Bayesian Approach to Model CD137 Signaling in Human M.tuberculosis in vitro Responses

Abstract Immune responses are qualitatively and quantitatively influenced by a complex network of receptor-ligand interactions. Among them, the CD137:CD137L pathway is known to modulate innate and adaptive human responses against Mycobacterium tuberculosis. However, the underlying mechanisms of this regulation remain unclear. In this work, we developed a Bayesian Computational Model (BCM) of in vitro CD137 signaling, devised to fit previously gathered experimental data. The BCM is fed with the data and the prior distribution of the model parameters and it returns theirposterior distribution and the model evidence, which allows comparing alternative signaling mechanisms. The BCM uses a coupled system of non-linear differential equations to describe the dynamics of Antigen Presenting Cells, Natural Killer and T Cells together with the interpheron (IFN)-c and tumor necrosis factor (TNF)-a levels in the media culture. Fast and complete mixing of the media is assumed. The prior distribution of the parameters that describe the dynamics of the immunological response was obtained from the literature and theoretical considerations Our BCM applies successively the Levenberg-Marquardt algorithm to find the maximum a posteriori likelihood (MAP); the Metropolis Markov Chain Monte Carlo method to approximate the posterior distribution of the parameters and Thermodynamic Integration to calculate the evidence of alternative hypothesis. Bayes factors provided decisive evidence favoring direct CD137 signaling on T cells. Moreover, the posterior distribution of the parameters that describe the CD137 signaling showed that the regulation of IFNc levels is based more on T cells survival than on direct induction. Furthermore, the mechanisms that account for the effect of CD137 signaling on TNF-a production were based on a decrease of TNF-a production by APC and, perhaps, on the increase in APC apoptosis. BCM proved to be a useful tool to gain insight on the mechanisms of CD137 signaling during human response against Mycobacterium tuberculosis.Fil: Darío A Fernández Do Porto. UNIV.DE BUENOS AIRES. FAC.DE CS.EXACTAS Y NATURALES. UNIV.DE BUENOS AIRES. FAC.DE CS.EXACTAS Y NATURALES. INST QUIM FISICA D/L/MATERIALES MED AMB Y ENERG.Fil: Jerónimo Auzmendi. UNIV.DE BUENOS AIRES. FAC.DE CS.EXACTAS Y NATURALES. INST QUIM FISICA D/L/MATERIALES MED AMB Y ENERG.Fil: Delfina Peña. UNIV.DE BUENOS AIRES. FAC.DE CS.EXACTAS Y NATURALES. CONSEJO NAC.DE INVEST.CIENTIF.Y TECNICAS. OFICINA DE COORDINACION ADMINISTRATIVA CIUDAD UNIVERSITARIA. INSTITUTO DE QUIMICA BIOLOGICA DE LA FACULTAD DE CS. EXACTAS Y NATURALES. UNIV.DE BUENOS AIRES. FAC.DE CS.EXACTAS Y NATURALES. DTO.DE QUIMICA BIOLOGICA.Fil: Veronica E Garcia. CONSEJO NAC.DE INVEST.CIENTIF.Y TECNICAS. OFICINA DE COORDINACION ADMINISTRATIVA CIUDAD UNIVERSITARIA. INSTITUTO DE QUIMICA BIOLOGICA DE LA FACULTAD DE CS. EXACTAS Y NATURALES.Fil: Luciano Moffatt. UNIV.DE BUENOS AIRES. FAC.DE CS.EXACTAS Y NATURALES. INST QUIM FISICA D/L/MATERIALES MED AMB Y ENERG

High-throughput fluorescence assays for ion channels and GPCRs

Ca2+, Na+ and K+- permeable ion channels as well as GPCRs linked to Ca2+ release are important drug targets. Accordingly, high-throughput fluorescence plate reader assays have contributed substantially to drug discovery efforts and pharmacological characterization of these receptors and ion channels. This chapter describes some of the basic properties of the fluorescent dyes facilitating these assay approaches as well as general methods for establishment and optimisation of fluorescence assays for ion channels and Gq-coupled GPCRs