RESUMEN
BNT162b2, a nucleoside-modified mRNA formulated in lipid nanoparticles that encodes the SARS-CoV-2 spike glycoprotein (S) stabilized in its prefusion conformation, has demonstrated 95% efficacy in preventing COVID-191. Here we extend a previous phase-I/II trial report2 by presenting data on the immune response induced by BNT162b2 prime-boost vaccination from an additional phase-I/II trial in healthy adults (18-55 years old). BNT162b2 elicited strong antibody responses: at one week after the boost, SARS-CoV-2 serum geometric mean 50% neutralizing titres were up to 3.3-fold above those observed in samples from individuals who had recovered from COVID-19. Sera elicited by BNT162b2 neutralized 22 pseudoviruses bearing the S of different SARS-CoV-2 variants. Most participants had a strong response of IFNγ+ or IL-2+ CD8+ and CD4+ T helper type 1 cells, which was detectable throughout the full observation period of nine weeks following the boost. Using peptide-MHC multimer technology, we identified several BNT162b2-induced epitopes that were presented by frequent MHC alleles and conserved in mutant strains. One week after the boost, epitope-specific CD8+ T cells of the early-differentiated effector-memory phenotype comprised 0.02-2.92% of total circulating CD8+ T cells and were detectable (0.01-0.28%) eight weeks later. In summary, BNT162b2 elicits an adaptive humoral and poly-specific cellular immune response against epitopes that are conserved in a broad range of variants, at well-tolerated doses.
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Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Vacunas contra la COVID-19/inmunología , COVID-19/inmunología , SARS-CoV-2/inmunología , Linfocitos T/inmunología , Adolescente , Adulto , Vacuna BNT162 , Linfocitos T CD8-positivos/inmunología , COVID-19/virología , Vacunas contra la COVID-19/administración & dosificación , Vacunas contra la COVID-19/efectos adversos , Epítopos de Linfocito T/inmunología , Femenino , Humanos , Inmunoglobulina G/inmunología , Memoria Inmunológica , Interferón gamma/inmunología , Interleucina-2/inmunología , Masculino , Persona de Mediana Edad , SARS-CoV-2/química , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Células TH1/inmunología , Adulto JovenRESUMEN
To promote drug delivery to exact sites and cell types, the surface of nanocarriers is functionalized with targeting antibodies or ligands, typically coupled by covalent chemistry. Once the nanocarrier is exposed to biological fluid such as plasma, however, its surface is inevitably covered with various biomolecules forming the protein corona, which masks the targeting ability of the nanoparticle. Here, we show that we can use a pre-adsorption process to attach targeting antibodies to the surface of the nanocarrier. Pre-adsorbed antibodies remain functional and are not completely exchanged or covered by the biomolecular corona, whereas coupled antibodies are more affected by this shielding. We conclude that pre-adsorption is potentially a versatile, efficient and rapid method of attaching targeting moieties to the surface of nanocarriers.
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Anticuerpos , Células Dendríticas/metabolismo , Portadores de Fármacos , Nanopartículas de Magnetita/química , Corona de Proteínas/química , Anticuerpos/química , Anticuerpos/farmacología , Células Dendríticas/citología , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacología , Humanos , Poliestirenos/químicaRESUMEN
HYPOTHESIS: Carbon dot systems are highly surface sensitive fluorescent nanomaterials. In the presence of specific molecules or ions, the fluorescence properties can be strongly influenced. Often their fluorescent properties are activated or strongly enhanced through passivation agents such as polymer coatings. While several passivating polymers have been directly attached to the carbon dot systems, the interaction of carbon dot systems with the polymer surface of colloids has not been investigated as a way to activate or enhance the photoluminescent properties. Here, we show for the first time that the interaction of carbon dot systems with polymer colloids can strongly enhance the fluorescent properties of the carbon dot systems. EXPERIMENTS: To introduce carbon dot - polymer nanoparticle interactions, carbon dots are either generated directly in a microwave assisted synthesis in the presence of negatively charged polystyrene nanoparticles (in situ) or synthesized in the microwave separately and mixed afterwards with polymer nanoparticles (mixing). For the carbon dot system synthesis, chitosan, 1,2-ethylenediamine, and acetic acid are used as precursors. The produced carbon dot - polymer nanoparticle system are characterized by scanning electron microscopy, transmission electron microscopy, and flow cytometry measurements, and their interaction is assessed by fluorescence spectroscopy and fluorescence lifetime measurements. FINDINGS: We show that depending on the synthesis route (in situ or mixing), the carbon dot systems are either covalently attached (in situ) or electrostatically bound (mixing) to the surface of the nanoparticles. Regardless of the preparation methods of the investigated carbon dot - polymer nanoparticle system and the interaction (chemical or physical) with the surface, the fluorescence intensity is strongly enhanced and the fluorescence lifetime prolonged. These findings indicate a stabilization of the radiative trap states of carbon dot systems through interaction with the surface of the particles.
RESUMEN
PURPOSE: Head and neck squamous cell carcinoma (HNSCC) cell lines with cytoplasmically sequestered mutant p53 (p53(mut_c)) are frequently more resistant to cisplatin (CDDP) than cells with mutant but nuclear p53 (p53(mut_n)). The aim of the study was to identify underlying mechanisms implicated in CDDP resistance of HNSCC cells carrying cytoplasmic p53(mut). METHODS: Microarray analysis, quantitative reverse transcription polymerase chain reaction, Western blot analysis and immunocytochemistry were used to identify and evaluate candidate genes involved in CDDP resistance of p53(mut_c) cells. RNAi knockdown or treatment with inhibitors together with flow cytometry-based methods was used for functional assessment of the identified candidate genes. Cellular metabolic activity was assessed with the XTT assay, and the redox capacity of cells was evaluated by measuring cellular glutathione (GSH) levels. RESULTS: Upregulation of ABCC2 and ABCG2 transporters was observed in CDDP-resistant p53(mut_c) HNSCC cells. Furthermore, p53(mut_c) cells exhibited a pronounced side population that could be suppressed by RNAi knockdown of ABCG2 as well as treatment with the ATP-binding-cassette transporter inhibitors imatinib, MK571 and tariquidar. Metabolic activity and cellular GSH levels were higher in CDDP-resistant p53(mut_c) cells, consistent with a higher capacity to fend off cytotoxic oxidative effects such as those caused by CDDP treatment. Finally, ABCC2/G2 inhibition of HNSCC cells with MK571 markedly enhanced CDDP sensitivity of HNSCC cells. CONCLUSIONS: The observations in this study point to a major role of p53(mut_c) in conferring a stem cell like phenotype to HNSCC cells that is associated with ABCC2/G2 overexpression, high GSH and metabolic activity levels as well as CDDP resistance.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Antineoplásicos/farmacología , Carcinoma de Células Escamosas/metabolismo , Cisplatino/farmacología , Citoplasma/metabolismo , Resistencia a Antineoplásicos , Glutatión/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Mutación , Proteína p53 Supresora de Tumor/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Western Blotting , Carcinoma de Células Escamosas/tratamiento farmacológico , Línea Celular Tumoral , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Humanos , Inmunohistoquímica , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia ArribaRESUMEN
Objective response rates to standard chemotherapeutic regimens remain low in pancreatic cancer. Subpopulations of cells have been identified in various solid tumors which express stem cell-associated markers and are associated with increased resistance against radiochemotherapy. We investigated the expression of stem cell genes and markers of epithelial-mesenchymal transition in pancreatic cancer cells that survived high concentrations of gemcitabine treatment. Capan-1 and Panc-1 cells were continuously incubated with 1 and 10 µM gemcitabine. Surviving cells were collected after 1, 3 and 6 days. Expression of PDX-1, SHH, CD24, CD44, CD133, EpCAM, CBX7, OCT4, SNAIL, SLUG, TWIST, Ki-67, E-cadherin, ß-catenin and vimentin were quantified by qPCR or immunocytochemistry. Migration was assessed by woundhealing assay. SHH was knocked down using RNA interference. Five primary pancreatic cancer cell lines were used to validate the qPCR results. All investigated genes were upregulated after 6 days of gemcitabine incubation. Highest relative expression levels were observed for OCT4 (13.4-fold), CD24 (47.3-fold) and EpCAM (15.9-fold) in Capan-1 and PDX-1 (13.3fold), SHH (24.1-fold), CD44 (17.4-fold), CD133 (20.2-fold) and SLUG (15.2-fold) in Panc-1 cells. Distinct upregulation patterns were observed in the primary cells. Migration was increased in Panc-1 cells and changes in the expression of E-cadherin and ß-catenin were typical of epithelial-mesenchymal transition in both cell lines. SHH knockdown reduced IC(50) from 30.1 to 27.6 nM in Capan-1 while it strongly inhibited proli-feration in Panc-1 cells. Cells surviving high-dose gemcitabine treatment express increased levels of stem cell genes, show characteristics associated with epithelial-mesenchymal transition and retain their proliferative capacity.