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. 2015 Feb 3;9(2):e0003459.
doi: 10.1371/journal.pntd.0003459. eCollection 2015 Feb.

Platelet activating factor contributes to vascular leak in acute dengue infection

Affiliations

Platelet activating factor contributes to vascular leak in acute dengue infection

Chandima Jeewandara et al. PLoS Negl Trop Dis. .

Abstract

Background: Although plasma leakage is the hallmark of severe dengue infections, the factors that cause increased vascular permeability have not been identified. As platelet activating factor (PAF) is associated with an increase in vascular permeability in other diseases, we set out to investigate its role in acute dengue infection.

Materials and methods: PAF levels were initially assessed in 25 patients with acute dengue infection to determine if they were increased in acute dengue. For investigation of the kinetics of PAF, serial PAF values were assessed in 36 patients. The effect of dengue serum on tight junction protein ZO-1 was determined by using human endothelial cell lines (HUVECs). The effect of dengue serum on and trans-endothelial resistance (TEER) was also measured on HUVECs.

Results: PAF levels were significantly higher in patients with acute dengue (n = 25; p = 0.001) when compared to healthy individuals (n = 12). In further investigation of the kinetics of PAF in serial blood samples of patients (n = 36), PAF levels rose just before the onset of the critical phase. PAF levels were significantly higher in patients with evidence of vascular leak throughout the course of the illness when compared to those with milder disease. Serum from patients with dengue significantly down-regulated expression of tight junction protein, ZO-1 (p = 0.004), HUVECs. This was significantly inhibited (p = 0.004) by use of a PAF receptor (PAFR) blocker. Serum from dengue patients also significantly reduced TEER and this reduction was also significantly (p = 0.02) inhibited by prior incubation with the PAFR blocker.

Conclusion: Our results suggest the PAF is likely to be playing a significant role in inducing vascular leak in acute dengue infection which offers a potential target for therapeutic intervention.

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Conflict of interest statement

All authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. PAF, PAF-AH and PAF-R levels in patients and healthy individuals.
A: PAF levels in sera of patients with DHF (n = 16), DF (n = 9) and healthy individuals (n = 12). The bars represent the mean and the standard error of mean; p = 0.002. B: PAF-AH levels in sera of patients with DHF (n = 16), DF (n = 9) and healthy individuals (n = 12). The bars represent the mean and the standard error of mean; p<0.0001. C: PAFR levels in sera of patients with DHF (n = 16), DF (n = 9) and healthy individuals (n = 12). The bars represent the mean and the standard error of mean; p = 0.33.
Figure 2
Figure 2. Kinetics of PAF.
Levels of PAF were measured twice a day throughout the course of illness in patients with DF and DHF from the time of admission to the ward until time of discharge. ** indicate time points where the PAF values were significantly higher in patients with DHF when compared to those with DF.
Figure 3
Figure 3. Effect of PAF and PAFR blocker on expression of ZO-1.
A: ZO-1 expression in HUVECs was evaluated with different concentrations of PAF. ZO-1 expression was compared in untreated HUVECs, with HUVECs incubated with 100ng/ml PAF; 200ng/ml PAF and 500ng/ml PAF. B: The differences in ZO-1 expression were evaluated in HUVECs treated with different concentrations of PAF. The HUEVECs were pretreated with a PAFR blocker for one hour prior to been treated with different concentrations of PAF. C: ZO-1 expression was evaluated in HUVECs that were treated with media alone (untreated) compared to HUVECs treated with 500ng/ml PAFR blocker alone; treated with dengue patient serum (DS) and pre-treated with a PAFR blocker (PAFRA) prior to treatment with dengue patient serum (DS)
Figure 4
Figure 4. Immunofluorescence analysis of tight junction protein ZO-1 on HUVECs.
Images of HUVECs incubated with EGM, dengue serum and pre-treated with a PAFR blocker prior to addition of dengue serum from 3 patients are shown. Cells were treated with A – Endothelial growth medium only; B- treated with dengue patient serum (n = 3); C- HUVEC pre-treated a PAFR blocker before addition of dengue serum (n = 3. All assays were done in duplicate and repeated twice. Five separate images per condition were analysed quantitatively. Arrows indicate the ZO-1 staining.
Figure 5
Figure 5. Changed in the trans-endothelial electrical resistance with dengue sera and the effect of PAFR blocker.
Change in TEER with dengue serum (DS; n = 9); with DS following pretreatment with a PAFR blocker (PAFRA n = 9); with control serum (n = 9); with in control serum following pretreatment with a PAFR blocker (n = 9).

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