A monoclonal antibody, mNI-58A, against CD11a induces homotypic cell aggregation and promotes interferon-γ production by the human NK-like cell line, KHYG-1

我々が開発したCD11a(LFA-1α)モノクローナル抗体(mNI-58A)のヒトNK様細胞株(KHYG-1)に与える影響を検討したところ、mNI-58A抗体はKHYG-1細胞に対して細胞間凝集を特異的に誘導した。この細胞間凝集反応は、conventional protein kinase C (cPKC)抑制剤であるGo6976とnovel protein kinase C (nPKC)抑制剤であるRottlerinでブロックされ、その効果はRottlerinの方がGo6976より強かった。また、mNI-58A抗体はKHYG-1細胞にインターフェロンγの産生能も増強させた。以上の結果は、mNI-58A抗体がヒト免疫応答におけるNK細胞の機能を解析する上で非常に有益な抗体であることを示唆している。A monoclonal antibody (mAb), designated as mNI-58A, against CD11a (leukocyte function-associated antigen-1 α; LFA-1α) was established in our laboratories by immunizing mice with the lipopolysaccharide (LPS)-stimulated human monocyte-like cell line, U937. This mAb specifically induced homotypic cell aggregation of the human NH-like cell line, KHYG-1.This mNI58A-induced homotypic cell aggregation was markedly blocked by the addition of an optional concentration of a conventional protein kinase C (cPKC) inhibitor, Go6976, and was completely blocked by the addition of an optimal concentration of a novel protein kinase C (nPKC); a PKC delta isoenzyme) inhibitor, Rottlerin. Interestingly, KHYG-1 cells effectively promoted interferon-γ (IFN-γ) production in the culture supernatants of the homotypic cell aggregations of KHYG-1 cells induced by mNI-58A. These findings strongly indicate that CD11a mAb (mNI-58A) has some unique properties and many be useful for analyzing the cell-to-cell interactions of NK cells in several human immune responses

Analysis of the salivary levels of immune biomarkers after inhalation of a steam mixed gas containing hydrogen gas

スイソニアから発生する水素ガス（濃度0.1～0.3％）を含む蒸気混合ガス“XEN”を鼻カニューラで吸入した。吸入後、唾液中の免疫バイオマーカーの動態を酵素抗体法（enzyme immunoassay：EIA）で解析した。その結果、“XEN”吸入15分後、唾液中のインターロイキン-1β（IL-1β）の濃度が吸入前と比較して有意に増加した（P<0.01）。一方、唾液中の可溶性CD44分子（sCD44）は吸入前後で有意な差は認められなかった。以上の結果は、“XEN”が鼻粘膜免疫系を活性化し、生体の免疫力を増強させる作用があることを示唆するものである。In this preliminary study, we examined the effects of inhalation of a steam mixed gas containinghydrogen gas on the salivary levels of immune biomarkers, especially interleukin-1????and soluble-formCD44, in healthy adult volunteers. This gas was generated by the decomposition of superheated steamusing a machine (Suisonia) manufactured by Earth Engineering Co., and was designated as“XEN”in ourlaboratory. The salivary levels of interleukin-1????and soluble-form CD44 were measured by enzymeimmunoassay 15 min after the subjects had inhaled “XEN” via a nasal cannula. The results revealed asignificant increase in salivary interleukin-1????levels (P < 0.01), but no significant change (P = 0.093) insalivary soluble-form CD44 levels at 15 min after“XEN”inhalation, compared to the values measuredbefore inhalation. These findings indicate that “XEN”rapidly induces salivary interleukin-1????secretion andthat elevated salivary interleukin-1????levels after“XEN”inhalation may reflect the activation of bothnatural and acquired immune responses in the living body

Modulation of CD93 molecule in a human monocyte-like cell line (U937) treated with nickel

ニッケルで処理されたヒト単球系細胞株(U937)におけるCD93 分子の動態を解析した。ニッケル処理はU937 細胞に対してインターロイキン-8(IL-8) の産生を増強しながらアポトーシスを誘導した。また、ニッケル処理によりU937 細胞表面上のCD93 分子(mCD93)の発現は有意に減少し、可溶性CD93 分子(sCD93)の産生は有意に増強した。以上の結果は、CD93 分子の動態が金属(ニッケル)アレルギーの発症メカニズムを解析するための新たなバイオマーカーに成り得ることを示唆している。In this study, we demonstrated the modulation of CD93 molecules (both membrane-bound and soluble-form) in a human monocyte-like cell line, U937 treated with nickel (Ni2+) to model contact hypersensitivity (CHS), such as metal allergy. Ni2+ induced the apoptosis of the U937 cells accompanied by the enhanced secretion of an inflammatory cytokine, interleukin-8 (IL-8). The percentages and mean fluorescence intensities (MFIs) of membrane-bound CD93 (mCD93) expressed on U937 cells were significantly decreased after treatment with Ni2+ when examined using flow cytometry with a CD93 monoclonal antibody (mAb) (mNI-11), which was established in our laboratories. In contrast, the secretion of soluble-form CD93 (sCD93) from U937 cells increased markedly after treatment with Ni2+. Taken together, these findings suggest that the modulation of CD93 molecules (both mCD93 and sCD93) might serve as a new biomarker for analyzing the inflammatory reaction in CHS induced by Ni2+

Identification of CD93 expression on hematopoietic stem cells in human neonatal umbilical cord blood cells

臍帯血由来造血幹細胞表面上のCD93の発現を我々が開発したCD93モノクローナル抗体(mNI-11)、既存のCD34抗体、CD45抗体を組み合わせたフローサイトメトリー法で解析した。その結果、臍帯血由来造血幹細胞(CD34+細胞およびCD34+CD45dim+細胞)表面上にはCD93が発現していることが分かった。以上の結果は、CD93が未熟な造血幹細胞の新たな細胞表面マーカーに成り得ると共に、造血幹細胞におけるCD93の免疫学的な機能解析に非常に有益な情報と考えられる。Human CD93 is a heavily O-glycosylated type I transmembrane protein consisting of unique C-type lectin domains (CTLDs) containing glycoprotein. CD93 is mainly expressed on myeloid cells (monocytes and granulocytes) and endothelial cells. However, the expression patterns of CD93 on various other kinds of cells are not well understood. In this study, we found that CD93 was recognized by a CD93 monoclonal antibody (mAb) (mNI-11) that was established in our laboratories and was expressed on a broad hematopoietic stem cell population (CD34+ cells) from human neonatal umbilical cord blood cells (UCBCs), as shown using a two-color flow cytometric analysis. In addition, the CD93 recognized by mNI-11 was also expressed on a narrow hematopoietic stem cell population(CD34+CD45dim+ cells) in which the non-specific reactivity of CD34 mAb from human neonatal UCBCs was excluded using a three-color flow cytometric analysis. Taken together, these results provide the first evidence concerning the identification of CD93 expression on hematopoietic stem cells. These cell populations (CD34+CD93+ and CD34+CD45dim+CD93+ cells) in human neonatal UCBCs are thought to have an important role in cell biology, transplantation, and immature/mature immune responses

Secretion of soluble-form CD93 from human endothelial progenitor cells

ヒト血管内皮前駆細胞(Human endothelial progenitor cells :HEPCs)から分泌される可溶性CD93(sCD93)分子の動態を自主開発した抗ヒトCD93 抗体(mNI-11)を用いて解析した。HEPCs は低濃度であるが、sCD93 分子を培養液中に自然分泌していることがわかった。HEPCs をPKC 活性剤であるPMAで処理したところ、sCD93 分子の分泌は有意に増強した(P < 0.01)。また、PMA 処理の初期段階からsCD93 分泌の増強は認められた。次に、PMA 処理HEPCs にPKC 活性阻害剤であるGo6976 を添加したところ、sCD93 分子の分泌は有意に抑制された(P < 0.01)。一方、HEPCs をアクチンフィラメントの脱重合剤(サイトカラシンE)で処理したところ、sCD93 分子の分泌が有意に増強した(P < 0.01)。HEPCs から分泌されるsCD93 分子は、PKC によるリン酸化とアクチンフィラメントの脱重合が密接に関与していることがわかった。In this study, we examined the secretion of soluble-form CD93 (sCD93) from human endothelial progenitor cells (HEPCs) expressing cell surface CD93 by enzyme-linked immunoassay (EIA) using CD93 monoclonal antibody (mAb) (mNI-11) established in our laboratories. We found that a small amount of sCD93 was spontaneously secreted into the culture supernatants of HEPCs. Furthermore, significantly (P < 0.01) enhanced secretion of sCD93 was found in the culture supernatants of HEPCs treated with phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, which was significantly (P < 0.01) suppressed by treatment of the cells with the protein kinase (PKC) inhibitor Go6976. Interestingly, depolymerization of the actin filaments in the cells by cytochalasin E (CyE) also significantly (P < 0.01) enhanced the sCD93 secretion into the culture supernatants of HEPCs. Taken together, these findings suggest that the sCD93 might serve as a new biomarker for analyzing the biological and immunological functions of HEPCs

Serum levels of soluble CD93 in patients with chronic renal failure

慢性腎不全（CRF）患者における可溶性CD93（sCD93）の血清レベルを自主開発した抗ヒトCD93抗体（mNI-11）とオリジナル構築酵素抗体法（EIAキット）を用いて検討した。その結果、CRF患者血清中のsCD93は健康人と比較して有意に高値を示した。また、血清中インターロイキン-6（IL-6）もCRF患者は健康人に比較して有意に高値を示した。CRF患者血清中のsCD93と腎機能マーカーである尿素窒素、血清クレアチニン、血清シスタチンCとの相関を解析したところ、いずれも強い相関を示した。特に血清シスタチンCとはより強い相関を示した（r＝0.903、P＜0.001）。以上の結果から、CRF患者血清中のsCD93は、特に血清シスタチンCとの相関性がより強いことから、早期の腎障害を検出する新規のバイオマーカーになることがわかった。In this clinical study, we examined the serum levels of soluble CD93 (sCD93) in patients with chronic renal failure (CRF) by an originally constructed enzyme-linked immunoassay (EIA) using a CD93 monoclonal antibody (mAb) (mNI-11) established in our laboratories. The serum concentrations of sCD93 in patients with CRF were significantly higher (P < 0.001) than those in normal healthy controls. Furthermore, the serum levels of interleukin-6 (IL-6) were also significantly higher in the patients with CRF (P < 0.05) than in the normal healthy controls. In addition, the serum levels of sCD93 were strongly correlated with the blood urea nitrogen (BUN) (r = 0.797, P < 0.001), serum creatinine (r = 0.784, P < 0.001) and serum cystatin C (r = 0.913, P < 0.001). Taken together, these findings suggest that the serum sCD93 may serve as a new biomarker for analyzing or measuring renal function

Actions of Taimatsu fermented rice germ solution on human immune responses

本研究では、我々が独自に開発したGABA およびIP6 含有たいまつ米胚芽発酵液(T-FRGS)のヒト末梢血単核球(PBMCs) に対する形態変化およびサイトカイン産生能を解析した。その結果、T-FRGS はPBMCs に形態変化(細胞間凝集)を誘導した。さらに、Enzyme immunoassay (EIA) を用いた解析から、T-FRGS はPBMCs からIL-1β、IL-6、IL-8、TNF-α、GM-CSF、G-CSF の産生を有意に増強させた(P<0.001)。以上の結果から、T-FRGS にはヒト免疫応答を増強させる作用があることがわかった。In this study, we examined the immunological actions of Taimatsu fermented rice germ solution (designated as T-FRGS), containing gamma-aminobutyric acid (GABA) and inositol hexaphosphate (IP6). The morphology of peripheral blood mononuclear cells (PBMCs) from normal healthy donors cultured with T-FRGS was dramatically altered (inducement of cell aggregation) as compared with that of the control cells (cultured without T-FRGS) at 24 hrs. In addition, the PBMCs from normal healthy donors cultured with T-FRGS showed strongly enhanced ability to produce some cytokines, such as interleukin (IL)-1 β ,IL-6, IL-8, tumor necrosis factor-α (TNF-α), granulocyte-macrophage colony stimulating factor (GM-CSF) and granulocyte colony stimulating factor (G-CSF) but not others, such as IL-2 or IL-12. Taken together, these findings clearly indicate that T-FRGS has stimulatory effects on some human immune responses

Enhancement of CD93 expression and interleukin-8 (IL-8) production in the human monocyte-like cell line U937 in response to Taimatsu fermented rice germ gamma-amino butyric acid (GABA)

たいまつ米胚芽発酵GABAによる単球系細胞株(U937)表面上のCD93抗原の発現をモノクローナル抗体を用いた酵素抗体法およびフローサイトメトリー法で解析した。その結果、たいまつ米胚芽発酵GABAで培養したU937細胞はCD93(C1qRp)の発現が有意に増強した。また、CD93の発現増強はprotein kinase C(PKC)阻害剤であるGo6976で抑制された。さらに、たいまつ米胚芽発酵GABAで培養したU937細胞はインターロイキン-8(IL-8)の産生も有意に増強した。以上の結果から、たいまつ米胚芽発酵GABAにはヒトの免疫応答を増強する作用があることがわかった。We examined the modulation of CD93 (receptor for complement component 1, subcomponent qphagocytosis; C1qRp) expression in the human monocyte-like cell line U937 cultured with Taimatsufermented rice germ gamma-amino butyric acid (GABA) using CD93 monoclonal antibodies (mAbs), aglutaraldehyde-fixed cellular enzyme immunoassay (GCEIA), and flow cytometry. The expression ofCD93 in the U937 cells cultured with Taimatsu fermented rice germ GABA was significantly enhanced (P<0.01), compared with that in the control cells (cultured without Taimatsu fermented rice germ GABA) at 24 hrs. This enhancement of CD93 expression was dose-dependent and timedependent. The enhancement of CD93 expression was markedly blocked in the presence of the protein kinase C (PKC) inhibitor Go6976. In addition, the U937 cells cultured with Taimatsu fermented rice germ GABA significantly enhanced (P<0.01) the production of interleukin-8 (IL-8) in the culture supernatants. Together, these findings strongly indicated that Taimatsu fermented rice germ GABA has immunopotentiator effects on some human immune responses

Inhibition of tumor-associated antigens secreted from cancer cell lines by Taimatsu fermented rice germ solution containing inositol hexaphosphate (IP6)

本研究では、我々が開発したIP6含有たいまつ米胚芽発酵液(T-IP6)の癌細胞株(MKN-45およびHepG2)から分泌される腫瘍関連抗原(CEAまたはPIVKA-II)に対する影響を化学発光および電気化学発光酵素免疫測定法で解析した。その結果、T-IP6で培養したMKN-45およびHepG2細胞から分泌されるCEAまたはPIVKA-IIは、細胞の形態変化を伴いながら有意に抑制された(P<0.01)。また、T-IP6によるCEAまたはPIVKA-IIの抑制作用は濃度依存性であった(P<0.05)。以上の結果から、T-IP6にはヒトの癌細胞を正常化させる作用があることがわかった。Inositol hexaphosphate (IP6), a naturally polyphosphorylated carbohydrate, has been reported to have significant anticancer activity against numerous tumors, such as colon, prostate, breast, liver and rhabdomyosarcomas. Recently, we established a new IP6-containing solution, designated as T-IP6, produced by Taimatsu fermented rice germ. In this study, we examined the inhibitory effects of tumor-associated antigens secreted from a gastric cancer cell line (MKN-45) and a hepatocellular carcinoma cell line (HepG2) exposed to T-IP6. The tumor-associated antigens, such ascarcinoembryonic antigen (CEA) and protein induced by vitamin K absence or antagonists-II (PIVKAII), secreted from these cancer cell lines cultured with T-IP6 were significantly inhibited (P<0.01), compared with those in control cells (cultured without T-IP6) at 72 hrs, and this inhibition was accompanied by morphological changes, respectively. In addition, T-IP6 inhibited CEA and PIVKA-II in a dose-dependent manner. These findings suggest that T-IP6 has an immunoinhibitory and normalization effect in human cancer cells

Inhalation of a steam-mixed gas containing hydrogen gas enhances the salivary titers of immunoglobulin A against pathogenic hemagglutinin antigens derived from influenza virus strains in healthy volunteers

　水素発生装置（スイソニア）から発生する水素ガス（濃度0.1%～ 0.3%）を含む蒸気混合ガスXENを鼻カニューラで吸入した。吸入後、A型およびB型インフルエンザウイルス由来のhemagglutinin（HA）抗原に対する唾液中IgA2抗体価を自主開発の酵素免疫測定法で解析した。その結果、XEN吸入15分後、A型インフルエンザウイルス株（A/California/7/2009/H1N1とA/Hong Kong/4801/2014/H3N2）由来のHA抗原に対する唾液中IgA2抗体価は、吸入前と比較して有意に増加した（P=0.028およびP=0.047）。さらに、XEN吸入15分後、A型とB型インフルエンザウイルス株（A/Singapore/GP1908/2015/H1N1、A/HongKong/4801/2014/H3N2、B/Texas/2/2013/Victoria system、B/Phuket/1307/2013 /Yamagata system）由来のHA抗原（ワクチンタイプ）に対する唾液中IgA2抗体価も吸入前と比較して有意に増加した（P=0.039）。以上の結果から、XENにはHA抗原に対する唾液中IgA2抗体価の増強作用があることがわかった。In a previous study, we demonstrated a significant increase in the salivary interleukin-1β (IL-1β) levelsfollowing the administration, via inhalation, of a steam-mixed gas containing hydrogen gas (designated asXEN) in healthy volunteers. In this study, we examined the salivary titers of immunoglobulin A2 (IgA2)directed against pathogenic hemagglutinin (HA) antigens derived from influenza virus strains(hereinafter, shortened to IgA2) after XEN administration by inhalation via a nasal cannula for 15 min inhealthy volunteers. The salivary titers of IgA2 directed against HA antigens were measured using anoriginally constructed enzyme immunoassay system. The results indicated significant increases of thesalivary IgA2 directed against the HA antigens derived from the following influenza virus strains after15 min of XEN inhalation, as compared to the titers recorded prior to the inhalation: A/California/7/2009/H1N1 (P = 0.028), A/Hong Kong/4801/2014/H3N2 (P = 0.047), vaccine-type containingHA antigens derived from influenza virus strains, A/Singapore/GP1908/2015/H1N1, A/HongKong/4801/2014/H3N2, B/Texas/2/2013/Victoria system, and B/Phuket/1307/2013 /Yamagata system(P = 0.039). Taken together, XEN rapidly enhanced the salivary titers of IgA2 directed against HAantigens derived from influenza viruses, which may imply a role in the protection against influenza virusinfection by activation of humoral immunity (IgA2 action) in the whole body via the nasal mucosalimmune system