Venn diagrams showing the numbers of AS-positive patients from Lithuanian study group sensitive to one or more allergens of the same type.

<p>(A) AS to pollens. (B) AS to animal dander. (C) AS to moulds.</p

Pie diagram showing numbers of AS cases from Lithuanian study group sensitive to indicated allergens.

<p>Pie diagram showing numbers of AS cases from Lithuanian study group sensitive to indicated allergens.</p

Inhibition of Influenza A Virus Infection <i>in Vitro</i> by Saliphenylhalamide-Loaded Porous Silicon Nanoparticles

Influenza A viruses (IAVs) cause recurrent epidemics in humans, with serious threat of lethal worldwide pandemics. The occurrence of antiviral-resistant virus strains and the emergence of highly pathogenic influenza viruses have triggered an urgent need to develop new anti-IAV treatments. One compound found to inhibit IAV, and other virus infections, is saliphenylhalamide (SaliPhe). SaliPhe targets host vacuolar-ATPase and inhibits acidification of endosomes, a process needed for productive virus infection. The major obstacle for the further development of SaliPhe as antiviral drug has been its poor solubility. Here, we investigated the possibility to increase SaliPhe solubility by loading the compound in thermally hydrocarbonized porous silicon (THCPSi) nanoparticles. SaliPhe-loaded nanoparticles were further investigated for the ability to inhibit influenza A infection in human retinal pigment epithelium and Madin-Darby canine kidney cells, and we show that upon release from THCPSi, SaliPhe inhibited IAV infection <i>in vitro</i> and reduced the amount of progeny virus in IAV-infected cells. Overall, the PSi-based nanosystem exhibited increased dissolution of the investigated anti-IAV drug SaliPhe and displayed excellent <i>in vitro</i> stability, low cytotoxicity, and remarkable reduction of viral load in the absence of organic solvents. This proof-of-principle study indicates that PSi nanoparticles could be used for efficient delivery of antivirals to infected cells

KSHV reactivation in iSLK.219 cells induces a G2 cell-cycle arrest.

<p>(A) Development of a high-content, image-based cell-cycle progression analysis based on the levels of phosphorylation of the histone H3 on serine-10 (pH3-S10). Automated image analysis was used to quantify the fluorescence intensity of each nucleus after immunofluorescence staining of pH3-S10, and to assign the corresponding cell to a specific stage of the cell cycle (<i>a</i> for G1/S, <i>b-e</i> for G2 or <i>f-g</i> for M). (B) Representative images of iSLK cells treated with DMSO or TPA/Dox for 20h and analyzed for cell cycle progression by pH3-S10 staining (green) and reactivation by RFP (red). Italics letters indicate different stages of the cell cycle (compare to (A)). The inset shows a magnification of the cell pointed by the white arrow head. (C-D) Quantification of the number of cells in M- (C) or G2-phase (D) in iSLK.219 cells treated with DMSO, TPA/Dox or TPAas. Values are the mean and SD of three independent experiments. The total number of cells in each sample was set as 100%. In each repetition, more than 1800 cells were analyzed. * p<0.05. (E) Images of pH3-S10 in iSLK.219 cells transfected with siRNAs against p21 (si-p21) or non-targeting controls (si-Ctrl), and reactivated with TPA/Dox for 20 hours. (F) Higher magnification images of cells in the respective white-box areas in panel (E). (G) Quantifications of the number of cells in M- or G2-phase in iSLK.219 cells treated as in (E). Values are normalized to the respective non-induced (DMSO) controls set to one (dotted red line). Error bars represent the SD of three independent experiments. * p<0.05.</p

During lytic reactivation iSLK.219 cells 'by-pass' the G1-checkpoint and accumulate high levels of DNA damage.

<p>(A) Schematic representation of the Nutlin pretreatment experiment. iSLK.219 cells were treated with Nutlin 18h before reactivation with TPA/Dox. Cells were fixed 18h later and processed for immunofluorescence and high-content imaging. (B) Fluorescence images of iSLK.219 cells treated as in (A) and processed for immunofluorescence using antibodies against pH3 S10 (green). Lytic reactivation was monitored by RFP (red) expression, nuclei were stained with Hoechst. (C) Quantification of cell cycle progression by image analysis from the experiment shown in (B). In each condition, the values are normalized to the total number of cells (set as one). The cell cycle arrest induced by the different treatments is summarized in the scheme above the chart. (D-E) Representative fluorescence images of iSLK.219 cells reactivated with TPA/Dox for 24 h and processed for immunofluorescence staining of two markers of DNA damage response, phosphorylated ATM (pATM, D) and phosphorylated Chk1 (pChk1, E). The insets show higher magnifications of cells from the respective white-boxed areas. Nuclei are stained with Hoechst and virus reactivation monitored by RFP expression. (F) Quantification of the median fluorescence intensity of indicated markers of DNA damage in cells treated as indicated for 24 h or 48 h and processed for immunofluorescence analysis. For each marker, values were normalized to the median fluorescence intensities obtained from iSLK.219 cells treated with Etoposide (6.25 μM) for the same times (100%, red-dashed line). Error bars represent the SD of three independent experiments. (G) The fluorescence intensity of indicated DNA damage response was quantified as in (F) in cells treated with TPA or NaB for 24 h.</p

p53 and p21 are required for efficient virus lytic gene expression.

<p>(A) Fluorescence images of lytic gene expression (RFP, red) in iSLK.219 co-treated with TPA/Dox and DMSO or Nutlin for 24 h. Nuclei are stained with Hoechst (grey). (B) Quantification of the median RFP intensity from the experiment shown in (A). Values represent the mean of three independent experiments. Error bars indicate the SD. * p<0.05. (C) Fluorescence images of RFP (Red) expression in iSLK.219 cells transduced for 48 h with lentiviruses expressing shRNA against p53 (sh1-p53) or non-targeting controls (sh-Ctrl), and reactivated with TPA/Dox for 18 h. Nuclei are stained with Hoechst (grey). (D) Quantification of the median RFP intensity from the experiment shown in (C). A second, independent shRNA against p53 (sh2-p53) was also included. The values are the mean of three independent repetitions. Error bars represent the SD. The efficiency of p53 depletion was monitored in parallel experiments by WB (right panel). * p<0.05. (E) Images of RFP (Red) expression in iSLK.219 cells transfected with siRNAs against p21 (si-p21) or non-targeting controls (si-Ctrl), and treated with TPA/Dox for 18 h. Nuclei are stained with Hoechst (grey). (F) Quantification of the median RFP intensity from cells treated as in (E). The values are the mean of three independent repetitions. Error bars represent the SD. Confirmation of the p21 depletion by WB is shown in the right panel. * p<0.05. (G) Quantification of lytic gene expression in iSLK.219 cells treated as in (E) and processed for immunofluorescence and image analysis. Values are normalized to the respective nonspecific controls (si-Ctrl) set to one (dotted red line). Each value represents the mean of three independent experiments and associated SD. * p<0.05</p

siRNA screen identifies MDM2 as a novel regulator of KSHV reactivation.

<p>(A) Fluorescence images of iSLK.219 cells treated with indicated siRNAs. Virus reactivation was induced 48h after siRNA transfection by a low-dose (0.05 μg/ml) doxycycline treatment and the extent of reactivation measured by detection of RFP positive cells using high-content fluorescence imaging and automated image analysis. Cells were visualized with Hoechst 33342 (Nuclei, blue), GFP (marker of latency, green) and RFP (marker of reactivation, red). (B) Quantification of the experiment shown in (A). The results are the average of three independent experiments. The error bars represent the standard error of the mean (SEM). * p<0.05. (C) qRT-PCR analysis of ORF50, ORF57, vGPCR and K8.1 transcripts from latent, DMSO-treated BC-3 cells transduced for 72 h with lentiviruses expressing nonspecific control (shCtrl) or MDM2 targeting shRNAs (shMDM2). The relative mRNA levels obtained from the shMDM2 treated cells were normalized to the corresponding values from shCtrl treated cells. (D) qRT-PCR analysis of ORF50, ORF57, vGPCR and K8.1 transcripts from BC-3 treated with shRNA as in (C) and reactivated with TPA for indicated times. Values normalized as in (C). The error bars in (C) and (D) represent the SEM of three independent experiments. * p<0.05</p

p21 depletion impairs lytic gene expression in PEL cells.

<p>(A-B) Quantification of p21 mRNA levels by qRT-PCR during a time course of TPA or doxyxyclin induced reactivation in the BC-3 and BCBL1_RTA PEL cells, respectively, stably expressing non specific sh-Ctrl. (C) WB analysis of p21 levels in whole cell extracts from PEL cells stably expressing indicated shRNAs and induced to reactivation with TPA (BC-3) or Dox (BCBL1_RTA) for 24 h. GAPDH was used as a loading control. (D) Quantification of ORF73 (LANA) mRNA levels by qRT-PCR in PEL treated as in (C). Values represent the average and SEM of three replicates, and are normalized to the shCtrl set to one (red dashed line). (E-F) Quantification of transcription of the indicated lytic genes in PEL cells stably expressing sh-Scr, sh1-p21 or sh2-p21 and induced by TPA for 24h (BC-3, E) or Dox for 48h (BCBL1_RTA, F). The values represent the mean and SDM of three independent experiments, and are normalized to sh-Ctrl set to one (red dashed line). *p<0.05. (G-H) Quantification of cell number to monitor virus replication induced cytopathic effect in PEL cells expressing indicated shRNAs and induced to lytic reactivation by TPA (BC-3, G) or Dox (BCBL1_RTA, H). The values represent the mean and SDM of three independent experiment.</p

Viral reactivation leads to a p53 response in PEL cells.

<p>(A) BC-3 cells treated with TPA (red line) or Nutlin (blue line) for indicated times were processed for ChIP-seq analysis using an antibody against p53. Each chart represents the sequencing signal (ChIP-seq peak) obtained after immunoprecipitation with the p53 (blue and red lines) or nonspecific IgG antibodies (light blue and green lines) for the indicated cellular genes. The known function of each gene is indicated at the bottom of each panel. (B) The sequencing signal of BC-3 cells treated as in (A) is shown for the whole KSHV genome. The inset is an enlarged view of the DNA region coding for the RTA gene (black dashed box). (C) Using the MEME software, the same <i>consensus</i> binding-motif of p53 was identified <i>de novo</i> from the ChIP-seq results of BC-3 cells treated with Nutlin (8 h) or TPA (24 h). (D) BC-3 cells were treated with TPA for or Nutlin for indicated times and processed for WB using antibodies against p53, p21 and tubulin. (E) BC-3 or iSLK.219 cells treated with vehicle (DMSO) or indicated inducers for 24 h were processed for WB using antibodies against p21 and GAPDH. (F) iSLK.219 cells were treated with indicated sh- or siRNAs for 48 h and then incubated with DMSO, Nutlin or TPA/Dox for 4 h before WB analysis using antibodies against p53, p21 and GAPDH.</p