Desarrollo y evaluación de KIT NAPPA Array para su uso como plataforma de secreening de fármacos

Comunicaciones a congreso

Functional proteomics approaches for high throuput determination of CKIT and small molecules

Comunicaciones a congreso

A simple immunoassay for extracellular vesicle liquid biopsy in microliters of non-processed plasma

Background: Extracellular vesicles (EVs), released by most cell types, provide an excellent source of biomarkers in biological fluids. However, in order to perform validation studies and screenings of patient samples, it is still necessary to develop general techniques permitting rapid handling of small amounts of biological samples from large numbers of donors. Results: Here we describe a method that, using just a few microliters of patient’s plasma, identifies tumour markers exposed on EVs. Studying physico-chemical properties of EVs in solution, we demonstrate that they behave as stable colloidal suspensions and therefore, in immunocapture assays, many of them are unable to interact with a stationary functionalised surface. Using flocculation methods, like those used to destabilize colloids, we demonstrate that cationic polymers increase EV ζ-potential, diameter, and sedimentation coefficient and thus, allow a more efficient capture on antibody-coated surfaces by both ELISA and bead-assisted flow cytometry. These findings led to optimization of a protocol in microtiter plates allowing effective immunocapture of EVs, directly in plasma without previous ultracentrifugation or other EV enrichment. The method, easily adaptable to any laboratory, has been validated using plasma from lung cancer patients in which the epithelial cell marker EpCAM has been detected on EVs. Conclusions: This optimized high throughput, easy to automate, technology allows screening of large numbers of patients to phenotype tumour markers in circulating EVs, breaking barriers for the validation of proposed EV biomarkers and the discovery of new one

Evaluación del efecto acaricida de Momordica charantia, Megaskepasma erythrochlamys y Gliricidia sepium sobre Rhipicephalus microplus

Objective. The acaricidal activity of Momordica charantia (Mc), Megaskepasma erythrochlamys (Me) and Gliricidia sepium (Gs) on Rhipicephalus microplus (Rm) was evaluated. Materials and methods. Preliminary phytochemical analysis of leaves of the methanolic extract of Mc (EMc), the ethanolic extract of Me (EMe) and the acetone extract of Gs (EGs) were carried out through the technique of colorimetry and thin layer chromatography (CCD). The acaricidal activity was performed through in-vitro tests using the larval immersion test (LIT) and the adult immersion test (AIT). For in-situ tests, grazing cattle naturally infested with ticks were used, using the LC50 obtained from the in-vitro AIT tests; later the teleogines were taken to incubation to evaluate their reproductive capacity. Results. The presence of several groups of secondary metabolites of acaricidal interest was determined. The acaricidal effect of the extracts of the plants on teleogines was demonstrated; although only EGs showed larvicidal activity. Extracts at 160 mg/mL affected the life cycle of Rm by inhibiting ovoposition in 46.9%, 66.1% and 84.03% (p<0.05) for EGs, EMc and EMe, respectively. On the other hand, the in-situ tests showed a significant difference (p<0.05) between the treatment of EMc and EMe with respect to the control groups. Conclusions. The results obtained are promising to strengthen the possibility of linking the extracts of these plants into integrated plans for the control of ticks in cattle systems.  Objetivo. Se evaluó la actividad acaricida de Momordica charantia (Mc), Megaskepasma erythrochlamys (Me) y Gliricidia sepium (Gs) sobre Rhipicephalus microplus (Rm). Materiales y métodos. Se realizó la marcha fitoquímica preliminar de hojas del extracto metanólico de Mc (EMc), del extracto etanólico de Me (EMe) y del extracto acetónico de Gs (EGs) a través de la técnica de colorimetría y cromatografía en capa delgada (CCD).  La actividad acaricida se realizó a través de pruebas in-vitro utilizando la prueba de inmersión de larvas  (LIT) y la prueba de inmersión de adultos (AIT). Para las pruebas in-situ se usaron bovinos en pastoreo infestados naturalmente con garrapatas, utilizando las CL50 obtenidas en las pruebas in-vitro AIT; posteriormente las teleoginas se llevaron  a incubación para evaluar su capacidad reproductiva. Resultados. Se determinó la presencia de varios grupos de metabolitos secundarios de interés acaricida. Se demostró el efecto acaricida de los extractos de las plantas sobre teleoginas; aunque sólo EGs mostró actividad larvicida. Los extractos a 160 mg/mL afectaron el ciclo de vida de Rm inhibiendo la ovoposición en un 46.9%, 66.1% y 84.03% (p<0.05) para EGs, EMc y EMe, respectivamente. Por otro lado, en las pruebas in situ se observó diferencia significativa (p<0.05) entre el tratamiento de EMc y EMe respecto a los grupos controles. Conclusiones. Los resultados obtenidos son prometedores para fortalecer la posibilidad de vinculación de los extractos de estas plantas dentro de planes integrados de control de garrapatas en sistemas de producción de bovinos

Deciphering biomarkers for leptomeningeal metastasis in malignant hemopathies (Lymphoma/Leukemia) patients by comprehensive multipronged proteomics characterization of cerebrospinal fluid

In the present work, leptomeningeal disease, a very destructive form of systemic cancer, was characterized from several proteomics points of view. This pathology involves the invasion of the leptomeninges by malignant tumor cells. The tumor spreads to the central nervous system through the cerebrospinal fluid (CSF) and has a very grim prognosis; the average life expectancy of patients who suffer it does not exceed 3 months. The early diagnosis of leptomeningeal disease is a challenge because, in most of the cases, it is an asymptomatic pathology. When the symptoms are clear, the disease is already in the very advanced stages and life expectancy is low. Consequently, there is a pressing need to determine useful CSF proteins to help in the diagnosis and/or prognosis of this disease. For this purpose, a systematic and exhaustive proteomics characterization of CSF by multipronged proteomics approaches was performed to determine different protein profiles as potential biomarkers. Proteins such as PTPRC, SERPINC1, sCD44, sCD14, ANPEP, SPP1, FCGR1A, C9, sCD19, and sCD34, among others, and their functional analysis, reveals that most of them are linked to the pathology and are not detected on normal CSF. Finally, a panel of biomarkers was verified by a prediction model for leptomeningeal disease, showing new insights into the research for potential biomarkers that are easy to translate into the clinic for the diagnosis of this devastating disease.We gratefully acknowledge financial support from the Spanish Health Institute, Carlos III (ISCIII), for the grants: FIS PI14/01538, FIS PI17/01930 and CB16/12/00400. We also acknowledge Fondos FEDER (EU) and Junta Castilla-León (COVID-19 grant COV20EDU/00187). The Proteomics Unit belongs to ProteoRed, PRB3-ISCIII, supported by grant PT17/0019/0023 of the PE I + D + I2017-2020, funded by ISCIII and FEDER—Norma Galicia is supported by the CONACYT Program. P. Juanes-Velasco is supported by JCYL PhD Program “Nos Impulsa-JCYL” and scholarship JCYLEDU/601/2020

MAPK inhibitors dynamically affect melanoma release of immune NKG2D-ligands, as soluble protein and extracellular vesicle-associated

Metastatic melanoma presents, in many cases, oncogenic mutations in BRAF, a MAPK involved in proliferation of tumour cells. BRAF inhibitors, used as therapy in patients with these mutations, often lead to tumour resistance and, thus, the use of MEK inhibitors was introduced in clinics. BRAFi/MEKi, a combination that has modestly increased overall survival in patients, has been proven to differentially affect immune ligands, such as NKG2D-ligands, in drug-sensitive vs. drug-resistant cells. However, the fact that NKG2D-ligands can be released as soluble molecules or in extracellular vesicles represents an additional level of complexity that has not been explored. Here we demonstrate that inhibition of MAPK using MEKi, and the combination of BRAFi with MEKi in vitro, modulates NKG2D-ligands in BRAF-mutant and WT melanoma cells, together with other NK activating ligands. These observations reinforce a role of the immune system in the generation of resistance to directed therapies and support the potential benefit of MAPK inhibition in combination with immunotherapies. Both soluble and EV-associated NKG2D-ligands, generally decreased in BRAF-mutant melanoma cell supernatants after MAPKi in vitro, replicating cell surface expression. Because potential NKG2D-ligand fluctuation during MAPKi treatment could have different consequences for the immune response, a pilot study to measure NKG2D-ligand variation in plasma or serum from metastatic melanoma patients, at different time points during MAPKi treatment, was performed. Not all NKG2D-ligands were equally detected. Further, EV detection did not parallel soluble protein. Altogether, our data confirm the heterogeneity between melanoma lesions, and suggest testing several NKG2D-ligands and other melanoma antigens in serum, both as soluble or vesicle-released proteins, to help classifying immune competence of patients

Comprehensive and systematic characterization of multi-functionalized cisplatin nano-conjugate: from the chemistry and proteomic biocompatibility to the animal model

Background Nowadays, nanoparticles (NPs) have evolved as multifunctional systems combining different custom anchorages which opens a wide range of applications in biomedical research. Thus, their pharmacological involvements require more comprehensive analysis and novel nanodrugs should be characterized by both chemically and biological point of view. Within the wide variety of biocompatible nanosystems, iron oxide nanoparticles (IONPs) present mostly of the required features which make them suitable for multifunctional NPs with many biopharmaceutical applications. Results Cisplatin-IONPs and different functionalization stages have been broadly evaluated. The potential application of these nanodrugs in onco-therapies has been assessed by studying in vitro biocompatibility (interactions with environment) by proteomics characterization the determination of protein corona in different proximal fluids (human plasma, rabbit plasma and fetal bovine serum),. Moreover, protein labeling and LC–MS/MS analysis provided more than 4000 proteins de novo synthetized as consequence of the nanodrugs presence defending cell signaling in different tumor cell types (data available via ProteomeXchanges with identified PXD026615). Further in vivo studies have provided a more integrative view of the biopharmaceutical perspectives of IONPs. Conclusions Pharmacological proteomic profile different behavior between species and different affinity of protein coating layers (soft and hard corona). Also, intracellular signaling exposed differences between tumor cell lines studied. First approaches in animal model reveal the potential of theses NPs as drug delivery vehicles and confirm cisplatin compounds as strengthened antitumoral agents

Bead-assisted SARS-CoV-2 multi-antigen serological test allows effective identification of patients

Many new aspects of COVID-19 disease, including different clinical manifestations, have been identified during the pandemic. The wide array of symptoms and variation in disease severity after SARS-CoV-2 infection might be related to heterogeneity in the immune responses of different patients. Here we describe a new method for a simple multi-antigen serological test that generates a full picture of seroconversion in a single reaction. The assay is based on the detection by flow cytometry of multiple immunoglobulin classes (isotypes) specific for four SARS-CoV-2 antigens: the Spike glycoprotein (one of the highly immunogenic proteins), its RBD fragment (the major target for neutralising antibodies), the nucleocapsid protein and the main cysteine-like protease. Until now, most diagnostic serological tests measured antibodies to only one antigen and some patients seemed to not make any antibody response. Our data reveal that while most patients respond against all the viral antigens tested, others show a marked bias to make antibodies against either proteins exposed on the viral particle or those released after cellular infection. Combining all the four antigens and using machine learning techniques, it was possible to clearly discriminate between patients and healthy controls with 100% confidence. Further, combination of antigens and different immunoglobulin isotypes in this multi-antigen assay improved the classification of patients with mild and severe disease. Introduction of this method will facilitate massive screenings of patients to evaluate their immune response. It could also support vaccination campaigns both to select non-immune individuals and to distinguish infected patients from vaccine responders.This work was supported by the Spanish National Research Council (CSIC, project numbers 202020E079 and CSIC-COVID19-028) and grants from Madrid Regional Government “IMMUNOTHERCAN” [S2017/BMD-3733-2 (MVG)]; the Spanish Ministry of Science and Innovation [(MCIU/AEI/FEDER, EU): RTI2018-093569-B-I00 (MVG), SAF2017-82940-R (JMRF), SAF2017-83265-R (HTR); SAF2017-82886-R (FSM)]; Health Institute Carlos III (ISCIII) [RETICS Program RD16/0012/0006; RIER (JMRF); PI19/00549 (AA)]. The study was also funded by “La Caixa Banking Foundation” (HR17-00016 to FSM) and Fondo Supera COVID (CRUE-Banco de Santander) to FSM.N

Single-reaction multi-antigen serological test for comprehensive evaluation of SARS-CoV-2 patients by flow cytometry

Here, we describe a new, simple, highly multiplexed serological test that generates a more complete picture of seroconversion than single antigen-based assays. Flow cytometry is used to detect multiple Ig isotypes binding to four SARS-CoV-2 antigens: the Spike glycoprotein, its RBD fragment (the main target for neutralizing antibodies), the nucleocapsid protein, and the main cysteine-like protease in a single reaction. Until now, most diagnostic serological tests measured antibodies to only one antigen and in some laboratory-confirmed patients no SARS-CoV-2-specific antibodies could be detected. Our data reveal that while most patients respond against all the viral antigens tested, others show a marked bias to make antibodies against either proteins exposed on the viral particle or those released after cellular infection. With this assay, it was possible to discriminate between patients and healthy controls with 100% confidence. Analysing the response of multiple Ig isotypes to the four antigens in combination may also help to establish a correlation with the severity degree of disease. A more detailed description of the immune responses of different patients to SARS-CoV-2 virus might provide insight into the wide array of clinical presentations of COVID-19.This work was supported by: Spanish National Research Council (CSIC-202020E079, CSIC-COVID19-028); Madrid Regional Government “IMMUNOTHERCAN” [S2017/BMD-3733-2 (MVG)]; Spanish Ministry of Science and Innovation [(MCIU/AEI/FEDER, EU, RTI2018-093569-B-I00 (MVG), SAF2017-82940-R (JMRF), SAF2017-83265-R (HTR); SAF2017-82886-R (FSM)]; Health Institute Carlos III (ISCIII) [RETICS Program RD16/0012/0006; RIER (EMGC); PI19/00549 (AA)]; “La Caixa Bank Foundation” (HR17-00016), Fondo Supera COVID (CRUE-Banco de Santander), both to FSM.Peer reviewe

Evaluation of chemically modified carrier proteins for developing monoclonal antibodies against a clinically relevant mutation of cKIT

et al.In this report we show that combining double-chemically modified carrier proteins and hetero-functional cross-linkers allows preparing tailor-made hapten-protein carrier conjugates. Accordingly, a new carrier protein has been designed where carboxylic groups were transformed into highly reactive primary amino groups by reaction with ethylendiamine after activation with EDCI. The aminated protein carrier is then modified by different cross-linkers (hyper-activated proteins) at different conditions in order to control the conjugation ratio from 1 to > 12 molecules of hapten per carrier protein. Finally, this novel strategy has been successfully used to develop antibodies against a short specific peptide corresponding to a point mutation (D816V) of cKIT, which is a clinically relevant mutation related to mastocytosis and gastrointestinal stroma tumor. © 2012 Elsevier B.V.We gratefully acknowledge financial support from Health Institute Carlos III of Spain (ISCIII, FIS PI081884) and JCYL-SAN10. María Gonzalez-Gonzalez is supported by a PhD Scholarship of ISCIII FI08/00721.Peer Reviewe