HIGHLIGHTS
IgG and IgA from normal human serum can recognize E. coli antigens and LPS.
The PBMC proliferation assay was induced by E. coli antigens and by LPS.
Mucosal antigens may induce local and systemic responses.
Abstract
Among the microorganisms that make up the intestinal microbiota, stands out Escherichia coli, which has as main ecological niche, the large human intestine. Its importance stands out in being part of the pioneer’s commensal microorganisms on the colonization of the intestinal mucosa and its pathogenic role causing extra and intra intestinal diseases. The aim of the study was to evaluate the antibody production and proliferative response of Peripheral Blood Mononuclear Cells (PBMC) to E. coli antigens. The bacteria were grown on Brain Heart Infusion broth medium at 35 ºC for 72 hours. Pellet bacteria were lysed for one hour at room temperature with an 8M sodium guanidine solution. After spin and dialysis, the protein antigens were measured in the supernatant by protein assay. The antigens were characterized by polyacrylamide gel electrophoresis and the antigenic profile by western blotting. The presence of specific IgG and IgA antibodies were evaluated using thirty normal human sera by an indirect ELISA. The response of PBMC to E. coli antigens was assessed by MTT metabolization. The results demonstrated that the antigens were composed of proteins of different sizes and they were recognized by antibodies present in normal human serum. Human sera presented high titers of IgG and IgA antibodies to E. coli antigens when compared to the results of lipopolysaccharide. We also showed that total E. coli antigens induced PBMC proliferation at different antigen concentrations. Taken together the results suggest that the antigens from E. coli can induce local and systemic immune responses.
Keywords:
Antibody response; Escherichia coli; Gastrointestinal Microbiota; MTT; PBMC proliferation; Viability
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A: Lane 1A corresponds to E. coli antigen proteins (strain ATCC 25922) and lane 2A is the molecular weight standard (200, 150, 66, 29 kDa). 12% polyacrylamide gel stained with Coomassie blue R-250.B: Lane 1B corresponds to the molecular weight standard and lane 2B the E. coli protein profile recognized by a pool of seven normal human sera. The profile of proteins recognized by human sera was revealed using the DAB method.
PBMC: 2x105 cells/well. MTT: 5 mg/mL was added to the cultures 4 horas before the end of the culture period. The formazan crystals were solubilized with the addition of 200 μL of DMSO. The absorbance reading was taken at 570 nm. Statistical analysis was performed using a one-way analysis, followed by Bonferroni post-test.