Bacteriemias por Pseudomonas Aeruginosa : análisis comparativo

Objetivos: Analizar características clínicas y morbimortalidad de las bacteriemias por Pseudomonas aeruginosa comparadas con Klebsiella spp en pacientes hospitalizados en un Servicio de Clínica Médica de adultos. Material y métodos: Estudio protocolizado, descriptivo, observacional de 15 años de duración. Criterio de inclusión: 2 o más hemocultivos positivos para el germen. Los datos fueron procesados en EPI Info 6.04. El criterio de significación se estableció para un error alfa menor del 5%. Resultados: Se detectaron en el período de estudio 282 bacteriemias por bacilos gram negativos de las cuales 19 fueron por Pseudomonas aeruginosa (6.7%) y 76 por Klebsiella (26.9%). No se encontraron diferencias significativas entre ambas en cuanto a edad media [53.9 años (DS±17.9 ) vs 58.7 años (DS±15.2)], sexo (femenino: 26.3 vs 38.2%) ni complicaciones (77.8 vs 77.3%). La presencia de neutropenia (52.6 vs 9.2%)(p<0.0001), comorbilidad mayor (94.7 vs 68.4%)(p<0.05), neoplasias (47.4 vs 22.4%)(p<0.05), uso de corticoides (21.1 vs 3.9%)(p<0.05), e inmunosupresores (31.6 vs 7.9%)(p<0.01), trombocitopenia (77.7 vs 49.3%) (p<0.05) y leucopenia (52.6 vs 21.3%)(p<0.01) fueron más frecuentes en las bacteriemias por P. aeruginosa. Resultó más frecuente la hipoalbuminemia (88.5 vs 37.5%)(p<0.001) en las bacteriemias por Klebsiella spp. No se encontraron diferencias significativas en cuanto a puerta de entrada conocida (78.9 vs 77.6%), anemia (84.2 vs 71.2%), complicaciones infecciosas (84.2 vs 73.7%), descompensación de comórbidas (55.6 vs 51.3%) y encefalopatía (36.8 vs 57.9%)(pNS). La mortalidad precoz (dentro de los 7 días) fue significativamente mayor en el grupo de las bacteriemias por P.aeruginosa (57.1 vs 12%) (p<0.01), sin diferencias en la mortalidad global (36.8 vs 32.9%) (pNS). Conclusiones: Las bacteriemias por P.aeruginosa comparadas con las producidas por Klebsiella spp. se asociaron significativamente a mayor frecuencia de neoplasias, leucopenia, trombocitopenia y neutropenia, comorbilidad mayor, uso de corticoides e inmunosupresores, y a mortalidad precoz.

Enfermedad de Creutzfeld Jakob, desafío diagnóstico : reporte de dos casos en la ciudad de Mendoza

La enfermedad de Creutzfeldt-Jakob (ECJ) es una afección neurodege-nerativa rápidamente progresiva y mortal producida por priones. Es la más común de las encefalopatías espongiformes. La hipótesis prevalente sugiere que se inicia y propaga por conversión de una proteína priónica normal (PrP) en una isoforma conformacional anormal (PrPreS), que se acumula en el cerebro causando destrucción neuronal. Se reporta el caso de dos pacientes asistido en el Servicio de Neurología del Hospital Lagomaggiore de la ciudad de Mendoza en el período de 2 años: mujer de 49 años con cuadro de deterioro cognitivo rápidamente progresivo asociado a trastorno de la marcha y ataxia de 2 meses de evolución; y varón de 54 años con conductas inapropiadas y desorientación temporo-espacial de 20 días de evolución. Ambos con EEG compatible y proteína 14.3.3 positiva, falleciendo semanas después del diagnóstico. Se discute ambos casos, los cuales representaron un desafío diagnóstico dada la baja frecuencia y escaso reconocimiento de esta entidad en nuestro medio, debiendo debe ser considerado en pacientes que evolucionan a una demencia rápidamente progresiva.

Inhibition of protease-resistant prion protein formation by porphyrins and phthalocyanines

A central aspect of pathogenesis in the transmissible spongiform encephalopathies or prion diseases is the conversion of normal protease-sensitive prion protein (PrP-sen) to the abnormal protease-resistant form, PrP-res. Here we identify porphyrins and phthalocyanines as inhibitors of PrP-res accumulation. The most potent of these tetrapyrroles had IC50 values of 0.5–1 μM in scrapie-infected mouse neuroblastoma (ScNB) cell cultures. Inhibition was observed without effects on protein biosynthesis in general or PrP-sen biosynthesis in particular. Tetrapyrroles also inhibited PrP-res formation in a cell-free reaction composed predominantly of hamster PrP-res and PrP-sen. Inhibitors were found among phthalocyanines, deuteroporphyrins IX, and meso-substituted porphines; examples included compounds containing anionic, neutral protic, and cationic peripheral substituents and various metals. We conclude that certain tetrapyrroles specifically inhibit the conversion of PrP-sen to PrP-res without apparent cytotoxic effects. The inhibition observed in the cell-free conversion reaction suggests that the mechanism involved direct interactions of the tetrapyrrole with PrP-res and/or PrP-sen. These findings introduce a new class of inhibitors of PrP-res formation that represents a potential source of therapeutic agents for transmissible spongiform encephalopathies.

Normal host prion protein (PrPC) is required for scrapie spread within the central nervous system

Mice devoid of PrPC (Prnpo/o) are resistant to scrapie and do not allow propagation of the infectious agent (prion). PrPC-expressing neuroectodermal tissue grafted into Prnpo/o brains but not the surrounding tissue consistently exhibits scrapie-specific pathology and allows prion replication after inoculation. Scrapie prions administered intraocularly into wild-type mice spread efficiently to the central nervous system within 16 weeks. To determine whether PrPC is required for scrapie spread, we inoculated prions intraocularly into Prnpo/o mice containing a PrP-overexpressing neurograft. Neither encephalopathy nor protease-resistant PrP (PrPSc) were detected in the grafts for up to 66 weeks. Because grafted PrP-expressing cells elicited an immune response that might have interfered with prion spread, we generated Prnpo/o mice immunotolerant to PrP and engrafted them with PrP-producing neuroectodermal tissue. Again, intraocular inoculation did not lead to disease in the PrP-producing graft. These results demonstrate that PrP is necessary for prion spread along neural pathways.

Epithelial and endothelial expression of the green fluorescent protein reporter gene under the control of bovine prion protein (PrP) gene regulatory sequences in transgenic mice

The expression of the cellular form of the prion protein (PrPc) gene is required for prion replication and neuroinvasion in transmissible spongiform encephalopathies. The identification of the cell types expressing PrPc is necessary to understanding how the agent replicates and spreads from peripheral sites to the central nervous system. To determine the nature of the cell types expressing PrPc, a green fluorescent protein reporter gene was expressed in transgenic mice under the control of 6.9 kb of the bovine PrP gene regulatory sequences. It was shown that the bovine PrP gene is expressed as two populations of mRNA differing by alternative splicing of one 115-bp 5′ untranslated exon in 17 different bovine tissues. The analysis of transgenic mice showed reporter gene expression in some cells that have been identified as expressing PrP, such as cerebellar Purkinje cells, lymphocytes, and keratinocytes. In addition, expression of green fluorescent protein was observed in the plexus of the enteric nervous system and in a restricted subset of cells not yet clearly identified as expressing PrP: the epithelial cells of the thymic medullary and the endothelial cells of both the mucosal capillaries of the intestine and the renal capillaries. These data provide valuable information on the distribution of PrPc at the cellular level and argue for roles of the epithelial and endothelial cells in the spread of infection from the periphery to the brain. Moreover, the transgenic mice described in this paper provide a model that will allow for the study of the transcriptional activity of the PrP gene promoter in response to scrapie infection.

A scrapie-like unfolding intermediate of the prion protein domain PrP(121–231) induced by acidic pH

The infectious agent of transmissible spongiform encephalopathies is believed to consist of an oligomeric isoform, PrPSc, of the monomeric cellular prion protein, PrPC. The conversion of PrPC to PrPSc is characterized by a decrease in α-helical structure, an increase in β-sheet content, and the formation of PrPSc amyloid. Whereas the N-terminal part of PrPC comprising residues 23–120 is flexibly disordered, its C-terminal part, PrP(121–231), forms a globular domain with three α-helices and a small β-sheet. Because the segment of residues 90–231 is protease-resistant in PrPSc, it is most likely structured in the PrPSc form. The conformational change of the segment containing residues 90–120 thus constitutes the minimal structural difference between PrPC and a PrPSc monomer. To test whether PrP(121–231) is also capable to undergo conformational transitions, we analyzed its urea-dependent unfolding transitions at neutral and acidic pH. We identified an equilibrium unfolding intermediate of PrP(121–231) that is exclusively populated at acidic pH and shows spectral characteristics of a β-sheet protein. The intermediate is in rapid equilibrium with native PrP(121–231), significantly populated in the absence of urea at pH 4.0, and may have important implications for the presumed formation of PrPSc during endocytosis.

Chaperone-supervised conversion of prion protein to its protease-resistant form

Transmissible spongiform encephalopathies (TSEs) are lethal, infectious disorders of the mammalian nervous system. A TSE hallmark is the conversion of the cellular protein PrPC to disease-associated PrPSc (named for scrapie, the first known TSE). PrPC is protease-sensitive, monomeric, detergent soluble, and primarily α-helical; PrPSc is protease-resistant, polymerized, detergent insoluble, and rich in β-sheet. The “protein-only” hypothesis posits that PrPSc is the infectious TSE agent that directly converts host-encoded PrPC to fresh PrPSc, harming neurons and creating new agents of infection. To gain insight on the conformational transitions of PrP, we tested the ability of several protein chaperones, which supervise the conformational transitions of proteins in diverse ways, to affect conversion of PrPC to its protease-resistant state. None affected conversion in the absence of pre-existing PrPSc. In its presence, only two, GroEL and Hsp104 (heat shock protein 104), significantly affected conversion. Both promoted it, but the reaction characteristics of conversions with the two chaperones were distinct. In contrast, chemical chaperones inhibited conversion. Our findings provide new mechanistic insights into nature of PrP conversions, and provide a new set of tools for studying the process underlying TSE pathogenesis.

kappa opioid receptors in human microglia downregulate human immunodeficiency virus 1 expression.

Microglial cells, the resident macrophages of the brain, play an important role in the neuropathogenesis of human immunodeficiency virus type 1 (HIV-1), and recent studies suggest that opioid peptides regulate the function of macrophages from somatic tissues. We report herein the presence of kappa opioid receptors (KORs) in human fetal microglia and inhibition of HIV-1 expression in acutely infected microglial cell cultures treated with KOR ligands. Using reverse transcriptase-polymerase chain reaction and sequencing analyses, we found that mRNA for the KOR was constitutively expressed in microglia and determined that the nucleotide sequence of the open reading frame was identical to that of the human brain KOR gene. The expression of KOR in microglial cells was confirmed by membrane binding of [3H]U69,593, a kappa-selective ligand, and by indirect immunofluorescence. Treatment of microglial cell cultures with U50,488 or U69,593 resulted in a dose-dependent inhibition of expression of the monocytotropic HIV-1 SF162 strain. This antiviral effect of the kappa ligands was blocked by the specific KOR antagonist, nor-binaltrophimine. These findings suggest that kappa opioid agonists have immunomodulatory activity in the brain, and that these compounds could have potential in the treatment of HIV-1-associated encephalopathy.

Vascular variant of prion protein cerebral amyloidosis with tau-positive neurofibrillary tangles: the phenotype of the stop codon 145 mutation in PRNP.

Deposition of PrP amyloid in cerebral vessels in conjunction with neurofibrillary lesions is the neuropathologic hallmark of the dementia associated with a stop mutation at codon 145 of PRNP, the gene encoding the prion protein (PrP). In this disorder, the vascular amyloid in tissue sections and the approximately 7.5-kDa fragment extracted from amyloid are labeled by antibodies to epitopes located in the PrP sequence including amino acids 90-147. Amyloid-laden vessels are also labeled by antibodies against the C terminus, suggesting that PrP from the normal allele is involved in the pathologic process. Abundant neurofibrillary lesions are present in the cerebral gray matter. They are composed of paired helical filaments, are labeled with antibodies that recognize multiple phosphorylation sites in tau protein, and are similar to those observed in Alzheimer disease. A PrP cerebral amyloid angiopathy has not been reported in diseases caused by PRNP mutations or in human transmissible spongiform encephalopathies; we propose to name this phenotype PrP cerebral amyloid angiopathy (PrP-CAA).

Molecular basis of human mitochondrial very-long-chain acyl-CoA dehydrogenase deficiency causing cardiomyopathy and sudden death in childhood.

beta-Oxidation of long-chain fatty acids provides the major source of energy in the heart. Defects in enzymes of the beta-oxidation pathway cause sudden, unexplained death in childhood, acute hepatic encephalopathy or liver failure, skeletal myopathy, and cardiomyopathy. Very-long-chain acyl-CoA dehydrogenase [VLCAD; very-long-chain-acyl-CoA:(acceptor) 2,3-oxidoreductase, EC 1.3.99.13] catalyzes the first step in beta-oxidation. We have isolated the human VLCAD cDNA and gene and determined the complete nucleotide sequences. Polymerase chain reaction amplification of VLCAD mRNA and genomic exons defined the molecular defects in two patients with VLCAD deficiency who presented with unexplained cardiac arrest and cardiomyopathy. In one, a homozygous mutation in the consensus dinucleotide of the donor splice site (g+1-->a) was associated with universal skipping of the prior exon (exon 11). The second patient was a compound heterozygote, with a missense mutation, C1837-->T, changing the arginine at residue 613 to tryptophan on one allele and a single base deletion at the intron-exon 6 boundary as the second mutation. This initial delineation of human mutations in VLCAD suggests that VLCAD deficiency reduces myocardial fatty acid beta-oxidation and energy production and is associated with cardiomyopathy and sudden death in childhood.

Species specificity in the cell-free conversion of prion protein to protease-resistant forms: a model for the scrapie species barrier.

Scrapie is a transmissible neurodegenerative disease that appears to result from an accumulation in the brain of an abnormal protease-resistant isoform of prion protein (PrP) called PrPsc. Conversion of the normal, protease-sensitive form of PrP (PrPc) to protease-resistant forms like PrPsc has been demonstrated in a cell-free reaction composed largely of hamster PrPc and PrPsc. We now report studies of the species specificity of this cell-free reaction using mouse, hamster, and chimeric PrP molecules. Combinations of hamster PrPc with hamster PrPsc and mouse PrPc with mouse PrPsc resulted in the conversion of PrPc to protease-resistant forms. Protease-resistant PrP species were also generated in the nonhomologous reaction of hamster PrPc with mouse PrPsc, but little conversion was observed in the reciprocal reaction. Glycosylation of the PrPc precursors was not required for species specificity in the conversion reaction. The relative conversion efficiencies correlated with the relative transmissibilities of these strains of scrapie between mice and hamsters. Conversion experiments performed with chimeric mouse/hamster PrPc precursors indicated that differences between PrPc and PrPsc at residues 139, 155, and 170 affected the conversion efficiency and the size of the resultant protease-resistant PrP species. We conclude that there is species specificity in the cell-free interactions that lead to the conversion of PrPc to protease-resistant forms. This specificity may be the molecular basis for the barriers to interspecies transmission of scrapie and other transmissible spongiform encephalopathies in vivo.

Encefalopatia tardia da entoxicação por monóxido de carbono : case report e revisão de literatura

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Metabolomics analysis in rats with thiamine deficiency identifies key metabolites in vulnerable brain regions and suggests neural stem progenitor cells play a role in ameliorating metabolic dysfunction

La documentation scientifique fait état de la présence, chez l’adulte, de cellules souches et progénitrices neurales (CSPN) endogènes dans les zones sous-ventriculaire et sous-granulaire du cerveau ainsi que dans le gyrus denté de l’hippocampe. De plus, un postulat selon lequel il serait également possible de retrouver ce type de cellules dans la moelle épinière et le néocortex des mammifères adultes a été énoncé. L’encéphalopathie de Wernicke, un trouble neurologique grave toutefois réversible qui entraîne un dysfonctionnement, voire une défaillance du cerveau, est causée principalement par une carence importante en thiamine (CT). Des observations récentes laissent envisager que les facteurs en cause dans la prolifération et la différenciation des CSPN pourraient également jouer un rôle important lors d’un épisode de CT. L’hypothèse, selon laquelle l’identification de nouveaux métabolites entrant dans le mécanisme ou la séquence de réactions se soldant en une CT pourraient en faciliter la compréhension, a été émise au moyen d'une démarche en cours permettant d’établir le profil des modifications métaboliques qui surviennent en de telles situations. Cette approche a été utilisée pour constater les changements métaboliques survenus au niveau du foyer cérébral dans un modèle de rats déficients en thiamine (rats DT), particulièrement au niveau du thalamus et du colliculus inférieur (CI). La greffe de CSPN a quant à elle été envisagée afin d’apporter de nouvelles informations sur la participation des CSPN lors d’un épisode de CT et de déterminer les bénéfices thérapeutiques potentiels offerts par cette intervention. Les sujets de l’étude étaient répartis en quatre groupes expérimentaux : un premier groupe constitué de rats dont la CT était induite par la pyrithiamine (rats DTiP), un deuxième groupe constitué de rats-contrôles nourris ensemble (« pair-fed control rats » ou rats PFC) ainsi que deux groupes de rats ayant subi une greffe de CSPN, soit un groupe de rats DTiP greffés et un dernier groupe constitué de rats-contrôles (rats PFC) greffés. Les échantillons de foyers cérébraux (thalamus et CI) des quatre groupes de rats ont été prélevés et soumis à des analyses métabolomiques non ciblées ainsi qu’à une analyse visuelle par microscopie à balayage électronique (SEM). Une variété de métabolites-clés a été observée chez les groupes de rats déficients en thiamine (rats DTiP) en plus de plusieurs métabolites dont la documentation ne faisait pas mention. On a notamment constaté la présence d’acides biliaires, d’acide cynurénique et d’acide 1,9— diméthylurique dans le thalamus, alors que la présence de taurine et de carnosine a été observée dans le colliculus inférieur. L’étude a de plus démontré une possible implication des CSPN endogènes dans les foyers cérébraux du thalamus et du colliculus inférieur en identifiant les métabolites-clés ciblant les CSPN. Enfin, les analyses par SEM ont montré une amélioration notable des tissus à la suite de la greffe de CSPN. Ces constatations suggèrent que l’utilisation de CSPN pourrait s’avérer une avenue thérapeutique intéressante pour soulager la dégénérescence symptomatique liée à une grave carence en thiamine chez l’humain.

Investigation du rôle de Myo9b dans la migration des interneurones GABAergiques corticaux

Les encéphalopathies épileptogènes sont des maladies graves de l’enfance associant une épilepsie, souvent réfractaire, et un retard de développement. Les mécanismes sous-tendant ces maladies sont peu connus. Cependant, nous postulons que ces épilepsies puissent être causées par une dysfonction du réseau inhibiteur. En effet, des défauts de migration ou de maturation des interneurones GABAergiques (INs) corticaux induisent l’épilepsie, tant chez l’humain que chez la souris. Dans le but d’étudier les causes génétiques des encéphalopathies épileptogènes sporadiques inexpliquées, le laboratoire de la Dre Rossignol a procédé au séquençage d’exome entier d’une cohorte d’enfants atteints. Cela a permis d’identifier, chez un patient, une nouvelle mutation de novo, possiblement pathogène, dans le gène MYO9b. MYO9b est impliqué dans la migration de cellules immunitaires et cancéreuses et est exprimée durant le développement cérébral. Nous émettons l’hypothèse voulant que MYO9b puisse être importante pour la migration des INs corticaux. Les résultats présentés dans ce mémoire démontrent que Myo9b est exprimé dès le stade embryonnaire par les progéniteurs des INs corticaux et que son expression se restreint aux INs dans le cortex mature. De plus, nous démontrons que la répression ex vivo de Myo9b sélectivement dans les INs au sein de tranches corticales organotypiques embryonnaires mène à des défauts morphologiques majeurs de ces cellules en migration. En effet, ces cellules présentent une morphologie multipolaire et des neurites rostraux plus longs et plus complexes. Ces changements morphologiques pourraient avoir un impact majeur sur la migration des INs et ainsi perturber le développement des réseaux inhibiteurs.

Thiamine Deficiency Results in Downregulation of the GLAST Glutamate Transporter in Cultured Astrocytes

Pyrithiamine-induced thiamine deficiency (TD) is a well-established model of Wernicke's encephalopathy in which a glutamate-mediated excitotoxic mechanism may play an important role in determining selective vulnerability. In order to examine this possibility, cultured astrocytes were exposed to TD and effects on glutamate transport and metabolic function were studied. TD led to decreases in cellular levels of thiamine and thiamine diphosphate (TDP) after 24 h of treatment and decreased activities of the TDP-dependent enzymes alpha-ketoglutarate dehydrogenase and transketolase after 4 and 7 days, respectively. TD treatment for 10 days led to a reversible decrease in the uptake of [H-3]-D-aspartate, a nonmetabolizable analogue of glutamate. Kinetic analysis revealed that the uptake inhibition was caused by a 47% decrease in the V-max for uptake of [H-3]-D-aspartate, with no change in the K-m value. Immunoblotting showed that this decrease in uptake was due to an 81% downregulation of the astrocyte-specific GLAST glutamate transporter. Loss of uptake activity and GLAST protein were blocked by treatment with the protein kinase C inhibitor H7, while exposure to DCG IV, a group II metabotropic glutamate receptor (mGluR) agonist, resulted in improvement of [H-3]-D-aspartate uptake and a partial reversal of transporter downregulation. These results are consistent with our recent in vivo findings of a loss of astrocytic glutamate transporters in TD and provide evidence that TD conditions may increase phosphorylation. of GLAST, contributing to its downregulation. In addition, manipulation of group II mGluR activity may provide an important strategy in the treatment of this disorder. (C) 2003 Wiley-Liss, Inc.