Microarray-basierte Genexpressionsanalyse zur Untersuchung immunologisch relevanter Vorgänge in Tumoren

Molecularly defined immunotherapies against cancer require the identification of appropriate tumor associated antigens (TAAs). Against this background, patient-individual TAAs for renal cell carcinoma were identified by whole-genome mRNA expression analysis of tumors, autologous normal kidney, and a selection of other normal tissues using oligonucleotide microarrays. These results were applied to HLA ligands simultaneously identified on the patients' tumors in order to define peptide vaccines aiming at the generation of anti-tumor cytotoxic T lymphocyte (CTL) responses. In addition, gene expression analysis was used to demonstrate that the overall expression profiles of metastases are closely related to the primary tumor. This similarity justifies the approach of TAA identification using resected primary RCC tumors in order to target metastases with the so defined vaccination cocktail. This approach for TAA identification was also applied to colorectal carcinoma, which is a heterogeneous tumor species requiring the careful isolation of tumor cells and corresponding normal epithelial cells in order to obtain tumor cell-specific expression data. Laser microdissection followed by mRNA amplification was employed to generate microarray data and identify candidate TAAs. Impaired RNA quality is a frequent problem in this setting but it could be shown that moderate levels of degradation may still lead to meaningful microarray results. So far, it was unclear whether MHC class II positive tumors are able to directly present peptides of TAAs to CD4+ T helper cells. We could show that autophagy is a process enabling the presentation of MHC-II peptides from intracellular source proteins in general, thereby providing a mechanism for the potential recognition of tumors by CD4+ T cells. These results show how TAAs on MHC-II might arise and illustrates how comprehensive gene expression analysis may contribute to the elucidation of such a fundamental process, which likely has important implications for tumor immunology and cancer immunotherapy.Jede molekular definierte Immuntherapie gegen Krebs erfordert die Identifizierung geeigneter tumor-assoziierter Antigene (TAAs). Vor diesem Hintergrund wurden patientenindividuelle TAAs beim Nierenzellkarzinom identifiziert. Dazu kam eine genomweite mRNA-Expressionsanalyse von Tumoren, autologem Nierennormalgewebe und einer Auswahl anderer Normalgewebe mittels Oligonukleotid-Microarrays zum Einsatz. Diese Ergebnisse wurden mit HLA-Liganden in Beziehung gesetzt, die zugleich auf Tumoren der Patienten identifiziert wurden, um Peptidvakzine zur Erzeugung einer gegen den Tumor gerichteten Immunantwort durch cytotoxische T-Lymphocyten (CTLs) zu definieren. Zusätzlich wurde mit Hilfe der Genexpressionsanalyse gezeigt, dass die Gesamtexpressionsprofile von Metastasen denen eines Primärtumors sehr ähnlich sind. Diese Ähnlichkeit rechtfertigt die Identifizierung von TAAs auf operativ entfernten Primärtumoren, um mit dem so definierten Vakzin Metastasen anzugreifen. Diese Strategie zur Identifizierung von TAAs wurde auch auf das kolorektale Karzinom angewandt. Es stellt eine heterogene Tumorspezies dar, die eine sorgfältige Isolierung von Tumor- und korrespondierenden normalen Epithelzellen erfordert, um tumorzellspezifische Daten zu ermöglichen. Deshalb wurde ein Verfahren zur lasergestützten Mikrodissektion eingesetzt, um Microarray-Daten zu erhalten und potenzielle TAAs zu identifizieren. In diesem Zusammenhang tritt häufig das Problem auf, dass die gewonnene RNA in ihrer Qualität beeinträchtigt ist. Es konnte aber gezeigt werden, dass ein gewisses Maß an RNA-Abbau immer noch zu aussagekräftigen Microarray-Daten führen kann. Bisher war unklar, ob MHC Klasse II-positive Tumore Peptide von TAAs direkt CD4+ T-Helferzellen präsentieren können. Wir konnten zeigen, dass Autophagie ein Vorgang ist, der die Präsentation von MHC II-Peptiden aus intrazellulären Quellproteinen allgemein ermöglicht. Dies könnte ein Mechanismus für die Erkennung von Tumoren durch CD4+ T-Zellen sein. Diese Ergebnisse zeigen, wie TAAs auf MHC II entstehen könnten und liefern ein Beispiel dafür, wie umfangreiche Genexpressionsanalysen zur Aufklärung solch eines grund-legenden Prozesses beitragen können, der möglicherweise wichtige Auswirkungen für Tumorimmunologie und Krebsimmuntherapie haben könnte

CD8+ T cells specific for a potential HLA-A*0201 epitope from Chlamydophila pneumoniae are present in the PBMCs from infected patients

Infection with the common pathogen Chlamydophila pneumoniae (Cpn, previously Chlamydia pneumoniae) has a high prevalence in patients suffering from arteriosclerosis and may trigger or contribute to heart disease. In mice, CD8-positive T cells are critical for the eradication of the infection and the development of immune memory against Cpn. Although several H2-class I epitopes have been described, no HLA-class I-associated peptides from Cpn are known. In order to define HLA-A*0201 epitopes from Cpn, we focused on the bacterial heat shock proteins (HSP) 60 and 70 which are known to be recognized by the immune system. Using epitope prediction, peptide binding studies and peptide-specific CTLs from HLA-A2 transgenic mice, we could define a potential HSP-70-derived epitope. The study of PBMCs from Cpn-infected individuals using fluorescent MHC tetramers revealed that some patients have CD8+ T cells capable of recognizing the Cpn HSP-70 HLA-A*0201 epitope. Our studies pave the way to the immunomonitoring of the anti-Cpn CTL immune response present in patients suffering from different diseases potentially linked to Cpn or anti-Cpn immunit

Exploiting the glioblastoma peptidome to discover novel tumour-associated antigens for immunotherapy

Peptides presented at the cell surface reflect the protein content of the cell; those on HLA class I molecules comprise the critical peptidome elements interacting with CD8 T lymphocytes. We hypothesize that peptidomes from ex vivo tumour samples encompass immunogenic tumour antigens. Here, we uncover >6000 HLA-bound peptides from HLA-A*02+ glioblastoma, of which over 3000 were restricted by HLA-A*02. We prioritized in-depth investigation of 10 glioblastoma-associated antigens based on high expression in tumours, very low or absent expression in healthy tissues, implication in gliomagenesis and immunogenicity. Patients with glioblastoma showed no T cell tolerance to these peptides. Moreover, we demonstrated specific lysis of tumour cells by patients' CD8+ T cells in vitro. In vivo, glioblastoma-specific CD8+ T cells were present at the tumour site. Overall, our data show the physiological relevance of the peptidome approach and provide a critical advance for designing a rational glioblastoma immunotherapy. The peptides identified in our study are currently being tested as a multipeptide vaccine (IMA950) in patients with glioblastom

The development of a multidisciplinary fall risk evaluation tool for demented nursing home patients in the Netherlands

BACKGROUND: Demented nursing home patients are at high risk for falls. Falls and associated injuries can have a considerable influence on the autonomy and quality of life of patients. The prevention of falls among demented patients is therefore an important issue. In order to intervene in an efficient way in this group of patients, it is important to systematically evaluate the fall risk profile of each individual patient so that for each patient tailor-made preventive measures can be taken. Therefore, the objective of the present study is to develop a feasible and evidence based multidisciplinary fall risk evaluation tool to be used for tailoring preventive interventions to the needs of individual demented patients. METHODS: To develop this multidisciplinary fall risk evaluation tool we have chosen to combine scientific evidence on the one hand and experts' opinions on the other hand. Firstly, relevant risk factors for falling in elderly persons were gathered from the literature. Secondly, a group of Dutch experts in the field of falls and fall prevention in the elderly were consulted to judge the suitability of these risk factors for use in a multidisciplinary fall risk evaluation tool for demented nursing home patients. Thirdly, in order to generate a compact list of the most relevant risk factors for falling in demented elderly, all risk factors had to fulfill a set of criteria indicating their relevance for this specific target population. Lastly the final list of risk factors resulting from the above mentioned procedure was presented to the expert group. The members were also asked to give their opinion about the practical use of the tool. RESULTS: The multidisciplinary fall risk evaluation tool we developed includes the following items: previous falls, use of medication, locomotor functions, and (correct) choice and use of assistive and protective devices. The tool is developed for the multidisciplinary teams of the nursing homes. CONCLUSION: This evidence and practice based multidisciplinary fall risk evaluation tool targets the preventive interventions aimed to prevent falls and their negative consequences in demented nursing home patients

Non-pharmacological management of osteoporosis: a consensus of the Belgian Bone Club

This consensus article reviews the various aspects of the non-pharmacological management of osteoporosis, including the effects of nutriments, physical exercise, lifestyle, fall prevention, and hip protectors. Vertebroplasty is also briefly reviewed. Non-pharmacological management of osteoporosis is a broad concept. It must be viewed as an essential part of the prevention of fractures from childhood through adulthood and the old age. The topic also includes surgical procedures for the treatment of peripheral and vertebral fractures and the post-fracture rehabilitation. The present document is the result of a consensus, based on a systematic review and a critical appraisal of the literature. Diets deficient in calcium, proteins or vitamin D impair skeletal integrity. The effect of other nutriments is less clear, although an excessive consumption of sodium, caffeine, or fibres exerts negative effects on calcium balance. The deleterious effects of tobacco, excessive alcohol consumption and a low BMI are well accepted. Physical activity is of primary importance to reach optimal peak bone mass but, if numerous studies have shown the beneficial effects of various types of exercise on bone mass, fracture data as an endpoint are scanty. Fall prevention strategies are especially efficient in the community setting, but less evidence is available about their effectiveness in preventing fall-related injuries and fractures. The efficacy of hip protectors remains controversial. This is also true for vertebroplasty and kyphoplasty. Several randomized controlled studies had reported a short-term advantage of vertebroplasty over medical treatment for pain relief, but these findings have been questioned by recent sham-controlled randomized clinical studies

Common variants in Alzheimer’s disease and risk stratification by polygenic risk scores

Funder: Funder: Fundación bancaria ‘La Caixa’ Number: LCF/PR/PR16/51110003 Funder: Grifols SA Number: LCF/PR/PR16/51110003 Funder: European Union/EFPIA Innovative Medicines Initiative Joint Number: 115975 Funder: JPco-fuND FP-829-029 Number: 733051061Genetic discoveries of Alzheimer's disease are the drivers of our understanding, and together with polygenetic risk stratification can contribute towards planning of feasible and efficient preventive and curative clinical trials. We first perform a large genetic association study by merging all available case-control datasets and by-proxy study results (discovery n = 409,435 and validation size n = 58,190). Here, we add six variants associated with Alzheimer's disease risk (near APP, CHRNE, PRKD3/NDUFAF7, PLCG2 and two exonic variants in the SHARPIN gene). Assessment of the polygenic risk score and stratifying by APOE reveal a 4 to 5.5 years difference in median age at onset of Alzheimer's disease patients in APOE ɛ4 carriers. Because of this study, the underlying mechanisms of APP can be studied to refine the amyloid cascade and the polygenic risk score provides a tool to select individuals at high risk of Alzheimer's disease

Will climate mitigation ambitions lead to carbon neutrality? An analysis of the local-level plans of 327 cities in the EU

Cities across the globe recognise their role in climate mitigation and are acting to reduce carbon emissions. Knowing whether cities set ambitious climate and energy targets is critical for determining their contribution towards the global 1.5 °C target, partly because it helps to identify areas where further action is necessary. This paper presents a comparative analysis of the mitigation targets of 327 European cities, as declared in their local climate plans. The sample encompasses over 25% of the EU population and includes cities of all sizes across all Member States, plus the UK. The study analyses whether the type of plan, city size, membership of climate networks, and its regional location are associated with different levels of mitigation ambition. Results reveal that 78% of the cities have a GHG emissions reduction target. However, with an average target of 47%, European cities are not on track to reach the Paris Agreement: they need to roughly double their ambitions and efforts. Some cities are ambitious, e.g. 25% of our sample (81) aim to reach carbon neutrality, with the earliest target date being 2020.90% of these cities are members of the Climate Alliance and 75% of the Covenant of Mayors. City size is the strongest predictor for carbon neutrality, whilst climate network(s) membership, combining adaptation and mitigation into a single strategy, and local motivation also play a role. The methods, data, results and analysis of this study can serve as a reference and baseline for tracking climate mitigation ambitions across European and global cities

Common variants in Alzheimer's disease and risk stratification by polygenic risk scores.

Funder: Funder: Fundación bancaria ‘La Caixa’ Number: LCF/PR/PR16/51110003 Funder: Grifols SA Number: LCF/PR/PR16/51110003 Funder: European Union/EFPIA Innovative Medicines Initiative Joint Number: 115975 Funder: JPco-fuND FP-829-029 Number: 733051061Genetic discoveries of Alzheimer's disease are the drivers of our understanding, and together with polygenetic risk stratification can contribute towards planning of feasible and efficient preventive and curative clinical trials. We first perform a large genetic association study by merging all available case-control datasets and by-proxy study results (discovery n = 409,435 and validation size n = 58,190). Here, we add six variants associated with Alzheimer's disease risk (near APP, CHRNE, PRKD3/NDUFAF7, PLCG2 and two exonic variants in the SHARPIN gene). Assessment of the polygenic risk score and stratifying by APOE reveal a 4 to 5.5 years difference in median age at onset of Alzheimer's disease patients in APOE ɛ4 carriers. Because of this study, the underlying mechanisms of APP can be studied to refine the amyloid cascade and the polygenic risk score provides a tool to select individuals at high risk of Alzheimer's disease