Patogenia de mutantes de Salmonella Typhimurium en dos modelos experimentales in vivo / Pathogenesis of Salmonella Typhimurium mutants in two experimental models in vivo

Con la finalidad de evaluar la patogenia en cepas de Salmonella Typhimurium con mutaciones en los genes invG/invE de la Isla de Patogenicidad de Salmonella 1 (SPI-1) y de los genes ssaJ/ssaK en la SPI-2, se evaluaron los modelos asa intestinal ligada de ratón asociado a la observación de los tejidos por microscopía electrónica de transmisión (MET) y la producción de salmonelosis sistémica en ratón. Para ello, se utilizaron seis cepas de Salmonella: S. Typhimurium SL-1344 (cepa control) y sus derivadas mutantes: ∆invEG S. Typhimurium SL-1344 (mutante en SPI-1) y ∆ssaJK S. Typhimurium SL-1344 (mutante en SPI-2), S. Typhimurium (cepa clínica) y sus derivadas mutantes: ∆invEG S. Typhimurium y ∆ssaJK S. Typhimurium. Los resultados de MET permitieron verificar las alteraciones morfológicas del epitelio intestinal en el ratón infectado con cepas de Salmonella cuyos genes de patogenicidad estaban intactos. Fue comprobada la pérdida del poder invasivo solo en las cepas mutadas en la SPI-1. A través del modelo de salmonelosis sistémica en ratón se pudo comprobar la pérdida de la capacidad de diseminación en ambas mutantes. En conclusión los modelos permitieron verificar la importancia que tienen los genes invG/invE de la SPI-1 y ssaJ/ssaK de la SPI-2 en la patogenia de la salmonelosis, utilizando como modelo experimental de infección ratones BALB/c. Se sugieren estos modelos in vivo para evaluar mutantes de genes implicados en la patogenia de Salmonella, ya que representan una herramienta importante para la comprensión de la interacción Salmonella-hospedero(AU)

With the aim of evaluate the pathogenesis in Salmonella Typhimurium strains with mutations in genes invG/invE of Salmonella Pathogenicity Island 1 (SPI-1) and genes ssaJ/ssaK in the SPI-2 models were evaluated ligated intestinal loop associated mouse tissues by observation by transmission electron microscopy (TEM) and the production of mouse systemic salmonellosis. For this, we used six Salmonella strains: S. Typhimurium SL-1344 (control strain) and its derived mutants: ΔinvEG S. Typhimurium SL-1344 (mutant in SPI-1) and ΔssaJK S. Typhimurium SL-1344 (mutant in SPI-2), S. Typhimurium (clinical isolate) and its derived mutants: ΔinvEG S.Typhimurium and ΔssaJK S. Typhimurium. TEM results allowed us to verify the morphological alterations of the intestinal epithelium in mice infected with Salmonella strains whose pathogenicity genes were intact. It was proven invasive power loss only in strains mutated in the SPI-1. Through systemic salmonellosis model mouse we noted the loss of the ability to spread in both mutants. In conclusion, the models allowed us to verify the importance of the invG/invE genes of SPI-1 and ssaJ/ssaK of SPI-2 in the pathogenesis of salmonellosis, using BALB/c mice as an experimental model of infection. These in vivo models are suggested to evaluate mutants of genes involved in the pathogenesis of Salmonella, since they represent an important tool for the understanding of the Salmonella-host interaction(AU)

Developmental exposure to mancozeb induced neurochemical and morphological alterations in adult male mouse hypothalamus.

Mancozeb, a dithiocarbamate widely used in agriculture, is considered a developmental hazard in humans; however, more evidences are still needed concerning the consequences of chronic exposure to this pesticide. Mancozeb neurotoxicity in developing mouse hypothalamus was evaluated by subchronic exposure of male Mus musculus mice to low and high doses of mancozeb (30 and 90 mg/kg body weight, respectively) from late neonatal until adolescence. Variations in hypothalamic amino acid neurotransmitter levels and changes in histological as well as cytological characteristics were analyzed in young adult experimental mice and compared with control. A dose-dependent increase in excitation/ inhibition ratio was observed in mancozeb-exposed hypothalamus, indicating an overall state of excitoxicity. Histopathological and ultrastructural studies showed increased apoptosis, neuroinflammation and demyelination, demonstrating mancozeb-induced cytotoxicity in hypothalamic neurosecretory cells. In summary, both neurochemical and morphological data revealed mancozeb-induced alterations during development of hypothalamic circuitry that are critical for maturation of the neuroendocrine system.

α2- and ß-adrenoceptors involvement in nortriptyline modulation of auditory sustained attention and impulsivity.

RATIONALE: The catecholamine innervation of the prefrontal cortex controls attentional focus and inhibits inappropriate behavioral responses. The mechanism of action with which norepinephrine (NE) reuptake inhibitors modulate these cognitive functions has not been fully investigated. OBJECTIVE: We investigated the effect of systemic administration of the NE reuptake blocker nortriptyline (NT) on attention and impulsivity using an auditory sustained attention task. The task was designed to assess impulsive behavior and the maintenance of attentional focus to an auditory stimulus presented at interresponse time durations (IRT) between 5 and 80 s. RESULTS: NT (2.0 but not 3.0 mg/kg) improved sustained attention and decreased the percentage of premature responses without changing their latency. To better understand the adrenergic component of NT action, we tested the effect of noradrenergic receptor antagonists alone or together with NT. The α(2)-receptor antagonist yohimbine, the α(1)-receptor antagonist prazosin, or the ß-receptor antagonist propranolol alone did not significantly affect attentive performance or premature responses. However, the beneficial effects of NT on sustained attention and premature responses were attenuated by pretreatment with either yohimbine or propranolol. On the contrary, prazosin did not affect the NT-mediated improvement in sustained attention. CONCLUSIONS: We conclude that sustained attention displays an inverse U-shaped dependence on NT, mediated-at least in part-by α(2)- and ß-adrenoceptors. We speculate that low doses of NT improve performance by maximizing the phasic release of NE, while higher doses of NT would elevate tonic levels of NE, thus producing suboptimal levels of phasically released NE.

Investigación de blastocystis sp. en agua de consumo humano en una población escolar de la zona rural del estado Mérida, Venezuela / Blastocystis sp. in drinking water at a rural school in Mérida, Venezuela

Para detectar la presencia de Blastocystis sp. en el agua de consumo de una escuela rural del estado Mérida, se recolectaron 36 muestras provenientes de la fuente natural que surte el tanque de distribución, del tanque y de los grifos, durante los meses de Septiembre 2008, Febrero y Diciembre del 2009 y Mayo 2010. El procesamiento de las muestras se realizó siguiendo la técnica propuesta por Suresh y col. (2005). Los resultados sugieren que el agua de consumo estaba exenta de Blastocystis o que la cantidad de quistes viables presentes en las muestras fue muy baja, lo que no permitió lograr la recuperación del parásito en el cultivo. Por ello se requiere el uso de técnicas más sensibles que permitan detectar cantidades bajas del microorganismo en aguas de consumo humano. El estudio de los hábitos de consumo de agua de la población estudiantil de la escuela rural, mostró que más de la mitad de la población consume agua hervida, esto probablemente se deba a que están conscientes que el agua a la cual tienen acceso proviene de una fuente natural no tratada

In order to determine the presence of Blastocystis sp. in drinking water at a rural school in Merida, 36 water samples were collected from natural sources that feed the distribution tank and faucets during the months of September, 2008, February and December, 2009, and May, 2010. Samples were processed using the technique proposed by Suresh et al. (2005). All samples were negative. These results suggest that the water was exempt from Blastocystis or that the quantity of viable cysts present in the samples was very low, not permitting reproduction in the culture. This report suggests the importance of using more sensitive techniques to detect low amounts of the organismin drinking water. The study of student water consumption habits at the rural school showed that more than half the population consume boiled water; probably, they are aware that the water source has not been treated for drinking

Experimental toxicity of Aeromonas spp. In mouse’s small intestine: Ultrastructural aspects / Aspectos ultraestructurales de la toxicidad experimental producida por Aeromonas spp. En el intestino delgado de ratón / Aspectos ultra-estruturais da toxicidade experimental produzida por Aeromonas spp. No intestino delgado de rato

Ultrastructural aspects of mouse small intestinal tissue cultures infected with Aeromonas spp. strains are described. High resolution light and transmission electron microscopy were used to assess the bacterial pathogenic mechanism, the ultrastructural changes that take place during the colonization of the intestinal tract and the interaction of Aeromonas spp. with the host epithelium. After 24h of culture, chains of vesicles were seen on the outer surface of the Aeromonas’ membrane. The vesicles were also found on the enterocyte surface. After 48h of culture, lysis of the epithelial intestinal cells, mononuclear phagocytic cells, phagocytic eosinophils and phagocyted Aeromonas were observed.

Se describen aspectos ultraestructurales del tejido intestinal de ratón cultivado e infectado con Aeromonas spp. Se utilizó microscopía de luz de alta resolución y microscopía electrónica de transmisión para evaluar los mecanismos patogénicos bacterianos, los cambios ultraestructurales que ocurren durante la colonización del tracto intestinal por Aeromonas spp. y la interacción de éstas con el epitelio huésped. En los cultivos de 24h se observaron vesículas distribuidas en cadenas sobre la superficie de la membrana externa de las Aeromonas. Estas vesículas se observaron unidas a la superficie del enterocito. En los cultivos de 48h se observó lisis de la superficie epitelial del intestino, migración de células fagocíticas mononucleares y presencia en la cavidad intestinal de eosinófilos fagocíticos, algunos conteniendo Aeromonas en su interior.

Descrevem-se aspectos ultra-estruturais do tecido intestinal de rato cultivado e infectado com Aeromonas spp. Utilizaram-se microscopia de luz de alta resolução e microscopia eletrônica de transmissão para avaliar os mecanismos patogênicos bacterianos, As mudanças ultra-estruturais que ocorrem durante a colonização do tracto intestinal por Aeromonas spp. e a interação destas com o epitélio hospede. Nos cultivos de 24h se observaram vesículas distribuídas em cadeias sobre a superfície da membrana externa das Aeromonas. Estas vesículas se observaram unidas à superfície do enterócito. Nos cultivos de 48h se observou lise da superfície epitelial do intestino, migração de células fagocíticas mononucleares e presença na cavidade intestinal de eosinófilos fagocíticos, alguns contendo Aeromonas no seu interior.

Effects of aeromonas caviae co-cultured in mouse small intestine

Se describen los hallazgos ultraestructurales producidos por la interacción de dos cepas de Aeromonas caviae con la mucosa intestinal de ratón. Una de las cepas fue aislada de un paciente asintomático y la otra de un paciente con diarrea. Ambas fueron inoculadas por separado en pequeños cilindros cerrados de intestino delgado de ratón, los cuales fueron incubados en medio de cultivo de Eagle. Los cilindros intestinales fueron divididos en dos grupos, uno fue cultivado por 24h y el otro por 48h. Muestras de estos cultivos fueron procesadas para microscopía de luz de alta resolución y microscopía electrónica de transmisión. La cepa aislada del paciente asintomático mostró alteraciones menores en la mucosa intestinal, de acuerdo a los diferentes períodos de incubación; mientras que la muestra obtenida del paciente con diarrea produjo siempre severos efectos enteropatológicos

Histotypic mouse parietal cortex cultures: excitation/inhibition ratio and ultrastructural analysis.

Primary cultures of mouse parietal cortex, prepared between postnatal day 3 (P3) and P9, were studied using transmission electron microscopy and HPLC of excitatory (aspartate and glutamate) and inhibitory neurotransmitters (glycine, GABA and taurine) to determine their morphological and functional development. Relations between excitation and inhibition (E/I) were contrasted with ultrastructural features over the time course of in vitro development. After 6 days in vitro, cultured parietal cortex neurons prepared from mice at P3 had immature morphological characteristics, whereas P5 cultures showed a more developed histological structure but still with scarce synapses. The acquirement of histotypic characteristics was seen in P7 cultures, which contained numerous symmetric and asymmetric synaptic contacts. On P9, the cultures showed signs of tissue damage. In terms of neurotransmitter levels and E/I ratios, P7 cultures had relatively low E/I ratio as compared with the rest of the cultures prepared before or after P7. These results demonstrated that the development of inhibitory synaptic transmission, as indicated in the fall of E/I ratio, marked the maturation of cerebral cortical tissue and that the critical period to obtain histotypic cultures of mouse parietal cerebral cortex coincides between P5 and P7. This work provides useful information regarding the balance between excitation and inhibition as an indicative parameter for in vitro nerve cell survival, differentiation and maturation and reinforces the great value of histotypic cultures in the study of central nervous system development.

Effects of prenatal paraquat and mancozeb exposure on amino acid synaptic transmission in developing mouse cerebellar cortex.

The goal of this study was to analyze the effects of prenatal exposure to the pesticides paraquat (PQ) and mancozeb (MZ) on the development of synaptic transmission in mouse cerebellar cortex. Pregnant NMRI mice were treated with either saline, 10 mg/kg PQ, 30 mg/kg MZ or the combination of PQ + MZ, between gestational days 12 (E12) and E20. Variation in the levels of amino acid neurotransmitters was determined by HPLC, between postnatal day 1 (P1) and P30. Motor coordination was assessed by locomotor activity evaluation of control and experimental pups at P14, P21 and P30. Significant reductions in the levels of excitatory neurotransmitters, aspartate and glutamate, were observed in PQ-, MZ- or combined PQ + MZ-exposed pups, with respect to control, during peak periods of excitatory innervation of Purkinje cells: between P2-P5 and P11-P15. However, at P30, lower aspartate contents, in contrast with increased glutamate levels, were detected in all experimental groups. During the first two postnatal weeks, delays in GABA and glycine ontogenesis were observed in PQ- and PQ + MZ-exposed pups, whereas notable decrements in GABA and glycine levels were seen in PQ + MZ-exposed animals. Decreased taurine contents were detected at P3 and P11 in PQ- and PQ + MZ-exposed mice. Pups in different experimental groups all showed hyperactivity at P14 and then exhibited reduced locomotor activity at P30. Taken together, our results indicate that prenatal exposure to either PQ or MZ or the combination of both could alter the chronology and magnitude of synaptic transmission in developing mouse cerebellar cortex.

Prenatal and postnatal contents of amino acid neurotransmitters in mouse parietal cortex.

This study documents the variation in the amino acid neurotransmitter contents during mouse parietal cortex development, from embryonic day 13 (E13) until young adulthood, between postnatal day 21 (P21) and P30. Taurine, an inhibitory neurotransmitter and neuromodulator, is the most abundant neurotransmitter in the developing neocortex, whereas, at the adult stage, glutamate is the more prominent neurotransmitter playing an excitatory role, and GABA is the major inhibitory neurotransmitter. During the proliferative stage of neurogenesis in the mouse cerebral cortex, between E13 and E17, relatively high levels of glutamate, aspartate, taurine and glycine were detected, consistent with a possible trophic influence of these neurotransmitters during cortical development prior to synaptogenesis. Between E17 and E19, a significant decline in the contents of these neurotransmitters was observed, consistent with earlier reports of cell death in the ventricular and subventricular zones during this stage of development. During the perinatal period, a progressive increment in glutamate level was seen between E21 and P5, and then the values remained constant until the second postnatal week. Glutamate also decreased by about 25% between P11 and P15, on the other hand, aspartate diminished by about 20% between P7 and P9. These results were consistent with previous reports of histogenetic cell death during the first 2 postnatal weeks in mouse neocortex. GABA increased from the embryonic period until young adulthood, in contrast, the glycine content decreased; thus, in the adult parietal cortex, the GABA content was about 2.6-fold higher than that of glycine. During the first postnatal week, the concentrations of glutamate and GABA showed significant increments between P0 and P5, while those of aspartate and glycine remained constant. During this period, amino acids are predominantly excitatory and the cerebral cortex is vulnerable to epileptiform activity; the significant increment in taurine content between P0 and P3 suggests a neuroprotective action of taurine against excitotoxicity. At P15, coinciding with the period of maximum cortical synaptogenesis, significant increments in GABA and glycine contents were observed which could be related to the maturation of inhibitory synaptic transmission. At the young adult stage, there was a rise in the levels of both excitatory neurotransmitters, glutamate and aspartate, and a significant reduction in the contents of all three inhibitory neurotransmitters, GABA, glycine and taurine.

Levels of amino acid neurotransmitters during neurogenesis and in histotypic cultures of mouse spinal cord.

The development of spinal cord interneurons and the formation of interneuronal synaptic connections has received little attention; the most comprehensively studied developing circuit has been the connection between motoneurons and the muscle they innervate. All motoneurons are cholinergic whereas spinal interneurons are mostly glutamatergic, glycinergic or GABAergic neurons. In this study, we show quantitative data, obtained by high-pressure liquid chromatography (HPLC), on the levels of amino acid neurotransmitters during mouse spinal cord neurogenesis, from embryonic day (E) 12 until postnatal day (P) 30. At E12, high levels of glutamate, glycine and taurine were already detected but between E16 and P3, significant increments in their contents were observed, indicating the occurrence of maximum synaptogenesis during this period. Important reductions in their contents were also observed in two stages: between E12-E16 and P3-P7. These results suggest that the apoptotic death of interneurons and motoneurons in the developing brain or the synapse refinement of neural circuitry during maturation reduced the number of synapses, thereby decreasing the levels of neurotransmitters. The contents of these neurotransmitters were also analyzed in primary cultures of mouse spinal cord prepared from embryos between E13 and E19. As deduced from light microscopy, ultrastructural studies, as well as results from HPLC analysis, the cultures derived from E15-E16 embryos showed the highest degree of histotypic features and neurotransmitter contents comparable with those obtained in situ. Although glycine, GABA and taurine levels reached about 80-90% of normal in situ values, the contents of aspartate and glutamate were lower by about 40%, which could be mainly due to deafferentation of both sensory and supraspinal afferent axon terminals. These results indicate that intrinsic synaptic circuits can be maintained in histotypic spinal cord cultures prepared from E15-E16 mouse embryos. Histotypic cultures of the spinal cord will serve as a good model for studies on the pathophysiology of amino-acid-based neurotransmission and repair strategies in many CNS disorders.