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1.
PLoS One ; 16(2): e0246482, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33544781

RESUMO

The emergence and global spread of extended-spectrum or AmpC ß-lactamase (ESBL/AmpC)-producing Enterobacteriaceae in companion animals have led to the hypothesis that companion animals might be reservoirs for cross-species transmission because of their close contact with humans. However, current knowledge in this field is limited; therefore, the role of companion animals in cross-species transmission remains to be elucidated. Herein, we studied ESBL/AmpC-producing Enterobacteriaceae, Escherichia coli in particular, isolated from extraintestinal sites and feces of companion dogs. Whole-genome sequencing analysis revealed that (i) extraintestinal E. coli isolates were most closely related to those isolated from feces from the same dog, (ii) chromosomal sequences in the ST131/C1-M27 clade isolated from companion dogs were highly similar to those in the ST131/C1-M27 clade of human origin, (iii) certain plasmids, such as IncFII/pMLST F1:A2:B20/blaCTX-M-27, IncI1/pMLST16/blaCTX-M-15, or IncI1/blaCMY-2 from dog-derived E. coli isolates, shared high homology with those from several human-derived Enterobacteriaceae, (iv) chromosomal blaCTX-M-14 was identified in the ST38 isolate from a companion dog, and (v) eight out of 14 tested ESBL/AmpC-producing E. coli isolates (i.e., ST131, ST68, ST405, and ST998) belonged to the human extraintestinal pathogenic E. coli (ExPEC) group. All of the bla-coding plasmids that were sequenced genome-wide were capable of horizontal transfer. These results suggest that companion dogs can spread ESBL/AmpC-producing ExPEC via their feces. Furthermore, at least some ESBL/AmpC-producing ExPECs and bla-coding plasmids can be transmitted between humans and companion dogs. Thus, companion dogs can act as an important reservoir for ESBL/AmpC-producing E. coli in the community.

2.
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.

3.
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.

4.
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.

5.
J Vet Med Sci ; 82(6): 759-763, 2020 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-32295995

RESUMO

Human patients with inflammatory bowel disease may have poor prognosis with hypozincemia. However, there are limited data on zinc concentrations in the blood of dogs with lymphocytic-plasmacytic enteritis (LPE). The purpose of this study was to investigate the serum zinc concentration in dogs with LPE and its influence on disease severity and prognosis. Thirty-five dogs with LPE were recruited. Serum zinc concentration was measured using atomic absorption spectrometry. Hypozincemia was observed in 18/35 (51%) dogs with LPE. Serum zinc concentration was inversely correlated with histological and clinical severities. Overall survivals were significantly shorter in dogs with hypozincemia than in those without it. These findings suggest that serum zinc concentration is a useful biomarker for LPE severity and prognosis in dogs.

6.
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.

7.
J Vet Med Sci ; 82(6): 704-706, 2020 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-32249255

RESUMO

A 16 years old neutered male Miniature Dachshund with 1-year history of repetitive administration of zonisamide for treatment of epileptic seizure was presented for vomiting, anorexia and diarrhea. Serum biochemistry showed a markedly elevated ALP level. The dog died 6 days after the presentation and a necropsy was performed. Histopathologically, random, focal to extensive necrosis, formation of regenerative hepatocellular nodules surrounded by fibrous septa and proliferation of bile ducts were seen in the liver. From these findings, the hepatic lesion was diagnosed as hepatocellular necrosis with prominent regenerative reactions due to the chronic persistent liver injury. Hepatic lesions were considered to be induced by zonisamide, based on the history of continuous administration, and clinical and histopathological findings.

8.
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.

9.
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.

10.
Arch Virol ; 165(1): 157-167, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31748876

RESUMO

Endogenous retroviruses of domestic cats (ERV-DCs) are members of the genus Gammaretrovirus that infect domestic cats (Felis silvestris catus). Uniquely, domestic cats harbor replication-competent proviruses such as ERV-DC10 (ERV-DC18) and ERV-DC14 (xenotropic and nonecotropic viruses, respectively). The purpose of this study was to assess invasion by two distinct infectious ERV-DCs, ERV-DC10 and ERV-DC14, in domestic cats. Of a total sample of 1646 cats, 568 animals (34.5%) were positive for ERV-DC10 (heterozygous: 377; homozygous: 191), 68 animals (4.1%) were positive for ERV-DC14 (heterozygous: 67; homozygous: 1), and 10 animals (0.6%) were positive for both ERV-DC10 and ERV-DC14. ERV-DC10 and ERV-DC14 were detected in domestic cats in Japan as well as in Tanzania, Sri Lanka, Vietnam, South Korea and Spain. Breeding cats, including Singapura, Norwegian Forest and Ragdoll cats, showed high frequencies of ERV-DC10 (60-100%). By contrast, ERV-DC14 was detected at low frequency in breeding cats. Our results suggest that ERV-DC10 is widely distributed while ERV-DC14 is maintained in a minor population of cats. Thus, ERV-DC10 and ERV-DC14 have invaded cat populations independently.


Assuntos
Gammaretrovirus/classificação , Técnicas de Genotipagem/métodos , Infecções por Retroviridae/epidemiologia , Animais , Animais Domésticos , Ásia , Cruzamento , Gatos , Gammaretrovirus/genética , Gammaretrovirus/isolamento & purificação , Noruega , Filogenia , Filogeografia , Infecções por Retroviridae/virologia , Espanha , Tanzânia
11.
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
12.
Vet Immunol Immunopathol ; 210: 15-22, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30947975

RESUMO

Toll-like receptor 4 (TLR4), nucleotide-binding oligomerization domain 2 (NOD2), and TNF-α play important roles in human inflammatory bowel diseases. The aim of this study was to elucidate the relationship between Toll-like receptor 4, NOD2, and TNF-α and the severity of chronic gastrointestinal diseases in dogs. We examined the expression levels of TLR4, NOD2, and TNF-α in the stomach, duodenum, ileum, colon, and rectum obtained from 21 dogs with chronic gastrointestinal disease, including inflammatory bowel disease, high-grade lymphoma, food responsive enteropathy, chronic pancreatitis, low-grade lymphoma, inflammatory colorectal polyp, and chronic colitis. Next, we demonstrated whether there is good correlation between the expression levels of TLR4, NOD2, and TNF-α and the histopathological analysis of each sample. We found that the level of TLR4 expression in the ileum of dogs with chronic gastrointestinal disease was positively associated with the histopathological severity. We also found that the level of NOD2 expression in the duodenum, stomach, and rectum was positively associated with the histopathological severity. However, there was no correlation between TNF-α expression in the 5 regions tested in this study and the histopathological severity. These findings indicate that TLR4 and NOD2 are remarkably associated with the severity of chronic gastrointestinal disease in dogs.


Assuntos
Gastroenteropatias/imunologia , Gastroenteropatias/patologia , Proteína Adaptadora de Sinalização NOD2/genética , Receptor 4 Toll-Like/genética , Animais , Biópsia , Doença Crônica , Colo/imunologia , Colo/patologia , Cães , Duodeno/imunologia , Duodeno/patologia , Feminino , Masculino , Índice de Gravidade de Doença , Transdução de Sinais , Estômago/imunologia , Estômago/patologia , Fator de Necrose Tumoral alfa/genética
13.
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
14.
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
15.
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
16.
J Vet Med Sci ; 80(3): 532-535, 2018 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-29415921

RESUMO

It is currently unclear how mechanical micro-vibration affects the in vitro culture of embryos in Japanese Black cow. In the experimental groups, immature oocytes and fertilized embryos were cultured using the micro-vibration culture system with the vibration set for 5 sec at intervals of 60 min and frequency of 20, 40 or 80 Hz, respectively, during in vitro maturation and in vitro development. Compared with the control group, the rate of blastocyst development significantly increased in the 40 Hz group. In addition, the number of blastocyst cells reduced significantly in the 80 Hz group. In conclusion, the development of blastocysts in cows is facilitated by providing moderate mechanical micro-vibration to immature oocytes and embryos during the in vitro maturation and in vitro development.


Assuntos
Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário , Fertilização In Vitro/veterinária , Oócitos/crescimento & desenvolvimento , Estimulação Física , Vibração , Animais , Blastocisto , Bovinos , Feminino
17.
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
18.
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
19.
J Med Microbiol ; 66(8): 1085-1091, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28749329

RESUMO

PURPOSE: The aim of this study was to assess the in vitro efficacy of candidate antimicrobials against extended-spectrum ß-lactamase (ESBL)-producing isolates of extraintestinal pathogenic Escherichia coli (ExPEC) from companion animals. METHODOLOGY: A total of 90 ESBL-producing ExPEC isolates from dogs and cats were tested for susceptibility to 16 antimicrobials with the agar dilution method. We also identified the ESBLs and AmpC ß-lactamases of these isolates with PCR and DNA sequencing.Results/Key findings. All isolates were susceptible to meropenem, tebipenem and amikacin (AMK), and various proportions were susceptible to latamoxef (LMX, 97.8 %), fosfomycin (FOM, 97.8 %), faropenem (FPM, 96.7 %), nitrofurantoin (NFT, 96.7 %), flomoxef (FMX, 93.3 %), piperacillin/tazobactam (PTZ, 92.2 %), cefmetazole (CMZ, 91.1 %), chloramphenicol (80.0 %), trimethoprim/sulfamethoxazole (64.4 %), amoxicillin/clavulanic acid (63.3 %), ceftibuten (60.0 %), tetracycline (52.2 %) and enrofloxacin (10.0 %). A genetic analysis showed that 83 of the 90 (92.2 %) isolates were positive for CTX-M-type genes: CTX-M-14 (n=26), CTX-M-27 (n=20), CTX-M-55 (n=17), CTX-M-15 (n=12), CTX-M-2 (n=5), CTX-M-24 (n=2), CTX-M-104 (n=2) and CTX-M-3 (n=1). Eight isolates also expressed AmpC ß-lactamase phenotypes. CONCLUSION: This study demonstrates that the susceptibility rates to PTZ, CMZ, LMX, AMK, FOM, FPM, NFT and FMX were similar to those to carbapenems (>90 %), implying that these drugs are available alternatives to carbapenems for the treatment of companion animals infected with ExPEC-producing CTX-M-type ESBLs. Further in vivo studies of the effective use of these antimicrobials are required.


Assuntos
Antibacterianos/farmacologia , Doenças do Gato/microbiologia , Doenças do Cão/microbiologia , Infecções por Escherichia coli/veterinária , Escherichia coli Extraintestinal Patogênica/efeitos dos fármacos , Animais , Doenças do Gato/tratamento farmacológico , Gatos , Doenças do Cão/tratamento farmacológico , Cães , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli Extraintestinal Patogênica/enzimologia , Escherichia coli Extraintestinal Patogênica/genética , Escherichia coli Extraintestinal Patogênica/isolamento & purificação , Testes de Sensibilidade Microbiana , beta-Lactamases/genética , beta-Lactamases/metabolismo
20.
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
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