RESUMO
This work reports the first dual magnetic beads (MBs) assisted immunoplatform for the simultaneous determination of BAFF (B cell activation factor) and APRIL (a proliferation-induced ligand), two cytokines related to immunity and tumour invasion, growth and metastasis. The electrochemical immunoplatform involves sandwich-type immunoassays implemented on magnetic microparticles functionalized with neutravidin (NA-MBs) or carboxylic groups (HOOC-MBs), and amperometric detection (Eapp = - 0.20 V vs. Ag pseudo-reference electrode) at screen-printed dual carbon electrodes (SPdCE) using the H2O2/hydroquinone (HQ) system. The developed immunosensors provide improved sensitivity, with LOD values of 0.33 and 16.4 pg mL-1 for BAFF and APRIL, respectively, and much shorter assay time that those claimed for ELISA kits and allow their simultaneous determination. The dual immunosensor permits discrimination between healthy individuals and patients diagnosed with Systemic Lupus Erythematosus (SLE) or colorectal cancer (CRC) through the determination of both cytokines in 100-times diluted human sera with results in agreement with those provided by the individual ELISA methodologies.
Assuntos
Doenças Autoimunes , Técnicas Biossensoriais , Citocinas , Neoplasias , Doenças Autoimunes/diagnóstico , Técnicas Biossensoriais/métodos , Citocinas/análise , Técnicas Eletroquímicas , Humanos , Peróxido de Hidrogênio , Imunoensaio/métodos , Neoplasias/diagnósticoRESUMO
This paper describes the reliable determination of progesterone (P4) in undiluted saliva making use of a disposable amperometric immunosensors implemented on low-cost and portable device/potentiostat constructed with commercial-off-the-shelf (COTS) components. The immunosensor allows the fast (45â¯min), selective and sensitive determination (5â¯pgâ¯mL-1 LOD) of P4 using amperometry in stirred solutions. The immunosensor was coupled to the COTS-based potentiostat and amperometry was made into drops of quiescent solutions. No significant differences were apparent between the analytical performance achieved with the immunosensor for P4 using both a conventional and the COST-based potentiostats. The practical applicability of the immunosensor coupled with the COTS-based potentiostat was demonstrated by determining the endogenous P4 content in different undiluted saliva samples with highly variable endogenous contents of the target hormone. The obtained results were in good agreement with those provided by the conventional ELISA methodology and with the contents reported in the literature for samples with similar characteristics. This validated the combined device for the reliable and minimally invasive determination of the target hormone involving a very simple protocol and taking only 45â¯min.
Assuntos
Técnicas Eletroquímicas/instrumentação , Imunoensaio/instrumentação , Progesterona/análise , Saliva/química , Anticorpos/imunologia , Técnicas Eletroquímicas/métodos , Feminino , Humanos , Imunoensaio/métodos , Limite de Detecção , Fenômenos Magnéticos , Masculino , Ciclo Menstrual/metabolismo , Progesterona/imunologia , Progesterona/metabolismoAssuntos
Calcineurina/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Fosforilação/fisiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Células Precursoras de Linfócitos T/metabolismo , Fator de Transcrição STAT3/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Células Cultivadas , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células Mieloides/metabolismo , Proteoma/metabolismo , Transcriptoma/fisiologiaAssuntos
Canais de Potássio Éter-A-Go-Go/genética , Recidiva Local de Neoplasia/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/classificação , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Pré-Escolar , Estudos de Coortes , Feminino , Humanos , Lactente , Masculino , Recidiva Local de Neoplasia/genética , Pediatria , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The serine/threonine kinase AMP-activated protein kinase (AMPK) and its downstream effectors, including endothelial nitric oxide synthase and BCL-2, are hyperactivated in B-cell precursor-acute lymphoblastic leukemia (BCP-ALL) cells with MLL gene rearrangements. We investigated the role of activated AMPK in supporting leukemic cell survival and evaluated AMPK as a potential drug target. Exposure of leukemic cells to the commercial AMPK inhibitor compound C resulted in massive apoptosis only in cells with MLL gene rearrangements. These results were confirmed by targeting AMPK with specific short hairpin RNAs. Compound C-induced apoptosis was associated with mitochondrial membrane depolarization, reactive oxygen species production, cytochrome c release and caspases cleavage, indicating intrinsic apoptosis pathway activation. Treatment with low concentrations of compound C resulted in a strong antileukemic activity, together with cytochrome c release and cleavage of caspases and poly(ADP-ribose) polymerase, also in MLL-rearranged primary BCP-ALL samples. Moreover, AMPK inhibition in MLL-rearranged cell lines synergistically enhanced the antiproliferative effects of vincristine, daunorubicin, cytarabine, dexamethasone and L-asparaginase in most of the evaluated conditions. Taken together, these results indicate that the activation of the AMPK pathway directly contributes to the survival of MLL-rearranged BCP-ALL cells and AMPK inhibitors could represent a new therapeutic strategy for this high-risk leukemia.