2 research outputs found
Circulating EZH2-positive T cells are decreased in multiple sclerosis patients
BACKGROUND: Recent studies in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis (MS), suggest an involvement of the histone methyltransferase enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) in important processes such as cell adhesion and migration. METHODS: Here, we aimed to expand these initial observations by investigating the role of EZH2 in MS. mRNA expression levels for EZH2 were measured by real-time PCR in peripheral blood mononuclear cells (PBMC) from 121 MS patients (62 untreated and 59 receiving treatment) and 24 healthy controls. RESULTS: EZH2 expression levels were decreased in PBMC from untreated patients compared to that from controls, and treatment significantly upregulated EZH2 expression. Expression of miR-124 was increased in MS patients compared to controls. Blood immunophenotyping revealed EZH2 expression mostly restricted to CD4+ and CD8+ T cells, and circulating EZH2+ CD4+ and CD8+ T cells were decreased in untreated MS patients compared to controls. CD8+ T cells expressing EZH2 exhibited a predominant central memory phenotype, whereas EZH2+ CD4+ T cells were of effector memory nature, and both T cell subsets produced TNF-Îą. EZH2+ T cells were enriched in the cerebrospinal fluid compartment compared to blood and were found in chronic active lesions from MS patients. EZH2 inhibition and microarray analysis in PBMC was associated with significant downregulation of key T cell adhesion molecules. CONCLUSION: These findings suggest a role of EZH2 in the migration of T cells in MS patients. The observation of TNF-Îą expression by CD4+ and CD8+ T cells expressing EZH2 warrants additional studies to explore more in depth the pathogenic potential of EZH2+-positive cells in MS
AnĂ lisi de biomarcadors neuropeptĂdics en fluids biològics per SPE-CE-MS
[cat]La determinaciĂł de biomarcadors peptĂdics en fluids biològics resulta de gran interès pel diagnòstic, la monitoritzaciĂł i el pronòstic de nombrosos desordres. En aquesta tesi doctoral sâhan explorat diverses metodologies dâextracciĂł en fase sòlida acoblada en lĂnia amb lâelectroforesi capilâ˘lar amb detecciĂł per espectrometria de masses (SPE-CE-MS) per a lâanĂ lisi de biomarcadors peptĂdics, que es troben a baixa concentraciĂł en matrius complexes. Sâhan estudiat principalment pèptids opiacis que estan relacionats amb desordres neurològics i sĂndromes de dolor crònic. Sâhan avaluat diverses fases estacionĂ ries comercials per a lâanĂ lisi de pèptids opiacis per SPE-CE emprant preconcentradors amb frits i la fase estacionĂ ria C18 ĂŠs la que ha proporcionat millors resultats per a lâanĂ lisi de mostres de plasma humĂ . La saturaciĂł del preconcentrador sâha previngut mitjançant un tractament de mostra previ que consta de dues etapes, una precipitaciĂł amb acetonitril seguida dâuna filtraciĂł per centrifugaciĂł. Com que lâetapa de filtraciĂł ĂŠs crĂtica per tal dâobtenir les mĂ ximes recuperacions dels anĂ lits i eliminar la matriu de la mostra, sâhan provat filtres de diversos talls moleculars i diverses condicions de filtraciĂł. Lâacoblament de la SPE-CE a un espectròmetre de masses amb analitzador de temps de vol (TOF-MS) mitjançant una interfase amb lĂquid auxiliar convencional ha permès disminuir els LOD per a la majoria dels pèptids opiacis en mostres de plasma humĂ . Per tal de poder detectar concentracions inferiors, sâha demostrat que la combinaciĂł de la SPE-CE amb la tècnica de separaciĂł electroforètica isotacoforesi transitòria (tITP) ĂŠs una bona alternativa. TambĂŠ, sâha comparat el comportament de preconcentradors amb i sense frits per a lâanĂ lisi de pèptids opiacis per C18-SPE-CE-UV i C18-SPE-CE-TOF-MS. Sâha avaluat un prototip recentment desenvolupat dâuna interfase nanoelectroesprai (nanoESI) sense lĂquid auxiliar basada en una punta porosa per a lâanĂ lisi de pèptids opiacis per CE-MS i tambĂŠ, sâha establert una metodologia de C18-SPE-CE-TOF-MS emprant un innovador preconcentrador sense frits compatible amb les dimensions especĂfiques del capilâ˘lar de separaciĂł requerit per a la interfase nanoESI sense lĂquid auxiliar. Una altra possibilitat per a millorar la detecciĂł de pèptids ĂŠs utilitzar fases estacionĂ ries amb una selectivitat mĂŠs elevada que el C18, com les fases estacionĂ ries dâimmunoafinitat. Sâhan explorat dos procediments de preparaciĂł dâaquest tipus de fases estacionĂ ries: la immobilitzaciĂł dâanticossos intactes oxidats (immunoglobulina G, IgG) a travĂŠs dels carbohidrats a partĂcules de sĂlice activades amb grups hidrazida i la immobilitzaciĂł de fragments dâIgG (fragment dâuniĂł a lâantigen, Fabâ) a partĂcules de sĂlice activades amb grups succinimidil. Seguint els procediments establerts, sâha preparat una fase estacionĂ ria amb IgG i una altra amb Fabâ contra les endomorfines 1 i 2 (End1 i End2). Sâhan optimitzat i avaluat metodologies dâIA-SPE-CE-TOF-MS per a lâanĂ lisi de mescles de patrons dâEnd1 i dâEnd2 i sâhan analitzat mostres de plasma humĂ . TambĂŠ, sâha explorat lâaplicabilitat dâuna fase estacionĂ ria dâimmunoafinitat amb IgG per a lâanĂ lisi de biomolècules dâelevat pes molecular per IA-SPE-CE-TOF-MS emprant la glicoproteĂŻna transferrina com a model.[eng]
The determination of peptide biomarkers in biological fluids has become crucial for the diagnosis, monitoring and prognosis of numerous disorders. This doctoral thesis explores several on-line solid-phase extraction capillary electrophoresis mass spectrometry (SPE-CE-MS) methodologies for the analysis of peptide biomarkers that are found at low concentration in complex matrices, such as opioid peptides. Several commercial sorbents have been compared for the analysis of opioid peptides by SPE-CE using microcartridges with frits and the C18 sorbent has provided the best results for the analysis of human plasma samples. The saturation of the on-line SPE microcartridge has been prevented by a double-step sample clean-up pretreatment that consists of precipitation with acetonitrile and centrifugal filtration. The coupling of SPE-CE with a time-of-flight mass spectrometer (TOF-MS) by a conventional sheathflow interface has provided better LODs. In order to detect lower concentrations, the combination of SPE-CE with the electrophoretic preconcentration technique transient isotachophoresis (tITP) has demonstrated to be a good alternative. The performances of frit and fritless microcartridges have been compared for the analysis of opioid peptides by C18-SPE-CE-UV and C18-SPE-CE-TOF-MS. A recently developed sheathless nanoelectroespray (nanoESI) interface based on a porous tip has also been evaluated for the analysis of opioid peptides by CE-TOF-MS and a C18-SPE-CE-TOF-MS using a novel fritless microcartridge. Finally, two different procedures for the preparation of immunoaffinity sorbents (IA) have been explored: the immobilization of intact antibodies (immunoglobulin G, IgG) or IgG fragments (antigen binding fragment, Fabâ) to silica particles. Two IA sorbents against Endomorphins 1 and 2 have been prepared following the established procedures. IA-SPE-CE-TOF-MS methodologies have been optimized and evaluated for the analysis of standards solutions of End1 and End2 and human plasma samples. The suitability of an immunoaffinity sorbent with IgG has also been explored for the analysis of large biomolecules by IA-SPE-CE-TOF-MS using the glycoprotein transferrin as a model