Neutrophil Activation and Early Features of NET Formation Are Associated With Dengue Virus Infection in Human

The involvement of the immune system in the protection and pathology of natural dengue virus (DENV) has been extensively studied. However, despite studies that have referred to activation of neutrophils in DENV infections, the exact roles of neutrophils remain elusive. Here, we explored the phenotypic and functional responses of neutrophils in a cohort of adult dengue patients. Results indicated that during an acute DENV infection, neutrophils up-regulate CD66b expression, and produce a more robust respiratory response as compared with that in convalescent or healthy individuals; this confirmed in vivo neutrophil activation during DENV infection. Spontaneous decondensation of nuclei, an early event of neutrophil extracellular trap (NET) formation, was also markedly increased in cells isolated from DENV-infected patients during the acute phase of the infection. In vitro incubation of NETs with DENV-2 virus significantly decreased DENV infectivity. Interestingly, increased levels of NET components were found in the serum of patients with more severe disease form—dengue hemorrhagic fever (DHF), but not uncomplicated dengue fever, during the acute phase of the infection. Levels of pro-inflammatory cytokines IL-8 and TNFα were also increased in DHF patients as compared with those in healthy and DF subjects. This suggested that NETs may play dual roles during DENV infection. The increased ability for NET formation during acute DENV infection appeared to be independent of PAD4-mediated histone H3 hyper-citrullination. Our study suggests that neutrophils are involved in immunological responses to DENV infection

Bacteria Interface Pickering Emulsions Stabilized by Self-assembled Bacteria–Chitosan Network

An oil-in-water Pickering emulsion stabilized by biobased material based on a bacteria–chitosan network (BCN) was developed for the first time in this study. The formation of self-assembled BCN was possible due to the electrostatic interaction between negatively charged bacterial cells and polycationic chitosan. The BCN was proven to stabilize the tetradecane/water interface, promoting formation of highly stable oil-in-water emulsion (o/w emulsion). We characterized and visualized the BCN stabilized o/w emulsions by scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM). Due to the sustainability and low environmental impact of chitosan, the BCN-based emulsions open up opportunities for the development of an environmental friendly new interface material as well as the novel type of microreactor utilizing bacterial cells network

Neutrophil Activation and Early Features of NET Formation Are Associated With Dengue Virus Infection in Human

International audienceThe involvement of the immune system in the protection and pathology of natural dengue virus (DENV) has been extensively studied. However, despite studies that have referred to activation of neutrophils in DENV infections, the exact roles of neutrophils remain elusive. Here, we explored the phenotypic and functional responses of neutrophils in a cohort of adult dengue patients. Results indicated that during an acute DENV infection, neutrophils up-regulate CD66b expression, and produce a more robust respiratory response as compared with that in convalescent or healthy individuals; this confirmed in vivo neutrophil activation during DENV infection. Spontaneous decondensation of nuclei, an early event of neutrophil extracellular trap (NET) formation, was also markedly increased in cells isolated from DENV-infected patients during the acute phase of the infection. In vitro incubation of NETs with DENV-2 virus significantly decreased DENV infectivity. Interestingly, increased levels of NET components were found in the serum of patients with more severe disease form-dengue hemorrhagic fever (DHF), but not uncomplicated dengue fever, during the acute phase of the infection. Levels of pro-inflammatory cytokines IL-8 and TNFα were also increased in DHF patients as compared with those in healthy and DF subjects. This suggested that NETs may play dual roles during DENV infection. The increased ability for NET formation during acute DENV infection appeared to be independent of PAD4-mediated histone H3 hyper-citrullination. Our study suggests that neutrophils are involved in immunological responses to DENV infection

Comparison between real-time RT-PCR (X-axis) and RT-RPA (Y-axis) for the detection of DENV in 31 clinical samples in Senegal.

<p>Linear regression analysis of real-time RT-PCR cycle threshold values (Ct, X-axis) and RT-RPA threshold time in minutes (TT, Y-axis) were determined by PRISM (R² = 0.39). The RT-RPA is much faster than the real-time RT-PCR even with samples with high Ct value.</p

List of viral RNA tested.

<p>DENV1-3 RT-RPA assay detected DENV1-3 but not other viral RNA. DENV4 RT-RPA identified DENV2 and DENV4.</p><p>+, positive;-, negative.</p><p>List of viral RNA tested.</p

Differentiation between specific and non-specific signals of the RT-RPA assay.

<p>A and C are real-time fluorescence intensity; B and D are the 1^st derivative analysis. Specific DNA amplification represented by progressive fluorescence development in both views, A and B, while non-specific not. Black line shows specific amplification where blue line shows no amplification.</p

Reproducibility of DENV RT-RPA assays.

<p>A, DENV1-3 and B, DENV4 RT-RPA assays. Semi-logarithmic regression of the data collected from eight DENV RT-RPA test runs on the RNA standard using PRISM. Both assays yielded results between 3–7 minutes. In DENV1-3 RT-RPA assay, 10⁷–10³ RNA molecules were detected 8 out of 8 runs, 10² in 1 out of 8 and 10 copies was not identified. In DENV4 RT-RPA assay, 10⁷–10² RNA molecules were detected 8 out of 8 runs and 10 copies in 6/ out of 8. In Fig 4B, the value for 10 RNA copies was consistently 5.3 minutes in all 6 cases.</p

Analytical sensitivity of DENV RT-RPA assays.

<p>A, DENV1-3 and B, DENV4 RT-RPA assays. Fluorescence development via real-time detection in one RT-RPA run by using a dilution range of 10⁷–10¹ RNA molecules/μl of the DENV1-3 and DENV4 RNA molecular standards (Graph generated by ESEquant tubescanner studio software). The sensitivity was 100 and 10 RNA copies for DENV1-3 and DENV4 RT-RPA, respectively. Data of 8 RT-RPA runs is compiled in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129682#pone.0129682.g004" target="_blank">Fig 4</a>. The signal for ten RNA copies is very weak. The box in the lower right corner of Fig 3B magnifies the fluorescence signals for the ten RNA copies and the negative control. 10⁷ represented by black line; 10⁶, gray; 10⁵, red; 10⁴, blue; 10³, green; 10², cyan; 10¹, dark khaki; negative control, orange.</p

Performance of DENV RT-RPA assays on spiked plasma samples.

<p>A, sample spiked with DENV1; B, DENV2; C, DENV3; D, DENV4. DENV serotypes 1–4 were spiked into plasma samples. Serial dilutions of each of the spiked sample were tested simultaneously with real-time RT-PCR and RT-RPA assays (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129682#pone.0129682.s009" target="_blank">S2 Table</a>). Limits of detection in RT-RPA assays were 237, 618, 363, and 383 RNA copies of DENV serotypes 1, 2, 3, and 4, respectively.</p

Sequence of primers and probes for the construction of molecular standards, real-time RT-PCR and RT-RPA assays.

<p>Bp, base pair; DENV1-3-STD-UP/DP are for DENV1-3 RT-PCR; DENV4-STD-UP/DP are for DENV4 RT-PCR; DENV-PCR-FP/RP/P, real-time RT-PCR primers and Taqman probe (FAM/TAMRA); DENV1-3-RPA-FP, forward and reverse primers for DENV1-3 RT-RPA; DENV4-RPA-FP, forward and reverse primers for DENV4 RT-RPA; DENV-RPA-P, exo-probe; BHQ1-dT: thymidine nucleotide carrying Blackhole quencher1, THF: tetrahydrofuran spacer, FAM-dT: thymidine nucleotide carrying Fluorescein. N/A, non applicable.</p><p>Sequence of primers and probes for the construction of molecular standards, real-time RT-PCR and RT-RPA assays.</p