Banna Minipig Research Articles

A RAPID METHOD FOR DETECTION OF THE COPY NUMBER OF PORCINE ENDOGENOUS RETROVIRUS IN SWINE

ABSTRACT Pig‐to‐human xenotransplantation is associated with the risk of porcine endogenous retrovirus (PERV) transmission. It was assumed that copy number of PERV could increase during inbreeding. To detect the copy number of PERV of Banna minipig inbred line (BMI) and three other breeds of Chinese pigs, real‐time quantitative polymerase chain reaction (RT‐QPCR, SYBR Green I ) was performed in this study. The results demonstrated that BMI pigs contained higher copy numbers of PERV gag, pol and envB genes than other tested domestic pigs, which greatly suggested that copy numbers had increased during inbreeding. Domestic pigs outbreeding and from different regions had similar copy numbers of PERV in genome. Thus, selective outbreeding of pigs of remote origin might be a feasible means to reduce PERV load in organ donors. RT‐QPCR (SYBR Green I) offered a rapid and accurate method for the detection of the copy number of most relevant retroviruses and was especially suitable for high‐throughput screen.

Expressed sequence tags analysis of a liver tissue cDNA library from a highly inbred minipig line

Porcine liver performing efficient physiological functions in the human body is prerequisite for successful liver xenotransplantation. However, the protein differences between pig and human remain largely unexplored. Therefore, we investigated the liver expression profile of a highly inbred minipig line. A cDNA library was constructed from liver tissue of an inbred Banna minipig. Two hundred randomly selected clones were sequenced then analysed by BLAST programme. Alignments of the sequences showed 44% encoded previously known porcine genes. Among the 56% unknown genes, sequences of 72 clones had high similarities with known genes of other species and the similarities to human were mostly above 0.80. The other 40 clones showing no similarity to genes in National Centre for Biotechnology Information are newly discovered, expressed sequence tags specific to liver of inbred Banna minipig. Twenty-two of the 200 clones had full length encoding regions, 38 complete 5' terminal sequences and 140 complete 3' terminal sequences. These newly discovered expression sequences may be an important resource for research involving physiological characteristics and medical usage of inbred pigs and contribute to matching studies in xenotransplantation.

Immunogenicity and Immune Modulation of Osteogenic Differentiated Mesenchymal Stem Cells From Banna Minipig Inbred Line

Mesenchymal stem cells (MSCs) represent a good choice for cell transplantation due to their multilineage differentiation ability and low immunogenicity. Our previous in vitro studies indicated that undifferentiated swine MSCs show low immunogenicity suppressing the proliferative responses of human peripheral blood lymphocyte to several antigens. In this study, we investigated the immunogenicity and immune modulation ability of osteogenic differentiated MSCs. SLA class I (P1, P14) was detectable by reverse-transcriptase polymerase chain reaction on both differentiated and undifferentiated MSCs. SLA class II (SLA-DRA, SLA-DQA) was only detectable on differentiated MSCs mixed lymphocyte reaction assays demonstrated that both differentiated and undifferentiated MSCs failed to stimulate proliferative responses by human peripheral blood lymphocytes (hPBLs). Furthermore, as undifferentiated MSCs, osteogenic differentiated MSCs also suppressed hPBL proliferation to phytohemaglutinin.

Phylogenetic Analysis of Porcine Endogenous Retroviruses Expressed in Chinese Pigs Based on Envelope Sequences

The promise of successful clinical xenotransplantation is now offset by the potential risk of transmission of porcine endogenous retrovirus (PERV). PERV consists of three subtypes according to the varieties of env sequences. We analyzed PERV subtypes in two species of Chinese pigs (Banna minipig inbred, BMI, and Wu-Zhi-Shan pig, WZSP). Positive A and B were detected while positive C was absent in the analyzed Chinese pigs. The polymerase chain reaction products were then cloned into a pGEM-T vector system and sequenced. Phylogenetic trees were constructed from the translated amino acids of PERVs and other type C and type D retrovirus, as well as the lentivirus in the GeneBank. The results suggested that PERV-A and PERV-B that exist in Chinese pig genomes share similarities with other PERV from the GeneBank and some type C retroviruses, including lymphotropic, leukemic and endogenous retroviruses.

Construction and Characterization of a cDNA Library from Liver Tissue of Chinese Banna Minipig Inbred Line

A xenograft that performs efficient functions is an essential premise for successful xenotransplantation. Our early study indicated that Chinese Banna minipig inbred line (BMI) was an ideal xenograft donor. However, the activities of some proteins synthesized by the BMI liver are different from the human, which could lead to functional disorders in coagulation, fibrinolysis, and anticoagulation after liver xenotransplantation. Therefore, it is important to investigate the genetic background of protein incompatibility and to provide new strategies for gene manipulation. In this study we constructed a cDNA expression library using BMI liver tissue to obtain an understanding of nucleic acid and protein differences between the two species. We extracted total RNA and purified mRNA of the liver tissue from one of the sixteenth inbred generation of BMI/JS 151 substrain. After double-strand cDNA synthesis, we fractionated it on a CHROMA APIN-400 column; ligated the longer than 500bp cDNA into a ZAP Express Vector; and performed a λ: phage packaging reaction, library amplification, and titer. We randomly picked 12 plaques and tested the length of inserts. The titers of the primary and amplified libraries were 1.0 × 10 6 pfu/mL and 5.0 × 10 9 pfu/mL, respectively. The percentages of recombinants were 97.0% in the primary library and 98.0% in the amplified library. The lengths of most inserts were between 750 bp and 2.0 kb. Thus, we successfully constructed a cDNA expression library from BMI liver tissue. Using the library, we hope to get a full-length cDNA of some important genes and conduct further studies on porcine liver function in xenotransplantation.

In Vivo Screening of Porcine Endogenous Retrovirus in Chinese Banna Minipig Inbred

The risk of porcine endogenous retrovirus (PERV) infection is one of the major barriers in clinical trials of pig-to-human xenotransplantation. Previous experiments showed that PERV could infect many types of human and nonhuman primate cells, but there is no reported evidence of in vivo infection. In this study, extracted genomic DNA from tissues of seventeen pigs was analyzed using specific sequence primers for gag, pol, and env. The results suggested that PERV exist in the genomes of all tissues. A subtype analysis indicated that PERV-A and PERV-B were in the tissue genome with no positive PERV-C. A greater understanding of the properties of PERV in different pig tissues is necessary to evaluate the risk posed by PERV.

Variation of Host Cell Tropism of Porcine Endogenous Retroviruses Expressed in Chinese Banna Minipig Inbred

A serious donor-organ shortage urges the use of animal donors to treat a wide appropriate variety of major health problems including organ failure and diabetes. However, the promise of clinical xenotransplantation is offset at the present time by the potential of a public health risk due to the cross-species transmission of pathogens from animal donors to human patients. In particular, the transmission of porcine endogenous retrovirus (PERV) is a major concern. In this study, cell tropism of PERV was tested by in vitro infection of human primary cells and cell lines. Coculture of PERV supernatant derived from PK15 with human primary cells and cell lines resulted in the transfer and expression of PERV-specific sequences and the establishment of a productive infection. In the detection of tropism variation of PERV in pigs, 293 cells were cocultured with mitogenic-activated and lethally irradiated PBMC from 12 Banna minipig inbred (BMI). The results were that six coculture groups were PERV-positive. However, infectious virus was not detected when activated PBMC from the other 7 pigs were cocultivated with human cells known to be permissive for PERV, which indicated a tropism variation among the tested individuals. All these findings demonstrate that the presence of endogenous viruses in source animals needs to be carefully considered when the infectious disease potential of xenotransplantation is being assessed.

Comparison of Hepatic Coagulant, Fibrinolytic, and Anticoagulant Functions Between Banna Minipig Inbred Line and Humans

As an ideal candidate for xenotransplantation, the compatibility of physiological porcine organs with those of humans is an essential premise. In this study, we analyzed hepatic coagulant, fibrinolytic, and anticoagulant functions between Banna Minipig Inbreds (BMIs) and humans to evaluate such hepatic compatibility. BMI factors II, V, VII, X, and XII were added to the corresponding factor-deficient human plasma to determine prothrombin times (PT) and activated partial thromboplastin times (APTT). Human tissue plasminogen activator (t-PA) was added to both BMI and human plasma to determine plasmin activity. The antithrombin-III (AT-III) activity of plasma was analyzed with the STA-Stago autoanalyzer using an AT-III assay kit. Both PT and APTT were reduced but within normal parameters when BMI factors II, V, VII, X, and XII were added to the corresponding factor-deficient human plasma. The activities of BMI coagulation factors II, V, VII, X, and XII were 3.2, 3.7, 4.7, 2.9, and 4.5 times those of humans, respectively. The activity of plasmin was significantly higher in BMI plasma than in humans when human t-PA was added to both. The normal range of human AT-III activity was 90-108% while BMI AT-III was 124.50 +/- 2.38%. The activities of coagulation factors and AT-III were higher in BMIs than in humans. BMI coagulation factors XII, VII, and X trigger human intrinsic, extrinsic, and common pathways, respectively, which functioned normally. In addition, BMI plasminogen could be activated by human t-PA.

Suppression of human peripheral blood lymphocyte proliferation by immortalized mesenchymal stem cells derived from bone marrow of Banna Minipig inbred-line

This study sought to investigate whether mesenchymal stem cells (MSC) derived from Banna Minipig Inbred-line (BMI-MSC) suppressed human peripheral blood lymphocyte (hPBLs) proliferation in a one-way mixed lymphocyte reaction system. BMI-MSC failed to stimulate proliferative responses by hPBLs, which were activated by allogenic endothelial cells, BMI-PBLs and non-specific mitogenic stimuli. Furthermore, BMI-MSC also suppressed proliferation of hPBLs stimulated by mismatched allogenic, as well as xenogenic PBLs, and the mitogenic stimulus ConA. The suppression occurred in dose-dependent fashion when the ratio of hPBLs to BMI-MSC varied from 1 to 5 fold; fewer, BMI-MSC (0.001 to 0.01 times) showed no obvious suppression. When BMI-MSC were added to hPBLs stimulated for 72 hours, the proliferative suppression was still evident. Addition of anti-FasL or anti-TGF-β1 antibody attenuated the proliferative suppression, while antibody against IL-10 had no effect on it. Further immunofluorescence analysis demonstrated that FasL and TGF-β1 constitutively expressed BMI-MSC. These findings suggest that BMI-MSC suppress hPBLs proliferation relying on FasL and TGF-β1 mediated pathways.

Phylogenetic analysis of porcine endogenous retrovirus variation in three Chinese pigs

PCR amplification was performed on genomic DNA extracted from peripheral blood lymphocytes of three species of Chinese pigs (Banna minipig inbreed [BMI], Wu-Zhi-Shan pig [WZSP], and Nei jiang pig [NJP]), using primers corresponding to the highly conserved regions of polymerase ( pol) gene. Extracted PCR products were then cloned in a pGEM-T vector. Phylogenetic analysis of the nucleotide sequences of BMI-PERV, NJJP-PERV, and WZSP-PERV revealed them to be a novel category of PERV. In comparison to other type C retrovirus and lentivirus, their amino acid sequenced show about 30% to 57.7% identities. Our previous research demonstrated that PERV in the three pigs was highly expressed. It appears likely that functional loci encoding these novel PERV sequences exist, but this remains to be established. The novel sequences described in this report will allow such investigations to be actively pursued.

Activation of human coagulation system by liver-derived clotting factors of banna minipig inbred line

The liver synthesizes most of the coagulation factors that play a major role in arresting hemorrhage. Matching hepatic coagulation factors is an important premise in successful xenotransplantation. As a unique inbred pig, the Banna minipig inbred (BMI) animals have a huge potential value for pig-to-human xenotransplantation, due to its clear genetic background and tiny interindividual differences. Whether the coagulation factors synthesized by porcine liver can trigger human clotting pathways has not been reported. This study focused on the activities of BMI coagulant factors synthesized exclusively by the liver to activate human clotting pathways. In these experiments we prepared coagulant factors II, V, VII, X, and XII synthesized exclusively by liver from BMI and humans. The factors were used in common correction tests, added to the corresponding factor-deficient human plasma to determine prothrombin times or activated partial thromboplastin time, thereby calculating BMI and human coagulant factor activities. BMI clotting factors XII, VII, and X triggered human intrinsic, extrinsic, and common pathways, respectively. BMI clotting factors II, V, VII, X, and XII activities were 3.2-, 3.7-, 4.7-, 2.9-, and 4.5-fold as potent as those from humans.

Study of renal function matching between banna minipig inbred line and human

Pigs have been thought to be ideal candidates for xenotransplant donors. However besides the immunological barrier, physiological and pharmacokinetic comparabilities of kidney function between donor animals and humans are important factors for successful xenotransplantation. As a unique large inbred animal, Banna Minipig Inbred (BMI) has been reported to be a potential large animal suitable for xenotransplantation. However, its physical and pharmacokinetic compatibilities with humans have not been documented. The purpose of this investigation was to measure renal routine function, glomerular filtration rate (GFR), renal blood flow (ERPF), and drug metabolism to evaluate comparability to humans. The results suggested that the renal function of BMI was similar to that of humans to eliminate nonprotein nitrogenous end products of metabolism. Pharmacokinetics of p-aminohippurate (PAH) and inulin—the most widely used methods to assess ERPF and GFR—showed that BMI showed lower values than humans for GFR, but similar function to humans in ERPF. The pharmacokinetics of cefazolin; a widely used model drug to study kidney drug metabolizing capacity, showed greater overall renal drug elimination of BMI than of humans. These results suggested that BMI did show comparable data to human renal function.

Screening and analysis of porcine endogenous retrovirus in Chinese banna minipig inbred line

Pigs have been the most likely animal as the source of cells, tissues, and organs for xenotransplantation. But the use of pigs in xenotransplantation is associated with the risk of porcine endogenous retrovirus (PERV) transmission. Previous studies have identified that the proviruses are integrated into the genome of normal pigs and that virus particles released from the porcine cells can infect human cells in vitro. As a unique inbred pig, Banna minipig inbred (BMI) has a huge potential value for xenotransplantation and medical research. It has been the focal experimental animal for pig-to-human xenotransplantation in China, due to its clear genetic background and tiny individual differences. To evaluate whether the potential risk of PERV exists in inbred pigs, a series of screening experiments were performed herein. The results of PCR with primers specific for gag, pol, and env showed that proviruses existed in the genome of BMI, and the PERV subtypes were PERV-A and PERV-B. PERV mRNA was expressed functionally in BMI. Positive results of an RT assay identified that PERV in BMI had potential infectivity, but the concentration of PERV reverse transcriptase in BMI was almost 20 times lower than that of HIV. These results suggested that gag, pol and env genes of PERV were not lost during inbreeding, which created favorable conditions to produce viral particles that could possibly infect human cells in xenotransplantation.

Study of hepatic function matching between banna minipig inbred and humans

As a unique inbred pig Banna minipig inbred (BMI) is potentially suitable for pig-to-human xenotransplantation due to its clear genetic background and minor interindividual differences. Previous studies of BMI have focused on immunological barriers between BMI and humans. However, a comparison of liver function between donor animals and humans is an essential premise for successful xenotransplantation. In this study, we investigated routine hepatic functions, protein electrophoresis, and drug metabolism to compare capacity of liver synthesis, metabolism, and drainage between BMI and humans. The results showed no significant differences in the concentrations of albumin and globulin synthesized in the liver (α1, α2, and β-globulin). Serum enzyme activities in BMI were higher than those in humans, and levels of total bilirubin and direct-reacting bilirubin of BMI were lower than those of humans. In BMI, the clearance of antipyrine, a widely used model drug to study hepatic drug metabolism, was 16 times greater than that by humans, with a mean residual time of antipyrine in BMI, one-tenth of that in human. These findings suggested that BMI livers are similar to humans in albumin and α, β-globulin synthesis, but stronger in bilirubin elimination, enzyme activity, and drug metabolism. BMI livers may have stronger functions compared with those of humans. No incompatibility was identified in hepatic function between BMI and humans.

Phylogenetic relationship of porcine endogenous retrovirus (PERV) in Chinese pigs with some type C retroviruses

PCR amplification of proviral DNA extracted from peripheral blood lymphocytes of three Chinese pigs (Banna minipig inbreed (BMI), Wu-Zhi-Shan pig (WZSP) and Neijiang pig (NJP)), using primers corresponding to highly conserved regions of reverse transcriptase (RT) of pol gene and nucleocapsid sequence of gag gene. PCR products were then extracted and cloned into pGEM-T vector. Phylogenetic analysis of the nucleotide sequences of PERV-BMI, PERV-WZSP and PERV-WZSP revealed that they were of retroviral origin. Phylogenetic trees were constructed from the translated amino acids of PERVs and other type C retrovirus, as well as lentivirus of GenBank. The research demonstrated that PERVs of Chinese pigs and other PERVs were closely related to other pathogenic type C retroviruses. From the gag analysis, a novel subgroup of PERV was identified and this novel sequence described in this report would allow such investigation to be actively pursued.

SUPPRESSION OF HUMAN PERIPHERAL BLOOD LYMPHOCYTE PROLIFERATION BY IMMORTALIZED MESENCHYMAL STEM CELLS FROM AN INBRED PORCINE BREEDING

P1100 Aims: Use mesenchymal stem cells (MSC) from Banna Minipig Inbred Line (BMI) (the 18th generation of subline JS) to investigate its suppression effects on human peripheral blood lymphocyte proliferation and explore the possible mechanism underlying. Methods: MSCs from BMI (BMI-MSC) were immortalized by introducing simian virus 40 large T antigen gene (SV40 gene). Immunogenic and immunosupressive properties of BMI-MSC were investigated through one-way mixed lymphocyte reactions (MLR) using MTS assay to measure the proliferative activity of hPBLs. Results: BMI-MSC failed to elicit a proliferative response when cultured with hPBLs (Fig.1) and the viability of stimulated hPBLs were suppressed in a dose dependent fashion after 1 to 5 times xenogenic BMI-MSCs were added to ConA, PHA, allogeneic or xenogenic PBLs stimulated hPBLs(Fig.2). Furthermore, addition of anti-FasL or anti-TGF-β1, but not IL-10, could attenuated the inhibition activity of BMI-MSC in a dose dependent fashion(Fig.3). The expression of FasL and TGF-β1 of BMI-MSC were confirmed by Immunofluorescence analysis under laser confocal microscope (LCM).Figure 1. BMI-MSC failed to elicit a proliferative response when cultured with xenogenic hPBLs.Figure 2. Dose dependence inhibitory effect of immortalized BMI-MSCs on hPBLs proliferation induced by 10μg/ml ConA or 0.5%PHA.Figure 3. Modulation of PBLs proliferation by antibodies reacting with MSC derived cytokines.Conclusions: Our data suggested that BMI-MSC have significant immunosupressive effect on xenogenic hPBLs proliferation which might partially due to FasL and TGF-β1 mediated apoptosis. Engineered BMI-MSC might be a protencial seed cell in tissue engineering and immunomodulator in hematopietic as well as solid organ transplantation.