Lag-response association specific to rainfall category 2(0 mm

<p>Lag-response association specific to rainfall category 2(0 mm</p

Seasonal variation in daily hand, foot and mouth disease cases and meteorological variables in Hong Kong, 2008–2011.

<p>Seasonal variation in daily hand, foot and mouth disease cases and meteorological variables in Hong Kong, 2008–2011.</p

DC-SIGN-mediated and clathrin-dependent entry of LV-SVGmu.

<p>(A) Viruses binding to DC-SIGN-expressing cells. 293T/hDC-SIGN cells were incubated with GFP-Vpr-labeled LV-SVGmu particles (green) for 30 min at 4°C, fixed, and immunostained with anti-DC-SIGN antibody (red). (B) Real-time monitoring of virus internalization mediated by the DC-SIGN receptor. 293T cells seeded on a glass-bottom dish were transiently transfected with DsRed-DC-SIGN (red). At 24 h post-transfection, the cells were incubated with GFP-Vpr-labeled LV-SVGmu particles (green) for 30 min at 4°C and were then warmed to 37°C to initiate virus internalization. Confocal time-lapse images were then recorded. Selected frames of the real-time imaging are presented. (C) 293T/hDC-SIGN cells were incubated with GFP-Vpr-labeled LV-SVGmu viruses (green) for 30 min at 4°C to synchronize infection and were then shifted to 37°C for 5 min. After incubation, cells were fixed, permeabilized and immunostained with antibody against caveolin-1 (red) or clathrin (red). The boxed regions are enlarged in the bottom panels. Scale bar represents 5 µm. (D) Quantification of GFP-Vpr-labeled LV-SVGmu particles colocalized with clathrin signals. Overlap coefficents were calculated using Mander’s overlap coefficient in Nikon NIS-Elements software by viewing more than 40 cells. (E) The effect of inhibitory drugs on LV-SVGmu or LV-SFV transduction. 293T/hDC-SIGN cells were preincubated with chlorpromazine (CPZ, 30 nM) or filipin (15 nM) for 30 min at 37°C. Cells were then infected with LV-SVGmu or LV-SFV encoding a reporter GFP gene for 90 min in the presence of drugs. After an additional 3 h of incubation, the medium was replaced with fresh media, and the percentage of GFP-positive cells was analyzed by flow cytometry after 72 h. All of the data were then normalized based on the actual percentage of GFP-positive cells of the no drug treatment group of LV-SVGmu (35.2±0.028%) or LV-SFV (33.5±0.34%). All data are shown as the means of triplicate experiments. Asterisk indicates comparison to the no drug treatment group (*<i>P</i><0.05, **<i>P</i><0.01). (F) Functional involvement of dynamin in LV-SVGmu transduction. 293T/hDC-SIGN cells were transiently transfected with the wild-type or dominant-mutant form of dynamin I (K44) for 24 h, then infected with LV-SVGmu. The percentage of GFP-positive cells was analyzed by flow cytometry at 72 h post-infection. The data are presented as the mean values ± SD (n >3). The data were normalized based on the actual percentage of GFP-positive cells of Dyn-WT-expressing cells infected by LV-SVGmu (30.9±3.09%).</p

Three-dimensional plots of relative risks along daily mean temperature (A), relative humidity (B), square root of wind speed (C) and total daily solar radiation (D) and their corresponding lags.

<p>Three-dimensional plots of relative risks along daily mean temperature (A), relative humidity (B), square root of wind speed (C) and total daily solar radiation (D) and their corresponding lags.</p

Cumulative effects of different meteorological factors on hand, foot and mouth disease by different days of lag.

<p>Cumulative effects of different meteorological factors on hand, foot and mouth disease by different days of lag.</p

PSCA-specific T cell response after a single dose of <i>in vivo</i> immunization with DCLV-PSCA.

<p>(A) Male C57BL/6 mice were immunized with 6×10⁷ TU of DCLV-PSCA through different administration routes: intraperitoneal space (i.p.), subcutaneous area (s.c.), intramuscular area (i.m.), footpad (f.p.), or intradermal (the base of tail, i.d.). One immunization group was included as a negative control. Two weeks after immunization, splenocytes from mice were harvested and analyzed for the presence of PSCA-specific CD8⁺ T cells by restimulating splenocytes with a PSCA peptide (PSCA_83-91), followed by intracellular staining for IFN-γ and surface staining for CD8. Percentage of IFN-γ-secreting CD8⁺ T cells is indicated. (B) Statistical comparison of immunization elicited by administration of DCLV-PSCA among different administration routes. (C) Male C57BL/6 mice were immunized with different doses of DCLV-PSCA vectors (0, 2, 10, 40 and 80 million TU) at the base of tail. Two weeks post-vaccination, PSCA-specific CD8⁺ T cells from the spleen were analyzed by restimulating with the peptide PSCA_83-91, followed by intracellular staining for IFN-γ. (D) Production of PSCA-specific IFN-γ-secreting cells from both spleen (SP) and inguinal lymph node (LN) was evaluated by restimulation with the PSCA_83-91 peptide, followed by ELISPOT analysis for IFN-γ. (E) Production of PSCA-specific IL-2 from splenocytes (with CD8⁺ T cells depleted) was measured by restimulation with 293T cell lysate transfected to express PSCA, followed by the ELISPOT analysis for IL-2. (**: <i>P</i><0.01; *: <i>P</i><0.05; One-way ANOVA followed by Bonferroni's multiple comparison test. Error bars represent SD.) All experiments were repeated three times and the representative data is shown.</p

Cumulative relative risks of hand, foot and mouth disease hospitalization as a function of meteorological variables for a time lag of 1 week (mean temperature (A) and relative humidity (B)) and 2 weeks (square root of wind speed (C) and total solar radiation (D)).

<p>Cumulative relative risks of hand, foot and mouth disease hospitalization as a function of meteorological variables for a time lag of 1 week (mean temperature (A) and relative humidity (B)) and 2 weeks (square root of wind speed (C) and total solar radiation (D)).</p

CD8⁺/CD4⁺ T cell-dependent immune protection against TRAMP-C1 tumors induced by DC-LV-PSCA immunization.

<p>(A) Infiltration of T cells into tumor tissues. TRAMP-C1 tumors from tumor-bearing mice were excised 3 weeks post-immunization, paraffin-embedded, and stained for immunofluorescence-conjugated CD3, CD4 and CD8 antibody (green color as indicated by white arrows) together with nuclear staining (red color). Representative images showing CD4⁺ and CD8⁺ T cells infiltrated to tumor tissues from DCLV-PSCA-immunized mice as compared to those of DCLV-Null-immunized mice. (B) Four groups of male C57BL/6 mice (n = 8 for each group) were transplanted with 5×10⁵ TRAMP-C1 cells subcutaneously at day 0. Fourteen days later, 3 groups were immunized with DCLV-PSCA, while the other group was immunized with mock vector DCLV-Null. Two groups of mice from the DCLV-PSCA-immunized groups were subjected to CD4⁺ or CD8⁺ T cell depletion by injecting CD4- or CD8-depletion antibody intraperitoneally. (C) Tumor volume for each group of mice was monitored. Error bars represent SEM. All experiments were repeated twice and the representative data is shown.</p

The role of autophagy and lysosome network in intracellular trafficking of LV-SVGmu.

<p>(A) Imaging the colocalization of autophages and lysosomes with LV-SVGmu particles with or without rapamycin treatment. 293T/hDC-SIGN cells treated or untreated by rapamycin were incubated with GFP-Vpr-labeled LV-SVGmu (green) for 30 min at 4°C to synchronize infection and were then shifted to 37°C for different indicated incubation periods. Subsequently, the cells were fixed, permeabilized and immunostained with antibodies against lysosomes (Lamp-1, red) and autophagy (LC-3, blue). The boxed regions are enlarged in the bottom panels. Scale bar represents 5 µm. (B) Quantification data on the colocalization of viral particles with autophagy-lysosome double-positive structure or autophagy alone in cells after rapamycin treatment. Overlap coefficients were calculated using Mander’s overlap coefficient in Nikon NIS-Elements software by viewing more than 40 cells at each time point. (C) The effect of autophagy activity on LV-SVGmu transduction. 293T/hDC-SIGN cells were preincubated with 3-MA (5 mM) or rapamycin (1 µM) for 30 min at 37°C. Then cells were infected with LV-SVGmu carrying a reporter GFP gene for 90 min in the presence of drugs. After an additional 3 h of incubation, the medium was replaced with fresh media, and the percentage of GFP-positive cells was analyzed by flow cytometry after 72 h. All of the data were then normalized based on the actual percentage of GFP-positive cells of the no drug treatment group of LV-SVGmu (25.8±1.14%). All data are shown as the means of triplicate experiments.</p

The ability of DCLV-PSCA immunization to suppress lung metastases.

<p>(A) Male C57BL/6 mice were immunized with DCLV-PSCA or DCLV-Null as a mock control. Ten days later, mice were challenged with 0.2 million B10-F10-PSCA cells by intravenous injection through tail vein. Two weeks later, mice were sacrificed, and macroscopic views of the lungs were shown. (B) Microscopic H&E staining (20×) of lung tissue samples from mice immunized with DCLV-PSCA or DCLV-Null. (C) Statistical quantification of melanoma lung metastases (number of black nodules on the lungs) of immunized mice; similar immunization, but with the original B16-F10 melanoma metastases included as a control. (**: <i>P</i><0.01 and n/s: not statistically significant; One-way ANOVA followed by Bonferroni's multiple comparison test. Error bars represent SD, n = 4). All experiments were repeated twice and the representative data is shown.</p