102, L103 and L109 in the α2 domain. The Hp-16.0 haplotype swine possess only SLA-1*0401, which has the mAb epitope, while other haplotypes possess 0 to 3 SLA classical class I loci with the mAb epitopes. When PBMCs from SLA-1*0401 homozygous pigs were stimulated, the SLA-1*0401 mRNA expression level increased until 24 hrs and decreased at 48 hrs. The kinetics of the interferon regulatory transcription factor-1 (IRF-1) mRNA level were similar to those of the SLA-1*0401 mRNA. However, the surface protein expression level continued to increase until 72 hrs. Similar results were observed in the Hp-10.0 pigs with three mAb epitopes. These results suggest that TSST-1 stimulation induced both mRNA and surface protein expression of class I SLA in the swine PBMCs differentially and that the surface protein level was sustained independently of mRNA regulation.

MicroRNA Transcriptome of Poly I:C-Stimulated Peripheral Blood Mononuclear Cells Reveals Evidence for MicroRNAs in Regulating Host Response to RNA Viruses in Pigs.

MicroRNAs (miRNAs) are one family of small noncoding RNAs that function to modulate the activity of specific mRNA targets in animals. To understand the role of miRNAs in regulating genes involved in the host immune response to RNA viruses, we profiled and characterized the miRNAs of swine peripheral blood mononuclear cells (PBMC) stimulated with poly I:C, a synthetic dsRNA analog, by miRNA-sequencing (miRNA-seq). We identified a total of 905 miRNAs, of which 503 miRNAs were firstly exploited herein with no annotation in the latest miRBase 21.0. Expression analysis demonstrated that poly I:C stimulation can elicit significantly differentially expressed (DE) miRNAs in Dapulian (n = 20), one Chinese indigenous breed, as well as Landrace (n = 23). By integrating the mRNA expression profiles of the same sample with miRNA profiles, we carried out function analyses of the target genes of these DE miRNAs, with the results indicating that target genes were most enriched in some immune-related pathways and gene ontology (GO) terms, suggesting that DE miRNAs play an important role in the regulation of host to poly I:C stimulation. Furthermore, we also detected 43 and 61 significantly DE miRNAs between the two breeds in the control sample groups and poly I:C stimulation groups, respectively, which may be involved in regulation of the different characteristics of the two breeds. This study describes for the first time the PBMC miRNA transcriptomic response to poly I:C stimulation in pigs, which not only contributes to a broad view of the pig miRNAome but improves our understanding of miRNA function in regulating host immune response to RNA viruses.

Analysis of the Immuno-enhancing effects of Biophoton Energy Projector on Swine Peripheral Blood Mononuclear Cells

Laboratory of Veterinary Pharmacology, College of Veterinary Medicine, Jeju National University, Jeju 63243, Republic of Korea 3) Abstract: In photobiophysics, biophoton means a kind of biological energy which enhances most metabolisms of the body. To investigate the immuno-enhancing effects of biophoton energy projector (BEP) producing light energy, pigs were irradiated with BEP for 8 weeks. Swine peripheral blood mononuclear cells (sPBMCs) were isolated from the blood of irradiated pigs. In this study, the antigen uptake and mitochondrial membrane potential of sPBMCs were measured by flow cytometric analysis. The irradiation of BEP increased the antigen uptake of sPBMCs. For functional analysis, the production of Bordetella bronchiseptica-specific IgG, measured using antigen-specific ELISA, was increased during the period of BEP irradiation. Taken together, the results suggest that the irradiation of BEP has immune-enhancing effects on sPBMCs.

Interactive host cells related to Mycoplasma suis α-enolase by yeast two-hybrid analysis

Mycoplasma suis belongs to the haemotrophic mycoplasmas, which colonise the red blood cells of a wide range of vertebrates. Adhesion to red blood cells is the crucial step in the unique lifecycle of M. suis. In addition to MSG1 protein, α-enolase is the second adhesion protein of M. suis, and may be involved in the adhesion of M. suis to porcine red blood cells (RBC). To simulate the environment of the RBC, we established the cDNA library of swine peripheral blood mononuclear cells (PBMC). The yeast two-hybrid (Y2H) system was adopted to screen α-enolase interactive proteins in the PBMC line. Alignment with the NCBI database revealed four interactive proteins: beta-actin, 60S ribosomal protein L11, clusterin precursor and endonuclease/reverse transcriptase. However, the M. suis α-enolase interactive proteins in the PBMC cDNA library obtained in the current study provide valuable information about the host cell interactions of the M. suis α-enolase protein.

Modulation of the release of Ang-2 in experimental endotoxic shock by a species-specific circulating factor

ObjectivesTo study the modulation of the release of angiopoietin-2 (Ang-2) in experimental endotoxic shock. MethodsTwelve pigs were studied; eight became septic after the intravenous infusion of lipopolysaccharide (LPS) of Escherichia coli O55:B5. The concentrations of LPS, angiopoietin-2 (Ang-2), tumour necrosis factor-alpha (TNFα) and malondialdehyde (MDA) were measured soon after the LPS infusion in the serum samples from the pulmonary and systemic circulation. Peripheral blood mononuclear cells (PBMCs) were isolated from two healthy swine, from two healthy human donors and from four patients with septic shock. The PBMCs were cultured with the serum of the septic animals in the presence or absence of polymyxin B. Concentrations of Ang-2 and TNFα were measured in supernatants. ResultsSerum Ang-2 was higher in the systemic circulation than in the pulmonary circulation. Increased Ang-2 release was noted in swine PBMCs in the presence of polymyxin B. A reciprocal decrease in TNFα release was observed, typically after incubation with serum sampled from the pulmonary circulation. ConclusionThere is evidence for a circulating factor that primes Ang-2 release from blood monocytes in the event of septic shock. The finding indicates a possible site of interference within the septic shock cascade.

Porcine MHC classical class I genes are coordinately expressed in superantigen-activated mononuclear cells

The expression of the major histocompatibility complex (MHC) classical class I genes is important for the adaptive immune response to target virus-infected cells and cancer cells. The up-regulation of the MHC is achieved by hormonal/cytokine signals including IFN-γ-inducible elements. The swine leukocyte antigen (SLA), the MHC class I region of pigs, consists of the duplicated classical class I genes, SLA-1, SLA-2 and SLA-3, but the molecular mechanisms involved in their up-regulation after T cell stimulation have not been fully elucidated. In order to better understand some of the putative regulatory mechanisms of SLA class I gene expression in activated T cells, we examined the coordinated expression of the SLA classical class I, IFN-γ and interferon regulatory factor-1 (IRF-1) genes in the peripheral blood mononuclear cells (PBMCs) of SLA homozygous Clawn miniature swine stimulated for 72h with either IFN-γ or an enterotoxin produced by Staphylococcus aureus. This enterotoxin, toxic shock syndrome-1 (TSST-1), is known to act as a superantigen (sAG) to activate the T cells in various vertebrate species. We showed by using mAbs and flow cytometry that the CD4+CD25+ cell number of swine PBMCs was also increased by TSST-1 and to a lesser degree by IFN-γ. Time course analyses of the expression of the IFN-γ, IRF-1 and the three classical class I genes, SLA-1, SLA-2, and SLA-3, in PBMCs by quantitative real-time PCR revealed a transitory response to TSST-1 or IFN-γ stimulation. The IFN-γ mRNA levels in the PBMCs were continuously up-regulated over the first 48h by TSST-1 or IFN-γ. In contrast, SLA class I expression moderately increased at 24h and then decreased to a baseline level or less at 72h of IFN-γ or TSST-1 stimulation. The three classical SLA class I genes showed similar expression kinetics, although SLA-3 mRNA level was consistently lower than those of SLA-1 and -2. The expression of IRF-1, a modulator of SLA expression, showed similar kinetics to those of the three classical SLA class I genes. The expression profiles detected by flow cytometry of the SLA molecules on the cell surface of PBMCs were maintained at a consistently high level during cell stimulation with either TSST-1 or IFN-γ, which was distinct from the kinetics of mRNA expression. These results showed that miniature swine SLA class I mRNA expression was effectively and equally up-regulated among the three loci and coordinately with IRF-1 gene expression after stimulation of T cell activation by sAG or IFN-γ.

Construction of Swine-Specific CpG Motif Enriched Plasmid and the Study of Its Immunostimulatory Effects Both <i>In Vitro</i> and <i>In Vivo</i>

A swine-specific CpG motif enriched plasmid (pUC18-CpG) was constructed in this study. Its immunostimulant property was tested in vitro via lymphocyte transformation assay using swine peripheral blood mononuclear cells (PBMCs). The recombinant plasmid showed higher Stimulation Index (SI) compared to the positive control (LPS). In a following animal experiment, pUC18-CpG was co-administered with a commercial swine FMD killed vaccine. Animals in the pUC18-CpG adjuvanted groups showed much higher antibody titers during the vaccination period.

Changes in the porcine peripheral blood mononuclear cell proteome induced by infection with highly virulent classical swine fever virus

Leukopenia and immunosuppression are characteristic clinical manifestations of classical swine fever and peripheral blood mononuclear cells (PBMCs) are major targets of classical swine fever virus. To investigate proteomic expression changes in swine PBMCs during lethal CSFV infection, proteins of PBMCs from five lethally CSFV-infected pigs were resolved by two-dimensional electrophoresis followed by mass spectrometry. Quantitative intensity analysis revealed that 66 protein spots showed altered expression, 44 of which were identified as 34 unique proteins by MALDI-TOF-MS/MS. Cellular functions of these proteins included cytoskeletal, energy metabolism, protein translation and processing, antioxidative stress, heat shock and blood clotting. Western blot analysis confirmed the upregulation of annexin A1 and downregulation of cofilin. Identification of these changed levels of expression provides an understanding at the molecular level of the response of in vivo target cells to CSFV infection and of the pathogenic mechanisms of leukopenia and immunosuppression induced by the virus.

Effects of Interferon-α on the Inflammatory Response of Swine Peripheral Blood Mononuclear Cells

Interferon-α (IFN-α) at low concentrations had been previously shown to control the expression of inflammatory cytokine genes in swine pulmonary alveolar macrophages. In the first part of this study, cultured swine peripheral blood mononuclear cells (PBMCs) were supplemented with IFN-α at low/moderate concentrations, and then stimulated with lipopolysaccharide (LPS). The expression of IFN-α, IFN-γ, IL-1β, TNF-α, and IL-6 genes was determined by real-time PCR. IFN-α at low/moderate concentrations did not significantly reduce the expression of any cytokine gene under study, with clear trends though to a concentration-dependent reduction of IL-1β gene expression and to a concentration-dependent increase of IFN-γ gene expression. In vivo, orally administered IFN-α was shown instead to modulate the inflammatory response to early weaning in uncultured PBMCs of specific pathogen-free piglets. As opposed to the in vitro model, the oral IFN-α treatment reduced after weaning the expression of the IFN-γ gene (P < 0.08) and increased that of the IL-1β gene (P < 0.05). There was also a trend to a reduced expression of both IL-6 and TNF-α. The above modulation of cytokine genes expression and the greater daily mean weight gain of treated piglets highlight important regulatory properties of oral IFN-α in the response to the weaning stress.

The cytotoxicity to leukemia cells and antiviral effects of Isatis indigotica extracts on pseudorabies virus

Aim of the study Isatis indigotica ( I. indigotica), Cruciferae, has been used in Chinese medicine for anti-leukemia and anti-severe acute respiratory syndrome (SARS). The aim of this study was to evaluate the cytotoxicity of Isatis indigotica extracts on human leukemia cell line (HL-60) and the antiviral activity on swine pseudorabies virus (PrV) in in vitro assays. Materials and methods Extracts and derived fractions of Isatis indigotica were prepared from root (R) and leaf (L) using methanol (M), ethyl acetate (E) and distilled water (D). The cytotoxic effect of extracts on swine peripheral blood mononuclear cells (PBMCs) and HL-60 was assessed by MTT method. The cytopathic effect (CPE) reduction, plaque reduction and inhibition assays on viral replication, and virucidal activity were further conducted to investigate the anti-PrV activity. Results Indirubin, one of the biological active compounds of Isatis indigotica, had the most significant cytotoxicity on HL-60 cells and inhibitory effect on PrV replication. Extracts from roots and leaves of Isatis indigotica also presented CPE inhibition either before or after infection of PrV on porcine kidney (PK-15) cells. Leaf extracts had better virucidal activity than roots, and ethyl acetate extracts exhibited the highest efficacy among extracts tested. Conclusion Isatis indigotica posses a valuable virucidal effect in disease control of pseudorabies virus infection in swine.

Fumonisin B1 alters cell cycle progression and interleukin‐2 synthesis in swine peripheral blood mononuclear cells

Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium verticillioides, a fungus that commonly contaminates maize. In the present study, we investigated the effects of FB1 on swine peripheral blood mononuclear cells (PBMC) by measuring cell proliferation, cell cycle progression and interleukin (IL)-2 production. Forty-eight hours after treatment in vitro, FB1 induced a decrease of PBMC proliferation as measured by cell counting and dehydrogenase enzyme activity. This effect was observed starting with 10 microM FB1. The effect of FB1 on cell cycle progression was analyzed by flow cytometry. Incubation of PBMC with FB1 increased the percentage of cells blocked in G0/G1 phase of the cell cycle. Treatment with 200 microM FB1 induces a high blockade of the cell cycle, with 92.4% of cells in G0/G1 phase. This blockade was observed in all lymphocyte subsets tested (CD2+, CD4+, CD8+ and Ig+) as evidenced by dual staining for DNA and membrane surface molecules. A significant decrease of IL2 production was also observed in the supernatants of ConA stimulated PBMC treated with 100 or 200 microM FB1. In conclusion, these data suggest that FB1 may affect immune functions by inhibiting lymphocyte proliferation and IL2 production, suggesting a possible role of FB1 exposure during infectious disease and cancer.

Effects of CpG-B ODN on the protein expression profile of swine PBMC

The CpG motif within bacterial DNA is a potent immuno-stimulatory moiety. Here, using a 2-D electrophoretic approach, we investigated the effect of synthetic oligodeoxynucleotide containing a B type CpG motif (CpG-B ODN) on the protein expression profile of swine peripheral blood mononuclear cells (PBMC). We found that several proteins including spondin 1, N-acetolactate alpha linked acidic dipeptidase; V kappa light chain, T cell receptor variable alpha chain, heat shock protein (Hsp) 60, Hsp70, KIAA0857 protein, and PNAS-146 were up-regulated in PBMC by CpG-B ODN stimulation. Further studies showed that CpG-B ODN-mediated Hsp60, Hsp70 and Hsp90 expressions were closely associated with the TLR9 signalling pathway. Pretreatment with inhibitors of Hsp70, Hsp90 and TLR9 all blocked the CpG-B ODN-mediated anti-apoptotic effect in swine PBMC. These results suggest that CpG-B ODN treatment of swine PBMC may enhance the expression of biologically active proteins, notably spondin 1, V kappa light chain, T cell receptor variable alpha chain and Hsps, which may play an important role in CpG-B ODN-mediated activation of immune responses and enhancement of swine PBMC survival.

The hDAF Exogene Protects Swine Endothelial and Peripheral Blood Mononuclear Cells From Xenoreactive Antibody Mediated Cytotoxicity in hDAF Transgenic Pigs

Xenoreactive antibody-induced complement activation and cytotoxicity poses a major obstracle to xenograft survival in humans. Previously, we have generated transgenic pigs carrying the hDAF exogene to help overcome this problem. In this study, we examined whether the hDAF exogene in various swine cells shows an equally protective effect for the complement-mediated cytotoxicity induced by human xenoreactive antibodies. Pig peripheral blood mononuclear cells (PBMCs) and aortic endothelial cells (PAECs) were used as targets. Fresh human serum was harvested from a single healthy human donor as the source of human xenoreactive antibodies and complement. The target cells cocultured with medium containing various concentrations of human serum for 24 hours were evaluated using the 3-[4,5-dimethylthiaolyl]-2,5-diphenyl-tetrazolium bromide assay for cellular viability. We observed that xenoreactive antibody plus human complement-mediated cellular cytoxicity dose-dependently correlated with the concentration of human serum in the culture medium. As compared with the PBMCs from the normal pigs, PBMCs from hDAF transgenic pigs showed significantly better survival after treatment with human serum ( P < .05). Similarly, the survival of PAECs from the hDAF transgenic pig were also significantly higher than that from normal pigs ( P < .05). Our data demonstrated a protective effect from human xenoreactive antibodies and complement-mediated cytoxicity of hDAF exogenes in pig PBMCs and PAECs. These observations support the clinical value of the hDAF transgenic pig as an organ donor in xenotransplantation.

Effects of IFN-α on the Inflammatory Response of Swine Leukocytes to Bacterial Endotoxin

Because low-dose interferon-alpha (IFN-alpha) treatment had proved effective in several models of chronic inflammation and autoimmune disease, a possible role of IFN-alpha in modulating the response of swine leukocytes to bacterial endotoxin was investigated in this study. Exposure of swine peripheral blood mononuclear cells (PBMC) to low concentrations of human IFN-alpha caused a strong, dose-dependent decrease in CD14 expression, the lowest level being observed at 5 U/ml IFN-alpha. This result was confirmed if PBMC were later exposed to purified lipopolysaccharide (LPS). A 10-fold lower IFN-alpha concentration (0.5 U/ml) caused the largest reduction of tumor necrosis factor-alpha (TNF-alpha) accumulation in the medium of pulmonary alveolar macrophages (PAM), stimulated with bacterial LPS. At 0.5 U/ml, the expression of the TNF-alpha gene in PAM was also strongly reduced, as opposed to cells pretreated with 50 U/ml IFN-alpha. In contrast, expression of the interleukin-1beta (IL- 1beta) gene was stimulated and that of the IL-6 gene was not significantly affected at both IFN-alpha concentrations. Results point to an important role of IFN-alpha in control of the inflammatory response to bacterial endotoxin in pigs.

Identification of exogenous forms of human-tropic porcine endogenous retrovirus in miniature Swine.

The replication of porcine endogenous retrovirus subgroup A (PERV-A) and PERV-B in certain human cell lines indicates that PERV may pose an infectious risk in clinical xenotransplantation. We have previously reported that human-tropic PERVs isolated from infected human cells following cocultivation with miniature swine peripheral blood mononuclear cells (PBMC) are recombinants of PERV-A with PERV-C. Here, we report that these recombinants are exogenous viruses in miniature swine; i.e., they are not present in the germ line DNA. These viruses were invariably present in miniature swine that transmitted PERV to human cells and were also identified in some miniature swine that lacked this ability. These data, together with the demonstration of the absence of both replication-competent PERV-A and recombinant PERV-A/C loci in the genome of miniature swine (L. Scobie, S. Taylor, J. C. Wood, K. M. Suling, G. Quinn, C. Patience, H.-J. Schuurman, and D. E. Onions, J. Virol. 78:2502-2509, 2004), indicate that exogenous PERV is the principal source of human-tropic virus in these animals. Interestingly, strong expression of PERV-C in PBMC correlated with an ability of the PBMC to transmit PERV-A/C recombinants in vitro, indicating that PERV-C may be an important factor affecting the production of human-tropic PERV. In light of these observations, the safety of clinical xenotransplantation from miniature swine will be most enhanced by the utilization of source animals that do not transmit PERV to either human or porcine cells. Such animals were identified within the miniature swine herd and may further enhance the safety of clinical xenotransplantation.

Molecular cloning and chromosomal assignment to SSC12p13→p11 of swine chemokine receptor CCR7

We cloned a gene encoding the swine chemokine (C-C motif) receptor 7 (CCR7) and clarified its genomic structure and chromosomal assignment. The ORF and deduced amino-acid sequence were highly conserved with human and mouse CCR7. The swine CCR7 gene was mapped to SSC12p13→p11 by FISH analysis. Stimulation of swine peripheral blood mononuclear cells by IL-12 and IL-18, considered potent inducers of Th1 cells from analyses in humans and mice, downregulated the expression of CCR7. This is the first report of the molecular cloning, chromosomal assignment and characterization of a chemokine receptor in swine.