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Colistin is considered the last resort for treating infections caused by multidrug-resistant bacteria. However, the spread of the plasmid-borne colistin-resistance gene mcr-1 has become a public health threat. In this study, we identified mcr-1-harboring Leclercia adecarboxylata strain (WWCOL-134) isolated from wastewater in Seoul. The strain had a colistin MIC value of 2 µg/ml and was resistant to cefotaxime, gentamicin, tetracycline, trimethoprim, and sulfamethoxazole. The mcr-1 gene, along with an array of resistance genes, was located on a 236-kb plasmid (pCOL134-1), which contained the typical IncHI2 backbone of reported mcr-1-carrying plasmids, and was transferred to an Escherichia coli strain by conjugation. To the best of our knowledge, this is the first study to report the emergence of mcr-1-harboring Leclercia sp. isolate. Our findings demonstrate the ongoing spread of colistin resistance among Enterobacterales species, emphasizing the need for surveillance of antimicrobial resistance in wastewater environments.
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OBJECTIVE: Maintaining a constant core body temperature is essential to homeothermic vertebrate survival. Adaptive thermogenesis in brown adipose tissue and skeletal muscle is the primary mechanism of adjustment to an external stimulus such as cold exposure. Recently, several reports have revealed that the liver can play a role as a metabolic hub during adaptive thermogenesis. In this study, we suggest that the liver plays a novel role in secreting thermogenic factors in adaptive thermogenesis. Bone morphogenetic protein 9 (BMP9) is a hepatokine that regulates many biological processes, including osteogenesis, chondrogenesis, hematopoiesis, and angiogenesis. Previously, BMP9 was suggested to affect preadipocyte proliferation and differentiation. However, the conditions and mechanisms underlying hepatic expression and secretion and adipose tissue browning of BMP9 remain largely unknown. In this study, we investigated the physiological conditions for secretion and the regulatory mechanism of hepatic Bmp9 expression and the molecular mechanism by which BMP9 induces thermogenic gene program activation in adipose tissue. Here, we also present the pharmacological effects of BMP9 on a high-fat-induced obese mouse model. METHODS: To investigate the adaptive thermogenic role of BMP9 in vivo, we challenged mice with cold temperature exposure for 3 weeks and then examined the BMP9 plasma concentration and hepatic expression level. The cellular mechanism of hepatic Bmp9 expression under cold exposure was explored through promoter analysis. To identify the role of BMP9 in the differentiation of brown and beige adipocytes, we treated pluripotent stem cells and inguinal white adipose tissue (iWAT)-derived stromal-vascular (SV) cells with BMP9, and brown adipogenesis was monitored by examining thermogenic gene expression and signaling pathways. Furthermore, to evaluate the effect of BMP9 on diet-induced obesity, changes in body composition and glucose tolerance were analyzed in mice administered recombinant BMP9 (rBMP9) for 8 weeks. RESULTS: Hepatic Bmp9 expression and plasma levels in mice were significantly increased after 3 weeks of cold exposure. Bmp9 mRNA expression in the liver was regulated by transcriptional activation induced by cAMP response-element binding protein (CREB) and CREB-binding protein (CBP) on the Bmp9 promoter. Treatment with BMP9 promoted the differentiation of multipotent stem cells and iWAT-derived SV cells into beige adipocytes, as indicated by the increased expression of brown adipocyte and mitochondrial biogenesis markers. Notably, activation of the mothers against decapentaplegic homolog 1 (Smad1) and p44/p42 mitogen-activated protein kinase (MAPK) pathways was required for the induction of uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC1α) expression in BMP9-induced differentiation of SVs into beige adipocytes. The administration of rBMP9 in vivo also induced browning markers in white adipose tissue. In high-fat diet-induced obese mice, rBMP9 administration conferred protection against obesity and enhanced glucose tolerance. CONCLUSIONS: BMP9 is a hepatokine regulated by cold-activated CREB and CBP and enhances glucose and fat metabolism by promoting the activation of the thermogenic gene program in adipocytes. These data implicate BMP9 as a potential pharmacological tool for protecting against obesity and type 2 diabetes.
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Diabetes Mellitus Tipo 2 , Factor 2 de Diferenciación de Crecimiento/metabolismo , Adipocitos Marrones/metabolismo , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/metabolismo , Animales , Frío , Diabetes Mellitus Tipo 2/metabolismo , Glucosa/metabolismo , Factor 2 de Diferenciación de Crecimiento/farmacología , Ratones , Ratones Endogámicos C57BL , Obesidad/metabolismo , Termogénesis/genética , Proteína Desacopladora 1/metabolismoRESUMEN
The biological role of BMP-9 signaling in liver cancer remains dubious. To explore the potential use of BMP-9 signaling for anti-cancer therapy, we used recombinant human BMP-9, which we referred to as MB109, to study the effect on growth of fifteen hepatocellular carcinoma (HCC) cell lines. MB109 effectively inhibits the proliferation of nine HCC cells in vitro. The anti-proliferative effect was found to be induced by turning on p21 signaling, which caused survivin suppression and G0/G1 cell cycle arrest. ID3 was identified to be the mediator of the MB109-induced p21 expression. Blocking the activity of p38 MAPK diminished ID3 and p21 expression, indicating that MB109 signals through a p38 MAPK/ID3/p21 pathway to arrest cell cycle progression. Moreover, prolonged MB109 treatment suppressed the expression of five prominent liver cancer stem cell (LCSC) markers, including CD44, CD90, AFP, GPC3 and ANPEP. Xenograft model confirmed the anti-tumor and LCSC-suppression capability of MB109 in vivo. Contrary to ongoing efforts of suppressing BMP-9 signaling to inhibit angiogenesis of cancer tissue, these results demonstrate an unexpected therapeutic potential of MB109 to stimulate BMP-9 signaling for anti-cancer therapies.
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Carcinoma Hepatocelular/metabolismo , Factor 2 de Diferenciación de Crecimiento/metabolismo , Neoplasias Hepáticas/metabolismo , Células Madre Neoplásicas/metabolismo , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Modelos Animales de Enfermedad , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Factor 2 de Diferenciación de Crecimiento/genética , Factor 2 de Diferenciación de Crecimiento/farmacología , Humanos , Proteínas Inhibidoras de la Diferenciación/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Ratones , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Fosforilación , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND: The development of chemical refolding of transforming growth factor-beta (TGF-ß) superfamily ligands has been instrumental to produce the recombinant proteins for biochemical studies and exploring the potential of protein therapeutics. The osteogenic human bone morphogenetic protein-2 (hBMP-2) and its Drosophila DPP homolog were the early successful cases of refolding into functional form. Despite the similarity in their three dimensional structure and amino acid sequences, several other TGF-ß superfamily ligands could not be refolded readily by the same methods. RESULTS: Here, we report a comprehensive study on the variables of a rapid-dilution refolding method, including the concentrations of protein, salt, detergent and redox agents, pH, refolding duration and the presence of aggregation suppressors and host-cell contaminants, in order to identify the optimal condition to refold human BMP-9 (hBMP-9). To produce a recombinant form of hBMP-9 in E. coli cells, a synthetic codon-optimized gene was designed to encode the mature domain of hBMP-9 (Ser320 - Arg429) directly behind the first methionine, which we herein referred to as MB109. An effective purification scheme was also developed to purify the refolded MB109 to homogeneity with a final yield of 7.8 mg from 100 mg of chromatography-purified inclusion bodies as a starting material. The chemically refolded MB109 binds to ALK1, ActRIIb and BMPRII receptors with relatively high affinity as compared to other Type I and Type II receptors based on surface plasmon resonance analysis. Smad1-dependent luciferase assay in C2C12 cells shows that the MB109 has an EC50 of 0.61 ng/mL (25 pM), which is nearly the same as hBMP-9. CONCLUSION: MB109 is prone to be refolded as non-functional dimer and higher order multimers in most of the conditions tested, but bioactive MB109 dimer can be refolded with high efficiency in a narrow window, which is strongly dependent on the pH, refolding duration, the presence of aggregation suppressors and the concentrations of protein, salt and detergent. These results add to the current understanding of producing recombinant TGF-ß superfamily ligands in the microbial E. coli system. An application of the technique to produce a large number of synthetic TGF-ß chimeras for activity screen is also discussed.
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Escherichia coli/metabolismo , Factor 2 de Diferenciación de Crecimiento/metabolismo , Cuerpos de Inclusión/metabolismo , Secuencia de Aminoácidos , Factor 2 de Diferenciación de Crecimiento/química , Factor 2 de Diferenciación de Crecimiento/genética , Humanos , Concentración de Iones de Hidrógeno , Cuerpos de Inclusión/química , Datos de Secuencia Molecular , Oxidantes/química , Replegamiento Proteico , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Sustancias Reductoras/química , Sales (Química)/químicaRESUMEN
This work was first development of a delivery system capable of maintaining a sustained release of protein drugs at specific sites by using potentially biocompatible porcine articular cartilage. The prepared porcine articular cartilage powder (PCP) was easily soluble in phosphate-buffered saline. The PCP suspension easily entrapped bovine serum albumin-fluorescein isothiocyanate (BSA-FITC) in pharmaceutical formulations at room temperature. The aggregation of PCP and BSA-FITC was confirmed by dynamic light scattering. When the BSA-FITC-loaded PCP suspension was subcutaneously injected into rats, it gelled and formed an interconnecting three-dimensional PCP structure that allowed BSA to penetrate through it. The amount of BSA-FITC released from the PCP hydrogel was determined in rat plasma and monitored by real-time in vivo molecular imaging. The data indicated sustained release of BSA-FITC for 20 days in vivo. In addition, the PCP hydrogel induced a slight inflammatory response. In conclusion, we showed that the PCP hydrogel could serve as a minimally invasive therapeutics depot.
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Materiales Biocompatibles/química , Cartílago Articular/química , Portadores de Fármacos , Matriz Extracelular/química , Animales , Materiales Biocompatibles/administración & dosificación , Materiales Biocompatibles/toxicidad , Preparaciones de Acción Retardada , Fluoresceína-5-Isotiocianato/administración & dosificación , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/química , Hidrogeles , Inflamación/inducido químicamente , Inyecciones Subcutáneas , Luz , Polvos , Ratas , Ratas Sprague-Dawley , Dispersión de Radiación , Albúmina Sérica Bovina/administración & dosificación , Albúmina Sérica Bovina/química , Solubilidad , Tecnología Farmacéutica/métodos , Temperatura , Factores de Tiempo , ViscosidadRESUMEN
Methoxy polyethylene glycol-poly(ε-caprolactone) (MPEG-PCL; MP) diblock copolymers undergo a solution-to-gel phase transition at body temperature and serve as ideal biomaterials for drug delivery and tissue engineering. Here, we examined the potential use of a chondrocyte-loaded MP solution as an injectable, in situ-forming hydrogel for cartilage regeneration. The chondrocyte-MP solution underwent a temperature-dependent solution-to-gel phase transition in vitro, as shown by an increase in viscosity from 1 cP at 20-30 °C to 1.6 × 105 cP at 37 °C. The chondrocytes readily attached to and proliferated on the MP hydrogel in vitro. The chondrocyte-MP solution transitioned to a hydrogel immediately after subcutaneous injection into mice, and formed an interconnected pore structure required to support the growth, proliferation, and differentiation of the chondrocytes. The chondrocyte-MP hydrogels formed cartilage in vivo, as shown by the histological and immunohistochemical staining of glycosaminoglycans, proteoglycans, and type II collagen, the major components of cartilage. Cartilage formation increased with hydrogel implantation time, and the expression of glycosaminoglycans, and type II collagen reached maximal levels at 6 weeks post-implantation. Collectively, these data suggest that in situ-forming chondrocyte-MP hydrogels have potential as non-invasive alternatives for tissue-engineered cartilage formation.
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In this study, we used a chitosan hydrogel as a 3-dimensional substrate for the attachment, proliferation, and differentiation of rat muscle-derived stem cells (rMDSCs) in the presence of valproic acid (VA). Chitosan solutions containing glycerol phosphate disodium salt form a hydrogel at body temperature. The chitosan hydrogel exhibited a porous 3-dimensional network that allowed the culture medium to penetrate. The chitosan hydrogel acted as a suitable biocompatible substrate for the attachment and proliferation of rMDSCs. On chitosan hydrogel in the presence of VA, rMDSCs exhibited higher expression of the neural markers, neuron-specific enolase (NSE) and beta tubulin III (Tuj-1), the oligodendrocyte marker, oligodendrocyte transcription factor 2 (Olig-2), and the astrocyte marker, glial fibrillary acidic protein (GFAP) than those in the absence of VA. Our results suggest that rMDSCs on a chitosan hydrogel in the presence of VA can differentiate into cells with a neural-like phenotype.
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Diferenciación Celular/efectos de los fármacos , Quitosano/química , Hidrogeles/química , Hidrogeles/farmacología , Músculos/citología , Neuronas/citología , Células Madre/citología , Animales , Adhesión Celular/efectos de los fármacos , Glicerol/química , Neurogénesis/efectos de los fármacos , Ratas , Células Madre/efectos de los fármacos , Ácido Valproico/químicaRESUMEN
The present study employed a combinatorial strategy using poly(D,L-lactide-co-glycolide) (PLGA) scaffolds seeded with human mesenchymal stem cells (hMSCs) to promote cell survival, differentiation, and neurological function in a completely transected spinal cord injury (SCI) model. The SCI model was prepared by complete removal of a 2-mm length of spinal cord in the eighth-to-ninth spinal vertebra, a procedure that resulted in bilateral hindlimb paralysis. PLGA scaffolds 2 mm in length without hMSCs (control) or with different numbers of hMSCs (1 × 10(5), 2 × 10(4), and 4 × 10(3)) were fitted into the completely transected spinal cord. Rats implanted with hMSCs received Basso-Beattie-Bresnahan scores for hindlimb locomotion of about 5, compared with ~2 for animals in the control group. The amplitude of motor-evoked potentials (MEPs) averaged 200-300 µV in all hMSC-implanted SCR model rats. In contrast, the amplitude of MEPs in control group animals averaged 135 µV at 4 weeks and then declined to 100 µV at 8 weeks. These results demonstrate functional recovery in a completely transected SCI model under conditions that exclude self-recovery. hMSCs were detected at the implanted site 4 and 8 weeks after transplantation, indicating in vivo survival of implanted hMSCs. Immunohistochemical staining revealed differentiation of implanted hMSCs into nerve cells, and immunostained images showed clear evidence for axonal regeneration only in hMSC-seeded PLGA scaffolds. Collectively, our results indicate that hMSC-seeded PLGA scaffolds induced nerve regeneration in a completely transected SCI model, a finding that should have significant implications for the feasibility of therapeutic and clinical hMSC-delivery using three-dimensional scaffolds, especially in the context of complete spinal cord transection.
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Células Madre Mesenquimatosas/citología , Traumatismos de la Médula Espinal/terapia , Andamios del Tejido/química , Animales , Células Cultivadas , Electrofisiología , Femenino , Humanos , Ácido Láctico/química , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Ratas Endogámicas F344RESUMEN
OBJECTIVE: To investigate the cognitive effect of levetiracetam (LEV) monotherapy with quantitative electroencephalogram (EEG) analysis and neuropsychological (NP) tests. METHODS: Twenty-two drug-naïve epilepsy patients were enrolled. EEG recordings were performed before and after LEV therapy. Relative power of discrete frequency bands was computed, as well as alpha peak frequency (APF) at occipital electrodes. Eighteen patients performed a battery of NP tests twice across LEV treatment. RESULTS: LEV therapy decreased the power of delta (1-3 Hz, p<0.01) and theta (3-7 Hz, p<0.05) bands and increased that of alpha-2 (10-13 Hz, p<0.05) and beta-2 (19-24 Hz, p<0.05) bands. Region-specific spectral change was observed: delta power change was significant in fronto-polar region, theta in anterior region, alpha-2 in broad region, and beta-2 in left fronto-central region. APF change was not significant. Improvement in diverse NP tests requiring attention, working memory, language and executive function was observed. Change in theta, alpha-2, and beta-2 power was correlated with improvement in several NP tests. CONCLUSIONS: Our data suggest LEV is associated with acceleration of background EEG frequencies and improved cognitive function. Change in frequency band power could predict improvement in several cognitive domains across LEV therapy. SIGNIFICANCE: Combined study of quantitative EEG analysis and NP tests can be useful in identifying cognitive effect of antiepileptic drugs.
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Anticonvulsivantes/uso terapéutico , Ondas Encefálicas/efectos de los fármacos , Cognición/efectos de los fármacos , Electroencefalografía , Epilepsia/fisiopatología , Piracetam/análogos & derivados , Adolescente , Adulto , Anticonvulsivantes/farmacología , Mapeo Encefálico , Epilepsia/tratamiento farmacológico , Función Ejecutiva/efectos de los fármacos , Femenino , Estudios de Seguimiento , Análisis de Fourier , Humanos , Levetiracetam , Modelos Lineales , Masculino , Persona de Mediana Edad , Pruebas Neuropsicológicas , Piracetam/farmacología , Piracetam/uso terapéutico , Estudios Retrospectivos , Aprendizaje Verbal/efectos de los fármacos , Adulto JovenRESUMEN
We aimed to develop a delivery system capable of maintaining a sustained release of protein drugs at specific sites using potentially biocompatible biomaterials. Here, we used bovine serum albumin (BSA) as a test protein to explore the potential utility of an injectable small intestine submucosa (SIS) as a depot for protein drugs. The prepared SIS powder was dispersed in PBS. The SIS suspension easily entrapped BSA in pharmaceutical formulations at room temperature. When this was suspension subcutaneously injected into rats, it gelled, forming an interconnecting three-dimensional network SIS structure to allow BSA to penetrate through it. The amount of BSA-FITC released from the SIS gel was determined in rat plasma and monitored by real-time in vivo molecular imaging. The data indicated the sustained release of BSA-FITC for 30 days in vivo. In addition, SIS gel provoked little inflammatory response. Collectively, our results show that the SIS gel described here could serve as a minimally invasive therapeutics depot with numerous benefits compared to other injectable biomaterials.
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Materiales Biocompatibles , Portadores de Fármacos/farmacocinética , Fluoresceína-5-Isotiocianato/análogos & derivados , Yeyuno , Albúmina Sérica Bovina/farmacocinética , Animales , Disponibilidad Biológica , Preparaciones de Acción Retardada , Portadores de Fármacos/administración & dosificación , Emulsiones , Fluoresceína-5-Isotiocianato/administración & dosificación , Fluoresceína-5-Isotiocianato/farmacocinética , Geles , Inyecciones Subcutáneas , Masculino , Ratones , Ratones Desnudos , Microscopía Electrónica de Rastreo , Ratas , Ratas Sprague-Dawley , Albúmina Sérica Bovina/administración & dosificaciónRESUMEN
Microbial phytases are widely used as feed additive to increase phytate phosphorus utilization and to reduce fecal phytates and inorganic phosphate (iP) outputs. To facilitate the process of application, we engineered an Escherichia coli appA phytase gene into the chloroplast genome of the model microalga, Chlamydomonas reinhardtii, and isolated homoplasmic plastid transformants. The catalytic activity of the recombinant E. coli AppA can be directly detected in the whole-cell lysate, termed Chlasate, prepared by freeze-drying the transgenic cell paste with liquid nitrogen. The E. coli AppA in the Chlasate has a pH and temperature optima of 4.5 and 60°C, respectively, which are similar to those described in the literature. The phytase-expressed Chlasate contains 10 phytase units per gram dry matter at pH 4.5 and 37°C. Using this transgenic Chlasate at 500 U/kg of diet for young broiler chicks, the fecal phytate excretion was reduced, and the iP was increased by 43% and 41%, respectively, as compared to those of the chicks fed with only the basal diet. The effectiveness of the Chlasate to break down the dietary phytates is compatible with the commercial Natuphos fungal phytase. Our data provide the first evidence of functional expression of microbial phytase in microalgae and demonstrate the proof of concept of using transgenic microalgae as a food additive to deliver dietary enzymes with no need of protein purification.
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6-Fitasa/metabolismo , Fosfatasa Ácida/metabolismo , Chlamydomonas reinhardtii/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Microalgas , Ácido Fítico/metabolismo , 6-Fitasa/administración & dosificación , 6-Fitasa/genética , Fosfatasa Ácida/administración & dosificación , Fosfatasa Ácida/genética , Alimentación Animal , Animales , Pollos/metabolismo , Cloroplastos/genética , Digestión/genética , Escherichia coli/genética , Proteínas de Escherichia coli/administración & dosificación , Proteínas de Escherichia coli/genética , Ingeniería Genética , Estiércol/microbiología , Microalgas/enzimología , Microalgas/genética , Microalgas/metabolismo , Reacción en Cadena de la PolimerasaRESUMEN
The effectiveness of systemically administered anticancer treatments is limited by difficulties in achieving therapeutic doses within tumors, a problem that is complicated by dose-limiting side effects to normal tissue. This work examined injectable in situ-forming gels as a localized drug-delivery system. An MPEG-PCL (MP) solution containing doxorubicin (Dox) existed in an emulsion-sol state at room temperature and rapidly gelled in vitro and in vivo at body temperature. The release of Dox from Dox-loaded MP gels was sustained in vitro over 20 days after an initial burst, indicating that the MP gel acted as a drug depot. Dox-loaded MP gels exhibited remarkable in vitro anti-proliferative activities against B16F10 cancer cells. In vivo experiments employing B16F10 cancer cell xenograft-bearing mice showed that a single intratumoral injection of Dox-loaded MP gel inhibited the growth of tumors as effectively as repeated injections of free Dox, and more effectively than a single dose of free Dox, or saline or gel alone. Consistent with the observed suppression of tumor growth, intratumorally injected free Dox or Dox released from Dox-loaded MP gels caused apoptosis of tumor cells. The tumor biodistribution of free Dox after 1 day was â¼90%, which dropped to â¼15% after 4 days. The biodistribution of Dox following a single injection of Dox-loaded MP gel was also â¼90% on day 1, but remained at â¼13%, even after 15 days. Only a small amount of Dox was found in other organ tissues following intratumoral injection, implying fewer off-target side effects.
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Antibióticos Antineoplásicos , Doxorrubicina , Geles/química , Neoplasias/tratamiento farmacológico , Poliésteres , Polietilenglicoles , Polímeros , Animales , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/metabolismo , Materiales Biocompatibles/química , Materiales Biocompatibles/metabolismo , Doxorrubicina/administración & dosificación , Doxorrubicina/uso terapéutico , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Sistemas de Liberación de Medicamentos , Humanos , Inyecciones , Ensayo de Materiales , Ratones , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Neoplasias/patología , Poliésteres/química , Poliésteres/metabolismo , Polietilenglicoles/química , Polietilenglicoles/metabolismo , Polímeros/química , Polímeros/metabolismo , Distribución Tisular , Trasplante HeterólogoRESUMEN
UNLABELLED: Epithelial cell adhesion molecule (EpCAM) is a surface marker on human hepatic stem/progenitor cells that is reported as absent on mature hepatocytes. However, it has also been noted that in cirrhotic livers of diverse causes, many hepatocytes have EpCAM surface expression; this may represent aberrant EpCAM expression in injured hepatocytes or, as we now hypothesize, persistence of EpCAM in hepatocytes that have recently derived from hepatobiliary progenitors. To evaluate this concept, we investigated patterns of EpCAM expression in hepatobiliary cell compartments of liver biopsy specimens from patients with all stages of chronic hepatitis B and C, studying proliferation, senescence and telomere lengths. We found that EpCAM(+) hepatocytes were rare in early stages of disease, became increasingly prominent in later stages in parallel with the emergence of ductular reactions, and were consistently arrayed around the periphery of cords of keratin 19(+) hepatobiliary cells of the ductular reaction, with which they shared EpCAM expression. Proliferating cell nuclear antigen (proliferation marker) and p21 (senescence marker) were both higher in hepatocytes in cirrhosis than in normal livers, but ductular reaction hepatobiliary cells had the highest proliferation rate, in keeping with being stem/progenitor cell-derived transit amplifying cells. Telomere lengths in EpCAM(+) hepatocytes in cirrhosis were higher than EpCAM(-) hepatocytes (P < 0.046), and relatively shorter than those in the corresponding ductular reaction hepatobiliary cells (P = 0.057). CONCLUSION: These morphologic, topographic, immunophenotypic, and molecular data support the concept that EpCAM(+) hepatocytes in chronic viral hepatitis are recent progeny of the hepatobiliary stem/progenitor cell compartment through intermediates of the transit amplifying, ductular reaction hepatobiliary cells.
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Antígenos de Neoplasias/metabolismo , Moléculas de Adhesión Celular/metabolismo , Hepatocitos/metabolismo , Células Madre/citología , Proliferación Celular , Senescencia Celular , Molécula de Adhesión Celular Epitelial , Hepatitis B Crónica/metabolismo , Hepatitis B Crónica/patología , Hepatitis C Crónica/metabolismo , Hepatitis C Crónica/patología , Hepatocitos/citología , Hepatocitos/patología , Humanos , Hígado/citología , Cirrosis Hepática/patología , Telómero/metabolismoRESUMEN
UNLABELLED: Large liver cell change (LLCC) refers to microscopic lesions often found in various chronic liver diseases; however, its nature is still controversial. Thirty-four formalin-fixed and 19 fresh frozen hepatitis B virus (HBV)-related cirrhosis samples were examined for the presence of LLCC, small liver cell change (SLCC), and hepatocellular carcinoma (HCC). The cell cycle checkpoint status (p21, p27, p16, Tp53), cell dynamics (proliferating cell nuclear antigen, Ki-67, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling, M30), DNA damage (gamma-H2AX [H2A histone family, member X]), telomere lengths, chromosomal instability (micronuclei index), and senescence-associated beta-galactosidase (SA-beta-Gal) activity were evaluated using an in situ approach and compared to those in normal liver (n = 5) and liver with chronic cholestasis (34 cases of hepatolithiasis and three cases of primary biliary cirrhosis). In HBV-related cirrhosis, the p21, p27, and p16 cell cycle checkpoint markers were activated in normal-looking cirrhotic hepatocytes (NLCH), but diminished gradually from LLCC, SLCC, to HCC, with an increase in Tp53 expression. There was a general decrease in telomere length from NLCH, LLCC, SLCC, to HCC. Micronuclei, gamma-H2AX foci, and net cellular gain were significantly increased from normal hepatocytes, NLCH, LLCC, SLCC, to HCC. The SA-beta-Gal activity was weaker in LLCC compared to NLCH and absent in SLCC and HCC. In contrast, cholestatic LLCC showed retained expression of cell cycle checkpoint markers and decreased net cellular gain compared to adjacent normal-looking hepatocytes. HBV-related LLCC showed significantly higher Tp53 labeling index, gamma-H2AX labeling index, and micronuclei index; shorter telomere length; decreased SA-beta-Gal activity; and increased net cellular gain compared to cholestatic LLCC. CONCLUSION: The nature of LLCC is rather heterogeneous depending on the biological setting. The characteristics of HBV-related LLCC are more consistent with dysplastic rather than merely reactive hepatocytes, whereas cholestatic LLCC more likely represents reactive change with more stringent cell cycle checkpoint control.
Asunto(s)
Hepatitis B Crónica/patología , Cirrosis Hepática/patología , Adulto , Anciano , Apoptosis , Carcinoma Hepatocelular/patología , Ciclo Celular , Proliferación Celular , Senescencia Celular/fisiología , Colestasis Intrahepática/metabolismo , Colestasis Intrahepática/patología , Inestabilidad Cromosómica , Daño del ADN , Femenino , Hepatitis B Crónica/metabolismo , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Cirrosis Hepática/metabolismo , Neoplasias Hepáticas/patología , Persona de Mediana Edad , Telómero/química , beta-Galactosidasa/análisisRESUMEN
Systemic analysis for chromosomal instability and inactivation of cell cycle checkpoints are scarce during hepatocarcinogenesis. We studied 24 patients with chronic B viral cirrhosis including 30 cirrhotic regenerative nodules, 35 low-grade dysplastic nodules, 15 high-grade dysplastic nodules, 7 dysplastic nodules with hepatocellular carcinoma foci, and 18 hepatocellular carcinomas. Eight normal livers were studied as the control group. Telomere length and micronuclei were detected by Southern blot and Feulgen-fast green dyeing technique, respectively, and p21(WAF1/CIP1) expression was studied by immunohistochemistry. Micronuclei >1 per 3000 hepatocytes were found in 17% of low-grade dysplastic nodules, 87% of high-grade dysplastic nodules, and 100% of high-grade dysplastic nodules with hepatocellular carcinoma foci and hepatocellular carcinomas in contrast to those of all normal livers, and 90% of cirrhosis showed no micronuclei. The micronuclei index showed a gradual increase during hepatocarcinogenesis and there was a significant increase between cirrhosis and low-grade dysplastic nodules, low-grade dysplastic nodules and high-grade dysplastic nodules, and high-grade dysplastic nodules and hepatocellular carcinomas. Telomere length showed a gradual shortening during hepatocarcinogenesis and a significant reduction was found in high-grade dysplastic nodules (P=0.024) and hepatocellular carcinomas (P=0.031) compared with normal and cirrhotic livers. The micronuclei index was correlated with telomere shortening (P=0.016). The p21(WAF1/CIP1) labeling index was significantly higher in cirrhosis than in normal livers (P=0.024) and markedly decreased in low-grade dysplastic nodules, high-grade dysplastic nodules, and hepatocellular carcinomas compared with cirrhosis (P<0.05). The p21(WAF1/CIP1) labeling index was associated with telomere length (P<0.001) but not micronuclei index. This study shows that telomere shortening, chromosomal instability, and inactivation of p21(WAF1/CIP1) checkpoint function occur in low-grade dysplastic nodules as well as in high-grade dysplastic nodules, and their cooperation is considered to be critical for malignant transformation during hepatitis B virus associated-multistep hepatocarcinogenesis.
Asunto(s)
Carcinoma Hepatocelular/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Hepatitis B Crónica/genética , Neoplasias Hepáticas/genética , Lesiones Precancerosas/genética , Telómero/patología , Adulto , Southern Blotting , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Inestabilidad Cromosómica , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Silenciador del Gen , Hepatitis B Crónica/complicaciones , Hepatitis B Crónica/patología , Humanos , Inmunohistoquímica , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Masculino , Micronúcleos con Defecto Cromosómico , Persona de Mediana Edad , Lesiones Precancerosas/patología , Lesiones Precancerosas/virologíaRESUMEN
Hepatitis B virus X protein (HBx) is involved in viral metabolism and progression of liver disease. Iron metabolism plays a significant role in liver disease. In this report, to elucidate the relationship between iron metabolism and HBx, we established the Huh7 cell lines in which HBx was stably expressed (Huh7-HBx). In Huh7-HBx, we observed that transferrin receptor 1 (TfR1) expression decreased and ferritin heavy chain (FtH) expression increased as well as reactive oxygen species (ROS) level increased. We also found that these modulations were caused by the downregulation of iron regulatory protein 1 (IRP1). Furthermore, the levels of total iron and labile iron pool (LIP) were altered in Huh7-HBx. In addition, antioxidant N-acetylcystein (NaC) increased IRP1 expression by depleting HBx-induced ROS. We also confirmed these alterations of TfR1 and FtH in the primary hepatocytes of HBx transgenic mice and in HepG2.2.15 cells that constitutively replicate the intact HBV genome. In conclusion, these results suggest that HBx modulates iron metabolism via ROS leading to pathological status in liver diseases.
Asunto(s)
Regulación Viral de la Expresión Génica , Proteína 1 Reguladora de Hierro/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transactivadores/metabolismo , Animales , Antígenos CD/metabolismo , Apoferritinas/metabolismo , Células Cultivadas , Ferritinas , Hepatocitos , Humanos , Hierro/metabolismo , Proteína 1 Reguladora de Hierro/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de Transferrina/metabolismo , Proteínas Reguladoras y Accesorias ViralesRESUMEN
Human PinX1 involves in regulation of telomere length. Here, we describe the function of a rat homolog of PinX1. Rat PinX1 (rPinX1) was cloned from WB-F344, a rat hepatic stem-like epithelial cell. It encodes a protein of 331 amino acids with 70% homology to human PinX1 and 91% homology to mouse. Northern analysis revealed that rPinX1 is expressed in both somatic and germ tissues, most abundantly in heart, liver and testis. Co-localization with a nucleolar protein, fibrillarin, showed that rPinX1 resides in the nucleolus. Analysis of truncated mutants revealed that an internal K,E/D region seems to be important for nucleolar localization. A stable cell line expressing rPinX1 was established in NIH3T3, a mouse-transformed embryonic fibroblast cell line, and stable cells were subcultured for more than 150 population doublings. The growth of stable rPinX1 cells slowed down at late passages, and a fraction of these cells exhibited increased size and stained positively for senescence-associated beta-galactosidase. Overexpression of rPinX1 in NIH3T3 cells resulted in gradual telomere shortening over successive passages. However, the telomeric 3' overhang was not altered by PinX1 expression. This study demonstrates that a rat homolog of human PinX1 is a nucleolar protein, and that overexpression of rPinX1 induces cellular senescence and telomere shortening, but has no effect on 3' overhang length. The function of PinX1 in regulating telomere length is conserved in rodents, and this study may provide insight into the mechanism by which a nucleolar protein can regulate telomere length.