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1.
Stem Cells Dev ; 2021 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-33528297

RESUMO

Mesenchymal stem cells (MSCs) isolated from adipose tissue (adipose-derived stem cells, ADSCs) are considered one of the most promising cell types for applications in regenerative medicine. However, the regenerative potency of ADSCs may vary due to heterogeneity. Long-term trypsin treatment (LTT) is known to significantly concentrate multilineage-differentiating stress-enduring (Muse) cells from human MSCs. In this study, we aimed to generate cells with high stem cell potency from canine ADSCs using LTT. After 16-hour of treatment with trypsin, surviving ADSCs (LTT-tolerant cells) had significantly enhanced expression of stage-specific embryonic antigen (SSEA)-1, a mouse embryonic stem cell marker, and fucosyltransferase 9, one of several fucosyltransferases for SSEA-1 biosynthesis. However, LTT-tolerant cells did not enhance the expression of SSEA-3, a known human Muse cell marker. LTT-tolerant cells, however, showed significantly higher self-renewal capacity in the colony-forming unit fibroblast assay than ADSCs. Additionally, the LTT-tolerant cells formed cell clusters similar to embryoid bodies and expressed undifferentiated markers. Moreover, these cells differentiated into cells of all three germ layers and showed significantly higher levels of α 2-6 sialic acid (Sia)-specific lectins, known as differentiation potential markers of human MSCs, than ADSCs. LTT-tolerant cells had a normal karyotype and had low telomerase activity, showing little carcinogenetic potency. LTT-tolerant cells also showed significantly increased activity of transmigration in the presence of chemoattractants and had increased expression of migration-related genes compared to ADSCs. In addition, LTT-tolerant cells had stronger suppressive activity against mitogen-stimulated lymphocyte proliferation than ADSCs. Overall, these results indicated that the LTT-tolerant cells in canine ADSCs have similar properties as human Muse cells (although one of the undifferentiated markers is different) and are expected to be a promising tool for regenerative therapy in dogs.

2.
Stem Cells Dev ; 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-33256572

RESUMO

Forced co-expression of the transcription factors Oct3/4, Klf4, Sox2, and c-Myc reprograms somatic cells into pluripotent stem cells (PSCs). Such induced PSCs (iPSCs) can generate any cell type of the adult body or indefinitely proliferate without losing their potential. Accordingly, iPSCs can serve as an unlimited cell source for the development of various disease models and regenerative therapies for animals as well as humans. Although canine peripheral blood mononuclear cells (PBMCs) can be easily obtained, they have a very low iPSC-reprogramming efficiency. In this study, we determined the reprogramming efficiency of canine PBMCs under several conditions involving three types of media supplemented with small-molecule compounds. We found that canine iPSCs (ciPSCs) could be efficiently generated from PBMCs using N2B27 medium supplemented with leukemia inhibitory factor (LIF), basic fibroblast growth factor, and a small-molecule cocktail (Y-27632, PD0325901, CHIR99021, A-83-01, Forskolin, and L-ascorbic acid). We generated five ciPSC lines that could be maintained in StemFit® medium supplemented with LIF. The SeVdp(KOSM)302L vectors were appropriately silenced in four ciPSC lines. Of the two lines characterized, both were positive for alkaline phosphatase activity and expressed pluripotency markers, including the Oct3/4, Sox2, and Nanog transcripts, as well as the OCT3/4 and NANOG proteins, and the SSEA-1 carbohydrate antigen. The ciPSCs could form embryoid bodies and differentiate into the three germ layers, as indicated by marker gene and protein expression. Furthermore, one ciPSC line formed teratomas comprising several tissues from every germ layer. Our ciPSC lines maintained a normal karyotype even after multiple passages. Moreover, our new reprograming method was able to generate ciPSCs from multiple donors' PBMCs. In conclusion, we developed an easy and efficient strategy for the generation of footprint-free ciPSCs from PBMCs. We believe that this strategy can be useful for disease modeling and regenerative medicine in the veterinary field.

3.
Mol Reprod Dev ; 87(6): 663-665, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32424848

RESUMO

Using auto-erasable Sendai virus vector, we generated ciPSC line. After several passages, virus was not present in ciPSCs by RT-PCR. ciPSCs from canine PBMCs had pluripotent state, differentiated all three germ layers in vitro, and had normal 78 XX karyotype. These results proved that PBMCs were one of the good cell sources to generate ciPSC lines from companion and patient dogs.

4.
J Vet Med Sci ; 82(5): 668-672, 2020 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-32249241

RESUMO

We examined the paracrine action of canine mesenchymal stromal cells (MSCs) derived from bone marrow on the survival and differentiation of neural stem cells (NSCs) in vitro. MSCs were collected from the proximal end of the diaphysis of femur of healthy beagle dogs. The 70-80% confluent MSCs were re-fed with serum-free DMEM. The MSCs were incubated for 48 hr and the supernatant was collected as the conditioned medium (MSC-CM). The survival rate of NSCs in MSC-CM was significantly greater than in the medium without MSC-CM. The percentage of differentiated neurons and neurite length in MSC-CM was also significantly higher than in the medium without MSC-CM. These results suggested that canine MSC-CM promotes stem cell survival and neural differentiation of NSCs.

5.
FASEB Bioadv ; 2(1): 5-17, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32123853

RESUMO

The tumor microenvironment strongly influences clinical outcomes of immunotherapy. By transfecting genes of relevant cytokines into tumor cells, we sought to manipulate the microenvironment so as to elicit activation of T helper type 1 (Th1) responses and the maturation of dendritic cells (DCs). Using a synthetic vehicle, the efficiency of in vivo transfection of GFP-cDNA into tumor cells was about 7.5% by intratumoral injection and about 11.5% by intravenous injection. Survival was significantly improved by both intratumoral and intravenous injection of the plasmid containing cDNA of interferon-gamma, followed by intratumoral injection of DCs presenting the tumor antigens. Also, tumor growth was inhibited by these treatments. A more significant effect on survival and tumor growth inhibition was observed following injection of the plasmid containing cDNA of CD40 ligand, which is a potent inducer of DC-maturation. Furthermore, the co-injection of both IFNγ- and CD40 ligand-encoding cDNA-plasmids, followed by DC treatment, gave rise to further marked and enhancement, including 100% survival and more than 50% complete remission. This treatment regimen elicited significant increases in mature DCs and types of cells contributing to Th1 responses, and significant decreases in immune suppressor cells in the tumor. In the spleen, the treatment significantly increased activities of tumor-specific killer and natural killer cells, but no alteration was observed in mature DCs or suppressor cells. These results indicate that transfection of these cytokine genes into tumor cells significantly alter the tumor microenvironment and improve the therapeutic results of DC-based immunotherapy.

6.
Theriogenology ; 147: 71-76, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32126383

RESUMO

Freeze drying has been developed as a new sperm preservation method that eliminates the necessity of using liquid nitrogen. An advantage of freeze-dried sperm is that it can be stored at 4 °C and transported at room temperature. To develop assisted reproductive techniques (ARTs) for domestic cats, we evaluated the effect of the freeze-dry procedure on cat sperm DNA by analyzing DNA integrity (experiment 1) and by generating cat embryos using freeze-dried sperm that had been preserved for several months (experiment 2). In experiment 1, the rate of DNA damage to freeze-dried sperm was not significantly different than that of sperm cryopreserved with liquid nitrogen (P > 0.05). In experiment 2, the proportions of cleaved embryos, morulae, and blastocysts and the cell number of blastocysts did not differ between experimental groups in which fresh sperm and freeze-dried sperm were used (P > 0.05). In addition, we generated feline blastocysts using freeze-dried sperm stored for 1-5 months. These results support an expansion of the repertoire of ARTs that are potentially applicable to both domestic and endangered species of cats.

7.
ACS Chem Biol ; 15(2): 360-368, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-31841301

RESUMO

Molecular-targeting peptides and mini-proteins are promising alternatives to antibodies in a wide range of applications in bioscience and medicine. We have developed a helix-loop-helix (HLH) peptide as an alternative to antibodies to inhibit specific protein interactions. Cytotoxic T lymphocyte antigen-4 (CTLA-4) downregulates immune responses of cytotoxic T-cells by interaction with B7-1, a co-stimulatory molecule expressed on antigen presenting cells (APCs). To induce immune stimulatory activity, we used directed evolution methods to generate a HLH peptide that binds to CTLA-4, inhibiting the CTLA-4-B7-1 interaction and inducing immune stimulatory activity. Yeast-displayed libraries of HLH peptides were constructed and screened against CTLA-4 and identified the binding peptide Y-2, which exhibits a moderate affinity. The affinity of Y-2 was improved by in vitro affinity maturation to afford a stronger binder, ERY2-4. Peptide ERY2-4 specifically bound to CTLA-4 with a KD of 196.8 ± 2.3 nM, comparable to the affinity of the CTLA-4-B7-1 interaction. Furthermore, ERY2-4 inhibited the CTLA-4-B7-1 interaction with an IC50 of 1.1 ± 0.03 µM and blocked the interaction between CTLA-4 and dendritic cells (DCs) presenting B7 on their surface. Importantly, ERY2-4 showed no cross-reactivity against CD28, suggesting it does not suppress T-cell activation. Finally, in a mixed lymphocyte reaction assay with DCs and T cells, ERY2-4 enhanced an allogeneic lymphocyte response. Since CTLA-4 is a critical immune checkpoint for restricting the cancer immune response, this inhibitory HLH peptide represents a new class of drug candidates for immunotherapy.

8.
J Vet Med Sci ; 81(12): 1842-1849, 2019 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-31666444

RESUMO

A cat was referred because of diffuse parenchymal lung disease. Close examinations revealed a swollen abdominal lymph node and multiple nodules of the liver. Mycobacterium avium subspecies hominissuis infection was confirmed by culture and single nucleotide polymorphism analysis of samples recovered from the liver and bronchoalveolar lavage. After administration of combination antibiotics for 6 months, culture results were negative. Though atonic seizures were observed during the treatment, it disappeared after isoniazid discontinuation and pyridoxal phosphate administration. On day 771 of illness, no clinical signs, lung diseases, or obvious swelling of lymph nodes was observed. This is the first report to confirm Mycobacterium avium subspecies hominissuis infection in cats through gene analysis and to completely cure it with combination antibiotics.


Assuntos
Doenças do Gato/microbiologia , Pneumopatias/veterinária , Mycobacterium avium/isolamento & purificação , Tuberculose/veterinária , Animais , Antibacterianos/uso terapêutico , Gatos , Quimioterapia Combinada , Isoniazida/efeitos adversos , Isoniazida/uso terapêutico , Fígado/microbiologia , Pneumopatias/tratamento farmacológico , Pneumopatias/microbiologia , Masculino , Mycobacterium avium/genética , Polimorfismo de Nucleotídeo Único , Fosfato de Piridoxal/uso terapêutico , Convulsões/induzido quimicamente , Convulsões/veterinária , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia
9.
J Vet Med Sci ; 81(4): 629-635, 2019 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-30787208

RESUMO

Feline embryo development was examined for 7 days after fertilization using commercially available human media supplemented with 0.3% bovine serum albumin (BSA) or 5% fetal bovine serum (FBS). Cumulus-oocyte complexes were categorized as Grades 1, 2, and 3 according to morphology. Only-One Medium (OM) was used for in vitro culture (IVC) in OM + BSA, OM + FBS, and OM + BSA/FBS, with BSA supplementation for the first 2 days and FBS for the subsequent 5 days. Embryos cultured in Early Culture Medium (1-2 days) and Blastocyst Medium (3-7 days) were defined as EB + BSA and EB + BSA/FBS. The developmental rate until the blastocyst stage of Grade 1 and 2 oocytes cultured in OM + BSA/FBS was higher than for the other groups and was significantly higher than for the OM + BSA and EB + BSA groups (P<0.01). Grade 3 oocytes cultured in OM + BSA/FBS also showed the greatest proportion of blastocyst formation. However, FBS supplementation throughout the IVC period reduced blastocyst number. The percentage of 2 pronuclei after fertilization as well as blastocyst cell number were significantly higher in Grade 1 and 2 than Grade 3 oocytes when cultured in OM + BSA/FBS (P<0.05). These results indicate that commercially available OM supplemented with BSA for the first 2 days of culture and FBS for the subsequent 5 days is suitable for feline embryo development until the blastocyst stage.


Assuntos
Gatos/embriologia , Meios de Cultura , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário , Soroalbumina Bovina , Animais , Blastocisto , Meios de Cultura/química , Feminino , Fertilização In Vitro , Humanos , Masculino
10.
J Reprod Dev ; 65(3): 245-250, 2019 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-30773507

RESUMO

Piezo-actuated intracytoplasmic sperm injection (Piezo-ICSI) is used as an efficient in vitro fertilization method with various animals. With this method, elongated spermatids are collected from testicular tissues and are easier to obtain from animals that unexpectedly die than ejaculate sperm. Additionally, elongated spermatid injection often results in the development of embryos and offspring. To develop assisted reproductive techniques (ARTs) for domestic cats, we examined the effects of oocyte activation on cleavage and embryo development after Piezo-ICSI with motile sperm (experiment 1) and after Piezo-ICSI with either testicular sperm or elongated spermatids (experiment 2). In experiment 1, the proportions of cleaved embryos, morulas, and blastocysts following Piezo-ICSI with ethanol activation were significantly higher (P < 0.05) than in the non-activated groups. However, the proportion of blastocysts and the blastocyst quality did not differ significantly (P > 0.05) between the ethanol-activated and non-activated groups. In experiment 2, the cleavage frequencies of oocytes after Piezo-ICSI of testicular sperm or elongated spermatids and ethanol activation were higher (P < 0.05) than that of oocytes in the non-activated group, but the occurrence of blastocyst formation and quality of blastocysts did not differ between the activated and non-activated groups. In summary, cat embryos can be produced by Piezo-actuated microinjection of elongated spermatids. Ethanol activation increased the frequency of cleavage, but it affected neither the occurrence of blastocyst development nor the quality of blastocysts. These results represent an expansion in the repertoire of ARTs that are potentially applicable to both domestic and endangered species of cats.


Assuntos
Oócitos/citologia , Injeções de Esperma Intracitoplásmicas/veterinária , Espermátides/fisiologia , Espermatozoides/fisiologia , Animais , Blastocisto/citologia , Gatos , Fase de Clivagem do Zigoto , Criopreservação , Desenvolvimento Embrionário , Feminino , Fertilização In Vitro , Masculino , Microinjeções , Ovário/citologia , Testículo/citologia
11.
Stem Cells Dev ; 27(22): 1577-1586, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30215317

RESUMO

Canine induced pluripotent stem cells (ciPSCs) can be used in regenerative medicine. However, there are no reports on the generation of genome integration-free and completely exogenous gene-silenced (footprint free) ciPSCs that are tolerant to enzymatic single-cell passage. In this study, we reprogrammed canine embryonic fibroblasts using the auto-erasable replication-defective and persistent Sendai virus vector, SeVdp(KOSM)302L, and generated two ciPSC lines. The ciPSCs were positive for pluripotent markers, including alkaline phosphatase activity as well as OCT3/4, SOX2, and NANOG transcripts, and NANOG, stage-specific embryonic antigen-1, and partial TRA-1-60 protein expression, even after SeVdp(KOSM)302L removal. The ciPSCs were induced to differentiate into all the three germ layers as embryoid bodies in vitro and as teratomas in vivo. Furthermore, SeVdp(KOSM)302L-free ciPSCs maintained a normal karyotype even after repeated enzymatic single-cell passaging. Therefore, to our knowledge, for the first time, we demonstrated the generation of footprint-free and high-quality ciPSCs that can be passaged at the single-cell stage using enzymatic methods. Our method for generation of ciPSCs is a good step toward the development of clinical application of ciPSCs.


Assuntos
Diferenciação Celular/genética , Corpos Embrioides/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Vírus Sendai/genética , Animais , Reprogramação Celular/genética , Cães , Fibroblastos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Vetores Genéticos/genética , Humanos
12.
Cancer Sci ; 109(5): 1319-1329, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29575556

RESUMO

For a successful tumor vaccine, it is necessary to develop effective immuno-adjuvants and identify specific tumor antigens. Tumor cells obtained from surgical or biopsy tissues are a good source of tumor antigens but, unlike bacteria, they do not induce strong immune responses. Here, we designed 2 novel lipopeptides that coat tumor cell surfaces and mimic bacterial components. Tumor cells coated with these lipopeptides (called bacteria-mimicking tumor cells [BMTC]) were prepared and their efficacy as a tumor vaccine examined. Natural bacterial lipopeptides act as ligands for toll-like receptor 2 (TLR2) and activate dendritic cells (DC). To increase the affinity of the developed lipopeptides for the negatively charged plasma membrane, a cationic polypeptide was connected to Pam2Cys (P2C), which is the basic structure of the TLR2 ligand. This increased the non-specific binding affinity of the peptides for the cell surface. Two such lipopeptides, P2CSK11 (containing 1 serine and 11 lysine residues) and P2CSR11 (containing 1 serine and 11 arginine residues) bound to irradiated tumor cells via the long cationic polypeptides more efficiently than the natural lipopeptide MALP2 (P2C-GNNDESNISFKEK) or a synthetic lipopeptide P2CSK4 (a short cationic polypeptide containing 1 serine and 4 lysines). BMTC coated with P2CSR11 or P2CSK11 were efficiently phagocytosed by DC and induced antigen cross-presentation in vitro. They also induced effective tumor-specific cytotoxic T cell responses and inhibited tumor growth in in vivo mouse models. P2CSR11 activated DC but induced less inflammation-inducing cytokines/interferons than other lipopeptides. Thus, P2CSR11 is a strong candidate antigen-specific immuno-adjuvant, with few adverse effects.


Assuntos
Vacinas Anticâncer/administração & dosagem , Lipopeptídeos/administração & dosagem , Neoplasias/tratamento farmacológico , Receptor 2 Toll-Like/imunologia , Animais , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/farmacologia , Células Dendríticas/imunologia , Células Dendríticas/fisiologia , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Ligantes , Lipopeptídeos/imunologia , Lipopeptídeos/farmacologia , Camundongos , Neoplasias/imunologia , Fagocitose , Ensaios Antitumorais Modelo de Xenoenxerto
13.
J Vet Med Sci ; 80(2): 190-196, 2018 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-29311492

RESUMO

By using a complex of DNA, polyethylenimine and chondroitin sulfate, the in vivo transfection of early secretory antigenic target-6 (ESAT-6) gene into tumor cells was found to cause significant suppression of the tumor growth. In order to apply the method in clinical cancer treatment in dogs and cats, mechanisms underlying the suppressive effects were investigated in a tumor-bearing mouse model. The transfection efficiency was only about 10%, but the transfection of ESAT-6 DNA nevertheless induced systemic immune responses against ESAT-6. By triple injection of ESAT-6 DNA at three day intervals, the tumor was significantly reduced and almost disappeared by 5 days after the start of treatment, and did not increase for more than 15 days after the final treatment. In the immunohistochemistry, a larger number of dendritic cells (DCs)/macrophages expressing ionized calcium-binding adapter molecule 1 and CD3+ T cells was observed in tumors treated with ESAT-6 DNA, and their population further increased significantly by day 5. Moreover, the amount of tumor necrosis factor, which is an apoptosis-inducing factor produced mainly by DCs/macrophages, was greater in the ESAT-6 DNA treated tumors than in controls, and increased with repeat of the treatment. These results indicate that in vivo transfection of ESAT-6 DNA into tumor cells elicits significant inhibition of tumor growth by inducing potent activity of innate immunity mediated by DCs/macrophages, which may be followed by adaptive immunity against tumor associated antigens, elicited by the costimulation with ESAT-6 antigen.


Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Células Dendríticas/imunologia , Imunidade Inata/imunologia , Macrófagos/imunologia , Neoplasias Experimentais/terapia , Transfecção/métodos , Animais , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias
14.
Biochem Biophys Res Commun ; 495(3): 2165-2170, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29258821

RESUMO

To investigate the role of nitric oxide (NO)/reactive oxygen species (ROS) redox signaling in Parkinson's disease-like neurotoxicity, we used 1-methyl-4-phenylpyridinium (MPP+) treatment (a model of Parkinson's disease). We show that MPP+-induced neurotoxicity was dependent on ROS from neuronal NO synthase (nNOS) in nNOS-expressing PC12 cells (NPC12 cells) and rat cerebellar granule neurons (CGNs). Following MPP+ treatment, we found production of 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), a second messenger in the NO/ROS redox signaling pathway, in NPC12 cells and rat CGNs, that subsequently induced S-guanylation and activation of H-Ras. Additionally, following MPP+ treatment, extracellular signal-related kinase (ERK) phosphorylation was enhanced. Treatment with a mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor attenuated MPP+-induced ERK phosphorylation and neurotoxicity. In conclusion, we demonstrate for the first time that NO/ROS redox signaling via 8-nitro-cGMP is involved in MPP+-induced neurotoxicity and that 8-nitro-cGMP activates H-Ras/ERK signaling. Our results indicate a novel mechanism underlying MPP+-induced neurotoxicity, and therefore contribute novel insights to the mechanisms underlying Parkinson's disease.


Assuntos
1-Metil-4-fenilpiridínio , Cerebelo/metabolismo , GMP Cíclico/análogos & derivados , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Transtornos Parkinsonianos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Células Cultivadas , Cerebelo/efeitos dos fármacos , Cerebelo/patologia , GMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neurotoxinas , Células PC12 , Transtornos Parkinsonianos/induzido quimicamente , Ratos
15.
PLoS One ; 12(11): e0188738, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29190690

RESUMO

Although dendritic cell (DC)-based immunotherapy shows little toxicity, improvements should be necessary to obtain satisfactory clinical outcome. Using interferon-gamma injection along with DCs, we previously obtained significant clinical responses against small or early stage malignant tumors in dogs. However, improvement was necessary to be effective to largely developed or metastatic tumors. To obtain effective methods applicable to those tumors, we herein used a DC-targeting Toll-like receptor ligand, h11c, and examined the therapeutic effects in murine subcutaneous and visceral tumor models and also in the clinical treatment of canine cancers. In murine experiments, most and significant inhibition of tumor growth and extended survival was observed in the group treated with the combination of h11c-activated DCs in combination with interferon-gamma and a cyclooxygenase2 inhibitor. Both monocytic and granulocytic myeloid-derived suppressor cells were significantly reduced by the combined treatment. Following the successful results in mice, the combined treatment was examined against canine cancers, which spontaneously generated like as those in human. The combined treatment elicited significant clinical responses against a nonepithelial malignant tumor and a malignant fibrous histiocytoma. The treatment was also successful against a bone-metastasis of squamous cell carcinoma. In the successful cases, the marked increase of tumor-responding T cells and decrease of myeloid-derived suppressor cells and regulatory T cells was observed in their peripheral blood. Although the combined treatment permitted the growth of lung cancer of renal carcinoma-metastasis, the marked elevated and long-term maintaining of the tumor-responding T cells was observed in the patient dog. Overall, the combined treatment gave rise to emphatic amelioration in DC-based cancer therapy.


Assuntos
Células Dendríticas/imunologia , Imunoterapia , Neoplasias/terapia , Receptores Toll-Like/metabolismo , Animais , Doenças do Cão/terapia , Cães , Ligantes , Camundongos , Camundongos Endogâmicos , Neoplasias/veterinária
16.
Stem Cells Dev ; 26(15): 1111-1120, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28474540

RESUMO

Extraembryonic endoderm (XEN) cells are stem cell lines derived from primitive endoderm cells of inner cell mass in blastocysts. These cells have self-renewal properties and differentiate into visceral endoderm (VE) and parietal endoderm (PE) of the yolk sac. Recently, it has been reported that XEN cells can contribute to fetal embryonic endoderm, and their unique potency has been evaluated. In this study, we have described the induction and characterization of new canine stem cell lines that closely resemble to XEN cells. These cells, which we designated canine induced XEN (ciXEN)-like cells, were induced from canine embryonic fibroblasts by introducing four transgenes. ciXEN-like cells expressed XEN markers, which could be maintained over 50 passages in N2B27 medium supplemented with inhibitors of mitogen-activated protein kinase p38 and transforming growth factor-beta 1. Our ciXEN-like cells were maintained without transgene expression and exhibited upregulated expression of VE and PE markers in feeder-free conditions. The cells differentiated from ciXEN-like cells using a coculture system showed multiple nuclei and expressed albumin protein, similar to characteristics of hepatocytes. Furthermore, these cells expressed the adult hepatocyte marker, CYP3A4. Interestingly, these cells also formed a net structure expressing the bile epithelium capillary marker, multidrug resistance-associated protein 2. Thus, we have demonstrated the induction of a new canine stem cell line, ciXEN-like cells, which could form an embryonic endodermal cell layer. Our ciXEN-like cells may be a helpful tool to study the canine embryo development and represent a promising cell source for proceeding human and canine regenerative medicine.


Assuntos
Técnicas de Cultura de Células/métodos , Embrião de Mamíferos/citologia , Endoderma/citologia , Membranas Extraembrionárias/citologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem Celular , Cães , Células Alimentadoras/citologia , Regulação da Expressão Gênica , Fator 3-beta Nuclear de Hepatócito/metabolismo , Camundongos , Células Estromais/citologia , Células Estromais/metabolismo , Transgenes
17.
Mol Reprod Dev ; 84(4): 329-339, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28240438

RESUMO

Canine induced pluripotent stem cells (ciPSCs) are an attractive source for regenerative veterinary medicine, and may also serve as a disease model for human regenerative medicine. Extending the application of ciPSCs from bench to bedside, however, requires resolving many issues. We generated ciPSCs expressing doxycycline-inducible murine Oct3/4 (Pou5f1), Sox2, Klf4, and c-Myc, which were introduced using lentiviral vectors. The resultant ciPSCs required doxycycline to proliferate in the undifferentiated state. Those ciPSC colonies exhibiting basic fibroblast growth factor (bFGF)-dependent proliferation were dissociated into single cells for passaging, and were maintained on a Matrigel-coated dish without feeder cells in a serum-free medium. The established ciPSCs had the ability to differentiate into three germ layers, via formation of embryoid bodies, as well as into cells expressing the same markers as mesenchymal stem cells. These ciPSCs may thus serve as a suitable source of pluripotent stem cell lines for regenerative veterinary medicine, with fewer concerns of contamination from unknown animal components.


Assuntos
Proliferação de Células , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Animais , Cães , Células Alimentadoras , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Mesenquimais/citologia , Camundongos , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética
18.
Biotechnol Lett ; 38(11): 1857-1866, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27484689

RESUMO

OBJECTIVES: To examine the potential of exosomes derived from the tumor cells, which had been genetically modified to express a Mycobacterium tuberculosis antigen, as a cancer vaccine aimed at overcoming the weak immunogenicity of tumor antigens. RESULTS: We transfected B16 melanoma cells with a plasmid encoding the M. tuberculosis antigen, early secretory antigenic target-6 (ESAT-6). The secreted exosomes bearing both tumor-associated antigens and the pathogenic antigen (or their epitopes) were collected. When the exosomes were injected into foot pads of mice, they significantly (p < 0.05) evoked cellular immunity against both ESAT-6, and B16 tumor cells. Intra-tumoral injection of the exosomes significantly suppressed (p < 0.001) tumor growth in syngeneic B16 tumor-bearing mice, while the exosomes derived from the non-transfected B16 cells showed no effect on tumor growth, although both exosomes should have similar tumor antigens. CONCLUSIONS: Exosomes bearing both tumor antigens and the M. tuberculosis antigen (or their epitopes) have a high potential as a candidate for cancer vaccine to overcome the immune escape by tumor cells.


Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Vacinas Anticâncer/administração & dosagem , Exossomos/metabolismo , Melanoma Experimental/tratamento farmacológico , Animais , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Antígenos de Neoplasias/imunologia , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Exossomos/genética , Exossomos/imunologia , Imunoterapia , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Camundongos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/imunologia , Transfecção
19.
Biomaterials ; 67: 214-24, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26222284

RESUMO

Potentiation of pH-sensitive liposome-based antigen carriers with IFN-γ gene lipoplexes was attempted to achieve efficient induction of tumor-specific immunity. 3-Methylglutarylated poly(glycidol) (MGluPG)-modified liposomes and cationic liposomes were used, respectively, for the delivery of antigenic protein ovalbumin (OVA) and IFN-γ-encoding plasmid DNA (pDNA). The MGluPG-modified liposomes and the cationic liposome-pDNA complexes (lipoplexes) formed hybrid complexes via electrostatic interactions after their mixing in aqueous solutions. The hybrid complexes co-delivered OVA and IFN-γ-encoding pDNA into DC2.4 cells, a murine dendritic cell line, as was the case of MGluPG-modified liposomes for OVA or the lipoplexes for pDNA. Both the lipoplexes and the hybrid complexes transfected DC2.4 cells and induced IFN-γ protein production, but transfection activities of the hybrid complexes were lower than those of the parent lipoplexes. Subcutaneous administration of hybrid complexes to mice bearing E.G7-OVA tumor reduced tumor volumes, which might result from the induction of OVA-specific cytotoxic T lymphocytes (CTLs). However, the hybrid complex-induced antitumor effect was the same level of the MGluPG-modified liposome-mediated antitumor immunity. In contrast, an extremely strong antitumor immune response was derived when these liposomes and lipoplexes without complexation were injected subcutaneously at the same site of tumor-bearing mice. Immunohistochemical analysis of tumor sections revealed that immunization through the liposome-lipoplex combination promoted the infiltration of CTLs to tumors at an early stage of treatment compared with liposomes, resulting in strong therapeutic effects.


Assuntos
Antígenos/imunologia , Sistemas de Liberação de Medicamentos , Imunoterapia , Interferon gama/genética , Neoplasias/imunologia , Neoplasias/terapia , Polímeros/química , Animais , Linhagem Celular Tumoral , Dendritos/metabolismo , Eletroforese , Feminino , Imunofluorescência , Concentração de Íons de Hidrogênio , Interferon gama/biossíntese , Lipossomos/química , Camundongos Endogâmicos C57BL , Microscopia Confocal , Neoplasias/patologia , Ovalbumina/imunologia , Tamanho da Partícula , Propilenoglicóis/química , Baço/imunologia , Eletricidade Estática , Tela Subcutânea , Linfócitos T Citotóxicos/imunologia
20.
Pharmaceutics ; 7(3): 152-64, 2015 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-26213961

RESUMO

We have reported that ternary complexes of plasmid DNA with conventional linear polyethylenimine (l-PEI) and certain polyanions were very stably dispersed, and, with no cryoprotectant, they could be freeze-dried and re-hydrated without the loss of transfection ability. These properties enabled the preparation of a concentrated suspension of very small pDNA complex, by preparing the complexes at highly diluted conditions, followed by condensation via lyophilization-and-rehydration procedure. Recently, a high potency linear polyethylenimine having no residual protective groups, i.e., Polyethylenimine "Max" (PEI "Max"), is available, which has been reported to induce much higher gene expression than conventional l-PEI. We tried to prepare the small DNA/PEI "Max"/polyanion complexes by a similar freeze-drying method. Small complex particles could be obtained without apparent aggregation, but transfection activity of the rehydrated complexes was severely reduced. Complex-preparation conditions were investigated in details to achieve the freeze-dried DNA/PEI "Max"/polyanion small ternary complexes with high transfection efficiency. DNA/PEI "Max"/polyanion complexes containing cytokine-coding plasmids were then prepared, and their anti-tumor therapeutic efficacy was examined in tumor-bearing mice.

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