Innate immune signaling via endogenous TLR4 ligands plays important roles in pathogenesis of metabolic disorders. Recently, we found that ganglioside GM3, one of glycosphingopilids, acts as an endogenous TLR4 modulator. GM3 ganglioside in human serum is comprised of a variety of fatty acids including long-chain (LCFA), very long-chain (VLCFA), and those with modifications such asω-9 desaturation and α-hydroxylation. VLCFA GM3 synergistically and selectively enhanced TLR4 activation by LPS and HMGB1, and in contrast, LCFA- and unsaturated VLCFA GM3 inhibited TLR4 activation. In metabolic disorders, serum VLCFA GM3 increased, while LCFA GM3 decreased, indicating the proinflammatory shift of GM3 species. VLCFA-and α-hydroxyl GM3 increased in the adipose tissue of obese mice, and the increase was attenuated in TLR4-mutant mice, implying that TLR4 signaling itself is involved in production of VLCFA GM3. Our findings suggest that serum GM3 species modulate TLR4 signaling, and the increase of VLCFA GM3 is a risk factor for TLR4-mediated disease progression.

 Endotoxin in the bloodstream presents a severe health risk already in small doses. Thus endotoxin tests are extremely important and mandatory for release of parenteral administered drugs. For detection of endotoxin, Limulus-based methods are the gold standard. Many Drug Products however cause interference with such detection methods. Very often this interference can be overcome by dilution, but not in the case of Low Endotoxin Recovery (LER). Excipients used for drug product formulations like citrate buffer in combination with polysorbates or even the active pharmaceutical ingredient itself are able to cause LER. This effect leads to failure in determination of correct endotoxin contaminations. A controversial discussion about the relevance of LER and the setup of these studies is ongoing. Here we present a review of the molecular mechanism behind LER and the factors that influence this effect. The importance of standardized protocols for LER studies to produce comparable results is summarized and an outlook for dedicated sample treatments that are able to overcome the LER effect is given.

 Low Endotoxin Recovery (LER) is a phenomenon of endotoxin activity decrease in a matrix containing a chelating agent and a detergent, and is a controversial topic in the biopharmaceutical field. LER raised a questions on the target of the Bacterial Endotoxins Test (BET). This includes endotoxin activity change by samples and undetectable endotoxin in samples. We have been measuring endotoxin activity, not the amount of endotoxin in a sample by the BET. Endotoxin activity in a sample can be changed by a different condition, like LER. There may be a status of endotoxin that cannot be detected by the BET. There was difference in LER resistance between purified endotoxin and naturally occurring endotoxin. Although there may be the highest endotoxin activity in a sample under a different condition, the highest activity is not the target of the BET. The endotoxin in a sample should be measured as is. There may be undetectable endotoxin, but we do not have enough information to discuss about its effects on human health. This should be studied in the future.

 We released the Bioluminescence Endotoxin Analyzer (Luminutes-ET) for hemodialysis facilities in April 2017, which uses a new detection method combining LAL reaction and bioluminescence. The bioluminescence method has superior Signal/Noise ratio, allowing fast and highly sensitive measurements of up to 0.0003EU/mL in under 20 minutes.

 This detection method applies the mutant firefly luciferase discovered by Kuroda laboratory at Hiroshima University which is 10 times higher in luminescence intensity compared to the wild-type. We designed the analyzer and reagent kit to significantly reduce measurement errors and lyophilized the reagent for long-term stability.

 The luciferase developed by Kuroda laboratory was not suitable for detecting endotoxin in dialysis fluid as its luminescence reaction was affected by the Na^＋ in dialysate solutions. We improved on this luciferase to prevent the effect of Na^＋ on its reaction. As a result, endotoxin detection in deionized water, i.e. dialysis water, and dialysis fluid now uses the same calibration curve.

 Endotoxin measurement using this bioluminescence method was evaluated ; its accuracy and measurement validated at six hemodialysis facilities based on the common guidelines set forth by Japanese Society for Hemodiafiltration. The validity of its calibration curve, blank tests, reaction interfering factor tests, detection limits, and quantitation limit of dialysate measurement were assessed. In conclusion, the bioluminescence method’s fast and highly sensitive measurements were verified, and was confirmed that it is fit for biological contamination control in dialysis fluid.

 Marine organisms produce second metabolites with unique chemical structures because of their complexed symbiotic systems different from that of land organisms.

 Unique glycosphingolipids with α-galactosylceramide structure, agelasphins, were obtained from Okinawan marine sponge Agelas mauritianus by the in vivo antitumor substance screening system. Agelasphines and the developmental candidate, KRN7000 (α-GalCer), markedly showed antitumor activity against tumor bearing mouse by activating host's immune system. The discovery of agelasphins and the development of KRN7000 revealed the detail of NKT cell functions. Unfortunately, development of KRN7000 intended to the antitumor drug by the systemic injection was abandoned because of several limitations. Now it is strongly expected that agelasphin or its derivative to be developed as a novel immunomodulator by conquering the limitations.

 Invariant natural killer T (iNKT) cells are a unique lymphocyte subpopulation that possess an invariant T cell receptor (TCR) and recognize glycolipid antigens, such as α-Galactosylceramide (αGalCer), presenting on CD1d. Activated iNKT cells show a direct and indirect anti-tumor effect by producing effector molecules and cytokines that activate other immune cells, including NK cells and cytotoxic T cells. We are currently focusing on the development of immunotherapy targeting iNKT cells and have conducted early-phase clinical trials for non-small-cell lung cancer (NSCLC). Previous clinical studies have shown that the intravenous injection of αGalCer-pulsed dendritic cells (DCs) induced the activation of endogenous iNKT cells and iNKT cell-dependent responses. Furthermore, an increase in the number of IFN-γ-producing cells among peripheral blood mononuclear cells has been shown to be associated with a prolonged survival. A dramatic infiltration of iNKT cells in the tumor microenvironment was also observed after the injection of αGalCer-pulsed DCs. Based on these results, a phase Ⅱ clinical trials of αGalCer-pulsed DCs for NSCLC were desiged as an Advanced Medical Technology and approved by the Japanese Ministry of Health, Labor and Welfare. Patients with advanced or recurrent NSCLC who had received first-line chemotherapy underwent intravenous injection of α-GalCer-pulsed DCs. αGalCer-pulsed DCs were found to be well-tolerated and prolonged the overall survival. We also discuss future potential combination therapies of iNKT cell-based immunotherapy to achieve enhanced anti-tumor activity and provide better treatment options for patients with advanced NSCLC.

 Most of the current depression drugs have been developed based on the monoamine hypothesis. However, about 30% of patients indicate resistance to medication, and patients with relatively mild depression get only a small benefit from antidepressants. In addition, although an increase in monoamine concentration in synaptic gaps by monoamine transporter inhibition occurs within a relatively short time, it takes about six weeks to show an antidepressant effect in actual clinical settings. There are cases in which an antidepressant effect is observed for drugs that do not regulate the amount of monoamine. These facts suggest the presence of a variety of pathophysiologies in depression and depressive symptoms. Recently, a relationship between the onset of depression and the expression levels of immune-related molecules such as cytokines in the blood and the brain derived from patients with depression has been pointed out. Although there is so far no medication targeting neuroinflammation, many recent studies have shown that inflammation is not negligible and a significant factor in the pathogenesis of depression. Therefore, it is meaningful to focus on inflammation for elucidating the pathogenesis and developing medications. In this paper, we describe the pathogenesis pathways known to be involved in the inflammation, the serotonin hypothesis, hypothalamic-pituitary-adrenal axis hypothesis, and neurodegeneration/neurogenesis hypothesis and describe the applications to therapy and preventions based on them.

 Adjuvants enhance immune system during vaccination. Among FDA-approved adjuvants, aluminum salts are most commonly used for vaccines. Although aluminum salts enhance antibody production, they show a limited effect for the cell-mediated immune response. Thus, further development of adjuvants inducing T cell mediated immuno-responses are demanded. Toll-like receptors (TLRs) are immune-related receptors that recognize specific pathogen-associated molecular patterns and play important roles in the activation of innate immunity, which is crucial to shape adaptive immunity. Studies using TLR ligands as novel adjuvants for anti-microbial and anti-cancer immunotherapies have therefore attracted much attention. Among them, a low molecular weight TLR7 ligand, Imiquimod, has been approved for clinical use, but its use is restricted only for local administration due to unwanted adverse effects. Since TLR7 is mainly located in the endosomal compartment of immune cells, efficient transport of the ligand into the cell is important for activating TLR7. Our previous work indicated that the conjugation of a low molecular weight TLR7 ligand with serum albumin and polysaccharides can greatly enhance its potency. In this study, we examined gold nanoparticles (GNPs) as carriers, as GNPs are less toxic and can immobilize multiple molecules including antigens for pathogens and tumors. Furthermore, α-mannose for targeting antigen presenting cells was also examined for the efficient delivery of GNPs. In this paper, we describe preparation of a low molecular weight TLR7 ligand and α-mannose immobilized GNPs and its in vitro and in vivo immunostimulatory activities.

 Interleukin (IL) -29 is a cytokine belonging to the type Ⅲ interferon family, which regulates a similar set of genes as type Ⅰ interferons. Although type Ⅰ interferons act globally, type Ⅲ interferons primarily target epithelial cells and protect them against the frequent viral attacks that are common for barrier tissues. The antiviral effects of IL-29 have been demonstrated at barrier surfaces in the respiratory and gastrointestinal tracts, liver, blood-brain barrier, and skin, but it remains unknown whether IL-29 exhibits these effects in oral epithelial cells. In this study, we found that the functional IL-29 receptor, interferon-lambda receptor 1, is expressed in epithelial cells from both human oral mucosa and gingiva, but not in human gingival fibroblasts. Although IL-29 stimulation did not induce pro-inflammatory cytokine mRNA expression, such as IL-6 and IL-8, it did induce retinoic acid-inducible gene (RIG) -I and interferon gamma-inducible protein 16 (IFI-16) production via a signal transducers and activator of transcription 1 (STAT1) -dependent pathway in gingival epithelial cells. RIG-I and IFI-16 sense viral nucleic acids, and the stimulation of these receptors induces interferon beta production. Moreover, we confirmed that the augmenting effects of IL-29 on 5’triphosphate double-stranded RNA (a synthetic ligand for RIG-I) -induced interferon beta production in gingival epithelial cells. These data suggest the therapeutic potential of IL-29 for preventing viral infections in the oral mucosa.

 CD1d, one of the lipid antigen-presenting proteins, binds to a glycolipid ligand and forms CD1d-ligand complex, which is recognized by NKT cells and induces the secretion of various cytokines including Th1 and Th2 cytokines. The cytokines are known to control immune responses : Th1 cytokines (e. g. IFN-γ) are involved in cell-mediated immunity such as tumor clearance and protection against infection, and Th2 cytokines (e. g. IL-4) are associated with humoral immunity such as allergies and promotion of antibody production. Recent studies revealed that the balance of the cytokines released by NKT cells depends on the CD1d ligand structures. α-GalCer (KRN7000) is a representative ligand and has potent activity to induce both Th1 and Th2 cytokines. On the other hand, OCH is known as a Th2-selective CD1d ligand, and several clinical trials for the ligand in patients with multiple sclerosis and Crohn's disease are ongoing. However, few studies of potent Th2-selective CD1d ligands have been reported compared with Th1-selective ones, and the detailed mechanism of cytokine balance regulation remains unclear. Therefore, the development of potent Th2-selective ligands and elucidation of their biasing mechanism are required. In this article, we review the reported Th2-biased CD1d ligands and the cellular imaging with Th2-biased lipid-modified CD1d ligands for understanding of Th1/Th2 selectivity.

 Late stages of lipid A biosynthesis of Escherichia coli are transfer reactions of lauric acid (C_{12 : 0}) and myristic acid (C_{14 : 0}) to the hydroxyl group of 3-hydroxy-myristic acid (3-OH-C_{14 : 0}). In the previous study we constructed the mutant strains with disrupted C_{12 : 0}-transferase and C_{14 : 0}-transferase genes, and used those mutant strains for the modification of lipid A by the introduction of foreign acyltransferase genes. In the study reviewed here, C_{14 : 0}-transferase gene (lpxL2) of Klebsiella pneumoniae was cloned, and introduced to the mutant strains by transformation to modify the lipid A structure. LPS preparations of the transformants were analyzed through chemical modification and MALDI-TOF mass spectrometry, and were proved to have the lipid A with one C_{14 : 0}, or two C_{14 : 0}, one of which replaced C_{12 : 0} bound to 3-OH-C_{14 : 0} at the C2-position of the non-reducing end glucosamine. The IL-6 inducing activity of the LPS with C_{14 : 0} was measured, and compared with that of the original LPS with C_{12 : 0}. The activity of LPS with C_{14 : 0} was found to be comparable with that of LPS with C_{12 : 0}, suggesting that C_{14 : 0} can replace C_{12 : 0} without changing the immunostimulating activity of lipid A.

 Outer membrane vesicles (OMVs) are mainly composed of lipopolysaccharide (LPS), phospholipids, and outer membrane and periplasmic proteins. Recently OMV vaccines have been developed, because their LPS act as adjuvant. However, attenuation of the toxicity of typical LPS is necessary to reduce adverse effects of OMV vaccine. Previously we found that acetic acid bacteria Acetobacter pasteurianus produces low immunostimulatory LPS. In this study, we separated OMVs from A. pasteurianus and characterized their immunostimulatory effects. A. pasteurianus NBRC 3283 were grown at 27℃ in 804 broth. Vesicle secretion from the cell was observed after 2 days in culture by TEM imaging. The vesicles were separated from culture supernatants after 7 days in culture by ultracentrifugation. We found that the precipitation contains vesicles which can be purified by OptiPrep density gradient centrifugation. Since the vesicles composed of LPS and outer membrane proteins, we concluded they are OMVs and designated as Ap-OMV. We further found that Ap-OMV stimulated TLR2 and weakly TLR4 in TLR expressing cells and TNF-α production in J774A. 1 cells. Furthermore the OMV-like vesicles were also found in Japanese black vinegar, kurozu. These data suggest that A. pasteurianus produce LPS-containing OMVs and can stimulate innate immune system.

 Hemolymph coagulation in horseshoe crabs is triggered by the autocatalytic activation of a lipopolysaccharide (LPS) -sensitive serine protease zymogen factor C through its transition state (factor C^＊). However, the existence of factor C^＊ is only speculative, and it remains unknown whether the autocatalytic cleavage of the Phe⁷³⁷-Ile⁷³⁸ bond (the F737 site) of factor C^＊ required for the conversion to an active form α-factor C occurs intramolecularly or intermolecularly. We show that the F737 site of a catalytic Ser⁹⁴¹-deficient mutant of factor C is cleaved by an F737 site-uncleavable mutant in the presence of LPS. These data clearly indicate the existence of factor C^＊ without cleavage of the F737 site. We also found the following facts : (1) the autocatalytic cleavage at the F737 site of factor C^＊ occurs intermolecularly on the LPS surface ; (2) factor C^＊ does not exhibit intrinsic chymotryptic activity against the F737 site during the autocatalytic activation, and (3) LPS is required not only to complete the substrate-binding site and oxyanion hole of factor C^＊ but also to allow the F737 site to be cleaved.

 Mast cells play an important role in the innate immune responses to bacterial infections as the first line of defense such as in the skin and mucosa. Mast cells can produce extracellular traps to kill bacteria by trapping pathogens. Mast cell extracellular traps (MCETs) are composed of web-like DNA fibers that contain bactericidal substances such as DNA, histones, tryptase, and antimicrobial peptides. At present, it is unknown whether the induction of inflammation in periodontal diseases is due to MCETs induced by periodontal bacteria. We investigated the role of mast cells in the induction of MCET production following infection with Fusobacterium nucleatum, a Gram-negative anaerobic bacterium associated with periodontal disease. We found that mast cells produced MCETs in response to F. nucleatum infection. Furthermore, the MCETs highly expressed macrophage migration inhibitory factor (MIF). Of note, the level of MIF expressed in the MCETs was inhibited by taurolidine, an LPS antagonist. We next investigated whether MCETs can induce inflammatory responses in monocytes. The MCETs induced the production of IL-1β, IL-6, and IL-8 by monocytes. The production of IL-1β, IL-6, and IL-8 was inhibited by an MIF inhibitor. These findings suggest that MCETs produced by mast cells in response to F. nucleatum infection induce proinflammatory cytokine production by monocytes, which may lead to the chronic inflammation observed in periodontal diseases.

 Neutrophils release microvesicles (ectosomes) upon stimulation. Interestingly, ectosome level is elevated in sepsis survivors. Previously, we revealed that LL-37, a human cathelicidin antimicrobial peptide, improves the survival of a murine cecal ligation and puncture (CLP) sepsis model. Thus, in the present study, we elucidated the action of LL-37 on sepsis, by focusing on the effect of LL-37 on ectosome release in the CLP model. The results demonstrated that the ectosome level was elevated in CLP mice, and the level was further enhanced by the LL-37-administration, accompanied with the reduced bacterial load. Importantly, ectosome-containing microvesicles isolated from LL-37-injected CLP mice contained higher amounts of antimicrobial proteins/peptides (such as lactoferrin and murine cathelicidin-related antimicrobial peptide), and exhibited higher antibacterial activity, compared with those from PBS-injected CLP mice, suggesting that LL-37 induces the release of ectosomes with antibacterial potential in vivo. In fact, LL-37 stimulated mouse bone marrow neutrophils to release ectosomes ex vivo, and the LL-37-induced ectosomes possessed the antibacterial activity. Furthermore, the administration of LL-37-induced ectosomes reduced the bacterial load and improved the survival of CLP mice. Together these observations suggest that LL-37 induces the release of ectosome-containing antimicrobial microvesicles in CLP mice, thereby reducing the bacterial load and protecting mice from lethal septic condition.

 IgE is known to play a key role in allergy. Mast cells bind IgE via the Fc receptor, FcεRI, and secrete inflammatory mediators via the recognition of allergens bound with IgE. Therefore, IgE is a major target for therapeutic treatment. Previous reports have demonstrated that oligomannose on IgE could be a new target to inhibit allergen function. However, the specific enzyme that modulates IgE for allergy treatment is not yet known. Here, we found that commercial receptor destroying enzyme (RDE) from Vibrio cholerae culture fluid can specifically modulate IgE, not IgG, and inactivate the initiation of anaphylaxis. RDE-treated IgE was unable to find the binding site of bone marrow derived-mast cells (BMMCs), followed by a reduction in the release of histamine and cytokines. We also confirmed that RDE-treated IgE could not induce passive cutaneous anaphylaxis (PCA) in mouse ears. From these results, we consider that RDE modulates the structure of IgE, rendering it unable to cause allergy. To reveal the function of RDE, we focused on the relationship of the modulation and glycosylation of IgE using lectin microarray analysis. We found that RDE-treated IgE significantly reduced the binding to Lycopersicon esculentum lectin (LEL) and Phaseolus vulgaris leucoagglutinin (PHA-L). These results suggest that RDE specifically modulates branched glycans on IgE, which is then rendered unable to induce an allergic response. These findings could be used in the development of a new drug to inhibit the function of IgE.

 The bee venom (BV) is the secretion which is produced by a needle device for protection the bee from an enemy. However, BV has been applied to the folk medicine for various diseases because it is included many enzymes which are containing anti-inflammatory or anti-cancer action. Above all, Phospholipase A2 (PLA2) is a hydrolytic enzyme which cleaves membrane phospholipids, and in bee venom occupying up to 12%. PLA2 has been analyzed in greatest detail. This mini review sets out the latest scientific evidence concerning the therapeutic effects of PLA2 in the context of diseases and provides a detailed description of the mechanisms.