Organic polymers S2218600, S2429901 and S2219200 (referred to as Polymer 1, Polymer 2 and Polymer 3, respectively) of varying toxic potential, designed for use in cosmetic aerosols, were used as model substances to predict inflammatory potential. In vivo inflammogenic potential was evaluated by assessment of inflammatory cell profile (alveolar macrophage (AM), polymorphonuclear neutrophil (PMN)) of broncho-alveolar lavage fluid (BAL) 24hrs after a single instillation of either 0.5 mg or 2 mg polymer in Sprague Dawley rats. Pro-inflammatory Minusil particles and non-inflammatory titanium dioxide (TiO2) particles were used as controls. For comparison, cultured rat NR8383 AM-like cells, human THP-1 monocyte cells or human monocyte derived macrophages were treated with polymer for 24 h and supernatants analysed for indicators of cytotoxicity and inflammatory mediator release. In addition, after 6 h treatment, gene changes in the rat lung tissue and also in the rat NR8383 alveolar macrophage cell line were assessed using microarray to analyse the entire rat genome. The in vivo studies showed that Polymer 1, Polymer 3 and Minusil caused significant PMN influx into BAL. Polymer 3 and Minusil caused a significant increase in AM number in BAL. Polymer 2 and TiO2 had no effect on BAL cell profile. BAL tumour necrosis factor-α (TNFα) and macrophage inflammatory protein-2 (MIP-2) levels were significantly increased following instillation of Polymer 3 and Minusil. Thus the polymers and particles were ranked for potential to cause pulmonary inflammation: Polymer 3 > Minusil > Polymer 1 > Polymer 2 > TiO2. In vitro studies using cultured rat NR8383 AM-like cells showed that the polymers and particles could be ranked similarly for cytotoxic potential and their ability to stimulate the release of both TNFα and MIP-2. Cultured human monocyte derived macrophages detected the pro-inflammatory abilities of Polymer 3, as measured by cytotoxic potential and ability to stimulate TNFα, interleukin-8 (IL-8) and macrophage inflammatory protein-1α (MIP-1α), however, did not detect the pro-inflammatory abilities of Polymer 1. Cultured human THP-1 cells predicted the pro-inflammatory effects of Polymer 3 in rat lungs using the cytotoxicity assay and by changes in IL-1β, MIP-1α and IL-10 levels. The human THP-1 cell line did not predict the pro-inflammatory effects of Polymer 1 that were observed the rat lungs. Electron spin resonance (ESR) detected free radicals produced by the pro-inflammatory polymers and particles which had the ability to break bonds in super-coiled DNA and deplete intracellular glutathione (GSH). Microarray analysis of the canonical pathways activated by the pro-inflammatory polymers, Polymer 1 and Polymer 3, showed that 3 similar pathways were significantly activated in the instilled rats and the rat NR8383 AM-like cells following treatment. ‘Xenobiotic metabolism’, ‘IL-10 signalling’ and ‘leukocyte extravasation signalling’ pathways were significantly changed by the pro-inflammatory polymers. Use of these cell model alternatives in an industrial setting will refine and reduce in vivo testing and as these models are further developed and used alongside future new alternatives they will provide a substantial contribution towards the replacement of animal testing.

Les processus de neuro-inflammation jouent un rôle majeur dans la maladie d'Alzheimer (MA). Des études génétiques récentes démontrent cette association entre neuro-inflammation et MA et impliquent notamment un gène, TREM2, qui code pour un récepteur exprimé à la surface de la microglie. La tauopathie est une lésion caractéristique de la MA. Elle se traduit par l’hyperphosphorylation et l’agrégation intraneuronale de la protéine Tau. Les travaux sur le rôle de TREM2 dans le développement de la pathologie Tau sont peu nombreux et donnent des résultats contradictoiresAinsi, l’objectif de ma thèse est d’étudier le rôle de la neuroinflammation et de TREM2 dans la progression de la tauopathie, dans deux modèles différents. Le premier est obtenu par injection stéréotaxique de vecteurs AAV dans la couche CA1 de l’hippocampe de souris déficientes ou non en TREM2. Ces vecteurs entrainent la surexpression de différentes formes de la protéine Tau humaine et permettent de récapituler les différents stades de la tauopathie.Nous avons en parallèle utilisé un modèle transgénique plus progressif de tauopathie, la souris THY-Tau22, afin d’étudier l’influence du stade de la pathologie dans l’effet provoqué par une déficience en TREM2 sur l’évolution de la pathologie. Notre étude a mis en évidence la toxicité des formes solubles de Tau dans le modèle AAV par rapport à ses formes agrégées. Le modèle transgénique THY-Tau22 nous a permis de mettre en évidence une augmentation des lésions tauopathiques dans les souris déficientes en TREM2 par rapport aux souris qui ne le sont pas, uniquement à un stade avancé. Cela suggère que, à l’instar des modèles amyloïdes, l’effet de la déficience en TREM2 sur le décours de la tauopathie est différent en fonction du stade considéré. / Neuroinflammation processes appear to play a major role in Alzheimer's disease (AD). Recent genetic studies support this correlation between neuroinflammation and AD and include a gene, TREM2, expressed on microglial surface. Tauopathy is a characteristic lesion of AD. It results in hyperphosphorylation and intraneuronal aggregation of Tau protein. In the literature, only few articles describe the role of TREM2 in the development of Tau pathology, and they report contradictory results. We therefore do not know for sure whether a deficiency in TREM2 has a deleterious effect or not on tauopathy. Thus, the goal of my thesis is to study the role of neuroinflammation and TREM2 in the progression of tauopathy, in two different models. The first is obtained by stereotaxic injection of AAV vectors into the CA1 layer of the hippocampus of TREM2-deficient or non-deficient mice. These vectors lead to the overexpression of different forms of the human tau protein, thus making it possible to recapitulate the different tauopathy stages.In parallel, we used a more progressive trangenic model of tauopathy, the THY-Tau22 mouse, to study the influence of TREM2 deficiency at different stage of the pathology. Our study demonstrated the toxicity of Tau soluble forms in the AAV model compared to its aggregated forms. The THY-Tau22 transgenic model allowed us to demonstrate an increase in tauopathic lesions in TREM2 deficient mice compared to wild type mice, at late stage only. This suggests that, similar to amyloid models, the effect of TREM2 deficiency on the course of tauopathy is influenced by the stage of the disease.

Cancer is responsible for upward of 13% of human deaths. Contemporary chemotherapy of disseminated cancer is often thwarted by dose limiting systemic toxicity and by multi-drug resistance (MDR). Riminophenazines are a novel class of potential anticancer agents that possess a potent multi-mechanistic antineoplastic action. Apart from their broad action against intrinsic, non-classical resistance, Riminophenazines inhibit the action of Pgp and hypothetically all ABC transporters demonstrating their great utility against classical MDR. Considering that combination chemotherapy is the norm, the vision directing R&D efforts was that Riminophenazines could be used with benefit within many standard chemotherapeutic regimes. The strategic intent of this project was to attain improved therapeutic benefit for patients through gains in both pharmaco dynamic and pharmacokinetic specificity for cancer cells over what is currently available. Tactically, this was driven through the use of synergistic Fixed-Ratio Drug Combinations (FRDC) encapsulated within tumour-targeting Nanoparticulate Drug Delivery Systems (NDDS). Long-term aims of this R&D project were to: 1) Screen FRDC of clofazimine (B663) and the lead derivative (B4125) with etoposide, paclitaxel and vinblastine for synergistic drug interactions in vitro. 2) Design, assemble and characterize a novel nanoparticulate, synergistic, anticancer co-formulation. 3) Evaluate the in vivo safety and efficacy of the developed product/s in accordance with international regulatory guidelines. Using the median effect and combination index equations, impressive in vitro synergistic drug interactions (CI<1) were shown for various FRDC of the three standard chemotherapeutics tested (etoposide, paclitaxel and vinblastine) in combination with either B663 or B4125 against MDR neoplastic cell cultures. Considering in vitro results and with the view to advance quickly to clinical studies, the already approved clofazimine (B663) was elected as the combination partner for paclitaxel (PTX). Considering the potency and wide action of PTX, a novel coformulation (designed to circumvent drug resistance) has the potential to greatly impact upon virtually all cancer types, particularly if selectively delivered through innovative delivery systems and loco-regional administration. A passively tumour targeting, micellular NDDS system called Riminocelles™ that encapsulates a synergistic FRDC of B663 and PTX has been designed, assembled using thin film hydration methods and characterized in terms of drug loading, particle size, zeta potential, CMC and drug retention under sink conditions. An acute toxicity and a GLP repeat dose toxicity study confirmed Riminocelles to be well tolerated and safe at clinically relevant dosages whilst Taxol® (QDx7) produced statistically significant (P<0.05) weight loss within 14 days. The same study demonstrated statistically significant (P<0.05) tumour growth delays superior to that of Taxol at an equivalent PTX dosage of 10 mg/kg. Importantly, all components (amphiphiles and drugs) used in assembly of Riminocelles are already individually approved for medicinal use - this promotes accelerated development towards advanced clinical trials and successful registration. Although these results are very promising (outperforming Taxol), this system was however found in a pharmacokinetic study to suffer from in vivo thermodynamic instability due to the high concentration (abundance) of albumin present in plasma. For this reason, in vivo longevity within circulation, permitting passive tumour accumulation was not fully realized. A second NDDS called the RiminoPLUS™ imaging system was additionally developed. This lipopolymeric nanoemulsion system has successfully entrapped Lipiodol® Ultra fluid (an oil based contrast agent) within the hydrophobic core of a monodisperse particle population with a size of roughly 100 nm and a stability of one week. This formulation is therefore thought capable of CT imaging of tumour tissue and drug targeting after either intravenous or loco-regional injection. In vivo proof of the imaging concept is warranted. The results of this study serve to highlight the great potential of in vitro optimized synergistic FRDC against drug resistant cancers. Lipopolymeric micelles are an effective way to formulate multiple hydrophobic drugs for intravenous administration and present a means by which cancer can be readily targeted; provided that the delivery system possess the prerequisite in vivo stability and surface attributes. Further experiments exploring synergistic drug and biological combinations as well as “intelligent” NDDS actively guided through specific molecular recognition are called for. / Thesis (PhD)--University of Pretoria, 2012. / Pharmacology / unrestricted

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with worsening respiratory symptoms and physiological impairment. Pulmonary fibrosis is a chronic lung disease characterized by forming scar tissue (fibrosis) in the lungs. Alternatively activated macrophages (M2) known as pro-fibrotic are known to contribute to the fibrotic remodeling of the lung. In addition to the polarization of slices from naïve to pro-fibrotic, the addition of anti-fibrotic therapeutics reprogram slices back to a naïve condition. To polarize the slices, naïve slices are incubated with a previously investigated method in the lab known as the polarization cocktail. The polarization cocktail can be achieved by adding of IL-4, IL-6 and IL-13 to naïve(M0) slices in the Precision Cut lung slice (PCLS) model. For the therapeutic model, slices are incubated with the polarization cocktail and subsequently with the therapeutic. Our results have shown that the precision cut lung slice model can mimic previously investigated in-vivo experiments with the polarization cocktail. Secondly, the addition of therapeutics result in the slices exhibiting lower amounts of M2 markers and arginase activity concluding the model is suitable for the polarization and reprogramming of macrophages. / Thesis / Master of Science in Medical Sciences (MSMS)

Le lymphome à cellules du manteau (LCM) est une hémopathie maligne B mature, appartenant à la famille des lymphomes non hodgkiniens. Le LCM est caractérisé par la translocation t(11;14)(q13;q32) qui provoque une expression aberrante de cycline D1. C’est une pathologie rare mais à haut risque de rechute, et qui reste le plus souvent incurable suite à l’apparition de clones chimiorésistants. L’acquisition de résistance est intimement liée aux interactions entre les cellules tumorales et leur microenvironnement. Afin de mimer de la manière la plus pertinente possible ces interactions, nous avons mis en place un modèle murin de xénogreffe en utilisant les lignées cellulaires de LCM JeKo1, REC1, Z138 et Granta-519 que nous avons modifiées afin qu’elles expriment un fluorophore (GFP ou m-cherry) et/ou le gène codant pour la luciférase. Après injection aux souris du substrat de la luciférase, la luciférine, nous sommes en mesure de suivre au cours du temps la progression tumorale. Nous pouvons également évaluer le degré d’infiltration tumorale dans la moelle osseuse, la rate, le cerveau et le sang après euthanasie des animaux, par des techniques de cytométrie en flux et d’immunocytochimie. Ce modèle nous a permis de montrer l’intérêt thérapeutique d’un inhibiteur de l’exportine 1 (XPO1) : le KPT 330 (ou selinexor) qui est capable de contenir cycline D1 uniquement au niveau nucléaire. Nous avons montré que la localisation subcellulaire de cycline D1, est retrouvée majoritairement cytoplasmique dans certaines lignées cellulaires de LCM (2/7) et chez un certain nombre de patients (6/42, 14%), et est associée à un fort potentiel d’invasion, de migration et à un phénotype agressif. Par ailleurs, grâce à ce modèle, nous avons pu objectiver le manque d’efficacité in vivo d’agonistes aux récepteurs aux œstrogènes de type β (ER β). Ces récepteurs, présents sur les lymphocytes B étaient supposés inhiber la prolifération cellulaire et provoquer la mort des cellules par apoptose. L’utilisation de deux agonistes des ER β, le diarylpropionitrile (DPN) et l’ERB-041 a montré une absence d’effet de ces molécules, lorsque les cellules tumorales sont au contact de leur microenvironnement. D’autre part, afin de mieux comprendre les mécanismes de résistance aux chimiothérapies, nous avons étudié la résistance de la lignée cellulaire REC-1 traitée par des agents génotoxiques. Nous avons montré que cette lignée présentait une anomalie de dégradation de cycline D1 associée à une activité diminuée du protéasome 26S. Enfin, nous avons montré dans des travaux préliminaires que la protéine fused in sarcoma (FUS) pourrait, lorsqu’elle est associée à cycline D1, être capable de réguler les voies de réparation des dommages à l’ADN. Des anomalies de ces voies induisent une grande instabilité génétique responsable de l’échappement des tumeurs aux traitements, le ciblage de FUS pourrait par conséquent présenter un intérêt thérapeutique.Pris dans leur ensemble, ces résultats permettent de renforcer ou d’infirmer l’intérêt de certaines cibles thérapeutiques dans l’espoir de pouvoir continuer à améliorer la prise en charge des patients. Ils fournissent également un outil pour l’évaluation de nouvelles molécules dans un modèle murin prenant en compte les interactions entre la cellule tumorale et son microenvironnement. / Mantle cell lymphoma (MCL) is a mature malignant hemopathy, belonging to the non-Hodgkin's lymphoma family. The MCL is characterized by the translocation t(11;14)(q13;q32) which causes an aberrant expression of cyclin D1. It is a rare disease but at high risk of relapse, and it is most often incurable due to the appearance of chemoresistant clones. The acquisition of resistance is intimately linked to the interactions between the tumor cells and their microenvironment. In order to mimic, in the most relevant way, these interactions, we have implemented a mouse xenograft model using the MCL cell lines JeKo1, REC1, Z138 and Granta-519 which we have modified so that they express a fluorophore (GFP or m-cherry) and / or the gene encoding the luciferase. After injection to the mice of the luciferase substrate, luciferin, we are able to follow over time the tumor progression. We can also assess the degree of tumor infiltration in bone marrow, spleen, brain and blood after euthanasia of animals, by flow cytometry and immunocytochemistry. This model allowed us to show the therapeutic interest of an inhibitor of exportin 1 (XPO1): the KPT 330 (or selinexor) which is able to contain cyclin D1 only on the nuclear level. We have shown that the subcellular localization of cyclin D1 is mainly cytoplasmic in some LCM (2/7) cell lines and in a number of patients (6/42, 14%), and is associated with a high potential Invasion, migration and an aggressive phenotype. Moreover, thanks to this model, we have been able to objectify the in vivo lack of efficacy of agonists to β-type estrogen receptors (ER β). These receptors, present on B lymphocytes, were thought to inhibit cell proliferation and cause cell death by apoptosis. The use of two ER β agonists, diarylpropionitrile (DPN) and ERB-041 showed an absence of effect of these molecules, when the tumor cells are in contact with their microenvironment. On the other hand, in order to better understand the mechanisms of resistance to chemotherapies, we studied the resistance of the REC-1 cell line treated with genotoxic agents. We have shown that this line has an abnormality of cyclin D1 degradation associated with decreased activity of the 26S proteasome. Finally, we have shown in preliminary work that the fused in sarcoma protein (FUS) could, when associated with cyclin D1, be able to regulate the repair pathways of DNA damage. Abnormalities of these pathways induce a great genetic instability responsible for the escape of tumors to treatments, the targeting of FUS could therefore be of therapeutic interest.Taken as a whole, these results reinforce or invalidate the interest of certain therapeutic targets in the hope of continuing to improve the management of patients. They also provide a tool for evaluating new molecules in a murine model that takes into account the interactions between the tumor cell and its microenvironment.