Immunity to Trop-1, a newly identified breast cancer antigen, inhibits the growth of breast cancer in mice.

This study describes the immunotherapeutic properties of vaccines that encode tumor-associated calcium signal transducer-1 (Trop-1), a newly identified breast cancer antigen, in mice with breast cancer. Previously we found that Trop-1 was over-expressed in cellular breast cancer vaccines that were highly enriched for cells that induced therapeutic CTL-mediated immune responses in mice with breast cancer, as compared with non-enriched vaccines. In this study, to determine if the expression of Trop-1 by cells in the enriched vaccine was responsible for its therapeutic benefits, an expression plasmid that specified the Trop-1 gene was transfected into the LM fibroblast cells, which was then used as a vaccine. To augment their immunogenic properties, the fibroblasts were genetically modified before Trop-1 DNA-transfer to secrete IL-2 and to express allogeneic MHC class I H-2K(b)-determinants. Mice with established breast cancer treated solely by immunization with fibroblasts modified to express Trop-1 developed CD8(+) cell-mediated immunity to the breast cancer cells. The immunity was sufficient to prolong the survival of mice with established breast cancer. In some instances, the immunity was sufficient to result in rejection of the tumor; the mice remained tumor free more than 60 days.

Gene transfer of AIMP1 and B7.1 into epitope-loaded, fibroblasts induces tumor-specific CTL immunity, and prolongs the survival period of tumor-bearing mice.

T helper type 1 (Th1) cell-mediated immune responses play various roles in cellular immunity, including inducing cytotoxic T lymphocytes (CTLs) and they have been shown to be crucial in cancer immunotherapy. Previously, we found that aminoacyl-tRNA synthetase-interacting multifunctional protein 1 (AIMP1) stimulated antigen-presenting cells to secrete IL-12, leading to enhanced Th1 cell responses. In this study, as a way of enhancing antigen-specific Th1 responses, mouse fibroblasts (H-2(b)) were genetically modified to express an AIMP1 and a costimulatory B7.1 (Fb/AIMP1/B7.1). Fb/AIMP1/B7.1 cells were then loaded with an ovalbumin epitope as a model antigen (Fb/AIMP1/B7.1/OVA), and tested to determine if they induced OVA-specific CTLs in C57BL/6 mice (H-2(b)). Immunization with Fb/AIMP1/B7.1/OVA cells induced strong cytotoxic activities against OVA-expressing EG7 tumor cells, but not against other H-2(b) tumor cells. The levels of the cytotoxic response in the immunized mice with Fb/AIMP1/B7.1/OVA cells were significantly higher than the responses in mice immunized with other cell constructs. CD8(+) T cells were a major cell-type of OVA-specific antitumor immunity induced by Fb/AIMP1/B7.1/OVA cells. Furthermore, treatment with Fb/AIMP1/B7.1/OVA cells significantly prolonged the survival period of EG7 tumor-bearing mice. These results indicate that AIMP1-secreting, epitope-loaded fibroblasts efficiently induce antigen-specific CTL responses in mice.

An optimized protocol of a human-to-cattle interspecies somatic cell nuclear transfer.

Human somatic cells were transferred into cattle enucleated oocytes, and a prospective, randomized study was designed to optimize donor cell preparation, fusion medium, and culture method. As a result, improved development of interspecies embryos was achieved by employing serum-starved cord fibroblasts, with Ca(2+)/Mg(2+)-free fusion medium and a serum-free medium being used for bovine embryos.

Promoting effect of amino acids added to a chemically defined medium on blastocyst formation and blastomere proliferation of bovine embryos cultured in vitro.

This study was conducted to elucidate the role of amino acids added singly or in groups to a chemically defined culture medium in blastocyst formation and blastomere proliferation of bovine embryos. Embryos were generated by in vitro fertilization, and blastocyst formation and hatching, and blastomere number of blastocysts were subsequently monitored after the culture of embryos in synthetic oviduct fluid medium (SOFM). First, one of four non-essential amino acids (asparagine, aspartate, glutamate or serine) was added to SOFM and, compared with no addition, a significant (P <0.05) increase in blastocyst formation was found after the addition of asparagine, aspartate, or glutamate (35-42% versus 22%). Second, one of four essential amino acids (arginine, cystine, isoleucine or leucine) was added and arginine or isoleucine greatly improved blastocyst formation (30-36% versus 16%). Third, the addition of five stimulatory amino acids (aspartate, asparagine, glutamate, arginine and isoleucine) to SOFM significantly improved blastocyst formation compared with no addition (12% versus 21%) and such value was similar to that obtained after the addition of 19 amino acids consisting of MEM amino acid solutions (21-27%). However, five amino acids yielded fewer hatched blastocysts than 19 amino acids. Finally, although five amino acids yielded more cell number of blastocysts than no addition (93 versus 74 cells per blastocyst), it was lower than that from 19 amino acids (131 cells per blastocyst). In conclusion, either single or combined addition of asparagine, aspartate, glutamate, arginine and isoleucine stimulated blastocyst formation, while other amino acids might be necessary for further stimulating blastomere proliferation and blastocyst hatching.

Blastocyst formation, karyotype, and mitochondrial DNA of interspecies embryos derived from nuclear transfer of human cord fibroblasts into enucleated bovine oocytes.

OBJECTIVE: To establish an interspecies somatic cell nuclear transfer (iSCNT) technique for deriving blastocysts having human chromosome complements without sacrificing human oocytes. DESIGN: Prospective, randomized study undertaken in vitro. SETTING: University-affiliated hospital and laboratory, Seoul National University. PATIENT(S): Postpartum women with natural spontaneous vaginal delivery. INTERVENTION(S): Human cord fibroblasts were retrieved from five postpartum women from whom informed consent was obtained. After subculture and cryopreservation, serum-starved cells were transferred into enucleated bovine oocytes. MAIN OUTCOME MEASURE(S): Embryo development, karyotype, and the presence of mitochondrial DNA (mtDNA). RESULT(S): A total 1,742 oocytes were provided for iSCNT and results showed that both fibroblast batch and reconstruction method significantly affected iSCNT outcome. An iSCNT using a single DC pulse of 1.9-2.1 kV/cm for 20 microseconds yielded better rates of fusion (30%-56%) and cleavage (36%) than the other iSCNT protocols. Four to 9% interspecies embryos produced with the optimized method developed to morulae or blastocysts after cultured in a serum-free medium. Results from karyotyping demonstrated that 56% of interspecies embryos evaluated had human chromosome complements. In polymerase chain reaction (PCR) analysis of a single embryo, both human and bovine mtDNAs were detected until the 16-cell stage, whereas only the bovine mtDNA was found beyond the morula stage. CONCLUSION(S): An iSCNT using human cord fibroblasts and bovine oocytes can yield blastocysts and the results of karyotyping and mtDNA analysis confirmed the feasibility of the iSCNT technique.