Update on the treatment of endemic hemolytic uremic syndrome. Pathogenesis and treatment of the most severe systemic complication of infections by Shiga toxin-producing Escherichia coli

La forma típica o post-diarreica del síndrome urémico hemolítico (SUH) es la complicación más grave de las infecciones por cepas de Escherichia coli productoras de toxina Shiga (STEC). En la Argentina el SUH es un problema crítico de salud pública, ya que representa la principal causa de falla renal aguda en la infancia, la segunda causa de falla renal crónica, y aporta el 20% de los casos de transplante renal durante la infancia y la adolescencia. A pesar de los avances en el conocimiento de su patogénesis, el único tratamiento actual de los pacientes con SUH es de sostén, y no existen terapias específicas ni preventivas. En la presente revisión expondremos los conocimientos básicos de los mecanismos patogénicos y discutiremos los enfoques terapéuticos tradicionales e innovadores, con especial foco en la situación nacional y los aportes hechos por grupos de la Argentina.The typical form of hemolytic uremic syndrome (HUS) is the major complication of Shiga toxin-producing Escherichia coli (STEC) infections. HUS is a critical health problem in Argentina since it is the main cause of acute renal failure in children and the second cause of chronic renal failure, giving account for 20% of renal transplants in children and adolescents in our country. In spite of the extensive research in the field, the mainstay of treatment for patients with HUS is supportive therapy, and there are no specific therapies preventing or ameliorating the disease course. In this review, we present the current knowledge about pathogenic mechanisms and discuss traditional and innovative therapeutic approaches, with special focus in national status and contributions made by Argentinean groups.Fil: Fernández Brando, Romina Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Bentancor, Leticia Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Mejias, María Pilar. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Panek, Cecilia Analía. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Cabrera, Gabriel Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Exeni, Ramón A.. Municipio de La Matanza. Hospital Municipal del Niño San Justo; ArgentinaFil: Palermo, Marina Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

<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

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

Recombinant plasmid constructs.

<p>pStx2 is the pGEM-T plasmid with the <i>stx2</i> locus cloned in the same direction that <i>lacZ</i> promoter. pr1-eGFP and pr7-eGFP are reporter plasmids with the region pr1 or pr7 driving eGFP expression, and Δpr-eGFP is a construction lacking the wild CMV promoter and was used as negative control. Linear pr1-eGFP and pr7-eGFP were obtained after digestion with PciI. In pr1ΔTATA-eGFP and pr7ΔTATA-eGFP TATA Box sequence has been replaced by BamHI restriction site. <i>Stx2</i> linear DNA was obtained after digestion with EcoRI.</p

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

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