Yu-Li Lin

Polysaccharide Purified from Ganoderma lucidum Induces Gene Expression Changes in Human Dendritic Cells and Promotes T Helper 1 Immune Response in BALB/c Mice

Ganoderma lucidum is a medicinal mushroom in China and other Asian countries. The polysaccharide from G. lucidum (PS-G) is a branched (1→6)-β-d-glucan moiety. In this study, we examined the effects of PS-G on human monocyte-derived dendritic cells (DCs) with microarray analysis by Human Genome U133 Plus 2.0 GeneChip. In comparing mean signal values between PS-G-treated DCs with untreated DCs, 3477 (17%) probe sets were up-regulated, and 4418 (19%) probe sets were down-regulated after PS-G treatment. These results demonstrate that genes associated with phagocytosis (CD36, CD206, and CD209) are decreased and genes associated with proinflammatory chemokines (CCL20, CCL5, and CCL19), cytokines [interleukin (IL)-27, IL-23A, IL-12A, and IL-12B], and costimulatory molecules (CD40, CD54, CD80, and CD86) are increased. To confirm the microarray data, we further investigated the effect of PS-G on antigen-specific antibody and cytokine production in BALB/c mice. Immunization with ovalbumin (OVA)/PS-G showed that the anti-OVA IgG2a levels were significantly increased compared with OVA alone in BALB/c mice. Together, our data demonstrate that PS-G could effectively promote the activation and maturation of immature DCs, preferring a T helper 1 response. Furthermore, the results also demonstrate that the data from microarray analysis could be correlated with the in vivo effect of the immune-enhancing compound.

Enterovirus type 71 neutralizing antibodies in the serum of macaque monkeys immunized with EV71 virus-like particles.

Enterovirus type 71 (EV71) is a virulent form of enteroviruses causing hospitalizations for children less than three years of age. Currently there are no anti-viral therapies or vaccines available for EV71. Due to the high risk of poliomyelitis-like paralysis and fatal encephalitis, an effective vaccine to EV71 could potentially prevent virus-induced morbidity and mortality. In this study, we first tested a potential EV71 vaccine candidate based on virus-like particles (VLP). We vaccinated macaque monkeys to validate the immunogenicity of the VLP vaccine to EV71. We detected the VLP or EV71-specific antibodies, neutralization titers, ELISPOT, and T cell response to find their immune responses to EV71. When the VLP vaccine adjuvanted with alum was given to macaque monkeys, these monkeys developed both specific humoral and cellular immune responses to EV71. Despite lower neutralizing antibodies to EV71 were found in sera of VLP-immunized monkeys than monkeys vaccinated with inactivated EV71, VLP-based vaccine generated a memory immune response to EV71. Hence, VLP-based EV71 vaccine is a potential vaccine against EV71 infection.

Enterovirus 71 virus-like particle vaccine: Improved production conditions for enhanced yield

To develop the enterovirus 71 (EV71) vaccine, we previously constructed a recombinant baculovirus (Bac-P1-3CD) co-expressing EV71 P1 (under polyhedrin promoter) and 3CD (under p10 promoter) proteins, which caused P1 cleavage by 3CD protease and self-assembly of virus-like particles (VLPs) in Sf-9 cells. Assuming that reducing the 3CD expression can alleviate the competition with P1 expression and elevate the VLPs yield, hereby we constructed Bac-P1-C3CD and Bac-P1-I3CD expressing 3CD under weaker CMV and IE-1 promoters, respectively. Western blot and ELISA analyses revealed that Bac-P1-C3CD and Bac-P1-I3CD led to the VLPs release into the supernatant and enhanced the extracellular VLPs yield in Sf-9 cells, but gave poor VLPs production in High Five™ (Hi-5) cells. By optimizing the process parameters including host cells, cell density, culture mode and dissolved oxygen (DO), the best extracellular VLPs yield was achieved by infecting Sf-9 cells (4 × 10(6)cells/mL) cultured in the bioreactor (DO=30%) with Bac-P1-C3CD, which approached ≈64.3mg/L and represented a ≈43-fold increase over the yield (1.5mg/L) attained using the old process (Bac-P1-3CD infection of Sf-9 cells in the spinner flasks). The resultant VLPs not only resembled the VLPs produced from Bac-P1-3CD infection in density, size and shape, but also induced potent antibody responses in mouse models. The antibodies neutralized EV71 strains of homologous and heterologous genogroups, implicating the potential of the VLPs to confer cross-protection for the prevention of future epidemics. Altogether, Bac-P1-C3CD and the bioprocess render mass production more economical, obviate the need for cell lysis and hold promise for future industrial vaccine production.

Levamisole enhances immune response by affecting the activation and maturation of human monocyte‐derived dendritic cells

Levamisole is a synthetic phenylimidazolthiazole that was first introduced in 1966 as an anti‐helmintic agent. Current studies have been focused upon its effect on immune response and on cancer treatment. We examined the molecular mechanisms of levamisole in the activation and maturation of human monocyte‐derived dendritic cells (DC) and human T cells. Treatment of DC with levamisole increased the presentation of CD80, CD86, CD83 and human leucocyte antigen D‐related (HLA‐DR) molecules on the cell membrane, as well as the production of interleukin (IL)‐12 p40 and IL‐10. Levamisole‐treated human DC also enhanced T cell activation towards type 1 T helper immune response by inducing interferon‐γ secretion. Neutralization with antibodies against Toll‐like receptor (TLR)‐2 inhibited levamisole‐induced production of IL‐12 p40 and IL‐10, suggesting a vital role for TLR‐2 in signalling DC upon incubation with levamisole. The inhibition of nuclear factor‐κB, extracellular signal‐regulated kinases 1/2 or c‐Jun N‐terminal kinases pathways also prevented the effects of levamisole on DC in producing IL‐12 p40 or IL‐10. Taken together, levamisole could enhance immune response towards T helper 1 development through the activation of dendritic cells or T cell aspects.

Circulating IgA from acute stage of childhood Henoch‐Schönlein purpura can enhance endothelial interleukin (IL)‐8 production through MEK/ERK signalling pathway

Recently, sera from children with active Henoch‐Schönlein purpura (HSP) have been found to enhance interleukin (IL)‐8 production by human umbilical venous endothelial cells (HUVEC). To further determine the possible factor with the ability to enhance endothelial IL‐8 production in sera from acute stage of HSP, 10 children with HSP at the acute stage and 10 healthy controls were enrolled. IgA antiendothelial cell antibodies (AECA) were detected by cell‐based ELISA. Active sera with or without pretreatment with anti‐human IgA antibody, sera of controls, and immunoglobulin A (IgA) derived from sera were used to stimulate the HUVEC. The ability of these factors to enhance endothelial IL‐8 production was evaluated. Furthermore, signalling pathways were also assayed by different inhibitors, and confirmed by immunoblotting. Serum levels of IgA AECA in HPS patients at the acute stage were significantly higher than in controls (Pu2003<u20030·001). The active sera could enhance endothelial IL‐8 production (Pu2003=u20030·004, compared with control sera), and the ability of these sera was mostly abolished when pretreated with fixed anti‐human IgA antibody. The supernatant IL‐8 levels of endothelial cells stimulated by IgA derived from acute stage of HSP were statistically higher than controls (Pu2003<u20030·001). PD98059, an inhibitor of ERK phosphorylation, significantly reduced IgA AECA‐stimulated endothelial IL‐8. IgA AECA also enhanced the phosphorylation of ERK1 with a time‐dependent manner. Together with these findings, it is concluded that IgA AECA derived from acute stage of HSP may bind to endothelial and enhance endothelial cells to produce IL‐8 via MEK/REK signalling pathway.

Caffeic acid phenethyl ester inhibits nuclear factor-κB and protein kinase B signalling pathways and induces caspase-3 expression in primary human CD4+ T cells

Caffeic acid phenethyl ester (CAPE), an active component in propolis, is known to have anti‐tumour, anti‐inflammatory and anti‐oxidant properties. In this study, the effects of CAPE on the functions of primary human CD4+ T cells were evaluated in vitro. CAPE significantly suppressed interferon (IFN)‐γ and interleukin (IL)‐5 production and proliferation of CD4+ T cells stimulated by soluble anti‐CD3 and anti‐CD28 monoclonal antibodies in both healthy subjects and asthmatic patients. CAPE inhibited nuclear factor (NF)‐κB activation and protein kinase B (Akt) phosphorylation, but not p38 mitogen‐activated protein kinase (MAPK) phosphorylation in T cells. CAPE also induced active caspase‐3 expression in CD4+ T cells; CCR4+CD4+ T cells were more sensitive to CAPE induction than CXCR3+CD4+ T cells. Together, these results indicate that CAPE inhibits cytokine production and proliferation of T cells, which might be related to the NF‐κB and Akt signalling pathways, and that CCR4+CD4+ T cells are more sensitive to CAPE inhibition. This study provides a new insight into the mechanisms of CAPE for immune regulation and a rationale for the use of propolis for the treatment of allergic disorders.

Immuno-modulatory activity of Ganoderma lucidum-derived polysacharide on human monocytoid dendritic cells pulsed with Der p 1 allergen

BackgroundGanoderma lucidum-derived polysaccharide (PS-G) can rapidly and effectively promote the activation and maturation of immature dendritic cells (DCs), suggesting that PS-G possesses the capacity to regulate immune responses. This study aimed to clarify the immunologic effect of PS-G on monocyte-derived dendritic cells (MD-DCs) from asthmatic children allergic to house dust mites. The MD-DCs were stimulated for 24 h with the related allergen, Der p 1, in the presence or absence of PS-G. Cell surface markers and phagocytic capacity were assessed by FACS analysis, and key polarizing cytokines (IL-12 p40, IL-12 p70, IL-6, IL-23, and IL-10) were quantified. The subsequent regulatory effect of pulsed MD-DCs on naïve T cells was evaluated by determining the T-cell cytokine profile.ResultsPS-G induced the maturation of MD-DCs and decreased phagocytic capacity, even if pulsed with Der p 1. After incubation with PS-G and Der p 1, MD-DCs produced higher amounts of IL-12 p70, IL-12 p40, IL-6, IL-23, and IL10 than Der p 1-pulsed DCs. Furthermore, type 1 helper T (Th1) cell cytokine (INF-γ) production was highly increased when naïve autologous T cells were co-cultured with Der p 1-pulsed MD-DCs. Naïve T cells stimulated by MD-DCs pulsed with Der p 1 failed to produce proliferation of T-cells, whereas the addition of PS-G to Der p 1 induced a significant proliferation of T-cells similar to that observed with PS-G alone.ConclusionThe presence of PS-G in an allergen pulse promoted allergic MD-DCs to produce IL-12 p70, IL-12 p40, IL-6, IL-23, and IL-10, and exerted an effect on shifting the immune balance towards Th1 in children with allergic asthma.

Functional impairment of dendritic cells in patients infected with hepatitis C virus genotype 1 who failed peginterferon plus ribavirin therapy.

Although chronic hepatitis C patients have a lower frequency and functions of dendritic cells (DCs) than healthy subjects, little is known about the serial changes in frequency and functions of DCs following anti‐viral treatment and the relationship with treatment outcomes. Twenty patients with hepatitis C virus genotype 1 receiving peginterferon (PEG‐IFN) and ribavirin for 24 weeks were enrolled. The frequency and functions of DCs were assayed at baseline and 24 weeks post‐treatment. Ten sex and age‐matched healthy adults served as controls. Nineteen of the 20 chronic hepatitis C patients completed 24 weeks of combination therapy. Fifteen patients achieved rapid virologic response and 12 achieved sustained virologic response (SVR). The baseline frequency of peripheral blood myeloid DCs and plasmacytoid DCs was significantly lower in chronic hepatitis C patients than in healthy controls. In patients who achieved SVR, the frequency of DCs subsets at the end of follow‐up increased to a level comparable to healthy controls. Although no functional defects of DCs was found in chronic hepatitis C patients in comparison with healthy controls, in patients without SVR had a lower CD83 expression and higher interleukin‐10 production of DCs than SVR patients. The results suggest that low CD83 expression and high IL‐10 production of DCs at the baseline may predict a poor virologic response to 24‐week PEG‐IFN plus ribavirin therapy in HCV genotype 1 patients. J. Med. Virol. 83:1212–1220, 2011.

Hepatitis B virus surface antigen can activate human monocyte‐derived dendritic cells by nuclear factor kappa B and p38 mitogen‐activated protein kinase mediated signaling

Hepatitis B virus Ag (HBsAg), a major antigen of hepatitis B virus (HBV), is also a vaccine component for prevention of HBV infection. Dendritic cells (DCs) of HBV carriers reportedly exhibit functional impairment. In this study, the aim was to investigate the effect of HBsAg on activation of human monocyte‐derived dendritic cells (MD‐DCs), and the subsequent signal transduction pathway. Treatment of MD‐DCs with HBsAg resulted in enhanced cell surface expression of cluster of differentiation 80, CD83, CD86 and major histocompatibility complex class II, and increased interleukin (IL)‐12 p40, IL‐12p70, and IL‐10 production. Furthermore, HBsAg treatment of MD‐DCs with HBsAg resulted in enhanced T cell‐stimulatory capacity and increased T cell secretion of interferon and IL‐10. The pathway of MD‐DCs activation by HBsAg was further investigated in the present study. Inhibition of nuclear factor (NF)‐kappa B (κB) by helenalin and p38 mitogen‐activated protein kinase (MAPK) by SB203580 prevented production of IL‐12 p40, IL‐12 p70, and IL‐10. HBsAg also augmented MAPK phosphorylation. Thus, cytokine secretion of human MD‐DCs by HBsAg is blocked by inhibition of the NF‐κB and p38 MAPK pathways. Likewise, decreased inhibition of kappa B alpha concentrations and MAPK phosphorylation are critical for MD‐DC maturation by HBsAg. These findings may provide a strategy for improving the prophylactic and therapeutic efficacy of vaccines and tumor therapies that utilize these pathways.

Hepatitis B virus surface antigen can activate dendritic cells and modulate T helper type immune response

Hepatitis B virus surface antigen (HBsAg) is a major antigen of hepatitis B virus (HBV). Dendritic cells (DC) of HBV carriers have been reported to exhibit functional impairment. In this study, the role of HBsAg on mice bone marrow‐derived dendritic cells and immune responses in vivo was studied. The immune modulatory function of HBsAg was explored by using mice bone marrow‐derived dendritic cells in vitro and also by examining an ovalbumin (OVA) specific immune response in vivo. Treatment of dendritic cells with HBsAg resulted in enhanced cell surface expression of cluster of differentiation (CD) 80, CD83, CD86, and major histocompatibility complex (MHC) class II, and enhanced production of interleukin (IL)‐12 p40 and IL‐12 p70. Treatment of dendritic cells with HBsAg resulted in decreased T cell secretion of IL‐5 by OVA stimulation. In addition, the results showed stronger OVA‐specific immunoglobulin (Ig) M and weaker IgG responses in mice sera when they had been immunized with OVA and co‐injected with HBsAg. It was also found that the mice exhibited significant enhancement of anti‐OVA IgG2a antibody (Ab), as well as marked inhibition of IgG1 Ab production. In cellular immune responses, IL‐5 production was significantly decreased and interferon (IFN)‐γ increased in the group co‐injected with HBsAg. On the other hand, the induction of lymphoproliferative response to OVA stimulation in spleen cells was decreased in the HBsAg co‐injected group. These results demonstrate that HBsAg can affect the differentiation of T helper (Th) cells, which might provide a strategy for improving its prophylactic and therapeutic efficacy.