Effects of WPCD on the splenocyte proliferation.

<p>Single splenocyte was isolated from mice immunized on day 21 after the first vaccination and splenocyte proliferation was determined by the MTT assay and expressed as SI. OVA-antigen stimulated splenocyte proliferation <b>(A)</b>. OVA^323-339-antigen stimulated splenocyte proliferation <b>(B).</b> ConA-mitogen stimulated splenocyte proliferation <b>(C)</b>. LPS-mitogen splenocyte proliferation <b>(D)</b>. Data are expressed as mean ± SD (n = 5) from 3 independent experiments. *<i>P</i> < 0.05, ** <i>P</i> < 0.01 compared with the OVA group; ^#<i>P</i> < 0.05, ^##<i>P</i> < 0.01 compared with the OVA/Alum group.</p

Effects of WPCD on cytokine production and functional activation of DCs <i>in vitro</i>.

<p>After DCs were treated with different concentrations of WPCD for 12 h, the supernatant was collected and the productions of IL-12 and TNF-α were detected by ELISA. The concentrations of IL-12 <b>(A)</b> and TNF-α <b>(B)</b> were shown. An allogenic T cell proliferation of WPCD-treated DCs was analyzed by MLR. Allogenic T cells were co-cultured for 3 days at the indicated ratio with DCs which had been cultured for 12 h in triplicate <b>(C and D)</b>. MTT was added into the cultures for final 4-h treatment and the T cell proliferation was then determined by ELISA reader. Data represent mean ±SD (n = 3) from 3 independent experiments.*<i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001 compared with the untreated DCs group.</p

Effects of WPCD upon DCs phenotypic maturation <i>in vitro</i>.

<p>BM-DCs from C57BL/6 mice were treated with different concentrations of WPCD for 12 h. LPS was used as the positive control. Then, the expression levels of CD11c, CD40, CD80, CD86, and MHC-II in cells were analyzed. Phenotypic maturation of BM-DCs was induced by PBS, LPS and WPCD. The mean fluorescence intensity values (MFI) of CD40 <b>(A)</b>, CD80 <b>(B)</b>, CD86 <b>(C)</b>, and MHC-II <b>(D)</b> were shown. Data represent mean ± SD (n = 3) from 3 independent experiments. *<i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001 compared with the untreated DCs group.</p

Effects of subcutaneous administration of WPCD on the mean body weight.

<p>Effects of subcutaneous administration of WPCD on the mean body weight.</p

Effects of WPCD on OVA-specific IgG and IgG subclass.

<p>Effects of WPCD on OVA-specific IgG, IgG₁ and IgG_2a antibody in serum were assessed by ELISA. IgG titer on Days 14, 21, 35, and 49 after the first vaccination <b>(A)</b>, the antibody levels of IgG₁ and IgG_2a on Day 35 after the first vaccination <b>(B).</b> Data represent mean ± SD (n = 5) from 3 independent experiments. *<i>P</i>< 0.05, **<i>P</i>< 0.01, ***<i>P</i>< 0.001 compared with the OVA group; ^##<i>P</i>< 0.01, ^###<i>P</i> < 0.001 compared with the OVA/Alum group.</p

Evaluation of the oral acute toxicity of WPCD.

<p>Evaluation of the oral acute toxicity of WPCD.</p

Immunostimulatory activity of water-extractable polysaccharides from <i>Cistanche deserticola</i> as a plant adjuvant <i>in vitro</i> and <i>in vivo</i>

<div><p>A safe and effective vaccine adjuvant is important in modern vaccines. Various Chinese herbal polysaccharides can activate the immune system. <i>Cistanche deserticola</i> (CD) is a traditional Chinese herb and an adjuvant candidate. Here, we confirmed that water-extractable polysaccharides of CD (WPCD) could modulate immune responses <i>in vitro</i> and <i>in vivo</i>. In a dose-dependent manner, WPCD significantly promoted the maturation and function of murine marrow-derived dendritic cells (BM-DCs) through up-regulating the expression levels of MHC-II, CD86, CD80, and CD40, allogenic T cell proliferation, and the yields of IL-12 and TNF-α via toll-like receptor4 (TLR4), as indicated by <i>in vitro</i> experiments. In addition, its immunomodulatory activity was also observed in mice. WPCD effectively improved the titers of IgG, IgG₁ and IgG_2a and markedly enhanced the proliferation of T and B cells, the production of IFN-γ and IL-4 in CD4⁺ T cells and the expression level of IFN-γ in CD8⁺ T cells better than Alum. Furthermore, WPCD could markedly up-regulate the expression levels of CD40 and CD80 on DCs in spleen and down-regulate the Treg frequency. The study suggests that polysaccharides of <i>Cistanche deserticola</i> are a safe and effective vaccine adjuvant for eliciting both humoral immunity and cellular immunity by activating DCs via TLR4 signaling pathway.</p></div

Effects of WPCD on BM-DCs function through TLR4-mediated pathway.

<p>After pretreatment with 5 μM of TAK-242 for 1 h, BM-DCs were treated with 100 ng/mL LPS or 100 and 200 μg/mL WPCD for 12 h in the presence or absence of Golgi Stop. Then CD40, CD86, TNF-a and IL-12 expressions were detected by FACs or ELISA. The MFI values of CD40 <b>(A)</b> and CD86 <b>(B)</b>, the yields of TNF-a <b>(C)</b>, and IL-12 <b>(D)</b> were shown. Data represent mean ± SD (n = 3) from 3 independent experiments.*<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001 compared with the untreated DCs group.</p