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Hemolytic Uremic Syndrome (HUS) associated with Shiga-toxigenic Escherichia coli (STEC) infections is the principal cause of acute renal injury in pediatric age groups. Shiga toxin type 2 (Stx2) has in vitro cytotoxic effects on kidney cells, including human glomerular endothelial (HGEC) and Vero cells. Neither a licensed vaccine nor effective therapy for HUS is available for humans. Recombinant antibodies against Stx2, produced in bacteria, appeared as the utmost tool to prevent HUS. Therefore, in this work, a recombinant FabF8:Stx2 was selected from a human Fab antibody library by phage display, characterized, and analyzed for its ability to neutralize the Stx activity from different STEC-Stx2 and Stx1/Stx2 producing strains in a gold standard Vero cell assay, and the Stx2 cytotoxic effects on primary cultures of HGEC. This recombinant Fab showed a dissociation constant of 13.8 nM and a half maximum effective concentration (EC50) of 160 ng/mL to Stx2. Additionally, FabF8:Stx2 neutralized, in different percentages, the cytotoxic effects of Stx2 and Stx1/2 from different STEC strains on Vero cells. Moreover, it significantly prevented the deleterious effects of Stx2 in a dose-dependent manner (up to 83%) in HGEC and protected this cell up to 90% from apoptosis and necrosis. Therefore, this novel and simple anti-Stx2 biomolecule will allow further investigation as a new therapeutic option that could improve STEC and HUS patient outcomes.

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Shiga toxin-producing E. coli (STEC) produces Stx1 and/or Stx2, and Subtilase cytotoxin (SubAB). Since these toxins may be present simultaneously during STEC infections, the purpose of this work was to study the co-action of Stx2 and SubAB. Stx2 + SubAB was assayed in vitro on monocultures and cocultures of human glomerular endothelial cells (HGEC) with a human proximal tubular epithelial cell line (HK-2) and in vivo in mice after weaning. The effects in vitro of both toxins, co-incubated and individually, were similar, showing that Stx2 and SubAB contribute similarly to renal cell damage. However, in vivo, co-injection of toxins lethal doses reduced the survival time of mice by 24 h and mice also suffered a strong decrease in the body weight associated with a lowered food intake. Co-injected mice also exhibited more severe histological renal alterations and a worsening in renal function that was not as evident in mice treated with each toxin separately. Furthermore, co-treatment induced numerous erythrocyte morphological alterations and an increase of free hemoglobin. This work shows, for the first time, the in vivo effects of Stx2 and SubAB acting together and provides valuable information about their contribution to the damage caused in STEC infections.

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PURPOSE: γδ T lymphocytes are non-conventional T cells that participate in protective immunity and tumor surveillance. In healthy humans, the main subset of circulating γδ T cells express the TCRVγ9Vδ2. This subset responds to non-peptide prenyl-pyrophosphate antigens such as (E)-4-hydroxy-3-methyl-but-enyl pyrophosphate (HMBPP). This unique feature of Vγ9Vδ2 T cells makes them a candidate for anti-tumor immunotherapy. In this study, we investigated the response of HMBPP-activated Vγ9Vδ2 T lymphocytes to glioblastoma multiforme (GBM) cells. METHODS: Human purified γδ T cells were stimulated with HMBPP (1 µM) and incubated with GBM cells (U251, U373 and primary GBM cultures) or their conditioned medium. After overnight incubation, expression of CD69 and perforin was evaluated by flow cytometry and cytokines production by ELISA. As well, we performed a meta-analysis of transcriptomic data obtained from The Cancer Genome Atlas. RESULTS: HMBPP-stimulated γδ T cells cultured with GBM or its conditioned medium increased CD69, intracellular perforin, IFN-γ, and TNF-α production. A meta-analysis of transcriptomic data showed that GBM patients display better overall survival when mRNA TRGV9, the Vγ9 chain-encoding gene, was expressed in high levels. Moreover, its expression was higher in low-grade GBM compared to GBM. Interestingly, there was an association between γδ T cell infiltrates and TNF-α expression in the tumor microenvironment. CONCLUSION: GBM cells enhanced Th1-like profile differentiation in phosphoantigen-stimulated γδ T cells. Our results reinforce data that have demonstrated the implication of Vγ9Vδ2 T cells in the control of GBM, and this knowledge is fundamental to the development of immunotherapeutic protocols to treat GBM based on γδ T cells.

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Hemolytic Uremic Syndrome (HUS) associated with Shiga-toxigenic Escherichia coli (STEC) infections is the principal cause of acute renal injury in pediatric age groups. Shiga toxin type 2 (Stx2) has in vitro cytotoxic effects on kidney cells, including human glomerular endothelial (HGEC) and Vero cells. Neither a licensed vaccine nor effective therapy for HUS is available for humans. Recombinant antibodies against Stx2, produced in bacteria, appeared as the utmost tool to prevent HUS. Therefore, in this work, a recombinant FabF8:Stx2 was selected from a human Fab antibody library by phage display, characterized, and analyzed for its ability to neutralize the Stx activity from different STEC-Stx2 and Stx1/Stx2 producing strains in a gold standard Vero cell assay, and the Stx2 cytotoxic effects on primary cultures of HGEC. This recombinant Fab showed a dissociation constant of 13.8 nM and a half maximum effective concentration (EC50) of 160 ng/mL to Stx2. Additionally, FabF8:Stx2 neutralized, in different percentages, the cytotoxic effects of Stx2 and Stx1/2 from different STEC strains on Vero cells. Moreover, it significantly prevented the deleterious effects of Stx2 in a dose-dependent manner (up to 83%) in HGEC and protected this cell up to 90% from apoptosis and necrosis. Therefore, this novel and simple anti-Stx2 biomolecule will allow further investigation as a new therapeutic option that could improve STEC and HUS patient outcomes.

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