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Pesquisa : D09.698.330 [Categoria DeCS]
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[PMID]:29304068
[Au] Autor:Drake AC; Lee Y; Burgess EM; Karlsson JOM; Eroglu A; Higgins AZ
[Ad] Endereço:School of Chemical, Biological and Environmental Engineering, Oregon State University, Corvallis, Oregon, United States of America.
[Ti] Título:Effect of water content on the glass transition temperature of mixtures of sugars, polymers, and penetrating cryoprotectants in physiological buffer.
[So] Source:PLoS One;13(1):e0190713, 2018.
[Is] ISSN:1932-6203
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Long-term storage of viable mammalian cells is important for applications ranging from in vitro fertilization to cell therapy. Cryopreservation is currently the most common approach, but storage in liquid nitrogen is relatively costly and the requirement for low temperatures during shipping is inconvenient. Desiccation is an alternative strategy with the potential to enable viable cell preservation at more convenient storage temperatures without the need for liquid nitrogen. To achieve stability during storage in the dried state it is necessary to remove enough water that the remaining matrix forms a non-crystalline glassy solid. Thus, the glass transition temperature is a key parameter for design of cell desiccation procedures. In this study, we have investigated the effects of moisture content on the glass transition temperature (Tg) of mixtures of sugars (trehalose or raffinose), polymers (polyvinylpyrrolidone or Ficoll), penetrating cryoprotectants (ethylene glycol, propylene glycol, or dimethyl sulfoxide), and phosphate buffered saline (PBS) solutes. Aqueous solutions were dried to different moisture contents by equilibration with saturated salt solutions, or by baking at 95°C. The glass transition temperatures of the dehydrated samples were then measured by differential scanning calorimetry. As expected, Tg increased with decreasing moisture content. For example, in a desiccation medium containing 0.1 M trehalose in PBS, Tg ranged from about 360 K for a completely dry sample to about 220 K at a water mass fraction of 0.4. Addition of polymers to the solutions increased Tg, while addition of penetrating cryoprotectants decreased Tg. Our results provide insight into the relationship between relative humidity, moisture content and glass transition temperature for cell desiccation solutions containing sugars, polymers and penetrating cryoprotectants.
[Mh] Termos MeSH primário: Crioprotetores/química
Polímeros/química
Açúcares/química
Temperatura de Transição
Água/química
[Mh] Termos MeSH secundário: Tampões (Química)
Varredura Diferencial de Calorimetria
Criopreservação/métodos
Dessecação/métodos
Dimetil Sulfóxido/química
Etilenoglicol/química
Ficoll/química
Vidro/química
Modelos Teóricos
Povidona/química
Propilenoglicol/química
Rafinose/química
Soluções/química
Trealose/química
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, N.I.H., EXTRAMURAL
[Nm] Nome de substância:
0 (Buffers); 0 (Cryoprotective Agents); 0 (Polymers); 0 (Solutions); 0 (Sugars); 059QF0KO0R (Water); 25702-74-3 (Ficoll); 6DC9Q167V3 (Propylene Glycol); B8WCK70T7I (Trehalose); FC72KVT52F (Ethylene Glycol); FZ989GH94E (Povidone); N5O3QU595M (Raffinose); YOW8V9698H (Dimethyl Sulfoxide)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180205
[Lr] Data última revisão:
180205
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:180106
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pone.0190713


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[PMID]:29054581
[Au] Autor:Eronina TB; Mikhaylova VV; Chebotareva NA; Borzova VA; Yudin IK; Kurganov BI
[Ad] Endereço:Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, 33, bld. 2, Leninsky Ave., Moscow 119071, Russia. Electronic address: eronina@inbi.ras.ru.
[Ti] Título:Mechanism of aggregation of UV-irradiated glycogen phosphorylase b at a low temperature in the presence of crowders and trimethylamine N-oxide.
[So] Source:Biophys Chem;232:12-21, 2018 01.
[Is] ISSN:1873-4200
[Cp] País de publicação:Netherlands
[La] Idioma:eng
[Ab] Resumo:To characterize the initial stages of protein aggregation, the kinetics of aggregation of UV-irradiated glycogen phosphorylase b (UV-Phb) was studied under conditions when the aggregation proceeded at a low rate (10°C, 0.03M Hepes buffer, pH6.8, containing 0.1M NaCl). Aggregation of UV-Phb was induced by polyethylene glycol and Ficoll-70, acting as crowders, or a natural osmolyte trimethylamine N-oxide (TMAO). It has been shown that the initial rate of the stage of aggregate growth is proportional to the protein concentration squared, suggesting that the order of aggregation with respect to the protein is equal to two. It has been concluded that the aggregation mechanism of UV-Phb at 10°C in the presence of crowders includes the nucleation stage and stages of protein aggregate growth (the basic aggregation pathway). The aggregation mechanism is complicated in the presence of TMAO, and the stage of aggregate-aggregate assembly induced by TMAO should be added to the basic aggregation pathway. It has been shown that the ability of TMAO at a low concentration (0.05M) to induce aggregation of UV-Phb is due to the decrease in the absolute value of zeta potential of the protein in the presence of TMAO.
[Mh] Termos MeSH primário: Inibidores Enzimáticos/farmacologia
Ficoll/farmacologia
Glicogênio Fosforilase Muscular/antagonistas & inibidores
Metilaminas/farmacologia
Polietilenoglicóis/farmacologia
Temperatura Ambiente
[Mh] Termos MeSH secundário: Animais
Difusão Dinâmica da Luz
Inibidores Enzimáticos/química
Ficoll/química
Glicogênio Fosforilase Muscular/isolamento & purificação
Glicogênio Fosforilase Muscular/metabolismo
Cinética
Metilaminas/química
Polietilenoglicóis/química
Agregados Proteicos/efeitos dos fármacos
Coelhos
Raios Ultravioleta
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Enzyme Inhibitors); 0 (Methylamines); 0 (Protein Aggregates); 25702-74-3 (Ficoll); 30IQX730WE (Polyethylene Glycols); EC 2.4.1.- (Glycogen Phosphorylase, Muscle Form); FLD0K1SJ1A (trimethyloxamine)
[Em] Mês de entrada:1711
[Cu] Atualização por classe:171213
[Lr] Data última revisão:
171213
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171022
[St] Status:MEDLINE


  3 / 1006 MEDLINE  
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[PMID]:28425679
[Au] Autor:Rusinga FI; Weis DD
[Ad] Endereço:Department of Chemistry and R. N. Adams Institute for Bioanalytical Chemistry, University of Kansas, Lawrence, Kansas, USA.
[Ti] Título:Soft interactions and volume exclusion by polymeric crowders can stabilize or destabilize transient structure in disordered proteins depending on polymer concentration.
[So] Source:Proteins;85(8):1468-1479, 2017 Aug.
[Is] ISSN:1097-0134
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The effects of macromolecular crowding on the transient structure of intrinsically disordered proteins is not well-understood. Crowding by biological molecules inside cells could modulate transient structure and alter IDP function. Volume exclusion theory and observations of structured proteins suggest that IDP transient structure would be stabilized by macromolecular crowding. Amide hydrogen exchange (HX) of IDPs in highly concentrated polymer solutions would provide valuable insights into IDP transient structure under crowded conditions. Here, we have used mass spectrometry to measure HX by a transiently helical random coil domain of the activator of thyroid and retinoid receptor (ACTR) in solutions containing 300 g L and 400 g L of Ficoll, a synthetic polysaccharide, using a recently-developed strong cation exchange-based cleanup method [Rusinga, et al., Anal Chem 2017;89:1275-1282]. Transiently helical regions of ACTR exchanged faster in 300 g L Ficoll than in dilute buffer. In contrast, one transient helix exchanged more slowly in 400 g L Ficoll. Nonspecific interactions destabilize ACTR helicity in 300 g L Ficoll because ACTR engages with the Ficoll polymer mesh. In contrast, 400 g L Ficoll is a semi-dilute solution where ACTR cannot engage the Ficoll mesh. At this higher concentration, volume exclusion stabilizes ACTR helicity because ACTR is compacted in interstitial spaces between Ficoll molecules. Our results suggest that the interplay between nonspecific interactions and volume exclusion in different cellular compartments could modulate IDP function by altering the stability of IDP transient structures. Proteins 2017; 85:1468-1479. © 2017 Wiley Periodicals, Inc.
[Mh] Termos MeSH primário: Proteína de Ligação a CREB/química
Ficoll/química
Hidrogênio/química
Proteínas Intrinsicamente Desordenadas/química
Coativador 3 de Receptor Nuclear/química
Proteínas Recombinantes de Fusão/química
[Mh] Termos MeSH secundário: Sequência de Aminoácidos
Animais
Proteína de Ligação a CREB/genética
Clonagem Molecular
Medição da Troca de Deutério
Escherichia coli/genética
Escherichia coli/metabolismo
Expressão Gênica
Seres Humanos
Proteínas Intrinsicamente Desordenadas/genética
Espectrometria de Massas
Camundongos
Coativador 3 de Receptor Nuclear/genética
Conformação Proteica em alfa-Hélice
Dobramento de Proteína
Estabilidade Proteica
Proteínas Recombinantes de Fusão/genética
Proteínas Recombinantes/química
Proteínas Recombinantes/genética
Proteínas Recombinantes/metabolismo
Alinhamento de Sequência
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Intrinsically Disordered Proteins); 0 (Recombinant Fusion Proteins); 0 (Recombinant Proteins); 25702-74-3 (Ficoll); 7YNJ3PO35Z (Hydrogen); EC 2.3.1.48 (CREB-Binding Protein); EC 2.3.1.48 (Crebbp protein, mouse); EC 2.3.1.48 (NCOA3 protein, human); EC 2.3.1.48 (Nuclear Receptor Coactivator 3)
[Em] Mês de entrada:1708
[Cu] Atualização por classe:170825
[Lr] Data última revisão:
170825
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170421
[St] Status:MEDLINE
[do] DOI:10.1002/prot.25307


  4 / 1006 MEDLINE  
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[PMID]:28353249
[Au] Autor:Rahmoune H; Guest PC
[Ad] Endereço:Department of Chemical Engineering and Biotechnology, University of Cambridge, CB2 3RA, Cambridge, UK. hr288@cam.ac.uk.
[Ti] Título:Preparation of Peripheral Blood Mononuclear Cells (PBMCs) as a Model for Proteomic Studies of Psychiatric Disorders.
[So] Source:Adv Exp Med Biol;974:299-303, 2017.
[Is] ISSN:0065-2598
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Peripheral blood mononuclear cells (PBMCs) have been used as a surrogate model of brain function in studies of psychiatric disorders, such as schizophrenia. This chapter describes the preparation of PBMCs from whole blood using a density gradient/cell culture protocol. This includes collection of the PBMC culture media and preparation of cell extracts in order to provide the raw materials for proteomic analyses.
[Mh] Termos MeSH primário: Proteínas Sanguíneas/análise
Separação Celular/métodos
Leucócitos Mononucleares/metabolismo
Transtornos Mentais/sangue
Proteômica/métodos
[Mh] Termos MeSH secundário: Células Cultivadas
Centrifugação
Ficoll
Seres Humanos
Transtornos Mentais/patologia
Esquizofrenia/sangue
Esquizofrenia/patologia
Manejo de Espécimes/métodos
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Blood Proteins); 25702-74-3 (Ficoll)
[Em] Mês de entrada:1709
[Cu] Atualização por classe:170928
[Lr] Data última revisão:
170928
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170330
[St] Status:MEDLINE
[do] DOI:10.1007/978-3-319-52479-5_28


  5 / 1006 MEDLINE  
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[PMID]:28267209
[Au] Autor:Munari F; Bortot A; Zanzoni S; D'Onofrio M; Fushman D; Assfalg M
[Ad] Endereço:Department of Biotechnology, University of Verona, Italy.
[Ti] Título:Identification of primary and secondary UBA footprints on the surface of ubiquitin in cell-mimicking crowded solution.
[So] Source:FEBS Lett;591(7):979-990, 2017 Apr.
[Is] ISSN:1873-3468
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Despite significant advancements in our understanding of ubiquitin-mediated signaling, the influence of the intracellular environment on the formation of transient ubiquitin-partner complexes remains poorly explored. In our work, we introduce macromolecular crowding as a first level of complexity toward the imitation of a cellular environment in the study of such interactions. Using NMR spectroscopy, we find that the stereospecific complex of ubiquitin and the ubiquitin-associated domain (UBA) is minimally perturbed by the crowding agent Ficoll. However, in addition to the primary canonical recognition patch on ubiquitin, secondary patches are identified, indicating that in cell-mimicking crowded solution, UBA contacts ubiquitin at multiple sites.
[Mh] Termos MeSH primário: Aminoácidos/química
Espectroscopia de Ressonância Magnética/métodos
Domínios Proteicos
Ubiquitina/química
[Mh] Termos MeSH secundário: Algoritmos
Aminoácidos/genética
Aminoácidos/metabolismo
Sítios de Ligação/genética
Ficoll/química
Seres Humanos
Modelos Moleculares
Ligação Proteica
Estrutura Secundária de Proteína
Soluções/química
Estereoisomerismo
Propriedades de Superfície
Ubiquitina/genética
Ubiquitina/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Amino Acids); 0 (Solutions); 0 (Ubiquitin); 25702-74-3 (Ficoll)
[Em] Mês de entrada:1706
[Cu] Atualização por classe:170601
[Lr] Data última revisão:
170601
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170308
[St] Status:MEDLINE
[do] DOI:10.1002/1873-3468.12615


  6 / 1006 MEDLINE  
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[PMID]:28102665
[Au] Autor:Guseman AJ; Pielak GJ
[Ad] Endereço:Department of Chemistry, ‡Department of Biochemistry and Biophysics, and §Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.
[Ti] Título:Cosolute and Crowding Effects on a Side-By-Side Protein Dimer.
[So] Source:Biochemistry;56(7):971-976, 2017 Feb 21.
[Is] ISSN:1520-4995
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The effects of small (∼10 Da) and larger (>10 Da) cosolutes on the equilibrium stability of monomeric globular proteins are broadly understood, excluding volume stabilizes proteins and chemical interactions are stabilizing when repulsive, but destabilizing when attractive. Proteins, however, rarely work alone. Here, we investigate the effects of small and large cosolutes on the equilibrium stability of the simplest defined protein-protein interactions, the side-by-side homodimer formed by the A34F variant of the 56-residue B1 domain of protein G. We used F nuclear magnetic resonance spectroscopy to quantify the effects of urea, trimethylamine oxide, Ficoll, and more physiologically relevant cosolutes on the dimer dissociation constant. The data reveal the same stabilizing and destabilizing influences from chemical interactions as observed in studies of protein stability. Results with more physiologically relevant molecules such as bovine serum albumin, lysozyme, and reconstituted Escherichia coli cytosol reflect the importance of chemical interactions between these cosolutes and the test protein. Our study serves as a stepping-stone to a more complete understanding of crowding effects on protein-protein interactions.
[Mh] Termos MeSH primário: Multimerização Proteica
Receptores de GABA-B/química
Receptores de GABA-B/metabolismo
[Mh] Termos MeSH secundário: Citosol/química
Escherichia coli/citologia
Ficoll/química
Flúor
Metilaminas/química
Modelos Moleculares
Muramidase/química
Ressonância Magnética Nuclear Biomolecular
Estabilidade Proteica
Receptores de GABA-B/genética
Soroalbumina Bovina/química
Ureia/química
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (GABA type B receptor, subunit 1); 0 (Methylamines); 0 (Receptors, GABA-B); 25702-74-3 (Ficoll); 27432CM55Q (Serum Albumin, Bovine); 284SYP0193 (Fluorine); 8W8T17847W (Urea); EC 3.2.1.17 (Muramidase); FLD0K1SJ1A (trimethyloxamine)
[Em] Mês de entrada:1705
[Cu] Atualização por classe:171116
[Lr] Data última revisão:
171116
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170120
[St] Status:MEDLINE
[do] DOI:10.1021/acs.biochem.6b01251


  7 / 1006 MEDLINE  
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[PMID]:28034522
[Au] Autor:Kaur I; Zulovich JM; Gonzalez M; McGee KM; Ponweera N; Thandi D; Alvarez EF; Annandale K; Flagge F; Rezvani K; Shpall E
[Ad] Endereço:Stem Cell Transplantation and Cellular Therapy, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA. Electronic address: ikaur@mdanderson.org.
[Ti] Título:Comparison of two methodologies for the enrichment of mononuclear cells from thawed cord blood products: The automated Sepax system versus the manual Ficoll method.
[So] Source:Cytotherapy;19(3):433-439, 2017 Mar.
[Is] ISSN:1477-2566
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:BACKGROUND AIMS: Umbilical cord blood (CB) is being used as a source of hematopoietic stem cells (HSCs) and immune cells to treat many disorders. Because these cells are present in low numbers in CB, investigators have developed strategies to expand HSCs and other immune cells such as natural killer (NK) cells. The initial step in this process is to enrich mononuclear cells (MNCs) while depleting unwanted cells. The manual method of MNC enrichment is routinely used by many centers; however, it is an open system, time-consuming and operator dependent. For clinical manufacturing, it is important to have a closed system to avoid microbial contamination. METHODS: In this study, we optimized an automated, closed system (Sepax) for enriching MNCs from cryopreserved CB units. RESULTS: Using Sepax, we observed higher recovery of total nucleated cells (TNC), CD34 cells, NK cells and monocytes when compared to manual enrichment, despite similar TNC and CD34 viability with the two methods. Even though the depletion of red blood cells, granulocytes and platelets was superior using the manual method, significantly higher CFU-GM were obtained in MNCs enriched using Sepax compared to the manual method. This is likely related to the fact that the automated Sepax significantly shortened the processing time (Sepax: 74 - 175 minutes versus manual method: 180 - 290 minutes). The use of DNAse and MgCl during the Sepax thaw and wash procedure prevents clumping of cells and loss of viability, resulting in improved post-thaw cell recovery. DISCUSSION: We optimized enrichment of MNCs from cryopreserved CB products in a closed system using the Sepax which is a walk away and automated processing system.
[Mh] Termos MeSH primário: Separação Celular/instrumentação
Separação Celular/métodos
Eritrócitos/citologia
Sangue Fetal/citologia
Ficoll/química
[Mh] Termos MeSH secundário: Técnicas de Cultura de Células
Proliferação Celular
Sobrevivência Celular
Criopreservação
Eritrócitos/fisiologia
Citometria de Fluxo
Congelamento/efeitos adversos
Granulócitos/citologia
Granulócitos/fisiologia
Células-Tronco Hematopoéticas/citologia
Células-Tronco Hematopoéticas/fisiologia
Seres Humanos
Monócitos/citologia
Monócitos/fisiologia
[Pt] Tipo de publicação:COMPARATIVE STUDY; EVALUATION STUDIES; JOURNAL ARTICLE
[Nm] Nome de substância:
25702-74-3 (Ficoll)
[Em] Mês de entrada:1708
[Cu] Atualização por classe:170802
[Lr] Data última revisão:
170802
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161231
[St] Status:MEDLINE


  8 / 1006 MEDLINE  
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[PMID]:28024644
[Au] Autor:Gtari W; Bey H; Aschi A; Bitri L; Othman T
[Ad] Endereço:Université de Tunis El Manar, Faculté des Sciences de Tunis, LR99ES16 Laboratoire Physique de la Matière Molle et de la Modélisation Électromagnétique, 2092 Tunis, Tunisia.
[Ti] Título:Impact of macromolecular crowding on structure and properties of pepsin and trypsin.
[So] Source:Mater Sci Eng C Mater Biol Appl;72:98-105, 2017 Mar 01.
[Is] ISSN:1873-0191
[Cp] País de publicação:Netherlands
[La] Idioma:eng
[Ab] Resumo:The change of conformation of pepsin and trypsin in absence and presence of a high molecular crowding agent has been characterized using dynamic light scattering (DLS). Structural properties were investigated as a function of chemical denaturant concentrations, the guanidine hydrochloride (GdmCl). The results showed that Ficoll 400, macromolecular crowding, has a strong effect on the chemical denaturation process of these two proteins. The changes of measured hydrodynamic radius are attributed to the enhancement effect of the crowder agent due to the excluded volume effects. The data proved that the large size of a macromolecular crowder plays a crucial role on the conformation of a protein in its unfolded states. The values of interactions Parameter k of complex particles and a number of proteins n attached on the Ficoll 400 measured in different GdmCl concentrations. The effect of aging on the structure of complex are studied by small angle light scattering (SALS).
[Mh] Termos MeSH primário: Pepsina A/química
Tripsina/química
[Mh] Termos MeSH secundário: Difusão Dinâmica da Luz
Ficoll/química
Guanidina/química
Hidrodinâmica
Cinética
Pepsina A/metabolismo
Desnaturação Proteica
Estrutura Terciária de Proteína
Espalhamento a Baixo Ângulo
Eletricidade Estática
Tripsina/metabolismo
Difração de Raios X
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
25702-74-3 (Ficoll); EC 3.4.21.4 (Trypsin); EC 3.4.23.1 (Pepsin A); JU58VJ6Y3B (Guanidine)
[Em] Mês de entrada:1704
[Cu] Atualização por classe:170428
[Lr] Data última revisão:
170428
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161228
[St] Status:MEDLINE


  9 / 1006 MEDLINE  
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[PMID]:27490854
[Au] Autor:Pelák O; Kuzílková D; Thürner D; Kiene ML; Stanar K; Stuchlý J; Vásková M; Starý J; Hrusák O; Stadler H; Kalina T
[Ad] Endereço:CLIP - Childhood Leukemia Investigation Prague, Department of Pediatric Hematology and Oncology, 2nd Faculty of Medicine, Charles University Prague and University Hospital Motol, Prague, Czech Republic.
[Ti] Título:Lymphocyte enrichment using CD81-targeted immunoaffinity matrix.
[So] Source:Cytometry A;91(1):62-72, 2017 Jan.
[Is] ISSN:1552-4930
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:In mass cytometry, the isolation of pure lymphocytes is very important to obtain reproducible results and to shorten the time spent on data acquisition. To prepare highly purified cell suspensions of peripheral blood lymphocytes for further analysis on mass cytometer, we used the new CD81+ immune affinity chromatography cell isolation approach. Using 21 metal conjugated antibodies in a single tube we were able to identify all basic cell subsets and compare their relative abundance in final products obtained by density gradient (Ficoll-Paque) and immune affinity chromatography (CD81+ T-catch™) isolation approach. We show that T-catch isolation approach results in purer final product than Ficoll-Paque (P values 0.0156), with fewer platelets bound to target cells. As a result acquisition time of 10 nucleated cells was 3.5 shorter. We then applied unsupervised high dimensional analysis viSNE algorithm to compare the two isolation protocols, which allowed us to evaluate the contribution of unsupervised analysis over supervised manual gating. ViSNE algorithm effectively characterized almost all supervised cell subsets. Moreover, viSNE uncovered previously overseen cell subsets and showed inaccuracies in Maxpar™ Human peripheral blood phenotyping panel kit recommended gating strategy. These findings emphasize the use of unsupervised analysis tools in parallel with conventional gating strategy to mine the complete information from a set of samples. They also stress the importance of the impurity removal to sensitively detect rare cell populations in unsupervised analysis. © 2016 International Society for Advancement of Cytometry.
[Mh] Termos MeSH primário: Separação Celular/métodos
Citometria por Imagem/métodos
Leucócitos Mononucleares/citologia
Linfócitos/citologia
[Mh] Termos MeSH secundário: Anticorpos/química
Anticorpos/imunologia
Sobrevivência Celular/imunologia
Ficoll/química
Seres Humanos
Linfócitos/imunologia
Tetraspanina 28/química
Tetraspanina 28/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Antibodies); 0 (Tetraspanin 28); 25702-74-3 (Ficoll)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171116
[Lr] Data última revisão:
171116
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:160805
[St] Status:MEDLINE
[do] DOI:10.1002/cyto.a.22918


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[PMID]:27967303
[Au] Autor:Kwast L; Aida T; Fiechter D; Kruijssen L; Bleumink R; Boon L; Ludwig I; Pieters R
[Ad] Endereço:a Division of Toxicology , Institute for Risk Assessment Sciences (IRAS), Utrecht University , Utrecht , The Netherlands.
[Ti] Título:Immune responses induced by diclofenac or carbamazepine in an oral exposure model using TNP-Ficoll as reporter antigen.
[So] Source:J Immunotoxicol;13(6):918-926, 2016 Nov.
[Is] ISSN:1547-6901
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Immune-mediated drug hypersensitivity reactions (IDHR) may result from immuno-sensitization to a drug-induced neo-antigen. They rarely occur in patients and are usually not predicted preclinically using standard toxicity studies. To assess the potential of a drug to induce T-cell sensitization, trinitrophenyl (TNP)-Ficoll was used here as a bystander antigen in animal experiments. TNP-Ficoll will only elicit TNP-specific IgG antibodies in the presence of non-cognate T-cell help. Therefore, the presence of TNP-specific IgG antibodies after co-injection of drug and TNP-Ficoll was indicative of T-cell sensitization potential. This TNP-Ficoll-approach was used here to characterize T-cell help induced by oral exposure to diclofenac (DF) or carbamazepine (CMZ). DF or CMZ was administered orally to BALB/c mice and after 3 w, the mice were challenged in a hind paw with TNP-Ficoll and a dose of the drug that by itself does only elicit a sub-optimal popliteal lymph node assay (PLNA) response. T-cell-dependent responses were then evaluated in paw-draining popliteal lymph nodes (PLN). Also, shortly after oral exposure, mesenteric lymph nodes (MLN) were excised for evaluation of local responses. Both drugs were able to increase PLN cellularity and TNP-specific IgG production after challenge. Both DF and CMZ stimulated CD4 and CD8 T-cells and caused shifts of the subsets toward an effector phenotype. DF, but not CMZ, appeared to stimulate interferon (IFN)-γ production. Remarkably, depletion of CD8 , but not CD4 , T-cells reduced TNP-specific IgG production, and was more pronounced in CMZ- than in DF-exposed animals. Local responses in the MLN caused by DF or CMZ also showed shifts of CD4 and CD8 -cells toward a memory phenotype. Together, the data indicate that oral exposure to CMZ and DF differentially induced neo-antigen-specific T-cell reactions in the PLNA.
[Mh] Termos MeSH primário: Anti-Inflamatórios não Esteroides/efeitos adversos
Carbamazepina/efeitos adversos
Diclofenaco/efeitos adversos
Hipersensibilidade a Drogas/imunologia
Linfócitos T/imunologia
[Mh] Termos MeSH secundário: Administração Oral
Animais
Anti-Inflamatórios não Esteroides/uso terapêutico
Antígenos/imunologia
Carbamazepina/uso terapêutico
Diclofenaco/uso terapêutico
Ficoll/análogos & derivados
Ficoll/imunologia
Imunoglobulina G/metabolismo
Interferon gama/metabolismo
Linfonodos/patologia
Ativação Linfocitária
Camundongos
Camundongos Endogâmicos BALB C
Trinitrobenzenos/imunologia
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Anti-Inflammatory Agents, Non-Steroidal); 0 (Antigens); 0 (Immunoglobulin G); 0 (TNP-ficoll); 0 (Trinitrobenzenes); 144O8QL0L1 (Diclofenac); 25702-74-3 (Ficoll); 33CM23913M (Carbamazepine); 82115-62-6 (Interferon-gamma)
[Em] Mês de entrada:1709
[Cu] Atualização por classe:170929
[Lr] Data última revisão:
170929
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161215
[St] Status:MEDLINE



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