Cordyceps militaris fruit body activates myeloid dendritic cells via a Dectin-1-mediated pathway.

Cordyceps militaris, an entomopathogenic fungus, has been traditionally used in East Asian medicine. Recent research indicates that the fruit bodies of C. militaris are rich in bioactive compounds, such as polysaccharides and nucleosides, which may offer health benefits. However, the specific components responsible for its immunostimulatory effects and the mechanisms involved remain unclear. This study explored the immunomodulatory activity of a fruit body extract from C. militaris, named Ryukyu-kaso (RK), and examined the effect of the ß-glucan receptor Dectin-1 on bone marrow-derived dendritic cells (BMDCs). Our results demonstrated that RK, which contains 1,3-ß-glucan, effectively stimulated BMDCs to secrete pro-inflammatory and immunoregulatory cytokines and upregulated surface markers indicative of maturation and activation. Notably, these immunostimulatory effects were completely absent in BMDCs derived from Dectin-1-knockout mice, confirming that Dectin-1 is crucial for RK-induced immunomodulation. These findings provide new insights into the immunostimulatory mechanisms of C. militaris and underscore the potential of RK as a natural immunomodulatory agent for various therapeutic applications.

Addition of Mucoadhesive Agent to Enzymatically Polymerized Caffeic Acid-Based Nasal Vaccine Formulation Attenuates Antigen-Specific Antibody Responses in Mice.

Mucosal vaccination is a promising strategy for combating infectious diseases caused by pathogenic microbes, as it can generate antigen-specific immune responses in both systemic and mucosal compartments. In our recent study, we developed a nasal vaccine system for Streptococcus pneumoniae infections in mice using enzymatically polymerized polyphenols such as caffeic acid. However, the efficacy of this mucosal vaccine system is approximately 70%, indicating a need for improvement. To address this issue, we hypothesized that incorporating a mucoadhesive agent that enhances mucosal absorption into a polyphenol-based mucosal vaccine system would improve vaccine efficacy. Contrary to our expectations, we found that adding a mucoadhesive agent, hydrophobically modified hydroxypropylmethylcellulose, to the vaccine system reduced the stimulation of antigen-specific antibody responses in both the mucosal (more than 90% reduction; P < 0.05) and systemic compartments (more than 80% reduction; P < 0.05). Although the addition of the mucoadhesive agent may have interfered with the interaction between the mucosal epithelium and the vaccine system, the underlying mechanism remains unclear, and further research is needed to fully understand the mechanisms involved.

Polymeric Caffeic Acid Acts as an Antigen Delivery Carrier for Mucosal Vaccine Formulation by Forming a Complex with an Antigenic Protein.

The development of mucosal vaccines, which can generate antigen-specific immune responses in both the systemic and mucosal compartments, has been recognized as an effective strategy for combating infectious diseases caused by pathogenic microbes. Our recent research has focused on creating a nasal vaccine system in mice using enzymatically polymerized caffeic acid (pCA). However, we do not yet understand the molecular mechanisms by which pCA stimulates antigen-specific mucosal immune responses. In this study, we hypothesized that pCA might activate mucosal immunity at the site of administration based on our previous findings that pCA possesses immune-activating properties. However, contrary to our initial hypothesis, the intranasal administration of pCA did not enhance the expression of various genes involved in mucosal immune responses, including the enhancement of IgA responses. Therefore, we investigated whether pCA forms a complex with antigenic proteins and enhances antigen delivery to mucosal dendritic cells located in the lamina propria beneath the mucosal epithelial layer. Data from gel filtration chromatography indicated that pCA forms a complex with the antigenic protein ovalbumin (OVA). Furthermore, we examined the promotion of OVA delivery to nasal mucosal dendritic cells (mDCs) after the intranasal administration of pCA in combination with OVA and found that OVA uptake by mDCs was increased. Therefore, the data from gel filtration chromatography and flow cytometry imply that pCA enhances antigen-specific antibody production in both mucosal and systemic compartments by serving as an antigen-delivery vehicle.

Development of a concise and reliable method for quantifying the antibody loaded onto lipid nanoparticles modified with Herceptin.

Antibodies are essential components of the immune system with a wide range of molecular targets. They have been recognized as modalities for treating several diseases and more than 130 approved antibody-based therapeutics are available for clinical use. However, limitations remain associated with its efficacy, tissue permeability, and safety, especially in cancer treatment. Nanoparticles, particularly those responsive to external stimuli, have shown promise in improving the efficacy of antibody-based therapeutics and tissue-selective delivery. In this study, we developed a reliable and accurate method for quantifying the amount of antibody loaded onto lipid nanoparticles modified with Herceptin® (Trastuzumab), an antibody-based therapeutic used to treat HER2-positive cancers, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining. This method proved to be a suitable alternative to commonly used protein quantification techniques, which are limited by lipid interference present in the samples. Furthermore, the amount of Herceptin modified on the liposomes, measured by this method, was confirmed by Herceptin's antibody-dependent cell-mediated cytotoxicity activity. Our results demonstrate the potential of this method as a critical tool for developing tissue-selective antibody delivery systems, leading to improved efficacy and reduced side effects of antibody-based therapeutics.

Development of a Gene and Nucleic Acid Delivery System for Skeletal Muscle Administration via Limb Perfusion Using Nanobubbles and Ultrasound.

Strategies for gene and nucleic acid delivery to skeletal muscles have been extensively explored to treat Duchenne muscular dystrophy (DMD) and other neuromuscular diseases. Of these, effective intravascular delivery of naked plasmid DNA (pDNA) and nucleic acids into muscles is an attractive approach, given the high capillary density in close contact with myofibers. We developed lipid-based nanobubbles (NBs) using polyethylene-glycol-modified liposomes and an echo-contrast gas and found that these NBs could improve tissue permeability by ultrasound (US)-induced cavitation. Herein, we delivered naked pDNA or antisense phosphorodiamidate morpholino oligomers (PMOs) into the regional hindlimb muscle via limb perfusion using NBs and US exposure. pDNA encoding the luciferase gene was injected with NBs via limb perfusion into normal mice with application of US. High luciferase activity was achieved in a wide area of the limb muscle. DMD model mice were administered PMOs, designed to skip the mutated exon 23 of the dystrophin gene, with NBs via intravenous limb perfusion, followed by US exposure. The number of dystrophin-positive fibers increased in the muscles of mdx mice. Combining NBs and US exposure, which can be widely delivered to the hind limb muscles via the limb vein, could be an effective therapeutic approach for DMD and other neuromuscular disorders.

Intranasal administration of sodium nitroprusside augments antigen-specific mucosal and systemic antibody production in mice.

The coronavirus disease 2019, i.e., the COVID-19 pandemic, caused by a highly virulent and transmissible pathogen, has profoundly impacted global society. One approach to combat infectious diseases caused by pathogenic microbes is using mucosal vaccines, which can induce antigen-specific immune responses at both the mucosal and systemic sites. Despite its potential, the clinical implementation of mucosal vaccination is hampered by the lack of safe and effective mucosal adjuvants. Therefore, developing safe and effective mucosal adjuvants is essential for the fight against infectious diseases and the widespread clinical use of mucosal vaccines. In this study, we demonstrated the potent mucosal adjuvant effects of intranasal administration of sodium nitroprusside (SNP), a known nitric oxide (NO) donor, in mice. The results showed that intranasal administration of ovalbumin (OVA) in combination with SNP induced the production of OVA-specific immunoglobulin A in the mucosa and increased serum immunoglobulin G1 levels, indicating a T helper-2 (Th2)-type immune response. However, an analog of SNP, sodium ferrocyanide, which does not generate NO, failed to show any adjuvant effects, suggesting the critical role of NO generation in activating an immune response. In addition, SNPs facilitated the delivery of antigens to the lamina propria, where antigen-presenting cells are located, when co-administered with antigens, and also transiently elicited the expression of interleukin-6, interleukin-1ß, granulocyte colony-stimulating factor, C-X-C motif chemokine ligand 1, and C-X-C motif chemokine ligand 2 in nasal tissue. These result suggest that SNP is a dual-functional formulation with antigen delivery capabilities to the lamina propria and the capacity to activate innate immunity. In summary, these results demonstrate the ability of SNP to induce immune responses via an antigen-specific Th2-type response, making it a promising candidate for further development as a mucosal vaccine formulation against infectious diseases.

Refinement and complete solution NMR analysis of the structure of a 6-branched 1,3-ß-D-glucan (OL-2) isolate from Omphalialapidescens.

OL-2 is a water-soluble ß-glucan produced by Omphalia lapidescens. This versatile glucan has potential applications in various industries, including food, cosmetics, and pharmaceuticals. In addition, OL-2 is known for its promising applications as a biomaterial and drug, owing to its reported antitumor and antiseptic properties. Although the biological activities of ß-glucans vary depending on their primary structure, holistic clarification of OL-2 via solution NMR spectroscopy to ascertain its complete and unambiguous structure has not yet been achieved. In this study, a chain of solution NMR techniques, such as correlation spectroscopy, total correlation spectroscopy (TOCSY), nuclear Overhauser effect and exchange spectroscopy, 13C-edited heteronuclear single quantum coherence (HSQC), HSQC-TOCSY, heteronuclear multiple bond correlation, and heteronuclear 2-bond correlation pulse sequences were used to unambiguously assign all 1H and 13C atoms in OL-2. Based on our investigation, OL-2 consists of a 1,3-ß-glucan backbone chain decorated with a single 6-branched ß-glucosyl side unit on every fourth residue.

Potentiation of Antitumor Activity by Antibody Drugs and Mushroom-Derived ß-Glucans in Natural Killer Cell-Mediated Tumoricidal Activities against Non-Hodgkin's B-Cell Lymphoma.

ß-glucans are polysaccharides that activate innate immunity. We herein investigated whether P-glucans promote the immunological effects of antibody drugs against malignant tumor cells using human peripheral blood mononuclear cells (PBMCs). Rituximab bound to CD20-specific lymphoma and exhibited cytotoxic activity in the presence of human mononuclear cells, but not neutrophils. The addition of Sparassis crispa (cauliflower mushroom)-derived ß-glucan (SCG) and granulocyte macrophage colony-stimulating factor (GM-CSF) to co-cultures of PBMCs and Raji lymphoma cells further promoted antibody-dependent cell-mediated cytotoxicity (ADCC). The GM-CSF treatment increased ß-glucan receptor expression on adherent cells in PBMCs. A co-stimulation with GM-CSF and SCG of PBMCs induced an increase in the number of spreading cells and the activation of natural killer (NK) cells. The enhancement in ADCC was abolished by the removal of NK cells, indicating that SCG and GM-CSF increased ADCC against lymphoma by activating ß-glucan receptor-expressing cells in PBMCs and enhancing NK cell activity. The synergistic mechanisms of action of mushroom-derived ß-glucans and biopharmaceuticals, including recombinant cytokines and antibodies, in the treatment of malignant tumor cells provide important insights into the clinical efficacy of ß-glucans from mushrooms.

Involvement of splenic marginal zone macrophages in the recognition of systemically administered phosphatidylserine-coated liposomes in mice.

Autoimmune diseases present a significant clinical problem, highlighting the need for the development of novel or improved therapeutic methods. One of the factors that causes autoimmune diseases is a defect in the clearance of apoptotic cells by phagocytes. Thus, improved apoptotic cell processing has been considered as a strategy to treat autoimmune diseases. However, therapeutic strategies focusing on apoptotic cell clearance have not been approved till date. We have reported that liposomes composed of phosphatidylserine (PS liposomes) exhibit anti-inflammatory or immunosuppressive effects in macrophages. A PS liposome display PS on its surface, which plays a crucial role in the phagocytosis of apoptotic cells by marginal zone macrophages (MZMs), a key player in the clearance of apoptotic cells, by recognizing PS exposed on the surface of apoptotic cells. Therefore, we hypothesized that PS liposomes could be used as "antigen delivery vesicles" to act as a substitute for apoptotic cells in the treatment of autoimmune diseases. In this study, we showed that systemically administered PS liposomes accumulated in the marginal zone of the spleen due to recognition of surface-displayed PS by MZMs because it was observed that liposomes without PS did not accumulate in the marginal zone. In conclusion, PS liposomes may be useful vehicles to function as active agents and/or antigens against autoimmune diseases.

Role of Interleukin-6 in the Antigen-Specific Mucosal Immunoglobulin A Responses Induced by CpG Oligodeoxynucleotide-Loaded Cationic Liposomes.

An advantage of mucosal vaccines over conventional parenteral vaccines is that they can induce protective immune responses not only at mucosal surfaces but also in systemic compartments. Despite this advantage, few live attenuated or inactivated mucosal vaccines have been developed and applied clinically. We recently showed that the intranasal immunization of ovalbumin (OVA) with class B synthetic oligodeoxynucleotides (ODNs) containing immunostimulatory CpG motif (CpG ODN)-loaded cationic liposomes synergistically exerted both antigen-specific mucosal immunoglobulin A (IgA) and systemic immunoglobulin G (IgG) responses in mice. However, the mechanism underlying the mucosal adjuvant activity of CpG ODN-loaded liposomes remains unknown. In the present study, we showed that the intranasal administration of CpG ODN-loaded cationic liposomes elicited interleukin (IL)-6 release in nasal tissues. Additionally, pre-treatment with an anti-IL-6 receptor (IL-6R) antibody attenuated antigen-specific nasal IgA production but not serum IgG responses. Furthermore, the intranasal administration of OVA and CpG ODN-loaded cationic liposomes increased the number of IgA+/CD138+ plasma cells and IgA+/B220+ B cells in the nasal passages. This increase was markedly suppressed by pre-treatment with anti-IL-6R blocking antibody. In conclusion, IL-6 released by CpG ODN-loaded cationic liposomes at the site of administration may play a role in the induction of antigen-specific IgA responses by promoting differentiation into IgA+ plasma cells for IgA secretion from B cells.