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The administration of mesenchymal stem cells (MSCs) has a positive effect on wound healing; however, the lack of adequate MSC engraftment at the wound site is a major limiting factor in current MSC-based therapies. In this study, a biosheet prepared using in-body tissue architecture (iBTA) was used as a material to address these problems. This study aimed to assess and evaluate whether biosheets containing somatic stem cells would affect the wound healing process in dogs. Biosheets were prepared by subcutaneously embedding molds in beagles. These were then evaluated grossly and histologically, and the mRNA expression of inflammatory cytokines, interleukins, and Nanog was examined in some biosheets. Skin defects were created on the skin of the beagles to which the biosheets were applied. The wound healing processes of the biosheet and control (no biosheet application) groups were compared for 8 weeks. Nanog mRNA was expressed in the biosheets, and SSEA4/CD105 positive cells were observed histologically. Although the wound contraction rates differed significantly in the first week, the biosheet group tended to heal faster than the control group. This study revealed that biosheets containing somatic stem cells may have a positive effect on wound healing.
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BACKGROUND: Mammalian sirtuins are homologs to the yeast silent information regulator 2 (Sir2), which is an NAD-dependent deacetylase. Sirtuins are comprised of 7 proteins, and each has different target proteins. Sirtuin 1 (SIRT1) plays important roles in maintaining metabolic functions and immune responses, and SIRT3 protects cells from oxidative stress-induced cell death. Both SIRT1 and SIRT3 are regulated by metabolic status and aging. Hence, SIRT1 and SIRT3 have been researched in metabolic diseases, such as type 2 diabetes mellitus (DM), fatty liver, and heart diseases. Although these diseases have been increasing, there is little information about relation between the diseases and SIRT1 and SIRT3 in cats. Therefore we cloned SIRT1 and SIRT3 cDNA, examined mRNA expression in cat tissues, and investigated the changes in SIRT1 and SIRT3 mRNA expression in peripheral blood leukocyte of cats fed on HFD for 6 weeks. RESULTS: Cat SIRT1 and SIRT3 contained a catalytic core region and showed high sequence homology with other vertebrate SIRT1 (>61.3%) and SIRT3 (>65.9%) amino acids. Real-time polymerase chain reaction analyses revealed that high expression levels were observed in the liver and skeletal muscle for SIRT1 and in the heart for SIRT3 in cats. In addition, both cat SIRT1 and SIRT3 expression levels in the pancreas were different between individuals. Cat SIRT1 mRNA expression in peripheral blood leukocytes was significantly elevated in obese cats fed on HFD (P < 0.05). CONCLUSIONS: Cat SIRT1 and SIRT3 genes are highly conserved among vertebrates, and HFD feeding may be related to SIRT1 mRNA expression mechanisms in cat peripheral blood leukocytes.
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Ração Animal/análise , Dieta/veterinária , Gorduras na Dieta/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Sirtuína 1/metabolismo , Sirtuína 3/metabolismo , Fenômenos Fisiológicos da Nutrição Animal , Animais , Gatos , Clonagem Molecular , DNA Complementar/metabolismo , Gorduras na Dieta/administração & dosagem , Fígado/metabolismo , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Sirtuína 1/genética , Sirtuína 3/genéticaRESUMO
Insulin is a critical hormone in the regulation of blood glucose levels and is produced exclusively by pancreatic islet beta-cells. Insulin deficiency due to reduced pancreatic islet beta-cell number underlies the progression of diabetes mellitus, prompting efforts to develop beta-cell replacement therapies. However, precise information on beta-cell replacement and differentiation in canines is limited. In this study, we established insulin-producing cells from bone marrow derived mesenchymal stem cells transiently expressing canine pancreatic and duodenal homeobox 1 (Pdx1), beta cell transactivator 2 (Beta2) and V-maf avian musculoaponeurotic fibrosarcoma oncogene homolog A (Mafa) using a gene transfer technique. Real-time PCR analysis revealed an increase in insulin mRNA expression of transfected cells. And ELISA revealed that insulin protein expressed was detected in cytoplasmic fraction. Insulin immunostaining analysis was performed and observed in cytoplasmic fraction. These results suggest that co-transfection of Pdx1, Beta2 and Mafa induce insulin production in canine BMSCs. Our findings provide a clue to basic research into the mechanisms underlying insulin production in the canines.
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Insulina/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Animais , Medula Óssea/metabolismo , Diferenciação Celular/fisiologia , Cães , Ensaio de Imunoadsorção Enzimática , Feminino , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica , Reação em Cadeia da Polimerase em Tempo Real , Transativadores/genética , Transativadores/metabolismoRESUMO
BACKGROUND: Bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs) are potential cellular sources of therapeutic stem cells. MSCs are a multipotent population of cells capable of differentiating into a number of mesodermal lineages. Treatment using MSCs appears to be a helpful approach for structural restoration in regenerative medicine. Correct identification of these cells is necessary, but there is inadequate information on the MSC profile of cell surface markers and mRNA expression in dogs. In this study, we performed molecular characterization of canine BM-MSCs and AT-MSCs using immunological and mRNA expression analysis. RESULTS: Samples were confirmed to be multipotent based on their osteogenic and adipogenic differentiation. And these cells were checked as stem cell, hematopoietic and embryonic stem cell (ESC) markers by flow cytometry. BM- and AT-MSCs showed high expression of CD29 and CD44, moderate expression of CD90, and were negative for CD34, CD45, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. SSEA-1 was expressed at very low levels in AT-MSCs. Quantitative real-time PCR (qRT-PCR) revealed expression of Oct3/4, Sox2, and Nanog in BM- and AT-MSCs. There was no significant difference in expression of Oct3/4 and Sox2 between BM-MSCs and AT-MSCs. However, Nanog expression was 2.5-fold higher in AT-MSCs than in BM-MSCs. Using immunocytochemical analysis, Oct3/4 and Sox2 proteins were observed in BM- and AT-MSCs. CONCLUSION: Our results provide fundamental information to enable for more reproducible and reliable quality control in the identification of canine BM-MSCs and AT-MSCs by protein and mRNA expression analysis.
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Tecido Adiposo/citologia , Células da Medula Óssea/citologia , Células-Tronco Mesenquimais/citologia , Tecido Adiposo/fisiologia , Animais , Biomarcadores , Células da Medula Óssea/fisiologia , Técnicas de Cultura de Células , Diferenciação Celular , Cães , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica/fisiologia , Imuno-Histoquímica , Células-Tronco Mesenquimais/fisiologiaRESUMO
BACKGROUND: Salmonella contamination of raw meat-based diets (RMBDs) for pets poses a major public health concern but has not been investigated in Japan. OBJECTIVE: To investigate Salmonella contamination in RMBDs for dogs marketed in Japan and the anti-microbial resistance profiles of the Salmonella isolates. METHODS: Sixty commercial RMBD samples were collected in the Okayama and Osaka Prefectures, Japan, between December 2016 and March 2017. The obtained Salmonella isolates were serotyped, their anti-microbial resistance patterns were determined, and the anti-microbial-resistant isolates were screened for the presence of resistance genes by polymerase chain reaction. RESULTS: Salmonella enterica subsp. enterica was detected in seven of the 60 RMBD samples. Among them, five isolates were identified as S. Infantis (n = 3), S. Typhimurium (n = 1) and S. Schwarzengrund (n = 1), while the serotypes of two isolates were unable to be identified. All isolates were susceptible to ampicillin, cefazolin, cefotaxime and gentamycin. Two isolates were resistant to more than one anti-microbial agent; one of the S. Infantis isolates was resistant to streptomycin, kanamycin, tetracycline and trimethoprim, while the S. Typhimurium isolate was resistant to nalidixic acid, ciprofloxacin and chloramphenicol. The S. Schwarzengrund isolate was resistant to tetracycline. Additionally, the S. Typhimurium isolate harboured the anti-microbial resistance gene gyrA with a mutation corresponding to Ser-83âPhe amino acid substitution. CONCLUSION: The study findings suggest that RMBDs for dogs marketed in Japan can be a potential source of Salmonella infection for dogs and humans including infections caused by quinolone-resistant isolates.
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Farmacorresistência Bacteriana Múltipla , Salmonella enterica , Ração Animal , Animais , Antibacterianos/farmacologia , Cães , Farmacorresistência Bacteriana Múltipla/genética , Japão , Carne , Salmonella , Salmonella enterica/genética , TetraciclinasRESUMO
BACKGROUND AND AIM: Mesenchymal stem cells (MSCs), which have multi-lineage differentiation potentials, are a promising source for regenerative medicine. However, the focus of study of MSCs is shifting from the characterization of the differentiation potential to their secretion potential for cell transplantation. Tissue regeneration and the attenuation of immune responses are thought to be affected by the secretion of multiple growth factors and cytokines by MSCs. However, the secretion potential of MSCs profiling remains incompletely characterized. In this study, we focused on the secretion ability related and protein mRNA expression of dog adipose tissue-derived MSCs (AT-MSC), bone marrow (BM)-derived MSCs, and BM-derived mononuclear cells (BM-MNC). MATERIALS AND METHODS: Real-time polymerase chain reaction analyses revealed mRNA expression of nine growth factors and seven interleukins in these types of cells and three growth factors protein expression were determined using Enzyme-linked immunosorbent assay. RESULTS: For the BM-MNC growth factors, the mRNA expression of transforming growth factor-ß (TGF-ß) was the highest. For the BM-derived MSC (BM-MSC) and AT-MSC growth factors, the mRNA expression of vascular endothelial growth factor (VEGF) was highest. BM-MSCs and AT-MSCs showed similar expression profiles. In contrast, BM-MNCs showed unique expression profiles for hepatocyte growth factor and epidermal growth factor. The three types of cells showed a similar expression of TGF-ß. CONCLUSION: We conclude that expression of cytokine proteins and mRNAs suggests involvement in tissue repair and protection.
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Immunotherapy improves both survival and remission rates after cancer surgery in humans, but its veterinary use has been limited. We determined the safety and feasibility of lymphokine-activated killer (LAK) cell transplantation in two aged cats that had undergone surgery for malignancy. Case 1 involved an 18-year-old male Japanese domestic cat. The cat exhibited appetite loss and poor physical activity after the surgical excision of oral squamous cell carcinoma followed by four sessions of radiotherapy, and the owner strongly requested immunotherapy for preventing further deterioration in the animal's quality of life (QOL). We subsequently administered LAK cells three times during a 2-month period. Case 2 involved a 20-year-old female Japanese domestic cat who had undergone mammectomy after a diagnosis of breast adenocarcinoma. The owner strongly requested immunotherapy for QOL maintenance. We administered LAK cells four times over a period of 5 months. Autologous peripheral blood mononuclear cells (PBMCs) fractionated using density gradient centrifugation were cultured in the media containing a high concentration of interleukin-2 and supplemented with 2.5% fetal calf serum. The derived LAK cells were centrifuged, suspended in 10 ml of saline containing 1% of the subject's own blood, and infused into the cephalic vein of the cats over 30 min. The composition ratios of CD3, CD4, CD8, and CD21 were evaluated by flow cytometry. Bacterial culture and endotoxin testing for a sample of LAK cells showed negative results in both the cases. The leukocyte and erythrocyte counts and the body temperature were assessed on days 7, 14, and 21 after the transfusion. No abnormal signs were observed in either case, which confirmed the safety of the procedure. QOL scores showed no significant changes after the treatment, and the body temperature remained steady throughout the treatment. The findings from these cases suggest that the transplantation of LAK cells derived from PBMCs may be safe and feasible for use in cats, regardless of their age.
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Doenças do Gato/cirurgia , Doenças do Gato/terapia , Imunoterapia/veterinária , Células Matadoras Ativadas por Linfocina/transplante , Adenocarcinoma/cirurgia , Animais , Neoplasias da Mama/cirurgia , Carcinoma de Células Escamosas/cirurgia , Gatos , Feminino , Leucócitos Mononucleares/fisiologia , Masculino , Neoplasias Bucais/cirurgiaRESUMO
Nuclear factor κB (NF-κB) is a key factor in the development of chronic inflammation and is deeply involved in age-related and metabolic diseases development. These diseases have become a serious problem in cats. Sirtuin 1 (SIRT1) is associated with aging and metabolism through maintaining inflammation via NF-κB. In addition, fibroblasts are considered an important factor in the development of chronic inflammation. Therefore, we aimed to examine the effect of cat SIRT1 (cSIRT1) on NF-κB in cat fibroblast cells. The up-regulation of NF-κB transcriptional activity and pro-inflammatory cytokine mRNA expression by p65 subunit of NF-κB and lipopolysaccharide was suppressed by cSIRT1 in cat fibroblast cells. Our findings show that cSIRT1 is involved in the suppression of inflammation in cat fibroblast cells.
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Gatos , Fibroblastos/efeitos dos fármacos , NF-kappa B/metabolismo , Sirtuína 1/farmacologia , Animais , Células Cultivadas , Citocinas , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação , NF-kappa B/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica , Regulação para CimaRESUMO
Nuclear factor kappa B (NF-κB) plays an important role in the immune system. The p65 subunit is an important part of NF-κB unit, and studies of dog and cat p65 subunits of NF-κB (dp65 and cp65) are important in understanding their immune function. In this study, we described the molecular characterization of dp65 and cp65. The dp65 and cp65 complementary DNA encoded 542 and 555 amino acids, respectively, showing a high sequence homology with the mammalian p65 subunit (>87.5%). Quantitative polymerase chain reaction revealed that the p65 messenger RNA is highly expressed in the dog stomach and cat heart and adipose tissue. Functional NF-κB promoter-luciferase reporter vectors revealed that our isolated dp65 and cp65 cDNA encodes a functionally active protein. Transiently expressed dp65 and cp65 up-regulated pro-inflammatory cytokine expression levels in dog and cat, respectively. These findings suggest that dp65 and cp65 play important roles in regulating immune function.
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Gatos/genética , Cães/genética , NF-kappa B/genética , Regulação para Cima , Animais , Gatos/metabolismo , Cães/metabolismo , Dados de Sequência Molecular , NF-kappa B/metabolismo , Especificidade de Órgãos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Análise de Sequência de DNA/veterináriaRESUMO
OBJECTIVES: In dogs with deep analgesia caused by acute spinal cord injury from thoracolumbar disk herniation, autologous bone marrow mononuclear cell transplant may improve recovery. The purpose of the present study was to evaluate autologous bone marrow mononuclear cell transplant in a dog that had paraplegia and deep analgesia caused by chronic spinal cord injury. MATERIALS AND METHODS: Autologous bone marrow mononuclear cell transplant was performed in a dog having paraplegia and analgesia for 3 years that was caused by a chronic spinal cord injury secondary to Hansen type I thoracolumbar disk herniation. Functional recovery was evaluated with electrophysiologic studies and the Texas Spinal Cord Injury Scale. RESULTS: Somatosensory evoked potentials were absent before transplant but were detected after transplant. Functional improvement was noted (Texas Spinal Cord Injury Scale: before transplant, 0; after transplant, 6). No adverse events were observed. CONCLUSIONS: Autologous bone marrow mononuclear cell transplant into the subarachnoid space may be a safe and beneficial treatment for chronic spinal cord injury in dogs.
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Transplante de Medula Óssea/veterinária , Descompressão Cirúrgica/veterinária , Doenças do Cão/cirurgia , Deslocamento do Disco Intervertebral/veterinária , Traumatismos da Medula Espinal/veterinária , Animais , Biópsia/veterinária , Doença Crônica , Discotomia/veterinária , Doenças do Cão/diagnóstico , Cães , Potenciais Somatossensoriais Evocados , Deslocamento do Disco Intervertebral/diagnóstico , Deslocamento do Disco Intervertebral/fisiopatologia , Deslocamento do Disco Intervertebral/cirurgia , Imageamento por Ressonância Magnética/veterinária , Atividade Motora , Exame Neurológico/veterinária , Recuperação de Função Fisiológica , Traumatismos da Medula Espinal/diagnóstico , Traumatismos da Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/cirurgia , Tomografia Computadorizada por Raios X/veterinária , Transplante Autólogo/veterinária , Resultado do TratamentoRESUMO
Pancreatic and duodenal homeobox 1 (Pdx-1) is a critical insulin transcription factor expressed by pancreatic ß-cells, and is crucial in the early stage of pancreas development. Unfortunately, nothing concerning Pdx-1 in canine has been elucidated yet. In this study, full length canine Pdx-1 cDNA was cloned and it was 1498 bp in length, consisting of a 99 bp 5'-untranslated region (UTR), a 849 bp coding region, and a 550 bp 3'-UTR region. A deduced 282 amino acid sequence of canine PDX-1 displayed high overall sequence identity with human, bovine, and mouse PDX-1. qRT-PCR analysis revealed that a high level of Pdx1 mRNA expression is exists in the duodenum and pancreas of canines. In addition, functional canine insulin promoter-luciferase reporter constructs with various canine insulin promoter region fragments revealed that our Pdx-1 isolated cDNA sequence encodes for a functionally active PDX-1 protein. Significant promoter activity was observed within the -583 bp 5'-upstream region of canine insulin gene with Chinese hamster ovary cells. In addition, Pdx-1 appears to have a synergistic effect with beta cell transactivator 2 (BETA2) and V-maf avian musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), which also have important roles in the activation of the insulin gene promoter. Our results confirm that similar to humans, Pdx1 plays an important role in expression of insulin gene in canines.
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DNA Complementar/genética , Proteínas de Homeodomínio/metabolismo , RNA Mensageiro/metabolismo , Transativadores/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Cães , Regulação da Expressão Gênica/fisiologia , Proteínas de Homeodomínio/genética , Insulina/genética , Insulina/metabolismo , Fatores de Transcrição Maf Maior/genética , Fatores de Transcrição Maf Maior/metabolismo , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Transativadores/genéticaRESUMO
The cyclin-dependent kinase 5 regulatory subunit-associated protein 1-like 1 (CDKAL1) gene encodes methylthiotransferase, and the gene contains risk variants for type 2 diabetes in humans. In this study, we performed complementary DNA cloning for Cdkal1 in the cat and dog and characterized the tissue expression profiles of its messenger RNA. Cat and dog Cdkal1 complementary DNA encoded 576 and 578 amino acids, showing very high sequence homology to mammalian CDKAL1 (>88.4%). Real-time polymerase chain reaction analyses revealed that Cdkal1 messenger RNA is highly expressed in smooth muscle and that tissue distribution of Cdkal1 is similar in cats and dogs. Genotyping analysis of single-nucleotide polymorphism for cat Cdkal1 revealed that obese cats had different tendencies from normal cats. These findings suggest that the cat and dog Cdkal1 gene is highly conserved among mammals and that cat Cdkal1 may be a candidate marker for genetic diagnosis of obesity.
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The effect of overweight status on the expression of SREBP-1c and downstream lipogenic genes, such as ATP citrate lyase (ACL) and fatty acid synthase (FAS), in abdominal adipose and liver tissues was determined in cats using a diet-induced weight gain model. ACL and SREBP-1c mRNA expression was significantly reduced (â¼65% and 20%, respectively) in liver tissue, whereas FAS and SREBP-1c expression was significantly increased (â¼80% and 45%, respectively) in abdominal omental adipose tissue of overweight animals as compared to healthy animals. Additionally, ACL, FAS, and SREBP-1c expression was significantly reduced by â¼50%, 75%, and 70%, respectively, in abdominal subcutaneous adipose tissue of overweight animals. Omental adipose tissue appeared to foster, whereas subcutaneous adipose and liver tissues appeared to defer lipid storage based on differences in SREBP-1c mRNA expression. Overall, reduced lipogenic gene mRNA expression patterns support the hypothesis that SREBP-1c expression is reduced in overweight and possibly obese cats, reflecting down-regulation of the lipogenic pathway to prevent further fat accumulation and weight gain.