Targeting of the C-Type Lectin Receptor Langerin Using Bifunctional Mannosylated Antigens

Langerhans cells (LCs) are antigen-presenting cells that reside in the skin. They uniquely express high levels of the C-type lectin receptor Langerin (CD207), which is an attractive target for antigen delivery in immunotherapeutic vaccination strategies against cancer. We here assess a library of 20 synthetic, well-defined mannoside clusters, built up from one, two, and three of six monomannosides, dimannosides, or trimannosides, appended to an oligopeptide backbone, for binding with Langerin using surface plasmon resonance and flow cytometric quantification. It is found that Langerin binding affinity increases with increasing number of mannosides. Hexavalent presentation of the mannosides resulted in binding affinities ranging from 3 to 12 mu M. Trivalent presentation of the dimannosides and trimannosides led to Langerin affinity in the same range. The model melanoma gp100 antigenic peptide was subsequently equipped with a hexavalent cluster of the dimannosides and trimannosides as targeting moieties. Surprisingly, although the bifunctional conjugates were taken up in LCs in a Langerin-dependent manner, limited antigen presentation to cytotoxic T cells was observed. These results indicate that targeting glycan moieties on immunotherapeutic vaccines should not only be validated for target binding, but also on the continued effects on biology, such as antigen presentation to both CD8(+)and CD4(+)T cells.Bio-organic Synthesi

Functional Characterization of Cultured Keratinocytes after Acute Cutaneous Burn Injury

In addition to forming the epithelial barrier against the outside environment keratinocytes are immunologically active cells. In the treatment of severely burned skin, cryoconserved keratinocyte allografts gain in importance. It has been proposed that these allografts accelerate wound healing also due to the expression of a favourable--keratinocyte-derived--cytokine and growth factor milieu. In this study the morphology and cytokine expression profile of keratinocytes from skin after acute burn injury was compared to non-burned skin. Skin samples were obtained from patients after severe burn injury and healthy controls. Cells were cultured and secretion of selected inflammatory mediators was quantified using Bioplex Immunoassays. Immunohistochemistry was performed to analyse further functional and morphologic parameters. Histology revealed increased terminal differentiation of keratinocytes (CK10, CK11) in allografts from non-burned skin compared to a higher portion of proliferative cells (CK5, vimentin) in acute burn injury. Increased levels of IL-1α, IL-2, IL-4, IL-10, IFN-γ and TNFα could be detected in culture media of burn injury skin cultures. Both culture groups contained large amounts of IL-1RA. IL-6 and GM-CSF were increased during the first 15 days of culture of burned skin compared to control skin. Levels of VEGF, FGF-basic, TGF-ß und G-CSF were high in both but not significantly different. Cryoconservation led to a diminished mediator synthesis except for higher levels of intracellular IL-1α and IL-1ß. Skin allografts from non-burned skin show a different secretion pattern of keratinocyte-derived cytokines and inflammatory mediators compared to keratinocytes after burn injury. As these secreted molecules exert auto- and paracrine effects and subsequently contribute to healing and barrier restoration after acute burn injury therapies affecting this specific cytokine/growth factor micromilieu could be beneficial in burned patients

Novelty Detection in Time Series

This report discusses novelty detection in time series. Particularly novelty detection with clustering and time series prediction using a neural network. The question discussed in this report is weather a clustering of static relations or the modelling of dynamics in time series provides a better approach for novelty detection.This question originates from a problem of novelty detection in radiotelescope data gathered by the NFRAJAstron and is posed as a hypothesis after introducing novelty detection in chapter 1.To answer the question we discuss how to handle time series, specially how to extract information from time series and what kind of novelties can be present in time series. Also unportant are measures for the quality of a novelty detection system. These topics are discussed in chapter 2.Another necessary ingredient for answering the question are implementations of the two approaches. The Kohonen self—organizing feature map is chosen as the most appropriate implementation for novelty detection based on clustering static information of a time series system, while a Tapped Delay Line Multilayer Perceptron is used for novelty detection based on time series prediction using dynamic information. The background of the used neural network architectures and experimental results of these two neural network methods are discussed in chapters 3 and 4. The results of the experiments and some of the discussed quality measures are used to give the final answer to the hypothesis stated in chapter 1. This answer is presented in chapter 5 together with recommendations for further research.

Detectie van antilichamen gericht tegen polymeren (APA) in serum van vrouwen met siliconen borstimplantaten. Introductie en reproduceerbaarheid van de test in het RIVM

Siliconen bevattende borst implantaten (SBI) worden veelvuldig gebruikt om medische en kosmetische redenen. De laatste tijd is een discussie op gang gekomen dat het aanwezig zijn van een siliconen borstimplantaat mogelijk kan leiden tot gezondheidsklachten. Recentelijk is een laboratoriumtest beschreven, die de APA-test genoemd wordt (Antipolymeer Antilichaam), die antilichamen in het serum gericht tegen polymeren detecteert (Tenenbaum et al, Lancet 1997; 349: 449-454). De hoogte van het antistofgehalte bleek gecorreleerd te zijn aan de ernst van de klachten bij vrouwen met een siliconen borstimplantaat. In het rapport wordt de introductie en reproduceerbaarheid van de APA-test in het RIVM beschreven. De resultaten verkregen in het RIVM werden vergeleken met de resultaten behaald door 'Autoimmune Technologies, LLC' (New Orleans, USA) (producent en patenthouder van de test). Referentie en test monsters werden verkregen van Autoimmune Technologies LCC. Vervanging van de door 'Autoimmune Technologies' bijgeleverde chemicalien door chemicalien van lokale leveranciers gaf dezelfde resultaten in de APA-test. Concluderend kan gezegd worden dat de APA-test succesvol geintroduceerd is in het RIVM. De variatie binnen de test, in zesvoud uitgevoerd op een dag en tussen verschillende testen uitgevoerd op zes verschillende dagen, lagen binnen een acceptabele marge. Om deze reden kan worden geconcludeerd dat de APA-test reproduceerbare uitslagen geeft en geschikt is om te gebruiken voor het onderzoek naar de aanwezigheid van antipolymeer antistoffen in serum van vrouwen met een siliconen borstimplantaat.Silicone breast implants (SBI) are widely used in women both for medical and cosmetic reasons. During the last decade there has been much debate whether the use of SBI is related to health complaints. Recently a laboratory assay has been described, designated the APA-assay ( Anti-Polymer Antibody), which detects antibodies directed to a polymer in serum of patients with SBI (Tenenbaum et al, 1997). The level of antipolymer antibodies was described to be associated to the severity of the clinical symptoms of SBI recipients. The report describes the introduction and performance of the APA-assay at the RIVM. The results obtained at the RIVM were compared to the results obtained by Autoimmune Technologies LLC, (New Orleans, USA) (manufacturer and patent holder of the assay). Reference and test samples were provided by Autoimmune Technologies LCC. Substitution of the chemicals supplied by Autoimmune Technologies, by chemicals obtained from local suppliers had no impact, simmilar results were observed using both sets of chemicals. In conclusion, the APA-assay was successfully introduced in our laboratory. Intra-assay variability and inter-assay variability were found to be within an acceptable range. Hence, the APA-assay gives reproducible results, and can be used for the evaluation of the presence of antipolymer antibodies in serum of women with a silicone breast implant.VWS/IG