Abstract
Although the etiology of multiple sclerosis is not yet understood, it is accepted that its pathogenesis involves both autoimmune and neurodegenerative processes, in which the role of autoreactive T-cells has been elucidated. Instead, the contribution of humoral response is still unclear, even if the presence of intrathecal antibodies and B-cells follicle-like structures in meninges of patients has been demonstrated. Several myelin and non-myelin antigens have been identified, but none has been validated as humoral biomarker. In particular autoantibodies against myelin proteins have been found also in healthy individuals, whereas non-myelin antigens have been implicated in neurodegenerative phase of the disease. To provide further putative autoantigens of multiple sclerosis, we investigated the antigen specificity of immunoglobulins present both in sera and in cerebrospinal fluid of patients using phage display technology in a new improved format. A human brain cDNA phage display library was constructed and enriched for open-read-frame fragments. This library was selected against pooled and purified immunoglobulins from cerebrospinal fluid and sera of multiple sclerosis patients. The antigen library was also screened against an antibody scFv library obtained from RNA of B cells purified from the cerebrospinal fluid of two relapsing remitting patients. From all biopanning a complex of 14 antigens were identified; in particular, one of these antigens, corresponding to DDX24 protein, was present in all selections. The ability of more frequently isolated antigens to discriminate between sera from patients with multiple sclerosis or other neurological diseases was investigated. The more promising novel candidate autoantigens were DDX24 and TCERG1. Both are implicated in RNA modification and regulation which can be altered in neurodegenerative processes. Therefore, we propose that they could be a marker of a particular disease activity state.
Highlights
We have previously described a novel system whereby, using oriented cloning of cDNA fragments upstream the gene for the β-lactamase followed by selection with ampicillin, we were able to generate ORF-enriched cDNA phage display libraries using the phagemid vector pEP2 [24]
New non-myelin biomarkers for multiple sclerosis library, 15 randomly picked clones were sequenced and identified through data bank screening, on http://blast.ncbi.nlm.nih.gov/ (S1 Table). Both cytoplasmic and nuclear protein were represented in the library which included, as expected, proteins expressed by the nervous tissue such as “γEnolase”, the “Neurofilament 3”, the “Brain expressed X-linked 1” and the “Vesicle-associated membrane protein 1”
In the past 40 years, the research of the antigens eliciting the B cell response in Multiple sclerosis (MS) has been the aim of many studies as their identification would be extremely helpful for both a better understanding of MS pathogenesis and its diagnosis
Summary
Multiple sclerosis (MS) is a chronic neuroinflammatory disease of the central nervous system (CNS) characterized by the presence of areas of demyelination and neuroaxonal loss (lesion). Sequence analysis of V segments of Igs from single-cell demonstrates that clonal expansion is a prominent feature in MS [7,8,9], suggesting that a specific antigen or group of antigens is driving CNS B-cell activity in MS. These evidences are further supported by the presence of lymphoid follicle-like structures, observed in the cerebral meninges of some MS patients [10,11].
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