Detection of plasma anti-lipopolysaccharide (LPS)antibodies against enterohemorrhagic Escherichia coli(EHEC) in asymptomatic kindergarten teachers fromBuenos Aires province

Resumen En Argentina, el síndrome urémico hemolítico causado por Escherichia coli enterohemorrágica (EHEC) tiene la más alta incidencia del mundo. Las infecciones por EHEC tienen un comportamiento endemoepidémico y causan del 20 al 30% de los síndromes de diarrea sanguinolenta en niños menores de 5 años. En el período 2016-2020, se notificaron 272 nuevos casos por año al Sistema de Vigilancia de Salud Nacional. Múltiples factores son responsables de la alta incidencia de SUH en Argentina, incluyendo la transmisión persona-persona. Con el objetivo de detectar posibles portadores asintomáticos de EHEC, realizamos un estudio preliminar de la frecuencia de anticuerpos antilipopolisacáridos contra los serotipos de EHEC más prevalentes en Argentina. El estudio se realizó con muestras de plasma obtenidas de 61 maestras y maestros de jardines de infantes de 26 instituciones del distrito de José C. Paz, localizado en el área suburbana de la provincia de Buenos Aires, Argentina. El 51% de las muestras presentaron anticuerpos contra los serotipos de lipopolisacáridos O157, O145, O121 y O103; el 6,4% de las muestras positivas tuvieron el isotipo IgM (n=2), el 61,3% el isotipo IgG (n=19) y el 32,3% los isotipos IgM e IgG (n=10). Dado que los anticuerpos antilipopolisacáridos presentan usualmente una duración corta, la detección de IgM específica podría indicar una infección reciente. Además, el alto porcentaje de muestras positivas hallado podría indicar una exposición frecuente a las cepas de EHEC en la cohorte estudiada. Asimismo, la gran población de adultos portadores asintomáticos de estas cepas patógenas podría contribuir al comportamiento endémico, a través de la transmisión persona-persona. El perfeccionamiento de programas educacionales continuos en jardines de infantes podría constituir una medida importante para reducir los casos de síndrome urémico hemolítico, no solo en Argentina, sino también en el mundo.

Abstract In Argentina, hemolytic uremic syndrome (HUS) caused by EHEC has the highest incidence in the world. EHEC infection has an endemo-epidemic behavior, causing 20-30% of acute bloody diarrhea syndrome in children under 5 years old. In the period 2016-2020, 272 new cases per year were notified to the National Health Surveillance System. Multiple factors are responsible for HUS incidence in Argentina including person-to-person transmission. In order to detect possible EHEC carriers, we carried out a preliminary study of the frequency of kindergarten teachers with anti-LPS antibodies against the most prevalent EHEC serotypes in Argentina. We analyzed 61 kindergarten teachers from 26 institutions from José C. Paz district, located in the suburban area of Buenos Aires province, Argentina. Fifty-one percent of the plasma samples had antibodies against O157, O145, O121 and O103 LPS: 6.4% of the positive samples had IgM isotype (n=2), 61.3% IgG isotype (n=19) and 32.3% IgM and IgG (n=10). Given that antibodies against LPS antigens are usually short-lived specific IgM detection may indicate a recent infection. In addition, the high percentage of positive samples may indicate a frequent exposure to EHEC strains in the cohort studied, as well as the existence of a large non-symptomatic population of adults carrying pathogenic strains that could contribute to the endemic behavior through person-to-person transmission. The improvement of continuous educational programs in kindergarten institutions could be a mandatory measure to reduce HUS cases not only in Argentina but also globally.

Obtención de nueva información sobre el síndrome urémico hemolítico mediante minería de texto / Obtaining new information on hemolytic uremic syndrome by text mining

Resumen El síndrome urémico hemolítico (SUH) está caracterizado por microangiopatía trombótica, anemia hemolítica, trombocitopenia e insuficiencia renal aguda. Puede causar desde secuelas permanentes hasta muerte, principalmente en niños. En este trabajo, utilizando minería de textos (MT), se analizó el texto explícito e implícito de 16 192 artículos científicos originales sobre SUH indexados en la base de datos de Europe PMC. Los objetivos fueron examinar comportamientos, realizar seguimiento de tendencias, hacer predicciones y cruzar datos con otras fuentes de información. Para el análisis se utilizaron -entre otras herramientas infor máticas- flujos de trabajo (FT) especialmente desarrollados en la plataforma KNIME. La MT sobre las palabras de los resúmenes de las publicaciones permitió: detectar asociaciones no descritas entre eventos relacionados con SUH; extraer información subyacente; hacer agrupamientos temáticos mediante algoritmos no supervisados; realizar predicciones sobre el curso de las investigaciones asociadas al tema. Tanto el abordaje como los FT desarrollados para realizar Ciencia de Datos sobre SUH pueden aplicarse a otros temas biomédicos y a otras bases de datos científicos, permitiendo analizar aspectos relevantes en el campo de la salud humana para me jorar la investigación, la prevención y el tratamiento de múltiples enfermedades.

Abstract Hemolytic uremic syndrome (HUS) is characterized by thrombotic microangiopathy, hemolytic anemia, thrombocytopenia and acute renal failure. It can cause from permanent sequelae to death, mainly in children. In this work, using text mining (TM), we analyzed the explicit and implicit text of 16 192 original scientific articles on HUS indexed in the Europe PMC database. The objectives were to examine behaviors, track trends, and make predictions and cross-check data with other sources of information. For the analysis we used -among other computational tools- specially developed workflows (WF) in the KNIME platform. The TM on the words of the abstracts of the publications made it possible to: detect undescribed associations between events related to HUS; extract underly ing information; make thematic clustering using unsupervised algorithms; make forecasting about the course of research associated with the topic. Both the approach and the WFs developed to perform Data Science on HUS can be applied to other biomedical topics and other scientific databases, making it possible to analyze relevant aspects in the field of human health to improve research, prevention and treatment of multiples diseases.

Eliglustat prevents Shiga toxin 2 cytotoxic effects in human renal tubular epithelial cells.

BACKGROUND: Shiga toxin-producing Escherichia coli is responsible for post-diarrheal (D+) hemolytic uremic syndrome (HUS), which is a cause of acute renal failure in children. The glycolipid globotriaosylceramide (Gb3) is the main receptor for Shiga toxin (Stx) in kidney target cells. Eliglustat (EG) is a specific and potent inhibitor of glucosylceramide synthase, first step of glycosphingolipid biosynthesis, actually used for the treatment of Gaucher's disease. The aim of the present work was to evaluate the efficiency of EG in preventing the damage caused by Stx2 in human renal epithelial cells. METHODS: Human renal tubular epithelial cell (HRTEC) primary cultures were pre-treated with different dilutions of EG followed by co-incubation with EG and Stx2 at different times, and cell viability, proliferation, apoptosis, tubulogenesis, and Gb3 expression were assessed. RESULTS: In HRTEC, pre-treatments with 50 nmol/L EG for 24 h, or 500 nmol/L EG for 6 h, reduced Gb3 expression and totally prevented the effects of Stx2 on cell viability, proliferation, and apoptosis. EG treatment also allowed the development of tubulogenesis in 3D-HRTEC exposed to Stx2. CONCLUSIONS: EG could be a potential therapeutic drug for the prevention of acute kidney injury caused by Stx2. IMPACT: For the first time, we have demonstrated that Eliglustat prevents Shiga toxin 2 cytotoxic effects on human renal epithelia, by reducing the expression of the toxin receptor globotriaosylceramide. The present work also shows that Eliglustat prevents Shiga toxin 2 effects on tubulogenesis of renal epithelial cells. Eliglustat, actually used for the treatment of patients with Gaucher's disease, could be a therapeutic strategy to prevent the renal damage caused by Shiga toxin.

The Deleterious Effects of Shiga Toxin Type 2 Are Neutralized In Vitro by FabF8:Stx2 Recombinant Monoclonal Antibody.

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.

Combined Action of Shiga Toxin Type 2 and Subtilase Cytotoxin in the Pathogenesis of Hemolytic Uremic Syndrome.

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.

Protein Concentrates on Tepary Bean (Phaseolus acutifolius Gray) as a Functional Ingredient: In silico Docking of Tepary Bean Lectin to Peroxisome Proliferator-Activated Receptor Gamma.

The tepary bean (Phaseolus acutifolius Gray) is a US-Mexico frontier native crop, produces high yields in agriculture, and needs to be reconsidered because of its nutritional and functional properties. This study aimed to determine the technological and nutritional properties of flours and protein concentrates of tepary bean, besides determining an in silico agonist effect of tepary bean lectin to peroxisome proliferator-activated receptor gamma (PPAR-γ). We evaluated the technological properties of raw samples (tepary flour and tepary protein concentrate) and cooked samples (tepary flour and tepary protein concentrate). The flours present a significant difference (p < 0.05) concerning protein concentrates in water absorption and oil absorption capacity. The raw samples' emulsifying capacity was higher than that reported in the literature for other legumes, but not the cooked samples. The samples' foaming capacity had no significant difference in treatments (p > 0.05), and cooked tepary bean protein concentrate presented complete gelation at a lower concentration (2%). Nutritionally, raw samples present a protein percentage of 23.46 ± 0.06 and 71.38 ± 0.44 and cooked samples present a protein percentage of 25.27 ± 0.04 and 62.69 ± 0.14; a chemical score of 72, 86, 82, and 72; in vitro protein digestibility (%) = 48.20 ± 0.31, 49.80 ± 0.80, 61.77 ± 1.70, and 63.61 ± 4.19; and C-PER = 0.86, 1.34, 1.93, and 1.81, respectively. All the samples showed methionine + cysteine as the limiting amino acid. All these nutritional data are very similar to the common bean (Phaseolus vulgaris). SDS-PAGE preserves the lectin fraction in both protein concentrates. The in silico study of tepary lectin (PDB: 6tt9) shows that there were seven peptides that presented values below -120 kcal/mol: PEW, VSVGF, PSQK, TTPW, ATSF, ITY, and TSF, with VSVGF, PSQK, and PEW having the highest affinity for active sites of the PAPRγ receptor (binding energies from -5.32 to -7.04 kcal/mol). These peptides could show antiadipogenic or antidiabetic activity based on the intermolecular bond energies and open an interesting research item.

The limited knowledge of placental damage due to neglected infections: ongoing problems in Latin America.

The placenta works as a selective barrier, protecting the fetus from potential infections that may affect the maternal organism during pregnancy. In this review, we will discuss several challenging infections that are common within Latin American countries and that may affect the maternal-fetal interface and pose risks to fetal development. Specifically, we will focus on emerging infectious diseases including the arboviruses, malaria, leishmaniasis, and the bacterial foodborne disease caused by Shiga toxin-producing Escherichia coli. We will also highlight some topics of interest currently being studied by research groups that comprise an international effort aimed at filling the knowledge gaps in this field. These topics address the relationship between exposure to microorganisms and placental abnormalities, congenital anomalies, and complications of pregnancy. ABBREVIATIONS: ADE: antibody-dependent enhancement; CCL2: monocyte chemoattractant protein-1; CCL3: macrophage inflammatory protein-1 α; CCL5: chemokine (C-C motif) ligand 5; CHIKV: chikungunya virus; DCL: diffuse cutaneous leishmaniasis; DENV: dengue virus; Gb3: glycolipid globotriaosylceramyde; HIF: hypoxia-inducible factor; HUS: hemolytic uremic syndrome; IFN: interferon; Ig: immunoglobulins; IL: interleukin; IUGR: intrauterine growth restriction; LCL: localized cutaneous leishmaniasis; LPS: lipopolysaccharid; MCL: mucocutaneous leishmaniasis; NO: nitric oxide; PCR: polymerase chain reaction; PGF: placental growth factor; PM: placental malaria; RIVATREM: Red Iberoamericana de Alteraciones Vasculares em transtornos del Embarazo; sVEGFR: soluble vascular endothelial growth factor receptor; STEC: shiga toxin-producing Escherichia coli; stx: shiga toxin protein; TNF: tumor necrosis factor; TOAS: T cell original antigenic sin; Var2CSA: variant surface antigen 2-CSA; VEGF: vascular endothelial growth factor; VL: visceral leishmaniasis; WHO: world health organization; YFV: yellow fever virus; ZIKV: Zika virus.

Crosstalk between Human Microvascular Endothelial Cells and Tubular Epithelial Cells Modulates Pro-Inflammatory Responses Induced by Shiga Toxin Type 2 and Subtilase Cytotoxin.

Hemolytic uremic syndrome (HUS) is a consequence of Shiga toxin (Stx)-producing Escherichia coli (STEC) infection and is the most frequent cause of acute renal failure (ARF) in children. Subtilase cytotoxin (SubAB) has also been associated with HUS pathogenesis. We previously reported that Stx2 and SubAB cause different effects on co-cultures of human renal microvascular endothelial cells (HGEC) and human proximal tubular epithelial cells (HK-2) relative to HGEC and HK-2 monocultures. In this work we have analyzed the secretion of pro-inflammatory cytokines by co-cultures compared to monocultures exposed or not to Stx2, SubAB, and Stx2+SubAB. Under basal conditions, IL-6, IL-8 and TNF-α secretion was different between monocultures and co-cultures. After toxin treatments, high concentrations of Stx2 and SubAB decreased cytokine secretion by HGEC monocultures, but in contrast, low toxin concentrations increased their release. Toxins did not modulate the cytokine secretion by HK-2 monocultures, but increased their release in the HK-2 co-culture compartment. In addition, HK-2 monocultures were stimulated to release IL-8 after incubation with HGEC conditioned media. Finally, Stx2 and SubAB were detected in HGEC and HK-2 cells from the co-cultures. This work describes, for the first time, the inflammatory responses induced by Stx2 and SubAB, in a crosstalk model of renal endothelial and epithelial cells.

Immunization of pregnant cows with Shiga toxin-2 induces high levels of specific colostral antibodies and lactoferrin able to neutralize E. coli O157:H7 pathogenicity.

E. coli O157:H7 is a foodborne pathogen responsible for bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). The objective of the present work was to evaluate the ability of colostral IgG obtained from Stx2-immunized cows to prevent against E. coli O157:H7 infection and Stx2 cytotoxicity. Hyperimmune colostrum (HC) was obtained from cows intramuscularly immunized with inactivated Stx2 or vehicle for controls. Colostral IgG was purified by affinity chromatography. Specific IgG antibodies against Stx2 and bovine lactoferrin (bLF) levels in HC and the corresponding IgG (HC-IgG/bLF) were determined by ELISA. The protective effects of HC-IgG/bLF against Stx2 cytotoxicity and adhesion of E. coli O157:H7 and its Stx2-negative mutant were analyzed in HCT-8 cells. HC-IgG/bLF prevention against E. coli O157:H7 was studied in human colon and rat colon loops. Protection against a lethal dose of E. coli O157:H7 was evaluated in a weaned mice model. HC-IgG/bLF showed high anti-Stx2 titers and high bLF levels that were able to neutralize the cytotoxic effects of Stx2 in vitro and in vivo. Furthermore, HC-IgG/bLF avoided the inhibition of water absorption induced by E. coli O157:H7 in human colon and also the pathogenicity of E. coli O157:H7 and E. coli O157:H7Δstx2 in rat colon loops. Finally, HC-IgG/bLF prevented in a 100% the lethality caused by E. coli O157:H7 in a weaned mice model. Our study suggests that HC-IgG/bLF have protective effects against E. coli O157:H7 infection. These beneficial effects may be due to specific anti-Stx2 neutralizing antibodies in combination with high bLF levels. These results allow us to consider HC-IgG/bLF as a nutraceutical tool which could be used in combination with balanced supportive diets to prevent HUS. However further studies are required before recommendations can be made for therapeutic and clinical applications.

Soluble CD40 Ligand and Oxidative Response Are Reciprocally Stimulated during Shiga Toxin-Associated Hemolytic Uremic Syndrome.

Shiga toxin (Stx), produced by Escherichia coli, is the main pathogenic factor of diarrhea-associated hemolytic uremic syndrome (HUS), which is characterized by the obstruction of renal microvasculature by platelet-fibrin thrombi. It is well known that the oxidative imbalance generated by Stx induces platelet activation, contributing to thrombus formation. Moreover, activated platelets release soluble CD40 ligand (sCD40L), which in turn contributes to oxidative imbalance, triggering the release of reactive oxidative species (ROS) on various cellular types. The aim of this work was to determine if the interaction between the oxidative response and platelet-derived sCD40L, as consequence of Stx-induced endothelium damage, participates in the pathogenic mechanism during HUS. Activated human glomerular endothelial cells (HGEC) by Stx2 induced platelets to adhere to them. Although platelet adhesion did not contribute to endothelial damage, high levels of sCD40L were released to the medium. The release of sCD40L by activated platelets was inhibited by antioxidant treatment. Furthermore, we found increased levels of sCD40L in plasma from HUS patients, which were also able to trigger the respiratory burst in monocytes in a sCD40L-dependent manner. Thus, we concluded that platelet-derived sCD40L and the oxidative response are reciprocally stimulated during Stx2-associated HUS. This process may contribute to the evolution of glomerular occlusion and the microangiopathic lesions.

Ouabain Protects Human Renal Cells against the Cytotoxic Effects of Shiga Toxin Type 2 and Subtilase Cytotoxin.

Hemolytic uremic syndrome (HUS) is one of the most common causes of acute renal failure in children. The majority of cases are associated with Shiga toxin (Stx)-producing Escherichia coli (STEC). In Argentina, HUS is endemic and presents the highest incidence rate in the world. STEC strains expressing Stx type 2 (Stx2) are responsible for the most severe cases of this pathology. Subtilase cytotoxin (SubAB) is another STEC virulence factor that may contribute to HUS pathogenesis. To date, neither a licensed vaccine nor effective therapy for HUS is available for humans. Considering that Ouabain (OUA) may prevent the apoptosis process, in this study we evaluated if OUA is able to avoid the damage caused by Stx2 and SubAB on human glomerular endothelial cells (HGEC) and the human proximal tubule epithelial cell (HK-2) line. HGEC and HK-2 were pretreated with OUA and then incubated with the toxins. OUA protected the HGEC viability from Stx2 and SubAB cytotoxic effects, and also prevented the HK-2 viability from Stx2 effects. The protective action of OUA on HGEC and HK-2 was associated with a decrease in apoptosis and an increase in cell proliferation. Our data provide evidence that OUA could be considered as a therapeutic strategy to avoid the renal damage that precedes HUS.

Microbiological and serological control of Escherichia coli O157: H7 in kindergarten staff in Buenos Aires city and suburban areas

Shiga toxin (Stx)-producing Escherichia coli (STEC) infections are implicated in the development of the life-threatening hemolytic-uremic syndrome (HUS). Despite the magnitude of the social and economic problems caused by HUS, no licensed vaccine or effective therapy is currently available for human use. Prevention of STEC infections continues being the most important measure to reduce HUS incidence. This is especially true for Argentina where HUS incidence among children is extremely high and shows an endemic pattern. The aim of this work was to investigate serologically adult staff of kindergartens in Buenos Aires city and suburban areas in order to detect possible carriers, and to educate personnel about good practices to reduce HUS transmission. We also assessed the microbiological quality of water and meal samples from the same kindergartens. We tested 67 healthy adults, 13 water supplies and 6 meals belonging to 6 public kindergartens. We analysed hand swabs for isolation of STEC and serum samples for the presence of antibodies against Stx and lipopolysaccharide (LPS) of O157 serogroup. We identified 46 Stx2-positive individuals, but only 7 for O157 LPS. No presence of STEC pathogens was detected in hands of staff, water or meal samples.

Las infecciones bacterianas con Escherichia coli productor de toxina Shiga (Stx) (STEC) están implicadas en el desarrollo del síndrome urémico hemolítico (SUH). A pesar de la magnitud del problema social y económico causado por el SUH, actualmente no existe un tratamiento específico o una vacuna eficaz para uso humano. Por lo tanto, la prevención de las infecciones por STEC es la tarea central para reducir la incidencia del SUH. Esto es especialmente cierto para Argentina en donde el SUH muestra un comportamiento endémico y presenta una incidencia extremadamente alta entre los niños. En efecto, la mediana de casos notificados en menores de 5 años para el periodo 2010-2015 fue 306, mientras que la tasa de notificación fue 8.5 casos cada 100 000 menores/año (http://www.msal.gob.ar/images/stories/boletines/boletin_integrado_vigilancia_N335-SE45.pdf). El objetivo de este trabajo fue analizar serológicamente al personal adulto de jardines de infantes de la ciudad de Buenos Aires y el área suburbana con el fin de detectar portadores, y brindarles formación sobre las buenas prácticas para reducir la transmisión de infecciones con STEC y así evitar el SUH. También se evaluó la calidad microbiológica de las muestras de agua y de la comida elaborada en los mismos jardines. Hemos estudiado 67 adultos, a través del hisopado de manos para la búsqueda de STEC y suero para la presencia de anticuerpos contra Stx y el lipopolisacárido (LPS) de serogrupo O157. También se analizaron 13 suministros de agua y 6 muestras de comida pertenecientes a 6 jardines de infantes públicos. Se identificaron 46 individuos positivos para Stx2, pero solo 7 para LPS-O157. No se detectó presencia de patógenos STEC en las muestras de las manos del personal, ni en los reservorios de agua o muestras de comida.

Comparative Characterization of Shiga Toxin Type 2 and Subtilase Cytotoxin Effects on Human Renal Epithelial and Endothelial Cells Grown in Monolayer and Bilayer Conditions.

Postdiarrheal hemolytic uremic syndrome (HUS) affects children under 5 years old and is responsible for the development of acute and chronic renal failure, particularly in Argentina. This pathology is a complication of Shiga toxin (Stx)-producing Escherichia coli infection and renal damage is attributed to Stx types 1 and 2 (Stx1, Stx2) produced by Escherichia coli O157:H7 and many other STEC serotypes. It has been reported the production of Subtilase cytotoxin (SubAB) by non-O157 STEC isolated from cases of childhood diarrhea. Therefore, it is proposed that SubAB may contribute to HUS pathogenesis. The human kidney is the most affected organ because very Stx-sensitive cells express high amounts of biologically active receptor. In this study, we investigated the effects of Stx2 and SubAB on primary cultures of human glomerular endothelial cells (HGEC) and on a human tubular epithelial cell line (HK-2) in monoculture and coculture conditions. We have established the coculture as a human renal proximal tubule model to study water absorption and cytotoxicity in the presence of Stx2 and SubAB. We obtained and characterized cocultures of HGEC and HK-2. Under basal conditions, HGEC monolayers exhibited the lowest electrical resistance (TEER) and the highest water permeability, while the HGEC/HK-2 bilayers showed the highest TEER and the lowest water permeability. In addition, at times as short as 20-30 minutes, Stx2 and SubAB caused the inhibition of water absorption across HK-2 and HGEC monolayers and this effect was not related to a decrease in cell viability. However, toxins did not have inhibitory effects on water movement across HGEC/HK-2 bilayers. After 72 h, Stx2 inhibited the cell viability of HGEC and HK-2 monolayers, but these effects were attenuated in HGEC/HK-2 bilayers. On the other hand, SubAB cytotoxicity shows a tendency to be attenuated by the bilayers. Our data provide evidence about the different effects of these toxins on the bilayers respect to the monolayers. This in vitro model of communication between human renal microvascular endothelial cells and human proximal tubular epithelial cells is a representative model of the human proximal tubule to study the effects of Stx2 and SubAB related to the development of HUS.

Clade 8 and Clade 6 Strains of Escherichia coli O157:H7 from Cattle in Argentina have Hypervirulent-Like Phenotypes.

The hemolytic uremic syndrome (HUS) whose main causative agent is enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a disease that mainly affects children under 5 years of age. Argentina is the country with the highest incidence of HUS in the world. Cattle are a major reservoir and source of infection with E. coli O157:H7. To date, the epidemiological factors that contribute to its prevalence are poorly understood. Single nucleotide polymorphism (SNP) typing has helped to define nine E. coli O157:H7 clades and the clade 8 strains were associated with most of the cases of severe disease. In this study, eight randomly selected isolates of EHEC O157:H7 from cattle in Argentina were studied as well as two human isolates. Four of them were classified as clade 8 through the screening for 23 SNPs; the two human isolates grouped in this clade as well, while two strains were closely related to strains representing clade 6. To assess the pathogenicity of these strains, we assayed correlates of virulence. Shiga toxin production was determined by an ELISA kit. Four strains were high producers and one of these strains that belonged to a novel genotype showed high verocytotoxic activity in cultured cells. Also, these clade 8 and 6 strains showed high RBC lysis and adherence to epithelial cells. One of the clade 6 strains showed stronger inhibition of normal water absorption than E. coli O157:H7 EDL933 in human colonic explants. In addition, two of the strains showing high levels of Stx2 production and RBC lysis activity were associated with lethality and uremia in a mouse model. Consequently, circulation of such strains in cattle may partially contribute to the high incidence of HUS in Argentina.

Phase 1/1B trial of the heat shock protein 90 inhibitor NVP-AUY922 as monotherapy or in combination with bortezomib in patients with relapsed or refractory multiple myeloma.

BACKGROUND: NVP-AUY922 (AUY; Luminespib) with or without bortezomib showed preclinical activity against multiple myeloma (MM) cells. This phase 1/1B study assessed NVP-AUY922 alone and with bortezomib in patients with relapsed or refractory MM. METHODS: Dose escalation was guided by an adaptive Bayesian logistic regression model. In phase 1, patients who progressed after 2 to 4 prior therapies received NVP-AUY922 intravenously once weekly. In phase 1B, patients who progressed after 2 or fewer prior therapies received NVP-AUY922 plus bortezomib. The primary objective was to determine the maximum tolerated dose (MTD) of NVP-AUY922. RESULTS: Twenty-four patients received NVP-AUY922 monotherapy at doses of 8 to 70 mg/m(2) . One dose-limiting toxicity (DLT) was observed (grade 3 blurred vision at 70 mg/m(2) ); no MTD was reached. The recommended phase 2 dose was 70 mg/m(2) . The most frequent drug-related adverse events (AEs) were diarrhea, nausea, and ocular toxicities. Grade 3/4 AEs were uncommon (<10%). Eight patients discontinued treatment because of AEs; 5 had ocular toxicities (≥45 mg/m(2) ). The best response was stable disease in 66.7% of the patients. There were no partial or complete responses. Five patients received NVP-AUY922 (which was started at 50 mg/m(2) ) plus bortezomib (1.3 mg/m(2) ). Three of these patients experienced DLT. No further dose escalation was performed; the MTD for NVP-AUY922 plus bortezomib was not established. CONCLUSIONS: This study showed disease stabilization with NVP-AUY922 in patients with relapsed or refractory MM. The MTD for NVP-AUY922 was not reached, but reversible ocular toxicity has been reported at high dose levels. Bortezomib at the standard recommended dose plus NVP-AUY922 was not tolerated.

89Zr-trastuzumab and 89Zr-bevacizumab PET to evaluate the effect of the HSP90 inhibitor NVP-AUY922 in metastatic breast cancer patients.

PURPOSE: HSP90 chaperones have key client proteins that are involved in all hallmarks of breast cancer growth and progression. The primary aim of this clinical trial was to evaluate the feasibility of using (89)Zr-trastuzumab PET (for HER2-positive breast cancer) or (89)Zr-bevacizumab PET [for estrogen receptor (ER)-positive breast cancer] to determine in vivo degradation of client proteins caused by the novel HSP90 inhibitor NVP-AUY922. EXPERIMENTAL DESIGN: Of note, 70 mg/m(2) NVP-AUY922 was administered intravenously in a weekly schedule to patients with advanced HER2 or ER-positive breast cancer. Biomarker analysis consisted of serial PET imaging with 2[18F]fluoro-2-deoxy-D-glucose (FDG), (89)Zr-trastuzumab, or (89)Zr-bevacizumab. Response evaluation was performed according to RECIST1.0. FDG, (89)Zr-trastuzumab, and (89)Zr-bevacizumab distributions were scored visually and quantitatively by calculating the maximum standardized uptake values (SUVmax). In blood samples, serial HSP70 levels, extracellular form of HER2 (HER2-ECD), and pharmacokinetic and pharmacodynamic parameters were measured. RESULTS: Sixteen patients (ten HER2-positive and six ER-positive tumors) were included. One partial response was observed; seven patients showed stable disease. SUVmax change in individual tumor lesions on baseline versus 3 weeks (89)Zr-trastuzumab PET was heterogeneous and related to size change on CT after 8 weeks treatment (r(2) = 0.69; P = 0.006). Tumor response on (89)Zr-bevacizumab PET and FDG-PET was not correlated with CT response. CONCLUSIONS: NVP-AUY922 showed proof-of-concept clinical response in HER2-amplified metastatic breast cancer. Early change on (89)Zr-trastuzumab PET was positively associated with change in size of individual lesions assessed by CT.

Effects of Escherichia coli subtilase cytotoxin and Shiga toxin 2 on primary cultures of human renal tubular epithelial cells.

Shiga toxin (Stx)-producing Escherichia coli (STEC) cause post-diarrhea Hemolytic Uremic Syndrome (HUS), which is the most common cause of acute renal failure in children in many parts of the world. Several non-O157 STEC strains also produce Subtilase cytotoxin (SubAB) that may contribute to HUS pathogenesis. The aim of the present work was to examine the cytotoxic effects of SubAB on primary cultures of human cortical renal tubular epithelial cells (HRTEC) and compare its effects with those produced by Shiga toxin type 2 (Stx2), in order to evaluate their contribution to renal injury in HUS. For this purpose, cell viability, proliferation rate, and apoptosis were assayed on HRTEC incubated with SubAB and/or Stx2 toxins. SubAB significantly reduced cell viability and cell proliferation rate, as well as stimulating cell apoptosis in HRTEC cultures in a time dependent manner. However, HRTEC cultures were significantly more sensitive to the cytotoxic effects of Stx2 than those produced by SubAB. No synergism was observed when HRTEC were co-incubated with both SubAB and Stx2. When HRTEC were incubated with the inactive SubAA272B toxin, results were similar to those in untreated control cells. Similar stimulation of apoptosis was observed in Vero cells incubated with SubAB or/and Stx2, compared to HRTEC. In conclusion, primary cultures of HRTEC are significantly sensitive to the cytotoxic effects of SubAB, although, in a lesser extent compared to Stx2.

Action of shiga toxin type-2 and subtilase cytotoxin on human microvascular endothelial cells.

The hemolytic uremic syndrome (HUS) associated with diarrhea is a complication of Shiga toxin (Stx)-producing Escherichia coli (STEC) infection. In Argentina, HUS is endemic and responsible for acute and chronic renal failure in children younger than 5 years old. The human kidney is the most affected organ due to the presence of very Stx-sensitive cells, such as microvascular endothelial cells. Recently, Subtilase cytotoxin (SubAB) was proposed as a new toxin that may contribute to HUS pathogenesis, although its action on human glomerular endothelial cells (HGEC) has not been described yet. In this study, we compared the effects of SubAB with those caused by Stx2 on primary cultures of HGEC isolated from fragments of human pediatric renal cortex. HGEC were characterized as endothelial since they expressed von Willebrand factor (VWF) and platelet/endothelial cell adhesion molecule 1 (PECAM-1). HGEC also expressed the globotriaosylceramide (Gb3) receptor for Stx2. Both, Stx2 and SubAB induced swelling and detachment of HGEC and the consequent decrease in cell viability in a time-dependent manner. Preincubation of HGEC with C-9 -a competitive inhibitor of Gb3 synthesis-protected HGEC from Stx2 but not from SubAB cytotoxic effects. Stx2 increased apoptosis in a time-dependent manner while SubAB increased apoptosis at 4 and 6 h but decreased at 24 h. The apoptosis induced by SubAB relative to Stx2 was higher at 4 and 6 h, but lower at 24 h. Furthermore, necrosis caused by Stx2 was significantly higher than that induced by SubAB at all the time points evaluated. Our data provide evidence for the first time how SubAB could cooperate with the development of endothelial damage characteristic of HUS pathogenesis.

First-in-human phase I dose-escalation study of the HSP90 inhibitor AUY922 in patients with advanced solid tumors.

PURPOSE: A phase I study was conducted with the primary objective of determining the maximum tolerated dose (MTD) of AUY922 in patients with advanced solid tumors. Secondary objectives included characterization of the safety, pharmacokinetic, and pharmacodynamic profiles. PATIENTS AND METHODS: Patients with advanced solid tumors received 1-hour i.v. infusions of AUY922 once a week in a 28-day cycle. An adaptive Bayesian logistic regression model that employed observed dose-limiting toxicities (DLT) in the first treatment cycle was used to guide dose-escalation decisions, with the established MTD to be used in phase II studies. RESULTS: One hundred and one patients were enrolled and explored at doses in the range of 2 to 70 mg/m(2). DLTs occurred in 8 patients (22-70 mg/m(2)) and included diarrhea, asthenia/fatigue, anorexia, atrial flutter, and visual symptoms. At 70 mg/m(2), the AUY922 concentration achieved was consistent with active concentrations in a range of xenograft models. There was evidence of target inhibition in peripheral blood mononuclear cells (HSP70 induction) and tumor (client protein depletion and reduction of metabolic activity by (18)F-FDG PET). The recommended phase II dose (RP2D) of 70 mg/m(2) was proposed on the basis of toxicity and pharmacokinetic and pharmacodynamic profiles. CONCLUSIONS: At the RP2D of 70 mg/m(2), AUY922 exhibited acceptable tolerability, and phase II single-agent and combination studies have been initiated in patients with HER2-positive breast, gastric, and non-small cell lung cancers.

Effect of coiled-coil peptides on the function of the type III secretion system-dependent activity of enterohemorragic Escherichia coli O157:H7 and Citrobacter rodentium.

Many animal and human pathogenic Gram-negative bacteria such as Salmonella, Yersinia, enterohemorrhagic Escherichia coli (EHEC), and enteropathogenic Escherichia coli (EPEC) possess a type III secretion system (TTSS) that is used to deliver virulence proteins directly into the host cell. Recent evidence has suggested that CoilA and CoilB, two synthetic peptides corresponding to coiled-coil domains of the translocator protein EspA, are effective in inhibiting the action of TTSS from EPEC. In the current study, the action of these coiled-coil peptides on the TTSS of EHEC O157:H7 and Citrobacter rodentium was examined. CoilA and CoilB showed to be effective in reducing the red blood cell lysis mediated by EHEC O157:H7 and the in vitro secretion of translocator proteins EspB and EspD by EHEC O157:H7 and EspD by C. rodentium. Treatment of mice with CoilA and CoilB peptides prevented colon damage when the animals were inoculated with C. rodentium. Colon samples of the non-treated group showed areas with loss of superficial epithelium, damaged cells, and endoluminal mononuclear inflammatory infiltrate, consistent with histological lesions induced by C. rodentium, whereas mice treated with the synthetic peptides displayed normal surface epithelium showing a similar structure as the uninfected control group. These encouraging results prompt us to test coiled-coil peptides as treatment or vaccines in other models of bacterial infections in future work.