Human Glomerular Endothelial Cells Treated With Shiga Toxin Type 2 Activate γδ T Lymphocytes.

The hemolytic uremic syndrome associated with diarrhea, a consequence of Shiga toxin (Stx)-producing Escherichia coli infection, is a common cause of pediatric acute renal failure in Argentina. Stx type 2a (Stx2a) causes direct damage to renal cells and induces local inflammatory responses that involve secretion of inflammatory mediators and the recruitment of innate immune cells. γδ T cells constitute a subset of T lymphocytes, which act as early sensors of cellular stress and infection. They can exert cytotoxicity against infected and transformed cells, and produce cytokines and chemokines. In this study, we investigated the activation of human peripheral γδ T cells in response to the incubation with Stx2a-stimulated human glomerular endothelial cells (HGEC) or their conditioned medium, by analyzing in γδ T lymphocytes, the expression of CD69, CD107a, and perforin, and the production of TNF-α and IFN-γ. In addition, we evaluated by confocal microscopy the contact between γδ T cells and HGEC. This analysis showed an augmentation in cellular interactions in the presence of Stx2a-stimulated HGEC compared to untreated HGEC. Furthermore, we observed an increase in cytokine production and CD107a expression, together with a decrease in intracellular perforin when γδ T cells were incubated with Stx2a-treated HGEC or their conditioned medium. Interestingly, the blocking of TNF-α by Etanercept reversed the changes in the parameters measured in γδ T cells incubated with Stx2a-treated HGEC supernatants. Altogether, our results suggest that soluble factors released by Stx2a-stimulated HGEC modulate the activation of γδ T cells, being TNF-α a key player during this process.

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.

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.

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.

Immunization with BLS-Stx2B chimera totally protects dams from early pregnancy loss induced by Shiga toxin type 2 (Stx2) and confers anti-Stx2 immunity to the offspring.

Shiga toxin producing Escherichia coli (STEC) are bacterial pathogens involved in food-borne diseases. Shiga toxin (Stx) is the main virulence factor of STEC and is responsible for systemic complications including Hemolytic Uremic Syndrome (HUS). It has been previously demonstrated that Shiga toxin type 2 (Stx2) induces pregnancy loss in rats in early stage of pregnancy. The main purpose of this study was to determine if an active immunization prevents Stx2 mediated pregnancy loss and confers passive protective immunity to the offspring. For that purpose Sprague Dawley female rats were immunized with the chimera based on the enzyme lumazine synthase from Brucella spp. (BLS) and the B subunit of Shiga toxin 2 (Stx2B) named BLS-Stx2B. After immunization females were mated with males. At day 8 of gestation, dams were challenged intraperitoneally with a sublethal and abortifacient dose of Stx2. The immunization induced high anti-Stx2B-specific antibody titers in sera and most important, prevented pregnancy loss. Pups born and breastfeed by immunized dams had high anti-Stx2B-specific antibody titers in sera. Cross-fostering experiments indicated that passive protective immunity against Stx2 was transmitted through lactation. These results indicate that immunization of adult female rats with BLS-Stx2B prevents Stx2-induced pregnancy loss and confers anti Stx2 protective immunity to the offspring.

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.

Impairing squamous differentiation by Klf4 deletion is sufficient to initiate tongue carcinoma development upon K-Ras activation in mice.

Oral squamous cell carcinoma (SCC) is among the most prevalent cancers in the world and is characterized by high morbidity and few therapeutic options. Like most cancers, oral SCC arises from a multistep process involving alterations of genes responsible for balancing proliferation and differentiation. Among these, Krϋppel-like factor 4 (Klf4) suppresses cell proliferation and promotes differentiation and thus helps to maintain epithelial homeostasis. However, the prevailing role of Klf4 in maintenance of normal homeostasis in oral epithelium has not been established in vivo. Here, we used an inducible oral-specific mice model to selectively ablate Klf4 in the oral cavity. We generated K14-CreER(Tam)/Klf4 (f/f) mice that survived to adulthood and did not present overt phenotype. However, histologically these mice showed dysplastic lesions, increased cell proliferation and abnormal differentiation in the tongue 4 months after induction, supporting a homeostatic role of Klf4 in the oral epithelia. Furthermore, by breeding these mutants with a transgenic line expressing at endogenous levels K-ras (G12D), we assessed the role of disrupting differentiation gene programs to the carcinogenesis process. The K14-CreER(TAM)/K-ras (G12D)/Klf4 (-) (/-) mice rapidly develop oral SCC in the tongue. Thus, our findings support the emerging notion that activation of differentiating gene programs may represent a barrier preventing carcinogenesis in epithelial cells harboring oncogenic mutations, and thus that molecules acting upstream and downstream of Klf4 may represent components of a novel tumor-suppressive pathway.

Linfoma de Burkitt abdominal: presentación de caso y revisión de la literatura / Abdominal burkitt lymphoma

El Linfoma de Burkitt (LB) es un tipo de linfoma no Hodgkin, infrecuente, que afecta principalmente a niños y adolescentes. Dada la escasa información disponible, nuestra intención es describir los hallazgos imagenológicos de L.B. Se realizó un análisis retrospectivo de la presentación clínica e imagenológica en un paciente varón de 4 años de edad, confirmado por biopsia. Combinación de distintos métodos de imagen. Rx negativas. La ecografía mostró hepatomegalia moderada con múltiples nódulos hipoecoicos, y líquido libre en cavidad abdominal. TC demostró hepatomegalia, mayor número y tamaño de nódulos sólidos (debido a su comportamiento densitométrico y realce postcontraste), escasa cantidad de ascitis y aumento de la densidad de la grasa del mesenterio. RMI caracterizó y puso en evidencia con mayor precisión los hallazgos por Ecografía y tomografía computada. El LB es una rara entidad que necesita de la clínica y la combinación de varios métodos de imagen para aproximarse a la sospecha diagnóstica y representa un gran desafío

Linfoma de Burkitt abdominal: presentación de caso y revisión de la literatura / Abdominal burkitt lymphoma

El Linfoma de Burkitt (LB) es un tipo de linfoma no Hodgkin, infrecuente, que afecta principalmente a niños y adolescentes. Dada la escasa información disponible, nuestra intención es describir los hallazgos imagenológicos de L.B. Se realizó un análisis retrospectivo de la presentación clínica e imagenológica en un paciente varón de 4 años de edad, confirmado por biopsia. Combinación de distintos métodos de imagen. Rx negativas. La ecografía mostró hepatomegalia moderada con múltiples nódulos hipoecoicos, y líquido libre en cavidad abdominal. TC demostró hepatomegalia, mayor número y tamaño de nódulos sólidos (debido a su comportamiento densitométrico y realce postcontraste), escasa cantidad de ascitis y aumento de la densidad de la grasa del mesenterio. RMI caracterizó y puso en evidencia con mayor precisión los hallazgos por Ecografía y tomografía computada. El LB es una rara entidad que necesita de la clínica y la combinación de varios métodos de imagen para aproximarse a la sospecha diagnóstica y representa un gran desafío (AU)

Tromboflebitis de la vena cava inferior en el puerperio / Postpartum inferior vena cava thrombophlebitis

La trombosis es la patología intraluminal más frecuente de la vena cava inferior (VCI) habitualmente es el resultado de la propagación del trombo que tendrá origen en las venas de la pelvis, o del hígado, o del riñón o de los miembros inferiores; los trombos podrán ser cruóricos o tumorales; cuando se agrega inflamación e infección de la vena, estaremos en presencia de tromboflebitis. El diagnóstico se basa en 2 pilares de relevancia, que son: la clínica y el diagnóstico por imágenes. El tratamiento puede ser médico, quirúrgico o ambos. En éste caso comprobaremos la preponderancia de las imágenes (TC y RM) para identificar y diagnosticar dicha entidad (AU)

Sialodoquitis: a propósito de un caso / Sialodochitis: a case report

El término sialodoquitis se refiere a la inflamación y dilatación del conducto excretor principal de la glándula parótida. Se presenta la historia clínica de un paciente de 46 años con sialodoquitis, patología infrecuente en nuestro medio, donde los métodos de diagnóstico por imágenes contribuyeron de manera importante en el esclarecimiento del caso. Los estudios realizados fueron: ecografía bidimensional, sialografía bajo control de TV y sialografía TC de la región parotídea (AU)

Tromboflebitis de la vena cava inferior en el puerperio / Postpartum inferior vena cava thrombophlebitis

La trombosis es la patología intraluminal más frecuente de la vena cava inferior (VCI) habitualmente es el resultado de la propagación del trombo que tendrá origen en las venas de la pelvis, o del hígado, o del riñón o de los miembros inferiores; los trombos podrán ser cruóricos o tumorales; cuando se agrega inflamación e infección de la vena, estaremos en presencia de tromboflebitis. El diagnóstico se basa en 2 pilares de relevancia, que son: la clínica y el diagnóstico por imágenes. El tratamiento puede ser médico, quirúrgico o ambos. En éste caso comprobaremos la preponderancia de las imágenes (TC y RM) para identificar y diagnosticar dicha entidad

Sialodoquitis: a propósito de un caso / Sialodochitis: a case report

El término sialodoquitis se refiere a la inflamación y dilatación del conducto excretor principal de la glándula parótida. Se presenta la historia clínica de un paciente de 46 años con sialodoquitis, patología infrecuente en nuestro medio, donde los métodos de diagnóstico por imágenes contribuyeron de manera importante en el esclarecimiento del caso. Los estudios realizados fueron: ecografía bidimensional, sialografía bajo control de TV y sialografía TC de la región parotídea