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Human hepatocyte culture system represents by far the most physiologically relevant model for our understanding of liver biology and diseases; however, its versatility has been limited due to the rapid and progressive loss of genuine characteristics, indicating the inadequacy of in vitro milieu for fate maintenance. This study, therefore, is designed to define environmental requirements necessary to sustain the homeostasis of terminally differentiated hepatocytes. Our study reveals that the supplementation of dimethyl sulfoxide (DMSO) is indispensable in mitigating fate deterioration and promoting adaptation to the in vitro environment, resulting in the restoration of tight cell-cell contact, cellular architecture, and polarity. The morphological recovery was overall accompanied by the restoration of hepatocyte marker gene expression, highlighting the interdependence between the cellular architecture and the maintenance of cell fate. However, beyond the recovery phase culture, DMSO supplementation is deemed detrimental due to the potent inhibitory effect on a multitude of hepatocyte functionalities while its withdrawal results in the loss of cell fate. In search of DMSO substitute, our screening of organic substances led to the identification of dimethyl sulfone (DMSO2), which supports the long-term maintenance of proper morphology, marker gene expression, and hepatocytic functions. Moreover, hepatocytes maintained DMSO2 exhibited clinically relevant toxicity in response to prolonged exposure to xenobiotics as well as alcohol. These observations suggest that the stepwise culture configuration consisting of the consecutive supplementation of DMSO and DMSO2 confers the microenvironment essential for the fate and functional maintenance of terminally differentiated human hepatocytes.
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Dimetilsulfóxido , Hepatocitos , Humanos , Dimetilsulfóxido/farmacología , Hepatocitos/metabolismo , Hígado/metabolismo , Diferenciación Celular , Células CultivadasRESUMEN
BACKGROUND AND AIMS: Lipoprotein Z (LP-Z) is an abnormal free cholesterol (FC)-enriched LDL-like particle discovered from patients with cholestatic liver disease. This study aims to define the diagnostic value of LP-Z in alcohol-associated hepatitis (AH) and interrogate the biology behind its formation. APPROACH AND RESULTS: We measured serum levels of LP-Z using nuclear magnetic resonance spectroscopy, a well-established clinical assay. Serum levels of LP-Z were significantly elevated in four AH cohorts compared with control groups, including heavy drinkers and patients with cirrhosis. We defined a Z-index, calculated by the ratio of LP-Z to total apolipoprotein B-containing lipoproteins, representing the degree of deviation from normal VLDL metabolism. A high Z-index was associated with 90-day mortality independent from the Model for End-Stage Liver Disease (MELD) and provided added prognosticative value. Both a Z-index ≤ 0.6 and a decline of Z-index by ≥0.1 in 2 weeks predicted 90-day survival. RNA-sequencing analyses of liver tissues demonstrated an inverse association in the expression of enzymes responsible for the extrahepatic conversion of VLDL to LDL and AH disease severity, which was further confirmed by the measurement of serum enzyme activity. To evaluate whether the FC in LP-Z could contribute to the pathogenesis of AH, we found significantly altered FC levels in liver explant of patients with AH. Furthermore, FC in reconstituted LP-Z particles caused direct toxicity to human hepatocytes in a concentration-dependent manner, supporting a pathogenic role of FC in LP-Z. CONCLUSIONS: Impaired lipoprotein metabolism in AH leads to the accumulation of LP-Z in the circulation, which is hepatotoxic from excessive FC. A Z-index ≤ 0.6 predicts 90-day survival independent from conventional biomarkers for disease prognostication.
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Enfermedad Hepática en Estado Terminal , Hepatitis Alcohólica , Apolipoproteínas B , Colesterol , Humanos , Lipoproteína(a) , Lipoproteínas , Índice de Severidad de la EnfermedadRESUMEN
Ornithine transcarbamylase deficiency (OTCD) is a metabolic and genetic disease caused by dysfunction of the hepatocytic urea cycle. To develop new drugs or therapies for OTCD, it is ideal to use models that are more closely related to human metabolism and pathology. Primary human hepatocytes (HHs) isolated from two patients (a 6-month-old boy and a 5-year-old girl) and a healthy donor were transplanted into host mice (hemi-, hetero-OTCD mice, and control mice, respectively). HHs were isolated from these mice and used for serial transplantation into the next host mouse or for in vitro experiments. Histological, biochemical, and enzyme activity analyses were performed. Cultured HHs were treated with ammonium chloride or therapeutic drugs. Replacement rates exceeded 80% after serial transplantation in both OTCD mice. These highly humanized OTCD mice showed characteristics similar to OTCD patients that included increased blood ammonia levels and urine orotic acid levels enhanced by allopurinol. Hemi-OTCD mice showed defects in OTC expression and significantly low enzymatic activities, while hetero-OTCD mice showed residual OTC expression and activities. A reduction in ammonium metabolism was observed in cultured HHs from OTCD mice, and treatment with the therapeutic drug reduced the ammonia levels in the culture medium. In conclusion, we established in vivo OTC mouse models with hemi- and hetero-patient HHs. HHs isolated from the mice were useful as an in vitro model of OTCD. These OTC models could be a source of valuable patient-derived hepatocytes that would enable large scale and reproducible experiments using the same donor.
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Hepatocitos/trasplante , Enfermedad por Deficiencia de Ornitina Carbamoiltransferasa/terapia , Ornitina Carbamoiltransferasa/genética , Amoníaco/sangre , Animales , Preescolar , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Hepatocitos/química , Hepatocitos/citología , Humanos , Lactante , Masculino , Ratones , Enfermedad por Deficiencia de Ornitina Carbamoiltransferasa/genética , Ácido Orótico/orinaRESUMEN
Advancement in both bioengineering and cell biology of the liver led to the establishment of the first-generation humanized liver chimeric mouse (HLCM) model in 2001. The HLCM system was initially developed to satisfy the necessity for a convenient and physiologically representative small animal model for studies of hepatitis B virus and hepatitis C virus infection. Over the last two decades, the HLCM system has substantially evolved in quality, production capacity, and utility, thereby growing its versatility beyond the study of viral hepatitis. Hence, it has been increasingly employed for a variety of applications including, but not limited to, the investigation of drug metabolism and pharmacokinetics and stem cell biology. To date, more than a dozen distinctive HLCM systems have been established, and each model system has similarities as well as unique characteristics, which are often perplexing for end-users. Thus, this review aims to summarize the history, evolution, advantages, and pitfalls of each model system with the goal of providing comprehensive information that is necessary for researchers to implement the ideal HLCM system for their purposes. Furthermore, this review article summarizes the contribution of HLCM and its derivatives to our mechanistic understanding of various human liver diseases, its potential for novel applications, and its current limitations.
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Modelos Animales de Enfermedad , Hígado Artificial , Animales , Humanos , Hepatopatías/tratamiento farmacológico , Hepatopatías/virología , RatonesRESUMEN
Drug-induced liver injury (DILI) is a common side effect of several medications and is considered a major factor responsible for the discontinuation of drugs during their development. Cholestasis is a DILI that results from impairment of bile acid transporters, such as the bile salt export pump (BSEP), leading to accumulation of bile acids. Both in vitro and in vivo studies are required to predict the risk of drug-induced cholestasis. In the present study, we used chimeric mice with humanized liver as a model to study drug-induced cholestasis. Administration of a single dose of ketoconazole or rifampicin, known to potentially cause cholestasis by inhibiting BSEP, did not result in elevated levels of alkaline phosphatase (ALP), which are known hepatic biomarkers. The concentration of taurodeoxycholic acid increased in the liver after ketoconazole administration, whereas rifampicin resulted in increased tauromuricholic acid and taurocholic acid (TCA) levels in the liver and plasma. Furthermore, rifampicin resulted in an increase in the uniform distribution of a compound with m/z 514.3, presumed as TCA through imaging mass spectrometry. The mRNA levels of bile acid-related genes were also altered after treatment with ketoconazole or rifampicin. We believe these observations to be a part of a feedback mechanism to decrease bile acid concentrations. The changes in bile acid concentrations results may reflect the initial responses of the human body to cholestasis. Furthermore, these findings may contribute to the screening of drug candidates, thereby avoiding drug-induced cholestasis during clinical trials and drug development.
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Ácidos y Sales Biliares/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Colestasis/metabolismo , Cetoconazol/efectos adversos , Hígado/efectos de los fármacos , Rifampin/efectos adversos , Alanina Transaminasa/sangre , Fosfatasa Alcalina/sangre , Animales , Aspartato Aminotransferasas/sangre , Ácidos y Sales Biliares/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas/sangre , Colestasis/sangre , Colestasis/inducido químicamente , Humanos , Cetoconazol/sangre , Cetoconazol/farmacocinética , Hígado/metabolismo , Masculino , Ratones , Rifampin/sangre , Rifampin/farmacocinéticaRESUMEN
BACKGROUND: The role of PPARα in gene regulation in mouse liver is well characterized. However, less is known about the role of PPARα in human liver. The aim of the present study was to better characterize the impact of PPARα activation on gene regulation in human liver. To that end, chimeric mice containing hepatocyte humanized livers were given an oral dose of 300 mg/kg fenofibrate daily for 4 days. Livers were collected and analyzed by hematoxilin and eosin staining, qPCR, and transcriptomics. Transcriptomics data were compared with existing datasets on PPARα activation in normal mouse liver, human primary hepatocytes, and human precision cut liver slices. RESULTS: Of the different human liver models, the gene expression profile of hepatocyte humanized livers most closely resembled actual human liver. In the hepatocyte humanized mouse livers, the human hepatocytes exhibited excessive lipid accumulation. Fenofibrate increased the size of the mouse but not human hepatocytes, and tended to reduce steatosis in the human hepatocytes. Quantitative PCR indicated that induction of PPARα targets by fenofibrate was less pronounced in the human hepatocytes than in the residual mouse hepatocytes. Transcriptomics analysis indicated that, after filtering, a total of 282 genes was significantly different between fenofibrate- and control-treated mice (P < 0.01). 123 genes were significantly lower and 159 genes significantly higher in the fenofibrate-treated mice, including many established PPARα targets such as FABP1, HADHB, HADHA, VNN1, PLIN2, ACADVL and HMGCS2. According to gene set enrichment analysis, fenofibrate upregulated interferon/cytokine signaling-related pathways in hepatocyte humanized liver, but downregulated these pathways in normal mouse liver. Also, fenofibrate downregulated pathways related to DNA synthesis in hepatocyte humanized liver but not in normal mouse liver. CONCLUSION: The results support the major role of PPARα in regulating hepatic lipid metabolism, and underscore the more modest effect of PPARα activation on gene regulation in human liver compared to mouse liver. The data suggest that PPARα may have a suppressive effect on DNA synthesis in human liver, and a stimulatory effect on interferon/cytokine signalling.
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Quimera , Fenofibrato/farmacología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hígado/citología , PPAR alfa/agonistas , Transcriptoma/efectos de los fármacos , Animales , Humanos , RatonesRESUMEN
BACKGROUND: Nonimmune-mediated glomerulonephropathies are rarely reported in domestic animals with the exception of amyloidosis. Here we describe the pathological features and clinical course of a feline with protein-losing nonimmune-mediated glomerulonephropathy characterized by segmental glomerulosclerosis and severe podocyte injury. CASE PRESENTATION: A castrated male Japanese domestic cat aged 3 years and 8 months had hypertension, persistent proteinuria, and azotemia. Microscopic examination of a renal biopsy revealed many glomeruli with adhesion to the Bowman's capsule and segmental sclerosis. The most characteristic ultrastructural glomerular feature was severe podocyte foot process effacement. No electron-dense deposits were observed. Immunofluorescence revealed no immune deposits, but abnormal expression of nephrin and podocin was detected in the glomeruli. These findings resemble those of human focal segmental glomerulosclerosis. The cat temporarily responded to treatment with angiotensin-converting enzyme inhibitors and prednisolone administration but died of progressive renal failure 32 months after biopsy. CONCLUSIONS: The cat was diagnosed with nonimmune mediated glomerulonephropathy because of the absence of immune deposits and severe podocyte injury. To our knowledge, this is the first report of nonimmune-mediated glomerulonephropathy in a cat resembling human focal segmental glomerulosclerosis.
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Enfermedades de los Gatos/patología , Glomerulonefritis/veterinaria , Glomeruloesclerosis Focal y Segmentaria/veterinaria , Animales , Nitrógeno de la Urea Sanguínea , Gatos , Creatinina/sangre , Técnica del Anticuerpo Fluorescente/veterinaria , Glomerulonefritis/patología , Glomeruloesclerosis Focal y Segmentaria/patología , Riñón/patología , Riñón/ultraestructura , Glomérulos Renales/patología , Glomérulos Renales/ultraestructura , Masculino , Microscopía Electrónica de Transmisión/veterinariaRESUMEN
Glomerular lesions including membranoproliferative glomerulonephritis occur spontaneously in aged mice, but they are rare in young animals. In our laboratory, spontaneous glomerulonephritis was observed in an 8-week-old male Crj:CD1 (ICR) mouse. Macroscopically, the bilateral kidneys were discolored, but no edema or ascites was observed. Glomerular lesions were characterized by a thickening of capillary walls, a double-contoured basement membrane and mesangial expansion due to increased amounts of matrix. Ultrastructurally, mesangial interposition in the capillary wall and subendothelial deposition of basement membrane-like material were observed. No evidence of immune complex deposition or amyloid was found. On the basis of the observed clinical pathology and histopathology, a secondary form of glomerular lesion was excluded. The glomerular lesion was compatible with glomerulonephritis in a young Crj:CD1 (ICR) mouse.
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BACKGROUND AND AIMS: The mitochondrial translocator protein (TSPO, 18 kDa) is pivotal in binding cholesterol and facilitating its transfer from the outer to the inner mitochondrial membrane. Atriol is a TSPO ligand disrupting cholesterol binding by targeting the cholesterol-recognition amino acid consensus domain. Prior research has shown that TSPO deficiency improved metabolic-associated steatohepatitis (MASH). We hypothesized that Atriol may have the potential to alleviate MASH. METHODS AND RESULTS: In vitro cell culture studies revealed that Atriol treatment effectively mitigated MASH by restoring mitochondrial function, inhibiting the NF-κB signaling pathway, and reducing hepatic stellate cell (HSC) activation. SD male rats were fed a GAN diet for 10 months to induce MASH. During the final two weeks of feeding, rats received intraperitoneal Atriol administration daily. Atriol treatment significantly ameliorated MASH by reducing lipid accumulation, diminishing hepatic lobular inflammation and fibrosis, decreasing cell death, and inhibiting excessive bile acid synthesis. Moreover, Atriol restored mitochondrial function in primary hepatocytes isolated from MASH rats. In search of the mechanism(s) governing these effects, we found that Atriol downregulated the proinflammatory chemokine CXCL1 through the NF-κB signaling pathway or via myeloperoxidase (MPO) in HSCs and Kupffer cells. Additionally, in vitro, studies further suggested that CXCL1 treatment induced dysfunctional mitochondria, inflammation, HSCs activation, and macrophage migration, whereas Atriol countered these effects. Finally, the mitigating effects of Atriol on MASH were reproduced by pharmacological inhibition of NF-κB or MPO and neutralization of CXCL1. CONCLUSION: Atriol ameliorates MASH both in vitro and in vivo, demonstrating its potential therapeutic benefits in managing MASH.
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Hepatitis delta virus (HDV), a satellite virus of HBV, is regarded as the most severe type of hepatitis virus because of the substantial morbidity and mortality. The IFN system is the first line of defense against viral infections and an essential element of antiviral immunity; however, the role of the hepatic IFN system in controlling HBV-HDV infection remains poorly understood. Herein, we showed that HDV infection of human hepatocytes induced a potent and persistent activation of the IFN system whereas HBV was inert in triggering hepatic antiviral response. Moreover, we demonstrated that HDV-induced constitutive activation of the hepatic IFN system resulted in a potent suppression of HBV while modestly inhibiting HDV. Thus, these pathogens are equipped with distinctive immunogenicity and varying sensitivity to the antiviral effectors of IFN, leading to the establishment of a paradoxical mode of viral interference wherein HDV, the superinfectant, outcompetes HBV, the primary pathogen. Furthermore, our study revealed that HDV-induced constitutive IFN system activation led to a state of IFN refractoriness, rendering therapeutic IFNs ineffective. The present study provides potentially novel insights into the role of the hepatic IFN system in regulating HBV-HDV infection dynamics and its therapeutic implications through elucidating the molecular basis underlying the inefficacy of IFN-based antiviral strategies against HBV-HDV infection.
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Virus de la Hepatitis B , Virus de la Hepatitis Delta , Humanos , Virus de la Hepatitis Delta/fisiología , Hepatocitos , Replicación Viral , Antivirales/farmacología , Antivirales/uso terapéuticoRESUMEN
The ability to maintain functional hepatocytes has important implications for bioartificial liver development, cell-based therapies, drug screening, and tissue engineering. Several approaches can be used to restore hepatocyte function in vitro, including coating a culture substrate with extracellular matrix (ECM), encapsulating cells within biomimetic gels (Collagen- or Matrigel-based), or co-cultivation with other cells. This paper describes the use of bioactive heparin-based core-shell microcapsules to form and cultivate hepatocyte spheroids. These microcapsules are comprised of an aqueous core that facilitates hepatocyte aggregation into spheroids and a heparin hydrogel shell that binds and releases growth factors. We demonstrate that bioactive microcapsules retain and release endogenous signals thus enhancing the function of encapsulated hepatocytes. We also demonstrate that hepatic function may be further enhanced by loading exogenous hepatocyte growth factor (HGF) into microcapsules and inhibiting transforming growth factor (TGF)-ß1 signaling. Overall, bioactive microcapsules described here represent a promising new strategy for the encapsulation and maintenance of primary hepatocytes and will be beneficial for liver tissue engineering, regenerative medicine, and drug testing applications.
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Nonalcoholic fatty liver disease/steatohepatitis (NAFLD/NASH) is the most common liver disorder in developed countries. Although many new therapeutics for NASH are present in the drug development pipeline, there are still no approved drugs. One of the reasons that makes NASH drug development challenging is the lack of appropriate animal NASH models that resolve issues arising from inter-species differences between humans and rodents. In the present study, we developed a choline-deficient, L-amino-acid-defined, high-fat-diet (CDAHFD)-induced human NASH model using human liver chimeric mice. We demonstrated human hepatocyte injury by an elevation of plasma human alanine aminotransferase 1 in mice fed CDAHFD. Histological analysis showed that CDAHFD feeding induced similar histological changes to human NASH patients, including ballooning, inflammation, apoptosis, regeneration of human hepatocytes, and pericellular and perisinusoidal fibrosis. The chimeric mice fed CDAHFD were treated with a peroxisome-proliferator-activated receptor α/δ agonist, Elafibranor. Elafibranor ameliorated steatosis, ballooning of hepatocytes, and preserved fibrosis progression. We developed a novel humanized NASH model that can elucidate pathophysiological mechanisms and predict therapeutic efficacy in human NASH. This model will be useful in exploring new drugs and biomarkers in the early stages of human NASH.
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Interferons (IFNs) suppress viral infection through the induction of >400 interferon-stimulated genes (ISGs). Among ISGs, IFN-induced protein with tetratricopeptide repeats (IFITs) is one of the most potent and well-characterized ISGs. IFIT family consists of 4 cluster genes. It has been suggested that the antiviral action of each IFIT employs distinct mechanisms. In addition, it has been shown that each IFIT exhibits its antiviral properties partially in a pathogen-specific manner. To date, the expression profile of IFITs in the liver, as well as the antiviral potency of the individual IFITs in the regulation of hepatitis C virus (HCV) infection, is not yet fully defined. Our previous study found that the expression of hepatic IFITs is well correlated with the outcome of IFN-based antiviral therapy. This study explored the significance of each IFIT in the suppression of HCV. Our in vitro and in vivo studies with humanized liver chimeric mouse system revealed that IFIT1, 2, and 3/4 play an important role in the suppression of HCV. In addition, our in vitro experiment found that all IFITs possess a comparable anti-HCV potency. Follow-up studies collectively indicated that IFITs suppress HCV likely through 2 distinct mechanisms: (1) inhibition of internal ribosome entry site-dependent viral protein translation initiation complex according to experiments with bicistronic reporter assay as well as confocal microscopic analyses and (2) sequestration of viral genome based on an experiment using replication defective viral genome. In conclusion, our study defined the importance of IFITs in the regulation of HCV and also suggested the multifaceted antiviral actions.
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Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Hepatitis C/tratamiento farmacológico , Interferones/farmacología , Línea Celular , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatitis C/metabolismo , Humanos , Interferones/genética , Repeticiones de Tetratricopéptidos , Replicación Viral/efectos de los fármacos , Replicación Viral/genéticaRESUMEN
Atopic dermatitis (AD) is a chronic, pruritic, and allergic skin disease in humans and animals, particularly dogs. Canine AD (cAD) has received attention as a spontaneous atopic animal model because domesticated dogs inhabit a human environment, and cAD shares several clinicopathological features with human AD (hAD). In hAD, periostin (PO) is suggested to play a critical role in the enhancement and chronicity of allergic skin inflammation; however, PO involvement in the pathogenesis of cAD is unknown. Here we aimed to clarify PO involvement in the pathophysiology of cAD and focused on the inducing factor and function of PO in canine atopic skin. Using double-labeled in situ hybridization (ISH), interleukin (IL)-13 mRNA-positive cells were detected near the keratinocytes and dermal fibroblasts expressing PO mRNA in atopic skin. Using an in vitro assay, IL-13 induced PO gene expression in both canine dermal fibroblasts and keratinocytes. PO enhanced in vitro growth of canine keratinocytes. Moreover, among PO-induced genes in cultured canine keratinocytes detected using a microarray, we identified IL-25 as a possible mediator in canine atopic skin. In addition, real time polymerase chain reaction (PCR) analysis revealed upregulation of IL-25 gene expression in PO-stimulated keratinocytes. These data suggest that IL-13 possibly derived from T helper 2 (Th2) cells stimulates PO production in both keratinocytes and fibroblasts, and then PO may play a critical role in the pathophysiology of cAD, particularly in the enhancement and chronicity of skin lesions via IL-25.
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Moléculas de Adhesión Celular/metabolismo , Dermatitis Atópica/veterinaria , Enfermedades de los Perros/patología , Animales , Moléculas de Adhesión Celular/genética , Dermatitis Atópica/metabolismo , Dermatitis Atópica/fisiopatología , Enfermedades de los Perros/metabolismo , Perros , Femenino , Fibroblastos/metabolismo , Expresión Génica , Interleucina-13/metabolismo , Interleucina-17/metabolismo , Queratinocitos/metabolismo , Masculino , ARN Mensajero/metabolismo , Piel/metabolismo , Piel/patologíaRESUMEN
Canine squamous cell carcinoma (SCC) shows highly invasive and locally destructive growth. In animal models and human cancer cases, periostin plays a critical role in the enhancement of cancer growth; however, the mechanism of involvement in canine cancers remains unknown. The aim of this study was to examine the involvement of periostin in the pathophysiology of SCC in dogs. We examined the localization of periostin and periostin-producing cells in 20 SCC and three squamous papilloma specimens. Furthermore, we focused on transforming growth factor (TGF)-ß1, which was assumed to be an inducing factor of periostin, using culture cells. By immunohistochemistry, limited periostin expression in the stroma was observed in all squamous papillomas. In SCC, periostin protein diffusely expressed at the tumor invasion front of cancer growth. In situ hybridization revealed that periostin mRNA was expressed in the stromal fibroblasts in SCC. In vitro analysis determined that canine SCC cells expressed significantly higher levels of TGF-ß1 mRNA compared with canine keratinocytes. In addition, recombinant TGF-ß1 induced secretion of periostin from cultured dermal fibroblasts. These data suggest that periostin produced by stromal fibroblasts may be involved in the pathophysiology of canine SCC. TGF-ß1 derived from SCC cells may stimulate fibroblasts to produce periostin.
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Carcinoma de Células Escamosas/veterinaria , Moléculas de Adhesión Celular/metabolismo , Enfermedades de los Perros/metabolismo , Animales , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Enfermedades de los Perros/patología , Perros , Femenino , Fibroblastos/metabolismo , Hibridación in Situ , Masculino , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
As aldehyde oxidase (AOX) plays an emerging role in drug metabolism, understanding its significance for drug-drug interactions (DDI) is important. Therefore, we tested 10 compounds for species-specific and substrate-dependent differences in the inhibitory effect of AOX activity using genetically engineered HEK293 cells over-expressing human AOX1, mouse AOX1 or mouse AOX3. The IC50 values of 10 potential inhibitors of the three AOX enzymes were determined using phthalazine and O6-benzylguanine as substrates. 17ß-Estradiol, menadione, norharmane and raloxifene exhibited marked differences in inhibitory effects between the human and mouse AOX isoforms when the phthalazine substrate was used. Some of the compounds tested exhibited substrate-dependent differences in their inhibitory effects. Docking simulations with human AOX1 and mouse AOX3 were conducted for six representative inhibitors. The rank order of the minimum binding energy reflected the order of the corresponding IC50 values. We also evaluated the potential DDI between an AOX substrate (O6-benzylguanine) and an inhibitor (hydralazine) using chimeric mice with humanized livers. Pretreatment of hydralazine increased the maximum plasma concentration (Cmax) and the area under the plasma concentration-time curve (AUC0-24) of O6-benzylguanine compared to single administration. Our in vitro data indicate species-specific and substrate-dependent differences in the inhibitory effects on AOX activity. Our in vivo data demonstrate the existence of a DDI which may be of relevance in the clinical context.
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Activación Metabólica/efectos de los fármacos , Aldehído Oxidasa/antagonistas & inhibidores , Aldehído Oxidorreductasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Activación Metabólica/fisiología , Aldehído Oxidasa/metabolismo , Aldehído Oxidorreductasas/metabolismo , Animales , Quimera , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas/fisiología , Inhibidores Enzimáticos/metabolismo , Células HEK293 , Humanos , Hígado/efectos de los fármacos , Hígado/enzimología , Ratones , Ratones SCID , Preparaciones Farmacéuticas/metabolismo , Ftalazinas/metabolismo , Ftalazinas/farmacologíaRESUMEN
The histopathological, immunohistochemical, and ultrastructural morphologic characteristics of a tumor in the subcutaneous tissue of the chest of a 19-year-old female Japanese macaque were investigated. Consequently, the mass was diagnosed as a malignant mast cell tumor (MCT). Tumors were present in both mammary gland portions of the anterior thorax. Both tumors showed the same histopathological findings. The tumor tissue was defined by the presence of delicate connective tissue, and the tumor cells grew in a cord-like or cobblestone pattern. The tumor cell cytoplasm was very clear. The nuclei were relatively uniform and the cells showed a low nucleus-cytoplasm ratio. The cytoplasmic granules stained blue with Alcian blue and eosinophils had infiltrated into the tumor tissue. Furthermore, immunohistochemical analysis revealed that the tumor cell membrane was positive for the anti-c-kit antibody. In ultrastructural morphologic analyses, all tumor cells showed a rich cytoplasm and, occasionally, granules wrapped in a limiting membrane of high electron density. The tumor cells had metastasized to the axillary lymph nodes, the kidney, and the peritoneum. Based on these results, the mass was diagnosed as a malignant MCT originating from the subcutaneous tissue of the chest. Since cases of MCTs in macaques are very rare, this report presents important new knowledge of neoplastic lesions in this species.
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Macaca , Mastocitos/patología , Mastocitosis/veterinaria , Enfermedades de los Monos/patología , Neoplasias Torácicas/veterinaria , Animales , Femenino , Neoplasias Renales/secundario , Neoplasias Renales/veterinaria , Mastocitosis/patología , Mastocitosis/secundario , Neoplasias Peritoneales/secundario , Neoplasias Peritoneales/veterinaria , Tejido Subcutáneo/patología , Neoplasias Torácicas/patología , Neoplasias Torácicas/secundarioRESUMEN
Phospholipase A2 receptor (PLA2R) expressed in human podocytes has been highlighted as a causative autoantigen of human idiopathic membranous nephropathy. However, its expression was found to be minimal or absent in murine and rat podocytes. In this study, immunofluorescence revealed the expression of PLA2R in the glomerular podocytes in the kidney tissue sections of dogs. We then attempted to culture canine podocytes and investigate the expression of PLA2R in these cells. Glomeruli were isolated from dog kidneys and cultured to obtain podocytes using nylon mesh-based isolation method as followed for isolating rat podocytes. The cultured cells expressed PLA2R mRNA and protein in addition to other podocyte markers (synaptopodin, podocin and nephrin). These results indicate that the canine podocytes express PLA2R.
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Podocitos/química , Receptores de Fosfolipasa A2/análisis , Animales , Células Cultivadas , Perros/metabolismo , Técnica del Anticuerpo Fluorescente/veterinaria , Microscopía de Contraste de Fase/veterinaria , Podocitos/citología , Receptores de Fosfolipasa A2/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinariaRESUMEN
Two solitary and minute tumors of 1 and 1.5 mm diameter were identified by microscopy in the left fourth mammary gland of a 13-year-old female Labrador Retriever dog, in addition to multiple mammary gland tumors. The former tumors were well circumscribed and were composed of small-to-large polyhedral neoplastic oncocytes with finely granular eosinophilic cytoplasm, and were arranged in solid nests separated by fine fibrovascular septa. Scattered lumina of variable sizes containing eosinophilic secretory material were evident. Cellular atypia was minimal, and no mitotic figures were visible. One tumor had several oncocytic cellular foci revealing cellular transition, with perivascular pseudorosettes consisting of columnar epithelial cells surrounding the fine vasculature. Scattered foci of mammary acinar cell hyperplasia showing oncocytic metaplasia were also observed. Immunohistochemically, the cytoplasm of neoplastic cells of the 2 microtumors showed diffuse immunoreactivity to anti-cytokeratin antibody AE1/AE3, and finely granular immunoreactivity for 60-kDa heat shock protein, mitochondrial membrane ATP synthase complex V beta subunit, and chromogranin A. One tumor also had oncocytic cellular foci forming perivascular pseudorosettes showing cellular membrane immunoreactivity for neural cell adhesion molecule. The tumors were negative for smooth muscle actin, neuron-specific enolase, vimentin, desmin, S100, and synaptophysin. Ultrastructural observation confirmed the abundant mitochondria in the cytoplasm of both neoplastic and hyperplastic cells, the former cells also having neuroendocrine granule-like electron-dense bodies. From these results, our case was diagnosed with mammary oncocytomas accompanied by neuroendocrine differentiation. Scattered foci of mammary oncocytosis might be related to the multicentric occurrence of these oncocytomas.
Asunto(s)
Células Acinares/patología , Adenoma Oxifílico/veterinaria , Enfermedades de los Perros/diagnóstico , Neoplasias Mamarias Animales/diagnóstico , Adenoma Oxifílico/diagnóstico , Adenoma Oxifílico/patología , Animales , Diagnóstico Diferencial , Enfermedades de los Perros/patología , Perros , Femenino , Hiperplasia/diagnóstico , Hiperplasia/patología , Hiperplasia/veterinaria , Neoplasias Mamarias Animales/patología , VimentinaRESUMEN
Cutaneous epitheliotropic T-cell lymphoma (CETL) is characterized by neoplastic T-cell infiltration of the epidermis, adnexal structures, and oral mucosa. The objective of this report was to describe the pathological findings of a canine case of terminal-stage CETL. A 10-year-old, mixed-breed, neutered male dog was presented with erosion of the oral mucosa and mucocutaneous junction. The dog was diagnosed with CETL with no evidence of metastasis. Despite chemotherapy, the dog was re-presented with oral pain, vomiting, and diarrhea, and died 17 months after the first visit to the hospital. A complete autopsy was performed. Histologic examination of the primary lesion and systemic organs was performed. Gross examination revealed an advanced-stage oral lesion. Distinct tumor formation was not observed in the primary sites and systemic organs. Histologically, the primary oral lesion was characterized by massive intraepithelial infiltration of a large number of neoplastic lymphocytes. The neoplastic cells in the metastatic sites also showed exclusive epitheliotropic proliferation in organs, including the trachea, tonsils, esophagus, stomach, small intestine, colon, anal mucosa, liver, pancreas, kidneys, urinary bladder, prostate gland, ear canals, and auricular and ventral skin. Immunohistochemically, the neoplastic cells were positive for CD3 and negative for CD20 as well as CD79α, supporting a diagnosis of CETL with systemic dissemination. In canine CETL with systemic signs, systemic metastasis should be considered even without evident mass formation. Neoplastic lymphocytes of CETL showed distinct epitheliotropism even in the systemic metastatic sites.