Dengue virus infection usually courses as a benign self-limited fever, called dengue fever. However, on occasions it can progress to a life-threatening complication known as severe dengue (SD). A hallmark of SD is a sharp increase in vascular permeability. Secondary infections are considered a risk factor to develop SD, presumably through a mechanism called Antibody-Dependent Enhancement (ADE) of infection in cells with the capacity to bind antigen-antibody complexes, such as macrophages, and to trigger a subsequent aberrant cytokine response. The massive release of cytokine from macrophages has been postulated to cause changes in vascular permeability. The vascular permeability assay presented in this protocol is designed to assess whether any compound or cell-secreted product or soluble factor present in sera from patients may induce plasma leakage in mice. This test was used in the laboratory to determine whether cytokines and soluble factors produced in vitro by macrophages infected with dengue virus or dengue virus in the presence of facilitating antibodies are able to induce plasma leakage in vivo. Macrophages were infected with dengue virus or dengue virus in the presence of facilitating antibodies for 48 h. After this time, the conditioned supernatant containing cytokines and soluble factors released by the macrophages were collected and inoculated intraperitoneally into CD-1 mice. Twenty four hours after the first inoculation, mice were reinoculated with a second dose with Evans blue dye. After another 24 h, mice were euthanized and the amount of Evans blue present in the blood and lung was determined by spectrophotometric analysis. The assay was able to show differences in the capacity of the conditioned media to induce vascular permeability changes in the inoculated animals (Puerta-Guardo et al., 2013).
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