Echo30 infection induced TRIO-RhoA signaling activation in neuronal cell.

<p>Human neuroblastoma SK-N-SH cells at 80% confluence were infected with Echo30 with 2% FBS contained MEM at 48 hrs. (A) Confirmation results of 2-DE using immunoblot blot analysis. TRIO protein expression was significantly increased in Echo30-infected SK-N-SH cells. (B) Immnoblot analysis for RhoA signaling. Echo30 infection induced activation of RhoA and RhoA targeted signaling in SK-N-SH cell. All data are representative of three independent experiments.</p

Echo30 infection induced neuronal cell death.

<p>Human neuroblastoma SK-N-SH cells at 80% confluence were infected with Echo30 with 2% FBS contained MEM at 48 hrs. (A) The effect of Echo30 infection on SK-N-SH cells. Echo30 infected SK-N-SH cells significantly increase cytopathic effect. (B) Measurement of cell viability by WST-1 assay. The viability of SK-N-SH cells was decreased by Echo30 infection (mean±S.E.M, *<i>p</i><0.05). (C) Measurement of sub-G1 phase using flow cytometry analysis. Echo30 infection increase sub-G1 phase at cell cycle distribution. (D) Expression of apoptotic proteins using immunoblot analysis. The expression level of pro-apoptotic protein was increased by Echo30 infection. All data are representative of three independent experiments.</p

Identification of differentially expressed proteins in Echo30-infected SK-N-SH cells at 48 hrs by MALDI-TOF.

*<p>n/a⁺: Detected only in SK-N-SH cells infected with Echo30.</p

Protein identification of differentially expressed cellular proteins in Echo30-infected SK-N-SH cells at 48 hrs by two-dimensional gel electrophoresis (2-DE).

<p>(A) The arrows indicated the protein spots identified as differentially regulated at greater than four-fold changes (<i>p</i><0.05). (B) The spot density was represented relative to the control (non-infected SK-N-SH cells).</p

Echo30 infection enhanced NO production through TRIO-RhoA signaling activation in neuronal cells.

<p>(A) Echo30-infected SK-N-SH cells increase NOS family (eNOS, iNOS, nNOS) protein levels using immunoblot analysis. Echo30 infection enhanced NOS family protein levels. (B) Measurement of cellular NO production by griess reagent system. Echo30-infected SK-N-SH cells significantly increase cellular NO level (mean±S.E.M, *<i>p</i><0.05). (C) The effect of Rho kinase inhibitor Y27632 on NOS family induction by Echo30 infection. Pre-treatment of cells with the Y27632 prevent NOS family induction by Echo30 infection. (D) The effect of Y27632 on NO production by Echo30 infection. Pre-treatment of cells with the Y27632 blocked NO production by Echo30 infection (mean±S.E.M, *<i>p</i><0.05). All data are representative of three independent experiments.</p

Rho kinase inhibitor prevented neuronal cell death by Echo30 infection.

<p>(A) The effect of Y27632 on the cytopathic effect by Echo30 infection. The pretreatment of Y27632 inhibited Echo30 infection-inducing cytopathic effect in SK-N-SH cells. (B) The effect of Y27632 on cell viability by Echo30 infection determined by WST-1 assay. Y27632 inhibited Echo30 infection-inducing SK-N-SH cell death. (mean±S.E.M, *<i>p</i><0.05). (C) The effect of Y27632 on cell cycle analysis by Echo30 infection using FACS analysis. Y27632 inhibited increase of sub-G1 phase by Echo30 infection in SK-N-SH cells. (D) The effect of Y27632 on RhoA signaling by Echo30 infection using immunoblot analysis. Y27632 prevented Echo30 infection induced activation of RhoA signaling. (E) Graphical representation of densitometric analysis of the immnoblot bands for TRIO-RhoA signaling (mean±S.E.M, **<i>p</i><0.05, *<i>p</i><0.1). All data are representative of three independent experiments.</p

An inactivated hand-foot-and-mouth disease vaccine using the enterovirus 71 (C4a) strain isolated from a Korean patient induces a strong immunogenic response in mice

<div><p>Enterovirus 71 (EV71) is a major causative agent of hand-foot-and-mouth disease (HFMD) frequently occurring in children. HFMD induced by EV71 can cause serious health problems and has been reported worldwide, particularly in the Asia-Pacific region. In this study, we assessed the immunogenicity of a formalin-inactivated HFMD vaccine using an EV71 strain (FI-EV71 C4a) isolated from a Korean patient. The vaccine candidate was evaluated in mice to determine the vaccination doses and vaccine schedules. BALB/c mice were intramuscularly administered 5, 10, or 20 μg FI-EV71 vaccine, followed by a booster 2 weeks later. EV71-specific antibodies and neutralizing antibodies were induced and maintained until the end of the experimental period in all vaccinated groups. To determine the effectiveness of adjuvant for the EV71 vaccine, three adjuvants, i.e., aluminium hydroxide gel, monophosphoryl lipid A, and polyinosinic-polycytidylic acid, were administered separately with the FI-EV71 vaccine to mice via the intramuscular route. Mice administered the FI-EV71 vaccine formulated with all three adjuvants induced a significantly increased antibody response compared with that of the single adjuvant groups. The vaccinated group with triple adjuvants exhibited more rapid induction of EV71-specific and neutralizing antibodies than the other groups. These results suggested that the role of adjuvant in inactivated vaccine was important for eliciting effective immune responses against EV71. In conclusion, our results showed that FI-EV71 was a potential candidate vaccine for prevention of EV71 infection.</p></div

EV71-specific cell mediated immune responses induced by the formalin-inactivated EV71 vaccine with diverse adjuvants.

<p>Splenocytes from vaccinated mice were isolated and stimulated with the EV71 vaccine strain. Supernatants of splenocytes were harvested at 48 h after stimulation and analyzed for cytokine levels using a multiplex system. Graphs show means + SEMs. *<i>p</i> < 0.01, using one-way ANOVA with Tukey’s post-hoc test.</p

EV71-specific immune responses in serum samples from mice vaccinated with the formalin-inactivated EV71 vaccine combined with diverse adjuvants.

<p>Mice were administered the inactivated EV71 vaccine mixed with 500 μg for alum, 2 μg for MPLA, and 10 μg for poly I:C adjuvant. (A) The lgG antibody titer against EV71 was measured by ELISA at 4, 8, 12 and 16 weeks. (B) IgG isotype analysis of EV71-specific IgGs in sera of mice at 8 weeks postvaccination. The IgG isotype graph shows means + SEMs. ***<i>p</i> < 0.0001 using one-way ANOVA with Tukey’s post-hoc test.</p

EV71-specific humoral immune responses induced by a formalin-inactivated EV71 vaccine in mice.

<p>Mice were administered inactivated EV71 vaccine mixed with 500 μg for alum, 2 μg for MPLA, and 10 μg for poly I:C adjuvant or PBS as a control. (A) Titers of total IgG antibodies against EV71 viral particles were determined by ELISAs. (B) The levels of neutralization antibodies against EV71 were measured by endpoint dilution of serum in neutralization assays.</p