Organotypic in vitro models of human cutaneous squamous cell carcinoma

Skin cancer is the most common type of cancer in fair-skinned populations. Cutaneous squamous cell carcinoma (SCC) comprises about 15% of all skin cancer diagnoses. Treatment associated with the high and rising prevalence of cutaneous SCC puts an increasingly high financial burden on society, marking a pressing need for advancements in skin cancer drug development. For screening of novel therapeutics, representative models of human cutaneous SCC are required. The aim of the research described in this thesis was to develop a representative in vitro model of human SCC for screening therapeutics, without the unnecessary use of animals. To this end, we generated three-dimensional in vitro SCC models in which the malignant epidermal cancer cells were either represented by intact primary human cutaneous or by established, spontaneously immortalized cutaneous SCC cell lines. The dermal microenvironment in our models was seeded with either primary normal human dermal fibroblasts or primary SCC-associated fibroblasts. In verifying human cutaneous SCC representation by these in vitro models, we focused on hyperproliferation, cytological and architectural atypia and invasion as three main features of primary SCC. The in vitro skin cancer models presented in this thesis add to the spectrum of available in vitro models for therapeutic screening.Stichting Stimuleringsfonds Alternatieven voor Proefdieren, Stichting Proefdiervrij, Animal Free Research, Aeon Astron Europe B.V., GlaxoSmithKline B.V., LEO Pharma B.V., Galderma AS Nederland, Louis Widmer Nederland B.V., Astellas Pharma B.V., Fagron B.V. and Merz Pharmaceuticals GmbHUBL - phd migration 201

Facing the phases of Mycobacterium tuberculosis : hunting for better TB vaccines

Currently, only one tuberculosis (TB) vaccine is available: Mycobacterium bovis Bacille Calmette-Gu_rin (BCG). This vaccine induces highly variable protection against pulmonary TB, the most common and contagious form of TB. There is an urgent need for an effective TB vaccine which is safe also in the immunocompromised host. The main focus of this thesis was to identify Mycobacterium tuberculosis (Mtb) infection phase related antigens and to evaluate these as potential antigens for TB vaccines. The studies presented in this thesis describe: (i) the immunogenic potential of two previously described sets of antigens; resuscitation promoting factor (Rpf) and dormancy regulon encoded (DosR) antigens, (ii) the identification and immunogenicity of a third set of antigens known as in vivo expressed Mtb (IVE-TB) antigens, (iii) the protective value of IVE-TB antigen Rv2034 and (iv) the analysis of Rv2034-specific T cell r esponses at the clonal level. Together, these data illustrate the vaccine potential of infection phase related antigens.UBL - phd migration 201

Phase Evolution and Li Diffusion in LATP Solid-State Electrolyte Synthesized via a Direct Heat-Cycling Method

Herein, the direct synthesis of phase-pure lithium aluminum titanium phosphate (Li_{1.3}Al_{0.3}Ti_{1.7}(PO_{4})_{3}, LATP) solid-electrolyte powder in 220 min and relatively low temperatures (850 °C) is achieved via a new (cyclic) fast heat treatment (c-FHT) route. The complex structural evolution highlights rate-limited lithium incorporation of intermediate metal phosphates formed prior to the final phase-pure LATP. The prepared LATP product powder displays similar bulk (2 × 10^{−10} cm^{2} s^{−1}) and local (3 × 10^{−10} cm^{2} s^{−1}) values for lithium diffusion coefficients (D_{Li}) characterized by electrochemical impedance spectroscopy and muon spin relaxation (μSR), respectively. The similarity between both D_{Li} values suggests excellent retention of inter- and intraparticle lithium diffusion, which is attributed to the absence of deleterious surface impurities such as AlPO4. A low-energy barrier (E_{a} = 73 meV) of lithium diffusion is also estimated from the μSR data

Assessment of left ventricular ejection fraction in patients eligible for ICD therapy: Discrepancy between cardiac magnetic resonance imaging and 2D echocardiography

OBJECTIVE: Implantable cardioverter defibrillators (ICD) and cardiac resynchronisation therapy (CRT) have substantially improved the survival of patients with cardiomyopathy. Eligibility for this therapy requires a left ventricular ejection fraction (LVEF) <35 %. This is largely based on studies using echocardiography. Cardiac magnetic resonance imaging (CMR) is increasingly utilised for LVEF assessment, but several studies have shown differences between LVEF assessed by CMR and echocardiography. The present study compared LVEF assessment by CMR and echocardiography in a heart failure population and evaluated effects on eligibility for device therapy. METHODS: 152 patients (106 male, mean age 65.5 ± 9.9 years) referred for device therapy were included. During evaluation of eligibility they underwent both CMR and echocardiographic LVEF assessment. CMR volumes were computed from a stack of short-axis images. Echocardiographic volumes were computed using Simpson’s biplane method. RESULTS: The study population demonstrated an underestimation of end-diastolic volume (EDV) and end-systolic volume (ESV) by echocardiography of 71 ± 53 ml (mean ± SD) and 70 ± 49 ml, respectively. This resulted in an overestimation of LVEF of 6.6 ± 8.3 % by echocardiography compared with CMR (echocardiographic LVEF 31.5 ± 8.7 % and CMR LVEF 24.9 ± 9.6 %). 28 % of patients had opposing outcomes of eligibility for cardiac device therapy depending on the imaging modality used. CONCLUSION: We found EDV and ESV to be underestimated by echocardiography, and LVEF assessed by CMR to be significantly smaller than by echocardiography. Applying an LVEF cut-off value of 35 %, CMR would significantly increase the number of patients eligible for device implantation. Therefore, LVEF cut-off values might need reassessment when using CMR

Study on serious road traffic injuries in the EU

The general objective of this study is to collect knowledge that will enable the future identification of measures for effective prevention of serious road traffic injuries. The specific objective is to provide fact-based analysis on the most common circumstances and types of road traffic crashes leading to serious injuries of MAIS3+ severity. More specifically, the study is directed at providing an understanding of the main circumstances and factors that affect the emergence of serious road traffic injuries, medically coded as MAIS3+, for the following road traffic modes in the EU: pedestrians, bicyclists, motorcyclists and car occupants

Giving the Green Light to Photochemical Uncaging of Large Biomolecules in High Vacuum

The isolation of biomolecules in a high vacuum enables experiments on fragile species in the absence of a perturbing environment. Since many molecular properties are influenced by local electric fields, here we seek to gain control over the number of charges on a biopolymer by photochemical uncaging. We present the design, modeling, and synthesis of photoactive molecular tags, their labeling to peptides and proteins as well as their photochemical validation in solution and in the gas phase. The tailored tags can be selectively cleaved off at a well-defined time and without the need for any external charge-transferring agents. The energy of a single or two green photons can already trigger the process, and it is soft enough to ensure the integrity of the released biomolecular cargo. We exploit differences in the cleavage pathways in solution and in vacuum and observe a surprising robustness in upscaling the approach from a model system to genuine proteins. The interaction wavelength of 532 nm is compatible with various biomolecular entities, such as oligonucleotides or oligosaccharides.</p

Analysis of host responses to Mycobacterium tuberculosis antigens in a multi-site study of subjects with different TB and HIV infection states in sub-Saharan Africa.

BACKGROUND: Tuberculosis (TB) remains a global health threat with 9 million new cases and 1.4 million deaths per year. In order to develop a protective vaccine, we need to define the antigens expressed by Mycobacterium tuberculosis (Mtb), which are relevant to protective immunity in high-endemic areas. METHODS: We analysed responses to 23 Mtb antigens in a total of 1247 subjects with different HIV and TB status across 5 geographically diverse sites in Africa (South Africa, The Gambia, Ethiopia, Malawi and Uganda). We used a 7-day whole blood assay followed by IFN-γ ELISA on the supernatants. Antigens included PPD, ESAT-6 and Ag85B (dominant antigens) together with novel resuscitation-promoting factors (rpf), reactivation proteins, latency (Mtb DosR regulon-encoded) antigens, starvation-induced antigens and secreted antigens. RESULTS: There was variation between sites in responses to the antigens, presumably due to underlying genetic and environmental differences. When results from all sites were combined, HIV- subjects with active TB showed significantly lower responses compared to both TST(-) and TST(+) contacts to latency antigens (Rv0569, Rv1733, Rv1735, Rv1737) and the rpf Rv0867; whilst responses to ESAT-6/CFP-10 fusion protein (EC), PPD, Rv2029, TB10.3, and TB10.4 were significantly higher in TST(+) contacts (LTBI) compared to TB and TST(-) contacts fewer differences were seen in subjects with HIV co-infection, with responses to the mitogen PHA significantly lower in subjects with active TB compared to those with LTBI and no difference with any antigen. CONCLUSIONS: Our multi-site study design for testing novel Mtb antigens revealed promising antigens for future vaccine development. The IFN-γ ELISA is a cheap and useful tool for screening potential antigenicity in subjects with different ethnic backgrounds and across a spectrum of TB and HIV infection states. Analysis of cytokines other than IFN-γ is currently on-going to determine correlates of protection, which may be useful for vaccine efficacy trials

The chemopreventive polyphenol Curcumin prevents hematogenous breast cancer metastases in immunodeficient mice

Dissemination of metastatic cells probably occurs long before diagnosis of the primary tumor. Metastasis during early phases of carcinogenesis in high risk patients is therefore a potential prevention target. The plant polyphenol Curcumin has been proposed for dietary prevention of cancer. We therefore examined its effects on the human breast cancer cell line MDA-MB-231 in vitro and in a mouse metastasis model. Curcumin strongly induces apoptosis in MDA- MB- 231 cells in correlation with reduced activation of the survival pathway NF kappa B, as a consequence of diminished I kappa B and p65 phosphorylation. Curcumin also reduces the expression of major matrix metalloproteinases (MMPs) due to reduced NF kappa B activity and transcriptional downregulation of AP-1. NF kappa B/p65 silencing is sufficient to downregulate c-jun and MMP expression. Reduced NF kappa B/AP-1 activity and MMP expression lead to diminished invasion through a reconstituted basement membrane and to a significantly lower number of lung metastases in immunodeficient mice after intercardiac injection of 231 cells (p=0.0035). 68% of Curcumin treated but only 17% of untreated animals showed no or very few lung metastases, most likely as a consequence of down-regulation of NF kappa B/AP-1 dependent MMP expression and direct apoptotic effects on circulating tumor cells but not on established metastases. Dietary chemoprevention of metastases appears therefore feasible. Copyright (c) 2007 S. Karger AG, Basel

Peripheral T Cell Cytokine Responses for Diagnosis of Active Tuberculosis

BACKGROUND: A test for diagnosis of active Tuberculosis (TB) from peripheral blood could tremendously improve clinical management of patients. METHODS: Of 178 prospectively enrolled patients with possible TB, 60 patients were diagnosed with pulmonary and 27 patients with extrapulmonary TB. The frequencies of Mycobacterium tuberculosis (MTB) specific CD4(+) T cells and CD8(+) T cells producing cytokines were assessed using overnight stimulation with purified protein derivate (PPD) or early secretory antigenic target (ESAT)-6, respectively. RESULTS: Among patients with active TB, an increased type 1 cytokine profile consisting of mainly CD4(+) T cell derived interferon (IFN)-γ was detectable. Despite contributing to the cytokine profile as a whole, the independent diagnostic performance of one cytokine producing T cells as well as polyfunctional T cells was poor. IFN-γ/Interleukin(IL)-2 cytokine ratios discriminated best between active TB and other diseases. CONCLUSION: T cells producing one cytokine and polyfunctional T cells have a limited role in diagnosis of active TB. The significant shift from a "memory type" to an "effector type" cytokine profile may be useful for further development of a rapid immune-diagnostic tool for active TB