Design And Synthesis of a Novel Potent Myelin Basic Protein Epitope 87−99 Cyclic Analogue: Enhanced Stability and Biological Properties of Mimics Render Them a Potentially New Class of Immunomodulators †

A cyclic analogue, [cyclo(87−99)MBP87-99], of the human immunodominant MBP87-99 epitope, was designed based on ROESY/NMR distance information and modeling data for linear epitope 87−99, taking into account T-cell (Phe89, Lys91, Pro96) and HLA (His88, Phe90, Ile93) contact side-chain information. The cyclic analogue was found to induce experimental allergic encephalomyelitis (EAE), to bind HLA-DR4, and to increase CD4 T-cell line proliferation, like that of the conformationally related linear MBP87-99 epitope peptide. The mutant cyclic peptides, the cyclo(91−99)[Ala96]MBP87-99 and the cyclo(87−99)[Arg91Ala96]MBP87-99, reported previously for suppressing, to a varying degree, autoimmune encephalomyelitis in a rat animal model, were found in this study to possess the following immunomodulatory properties:  (i) they suppressed the proliferation of a CD4 T-cell line raised from a multiple sclerosis patient, (ii) they scored the best in vitro TH2/TH1 cytokine ratio in peripheral blood mononuclear cell cultures derived from 13 multiple sclerosis patients, inducing IL-10 selectively, and (iii) they bound to HLA-DR4, first to be reported for cyclic MBP peptides. In addition, cyclic peptides were found to be more stable to lysosomal enzymes and Cathepsin B, D, and H, compared to their linear counterparts. Taken together, these data render cyclic mimics as putative drugs for treating multiple sclerosis and potentially other Th1-mediated autoimmune diseases

SERS-based monitoring of the intracellular pH in endothelial cells:the influence of the extracellular environment and tumour necrosis factor-alpha

The intracellular pH plays an important role in various cellular processes. In this work, we describe a method for monitoring of the intracellular pH in endothelial cells by using surface enhanced Raman spectroscopy (SERS) and 4-mercaptobenzoic acid (MBA) anchored to gold nanoparticles as pH-sensitive probes. Using the Raman microimaging technique, we analysed changes in intracellular pH induced by buffers with acid or alkaline pH, as well as in endothelial inflammation induced by tumour necrosis factor-alpha (TNF alpha). The targeted nanosensor enabled spatial pH measurements revealing distinct changes of the intracellular pH in endosomal compartments of the endothelium. Altogether, SERS-based analysis of intracellular pH proves to be a promising technique for a better understanding of intracellular pH regulation in various subcellular compartments.This work was supported by the National Center of Science (grant PRELUDIUM DEC-2012/05/N/ST4/00218) and by the European Union from the resources of the European Regional Development Fund under the Innovative Economy Programme (grant coordinated by JCET-UJ, no. POIG.01.01.02-00-069/09). We also thank the University of Edinburgh School of Chemistry for the Neil Campbell Travel Award for supporting LJ. We also thank Joanna Jalmuzna from the Department of Mathematics and Computer Sciences, Jagiellonian University in Krakow for fitting the calibration curve using Gnuplot software

Design and synthesis of a novel potent myelin basic protein epitope 87-99 cyclic analogue: enhanced stability and biological properties of mimics render them a potentially new class of immunomodulators

A cyclic analogue, [cyclo(87−99)MBP87-99], of the human immunodominant MBP87-99 epitope, was designed based on ROESY/NMR distance information and modeling data for linear epitope 87−99, taking into account T-cell (Phe89, Lys91, Pro96) and HLA (His88, Phe90, Ile93) contact side-chain information. The cyclic analogue was found to induce experimental allergic encephalomyelitis (EAE), to bind HLA-DR4, and to increase CD4 T-cell line proliferation, like that of the conformationally related linear MBP87-99 epitope peptide. The mutant cyclic peptides, the cyclo(91−99)[Ala96]MBP87-99 and the cyclo(87−99)[Arg91Ala96]MBP87-99, reported previously for suppressing, to a varying degree, autoimmune encephalomyelitis in a rat animal model, were found in this study to possess the following immunomodulatory properties:  (i) they suppressed the proliferation of a CD4 T-cell line raised from a multiple sclerosis patient, (ii) they scored the best in vitro TH2/TH1 cytokine ratio in peripheral blood mononuclear cell cultures derived from 13 multiple sclerosis patients, inducing IL-10 selectively, and (iii) they bound to HLA-DR4, first to be reported for cyclic MBP peptides. In addition, cyclic peptides were found to be more stable to lysosomal enzymes and Cathepsin B, D, and H, compared to their linear counterparts. Taken together, these data render cyclic mimics as putative drugs for treating multiple sclerosis and potentially other Th1-mediated autoimmune diseases