Shiga toxin targets the podocyte causing hemolytic uremic syndrome through endothelial complement activation.

BACKGROUND: Shiga toxin (Stx)-producing Escherichia coli hemolytic uremic syndrome (STEC-HUS) is the leading cause of acute kidney injury in children, with an associated mortality of up to 5%. The mechanisms underlying STEC-HUS and why the glomerular microvasculature is so susceptible to injury following systemic Stx infection are unclear. METHODS: Transgenic mice were engineered to express the Stx receptor (Gb3) exclusively in their kidney podocytes (Pod-Gb3) and challenged with systemic Stx. Human glomerular cell models and kidney biopsies from patients with STEC-HUS were also studied. FINDINGS: Stx-challenged Pod-Gb3 mice developed STEC-HUS. This was mediated by a reduction in podocyte vascular endothelial growth factor A (VEGF-A), which led to loss of glomerular endothelial cell (GEnC) glycocalyx, a reduction in GEnC inhibitory complement factor H binding, and local activation of the complement pathway. Early therapeutic inhibition of the terminal complement pathway with a C5 inhibitor rescued this podocyte-driven, Stx-induced HUS phenotype. CONCLUSIONS: This study potentially explains why systemic Stx exposure targets the glomerulus and supports the early use of terminal complement pathway inhibition in this devastating disease. FUNDING: This work was supported by the UK Medical Research Council (MRC) (grant nos. G0901987 and MR/K010492/1) and Kidney Research UK (grant nos. TF_007_20151127, RP42/2012, and SP/FSGS1/2013). The Mary Lyon Center is part of the MRC Harwell Institute and is funded by the MRC (A410).

Componentes alimentarios de potencial aplicación en la dietoterapia de enfermedades provocadas por cepas de escherichia coli productoras de toxina tipo shiga / Food components of potential application in the dietoterapia of diseases provoked by vine-stocks of escherichia coli producers of toxin type shiga

En Argentina, Escherichia coli O157:H7 es el serotipo prevalente asociado a grandes brotes y casos esporádicos de colitis hemorrágica y síndrome urémico hemolítico (SUH), siendo el país con mayor tasa de incidencia en el mundo con alrededor de 500 nuevos casos por año. El SUH representa la principal causa de falla renal aguda en la infancia, la segunda causa de falla renal crónica y el 20% de los casos de transplante renal durante la infancia y la adolescencia. Se ha demostrado que la habilidad de la cepa para causar la enfermedad está relacionada a la acción de factores de virulencia implicados en el proceso de colonización del epitelio intestinal con posterior reacción inflamatoria y por la capacidad de secretar toxinas VT1 y VT2 responsables del daño del endotelio vascular con el consecuente desarrollo de SUH. En la presente tesis doctoral se investigó desde la dietoterapia, extractos de origen vegetal y componentes de cepas probióticas potencialmente útiles en la inhibición de la colonización de E. coli O157:H7 a nivel intestinal como medida preventiva, e inhibición de la acción citotóxica de VT sobre células eucariotas. Se determinaron componentes fenólicos obtenidos a partir de la maceración de harina de algarroba (Prosopis alba) y café de mistol (Ziziphus mistol) en distintos solventes (alcohol, agua destilada, acetona y hexano). Los mismos fueron nalizados mediante marcha analítica fitoquímica, cromatografía en capa delgada y cuantificados por espectrofotometría. Por otro lado se obtuvieron componentes de leche fermentada con granos de kéfir.

SUMMARY: In Argentina, Escherichia coli O157:H7 is prevalent serotype associated with large outbreaks and sporadic cases of hemorrhagic colitis and hemolytic uremic syndrome (HUS), being the country with the highest incidence rate in the world with about 500 new cases per year. HUS is the leading cause of acute renal failure in infancy, the second leading cause of chronic renal failure and 20% of cases of renal transplantation during childhood and adolescence. It has been shown that the ability of the strain to cause disease is linked by the action of virulence factors required in the colonization of intestinal epithelium and subsequent inflammatory response and their ability to secrete toxins, VT1 and VT2 responsible for damage to the vascular endothelium with the subsequent development of HUS. In this thesis we investigated plant extracts and components of probiotic strains potentially useful in diet therapy to inhibit the colonization of E. coli O157:H7 in the intestine as a preventive measure, able to interfere the cytotoxic action of VT on eukaryotic cells. Phenolic compounds were identified, they were obtained from plant extracts of P. alba and Z. mistol macerated in different solvents (alcohol, distilled water, acetone and hexane). They were analyzed using phytochemical methods, thin layer chromatography and quantified by spectrophotometry. Furthermore components were obtained from fermented milk kefir grains.

Shiga toxin and its use in targeted cancer therapy and imaging.

Shiga and the Shiga-like toxins are related protein toxins produced by Shigella dysenteriae and certain strains of Escherichia coli. These toxins are composed of two non-covalently attached, modular parts: the A moiety (StxA) containing the enzymatically active A1 fragment, and the non-toxic, pentameric binding moiety (StxB). Stx binds specifically to the glycosphingolipid globotriaosylceramide (Gb3) at the surface of target cells and is then internalized by endocytosis. Subsequently, in toxin-sensitive cells, the Stx/Gb3 complex is transported in a retrograde manner via the Golgi apparatus to the endoplasmic reticulum, where the enzymatically active part of Stx is translocated to the cytosol, enabling it to irreversibly inhibit protein synthesis via modification of ribosomal 28S RNA. Whereas Gb3 shows a relatively restricted expression in normal human tissues, it has been reported to be highly expressed in many types of cancers. This review gives a brief introduction to Stx and its intracellular transport. Furthermore, after a description of Gb3 and the methods that are currently used to detect its cellular expression, we provide an updated overview of the published reports on Gb3 overexpression in human cancers. Finally, we discuss the possibility of utilizing Stx or StxB coupled to therapeutic compounds or contrast agents in targeted cancer therapy and imaging.

Selective CCR2-targeted macrophage depletion ameliorates experimental mesangioproliferative glomerulonephritis.

The CCL2/CCR2 chemokine/receptor axis directs the chemotaxis of infiltrating monocytes/macrophages and T cells and plays a pivotal role in tissue damage and fibrosis in kidney diseases. The eradication of the activated leucocytes should diminish the production of inflammatory mediators, limit tissue damage and ameliorate disease. A recombinant fusion protein (OPL-CCL2-LPM) comprised of the human CCL2 (monocyte chemoattractant protein-1) chemokine fused to a truncated form of the enzymatically active A1 domain of Shigella dysenteriae holotoxin (SA1) has been developed. The CCL2 portion binds specifically to CCR2-bearing leucocytes and the fusion protein enters the cells, where the SA1 moiety inhibits protein synthesis resulting in cell death. The compound was tested in a model of anti-thymocyte serum (ATS)-induced mesangioproliferative glomerulonephritis (ATS-GN). Male rats were injected with ATS on day 0 and treated intravenously with vehicle, 50 or 100 microg/kg of OPL-CCL2-LPM Q2D from days 2, 4, 6 and 8. Urine and blood were collected on days 0, 5 and 9. Animals were sacrificed on day 9. No treatment-related effects on body weight or signs of clinical toxicity were observed. Urine protein levels were decreased in treated animals. At the highest dose, histopathological analyses of kidney sections revealed maximum reductions of 36, 31, 30 and 24% for macrophage count, glomerular lesions, alpha-smooth muscle actin and fibronectin respectively. These results indicate a significant protective effect of OPL-CCL2-LPM in this model of nephritis.

Gene transfer-mediated pre-mRNA segmental trans-splicing as a strategy to deliver intracellular toxins for cancer therapy.

Virus-mediated transfer of genes coding for intracellular toxins holds promise for cancer therapy, but the inherent toxicity of such vectors make them a risk to normal tissues and a challenge to produce due to the intrinsic dilemma that expression of toxin molecules kills producer cells. We employed pre-mRNA segmental trans-splicing (STS), in which two engineered DNA fragments coding for 5' "donor" and 3' "acceptor" segments of a toxin gene, respectively, are expressed by viral vectors. When co-delivered to target cells, the two vectors generate two toxin pre-mRNA fragments which are spliced by the target cell machinery to produce functional mRNA and toxin. To test this approach, we used an enzymatic fragment of Shigatoxin1A1 (STX1A1) known to provoke apoptotic cell death. Two adenovirus vectors, Shigatoxin1A1 donor (AdStx1A1Do) and Shigatoxin1A1 acceptor (AdStx1A1Ac), respectively, were used to deliver the Stx1A1 gene fragments. HeLa, HEp2, and A549 cells transfected with AdStx1A1Do and AdStx1A1Ac had a dose-dependent reduction in viability and inhibition of protein synthesis. Intratumoral injection of AdStx1A1Do and AdStx1A1Ac into preexisting HeLa, Hep2, and A549 tumors in immunodeficient mice revealed significant inhibition of tumor growth. There was no evidence of liver damage, suggesting that there was no leakage of vector or toxin from the site of injection following intratumoral injection of AdStx1A1Do and AdStx1A1Ac. These results suggest that the obstacles preventing gene transfer of intracellular toxins for local cancer therapy could be overcome by pre-mRNA segmental trans-splicing.

Genetic medicine at the RNA level: modifications of the genetic repertoire for therapeutic purposes by pre-mRNA trans-splicing.

Gene therapy is conventionally carried out by transferring genetic material to the target cell where the exogenous gene is expressed using the endogenous transcription and translation machinery in parallel with the target cell genome. This review focuses on a new paradigm of gene therapy, the use of trans-splicing to modify the genetic repertoire at the pre-mRNA level to treat genetic and acquired disorders. Therapeutic trans-splicing can be used to alter coding domains, to create novel fusion proteins, to direct gene products to various cellular compartments, and to overcome some of the limitations to vector-derived gene transfer technology, including gene therapy with large genes or with genes coding for toxic proteins. To demonstrate the potential of therapeutic trans-splicing, eukaryotic cis-splicing and trans-splicing are reviewed, followed by a discussion of strategies of therapeutic pre-mRNA trans-splicing directed by exogenous gene transfer.