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1.
Biologicals ; 81: 101661, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36621353

RESUMO

The Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) collected historical data from 20 biopharmaceutical industry members on their experience with the in vivo adventitious virus test, the in vitro virus test, and the use of next generation sequencing (NGS) for viral safety. Over the past 20 years, only three positive in vivo adventitious virus test results were reported, and all were also detected in another concurrent assay. In more than three cases, data collected as a part of this study also found that the in vivo adventitious virus test had given a negative result for a sample that was later found to contain virus. Additionally, the in vivo adventitious virus test had experienced at least 21 false positives and had to be repeated an additional 21 times all while using more than 84,000 animals. These data support the consideration and need for alternative broad spectrum viral detection tests that are faster, more sensitive, more accurate, more specific, and more humane. NGS is one technology that may meet this need. Eighty one percent of survey respondents are either already actively using or exploring the use of NGS for viral safety. The risks and challenges of replacing in vivo adventitious virus testing with NGS are discussed. It is proposed to update the overall virus safety program for new biopharmaceutical products by replacing in vivo adventitious virus testing approaches with modern methodologies, such as NGS, that maintain or even improve the final safety of the product.


Assuntos
Produtos Biológicos , Vírus , Animais , Sequenciamento de Nucleotídeos em Larga Escala , Vírus/genética , Contaminação de Medicamentos/prevenção & controle
2.
J Clin Invest ; 132(12)2022 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-35536645

RESUMO

Caffeine is the most widely consumed psychoactive substance in the world. Strikingly, the molecular pathways engaged by its regular consumption remain unclear. We herein addressed the mechanisms associated with habitual (chronic) caffeine consumption in the mouse hippocampus using untargeted orthogonal omics techniques. Our results revealed that chronic caffeine exerts concerted pleiotropic effects in the hippocampus at the epigenomic, proteomic, and metabolomic levels. Caffeine lowered metabolism-related processes (e.g., at the level of metabolomics and gene expression) in bulk tissue, while it induced neuron-specific epigenetic changes at synaptic transmission/plasticity-related genes and increased experience-driven transcriptional activity. Altogether, these findings suggest that regular caffeine intake improves the signal-to-noise ratio during information encoding, in part through fine-tuning of metabolic genes, while boosting the salience of information processing during learning in neuronal circuits.


Assuntos
Cafeína , Proteômica , Animais , Cafeína/metabolismo , Cafeína/farmacologia , Hipocampo/metabolismo , Aprendizagem , Camundongos , Plasticidade Neuronal/fisiologia
3.
J Proteomics ; 216: 103670, 2020 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-31991189

RESUMO

The skin plays a crucial role in host defences against microbial attack and the innate cells must provide the immune system with sufficient information to organize these defences. This unique feature makes the skin a promising site for vaccine administration. Although cellular innate immune events during vaccination have been widely studied, initial events remain poorly understood. Our aim is to determine molecular biomarkers of skin innate reaction after intradermal (i.d.) immunization. Using an ex vivo human explant model from healthy donors, we investigated by NanoLC-MS/MS analysis and MALDI-MSI imaging, to detect innate molecular events (lipids, metabolites, proteins) few hours after i.d. administration of seasonal trivalent influenza vaccine (TIV). This multimodel approach allowed to identify early molecules differentially expressed in dermal and epidermal layers at 4 and 18 h after TIV immunization compared with control PBS. In the dermis, the most relevant network of proteins upregulated were related to cell-to-cell signalling and cell trafficking. The molecular signatures detected were associated with chemokines such as CXCL8, a chemoattractant of neutrophils. In the epidermis, the most relevant networks were associated with activation of antigen-presenting cells and related to CXCL10. Our study proposes a novel step-forward approach to identify biomarkers of skin innate reaction. SIGNIFICANCE: To our knowledge, there is no study analyzing innate molecular reaction to vaccines at the site of skin immunization. What is known on skin reaction is based on macroscopic (erythema, redness…), microscopic (epidermal and dermal tissues) and cellular events (inflammatory cell infiltrate). Therefore, we propose a multimodal approach to analyze molecular events at the site of vaccine injection on skin tissue. We identified early molecular networks involved biological functions such cell migration, cell-to-cell interaction and antigen presentation, validated by chemokine expression, in the epidermis and dermis, then could be used as early indicator of success in immunization.


Assuntos
Vacinas contra Influenza , Influenza Humana , Humanos , Vacinas contra Influenza/efeitos adversos , Influenza Humana/prevenção & controle , Injeções Intradérmicas , Estações do Ano , Espectrometria de Massas em Tandem , Vacinação/efeitos adversos
4.
Anal Chem ; 91(16): 10840-10848, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31355633

RESUMO

Visualizing the distributions of drugs and their metabolites is one of the key emerging application areas of matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) within pharmaceutical research. The success of a given MALDI-MSI experiment is ultimately determined by the ionization efficiency of the compounds of interest, which in many cases are too low to enable detection at relevant concentrations. In this work we have taken steps to address this challenge via the first application of laser-postionisation coupled with MALDI (so-called MALDI-2) to the analysis and imaging of pharmaceutical compounds. We demonstrate that MALDI-2 increased the signal intensities for 7 out of the 10 drug compounds analyzed by up to 2 orders of magnitude compared to conventional MALDI analysis. This gain in sensitivity enabled the distributions of drug compounds in both human cartilage and dog liver tissue to be visualized using MALDI-2, whereas little-to-no signal from tissue was obtained using conventional MALDI. This work demonstrates the vast potential of MALDI-2-MSI in pharmaceutical research and drug development and provides a valuable tool to broaden the application areas of MSI. Finally, in an effort to understand the ionization mechanism, we provide the first evidence that the preferential formation of [M + H]+ ions with MALDI-2 has no obvious correlation with the gas-phase proton affinity values of the analyte molecules, suggesting, as with MALDI, the occurrence of complex and yet to be elucidated ionization phenomena.


Assuntos
Lasers , Preparações Farmacêuticas/análise , Pesquisa Farmacêutica , Animais , Cartilagem/química , Cães , Humanos , Fígado/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
5.
J Pharm Biomed Anal ; 170: 220-227, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-30933897

RESUMO

Indoleamine-2,3-dioxygénase (IDO1) is an enzyme which converts tryptophan (Trp) into kynurenine (Kyn). Having a critical role in tumor immune escape by decreasing Trp and increasing Kyn levels in the microenvironment, IDO1 was one of the first targets for small molecules drug discovery in the field of immuno-oncology. A potent and selective IDO1 inhibitor such as Epacadostat (EPA) was shown to enhance the antitumor activity by restoring the immune system fitness. As exposure at the site of action and to its specific target are identified as the most important factors for success in drug discovery, the objective of this study was to explore the target exposure and intra-tumor pharmacodynamics effects of EPA drug on the tumor metabolism. To do so, we used both Quantitative Mass Spectrometry Imaging (QMSI) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) technologies in order to monitor drug and metabolites distribution and their endogenous quantity in the CT26 mouse tumor model. Target exposure analysis showed that almost 61% of EPA signal (26 µg/g) was concentrated within 38% of the entire tumor surface. Semi quantitative analysis of this region confirmed a positive correlation between IDO1 expression and EPA concentration. In parallel, pharmacodynamics analysis highlighted a response efficacy through Kyn/Trp ratio calculation that was shown decreasing after EPA treatment as noticed in treated CT26 tumors (-82%), plasma (-63%) and blood (-62%) compared to control samples. Finally, 15% and 85% of Kyn signal was found in regions with high and low EPA, respectively. In this study, using QMSI, we went further than only quantifying the metabolites and the drug, by estimating the pharmacological effect efficacy of the drug through a target exposure study handled in different regions of the tumor either expressing IDO1 or Kyn.


Assuntos
Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Oximas/farmacologia , Sulfonamidas/farmacologia , Animais , Cromatografia Líquida/métodos , Modelos Animais de Doenças , Cinurenina/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/metabolismo , Espectrometria de Massas em Tandem/métodos , Triptofano/metabolismo
6.
Anal Chem ; 91(9): 6266-6274, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30938516

RESUMO

The aim of this study was to assess potential sources of variability in quantitative imaging mass spectrometry (IMS) across multiple sites, analysts, and instruments. A sample from rat liver perfused with clozapine was distributed to three sites for analysis by three analysts using a predefined protocol to standardize the sample preparation, acquisition, and data analysis parameters. In addition, two commonly used approaches to IMS quantification, the mimetic tissue model and dilution series, were used to quantify clozapine and its major metabolite norclozapine in isolated perfused rat liver. The quantification was evaluated in terms of precision and accuracy with comparison to liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The results of this study showed that, across three analysts with six replicates each, both quantitative IMS methods achieved relative standard deviations in the low teens and accuracies of around 80% compared to LC-MS/MS quantification of adjacent tissue sections. The utility of a homogeneously coated stable-isotopically labeled standard (SIL) for normalization was appraised in terms of its potential to improve precision and accuracy of quantification as well as qualitatively reduce variability in the sample tissue images. SIL normalization had a larger influence on the dilution series, where the use of the internal standard was necessary to achieve accuracy and precision comparable to the non-normalized mimetic tissue model data. Normalization to the internal standard appeared most effective when the intensity ratio of the analyte to internal standard was approximately one, and thus precludes this method as a universal normalization approach for all ions in the acquisition.


Assuntos
Clozapina/análise , Fígado/química , Animais , Cromatografia Líquida , Clozapina/administração & dosagem , Clozapina/metabolismo , Marcação por Isótopo , Fígado/metabolismo , Masculino , Modelos Animais , Estrutura Molecular , Ratos , Ratos Wistar , Espectrometria de Massas em Tandem
7.
Mol Ther Methods Clin Dev ; 10: 17-28, 2018 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-30003117

RESUMO

Variants in the PLPP3 gene encoding for lipid phosphate phosphohydrolase 3 have been associated with susceptibility to atherosclerosis independently of classical risk factors. PLPP3 inactivates lysophosphatidic acid (LPA), a pro-inflammatory, pro-thrombotic product of phospholipase activity. Here we performed the first exploratory analysis of PLPP3, LPA, and LPA receptors (LPARs 1-6) in human atherosclerosis. PLPP3 transcript and protein were repressed when comparing plaques versus normal arteries and plaques from symptomatic versus asymptomatic patients, and they were negatively associated with risk of adverse cardiovascular events. PLPP3 localized to macrophages, smooth muscle, and endothelial cells (ECs) in plaques. LPAR 2, 5, and especially 6 showed increased expression in plaques, with LPAR6 localized in ECs and positively correlated to PLPP3. Utilizing in situ mass spectrometry imaging, LPA and its precursors were found in the plaque fibrous cap, co-localizing with PLPP3 and LPAR6. In vitro, PLPP3 silencing in ECs under LPA stimulation resulted in increased expression of adhesion molecules and cytokines. LPAR6 silencing inhibited LPA-induced cell activation, but not when PLPP3 was silenced simultaneously. Our results show that repression of PLPP3 plays a key role in atherosclerosis by promoting EC activation. Altogether, the PLPP3 pathway represents a suitable target for investigations into novel therapeutic approaches to ameliorate atherosclerosis.

8.
J Am Soc Mass Spectrom ; 28(12): 2635-2645, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28913742

RESUMO

Mass spectrometry imaging datasets are mostly analyzed in terms of average intensity in regions of interest. However, biological tissues have different morphologies with several sizes, shapes, and structures. The important biological information, contained in this highly heterogeneous cellular organization, could be hidden by analyzing the average intensities. Finding an analytical process of morphology would help to find such information, describe tissue model, and support identification of biomarkers. This study describes an informatics approach for the extraction and identification of mass spectrometry image features and its application to sample analysis and modeling. For the proof of concept, two different tissue types (healthy kidney and CT-26 xenograft tumor tissues) were imaged and analyzed. A mouse kidney model and tumor model were generated using morphometric - number of objects and total surface - information. The morphometric information was used to identify m/z that have a heterogeneous distribution. It seems to be a worthwhile pursuit as clonal heterogeneity in a tumor is of clinical relevance. This study provides a new approach to find biomarker or support tissue classification with more information. Graphical Abstract ᅟ.


Assuntos
Neoplasias Renais/patologia , Rim/patologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Biomarcadores Tumorais/análise , Linhagem Celular Tumoral , Processamento de Imagem Assistida por Computador/métodos , Rim/diagnóstico por imagem , Neoplasias Renais/diagnóstico por imagem , Camundongos Endogâmicos BALB C
9.
J Proteome Res ; 16(8): 2993-3001, 2017 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-28648079

RESUMO

Mass spectrometry imaging (MSI) is able to simultaneously record the distributions of hundreds of molecules directly from tissue. Rapid direct tissue analysis is essential for MSI in order to maintain spatial localization and acceptable measurement times. The absence of an explicit analyte separation/purification step means MSI lacks the depth of coverage of LC-MS/MS. In this work, we demonstrate how atmospheric pressure MALDI-MSI enables the same tissue section to be first analyzed by MSI, to identify regions of interest that exhibit distinct molecular signatures, followed by localized proteomics analysis using laser capture microdissection isolation and LC-MS/MS.


Assuntos
Imageamento Tridimensional/métodos , Microdissecção e Captura a Laser/métodos , Proteômica/métodos , Animais , Pressão Atmosférica , Cromatografia Líquida , Humanos , Análise Espacial , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas em Tandem , Fatores de Tempo , Distribuição Tecidual
10.
SLAS Discov ; 22(10): 1182-1192, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28557618

RESUMO

Inhibition of NK and effector T-cell functions and activation of regulatory cell populations are the main immunosuppressive effects of indoleamine-2,3-dioxygenase1 (IDO1). By converting tryptophan (Trp) into kynurenine (Kyn), IDO1 is involved in the immune response homeostasis, and its dysregulated expression is described in immune-related pathologies, as tumors that hijack it to evade immune destruction. Thereby, IDO1 inhibitors are being developed to stimulate antitumor immune responses. Existing and standard quantitation methods of IDO1 substrate and metabolite(s) are based on the total level of Trp and its metabolites determined by liquid chromatography tandem mass spectrometry analysis in human plasma, cerebrospinal fluid, and brain. Here, we describe the detection, localization, and absolute quantitation of Trp and Kyn by quantitative mass spectrometry imaging (qMSI) in transfected murine tumor models expressing various levels of IDO1. Myeloid, glycolysis metabolic signatures, and correlation between IDO1 expression and Trp to Kyn conversion are also shown. High-definition IDO1 and GCN2 immunostainings overlaid with Kyn molecular images underline the tumor metabolism and heterogeneity. The development of immunotherapies such as IDO1 inhibitors requires a deep understanding of the immune system, the interplay of cancer cells, and biomarker characterization. Our data underline that qMSI allows the study of the spatial distribution and quantitation of endogenous immune metabolites for biology and pharmacology studies.


Assuntos
Imageamento Tridimensional , Espectrometria de Massas , Neoplasias/imunologia , Neoplasias/metabolismo , Microambiente Tumoral/imunologia , Animais , Linhagem Celular Tumoral , Cinurenina/sangue , Camundongos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Triptofano/sangue
11.
J Am Soc Mass Spectrom ; 27(12): 2075-2079, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27752913

RESUMO

Atmospheric pressure MALDI on a Q-Exactive instrument was optimized for in-source decay and pseudo-MS3. The dependence of AP-MALDI ISD on the MALDI liquid matrix was investigated for peptides and proteins. The liquid matrices enabled long-life ISD signal, and exhibited high fragment ion yield and signal stability. Extensive a-, b-, c-, y-, and z-type fragment series were observed depending on the matrix used but were most extensive with 2,5-DHB. Complete sequence coverage of small peptide and intact protein-terminus sequence tags were obtained and confirmed using HCD as a pseudo-MS3 method. Graphical Abstract ᅟ.


Assuntos
Peptídeos/análise , Proteínas/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Sequência de Aminoácidos , Íons , Peptídeos/química , Proteínas/química
12.
MAbs ; 6(6): 1385-93, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25484065

RESUMO

Bevacizumab induces normalization of abnormal blood vessels, making them less leaky. By binding to vascular endothelial growth factor, it indirectly attacks the vascular tumor mass. The optimal delivery of targeted therapies including monoclonal antibodies or anti-angiogenesis drugs to the target tissue highly depends on the blood-brain barrier permeability. It is therefore critical to investigate how drugs effectively reach the tumor. In situ investigation of drug distribution could provide a better understanding of pharmacological agent action and optimize chemotherapies for solid tumors. We developed an imaging method coupled to protein identification using matrix-assisted laser desorption/ionization mass spectrometry. This approach monitored bevacizumab distribution within the brain structures, and especially within the tumor, without any labeling.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Inibidores da Angiogênese/farmacocinética , Inibidores da Angiogênese/uso terapêutico , Animais , Anticorpos Monoclonais Humanizados/farmacocinética , Bevacizumab , Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Monitoramento de Medicamentos/métodos , Feminino , Glioblastoma/metabolismo , Humanos , Masculino , Camundongos Nus , Palivizumab , Reprodutibilidade dos Testes , Distribuição Tecidual , Fator A de Crescimento do Endotélio Vascular/imunologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Proteomics ; 14(10): 1290-301, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24376047

RESUMO

Glioblastoma multiforme is one of the most common intracranial tumors encountered in adults. This tumor of very poor prognosis is associated with a median survival rate of approximately 14 months. One of the major issues to better understand the biology of these tumors and to optimize the therapy is to obtain the molecular structure of glioblastoma. MALDI molecular imaging enables location of molecules in tissues without labeling. However, molecular identification in situ is not an easy task. In this paper, we used MALDI imaging coupled to in-source decay to characterize markers of this pathology. We provided MALDI molecular images up to 30 µm spatial resolution of mouse brain tissue sections. MALDI images showed the heterogeneity of the glioblastoma. In the various zones and at various development stages of the tumor, using our top-down strategy, we identified several proteins. These proteins play key roles in tumorigenesis. Particular attention was given to the necrotic area with characterization of hemorrhage, one of the most important poor prognosis factors in glioblastoma.


Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Imagem Molecular/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Encéfalo/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Masculino , Camundongos , Camundongos Nus
14.
J Proteomics ; 79: 172-9, 2013 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-23279932

RESUMO

Identification of biomarkers is a major issue for enhancement of chemotherapies. The molecular characterization of tissues necessitates the identification of thousands of biomolecules each participating in physiopathological processes. MALDI in-source decay (ISD) fragmentation has already been proven to be effective for protein characterization. However, the difficulty to identify proteins from complex mixtures such as tissue sections can limit the applications of this technique. In this study, we evidenced that tubulin has an unusual fragmentation pathway in the MALDI source. This striking property allowed the detecting of several mouse brain tubulin isotypes simultaneously by simply using laser fragmentation. Tubulin isoforms are consistent markers of a bad prognosis of solid tumors and could be the target of targeted chemotherapies. Such a direct molecular printout of tubulin in tissues is a milestone that should be useful either at preclinical or clinical stage.


Assuntos
Biomarcadores Tumorais/isolamento & purificação , Tubulina (Proteína)/isolamento & purificação , Animais , Células HeLa , Humanos , Camundongos , Isoformas de Proteínas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
15.
Trends Biotechnol ; 30(9): 466-74, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22762968

RESUMO

Mass spectrometry (MS)-based technology provides label-free localization of molecules in tissue samples. Drugs, proteins, lipids and metabolites can easily be monitored in their environment. Resolution can be achieved down to the cellular level (10-20 µm) for conventional matrix-assisted laser desorption/ionization (MALDI) imaging, or even to the subcellular level for more complex technologies such as secondary ionization mass spectrometry (SIMS) imaging. One question remains: are we going to be able to investigate functional relationships between drugs and proteins and compare with localized phenomena? This review describes the various spatial levels of investigation offered by mass spectrometry imaging (MSI), and the advantages and disadvantages compared with other labeling technologies.


Assuntos
Imagem Molecular/métodos , Preparações Farmacêuticas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Histocitoquímica , Humanos , Preparações Farmacêuticas/metabolismo , Proteínas/química , Proteínas/metabolismo
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