Discussion
In this study we show that urinary LTE4 levels were significantly higher in early illness in patients, who proceed to develop DHF. The LTE4 levels continued to be higher during later stages of illness (day 5 to 6), in patients with DF and DHF and the levels during later stages of illness in patients with DHF were higher than those of early illness. Although the levels of urinary LTE4 was significantly higher in those who proceeded to develop DHF during early illness, urinary LTE4 levels did not appear to perform well as a biomarker due to the low AUC values. This is possibly due to the wide diurnal variation seen in the LTE4 levels in patients with both DF and DHF. Dirunal variation in both urinary LTE4 and histamine levels have been previously observed in patients with asthma and in normal individuals, although no specific pattern was observed, as in this study(26). We too did not observe a consistent pattern in the changes of urinary LTE4 in the morning compared to the afternoon. This is in contrast with changes observed with PAF, with the levels always being high in the early morning levels, compared to the afternoon levels(9, 13). Interestingly, similar changes were observed with viral loads between morning and afternoon samples, with a a 10-fold to 100-fold difference in some patients. Again, there was no particular pattern with the viral loads rising in some patients, while decreasing others and the changes in the viral loads did not show any relationship with the LTE4 or histamine levels.
Cysteinyl leukotrienes are synthesized from cells which posses LTC4 synthase, such as eosinophils, mast cells, basophils and macrophages(27). Many other enzymes act on the subsequent products LTC4, LTD4 and subsequently LTE4, of which the stable N-acetyl derivate is found in the urine(27). All these cells are readily infected by the DENV and are susceptible to enhanced infection and activation by the DENV in the presence of IgG antibodies which bind to the activating type of FcγRIIIA receptors(25, 28). Indeed it was shown that mast cells were significantly more activated in secondary dengue infections(12). Although LTE4 levels were not high in patients with secondary dengue infection, in this study, the levels were higher in those with DHF due to secondary dengue compared to those with DF. However, our results showed that overall the LTE4 levels were higher very early in illness, irrespective of serostatus and the degree of serum viraemia. Therefore, LTE4 production appears to be enhanced very early during infection, and increasing in those who develop DHF during the critical phase, suggesting that the pathway may contribute to the vascular leak. Montelukast is a cysteinly leukotriene receptor antagonist and is widely used to inhibit the action of leukotrienes in asthma and many other allergic diseases(29, 30). In dengue mouse models it was shown to reduce the extent of vascular leak(12). As montelukast is a relatively safe drug, it would be important to evaluate its efficacy in reducing dengue disease severity.
Mast cells are the main source of histamine although it can also be produced by basophils and the intestinal epithelium and other cells(31). Histamine has been shown to induce mast cells to release leukotrienes, cytokines and also increase vascular permeability(31). Although cross linking of the IgE bound to the high affinity FcεRI is the main trigger of histamine release from mast cells, complement components C3a, C5a and cytokines such as IL-33, IL-3, IL-18 and GM-CSF have been shown to induce synthesis and release(32). Although we found that histamine levels were significantly higher in patients with acute dengue, there was no difference in their levels in those with DF compared to those with DHF. In addition, the histamine levels fell in both patients with DF and DHF during the course of illness, which was in contrast to what was observed with urinary LTE4 levels. Interestingly, unlike LTE4 levels, the urinary histamine levels were always lower in the afternoon when compared to the morning, as seen with the variation of PAF(9, 13). We recently conducted a phase II, randomized, placebo controlled clinical trial with rupatadine to evaluate its efficacy in reducing vascular leak in acute dengue(2). Rupatadine significantly inhibited the effects of dengue sera on human endothelial cells lines by reducing the reduction in trans-endothelial resistance and a reduction is ZO-1 expression and also showed small but significant differences in the extent of fluid leakage and thrombocytopenia, when the drug was given early(2). Rupatadine is an antihistamine with PAF receptor antagonist effects(33). Therefore, it would be important to evaluate if some of the effects of rupatadine was also due to its antihistamine effects.