RESUMEN
Diabetic nephropathy (DN) is a major healthcare challenge for individuals with diabetes and associated with increased cardiovascular morbidity and mortality. The existing rodent models do not fully represent the complex course of the human disease. Hence, developing a translational model of diabetes that reproduces both the early and the advanced characteristics of DN and faithfully recapitulates the overall human pathology is an unmet need. Here, we introduce the Nile grass rat (NGR) as a novel model of DN and characterize key pathologies underlying DN. NGRs spontaneously developed insulin resistance, reactive hyperinsulinemia, and hyperglycemia. Diabetic NGRs evolved DN and the key histopathological aspects of the human advanced DN, including glomerular hypertrophy, infiltration of mononuclear cells, tubular dilatation, and atrophy. Enlargement of the glomerular tufts and the Bowman's capsule areas accompanied the expansion of the Bowman's space. Glomerular sclerosis, renal arteriolar hyalinosis, Kimmelsteil-Wilson nodular lesions, and protein cast formations in the kidneys of diabetic NGR occurred with DN. Diabetic kidneys displayed interstitial and glomerular fibrosis, key characteristics of late human pathology as well as thickening of the glomerular basement membrane and podocyte effacement. Signs of injury included glomerular lipid accumulation, significantly more apoptotic cells, and expression of KIM-1. Diabetic NGRs became hypertensive, a known risk factor for kidney dysfunction, and showed decreased glomerular filtration rate. Diabetic NGRs recapitulate the breadth of human DN pathology and reproduce the consequences of chronic kidney disease, including injury and loss of function of the kidney. Hence, NGR represents a robust model for studying DN-related complications and provides a new foundation for more detailed mechanistic studies of the genesis of nephropathy, and the development of new therapeutic approaches.
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Nefropatías Diabéticas , Modelos Animales de Enfermedad , Animales , Nefropatías Diabéticas/patología , Nefropatías Diabéticas/metabolismo , Ratas , Masculino , Humanos , Resistencia a la Insulina , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/complicaciones , Riñón/patología , Riñón/metabolismo , Glomérulos Renales/patología , Glomérulos Renales/metabolismoRESUMEN
BACKGROUND: This work elucidates the first cellular and molecular causes of cataractogenesis. Current paradigm presupposes elevated blood glucose as a prerequisite in diabetic cataractogenesis. Novel evidence in our model of diabetic cataract challenges this notion and introduces immune cell migration to the lens and epithelial-mesenchymal transformation (EMT) of lens epithelial cells (LECs) as underlying causes. METHODS: Paucity of suitable animal models has hampered mechanistic studies of diabetic cataract, as most studies were traditionally carried out in acutely induced hyperglycemic animals. We introduced diabetic cataract in the Nile grass rat (NGR) that spontaneously develops type 2 diabetes (T2D) and showed its closeness to the human condition. Specialized stereo microscopy with dual bright-field illumination revealed novel hyperreflective dot-like microlesions in the inner cortical regions of the lens. To study immune cell migration to the lens, we developed a unique in situ microscopy technique of the inner eye globe in combination with immunohistochemistry. RESULTS: Contrary to the existing paradigm, in about half of the animals, the newly introduced hyper reflective dot-like microlesions preceded hyperglycemia. Even though the animals were normoglycemic, we found significant changes in their oral glucose tolerance test (OGTT), indicative of the prediabetic stage. The microlesions were accompanied with significant immune cell migration from the ciliary bodies to the lens, as revealed in our novel in situ microscopy technique. Immune cells adhered to the lens surface, some traversed the lens capsule, and colocalized with apoptotic nuclei of the lens epithelial cells (LECs). Extracellular degradations, amorphous material accumulations, and changes in E-cadherin expressions showed epithelial-mesenchymal transformation (EMT) in LECs. Subsequently, lens fiber disintegration and cataract progression extended into cortical, posterior, and anterior subcapsular cataracts. CONCLUSIONS: Our results establish a novel role for immune cells in LEC transformation and death. The fact that cataract formation precedes hyperglycemia challenges the prevailing paradigm that glucose initiates or is necessary for initiation of the pathogenesis. Novel evidence shows that molecular and cellular complications of diabetes start during the prediabetic state. These results have foreseeable ramifications for early diagnosis, prevention and development of new treatment strategies in patients with diabetes.
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Catarata , Diabetes Mellitus Tipo 2 , Hiperglucemia , Cristalino , Humanos , Animales , Diabetes Mellitus Tipo 2/complicaciones , Murinae , Cristalino/metabolismo , Cristalino/patología , Catarata/etiología , Catarata/metabolismo , Catarata/patología , Hiperglucemia/complicaciones , Hiperglucemia/metabolismo , Hiperglucemia/patología , Células Epiteliales/metabolismoRESUMEN
Diabetes is a major risk factor for cataract, the leading cause of blindness worldwide. There is an unmet need for a realistic model of diabetic cataract for mechanistic and longitudinal studies, as existing models do not reflect key aspects of the complex human disease. Here, we introduce and characterize diabetic cataract in the Nile grass rat (NGR, Arvicanthis niloticus), an established model of metabolic syndrome and type 2 diabetes (T2D). We conducted a longitudinal study of cataract in over 88 NGRs in their non-diabetic, pre-diabetic, and diabetic stages of metabolism. Oral glucose tolerance test (OGTT) results distinguished the metabolic stages. Diverse cataract types were observed in the course of diabetes, including cortical, posterior subcapsular (PSC), and anterior subcapsular (ASC), all of which succeeded a characteristic dotted ring stage in all animals. The onset ages of diabetes and cataract were 44 ± 3 vs 29 ± 1 (P < .001) and 66 ± 5 vs 58 ± 6 (not significant) weeks in females and males, respectively. Histological analysis revealed fiber disorganization, vacuolar structures, and cellular proliferation and migration in cataractous lenses. The lens epithelial cells (LECs) in non-diabetic young NGRs expressed the stress marker GRP78, as did LECs and migrated cells in the lenses of diabetic animals. Elucidating mechanisms underlying LEC proliferation and migration will be clinically valuable in prevention and treatment of posterior capsule opacification, a dreaded complication of cataract surgery. Marked changes in N-cadherin expression emphasized a role for LEC integrity in cataractogenesis. Apoptotic cells were dispersed in the equatorial areas in early cataractogenesis. Our study reveals diverse cataract types that spontaneously develop in the diabetic NGR, and which uniquely mirror the cataract and its chronic course of development in individuals with diabetes. We provide mechanistic insights into early stages of diabetic cataract. These unique characteristics make NGR highly suited for mechanistic studies, especially in the context of metabolism, diabetes, and aging.
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Catarata/patología , Complicaciones de la Diabetes/patología , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Tipo 2/complicaciones , Células Epiteliales/patología , Cristalino/patología , Animales , Catarata/etiología , Movimiento Celular , Proliferación Celular , Complicaciones de la Diabetes/etiología , Chaperón BiP del Retículo Endoplásmico , Femenino , Estudios Longitudinales , Masculino , Fenotipo , RatasRESUMEN
Cancer stem cells (CSCs) are responsible for therapeutic resistance and recurrence in colorectal cancer. Despite advances in immunotherapy, the inability to specifically eradicate CSCs has led to treatment failure. Hence, identification of appropriate antigen sources is a major challenge in designing dendritic cell (DC)-based therapeutic strategies against CSCs. Here, in an in vitro model using the HT-29 colon cancer cell line, we explored the efficacy of DCs loaded with exosomes derived from CSC-enriched colonospheres (CSCenr -EXOs) as an antigen source in activating CSC-specific T-cell responses. HT-29 lysate, HT-29-EXOs and CSCenr lysate were independently assessed as separate antigen sources. Having confirmed CSCs enrichment in spheroids, CSCenr -EXOs were purified and characterized, and their impact on DC maturation was investigated. Finally, the impact of the antigen-pulsed DCs on the proliferation rate and also spheroid destructive capacity of autologous T cells was assessed. CSCenr -EXOs similar to other antigen groups had no suppressive/negative impacts on phenotypic maturation of DCs as judged by the expression level of costimulatory molecules. Notably, similar to CSCenr lysate, CSCenr -EXOs significantly increased the IL-12/IL-10 ratio in supernatants of mature DCs. CSCenr -EXO-loaded DCs effectively promoted T-cell proliferation. Importantly, T cells stimulated with CSCenr -EXOs disrupted spheroids' structure. Thus, CSCenr -EXOs present a novel and promising antigen source that in combination with conventional tumour bulk-derived antigens should be further explored in pre-clinical immunotherapeutic settings for the efficacy in hampering recurrence and metastatic spread.
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Células Dendríticas/inmunología , Exosomas/inmunología , Inmunoterapia/métodos , Células Madre Neoplásicas/inmunología , Esferoides Celulares/inmunología , Células Cultivadas , Células HT29 , Humanos , Interleucinas/metabolismo , Esferoides Celulares/citología , Linfocitos T/inmunologíaRESUMEN
In retinal degenerative disorders, when neural retinal cells are damaged, cell transplantation is one of the most promising therapeutic approaches. Optogenetic technology plays an essential role in the neural differentiation of stem cells via membrane depolarization. This study explored the efficacy of blue light stimulation in neuroretinal differentiation of Opto-mGluR6-engineered mouse retinal pigment epithelium (mRPE) and bone marrow mesenchymal stem cells (BMSCs). mRPE and BMSCs were selected for optogenetic study due to their capability to differentiate into retinal-specific neurons. BMSCs were isolated and phenotypically characterized by the expression of mesenchymal stem cell-specific markers, CD44 (99%) and CD105 (98.8%). mRPE culture identity was confirmed by expression of RPE-specific marker, RPE65, and epithelial cell marker, ZO-1. mRPE cells and BMSCs were transduced with AAV-MCS-IRES-EGFP-Opto-mGluR6 viral vector and stimulated for 5 days with blue light (470 nm). RNA and protein expression of Opto-mGluR6 were verified. Optogenetic stimulation-induced elevated intracellular Ca2+ levels in mRPE- and BMS-treated cells. Significant increase in cell growth rate and G1/S phase transition were detected in mRPE- and BMSCs-treated cultures. Pou4f1, Dlx2, Eomes, Barlh2, Neurod2, Neurod6, Rorb, Rxrg, Nr2f2, Ascl1, Hes5, and Sox8 were overexpressed in treated BMSCs and Barlh2, Rorb, and Sox8 were overexpressed in treated mRPE cells. Expression of Rho, Thy1, OPN1MW, Recoverin, and CRABP, as retinal-specific neuron markers, in mRPE and BMS cell cultures were demonstrated. Differentiation of ganglion, amacrine, photoreceptor cells, and bipolar and Muller precursors were determined in BMSCs-treated culture and were compared with mRPE. mRPE cells represented more abundant terminal Muller glial differentiation compared with BMSCs. Our results also demonstrated that optical stimulation increased the intracellular Ca2+ level and proliferation and differentiation of Opto-mGluR6-engineered BMSCs. It seems that optogenetic stimulation of mRPE- and BMSCs-engineered cells would be a potential therapeutic approach for retinal degenerative disorders.
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Diferenciación Celular , Células Madre Mesenquimatosas/metabolismo , Neuronas/metabolismo , Optogenética , Epitelio Pigmentado de la Retina/metabolismo , Animales , Línea Celular , Células Madre Mesenquimatosas/citología , Ratones , Neuronas/citología , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismo , Epitelio Pigmentado de la Retina/citologíaRESUMEN
Macrophages are the main infiltrating immune cells in choroidal neovascularization (CNV), a hallmark of the human wet, or neovascular age-related macular degeneration (AMD). Due to their plasticity and ability to adapt to the local microenvironment in a tissue-dependent manner, macrophages display polar functional phenotypes characterized by their cell surface markers and their cytokine profiles. We found accumulation of hemoglobin-scavenging cluster of differentiation 163 (CD163)(+) macrophages in laser-induced CNV lesions and higher expression of CD163(+) monocytes in the peripheral blood on day 7 post injury in mice. In comparison, CD80(+) macrophages did not differ with laser-injury in young or aged mice and did not significantly change in the peripheral blood of CNV mice. We examined the percentages of CD163(+), CD206(+), and CD80(+) monocytes in the peripheral blood of patients with wet AMD, patients with dry AMD, and in age-matched individuals without AMD as controls. Percentages of peripheral blood CD163(+) monocytes in both dry AMD (P < .001) and wet AMD (P < .05) were higher than in age-matched non-AMD controls, while there was no difference between the groups in the percentages of peripheral CD206(+) and CD80(+) monocytes. Further, serum level of soluble CD163 (sCD163) was elevated only in patients with wet AMD (P < .05). An examination of 40 cytokine levels across the study groups revealed that anti-VEGF treated patients with wet AMD, who showed no exudative signs on the day of blood drawing had a cytokine profile that was similar to that of non-AMD individuals. These results indicate that CD163 could be further evaluated for its potential as a useful marker of disease activity in patients with neovascular AMD. Future studies will address the origin and potential mechanistic role of CD163(+) macrophages in wet AMD pathologies of angiogenesis and leakage of blood components.
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Antígenos CD/sangre , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/sangre , Antígenos de Diferenciación Mielomonocítica/metabolismo , Monocitos/metabolismo , Receptores de Superficie Celular/sangre , Receptores de Superficie Celular/metabolismo , Degeneración Macular Húmeda/sangre , Degeneración Macular Húmeda/metabolismo , Anciano , Inhibidores de la Angiogénesis/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Neovascularización Coroidal/sangre , Neovascularización Coroidal/metabolismo , Femenino , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Monocitos/efectos de los fármacos , Retina/efectos de los fármacos , Retina/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Agudeza Visual/efectos de los fármacos , Agudeza Visual/fisiología , Degeneración Macular Húmeda/tratamiento farmacológicoRESUMEN
Aims: DLL4 of the Notch pathway is a key regulator of VEGF expression, which mediates tumor neovascularization and stem cell self-renewal in colorectal cancer (CRC). The authors investigated the association of DLL4 expression with the clinicopathological characteristics and survival outcomes of CRC patients. Methods: DLL4 expression level was evaluated in 199 CRC samples using immunohistochemistry analysis of tissue microarrays. Results: The high expression of DLL4 was inversely associated with distant metastasis (p < 0.029), tumor recurrence (p < 0.04) and longer overall survival following curative surgery compared with those with low DLL4 expression with 95% CI (log-rank test: p = 0.050). In univariate analysis, histological grade (hazard ratio: 3.859; 95% CI: 1.081-13.784; p = 0.038) was a strong prognostic risk factor, affecting the overall survival of CRC patients. Conclusion: The authors' results demonstrate that DLL4 expression might be considered a favorable prognostic factor for overall survival in CRC patients.
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Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Biomarcadores de Tumor/metabolismo , Proteínas de Unión al Calcio/biosíntesis , Neoplasias Colorrectales/patología , Adulto , Anciano , Citoplasma/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , PronósticoRESUMEN
Cell replacement is a promising therapy for degenerative diseases like age-related macular degeneration (AMD). Since the human retina lacks regeneration capacity, much attention has been directed toward persuading for cells that can differentiate into retinal neurons. In this report, we have investigated reprogramming of the human RPE cells and concerned the effect of donor age on the cellular fate as a critical determinant in reprogramming competence. We evaluated the effect of SOX2 over-expression in human neonatal and adult RPE cells in cultures. The coding region of human SOX2 gene was cloned into adeno-associated virus (AAV2) and primary culture of human neonatal/adult RPE cells were infected by recombinant virus. De-differentiation of RPE to neural/retinal progenitor cells was investigated by quantitative real-time PCR and ICC for neural/retinal progenitor cells' markers. Gene expression analysis showed 80-fold and 12-fold over-expression for SOX2 gene in infected neonatal and adult hRPE cells, respectively. The fold of increase for Nestin in neonatal and adult hRPE cells was 3.8-fold and 2.5-fold, respectively. PAX6 expression was increased threefold and 2.5-fold in neonatal/adult treated cultures. Howbeit, we could not detect rhodopsin, and CHX10 expression in neonatal hRPE cultures and expression of rhodopsin in adult hRPE cells. Results showed SOX2 induced human neonatal/adult RPE cells to de-differentiate toward retinal progenitor cells. However, the increased number of PAX6, CHX10, Thy1, and rhodopsin positive cells in adult hRPE treated cultures clearly indicated the considerable generation of neuro-retinal terminally differentiated cells.
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Desdiferenciación Celular , Reprogramación Celular , Dependovirus/genética , Células Epiteliales/metabolismo , Vectores Genéticos , Epitelio Pigmentado de la Retina/metabolismo , Factores de Transcripción SOXB1/metabolismo , Células Madre/metabolismo , Transducción Genética , Transfección/métodos , Adulto , Biomarcadores/metabolismo , Células Cultivadas , Técnicas de Reprogramación Celular , Regulación del Desarrollo de la Expresión Génica , Humanos , Recién Nacido , Fenotipo , Factores de Transcripción SOXB1/genéticaRESUMEN
The retinal pigment epithelium is a monolayer of highly specialized pigmented cells located between the neural retina and the Bruch's membrane of the choroid. RPE cells play a crucial role in the maintenance and function of the underlying photoreceptors. This study introduces a spontaneously arising human retinal pigment epithelial cell line, HRPE-2S, which was isolated from primary RPE cell culture of 2 days old male donor. We characterized morphology and functional properties of the new cell line. The immortalized cell line was maintained in culture for more than 70 passages and 240 divisions. The average doubling time of the cells was approximately 22 h and got freezed at 26th passage. The cell line expressed RPE-specific markers RPE65 and cell junction protein ZO1 as an epithelial cell marker. It also expressed CHX10, PAX6, Nestin, SOX2 as stem and retinal progenitor cell markers. Ki67 as a marker of cell proliferation was expressed in all HRPE-2S cells. It represented typical epithelial cobblestone morphology and did not phenotypically change through several passages. Stem cell-like aggregations (neurospheres) were observed in SEM microscopy. The cells represented high mitotic index. They could be viable under hypoxic conditions and serum deprivation. According to functional studies, the cell line exhibited stem cell-like behaviors with particular emphasis on its self-renewal capacity. LDH isoenzymes expression pattern confirmed the same cellular source for both of the HRPE-2S cells and primary RPE cells. Characteristics of HRPE-2S cells promise it as an in vitro model for RPE stem cell-based researches. J. Cell. Physiol. 232: 2626-2640, 2017. © 2016 Wiley Periodicals, Inc.
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Separación Celular/métodos , Células Epiteliales/fisiología , Epitelio Pigmentado de la Retina/fisiología , Células Madre/fisiología , Biomarcadores/metabolismo , Hipoxia de la Célula , Línea Celular Transformada , Movimiento Celular , Proliferación Celular , Autorrenovación de las Células , Forma de la Célula , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Proteínas de Homeodominio/metabolismo , Humanos , Recién Nacido , Antígeno Ki-67/metabolismo , Masculino , Microscopía Electrónica de Rastreo , Índice Mitótico , Nestina/metabolismo , Factor de Transcripción PAX6/metabolismo , Fenotipo , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/ultraestructura , Factores de Transcripción SOXB1/metabolismo , Esferoides Celulares , Células Madre/metabolismo , Células Madre/ultraestructura , Factores de Tiempo , Factores de Transcripción/metabolismo , Proteína de la Zonula Occludens-1/metabolismo , cis-trans-Isomerasas/metabolismoRESUMEN
Retinal pigmented epithelium (RPE), the outermost layer of the retina, has a key role in maintaining retinal cells' functions. Severity of the culture of RPE cells has exerted many limitations to both in vitro and in vivo studies and its therapeutic applications. Therefore, establishment of RPE cell lines with high proliferative potential can considerably improve study of RPE cell biology. Here we report generation of a spontaneously immortalized murine RPE cell line in primary mouse RPE cell culture. Founded colonized cells were picked up and expression of RPE and retinal progenitor cells' (RPC) markers were studied using immunocytochemistry (ICC). Emerged cells cultured over 35 passages and population doubling times in different serum concentrations were calculated. We also investigated the ability of cells for becoming transfected by calcium-phosphate method and for becoming infected by adeno-associated virus serotype 2 (AAV2) using flow cytometry. Data showed that the cobblestone constituent cells expressed RPE65, cytokeratin and ZO1 and moreover several progenitor markers such as Pax6, Sox2, Nestin and Chx10. It revealed that, despite primary RPE cells, the newly emerged cells were easily transfectable and were highly infectable when compared with HEK293T cells. Our data indicated that the emerged mouse RPE cell line pretended RPC-like phenotype and also simultaneously expressed RPE markers. It would be a promising model for leading studies on RPE and RPC cells and substantially confirmed the great RPE plasticity and its invaluable potential in research studies.
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Modelos Biológicos , Epitelio Pigmentado de la Retina/citología , Animales , Biomarcadores/metabolismo , Línea Celular , Proliferación Celular , Separación Celular , Forma de la Célula , Células Epiteliales/citología , Femenino , Células HEK293 , Humanos , Ratones , Células Madre/metabolismo , TransfecciónRESUMEN
Diabetic retinopathy (DR) is a debilitating organ manifestation of diabetes. Absent of early diagnosis and intervention, vision tends to drastically and irreversibly decline. Previously, we showed higher vascular endothelial growth factor receptor 2 (VEGFR-2) expression in diabetic microvessels, and the suitability of this molecule as a biomarker for early DR diagnosis. However, a hurdle to translation remained generation of biodegradable nanoprobes that are sufficiently bright for in vivo detection. Here, an adhesive fluorescent nanoprobe with high brightness was developed using biodegradable materials. To achieve that, a fluorophore with bulky hydrophobic groups was encapsulated in the nanoparticles to minimize fluorophore π-π stacking, which diminishes brightness at higher loading contents. The nanoprobe selectively targeted the VEGFR-2 under dynamic flow conditions. Upon systemic injection, the nanoprobes adhered in the retinal microvessels of diabetic mice and were visualized as bright spots in live retinal microscopy. Histology validated the in vivo results and showed binding of the nanoprobes to the microvascular endothelium and firmly adhering leukocytes. Leukocytes were found laden with nanoprobes, indicating the potential for payload transport across the blood-retinal barrier. Our results establish the translational potential of these newly generated nanoprobes in early diagnosis of DR.
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Técnicas Biosensibles , Diabetes Mellitus Experimental , Retinopatía Diabética , Ratones , Animales , Retinopatía Diabética/diagnóstico , Receptor 2 de Factores de Crecimiento Endotelial Vascular , Factor A de Crecimiento Endotelial VascularRESUMEN
Immunothrombosis, an inflammation-dependent activation of the coagulation cascade, leads to microthrombi formations in small vessels. It is a dreaded complication of COVID-19 and a major cause of respiratory failure. Due to their size and disseminated nature, microthrombi are currently undetectable. Here, noninvasive detection of a volatile reporter in the exhaled air is introduced for assessment of systemic immunothrombosis. A dendritic nanoprobe, containing high loading of a thrombin-sensitive substrate, is selectively cleaved by thrombin, resulting in release of a synthetic bioorthogonal volatile organic compound (VOC). The VOC is quantitated in the exhaled air biopsies via gas chromatography-mass spectrometry (GC-MS), allowing near real-time assessment of systemic immunothrombosis. The VOC detection can be further improved with more rapid and sensitive MS-based technologies. The amount of the VOC in the exhaled air decreases with resolution of the microvascular inflammation and intravascular fibrin depositions. Through conjugation of the thrombin-sensitive peptide with a rhodol derivative, a novel thrombin-sensitive fluorescent nanoprobe is developed for intravital visualization of thrombin activity in actively growing thrombi. These results establish unprecedented detection of thrombin activity in vivo, addressing this unmet medical need. This novel approach facilitates diagnosis of immunothrombosis in diseases such as diabetic complications, disseminated intravascular coagulation, and COVID-19.
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COVID-19 , Compuestos Orgánicos Volátiles , Humanos , Tromboinflamación , Trombina , Compuestos Orgánicos Volátiles/análisis , Biopsia , COVID-19/diagnósticoRESUMEN
Local stimuli differentiate monocytes into M2-like macrophages that mechanistically drive the pathologies in cancer and age-related macular degeneration (AMD). A photo-controlled nanodrug that halts macrophage polarization through Rho-associated kinase (ROCK) inhibition was developed. A small-molecule ROCK inhibitor, fasudil, was conjugated to a photo-responsive group and a short poly(ethylene glycol) (PEG) chain. This resulted in the novel amphiphilic prodrug, PEG-2-(4'-(di(prop-2-yn-1-yl)amino)-4-nitro-[1,1'-biphenyl]-yl)propan-1-ol (PANBP)-Fasudil, that spontaneously formed micelles. Ultraviolet (UV) irradiation of PEG-PANBP-Fasudil nanoparticles rapidly released fasudil. For visualization of linker degradation, a reporter nanoprobe was synthesized, in which 2-Me-4-OMe TokyoGreen (TG), a fluorophore that does not fluoresce in conjugation, was incorporated. Irradiation of nanoprobe-laden monocytes activated the reporter fluorophore. Cytokine stimulation differentiated monocytes into macrophages, while UV irradiation prevented polarization of PEG-PANBP-Fasudil nanoparticle-laden monocytes. Nanoarchitectonics-based design opens new possibilities for intracellular drug delivery and precise spatiotemporal immune cell modulation toward the development of new therapies.
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Profármacos , Quinasas Asociadas a rho , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Citocinas/metabolismo , Liberación de Fármacos , Mercaptoetanol , Micelas , Polietilenglicoles/metabolismoRESUMEN
Background: The octamer-binding transcription factor-4 (OCT4) is known as an established important regulator of pluripotency, as well as a genetic "master switch" in the self-renewal of embryonic stem and germ cells. OCT4B1, one of the three spliced variants of human OCT4, plays crucial roles in the regulation of pluripotency and stemness. Objectives: The present study developed a transgenic mouse model containing an OCT4B1-expressing construct under the transcriptional direction of mouse mammary tumor virus promoter (pMMTV) to evaluate the role of OCT4B1 in the function of male germ cells in terms of fertility potential. Additionally, the effect of ectopic OCT4B1 overexpression on endogenous OCT4 expression was examined in mouse embryonic stem cells (mESCs). Material and Methods: The pMMTV-OCT4B1cDNA construct was injected into the pronuclei of 0.5-day NMRI embryos. Transgenic mice were identified based on the PCR analysis of tail DNA. Further, Diff-Quik staining was applied to assess sperm morphology, while the other sperm parameters were analyzed through a conventional light microscopic evaluation according to World Health Organization (WHO) criteria. The fertility rate was scored by using in vitro frtilization (IVF) method. Furthermore, mESCs was electroporated with the OCT4B1cDNA-containing constructs, followed by analyzing through employing semi-quantitative RT-PCR and western blotting. Results: The results demonstrated the changes in sperm morphology, as well as a statistically significant decrease in the other sperm parameters (count, viability, and motility) and fertility rate (p<0.05) in the transgenic mice compared with the control group. The assessment of the cause of the embryonic stem cell (ESC) death following transfection revealed a significant reduction in the endogenous OCT4 expression at both mRNA and protein levels in the transfected mESCs compared to the control ones. Conclusion: In general, the in vivo results suggested a potential role of OCT4B1 in the spermatogenesis process. These results represented that the overexpression of OCT4B1 may induce its role in spermatogenesis and fertility rate by interfering endogenous OCT4 expression. However, further studies are required to clarify the mechanisms underlying OCT4B1 function.
RESUMEN
BACKGROUND: About 90% of cancer-related deaths are due to metastasis of cancer cells, and angiogenesis is a critical step in this process. sFLT01 is a novel fusion protein and a dual-targeting agent that neutralizes both VEGF and PlGF proangiogenic activities. GRP78 dual effect in tumor growth and angiogenesis could be activated under VEGF stimulation. The current study was designed to investigate the inhibitory impact of sFLT01 protein on VEGF/GRP78 axis. To this point, sFLT01 construct was synthesized, recombinant plasmid was expressed in eukaryotic host cells, sFLT01-HisTag protein was extracted and analyzed. The functional activity of sFLT01 on VEGF-enhanced tube formation and angiogenesis of HUVEC cells were examined. Eventually, the inhibitory impact of sFLT01 on growth, invasiveness, and migration of human prostate cancer cell line, DU145, was assessed. Real-time PCR evaluated the level of GRP78 and its effect on the downstream factors; matrix metallopeptidase proteins 2&9 (MMP2&9) along with tissue inhibitor of metalloproteinase proteins1&2 (TIMP1&2) under sFLT01 stimulation. RESULTS: According to the data, sFLT01 protein showed modulatory impact on proliferation, invasion, and migration of DU145 cells along with the potential of HUVECs angiogenesis. Real-Time PCR analysis depicted a significant downregulation in GRP78, MMP2 and MMP9 transcripts' levels, and a subsequent elevation of TIMP1 and TIMP2 expression under sFLT01 stimulation was detected. CONCLUSION: Overall, these data indicated that the inhibitory impact of sFLT01 on cancer cells growth and invasiveness could be mediated through the modulation of VEGF/GRP78/MMP2&9 axis and activation of TIMPs.
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Inhibidores de la Angiogénesis , Neoplasias de la Próstata/patología , Proteínas Recombinantes de Fusión , Inhibidores de la Angiogénesis/genética , Inhibidores de la Angiogénesis/aislamiento & purificación , Inhibidores de la Angiogénesis/farmacología , Línea Celular Tumoral , Movimiento Celular , Supervivencia Celular , Chaperón BiP del Retículo Endoplásmico , Células HEK293 , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Masculino , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Invasividad Neoplásica , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/farmacología , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
Identification of immunogenic tumor antigens that are efficiently processed and delivered by dendritic cells to prime the immune system and to induce an appropriate immune response is a research hotspot in the field of cancer vaccine development. High biosafety is an additional demand. Tumor-derived exosomes (TEXs) are nanosized lipid bilayer encapsulated vesicles that shuttle bioactive information to the tumor microenvironment facilitating tumor progression. However, accumulating evidence points toward the capacity of TEXs to efficiently stimulate immune responses against tumors provided they are appropriately administered. After briefly describing the function of exosomes in cancer biology and their communication with immune cells, we summarize in this review in vitro and preclinical studies eliciting the potency of TEXs in inducing effective anti-tumor responses and recently modified strategies further improving TEX-vaccination efficacy. We interpret the available data as TEXs becoming a lead in cancer vaccination based on tumor antigen-selective high immunogenicity.
Asunto(s)
Vacunas contra el Cáncer , Exosomas , Neoplasias , Antígenos de Neoplasias , Exosomas/inmunología , Humanos , Inmunoterapia , Neoplasias/terapia , Microambiente TumoralRESUMEN
Retinal degenerative diseases, due to the lack of regeneration systems and self-renewable cells, often lead to visual impairment. Pax6 is a pleiotropic transcription factor and its expression level determines self-renewal status or differentiation of retinal cells. Here, we investigated the fate of simultaneous induction of retinal ganglion cell death and Pax6 overexpression in retro-differentiation of retinal cells and their commitment to re-enter into the cell cycle. Induction of acute retinal ganglion cell death and generation of mouse experimental model was performed by N-methyl D-aspartic acid (NMDA) injection. Recombinant AAV2 virus harboring PAX6 cDNA and reporter gene was injected into untreated and model mouse eyes. Histological analyses, including IHC and retinal flatmounts immunostaining were performed. The number of Ki67+ cells was clearly increased in model mice, presumably due to NMDA treatment and regardless of Pax6 over-expression. Unlike previous studies, Ki67+ cells were found in GCL layer and interestingly ONL cells expressed Sox2 stemness marker after NMDA cytotoxicity. The potential of retinal cells for robust Ki67 expression, after injury, and expression of Sox2, confirmed their intrinsic plasticity and made a vivid prospect for retinal regenerative medicine.