RESUMO
Treatment (from 5 to 25 weeks of age) with a novel blocking monoclonal antibody, mAb I-10, directed against the plasma membrane (pm) form of LAMP-1, protected against development of autoimmune diabetes in the NOD mouse. A shorter course of treatment, i.e. from 5 to 12 weeks of age, significantly reduced the occurrence of insulitis as well as disease onset. Interfering with pm-LAMP-1 required continuous treatment as tolerance was not observed when treatment was stopped, and no higher proportion of cells with a T regulatory phenotype (e.g. CD4(+)CD25(+)) were induced. The mechanism appears to involve modulating a proinflammatory cytokine, as the proportion of pancreatic-infiltrating IFN-gamma-positive cells was significantly reduced in the mAb I-10-treated group. These results demonstrate an unexpected role for pm-LAMP-1 in autoimmune disease progression, and suggest that further investigation should be performed to understand how this molecule modulates IFN-gamma-driven responses.
Assuntos
Anticorpos Bloqueadores/imunologia , Anticorpos Monoclonais/imunologia , Antígenos CD/imunologia , Diabetes Mellitus Tipo 1/imunologia , Proteínas de Membrana/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais/farmacologia , Western Blotting , Diabetes Mellitus Tipo 1/prevenção & controle , Modelos Animais de Doenças , Eletroforese em Gel de Poliacrilamida , Feminino , Citometria de Fluxo , Interferon gama/biossíntese , Interferon gama/efeitos dos fármacos , Interferon gama/imunologia , Proteínas de Membrana Lisossomal , Proteínas de Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos NOD , Dados de Sequência Molecular , Pâncreas/efeitos dos fármacos , Pâncreas/imunologia , Células Th1/efeitos dos fármacos , Células Th1/imunologiaRESUMO
Fluorescence resonance energy transfer (FRET) is a technique used to measure the interaction between two molecules labeled with two different fluorophores (the donor and the acceptor) by the transfer of energy from the excited donor to the acceptor. In biological applications, this technique has become popular to qualitatively map protein-protein interactions, and in biophysical projects it is used as a quantitative measure for distances between a single donor and acceptor molecule. Numerous approaches can be found in the literature to quantify and map FRET, but the measures they provide are often difficult to interpret. We propose here a quantitative comparison of these methods by using a surface FRET system with controlled amounts of donor and acceptor fluorophores and controlled distances between them. We support the system with a Monte Carlo simulation of FRET, which provides reference values for the FRET efficiency under various experimental conditions. We validate a representative set of FRET efficiencies and indices calculated from the different methods with different experimental settings. Finally, we test their sensitivity and draw conclusions for the preparation of FRET experiments in more complex and less-controlled systems.