Tom G. Obrig

Response to Shiga toxin 1 and 2 in a baboon model of hemolytic uremic syndrome

Abstract.Post-diarrheal (D+) hemolytic uremic syndrome (HUS) is caused by Shiga-toxin (Stx)-producing Escherichia coli. There is epidemiological, cell culture, and mouse model evidence that Stx2-producing E. coli are more likely to cause HUS than strains that produce only Stx1, but this hypothesis has not been tested in a primate model of HUS. We have developed a baboon model of Stx-mediated HUS that was employed to compare the clinical, cytokine, and histological response to equal amounts of the two Shiga toxins. Animals given IV Stx2 developed progressive thrombocytopenia, hemolytic anemia, and azotemia, and urinary interleukin-6 levels rose significantly. Glomerular thrombotic microangiopathy was found at necropsy. Animals given Stx1 showed no cytokine response and no clinical, laboratory, or histological signs of HUS. Our findings from the primate model corroborate previous epidemiological, cell culture, and mouse model observations, and suggest that enteric infection with Stx2-producing E. coli is more likely to cause HUS than infection with organisms that produce only Stx1.

The Mouse Model of Amebic Colitis Reveals Mouse Strain Susceptibility to Infection and Exacerbation of Disease by CD4+ T Cells

Amebic colitis is an important worldwide parasitic disease for which there is not a well-established animal model. In this work we show that intracecal inoculation of Entamoeba histolytica trophozoites led to established infection in 60% of C3H mice, while C57BL/6 or BALB/c mice were resistant, including mice genetically deficient for IL-12, IFN-γ, or inducible NO synthase. Infection was a chronic and nonhealing cecitis that pathologically mirrored human disease. Characterization of the inflammation by gene chip analysis revealed abundant mast cell activity. Parasite-specific Ab and cellular proliferative responses were robust and marked by IL-4 and IL-13 production. Depletion of CD4+ cells significantly diminished both parasite burden and inflammation and correlated with decreased IL-4 and IL-13 production and loss of mast cell infiltration. This model reveals important immune factors that influence susceptibility to infection and demonstrates for the first time the pathologic contribution of the host immune response in amebiasis.

A Murine Model of HUS: Shiga Toxin with Lipopolysaccharide Mimics the Renal Damage and Physiologic Response of Human Disease

Hemolytic uremic syndrome (HUS), which is caused by Shiga toxin-producing Escherichia coli infection, is the leading cause of acute renal failure in children. At present, there is no complete small animal model of this disease. This study investigated a mouse model using intraperitoneal co-injection of purified Shiga toxin 2 (Stx2) plus LPS. Through microarray, biochemical, and histologic analysis, it was found to be a valid model of the human disease. Biochemical and microarray analysis of mouse kidneys revealed the Stx2 plus LPS challenge to be distinct from the effects of either agent alone. Microarrays identified differentially expressed genes that were demonstrated previously to play a role in this disease. Blood and serum analysis of these mice showed neutrophilia, thrombocytopenia, red cell hemolysis, and increased serum creatinine and blood urea nitrogen. In addition, histologic analysis and electron microscopy of mouse kidneys demonstrated glomerular fibrin deposition, red cell congestion, microthrombi formation, and glomerular ultrastructural changes. It was established that this C57BL/6 mouse is a complete model of HUS that includes the thrombocytopenia, hemolytic anemia, and renal failure that define the human disease. In addition, a time course of HUS disease progression that will be useful for identification of therapeutic targets and development of new treatments for HUS is described.

Hemolytic uremic syndrome: epidemiology, pathophysiology, and therapy

Shiga toxin (Stx)-producing Escherichia coli (STEC) were first linked to human disease in 1982 [1]. At that time an E. coli serotype (O157:H7) that had not previously been linked with human disease was found to be associated with outbreaks of hemorrhagic colitis [1]. Subsequent studies demonstrated that E. coli O157:H7 produced potent toxins that were genetically and physically very similar to Stx from Shigella dysenteriae type 1 [2]. The link between STEC infections and hemolytic uremic syndrome (HUS) was established in the 1980s in Canada by Karmali et al. [3] when they found that infection with various serotypes of STEC was strongly associated with the subsequent development of HUS. The most common E. coli serotype identified to cause hemorrhagic colitis and HUS in the United States was the single serotype – O157:H7. The reason this serotype is most commonly identified in STEC infections is that it is relatively easy to identify in clinical microbiology laboratories due to its inability to ferment sorbitol. Since the first description of O157:H7 as a human pathogen in 1983, it has become apparent that E. coli O157:H7 is but one of a much larger family of STEC whose primary virulence characteristic is the ability to produce Stxs [2, 3, 4]. STEC are clinically associated with both bloody and non-bloody diarrhea as well as systemic complications such as HUS [4]. It is now clear that there are at least 200 different types of STEC, of which approximately 60 have been associated with disease in humans [2, 4, 5]. Non-O157 STEC, especially E. coli O111, have been the cause of major outbreaks in both the United States and other parts of the world [6, 7]. However, apart from the occasional outbreak of nonO157 in the United States, the association of nonO157:H7 STEC with disease in this country is largely undetermined. Of note, the majority of laboratories do not routinely look for non-O157 STEC [8], suggesting that lack of diagnosis of non-O157:H7 disease is a failure of use of appropriate diagnostic techniques. Compatible with this hypothesis are at least two small studies in which non-O157:H7 STEC were routinely found in patients submitting stools for microbiological analysis in the United States. [9, 10]. Recent work from our laboratory in collaboration with multiple sites in the United States has attempted to determine the prevalence of both O157 and non-O157 STEC in selected sites in the United States. We have performed multi-site surveillance studies to determine the prevalence of O157 and non-O157 STEC in stool samples submitted to clinical microbiology laboratories for S.P. Andreoli is the editor of the Proceedings and H. Trachtman the associate editor. D.W.K. Acheson is the author of the section Microbiology and epidemiology of Shiga toxin-induced hemolytic uremic syndrome, R.L. Siegler is the author of the section Animal models of Stx-mediated HUS, T.G. Obrig is the author of the section Mechanisms of Stx-mediated cell injury, and H. Trachtman is author of the section New and future therapies for diarrhea-associated HUS.

Rapid Apoptosis Induced by Shiga Toxin in HeLa Cells

ABSTRACT Apoptosis was induced rapidly in HeLa cells after exposure to bacterial Shiga toxin (Stx1 and Stx2; 10 ng/ml). Approximately 60% of HeLa cells became apoptotic within 4 h as detected by DNA fragmentation, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and electron microscopy. Stx1-induced apoptosis required enzymatic activity of the Stx1A subunit, and apoptosis was not induced by the Stx2B subunit alone or by the anti-globotriaosylceramide antibody. This activity was also inhibited by brefeldin A, indicating the need for toxin processing through the Golgi apparatus. The intracellular pathway leading to apoptosis was further defined. Exposure of HeLa cells to Stx1 activated caspases 3, 6, 8, and 9, as measured both by an enzymatic assay with synthetic substrates and by detection of proteolytically activated forms of these caspases by Western immunoblotting. Preincubation of HeLa cells with substrate inhibitors of caspases 3, 6, and 8 protected the cells against Stx1-dependent apoptosis. These results led to a more detailed examination of the mitochondrial pathway of apoptosis. Apoptosis induced by Stx1 was accompanied by damage to mitochondrial membranes, measured as a reduced mitochondrial membrane potential, and increased release of cytochrome c from mitochondria at 3 to 4 h. Bid, an endogenous protein known to permeabilize mitochondrial membranes, was activated in a Stx1-dependent manner. Caspase-8 is known to activate Bid, and a specific inhibitor of caspase-8 prevented the mitochondrial damage. Although these data suggested that caspase-8-mediated cleavage of Bid with release of cytochrome c from mitochondria and activation of caspase-9 were responsible for the apoptosis, preincubation of HeLa cells with a specific inhibitor of caspase-9 did not protect against apoptosis. These results were explained by the discovery of a simultaneous Stx1-dependent increase in endogenous XIAP, a direct inhibitor of caspase-9. We conclude that the primary pathway of Stx1-induced apoptosis and DNA fragmentation in HeLa cells is unique and includes caspases 8, 6, and 3 but is independent of events in the mitochondrial pathway.

ZAK: a MAP3Kinase that transduces Shiga toxin- and ricin-induced proinflammatory cytokine expression.

Shiga toxins (Stxs) and ricin initiate damage to host cells by cleaving a single adenine residue on the α‐sarcin loop of the 28S ribosomal RNA. This molecular insult results in a cascade of intracellular events termed the ribotoxic stress response (RSR). Although Stxs and ricin have been shown to cause the RSR, the mitogen‐activated protein kinase kinase kinase (MAP3K) that transduces the signal from intoxicated ribosomes to activate SAPKinases has remained elusive. We show in vitro that DHP‐2 (7‐[3‐fluoro‐4‐aminophenyl‐(4‐(2‐pyridin‐2‐yl‐5,6‐dihydro‐4H‐pyrrolo[1,2‐b]pyrazol‐3‐yl))]‐quinoline), a zipper sterile‐α‐motif kinase (ZAK)‐specific inhibitor, blocks Stx2/ricin‐induced SAPKinase activation. Treatment of cells with DHP‐2 also blocks Stx2/ricin‐mediated upregulation of the proinflammatory cytokine interleukin‐8 and results in a modest but statistically significant improvement in cell viability following Stx2/ricin treatment. Finally we show that siRNA directed against the N‐terminus of ZAK diminishes Stx2/Ricin‐induced SAPKinase activation. Together, these data demonstrate that a ZAK isoform(s) is the MAP3Kinase that transduces the RSR. Therefore, ZAKα and/or β isoforms may act as potential therapeutic target(s) for treating Stx/ricin‐associated illnesses. Furthermore, a small molecule inhibitor like DHP‐2 may prove valuable in preventing the Stx/ricin‐induced proinflammatory and/or apoptotic effects that are thought to contribute to pathogenesis by Stx‐producing Escherichia coli and ricin.

Shiga Toxin 2 Affects the Central Nervous System through Receptor Globotriaosylceramide Localized to Neurons

Affinity-purified Shiga toxin (Stx) 2 given intraperitoneally to mice caused weight loss and hind-limb paralysis followed by death. Globotriaosylceramide (Gb(3)), the receptor for Stx2, was localized to neurons of the central nervous system (CNS) of normal mice. Gb3 was not found in astrocytes or endothelial cells of the CNS. In human cadaver CNS, we found Gb(3) in neurons and endothelial cells. Mouse Gb(3) localization was confirmed by immunoelectron microscopy. In Stx2-exposed mice, anti-Stx2-gold immunoreaction was positive in neurons. During paralysis, after Stx2 injection, multiple glial nuclei were observed surrounding motoneurons by electron microscopy. Also revealed was a lamellipodia-like process physically inhibiting the synaptic connection of motoneurons. Ca2+ imaging of cerebral astrocytic end-feet in Stx2-treated mouse brains suggested that the toxin increased neurotransmitter release from neurons. In this article, we propose that the neuron is a primary target of Stx2, affecting neuronal function and leading to paralysis.

Shiga Toxin 2 Targets the Murine Renal Collecting Duct Epithelium

ABSTRACT Hemolytic-uremic syndrome (HUS) caused by Shiga toxin-producing Escherichia coli infection is a leading cause of pediatric acute renal failure. Bacterial toxins produced in the gut enter the circulation and cause a systemic toxemia and targeted cell damage. It had been previously shown that injection of Shiga toxin 2 (Stx2) and lipopolysaccharide (LPS) caused signs and symptoms of HUS in mice, but the mechanism leading to renal failure remained uncharacterized. The current study elucidated that murine cells of the glomerular filtration barrier were unresponsive to Stx2 because they lacked the receptor glycosphingolipid globotriaosylceramide (Gb3) in vitro and in vivo. In contrast to the analogous human cells, Stx2 did not alter inflammatory kinase activity, cytokine release, or cell viability of the murine glomerular cells. However, murine renal cortical and medullary tubular cells expressed Gb3 and responded to Stx2 by undergoing apoptosis. Stx2-induced loss of functioning collecting ducts in vivo caused production of increased dilute urine, resulted in dehydration, and contributed to renal failure. Stx2-mediated renal dysfunction was ameliorated by administration of the nonselective caspase inhibitor Q-VD-OPH in vivo. Stx2 therefore targets the murine collecting duct, and this Stx2-induced injury can be blocked by inhibitors of apoptosis in vivo.

Monocyte Chemoattractant Protein 1, Macrophage Inflammatory Protein 1α, and RANTES Recruit Macrophages to the Kidney in a Mouse Model of Hemolytic-Uremic Syndrome

ABSTRACT The macrophage has previously been implicated in contributing to the renal inflammation associated with hemolytic-uremic syndrome (HUS). However, there is currently no in vivo model detailing the contribution of the renal macrophage to the kidney disease associated with HUS. Therefore, renal macrophage recruitment and inhibition of infiltrating renal macrophages were evaluated in an established HUS mouse model. Macrophage recruitment to the kidney was evident by immunohistochemistry 2 h after administration of purified Stx2 and peaked at 48 h postinjection. Mice administered a combination of Stx2 and lipopolysaccharide (LPS) showed increased macrophage recruitment to the kidney compared to mice treated with Stx2 or LPS alone. Monocyte chemoattractants were induced in the kidney, including monocyte chemoattractant protein 1 (MCP-1/CCL2), macrophage inflammatory protein 1α (MIP-1α/CCL3), and RANTES (CCL5), in a pattern that was coincident with macrophage infiltration as indicated by immunohistochemistry, protein, and RNA analyses. MCP-1 was the most abundant chemokine, MIP-1α was the least abundant, and RANTES levels were intermediate. Mice treated with MCP-1, MIP-1α, and RANTES neutralizing antibodies had a significant decrease in Stx2 plus LPS-induced macrophage accumulation in the kidney, indicating that these chemokines are required for macrophage recruitment. Furthermore, mice exposed to these three neutralizing antibodies had decreased fibrin deposition in their kidneys, implying that macrophages contribute to the renal damage associated with HUS.

Caspase and Bid Involvement in Clostridium difficile Toxin A-Induced Apoptosis and Modulation of Toxin A Effects by Glutamine and Alanyl-Glutamine In Vivo and In Vitro

ABSTRACT Clostridium difficile is the leading cause of nosocomial bacterial diarrhea. Glutamine and its stable and highly soluble derivative alanyl-glutamine, have been beneficial in models of intestinal injury. In this study, we extend our work on the mechanisms of Clostridium difficile toxin A (TxA)-induced apoptosis in human intestinal epithelial T84 cells and evaluate the effects of glutamine and alanyl-glutamine on TxA-induced apoptosis in vitro and disruption of ileal mucosa in vivo. T84 cells were incubated with TxA (100 ng/ml) in medium with or without glutamine or alanyl-glutamine (3 to 100 mM). Apoptosis was evaluated by DNA fragmentation in vitro and the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling method in vivo. Caspase and Bid involvement were investigated by Western blotting. Ligated rabbit ileal loops were used for the evaluation of intestinal secretion, mucosal disruption, and apoptosis. TxA induced caspases 6, 8, and 9 prior to caspase 3 activation in T84 cells and induced Bid cleavage by a caspase-independent mechanism. Glutamine or alanyl-glutamine significantly reduced TxA-induced apoptosis of T84 cells by 47% and inhibited activation of caspase 8. Both glutamine and alanyl-glutamine reduced TxA-induced ileal mucosal disruption and secretion. Altogether, we further delineated the apoptosis-signaling cascade induced by TxA in T84 cells and demonstrated the protective effects of glutamine and alanyl-glutamine. Glutamine and alanyl-glutamine inhibited the apoptosis of T84 cells by preventing caspase 8 activation and reduced TxA-induced intestinal secretion and disruption.