Background: Although the Luminex single antigen flow beads (SAFB) and the flow cytometry crossmatch (FCXM) are the most sensitive assays used for anti-HLA antibodies characterization in transplant... Show moreBackground: Although the Luminex single antigen flow beads (SAFB) and the flow cytometry crossmatch (FCXM) are the most sensitive assays used for anti-HLA antibodies characterization in transplant recipients, their semi quantitative fluorescence read-out is not closely linked to graft outcome.Methods: Surface plasmon resonance (SPR) was implemented to determine truly quantitative parameters of five human monoclonal anti-class I HLA antibodies (mAbs): first the active concentration and then the binding constants. The results were compared to those obtained with SAFB and T-cell FCXM (T-FCXM).Results: The five mAbs displayed different rate and equilibrium constants for their cognate antigens. No correlation was evidenced between SAFB MFI or T-FCXM ratio and the binding parameters measured by SPR. Some mAbs amino acid substitutions within the epitope that influenced SAFE MFI resulted in affinity variations evidenced by SPR.Conclusion: The SAFB MFI and T-FCXM ratio, both semi-quantitative parameters, only partially reflected the subtlety of the anti-HLA antibody/antigen interaction as it can be analyzed by SPR. Future clinical studies using SPR for anti-HLA antibodies characterization could bring novel insights into the understanding of HLA/anti-HLA interaction and therefore anti-HLA antibodies pathogenicity. Show less
This thesis describes new, non-ribose ligands for the human Adenosine A1 Receptor (hA1R). An introduction to the four adenosine receptors subtypes, their history and cloning, occurrence,... Show moreThis thesis describes new, non-ribose ligands for the human Adenosine A1 Receptor (hA1R). An introduction to the four adenosine receptors subtypes, their history and cloning, occurrence, functioning, trafficking and therapeutic potential is given in Chapter 1. The process of desensitization and internalization of adenosine receptors in cell systems, tissues and in vivo studies is described in Chapter 2. An overview of the current literature concerning desensitization and internalization of the adenosine receptors is given and the regulation of the different subtypes upon agonist binding is discussed. In Chapter 3, human adenosine A1 receptors fused to 351Cys mutated Gi_-subunits are used as tools to study inverse agonism. In addition, the enhancing effect of the allosteric modulator PD81,723 on agonist affinity is shown. The influence of allosteric modulators on the internalization of the A1R is detailed in Chapter 4. The synthesis, affinity and activity of twelve non-ribose ligands, all ___2-amino-4-(3 and/or 4-disubstituted phenyl)-6-(substituted)sulfanyl-pyridine-3,5-dicarbonitriles, are described in Chapter 5. Chapter 6 evaluates the characteristics of LUF6037 a representative of these 3,4-methylenedioxyphenyl substituted compounds. One of the other non-ribose agonists with high affinity for the A1R, LUF5834, was chosen to be radioactively labelled. Chapter 7 describes the evaluation of [3H]LUF5834 as a new agonist radioligand. Show less
