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• W130169008 abstract "Dendritic Cells (DCs) derived from human blood monocytes that have been nurtured inGM-CSF and IL-4, followed by maturation in a monocyte-conditioned medium, are themost potent APCs known. These DCs have many features of primary DCs, including theexpression of molecules that enhance antigen capture and selective receptors that guideDCs to and from several sites in the body, where they elicit the T cell mediated immuneresponse.For these features, immature DCs (iDC) loaded with tumor antigen and matured (mDC)with a standard cytokine cocktail, are used for therapeutic vaccination in clinical trials ofdifferent cancers.However, the efficacy of DCs in the development of immunocompetence is criticallyinfluenced by the type (whole lysate, proteins, peptides, mRNA), the amount and the timeof exposure of the tumor antigens used for loading in the presentation phase.The aim of the present study was to create instruments to acquire more information aboutDC antigen uptake and presentation mechanisms to improve the clinical efficacy of DCbasedvaccine.In particular, two different tumor antigen were studied: the monoclonal immunoglobulin(IgG or IgA) produced in Myeloma Multiple, and the whole lysate obtained from melanomatissues. These proteins were conjugated with fluorescent probe (FITC) to evaluate thekinetic of tumor antigen capturing process and its localization into DCs, by cytofluorimetricand fluorescence microscopy analysis, respectively.iDC pulsed with 100μg of IgG-FITC/106 cells were monitored from 2 to 22 hours afterloading. By the cytofluorimetric analysis it was observed that the monoclonal antibody wascompletely captured after 2 hours from pulsing, and was decreased into mDC in 5 hoursafter maturation stimulus.To monitor the lysate uptake, iDC were pulsed with 80μg of tumor lysate/106 cells, thenwere monitored in the 2h to 22 hours interval time after loading.Then, to reveal difference between increasing lysate concentration, iDC were loaded with20-40-80-100-200-400μg of tumor lysate/106 cells and monitored at 2-4-8-13h frompulsing.By the cytofluorimetric analysis, it was observed that, the 20-40-80-100μg uptake, after 8hours loading was completed reaching a plateau phase. For 200 and 400μg the meanfluorescence of cells increased until 13h from pulsing.The lysate localization into iDC was evaluated with conventional and confocalfluorescence microscopy analysis. In the 2h to 8h time interval from loading an intensiveand diffuse fluorescence was observed within the cytoplasmic compartment. Moreover,after 8h, the lysate fluorescence appeared to be organized in a restricted cloudy-shadedarea with a typical polarized aspect. In addition, small fluorescent spots clearly appearedwith an increment in the number and fluorescence intensity.The nature of these spot-like formations and cloudy area is now being investigateddetecting the colocalization of the fluorescence lysate and specific markers for lysosomes,autophagosomes, endoplasmic reticulum and MHCII positive vesicles." @default.
• W130169008 created "2016-06-24" @default.
• W130169008 creator A5002734384 @default.
• W130169008 date "2008-06-06" @default.
• W130169008 modified "2023-09-23" @default.
• W130169008 title "Marcatura di molecole biologiche a funzione antigenica per lo studio e la caratterizzazione di protocolli di vaccinoterapia in oncologia medica" @default.
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• W130169008 doi "https://doi.org/10.6092/unibo/amsdottorato/692" @default.
• W130169008 hasPublicationYear "2008" @default.
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