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BACKGROUND /OBJECTIVE: The phospholipid 1,2-dipalmitoyl-rac-glycero-3-phosphatidylethanolamine (PE) comprises two fatty acid chains: glycerol, phosphate, and ethanolamine. PE participates in critical cellular processes such as apoptosis and autophagy, which places it as a target for designing new therapeutic alternatives in diseases such as pulmonary fibrosis. Therefore, this study aimed obtain PE through a six-step organic synthesis pathway and determine its biological effect on apoptosis induction in normal human lung fibroblasts (NHLF). METHODOLOGY: The first step of the organic synthesis route began with protected glycerol that was benzylated at sn-3; later, it was deprotected to react with palmitic acid at sn-1, sn-2. To remove the benzyl group, hydrogenation was performed with palladium on carbon (Pd/C); subsequently, the molecule was phosphorylated in sn-3 with phosphorus oxychloride and triethylamine, and the intermediate was hydrolyzed in an acid medium to obtain the final compound. After PE synthesis, apoptosis assessment was performed: apoptosis was induced using exposure to annexin V-FITC/propidium iodide-ECD (PI) and quantified using flow cytometry. The experiments were performed in three NHLF cell lines with different concentrations of PE 10, 100 and 1000 µg/mL for 24 and 48 h. RESULTS: The PE obtained by organic synthesis presented a melting point of 190-192 °C, a purity of 95%, and a global yield of 8%. The evaluation of apoptosis with flow cytometry showed that at 24 h, exposure to PE 10, 100, and 1000 µg/mL induces early apoptosis in 19.42%- 25.54%, while late apoptosis was only significant P < 0.05 in cells challenged with 100 µg/mL PE. At 48 h, NHLF exposed to PE 10, 100, and 1000 µg/mL showed decreasing early apoptosis: 28.69-32.16%, 12.59-18.84%, and 10.91-12.61%, respectively. The rest of the NHLF exposed to PE showed late apoptosis: 12.03-16-42%, 11.04-15.94%, and 49.23-51.28%. Statistical analysis showed a significance P < 0.05 compared to the control. CONCLUSION: The organic synthesis route of PE allows obtaining rac-1,2-O-Dipalmitoyl-glycero-3-phosphoethanolamine (1), which showed an apoptotic effect on NHLF.
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Glicerol , Fosfatidiletanolaminas , Humanos , Fosfatidiletanolaminas/metabolismo , Apoptosis , Fibroblastos/metabolismo , Pulmón/metabolismoRESUMEN
OBJECTIVES: SSc is a devastating autoimmune disease characterized by fibrosis and obliterative vasculopathy affecting the skin and visceral organs. While the processes mediating excessive extracellular matrix deposition and fibroblast proliferation are clear, the exact link between autoimmunity and fibrosis remains elusive. Th17 cells have been proposed as critical drivers of profibrotic inflammation during SSc, but little is known about the immune components supporting their pathogenic role. Our aim was to determine cytokine responses of stimulated monocyte-derived dendritic cells (Mo-DCs) and to determine how they influence T-cell cytokine production in SSc. MATERIAL AND METHODS: Dendritic cells (DCs) activate and shape T cell differentiation by producing polarizing cytokines. Hence, we investigated the cytokine responses of monocyte-derived DCs (Mo-DCs) from patients with limited cutaneous SSc (lcSSc), diffuse cutaneous SSc (dcSSc) and healthy controls (HCs) after stimulation with toll-like receptor (TLR) agonists. Also, using co-culture assays, we analysed T cell subpopulations after contact with autologous TLR-activated Mo-DCs. RESULTS: In general, we observed an increased production of Th17-related cytokines like IL-1ß, IL-17F, IL-21 and IL-22 by SSc compared with HC Mo-DCs, with variations between lcSSc vs dcSSc and early- vs late-stage subgroups. Noticeably, we found a significant increment in IL-33 production by Mo-DCs in all SSc cases regardless of their clinical phenotype. Strikingly, T cells displayed Th2, Th17 and dual Th2-Th17 phenotypes after exposure to autologous TLR-stimulated Mo-DCs from SSc patients but not HCs. These changes were pronounced in individuals with early-stage dcSSc and less significant in the late-stage lcSSc subgroup. CONCLUSIONS: Our findings suggest that functional alterations of DCs promote immune mechanisms favouring the aberrant T cell polarization and profibrotic inflammation behind clinical SSc heterogeneity.
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Esclerodermia Sistémica , Humanos , Citocinas , Fibrosis , Células Dendríticas/patología , InflamaciónRESUMEN
Zinc oxide nanoparticles (ZnO NPs) are widely used in the cosmetic industry. They are nano-optical and nano-electrical devices, and their antimicrobial properties are applied in food packaging and medicine. ZnO NPs penetrate the body through inhalation, oral, and dermal exposure and spread through circulation to various systems and organs. Since the cardiovascular system is one of the most vulnerable systems, in this work, we studied ZnO NPs toxicity in H9c2 rat cardiomyoblasts. Cardiac cells were exposed to different concentrations of ZnO NPs, and then the morphology, proliferation, viability, mitochondrial membrane potential (ΔΨm), redox state, and protein expression were measured. Transmission electron microscopy (TEM) and hematoxylin-eosin (HE) staining showed strong morphological damage. ZnO NPs were not observed inside cells, suggesting that Zn2+ ions were internalized, causing the damage. ZnO NPs strongly inhibited cell proliferation and MTT reduction at 10 and 20 µg/cm2 after 72 h of treatment. ZnO NPs at 20 µg/cm2 elevated DCF fluorescence, indicating alterations in the cellular redox state associated with changes in ΔΨm and cell death. ZnO NPs also reduced the intracellular expression of troponin I and atrial natriuretic peptide. ZnO NPs are toxic for cardiac cells; therefore, consumption of products containing them could cause heart damage and the development of cardiovascular diseases.
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Nanopartículas del Metal , Nanopartículas , Óxido de Zinc , Ratas , Animales , Óxido de Zinc/química , Nanopartículas/toxicidad , Nanopartículas/química , Microscopía Electrónica de Transmisión , Oxidación-Reducción , Iones , Nanopartículas del Metal/toxicidad , Nanopartículas del Metal/químicaRESUMEN
Interferon-induced transmembrane (IFITM) proteins mediate protection against enveloped viruses by blocking membrane fusion at endosomes. IFITM1 and IFITM3 are crucial for protection against influenza, and various single nucleotide polymorphisms altering their function have been linked to disease susceptibility. However, bulk IFITM1 and IFITM3 mRNA expression dynamics and their correlation with clinical outcomes have not been extensively addressed in patients with respiratory infections. In this study, we evaluated the expression of IFITM1 and IFITM3 in peripheral leukocytes from healthy controls and individuals with severe pandemic influenza A(H1N1) or coronavirus disease 2019 (COVID-19). Comparisons between participants grouped according to their clinical characteristics, underlying disease, and outcomes showed that the downregulation of IFITM1 was a distinctive characteristic of severe pandemic influenza A(H1N1) that correlated with outcomes, including mortality. Conversely, increased IFITM3 expression was a common feature of severe pandemic influenza A(H1N1) and COVID-19. Using a high-dose murine model of infection, we confirmed not only the downregulation of IFITM1 but also of IFITM3 in the lungs of mice with severe influenza, as opposed to humans. Analyses in the comparative cohort also indicate the possible participation of IFITM3 in COVID-19. Our results add to the evidence supporting a protective function of IFITM proteins against viral respiratory infections in humans.
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Antígenos de Diferenciación , COVID-19 , Gripe Humana , Proteínas de la Membrana , Proteínas de Unión al ARN , Animales , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , COVID-19/genética , Humanos , Subtipo H1N1 del Virus de la Influenza A , Gripe Humana/genética , Leucocitos/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
BACKGROUND: Mesenchyme homeobox-2 (MEOX2)-mediated regulation of glioma-associated oncogene-1 (GLI1) has been associated with poor overall survival, conferring chemoresistance in lung cancer. However, the role of MEOX2/GLI1 in resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs)-based therapy remains unexplored in human lung cancer. METHODS: Functional assays using genetic silencing strategy by short hairpin RNAs, as well as cytotoxic (tetrazolium dye MTT) and clonogenic assays, were performed to evaluate MEOX2/GLI1-induced malignancy capacity in lung cancer cells. Further analysis performed includes western blot, qPCR and ChIP-qPCR assays to identify whether MEOX2/GLI1 promote EGFR/AKT/ERK activation, as well as EGFR overexpression through epigenetic mechanisms. Finally, preclinical tumour progression in vivo and progression-free disease interval analyses in patients treated with EGFR-TKI were included. RESULTS: Overexpressed MEOX2/GLI1 in both EGFR wild-type and EGFR/KRAS-mutated lung cancer cells were detected and involved in the activation/expression of EGFR/AKT/ERK biomarkers. In addition, MEOX2/GLI1 was shown to be involved in the increased proliferation of tumour cells and resistance capacity to cisplatin, EGFR-TKIs (erlotinib and AZD9291 'osimertinib'), AZD8542-SMO, and AZD6244-MEKK1/2. In addition, we identified that MEOX2/GLI1 promote lung tumour cells progression in vivo and are clinically associated with poorer progression-free disease intervals. Finally, both MEOX2 and GLI1 were detected to be epigenetically involved in EGFR expression by reducing both repressive markers polycomb-EZH2 and histone H3K27me3, but, particularly, increasing an activated histone profile H3K27Ac/H3K4me3 at EGFR-gene enhancer-promoter sequences that probably representing a novel EGFR-TKI-based therapy resistance mechanism. CONCLUSION: MEOX2/GLI1 promote resistance to cisplatin and EGFR-TKI-based therapy in lung cancer cells, modulating EGFR/AKT/ERK signalling pathway activation, as well as inducing an aberrant epigenetic modulation of the EGFR-gene expression in human lung cancer.
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Antineoplásicos/uso terapéutico , Epigénesis Genética/genética , Genes erbB-1/efectos de los fármacos , Neoplasias Pulmonares/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteína con Dedos de Zinc GLI1/genética , Antineoplásicos/farmacología , Línea Celular Tumoral , Progresión de la Enfermedad , HumanosRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by increased activation of fibroblasts/myofibroblasts. Previous reports have shown that IPF fibroblasts are resistant to apoptosis, but the mechanisms remain unclear. Since inhibition of the mitochondrial permeability transition pore (mPTP) has been implicated in the resistance to apoptosis, in this study, we analyzed the role of mitochondrial function and the mPTP on the apoptosis resistance of IPF fibroblasts under basal conditions and after mitomycin C-induced apoptosis. We measured the release of cytochrome c, mPTP opening, mitochondrial calcium release, oxygen consumption, mitochondrial membrane potential, ADP/ATP ratio, ATP concentration, and mitochondrial morphology. We found that IPF fibroblasts were resistant to mitomycin C-induced apoptosis and that calcium, a well-established activator of mPTP, is decreased as well as the release of pro-apoptotic proteins such as cytochrome c. Likewise, IPF fibroblasts showed decreased mitochondrial function, while mPTP was less sensitive to ionomycin-induced opening. Although IPF fibroblasts did not present changes in the mitochondrial membrane potential, we found a fragmented mitochondrial network with scarce, thinned, and disordered mitochondria with reduced ATP levels. Our findings demonstrate that IPF fibroblasts are resistant to mitomycin C-induced apoptosis and that altered mPTP opening contributes to this resistance. In addition, IPF fibroblasts show mitochondrial dysfunction evidenced by a decrease in respiratory parameters.
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Apoptosis , Fibroblastos/metabolismo , Fibrosis Pulmonar Idiopática/metabolismo , Mitocondrias/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial/metabolismo , Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Citocromos c/metabolismo , Fibroblastos/patología , Humanos , Fibrosis Pulmonar Idiopática/etiología , Fibrosis Pulmonar Idiopática/patología , Ionomicina , Mitocondrias/patología , Mitomicina , Oxígeno/metabolismo , Cultivo Primario de CélulasRESUMEN
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is a global health threat with the potential to cause severe disease manifestations in the lungs. Although COVID-19 has been extensively characterized clinically, the factors distinguishing SARS-CoV-2 from other respiratory viruses are unknown. Here, we compared the clinical, histopathological, and immunological characteristics of patients with COVID-19 and pandemic influenza A(H1N1). We observed a higher frequency of respiratory symptoms, increased tissue injury markers, and a histological pattern of alveolar pneumonia in pandemic influenza A(H1N1) patients. Conversely, dry cough, gastrointestinal symptoms and interstitial lung pathology were observed in COVID-19 cases. Pandemic influenza A(H1N1) was characterized by higher levels of IL-1RA, TNF-α, CCL3, G-CSF, APRIL, sTNF-R1, sTNF-R2, sCD30, and sCD163. Meanwhile, COVID-19 displayed an immune profile distinguished by increased Th1 (IL-12, IFN-γ) and Th2 (IL-4, IL-5, IL-10, IL-13) cytokine levels, along with IL-1ß, IL-6, CCL11, VEGF, TWEAK, TSLP, MMP-1, and MMP-3. Our data suggest that SARS-CoV-2 induces a dysbalanced polyfunctional inflammatory response that is different from the immune response against pandemic influenza A(H1N1). Furthermore, we demonstrated the diagnostic potential of some clinical and immune factors to differentiate both diseases. These findings might be relevant for the ongoing and future influenza seasons in the Northern Hemisphere, which are historically unique due to their convergence with the COVID-19 pandemic.
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COVID-19 , Citocinas , Subtipo H1N1 del Virus de la Influenza A , Gripe Humana , Metaloproteinasa 1 de la Matriz , Metaloproteinasa 3 de la Matriz , Receptores Inmunológicos , Adulto , Anciano , COVID-19/sangre , COVID-19/epidemiología , COVID-19/inmunología , Citocinas/sangre , Citocinas/inmunología , Femenino , Humanos , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Gripe Humana/sangre , Gripe Humana/epidemiología , Gripe Humana/inmunología , Masculino , Metaloproteinasa 1 de la Matriz/sangre , Metaloproteinasa 1 de la Matriz/inmunología , Metaloproteinasa 3 de la Matriz/sangre , Metaloproteinasa 3 de la Matriz/inmunología , Persona de Mediana Edad , Estudios Prospectivos , Receptores Inmunológicos/sangre , Receptores Inmunológicos/inmunología , Células TH1/inmunología , Células Th2/inmunologíaRESUMEN
[This corrects the article DOI: 10.3389/fimmu.2021.633297.].
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. In passages above ten and growing very actively, we observed that some human lung fibroblasts cultured under standard conditions were transformed into a lineage of epithelial-like cells (ELC). To systematically evaluate the possible mesenchymal-epithelial transition (MET) occurrence, fibroblasts were obtained from normal lungs and also from lungs affected by idiopathic interstitial diseases. When an unusual epithelial-like phenotypic change was observed, cultured cells were characterized by confocal immunofluorescence microscopy, immunoblotting, immunocytochemistry, cytofluorometry, gelatin zymography, RT-qPCR, and hybridization in a whole-transcript human microarray. Additionally, microvesicles fraction (MVs) from ELC and fibroblasts were used to induce MET, while the microRNAs (miRNAs) contained in the MVs were identified. Pattern-gene expression of the original fibroblasts and the derived ELC revealed profound changes, upregulating characteristic epithelial-cell genes and downregulating mesenchymal genes, with a marked increase of E-cadherin, cytokeratin, and ZO-1, and the loss of expression of α-SMA, collagen type I, and Thy-1 cell surface antigen (CD90). Fibroblasts, exposed to culture media or MVs from the ELC, acquired ELC phenotype. The miRNAs in MVs shown six expressed exclusively in fibroblasts, and three only in ELC; moreover, twelve miRNAs were differentially expressed between fibroblasts and ELC, all of them but one was overexpressed in fibroblasts. These findings suggest that the MET-like process can occur in human lung fibroblasts, either from normal or diseased lungs. However, the biological implication is unclear.
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Transición Epitelial-Mesenquimal , Fibroblastos/patología , Enfermedades Pulmonares Intersticiales/patología , Pulmón/patología , Actinas/metabolismo , Anciano , Biomarcadores/metabolismo , Micropartículas Derivadas de Células/metabolismo , Células Cultivadas , Análisis por Conglomerados , Colágeno Tipo I/metabolismo , Regulación hacia Abajo , Células Epiteliales/metabolismo , Células Epiteliales/patología , Transición Epitelial-Mesenquimal/genética , Fibroblastos/metabolismo , Humanos , Enfermedades Pulmonares Intersticiales/metabolismo , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Fenotipo , Antígenos Thy-1/metabolismoRESUMEN
The differentiation between influenza and coronavirus disease 2019 (COVID-19) could constitute a diagnostic challenge during the ongoing winter owing to their clinical similitude. Thus, novel biomarkers are required to enable making this distinction. Here, we evaluated whether the surfactant protein D (SP-D), a collectin produced at the alveolar epithelium with known immune properties, was useful to differentiate pandemic influenza A(H1N1) from COVID-19 in critically ill patients. Our results revealed high serum SP-D levels in patients with severe pandemic influenza but not those with COVID-19. This finding was validated in a separate cohort of mechanically ventilated patients with COVID-19 who also showed low plasma SP-D levels. However, plasma SP-D levels did not distinguish seasonal influenza from COVID-19 in mild-to-moderate disease. Finally, we found that high serum SP-D levels were associated with death and renal failure among severe pandemic influenza cases. Thus, our studies have identified SP-D as a unique biomarker expressed during severe pandemic influenza but not COVID-19.
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COVID-19/genética , Expresión Génica , Interacciones Huésped-Patógeno/genética , Subtipo H1N1 del Virus de la Influenza A , Gripe Humana/genética , Proteína D Asociada a Surfactante Pulmonar/genética , SARS-CoV-2 , Adulto , Anciano , Biomarcadores , COVID-19/sangre , COVID-19/diagnóstico , COVID-19/virología , Coinfección , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Gripe Humana/diagnóstico , Gripe Humana/virología , Masculino , Persona de Mediana Edad , Pronóstico , Proteína D Asociada a Surfactante Pulmonar/sangre , Índice de Severidad de la Enfermedad , Evaluación de Síntomas , Adulto JovenRESUMEN
The C-X-C motif chemokine ligand 17 (CXCL17) is chemotactic for myeloid cells, exhibits bactericidal activity, and exerts anti-viral functions. This chemokine is constitutively expressed in the respiratory tract, suggesting a role in lung defenses. However, little is known about the participation of CXCL17 against relevant respiratory pathogens in humans. Here, we evaluated the serum levels and lung tissue expression pattern of CXCL17 in a cohort of patients with severe pandemic influenza A(H1N1) from Mexico City. Peripheral blood samples obtained on admission and seven days after hospitalization were processed for determinations of serum CXCL17 levels by enzyme-linked immunosorbent assay (ELISA). The expression of CXCL17 was assessed by immunohistochemistry (IHQ) in lung autopsy specimens from patients that succumbed to the disease. Serum CXCL17 levels were also analyzed in two additional comparative cohorts of coronavirus disease 2019 (COVID-19) and pulmonary tuberculosis (TB) patients. Additionally, the expression of CXCL17 was tested in lung autopsy specimens from COVID-19 patients. A total of 122 patients were enrolled in the study, from which 68 had pandemic influenza A(H1N1), 24 had COVID-19, and 30 with PTB. CXCL17 was detected in post-mortem lung specimens from patients that died of pandemic influenza A(H1N1) and COVID-19. Interestingly, serum levels of CXCL17 were increased only in patients with pandemic influenza A(H1N1), but not COVID-19 and PTB. CXCL17 not only differentiated pandemic influenza A(H1N1) from other respiratory infections but showed prognostic value for influenza-associated mortality and renal failure in machine-learning algorithms and regression analyses. Using cell culture assays, we also identified that human alveolar A549 cells and peripheral blood monocyte-derived macrophages increase their CXCL17 production capacity after influenza A(H1N1) pdm09 virus infection. Our results for the first time demonstrate an induction of CXCL17 specifically during pandemic influenza A(H1N1), but not COVID-19 and PTB in humans. These findings could be of great utility to differentiate influenza and COVID-19 and to predict poor prognosis specially at settings of high incidence of pandemic A(H1N1). Future studies on the role of CXCL17 not only in severe pandemic influenza, but also in seasonal influenza, COVID-19, and PTB are required to validate our results.
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Biomarcadores/metabolismo , Quimiocinas CXC/metabolismo , Subtipo H1N1 del Virus de la Influenza A/fisiología , Gripe Humana/diagnóstico , Pulmón/metabolismo , Mycobacterium tuberculosis/fisiología , SARS-CoV-2/fisiología , Adulto , Anciano , COVID-19/diagnóstico , COVID-19/mortalidad , Quimiocinas CXC/genética , Quimiocinas CXC/inmunología , Estudios de Cohortes , Progresión de la Enfermedad , Femenino , Humanos , Gripe Humana/mortalidad , Pulmón/patología , Masculino , México , Persona de Mediana Edad , Pandemias , Evaluación del Resultado de la Atención al Paciente , Pronóstico , Análisis de Supervivencia , Tuberculosis Pulmonar/diagnóstico , Tuberculosis Pulmonar/mortalidad , Adulto JovenRESUMEN
The involvement of LncRNA SOX2-overlapping transcript (SOX2-OT), SOX2, and GLI-1 transcription factors in cancer has been well documented. Nonetheless, it is still unknown whether co-expressed SOX2-OT/SOX2 or SOX2-OT/SOX2/GLI-1 axes are epigenetically/transcriptionally involved in terms of resistance to oncology therapy and in poorer clinical outcomes for patients with lung cancer. We evaluated the role of SOX2-OT/SOX2 and SOX2-OT/SOX2/GLI-1 axes using RT-qPCR, western blot, immunofluorescence analyses, gene silencing, cellular cytotoxic, and ChIP-qPCR assays on human cell lines, solid lung malignant tumors, and normal lung tissue. We detected that the SOX2-OT/SOX2/GLI-1 axis promotes resistance to tyrosine kinase inhibitor (TKI)-erlotinib and cisplatin-based therapy. Evidence from this study show that SOX2-OT modulates the expression/activation of EGFR-pathway members AKT/ERK. Further, both SOX2-OT and GLI-1 genes are epigenetically regulated at their promoter sequences, in an LncRNA SOX2-OT-dependent manner, mainly through modifying the enrichment of the activation histone mark H3K4me3/H3K27Ac, versus the repressive histone mark H3K9me3/H3K27me3. In addition, we identified that inhibition of SOX2-OT and reduced expression of SOX2/GLI-1 sensitizes lung cancer cells to EGFR/TKI-erlotinib or cisplatin-based treatment. Finally, we show that high co-expression of SOX2-OT/SOX2 transcripts and SOX2/GLI-1 proteins appears to correlate with a poor clinical prognosis and lung malignant phenotype. Collectively, these results present evidence that LncRNA SOX2-OT modulates an orchestrated resistance mechanism, promoting poor prognosis and human lung malignancy through genetic, epigenetic, and post-translational mechanisms.
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Quinasas MAP Reguladas por Señal Extracelular/genética , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogénicas c-akt/genética , ARN Largo no Codificante/genética , Factores de Transcripción SOXB1/genética , Proteína con Dedos de Zinc GLI1/genética , Línea Celular Tumoral , Cisplatino/farmacología , Resistencia a Antineoplásicos , Epigénesis Genética , Clorhidrato de Erlotinib/farmacología , Histonas , Humanos , Neoplasias Pulmonares/diagnóstico , PronósticoRESUMEN
A hypoxic microenvironment is a hallmark in different types of tumors; this phenomenon participates in a metabolic alteration that confers resistance to treatments. Because of this, it was proposed that a combination of 2-methoxyestradiol (2-ME) and sodium dichloroacetate (DCA) could reduce this alteration, preventing proliferation through the reactivation of aerobic metabolism in lung adenocarcinoma cell line (A549). A549 cells were cultured in a hypoxic chamber at 1% O2 for 72 hours to determine the effect of this combination on growth, migration, and expression of hypoxia-inducible factors (HIFs) by immunofluorescence. The effect in the metabolism was evaluated by the determination of glucose/glutamine consumption and the lactate/glutamate production. The treatment of 2-ME (10 µM) in combination with DCA (40 mM) under hypoxic conditions showed an inhibitory effect on growth and migration. Notably, this reduction could be attributed to 2-ME, while DCA had a predominant effect on metabolic activity. Moreover, this combination decreases the signaling of HIF-3α and partially HIF-1α but not HIF-2α. The results of this study highlight the antitumor activity of the combination of 2-ME 10 µl/DCA 40 mM, even in hypoxic conditions.
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2-Metoxiestradiol/uso terapéutico , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Ácido Dicloroacético/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Hipoxia Tumoral , Microambiente Tumoral , 2-Metoxiestradiol/farmacología , Células A549 , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteínas Reguladoras de la Apoptosis/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ácido Dicloroacético/farmacología , Glucosa/metabolismo , Ácido Glutámico/metabolismo , Glutamina/metabolismo , Glucólisis/efectos de los fármacos , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ácido Láctico/metabolismo , Neoplasias Pulmonares/patología , Proteínas Represoras/metabolismo , Transducción de Señal/efectos de los fármacos , Hipoxia Tumoral/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Fibrosing diseases are causes of morbidity and mortality around the world, and they are characterized by excessive extracellular matrix (ECM) accumulation. The bHLH transcription factor scleraxis (SCX) regulates the synthesis of ECM proteins in heart fibrosis. SCX expression was evaluated in lung fibroblasts and tissue derived from fibrotic disease patients and healthy controls. We also measured SCX in sera from 57 healthy controls, and 56 Idiopathic Pulmonary Fibrosis (IPF), 40 Hypersensitivity Pneumonitis (HP), and 100 Systemic Sclerosis (SSc) patients. We report high SCX expression in fibroblasts and tissue from IPF patients versus controls. High SCX-serum levels were observed in IPF (0.663 ± 0.559 ng/mL, p < 0.01) and SSc (0.611 ± 0.296 ng/mL, p < 0.001), versus controls (0.351 ± 0.207 ng/mL) and HP (0.323 ± 0.323 ng/mL). Serum levels of the SCX heterodimerization partner, TCF3, did not associate with fibrotic illness. IPF patients with severely affected respiratory capacities and late-stage SSc patients presenting anti-topoisomerase I antibodies and interstitial lung disease showed the highest SCX-serum levels. SCX gain-of-function induced the expression of alpha-smooth muscle actin (α-SMA/ACTA2) in fibroblasts when co-overexpressed with TCF3. As late and severe stages of the fibrotic processes correlated with high circulating SCX, we postulate it as a candidate biomarker of fibrosis and a potential therapeutic target.
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Alveolitis Alérgica Extrínseca/sangre , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/sangre , Fibrosis Pulmonar Idiopática/sangre , Esclerodermia Sistémica/sangre , Adulto , Anciano , Alveolitis Alérgica Extrínseca/patología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/análisis , Biomarcadores/análisis , Biomarcadores/sangre , Células Cultivadas , Femenino , Fibroblastos/metabolismo , Humanos , Fibrosis Pulmonar Idiopática/patología , Pulmón/patología , Masculino , Persona de Mediana Edad , Esclerodermia Sistémica/patologíaRESUMEN
OBJECTIVES: To describe the kinetics of circulating cytokines and chemokines in humans with ZIKAV infection. METHODS: Serum levels of different immune mediators in patients with ZIKAV infection were measured at distinct stages of the disease, as well as in culture supernatants from human monocytes infected with a clinical ZIKAV isolate. We also looked for clinical features associated with specific immune signatures among symptomatic patients. RESULTS: We evaluated 23 ZIKAV-infected patients. Their mean age was 32 ± 8.3 years and 65% were female. ZIKAV patients showed elevated IL-9, IL-17A, and CXCL10 levels at acute stages of the disease. At day 28, levels of CCL4 and CCL5 were increased, whereas IL-1RA, CXCL8 and CCL2 were decreased. At baseline, IL-7 was increased among patients with headache, whereas CCL2, and CCL3 were decreased in patients with bleeding and rash, respectively. Our clinical ZIKAV isolate induced a broad immune response in monocytes that did not resemble the signature observed in ZIKAV patients. CONCLUSIONS: We showed a unique immune signature in our cohort of ZIKAV-infected patients. Our study may provide valuable evidence helpful to identify immune correlates of protection against ZIKAV.
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Quimiocinas/sangre , Citocinas/sangre , Infección por el Virus Zika/inmunología , Virus Zika/inmunología , Adulto , Estudios de Cohortes , Femenino , Humanos , Proteína Antagonista del Receptor de Interleucina 1/sangre , Interleucina-8/sangre , Masculino , México , Infección por el Virus Zika/sangre , Infección por el Virus Zika/virologíaRESUMEN
OBJECTIVES: To evaluate the performance of rapid influenza diagnostic tests (RIDT) and influenza vaccines' effectiveness (VE) during an outbreak setting. METHODS: We compared the performance of a RIDT with RT-PCR for influenza virus detection in influenza-like illness (ILI) patients enrolled during the 2016/17 season in Mexico City. Using the test-negative design, we estimated influenza VE in all participants and stratified by age, virus subtype, and vaccine type (trivalent vs quadrivalent inactivated vaccines). The protective value of some clinical variables was evaluated by regression analyses. RESULTS: We enrolled 592 patients. RT-PCR detected 93 cases of influenza A(H1N1)pdm09, 55 of AH3N2, 141 of B, and 13 A/B virus infections. RIDT showed 90.7% sensitivity and 95.7% specificity for influenza A virus detection, and 91.5% sensitivity and 95.3% specificity for influenza B virus detection. Overall VE was 33.2% (95% CI: 3.0-54.0; p = 0.02) against any laboratory-confirmed influenza infection. VE estimates against influenza B were higher for the quadrivalent vaccine. Immunization and occupational exposure were protective factors against influenza. CONCLUSIONS: The RIDT was useful to detect influenza cases during an outbreak setting. Effectiveness of 2016/17 influenza vaccines administered in Mexico was low but significant. Our data should be considered for future local epidemiological policies.
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Vacunas contra la Influenza/administración & dosificación , Gripe Humana/diagnóstico , Adolescente , Adulto , Niño , Pruebas Diagnósticas de Rutina/métodos , Brotes de Enfermedades , Femenino , Humanos , Inmunización , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Virus de la Influenza B/genética , Virus de la Influenza B/inmunología , Virus de la Influenza B/aislamiento & purificación , Vacunas contra la Influenza/inmunología , Gripe Humana/epidemiología , Gripe Humana/inmunología , Gripe Humana/prevención & control , Masculino , México/epidemiología , Persona de Mediana Edad , Estaciones del Año , Vacunación , Adulto JovenRESUMEN
Lung surfactant is a complex mixture of phospholipids and specific proteins but its role in the pathogenesis of interstitial lung diseases is not established. Herein, we analyzed the effects of three representative phospholipid components, that is, dipalmitoilphosphatidylcoline (DPPC), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE), on collagen expression, apoptosis and Ca2+ signaling in normal human lung fibroblasts (NHLF) and probed their effect in an experimental model of lung fibrosis. Collagen expression was measured with RT-PCR, apoptosis was measured by using either the APOPercentage assay kit (Biocolor Ltd., Northern Ireland, UK) or the Caspase-Glo 3/7 assay (Promega, Madison, WI, USA) and Ca2+ signaling by conventional epifluorescence imaging. The effect in vivo was tested in bleomycin-induced lung fibrosis in mice. DPPC and PG did not affect collagen expression, which was downregulated by PE. Furthermore, PE promoted apoptosis and induced a dose-dependent Ca2+ signal. PE-induced Ca2+ signal and apoptosis were both blocked by phospholipase C, endoplasmic reticulum pump and store-operated Ca2+ entry inhibition. PE-induced decrease in collagen expression was attenuated by blocking phospholipase C. Finally, surfactant enriched with PE and PE itself attenuated bleomycin-induced lung fibrosis and decreased the soluble collagen concentration in mice lungs. This study demonstrates that PE strongly contributes to the surfactant-induced inhibition of collagen expression in NHLF through a Ca2+ signal and that early administration of Beractant enriched with PE diminishes lung fibrosis in vivo.
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Bleomicina/efectos adversos , Fibroblastos/metabolismo , Fosfatidiletanolaminas/metabolismo , Fibrosis Pulmonar/etiología , Fibrosis Pulmonar/metabolismo , Animales , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Colágeno/genética , Colágeno/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Fosfatidiletanolaminas/farmacología , Fibrosis Pulmonar/tratamiento farmacológico , Fibrosis Pulmonar/patología , Surfactantes Pulmonares/metabolismoRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease characterized by epithelial cell activation, expansion of the fibroblast population and excessive extracellular matrix accumulation. The mechanisms are incompletely understood but evidence indicates that the deregulation of several proteases contributes to its pathogenesis. Transmembrane protease serine 4 (TMPRSS4) is a novel type II transmembrane serine protease that may promote migration and facilitate epithelial to mesenchymal transition (EMT), two critical processes in the pathogenesis of IPF. Thus, we hypothesized that over-expression of TMPRSS4 in the lung could promote the initiation and/or progression of IPF. In this study we first evaluated the expression and localization of TMPRSS4 in IPF lungs by real time PCR, western blot and immunohistochemistry. Then we examined the lung fibrotic response in wild-type and TMPRSS4 deficient mice using the bleomycin-induced lung injury model. We found that this protease is upregulated in IPF lungs, where was primarily expressed by epithelial and mast cells. Paralleling the findings in vivo, TMPRSS4 was expressed by alveolar and bronchial epithelial cells in vitro and unexpectedly, provoked an increase of E-cadherin. No expression was observed in normal human or IPF lung fibroblasts. The lung fibrotic response evaluated at 28 days after bleomycin injury was markedly attenuated in the haplodeficient and deficient TMPRSS4 mice. By morphology, a significant reduction of the fibrotic index was observed in KO and heterozygous mice which was confirmed by measurement of collagen content (hydroxyproline: WT: 164±21.1 µg/lung versus TMPRSS4 haploinsufficient: 110.2±14.3 µg/lung and TMPRSS4 deficient mice: 114.1±24.2 µg/lung (p<0.01). As in IPF, TMPRSS4 was also expressed in epithelial and mast cells. These findings indicate that TMPRSS4 is upregulated in IPF lungs and that may have a profibrotic role.
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Fibrosis Pulmonar Idiopática/genética , Lesión Pulmonar/genética , Proteínas de la Membrana/genética , Serina Endopeptidasas/genética , Regulación hacia Arriba , Animales , Bleomicina , Línea Celular , Transición Epitelial-Mesenquimal , Fibrosis , Humanos , Fibrosis Pulmonar Idiopática/metabolismo , Fibrosis Pulmonar Idiopática/patología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Lesión Pulmonar/inducido químicamente , Lesión Pulmonar/metabolismo , Lesión Pulmonar/patología , Proteínas de la Membrana/análisis , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Serina Endopeptidasas/análisis , Serina Endopeptidasas/metabolismoRESUMEN
Breast cancer is one of the most common neoplasias and the leading cause of cancer death in women worldwide. Its high mortality rate is linked to a great metastatic capacity associated with the epithelial-mesenchymal transition (EMT). During this process, a decrease in epithelial proteins expression and an increase of mesenchymal proteins are observed. On the other hand, it has been shown that dehydroepiandrosterone (DHEA), the most abundant steroid in human plasma, inhibits migration of breast cancer cells; however, the underlying mechanisms have not been elucidated. In this study, the in vitro effect of DHEA on the expression pattern of some EMT-related proteins, such as E-cadherin (epithelial), N-cadherin, vimentin and Snail (mesenchymal) was measured by Western blot and immunofluorescence in MDA-MB-231 breast cancer cells with invasive, metastatic and mesenchymal phenotype. Also, the in vivo effect of DHEA on xenograft tumor growth in nude mice (nu-/nu-) and on expression of the same epithelial and mesenchymal proteins in generated tumors was evaluated. We found that DHEA increased expression of E-cadherin and decreased N-cadherin, vimentin and Snail expression both in MD-MB-231 cells and in the formed tumors, possibly by DHEA-induced reversion of mesenchymal phenotype. These results were correlated with a tumor size reduction in mouse xenografts following DHEA administration either a week earlier or concurrent with breast cancer cells inoculation. In conclusion, DHEA could be useful in the treatment of breast cancer with mesenchymal phenotype.