FabC8 recombinant monoclonal antibodies avoid the deleterious effects of Shiga toxin type 2 on human microvascular endothelial cells.

FERNANDO DANIEL GOMEZ; LUZ, DANIELA; ROXANNE PIAZZA; PRESTA AGOSTINA

Ciudad Autónoma de Buenos Aires

Congreso; Reunión Anual de Biociencia 2020; 2020

Sociedad Argentina de Investigación Clinica

Hemolytic Uremic Syndrome (HUS) associated with Shiga‐toxigenic Escherichia coli(STEC) infections is the principal cause of acute renal injury in pediatric age groups in Argentina. Neither a licensed vaccine nor effective therapy for HUS is available forhumans. Previously, we demonstrated the in vitro cytotoxic effects of Shiga toxin type 2 (Stx2) on human glomerular endothelial cells (HGEC). Recently, recombinant antibodies against Stx2, produced in bacteria, were developed, and characterized. In this work, we studied the ability of anti-Stx2 FabC8 antibody to neutralize the Stx2 activity on primary cultures of HGEC. Cells were plated in 96-well plates and grown to confluence. Then, cells were treated in growth-arrested conditions for 72 h with different pre-incubations (1 h at 37ºC) o co-incubations of FabC8 with Stx2. Antibodies were used from 10 µg/ml to 0.001 µg/ml and Stx2 at the dilution required to kill 50% of cells (0.5 ng/ml). Finally, cellviability was assessed by neutral red uptake. In addition, cells were seeded on gelatincoated glass coverslips and then treated, as it was previously mentioned, with 1µg/mlFabC8 and 0.5 ng/ml Stx2, during 72 h. Cell morphology was analyzed by light microscopy after hematoxylin?eosin (H&E) stain. Cell counts were performed on fivefields of ×200 magnifications each. Cell area values were obtained using Image J software. Percentage of necrotic and apoptotic cells were established by fluorescence microscopy after staining with acridine orange/ethidium bromide.Under both conditions evaluated, FabC8 significantly neutralized, in a dose-dependent manner, the cytotoxic effects caused by 0.5 ng/ml Stx2 in HGEC (p