Dengue is an important health problem in tropical and sub-tropical areas. Although the pathogenesis of the disease has been studied intensively, some aspects remain not well understood. Genome-wide expression profile analysis on microarrays or 2D-electrophoresis protein difference analysis in combination with MALDI-TOF/TOF Mass Spectrometry have been used to search for biomarkers that could serve as prognostic tools or therapeutic targets for severe dengue. Proteins belonging to the complement pathway or involving in processes such as inflammation, signal transduction or translation/transcription have been identified but there is no clear consensus on their potential use in the severity prognostic
[14–17]. More recently, Brasier et al.
 described a new pre-fractionation method on acute dengue Venezuelan plasmas followed by a 2D-gel electrophoresis in tandem with LC-MS/MS and a non-parametric analysis. Previously unidentified biomarkers having biological significance in process underlying severe dengue such as capillary leakage or hepatic injury, have been found.
The plasma, which corresponds to the unclotted blood after removal of cellular components, has constant intimacy with different body parts and contains proteins released by diseased tissues
. The comparison of circulating proteins in plasma from patients developing different aspects of a same disease may lead to the discovery of biomarkers for diagnosis, prognosis and disease monitoring
. The present study aims to identify differences in circulating proteins during different degrees of severity of dengue virus acute infections.
Stable isotope protein labeling combined with Mass Spectrometry is a powerful tool to identify and relatively quantify proteins within complex protein mixtures such as tissue extracts or body fluids. The ICPLTM technique allows high-throughput quantitative proteome profiling on a global scale. It is used for protein profile comparison
Here, ICPLTM was used to compare mild and severe dengue plasmas for the identification of differentially expressed proteins. To increase the chance of finding low-abundance proteins the study was conducted on plasma depleted of the twenty most abundant proteins. For this purpose, it has previously been proven that immune-capture columns are useful
. However, the higher the number of depleted proteins is, the more important the drawback in removing associated proteins and peptides is. This drawback could have an impact on the list of identified markers of severity
For each pathology, the use of pool of five plasma specimen homogeneous in age, sex ratio, days of collect after onset of symptoms and serotype would limit the ICPL identification of idiotypic markers specific of each individual plasma sample. It would also promote the discovery of markers that should be common to all samples, such as severe dengue markers. Although sometimes criticized
, the use of a pool instead of several separate samples for proteomic analysis has been reported in recent studies
Three proteins, identified by mass spectrometry, were confirmed by ELISA to have significant higher expression levels in SD plasmas. These proteins were Leucine-Rich alpha-2 Glycoprotein 1, Ferritin and Vitamin D Binding-Protein.
The physiological function of LRG1 is still unknown. In 2002, O'Donnell et al.
 have suggested a physiological role in neutrophilic granulocytes. In 2006, Cummings et al.
 identified LRG1 as cytochrome c inhibitor in sera. More recently, Codina et al.
 have shown that LRG1 protects against Cytc-induced lymphotoxicity. LRG1 has also been shown to be involved in protein-protein interaction, signal transduction, cell adhesion and development. LRG1 was identified as a serum biomarker that identifies patients with heart failure
. In the literature, LRG1 has never been associated to the dengue severity. Interestingly, LRG1 has been shown to interact with the Fibronectin also identified in the present study as being over-expressed in SD plasmas
VitDBP is a 52 kDa protein that binds to vitamin D as well as its plasma metabolites and transports them to target tissues. VitDBP binds monomeric actin in addition to Vitamin D. The protein forms three domains. The structure of VitDBP is similar to the Human Serum Albumin
[32, 33]. It is presumed that the function of VitDBP binding actin is to clear up any actin that enters the blood stream as a result of cell injury. Its affinity for actin monomers is high
 and binding involves residues from all three domains
[32, 33]. VitDBP also participates to macrophage activation and chemotaxis. The protein has been already associated to the pathogenesis of dengue by a Two-dimensional difference gel electrophoresis (DiGE) analysis on Brazilian plasma specimens
Ferritin is a globular protein which serves to store iron in a non-toxic form, and to transport it to areas where it is required
. Free iron is toxic to cells because it catalyzes the formation of free radicals from reactive oxygen species
. Within cells, iron is stored in a protein complex as ferritin or hemosiderin. Ferritin concentrations increase drastically in the presence of an infection or with cancer
. The inflammatory response may cause ferritin to migrate from the plasma to the cells in order to deny iron to the infectious agent
. The concentration of ferritin has also been shown to increase in response to stresses such as anoxia
. In a previous study, high serum ferritin levels have been demonstrated by ELISA to be a biomarker of dengue hemorrhagic fever in Thai children
. Interestingly, proteins involved in inflammatory process have often been identified as potential markers of dengue severity
All these proteins are related to several functions: transportation, adhesion and iron storage and seem to participate in the preservation of the body homeostasis during dengue infection.