RESUMEN
The mammalian target of rapamycin (mTOR)-composed of multiple complexes, including mTOR complex 1/2 (mTORC1/2)-is a serine-threonine kinase that regulates embryonic development. The transcription factor, hypoxia-inducible factor-1α (HIF-1α), is also involved in embryonic development. As the relationship between mTOR and HIF-1α during embryonic development remains unclear, we investigated the relationship between the two using ex vivo submandibular salivary gland organ cultures. When the expression of HIF-1α increased under hypoxic conditions (1% O2), the expression of mTOR signaling pathway-related proteins decreased. Conversely, when the expression of HIF-1α decreased, the expression of mTOR signaling pathway-related proteins increased. These results indicate a strong relationship between HIF-1α and the mTOR signaling pathway. For the first time, we clarified that HIF-1α negatively regulates the mTOR signaling pathway and suppresses salivary gland development under 1% O2 using small molecules. Our research provides new insights into the relationship between HIF-1α and the mTOR signaling pathway in embryonic organ development.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia , Serina-Treonina Quinasas TOR , Femenino , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Embarazo , Glándulas Salivales/metabolismo , Transducción de Señal/fisiología , Sirolimus , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
BACKGROUND: Lobular dissecting hepatitis (LDH) is a rare form of canine liver cirrhosis that may be accompanied by portal hypertension in American Cocker Spaniels. In human patients with liver cirrhosis, portal vein thrombosis (PVT) is a common complication. However, PVT has not been reported in dogs with LDH. Herein, we describe the long-term follow-up of PVT in an American Cocker Spaniel with LDH. CASE PRESENTATION: An 8-year-old neutered male American Cocker Spaniel presented with a 1-month history of severe abdominal effusion. The dog was histopathologically diagnosed with LDH and treated with low-dose prednisolone on day 14. On day 115, computed tomography angiography (CTA) confirmed the presence of a thrombus in the portal vein. Therefore, the dog was subcutaneously administered with the anticoagulant dalteparin, and low-dose prednisolone was continued. As a follow-up for PVT, CTA examinations were performed on days 207, 515, 886, and 1168, and the dog's antithrombin and D-dimer levels were measured. Following anticoagulant therapy, the dog was confirmed to have gradually increased antithrombin activity and decreased D-dimer concentrations. In addition, although the thrombus was confirmed to be in the same area of the portal vein system by CTA, atrophy and increased CT values due to organization were observed during the follow-up period. The dog's condition remained stable without clinical signs until day 1112 when it developed hepatic encephalopathy. The dog died on day 1208. On postmortem examination, histopathologically, the liver showed marked bile duct hyperplasia and fibrosis with chronic thrombus in the portal vein. CONCLUSIONS: This case demonstrated that low-dose glucocorticoid combined with dalteparin allowed long-term follow-up of PVT in an American Cocker Spaniel with LDH.
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Dalteparina/uso terapéutico , Hepatitis/complicaciones , Vena Porta , Trombosis de la Vena/veterinaria , Animales , Anticoagulantes/uso terapéutico , Angiografía por Tomografía Computarizada , Enfermedades de los Perros/tratamiento farmacológico , Perros , Estudios de Seguimiento , Cirrosis Hepática/veterinaria , Masculino , Prednisolona/uso terapéutico , Trombosis de la Vena/complicaciones , Trombosis de la Vena/diagnóstico por imagenRESUMEN
BACKGROUND: Portal vein thrombosis (PVT) is a rare presentation in dogs with protein-losing enteropathy (PLE). Rivaroxaban, an oral, selective, direct factor Xa inhibitor, has not been reported to be administrated for canine PVT and the effect is unclear in dogs with PLE. CASE PRESENTATION: An 11-year-old Yorkshire Terrier presented with moderate ascites. The dog had severe hypoalbuminemia (1.2 g/dL), and a portal vein thrombus was confirmed on computed tomographic angiography (CTA). On endoscopic examination, it became apparent that the hypoalbuminemia was caused by PLE, which was consequent of lymphatic dilation and lymphoplasmacytic enteritis. Therefore, the dog was initially treated with oral administrations of spironolactone and clopidogrel, with dietary fat restriction. However, a follow-up CTA showed no changes in the ascites, thrombus, and portal vein to aorta (PV/Ao) ratio. Therefore, the dog was additionally prescribed rivaroxaban and low-dose prednisolone for the portal vein thrombus and hypoalbuminemia due to lymphoplasmacytic enteritis, respectively. Following the treatment, the PV/Ao ratio decreased because of a decrease in the thrombus and the ascites disappeared completely with an elevation of albumin concentration (1.9 g/dL). CONCLUSIONS: This case report demonstrated that oral administration of rivaroxaban combined with low-dose glucocorticoid was effective management for PVT in a dog with PLE.
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Enfermedades de los Perros/tratamiento farmacológico , Enteropatías Perdedoras de Proteínas/veterinaria , Rivaroxabán/uso terapéutico , Trombosis de la Vena/veterinaria , Administración Oral , Animales , Angiografía por Tomografía Computarizada/veterinaria , Perros , Inhibidores del Factor Xa/administración & dosificación , Inhibidores del Factor Xa/uso terapéutico , Femenino , Glucocorticoides/administración & dosificación , Glucocorticoides/uso terapéutico , Hipoalbuminemia/tratamiento farmacológico , Hipoalbuminemia/veterinaria , Vena Porta/diagnóstico por imagen , Vena Porta/patología , Prednisolona/administración & dosificación , Prednisolona/uso terapéutico , Rivaroxabán/administración & dosificación , Trombosis de la Vena/diagnóstico por imagen , Trombosis de la Vena/tratamiento farmacológicoRESUMEN
It has become anticipated that regenerative medicine will extend into the field of veterinary medicine as new treatments for various disorders. Although the use of allogeneic stem cells for tissue regeneration is more attractive than that of autologous cells in emergencies, the therapeutic potential of allogeneic transplantation is often limited by allo-immune responses inducing graft rejection. Therefore, a methodology for quantifying and monitoring alloreactive T cells is necessary for evaluating allo-immune responses. The mixed lymphocyte reaction (MLR) is widely used to evaluate T cell alloreactivity. In human, flow cytometric MLR with carboxyfluorescein diacetate succinimidyl ester has been established and used as a more useful assay than conventional MLR with radioisotope labeling. However, the available information about alloreactivity based on the differences of dog major histocompatibility complex (MHC) (dog leukocyte antigen, DLA) is quite limited in dog. In this paper, we describe our established flow cytometric MLR method that can quantify the T cell alloreactivity while distinguishing cell phenotypes in dog, and T cell alloreactivity among DLA-type matched pairs was significantly lower than DLA-mismatched pairs, suggesting that our developed flow cytometric MLR method is useful for quantifying T cell alloreactivity. In addition, we demonstrated the advantage of DLA homozygous cells as a donor (stimulator) for allogeneic transplantation. We also elucidated that the frequency of alloreactive T cell precursors was almost the same as that of mouse and human (1-10%). To our knowledge, this is the first report to focus on the degree of allo-immune responses in dog based on the differences of DLA polymorphisms.
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Citometría de Flujo/métodos , Histocompatibilidad , Prueba de Cultivo Mixto de Linfocitos/métodos , Complejo Mayor de Histocompatibilidad/genética , Complejo Mayor de Histocompatibilidad/inmunología , Polimorfismo Genético , Linfocitos T/inmunología , Animales , Perros , Haplotipos , Activación de Linfocitos/inmunologíaRESUMEN
The function of ARHGEF10, a known guanine nucleotide exchange factor (GEF) for RhoA with proposed roles in various diseases, is poorly understood. To understand the precise function of this protein, we raised a monoclonal antibody against ARHGEF10 and determined its localization in HeLa cells. ARHGEF10 was found to localize to vesicles containing Rab6 (of which there are three isoforms, Rab6a, Rab6b and Rab6c), Rab8 (of which there are two isoforms, Rab8a and Rab8b), and/or the secretion marker neuropeptide Y (NPY)-Venus in a Rab6-dependent manner. These vesicles were known to originate from the Golgi and contain secreted or membrane proteins. Ectopic expression of an N-terminal-truncated ARHGEF10 mutant led to the generation of large vesicle-like structures containing both Rab6 and Rab8. Additionally, small interfering (si)RNA-mediated knockdown of ARHGEF10 impaired the localization of Rab8 to these exocytotic vesicles. Furthermore, the invasiveness of MDA-MB231 cells was markedly decreased by knockdown of ARHGEF10, as well as of Rab8. From these results, we propose that ARHGEF10 acts in exocytosis and tumor invasion in a Rab8-dependent manner.
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Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Vesículas Secretoras/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Citoesqueleto de Actina/metabolismo , Anticuerpos Monoclonales/metabolismo , Especificidad de Anticuerpos/inmunología , Línea Celular Tumoral , Exocitosis , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Mutantes/metabolismo , Invasividad Neoplásica , Neuropéptido Y/metabolismo , Polimerizacion , Dominios Proteicos , Transporte de ProteínasRESUMEN
Hypofunction of the salivary gland causes several life-disrupting side effects such as dental caries, oral candidiasis, loss of taste, and swallowing disorders. No satisfactory therapy has been established to treat salivary hypofunction. Pilocarpine represents a potential treatment for dry mouth due to Sjögren's syndrome (SS). Although subjective improvement was consistently observed with pilocarpine therapy, the mechanism was unclear. In this study, we investigated the mechanism of recovery in salivation following treatment with pilocarpine. We first examined the effectiveness of pilocarpine in SS patients as quantified by the Saxon test and the visual analogue scale average. We found that salivation ability and subjective symptoms improved by continuous administration of pilocarpine. These results demonstrated that long-term medication for dry mouth patients was more effective. However, as the mechanism remained unclear, molecular biological mechanisms were analyzed based on the effects of continuous administration of pilocarpine using model mice. In the molecular biological analysis, continuous administration of pilocarpine was effective in both ICR and SS model mice. Gene and protein expression of muscarinic acetylcholine receptor 3 (M3R) increased in salivary glands following continuous administration of pilocarpine compared with single administration. Therefore, continuous administration of pilocarpine effectively induced M3R expression, thereby activating salivation.
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Agonistas Muscarínicos/uso terapéutico , Pilocarpina/uso terapéutico , Receptor Muscarínico M3/genética , Glándulas Salivales/efectos de los fármacos , Salivación/efectos de los fármacos , Síndrome de Sjögren/tratamiento farmacológico , Regulación hacia Arriba/efectos de los fármacos , Anciano , Animales , Femenino , Humanos , Masculino , Ratones Endogámicos ICR , Persona de Mediana Edad , Glándulas Salivales/metabolismo , Glándulas Salivales/fisiopatología , Síndrome de Sjögren/genética , Síndrome de Sjögren/fisiopatologíaRESUMEN
Development of the salivary gland is characterized by extensive branching morphogenesis and lumen formation, the latter of which is closely associated with differentiation into acinar and ductal cells. Although various molecules, including signaling and cell adhesion molecules, have been implicated in salivary gland development, transcription factors (TFs) regulating the expression of those molecules and morphological development of the gland are largely unknown. Here we show that knockdown of the epithelial TF, Grainyhead-like 2 (Grhl2), with siRNA in developing mouse submandibular salivary gland (SMG) cultured ex vivo resulted in retardation of epithelial development. This retardation was concomitant with suppression of gene expression for the cell adhesion molecules, such as E-cadherin and the extracellular protease inhibitor SPINT1, and with the disorganized deposition of the basal lamina protein laminin. ChIP-PCR demonstrated the binding of Grhl2 protein to the Spint1 gene in the SMG. Notably, addition of recombinant SPINT1 protein in cultured SMG overcame the suppressive effects of Grhl2 siRNA on epithelial development and laminin deposition. These findings show that Grhl2 regulation of SPINT1 expression controls salivary gland development.
Asunto(s)
Glicoproteínas de Membrana/metabolismo , Glándulas Salivales/metabolismo , Factores de Transcripción/metabolismo , Animales , Cadherinas/metabolismo , Diferenciación Celular , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica , Ratones , Organogénesis , Proteínas Inhibidoras de Proteinasas Secretoras , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/química , Glándulas Salivales/crecimiento & desarrollo , Transducción de Señal , Glándula Submandibular/embriologíaRESUMEN
Multi-parameter phenotypic profiling of small molecules is a powerful approach to their toxicity assessment and identifying potential mechanisms of actions. The present study demonstrates the application of image-based multi-parameter phenotypic profiling in MCF-7 cells to assess the overall toxicity and estrogenic activity of whole environmental water. Phenotypic profiling of 30 reference compounds and their complex mixtures was evaluated to investigate the cellular morphological outcomes to targeted biological pathways. Overall toxicity and estrogenic activity of environmental water samples were then evaluated by phenotypic analysis comparing with conventional bioassays and chemical analysis by multivariate analysis. The phenotypic analysis for reference compounds demonstrated that size and structure of cells related to biological processes like cell growth, death, and communication. The phenotypic alteration and nuclei intensity were selected as potential biomarkers to evaluate overall toxicity and estrogenic activities, respectively. The phenotypic profiles were associated with the chemical structure profiles in environmental water samples. Since the phenotypic parameters revealed multiple toxicity endpoints, it could provide more information that is relevant to assessing the toxicity of environmental water samples in compare with conventional bioassays. In conclusion, the image-based multi-parameters phenotypic analysis with MCF-7 cells provides a rapid and information-rich tool for toxicity evaluation and identification in whole water samples.
Asunto(s)
Contaminantes Químicos del Agua , Agua , Bioensayo , Monitoreo del Ambiente , Estrona , Humanos , Células MCF-7RESUMEN
The purpose of this study was to determine the utility of triple-phase helical computed tomography (CT) for differentiating canine hepatic masses. Seventy dogs with hepatic masses underwent triple-phase CT followed by surgical removal of the hepatic masses. Triple-phase helical CT scans for each dog included precontrast, arterial phase, portal venous phase, and delayed phase studies. The removed hepatic masses were histopathologically classified as hepatocellular carcinoma (n = 47), nodular hyperplasia (n = 14), and hepatic metastatic tumors (n = 9) in dogs. Of the 47 hepatocellular carcinomas, the most common CT findings included a heterogeneous pattern with hyper-, iso-, and hypoenhancement in both the arterial and portal venous phases (40/47, 85.1%). Of the 14 nodular hyperplasias, the most common CT findings were a homogeneous pattern with hyper- and isoenhancement in both the portal venous and delayed phases (13/14, 92.9%). Of nine hepatic metastatic tumors, the most common CT findings included a homogeneous hypoenhancement pattern in both the arterial and portal venous phases (8/9, 88.9%). In addition, 5 (55.6%) showed homogeneous hypoenhancement patterns in the delayed phase. Findings from our study indicated that triple-phase CT is a useful tool for preoperative differentiation of hepatocellular carcinoma, nodular hyperplasia, and hepatic metastatic tumors in dogs.
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Enfermedades de los Perros/diagnóstico por imagen , Hiperplasia/veterinaria , Neoplasias Hepáticas/veterinaria , Hígado/diagnóstico por imagen , Hígado/patología , Animales , Carcinoma Hepatocelular/diagnóstico por imagen , Carcinoma Hepatocelular/veterinaria , Perros , Femenino , Hiperplasia/diagnóstico por imagen , Neoplasias Hepáticas/diagnóstico por imagen , Masculino , Tomografía Computarizada Espiral/veterinariaRESUMEN
The salivary glands produce saliva and are important in maintaining oral health. Saliva keeps the mouth moist, cleanses the oral cavity, aids digestion, and has antibacterial properties. Saliva also helps in swallowing and speech. Investigating the development of the salivary glands is thus relevant in the context of both health and disease. Various cell culture methods have been used to study salivary gland development, including culturing cells in two dimensions (2D). Under physiological conditions, cells constantly interact with other cells and the extracellular matrix, which controls complex biological functions such as cell migration and apoptosis, and can modulate gene expression. Since many of these functions are not accurately represented or reproduced in 2D culture, the results of in vitro experiments using such culture methods are often not reflected in vivo. The use of 3D cultures, such as organ cultures, has helped address this issue and has emerged as a model that better reflects the in vivo physiological environment. Here, we describe a protocol for establishing submandibular salivary gland organ culture that is more concise and simpler than previous methods and includes the separation and dissection of the salivary glands. We also describe the use of environmental stress (hypoxic stimulation) and inhibitors (U0126, LY294002, and rapamycin) to elucidate signaling pathways involved in salivary gland development. This protocol can provide researchers with a simpler and more robust method of salivary gland organ culture, enabling analysis of organ-based signaling pathways to advance developmental biology research. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Submandibular salivary gland organ culture Basic Protocol 2: Analysis of salivary gland development in the presence of hypoxia and signaling pathway inhibitors Basic Protocol 3: Western blotting using submandibular salivary gland organ culture.
Asunto(s)
Glándulas Salivales , Glándula Submandibular , Animales , Ratones , Técnicas de Cultivo de Órganos , Saliva/metabolismo , Transducción de Señal , Glándula Submandibular/metabolismoRESUMEN
The transcription factor, hypoxia-inducible factor-1α (HIF-1α), has previously been shown to upregulate the expression of hypoxia-related genes, including erythropoietin (EPO). However, the role of hypoxia-inducible factor-1α in morphogenesis during salivary gland development is unclear. We investigated the function of HIF-1α in submandibular gland (SMG) organ cultures obtained from embryonic day 13.5 embryos from ICR female mice. Expression of HIF-1α, glucose transporter 1, and vascular endothelial growth factor was induced under hypoxia (5% O2 ). We further showed that BAY 87-2243-mediated inhibition of HIF-1α suppressed salivary gland development. Under severe hypoxia (1% O2 ), HIF-1α did not promote salivary gland development; this was due to suppression of cell proliferation and inhibition of the cell cycle and not because of autophagy and apoptosis. Additionally, using the inhibitor U0126, we verified that the ERK1/2 pathway is upstream of HIF-1α. Overall, we found that the HIF-1α signaling pathway plays a critical role in salivary gland development in ex vivo SMG organ cultures.
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Subunidad alfa del Factor 1 Inducible por Hipoxia , Factor A de Crecimiento Endotelial Vascular , Animales , Femenino , Ratones , Ratones Endogámicos ICR , Técnicas de Cultivo de Órganos , Glándulas Salivales/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: The renin-angiotensin-aldosterone system (RAAS) is activated in humans with portal hypertension (PH) associated with liver disease. However, involvement of RAAS in dogs with intrahepatic PH is not clear. OBJECTIVE: To measure plasma renin activity (PRA) and plasma aldosterone concentration (PAC) in dogs with PH (chronic hepatitis [CH] and primary hypoplasia of the portal vein [PHPV]), dogs with extrahepatic congenital portosystemic shunt (EH-CPSS), and healthy dogs and to determine whether the RAAS is activated in dogs with PH. ANIMALS: Twenty-seven dogs with acquired portosystemic collaterals (APSCs; 15 dogs with CH, 12 dogs with PHPV), 9 dogs with EH-CPSS, and 10 healthy dogs. METHODS: Retrospective study. Plasma renin activity and PAC were measured by radioimmunoassay. RESULTS: Plasma renin activity was significantly higher in the CH group (median, 4.4 ng/mL/h) than in the EH-CPSS (median, 1.0 ng/mL/h; P < .01) and the healthy (median, 1.1 ng/mL/h; P < .01) groups. No significant differences were found between the PHPV group (median, 2.2 ng/mL/h) and other groups. Plasma aldosterone concentration was significantly higher in the CH (median, 111.0 pg/mL) and PHPV (median, 89.5 pg/mL) groups than in the EH-CPSS (median, 1.0 pg/mL; P < .001, P < .01, respectively) and healthy (median, 14.5 pg/mL; P < .001, P < .05, respectively) groups. CONCLUSIONS AND CLINICAL IMPORTANCE: Activation of the RAAS contributes to the pathophysiology of intrahepatic PH in dogs, suggesting that spironolactone may not only be effective for the treatment of ascites but also for the suppression of intrahepatic PH.
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Aldosterona/sangre , Circulación Colateral , Hipertensión Portal/veterinaria , Sistema Porta/patología , Renina/sangre , Animales , Estudios de Casos y Controles , Enfermedades de los Perros , Perros , Femenino , Hepatitis Crónica/veterinaria , Hipertensión Portal/sangre , Hipertensión Portal/metabolismo , MasculinoRESUMEN
BACKGROUND: Most animals and organs have regenerative capabilities. Whether regeneration is a developmental process or a distinct phenomenon that is independent of development is debatable. METHOD: We examined the differences between developing and regenerating salivary glands using duct-ligation models. We performed morphological analyses comparing submandibular gland regeneration and development. To reveal the proliferation processes that occur during salivary gland regeneration and development, we counted the number of Ki67-positive cells over time. In addition, we examined the expression of the following markers: aquaporin 5, smooth muscle actin, cytokeratin 7, and tubulin beta 3. RESULT: The proliferation patterns seen during regeneration differed from those observed during development. Different salivary gland marker expression patterns were seen during development and regeneration. CONCLUSION: This study showed that regenerating salivary glands do not follow the same growth process as developing salivary glands.
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Regeneración , Glándula Submandibular/embriología , Glándula Submandibular/fisiología , Actinas/metabolismo , Animales , Acuaporina 5/metabolismo , Biomarcadores , Cadherinas/metabolismo , Femenino , Queratina-7/metabolismo , Antígeno Ki-67/metabolismo , Ligadura , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Tubulina (Proteína)/metabolismoRESUMEN
The transcription factor p63, a component of the p53 family, has important functions in development, homeostasis, and regeneration of epithelial tissues. However, the role of p63 in the regeneration of exocrine glands, including the salivary glands (SGs), has not been fully investigated. We investigated p63 expression in SG regeneration induced by duct ligation and irradiation. The expression of ΔNp63, a p63 isoform, increased and was colocalized with keratin 5 positive cells were myoepithelial cells. Furthermore, ΔNp63 expression was regulated by FGF7 stimulation via p38 MAPK phosphorylation and affected SG morphogenesis. These results suggest that ΔNp63 is essential for SG regeneration and may be a new target for regenerative treatment.
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Regeneración/efectos de la radiación , Glándulas Salivales/fisiología , Glándulas Salivales/efectos de la radiación , Transactivadores/genética , Regulación hacia Arriba/genética , Animales , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/efectos de la radiación , Femenino , Feto/metabolismo , Factor 7 de Crecimiento de Fibroblastos/metabolismo , Queratina-5/metabolismo , Ligadura , Ratones Endogámicos ICR , Fosforilación/efectos de la radiación , Glándulas Salivales/embriología , Regulación hacia Arriba/efectos de la radiación , Rayos X , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Transplantation medicine is used for the treatment of severe canine diseases, and the dog leukocyte antigen (DLA) is considered to be important in graft rejection. However, the utility of direct sequencing of both DLA classes I and II has not been assessed thoroughly. Eight healthy beagles with identified DLA genes were divided into two sets of four dogs, each including one donor and three recipients for skin transplantation. The following recipients were selected: one dog with a complete match, one with a haploidentical match, and one with a complete mismatch of the DLA gene with the donor. Full-thickness skin segments were obtained from each donor and transplanted to the recipients. A mixed lymphocyte reaction (MLR) assay was performed and analyzed by flow cytometry. Skin grafts of DLA haploidentical and mismatched pairs were grossly rejected within 14 days, whereas in fully matched DLA pairs, survival was as long as 21 days. Histopathological evaluation also showed moderate to severe lymphocytic infiltration and necrosis in DLA mismatched pairs. As seen in the MLR assay, the stimulation index of DLA mismatched pairs was significantly higher than that of fully matched DLA pairs in both sets (P<0.001). The allogeneic transplantation results suggested that it is possible to prolong transplant engraftment by completely matching the DLA genotype between the donor and recipient. Additionally, the MLR assay may be used as a simplified in vitro method to select donors.
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Perros/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Trasplante de Piel/veterinaria , Tolerancia al Trasplante/inmunología , Animales , Perros/genética , Femenino , Prueba de Histocompatibilidad/veterinaria , Masculino , Trasplante Homólogo/veterinariaRESUMEN
Computed tomographic (CT) angiography, the gold standard for diagnosing portal vein thrombosis (PVT) in humans, is poorly documented in dogs. Therefore, we retrospectively reviewed dogs with PVT diagnosed by CT angiography. Medical records of 13 client-owned dogs diagnosed with PVT by CT angiography were reviewed. All dogs had chronic PVT, and the most frequent clinical sign was vomiting (5/13), with pancreatitis the most frequent concurrent disease (6/13). All dogs tested for plasma D-dimer concentration (12/12) revealed elevated levels. On CT angiography, a thrombus was detected as a non-contrast enhancement structure in the portal vessel of 13 dogs. There was no evidence of complete obstruction of the portal vein in any of the dogs. The median luminal filling of the portal vein was 60.4%. The thrombus extension was variable among dogs, with a median of 34.9 mm. CT angiography identified the thrombus in the main portal vein of 12/13 dogs and multiple thrombus formation other than the main portal vein in 9/13 dogs. CT angiography provided specific information such as detecting the presence, location, and number of PVT in dogs. Therefore, CT angiography might be useful for the diagnosis and follow-up evaluation of PVT in dogs.
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Enfermedades de los Perros , Trombosis , Angiografía/veterinaria , Animales , Angiografía por Tomografía Computarizada/veterinaria , Enfermedades de los Perros/diagnóstico por imagen , Perros , Vena Porta/diagnóstico por imagen , Estudios Retrospectivos , Trombosis/veterinaria , Tomografía Computarizada por Rayos X/veterinariaRESUMEN
Blood hyaluronic acid (HA) concentration was measured in dogs with various liver diseases to determine its relationship with histological fibrosis of the liver. The blood HA concentration significantly increased in dogs with chronic liver diseases compared with extrahepatic diseases and control. Furthermore, the median blood HA concentration in dogs with liver cirrhosis (500 microg/l; range, 151-1970 microg/l) was significantly higher than dogs with non-cirrhotic liver diseases (153 microg/l; range, 15-477 microg/l). In histochemical analysis, HA was distributed primarily in the fibrotic area in dogs with chronic liver diseases. As a conclusion, the blood HA concentration was significantly increased in dogs with chronic liver diseases, especially those with cirrhosis. Measurement of the blood HA levels of dogs with suspected liver disease can be a useful diagnostic aid for canine cirrhosis.
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Enfermedades de los Perros/sangre , Ácido Hialurónico/sangre , Cirrosis Hepática/veterinaria , Animales , Biomarcadores/sangre , Perros , Femenino , Cirrosis Hepática/sangre , Cirrosis Hepática/patología , MasculinoRESUMEN
Development of the salivary gland is characterized by extensive branching morphogenesis. Although various molecules have been implicated in salivary gland development, the role of the mammalian target of rapamycin (mTOR) signalling pathway, including both mTOR complexes 1 and 2 (mTORC1 and 2), in salivary gland development is unknown. Here, we examined protein expression levels related to the mTOR signalling pathway using an ex vivo submandibular salivary gland (SMG) organ culture. We showed that branching buds in the salivary glands were substantially decreased and phosphorylation of mTORC1 signalling pathway related proteins (mTOR, p70 ribosomal protein S6 kinase 1 and eukaryotic initiation factor 4E-binding protein 1) was inhibited by rapamycin (an mTOR inhibitor). In addition, AKT, which is an upstream protein kinase of mTORC1 and is downstream of mTORC2, is inhibited by LY294002 (a phosphatidylinositol 3-kinase inhibitor), but not by rapamycin. Moreover, rapamycin-treated ICR neonatal mice exhibited a reduction in both body weight and salivary glands compared with vehicle-treated neonatal mice. The present data indicate that the mTOR signalling pathway, including both mTORC1 and mTORC2, plays a critical role in salivary gland development both in ex vivo SMG organ culture and ICR neonatal mice in vivo.
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Desarrollo Embrionario/genética , Glándulas Salivales/metabolismo , Glándula Submandibular/metabolismo , Serina-Treonina Quinasas TOR/genética , Animales , Animales Recién Nacidos , Cromonas/farmacología , Embrión de Mamíferos , Diana Mecanicista del Complejo 1 de la Rapamicina/efectos de los fármacos , Diana Mecanicista del Complejo 2 de la Rapamicina/efectos de los fármacos , Ratones , Morfogénesis/genética , Morfolinas/farmacología , Técnicas de Cultivo de Órganos , Fosforilación/efectos de los fármacos , Glándulas Salivales/crecimiento & desarrollo , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Glándula Submandibular/crecimiento & desarrolloRESUMEN
Matrix metalloproteinases (MMPs) play a pivotal role in tissue remodeling by degrading the extracellular matrix (ECM) components. This mechanism is implicated in a variety of physiological and pathological cellular processes including wound healing. One of the key proteins involved in this process is the proinflammatory cytokine interleukin-1ß (IL-1ß, which induces the expression of MMP-3 mRNA and the secretion of MMP-3 protein by dermal fibroblasts. In this study, we first investigated the contribution of activating transcription factor 2 (ATF-2) to IL-1ß-induced MMP-3 expression in dermal fibroblasts. Our results showed that in cells transfected with ATF-2 siRNA or treated with the ATF-2 inhibitor SBI-0087702, IL-1ß-induced MMP-3 mRNA expression was reduced. We also demonstrated that IL-1ß stimulates the phosphorylation of ATF-2. These observations suggest that ATF-2 plays an important role in IL-1ß-induced MMP-3 expression. Next, we investigated the role of MAPK signaling in ATF-2 activation. In cells treated with the extracellular signal-regulated kinase (ERK) inhibitor FR180240, as well as in cells transfected with ERK1 and ERK2 siRNAs, IL-1ß-induced MMP-3 mRNA expression was reduced. In addition, we showed that IL-1ß induced the phosphorylation of ERK1/2. These observations suggest that ERK1 and ERK2 are involved in IL-1ß-induced MMP-3 expression. However, ERK1 and ERK2 do seem to play different roles. While the ERK inhibitor FR180204 inhibited IL-1ß-induced ATF-2 phosphorylation, only in cells transfected with ERK1 siRNA, but not ERK2 siRNA, IL-1ß-induced ATF-2 phosphorylation was reduced. These findings suggest that the ERK1/ATF-2 signaling axis contributes to IL-1ß-induced MMP-3 expression in dermal fibroblasts.
Asunto(s)
Factor de Transcripción Activador 2/metabolismo , Fibroblastos/efectos de los fármacos , Interleucina-1beta/farmacología , Metaloproteinasa 3 de la Matriz/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Factor de Transcripción Activador 2/genética , Animales , Células Cultivadas , Dermis/citología , Perros , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Masculino , Metaloproteinasa 3 de la Matriz/genética , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
BACKGROUND: Chronic hepatitis (CH) in dogs is common and has the tendency to progress to liver cirrhosis (LC). Circulating microRNAs might have the potential as markers for disease progression. OBJECTIVES: To investigate whether concentration of specific microRNAs in serum correlate with the stage and grade of CH in Labrador Retrievers. ANIMALS: Twenty-two Labrador Retrievers with histological CH (n = 8), LC (n = 7), and normal liver (NL, n = 7). METHODS: In this retrospective study, serum concentrations of miR-122, miR-29a, miR-133a, miR-181b, and miR-17-5p were measured by quantitative real-time PCR and evaluated using univariate linear regression in dogs. A multivariate model was fit including the grade of hepatitis and the stage of fibrosis. RESULTS: Of the 5 microRNAs, only circulating miR-122 and miR-29a were significantly associated with the grade of hepatitis and the stage of fibrosis. A positive correlation was identified between the grade of hepatitis with miR-122 (rs = 0.79, P < .001) and miR-29a (rs = 0.78, P < .001). Both miR-122 (rs = 0.81, P < .001) and miR-29a (rs = 0.67, P < .001) showed a significant positive correlation with the stage of fibrosis. MiR-122 concentrations were significantly higher in the CH (P < .01) and LC groups (P < .001) compared to the NL group. MiR-29a concentrations were significantly higher in the CH (P < .001) and LC (P < .001) groups compared to the NL group. CONCLUSIONS AND CLINICAL IMPORTANCE: Circulating miR-122 and miR-29a concentrations might be useful for monitoring the response to treatment and progression of canine CH.