Study Of Proteomics In Cerebrospinal Fluid Of GBS Patients

Guillain-Barrésyndrome (GBS) is an immune-mediated inflammatory disease of the peripheral nerves, involving both the myelin sheath and the axons. GBS occurs worldwide and the incidence ranges from 0.6–4 per 100 000 per year. Even when patients are treated in well-equipped intensive care units, mortality rates are still 3–7%. Moreover, about 7–15% of patients have permanent neurological sequelae including bilateral footdrop, intrinsic hand muscle wasting, sensory ataxia, and dysesthesia, which severely affect human daily life. Increased protein without pleocytosis in cerebrospinal fluid (CSF) is a characteristic of GBS. CSF examination may be normal during the first days after the onset of symptoms, but protein levels usually increase after one week. It is, therefore, important to determine the cause and function of these abnormal levels of proteins that are realted to disease.In this study, we used two-dimensional difference gel electrophoresis (2-D DIGE) technique, in combination with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), to determine the abnormal levels of CSF proteins in GBS patients. CSF samples from five GBS and five control patients with other neurological disorder (headache with normal CSF examination) were collected. All CSF samples were precipitated with acetone and the salts were removed out by Clean-up kit. The prepared protein samples were quantified with 2D Quant kit, and then 50μg protein from every sample was minimally labelled with different fluorescent dyes. Cy3-, Cy5- and Cy2- labelled samples and internal standards were pooled and loaded on a pH gradient strip for isoelectric focusing, equilibrated twice then loaded on 12.5% polyacrylamide gels for the second electrophoresis. Then quantification and statistic analysis were carried out with DeCyder Differential In Gel Analysis software to find the differential expressed protein spots between GBS and control samples. Another two preparative gels of control and GBS groups were made with 600μg protein in the same condition. Spots of interest were excised and identified by MALDI-TOF MS. Peptide mass data were searched against National Center for Biotechnology Information (NCBI) and the results were further confirmed in the Swissprot protein database (www.us.expasy.org/sprot).Our data showed that three proteins haptoglobin, apolipoprotein A-IV and PRO2044 (unnamed protein) were markedly increased, however, the other three proteins transthyretin, apolipoprotein E and fibrinogen were significantly decreased in the CSF of GBS patients compared to controls using 2-D DIGE detection followed by MALDI-TOF MS. We delineated the possible roles by which the abnormal levels of proteins participate in the pathogenesis of GBS probably.Haptoglobin (Hpt) is a plasma protein with hemoglobin (Hb)-binding capacity and is also a positive acute-phase protein that functions as a bacteriostatic agent, an inhibitor of prostaglandin synthesis and angiogenesis. Hpt has been proposed to be involved in a highly interactive ensemble of lymphocytes, neutrophils, and monocytes participating in inflammatory processes. As described by Gutowski in 1998, haptoglobin levels might be a useful marker for the diagnosis of GBS. Our data further imply that haptoglobin related inflammation may be involved in the pathogenesis of GBS.ApoA-IV is synthesized primarily in the intestine then secreted into the plasma, and an acute inflammation can disturb the plasma apoA-IV concentration. The relationship between the level of apoA-IV and inflammation has never been described in humans to the best of our knowledge. The role of high level of apoA-IV in GBS CSF has not been reported previously which indicated that apoA-IV was probably related to inflammation in GBS.Transthyretin, a known negative acute phase protein, synthesized in liver cells and secreted into the CSF and plasma, was found significantly reduced in various acute liver disease, in the CSF of late stage Alzheimer's disease, and Amyotrophic lateral sclerosis patients. For the first time, we found that transthyretin was decreased in CSF of GBS, which might also provide a new negative marker for GBS.ApoE has multiple biological properties, in addition to its physiological role in cholesterol transport, such as apoE has immunomodulatory properties in vitro and in vivo. Previously, we reported that apoE acted as an inhibitor in experimental autoimmune neuritis (EAN), a model for GBS. Decreased CSF apoE can be found in multiple scleroses (MS), an analogous disease of the central nervous system (CNS). Since apoE is considered as a neurotrophic factor, consequently, any decrease in apoE level would contribute to the progression of neurological diseases, such as GBS.Fibrinogen is a plasma glycoprotein with the functions of healing wounds and inflammation. The fibrinogen level in plasma appears to reflect disease activity in acute GBS. Over 30% decrease in fibrinogen by plasma exchange was significantly associated with sustained neurological improvement in GBS. A less than 30% fall in fibrinogen level despite plasma exchange may indicate the necessity to continue therapy to maximize the benefit of treatment and in order to minimize the risk of relapse. These data provided evidences that fibrinogen may be a useful and easily identifiable marker for instructing GBS treatment.In conclusion, we identified six proteins (haptoglobin, apolipoprotein A-IV and PRO2044, as well as transthyretin, apoE and fibrinogen) that may be involved in the pathogenesis of GBS and provide potential markers for the diagnosis and evaluation of the treatment and prognosis of the disease.