RNA interference-mediated silencing of Foxo3 in antigen-presenting cells as a strategy for the enhancement of DNA vaccine potency.

The transcription factor Forkhead box O3 (Foxo3) has a critical role in suppressing the expansion of antigen-specific effector T-cell populations; hence, Foxo3 is a potential target for enhancing the antitumor immunity of cancer vaccines. In this report, we evaluated the potential of RNA interference (RNAi)-mediated silencing of Foxo3 in antigen-presenting cells as an adjuvant for HER2/neu DNA cancer vaccines. Bicistronic plasmids expressing the N-terminal extracellular domain of human HER-2/neu and the Foxo3 short hairpin RNA (hN'-neu-Foxo3 shRNA) or the scrambled control (hN'-neu-scramble shRNA) were subcutaneously injected into mice by gene gun administration to elicit antitumor immunity against p185neu-overexpressing MBT-2 bladder tumor cells. We found that mice treated with hN'-neu-Foxo3 shRNA showed greater reductions in tumor growth and longer survival times than mice treated with hN'-neu-scramble shRNA, indicating that the silencing of Foxo3 enhanced the antitumor efficacy of the HER-2/neu cancer vaccine. Cytotoxicity analyses further revealed that the Foxo3 shRNA-enhanced antitumor effect was associated with significant increases in the number of functional CD8(+) T cells and in the levels of cytotoxic T lymphocytes activity. Interleukin-6 was induced by hN'-neu-Foxo3 shRNA treatment but did not have a critical role in the antitumor effect of the hN'-neu-Foxo3 shRNA vaccine. Moreover, in vivo lymphocyte depletion analyses confirmed that the antitumor efficacy of the hN'-neu-Foxo3 shRNA vaccine depended on functional CD8(+) T cells. Finally, Foxo3 suppression was shown to markedly improve the effect of the HER-2/neu DNA vaccine in limiting the growth and lung metastases of MBT-2 cells. Overall, these results support RNAi-mediated silencing of Foxo3 as an effective strategy to enhance the therapeutic antitumor effect of HER-2/neu DNA vaccines against p185neu-positive tumors.

The impact of human ABO blood groups on human xenoreactive antibody-mediated cytotoxicity and the protective effect of human decay-accelerating factors exogene on swine endothelium.

UNLABELLED: Swine tissue can express antigens similar to human A/B blood types. We evaluated whether the variation in human blood type influences the human xenoreactive antibody-mediated cytotoxicity and modifies the protective effect of human decay-accelerating factor (hDAF) exogene, a complement activation regulator, on swine endothelium. METHODS: Pig aortic endothelial cells were harvested form normal and hDAF transgenic pigs. Cellular viability was evaluated with an MTT assay. RESULTS: As compared with that of other human blood types, human serum from blood type O donors induced more prominent cytotoxicity on swine endothelial cells both from hDAF transgenic or normal pigs (P < .05). In addition, this difference of xenoreactive antibody-induced cytotoxicity between treatment with O and other human blood type sera was more evident in hDAF transgenic swine endothelial cells than those of normal pigs (P < .05). The hDAF exogene can significantly protect the endothelial cells from human xenoreactive antibody-mediated cytotoxicty when treated with human serum from AB blood type (P < .05). Our data demonstrated that human ABO blood type significantly affected human xenoreactive antibody-induced cytotoxicity, which may modulate the protective effect of hDAF exogene expression on swine endothelial cells.

Human leukocyte antigen-DR matching improved skin graft survival from transgenic pigs to accommodate SCID mice reconstituted with human peripheral blood mononuclear cells.

The shortage of human organs has encouraged scientists to develop genetically modified pigs for xenotransplantation, such as CD55 or CD46, and CD59 transgenesis as well as alpha-galactosyl transferase gene knockouts. In allotransplantation, the match of human leukocyte antigen class II (HLA-II) may improve graft survival although the role of HLA-II in xenotransplantation is unknown. HLA-II transgenic pigs, including DP, DQ, and DR, have been successfully generated and HLA-DR15+ transgenic pig skin pieces grafted onto severe congenital immunodeficiency (SCID) mice reconstituted intraperitoneally with HLA-DR15+ or HLA-DR15(-) human peripheral blood mononuclear cells (hPBMCs). This study sought to develop an animal model to evaluate the effects of HLA-DR matching on xenograft survival. Human CD4+ and CD8+ were detected from days 7 to 29 after hPBMC reconstitution in SCID mice. Both CD4+ and CD8+ cells of HLA-DR15(-) reconstituted SCID mice were significantly higher at day 29 postgrafting compared with HLA-DR15+ reconstituted SCID mice. An HLA-DR15+ transgenic pig dermal graft survived and integrated into SCID mice reconstituted with hPBMCs/HLA-DR15+ as proven by the histopathological finding that the collagen layer remained intact with little lymphocytic response. In contrast, the transgenic pig dermal graft showed more collagen disruption as well as mild to moderate lymphocytic infiltration when reconstituted in an hPBMC/HLA-DR15(-) SCID mouse. The results suggested that HLA-DR matching eased xenograft rejection; however, it was not yet clear that the response was mediated by T cells.

Long-term survival of HLA-DR15+ pig skin in SCID mice after reconstitution with human peripheral blood mononuclear cells and under short-term immunosuppression.

In allotransplantation, donor-recipient human leukocyte antigen (HLA) matches improve graft survival. For studies of the role of donor-recipient HLA II matching on xenotransplantation, we successfully generated HLA-DR15+ transgenic pigs the the skins of which were transplanted to SCID mice, which were thereafter reconstituted with HLA-DR15+ or -DR15(-) hPBMC. Cyclosporine was given intraperitoneally to SCID mice for 12 days. Human T cells were observed in SCID mice after reconstitution. Mixed lymphocytes responses showed greater responses by HLA-DR15(-) human peripheral blood mononuclear cells (hPBMC) against HLA-DR15+ porcine PBMC. HLA-DR15+ porcine skins survived more than 100 days in all SCID mice. HLA-DR15+ porcine skins were rejected in all non-SCID (Balb/c) mice. The histologic pictures of transplanted HLA-DR15+ porcine skins showed surviving porcine epithelium in remodeling murine dermis and little lymphocyte infiltration into the murine dermis. The long-term survival of HLA-DR15+ pig skin in all hPBMC-SCID mice might be due to poor engraftment or function of reconstituted T cells. Further studies are needed to clarify the role of donor-recipient matching of HLA-DR15.

Transgenic cloned mice expressing enhanced green fluorescent protein generated by activation stimuli combined with 6-dimethylaminopurine.

Most studies of mouse cloning successfully achieved activation of the reconstructed oocytes by strontium (Sr) combined with cytochalasin B (CB) treatment. A protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), was used to inhibit the activity of maturation promoting factor for activation of oocytes, but it has never been successfully applied in mouse cloning. This study investigates the activation efficiency of 6-DMAP in mouse somatic cell nuclear transfer (SCNT). Higher parthenogenetic blastocyst rates (71-72%, p < 0.05) were achieved in the oocytes treated with Sr6D (10 mM Sr combined with 2 mM 6-DMAP for 4 h) and Sr6D + SrCB (Sr6D for 2 h then Sr combined with 5 mug/ml CB for another 2 h), and a higher rate of hatching and hatched blastocyst was observed in the Sr6D + SrCB group (31%, p < 0.01) compared with other treatment groups (1-8%). For mouse cloning, cumulus cells of enhanced green fluorescent protein (EGFP)-expressed ESC chimera F1 were used as donor nuclei. Following activation, better development of the cloned embryos was observed in Sr6D + SrCB treatment. Moreover, different media, i.e. KSOM-AA, MEM-alpha and MK, for culturing cloned embryos were also compared in this study. Better morula/blastocyst (40%) and blastocyst (29%) rates were achieved in the embryos cultured in MEM-alpha medium (p < 0.05). Consequently, four EGFP cloned mice were generated in the activation treatment containing 6-DMAP following embryo transfer. In conclusion, treatment with 6-DMAP in combination with other activation stimuli successfully activates mouse reconstructed oocytes and support full-term development of the transgenic SCNT cloned embryos.

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.

Application of xenogeneic anti-canine distemper virus antibodies in treatment of canine distemper puppies.

OBJECTIVE: The clinical feasibility of passive immunotherapy has not been demonstrated in dogs naturally infected with canine distemper. In this study, porcine anti-canine distemper virus IgG and F(ab')2 antibody fragments were used to treat infected puppies. METHODS: A total of 41 naturally infected puppies (age Äsix months) exhibiting severe respiratory signs, but lacking neurological signs, were enrolled in the study. Twenty-five puppies were treated with a combination of IgG or F(ab')2 antibody fragments (Group 1) and supportive therapy and 16 puppies received routine supportive care only (Group 2). RESULTS: The survival rate of dogs in Group 1 (19/25; 76%) was significantly higher than that in Group 2 (5/16; 31·3%) (P<0·05). During the therapy, 8 of the 25 dogs (32%) in Group 1 developed neurological signs versus 12 of the 16 dogs (75%) in Group 2 (P<0·05). Adverse reactions were limited to elevated body temperature in dogs that received IgG antibodies. CLINICAL SIGNIFICANCE: Porcine anti-canine distemper virus antibodies improved survival in puppies affected with canine distemper with minimal adverse effects. Therefore, this therapy could be considered for treatment of endangered animal species infected with canine distemper virus.

Effects of heme oxygenase-1 transgenic islets on transplantation.

Heme oxygenase-1 (HO-1) has been described as a protein capable of cytoprotection via radical scavenging and apoptosis prevention. The aim of this study was to analyze whether HO-1 overexpression in freshly isolated murine transgenic islets resulted in cell protection and improved in vivo functional performance after transplantation. We produced transgenic mice in which the human HO-1 transgene driven by chicken beta-actin promoter was expressed in the heart, liver, spleen, lung, kidney, muscle, intestine, and pancreas in Balb/c mice. One hundred fifty islets isolated from HO-1 transgenic and control Balb/c mice were syngeneically transplanted under the left kidney capsule of the streptozotocin-diabetic Balb/c mice. The recipients who underwent transplantation with HO-1 transgenic islets showed higher blood glucose than those with control islets at 4 weeks (320 +/- 25 vs 189 +/- 43 mg/dL; P < .05). Body weight was not significantly different between the 2 groups. Our data indicate transgenic islets with high HO-1 expression did not improve transplantation outcome.

cDNA cloning of pig testicular lactate dehydrogenase-C, thermal stability of the expressed enzyme, and polymorphism among strains.

Pig testicular lactate dehydrogenase-C (LDHC) cDNA was cloned and sequenced. The deduced sequence of 332 amino acids from pig LDHC shows 73% and 67% identity with that of pig LDHA (muscle) and LDHB (heart) respectively, whereas pig LDHA and LDHB isozymes shows 74% sequence identity. Pig and mouse LDHC cDNAs were subcloned into bacterial expression vector, and the expressed pig LDHC isozyme was shown to be as thermally stable as mouse LDHC isozyme. Pig genomic DNAs from Chinese Meishan, English Yorkshire, Danish Landrace and American Duroc were shown to exhibit polymorphic sites for restriction enzymes EcoRI, BamHI and PstI.

Synthesis of 60- and 72 kDa heat shock proteins in early porcine embryogenesis.

Proteins of selected embryonic stages were metabolically labeled with [(35)S]-methionine and analyzed by two-dimensional SDS-polyacrylamide gel electrophoresis (2-D PAGE) to study protein expression from 4- to 8-cell to blastocyst stage of porcine embryos. Two proteins with molecular weights of 60 and 72kDa were de novo synthesized during the 4- to 8-cell stage were the earliest that were detected. They were identified as HSP60 and HSP72 according to their locations on 2-D autoradiography and the immunoblotting result of anti-HSP 60 and HSP 72 antibodies of 1-cell stage of porcine embryos. In protein translation in early pig embryogenesis the timing of their synthesis suggests that HSP60 and HSP72 play significant roles as chaperones.

Generation of HLA-DP transgenic pigs for the study of xenotransplantation.

The shortage of human organs has prompted scientists to seek xenogeneic sources of donors. To date, DAF, MCP, and CD59 transgenic pigs have been generated to inhibit hyperacute rejection. However, besides hyperacute rejection, acute and chronic rejection must also be considered in the use of porcine organs for xenotransplantation. The role of HLA-II in transgenic xeno-organ transplantation remains to be elucidated. By microinjecting 1655 embryos, we have generated one stillborn HLA-DR and two live HLA-DP transgenic pigs: P113-7 (male, carrying one copy of exogene) and P113-8 (female, carrying 2-3 copies of exogenes). The gene status of the live transgenic pigs was confirmed by PCR, Southern blot, and PCR product sequencing analysis. The expression of transgenes in these transgenic pigs were confirmed by RT-PCR analysis and immunohistochemical staining of frozen sections of ear tissue.

Genetic polymorphisms, growth performance, hematological parameters, serum enzymes, and reproductive characteristics in phenotypically normal Landrace boars produced by somatic cell nuclear transfer.

Understanding the performances of cloned pigs and their offspring is critical to evaluate the practical applications of somatic cell nuclear transfer. In this study, genetic polymorphism, growth performance, hematological parameters, and reproduction characteristics of cloned Landrace boars were compared with those of controls. In addition, the growth performance of clone offspring was also evaluated. A total of 479 reconstructed embryos were transferred to five recipient pigs and resulted in the delivery of 14 piglets (overall cloning of 2.9%) from two litters. Analyses of microsatellite markers and polymorphisms of the specific genes confirmed that the 14 clones were genetically identical to the nuclear donor and maintained the desirable genotypes. Growth performance of five healthy, phenotypically normal cloned boars from one litter and eight of their male offspring did not differ from age, breed, and management-matched controls. Although some significant differences were observed between cloned and control boars in hematological and serum enzymes, most of these parameters were within the normal range. Cloned boars had less (P < 0.05) normal sperm in the ejaculated boars than in control boars (71.4% vs. 77.9%, respectively), but sperm production (ejaculate volume, sperm concentration, and total sperm) did not differ between these groups. In addition, use of frozen-thawed semen from cloned boars for insemination produced results that seemed comparable to a control. In conclusion, the present study reported that somatic cell nuclear transfer is effective in reproducing preferred genetic traits and has potential applications to conserve elite bloodlines in a routine pig breeding program.

Modest effects of Fas-ligand and heme oxygenase-1 double transgenic mouse islets on transplantation outcomes.

Interactions of Fas with its ligand (FasL) play an important role in the maintenance of immunologic homeostasis and peripheral tolerance. Heme oxygenase-1 (HO-1) is a protein capable of cytoprotection via radical scavenging and apoptosis prevention. The aim of this study was to test whether overexpression of FasL and HO-1 in murine islets resulted in cell protection and improved functional performance after transplantation. We first generated FasL and HO-1 double transgenic mice to investigate the protective effect of transgenic islets on transplantation. Islets were isolated from FasL and HO-1 double transgenic and nontransgenic Balb/c mice, for transplantation of 300 islets under the left kidney capsule of each streptozotocin-diabetic Balb/c mouse. During 6 weeks after transplantation, the blood glucose gradually decreased in recipients of double transgenic and nontransgenic islets. However, the decrease in blood glucose was more pronounced in the former (450 ± 16 mg/dL at day 0 to 302 ± 55 mg/dL at day 42; P = .01) than the latter (468 ± 17 mg/dL at day 0 to 379 ± 71 mg/dL at day 42; P = .24). The areas under the curve of intraperitoneal glucose tolerance tests at 2, 4, and 6 weeks were not significantly different between recipients of double transgenic and nontransgenic islets. The body weight increased in recipients of double transgenic islets (21.1 ± 1.4 g at day 0 to 26.2 ± 0.8 g at day 42; P = .0002) and nontransgenic islets (21.0 ± 1.4 g at day 0 to 25.1 ± 0.4 g at day 42; P = .0448). Our data suggested modest beneficial effects of transgenic islets with FasL and HO-1 overexpression for transplantation.

A survey of the attitudes of scientists toward xenotransplantation in Taiwan.

This study examined the attitudes of scientists in Taiwan's leading animal research institution toward xenotransplantation. The aim was primarily to evaluate the opinions of professionals in the biomedical field on key issues including ethical moral, legal, and regulatory issues raised by the biotechnology. A secondary objective was to identify potential factors that influenced opinions. A questionnaire-based survey was used to evaluate opinions. A test for internal consistency of the questionnaires to sample of 91 scientists was performed as well as a principal component analysis. We evaluated associations between variables using the nonparametric Kruskal-Wallis test. Among the subjects 85.2% thought that xenotransplantation can be more beneficial than harmful to human society and 94.3% believed that it is important to develop xenotransplantation. Also, 97.8% of participants believed that legislative guidelines should be adopted to regulate research in biotechnology. Gender was an influencing factor, whereas, variables such as religion, marital status, and age did not have obvious effects. Further studies on the general public are needed to detect other factors and to examine the attitude of nonprofessionals toward xenotransplantation.

An HDAC inhibitor enhances the antitumor activity of a CMV promoter-driven DNA vaccine.

The cytomegalovirus (CMV) promoter is considered to be one of the strongest promoters for driving the in vivo expression of genes encoded by DNA vaccines. However, the efficacy of DNA vaccines has so far been disappointing (particularly in humans), and this might be explained in part by histone deacetylase (HDAC)-mediated chromatin condensation. Hence, we sought to investigate whether increasing the expression of DNA vaccine antigens with the HDAC inhibitor OSU-HDAC42 would enhance the efficacy of DNA vaccines in vivo. A luciferase assay was used to determine the effects of OSU-HDAC42 on CMV promoter-driven DNA plasmids in vitro and in vivo. Three HDAC inhibitors were able to activate expression from the CMV promoter in NIH3T3 cells and MBT-2 bladder cancer cells. The expression of luciferase was significantly enhanced by co-administration of pCMV-luciferase and OSU-HDAC42 in mice. To explore whether OSU-HDAC42 could enhance the specific antitumor activity of a neu DNA vaccine driven by the CMV promoter, we evaluated therapeutic effects and immune responses in a mouse tumor natively overexpressing HER2/neu. Mice receiving OSU-HDAC42 in combination with the CMV-promoter neu DNA vaccine exhibited stronger antitumor effects than mice given the DNA vaccine only. In addition, a correlation between the antitumor effects and the specific cellular immune responses was observed in the mice receiving the DNA vaccine and OSU-HDAC42.

The in vitro protection of human decay accelerating factor and hDAF/heme oxygenase-1 transgenes in porcine aortic endothelial cells against sera of Formosan macaques.

To mitigate hyperacute rejection, pigs have been generated with alpha-Gal transferase gene knockout and transgenic expression of human decay accelerating factor (hDAF), MCP, and CD59. Additionally, heme-oxygenase-1 (HO-1) has been suggested to defend endothelial cells. Sera (MS) (0%, 1%, 5%, 10%, and 15%) from Formosan macaques (Macaca cyclopis, MC), an Old World monkey wildly populated in Taiwan, was used to test the protective in vitro, effects of hDAF or hDAF/hHO-1 on porcine aortic endothelial cells (pAEC) derived from hDAF(+), hDAF(+)/hHO-1(+), and hDAF(+)/hHO-1(-) and 1 nontransgenic pAEC. Ten percent human serum (HS) served as a positive control. When MS addition increased to 10% or 15%, all transgenic pAEC exhibited a greater survival than nontransgenic pAEC. Noticeably, 15% MS reduced survived to <10% versus >40% in nontransgenic and transgenic pAEC, respectively. These results revealed that hDAF exerted protective effects against MC complement activation. However, comparing with 10% MS and HS in pAEC of nontransgenic pigs, the survivability was higher in HS, suggesting that complement activation by MS was more toxic than that by HS. Furthermore, hDAF(+)/hHO-1(+) showed no further protection against effects of MS on transgenic pAEC.