Naohito Ohno - Academia.edu (original) (raw)

Papers by Naohito Ohno

Immunopharmacology and Immunotoxicology, Jul 30, 2010

We have previously obtained and elucidated the precise structure of a highly branched 1,3-β-D-glu... more We have previously obtained and elucidated the precise structure of a highly branched 1,3-β-D-glucan (with 6-monoglucopyranosyl side chains), Aureobasidium pullulans-fermented β-D-glucan (AP-FBG), from the fungus A. pullulans. However, the mechanism(s) of the effects of AP-FBG on in vitro mouse primary cells have not been analyzed in detail. Herein, we report that the induction of cytokines by AP-FBG was dependent on the existence of a granulocyte macrophage colony-stimulating factor (GM-CSF); this is similar way to be a typical 1,3-β-D-glucan from Sparassis crispa (SCG), which is a 1,3-β-D-glucopyranosyl backbone with single 1,6-β-D-glucopyranosyl side branching units every three residues. In other words, the production of cytokines in DBA/2-mouse-derived splenocytes by AP-FBG was completely hampered by an anti-GM-CSF neutralizing monoclonal antibody. Furthermore, the addition of exogenous GM-CSF to C57BL/6-derived splenocytes, which are less sensitive to AP-FBG, induced the production of cytokines by AP-FBG. Therefore, GM-CSF is indispensable for the induction of cytokines by AP-FBG in mouse-derived splenocytes. This finding has provided a new insight into our understanding of the actions of β-D-glucan but will also aid in the design and development of more effective β-D-glucan agents.

Carbohydrate Research, Jul 1, 2023

International Journal of Medicinal Mushrooms, 2023

Cells, 2020

Endothelial cellular stiffening has been observed not only in inflamed cultured endothelial cells... more Endothelial cellular stiffening has been observed not only in inflamed cultured endothelial cells but also in the endothelium of atherosclerotic regions, which is an underlying cause of monocyte adhesion and accumulation. Although recombinant soluble thrombomodulin (rsTM) has been reported to suppress the inflammatory response of endothelial cells, its role in regulating endothelial cellular stiffness remains unclear. The purpose of this study was to investigate the impact of anticoagulant rsTM on lipopolysaccharide (LPS)-induced endothelial cellular stiffening. We show that LPS increases endothelial cellular stiffness by using atomic force microscopy and that rsTM reduces LPS-induced cellular stiffening not only through the attenuation of actin fiber and focal adhesion formation but also via the improvement of gap junction functionality. Moreover, post-administration of rsTM, after LPS stimulation, attenuated LPS-induced cellular stiffening. We also found that endothelial cells reg...

International Journal of Molecular Sciences, 2021

To overcome the limitations of the Limulus amebocyte lysate (LAL) assay method for the diagnosis ... more To overcome the limitations of the Limulus amebocyte lysate (LAL) assay method for the diagnosis of invasive fungal infection, we applied a reaction system combining recombinant β-glucan binding proteins and a scanning single-molecule counting (SSMC) method. A novel (1→3)-β-D-glucan recognition protein (S-BGRP) and a (1→6)-β-glucanase mutant protein were prepared and tested for the binding of (1→6)-branched (1→3)-β-D-glucan from fungi. S-BGRP and (1→6)-β-glucanase mutant proteins reacted with β-glucan from Candida and Aspergillus spp. Although LAL cross-reacted with plant-derived β-glucans, the new detection system using the SSMC method showed low sensitivity to plant (1→3)-β-D-glucan, which significantly improved the appearance of false positives, a recognized problem with the LAL method. Measurement of β-glucan levels by the SSMC method using recombinant β-glucan-binding proteins may be useful for the diagnosis of fungal infections. This study shows that this detection system coul...

International Journal of Molecular Sciences, 2021

Because Japanese cedar pollen (JCP) contains beta-1,3-d-glucan (BG), there is concern that its li... more Because Japanese cedar pollen (JCP) contains beta-1,3-d-glucan (BG), there is concern that its lingering presence in the atmosphere, especially during its scattering period, may cause false positives in the factor-G-based Limulus amebocyte lysate (LAL) assay used to test for deep mycosis (i.e., G-test). Hence, we examined whether the LAL assay would react positively with substances contained in JCP by using the G-test to measure JCP particles and extracts. BG was purified from the JCP extract on a BG-specific affinity column, and the percentage extractability was measured using three different BG-specific quantitative methods. The G-test detected 0.4 pg BG in a single JCP particle and 10 fg from a single particle in the extract. The percentage extractability of JCP-derived BG was not significantly different among the three quantitative methods. As the JCP particles should technically have been removed during serum separation, they should be less likely to be a direct false-positive ...

Biological and Pharmaceutical Bulletin, 2020

Kawasaki disease (KD) is an inflammatory disease that was identified by Professor Tomisaku Kawasa... more Kawasaki disease (KD) is an inflammatory disease that was identified by Professor Tomisaku Kawasaki in 1961. Candida albicans-derived substances, such as the hot water extract of C. albicans (CADS) and Candida water-soluble fraction (CAWS), induced coronary vasculitis similar to KD in mice. An increasing proportion of deep-seated candidiasis cases are caused by non-albicans Candida and are often resistant to antifungal drugs. We herein investigated whether the hot water extract of C. krusei, inherently resistant to fluconazole, induces vasculitis in mice. Three strains of C. krusei, NBRC1395, NBRC1162, and NBRC10737, were cultured in natural (Y) and chemically defined (C) media and cell wall mannoprotein (MN) fractions were prepared by autoclaving cells (CKY1395MN, CKC1395MN, CKY1162MN, CKC1162MN, CKY10737MN, and CKC10737MN). All MN fractions reacted strongly with Concanavalin A (Con A) and dectin-2 and induced anaphylactoid shock in ICR mice. MNs induced severe coronary vasculitis in DBA/2 mice, resulting in cardiac hypertrophy. MNs also induced coronary vasculitis in C57Bl/6 mice. These results suggest that the MNs of non-albicans Candida, such as C. krusei, induce similar toxicity to those of C. albicans.

Recognition of (1→3)-β-d-glucans (BGs) by invertebrate β-1,3-d-glucan recognition protein (BGRP) ... more Recognition of (1→3)-β-d-glucans (BGs) by invertebrate β-1,3-d-glucan recognition protein (BGRP) plays a significant role in the activation of Toll pathway and prophenoloxidase systems in insect host defense against fungal invasion. To examine the structure diversity of BGRPs for the recognition of physiochemically different BGs, the binding specificity of BGRPs cloned from four different insects to structure different BGs was characterized using ELISA. Recombinant BGRPs expressed as Fc-fusion proteins of human IgG1 bound to the solid phase of BGs. Based on the binding specificities, the BGRPs were categorized into two groups with different ultrastructures and binding characters; one group specifically binds BGs with triple-helical conformation, while the other group recognizes BGs with disordered conformations like single-helical or partially opened triple helix. The BGRPs from the silkworm and the Indian meal moth bound to the BGs with a triple-helical structure, whereas BGRPs from the red flour beetle and yellow mealworm beetle showed no binding to triple-helical BGs, but bound to alkaline-treated BGs that have a partially opened triple-helical conformation. This evidence suggests that the insect BGRPs can distinguish between different conformations of BGs and are equipped for determining the diversity of BG structures.

Nutrients, 2019

Euglena gracilis EOD-1, a microalgal strain known for high yields of the β-1, 3-glucan paramylon,... more Euglena gracilis EOD-1, a microalgal strain known for high yields of the β-1, 3-glucan paramylon, is suggested to function as a dietary fiber and enhance immunity. Here, we aimed to investigate the effects of E. gracilis EOD-1 biomass (EOD1BM) ingestion on immunoglobulin A (IgA) antibody titers in saliva, its reactivity, and the health-related quality of life (QOL) in humans. Reacting human immunoglobulin preparations and saliva with paramylon granules revealed the presence of anti-paramylon antibodies in the blood and saliva. We conducted a placebo-controlled, double-blind, crossover study involving 13 healthy subjects who ingested the placebo or EOD1BM for 4 weeks. Saliva was collected from each subject before and after ingestion, and IgA titers and E. gracilis EOD-1 paramylon (EOD1PM) reactivity were compared. In the EOD1BM Ingestion group, the anti-EOD1PM IgA content and titer increased after EOD1BM ingestion. No such change was observed in the Placebo group. Furthermore, the he...

International Immunology, 2010

Calreticulin, a multifunctional calcium binding molecular chaperone, is a frequent autoantigen in... more Calreticulin, a multifunctional calcium binding molecular chaperone, is a frequent autoantigen in some gastrointestinal diseases. However, the reason for the elicitation of strong autoimmune response to this intracellular glycoprotein is not fully understood. In the present study, we analyzed the influence of bacterial and food antigens, and proinflammatory cytokine IFN-gamma (known to affect intestinal epithelium) on the expression, cellular localization, and posttranslation modification of calreticulin in human epithelial cell lines HT-29 and DLD-1. Employing fluorescence immunocytochemistry, proteomic, and glycomic analyses of subcellular fractions, we provided clear evidence that IFN-gamma, wheat gliadin (damaging jejunal mucosa of celiacs), and bacterial LPS cause up-regulation of calreticulin. IFN-gamma and gliadin had a strong synergistic effect on calreticulin overexpression, translocation to the external leaflet of plasma membrane and secretion. These events were accompanied by creation of truncated form of the molecule and its unique glycosylation. Upon stimulation, full-length calreticulin, and mainly its truncated form bearing sulfated glycans, was released into the supernatants of cultured epithelial cells. Based of these findings, we suggest that newly discovered (N-terminally truncated and unusually glycosylated) form of plasma-membrane and released calreticulin may contribute to the relatively frequent appearance of anti-calreticulin antibodies in sera of patients with gastrointestinal inflammatory and autoimmune diseases.

PloS one, 2012

High-molecular weight polymerised polyphenols have been shown to exhibit anti-influenza virus, an... more High-molecular weight polymerised polyphenols have been shown to exhibit anti-influenza virus, anti-HIV, and anti-cancer activities. The purpose of this study was to evaluate the immunomodulating activities of enzymatically polymerised polyphenols, and to clarify the underlying mechanisms of their effects. The cytokine-inducing activity of the enzymatically polymerised polyphenols derived from caffeic acid (CA), ferulic acid (FA), and p-coumaric acid (CoA) was investigated using murine splenocytes. Polymerised polyphenols, but not non-polymerised polyphenols, induced cytokine synthesis in murine splenocytes. Polymerised polyphenols induced several cytokines in murine splenocytes, with interferon-γ (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) being the most prominent. The underlying mechanisms of the effects of the polymerised polyphenols were then studied using neutralising antibodies and fluorescent-activated cell sorting (FACS) analysis. Our results show t...

International Journal of Vascular Medicine, 2012

It was reported previously that a Candida albicans water-soluble fraction (CAWS), including a man... more It was reported previously that a Candida albicans water-soluble fraction (CAWS), including a mannoprotein and β-glucan complex, has strong potency in inducing fatal necrotizing arteritis in DBA/2 mice. In this study, histopathological changes and cardiac function were investigated in this system. One mg/day of CAWS was given to DBA/2 mice via peritoneal injection for five days. The CAWS-treated DBA/2 mice were induced aortitis and died at an incidence of 100% within several weeks. Histological findings included stenosis in the left ventricular outflow tract (LVOT) and severe inflammatory changes of the aortic valve with fibrinoid necrosis. Cardiomegaly was observed and heart weight increased 1.62 fold (P < 0.01). Echocardiography revealed a severe reduction in contractility and dilatation of the cavity in the left ventricle (LV): LV fractional shortening (LVFS) decreased from 71% to 38% (P < 0.01), and the LV end-diastolic diameter (LVDd) increased from 2.21 mm to 3.26 mm (P < 0.01). The titer of BNP mRNA increased in the CAWS-treated group. Severe inflammatory changes resulting from CAWS brought about lethal LV dysfunction by aortic valve deformation with LVOT stenosis. This system is proposed as an easy and useful experimental model of heart failure because CAWS arteritis can be induced by CAWS injection alone.

Gene therapy, Jan 8, 2015

The CD40/CD154 co-stimulatory pathway is crucial in alloimmune response. We developed a novel sma... more The CD40/CD154 co-stimulatory pathway is crucial in alloimmune response. We developed a novel small interfering RNA (siRNA) delivery system with a poly-dA extension at the 5'-end of the siRNA sense strand that was stably incorporated into 1,3-β-glucan (schizophyllan, SPG). This was captured and incorporated into dendritic cells (DCs) through its receptor, Dectin-1, specifically silencing CD40 genes (siCD40) to exert immunoregulatory activity. siCD40/SPG-treated CBA mice permanently accepted B10 fully mismatched cardiac allografts. Consistent with graft survival, the infiltration of CD4(+), CD8(+) T cells into the graft was lower, and that the numbers of CD40(low)CD11c(+) DCs cells and CD4(+)Foxp3(+)cells were increased in both the graft and in the recipient spleen. In addition, naive CBA recipients given an adoptive transfer of splenocytes from the primary recipients with siCD40/SPG accepted a heart graft from donor-type B10, but not third-party Balb/c mice. In conclusion, the t...

Respiratory Research, 2009

Background: Although fungi have been implicated as initiating/deteriorating factors for allergic ... more Background: Although fungi have been implicated as initiating/deteriorating factors for allergic asthma, their contributing components have not been fully elucidated. We previously isolated soluble β-glucan from Candida albicans (CSBG) (Ohno et al., 2007). In the present study, the effects of CSBG exposure on airway immunopathology in the presence or absence of other immunogenic allergen was investigated in vivo, and their cellular mechanisms were analyzed both in vivo and in vitro. Methods: In vivo, ICR mice were divided into 4 experimental groups: vehicle, CSBG (25 μg/animal), ovalbumin (OVA: 2 μg/animal), and CSBG + OVA were repeatedly administered intratracheally. The bronchoalveolar lavage cellular profile, lung histology, levels of cytokines and chemokines in the lung homogenates, the expression pattern of antigen-presenting cell (APC)-related molecules in the lung digests, and serum immunoglobulin values were studied. In vitro, the impacts of CSBG (0-12.5 μg/ml) on the phenotype and function of immune cells such as splenocytes and bone marrow-derived dendritic cells (BMDCs) were evaluated in terms of cell proliferation, the surface expression of APC-related molecules, and OVA-mediated T-cell proliferating activity. Results: In vivo, repeated pulmonary exposure to CSBG induced neutrophilic airway inflammation in the absence of OVA, and markedly exacerbated OVA-related eosinophilic airway inflammation with mucus metaplasia in mice, which was concomitant with the amplified lung expression of Th2 cytokines and IL-17A and chemokines related to allergic response. Exposure to CSBG plus OVA increased the number of cells bearing MHC class II with or without CD80 in the lung compared to that of others. In vitro, CSBG significantly augmented splenocyte proliferation in the presence or absence of OVA. Further, CSBG increased the expression of APC-related molecules such as CD80, CD86, and DEC205 on BMDCs and amplified OVA-mediated T-cell proliferation through BMDCs. Conclusion: CSBG potentiates allergic airway inflammation with maladaptive Th immunity, and this potentiation was associated with the enhanced activation of APCs including DC.

Microbiology and Immunology, 2011

The biological effects of Candida metapsilosis water-soluble fraction (CMWS), prepared using a co... more The biological effects of Candida metapsilosis water-soluble fraction (CMWS), prepared using a completely synthesized medium, were examined to determine whether CMWS induces vasculitis similar to that seen in Kawasaki disease, and anaphylactoid shock, in mice. It was found that intraperitoneal injection of CMWS induces coronary arteritis and i.v. injection induces acute anaphylactoid shock in mice, similar to Candida albicans water-soluble fraction (CAWS)-induced arteritis and anaphylactoid shock. The mannan structure of the polysaccharide fraction was then analyzed by performing antiserum reactivity tests and nuclear magnetic resonance spectroscopy. The mannan structure was investigated because the present authors have recently found that the mannan moiety within the polysaccharide fraction might be responsible for these pathogenic activities. The structural analysis showed that the mannan structure within CMWS expresses α-mannan residues, but not β-mannan. In addition, the mannan structure of CMWS is quite similar to that of CAWS. The present findings indicate that the polysaccharide fraction from C. metapsilosis, which is mainly composed of mannan, contributes to coronary arteritis and acute shock, and that the mannan structure could be responsible for this pathogenicity.

Journal of Pharmacy and Pharmacology, 1996

Although it has been established that soluble glucan in fungi is important to host defence agains... more Although it has been established that soluble glucan in fungi is important to host defence against infection, the importance of insoluble glucans is not clear. We have examined the in-vivo immunopharmacological activity of the insoluble glucan, zymocel. Administration of zymocel increased peritoneal exudate cell number and spleen weight, and enhanced: phagocytic activity, hydrogen peroxide production, and nitric oxide production of peritoneal exudate cells; the extravascular release of Evans blue (which might reflect vascular permeability); lipopolysaccharide-triggered synthesis of tumour necrosis factor (TNF); and recovery of white blood cell number in cyclophosphamide-induced leukopenia. Zymocel also showed anti-tumour activity against sarcoma 180 in mice and also enhanced TNF synthesis and hydrogen peroxide production by macrophage-like cell line in-vitro, i.e. resulted in direct macrophage activation. These results show that zymocel shows varied immunopharmacological activity; i...

The Journal of Immunology, 2006

Macrophages and monocytes are activated by CpG DNA motifs to produce NO, which is enhanced dramat... more Macrophages and monocytes are activated by CpG DNA motifs to produce NO, which is enhanced dramatically by IFN-γ. We hypothesize that synergistic cellular responses to IFN-γ and CpG DNA are due to cross-talk between metabolic signaling pathways of leukocytes. Adherent RAW264.7 macrophages and human monocytes exhibited NAD(P)H autofluorescence oscillation periods of ∼20 s. IFN-γ increased the oscillatory amplitude, which was required for CpG DNA-mediated metabolic changes. These alterations in metabolic dynamics required the appropriate combinations of murine/human TLR9 and murine/human-specific CpG DNA. Other factors that also promoted an increase in metabolic oscillatory amplitude could substitute for IFN-γ. Because recent studies have shown that the metabolic frequency is coupled to the hexose monophosphate shunt, and the amplitude is coupled to the peroxidase cycle, we tested the hypothesis that myeloperoxidase (MPO) participates in IFN-γ priming for oxidant production. MPO inhib...

The Journal of Immunology, 2008

The mechanism of host cell recognition of Cryptococcus neoformans, an opportunistic fungal pathog... more The mechanism of host cell recognition of Cryptococcus neoformans, an opportunistic fungal pathogen in immunocompromised patients, remains poorly understood. In the present study, we asked whether the DNA of this yeast activates mouse bone marrow-derived myeloid dendritic cells (BM-DCs). BM-DCs released IL-12p40 and expressed CD40 upon stimulation with cryptococcal DNA, and the response was abolished by treatment with DNase, but not with RNase. IL-12p40 production and CD40 expression were attenuated by chloroquine, bafilomycin A, and inhibitory oligodeoxynucleotides (ODN) that suppressed the responses caused by CpG-ODN. Activation of BM-DCs by cryptococcal DNA was almost completely abrogated in TLR9 gene-disrupted (TLR9−/−) mice and MyD88−/− mice, similar to that by CpG-ODN. In addition, upon stimulation with whole yeast cells of acapsular C. neoformans, TLR9−/− BM-DCs produced a lower amount of IL-12p40 than those from wild-type mice, and TLR9−/− mice were more susceptible to pulmo...

The Journal of Immunology, 2009

Candida albicans water-soluble fraction (CAWS), a mannoprotein-β-glucan complex obtained from the... more Candida albicans water-soluble fraction (CAWS), a mannoprotein-β-glucan complex obtained from the culture supernatant of C. albicans NBRC1385, exhibits vasculitis-inducing activity (CAWS-vasculitis) in mice. The sensitivity to CAWS-vasculitis varies greatly among mouse strains. This study examined the factors contributing to or inhibiting CAWS-vasculitis using CAWS-vasculitis-resistant CBA/J mice and Bruton’s tyrosine kinase-deficient CBA/N mice, which is a CAWS-vasculitis-sensitive strain that has the same origin as CBA/J mice. After stimulation with various kinds of pathogen-associated molecular patterns, the production of inflammatory cytokines IL-6 and IFN-γ was induced in CBA/N mice, whereas that of immunosuppressive IL-10 was induced in CAWS-vasculitis-resistant CBA/J mice. Furthermore, the production of tissue inhibitor of metalloproteinase 1, an endogenous matrix metalloproteinase inhibitor, was observed in CBA/J mice. The results strongly suggest that the difference in the ...

Immunopharmacology and Immunotoxicology, Jul 30, 2010

We have previously obtained and elucidated the precise structure of a highly branched 1,3-β-D-glu... more We have previously obtained and elucidated the precise structure of a highly branched 1,3-β-D-glucan (with 6-monoglucopyranosyl side chains), Aureobasidium pullulans-fermented β-D-glucan (AP-FBG), from the fungus A. pullulans. However, the mechanism(s) of the effects of AP-FBG on in vitro mouse primary cells have not been analyzed in detail. Herein, we report that the induction of cytokines by AP-FBG was dependent on the existence of a granulocyte macrophage colony-stimulating factor (GM-CSF); this is similar way to be a typical 1,3-β-D-glucan from Sparassis crispa (SCG), which is a 1,3-β-D-glucopyranosyl backbone with single 1,6-β-D-glucopyranosyl side branching units every three residues. In other words, the production of cytokines in DBA/2-mouse-derived splenocytes by AP-FBG was completely hampered by an anti-GM-CSF neutralizing monoclonal antibody. Furthermore, the addition of exogenous GM-CSF to C57BL/6-derived splenocytes, which are less sensitive to AP-FBG, induced the production of cytokines by AP-FBG. Therefore, GM-CSF is indispensable for the induction of cytokines by AP-FBG in mouse-derived splenocytes. This finding has provided a new insight into our understanding of the actions of β-D-glucan but will also aid in the design and development of more effective β-D-glucan agents.

Carbohydrate Research, Jul 1, 2023

International Journal of Medicinal Mushrooms, 2023

Cells, 2020

Endothelial cellular stiffening has been observed not only in inflamed cultured endothelial cells... more Endothelial cellular stiffening has been observed not only in inflamed cultured endothelial cells but also in the endothelium of atherosclerotic regions, which is an underlying cause of monocyte adhesion and accumulation. Although recombinant soluble thrombomodulin (rsTM) has been reported to suppress the inflammatory response of endothelial cells, its role in regulating endothelial cellular stiffness remains unclear. The purpose of this study was to investigate the impact of anticoagulant rsTM on lipopolysaccharide (LPS)-induced endothelial cellular stiffening. We show that LPS increases endothelial cellular stiffness by using atomic force microscopy and that rsTM reduces LPS-induced cellular stiffening not only through the attenuation of actin fiber and focal adhesion formation but also via the improvement of gap junction functionality. Moreover, post-administration of rsTM, after LPS stimulation, attenuated LPS-induced cellular stiffening. We also found that endothelial cells reg...

International Journal of Molecular Sciences, 2021

To overcome the limitations of the Limulus amebocyte lysate (LAL) assay method for the diagnosis ... more To overcome the limitations of the Limulus amebocyte lysate (LAL) assay method for the diagnosis of invasive fungal infection, we applied a reaction system combining recombinant β-glucan binding proteins and a scanning single-molecule counting (SSMC) method. A novel (1→3)-β-D-glucan recognition protein (S-BGRP) and a (1→6)-β-glucanase mutant protein were prepared and tested for the binding of (1→6)-branched (1→3)-β-D-glucan from fungi. S-BGRP and (1→6)-β-glucanase mutant proteins reacted with β-glucan from Candida and Aspergillus spp. Although LAL cross-reacted with plant-derived β-glucans, the new detection system using the SSMC method showed low sensitivity to plant (1→3)-β-D-glucan, which significantly improved the appearance of false positives, a recognized problem with the LAL method. Measurement of β-glucan levels by the SSMC method using recombinant β-glucan-binding proteins may be useful for the diagnosis of fungal infections. This study shows that this detection system coul...

International Journal of Molecular Sciences, 2021

Because Japanese cedar pollen (JCP) contains beta-1,3-d-glucan (BG), there is concern that its li... more Because Japanese cedar pollen (JCP) contains beta-1,3-d-glucan (BG), there is concern that its lingering presence in the atmosphere, especially during its scattering period, may cause false positives in the factor-G-based Limulus amebocyte lysate (LAL) assay used to test for deep mycosis (i.e., G-test). Hence, we examined whether the LAL assay would react positively with substances contained in JCP by using the G-test to measure JCP particles and extracts. BG was purified from the JCP extract on a BG-specific affinity column, and the percentage extractability was measured using three different BG-specific quantitative methods. The G-test detected 0.4 pg BG in a single JCP particle and 10 fg from a single particle in the extract. The percentage extractability of JCP-derived BG was not significantly different among the three quantitative methods. As the JCP particles should technically have been removed during serum separation, they should be less likely to be a direct false-positive ...

Biological and Pharmaceutical Bulletin, 2020

Kawasaki disease (KD) is an inflammatory disease that was identified by Professor Tomisaku Kawasa... more Kawasaki disease (KD) is an inflammatory disease that was identified by Professor Tomisaku Kawasaki in 1961. Candida albicans-derived substances, such as the hot water extract of C. albicans (CADS) and Candida water-soluble fraction (CAWS), induced coronary vasculitis similar to KD in mice. An increasing proportion of deep-seated candidiasis cases are caused by non-albicans Candida and are often resistant to antifungal drugs. We herein investigated whether the hot water extract of C. krusei, inherently resistant to fluconazole, induces vasculitis in mice. Three strains of C. krusei, NBRC1395, NBRC1162, and NBRC10737, were cultured in natural (Y) and chemically defined (C) media and cell wall mannoprotein (MN) fractions were prepared by autoclaving cells (CKY1395MN, CKC1395MN, CKY1162MN, CKC1162MN, CKY10737MN, and CKC10737MN). All MN fractions reacted strongly with Concanavalin A (Con A) and dectin-2 and induced anaphylactoid shock in ICR mice. MNs induced severe coronary vasculitis in DBA/2 mice, resulting in cardiac hypertrophy. MNs also induced coronary vasculitis in C57Bl/6 mice. These results suggest that the MNs of non-albicans Candida, such as C. krusei, induce similar toxicity to those of C. albicans.

Recognition of (1→3)-β-d-glucans (BGs) by invertebrate β-1,3-d-glucan recognition protein (BGRP) ... more Recognition of (1→3)-β-d-glucans (BGs) by invertebrate β-1,3-d-glucan recognition protein (BGRP) plays a significant role in the activation of Toll pathway and prophenoloxidase systems in insect host defense against fungal invasion. To examine the structure diversity of BGRPs for the recognition of physiochemically different BGs, the binding specificity of BGRPs cloned from four different insects to structure different BGs was characterized using ELISA. Recombinant BGRPs expressed as Fc-fusion proteins of human IgG1 bound to the solid phase of BGs. Based on the binding specificities, the BGRPs were categorized into two groups with different ultrastructures and binding characters; one group specifically binds BGs with triple-helical conformation, while the other group recognizes BGs with disordered conformations like single-helical or partially opened triple helix. The BGRPs from the silkworm and the Indian meal moth bound to the BGs with a triple-helical structure, whereas BGRPs from the red flour beetle and yellow mealworm beetle showed no binding to triple-helical BGs, but bound to alkaline-treated BGs that have a partially opened triple-helical conformation. This evidence suggests that the insect BGRPs can distinguish between different conformations of BGs and are equipped for determining the diversity of BG structures.

Nutrients, 2019

Euglena gracilis EOD-1, a microalgal strain known for high yields of the β-1, 3-glucan paramylon,... more Euglena gracilis EOD-1, a microalgal strain known for high yields of the β-1, 3-glucan paramylon, is suggested to function as a dietary fiber and enhance immunity. Here, we aimed to investigate the effects of E. gracilis EOD-1 biomass (EOD1BM) ingestion on immunoglobulin A (IgA) antibody titers in saliva, its reactivity, and the health-related quality of life (QOL) in humans. Reacting human immunoglobulin preparations and saliva with paramylon granules revealed the presence of anti-paramylon antibodies in the blood and saliva. We conducted a placebo-controlled, double-blind, crossover study involving 13 healthy subjects who ingested the placebo or EOD1BM for 4 weeks. Saliva was collected from each subject before and after ingestion, and IgA titers and E. gracilis EOD-1 paramylon (EOD1PM) reactivity were compared. In the EOD1BM Ingestion group, the anti-EOD1PM IgA content and titer increased after EOD1BM ingestion. No such change was observed in the Placebo group. Furthermore, the he...

International Immunology, 2010

Calreticulin, a multifunctional calcium binding molecular chaperone, is a frequent autoantigen in... more Calreticulin, a multifunctional calcium binding molecular chaperone, is a frequent autoantigen in some gastrointestinal diseases. However, the reason for the elicitation of strong autoimmune response to this intracellular glycoprotein is not fully understood. In the present study, we analyzed the influence of bacterial and food antigens, and proinflammatory cytokine IFN-gamma (known to affect intestinal epithelium) on the expression, cellular localization, and posttranslation modification of calreticulin in human epithelial cell lines HT-29 and DLD-1. Employing fluorescence immunocytochemistry, proteomic, and glycomic analyses of subcellular fractions, we provided clear evidence that IFN-gamma, wheat gliadin (damaging jejunal mucosa of celiacs), and bacterial LPS cause up-regulation of calreticulin. IFN-gamma and gliadin had a strong synergistic effect on calreticulin overexpression, translocation to the external leaflet of plasma membrane and secretion. These events were accompanied by creation of truncated form of the molecule and its unique glycosylation. Upon stimulation, full-length calreticulin, and mainly its truncated form bearing sulfated glycans, was released into the supernatants of cultured epithelial cells. Based of these findings, we suggest that newly discovered (N-terminally truncated and unusually glycosylated) form of plasma-membrane and released calreticulin may contribute to the relatively frequent appearance of anti-calreticulin antibodies in sera of patients with gastrointestinal inflammatory and autoimmune diseases.

PloS one, 2012

High-molecular weight polymerised polyphenols have been shown to exhibit anti-influenza virus, an... more High-molecular weight polymerised polyphenols have been shown to exhibit anti-influenza virus, anti-HIV, and anti-cancer activities. The purpose of this study was to evaluate the immunomodulating activities of enzymatically polymerised polyphenols, and to clarify the underlying mechanisms of their effects. The cytokine-inducing activity of the enzymatically polymerised polyphenols derived from caffeic acid (CA), ferulic acid (FA), and p-coumaric acid (CoA) was investigated using murine splenocytes. Polymerised polyphenols, but not non-polymerised polyphenols, induced cytokine synthesis in murine splenocytes. Polymerised polyphenols induced several cytokines in murine splenocytes, with interferon-γ (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) being the most prominent. The underlying mechanisms of the effects of the polymerised polyphenols were then studied using neutralising antibodies and fluorescent-activated cell sorting (FACS) analysis. Our results show t...

International Journal of Vascular Medicine, 2012

It was reported previously that a Candida albicans water-soluble fraction (CAWS), including a man... more It was reported previously that a Candida albicans water-soluble fraction (CAWS), including a mannoprotein and β-glucan complex, has strong potency in inducing fatal necrotizing arteritis in DBA/2 mice. In this study, histopathological changes and cardiac function were investigated in this system. One mg/day of CAWS was given to DBA/2 mice via peritoneal injection for five days. The CAWS-treated DBA/2 mice were induced aortitis and died at an incidence of 100% within several weeks. Histological findings included stenosis in the left ventricular outflow tract (LVOT) and severe inflammatory changes of the aortic valve with fibrinoid necrosis. Cardiomegaly was observed and heart weight increased 1.62 fold (P < 0.01). Echocardiography revealed a severe reduction in contractility and dilatation of the cavity in the left ventricle (LV): LV fractional shortening (LVFS) decreased from 71% to 38% (P < 0.01), and the LV end-diastolic diameter (LVDd) increased from 2.21 mm to 3.26 mm (P < 0.01). The titer of BNP mRNA increased in the CAWS-treated group. Severe inflammatory changes resulting from CAWS brought about lethal LV dysfunction by aortic valve deformation with LVOT stenosis. This system is proposed as an easy and useful experimental model of heart failure because CAWS arteritis can be induced by CAWS injection alone.

Gene therapy, Jan 8, 2015

The CD40/CD154 co-stimulatory pathway is crucial in alloimmune response. We developed a novel sma... more The CD40/CD154 co-stimulatory pathway is crucial in alloimmune response. We developed a novel small interfering RNA (siRNA) delivery system with a poly-dA extension at the 5'-end of the siRNA sense strand that was stably incorporated into 1,3-β-glucan (schizophyllan, SPG). This was captured and incorporated into dendritic cells (DCs) through its receptor, Dectin-1, specifically silencing CD40 genes (siCD40) to exert immunoregulatory activity. siCD40/SPG-treated CBA mice permanently accepted B10 fully mismatched cardiac allografts. Consistent with graft survival, the infiltration of CD4(+), CD8(+) T cells into the graft was lower, and that the numbers of CD40(low)CD11c(+) DCs cells and CD4(+)Foxp3(+)cells were increased in both the graft and in the recipient spleen. In addition, naive CBA recipients given an adoptive transfer of splenocytes from the primary recipients with siCD40/SPG accepted a heart graft from donor-type B10, but not third-party Balb/c mice. In conclusion, the t...

Respiratory Research, 2009

Background: Although fungi have been implicated as initiating/deteriorating factors for allergic ... more Background: Although fungi have been implicated as initiating/deteriorating factors for allergic asthma, their contributing components have not been fully elucidated. We previously isolated soluble β-glucan from Candida albicans (CSBG) (Ohno et al., 2007). In the present study, the effects of CSBG exposure on airway immunopathology in the presence or absence of other immunogenic allergen was investigated in vivo, and their cellular mechanisms were analyzed both in vivo and in vitro. Methods: In vivo, ICR mice were divided into 4 experimental groups: vehicle, CSBG (25 μg/animal), ovalbumin (OVA: 2 μg/animal), and CSBG + OVA were repeatedly administered intratracheally. The bronchoalveolar lavage cellular profile, lung histology, levels of cytokines and chemokines in the lung homogenates, the expression pattern of antigen-presenting cell (APC)-related molecules in the lung digests, and serum immunoglobulin values were studied. In vitro, the impacts of CSBG (0-12.5 μg/ml) on the phenotype and function of immune cells such as splenocytes and bone marrow-derived dendritic cells (BMDCs) were evaluated in terms of cell proliferation, the surface expression of APC-related molecules, and OVA-mediated T-cell proliferating activity. Results: In vivo, repeated pulmonary exposure to CSBG induced neutrophilic airway inflammation in the absence of OVA, and markedly exacerbated OVA-related eosinophilic airway inflammation with mucus metaplasia in mice, which was concomitant with the amplified lung expression of Th2 cytokines and IL-17A and chemokines related to allergic response. Exposure to CSBG plus OVA increased the number of cells bearing MHC class II with or without CD80 in the lung compared to that of others. In vitro, CSBG significantly augmented splenocyte proliferation in the presence or absence of OVA. Further, CSBG increased the expression of APC-related molecules such as CD80, CD86, and DEC205 on BMDCs and amplified OVA-mediated T-cell proliferation through BMDCs. Conclusion: CSBG potentiates allergic airway inflammation with maladaptive Th immunity, and this potentiation was associated with the enhanced activation of APCs including DC.

Microbiology and Immunology, 2011

The biological effects of Candida metapsilosis water-soluble fraction (CMWS), prepared using a co... more The biological effects of Candida metapsilosis water-soluble fraction (CMWS), prepared using a completely synthesized medium, were examined to determine whether CMWS induces vasculitis similar to that seen in Kawasaki disease, and anaphylactoid shock, in mice. It was found that intraperitoneal injection of CMWS induces coronary arteritis and i.v. injection induces acute anaphylactoid shock in mice, similar to Candida albicans water-soluble fraction (CAWS)-induced arteritis and anaphylactoid shock. The mannan structure of the polysaccharide fraction was then analyzed by performing antiserum reactivity tests and nuclear magnetic resonance spectroscopy. The mannan structure was investigated because the present authors have recently found that the mannan moiety within the polysaccharide fraction might be responsible for these pathogenic activities. The structural analysis showed that the mannan structure within CMWS expresses α-mannan residues, but not β-mannan. In addition, the mannan structure of CMWS is quite similar to that of CAWS. The present findings indicate that the polysaccharide fraction from C. metapsilosis, which is mainly composed of mannan, contributes to coronary arteritis and acute shock, and that the mannan structure could be responsible for this pathogenicity.

Journal of Pharmacy and Pharmacology, 1996

Although it has been established that soluble glucan in fungi is important to host defence agains... more Although it has been established that soluble glucan in fungi is important to host defence against infection, the importance of insoluble glucans is not clear. We have examined the in-vivo immunopharmacological activity of the insoluble glucan, zymocel. Administration of zymocel increased peritoneal exudate cell number and spleen weight, and enhanced: phagocytic activity, hydrogen peroxide production, and nitric oxide production of peritoneal exudate cells; the extravascular release of Evans blue (which might reflect vascular permeability); lipopolysaccharide-triggered synthesis of tumour necrosis factor (TNF); and recovery of white blood cell number in cyclophosphamide-induced leukopenia. Zymocel also showed anti-tumour activity against sarcoma 180 in mice and also enhanced TNF synthesis and hydrogen peroxide production by macrophage-like cell line in-vitro, i.e. resulted in direct macrophage activation. These results show that zymocel shows varied immunopharmacological activity; i...

The Journal of Immunology, 2006

Macrophages and monocytes are activated by CpG DNA motifs to produce NO, which is enhanced dramat... more Macrophages and monocytes are activated by CpG DNA motifs to produce NO, which is enhanced dramatically by IFN-γ. We hypothesize that synergistic cellular responses to IFN-γ and CpG DNA are due to cross-talk between metabolic signaling pathways of leukocytes. Adherent RAW264.7 macrophages and human monocytes exhibited NAD(P)H autofluorescence oscillation periods of ∼20 s. IFN-γ increased the oscillatory amplitude, which was required for CpG DNA-mediated metabolic changes. These alterations in metabolic dynamics required the appropriate combinations of murine/human TLR9 and murine/human-specific CpG DNA. Other factors that also promoted an increase in metabolic oscillatory amplitude could substitute for IFN-γ. Because recent studies have shown that the metabolic frequency is coupled to the hexose monophosphate shunt, and the amplitude is coupled to the peroxidase cycle, we tested the hypothesis that myeloperoxidase (MPO) participates in IFN-γ priming for oxidant production. MPO inhib...

The Journal of Immunology, 2008

The mechanism of host cell recognition of Cryptococcus neoformans, an opportunistic fungal pathog... more The mechanism of host cell recognition of Cryptococcus neoformans, an opportunistic fungal pathogen in immunocompromised patients, remains poorly understood. In the present study, we asked whether the DNA of this yeast activates mouse bone marrow-derived myeloid dendritic cells (BM-DCs). BM-DCs released IL-12p40 and expressed CD40 upon stimulation with cryptococcal DNA, and the response was abolished by treatment with DNase, but not with RNase. IL-12p40 production and CD40 expression were attenuated by chloroquine, bafilomycin A, and inhibitory oligodeoxynucleotides (ODN) that suppressed the responses caused by CpG-ODN. Activation of BM-DCs by cryptococcal DNA was almost completely abrogated in TLR9 gene-disrupted (TLR9−/−) mice and MyD88−/− mice, similar to that by CpG-ODN. In addition, upon stimulation with whole yeast cells of acapsular C. neoformans, TLR9−/− BM-DCs produced a lower amount of IL-12p40 than those from wild-type mice, and TLR9−/− mice were more susceptible to pulmo...

The Journal of Immunology, 2009

Candida albicans water-soluble fraction (CAWS), a mannoprotein-β-glucan complex obtained from the... more Candida albicans water-soluble fraction (CAWS), a mannoprotein-β-glucan complex obtained from the culture supernatant of C. albicans NBRC1385, exhibits vasculitis-inducing activity (CAWS-vasculitis) in mice. The sensitivity to CAWS-vasculitis varies greatly among mouse strains. This study examined the factors contributing to or inhibiting CAWS-vasculitis using CAWS-vasculitis-resistant CBA/J mice and Bruton’s tyrosine kinase-deficient CBA/N mice, which is a CAWS-vasculitis-sensitive strain that has the same origin as CBA/J mice. After stimulation with various kinds of pathogen-associated molecular patterns, the production of inflammatory cytokines IL-6 and IFN-γ was induced in CBA/N mice, whereas that of immunosuppressive IL-10 was induced in CAWS-vasculitis-resistant CBA/J mice. Furthermore, the production of tissue inhibitor of metalloproteinase 1, an endogenous matrix metalloproteinase inhibitor, was observed in CBA/J mice. The results strongly suggest that the difference in the ...