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Exosomes are membrane-enclosed nanovesicles that shuttle active cargoes, such as circular RNAs (circRNAs) and microRNAs (miRNAs), between different cells. Human umbilical cord-derived mesenchymal stem cells (Hu-MSCs) can migrate to tumor sites and exert complex functions throughout tumor progression. In this study, we successfully isolated Hu-MSCs from human umbilical cords based on their surface marker expression. Hu-MSC-derived exosomes significantly reduced the invasion, migration, and proliferation of cholangiocarcinoma (CCA) cells. Furthermore, circ_0037104 was downregulated in CCA and inhibited the proliferation and metastasis of CCA cells. Then, we investigated the effect of Hu-MSC-derived exosomal circ_0037104 on CCA. Circ_0037104 mainly regulates miR-620 and enhances APAF1 expression, inhibiting CCA cell proliferation and metastasis. Overall, Hu-MSC exosomal circ_0037104 contributes to the progression and stemness of CCA cells via miR-620/APAF1. In conclusion, Hu-MSC-derived exosomal circ_0037104 sponges miR-620 directly and negatively targets APAF1 to suppress CCA.
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Neoplasias de los Conductos Biliares , Colangiocarcinoma , Células Madre Mesenquimatosas , MicroARNs , Humanos , Neoplasias de los Conductos Biliares/genética , Neoplasias de los Conductos Biliares/metabolismo , Neoplasias de los Conductos Biliares/patología , Conductos Biliares Intrahepáticos/metabolismo , Conductos Biliares Intrahepáticos/patología , Proliferación Celular , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patología , Células Madre Mesenquimatosas/metabolismo , MicroARNs/genética , MicroARNs/metabolismoRESUMEN
BACKGROUND: Thymosin alpha 1 (Tα1) had been used in the treatment of viral infections as an immune response modifier for many years. However, clinical benefits and the mechanism of Tα1 treatment for COVID-19 patients are still unclear. METHODS: We retrospectively reviewed the clinical outcomes of 76 severe COVID-19 cases admitted to 2 hospitals in Wuhan, China, from December 2019 to March 2020. The thymus output in peripheral blood mononuclear cells from COVID-19 patients was measured by T-cell receptor excision circles (TRECs). The levels of T-cell exhaustion markers programmed death-1 (PD-1) and T-cell immunoglobulin and mucin domain protein 3 (Tim-3) on CD8+ T cells were detected by flow cytometry. RESULTS: Compared with the untreated group, Tα1 treatment significantly reduced the mortality of severe COVID-19 patients (11.11% vs 30.00%, P = .044). Tα1 enhanced blood T-cell numbers in COVID-19 patients with severe lymphocytopenia. Under such conditions, Tα1 also successfully restored CD8+ and CD4+ T-cell numbers in elderly patients. Meanwhile, Tα1 reduced PD-1 and Tim-3 expression on CD8+ T cells from severe COVID-19 patients compared with untreated cases. It is of note that restoration of lymphocytopenia and acute exhaustion of T cells were roughly parallel to the rise of TRECs. CONCLUSIONS: Tα1 treatment significantly reduced mortality of severe COVID-19 patients. COVID-19 patients with counts of CD8+ T cells or CD4+ T cells in circulation less than 400/µL or 650/µL, respectively, gained more benefits from Tα1. Tα1 reversed T-cell exhaustion and recovered immune reconstitution through promoting thymus output during severe acute respiratory syndrome-coronavirus 2 infection.
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COVID-19/mortalidad , Linfopenia/metabolismo , SARS-CoV-2/patogenicidad , Timalfasina/metabolismo , Adulto , Anciano , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , COVID-19/virología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Timalfasina/genética , Timo/metabolismoRESUMEN
Viral fulminant hepatitis (FH) is a severe disease with high mortality resulting from excessive inflammation in the infected liver. Clinical interventions have been inefficient due to the lack of knowledge for inflammatory pathogenesis in the virus-infected liver. We show that wild-type mice infected with murine hepatitis virus strain-3 (MHV-3), a model for viral FH, manifest with severe disease and high mortality in association with a significant elevation in IL-1ß expression in the serum and liver. Whereas, the viral infection in IL-1ß receptor-I deficient (IL-1R1-/-) or IL-1R antagonist (IL-1Ra) treated mice, show reductions in virus replication, disease progress and mortality. IL-1R1 deficiency appears to debilitate the virus-induced fibrinogen-like protein-2 (FGL2) production in macrophages and CD45+Gr-1high neutrophil infiltration in the liver. The quick release of reactive oxygen species (ROS) by the infected macrophages suggests a plausible viral initiation of NLRP3 inflammasome activation. Further experiments show that mice deficient of p47phox, a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit that controls acute ROS production, present with reductions in NLRP3 inflammasome activation and subsequent IL-1ß secretion during viral infection, which appears to be responsible for acquiring resilience to viral FH. Moreover, viral infected animals in deficiencies of NLRP3 and Caspase-1, two essential components of the inflammasome complex, also have reduced IL-1ß induction along with ameliorated hepatitis. Our results demonstrate that the ROS/NLRP3/IL-1ß axis institutes an essential signaling pathway, which is over activated and directly causes the severe liver disease during viral infection, which sheds light on development of efficient treatments for human viral FH and other severe inflammatory diseases.
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Proteínas Portadoras/agonistas , Infecciones por Coronavirus/virología , Interacciones Huésped-Patógeno , Interleucina-1beta/agonistas , Hígado/virología , Virus de la Hepatitis Murina/fisiología , Receptores Tipo I de Interleucina-1/agonistas , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/metabolismo , Infecciones por Coronavirus/patología , Progresión de la Enfermedad , Fibrinógeno/metabolismo , Inmunidad Innata , Inflamasomas/inmunología , Inflamasomas/metabolismo , Proteína Antagonista del Receptor de Interleucina 1/metabolismo , Interleucina-1beta/antagonistas & inhibidores , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Hígado/inmunología , Hígado/metabolismo , Hígado/patología , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/ultraestructura , Macrófagos/virología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Virus de la Hepatitis Murina/efectos de los fármacos , Virus de la Hepatitis Murina/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR , Células RAW 264.7 , Especies Reactivas de Oxígeno/agonistas , Especies Reactivas de Oxígeno/metabolismo , Receptores Tipo I de Interleucina-1/antagonistas & inhibidores , Receptores Tipo I de Interleucina-1/genética , Receptores Tipo I de Interleucina-1/metabolismo , Proteínas Recombinantes/metabolismo , Transducción de Señal , Análisis de SupervivenciaRESUMEN
PURPOSE: To establish and evaluate a novel in vitro model of retinal ischemia, and to determine whether an autocrine pathway of retinal microvascular endothelial cells (RMVECs) by vascular endothelial growth factor (VEGF) signaling plays a role based on this model. METHODS: Primary RMVECs were isolated from the retinas of C57/BL6J rats and identified by an evaluation for FITC-marked CD31. The hypoxia models were established with the biobag at the time of 12, 24, 48 and 72 h, and evaluated with a blood-gas analyzer. The control groups were incubated under normoxic conditions for the same length of time. Cell proliferation was evaluated by the CCK-8 method. Apoptosis was assayed using a flow cytometry method. RNA and protein expressions of VEGF-A, VEGFR-2 and iNOs were analyzed by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. RESULTS: The results of blood-gas analysis showed that when the cultures were exposed to hypoxia for more than 2 h, the pO(2) was below 4.5 kPa, pCO(2) and pH shifted slightly. Real-time RT-PCR revealed that the expressions of VEGF-A, VEGFR-2 and iNOs mRNA in hypoxic groups increased in comparison to those in the normoxia groups (p < 0.01) and the expression of mRNA increased significantly in a time-dependent fashion in the hypoxic groups (p < 0.01), peaking at 48 h, and then decreasing. Western blot analysis revealed that the expression of relative proteins ranked in this order. CCK-8 analysis revealed that the proliferative capacity of RMVECs in the hypoxic groups was significantly higher than those in the normoxic groups at each time point (p < 0.05). At 48 h, the proliferative capacity was highest in the hypoxia groups (p < 0.05). Data acquisition from flow cytometry showed that cell survival rates in the hypoxic groups were higher than those in the normoxic groups and apoptosis rates dropped accordingly. The survival rate was highest at 48 h. CONCLUSION: These findings suggested that a novel in vitro model of retinal ischemia using the biobag had a good authenticity. According to the well-established in vitro hypoxia model by the biobag, RMVECs include the requisite elements for an autocrine pathway that may serve to amplify the angiogenic effects of VEGF.
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Comunicación Autocrina/fisiología , Modelos Animales de Enfermedad , Endotelio Vascular/metabolismo , Isquemia/metabolismo , Vasos Retinianos/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Apoptosis , Análisis de los Gases de la Sangre , Western Blotting , Hipoxia de la Célula/fisiología , Proliferación Celular , Células Cultivadas , Endotelio Vascular/patología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Isquemia/patología , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , ARN Mensajero/metabolismo , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Vasos Retinianos/patología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
OBJECTIVE: To investigate the association of angiotensinogen (AGT) gene polymorphisms and angiogenic factors with preeclampsia (PE) in Chinese women. METHODS: A study on Chinese women was performed. Detection of the M235T polymorphism of AGT gene was carried out by PCR. Using a χ² test, genotype and allele frequencies were compared in all groups. Maternal serum levels of vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) and soluble fms-like tyrosine kinase 1 (sFlt1) at gestation were compared between 92 women with PE and 100 controls by ELISA. RESULTS: Compared to the controls, the AGT homozygous of TT genotype in PE occurred significantly more frequently and the T allele was observed to occur more frequently than the M allele (p < 0.05). sFlt1 was present in high quantities in the serum of women with PE and was associated with low levels of free VEGF and PlGF (p < 0.05). Plasma sFlt1 levels are higher in PE patients with TT heterozygotes compared with MM homozygotes, but PIGF is lower (p < 0.05). Plasma VEGF concentrations showed no significant difference. CONCLUSIONS: Our study showed that AGT M235T polymorphism is associated with PE in Chinese women. Furthermore, the gene polymorphism of the components of the renin-angiotensin system may contribute to the concentration alterations of sFlt1, VEGF, and PlGF in maternal serum, which causes disordered vasculogenesis contributing to PE.
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Angiotensinógeno/genética , Preeclampsia/genética , Proteínas Gestacionales/genética , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Adulto , Alelos , Angiotensinógeno/sangre , Distribución de Chi-Cuadrado , China , ADN/química , ADN/genética , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Factor de Crecimiento Placentario , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido Simple , Preeclampsia/sangre , Embarazo , Proteínas Gestacionales/sangre , Receptor 1 de Factores de Crecimiento Endotelial Vascular/sangreRESUMEN
Objective: To determine the expression level of Sonic hedgehog (Shh) in the passage of hair follicle stem cells (HFSCs), analyze the effect of Shh overexpression on the proliferation activity of HFSCs, and explore the survival of HFSCs after Shh overexpression and its effect on hair follicle regeneration. Methods: Hair follicles from the normal area (H1 group) and alopecia area (H2 group) of the scalp donated by 20 female alopecia patients aged 40-50 years old were taken, and the middle part of the hair follicle was cut under the microscope to culture, and the primary HFSCs were obtained and passaged; the positive markers (CD29, CD71) and negative marker (CD34) on the surface of the fourth generation HFSCs were identified by flow cytometry. The two groups of HFSCs were transfected with Shh-overexpressed lentivirus. Flow cytometry and cell counting kit 8 assay were used to detect the cell cycle changes and cell proliferation of HFSCs before and after transfection, respectively. Then the HFSCs transfected with Shh lentivirus were transplanted subcutaneously into the back of nude mice as the experimental group, and the same amount of saline was injected as the control group. At 5 weeks after cell transplantation, the expression of Shh protein in the back skin tissue of nude mice was detected by Western blot. HE staining and immunofluorescence staining were used to compare the number of hair follicles and the survival of HFSCs between groups. Results: The isolated and cultured cells were fusiform and firmly attached to the wall; flow cytometry showed that CD29 and CD71 were highly expressed on the surface of the cells, while CD34 was lowly expressed, suggesting that the cultured cells were HFSCs. The results of real-time fluorescence quantitative PCR and Western blot showed that the expression levels of Shh protein and gene in the 4th, 7th, and 10th passages of cells in H1 and H2 groups decreased gradually with the prolongation of culture time in vitro. After overexpression of Shh, the proliferation activity of HFSCs in the two groups was significantly higher than that in the blank group (not transfected with lentivirus) and the negative control group (transfected with negative control lentivirus), and the proliferation activity of HFSCs in H1 group was significantly higher than that in H2 group before and after transfection, showing significant differences ( P<0.05). At 5 weeks after cell transplantation, Shh protein was stably expressed in the dorsal skin of each experimental group; the number of hair follicles and the expression levels of HFSCs markers (CD71, cytokeratin 15) in each experimental group were significantly higher than those in the control group, and the number of hair follicles and the expression levels of HFSCs markers in H1 group were significantly higher than those in H2 group, and the differences were significant ( P<0.05). Conclusion: Lentivirus-mediated Shh can be successfully transfected into HFSCs, the proliferation activity of HFSCs significantly increase after overexpression of Shh, which can secrete and express Shh continuously and stably, and promote hair follicle regeneration by combining the advantages of stem cells and Shh.
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Folículo Piloso , Proteínas Hedgehog , Animales , Femenino , Ratones , Alopecia/metabolismo , Alopecia/cirugía , Proteínas Hedgehog/genética , Ratones Desnudos , Regeneración , Células MadreRESUMEN
Following the publication of this paper, it was drawn to the Editor's attention by a concerned reader that the GAPDH control western blotting data shown in Fig. 1C, and other western blotting data included in Figs. 2D and 7C and D, were strikingly similar to data appearing in different form in other articles written by different authors at different research institutes. Owing to the fact that the contentious data in the above article were already under consideration for publication, or had already been published elsewhere, prior to its submission to Molecular Medicine Reports, the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a reply. The Editor apologizes to the readership for any inconvenience caused. [Molecular Medicine Reports 15: 20742082, 2017; DOI: 10.3892/mmr.2017.6257].
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Background: Glioblastoma (GBM) is adults' most common and fatally malignant brain tumor. The heterogeneity is the leading cause of treatment failure. However, the relationship between cellular heterogeneity, tumor microenvironment, and GBM progression is still elusive. Methods: Integrated analysis of single-cell RNA sequencing (scRNA-seq) and spatial transcriptome sequencing (stRNA-seq) of GBM were conducted to analyze the spatial tumor microenvironment. We investigated the subpopulation heterogeneity of malignant cells through gene set enrichment analyses, cell communications analyses, and pseudotime analyses. Significantly changed genes of the pseudotime analysis were screened to create a tumor progress-related gene risk score (TPRGRS) using Cox regression algorithms in the bulkRNA-sequencing(bulkRNA-seq) dataset. We combined the TPRGRS and clinical characteristics to predict the prognosis of patients with GBM. Furthermore, functional analysis was applied to uncover the underlying mechanisms of the TPRGRS. Results: GBM cells were accurately charted to their spatial locations and uncovered their spatial colocalization. The malignant cells were divided into five clusters with transcriptional and functional heterogeneity, including unclassified malignant cells and astrocyte-like, mesenchymal-like, oligodendrocytes-progenitor-like, and neural-progenitor-like malignant cells. Cell-cell communications analysis in scRNA-seq and stRNA-seq identified ligand-receptor pairs of the CXCL, EGF, FGF, and MIF signaling pathways as bridges implying that tumor microenvironment may cause malignant cells' transcriptomic adaptability and disease progression. Pseudotime analysis showed the differentiation trajectory of GBM cells from proneural to mesenchymal transition and identified genes or pathways that affect cell differentiation. TPRGRS could successfully divide patients with GBM in three datasets into high- and low-risk groups, which was proved to be a prognostic factor independent of routine clinicopathological characteristics. Functional analysis revealed the TPRGRS associated with growth factor binding, cytokine activity, signaling receptor activator activity functions, and oncogenic pathways. Further analysis revealed the association of the TPRGRS with gene mutations and immunity in GBM. Finally, the external datasets and qRT-PCR verified high expressions of the TPRGRS mRNAs in GBM cells. Conclusion: Our study provides novel insights into heterogeneity in GBM based on scRNA-seq and stRNA-seq data. Moreover, our study proposed a malignant cell transition-based TPRGRS through integrated analysis of bulkRNA-seq and scRNA-seq data, combined with the routine clinicopathological evaluation of tumors, which may provide more personalized drug regimens for GBM patients.
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Background: Excessive proliferation and activation of B cells, resulting in the production of various autoantibodies, is a crucial link and significant feature of the pathogenesis of systemic lupus erythematosus (SLE), as well as the pathological basis of systemic multiorgan damage. However, whether exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs-Exo) are involved in the immune regulation of SLE has not been clarified. Objectives: Therefore, our study aimed to investigate the efficacy of hucMSCs-Exo for treating SLE. Methods: hucMSCs-Exo and peripheral blood mononuclear cells (PBMCs) of SLE patients were cocultured in vitro, and B cell apoptosis, activation, proliferation, and inflammation levels were detected by flow cytometry. Subsequently, the expression level of miR-155 in B lymphocytes of SLE patients was detected by qRT-PCR, and the target gene relationship between miR-155 and SHIP-1 was found through bioinformatics and dual luciferase activity experiments, which verified the inhibition of miR-155 in B lymphocytes of SLE patients to regulate immunity. Results: We found that hucMSCs-Exo promoted B cell apoptosis, prevented B cell overactivation, and reduced inflammation. MicroRNA-155 (miR-155) has a powerful regulatory function in B cells. It was demonstrated that hucMSCs-Exo acts synergistically with miR-155 inhibitors to target SHIP-1 to B cells more effectively than exosomes alone. Conclusion: Our results provide insight into how hucMSCs-Exo regulates autoimmunity in patients with lupus and suggest targeting miR-155 for autoimmunity while protecting immunity.
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Exosomas , Lupus Eritematoso Sistémico , Células Madre Mesenquimatosas , MicroARNs , Humanos , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/metabolismo , Exosomas/metabolismo , Leucocitos Mononucleares/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Lupus Eritematoso Sistémico/terapia , Lupus Eritematoso Sistémico/metabolismo , Células Madre Mesenquimatosas/metabolismo , Cordón Umbilical/metabolismo , Inflamación/metabolismoRESUMEN
Introduction. Caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, coronavirus disease 2019 (COVID-19) has threatened global public health. Immune damage mechanisms are essential guidelines for clinical treatment and immune prevention.Hypothesis. The dysregulated type I interferon (IFN-I) responses, lymphocytopenia and hypercytokinemia during SARS-CoV-2 infection have been reported. However, whether there is a correlation between levels of IFN-I and the severity of COVID-19 has not been reported yet.Aim. To investigate the source of IFN-I and detect the exact roles of them in the pathogenesis of COVID-19.Methodology. Here ELISA was used to detect serum IFN-I (IFN-α and IFN-ß) for 137 cases with laboratory-confirmed COVID-19 admitted into one hospital in Wuhan from December 2019 to March 2020, and the relationships between IFN-α/ß concentrations and patients' clinical parameters were conducted by statistical analysis.Results. Both IFN-α and IFN-ß concentrations dramatically increased in COVID-19 patients, especially in old patients (>80 years) and severe cases. Statistical analysis demonstrated that serum IFN-α/ß concentrations were negatively correlated with the counts of total CD3+T, CD4+ and CD8+T cells, especially in critically ill cases. Moreover, serum IFN-α levels were positively correlated to IL-6 and TNF-α. Finally, immunofluorescent double staining showed that IFN-α and IFN-ß are major secretions from macrophages and dendritic cells (DCs) in lymph nodes from COVID-19 autopsies.Conclusion. These results demonstrate that macrophages and DCs are the main origination of IFN-I, and serum levels of IFN-I are positively associated with lymphopenia and cytokine storm, suggesting that IFN-α/ß deteriorated the severity of COVID-19. Anti-interferon or IFN-I signalling block drugs are needed to treat ICU patients.
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COVID-19 , Interferón Tipo I , Humanos , SARS-CoV-2 , Factor de Necrosis Tumoral alfaRESUMEN
PURPOSE: Natural killer (NK) cells are key players in the immune system, however, the exact mechanism of NK cell dysfunction during HBV infection remains poorly defined. METHODS: Hepatitis B envelope antigen-negative (HBeAg-, n = 19) chronic hepatitis B infection (CHB) patients, HBeAg-positive (HBeAg+, n = 20) CHB patients, HBV-related hepatocellular carcinoma (HBV-HCC, n = 12) patients and healthy blood donors (HD, n = 20), were enrolled in our study. The phenotype and function of the corresponding NK cells of these subjects were then determined. NK cells were cocultured with HBV to assess whether HBV influences the activation of STAT1. Receptors, proliferation, apoptosis rate, and cytotoxicity of NK-92 cells were detected after STAT1 overexpression and knockdown. The relationship between STAT1 and NKG2D promoter was determined by luciferase assay. RESULTS: The levels of NKG2D and STAT1 were the lowest in the HBV-HCC group compared with the HD group, followed by the HBeAg+ group and then the HBeAg- group, respectively. Interestingly, STAT1 levels were positively correlated with NKG2D expression and HBeAg status. Furthermore, STAT1 directly bound to the NKG2D promoter to regulate the transcription and expression of NKG2D. Finally, the results also suggested that knockdown of STAT1 can inhibit proliferation, increase apoptosis rate of NK-92 cells and impair cytotoxicity of NK-92 cells. CONCLUSION: STAT1 is correlated with NK cell dysfunction by downregulating NKG2D transcription in HBV-infected patients. Our findings demonstrate that STAT1 is an important and positive regulator of NK cells, which could provide a potential immunotherapy target for CHB.
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Carcinoma Hepatocelular , Hepatitis B Crónica , Neoplasias Hepáticas , Subfamilia K de Receptores Similares a Lectina de Células NK , Factor de Transcripción STAT1 , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virología , Antígenos e de la Hepatitis B/metabolismo , Virus de la Hepatitis B , Hepatitis B Crónica/genética , Células Asesinas Naturales , Neoplasias Hepáticas/virología , Subfamilia K de Receptores Similares a Lectina de Células NK/genética , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismoRESUMEN
Stem cell therapy is a promising strategy for the treatment of traumatic brain injury (TBI). However, animal experiments are needed to evaluate safety; in particular, to examine the immunogenicity and tumorigenicity of human umbilical cord mesenchymal stem cells (huMSCs) before clinical application. In this study, huMSCs were harvested from human amniotic membrane and umbilical cord vascular tissue. A rat model of TBI was established using the controlled cortical impact method. Starting from the third day after injury, the rats were injected with 10 µL of 5 × 106/mL huMSCs by cerebral stereotaxis or with 500 µL of 1 × 106/mL huMSCs via the tail vein for 3 successive days. huMSC transplantation decreased the serum levels of proinflammatory cytokines in rats with TBI and increased the serum levels of anti-inflammatory cytokines, thereby exhibiting good immunoregulatory function. The transplanted huMSCs were distributed in the liver, lung and brain injury sites. No abnormal proliferation or tumorigenesis was found in these organs up to 12 months after transplantation. The transplanted huMSCs negligibly proliferated in vivo, and apoptosis was gradually observed at later stages. These findings suggest that huMSC transplantation for the treatment of traumatic brain injury displays good safety. In addition, huMSCs exhibit good immunoregulatory function, which can help prevent and reduce secondary brain injury caused by the rapid release of inflammatory factors after TBI. This study was approved by the Ethics Committee of Wuhan General Hospital of PLA (approval No. 20160054) on November 1, 2016.
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BACKGROUND: Glioma is the most common human central nervous system tumour with a high degree of malignancy. Some Rab GTPases have significant effects on glioma. OBJECTIVE: This study aimed to investigate the effect of Rab3b (Rab GTPase3b) on human glioma cell proliferation and apoptosis by silencing Rab3b and to initially verify the value of Rab3b expression for the diagnosis and progression in human glioma. METHODS: Rab3b was silenced by siRNA transfection. Human glioma tissues and normal brain tissues adjacent to glioma were obtained by surgery. Rab3b, P53, Caspase 7, Bax, and Bim mRNA and protein expression levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. Cell proliferation was detected by the cell counting kit-8 assay, and the cell cycle and apoptosis were analysed using flow cytometry. RESULTS: Rab3b mRNA and protein expression in human glioma U251 and U87 cells were significantly downregulated after Rab3b silencing. Rab3b silencing inhibited glioma cell proliferation by promoting cell cycle arrest and induced apoptosis by upregulating the expression of apoptosis-related proteins. Rab3b expression in human glioma (n = 33) was significantly higher than that in normal brain tissues adjacent to glioma (n = 15). In addition, Rab3b expression levels in high-grade gliomas (WHO III-IV, n = 19) were also significantly higher than those in low-grade gliomas (WHO I-II, n = 14). CONCLUSION: Rab3b expression levels are significantly related to the progression of gliomas. Moreover, Rab3b silencing not only significantly inhibits cell proliferation in gliomas via cell cycle arrest but also promotes cell apoptosis by upregulating the expression levels of apoptosis-related proteins; however these preliminary in vitro results warrant validation on in vivo studies.
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Neoplasias Encefálicas , Glioma , Apoptosis , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Glioma/genética , HumanosRESUMEN
Since coronavirus disease 2019 (COVID-19) swept all over the world, several studies have shown the susceptibility of a patient with cancer to COVID-19. In this case, the removed glioblastoma multiforme (GBM)-adjacent (GBM-A), GBM-peritumor and GBM-central (GBM-C) tissues from a convalescent patient of COVID-19, who also suffered from glioblastoma meanwhile, together with GBM-A and GBM tissues from a patient without COVID-19 history as negative controls, were used for RNA ISH, electron microscopy observing and immunohistochemical staining of ACE2 and the virus antigen (N protein). The results of RNA ISH, electron microscopy observing showed that SARS-CoV-2 directly infects some cells within human GBM tissues and SARS-CoV-2 in GBM-C tissue still exists even when it is cleared elsewhere. Immunohistochemical staining of ACE2 and N protein showed that the expressions of ACE2 are significantly higher in specimens, including GBM-C tissue from COVID-19 patient than other types of tissue. The unique phenomenon suggests that the surgical protection level should be upgraded even if the patient is in a convalescent period and the pharyngeal swab tests show negative results. Furthermore, more attention should be paid to confirm whether the shelter-like phenomenon happens in other malignancies due to the similar microenvironment and high expression of ACE2 in some malignancies.
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Neoplasias Encefálicas/virología , COVID-19/metabolismo , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Glioblastoma/virología , SARS-CoV-2/metabolismo , Adulto , Enzima Convertidora de Angiotensina 2/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/cirugía , Neoplasias Encefálicas/ultraestructura , COVID-19/virología , Prueba de Ácido Nucleico para COVID-19 , Convalecencia , Glioblastoma/metabolismo , Glioblastoma/cirugía , Glioblastoma/ultraestructura , Humanos , Hibridación in Situ , Masculino , Microscopía Electrónica de Transmisión , Fosfoproteínas/metabolismo , ARN Viral/metabolismo , Receptores de Coronavirus/metabolismo , SARS-CoV-2/ultraestructura , Virión/ultraestructuraRESUMEN
Stromal cell derived factor-1 (SDF-1) is a chemokine that plays a critical role in the homing of stem and progenitor cells, including endothelial progenitor cells (EPCs). However, little research has been undertaken to evaluate the roles of SDF-1 in the biological functions of EPCs and related signaling pathways. The present study aimed to investigate the biological functions of EPCs in response to SDF-1, as well as the underlying mechanisms. The effects of SDF-1 treatment on EPC proliferation, migration and tube formation were assessed by performing MTS, Transwell and in vitro tube formation assays, respectively. The phosphorylation status of Akt and ERK was evaluated by western blotting. The present results indicated that SDF-1 treatment enhanced EPC proliferation, migration and tube formation compared with the control group. Furthermore, SDF-1-induced EPC proliferation was significantly reduced following treatment with a C-X-C Motif Chemokine Receptor 4 antagonist (AMD3100), a PI3K inhibitor (LY294002) and the mitogen-activated protein kinase kinase inhibitor (MEK; PD98059). SDF-1-induced migration and angiogenesis were significantly suppressed by the PI3K inhibitor, but not the MEK inhibitor. Moreover, SDF-1 significantly increased the protein expression levels of phosphorylated (p)-Akt and p-ERK; however, SDF-1-induced effects on protein expression were suppressed by AMD3100, LY294002 and PD98059. Thus, SDF-1-induced EPC proliferation was mediated by activation of the Akt and ERK signaling pathways, whereas SDF-1-mediated EPC migration and tube formation only involved activation of the Akt signaling pathway.
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OBJECTIVES: Rapid, reliable and easy-to-implement diagnostics that can be adapted in early severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis are critical to combat the epidemic. SARS-CoV-2 nucleocapsid protein (NP) is an ideal target for viral antigen-based detection. A rapid and convenient method was developed based on fluorescence immunochromatographic (FIC) assay to detect the SARS-CoV-2 NP antigen. However, the accuracy of this diagnostic method needs to be examined. METHODS: This prospective study was carried out between 10 and 15 February 2020 in seven hospitals in Wuhan and one hospital in Chongqing, China. Participants with clinically suspected SARS-CoV-2 infection were enrolled. NP antigen testing by FIC assay and nucleic acid (NA) testing by real-time reverse transcriptase PCR (RT-PCR) were performed simultaneously in a blinded manner with the same nasopharyngeal swab sample. The diagnostic accuracy of NP antigen testing was calculated by taking NA testing of RT-PCR as the reference standard, in which samples with a cycle threshold (Ct) value of ≤40 were interpreted as positive for SARS-CoV-2. RESULTS: A total of 253 participants were enrolled; two participants were excluded from the analyses because of invalid NP testing results. Of 251 participants (99.2%) included in the diagnostic accuracy analysis, 201 (80.1%) had a Ct value of ≤40. With Ct value 40 as the cutoff of NA testing, the sensitivity, specificity and percentage agreement of the FIC assay was 75.6% (95% confidence interval, 69.0-81.3), 100% (95% confidence interval, 91.1-100) and 80.5% (95% confidence interval, 75.1-84.9) respectively. CONCLUSIONS: With RT-PCR assay as the reference standard, NP antigen testing by FIC assay shows high specificity and relatively high sensitivity in SARS-CoV-2 diagnosis in the early phase of infection.
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Antígenos Virales/análisis , Prueba de COVID-19/métodos , COVID-19/diagnóstico , Proteínas de la Nucleocápside de Coronavirus/análisis , SARS-CoV-2/aislamiento & purificación , Adolescente , Adulto , Anciano , Prueba de COVID-19/normas , Femenino , Humanos , Inmunoensayo , Masculino , Persona de Mediana Edad , Nasofaringe/virología , Fosfoproteínas/análisis , Estudios Prospectivos , ARN Viral/genética , SARS-CoV-2/genética , SARS-CoV-2/inmunología , Sensibilidad y Especificidad , Adulto JovenRESUMEN
While lymphocytopenia is a common characteristic of coronavirus disease 2019 (COVID-19), the mechanisms responsible for this lymphocyte depletion are unclear. Here, we retrospectively reviewed the clinical and immunological data from 18 fatal COVID-19 cases, results showed that these patients had severe lymphocytopenia, together with high serum levels of inflammatory cytokines (IL-6, IL-8 and IL-10), and elevation of many other mediators in routine laboratory tests, including C-reactive protein, lactate dehydrogenase, α-hydroxybutyrate dehydrogenase and natriuretic peptide type B. The spleens and hilar lymph nodes (LNs) from six additional COVID-19 patients with post-mortem examinations were also collected, histopathologic detection showed that both organs manifested severe tissue damage and lymphocyte apoptosis in these six cases. In situ hybridization assays illustrated that SARS-CoV-2 viral RNA accumulates in these tissues, and transmission electronic microscopy confirmed that coronavirus-like particles were visible in the LNs. SARS-CoV-2 Spike and Nucleocapsid protein (NP) accumulated in the spleens and LNs, and the NP antigen restricted in angiotensin-converting enzyme 2 (ACE2) positive macrophages and dendritic cells (DCs). Furthermore, SARS-CoV-2 triggered the transcription of Il6, Il8 and Il1b genes in infected primary macrophages and DCs in vitro, and SARS-CoV-2-NP+ macrophages and DCs also manifested high levels of IL-6 and IL-1ß, which might directly decimate human spleens and LNs and subsequently lead to lymphocytopenia in vivo. Collectively, these results demonstrated that SARS-CoV-2 induced lymphocytopenia by promoting systemic inflammation and direct neutralization in human spleen and LNs.
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COVID-19/inmunología , Ganglios Linfáticos/inmunología , Linfopenia/inmunología , SARS-CoV-2/inmunología , Bazo/inmunología , Enzima Convertidora de Angiotensina 2/inmunología , COVID-19/complicaciones , COVID-19/patología , Proteínas de la Nucleocápside de Coronavirus/inmunología , Citocinas/inmunología , Femenino , Humanos , Inflamación/inmunología , Inflamación/patología , Ganglios Linfáticos/ultraestructura , Linfopenia/etiología , Linfopenia/patología , Persona de Mediana Edad , Fosfoproteínas/inmunología , ARN Mensajero/inmunología , Estudios Retrospectivos , SARS-CoV-2/patogenicidad , SARS-CoV-2/ultraestructura , Bazo/ultraestructuraRESUMEN
It is unclear whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can directly infect human kidney, thus leading to acute kidney injury (AKI). Here, we perform a retrospective analysis of clinical parameters from 85 patients with laboratory-confirmed coronavirus disease 2019 (COVID-19); moreover, kidney histopathology from six additional COVID-19 patients with post-mortem examinations was performed. We find that 27% (23/85) of patients exhibited AKI. The elderly patients and cases with comorbidities (hypertension and heart failure) are more prone to develop AKI. Haematoxylin & eosin staining shows that the kidneys from COVID-19 autopsies have moderate to severe tubular damage. In situ hybridization assays illustrate that viral RNA accumulates in tubules. Immunohistochemistry shows nucleocapsid and spike protein deposits in the tubules, and immunofluorescence double staining shows that both antigens are restricted to the angiotensin converting enzyme-II-positive tubules. SARS-CoV-2 infection triggers the expression of hypoxic damage-associated molecules, including DP2 and prostaglandin D synthase in infected tubules. Moreover, it enhances CD68+ macrophages infiltration into the tubulointerstitium, and complement C5b-9 deposition on tubules is also observed. These results suggest that SARS-CoV-2 directly infects human kidney to mediate tubular pathogenesis and AKI.
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Lesión Renal Aguda/etiología , COVID-19/complicaciones , Túbulos Renales/virología , SARS-CoV-2/patogenicidad , Lesión Renal Aguda/epidemiología , Lesión Renal Aguda/patología , Lesión Renal Aguda/virología , Adulto , Anciano , Anciano de 80 o más Años , Enzima Convertidora de Angiotensina 2/metabolismo , Antígenos Virales/genética , Antígenos Virales/metabolismo , COVID-19/epidemiología , COVID-19/virología , China/epidemiología , Femenino , Humanos , Inmunidad Innata , Pruebas de Función Renal , Túbulos Renales/metabolismo , Túbulos Renales/patología , Masculino , Persona de Mediana Edad , Pandemias , Estudios Retrospectivos , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , Proteínas Virales/genética , Proteínas Virales/metabolismo , Adulto JovenRESUMEN
Magnesium transporter 1 (MAGT1) is a key protein that regulates the level of free Mg2+ in cells. Previous studies found that the downregulation of MAGT1 expression in CD8+T cells of HBV patients was correlated with the decrease of intracellular magnesium. However, the expression of MAGT1 mRNA in the CD8+T cells from HBV patients was not significantly altered, indicating that the change in MAGT1 expression was accomplished through posttranscriptional regulation. Through bioinformatics and qRT-PCR detection, miR-199a-5p was found to have a target gene relationship with MAGT1. The expression levels of miR-199a-5p and MAGT1 in HBV infection were evaluated. Lentivirus assays were used to analyze the effects of miR-199a-5p upregulation and downregulation on the MAGT1 expression level and the immune system. Results showed no significant change in the expression of MAGT1 mRNA in HBV-infected cell lines, but the expression of MAGT1 was downregulated. Additionally, the expression level of miR-199a-5p was significantly increased. To this end, we predicted a target relationship between miR-199a-5p and MAGT1 by using TargetScan and verified this relationship through a luciferase activity reporter gene assay. As a result, MAGT1 was found to be the direct target of miR-199a-5p. The targeted inhibition of MAGT1 induced by miR-199a-5p overexpression led to the immune function depletion of CD8+T cells in HBV patients. Downregulating the expression level of miR-199a-5p could effectively improve the functional depletion of CD8+T cells. These findings indicate that miR-199a-5p and MAGT1 could potentially be used as biomarkers for the diagnosis and treatment of chronic HBV infection.