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1.
Nat Commun ; 12(1): 2369, 2021 04 22.
Artículo en Inglés | MEDLINE | ID: mdl-33888691

RESUMEN

Photoactivatable molecules enable ablation of malignant cells under the control of light, yet current agents can be ineffective at early stages of disease when target cells are similar to healthy surrounding tissues. In this work, we describe a chemical platform based on amino-substituted benzoselenadiazoles to build photoactivatable probes that mimic native metabolites as indicators of disease onset and progression. Through a series of synthetic derivatives, we have identified the key chemical groups in the benzoselenadiazole scaffold responsible for its photodynamic activity, and subsequently designed photosensitive metabolic warheads to target cells associated with various diseases, including bacterial infections and cancer. We demonstrate that versatile benzoselenadiazole metabolites can selectively kill pathogenic cells - but not healthy cells - with high precision after exposure to non-toxic visible light, reducing any potential side effects in vivo. This chemical platform provides powerful tools to exploit cellular metabolic signatures for safer therapeutic and surgical approaches.


Asunto(s)
Infecciones Bacterianas/tratamiento farmacológico , Colorantes Fluorescentes/administración & dosificación , Glioblastoma/tratamiento farmacológico , Compuestos de Organoselenio/administración & dosificación , Fotoquimioterapia/métodos , Animales , Técnicas de Cocultivo , Colorantes Fluorescentes/efectos adversos , Colorantes Fluorescentes/química , Colorantes Fluorescentes/efectos de la radiación , Glioblastoma/patología , Humanos , Microscopía Intravital , Luz , Pruebas de Sensibilidad Microbiana , Microscopía Confocal , Microscopía Fluorescente , Compuestos de Organoselenio/efectos adversos , Compuestos de Organoselenio/química , Compuestos de Organoselenio/efectos de la radiación , Esferoides Celulares , Ensayos Antitumor por Modelo de Xenoinjerto , Pez Cebra
2.
Int J Mol Sci ; 22(6)2021 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-33804063

RESUMEN

Besides its insulinotropic actions on pancreatic ß cells, neuroprotective activities of glucagon-like peptide-1 (GLP-1) have attracted attention. The efficacy of a GLP-1 receptor (GLP-1R) agonist exendin-4 (Ex-4) for functional repair after sciatic nerve injury and amelioration of diabetic peripheral neuropathy (DPN) has been reported; however, the underlying mechanisms remain unclear. In this study, the bioactivities of Ex-4 on immortalized adult rat Schwann cells IFRS1 and adult rat dorsal root ganglion (DRG) neuron-IFRS1 co-culture system were investigated. Localization of GLP-1R in both DRG neurons and IFRS1 cells were confirmed using knockout-validated monoclonal Mab7F38 antibody. Treatment with 100 nM Ex-4 significantly enhanced survival/proliferation and migration of IFRS1 cells, as well as stimulated the movement of IFRS1 cells toward neurites emerging from DRG neuron cell bodies in the co-culture with the upregulation of myelin protein 22 and myelin protein zero. Because Ex-4 induced phosphorylation of serine/threonine-specific protein kinase AKT in these cells and its effects on DRG neurons and IFRS1 cells were attenuated by phosphatidyl inositol-3'-phosphate-kinase (PI3K) inhibitor LY294002, Ex-4 might act on both cells to activate PI3K/AKT signaling pathway, thereby promoting myelination in the co-culture. These findings imply the potential efficacy of Ex-4 toward DPN and other peripheral nerve lesions.


Asunto(s)
Neuropatías Diabéticas/tratamiento farmacológico , Exenatida/farmacología , Péptido 1 Similar al Glucagón/genética , Receptor del Péptido 1 Similar al Glucagón/genética , Animales , Movimiento Celular/genética , Supervivencia Celular/genética , Cromonas/farmacología , Técnicas de Cocultivo , Neuropatías Diabéticas/genética , Neuropatías Diabéticas/patología , Exenatida/genética , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/patología , Receptor del Péptido 1 Similar al Glucagón/agonistas , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Morfolinas/farmacología , Vaina de Mielina/genética , Vaina de Mielina/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Ratas , Células de Schwann/citología , Células de Schwann/efectos de los fármacos , Células de Schwann/metabolismo , Nervio Ciático/lesiones
3.
Int J Mol Sci ; 22(6)2021 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-33809077

RESUMEN

Pregnancy is a unique situation of physiological immunomodulation, as well as a strong Multiple Sclerosis (MS) disease modulator whose mechanisms are still unclear. Both maternal (decidua) and fetal (trophoblast) placental cells secrete extracellular vesicles (EVs), which are known to mediate cellular communication and modulate the maternal immune response. Their contribution to the MS disease course during pregnancy, however, is unexplored. Here, we provide a first phenotypic and functional characterization of EVs isolated from cultures of term placenta samples of women with MS, differentiating between decidua and trophoblast. In particular, we analyzed the expression profile of 37 surface proteins and tested the functional role of placental EVs on mono-cultures of CD14+ monocytes and co-cultures of CD4+ T and regulatory T (Treg) cells. Results indicated that placental EVs are enriched for surface markers typical of stem/progenitor cells, and that conditioning with EVs from samples of women with MS is associated to a moderate decrease in the expression of proinflammatory cytokines by activated monocytes and in the proliferation rate of activated T cells co-cultured with Tregs. Overall, our findings suggest an immunomodulatory potential of placental EVs from women with MS and set the stage for a promising research field aiming at elucidating their role in MS remission.


Asunto(s)
Vesículas Extracelulares/genética , Inmunidad/genética , Esclerosis Múltiple/genética , Proteoma/genética , Comunicación Celular/genética , Técnicas de Cocultivo , Citocinas/genética , Decidua/inmunología , Decidua/metabolismo , Vesículas Extracelulares/inmunología , Femenino , Humanos , Inmunomodulación/genética , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Placenta/inmunología , Placenta/metabolismo , Embarazo , Linfocitos T Reguladores/inmunología , Trofoblastos/inmunología , Trofoblastos/metabolismo
4.
Int J Mol Sci ; 22(6)2021 Mar 13.
Artículo en Inglés | MEDLINE | ID: mdl-33805833

RESUMEN

A large British study, with almost 3000 patients, identified diabetes as main risk factor for delayed and nonunion fracture healing, the treatment of which causes large costs for the health system. In the past years, much progress has been made to treat common complications in diabetics. However, there is still a lack of advanced strategies to treat diabetic bone diseases. To develop such therapeutic strategies, mechanisms leading to massive bone alterations in diabetics have to be well understood. We herein describe an in vitro model displaying bone metabolism frequently observed in diabetics. The model is based on osteoblastic SaOS-2 cells, which in direct coculture, stimulate THP-1 cells to form osteoclasts. While in conventional 2D cocultures formation of mineralized matrix is decreased under pre-/diabetic conditions, formation of mineralized matrix is increased in 3D cocultures. Furthermore, we demonstrate a matrix stability of the 3D carrier that is decreased under pre-/diabetic conditions, resembling the in vivo situation in type 2 diabetics. In summary, our results show that a 3D environment is required in this in vitro model to mimic alterations in bone metabolism characteristic for pre-/diabetes. The ability to measure both osteoblast and osteoclast function, and their effect on mineralization and stability of the 3D carrier offers the possibility to use this model also for other purposes, e.g., drug screenings.


Asunto(s)
Huesos/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Redes y Vías Metabólicas/genética , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Resorción Ósea/genética , Resorción Ósea/metabolismo , Resorción Ósea/patología , Huesos/patología , Calcificación Fisiológica/genética , Anhidrasa Carbónica II/genética , Anhidrasa Carbónica II/metabolismo , Catepsina K/genética , Catepsina K/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Técnicas de Cocultivo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Regulación de la Expresión Génica , Humanos , Modelos Biológicos , Osteoblastos/patología , Osteoclastos/patología , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Ligando RANK/genética , Ligando RANK/metabolismo , Células THP-1 , Fosfatasa Ácida Tartratorresistente/genética , Fosfatasa Ácida Tartratorresistente/metabolismo , Andamios del Tejido
5.
Anticancer Res ; 41(4): 1733-1744, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33813377

RESUMEN

BACKGROUND/AIM: We sought to identify the mechanisms of perineural invasion in pancreatic ductal adenocarcinoma (PDAC). MATERIALS AND METHODS: We utilized in vitro cancer cell-nerve co-culture models comprising human PDAC cell lines (MIA Paca2 and PANC-1) and a dorsal root ganglion (DRG) isolated from neonatal mice. We compared gene expression profiles between cell lines with/without DRG conditioned medium (DRG-CM) using RNA-sequencing (RNA-seq). RESULTS: Migration, invasion, and neurotropism were significantly enhanced in MIA Paca2 but not in PANC-1 cells co-cultured with DRGs. Among 285 genes which showed significant differences in expression levels between cell lines in RNA-seq, we focused on Ephrin receptor A4 (EPHA4), which was upregulated in MIA Paca2 cells treated with DRG-CM. The abilities of migration, invasion, and neurotropism enhanced by DRG co-culture were abolished when EPHA4 was knocked down by siRNA in MIA Paca2 cells. CONCLUSION: EPHA4 can be a potential target gene to regulate perineural invasion in PDAC cells.


Asunto(s)
Carcinoma Ductal Pancreático/metabolismo , Movimiento Celular , Ganglios Espinales/metabolismo , Neoplasias Pancreáticas/metabolismo , Comunicación Paracrina , Receptor EphA4/metabolismo , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Técnicas de Cocultivo , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones Endogámicos ICR , Invasividad Neoplásica , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Receptor EphA4/genética , Transducción de Señal
6.
Anticancer Res ; 41(4): 1811-1819, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33813386

RESUMEN

BACKGROUND/AIM: Glioblastoma is the most common cancer among primary brain tumors, however, its prognosis and treatment advances are very poor. Here, we investigated whether c-Met, FOLR1, and AXL proteins are promising targets for chimeric antigen receptor (CAR) T-cell therapy, for they are known to be over-expressed in a variety of solid tumors. MATERIALS AND METHODS: CAR constructs were prepared and CAR KHYG-1 cells targeting c-Met, FOLR1, or AXL were made by lentiviral transduction. The activity of CAR KHYG-1 cells against cancer cells was measured by cytokine secretion and cell lysis assays. RESULTS: c-Met and AXL were over-expressed in most glioblastoma cell lines (11/13), but not in neuroblastoma cell lines (0/8). FOLR1 was over-expressed only in one among 16 glioblastoma cell lines. Our antigen-specific CAR KHYG-1 cells eradicated target positive glioblastoma cells selectively. CONCLUSION: Anti-c-Met and anti-AXL CAR NK or T cells could be effective in glioblastoma cells.


Asunto(s)
Neoplasias Encefálicas/terapia , Glioblastoma/terapia , Inmunoterapia Adoptiva , Células Asesinas Naturales/inmunología , Proteínas Proto-Oncogénicas c-met/inmunología , Proteínas Proto-Oncogénicas/inmunología , Proteínas Tirosina Quinasas Receptoras/inmunología , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Técnicas de Cocultivo , Citocinas/metabolismo , Citotoxicidad Inmunológica , Receptor 1 de Folato/inmunología , Receptor 1 de Folato/metabolismo , Glioblastoma/inmunología , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Células Jurkat , Células Asesinas Naturales/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T/metabolismo
7.
Molecules ; 26(6)2021 Mar 14.
Artículo en Inglés | MEDLINE | ID: mdl-33799468

RESUMEN

Recent reports highlighted the significant neuroprotective effects of thyronamines (TAMs), a class of endogenous thyroid hormone derivatives. In particular, 3-iodothyronamine (T1AM) has been shown to play a pleiotropic role in neurodegeneration by modulating energy metabolism and neurological functions in mice. However, the pharmacological response to T1AM might be influenced by tissue metabolism, which is known to convert T1AM into its catabolite 3-iodothyroacetic acid (TA1). Currently, several research groups are investigating the pharmacological effects of T1AM systemic administration in the search of novel therapeutic approaches for the treatment of interlinked pathologies, such as metabolic and neurodegenerative diseases (NDDs). A critical aspect in the development of new drugs for NDDs is to know their distribution in the brain, which is fundamentally related to their ability to cross the blood-brain barrier (BBB). To this end, in the present study we used the immortalized mouse brain endothelial cell line bEnd.3 to develop an in vitro model of BBB and evaluate T1AM and TA1 permeability. Both drugs, administered at 1 µM dose, were assayed by high-performance liquid chromatography coupled to mass spectrometry. Our results indicate that T1AM is able to efficiently cross the BBB, whereas TA1 is almost completely devoid of this property.


Asunto(s)
Encéfalo/metabolismo , Animales , Transporte Biológico/fisiología , Barrera Hematoencefálica/metabolismo , Línea Celular , Línea Celular Tumoral , Técnicas de Cocultivo/métodos , Células Endoteliales/metabolismo , Humanos , Ratones , Enfermedades Neurodegenerativas/tratamiento farmacológico , Fármacos Neuroprotectores/metabolismo , Permeabilidad/efectos de los fármacos , Tironinas/metabolismo
8.
Nat Genet ; 53(3): 332-341, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33649592

RESUMEN

Resistance to immune checkpoint inhibitors (ICIs) is a key challenge in cancer therapy. To elucidate underlying mechanisms, we developed Perturb-CITE-sequencing (Perturb-CITE-seq), enabling pooled clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 perturbations with single-cell transcriptome and protein readouts. In patient-derived melanoma cells and autologous tumor-infiltrating lymphocyte (TIL) co-cultures, we profiled transcriptomes and 20 proteins in ~218,000 cells under ~750 perturbations associated with cancer cell-intrinsic ICI resistance (ICR). We recover known mechanisms of resistance, including defects in the interferon-γ (IFN-γ)-JAK/STAT and antigen-presentation pathways in RNA, protein and perturbation space, and new ones, including loss/downregulation of CD58. Loss of CD58 conferred immune evasion in multiple co-culture models and was downregulated in tumors of melanoma patients with ICR. CD58 protein expression was not induced by IFN-γ signaling, and CD58 loss conferred immune evasion without compromising major histocompatibility complex (MHC) expression, suggesting that it acts orthogonally to known mechanisms of ICR. This work provides a framework for the deciphering of complex mechanisms by large-scale perturbation screens with multimodal, single-cell readouts, and discovers potentially clinically relevant mechanisms of immune evasion.


Asunto(s)
Antígenos CD58/inmunología , Resistencia a Antineoplásicos/inmunología , Melanoma/patología , Análisis de la Célula Individual/métodos , Escape del Tumor , Antígenos CD58/genética , Antígenos CD58/metabolismo , Sistemas CRISPR-Cas , Técnicas de Cocultivo , Biología Computacional/métodos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Epítopos/genética , Técnicas de Inactivación de Genes , Humanos , Interferón gamma/inmunología , Interferón gamma/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Melanoma/tratamiento farmacológico , Melanoma/inmunología , Análisis de Secuencia de ARN , Escape del Tumor/genética
9.
Nature ; 592(7852): 138-143, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33731925

RESUMEN

A variety of species of bacteria are known to colonize human tumours1-11, proliferate within them and modulate immune function, which ultimately affects the survival of patients with cancer and their responses to treatment12-14. However, it is not known whether antigens derived from intracellular bacteria are presented by the human leukocyte antigen class I and II (HLA-I and HLA-II, respectively) molecules of tumour cells, or whether such antigens elicit a tumour-infiltrating T cell immune response. Here we used 16S rRNA gene sequencing and HLA peptidomics to identify a peptide repertoire derived from intracellular bacteria that was presented on HLA-I and HLA-II molecules in melanoma tumours. Our analysis of 17 melanoma metastases (derived from 9 patients) revealed 248 and 35 unique HLA-I and HLA-II peptides, respectively, that were derived from 41 species of bacteria. We identified recurrent bacterial peptides in tumours from different patients, as well as in different tumours from the same patient. Our study reveals that peptides derived from intracellular bacteria can be presented by tumour cells and elicit immune reactivity, and thus provides insight into a mechanism by which bacteria influence activation of the immune system and responses to therapy.


Asunto(s)
Antígenos Bacterianos/análisis , Antígenos Bacterianos/inmunología , Bacterias/inmunología , Antígenos HLA/inmunología , Melanoma/inmunología , Melanoma/microbiología , Péptidos/análisis , Péptidos/inmunología , Presentación de Antígeno , Bacterias/clasificación , Bacterias/genética , Línea Celular Tumoral , Técnicas de Cocultivo , Antígenos HLA/análisis , Humanos , Linfocitos Infiltrantes de Tumor/citología , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/patología , Metástasis de la Neoplasia/inmunología , Filogenia , ARN Ribosómico 16S/genética
10.
Int J Mol Sci ; 22(4)2021 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-33671869

RESUMEN

Cancer-associated fibroblasts (CAFs) are one of the most abundant and critical components of the tumor stroma. CAFs can impact many important steps of cancerogenesis and may also influence treatment resistance. Some of these effects need the direct contact of CAFs and cancer cells, while some involve paracrine signals. In this study, we investigated the ability of head and neck squamous cell carcinomas (HNSCC) patient-derived CAFs to promote or inhibit the colony-forming ability of HNSCC cells. The effect of cisplatin on this promoting or inhibiting influence was also studied. The subsequent analysis focused on changes in the expression of genes associated with cancer progression. We found that cisplatin response in model HNSCC cancer cells was modified by coculture with CAFs, was CAF-specific, and different patient-derived CAFs had a different "sensitizing ratio". Increased expression of VEGFA, PGE2S, COX2, EGFR, and NANOG in cancer cells was characteristic for the increase of resistance. On the other hand, CCL2 expression was associated with sensitizing effect. Significantly higher amounts of cisplatin were found in CAFs derived from patients who subsequently experienced a recurrence. In conclusion, our results showed that CAFs could promote and/or inhibit colony-forming capability and cisplatin resistance in HNSCC cells via paracrine effects and subsequent changes in gene expression of cancer-associated genes in cancer cells.


Asunto(s)
Antineoplásicos/farmacología , Fibroblastos Asociados al Cáncer/efectos de los fármacos , Fibroblastos Asociados al Cáncer/metabolismo , Cisplatino/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Técnicas de Cocultivo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias de Cabeza y Cuello/patología , Humanos , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/patología , Comunicación Paracrina/efectos de los fármacos , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Ensayo de Tumor de Célula Madre
11.
J Vis Exp ; (168)2021 02 24.
Artículo en Inglés | MEDLINE | ID: mdl-33720119

RESUMEN

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.


Asunto(s)
Neoplasias Encefálicas/patología , Comunicación Celular , Movimiento Celular , Técnicas de Cocultivo/métodos , Glioblastoma/patología , Células Madre Neoplásicas/patología , Neuronas/patología , Animales , Comunicación Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Rastreo Celular , Glioma/patología , Humanos , Procesamiento de Imagen Asistido por Computador , Laminina/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Neuronas/efectos de los fármacos , Ratas , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/patología
12.
Int J Mol Sci ; 22(4)2021 Feb 21.
Artículo en Inglés | MEDLINE | ID: mdl-33670029

RESUMEN

Hair follicle morphogenesis is heavily dependent on reciprocal, sequential, and epithelial-mesenchymal interaction (EMI) between epidermal stem cells and the specialized cells of the underlying mesenchyme, which aggregate to form the dermal condensate (DC) and will later become the dermal papilla (DP). Similar models were developed with a co-culture of keratinocytes and DP cells. Previous studies have demonstrated that co-culture with keratinocytes maintains the in vivo characteristics of the DP. However, it is often challenging to develop three-dimensional (3D) DP and keratinocyte co-culture models for long term in vitro studies, due to the poor intercellular adherence between keratinocytes. Keratinocytes exhibit exfoliative behavior, and the integrity of the DP and keratinocyte co-cultured spheroids cannot be maintained over prolonged culture. Short durations of culture are unable to sufficiently allow the differentiation and re-programming of the keratinocytes into hair follicular fate by the DP. In this study, we explored a microgel array approach fabricated with two different hydrogel systems. Using poly (ethylene glycol) diacrylate (PEGDA) and gelatin methacrylate (GelMA), we compare their effects on maintaining the integrity of the cultures and their expression of important genes responsible for hair follicle morphogenesis, namely Wnt10A, Wnt10B, and Shh, over prolonged duration. We discovered that low attachment surfaces such as PEGDA result in the exfoliation of keratinocytes and were not suitable for long-term culture. GelMA, on the hand, was able to sustain the integrity of co-cultures and showed higher expression of the morphogens overtime.


Asunto(s)
Dermis/citología , Queratinocitos/citología , Microgeles/química , Polietilenglicoles/farmacología , Adhesión Celular/efectos de los fármacos , Agregación Celular/efectos de los fármacos , Línea Celular , Técnicas de Cocultivo , Proteínas Fluorescentes Verdes/metabolismo , /efectos de los fármacos , Humanos , Hidrogeles/farmacología , Proteínas Luminiscentes/metabolismo , Esferoides Celulares/citología , Esferoides Celulares/efectos de los fármacos , Proteínas Wnt/metabolismo
13.
Cell Prolif ; 54(4): e13022, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33686740

RESUMEN

OBJECTIVES: This study aimed to investigate the protective effect of SCARF1 on acute rejection (AR), phagocytic clearance of Kupffer cells (KCs), M2 polarization and the exact mechanism underlying these processes. METHODS: AAV was transfected into the portal vein of rats, and AR and immune tolerance (IT) models of liver transplantation were established. Liver tissue and blood samples were collected. The level of SCARF1 was detected via WB and immunohistochemical staining. Pathological changes in liver tissue were detected using HE staining. Apoptotic cells were detected using TUNEL staining. KC polarization was assessed via immunohistochemical staining. Primary KCs were isolated and co-cultured with apoptotic T lymphocytes. Phagocytosis of apoptotic cells and polarization of KCs were both detected using immunofluorescence. Calcium concentration was determined using immunofluorescence and a fluorescence microplate reader. The levels of PI3K, p-AKT and P-STAT3 were assessed via WB and immunofluorescence. RESULTS: Compared to the IT group, the level of SCARF1 was significantly decreased in the AR group. Overexpression of SCARF1 in KCs improved AR and liver function markers. Enhanced phagocytosis mediated by SCARF1 is beneficial for improving the apoptotic clearance of AR and promoting M2 polarization of KCs. SCARF1-mediated enhancement of phagocytosis promotes increased calcium concentration in KCs, thus further activating the PI3K-AKT-STAT3 signalling pathway. CONCLUSIONS: SCARF1 promotes the M2 polarization of KCs by promoting phagocytosis through the calcium-dependent PI3K-AKT-STAT3 signalling pathway.


Asunto(s)
Calcio/metabolismo , Trasplante de Hígado , Receptores Depuradores de Clase F/metabolismo , Transducción de Señal , Animales , Apoptosis , Polaridad Celular , Células Cultivadas , Técnicas de Cocultivo , Macrófagos del Hígado/citología , Macrófagos del Hígado/metabolismo , Hígado/metabolismo , Hígado/patología , Masculino , Fagocitosis , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Endogámicas Lew , Factor de Transcripción STAT3/metabolismo , Receptores Depuradores de Clase F/genética , Linfocitos T/citología , Linfocitos T/metabolismo
14.
Methods Mol Biol ; 2265: 111-118, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704709

RESUMEN

Within the adaptive and innate immune system, effector lymphocytes known as cytotoxic T cells (CTLs) or natural killer (NK) cells play an essential role in host defense against tumor cells and virally infected cells. Here we describe a flow cytometry-based method to quantify CTLs or NK cell cytotoxic activity against melanoma cells. In this assay, spleen cells, peripheral blood mononuclear cells (PBMCs), or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line. The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells. After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications.


Asunto(s)
Pruebas Inmunológicas de Citotoxicidad/métodos , Citometría de Flujo/métodos , Células Asesinas Naturales/inmunología , Melanoma Experimental/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Técnicas de Cultivo de Célula/métodos , Muerte Celular/inmunología , Línea Celular Tumoral , Técnicas de Cocultivo/métodos , Femenino , Colorantes Fluorescentes , Humanos , Leucocitos Mononucleares/inmunología , Ratones , Bazo/citología , Bazo/inmunología
15.
Methods Mol Biol ; 2265: 141-154, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704712

RESUMEN

Three-dimensional (3D) cell culture has allowed a deeper understanding of complex pathological and physiological processes, overcoming some of the limitations of 2D cell culture on plastic and avoiding the costs and ethical issues related to experiments involving animals. Here we describe a protocol to embed single melanoma cells alone or together with primary human lymphatic endothelial cells in a 3D cross-linked matrix, to investigate the invasion and molecular crosstalk between these two cell types, respectively. After fixation and staining with antibodies and fluorescent conjugates, phenotypic changes in both cell types can be specifically analyzed by confocal microscopy.


Asunto(s)
Técnicas de Cocultivo/métodos , Células Endoteliales/metabolismo , Melanoma/metabolismo , Esferoides Celulares/metabolismo , Línea Celular Tumoral , Células Endoteliales/citología , Técnica del Anticuerpo Fluorescente/métodos , Humanos , Melanoma/patología , Microscopía Confocal , Invasividad Neoplásica
16.
Methods Mol Biol ; 2265: 173-183, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704714

RESUMEN

Most currently available three-dimensional melanoma models have either focused on simplicity or were optimized for physiological relevance. Accordingly, these paradigms have been either composed of malignant cells only or they were sophisticated human skin equivalents featuring multiple cell types and skin-like organization. Here, an intermediate spheroid-based assay system is presented, which uses tri-cultures of human CCD-1137Sk fibroblasts, HaCaT keratinocytes, and SK-MEL-28 melanoma cells. Being made of cell lines, these spheroids can be reliably reproduced without any special equipment using standard culture procedures, and they feature different aspects of skin and early stage melanoma. Therefore, this kind of model can be useful for lead-compound testing or addressing fundamental principles of early melanoma formation.


Asunto(s)
Antineoplásicos/farmacología , Técnicas de Cocultivo/métodos , Fibroblastos/efectos de los fármacos , Queratinocitos/efectos de los fármacos , Melanoma/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Esferoides Celulares/metabolismo , Línea Celular Tumoral , Docetaxel/farmacología , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Melanoma/metabolismo , Melanoma/patología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología
17.
Int J Mol Sci ; 22(4)2021 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-33669494

RESUMEN

Campylobacter concisus is a human-pathogenic bacterium of the gastrointestinal tract. This study aimed at the contribution of the mucosal immune system in the context of intestinal epithelial barrier dysfunction induced by C. concisus. As an experimental leaky gut model, we used in vitro co-cultures of colonic epithelial cell monolayers (HT-29/B6-GR/MR) with M1-macrophage-like THP-1 cells on the basal side. Forty-eight hours after C. concisus infection, the decrease in the transepithelial electrical resistance in cell monolayers was more pronounced in co-culture condition and 22 ± 2% (p < 0.001) higher than the monoculture condition without THP-1 cells. Concomitantly, we observed a reduction in the expression of the tight junction proteins occludin and tricellulin. We also detected a profound increase in 4 kDa FITC-dextran permeability in C. concisus-infected cell monolayers only in co-culture conditions. This is explained by loss of tricellulin from tricellular tight junctions (tTJs) after C. concisus infection. As an underlying mechanism, we observed an inflammatory response after C. concisus infection through pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6) released from THP-1 cells in the co-culture condition. In conclusion, the activation of subepithelial immune cells exacerbates colonic epithelial barrier dysfunction by C. concisus through tricellulin disruption in tTJs, leading to increased antigen permeability (leaky gut concept).


Asunto(s)
Campylobacter/inmunología , Células Epiteliales/inmunología , Células Epiteliales/microbiología , Apoptosis , Línea Celular , Supervivencia Celular , Técnicas de Cocultivo , Impedancia Eléctrica , Células Epiteliales/patología , Humanos , Inflamación/patología , Intestinos/microbiología , Intestinos/patología , Macrófagos/metabolismo , Modelos Biológicos , Ocludina/metabolismo , Fracciones Subcelulares/metabolismo , Uniones Estrechas/metabolismo
18.
J Vis Exp ; (168)2021 02 28.
Artículo en Inglés | MEDLINE | ID: mdl-33720120

RESUMEN

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The significance of innate immunity signaling in viral infections has increased substantially as patients with respiratory infections who exhibit high innate T cell activation show a better disease outcome. Hence, dissecting these early innate immune interactions allows the elucidation of the processes that govern them and may facilitate the development of potential therapeutic targets and strategies for dampening or even preventing early progression of viral infections. This protocol details a versatile model that can be used to study early crosstalk, interactions, and activation of innate immune cells from factors secreted by virally infected airway epithelial cells. Using an H3N2 influenza virus (A/Aichi/2/1968) as the representative virus model, innate cell activation of co-cultured peripheral blood mononuclear cells (PBMCs) has been analyzed using flow cytometry to investigate the subsets of cells that are activated by the soluble factors released from the epithelium in response to the viral infection. The results demonstrate the gating strategy for differentiating the subsets of cells and reveal the clear differences between the activated populations of PBMCs and their crosstalk with the control and infected epithelium. The activated subsets can then be further analyzed to determine their functions as well as molecular changes specific to the cells. Findings from such a crosstalk investigation may uncover factors that are important for the activation of vital innate cell populations, which are beneficial in controlling and suppressing the progression of viral infection. Furthermore, these factors can be universally applied to different viral diseases, especially to newly emerging viruses, to dampen the impact of such viruses when they first circulate in naïve human populations.


Asunto(s)
Inmunidad Innata , Subtipo H3N2 del Virus de la Influenza A/inmunología , Gripe Humana/inmunología , Gripe Humana/virología , Modelos Biológicos , Células 3T3 , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Impedancia Eléctrica , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Células Nutrientes/citología , Humanos , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/virología , Ratones , Mitomicina/farmacología , Mucina 5AC/metabolismo , Mucosa Nasal/patología , Tubulina (Proteína)/metabolismo
19.
Int J Mol Sci ; 22(4)2021 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-33672854

RESUMEN

Enteric glial cells (EGCs) of the enteric nervous system are critically involved in the maintenance of intestinal epithelial barrier function (IEB). The underlying mechanisms remain undefined. Glial cell line-derived neurotrophic factor (GDNF) contributes to IEB maturation and may therefore be the predominant mediator of this process by EGCs. Using GFAPcre x Ai14floxed mice to isolate EGCs by Fluorescence-activated cell sorting (FACS), we confirmed that they synthesize GDNF in vivo as well as in primary cultures demonstrating that EGCs are a rich source of GDNF in vivo and in vitro. Co-culture of EGCs with Caco2 cells resulted in IEB maturation which was abrogated when GDNF was either depleted from EGC supernatants, or knocked down in EGCs or when the GDNF receptor RET was blocked. Further, TNFα-induced loss of IEB function in Caco2 cells and in organoids was attenuated by EGC supernatants or by recombinant GDNF. These barrier-protective effects were blunted when using supernatants from GDNF-deficient EGCs or by RET receptor blockade. Together, our data show that EGCs produce GDNF to maintain IEB function in vitro through the RET receptor.


Asunto(s)
Sistema Nervioso Entérico/metabolismo , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Mucosa Intestinal/metabolismo , Neuroglía/metabolismo , Animales , Células CACO-2 , Células Cultivadas , Técnicas de Cocultivo , Medios de Cultivo Condicionados/farmacología , Sistema Nervioso Entérico/efectos de los fármacos , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Factor Neurotrófico Derivado de la Línea Celular Glial/farmacología , Humanos , Mucosa Intestinal/efectos de los fármacos , Intestino Delgado/citología , Intestino Delgado/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neuroglía/efectos de los fármacos , Permeabilidad/efectos de los fármacos , Proteínas Recombinantes/farmacología , Factor de Necrosis Tumoral alfa/farmacología
20.
Bioresour Technol ; 330: 125008, 2021 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-33773267

RESUMEN

The treatment of wastewater by microalgae has been studied and proved to be effective through previous studies. Due to the small size of microalgae, how to efficiently harvest microalgae from wastewater is a crucial factor restricting the development of algal technologies. Fungi-assisted microalgae bio-flocculation for microalgae harvesting and wastewater treatment simultaneously, which was overlooked previously, has attracted increasing attention in the recent decade due to its low cost and high efficiency. This review found that fungal hyphae and microalgae can stick together due to electrostatic neutralization, surface protein interaction, and exopolysaccharide adhesion in the co-culture process, realizing co-pelletization of microalgae and fungi, which is conducive to microalgae harvesting. Besides, the combination of fungi and microalgae has a complementary effect on pollutant removal from wastewaters. The co-culture of fungi-microalgae has excellent development prospects with both environmental and economic benefits, and it is expected to be applied on an industrial scale.


Asunto(s)
Microalgas , Biomasa , Técnicas de Cocultivo , Floculación , Hongos , Aguas Residuales
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