Enhancement and regulation effect of myrcene on antibody response in immunization with ovalbumin and Ag85B in mice.

BACKGROUND: MF59, which is an adjuvant belonging to C30 member of the terpene family, is a T helper type-2 (Th2)-biased immune enhancer. Our previous studies showed that pyriproxyfen, a member of the terpene family with fewer carbon atoms (C20) than MF59, enhanced active T helper type-1 (Th1)-biased immune responses. OBJECTIVE: This study was performed to investigate the enhancement of antigen-specific immune responses by myrcene, a member of the terpene family with fewer carbon atoms (C10) than pyriproxyfen. METHOD: Ovalbumin (OVA) was used as an antigen to determine the effects of myrcene on the immune response. The IgG subtypes and cytokines induced by immunization of OVA with or without myrcene were monitored. Thereafter, we determined the effects of myrcene in the immune response against Ag85B, which is a dominant protective antigen for tuberculosis. RESULTS: The results showed that 0.8 mg/dose of myrcene enhanced antigen-specific total IgG immune response to OVA. Direct mixing of the antigen with myrcene was required for the enhancement of antibody production. Myrcene increased OVA-specific IgG2a titer, suggesting induction of Th1-immune response. The level of Th1 cytokines, IFN-γ was increased at 8 weeks after immunization, although IL-13 was also increased at the same time point. However, finally myrcene was found to increase Ag85B-specific total IgG titers at 5 weeks and specific IgG2a titer was increased at both 5 and 8 weeks. The results suggested that myrcene could enhance Th1 immune response. CONCLUSIONS: Myrcene enhanced specific immune responses against OVA and Ag85B. This study suggested the tendency of the enhancement of Th1 immune response by myrcene.

Recombinant fusion protein of cholera toxin B subunit with YVAD secreted by Lactobacillus casei inhibits lipopolysaccharide-induced caspase-1 activation and subsequent IL-1 beta secretion in Caco-2 cells.

BACKGROUND: Lactobacillus species are used as bacterial vectors to deliver functional peptides to the intestine because they are delivered live to the intestine, colonize the mucosal surface, and continue to produce the desired protein. Previously, we generated a recombinant Lactobacillus casei secreting the cholera toxin B subunit (CTB), which can translocate into intestinal epithelial cells (IECs) through GM1 ganglioside. Recombinant fusion proteins of CTB with functional peptides have been used as carriers for the delivery of these peptides to IECs because of the high cell permeation capacity of recombinant CTB (rCTB). However, there have been no reports of rCTB fused with peptides expressed or secreted by Lactobacillus species. In this study, we constructed L. casei secreting a recombinant fusion protein of CTB with YVAD (rCTB-YVAD). YVAD is a tetrapeptide (tyrosine-valine-alanine-aspartic acid) that specifically inhibits caspase-1, which catalyzes the production of interleukin (IL)-1ß, an inflammatory cytokine, from its inactive precursor. Here, we examined whether rCTB-YVAD secreted by L. casei binds to GM1 ganglioside and inhibits caspase-1 activation in Caco-2 cells used as a model of IECs. RESULTS: We constructed the rCTB-YVAD secretion vector pSCTB-YVAD by modifying the rCTB secretion vector pSCTB. L. casei secreting rCTB-YVAD was generated by transformation with pSCTB-YVAD. Both the culture supernatant of pSCTB-YVAD-transformed L. casei and purified rCTB-YVAD bound to GM1 ganglioside, as did the culture supernatant of pSCTB-transformed L. casei and purified rCTB. Interestingly, although both purified rCTB-YVAD and rCTB translocated into Caco-2 cells, regardless of lipopolysaccharide (LPS), only purified rCTB-YVAD but not rCTB inhibited LPS-induced caspase-1 activation and subsequent IL-1ß secretion in Caco-2 cells, without affecting cell viability. CONCLUSIONS: The rCTB protein fused to a functional peptide secreted by L. casei can bind to GM1 ganglioside, like rCTB, and recombinant YVAD secreted by L. casei may exert anti-inflammatory effects in the intestine. Therefore, rCTB secreted by L. casei has potential utility as a vector for the delivery of YVAD to IECs.

Expression and secretion of cholera toxin B subunit in lactobacilli.

Lactic acid bacteria (LAB) are used in various fields, including in food and medical supplies. There has been a great deal of research into vaccine development using LAB as carriers due to their "generally recognized as safe" status. Cholera is an infectious disease that causes diarrhea due to cholera toxin (CT) produced by Vibrio cholerae. The pentameric cholera toxin B (CTB) subunit has no toxicity, and is used as an antigen in cholera vaccines and as a delivery molecule in vaccines to various diseases. In this study, we generated recombinant LAB expressing and secreting CTB. Here, we first report that CTB expressed and secreted from LAB bound to GM1 ganglioside. The secreted CTB was purified, and its immunogenicity was determined by intranasal administration into mice. The results of the present study suggested that it may be useful as the basis of a new oral cholera vaccine combining LAB and CTB.

Pyriproxyfen enhances the immunoglobulin G immune response in mice.

Pyriproxyfen is a juvenile hormone mimic of vital importance for insect development with little risk to humans. This study was performed to investigate whether large doses of pyriproxyfen affect the immune response in mammals. Mice were immunized thrice with ovalbumin in 5% ethanol, with or without pyriproxyfen or alum. Large doses of pyriproxyfen (9 or 15 mM) significantly enhanced specific total IgG immune response. This enhancement was no longer present 24 hr after treatment with pyriproxyfen. These results suggest that pyriproxyfen is a safe chemical. Moreover, pyriproxyfen induced higher titers of IgG2a and enhanced tumor necrosis factor-alpha and gamma-interferon responses whereas alum induced IgG1 with enhanced interleukin-4 and -10. These observations indicate that the mechanism of immune enhancement by pyriproxyfen may differ from that of alum.

Differences in TLR9-dependent inhibitory effects of H(2)O(2)-induced IL-8 secretion and NF-kappa B/I kappa B-alpha system activation by genomic DNA from five Lactobacillus species.

Lactic acid bacteria (LAB) show anti-inflammatory effects, and their genomic DNA was identified as one of the anti-inflammatory components. Despite the differences in anti-inflammatory effects between live LAB dependent not only on genus but also species, this effect has not been compared at the genomic DNA level. We compared the anti-inflammatory effects of the genomic DNA from five Lactobacillus species-Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus gasseri, Lactobacillus plantarum, and Lactobacillus reuteri-using Caco-2 cells. To evaluate anti-inflammatory effects, decreases in H(2)O(2)-induced IL-8 secretion and inhibition of H(2)O(2)-induced NF-κB/IκB-α system activation were examined. All LAB genomic DNAs dose-dependently decreased H(2)O(2)-induced IL-8 secretion and inhibited H(2)O(2)-induced NF-κB/IκB-α system activation. Comparison of these effects between Lactobacillus species showed that the anti-inflammatory effects of L. acidophilus genomic DNA are lower than those of the other species. Furthermore, suppression of Toll-like receptor 9 (TLR9), a specific receptor of bacterial DNA, expression by RNAi abolished the decrease of H(2)O(2)-induced IL-8 secretion and inhibition of H(2)O(2)-induced NF-κB/IκB-α system activation by LAB genomic DNA. Our results demonstrated that the anti-inflammatory effects of genomic DNA differ between Lactobacillus species and TLR9 is one of the major pathways responsible for the anti-inflammatory effect of LAB genomic DNA.