An alkali-extracted neutral heteropolysaccharide from Phellinus nigricans used as an immunopotentiator in immunosuppressed mice by activating macrophages.

A neutral heteropolysaccharide (PNANb) was isolated with alkali (0.1 M NaOH) from mycelia of Phellinus nigricans, and the structure, immunostimulating activity and some of the underlying molecular mechanisms of action of PNANb were explored in the current study. PNANb (14.95 kDa) predominantly consisted of Gal, Glc, and Man with minor Fuc. GC-MS and NMR analyses indicated that the backbone of PNANb was mainly composed of 6-α-Galp, 2,6-α-Galp with minor 3,6-ß-Glcp, which was substituted with complex side chains at C-2 of 2,6-α-Galp and C-3 of 3,6-ß-Glcp. Notably, PNANb (50 or 100 mg/kg) possessed immunoprotective effects in cyclophosphamide (Cy)-induced immunosuppressed C57BL/6 mice, which was supported by evidence including the enhancement of spleen and thymus indices, levels of serum immunoglobulins (IgG, IgM) and cytokines (IFN-γ, IL-2, IL-4, IL-10), and macrophage activity. However, the immunostimulation effects of PNANb were decreased when macrophages were depleted, underscoring the essential role of macrophages in the beneficial effects of PNANb in Cy-induced immunosuppressed mice. Further investigations in vitro indicated that PNANb activated macrophages through MAPK/NF-κB signaling pathways mediated by Toll-like receptor 4. Therefore, PNANb can serve as a prospective immunopotentiator in immunosuppression.

An arabinogalactan extracted with alkali from Portulaca oleracea L. used as an immunopotentiator and a vaccine carrier in its conjugate to BSA.

A neutral polysaccharide (POPAN) from Portulaca oleracea L. was isolated with alkali and purified to obtain. HPLC analysis suggested POPAN (40.9 kDa) was mainly composed of Ara and Gal with traces of Glc and Man. GC-MS and 1D/2D NMR analysis confirmed POPAN was an arabinogalactan possessing a backbone mainly composing of (1 â†’ 3)-α-l-Araf-linked arabinan and (1 â†’ 4)-ß-d-Galp-linked galactan, which was different from structure characterization of typical arabinogalactan reported previously. Importantly, we conjugated POPAN to BSA (POPAN-BSA), and detected the potential and mechanism of POPAN as an adjuvant in POPAN-BSA. The results indicated, in contrast to BSA, POPAN-BSA induced the robust and persistent humoral response in addition to the cellular response with Th2-biased immunity response in mice. Further investigations of mechanism revealed effects of POPAN-BSA were a result of POPAN as the adjuvant to: 1) significantly activate DCs in vitro or in vivo including the upgraded expressions of costimulators, MHCs and cytokines; 2) greatly facilitated the capture of BSA. Overall, present studies demonstrated POPAN can be a potential adjuvant as an immunopotentiator and an antigen delivery vehicle in its conjugate to recombinant protein vaccines.

Chitosan hydrogel loaded with recombinant protein containing epitope C from HSP90 of Candida albicans induces protective immune responses against systemic candidiasis.

We reported previously a recombinant protein (rP-HSP90C) containing epitope C from heat shock protein 90 of Candida albicans mediates protective immune responses against systemic candidiasis. However, it exhibits weak immunogenicity. Therefore, we evaluated the potential and mechanisms of thermosensitive chitosan hydrogel (CH-HG) as an adjuvant in rP-HSP90C vaccine. CH-HG synthesized by ionic cross-linking showed buffering capacity and control-released rP-HSP90C in vitro. In comparison to naked rP-HSP90C, CH-HG-loaded rP-HSP90C (CH-HG/rP-HSP90C) not only evoked a long-lasting rP-HSP90C-specific IgG, but also enhanced Th1, Th2, Th17 responses and the ratio of Th1/Th2 in vivo; Meanwhile, CH-HG/rP-HSP90C provoked a stronger CTL response than rP-HSP90C. Notably, CH-HG increased the protective immune responses against systemic candidiasis in rP-HSP90C-immunized mice since CH-HG/rP-HSP90C enhanced the survival rate of infected mice, and diminished the CFUs in kidneys compared to rP-HSP90C, which were similar to that of QuilA. Further in vitro investigation displayed CH-HG upgraded the expressions of costimulators, MHCs and cytokines in BMDCs compared to rP-HSP90C；CH-HG also promoted cellular uptake, endosomal escape and "cross-presentation" of rP-HSP90C. In addition, it recruited immune cells at the injection site. Our study demonstrated that CH-HG can be an efficient adjuvant in fungal vaccines.

A polysaccharide isolated from the fruits of Physalis alkekengi L. induces RAW264.7 macrophages activation via TLR2 and TLR4-mediated MAPK and NF-κB signaling pathways.

Polysaccharides from Physalis alkekengi L. have been proven to possess many biological activities. In our previous study, a homogeneous polysaccharide (PPSB) was extracted and purified from the fruits of Physalis alkekengi L., and the structure characterization was analyzed. The present study aimed to investigate the effects of PPSB on RAW264.7 macrophage cells activation and the underlying molecular mechanism. PPSB could activate RAW264.7 cells by not only enhancing the pinocytic and phagocytic activity, but also promoting the production of NO, ROS, TNF-α, and IL-6 in RAW264.7 cells. Meanwhile, PPSB up-regulated the expression of major histocompatibility complex (MHC-I/II) and costimulatory molecules such as CD40, CD80 and CD86. Mechanism studies showed that PPSB induced the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) pathways. Moreover, the production of NO, TNF-α and IL-6 induced by PPSB in RAW264.7 cells were suppressed by specific MAPKs and NF-κB inhibitors. Further experiments with blocking antibodies demonstrated that the releases of NO, TNF-α and IL-6 and the activation of MAPKs and NF-κB induced by PPSB were decreased after TLR2 and TLR4 were blocked. Our date illustrated that PPSB was capable of activating the RAW264.7 cells via MAPKs and NF-κB signaling mediated by TLR2 and TLR4.