Development of screening assays to test novel integrin antagonists in allergic inflammation


Autoria(s): Spartà, Antonino Maria
Contribuinte(s)

Spampinato, Santi Mario

Data(s)

05/05/2009

Resumo

Aim of the research: to develop a prototype of homogeneous high-throughput screening (HTS) for identification of novel integrin antagonists for the treatment of ocular allergy and to better understand the mechanisms of action of integrin-mediated levocabastine antiallergic action. Results: This thesis provides evidence that adopting scintillation proximity assay (SPA) levocabastine (IC50=406 mM), but not the first-generation antihistamine chlorpheniramine, displaces [125I]fibronectin (FN) binding to human a4b1 integrin. This result is supported by flow cytometry analysis, where levocabastine antagonizes the binding of a primary antibody to integrin a4 expressed in Jurkat E6.1 cells. Levocabastine, but not chlorpheniramine, binds to a4b1 integrin and prevents eosinophil adhesion to VCAM-1, FN or human umbilical vein endothelial cells (HUVEC) cultured in vitro. Similarly, levocabastine affects aLb2/ICAM-1-mediated adhesion of Jurkat E6.1 cells. Analyzing the supernatant of TNF-a-treated (24h) eosinophilic cells (EoL-1), we report that levocabastine reduces the TNF-a-induced release of the cytokines IL-12p40, IL-8 and VEGF. Finally, in a model of allergic conjunctivitis, levocastine eye drops (0.05%) reduced the clinical aspects of the early and late phase reactions and the conjunctival expression of a4b1 integrin by reducing infiltrated eosinophils. Conclusions: SPA is a highly efficient, amenable to automation and robust binding assay to screen novel integrin antagonists in a HTS setting. We propose that blockade of integrinmediated cell adhesion might be a target of the anti-allergic action of levocabastine and may play a role in preventing eosinophil adhesion and infiltration in allergic conjunctivitis.

Formato

application/pdf

Identificador

http://amsdottorato.unibo.it/1613/1/FILE_COMPLETO_CON_INDICE_E_FRONTESPIZIO_130309.pdf

urn:nbn:it:unibo-1363

Spartà, Antonino Maria (2009) Development of screening assays to test novel integrin antagonists in allergic inflammation, [Dissertation thesis], Alma Mater Studiorum Università di Bologna. Dottorato di ricerca in Biotecnologie cellulari e molecolari <http://amsdottorato.unibo.it/view/dottorati/DOT210/>, 21 Ciclo. DOI 10.6092/unibo/amsdottorato/1613.

Idioma(s)

en

Publicador

Alma Mater Studiorum - Università di Bologna

Relação

http://amsdottorato.unibo.it/1613/

Direitos

info:eu-repo/semantics/openAccess

Palavras-Chave #BIO/14 Farmacologia
Tipo

Tesi di dottorato

NonPeerReviewed