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Vaccination is an important factor in public health. The recombinant bacillus Calmette Guérin (rBCG) vaccine, which expresses foreign antigens, is expected to be a superior vaccine against infectious diseases. Here, we report a new recombination platform in which the BCG Tokyo strain is transformed with nucleotide sequences encoding foreign protein fused with the MPB70 immunogenic protein precursor. By RNA-sequencing, mpb70 was found to be the most transcribed among all known genes of BCG Tokyo. Small oligopeptide, namely, polyhistidine tag, was able to be expressed in and secreted from rBCG through a process in which polyhistidine tag fused with intact MPB70 were transcribed by an mpb70 promoter. This methodology was applied to develop an rBCG expressing the receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2. Immunoblotting images and mass spectrometry data showed that RBD was also secreted from rBCG. Sera from mice vaccinated with the rBCG showed a tendency of weak neutralizing capacity. The secretion was retained even after a freeze-drying process. The freeze-dried rBCG was administered to and recovered from mice. Recovered rBCG kept secreting RBD. Collectively, our recombination platform offers stable secretion of foreign antigens and can be applied to the development of practical rBCGs.
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Vacuna BCG , Mycobacterium bovis , Animales , Ratones , Vacuna BCG/genética , Tokio , Mycobacterium bovis/genética , Activación de Linfocitos , Ingeniería Genética , Vacunas SintéticasRESUMEN
Background: In 2020, an incident involving spoiled salad dressing from a commercial source occurred. Upon opening the bottle, the contents exploded from gas that seemed to have fermented inside the bottle. For safety concerns, we sought to investigate the bacteria from the salad dressing in order to notify the company that made the product and relevant authorities. Methods: Anaerobic and carbon dioxide culture methods were used. To determine species of colonies, MALDI-TOF-MS and 16S rRNA whole sequencing were performed. Results: There were no colonies grown in anaerobic condition; however, we obtained three colonies from the carbon dioxide atmosphere. We determined the first colony as Alkalihalobacillus clausii (Bacillus clausii), the second as Bacillus spp. such as B. australimaris, B. safensis or B. safensis subsp. osmophilus and the third as B. paralicheniformis. Phylogenic tree analysis using the16S rRNA sequence revealed these colonies to be in a proximity of known gas-producing species. The NCBI database search revealed that a key gas production pathway gene, pyruvate formate-lyase (pfl), of which the gene product catalyzes pyruvate to formate conversion, exists in B. paralicheniformis. Formate dehydrogenase (FdhH) produces CO2 from formate that the coding gene fdhF positive bacteria can participate in gas production when formate is present in the culture. And we found fdhF from A. clausii, B. australimaris/B. safensis and B. paralicheniformis. Furthermore, under butanediol producing pathway, genes coding two enzymes involved in CO2 production, namely als and ald, existed in B. australimaris/B. safensis and B paralicheniformis, whereas A. clausii possessed als. Conclusion: Candidate species A. clausii, B. australimaris/B. safensis and B. paralicheniformis from spoiled salad dressing were thought to produce CO2 gas each from their own enzymes, or in combination, which caused the explosion upon opening. The endospore forming nature of Bacillus should alert us to be cautious when considering food producing process regulations where we need to thoroughly heat any product during manufacture in order to inactivate any bacteria as there is the possibility of this type of dangerous occurrence.
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Shiga toxin-producing Escherichia coli (STEC) causes proximal tubular defects in the kidney. However, factors altered by Shiga toxin (Stx) within the proximal tubules are yet to be shown. We determined Stx receptor Gb3 in murine and human kidneys and confirmed the receptor expression in the proximal tubules. Stx2-injected mouse kidney tissues and Stx2-treated human primary renal proximal tubular epithelial cell (RPTEC) were collected and microarray analysis was performed. We compared murine kidney and RPTEC arrays and selected common 58 genes that are differentially expressed vs. control (0 h, no toxin-treated). We found that the most highly expressed gene was GDF15, which may be involved in Stx2-induced weight loss. Genes associated with previously reported Stx2 activities such as src kinase Yes phosphorylation pathway activation, unfolded protein response (UPR) and ribotoxic stress response (RSR) showed differential expressions. Moreover, circadian clock genes were differentially expressed, suggesting Stx2-induced renal circadian rhythm disturbance. Circadian rhythm-regulated proximal tubular Na+-glucose transporter SGLT1 (SLC5A1) was down-regulated, indicating proximal tubular functional deterioration, and mice developed glucosuria confirming proximal tubular dysfunction. Stx2 alters gene expression in murine and human proximal tubules through known activities and newly investigated circadian rhythm disturbance, which may result in proximal tubular dysfunctions.
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Péptidos y Proteínas de Señalización del Ritmo Circadiano/metabolismo , Ritmo Circadiano , Regulación de la Expresión Génica/efectos de los fármacos , Túbulos Renales Proximales/efectos de los fármacos , Toxina Shiga II/toxicidad , Animales , Péptidos y Proteínas de Señalización del Ritmo Circadiano/genética , Células Epiteliales/efectos de los fármacos , Glucosuria/inducido químicamente , Humanos , Túbulos Renales Proximales/citología , Lipopolisacáridos/toxicidad , Ratones , Análisis por Matrices de ProteínasRESUMEN
Leptospirosis is a zoonosis caused by the pathogenic bacterium Leptospira. The Microscopic Agglutination Test (MAT) is widely used as the gold standard for diagnosis of leptospirosis. In this method, diluted patient serum is mixed with serotype-determined Leptospires, and the presence or absence of aggregation is determined under a dark-field microscope to calculate the antibody titer. Problems of the current MAT method are 1) a requirement of examining many specimens per sample, and 2) a need of distinguishing contaminants from true aggregates to accurately identify positivity. Therefore, increasing efficiency and accuracy are the key to refine MAT. It is possible to achieve efficiency and standardize accuracy at the same time by automating the decision-making process. In this study, we built an automatic identification algorithm of MAT using a machine learning method to determine agglutination within microscopic images. The machine learned the features from 316 positive and 230 negative MAT images created with sera of Leptospira-infected (positive) and non-infected (negative) hamsters, respectively. In addition to the acquired original images, wavelet-transformed images were also considered as features. We utilized a support vector machine (SVM) as a proposed decision method. We validated the trained SVMs with 210 positive and 154 negative images. When the features were obtained from original or wavelet-transformed images, all negative images were misjudged as positive, and the classification performance was very low with sensitivity of 1 and specificity of 0. In contrast, when the histograms of wavelet coefficients were used as features, the performance was greatly improved with sensitivity of 0.99 and specificity of 0.99. We confirmed that the current algorithm judges the positive or negative of agglutinations in MAT images and gives the further possibility of automatizing MAT procedure.
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Pruebas de Aglutinación/métodos , Interpretación de Imagen Asistida por Computador/métodos , Leptospirosis/diagnóstico por imagen , Algoritmos , Animales , Cricetinae , Sistemas de Apoyo a Decisiones Clínicas , Leptospirosis/inmunología , Masculino , Microscopía , Sensibilidad y Especificidad , Máquina de Vectores de Soporte , Análisis de OndículasRESUMEN
Nutrition and light-dark cycle influence rat testicular development. With 9% casein diet (low protein diet) under normal 12 h-12 h lighting cycles (9P), juvenile rat testes undergo normal growth. On the other hand, a low protein diet with constant darkness (D9P) results in a growth arrest of rat testes. Supplementation of cystine to the low protein diet under constant darkness (D9PC) had a tendency to increase testes weight, suggesting an improvement in growth suppression. Whether the growth suppression of testes in D9P is associated with suppression of spermatogenesis has not yet been shown. We aimed to determine the effect of a low protein diet and constant darkness with or without dietary cystine in testes using a histological technique. In the histological assessment, D9P testes showed a decreased number of seminiferous tubules with elongated spermatids, indicating a functional testicular defect in this group. However, cystine supplementation resulted in enhanced spermatogenesis versus control animals (D9PC vs. D9P) implying the importance of cystine to testicular development in this condition. Furthermore, serum testosterone concentration was increased in D9PC suggesting contribution of testosterone to ameliorate spermatogenesis. From these results, we conclude that cystine supplementation to a low protein diet under constant darkness promoted an increase in testosterone which in turn benefitted spermatogenesis.
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Cistina , Oscuridad/efectos adversos , Dieta con Restricción de Proteínas/efectos adversos , Espermatogénesis/efectos de los fármacos , Testosterona/metabolismo , Animales , Cistina/administración & dosificación , Cistina/farmacología , Proteínas en la Dieta/administración & dosificación , Proteínas en la Dieta/farmacología , Masculino , Tamaño de los Órganos/efectos de los fármacos , Ratas , Testículo/efectos de los fármacosRESUMEN
BACKGROUND: Choroid plexus (CP) is an important tissue not only to produce cerebrospinal fluid (CSF) but also to regulate substances that are secreted into or absorbed from CSF through blood-cerebrospinal fluid barrier (BCSFB) formed by CP epithelial cells (CPECs). CPECs display signs of deterioration in aged and diseased people. However, whether CPECs in hypercholesterolemic animals develop such damage is not known. METHODS: We used cholesterol-fed wild-type or Watanabe hereditary hyperlipidemic (WHHL) rabbits of identical age to determine CPEC changes in terms of morphology and protein expression/localization. RESULTS: Compared with non-cholesterol-fed control rabbits, prolonged exposure to cholesterol reduced CPEC height and increased lipofuscin levels in CPECs, indicating cellular damage. Expression of aquaporin 1 on the apical membranes of CPECs was diminished in cholesterol-exposed rabbits, implying a reduced CSF-producing function in the CP. The rabbit macrophage-specific antibody (RAM11) immunoreaction became positive in CPECs adjacent to foam cells, indicating an alteration in this cell type. CONCLUSION: Cholesterol insults from the circulation (which is reflected by foam-cell accumulation in the CP) induce CPEC dysfunction, and the latter seems to be enhanced by foam cells in hypercholesterolemic rabbits.
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Barrera Hematoencefálica/metabolismo , Plexo Coroideo/metabolismo , Células Epiteliales/citología , Hipercolesterolemia/metabolismo , Animales , Biomarcadores/metabolismo , Barrera Hematoencefálica/patología , Recuento de Células , Células Cultivadas , Hipercolesterolemia/patología , Masculino , Neuronas/metabolismo , ConejosRESUMEN
Aims: Inflammasome activation plays a pivotal role in many inflammatory diseases. Given that connexin (Cx) channels regulate numerous cellular events leading to inflammasome activation, we determined whether and how connexin affected inflammasome activation and inflammatory cell injury. Results: Exposure of mouse peritoneal macrophages (PMs) to lipopolysaccharide (LPS) plus ATP caused NLRP3 inflammasome activation, together with an increased connexin43 (Cx43). Inhibition of Cx43 blunted inflammasome activation. Consistently, PMs from the Cx43 heterozygous mouse (Cx43+/-) exhibited weak inflammasome activation, in comparison with those from the Cx43+/+ mouse. Further analysis revealed that inflammasome activation was preceded by an increased reactive oxygen species (ROS) production, nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase 2 (NOX2), protein carbonylation, and mitogen-activated protein kinase (MAPK) activation. Suppression of ROS with antioxidant, downregulation of NOX2 with small interfering RNA (siRNA), or inhibition of NADPH oxidase or MAPKs with inhibitors blocked Cx43 elevation and inflammasome activation. Intriguingly, suppression of Cx43 also blunted NOX2 expression, protein carbonylation, p38 phosphorylation, and inflammasome activation. In a model of acute renal injury induced by LPS, the Cx43+/- mouse exhibited a significantly lower level of blood interleukin-1ß (IL-1ß), blood urea nitrogen, and urinary protein, together with milder renal pathological changes and renal expression of NLRP3 and NOX4, as compared with the Cx43+/+ mouse. Moreover, inhibition of gap junctions suppressed IL-1ß- and tumor necrosis factor-α-induced expression of NOX4 in glomerular podocytes and tubular epithelial cells. Innovation and Conclusion: Our study indicates that Cx43 contributes to inflammasome activation and the progression of renal inflammatory cell injury through modulation of intracellular redox status. Cx43 could be a novel target for the treatment of certain inflammatory diseases.
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Lesión Renal Aguda/metabolismo , Conexina 43/metabolismo , Inflamasomas/metabolismo , Espacio Intracelular/metabolismo , Lipopolisacáridos , Estrés Oxidativo , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/patología , Animales , Aniones/análisis , Aniones/metabolismo , Línea Celular , Femenino , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/análisis , Especies Reactivas de Oxígeno/metabolismo , Superóxidos/análisis , Superóxidos/metabolismoRESUMEN
Shiga toxin-producing Escherichia coli (STEC) can cause central nervous system (CNS) damage resulting in paralysis, seizures, and coma. The key STEC virulence factors associated with systemic illness resulting in CNS impairment are Shiga toxins (Stx). While neurons express the Stx receptor globotriaosylceramide (Gb3) in vivo, direct toxicity to neurons by Stx has not been studied. We used murine neonatal neuron cultures to study the interaction of Shiga toxin type 2 (Stx2) with cell surface expressed Gb3. Single molecule imaging three dimensional STochastic Optical Reconstruction Microscopy-Total Internal Reflection Fluorescence (3D STORM-TIRF) allowed visualization and quantification of Stx2-Gb3 interactions. Furthermore, we demonstrate that Stx2 increases neuronal cytosolic Ca(2+), and NMDA-receptor inhibition blocks Stx2-induced Ca(2+) influx, suggesting that Stx2-mediates glutamate release. Phosphoinositide 3-kinase (PI3K)-specific inhibition by Wortmannin reduces Stx2-induced intracellular Ca(2+) indicating that the PI3K signaling pathway may be involved in Stx2-associated glutamate release, and that these pathways may contribute to CNS impairment associated with STEC infection.
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Shiga toxin-producing Escherichia coli (STEC) is a leading cause of childhood renal disease Hemolytic Uremic Syndrome (HUS). The involvement of renal cytokines and chemokines is suspected to play a critical role in disease progression. In current article, we tested the hypothesis that NKT cells are involved in Stx2-induced pathology in vivo. To address this hypothesis we compared Stx2 toxicity in WT and CD1 knockout (KO) mice. In CD1KO mice, which lack natural killer T (NKT) cells, Stx2-induced pathologies such as weight loss, renal failure, and death were delayed. In WT mice, Stx2-specific selective increase in urinary albumin occurs in later time points, and this was also delayed in NKT cell deficient mice. NKT cell-associated cytokines such as IL-2, IL-4, IFN-γ, and IL-17 were detected in kidney lysates of Stx2-injected WT mice with the peak around 36 h after Stx2 injection. In CD1KO, there was a delay in the kinetics, and increases in these cytokines were observed 60 h post Stx2 injection. These data suggest that NKT cells accelerate Stx2-induced pathology in mouse kidneys. To determine the mechanism by which NKT cells promote Stx2-associated disease, in vitro studies were performed using murine renal cells. We found that murine glomerular endothelial cells and podocytes express functional CD1d molecules and can present exogenous antigen to NKT cells. Moreover, we observed the direct interaction between Stx2 and the receptor Gb3 on the surface of mouse renal cells by 3D STORM-TIRF which provides single molecule imaging. Collectively, these data suggest that Stx2 binds to Gb3 on renal cells and leads to aberrant CD1d-mediated NKT cell activation. Therefore, strategies targeting NKT cells could have a significant impact on Stx2-associated renal pathology in STEC disease.
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Shiga toxin (Stx) is the primary cause of severe host responses including renal and central nervous system (CNS) disease in Shiga toxin-producing E. coli (STEC) infections. The interaction of Stx with different eukaryotic cell types is described. Host responses to Stx and bacterial lipopolysaccharide (LPS) are compared as related to the features of the STEC-associated Hemolytic Uremic Syndrome (HUS). Data derived from animal models of HUS and CNS disease, in vivo, and eukaryotic cells, in vitro, are evaluated in relation to HUS disease of humans.
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Enfermedades del Sistema Nervioso Central/microbiología , Infecciones por Escherichia coli/microbiología , Escherichia coli/metabolismo , Escherichia coli/patogenicidad , Síndrome Hemolítico-Urémico/microbiología , Toxinas Shiga/metabolismo , Factores de Virulencia/metabolismo , Animales , Enfermedades del Sistema Nervioso Central/patología , Técnicas Citológicas , Modelos Animales de Enfermedad , Infecciones por Escherichia coli/patología , Síndrome Hemolítico-Urémico/patología , Humanos , VirulenciaRESUMEN
Shiga toxin producing Escherichia coli (STEC) are a major cause of food-borne illness worldwide. However, a consensus regarding the role Shiga toxins play in the onset of diarrhea and hemorrhagic colitis (HC) is lacking. One of the obstacles to understanding the role of Shiga toxins to STEC-mediated intestinal pathology is a deficit in small animal models that perfectly mimic human disease. Infant rabbits have been previously used to study STEC and/or Shiga toxin-mediated intestinal inflammation and diarrhea. We demonstrate using infant rabbits that Shiga toxin-mediated intestinal damage requires A-subunit activity, and like the human colon, that of the infant rabbit expresses the Shiga toxin receptor Gb(3). We also demonstrate that Shiga toxin treatment of the infant rabbit results in apoptosis and activation of p38 within colonic tissues. Finally we demonstrate that the infant rabbit model may be used to test candidate therapeutics against Shiga toxin-mediated intestinal damage. While the p38 inhibitor SB203580 and the ZAK inhibitor DHP-2 were ineffective at preventing Shiga toxin-mediated damage to the colon, pretreatment of infant rabbits with the drug imatinib resulted in a decrease of Shiga toxin-mediated heterophil infiltration of the colon. Therefore, we propose that this model may be useful in elucidating mechanisms by which Shiga toxins could contribute to intestinal damage in the human.
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Benzamidas/metabolismo , Intestinos/efectos de los fármacos , Intestinos/patología , Piperazinas/metabolismo , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Quinasas/administración & dosificación , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirimidinas/metabolismo , Toxina Shiga II/toxicidad , Animales , Animales Recién Nacidos , Apoptosis , Mesilato de Imatinib , Quinasas Quinasa Quinasa PAM , Subunidades de Proteína/toxicidad , Conejos , Escherichia coli Shiga-Toxigénica/patogenicidad , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
We have shown previously that neurons in the mouse spinal cord express Gb(3). We show in this article that distribution of anti-Gb(3)-Ab reactivity occurs in many different types of neurons of different areas of the central nervous system (CNS). The immunoreactive neurons are in olfactory bulbs, cerebral cortex, hippocampus, striatum, amygdala, thalamus, hypothalamus, cerebellum, and medulla oblongata. In several different circumventricular organs where vessels do not have the blood-brain-barrier (BBB) structure, anti-Gb(3)-Ab is not positive for vessel structures, while neurons at these regions are positive. Also, within the ventricular area, ependymal cells in the third ventricle express Gb(3), as revealed by anti-Gb(3)-Ab staining and intensity analysis.
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In severe cases of the infectious disease by Shiga toxin-producing Escherichia coli (STEC), patients display renal dysfunction known as hemolytic uremic syndrome (HUS) and central nervous system (CNS) failure. Among those severe symptoms, patients with CNS dysfunction with HUS have a greater chance of getting severe sequelae and mortality than with HUS alone. Autopsy of the CNS shows mostly edema and hypoxic-ischemic changes, often with microhemorrhages. Magnetic resonance imaging (MRI) of brains of patients confirms hemorrhagic component involvement. This suggests the weakening of the blood-brain barrier (BBB) during the disease. Also, cerebrospinal fluid (CSF) analysis shows the weakening of the blood-CSF barrier. Although evidence of vascular involvement in CNS exists, the typical observation of microthrombosis in renal pathology is often absent in CNS. Importantly, there are people who develop CNS symptoms before the onset of HUS. This suggests direct involvement of Shiga toxin (Stx) in CNS disease which is in addition to renal involvement. The advantages of animal models are that Stx receptor expression in normal CNS tissue can be determined, and changes in histopathology, hematology, and serum and CSF contents can be analyzed at several different time points, which allow investigation of the nature of the disease. Importantly, in animal models with either STEC oral inoculation or purified Stx injection, paralysis of extremities is commonly observed. This shows the central role of Stx in CNS dysfunction in this disease. It is anticipated that precise mechanisms of Stx influence in the CNS will be delineated, and this information will lead to effective therapeutics in the near future.
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Infecciones Bacterianas del Sistema Nervioso Central , Sistema Nervioso Central/patología , Infecciones por Escherichia coli , Toxina Shiga/metabolismo , Escherichia coli Shiga-Toxigénica/patogenicidad , Animales , Infecciones Bacterianas del Sistema Nervioso Central/microbiología , Infecciones Bacterianas del Sistema Nervioso Central/patología , Infecciones Bacterianas del Sistema Nervioso Central/fisiopatología , Modelos Animales de Enfermedad , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/patología , Infecciones por Escherichia coli/fisiopatología , Humanos , Ratones , Conejos , RatasRESUMEN
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 (Gb(3)) 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 Gb(3) 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.
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Apoptosis/fisiología , Epitelio/patología , Túbulos Renales Colectores/patología , Toxina Shiga II/metabolismo , Animales , Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Western Blotting , Células Cultivadas , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Epitelio/inmunología , Epitelio/metabolismo , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/patología , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Túbulos Renales Colectores/inmunología , Túbulos Renales Colectores/metabolismo , Lipopolisacáridos/inmunología , Ratones , Toxina Shiga II/inmunología , Venas UmbilicalesRESUMEN
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.
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Sistema Nervioso Central/metabolismo , Neuronas/metabolismo , Toxina Shiga II/toxicidad , Trihexosilceramidas/metabolismo , Animales , Transporte Biológico , Calcio/metabolismo , Sistema Nervioso Central/efectos de los fármacos , Femenino , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Humanos , Masculino , Ratones , Neuronas Motoras/ultraestructura , Neuroglía/citología , Organismos Libres de Patógenos Específicos , Pérdida de PesoRESUMEN
Shiga toxin 1 (Stx1) and Stx2 produced by Escherichia coli O157 are known to be cytotoxic to Vero and HeLa cells by inhibiting protein synthesis and by inducing apoptosis. In the present study, we have demonstrated that 10 ng/ml Stx2 induced DNA fragmentation in human brain microvascular endothelial cells (HBMEC), with cleavage activation of caspase-3, -6, -8, and -9. A microarray approach used to search for apoptotic potential signals in response to Stx2 revealed that Stx2 treatment induced a marked upregulation of C/EBP homologous protein (CHOP)/growth arrest and DNA damage-inducible protein 153 (GADD153). Increased CHOP expression was dependent on enzymatically active Stx1. Knockdown of CHOP mRNA reduced the activation of caspase-3 and prevented apoptotic cell death. These results suggest that Stx2-induced apoptosis is mediated by CHOP in HBMEC and involves activation of both the intrinsic and extrinsic pathways of apoptosis.
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Apoptosis , Células Endoteliales/efectos de los fármacos , Células Endoteliales/microbiología , Toxina Shiga II/toxicidad , Factor de Transcripción CHOP/biosíntesis , Caspasas/metabolismo , Supervivencia Celular , Células Cultivadas , Cromatina/ultraestructura , Fragmentación del ADN , Perfilación de la Expresión Génica , Silenciador del Gen , Humanos , Microscopía Electrónica de Transmisión , Análisis de Secuencia por Matrices de Oligonucleótidos , Sintaxina 1/genética , Sintaxina 1/metabolismoRESUMEN
Shiga toxin-producing Escherichia coli causes hemolytic uremic syndrome, a constellation of disorders that includes kidney failure and central nervous system dysfunction. Shiga toxin binds the amphipathic, membrane-bound glycolipid globotriaosylceramide (Gb(3)) and uses it to enter host cells and ultimately cause cell death. Thus, cell types that express Gb(3) in target tissues should be recognized. The objective of this study was to determine whether immunohistologic detection of Gb(3) was affected by the method of tissue preparation. Tissue preparation included variations in fixation (immersion or perfusion) and processing (paraffin or frozen) steps; paraffin processing employed different dehydration solvents (acetone or ethanol). Perfusion-fixation in combination with frozen sections or acetone-dehydrated tissue for paraffin sections resulted in specific recognition of Gb(3) using immunohistochemical or immunofluorescent methods. In the mouse tissues studied, Gb(3) was associated with tubules in the kidney and neurons in the nervous system. On the other hand, Gb(3) localization to endothelial cells was determined to be an artifact generated due to immersion-fixation or tissue dehydration with ethanol. This finding was corroborated by glycolipid profiles from tissue subjected to dehydration; namely Gb(3) was subject to extraction by ethanol more than acetone during tissue dehydration. The results of this study show that tissue preparation is crucial to the persistence and preservation of the glycolipid Gb(3) in mouse tissue. These methods may serve as a basis for determining the localization of other amphipathic glycolipids in tissue.
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Técnica del Anticuerpo Fluorescente Directa/métodos , Secciones por Congelación/métodos , Túbulos Renales/química , Neuronas/química , Adhesión en Parafina/métodos , Trihexosilceramidas/análisis , Animales , Anticuerpos Monoclonales/inmunología , Síndrome Hemolítico-Urémico/metabolismo , Síndrome Hemolítico-Urémico/microbiología , Túbulos Renales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Toxinas Shiga/metabolismo , Escherichia coli Shiga-Toxigénica/metabolismo , Trihexosilceramidas/inmunología , Trihexosilceramidas/metabolismoRESUMEN
To evaluate antibody responses against lipopolysaccharide (LPS: O157, O26, and O111) in enterohemorrhagic Escherichia coli(EHEC) infection, sera of 24 schoolchildren associated with the Morioka outbreak in 1997 and of 74 sporadic patients suspected of having EHEC infection were examined. Using a positive standard serum, quantitative evaluation of LPS antibodies by an enzyme-linked immunosorbent assay (ELISA) was established. High levels of specific IgM and IgA antibodies against homologous E. coli LPS were present in the acute period and are characteristic of EHEC. This could be used for the serological diagnosis of EHEC infection, except for early infants and the elderly. In addition to the specific homologous response, multiple antibody responses against different serotypes other than those isolated were demonstrated in many cases by qualitative analysis using Western blotting.