Search

Infection with Shiga toxin-producing Escherichia coli (STEC) may cause hemorrhagic colitis, hemolytic uremic syndrome (HUS) and encephalopathy. The mortality rate derived from HUS adds up to 5% of the cases, and up to 40% when the central nervous system (CNS) is involved. In addition to the well-known deleterious effect of Stx, the gram-negative STEC releases lipopolysaccharides (LPS) and may induce a variety of inflammatory responses when released in the gut. Common clinical signs of severe CNS injury include sensorimotor, cognitive, emotional and/or autonomic alterations. In the last few years, a number of drugs have been experimentally employed to establish the pathogenesis of, prevent or treat CNS injury by STEC. The strategies in these approaches focus on: 1) inhibition of Stx production and release by STEC, 2) inhibition of Stx bloodstream transport, 3) inhibition of Stx entry into the CNS parenchyma, 4) blockade of deleterious Stx action in neural cells, and 5) inhibition of immune system activation and CNS inflammation. Fast diagnosis of STEC infection, as well as the establishment of early CNS biomarkers of damage, may be determinants of adequate neuropharmacological treatment in time.

Encephalopathy associated with hemolytic uremic syndrome is produced by enterohemorrhagic E. coli (EHEC) infection, which releases the virulence factors Shiga toxin (Stx) and lipopolysaccharide (LPS). Neurological compromise is a poor prognosis and mortality factor of the disease, and the thalamus is one of the brain areas most frequently affected. We have previously demonstrated the effectiveness of anti-inflammatory drugs to ameliorate the deleterious effects of these toxins. However, the thalamic production of cytokines involved in pro-inflammatory processes has not yet been acknowledged. The aim of this work attempts to determine whether systemic sublethal Stx2a or co-administration of Stx2a with LPS are able to rise a proinflammatory profile accompanying alterations of the neurovascular unit in anterior and lateral ventral nuclei of the thalamus (VA-VL) and motor behavior in mice. After 4 days of treatment, Stx2a affected the lectin-bound microvasculature distribution while increasing the expression of GFAP in reactive astrocytes and producing aberrant NeuN distribution in degenerative neurons. In addition, increased swimming latency was observed in a motor behavioral test. All these alterations were heightened when Stx2a was co-administered with LPS. The expression of pro-inflammatory cytokines TNFα, INF-γ and IL-2 was detected in VA-VL. All these effects were concomitant with increased expression of the Stx receptor globotriaosylceramide (Gb3), which hints at receptor involvement in the neuroinflammatory process as a key finding of this study. In conclusion, Stx2a to Gb3 may be determinant in triggering a neuroinflammatory event, which may resemble clinical outcomes and should thus be considered in the development of preventive strategies.

Shiga toxin 2 (Stx2) from enterohemorrhagic Escherichia coli (EHEC) produces hemorrhagic colitis, hemolytic uremic syndrome (HUS) and acute encephalopathy. The mortality rate in HUS increases significantly when the central nervous system (CNS) is involved. Besides, EHEC also releases lipopolysaccharide (LPS). Many reports have described cognitive dysfunctions in HUS patients, the hippocampus being one of the brain areas targeted by EHEC infection. In this context, a translational murine model of encephalopathy was employed to establish the deleterious effects of Stx2 and the contribution of LPS in the hippocampus. Results demonstrate that systemic administration of a sublethal dose of Stx2 reduced memory index and produced depression like behavior, pro-inflammatory cytokine release and NF-kB activation independent of the ERK 1/2 signaling pathway. On the other hand, LPS activated NF-kB dependent on ERK 1/2 signaling pathway. Cotreatment of Stx2 with LPS aggravated thepathologic state, while dexamethasone treatment succeeded in preventing behavioral alterations. Our present work suggests that the use of drugs such as corticosteroids or NF-kB signaling inhibitors may serve as neuroprotectors from EHEC infection. Fil: Berdasco, Clara Valentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Vasconcelos Esteves Pinto, Alipio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Blake, Mariano Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Correa, Fernando Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Longo, Nadia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Geogeghan, Patricia. Ministerio de Salud de la Nación. Administración Nacional de Laboratorios e Institutos de Salud; Argentina Fil: Cangelosi, Adriana. Ministerio de Salud de la Nación. Administración Nacional de Laboratorios e Institutos de Salud; Argentina Fil: Gironacci, Mariela Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Goldstein Raij, Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina

Clostridium perfringens epsilon toxin (ETX), the most potent toxin produced by this bacteria, plays a key role in the pathogenesis of enterotoxaemia in ruminants, causing brain edema and encephalomalacia. Studies of animals suffering from ETX intoxication describe severe neurological disorders that are thought to be the result of vasogenic brain edemas and indirect neuronal toxicity, killing oligodendrocytes but not astrocytes, microglia, or neurons in vitro. In this study, by means of intravenous and intracerebroventricular delivery of sub-lethal concentrations of ETX, the histological and ultrastructural changes of the brain were studied in rats and mice. Histological analysis showed degenerative changes in neurons from the cortex, hippocampus, striatum and hypothalamus. Ultrastructurally, necrotic neurons and apoptotic cells were observed in these same areas, among axons with accumulation of neurofilaments and demyelination as well as synaptic stripping. Lesions observed in the brain after sub-lethal exposure to ETX, result in permanent behavioral changes in animals surviving ETX exposure, as observed individually in several animals and assessed in the Inclined Plane Test and the Wire Hang Test. Pharmacological studies showed that dexamethasone and reserpine but not ketamine or riluzole were able to reduce the brain lesions and the lethality of ETX. Cytotoxicity was not observed upon neuronal primary cultures in vitro. Therefore, we hypothesize that ETX can affect the brain of animals independently of death, producing changes on neurons or glia as the result of complex interactions, independently of ETX-BBB interactions. Fil: Morris, Winston E.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina Fil: Goldstein Raij, Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: Redondo, Leandro Martin. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina Fil: Cangelosi, Adriana. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; Argentina Fil: Geoghegan, Patricia. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; Argentina Fil: Brocco, Marcela Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Loidl, Cesar Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentina Fil: Fernandez Miyakawa, Mariano Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina

Shiga toxin (Stx) produced by enterohemorrhagic E. coli produces hemolytic uremic syndrome and encephalopathies in patients, which can lead to either reversible or permanent neurological abnormalities, or even fatal cases depending on the degree of intoxication. It has been observed that the inflammatory component plays a decisive role in the severity of the disease. Therefore, the objective of this work was to evaluate the behavior of microglial cell primary cultures upon Stx2 exposure and heat shock or lipopolysaccharide challenges, as cues which modulate cellular environments, mimicking fever and inflammation states, respectively. In these contexts, activated microglial cells incorporated Stx2, increased their metabolism, phagocytic capacity, and pro-inflammatory profile. Stx2 uptake was associated to receptor globotriaosylceramide (Gb3)-pathway. Gb3 had three clearly distinguishable distribution patterns which varied according to different contexts. In addition, toxin uptake exhibited both a Gb3-dependent and a Gb3-independent binding depending on those contexts. Altogether, these results suggest a fundamental role for microglial cells in pro-inflammatory processes in encephalopathies due to Stx2 intoxication and highlight the impact of environmental cues.

Hemolytic uremic syndrome (HUS), principally caused by Shiga toxins (Stxs), is associated with Shiga toxin-producing Escherichia coli (STEC) infections. We previously reported Stx2 expression by host cells in vitro and in vivo. As the genes encoding the two Stx subunits are located in bacteriophage genomes, the aim of the current study was to evaluate the role of bacteriophage induction in HUS development in absence of an E. coli O157:H7 genomic background. Mice were inoculated with a non-pathogenic E. coli strain carrying the lysogenic bacteriophage 933W (C600Φ933W), and bacteriophage excision was induced by an antibiotic. The mice died 72 h after inoculation, having developed pathogenic damage typical of STEC infection. As well as renal and intestinal damage, markers of central nervous system (CNS) injury were observed, including aberrant immunolocalization of neuronal nuclei (NeuN) and increased expression of glial fibrillary acidic protein (GFAP). These results show that bacteriophage 933W without an E. coli O157:H7 background is capable of inducing the pathogenic damage associated with STEC infection. In addition, a novel mouse model was developed to evaluate therapeutic approaches focused on the bacteriophage as a new target. Fil: del Cogliano, Manuel Eugenio. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Vasconcelos Esteves Pinto, Alipio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Goldstein Raij, Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Zotta, Elsa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Ochoa, Federico Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina 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; Argentina Fil: Muniesa, Maite. Academia Nacional de Medicina de Buenos Aires; Argentina Fil: Ghiringhelli, Pablo Daniel. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: 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; Argentina Fil: Bentancor, Leticia Verónica. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Infections by Enterohemorrhagic Escherichia coli may cause in addition to hemolytic uremic syndrome neurological disorders which may lead to fatal outcomes in patients. The brain striatum is usually affected during this outcome. The aim of this study was to determine in this area the role of the microglia in pro-inflammatory events that may occur during Shiga toxin 2 intoxication and consequently to this, whether oligodendrocytes were being affected. In the present paper we demonstrated that anti-inflammatory treatments reduced deleterious effects in brain striatal cells exposed to Shiga toxin 2 and LPS. While dexamethasone treatment decreased microglial activation and recovered myelin integrity in the mice striatum, etanercept treatment decreased neuronal uptake of Stx2 in rat striatal neurons, improving the affected area from toxin-derived injury. In conclusion, microglial activation is related to pro-inflammatory events that may deteriorate the brain function during intoxication with Stx2 and LPS. Consequently, the role of anti-inflammatory agents in the treatment of EHEC-derived encephalopathy should be studied in clinical trials. Fil: Vasconcelos Esteves Pinto, Alipio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Berdasco, Clara Valentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Arenas Mosquera, David. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Cangelosi, Adriana. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; Argentina Fil: Geoghegan, Patricia A.. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; Argentina Fil: Nuñez, Myriam C.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Goldstein Raij, Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina

Background: Shiga toxin 2 from enterohemorrhagic Escherichia coli is the etiologic agent of bloody diarrhea, hemolytic uremic syndrome and derived encephalopathies that may result to death in patients. Being a Gram negative bacterium, lipopolysaccharide is also released. Particularly, the hippocampus has been found affected in patients intoxicated with Shiga toxin 2. In the current work, the deleterious effects of Shiga toxin 2 and lipopolysaccharide are investigated in detail in hippocampal cells for the first time in a translational murine model, providing conclusive evidences on how these toxins may damage in the observed clinic cases. Methods: Male NIH mice (25 g) were injected intravenously with saline solution, lipopolysaccharide, Shiga toxin 2 or a combination of Shiga toxin 2 with lipopolysaccharide. Brain water content assay was made to determine brain edema. Another set of animals were intracardially perfused with a fixative solution and their brains were subjected to immunofluorescence with lectins to determine the microvasculature profile, and anti-GFAP, anti-NeuN, anti-MBP and anti-Iba1 to study reactive astrocytes, neuronal damage, myelin dysarrangements and microglial state respectively. Finally, the Thiobarbituric Acid Reactive Substances Assay was made to determine lipid peroxidation. In all assays, statistical significance was performed using the One-way analysis of variance followed by Bonferroni post hoc test. Results: Systemic sublethal administration of Shiga toxin 2 increased the expressions of astrocytic GFAP and microglial Iba1, and decreased the expressions of endothelial glycocalyx, NeuN neurons from CA1 pyramidal layer and oligodendrocytic MBP myelin sheath from the fimbria of the hippocampus. In addition, increased interstitial fluids and Thiobarbituric Acid Reactive Substances-derived lipid peroxidation were also found. The observed outcomes were enhanced when sublethal administration of Shiga toxin 2 was co-administered together with lipopolysaccharide. Conclusion: Systemic sublethal administration of Shiga toxin 2 produced a deterioration of the cells that integrate the vascular unit displaying astrocytic and microglial reactive profiles, while edema and lipid peroxidation were also observed. The contribution of lipopolysaccharide to pathogenicity caused by Shiga toxin 2 resulted to enhance the observed hippocampal damage. Fil: Berdasco, Clara Valentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Vasconcelos Esteves Pinto, Alipio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Calabró López, María Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina Fil: Arenas Mosquera, David. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Cangelosi, Adriana. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán"; Argentina Fil: Geoghegan, Patricia. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán"; Argentina Fil: Evelson, Pablo Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina Fil: Goldstein Raij, Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina

Pathogenic Escherichia coli are known to be a common cause of diarrheal disease and a frequently occurring bacterial infection in children and adults in Latin America. Despite the effort to combat diarrheal infections, the south of the American continent remains a hot spot for infections and sequelae associated with the acquisition of one category of pathogenic E. coli, the Shiga toxin-producing E. coli (STEC). This review will focus on an overview of the prevalence of different STEC serotypes in human, animals and food products, focusing on recent reports from Latin America outlining the recent research progress achieved in this region to combat disease and endemicity in affected countries and to improve understanding on emerging serotypes and their virulence factors. Furthermore, this review will highlight the progress done in vaccine development and treatment and will also discuss the effort of the Latin American investigators to respond to the thread of STEC infections by establishing a multidisciplinary network of experts that are addressing STEC-associated animal, human and environmental health issues, while trying to reduce human disease. Regardless of the significant scientific contributions to understand and combat STEC infections worldwide, many significant challenges still exist and this review has focus in the Latin American efforts as an example of what can be accomplished when multiple groups have a common goal., Instituto de Genética Veterinaria

Shiga toxin 2 (Stx2) from enterohemorrhagic Escherichia coli (EHEC) causes bloody diarrhea and Hemolytic Uremic Syndrome (HUS) that may derive to fatal neurological outcomes. Neurological abnormalities in the striatum are frequently observed in affected patients and in studies with animal models while motor disorders are usually associated with pyramidal and extra pyramidal systems. A translational murine model of encephalopathy was employed to demonstrate that systemic administration of a sublethal dose of Stx2 damaged the striatal microvasculature and astrocytes, increase the blood brain barrier permeability and caused neuronal degeneration. All these events were aggravated by lipopolysaccharide (LPS). The injury observed in the striatum coincided with locomotor behavioral alterations. The anti-inflammatory Dexamethasone resulted to prevent the observed neurologic and clinical signs, proving to be an effective drug. Therefore, the present work demonstrates that: (i) systemic sub-lethal Stx2 damages the striatal neurovascular unit as it succeeds to pass through the blood brain barrier. (ii) This damage is aggravated by the contribution of LPS which is also produced and secreted by EHEC, and (iii) the observed neurological alterations may be prevented by an anti-inflammatory treatment. Fil: Vasconcelos Esteves Pinto, Alipio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Cangelosi, Adriana. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; Argentina Fil: Geoghegan, Patricia A.. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; Argentina Fil: Goldstein Raij, Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina

Shiga toxin producing Escherichia coli may damage the central nervous system before or concomitantly to manifested hemolytic–uremic syndrome symptoms. The cerebellum is frequently damaged during this syndrome, however, the deleterious effects of Shiga toxin 2 has never been integrally reported by ultrastructural, physiological and behavioral means. The aim of this study was to determine the cerebellar compromise after intravenous administration of a sub-lethal dose of Shiga toxin 2 by measuring the cerebellar blood–brain barrier permeability, behavioral task of cerebellar functionality (inclined plane test), and ultrastructural analysis (transmission electron microscope). Intravenous administration of vehicle (control group), sub-lethal dose of 0.5 and 1 ηg of Stx2 per mouse were tested for behavioral and ultrastructural studies. A set of three independent experiments were performed for each study (n = 6). Blood–brain barrier resulted damaged and consequently its permeability was significantly increased. Lower scores obtained in the inclined plane task denoted poor cerebellar functionality in comparison to their controls. The most significant lower score was obtained after 5 days of 1 ηg of toxin administration. Transmission electron microscope micrographs from the Stx2-treated groups showed neurons with a progressive neurodegenerative condition in a dose dependent manner. As sub-lethal intravenous Shiga toxin 2 altered the blood brain barrier permeability in the cerebellum the toxin penetrated the cerebellar parenchyma and produced cell damaged with significant functional implications in the test balance. Fil: D`Alessio, Luciana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos ; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia ; Argentina Fil: Vasconcelos Esteves Pinto, Alipio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Cangelosi, Adriana. Ministerio de Salud de la Nación. Administración Nacional de Laboratorios e Institutos de Salud; Argentina Fil: Geoghegan, Patricia A.. Ministerio de Salud de la Nación. Administración Nacional de Laboratorios e Institutos de Salud; Argentina Fil: Tironi Farinati, Alicia Carla Flavia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Brener, Gabriela J.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Goldstein Raij, Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina

Shiga toxin-producing Escherichia coli produces watery and hemorrhagic diarrhea, and hemolytic uremic syndrome (HUS) characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure. Central nervous system (CNS) complications are observed in around 30% of infant population with HUS. Common signs of severe CNS involvement leading to death include seizures, alteration of consciousness, hemiparesis, visual disturbances, and brain stem symptoms. The purpose of the present work was to study the effects of Shiga toxin 2 (Stx2) in the brain of rats intraperitoneally (i.p.) injected with a supernatant from recombinant E. coli expressing Stx2 (sStx2). Neurological alterations such as postural and motor abnormalities including lethargy, abnormal walking, and paralysis of hind legs, were observed in this experimental model of HUS in rats. Neuronal damage, as well as significant decrease in aquaporin 1 (AQP1) and aquaporin 4 (AQP4) expression levels were observed in the brain of rats, 2 days after sStx2 injection, compared to controls. Downregulation of aquaporin protein levels, and neuronal alterations, observed in brain of rats injected with sStx2, may be involved in edema formation and in neurological manifestations characteristic of HUS.

Shiga toxin 2 (Stx2)-producing enterohemorrhagic induced brain damage. Since a cerebroprotective action was reported for angiotensin (Ang)-(1–7), our aim was to investigate whether Ang-(1–7) protects from brain damage induced by Stx2-producing enterohemorrhagic Escherichia coli. The anterior hypothalamic area of adult male Wistar rats was injected with saline solution or Stx2 or Stx2 plus Ang-(1–7) or Stx2 plus Ang-(1–7) plus A779. Rats received a single injection of Stx2 at the beginning of the experiment, and Ang-(1–7), A779, or saline was administered daily in a single injection for 8 days. Cellular ultrastructural changes were analyzed by transmission electron microscopy. Stx2 induced neurodegeneration, axonal demyelination, alterations in synapse, and oligodendrocyte and astrocyte damage, accompanied by edema. Ang-(1–7) prevented neuronal damage triggered by the toxin in 55.6 ± 9.5% of the neurons and the Stx2-induced synapse dysfunction was reversed. In addition, Ang-(1–7) blocked Stx2-induced demyelination in 92 ± 4% of the axons. Oligodendrocyte damage caused by Stx2 was prevented by Ang-(1–7) but astrocytes were only partially protected by the peptide (38 ± 5% of astrocytes were preserved). Ang-(1–7) treatment resulted in 50% reduction in the number of activated microglial cells induced by Stx2, suggesting an anti-inflammatory action. All these beneficial effects elicited by Ang-(1–7) were blocked by the Mas receptor antagonist and thus it was concluded that Ang-(1–7) protects mainly neurons and oligodendrocytes, and partially astrocytes, in the central nervous system through Mas receptor stimulation. Fil: Goldstein Raij, Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Carden, Tomas Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Perez, María J.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Taira, Carlos Alberto. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Höcht, Christian. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología; Argentina Fil: Gironacci, Mariela Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina

ProThyrotropin-releasing hormone (proTRH) is a prohormone widely distributed in many areas of the brain. After biosynthesis, proTRH is subjected to post-translational processing to generate TRH and seven non-TRH peptides. Among these non-TRH sequences, we found previously that preproTRH178-199 could regulate the secretion of prolactin in suckled rats by their pups. Dopamine (DA), the main regulator of prolactin secretion, is produced in dopaminergic tyrosine hydroxylase (TH)-positive neurons in the hypothalamic arcuate nucleus (ARC). In this study we investigated whether prolactin release during the estrous sexual cycle is regulated by preproTRH178-199 through its effect on DA neurons of the ARC. We observed that biotinylated preproTRH178-199 bound to neurons in the ARC; this was higher during proestrus than during diestrus. Binding of preproTRH178-199 to DA neurons was seen only during proestrus in the ARC. Using primary neuronal hypothalamic cultures we found that preproTRH178-199 peptide decreased TH levels in a dose-responsive manner, whereas intra-ARC administration of preproTRH178-199 induced a 20-fold increase in plasma prolactin levels. Together, these results suggest a potential role for preproTRH178-199 in regulating dopaminergic neurons involved in the inhibition of pituitary prolactin release.

mce3 is one of the four mce operons in Mycobacterium tuberculosis that encode exported proteins with a probable role in the virulence of this bacterium. Upstream of mce3 there is a putative regulatory gene (Rv1963) that harbours a double tetR-family signature. To study the role of this putative regulatory gene in the transcriptional regulation of the mce3 operon, Mycobacterium smegmatis mc(2)155 and M. tuberculosis H37Rv strains that harboured gene fusions between the mce3 promoter region and the Escherichia coli lacZ gene, either containing or not containing the Rv1963 gene, were used. The presence of the Rv1963 gene in the strains greatly reduced beta-galactosidase activity, suggesting that the Rv1963-encoded protein is a transcriptional repressor of the mce3 operon. Expression of mce3 by recombinant M. tuberculosis was increased when it was grown in a macrophage-like cell line (J774), compared to the level of expression seen when the recombinant bacterium was grown under in vitro conditions. However, no lifting of repression was induced. The mce3 promoter was defined by deletion and cloning of the Rv1963-Rv1964 intergenic region in a 200 bp DNA fragment harbouring the region upstream of the Rv1964 start codon. Gel-shift experiments determined that the Rv1963-binding site was located in this region. These results indicate that the mce3 operon is transcriptionally regulated and that under certain, unknown, conditions repression of gene expression could be lifted.

Several evidences showed a cerebroprotective action for angiotensin (Ang) (1-7) but neither of them demonstrated its cellular target for this protective effect. Our aim was to investigate the cellular type protected by Ang-(1-7) by transmission electron microscopy in the model of brain damage induced by Shiga toxin 2 (Stx2)-producing enterohemorrhagic Escherichia Coli. Adult male Wistar rats were injected with saline solution or Stx2 or Stx2 plus Ang-(1-7) or Stx2 plus Ang-(1-7) plus A779 into the anterior hypothalamic area (AHA). Rats received a single injection of Stx2 at the beginning while Ang-(1-7), A779 or saline was given daily as a single injection during 8 days. Ultrastructural changes were analyzed by transmission electron microscopy. Stx2 induced neurodegeneration, axon demyelination, alterations in synapse and oligodendrocyte and astrocyte damage, accompanied with edema. Ang-(1-7) partially prevented neuronal damage: 55.6±9.5 % of the neurons were protected from the damage triggered by the toxin. In addition, Ang-(1-7) hampered the Stx2-induced demyelination in 92±4% of the axons. Oligodendrocyte damage caused by Stx2 was prevented by Ang-(1-7) but atrocytes were partially protected by the peptide (38±5 % of astrocytes were preserved). The Stx2-induced synapse dysfunction was reverted by Ang-(1-7). The number of activated microglial cells induced by Stx2 was reduced by 50% by Ang-(1-7) treatment (P

The RAS (renin-angiotensin system) is composed of two arms: the pressor arm containing AngII (angiotensin II)/ACE (angiotensin-converting enzyme)/AT1Rs (AngII type 1 receptors), and the depressor arm represented by Ang-(1-7) [angiotensin-(1-7)]/ACE2/Mas receptors. All of the components of the RAS are present in the brain. Within the brain, Ang-(1-7) contributes to the regulation of BP (blood pressure) by acting at regions that control cardiovascular function such that, when Ang-(1-7) is injected into the nucleus of the solitary tract, caudal ventrolateral medulla, paraventricular nucleus or anterior hypothalamic area, a reduction in BP occurs; however, when injected into the rostral ventrolateral medulla, Ang-(1-7) stimulates an increase in BP. In contrast with AngII, Ang-(1-7) improves baroreflex sensitivity and has an inhibitory neuromodulatory role in hypothalamic noradrenergic neurotransmission. Ang-(1-7) not only exerts effects related to BP regulation, but also acts as a cerebroprotective component of the RAS by reducing cerebral infarct size and neuronal apoptosis. In the present review, we provide an overview of effects elicited by Ang-(1-7) in the brain, which suggest a potential role for Ang-(1-7) in controlling the central development of hypertension. Fil: Gironacci, Mariela Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina Fil: Cerniello, Flavia Micaela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; Argentina Fil: Longo, Nadia Andrea. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; Argentina Fil: Goldstein Raij, Jorge. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Neurofisiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Cerrato, Bruno Diego. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; Argentina

Distribution of GAP-43 was studied in the retinas of rats after continuous illumination followed by different darkness periods. GAP-43 immunoreactivity was maximum in regenerating outer photoreceptor segments of rats kept in total darkness for 10 days, while in the inner plexiform layer, immunoreactivity was maximum immediately after illumination. Changes in GAP-43 expression could participate in retinal repair/regeneration after light-induced damage.

Shiga toxins (Stx) are the main agent responsible for the development of hemolytic-uremic syndrome (HUS), the most severe and life-threatening systemic complication of infection with enterohemorrhagic Escherichia coli (EHEC) strains. We previously described Stx2 expression by eukaryotic cells after they were transfected in vitro with the stx 2 gene cloned into a prokaryotic plasmid (pStx2). The aim of this study was to evaluate whether mammalian cells were also able to express Stx2 in vivo after pStx2 injection. Mice were inoculated by hydrodynamics-based transfection (HBT) with pStx2. We studied the survival, percentage of polymorphonuclear leukocytes in plasma, plasma urea levels, and histology of the kidneys and the brains of mice. Mice displayed a lethal dose-related response to pStx2. Stx2 mRNA was recovered from the liver, and Stx2 cytotoxic activity was observed in plasma of mice injected with pStx2. Stx2 was detected by immunofluorescence in the brains of mice inoculated with pStx2, and markers of central nervous system (CNS) damage were observed, including increased expression of glial fibrillary acidic protein (GFAP) and fragmentation of NeuN in neurons. Moreover, anti-Stx2B-immunized mice were protected against pStx2 inoculation. Our results show that Stx2 is expressed in vivo from the wild stx 2 gene, reproducing pathogenic damage induced by purified Stx2 or secondary to EHEC infection. IMPORTANCE Enterohemorrhagic Shiga toxin (Stx)-producing Escherichia coli (EHEC) infections are a serious public health problem, and Stx is the main pathogenic agent associated with typical hemolytic-uremic syndrome (HUS). In contrast to the detailed information describing the molecular basis for EHEC adherence to epithelial cells, very little is known about how Stx is released from bacteria in the gut, reaching its target tissues, mainly the kidney and central nervous system (CNS). In order to develop an efficient treatment for EHEC infections, it is necessary to understand the mechanisms involved in Stx expression. In this regard, the present study demonstrates that mammals can synthesize biologically active Stx using the natural promoter associated with the Stx-converting bacteriophage genome. These results could impact the comprehension of EHEC HUS, since local eukaryotic cells transduced and/or infected by bacteriophage encoding Stx2 could be an alternative source of Stx production.

The distribution of nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) reactivity and neuronal nitric oxide synthase immunoreactivity (nNOS-IR) was investigated in the rat retina during photoreceptor regeneration. Photoreceptor damage and the disappearance of a NADPHd reactive/nNOS-IR band corresponding to inner photoreceptor segments were observed after continuous exposure to light irradiation. Both events were reversible after 20 days of total darkness. Also a progressive decrease in the number and in the staining intensity of NADPHd reactivity in amacrine cells were found along the first 3-6 days of darkness stabilizing thereafter in both illuminated and control groups. However, staining intensity in the former group remained more elevated than in the latter one. NOS activity in the retina varies depending on functional and pathological states.

Ang-(1–7) [angiotensin-(1–7)] constitutes an important functional end-product of the RAS (renin–angiotensin system) endogenously formed from AngI (angiotensin I) or AngII (angiotensin II) through the catalytic activity of ACE2 (angiotensin-converting enzyme 2), prolyl carboxypeptidase, neutral endopeptidase or other endopeptidases. Ang-(1–7) lacks the pressor, dipsogenic or stimulatory effect on aldosterone release characteristic of AngII. In contrast, it produces vasodilation, natriuresis and diuresis, and inhibits angiogenesis and cell growth. At the central level, Ang-(1–7) acts at sites involved in the control of cardiovascular function, thus contributing to blood pressure regulation. This action may result from its inhibitory neuromodulatory action on NE [noradrenaline (norepinephrine)] levels at the synaptic cleft, i.e. Ang-(1–7) reduces NE release and synthesis, whereas it causes an increase in NE transporter expression, contributing in this way to central NE neuromodulation. Thus, by selective neurotransmitter release, Ang-(1–7) may contribute to the overall central cardiovascular effects. In the present review, we summarize the central effects of Ang-(1–7) and the mechanism by which the peptide modulates NE levels in the synaptic cleft. We also provide new evidences of its cerebroprotective role. Fil: Gironacci, Mariela Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; Argentina; Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Bioquímica Clínica; Argentina; Fil: Longo, Nadia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; Argentina; Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Bioquímica Clínica; Argentina; Fil: Goldstein Raij, Jorge. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Fisiopatogenia; Argentina; Fil: Cerrato, Bruno Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; Argentina; Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Bioquímica Clínica; Argentina

PURPOSE: To develop a time course study of vascularization and glial response to perinatal asphyxia in hypoxic-ischemic animals, and to evaluate hypothermia as possible protective treatment. METHODS: We used retinas of 7-, 15-, 21-, and 30-day-old male Sprague-Dawley rats that were exposed to perinatal asphyxia at either 37°C (PA) or 15°C (HYP). Born to term animals were used as controls (CTL). We evaluated the thickness of the most inner layers of the retina (IR), including internal limiting membrane, the retinal nerve fiber layer, and the ganglion cell layer; and studied glial development, neovascularization, adrenomedullin (AM), and VEGF by immunohistochemistry, immunofluorescence, and Western blot. RESULTS: A significant increment in IR thickness was observed in the PA group from postnatal day (PND) 15 on. This alteration was concordant with an increased number of new vessels and increased GFAP expression. The immunolocalization of GFAP in the internal limiting membrane and perivascular glia of the IR and in the inner processes of Müller cells was coexpressed with AM, which was also significantly increased from PND7 in PA animals. In addition, VEGF expression was immunolocalized in cells of the ganglion cell layer of the IR and this expression significantly increased in the PA group from PND15 on. The retinas of the HYP group did not show differences when compared with CTL at any age. CONCLUSIONS: This work demonstrates that aberrant angiogenesis and exacerbated gliosis seem to be responsible for the increased thickness of the inner retina as a consequence of perinatal asphyxia, and that hypothermia is able to prevent these alterations. Fil: Rey Funes, Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia ; Argentina Fil: Dorfman, Verónica Berta. Universidad Maimónides. Area de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Ibarra, Mariano Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia ; Argentina Fil: Peña, Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia ; Argentina Fil: Contartese, Daniela Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia ; Argentina Fil: Goldstein Raij, Jorge. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Acosta, Juan Manuel. Universidad Católica de Cuyo - Sede San Juan. Facultad de Ciencias Médicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Larráyoz, Ignacio M.. Centro de Investigación Biomédica de La Rioja; España Fil: Martínez Murillo, Ricardo. Consejo Superior de Investigaciones Cientificas; España. Instituto Cajal. Departamento de Neurobiología Molecular, Celular y del Desarrollo; España Fil: Martínez, Alfredo. Centro de Investigación Biomédica de La Rioja; España. Consejo Superior de Investigaciones Cientificas; España Fil: Loidl, Cesar Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia ; Argentina. Universidad Católica de Cuyo - Sede San Juan. Facultad de Ciencias Médicas; Argentina

Infection by Shiga toxin-producing Escherichia coli causes hemorrhagic colitis, hemolytic uremic syndrome (HUS), acute renal failure, and also central nervous system complications in around 30% of the children affected. Besides, neurological deficits are one of the most unrepairable and untreatable outcomes of HUS. Study of the striatum is relevant because basal ganglia are one of the brain areas most commonly affected in patients that have suffered from HUS and since the deleterious effects of a sub-lethal dose of Shiga toxin have never been studied in the striatum, the purpose of this study was to attempt to simulate an infection by Shiga toxin-producing E. coli in a murine model. To this end, intravenous administration of a sub-lethal dose of Shiga toxin 2 (0.5 ηg per mouse) was used and the correlation between neurological manifestations and ultrastructural changes in striatal brain cells was studied in detail. Neurological manifestations included significant motor behavior abnormalities in spontaneous motor activity, gait, pelvic elevation and hind limb activity eight days after administration of the toxin. Transmission electron microscopy revealed that the toxin caused early perivascular edema two days after administration, as well as significant damage in astrocytes four days after administration and significant damage in neurons and oligodendrocytes eight days after administration. Interrupted synapses and mast cell extravasation were also found eight days after administration of the toxin. We thus conclude that the chronological order of events observed in the striatum could explain the neurological disorders found eight days after administration of the toxin. Fil: Tironi Farinati, Alicia Carla Flavia. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Fisiopatogenia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Geoghegan, Patricia A.. Ministerio de Salud de la Nación. Administración Nacional de Laboratorios e Institutos de Salud; Argentina Fil: Cangelosi, Adriana. Ministerio de Salud de la Nación. Administración Nacional de Laboratorios e Institutos de Salud; Argentina Fil: Pinto, Alipio. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Fisiología. Laboratorio de Neurofisiopatología; Argentina Fil: Loidl, Cesar Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencias.; Fil: Goldstein, Jorge. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Fisiología. Laboratorio de Neurofisiopatología; Argentina

The correct name of the fourth author is: Carla Tironi-Farinati The correct citation is: Goldstein J, Morris WE, Loidl CF, Tironi-Farinati C, McClane BA, et al. (2009) Clostridium perfringens Epsilon Toxin Increases the Small Intestinal Permeability in Mice and Rats. PLoS ONE 4(9): e7065. doi:10.1371/journal.pone.0007065

Neurological damage caused by intoxication with Shiga toxin (Stx) from enterohemorrhagic Escherichia coli is the most unrepairable and untreatable outcome of Hemolytic Uremic Syndrome, and occurs in 30% of affected infants. In this work intracerebroventricular administration of Stx2 in rat brains significantly increased the expression of its receptor globotriaosylceramide (Gb3) in neuronal populations from striatum, hippocampus and cortex. Stx2 was immunodetected in neurons that expressed Gb3 after intracerebroventricular administration of the toxin. Confocal immunofluorescence of microtubule-associated protein 2 showed aberrant dendrites in neurons expressing increased Gb3. The pro-apoptotic Bax protein was concomitantly immunodetected in neurons and other cell populations from the same described areas including the hypothalamus. Confocal immunofluorescence showed that Gb3 colocalized also with glial fibrillary acidic protein only in reactive astrocytic processes, and not in vehicle-treated normal ones. Rats showed weight variation and motor deficits as compared to controls. We thus suggest that Stx2 induces the expression of Gb3 in neurons and triggers neuronal dysfunctions. Fil: Tironi Farinati, Alicia Carla Flavia. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Loidl, Cesar Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis; Argentina Fil: Boccoli, Javier. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas; Argentina Fil: Parma, Yanil Renee. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Fernandez Miyakawa, Mariano Enrique. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Goldstein Raij, Jorge. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Epsilon toxin is a potent neurotoxin produced by Clostridium perfringens types B and D, an anaerobic bacterium that causes enterotoxaemia in ruminants. In the affected animal, it causes oedema of the lungs and brain by damaging the endothelial cells, inducing physiological and morphological changes. Although it is believed to compromise the intestinal barrier, thus entering the gut vasculature, little is known about the mechanism underlying this process. This study characterizes the effects of epsilon toxin on fluid transport and bioelectrical parameters in the small intestine of mice and rats. The enteropooling and the intestinal loop tests, together with the single-pass perfusion assay and in vitro and ex vivo analysis in Ussing's chamber, were all used in combination with histological and ultrastructural analysis of mice and rat small intestine, challenged with or without C. perfringens epsilon toxin. Luminal epsilon toxin induced a time and concentration dependent intestinal fluid accumulation and fall of the transepithelial resistance. Although no evident histological changes were observed, opening of the mucosa tight junction in combination with apoptotic changes in the lamina propria were seen with transmission electron microscopy. These results indicate that C. perfringens epsilon toxin alters the intestinal permeability, predominantly by opening the mucosa tight junction, increasing its permeability to macromolecules, and inducing further degenerative changes in the lamina propria of the bowel.

Hemolytic uremic syndrome (HUS) is characterized by microangiopathic hemolytic anemia, plaquetopenia and kidney damage. It is the leading cause of acute renal failure in pediatric age and the second for chronic renal failure. Shiga toxin-producing Escherichia coli (STEC) is the first etiologic agent of HUS being its main reservoir cattle and transmitted via contaminated food. At present, there is no specific treatment to reduce the progression of HUS. The study of the mechanisms by which STEC infects and Shiga toxin induces HUS can help to find new strategies to prevent this disease.

Shiga toxin (Stx) from enterohemorrhagic Escherichia coli (STEC) is the main cause of hemorrhagic colitis which may derive into Hemolytic Uremic Syndrome (HUS) and acute encephalopathy, one of the major risk factors for infant death caused by the toxin. We have previously demonstrated that intracerebroventricular administration of Stx2 causes neuronal death and glial cell damage in rat brains. In the present work, we observed that the intracerebroventricular administration of Stx2 increased the expression of glial fibrillary acidic protein (GFAP) leading to astrogliosis. Confocal microscopy showed reactive astrocytes in contact with Stx2-containing neurons. Immunocolocalization of increased GFAP and Stx2 in astrocytes was also observed. This insult in the brain was correlated with changes in the expression and activity of neuronal nitric oxide synthase (nNOS) by using the NADPH-diaphorase histochemical technique (NADPH-d HT). A significant decrease in NOS/NADPH-d-positive neurons and NOS/NADPH-d activity was observed in cerebral cortex and striatum, whereas an opposite effect was found in the hypothalamic paraventricular nucleus. We concluded that the i.c.v. administration of Stx2 promotes a typical pattern of brain injury showing reactive astrocytes and an alteration in the number and activity of nNOS/NADPH-d. According to the functional state of nNOS/NADPH-d and to brain cell morphology data, it could be inferred that the i.c.v. administration of Stx2 leads to either a neurodegenerative or a neuroprotective mechanism in the affected brain areas. The present animal model resembles the encephalopathy developed in Hemolytic Uremic Syndrome (HUS) patients by STEC intoxication. Fil: Boccoli, Javier. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: Loidl, Cesar Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentina Fil: López, Juan José. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentina Fil: Pistone Creydt, Virginia. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Ibarra, Cristina Adriana. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Goldstein Raij, Jorge. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Shiga toxin (Stx) from enterohemorrhagic Escherichia coli (STEC) is the main cause of hemorrhagic colitis which may derive to hemolytic–uremic syndrome (HUS). HUS is characterized by acute renal failure, thrombocytopenia and microangiopathic hemolytic anemia. Mortality in the acute stage has been lower than 5% of total affected argentine children with endemic HUS. Common signs of severe CNS involvement leading to death included seizures, alteration of consciousness, hemiparesis, visual disturbances, and brainstem symptoms. The main purpose of the present work was to study the direct involvement of Stx2 in brain cells by intracerebroventricular (i.c.v.) administration of Stx2. Immunodetection of Stx2 was confirmed by immunoelectron cytochemistry in different subsets and compartments of affected caudate putamen cells of corpus striatum. Transmission electron microscopy (TEM) studies revealed apoptotic neurons, glial ultrastructural alterations and demyelinated fibers. The i.c.v. microinfusion was applied for the first time in rats to demonstrate the direct action of Stx2 in neurons and glial cells. The toxin may affect brain neuroglial cells without the involvement of proinflammatory or systemic neurotoxic elements. Fil: Goldstein Raij, Jorge. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Fisiopatogenia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Loidl, Cesar Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Pistone Creydt, Virginia. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Fisiopatogenia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Boccoli, Javier. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Fisiopatogenia; Argentina Fil: Ibarra, Cristina Adriana. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Fisiopatogenia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

In the last years, infection associated with Shiga toxin-producing Escherichia coli (STEC) and subsequent Hemolitic-Uremic Syndrome (HUS) became relevant as a public health since it was considered as one of the most important emergent patogen present in the food contaminated by cattle feces. STEC infection may be asymptomatic or begins with a watery diarrhea that may or may not progress to bloody diarrhea (hemorrhagic colitis) and HUS. In Argentina, HUS is the most common pediatric cause of acute renal insufficiency and the second cause of chronic renal failure. Up to now, STEC infection lacks of known effective treatment strategies that diminish risk of progression to HUS. The mechanisms by which Shiga toxin (Stx) induce HUS may help to find strategies to prevent or ameliorate HUS. In this article, recent progress that has contributed to understanding the disease pathogenesis of STEC is reviewed. New strategies to prevent further uptake of Shiga from the gut, either during the diarrheal phase or once HUS has developed are discussed.

Regulation of energy balance by leptin involves regulation of several neuropeptides, including thyrotropin-releasing hormone (TRH). Synthesized from a larger inactive precursor, its maturation requires proteolytic cleavage by prohormone convertases 1 and 2 (PC1 and PC2). Since this maturation in response to leptin requires prohormone processing, we hypothesized that leptin might regulate hypothalamic PC1 and PC2 expression, ultimately leading to coordinated processing of prohormones into mature peptides. Using hypothalamic neurons, we found that leptin stimulated PC1 and PC2 mRNA and protein expression and also increased PC1 and PC2 promoter activities in transfected 293T cells. Starvation of rats, leading to low serum leptin levels, decreased PC1 and PC2 gene and protein expression in the paraventricular nucleus (PVN) of the hypothalamus. Exogenous administration of leptin to fasted animals restored PC1 levels in the median eminence (ME) and the PVN to approximately the level found in fed control animals. Consistent with this regulation of PCs in the PVN, concentrations of TRH in the PVN and ME were substantially reduced in the fasted animals relative to the fed animals, and leptin reversed this decrease. Further analysis showed that proteolytic cleavage of pro-thyrotropin-releasing hormone (proTRH) at known PC cleavage sites was reduced by fasting and increased in animals given leptin. Combined, these findings suggest that leptin-dependent stimulation of hypothalamic TRH expression involves both activation of trh transcription and stimulation of PC1 and PC2 expression, which lead to enhanced processing of proTRH into mature TRH.

Adenylyl cyclase (AC) isoforms catalyze the synthesis of 3′,5′-cyclic AMP from ATP. These isoforms are critically involved in the regulation of gene transcription, metabolism, and ion channel activity among others. Nitric oxide (NO) is a gaseous product whose synthesis from L-arginine is catalyzed by the enzyme NO synthase. It has been well established that NO activates the enzyme guanylyl cyclase, but little has been reported on the effects of NO on other important second messengers, such as AC. In the present study, the effects of sodium nitroprusside (SNP), a nitric oxide-releasing compound, on COS-7 cells transfected with plasmids containing AC types I, II, V and VI were evaluated. Total inhibition (∼98.5%) of cAMP production was observed in COS-7 cells transfected with the AC I isoform and previously treated with SNP (10 mM) for 30 min, when stimulated with ionomycin. A high inhibition (∼76%) of cAMP production was also observed in COS-7 cells transfected with the AC VI isoform and previously treated with SNP (10 mM) for 30 min, when stimulated with forskolin. No effect on cAMP production was observed in cells transfected with AC isoforms II and V. Fil: Goldstein Raij, Jorge. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Fisiopatogenia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Ibarra, Cristina Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Fisiopatogenia; Argentina Fil: Silberstein, Claudia Marcela. Universidad de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Fisiopatogenia; Argentina

High K+ medium and glutamate elicited a significant [3H]-GABA release in the golden hamster retina. High K+ -induced GABA release was largely calcium-dependent, while the effect of glutamate was Ca2+ -independent. After replacing Na+ by Li+, glutamate-evoked [3H]-GABA release was abolished, while high K+ -evoked release remained unchanged. The effect of glutamate was completely blocked by DNQX but not by APV. Furthermore, kainate induced [3H]-GABA release, whereas NMDA was ineffective. Assessment of endogenous GABA efflux further confirmed results obtained for [3H]-GABA. GABA-like immunoreactivity was observed in amacrine cells, in neurons localized in ganglion cell layer, as well as in fibers and terminals at the inner plexiform layer. In addition a few horizontal cells showed GABA-like immunolabeling. The present results suggest the existence of at least two pools of GABA in the hamster retina, compatible with both vesicular and carrier-mediated mechanisms of transmitter release, being the amacrine cells the main gabaergic source in this tissue.

A detailed study about the distribution of nitric oxide synthase (NOS) isoforms, neuronal NOS (nNOS) and macrophagic NOS (mNOS), in normal rat retina was performed using immunocytochemistry by employing specific antibodies. The nNOS immunocytochemistry showed immunoreactive amacrine cells, fibres in inner and outer plexiform layers (IPL and OPL) and an immunostained band corresponding to inner photoreceptor segments (IPS). This was in agreement with NADPH-d histochemical results. mNOS immunoreactivity was found in cell somas localized in both, inner nuclear layer (INL) and ganglion cell layer (GCL), in slender Muller cell processes along IPL and GCL and also in the band corresponding to IPS. A different distribution of nNOS and mNOS was found in rat retina although both isoforms of NOS are co-localized in IPS.

Perinatal asphyxia (PA) produces changes in nitric oxide synthase (NOS) activity in neuronal and endothelial cells of the striatum and neocortex. The changes were examined using a histochemical NADPH-diaphorase (NADPH-d) staining method. Newborn rats were exposed to severe PA at 37 degrees C and other groups were subjected to severe PA under hypothermic condition (15 degrees C) for 20 or 100 min, respectively. Quantitative image analysis was performed on the striatum and neocortex in order to count cell number of reactive neurons and to compare the pattern of staining between the different groups of animals. Severe asphyctic pups showed an important neuronal loss in striatum and neocortex that was reduced by hypothermia. NADPH-d(+) neurons with reactive processes were found in the lateral zone of the striatum and neocortex in asphyctic pups. Controls and hypothermic striatum showed rounded cells without reactive process, while no cells were stained in cortex. There was also an increase in NADPH-d activity in endothelial cells in severe asphyctic pups in striatum and neocortex vs control and hypothermically treated animals. Our data evidenced that an inappropriate activation of NOS in neuronal and endothelial cells induced by PA is related to neuronal injury. Hypothermia inhibits neuronal injury and may be a valuable neuroprotective agent.