Shiga toxins (Stx1 and Stx2) produced by Shiga toxin-producing Escherichia coli (STEC) are potent AB5 type cytotoxins. This toxin family is characterized by a structure formed by two characteristic functional domains: the B subunit forms a pentamer responsible to bind to its receptor, GB3; while the A subunit has a catalytic action, whose mechanism is N-glycosidase, removing an adenine from the RNA 28S portion of the ribosome, which interrupts the protein synthesis and leads the cell death by apoptosis. The kidneys are most affected organ by the toxic effect of Stx, mainly because they express the receptor in great quantity. The most serious consequence of this intoxication is the development of hemolytic uremic syndrome (HUS), which affects from 5 to 15% of patients infected by STEC and may cause 10% of patient's death, thus, an important public health and food safety problem. The current therapies are not effective against this complication, and neutralization of Stx the most indicated treatment. The high specificity and affinity of antibodies made these molecules, promising tools to be used in the therapy of several diseases, moreover, from recombinant DNA technology, it is possible to develop antibody fragments as therapeutic tools. Employing such technology by Phage Display from a synthetic phage library, four Fab fragments of human recombinant antibodies (two against each Stx toxin) were obtained in preceding work, which were expressed in a heterologous manner in bacteria, LPS free. These different Fabs were previously characterized and proved to be efficient in their antigen recognition, showing high affinity against the target toxins and the ability to neutralize the cytotoxic effect in vitro, using the purified toxin in Vero cells assay (a gold standard assay for these toxins), making these antibody fragments possible as promising tools for neutralizing Stx and preventing SHU. In this context, the present work proposes to further characterize the neutralizing ability of these antibody molecules in human renal cell lines, one epithelial and another from proximal tubule (HEK and HK-2, respectively), using both the purified toxin and toxins produced by STEC strains isolated from human and from HUS cases, with the proposal to validate them before tests in animal models, in order to prove the ability of these molecules to neutralize the toxic effect and to prevent the sequels resulting from the Shiga toxins action.
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