Antibody Response to Shiga Toxins in Argentinean Children with Enteropathic Hemolytic Uremic Syndrome at Acute and Long-Term Follow-Up Periods

Shiga toxin (Stx)-producing Escherichia coli (STEC) infection is associated with a broad spectrum of clinical manifestations that include diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). Systemic Stx toxemia is considered to be central to the genesis of HUS. Distinct methods have been used to evaluate anti-Stx response for immunodiagnostic or epidemiological analysis of HUS cases. The development of enzyme-linked immunosorbent assay (ELISA) and western blot (WB) assay to detect the presence of specific antibodies to Stx has introduced important advantages for serodiagnosis of HUS. However, application of these methods for seroepidemiological studies in Argentina has been limited. The aim of this work was to develop an ELISA to detect antibodies against the B subunit of Stx2, and a WB to evaluate antibodies against both subunits of Stx2 and Stx1, in order to analyze the pertinence and effectiveness of these techniques in the Argentinean population. We studied 72 normal healthy children (NHC) and 105 HUS patients of the urban pediatric population from the surrounding area of Buenos Aires city. Using the WB method we detected 67% of plasma from NHC reactive for Stx2, but only 8% for Stx1. These results are in agreement with the broad circulation of Stx2-expressing STEC in Argentina and the endemic behavior of HUS in this country. Moreover, the simultaneous evaluation by the two methods allowed us to differentiate acute HUS patients from NHC with a great specificity and accuracy, in order to confirm the HUS etiology when pathogenic bacteria were not isolated from stools

Cytotoxicity on Vero Cells.

<p>Vero cells were incubated with purified Stx2 (1 CD50) or transfected with pStx2 plasmid. After 48 h, cells were stained with Crystal Violet and analyzed by optical microscopy. Representative pictures using 200X original magnification are shown. <b>A.</b> Non-treated Vero cells. <b>B.</b> Cells transfected with the pGEM-T plasmid. <b>C.</b> Cells transfected with the pStx2 plasmid. <b>D.</b> Cells incubated with purified Stx2.</p

GFP activity driven by linear reporter plasmid.

<p>293 T cells were transfected with pr1-eGFP or pr7-eGFP linearized with Pcil restriction enzyme. After 48 h, cells were analyzed by fluorescence microscopy using Nikon Eclipse TE2000 microscope equipped with a CCD camera, using 1000X magnification. Green fluorescence photos were taken with 400 ms of exposure and 3.2 of gain. Numbers 1, 2, 3, 4 correspond to images visualized with white light, green filter, merge between DAPI and green filter and merge between white light and green filter, respectively. <b>A.</b> 293 T cells transfected with the Δpr-eGFP plasmid. <b>B.</b> Cells transfected with linear pr1-eGFP. <b>C.</b> Cells transfected with linear pr7-eGFP.</p

<i>In silico</i> analysis of the <i>stx</i>2 sequence.

<p><b>A.</b> Seven regions (pr1-7) with high score for putative eukaryotic promoter sequences were found. The putative transcription start site (TSS, +1) detected with promoter prediction server is highlighted in gray <b>B.</b> The pr1-pr7 regions are indicated with dark gray boxes over the <i>stx2</i> gene. Putative mammalian transcription factor binding sites are indicated on the corresponding sequences of the pr1 and pr7 regions.</p

Transduction of THP-1 cells differentiated to macrophages by bacteriophage 933W.

<p>THP-1 cells PMA-differentiated to macrophages were transduced with φΔTOX-GFP. After 3 h, cells were analyzed by confocal microscopy, using 600X magnification. Green fluorescence photos were taken with 400 ms of exposure and 1 of gain. Numbers 1, 2, 3, 4 correspond to images visualized with white light, red filter, green filter and merge between green and red filter, respectively. <b>A.</b> Cells transduced with φΔTOX-GFP. <b>B.</b> Non-treated cells.</p

Neutralization of the Stx2 cytotoxic activity.

<p>Vero cells were incubated with a 1∶1600 dilution of cellular extracts (Panel A) or a 1∶400 dilution of culture supernatants (Panel B) derived from Vero cells transfected with pGEM-T (pGEM-T) or pStx2 (pStx2). As positive and negative controls, Vero cells were incubated with 1 CD50 of Stx2 (Stx2) or in medium (Vero cells), respectively. To evaluate the specificity of the cytotoxicity, cytotoxic samples were pre-incubated with mouse polyclonal anti-Stx2 antibodies (pStx2+Ab; Stx2+Ab). After 48 h, cells were stained with Crystal Violet and OD₅₉₅ was measured as detailed in Materials and Methods. One-way ANOVA (Tukey’s Multiple Comparison Test) was used to determine statistical significance between different samples.*P<0.05. **P<0.01. ***P<0.001.</p

Cytotoxicity on BHK cells.

<p>BHK cells were incubated with purified Stx2 (1 CD50) or transfected with the pStx2 plasmid. After 48 h, cells were stained with DAPI and phalloidin-TRITC and analyzed by fluorescence microscopy. Original magnification 600X. <b>A.</b> Non-treated BHK cells. <b>B.</b> Cells transfected with pGEM-T. <b>C.</b> Cells incubated with purified Stx2. <b>D.</b> Cells transfected with the pStx2 plasmid.</p