The potential physiological relevance of our results is supported by data obtained with plasma samples from dengue patients. To investigate whether DENV infection modulates the distribution of the intracellular isoforms of a-enolase as observed for the secreted isoforms, we performed Western blot analysis from two-dimensional gels of cell lysate samples from mock and DENV-infected cells. We observed five spots corresponding to a-enolase with the same pattern as observed for conditioned medium samples, i.e., spots with similar molecular weights but distinct isoeletric points. Comparative analysis of the corresponding spots from mock and infected cells revealed no differences in the distribution pattern of the isoforms. These results combined with those shown in Fig. 3 suggest that basic isoforms are addressed to secretion. To provide insight into the physiological relevance of a-enolase secretion in dengue diseases, we conducted preliminary experiments in which we analyzed a-enolase in albumin-depleted plasma samples from three healthy donors, three patients with non-severe dengue and three patients with dengue shock syndrome. The results indicated an increase in aenolase content in plasma of dengue patients. To compare the distribution of a-enolase isoforms between plasmas from a healthy donor and a DSS patient, we performed twodimensional electrophoresis followed by Western blot. The result revealed an alteration in the distribution profile of the a-enolase spots in these samples. Although a comprehensive study with larger number of subjects is required to Tofacitinib supply validate these results, our data suggest an interesting potential relevance for a-enolase role in dengue diseases. Comprehension of the effects of DENV infection on its target cells might provide important insights into dengue pathogenesis. Among the main cells infected by DENV in humans, hepatocytes play a central role. Several data obtained using liver specimens from fatal cases of dengue, animal models, and in vitro cell cultures show that liver is a major site for DENV replication. Also, liver enlargement and increase in the levels of plasma transaminases are among the criteria to diagnose severe dengue. Additionally, liver produces most of the plasmatic proteins and secretes several inflammatory mediators, which would not only affect organ function but also cause systemic effects, contributing to the increase in vascular permeability as well as to the coagulation disturbs that characterize severe cases of dengue. In this study, we report that DENV infection modulates a-enolase secretion by hepatic cells in a dose-dependent manner. For the first time, we show a positive correlation between DENV infection and secretion of a-enolase, indicating that a high viral load will lead to a burst of a-enolase secretion in hepatic cells. Alterations in a-enolase expression are frequently observed in many pathological conditions. Indeed, in a meta-analysis study that evaluated 169 two-dimensional electrophoresis based articles, a-enolase was identified as the second most often differentially expressed protein in humans, regardless of the tissue analyzed and experimental conditions used. Furthermore, it was the most frequently identified protein with altered expression in mouse and rats.