Caracterização de microvesículas extracelulares em pacientes com Leucemia Linfocítica Crônica e seu possível impacto no perfil de comprometimento imune

Abstract

Chronic lymphocytic leukemia (CLL) is a hematological malignancy with increased B lymphocytes mainly due to resistance to apoptosis and aberrant expression of CD5, in addition,an immunocompromised profile. Individual evolutionary changes in patients cannot be predicted. In this context, new tools that help evaluate the progression of the patient are important, among them extracellular microvesicles (EV). It has been well described how the fusion of the EVs can induce the expression genes, as well as they can be considered a factor to the resistance to treatments being a factor of alteration of the microenvironment. The hypothesis of this study is that the different stages of this disease would be associated with a different EV profile, and that the treatment would be able to modify this profile as well. These differences in the extracellular vesicles were assessed in the supernetant of the peripheral blood mononuclear cell supernatant from 28 LLC patients and 24 healthy controls cultured ex vivo. The aim was to evaluate profiles in size distribution by dynamic light scattering technology; the ability to interfere with CD3 expression by evaluating expression of the ã, ä, å, and æ chains by qPCR; in the immunophenotypic and protein profiles through flow cytometry and protein extraction respectively; and the amount of EV of that supernatant through nanoparticle tracing analysis technology; and possible variation of these parameters after exposure to the fludarabine. EV had no significant differences in size among patients, their clinical classifications and controls (p> 0.1). The expression of the CD3å,e CD3æ chains had decreased expression in the patients when compared to the control group (p = 0.014 and p = 0.008 respectively), whereas in the EV, chain expression was detected, but without statistical value. Immunophenotyping confirmed the profile of the predominance of EV of lymphocyte origin; the protein extraction suggests the separation of the clinical classification between Binet A and Binet B + C patients in the analysis of the molecular size profile of the proteins. The estimated EV concentration of the control, the non-exposed and exposed fludarabine EV in culture is 9.66e + 008 (+/- 0.35e + 007); 1.19e + 009 (+/- 0.73e + 007) and 7.61e + 008 (+/- 0.48e + 007) particles per ml, respectively. The increased number of EV in CLL indicates that microvesiculation may result from the pathogenesis of the disease without being related to staging. Expression of the CD3 chains in CLL patients are apparently decreased relative to controls, and EVs may be involved in modifying this expression profile; although absence of significant difference in the distribution of EV size in relation to staging and exposure to fludarabine, there is an apparent difference in relation to its concentrations.