Competitive ELISA for the identification of 35–55 myelin oligodendrocyte glycoprotein immunodominant epitope conjugated with mannan

Analogs of immunodominant myelin peptides involved in multiple sclerosis (MS: the most common autoimmune disease) have been extensively used to modify the immune response over the progression of the disease. The immunodominant 35–55 epitope of myelin oligodendrocyte glycoprotein (MOG_35–55) is an autoantigen appearing in MS and stimulates the encephalitogenic T cells, whereas mannan polysaccharide (Saccharomyces cerevisiae) is a carrier toward the mannose receptor of dendritic cells and macrophages. The conjugate of mannan-MOG_35–55 has been extensively studied for the inhibition of chronic experimental autoimmune encephalomyelitis (EAE: an animal model of MS) by inducing antigen-specific immune tolerance against the clinical symptoms of EAE in mice. Moreover, it presents a promising approach for the immunotherapy of MS under clinical investigation. In this study, a competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect the MOG_35–55 peptide that is conjugated to mannan. Intra- and inter-day assay experiments proved that the proposed ELISA methodology is accurate and reliable and could be used in the following applications: (i) to identify the peptide (antigen) while it is conjugated to mannan and (ii) to adequately address the alterations that the MOG_35–55 peptide may undergo when it is bound to mannan during production and stability studies.     

Synthesis and SAR development of quinoline analogs as novel P2X7 receptor antagonists

The P2X7 receptor (P2X7R) plays an important role in diverse conditions associated with tissue damage and inflammation, suggesting that the human P2X7R (hP2X7R) is an attractive therapeutic target. In the present study, the synthesis and structure-activity relationship (SAR) of a novel series of quinoline derivatives as P2X7R antagonists are described herein. These compounds exhibited mechanistic activity (YO PRO) in an engineered HEK293 expressing hP2X7R as well as a functional response (IL-1β) in human THP-1 (hTHP-1) cellular assays. Compound 19 was identified as the most promising compound in this series with excellent cellular potency, low liver microsomal clearance, good permeability and low efflux ratio. In addition, this compound also displayed good pharmacokinetic properties and acceptable brain permeability (K_p,uu of 0.37).     

And Yet it is Modified—Holding a Candle to the Dark Matter of White Matter

The multilamellar membrane myelin sheath of the CNS, that enwraps axons to facilitate saltatory conduction in higher vertebrates, is held together by myelin basic protein (MBP). Yet this generalization masks how enigmatic MBP is, much like cosmological “dark matter.” First, the casual use of the singular form for “protein” distracts that there are multiple, developmentally regulated “classic” splice isoforms ranging from 14 to 21.5 kDa, each with extensive PTMs. Second, the static image of MBP adhering two cytoplasmic leaflets of the oligodendrocyte membrane together in close apposition, suggests it to be inaccessible to modifying enzymes. And yet it is modified (to paraphrase Galileo's phrase on the earth's motion). In this issue of Proteomics, Sarg et al. apply an integrated CE–MS approach to investigate the PTMs of 18.5 kDa MBP from mouse brains of different ages. They identify new sites and types of modification, as well as confirming previously known PTMs. Innovative tools for unraveling the intricacies of the myelin basic proteome and how it organizes CNS myelin (much like basic histones organize chromatin), will help us understand white matter development and plasticity in health, during ageing, and in demyelinating diseases such as multiple sclerosis.     

Subunit asymmetry in the three-dimensional structure of a human CuZnSOD mutant found in familial amyotrophic lateral sclerosis.

The X-ray crystal structure of a human copper/zinc superoxide dismutase mutant (G37R CuZnSOD) found in some patients with the inherited form of Lou Gehrig's disease (FALS) has been determined to 1.9 angstroms resolution. The two SOD subunits have distinct environments in the crystal and are different in structure at their copper binding sites. One subunit (subunit[intact]) shows a four-coordinate ligand geometry of the copper ion, whereas the other subunit (subunit[broken]) shows a three-coordinate geometry of the copper ion. Also, subunit(intact) displays higher atomic displacement parameters for backbone atoms ((B) = 30 +/- 10 angstroms2) than subunit(broken) ((B) = 24 +/- 11 angstroms2). This structure is the first CuZnSOD to show large differences between the two subunits. Factors that may contribute to these differences are discussed and a possible link of a looser structure to FALS is suggested.     

HLA Class I allele frequencies in the Lebanese population

The highly polymorphic Human Leukocyte Antigen system encompasses different loci that have been studied in transplantation as well as diseases and population associated research. This study is the first and largest of its kind to describe the distribution of HLA-A, -B and -C alleles in Lebanon. Respectively, 1994, 1309 and 1163 Lebanese individuals referred for HLA typing and possible bone marrow/kidney donation were tested for HLA-A, HLA-B and HLA-C alleles using the polymerase chain reaction/Sequence specific priming (PCR-SSP) method. Our data were compared to that of several populations with interesting and common findings shared with the Moroccan, Jordanian, Tunisian, Omani, Korean, Chinese, Japanese, Peruan, Bulgarian, Irish, Polish, Spanish, Swiss, American, African and Brazilian populations. The following data concerning the Lebanese population will help future investigators to study the relation of HLA-A, -B and -C alleles with common diseases in Lebanon and will add to the available international literature. This new data will serve as a major reference report in the region.     

A new myelin protein, TPPP/p25, reduced in demyelinated lesions is enriched in cerebrospinal fluid of multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease with variable extent of remyelination coupled with the differentiation of oligodendrocytes, in which Tubulin Polymerization Promoting Protein/p25 (TPPP/p25) plays a crucial role. Previously we reported that the loss of TPPP/p25-positive oligodendrocytes in demyelinated lesions in the brain of MS patients could be a biomarker for MS [2]. In this work we tested the occurrence of TPPP/p25 in the cerebrospinal fluid (CSF) of MS patients, and by elaborating a sensitive assay for quantification of TPPP/p25 we showed that its level is significantly higher than in the case of non-MS patients. Patients with MS were diagnosed at the Department of Neurology, University of Szeged according to the clinical and laboratory diagnostic criteria of McDonald. In non-MS patients no significant pathological changes were detected on magnetic resonance imaging scans, while in MS patients multiple hyperintense T2 lesions in the white matter were detected. Kurtzke Expanded Disability Status Scale scores as well as IgG level and oligoclonal bands of MS patients were demonstrated. The sensitive assay elaborated in this study is based upon Western blot followed by chemiluminescent detection validated by human recombinant protein. The median TPPP/p25 contents in the CSF were 62.8 and 64.7 μg/L for patients with clinically isolated syndromes and relapsing remitting MS, respectively, while this value for non-MS patients was 27.9 μg/L. The enrichment of TPPP/p25 was independent of age, gender and the time period between lumbar puncture and relapse/shub. These data suggest that the TPPP/p25-based assay could be a powerful diagnostic test for MS patients.