Synergistic correlation between host angiogenin and dengue virus replication

DENV infection poses a major health concern globally and the pathophysiology relies heavily on host-cellular machinery. Although virus replication relies heavily on the host, the mechanistic details of DENV–host interaction is not fully characterized yet. Here, we are focusing on characterizing the mechanistic basis of virus-induced stress on the host cell. Specifically, we aim to characterize the role of the stress modulator ribonuclease Angiogenin during DENV infection. Our results suggested that the levels of Angiogenin are up-regulated in DENV-infected cells and the levels increase proportionately with DENV replication. Our efforts to knockdown Angiogenin using siRNA were unsuccessful in DENV-infected cells but not in mock-infected control. To further investigate the modulation between DENV replication and Angiogenin, we treated Huh7 cells with Ivermectin prior to DENV infection. Our results suggest a significant reduction in DENV replication specifically at the later stages as a consequence of Ivermectin treatment. Interestingly, Angiogenin levels were also found to be decreased proportionately. Our results suggest that Angiogenin modulation during DENV infection is important for DENV replication and pathogenesis.</p

Stat-5A transfection confers resistance to CD4⁺ T cells from tumor-induced death.

<p>A, Stat-5A (<i>left panel</i>) and Stat-5B (<i>right panel</i>) isoforms were immunoprecipitated from cell lysates using specific antibodies and then Western blotted with anti-phospho-tyrosine or anti-Stat-5A/Stat-5B antibodies to determine phosphorylation status of specific proteins. B, Jurkat T cells were transfected with control vector, wild-type <i>Stat-5A/Stat-5B</i>, C-terminal truncated <i>Stat-5A</i>₇₁₃/<i>Stat-5B</i>₇₁₈ or constitutively active <i>Stat-5A1*6</i> genes and were cultured in the presence of media alone or MCF-7 spent media (±theaflavins) for 48 h. Percent cell death (Annexin-V-PE⁺/7AAD⁺) was determined flow cytometrically. Values are mean±S.E.M. of three independent sets of experiments.</p

Re-confirmation of PGE₂ as the molecule behind tumor-induced perturbation in CD4⁺ T cell survival signaling.

<p>A, MCF-7 cells were treated with theaflavins or celecoxib or transfected with Cox-2-siRNA and the levels of Cox-2 and GAPDH (internal control) mRNA were determined by RT-PCR (<i>upper panel</i>). Western blot analysis was performed for the determination of levels of Cox-2 or α-Actin (internal control) proteins (<i>middle panel</i>). In parallel experiments the amount of tumor-secreted PGE₂ in the cell-free supernatant was determined by ELISA (<i>lower panel</i>). B, Purified CD4⁺ T cells were cultured in the presence of media alone or MCF-7-spent media (tumors were either pre-treated with 25 µg/ml theaflavins/3.5 ng/ml PGE₂/50 µM celecoxib or transfected with 300pmole Cox-2-siRNA) for 48 h. Expression levels of IL2Rγc and Bcl-2 as well as phosphorylation status of Jak-3/-Stat-5 were determined by Western blotting in which α-Actin was used as internal control (<i>upper panel</i>). In parallel experiments, flowcytometric determination of percent cell death (<i>lower panel</i>) was established. Values are mean±S.E.M. of three independent experiments.</p

Tumor-shed PGE₂ is responsible for CD4⁺ T cell apoptosis.

<p>A, Tumor-secreted PGE₂ over time in cell-free spent media of MCF-7 cells (control (○), Cox-2-siRNA-transfected (Δ) or theaflavin-treated (•) was determined by ELISA. B, Percent CD4⁺ T cell death (Annexin-V-PE⁺/7AAD⁺), induced by the spent media as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007382#pone-0007382-g002" target="_blank">Fig. 2A</a>, was plotted over time. Values are mean±S.E.M. of three independent sets of experiments.</p

Cell free tumor supernatant leads to CD4⁺ T cell depletion by inducing apoptosis.

<p>A, Purified human peripheral CD4⁺ T cells were cultured in the presence of media alone or cell-free MCF-7-spent media (±theaflavins, doses from 6.25 µg/ml to 50 µg/ml). After 48 hours, viable cell numbers were scored by Trypan Blue exclusion method. B, Graphical representation of percent apoptosis of CD4⁺ T cells (<i>left panel</i>) and Jurkat T cells (<i>right panel</i>). CD4⁺ T cells labelled with Annexin V-PE and 7AAD were analyzed flow cytometrically. Annexin V/7AAD-positive cells were regarded as apoptotic cells. Values are mean±S.E.M. of five independent sets of experiments.</p