Co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor increases human immunodeficiency virus-1 DNA vaccine-induced polyfunctional CD4+ T-cell responses

T-cell based vaccines against human immunodeficiency virus (HIV) generate specific responses that may limit both transmission and disease progression by controlling viral load. Broad, polyfunctional, and cytotoxic CD4+ T-cell responses have been associated with control of simian immunodeficiency virus/HIV-1 replication, supporting the inclusion of CD4+ T-cell epitopes in vaccine formulations. Plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) co-administration has been shown to induce potent CD4+ T-cell responses and to promote accelerated priming and increased migration of antigen-specific CD4+ T-cells. However, no study has shown whether co-immunisation with pGM-CSF enhances the number of vaccine-induced polyfunctional CD4+ T-cells. Our group has previously developed a DNA vaccine encoding conserved, multiple human leukocyte antigen (HLA)-DR binding HIV-1 subtype B peptides, which elicited broad, polyfunctional and long-lived CD4+ T-cell responses. Here, we show that pGM-CSF co-immunisation improved both magnitude and quality of vaccine-induced T-cell responses, particularly by increasing proliferating CD4+ T-cells that produce simultaneously interferon-γ, tumour necrosis factor-α and interleukin-2. Thus, we believe that the use of pGM-CSF may be helpful for vaccine strategies focused on the activation of anti-HIV CD4+ T-cell immunity.

Tratamento de diarreias neonatais em vitelos

Dissertação de Mestrado Integrado em Medicina Veterinária

Étude de l’impact de la prise de médicaments dans le traitement de l’arthrite juvénile sur les événements néfastes à l’accouchement chez la mère et son bébé

La plupart des femmes ayant été atteintes d’arthrite juvénile idiopathique (AJI) continuent de souffrir d’arthrite à l’âge adulte. Certains des médicaments utilisés dans le traitement de l’arthrite tels que les corticostéroïdes et les antiinflammatoires non stéroïdiens (AINS) ne sont pas recommandés durant la grossesse. Le but de ce mémoire est d’estimer l’impact de la prise de ces médicaments sur les événements néfastes à l’accouchement chez ces femmes et leur bébé. Des données administratives sur les prescriptions de médicaments et les hospitalisations d’une cohorte de 1756 femmes ayant souffert d’AJI sont utilisées. Elles ont permis de reconstruire l’historique de consommation de médicaments contre l’arthrite chez les femmes durant la grossesse et l’année précédente. Pour ce faire, deux sous-cohortes de femmes ayant souffert d’AJI ont été formées : une pour la période grossesse et une autre pour la grossesse et l’année précédant celle-ci. Les événements d’intérêt étaient : malformations congénitales, complications néonatales, complications maternelles et petit poids pour l’âge gestationnel. Les proportions de cas présentant l’un de ces événements variaient entre 11,52% et 37,08%. Les médicaments ont été modélisés en terme d’utilisation ou de durée totale de consommation durant la période d’étude. Pour chaque événement, des modèles logistiques ont été estimés pour mesurer l’association entre la prise de médicaments et l’événement, en ajustant pour des variables de confusion potentielles : hypertension avant la grossesse, âge à l’accouchement et obtention du diplôme de secondaire. La consommation de corticostéroïdes semble augmenter statistiquement significativement le risque de présenter des malformations congénitales mais n’avoir aucun impact sur les autres événements. Aucun lien statistiquement significatif n’a été observé entre la consommation de AINS et les événements d’intérêt.

Bionano Electronics: Magneto-Electric Nanoparticles for Drug Delivery, Brain Stimulation and Imaging Applications

Nanoparticles are often considered as efficient drug delivery vehicles for precisely dispensing the therapeutic payloads specifically to the diseased sites in the patient’s body, thereby minimizing the toxic side effects of the payloads on the healthy tissue. However, the fundamental physics that underlies the nanoparticles’ intrinsic interaction with the surrounding cells is inadequately elucidated. The ability of the nanoparticles to precisely control the release of its payloads externally (on-demand) without depending on the physiological conditions of the target sites has the potential to enable patient- and disease-specific nanomedicine, also known as Personalized NanoMedicine (PNM). In this dissertation, magneto-electric nanoparticles (MENs) were utilized for the first time to enable important functions, such as (i) field-controlled high-efficacy dissipation-free targeted drug delivery system and on-demand release at the sub-cellular level, (ii) non-invasive energy-efficient stimulation of deep brain tissue at body temperature, and (iii) a high-sensitivity contrasting agent to map the neuronal activity in the brain non-invasively. First, this dissertation specifically focuses on using MENs as energy-efficient and dissipation-free field-controlled nano-vehicle for targeted delivery and on-demand release of a anti-cancer Paclitaxel (Taxol) drug and a anti-HIV AZT 5’-triphosphate (AZTTP) drug from 30-nm MENs (CoFe2O4-BaTiO3) by applying low-energy DC and low-frequency (below 1000 Hz) AC fields to separate the functions of delivery and release, respectively. Second, this dissertation focuses on the use of MENs to non-invasively stimulate the deep brain neuronal activity via application of a low energy and low frequency external magnetic field to activate intrinsic electric dipoles at the cellular level through numerical simulations. Third, this dissertation describes the use of MENs to track the neuronal activities in the brain (non-invasively) using a magnetic resonance and a magnetic nanoparticle imaging by monitoring the changes in the magnetization of the MENs surrounding the neuronal tissue under different states. The potential therapeutic and diagnostic impact of this innovative and novel study is highly significant not only in HIV-AIDS, Cancer, Parkinson’s and Alzheimer’s disease but also in many CNS and other diseases, where the ability to remotely control targeted drug delivery/release, and diagnostics is the key.

Étude de l’impact de la prise de médicaments dans le traitement de l’arthrite juvénile sur les événements néfastes à l’accouchement chez la mère et son bébé

L'obtention des données a été subventionnée par CIORA (Canadian Initiative for Outcomes in Rheumatology Care). CIORA a aussi financé l'analyse des données effectuées par Justine Zehr. L'Initiative Canadienne Pour Des Resultats En Soins Rhumatologiques (ICORA) a financé l'obtention des données et une partie de l'analyse statistique présentée dans ce mémoire.

Treatment trials for neonatal seizures: the effect of design on sample size

Neonatal seizures are common in the neonatal intensive care unit. Clinicians treat these seizures with several anti-epileptic drugs (AEDs) to reduce seizures in a neonate. Current AEDs exhibit sub-optimal efficacy and several randomized control trials (RCT) of novel AEDs are planned. The aim of this study was to measure the influence of trial design on the required sample size of a RCT. We used seizure time courses from 41 term neonates with hypoxic ischaemic encephalopathy to build seizure treatment trial simulations. We used five outcome measures, three AED protocols, eight treatment delays from seizure onset (Td) and four levels of trial AED efficacy to simulate different RCTs. We performed power calculations for each RCT design and analysed the resultant sample size. We also assessed the rate of false positives, or placebo effect, in typical uncontrolled studies. We found that the false positive rate ranged from 5 to 85% of patients depending on RCT design. For controlled trials, the choice of outcome measure had the largest effect on sample size with median differences of 30.7 fold (IQR: 13.7–40.0) across a range of AED protocols, Td and trial AED efficacy (p<0.001). RCTs that compared the trial AED with positive controls required sample sizes with a median fold increase of 3.2 (IQR: 1.9–11.9; p<0.001). Delays in AED administration from seizure onset also increased the required sample size 2.1 fold (IQR: 1.7–2.9; p<0.001). Subgroup analysis showed that RCTs in neonates treated with hypothermia required a median fold increase in sample size of 2.6 (IQR: 2.4–3.0) compared to trials in normothermic neonates (p<0.001). These results show that RCT design has a profound influence on the required sample size. Trials that use a control group, appropriate outcome measure, and control for differences in Td between groups in analysis will be valid and minimise sample size.

Use of fluorescence lifetime imaging microscopy (FLIM) as a timer of cell cycle S phase

Incorporation of thymidine analogues in replicating DNA, coupled with antibody and fluorophore staining, allows analysis of cell proliferation, but is currently limited to monolayer cultures, fixed cells and end-point assays. We describe a simple microscopy imaging method for live real-time analysis of cell proliferation, S phase progression over several division cycles, effects of anti-proliferative drugs and other applications. It is based on the prominent (~ 1.7-fold) quenching of fluorescence lifetime of a common cell-permeable nuclear stain, Hoechst 33342 upon the incorporation of 5-bromo-2’-deoxyuridine (BrdU) in genomic DNA and detection by fluorescence lifetime imaging microscopy (FLIM). We show that quantitative and accurate FLIM technique allows high-content, multi-parametric dynamic analyses, far superior to the intensity-based imaging. We demonstrate its uses with monolayer cell cultures, complex 3D tissue models of tumor cell spheroids and intestinal organoids, and in physiological study with metformin treatment.

Revisión del uso de los medicamentos biosimilares vs. biológicos: implicaciones para la salud en Colombia.

Este documento tiene como objetivo describir las implicaciones para la salud con el uso de medicamentos biosimilares en comparación con los medicamentos biológicos en Colombia. Así mismo, describir el contexto normativo acerca del uso de medicamentos biosimilares, las recomendaciones y lineamientos sobre seguridad y efectividad del uso de medicamentos Biosimilares y Biológicos, partiendo de sus diferencias biomoleculares. Para esto, se desarrolló una revisión documental electrónica y manual de la literatura en bases de datos, revistas y libros limitada a términos MeSH. La selección de los artículos incluyo documentos completos publicados en revistas indexadas de los últimos 10 años, en español e inglés; la información recolectada se organizó para la construcción del presente documento. Concluyendo, se encontró que las patentes de muchos medicamentos biológicos han vencido o están próximas a caducar y varios biosimilares están desarrollándose y comercializándose incluso en países sin regulaciones estrictas. Los biosimilares nunca podrán ser iguales al original por su complejidad molecular, por ello debemos integrarlos a los sistemas de farmacovigilancia mejorando trazabilidad e identificando su origen mientras se establecen denominaciones comunes distinguibles. La evidencia actual sugiere que la regulación de medicamentos biosimilares debe ser evaluada y armonizada en todo el mundo.

Diclofenaco comparado con ibuprofeno para el cierre de Ductus arterioso persistente, en recién nacidos prematuros

Introducción: El Ductus arterioso persistente (DAP), es uno de los defectos congénitos cardiacos más comunes, requiere manejo farmacológico y/o quirúrgico; presenta complicaciones hemodinámicas, respiratorias y muerte. Los medicamentos de elección para su manejo son indometacina e ibuprofeno, pero su costo y accesibilidad llevo al uso de diclofenaco como alternativa de manejo en algunos hospitales. Objetivo: Comparar respuesta al tratamiento con diclofenaco vs ibuprofeno en cierre de DAP. Materiales y Métodos: Estudio observacional analítico retrospectivo, que compara los resultados obtenidos al usar Diclofenaco e Ibuprofeno para el cierre del DAP en recién nacidos pretérmino. Se recolecto información de pacientes hospitalizados en la Unidad Neonatal de un Hospital II nivel de Bogotá. Se revisaron las historias clínicas de pacientes de edad gestacional entre 24 y 36 semanas por Ballard con los criterios para diagnóstico de DAP y recibieron tratamiento farmacológico con una de las siguientes opciones: Ibuprofeno 10 mg/Kg dosis inicial después 5mg/Kg a las 24 48 horas, o Diclofenaco 0.2 mg/Kg dosis cada 12 horas tres dosis. Se comparó el Diclofenaco y el Ibuprofeno para el tratamiento farmacológico de DAP en recién nacidos prematuros. Resultados: Fueron evaluados 103 pacientes, el diagnóstico de DAP se realizó con ecocardiograma transtorácico, el 66.6 % de los pacientes presentó cierre farmacológico con Diclofenaco y 69 % con Ibuprofeno, La mortalidad fue de 17.65 % con Diclofenaco y 11.54 % con ibuprofeno; en ambos casos asociadas a la prematurez. Conclusiones: El éxito farmacológico fue similar en ambos grupos, el diclofenaco es una alternativa interesante cuando la terapia convencional no esté disponible.

Factores asociados a respuesta de abatacept subcutaneo en artritis reumatoide

INTRODUCCION Dado que la artritis reumatoide es la artropatía inflamatoria más frecuente en el mundo, siendo altamente discapacitante y causando gran impacto de alto costo, se busca ofrecer al paciente opciones terapéuticas y calidad de vida a través del establecimiento de un tratamiento oportuno y eficaz, teniendo presentes aquellos predictores de respuesta previo a instaurar determinada terapia. Existen pocos estudios que permitan establecer aquellos factores de adecuada respuesta para inicio de terapia biológica con abatacept, por lo cual en este estudio se busca determinar cuáles son esos posibles factores. METODOLOGIA Estudio analítico de tipo corte transversal de 94 pacientes con diagnóstico de AR, evaluados para determinar las posibles variables que influyen en la respuesta a terapia biológica con abatacept. Se incluyeron 67 de los 94 pacientes al modelo de regresión logística, que son aquellos pacientes en que fue posible medir la respuesta al tratamiento (respuesta EULAR) a través de la determinación del DAS 28 y así discriminar en dos grupos de comparación (respuesta y no respuesta). DISCUSION DE RESULTADOS La presencia de alta actividad de la enfermedad al inicio de la terapia biológica, aumenta la probabilidad de respuesta al tratamiento respecto al grupo con baja/moderada actividad de la enfermedad; OR 4,19 - IC 95%(1,18 – 14.9), (p 0,027). La ausencia de erosiones óseas aumenta la probabilidad de presentar adecuada respuesta a la terapia biológica respecto aquellos con erosiones, con un OR 3,1 (1,01-9,55), (p 0,048). Niveles de VSG y presencia de manifestaciones extra-articulares son otros datos de interés encontrados en el análisis bivariado. Respecto a las variables o características como predictores de respuesta al tratamiento con abatacept, se encuentran estudios que corroboran los hallazgos de este estudio, respecto al alto puntaje del DAS 28 al inicio de la terapia (9, 12). CONCLUSIONES Existen distintas variables que determinan la respuesta a los diferentes biológicos para manejo de AR. Es imprescindible evaluar dichos factores de manera individual con el fin de lograr de manera efectiva el control de la enfermedad y así mejorar la calidad de vida del individuo (medicina personalizada). Existen variables tales como la alta actividad de la enfermedad y la ausencia de erosiones como predictores de respuesta en la terapia con abatacept.

Epileptic and developmental encephalopathies: clinical and genetic study of adult patients attending a tertiary Epilepsy Centre

Objectives: To fully re-evaluate patients with early-onset epilepsy and intellectual disability with neurological, neurophysiological and neuropsychological examination in order to contribute to expanding the phenotypic spectrum of known epileptic encephalopathy (EE)-related genes and to identify novel genetic defects underlying EEs. Methods: We recruited patients with epilepsy and intellectual disability (ID) referring to our Epilepsy Centre. Patients underwent full clinical and neurophysiologic evaluation. When possible they underwent neuroradiologic investigations. Selected cases also underwent genetic analysis. Results: We recruited 200 patients (109 M, 91 F; mean age 36 years old). Mean age at epilepsy onset was 4 years old. The degree of ID was borderline in 4.5% of patients, mild in 25%, moderate in 38% and severe in 32.5%. EEG showed epileptiform abnormalities in 79.5% of patients. One hundred and thirty-one patients out of the 200 recruited (65.5%) did not have an aetiological diagnosis. All the patients underwent full clinical reassessment and when necessary they performed neuroradiologic and genetic investigations as well. We identified 35 patients with a genetic aetiology. In 8 cases a structural brain lesion was observed. In 33 patients, a genetic aetiology was identified. In 2 patients with drug-resistant seizures video-EEG allowed the identification of non-epileptic seizures, and in one patient we discontinued anti-epileptic drugs. In these patients, the aetiological diagnosis was made after 30 years (range 9-60 years) from the disease onset. Conclusions: In a population of 200 adult patients with epilepsy and ID, an aetiological cause was identified in 45 patients after 30 years from the disease onset. Aetiological diagnosis, especially if genetic, has significant positive implications for patients, even if it has been made after years from the beginning of the disease. Benefits include better-focused antiepileptic drug (AED) choice, sparing of further unnecessary investigations and improved knowledge of comorbidities.

Genomic and functional insights into the interactions between vaginal Lactobacillus strains and pathogens

The vaginal microbiota of healthy pre-menopausal women is typically dominated by one Lactobacillus species among L. crispatus, L. gasseri, L. jensenii and L. iners. Thanks to a series of antimicrobial activities, strains belonging to these species represent the first barrier against infections and maintain niche homeostasis. On the other hands, the increase abundance in pathogen species is associated with the onset of numerous diseases, leading also to an increase risk of other infections acquisition. The deciphering of factors which influence Lactobacillus survival, as well as the interactions between lactobacilli-pathogens and pathogens-pathogens represent an important topic of study for improving woman health and investigating effective probiotic strategies. Here, we investigated environmental factors and genetic traits that lead to the dominance of either L. crispatus or L. gasseri in the vaginal niche and the possible applications of liposomes loaded with L. gasseri biosurfactants for the treatment and prevention of Staphylococcus aureus biofilm infections. Furthermore, considering the increasing relevance acquired by bacterial extracellular vesicles (EVs) we analysed the role of EVs derived from vaginal lactobacilli and pathogens on both bacterial growth and HIV-1 infections. As a result, we reported for the first time i) common and species-specific genotypic and phenotypic features of L. crispatus and L. gasseri ii) significant antibiofilm activity of liposomes loading vaginal Lactobacillus biosurfactants against multi-drug resistant S. aureus strains iii) absence of growth regulation mediated by EVs derived from lactobacilli on pathogen cultures and vice versa iv) anti-HIV-1 activity of protein derived from L. gasseri EVs and unexpected antiviral effect of pathogen-derived EVs on HIV-1 infections in vitro. In conclusion, this PhD thesis explored characteristics and possible applications of vaginal lactobacilli for the human health, as well as promising antiviral effects of both lactobacilli and pathogen derived EVs.

Development of new chemical entities to target neuroinflammatory pathways.

Neuroinflammatory pathways are main culprits of neurodegenerative diseases' onset and progression, including Alzheimer’s disease (AD). On this basis, several anti-inflammatory drugs were repurposed in clinical trials. However, they have failed, probably because neuroinflammation is a complex network, still not fully understood. From these evidences, this thesis focused on the design and synthesis of new chemical entities as potential neuroinflammatory drugs or chemical probes. Projects 1 and 2 aimed to multi-target-directed ligand (MTDL) development to target neuroinflammation in AD. Polypharmacology by MTDLs is considered one of the most promising strategies to face the multifactorial nature of neurodegenerative diseases. Particularly, Project 1 took inspiration from a cromolyn-ibuprofen drug combination polypharmacological approach, which was recently investigated in AD clinical trials. Based on that, two cromolyn-(S)-ibuprofen codrug series were designed and synthesized. Parent drugs were combined via linking or fusing strategies in 1:2 or 1:1 ratio, by means of hydrolyzable bonds. Project 2 started from a still ongoing AD clinical trial on investigational drug neflamapimod. It is a selective inhibitor of p38α-MAPK, a kinase strictly involved in neuroinflammatory pathways. On the other side, rasagiline, an anti-Parkinson drug, was also repurposed as AD treatment. Indeed, rasagiline’s propargylamine fragment demonstrated to be responsible not only for the MAO-B selective inhibition, but also for the neuroprotective activity. Thus, to synergistically combine these two effects into single-molecules, a small set of neflamapimod-rasagiline hybrids was developed. In the end BMX, a poorly investigated kinase, which seems to be involved in pro-inflammatory mediator production, was explored for the development of new chemical probes. High-quality chemical probes are a powerful tool in target validation and starting points for the development of new drug candidates. Thus, Project 3 focused on the design and synthesis of two series of optimized BMX covalent inhibitors as selective chemical probes.

Longitudinal Micro CT-driven biomarkers as a tool to evaluate lung fibrosis progression and antifibrotic efficacy in a refined Bleomycin mouse model

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease with no curative pharmacological treatment. Animal models play an essential role in revealing molecular mechanisms involved in the pathogenesis of the disease. Bleomycin (BLM)-induced lung fibrosis is the most widely used and characterized model for anti-fibrotic drugs screening. However, several issues have been reported, such as the identification of an optimal BLM dose and administration scheme as well as gender-specificity. Moreover, the balance between disease resolution, an appropriate time window for therapeutic intervention and animal welfare remains critical aspects yet to be fully elucidated. In this thesis, Micro CT imaging has been used as a tool to identify the ideal BLM dose regimen to induce sustained lung fibrosis in mice as well as to assess the anti-fibrotic effect of Nintedanib (NINT) treatment upon this BLM administration regimen. In order to select the optimal BLM dose scheme, C57bl/6 male mice were treated with BLM via oropharyngeal aspiration (OA), following either double or triple BLM administration. The triple BLM administration resulted in the most promising scheme, able to balance disease resolution, appropriate time-window for therapeutic intervention and animal welfare. The fibrosis progression was longitudinally assessed by micro-CT every 7 days for 5 weeks after BLM administration and 5 animals were sacrificed at each timepoint for the BALF and histological evaluation. The antifibrotic effect of NINT was assessed following different treatment regimens in this model. Herein, we have developed an optimized mouse model of pulmonary fibrosis, enabling three weeks of the therapeutic window to screen putative anti-fibrotic drugs. micro-CT scanning, allowed us to monitor the progression of lung fibrosis and the therapeutical response longitudinally in the same subject, drastically reducing the number of animals involved in the experiment.

Vírus da Imunodeficiência Humana e AIDS

O objeto começa enfatizando que o HIV/Aids é ainda hoje um problema frequente e comum no dia a dia dos profissionais de saúde. Destaca que é importante que o médico saiba o diagnóstico, a abordagem inicial, a imunização, a instituição do esquema inicial de TARV e o manejo de situações que levam à baixa adesão ao tratamento. Aborda em uma apresentação como pode ser estabelecida a relação médico-paciente quando no atendimento. Detalha que o diagnóstico sorológico da infecção pelo HIV é baseado no desenvolvimento de anticorpos anti-HIV e aprofunda o tema. Ressalta como pode ser a abordagem inicial do adulto infectado pelo HIV e especifica as imunizações. Explica como manejar o tratamento. Conclui lembrando que, sobre a profilaxia de infecções oportunistas, a prevenção de infecção em pacientes portadores de HIV é medida de bastante efetividade que reduz a morbidade e a mortalidade, e identifica as situações que o médico de família deve institui-la. Unidade 6 do módulo 7 que compõe o Curso de Especialização em Saúde da Família.