Aislamiento de linfocitos B con receptor de tipo IgM específico para fosfatidilcolina de pacientes con Esclerosis Múltiple

Introduction and objective.Our group has demonstrated that serum IgM antibodies that recognize phosphatidylcholine (IgMPC) are elevated in 90% of patients with Multiple Sclerosis (MS). The 40% of these patients also present these antibodies at the central nervous system level, suffering a more aggressive evolution of the disease. Moreover, we also demonstrated the presence of IgM in the lesions co-localizing with complement factors and activated macrophages. These antibodies were associated with oligodendrocyte and axonal damage. These data support the main role of IgMPC antibodies in the physiopathology of MS. Therefore, the aim of our group is to isolate these lymphocytes responsible for the production of these specific antibodies to phosphatidylcholine by magnetic immunoselection. Methods. We obtained unilamellar phosphatidylcholine liposomes (PC-liposomes), loaded with the fluorescent molecule, carboxyfluorescein, using rotary evaporation. To purify and to assess the quality of the fluorescent PC-liposomes, we performed a gel filtration chromatography and disruption assay. Leucocytes from mouse peritoneum were isolated to obtain a high percentage of B-lymphocytes to PC, positive control. Then we developed a binding assay using fluorescent PCliposomes and mouse leucocytes, analyzed by flow cytometry. PC-liposomes loaded with magnetic iron nanoparticles were obtained using rotary evaporation. Immortalized human B lymphocytes obtained from peripheral blood samples were culture “in vitro”. We developed an immunomagnetic selection of immortalized human B lymphocytes to PC using magnetic PC-liposomes and MACS™ LS columns. Results. The filtration of the fluorescent PC-liposomes separated the components in three elution fractions containing, the first consisted of fluorescent PC-liposomes, the second of the buffer and the third contained the carboxyfluorescein not included in the liposomes. The disruption assay demonstrated a linear relation between the concentration of PC-liposomes and the fluorescence intensity. In addition, the addition of Triton-X induced the increase of the fluorescence. The binding assays demonstrated that the percentage of B lymphocytes to PC increased as the concentration of PCliposomes also increased. The percentage of positive PC-liposomes B lymphocytes were higher when the cells were incubated at 37º C than incubated at 4º. The analysis by light microscopy demonstrated the presence of B lymphocytes after the immunoselection of B lymphocytes to PC. We could observe a high number of clones in the flask containing B lymphocytes from MS patients compared with those containing B lymphocytes from controls when the cells were “in vitro” cultured for 7 days. Conclusion. We have developed a simple method for the generation of liposomes. Also, we have implemented the detection of lymphocytes with IgM type BCR specific for phosphatidylcholine by flow cytometry. It was possible to isolate B lymphocytes with specific IgM receptor for phosphatidylcholine despite being a minority population. This makes available the study of their phenotype, and the genotypic alterations that these cells may undergo. In addition, new prognostic and diagnostic biomarkers could be determined. These analyses are essential to clarity the role of these cells in the pathophysiology of the disease and to characterize new therapeutic targets