Synthetic Polymer and Microbubble Systems for Gene Delivery to the Brain

Thesis (Ph.D.)--University of Washington, 2016-08

Ischemic Stroke as the Initial Manifestation of Neurosyphilis in a Young Adult Patient Positive for Human Immunodeficiency Virus

A 31-year-old man with pontine infarction was referred to our hospital for further evaluation and treatment. At admission, his neurological examination was unremarkable. No lymphadenopathy or skin lesions were found. The Treponema pallidum haemagglutination test, rapid plasma regain test and fluorescent treponemal antibody absorption test of immunoglobulin G were positive in both serum and cerebrospinal fluid (CSF). CSF analysis showed lymphocytic pleocytosis. The patient had male-to-male sexual contact and was found to be HIV positive. Physicians should be aware that acute ischaemic stroke may be the first manifestation of neurosyphilis in a young adult, especially with HIV infection.

A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samples

Diagnostic techniques based on PCR have two major problems: false-positive reactions due to contamination with DNA fragments from previous PCRs (amplicons) and false-negative reactions caused by inhibitors that interfere with the PCR. We have improved our previously reported PCR based on the amplification of a fragment of the Mycobacterium tuberculosis complex-specific insertion element IS6110 with respect to both problems. False-positive reactions caused by amplicon contamination were prevented by the use of uracil-N-glycosylase and dUTP instead of dTTP. We selected a new set of primers outside the region spanned by the formerly used primers to avoid false-positive reactions caused by dTTP-containing amplicons still present in the laboratory. With this new primer set, 16 copies of the IS6110 insertion element, the equivalent of two bacteria, could be amplified 10(10) times in 40 cycles, resulting in a mean efficiency of 77% per cycle. To detect the presence of inhibitors of the Taq polymerase, which may cause false-negative reactions, part of each sample was spiked with M. tuberculosis DNA. The DNA purification method using guanidinium thiocyanate and diatoms effectively removed most or all inhibitors of the PCR. However, this was not suitable for blood samples, for which we developed a proteinase K treatment followed by phenol-chloroform extraction. This method permitted detection of 20 M. tuberculosis bacteria per ml of whole blood. Various laboratory procedures were introduced to reduce failure or inhibition of PCR and avoid DNA cross contamination. We have tested 218 different clinical specimens obtained from patients suspected of having tuberculosis. The samples included sputum (n=145), tissue biopsy samples (n=25), cerebrospinal fluid (n=15), blood (n=14), pleural fluid (n=9), feces, (n=7), fluid from fistulae (n=2), and pus from a wound (n=1). The results obtained by PCR were consistent with those obtained with culture, which is the "gold standard." We demonstrate that PCR is a useful technique for the rapid diagnosis of tuberculosis at various sites.

Guillain-Barré Syndrome with Absent Brainstem Reflexes: a Case Report

A 41-year-old man was admitted to an intensive care unit following respiratory arrest. One day prior to admission, he had complaints of nausea and pain involving lower limbs. On the night of admission he developed diplopia, dysphagia, and rapidly progressive quadriparesis. He developed respiratory failure requiring mechanical lung ventilation 24 hours later. On the fifth day of hospital stay the patient became comatose with absent brainstem reflexes and appeared to be brain dead. The cerebrospinal fluid showed albuminocytological dissociation. The electroencephalogram revealed an alpha rhythmical activity. The electrophysiological evaluation revealed an inexcitability of all nerves. Guillain-Barré syndrome was suspected. With supportive treatment the patient had a remarkable recovery and now is able to independently conduct his daily activities.

Increased CSF Aβ during the very early phase of cerebral Aβ deposition in mouse models

Abnormalities in brains of Alzheimer's disease (AD) patients are thought to start long before the first clinical symptoms emerge. The identification of affected individuals at this 'preclinical AD' stage relies on biomarkers such as decreased levels of the amyloid-β peptide (Aβ) in the cerebrospinal fluid (CSF) and positive amyloid positron emission tomography scans. However, there is little information on the longitudinal dynamics of CSF biomarkers, especially in the earliest disease stages when therapeutic interventions are likely most effective. To this end, we have studied CSF Aβ changes in three Aβ precursor protein transgenic mouse models, focusing our analysis on the initial Aβ deposition, which differs significantly among the models studied. Remarkably, while we confirmed the CSF Aβ decrease during the extended course of brain Aβ deposition, a 20-30% increase in CSF Aβ40 and Aβ42 was found around the time of the first Aβ plaque appearance in all models. The biphasic nature of this observed biomarker changes stresses the need for longitudinal biomarker studies in the clinical setting and the search for new 'preclinical AD' biomarkers at even earlier disease stages, by using both mice and human samples. Ultimately, our findings may open new perspectives in identifying subjects at risk for AD significantly earlier, and in improving the stratification of patients for preventive treatment strategies.

Neurofilaments and NFL-TBS.40-63 peptide penetrate oligodendrocytes through clathrin-dependent endocytosis to promote their growth and survival in vitro.

International audience

Estudo do papel da proteína multifuncional APE1/Ref-1 sobre a resposta inflamatória na meningite bacteriana

Despite advances in antibiotic therapy, bacterial meningitis (BM) remains with high mortality and morbidity rates in worldwide. One important mechanism associated to sequels during disease is the intense inflammatory response which promotes an oxidative burst and release of reactive oxygen species, consequently leading to cell death. Activation of DNA repair enzymes during oxidative stress has been demonstrated in several neurological disorders. APE1/Ref-1 is a multifunctional protein involved in DNA repair and plays a redox function on transcription factors such as NFkB and AP-1.The aim of this study was assess the role of APE1/Ref-1 on inflammatory response and the possibility of its modulation to reduce the sequels of the disease. Firstly it was performed an assay to measure cytokine in cerebrospinal fluid of patients with BM due to Streptococcus pneumoniae and Neisseriae meningitides. Further, a cellular model of inflammation was used to observe the effect of the inhibition of the endonuclease and redox activity of APE1/Ref-1 on cytokine levels. Additionally, APE1/Ref-1 expression in cortex and hippocampus of rat with MB after vitamin B6 treatment was evaluated. Altogether, results showed a similar profile of cytokines in the cerebrospinal fluid of patients from both pathogens, although IFNy showed higher expression in patients with BM caused by S. pneumoniae. On the other hand, inhibitors of APE1/Ref-1 reduced cytokine levels, mainly TNF-α. Reduction of oxidative stress markers was also observed after introduction of inhibitors in the LPS-stimulated cell. In the animal model, BM increased the expression of the protein APE1/Ref-1, while vitamin B6 promoted reduction. Thereby, this data rise important factors to be considered in pathogenesis of BM, e.g., IFNy can be used as prognostic factor during corticosteroid therapy, APE1/Ref-1 can be an important target to modulate the level of inflammation and VIII oxidative stress, and vitamin B6 seems modulates several proteins related to cell death. So, this study highlights a new understanding on the role of APE1/Ref-1 on the inflammation and the oxidative stress during inflammation condition

Análise do efeito de polimorfismos não-sinônimos em genes de reparo de DNA da via BER na resposta inflamatória da meningite

In vitro and in animal models, APE1, OGG1, and PARP-1 have been proposed as being involved with inflammatory response. In this work, we have investigated if the SNPs APE1 Asn148Glu, OGG1 Ser326Cys, and PARP-1 Val762Ala are associated to meningitis and also developed a system to enable the functional analysis of polymorphic proteins. Patients with bacterial meningitis (BM), aseptic meningitis (AM) and controls (non-infected) genotypes were investigated by PIRA-PCR or PCR-RFLP. DNA damages were detected in genomic DNA by Fpg treatment. IgG and IgA were measured from plasma and the cytokines and chemokines were measured from cerebrospinal fluid samples using Bio-Plex assays. The levels of NF-κB and c-Jun were measured in CSF by dot blot assays. A significant (P<0.05) increase in the frequency of APE1 148Glu allele in BM and AM patients was observed. A significant increase in the genotypes Asn/Asn in control group and Asn/Glu in BM group was also found. For the SNP OGG1 Ser326Cys, the genotype Cys/Cys was more frequent (P<0.05) in BM group. The frequency of PARP-1 Val/Val genotype was higher in control group (P<0.05). The occurrence of combined SNPs increased significantly in BM patients, indicating that these SNPs may be associated to the disease. Increasing in sensitive sites to Fpg was observed in carriers of APE1 148Glu allele or OGG1 326Cys allele, suggesting that SNPs affect DNA repair activity. Alterations in IgG production were observed in the presence of SNPs APE1Asn148Glu, OGG1Ser326Cys or PARP-1Val762Ala. Reductions in the levels ofIL-6, IL-1Ra, MCP-1/CCL2and IL-8/CXCL8 were observed in the presence of APE1148Glu allele in BM patients, however no differences were observed in the levels of NF-κB and c-Jun considering genotypes and analyzed groups. Using APE1 as model, a system to enable the analysis of cellular effects and functional characterization of polymorphic proteins was developed using strategies of cloning APE1 cDNA in pIRES2-EGFP vector, cellular transfection of the construction obtained, siRNA for endogenous APE1 and cellular cultures genotyping. In conclusion, we obtained evidences of an effect of SNPs in DNA repair genes on the regulation of immune response. This is a pioneering work in the field that shows association of BER variant enzymes with an infectious disease in human patients, suggesting that the SNPs analyzed may affect immune response and damage by oxidative stress level during brain infection. Considering these data, new approaches of functional characterization must be developed to better analysis and interactions of polymorphic proteins in response to this context

Preparo de amostras citológicas de efusões, lavados (broncoalveolar e peritoneal) e líquor pelo método “plasma-tromboplastina”

Dissertação (mestrado)—Universidade de Brasilia, Faculdade de Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde, 2016.

Potential protective role of reactive astrocytes in the periventricular parenchyma in congenital hydrocephalus

Background Cerebrospinal fluid accumulation in hydrocephalus produces an elevation of intraventricular pressure with pathological consequences on the periventricular brain parenchyma including ischemia, oedema, oxidative stress, and accumulation of metabolic waste products. Here we studied in the hyh mouse, an animal model of congenital hydrocephalus, the role of reactive astrocytes in this clinical degenerative condition. Materials and Methods Wild type and hydrocephalic hyh mice at 30 days of postnatal age were used. Three metabolites related to the oxidative and neurotoxic conditions were analysed in ex vivo samples (glutathione, glutamine and taurine) using High Resolution Magic Angle Spinning (HR-MAS). Glutathione synthetase and peroxidase, glutamine synthetase, kidney-type glutaminase (KGA), and taurine/taurine transporter were immunolocated in brain sections. Results Levels of the metabolites were remarkably higher in hydrocephalic conditions. Glutathione peroxidase and synthetase were both detected in the periventricular reactive astrocytes and neurons. Taurine was mostly found free in the periventricular parenchyma and in the reactive astrocytes, and the taurine transporter was mainly present in the neurons located in such regions. Glutamine synthetase was found in reactive astrocytes. Glutaminase was also detected in the reactive astrocytes and in periventricular neurons. These results suggest a possible protective response of reactive astrocytes against oxidative stress and neurotoxic conditions. Conclusions Astrocyte reaction seems to trigger an anti-oxidative and anti-neurotoxic response in order to ameliorate pathological damage in periventricular areas of the hydrocephalic mice.

Fratura traumática do osso temporal : diagnóstico, sequelas e tratamento

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

Reabilitação física de cães com hérnias de disco toracolombares

Dissertação de Mestrado Integrado em Medicina Veterinária

Safety and Efficacy of Intravenous Colistin in Neonates With Culture Proven Sepsis

Background: Although it is well described among adults, intravenous colistin use and its associated toxicities in newborns are poorly understood. Objectives: We present our experience of efficacy and safety of intravenous colistin in the treatment of sepsis in term and preterm neonates. Patients and Methods: The records of neonates who received colistin between January 2013 and February 2014 were retrospectively reviewed. All neonates with culture proven nosocomial infections due to multidrug resistant organisms and treated continuously with colistin for more than 72 hours were included in the study. Results: Patients were evaluated for clinical and microbiological response to the drug and its and side effects. Twelve newborn infants with mean 31.8 ± 3.5 weeks gestational age and median 1482 (810 - 3200) gram birth weight were included. 11/12 (91.7%) patients showed microbiological clearance with intravenous colistin. One patient who had recurrent cerebrospinal fluid positive culture was treated with intraventricular colistin. The major side effects observed was hyponatremia and hypokalemia in 2 (16.6%) patients, all infants required magnesium supplementation. Conclusions: Intravenous colistin administration appears to be safe and efficacious for multidrug-resistant gram-negative infections in neonates, including preterm infants. However, we believe that large prospective controlled studies are needed to confirm its efficacy and safety in neonates.

Caractérisation d'une entité neurologique émergente au sein du cheptel ovin québécois

Cette étude vise à caractériser le «crampage», une entité relativement nouvelle dans l’industrie ovine au Québec. Les signes cliniques se manifestent au pas, par une hyperflexion (hanche, grasset, jarret), d’un ou des deux membres pelviens. Cinq agneaux naturellement affectés et cinq agneaux appariés cliniquement normaux ont été soumis à des examens physique, neurologique et orthopédique, à des techniques d’imagerie avancée (tomodensitométrie, résonance magnétique), à des tests électrodiagnostiques (électromyogramme, vitesses de conduction nerveuse motrice et sensitive) puis à une nécropsie. Des hématologies, biochimies ainsi que des analyses du liquide céphalorachidien ont également été réalisées. Les résultats ont été comparés entre les groupes (affectés/cliniquement normaux). Il a été constaté à la tomodensitométrie que la surface du canal vertébral mesurée au niveau de la deuxième vertèbre lombaire était inférieure dans le groupe des agneaux affectés (p=0.045). Aucune répercussion n’a été constatée sur le segment de moelle épinière correspondant. La racine S2, quant à elle, était plus grêle dans le groupe des agneaux affectés (p=0.01). À l’issue de cette étude, une cause orthopédique, musculaire ou neurologique consécutive à une lésion structurale de la moelle épinière a été écartée. Il pourrait s‘agir d’une atteinte sensitive de la racine S2 altérant la sensation dans le membre affecté, toutefois, une anomalie fonctionnelle cérébrale ou de la moelle épinière dans le renflement lombaire, est également à considérer. Sans anomalie musculaire, l’appellation «crampage» est inexacte. Nous proposons de la remplacer par des termes plus descriptifs comme «syndrome d’hyperflexion» ou «high stepping gait».

Caractérisation d'une entité neurologique émergente au sein du cheptel ovin québécois

Cette étude vise à caractériser le «crampage», une entité relativement nouvelle dans l’industrie ovine au Québec. Les signes cliniques se manifestent au pas, par une hyperflexion (hanche, grasset, jarret), d’un ou des deux membres pelviens. Cinq agneaux naturellement affectés et cinq agneaux appariés cliniquement normaux ont été soumis à des examens physique, neurologique et orthopédique, à des techniques d’imagerie avancée (tomodensitométrie, résonance magnétique), à des tests électrodiagnostiques (électromyogramme, vitesses de conduction nerveuse motrice et sensitive) puis à une nécropsie. Des hématologies, biochimies ainsi que des analyses du liquide céphalorachidien ont également été réalisées. Les résultats ont été comparés entre les groupes (affectés/cliniquement normaux). Il a été constaté à la tomodensitométrie que la surface du canal vertébral mesurée au niveau de la deuxième vertèbre lombaire était inférieure dans le groupe des agneaux affectés (p=0.045). Aucune répercussion n’a été constatée sur le segment de moelle épinière correspondant. La racine S2, quant à elle, était plus grêle dans le groupe des agneaux affectés (p=0.01). À l’issue de cette étude, une cause orthopédique, musculaire ou neurologique consécutive à une lésion structurale de la moelle épinière a été écartée. Il pourrait s‘agir d’une atteinte sensitive de la racine S2 altérant la sensation dans le membre affecté, toutefois, une anomalie fonctionnelle cérébrale ou de la moelle épinière dans le renflement lombaire, est également à considérer. Sans anomalie musculaire, l’appellation «crampage» est inexacte. Nous proposons de la remplacer par des termes plus descriptifs comme «syndrome d’hyperflexion» ou «high stepping gait».