OVA-specific IgG1 antibody levels were less than those observed using the control immunogen. helper (Th) 1 and Th2 cells in a host’s immune response to infection.6 Cells designated as Th1 Melitracen hydrochloride are characterized by interleukin (IL)-2, IL-12, and interferon-gamma (IFN-) production, which also activates macrophages.5,7 In contrast, Th2 cells are characterized by IL-4, IL-5, IL-6, IL-10, and IL-13 synthesis, which further promotes humoral immunity.8C10 Currently, there are major CDF infectious diseases of humans and other animals that have no known cures, although the development of vaccines for many infectious diseases is increasingly being investigated.11 It has been reported that Melitracen hydrochloride the Th1 immune system is more effective than the Th2 immune system in combating disease-causing intracellular pathogens. Therefore, the development of an immunostimulator in a vaccine adjuvant for the induction of Th1 immunity has great potential for preventing infectious diseases. Given that safety is a paramount consideration in the development of such adjuvants, extracts from edible mushrooms that can also have immunostimulatory effects may provide a safe solution for vaccine development.1 However, to our knowledge, a few reports have evaluated the polarization of immune responses in mice inoculated with different extracts from the many edible mushrooms available. The aim of this study, therefore, was to evaluate the immune responses of mice administered hot-water extracts from one of the 15 different species of edible mushroom. In addition, we determined whether selected extracts could induce Th1 immunity Melitracen hydrochloride and act as potential immunostimulators for vaccine development. Fifteen different species of edible mushrooms were used to produce the following extracts: (Tsukuritake), (Himematsutake), (Kikurage), (Enokitake), (Maitake), (Yamabushitake), (Bunashimeji), (Shiitake), (Hatakeshimeji), (Amigasatake), (Nameko), (Tamogitake), (Eringi), (Hiratake), and (Hanabiratake). Each mushroom was dried using gentle airflow at 25C for 24?h, before being resuspended in sterile Milli-Q water (Millipore, MA, USA; 20?mg/mL), and homogenized using an ART-MICCRA D-8 homogenizer (setting C) for 5?min. The homogenate was then boiled for 2?h, and any solid particles and aggregates were removed by centrifugation at 12,000 for 20?min. The supernatant was filtered using a 0.45-m pore-size Millex-HV filter (Millipore), and subsequently used as a hot-water mushroom extract. Animals: Five-week-old female ddY mice were purchased from the Saitama Experimental Animals Supply Co., Ltd (Saitama, Japan). Experimental protocol was approved by the Animal Care and Use Committee of Nippon Veterinary and the Life Science University, Japan. Ten mice were inoculated intraperitoneally with 0.5?mL of the hot-water mushroom extract prepared as described earlier. A control group of mice were similarly inoculated with 0.5?mL of sterilized Milli Q water. Seven days after inoculation, whole blood was collected from the infra-axillary vein of each mouse.12 Serum was then separated from each blood sample and stored at ?80C until use. Cytokines IFN- and IL-4 were measured as markers of Th1 and Th2 cells, respectively, in the serum samples from mice inoculated with the individual mushroom extracts. Assays were performed using mouse IFN- and IL-4 ELISA kits (Pierce Endogen or Thermo Scientific, Rockford, IL, USA) according to each manufacturer’s instructions. Trial and control immunogens in oil-in-water (O/W) emulsions included ovalbumin (OVA; Kanto Chemical Co., Ltd., Tokyo, Japan) as an antigen, as well as the following: 10?g of squalane (Wake Pure Chemical Industries, Ltd., Osaka, Japan), 4?g of rheodol (HLB 7.1; Kao Co. Ltd., Tokyo, Japan), 2?g of glycerol (Wake Pure Chemical Industries, Ltd.), 1?mL of ovalbumin (0.1?mg/mL), and hot-water extract Melitracen hydrochloride (13?mL) as the trial immunogen or distilled water (13?mL) as the negative control. Both immunogens were inoculated intramuscularly (0.2?mL/mouse) into 10 mice. Blood was collected from the tail vein of each mouse weekly from 0 to 10 weeks postinoculation. OVA-specific IgG1 and IgG2a antibody levels were measured using enzyme-linked immunosorbent assays.