Two-dimensional SDS-PAGE fractionation of biological samples for biomarker discovery

Two-dimensional electrophoresis is still a very valuable tool in proteomics, due to its reproducibility and its ability to analyze complete proteins. However, due to its sensitivity to dynamic range issues, its most suitable use in the frame of biomarker discovery is not on very complex fluids such as plasma, but rather on more proximal, simpler fluids such as CSF, urine, or secretome samples. Here, we describe the complete workflow for the analysis of such dilute samples by two-dimensional electrophoresis, starting from sample concentration, then the two-dimensional electrophoresis step per se, ending with the protein detection by fluorescence

Functional diversity of chemokines and chemokine receptors in response to viral infection of the central nervous system.

Encounters with neurotropic viruses result in varied outcomes ranging from encephalitis, paralytic poliomyelitis or other serious consequences to relatively benign infection. One of the principal factors that control the outcome of infection is the localized tissue response and subsequent immune response directed against the invading toxic agent. It is the role of the immune system to contain and control the spread of virus infection in the central nervous system (CNS), and paradoxically, this response may also be pathologic. Chemokines are potent proinflammatory molecules whose expression within virally infected tissues is often associated with protection and/or pathology which correlates with migration and accumulation of immune cells. Indeed, studies with a neurotropic murine coronavirus, mouse hepatitis virus (MHV), have provided important insight into the functional roles of chemokines and chemokine receptors in participating in various aspects of host defense as well as disease development within the CNS. This chapter will highlight recent discoveries that have provided insight into the diverse biologic roles of chemokines and their receptors in coordinating immune responses following viral infection of the CNS

S100B as a potential biomarker and therapeutic target in multiple sclerosis

Multiple sclerosis (MS) pathology is characterized by neuroinflammation and demyelination. Recently, the inflammatory molecule S100B was identified in cerebrospinal fluid (CSF) and serum of MS patients. Although seen as an astrogliosis marker, lower/physiological levels of S100B are involved in oligodendrocyte differentiation/maturation. Nevertheless, increased S100B levels released upon injury may induce glial reactivity and oligodendrocyte demise, exacerbating tissue damage during an MS episode or delaying the following remyelination. Here, we aimed to unravel the functional role of S100B in the pathogenesis of MS. Elevated S100B levels were detected in the CSF of relapsing-remitting MS patients at diagnosis. Active demyelinating MS lesions showed increased expression of S100B and its receptor, the receptor for advanced glycation end products (RAGE), in the lesion area, while chronic active lesions displayed increased S100B in demyelinated areas with lower expression of RAGE in the rim. Interestingly, reactive astrocytes were identified as the predominant cellular source of S100B, whereas RAGE was expressed by activated microglia/macrophages. Using an ex vivo demyelinating model, cerebral organotypic slice cultures treated with lysophosphatidylcholine (LPC), we observed a marked elevation of S100B upon demyelination, which co-localized mostly with astrocytes. Inhibition of S100B action using a directed antibody reduced LPC-induced demyelination, prevented astrocyte reactivity and abrogated the expression of inflammatory and inflammasome-related molecules. Overall, high S100B expression in MS patient samples suggests its usefulness as a diagnostic biomarker for MS, while the beneficial outcome of its inhibition in our demyelinating model indicates S100B as an emerging therapeutic target in MS.This work was supported by Medal of Honor L’Oréal for Women in Science (FCT, UNESCO, L’Óreal) and innovation grant (Ordem dos Farmacêuticos) to AF, a post-doctoral grant from Fundação para a Ciência e Tecnologia (FCT-SFRH/BPD/96794/2013) and a DuPré Grant from the European Committee for Treatment and Research in Multiple Sclerosis (ECTRIMS) to AB, and by FCT-Pest- OE/SAU/UI4013 to iMed.ULisboa.info:eu-repo/semantics/publishedVersio

A lack of association between elevated serum levels of S100B protein and autoimmunity in autistic children

<p>Abstract</p> <p>Background</p> <p>S100B is a calcium-binding protein that is produced primarily by astrocytes. Increased serum S100B protein levels reflect neurological damage. Autoimmunity may have a role in the pathogenesis of autism in some patients. Autoantibodies may cross the blood-brain barrier and combine with brain tissue antigens, forming immune complexes and resulting in neurological damage. We are the first to investigate the relationship between serum levels of S100B protein, a marker of neuronal damage, and antiribosomal P protein antibodies in autistic children.</p> <p>Methods</p> <p>Serum S100B protein and antiribosomal P antibodies were measured in 64 autistic children in comparison to 46 matched healthy children.</p> <p>Results</p> <p>Autistic children had significantly higher serum S100B protein levels than healthy controls (<it>P </it>< 0.001). Children with severe autism had significantly higher serum S100B protein than patients with mild to moderate autism (<it>P </it>= 0.01). Increased serum levels of antiribosomal P antibodies were found in 40.6% of autistic children. There were no significant correlations between serum levels of S100B protein and antiribosomal P antibodies (<it>P </it>= 0.29).</p> <p>Conclusions</p> <p>S100B protein levels were elevated in autistic children and significantly correlated to autistic severity. This may indicate the presence of an underlying neuropathological condition in autistic patients. Antiribosomal P antibodies may not be a possible contributing factor to the elevated serum levels of S100B protein in some autistic children. However, further research is warranted to investigate the possible link between serum S100B protein levels and other autoantibodies, which are possible indicators of autoimmunity to central nervous system in autism.</p