Development of novel vaccine carriers

In this work, combined interdisciplinary research in the fields of Pharmaceutical Technology, (Bio)Materials Science, and Biology is presented to understand and promote lyotropic liquid crystalline dispersions as novel vaccine carriers. The work was initiated with a detailed development of lyotropic mesophases based on phytantriol. The aim was to gain knowledge on the phase behavior of the system and also on the structural impact of additives. Therefore, formulations with increasing degree of complexity were sequentially characterized. The study started establishing a phase diagram, in which bulk phases of phytantriol in excess of highly purified water were characterized, upon temperature increase, applying polarization microscopy and small angle X-ray scattering (SAXS). Subsequently, pursuing the development of a system with immunostimulatory properties, the impact of mannide monooleate on phytantriol-based mesophases was investigated. Mannide monooleate is an emulsifier widely used in adjuvant formulations and found to promote immune responses in emulsion form. Thus, progressively higher amounts of mannide monooleate were incorporated into the system and the structural modifications were monitored upon temperature increase. The phase diagram allowed for a deep understanding of the mesophase behavior and enabled the selection of a feasible phytantriol/mannide monooleate ratio for the subsequent investigations. In this way, a stable formulation, featuring inverse hexagonal structure was identified. Next, to enable dispersion of the characterized bulk and to produce a nanoparticulate system, different steric stabilizers were evaluated. Here, the selection criteria were based on the dispersion efficiency at low concentrations and on the impact on the internal structure of the particles. The last part of the formulation development was concerning the application and customization of the formed hexosomes. Thus, the particle loading with the model antigens ovalbumin and lysozyme was studied, as well as strategies to introduce new features to the particulate system in a controlled way, preserving the inverse hexagonal structure. Here, negatively and positively charged hexosomes could be prepared through the incorporation of charged phospholipids into the formulation. Additionally, a formulation based on phytantriol and mannide monooleate, which typically formed an inverse hexagonal structure was shown to self-assemble with bicontinuous cubic double diamond structure upon addition of octyl-β-D-glucopyranoside. The aim of the second part of this work was the verification of one of the most important hypothesis with respect to the biological performance of non-lamellar lyotropic liquid crystalline dispersions. More specifically, it is known that non-lamellar structures are involved in fusogenic processes of the cell membrane. For this reason, it was speculated that particles internally structured with non-lamellar phases, such as hexosomes, could have fusogenic properties. Thus, to verify this hypothesis and elucidate the cellular uptake mechanism of hexosomes, we investigated the interactions of these particles with cells and with models of the cell membrane. For this purpose, the established hexosomes, based on phytantriol/mannide monooleate, were fluorescently labeled and their toxicity in cultures of HeLa cells was determined. Series of uptake experiments were carried out after suppression of the major endocytic pathways using highly specific approaches (e.g. single and double knockdown of regulatory proteins), and also inhibition strategies of broader effect (e.g. temperature reduction to 4°C, hypotonic treatment, cytochalasin D treatment). Additionally, experiments using models of the cell membrane (e.g. phospholipid monolayers and bilayers) were performed. All the analyses were carried out with hexosomes and liposomes in parallel to enable the comparison between particles featuring non-lamellar and lamellar structures, respectively. The results revealed a steeper toxicity curve and faster internalization kinetics for hexosomes in comparison to liposomes. Interestingly, against the expectations, indications of fusogenic properties were not observed. However, strong evidences that hexosomes have an alternative cell entry pathway that bypasses standard endocytosis were identified. In contrast, liposomes appeared to enter the cells through well-known endocytic pathways, such as caveolae-mediated and clathrin- mediated endocytosis. The final part of this work consisted of the evaluation of the biological performance of hexosomes within the vaccination context. The experiments were performed in vitro and in vivo, in parallel with the already established and promising cationic liposomal formulation CAF04, which is based on dimethyldioctadecylammonium (DDA) and on a synthetic analogue of monomycoloyl glycerol (MMG-1). Since MMG-1 and the positive charge of DDA are considered the core of the success of this formulation, differently charged hexosomes containing MMG-1 were developed and included in the study. The development process was driven by the lessons learned in the first phase of the project with phytantriol/mannide monooleate systems. In this way, to create particles internally structured with inverse hexagonal phase, MMG-1 was combined to phytantriol. After the establishment of the formulations, the immunogenicity of plain particles (without antigen) was investigated in vitro in cultures of dendritic cells derived from human peripheral blood mononuclear cells. Through the evaluation of the expression of several surface markers, it was found that, while CAF04 liposomes induced the upregulation of the homing receptor CCR7, all hexosomal formulations tested did not provoke any sign of adjuvanticity. Considering that the cultures of dendritic cells represent a dramatic simplification of the immune system, further experiments were performed in vivo. Thus, mice were immunized with the formulations loaded with Chlamydia trachomatis major outer membrane protein (MOMP) antigen. The immunological output was analyzed through several parameters (e.g. T-cell activation, cytokine expression and release, polyfunctionality, specific serum IgG) and all of them revealed dramatic differences between the immunizations performed with the unadjuvanted antigen and antigen adjuvanted with either liposomes or hexosomes. In comparison to the unadjuvanted systems, liposomes and hexosomes were both able to increase the magnitude of the immune responses. However, the immune responses elicited by CAF04 liposomes and by hexosomes were very distinct, the first eliciting mainly cellular mediated responses and the second humoral responses. Throughout this work, it was shown that stable and robust lipid-based lyotropic liquid formulations, that allow customization without impairment of the internal structure, can be rationally designed. In addition, keeping track of the structural modifications induced by each additive (separately) was shown to be a key strategy to enable further optimization in advanced stages of the formulation development. Regarding the performance of hexosomes in the biological environment, we have obtained accurate results demonstrating, in vitro and in vivo, drastic differences in comparison to liposomes. This intriguing outcome was observed for different formulations and in completely independent experiments. Overall, the insights presented in this thesis show hexosomes as promising delivery systems with uncommon properties and an applicability potential that goes beyond the vaccination context

Chemistry and personalized medicine – the research and development future of Europe

Personalized medicine may represent a dramatic change of paradigm in the medium-term future. For a chemist, personalized medicine means the definition and understanding of any disease on molecular level for each individual or group of individuals (personalized diagnosis) ideally leading to the design of a drug that efficiently counteracts or prevents any molecular dysfunction, ie, a personalized drug without side effects. The interdisciplinary research required for personalized medicine should overcome a myriad of obstacles not the least being to find specific biomarkers and targets for each individual or group of individuals suffering from a given disease. Chemists enter then into action and will model/ design drugs and drug delivery pathways for a personalized therapy. They will either tap into the numerous drugs candidates, which were abandoned at some stage of clinical trials, or synthesize new drugs, mainly those “small molecules” mimicking the activity of natural products. This view has obvious economic, ethical, and social implications, beyond scientific challenges. All stakeholders will have to take them into account. Policy makers will have to examine all disciplines of regulatory science among which the thorny economics (cost-benefit analysis of specific research and development projects) are of paramount importance and critical to the development of personalized medicine

Chemistry and personalized medicine – the research and development future of Europe

Personalized medicine may represent a dramatic change of paradigm in the medium-term future. For a chemist, personalized medicine means the definition and understanding of any disease on molecular level for each individual or group of individuals (personalized diagnosis) ideally leading to the design of a drug that efficiently counteracts or prevents any molecular dysfunction, ie, a personalized drug without side effects. The interdisciplinary research required for personalized medicine should overcome a myriad of obstacles not the least being to find specific biomarkers and targets for each individual or group of individuals suffering from a given disease. Chemists enter then into action and will model/ design drugs and drug delivery pathways for a personalized therapy. They will either tap into the numerous drugs candidates, which were abandoned at some stage of clinical trials, or synthesize new drugs, mainly those “small molecules” mimicking the activity of natural products. This view has obvious economic, ethical, and social implications, beyond scientific challenges. All stakeholders will have to take them into account. Policy makers will have to examine all disciplines of regulatory science among which the thorny economics (cost-benefit analysis of specific research and development projects) are of paramount importance and critical to the development of personalized medicine

Hat eine Koinfektion mit GBV-C Auswirkungen auf die Klinik von HIV?

Die Auswirkungen einer Koinfektion auf den Krankheitsverlauf einer HIV-Infektion werden bis heute kontrovers diskutiert. Ebenso sind die Mechanismen, mit dem GBV-C den Progress einer HIV-Infektion beeinflussen kann bisher nicht vollständig verstanden. Um zu klären, welchen Einfluss eine GBV-C-Koinfektion auf den Verlauf einer HIVInfektion ausübt, wurde im Rahmen dieser Dissertation eine 564 Patienten umfassende Studienpopulation aus zwei europäischen HIV-1 Kohorten untersucht. Hierzu wurde in einer eingehenden epidemiologischen Analyse überprüft, ob eine Koinfektion mit GBV-C einen Effekt auf verschiedene klinische, immunologische und virologische Parameter wie CDCStadien, opportunistische Infektionen, CD4+ T-Zellzahl und HIV-Viruslast ausübt. Um ein möglichst vollständiges Bild über mögliche Wechselwirkungen einer HIV/GBV-CKoinfektion zu erhalten, wurden die verschiedenen Genotypen von GBV-C, die GBV-CViruslast und das Vorhandensein der GBV-C-E2-Antikörper mit berücksichtigt. Da die Progression einer HIV-Erkrankung unter anderem von dem Zytokinmilieu des betreffenden Patienten abhängt, wurde die Auswirkung einer GBV-C-Koinfektion auf die Serumspiegel von IL-12, IP-10 und IFN-γ mittels ELISA überprüft. Ebenso kann die Expression der HIV-Korezeptoren CCR5 und CXCR4 und deren Liganden MIP-1α, MIP-1ß und RANTES, einen Einfluss auf verschiedene Parameter einer HIVInfektion ausüben, da eine Herunterregulation von CCR5 bzw. CXCR4 zu einer verringerten Infizierbarkeit der CD4- bzw. CD8-positiven Zellen führen kann. Die extrazelluläre Expression der HIV-Korezeptoren wurde durchflusszytometrisch bestimmt, während die Expression der Liganden MIP-1a, MIP 1b mittels ELISA analysiert wurde. Weiterhin wurde die Induzierbarkeit von RANTES durch GBV-C-Viruspartikel mittels eines von uns entwickelten Stimulations-Assay untersucht. Die eingehende epidemiologische Analyse zeigte bei den 130 GBV-C koinfizierten Patienten einen von HAART unabhängigen statistisch signifikanten Zusammenhang zwischen der GBV-C-Viruslast mit der CD4+ T-Zellzahl/μl und ein ebenso deutlich signifikant inversen Zusammenhang zwischen der GBV-C-Viruslast mit der HIV-Viruslast. Analysen in Bezug auf eine mögliche Abhängigkeit biologischer Effekte vom GBV-C-Genotyp zeigten, dass Patienten, die mit einem GBV-C Genotyp 1 Isolat koinfiziert waren, gegenüber allen anderen HIV bzw. HIV/GBV-C Genotyp 2 koinfizierten Patienten eine signifikant erniedrigte CD4+ T-Zellzahl aufwiesen. Bei der Analyse der Zytokin-Serumspiegel wurden unabhängig von HAART signifikant niedrigere IFN-γ Serumspiegel bei GBV-C koinfizierten Patienten im Vergleich zu HIV monoinfizierten Patienten beobachtet. Interessanterweise korrelierten die von uns beobachteten niedrigen IFN-γ Serumspiegel mit niedrigeren HIV-Viruslasten. Zusätzlich konnten signifikant erhöhte IP-10 Serumspiegel bei GBV-C Genotyp 1 koinfizierten Patienten festgestellt werden, die möglicherweise über einen noch näher zu klärenden Mechanismus Apoptose von CD4+ T-Zellen auslösen könnte. Wir konnten beobachten, dass der HIV-Korezeptor CXCR4 auf CD4+ T-Zellen in HIV/GBVC koinfizierten Patienten unabhängig von einer HAART im Vergleich zu HIV monoinfizierten Patienten mit fortgeschrittener Immundefizienz herabgesenkt war, während bei HIV monoinfizierten Patienten mit fortgeschrittenem Immundefekt die CCR5 Oberflächenexpression erhöht war. Schließlich konnten wir in unserer in vitro Analyse zeigen, dass GBV-C in der Lage ist RANTES, ein Ligand des CCR5 Korezeptors, Genotyp-spezifisch zu induzieren. Hierbei war auffällig, dass GBV-C Genotyp 1 Isolate nicht in der Lage waren, RANTES zu induzieren. Diese Befunde sprechen für einen eher negativen Einfluss einer GBV-C Genotyp 1 Koinfektion. Die Hypothese, dass die Genotyp-abhängigen Effekte auf Sequenzunterschiede in der GBVC- E2 Region zurückzuführen sind, konnte nicht bestätigt werden. Aus unseren Beobachtungen lässt sich schlussfolgern, dass eine GBV-C Infektion einen positiven, von der GBV-C-Viruslast und dem GBV-C-Genotyp abhängigen, Einfluss auf die Klinik von HIV ausübt und das dieser Einfluss zumindest teilweise durch Pathomechanismen erklärbar ist, die auf das Zytokinmilieu von IFN-γ und IP-10, die Genotyp abhängige Induzierbarkeit von RANTES, sowie auf die Oberflächenexpression von CCR5 und CXCR4 wirken

L1₂ ordering and δ′ precipitation in Al-Cu-Li

The precipitation mechanism of the δ′ (Al₃Li) phase in Al-Li alloys has been controversially discussed in recent decades, specifically with respect to a conjectured congruent ordering process. However, kinetics in the Al-Li system does not allow to resolve the intermediate stages of precipitation and hence to experimentally clarify this issue. In this paper, we are revisiting the subject in ternary Al-Cu-Li alloys with pronouncedly slower kinetics, employing Transmission Electron Microscopy, High-Angle Annular Dark-Field Scanning Transmission Electron Microscopy, Differential Scanning Calorimetry and Atom Probe Tomography. The results show clear evidence for congruent ordering in a selected compositional range, revealing an already strongly L1₂ ordered microstructure after natural aging with a chemically homogeneous Li distribution and a decomposition of the alloy upon annealing at elevated temperatures. The presented study of the δ′ precipitation evaluates the reaction pathway of this process and compares it to the predictions of the Bragg-Williams-Gorsky model with respect to decomposition and ordering in this alloy system

Validation d'un modèle d'évaluation de la publicité pour système-expert

International audienceLa validité d'un système-expert devrait en conditionner l'acceptation. Nous vérifierons dans cet article la validité du système-expert ESWA, destiné à l'évaluation de la publicité, en nous appuyant sur des résultats «objectifs» d'études de marché. Le «modèle simultané d'évaluation de l'impact de la publicité» (ESWA), récemment développé, sera dans le même temps soumis à un test empirique. A l'aide d'une simulation de type Monte Carlo, nous déterminerons enfin un seuil d'interprétation des résultats de validation d'un modèle d'impact de la publicité qui se fonde sur les sciences du comportement. 37 000 données d'utilisateurs et faits déduits de l'ESWA ainsi que 11 000 personnes — interrogées dans le cadre d'une étude de marché — seront prises en compte par cette analyse

The transfer of ownership of movables in the context of international contracts of sale with specific reference to reservation of title clauses : a comparative study

Abstract: Please refer to full text to view abstractLL.M. (International Commercial Law