Human immunodeficiency virus type-1 (HIV-1) chronic infection causes millions of deaths each year. We previously developed a novel HIV-1 Gag-spe cific exosome (EXO)-targeted T cell-based vaccine (Gag-Texo) using ConAs...Human immunodeficiency virus type-1 (HIV-1) chronic infection causes millions of deaths each year. We previously developed a novel HIV-1 Gag-spe cific exosome (EXO)-targeted T cell-based vaccine (Gag-Texo) using ConAstimulated polyclonal CD8+T (ConA-T) cells armed with Gag-specific dendritic cell (DC)-released EXOs, and showed that Gag-Texo stimulated more efficient cytotoxic T lymphocyte (CTL) responses than DCs. Tat HIV-1 early regulatory protein possesses immunomodulatory and adjuvant properties. To enhance Gag-Texo immunogenicity, we generated Tat-engineered OVA/Tat Texo and Gag/Tat-Texo vaccines using ConA-T cells armed with EXOs release by DCs infected with recombinant OVA/Tat- and Gag/Tat-expressing adenoviruses (AdVOVA/Tat and AdVGag/Tat). We then assessed vaccination-stimulated CTL responses in naive mice, and therapeutic immunity in transgenic HLA-A2 mice bearing Gag/HLA-A2-expressing BL6-10OVA/A2 melanoma lung metastases. We demonstrate that the OVA/Tat-Texo vaccine enhances functional OVA-specific CTL responses, compared to the OVA-Texo vaccine, and broadens CTL responses recognizing the cryptic OVA epitope in C57BL/6 mice. Furthermore, we determine that the Gag/Tat-Texo not only stimulates more efficient CTL responses than Gag-Texo, but also induces enhanced therapeutic immunity. We show that, 30% of Gag/Tat-Texo-immunized mice are free of tumor lung-metastases, compared to all Gag-Texo-immunized mice displaying lung-metastasis. In addition, the average number of tumor lung metastases colonies (32/lung) in the Gag/Tat-Texo-immunized mice was also significantly lower than that (78/lung) observed in Gag-Texo-immunized mice. Taken together, this indicates that HIV-1 Gag/Tat-Texo capable of stimulating enhanced Gag-specific CTL responses and therapeutic immunity may become a new immunotherapeutic vaccine candidate for controlling virus in HIV-1 patients.展开更多
AIMTo determine the effect of overexpression of fibrinogen-like protein 2 (FGL2) on regulatory T cell (Treg) and effector T (Teff) cell function on T cell-induced colitis in Rag1<sup>-/-</sup> mice.METHODS...AIMTo determine the effect of overexpression of fibrinogen-like protein 2 (FGL2) on regulatory T cell (Treg) and effector T (Teff) cell function on T cell-induced colitis in Rag1<sup>-/-</sup> mice.METHODSTreg and Teff cells from fgl2<sup>-/-</sup>, fgl2<sup>+/+</sup>, and fgl2<sup>Tg</sup> mice were purified by FACS. They were studied in vitro for immunosuppressive activity and cell proliferation and in vivo for their effects on the development and prevention of T cell-induced colitis in Rag1<sup>-/-</sup> mice.RESULTSIn vitro, fgl2<sup>Tg</sup> Treg had enhanced immunosuppressive activity, and fgl2<sup>Tg</sup> Teff had reduced proliferation to alloantigen stimulation. Transfer of Teff from C57Bl/6J mice (fgl2<sup>+/+</sup>) into Rag1<sup>-/-</sup> mice produced both clinical and histologic colitis with dense infiltrates of CD3<sup>+</sup> T cells, crypt abscesses and loss of goblet cells. Fgl2<sup>Tg</sup> Treg prevented the development of T cell-induced colitis, whereas fgl2<sup>+/+</sup> and fgl2<sup>-/-</sup> Treg were only partially protective. In mice that received fgl2<sup>Tg</sup> Treg, the ratio of Foxp3<sup>+</sup> to CD3<sup>+</sup> cells was increased both in the colon and in mesenteric lymph nodes, and Teff cell proliferation as determined by staining with Ki67 was reduced. Teff cells from fgl2<sup>Tg</sup> mice did not produce colitis.CONCLUSIONHere we show that fgl2<sup>Tg</sup> Teff are hypoproliferative and do not induce colitis. We further demonstrate that fgl2<sup>Tg</sup> Treg prevent colitis in contrast to fgl2<sup>+/+</sup> Treg, which were only partially protective. These studies collectively provide a rationale for exploring the use of FGL2 or Treg expressing high levels of FGL2 in the treatment of inflammatory bowel disease.展开更多
BACKGROUND: Apoptosis plays an important role in central neural diseases and trauma. B-cell lymphoma/Leukemia-2 (Bcl-2) can inhibit apoptosis in a wide variety of cells including neurons. In this experiment, by stu...BACKGROUND: Apoptosis plays an important role in central neural diseases and trauma. B-cell lymphoma/Leukemia-2 (Bcl-2) can inhibit apoptosis in a wide variety of cells including neurons. In this experiment, by studying Bcl-2 over-expression transgenic (TG) mice subjected to spinal cord injury (SCI), we investigated whether Bcl-2 could reduce posttraumatic neuronal apoptosis, reduce the range of damage, and improve the behavioral functional recovery after contusive SCI.METHODS: Nine Bcl-2 TG mice and nine control mice were subjected to SCI of moderate severity at T10, with the use of weight dropping (WD) method (impact force 2.5×3.0 g/cm). At times up to 1 day, 7 days and 14 days after SCI, functional defi cits were evaluated with Basso, Beattie, and Bresnahan (BBB) scales, and apoptosis of neurons was investigated by using the TUNEL method. Another three control mice only underwent lamina opening, but were not subjected to SCI, to provide blank comparison.RESULTS: The mean functional scores for the control mice (5.05 ±0.35) were lower than those for the Bcl-2 TG mice (5.45 ±0.15), although the unpaired T-test revealed no signifi cant difference (P=0.67). On the other hand, the number of TUNEL positive neurons and integrated option density (IOD) scores for the Bcl-2 TG mice were both signifi cantly lower than those for the control mice (P〈0.05).CONCLUSIONS: This experiment suggests that overexpression of Bcl-2 may suppress neuronal apoptosis after SCI. Bcl-2 may be an important factor within the central nervous system that can relieve the damage after trauma.展开更多
Kindlin-2, an integrin-interacting protein, regulates breast cancer progression. However, currently, no animal model to study the role of Kindlin-2 in the carcinogenesis of mammary gland is available. We established a...Kindlin-2, an integrin-interacting protein, regulates breast cancer progression. However, currently, no animal model to study the role of Kindlin-2 in the carcinogenesis of mammary gland is available. We established a Kindlin-2 transgenic mouse model using a mammary gland-specific promoter, mammary tumor virus(MMTV) long terminal repeat(LTR). Kindlin-2 was overexpressed in the epithelial cells of the transgenic mice. The mammary gland ductal trees were found to grow faster in MMTV-Kindlin-2 transgenic mice than in control mice during puberty. Kindlin-2 promoted mammary gland growth as indicated by more numerous duct branches and larger lumens, and more alveoli were formed in the mammary glands during pregnancy under Kindlin-2 overexpression. Importantly, mammary gland-specific expression of Kindlin-2 induced tumor formation at the age of 55 weeks on average. Additionally, the levels of estrogen receptor and progesterone receptor were decreased, whereas human epidermal growth factor receptor 2 and β-catenin were upregulated in the Kindlin-2-induced mammary tumors. These findings demonstrated that Kindlin-2 induces mammary tumor formation via activation of the Wnt signaling pathway.展开更多
Fatty acid desaturase-2 (FAD2) introduces a double bond in position 12 in oleic acid (18:1) to form linoleic acid (18:2 n-6) in higher plants and microbes. A new transgenic expression cassette, containing CMV promoter...Fatty acid desaturase-2 (FAD2) introduces a double bond in position 12 in oleic acid (18:1) to form linoleic acid (18:2 n-6) in higher plants and microbes. A new transgenic expression cassette, containing CMV promoter/fad2 cDNA/SV40 polyA, was constructedto produce transgenic mice. Among 63 healthy offspring, 10 founders (15.9%) integrated the cotton fad2 transgene into their genomes, as demonstrated by PCR and Southern blotting analysis. All founder mice were fertile and heterozygous fad2 female and nontransgenic littermates were used for fatty acid analysis using gas chromatography. One fad2 transgenic line showed substantial differences in the fatty acid profiles and the level of linoleic acid was increased 19% (P<0.05) in transgenic muscles compared to their nontransgenic littermates. Moreover, it exhibited an 87% and a 9% increase (P<0.05) in arachidonic acid (20:4 n-6) in muscles and liver, compared to their nontransgenic littermates. The results indicate that the plant fad2 gene can be functionally expressed in transgenic mice and may playan active role in conversion of oleic acid into linoleic acid.展开更多
Objective To determine the function of, in vivo, renin and its role in the pathogenesis of hypertension. Methods A renin 2 gene restriction map was constructed by endonuclease digest ion and Southern blotting hy...Objective To determine the function of, in vivo, renin and its role in the pathogenesis of hypertension. Methods A renin 2 gene restriction map was constructed by endonuclease digest ion and Southern blotting hybridization. Transgenic rats were produced via micro injection method.Results The 24 kb fragments containing mouse full length ren 2 and it s flanking sequence were cleaved by single enzymes (EcoRⅠ, KpnⅠ and BamHⅠ) an d combined enzymes (EcoRⅠ/KpnⅠ, KpnⅠ/BamHⅠ and BamHⅠ/EcoRⅠ), respectively. The digests were electrophoresed in 0.8% agarose plates and transferred onto NC membranes. Radioactive 735 bp and 1400 bp probes obtained from half and full l ength renin 1 cDNA were used in southern blotting hybridization. According to t he electrophoresis and hybridization patterns, a ren 2 restriction map was cons tructed. 1603 fertilized rat ova after injection with purified 24 kb renin 2 ge ne were implanted into the oviducts of 81 pseudopregnant recipients in about 2 0 ova per female rat. 306 progenies were obtained from 50 foster mothers (averag e of pregnancies was 56.6%). 248 survived pups were identified by PCR analys is and Southern hybridization, and eight positive rats were found to be the transgenic rats (founder, F). All of them carried long fragments (24 kb) of renin 2 gene with normal blood pressure. Preliminary breeding and screening were carried out in the founder. Total survival pups (17.8%) and overall efficiencies (1%) were h arvested as the same as those reported in the literatures. A systemic observatio n and the problems occurred during production of transgenic rats were also descr ibed besides the technique procedure used in this study.Conclusions Mapping of full length murine ren 2 can be used in invest igation of the structure and function of the gene. The results denoted that the ren 2 tran sgenic rats were successfully established in this study and the technique used i n the production of transgenic rats was proved to be valid in leading to wide s pread application of transgenic technique to many other related researches.展开更多
文摘Human immunodeficiency virus type-1 (HIV-1) chronic infection causes millions of deaths each year. We previously developed a novel HIV-1 Gag-spe cific exosome (EXO)-targeted T cell-based vaccine (Gag-Texo) using ConAstimulated polyclonal CD8+T (ConA-T) cells armed with Gag-specific dendritic cell (DC)-released EXOs, and showed that Gag-Texo stimulated more efficient cytotoxic T lymphocyte (CTL) responses than DCs. Tat HIV-1 early regulatory protein possesses immunomodulatory and adjuvant properties. To enhance Gag-Texo immunogenicity, we generated Tat-engineered OVA/Tat Texo and Gag/Tat-Texo vaccines using ConA-T cells armed with EXOs release by DCs infected with recombinant OVA/Tat- and Gag/Tat-expressing adenoviruses (AdVOVA/Tat and AdVGag/Tat). We then assessed vaccination-stimulated CTL responses in naive mice, and therapeutic immunity in transgenic HLA-A2 mice bearing Gag/HLA-A2-expressing BL6-10OVA/A2 melanoma lung metastases. We demonstrate that the OVA/Tat-Texo vaccine enhances functional OVA-specific CTL responses, compared to the OVA-Texo vaccine, and broadens CTL responses recognizing the cryptic OVA epitope in C57BL/6 mice. Furthermore, we determine that the Gag/Tat-Texo not only stimulates more efficient CTL responses than Gag-Texo, but also induces enhanced therapeutic immunity. We show that, 30% of Gag/Tat-Texo-immunized mice are free of tumor lung-metastases, compared to all Gag-Texo-immunized mice displaying lung-metastasis. In addition, the average number of tumor lung metastases colonies (32/lung) in the Gag/Tat-Texo-immunized mice was also significantly lower than that (78/lung) observed in Gag-Texo-immunized mice. Taken together, this indicates that HIV-1 Gag/Tat-Texo capable of stimulating enhanced Gag-specific CTL responses and therapeutic immunity may become a new immunotherapeutic vaccine candidate for controlling virus in HIV-1 patients.
基金Supported by the Heart and Stroke Foundation of Canada,No.G-13-0002851the Canadian Institutes of Health Research Training Program in Regenerative Medicine to Bartczak A and Chruscinski Athe Ontario Graduate Scholarship in Science and Technology to Bartczak A
文摘AIMTo determine the effect of overexpression of fibrinogen-like protein 2 (FGL2) on regulatory T cell (Treg) and effector T (Teff) cell function on T cell-induced colitis in Rag1<sup>-/-</sup> mice.METHODSTreg and Teff cells from fgl2<sup>-/-</sup>, fgl2<sup>+/+</sup>, and fgl2<sup>Tg</sup> mice were purified by FACS. They were studied in vitro for immunosuppressive activity and cell proliferation and in vivo for their effects on the development and prevention of T cell-induced colitis in Rag1<sup>-/-</sup> mice.RESULTSIn vitro, fgl2<sup>Tg</sup> Treg had enhanced immunosuppressive activity, and fgl2<sup>Tg</sup> Teff had reduced proliferation to alloantigen stimulation. Transfer of Teff from C57Bl/6J mice (fgl2<sup>+/+</sup>) into Rag1<sup>-/-</sup> mice produced both clinical and histologic colitis with dense infiltrates of CD3<sup>+</sup> T cells, crypt abscesses and loss of goblet cells. Fgl2<sup>Tg</sup> Treg prevented the development of T cell-induced colitis, whereas fgl2<sup>+/+</sup> and fgl2<sup>-/-</sup> Treg were only partially protective. In mice that received fgl2<sup>Tg</sup> Treg, the ratio of Foxp3<sup>+</sup> to CD3<sup>+</sup> cells was increased both in the colon and in mesenteric lymph nodes, and Teff cell proliferation as determined by staining with Ki67 was reduced. Teff cells from fgl2<sup>Tg</sup> mice did not produce colitis.CONCLUSIONHere we show that fgl2<sup>Tg</sup> Teff are hypoproliferative and do not induce colitis. We further demonstrate that fgl2<sup>Tg</sup> Treg prevent colitis in contrast to fgl2<sup>+/+</sup> Treg, which were only partially protective. These studies collectively provide a rationale for exploring the use of FGL2 or Treg expressing high levels of FGL2 in the treatment of inflammatory bowel disease.
文摘BACKGROUND: Apoptosis plays an important role in central neural diseases and trauma. B-cell lymphoma/Leukemia-2 (Bcl-2) can inhibit apoptosis in a wide variety of cells including neurons. In this experiment, by studying Bcl-2 over-expression transgenic (TG) mice subjected to spinal cord injury (SCI), we investigated whether Bcl-2 could reduce posttraumatic neuronal apoptosis, reduce the range of damage, and improve the behavioral functional recovery after contusive SCI.METHODS: Nine Bcl-2 TG mice and nine control mice were subjected to SCI of moderate severity at T10, with the use of weight dropping (WD) method (impact force 2.5×3.0 g/cm). At times up to 1 day, 7 days and 14 days after SCI, functional defi cits were evaluated with Basso, Beattie, and Bresnahan (BBB) scales, and apoptosis of neurons was investigated by using the TUNEL method. Another three control mice only underwent lamina opening, but were not subjected to SCI, to provide blank comparison.RESULTS: The mean functional scores for the control mice (5.05 ±0.35) were lower than those for the Bcl-2 TG mice (5.45 ±0.15), although the unpaired T-test revealed no signifi cant difference (P=0.67). On the other hand, the number of TUNEL positive neurons and integrated option density (IOD) scores for the Bcl-2 TG mice were both signifi cantly lower than those for the control mice (P〈0.05).CONCLUSIONS: This experiment suggests that overexpression of Bcl-2 may suppress neuronal apoptosis after SCI. Bcl-2 may be an important factor within the central nervous system that can relieve the damage after trauma.
基金supported by grants from the Ministry of Science and Technology of China (2016YFC1302103, 2015CB553906, and 2013CB910501)the National Natural Science Foundation of China (81730071, 81230051, 81472734, and 31170711)+3 种基金the Beijing Natural Science Foundation (7120002 and 7171005)the 111 Project of the Ministry of Education, grants from Peking University (BMU20120314 and BMU20130364)a Leading Academic Discipline Project of Beijing Education Bureau to H.Z.supported by a grant from the National Natural Science Foundation of China (81773199) to J.Z
文摘Kindlin-2, an integrin-interacting protein, regulates breast cancer progression. However, currently, no animal model to study the role of Kindlin-2 in the carcinogenesis of mammary gland is available. We established a Kindlin-2 transgenic mouse model using a mammary gland-specific promoter, mammary tumor virus(MMTV) long terminal repeat(LTR). Kindlin-2 was overexpressed in the epithelial cells of the transgenic mice. The mammary gland ductal trees were found to grow faster in MMTV-Kindlin-2 transgenic mice than in control mice during puberty. Kindlin-2 promoted mammary gland growth as indicated by more numerous duct branches and larger lumens, and more alveoli were formed in the mammary glands during pregnancy under Kindlin-2 overexpression. Importantly, mammary gland-specific expression of Kindlin-2 induced tumor formation at the age of 55 weeks on average. Additionally, the levels of estrogen receptor and progesterone receptor were decreased, whereas human epidermal growth factor receptor 2 and β-catenin were upregulated in the Kindlin-2-induced mammary tumors. These findings demonstrated that Kindlin-2 induces mammary tumor formation via activation of the Wnt signaling pathway.
基金Supported by the National Basic Research and Development Program of China (Grant No 2004CB117500)the National Natural Science Foundation of China (Grant No 30571332)the National Major Special Project on New Varieties Cultivation for Transgenic Organisms (Grant No 2008ZX08008-003)
文摘Fatty acid desaturase-2 (FAD2) introduces a double bond in position 12 in oleic acid (18:1) to form linoleic acid (18:2 n-6) in higher plants and microbes. A new transgenic expression cassette, containing CMV promoter/fad2 cDNA/SV40 polyA, was constructedto produce transgenic mice. Among 63 healthy offspring, 10 founders (15.9%) integrated the cotton fad2 transgene into their genomes, as demonstrated by PCR and Southern blotting analysis. All founder mice were fertile and heterozygous fad2 female and nontransgenic littermates were used for fatty acid analysis using gas chromatography. One fad2 transgenic line showed substantial differences in the fatty acid profiles and the level of linoleic acid was increased 19% (P<0.05) in transgenic muscles compared to their nontransgenic littermates. Moreover, it exhibited an 87% and a 9% increase (P<0.05) in arachidonic acid (20:4 n-6) in muscles and liver, compared to their nontransgenic littermates. The results indicate that the plant fad2 gene can be functionally expressed in transgenic mice and may playan active role in conversion of oleic acid into linoleic acid.
文摘Objective To determine the function of, in vivo, renin and its role in the pathogenesis of hypertension. Methods A renin 2 gene restriction map was constructed by endonuclease digest ion and Southern blotting hybridization. Transgenic rats were produced via micro injection method.Results The 24 kb fragments containing mouse full length ren 2 and it s flanking sequence were cleaved by single enzymes (EcoRⅠ, KpnⅠ and BamHⅠ) an d combined enzymes (EcoRⅠ/KpnⅠ, KpnⅠ/BamHⅠ and BamHⅠ/EcoRⅠ), respectively. The digests were electrophoresed in 0.8% agarose plates and transferred onto NC membranes. Radioactive 735 bp and 1400 bp probes obtained from half and full l ength renin 1 cDNA were used in southern blotting hybridization. According to t he electrophoresis and hybridization patterns, a ren 2 restriction map was cons tructed. 1603 fertilized rat ova after injection with purified 24 kb renin 2 ge ne were implanted into the oviducts of 81 pseudopregnant recipients in about 2 0 ova per female rat. 306 progenies were obtained from 50 foster mothers (averag e of pregnancies was 56.6%). 248 survived pups were identified by PCR analys is and Southern hybridization, and eight positive rats were found to be the transgenic rats (founder, F). All of them carried long fragments (24 kb) of renin 2 gene with normal blood pressure. Preliminary breeding and screening were carried out in the founder. Total survival pups (17.8%) and overall efficiencies (1%) were h arvested as the same as those reported in the literatures. A systemic observatio n and the problems occurred during production of transgenic rats were also descr ibed besides the technique procedure used in this study.Conclusions Mapping of full length murine ren 2 can be used in invest igation of the structure and function of the gene. The results denoted that the ren 2 tran sgenic rats were successfully established in this study and the technique used i n the production of transgenic rats was proved to be valid in leading to wide s pread application of transgenic technique to many other related researches.