RESUMO
BACKGROUND: Detection of single cell epitopes has been a mainstay of immunophenotyping for over three decades, primarily using fluorescence techniques for quantitation. Fluorescence has broad overlapping spectra, limiting multiplexing abilities. METHODOLOGY/PRINCIPAL FINDINGS: To expand upon current detection systems, we developed a novel method for multi-color immuno-detection in single cells using "Composite Organic-Inorganic Nanoparticles" (COINs) Raman nanoparticles. COINs are Surface-Enhanced Raman Scattering (SERS) nanoparticles, with unique Raman spectra. To measure Raman spectra in single cells, we constructed an automated, compact, low noise and sensitive Raman microscopy device (Integrated Raman BioAnalyzer). Using this technology, we detected proteins expressed on the surface in single cells that distinguish T-cells among human blood cells. Finally, we measured intracellular phosphorylation of Stat1 (Y701) and Stat6 (Y641), with results comparable to flow cytometry. CONCLUSIONS/SIGNIFICANCE: Thus, we have demonstrated the practicality of applying COIN nanoparticles for measuring intracellular phosphorylation, offering new possibilities to expand on the current fluorescent technology used for immunoassays in single cells.
Assuntos
Antígenos de Superfície/análise , Células/metabolismo , Imunoensaio/métodos , Nanopartículas , Fosforilação , Análise Espectral Raman/métodos , Linhagem Celular , Humanos , Nanopartículas Metálicas , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT6/metabolismo , Linfócitos TRESUMO
Statins are a class of drugs that inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMGcoA) reductase, a critical enzyme in the mevalonate pathway. Several reports document that statins may prevent different human cancers. However, whether or not statins can prevent cancer is controversial due to discordant results. One possible explanation for these conflicting conclusions is that only some tumors or specific statins may be effective. Here, we demonstrate in an in vivo transgenic model in which atorvastatin reverses and prevents the onset of MYC-induced lymphomagenesis, but fails to reverse or prevent tumorigenesis in the presence of constitutively activated K-Ras (G12D). Using phosphoprotein fluorescence-activated cell sorter (FACS) analysis, atorvastatin treatment was found to result in the inactivation of the Ras and ERK1/2 signaling pathways associated with the dephosphorylation and inactivation of MYC. Correspondingly, tumors with a constitutively activated K-Ras (G12D) did not exhibit dephosphorylation of ERK1/2 and MYC. Atorvastatin's effects on MYC were specific to the inhibition of HMGcoA reductase, as treatment with mevalonate, the product of HMG-CoA reductase activity, abrogated these effects and inhibited the ability of atorvastatin to reverse or suppress tumorigenesis. Also, RNAi directed at HMGcoA reductase was sufficient to abrogate the neoplastic properties of MYC-induced tumors. Thus, atorvastatin, by inhibiting HMGcoA reductase, induces changes in phosphoprotein signaling that in turn prevent MYC-induced lymphomagenesis.
Assuntos
Transformação Celular Neoplásica/patologia , Ácidos Heptanoicos/farmacologia , Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Linfoma/metabolismo , Linfoma/prevenção & controle , Proteína Oncogênica p55(v-myc)/metabolismo , Pirróis/farmacologia , Animais , Atorvastatina , Sobrevivência Celular , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Citometria de Fluxo , Perfilação da Expressão Gênica , Glicina/genética , Glicina/metabolismo , Humanos , Linfoma/patologia , Camundongos , Camundongos Transgênicos , Mutação/genética , Proteína Oncogênica p55(v-myc)/genética , Fosfoproteínas/metabolismo , Fosforilação , Lesões Pré-Cancerosas/patologia , Transdução de Sinais , Taxa de Sobrevida , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
The diversity of autoimmune responses poses a formidable challenge to the development of antigen-specific tolerizing therapy. We developed 'myelin proteome' microarrays to profile the evolution of autoantibody responses in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS). Increased diversity of autoantibody responses in acute EAE predicted a more severe clinical course. Chronic EAE was associated with previously undescribed extensive intra- and intermolecular epitope spreading of autoreactive B-cell responses. Array analysis of autoantigens targeted in acute EAE was used to guide the choice of autoantigen cDNAs to be incorporated into expression plasmids so as to generate tolerizing vaccines. Tolerizing DNA vaccines encoding a greater number of array-determined myelin targets proved superior in treating established EAE and reduced epitope spreading of autoreactive B-cell responses. Proteomic monitoring of autoantibody responses provides a useful approach to monitor autoimmune disease and to develop and tailor disease- and patient-specific tolerizing DNA vaccines.
Assuntos
Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/imunologia , Imunoensaio/métodos , Bainha de Mielina/imunologia , Análise Serial de Proteínas/métodos , Vacinas de DNA/imunologia , Vacinas de DNA/uso terapêutico , Animais , Tolerância a Medicamentos , Encefalomielite Autoimune Experimental/diagnóstico , Camundongos , Esclerose Múltipla/diagnóstico , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/imunologia , Mapeamento de Interação de Proteínas/métodos , Resultado do TratamentoRESUMO
Statins, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, which are approved for cholesterol reduction, may also be beneficial in the treatment of inflammatory diseases. Atorvastatin (Lipitor) was tested in chronic and relapsing experimental autoimmune encephalomyelitis, a CD4(+) Th1-mediated central nervous system (CNS) demyelinating disease model of multiple sclerosis. Here we show that oral atorvastatin prevented or reversed chronic and relapsing paralysis. Atorvastatin induced STAT6 phosphorylation and secretion of Th2 cytokines (interleukin (IL)-4, IL-5 and IL-10) and transforming growth factor (TGF)-beta. Conversely, STAT4 phosphorylation was inhibited and secretion of Th1 cytokines (IL-2, IL-12, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha) was suppressed. Atorvastatin promoted differentiation of Th0 cells into Th2 cells. In adoptive transfer, these Th2 cells protected recipient mice from EAE induction. Atorvastatin reduced CNS infiltration and major histocompatibility complex (MHC) class II expression. Treatment of microglia inhibited IFN-gamma-inducible transcription at multiple MHC class II transactivator (CIITA) promoters and suppressed class II upregulation. Atorvastatin suppressed IFN-gamma-inducible expression of CD40, CD80 and CD86 co-stimulatory molecules. l-Mevalonate, the product of HMG-CoA reductase, reversed atorvastatin's effects on antigen-presenting cells (APC) and T cells. Atorvastatin treatment of either APC or T cells suppressed antigen-specific T-cell activation. Thus, atorvastatin has pleiotropic immunomodulatory effects involving both APC and T-cell compartments. Statins may be beneficial for multiple sclerosis and other Th1-mediated autoimmune diseases.