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

The Runx1 Transcription Factor Inhibits the Differentiation of Naive CD4+ T Cells into the Th2 Lineage by Repressing GATA3 Expression

Differentiation of naive CD4+ T cells into helper T (Th) cells is controlled by a combination of several transcriptional factors. In this study, we examined the functional role of the Runx1 transcription factor in Th cell differentiation. Naive T cells from transgenic mice expressing a dominant interfering form of Runx1 exhibited enhanced interleukin 4 production and efficient Th2 differentiation. In contrast, transduction of Runx1 into wild-type T cells caused a complete attenuation of Th2 differentiation and was accompanied by the cessation of GATA3 expression. Furthermore, endogenous expression of Runx1 in naive T cells declined after T cell receptor stimulation, at the same time that expression of GATA3 increased. We conclude that Runx1 plays a novel role as a negative regulator of GATA3 expression, thereby inhibiting the Th2 cell differentiation

Fucose-Containing Sulfated Polysaccharides from Brown Seaweeds Inhibit Proliferation of Melanoma Cells and Induce Apoptosis by Activation of Caspase-3 in Vitro

Fucose-containing sulfated polysaccharides (FCSPs) extracted from seaweeds, especially brown macro-algae, are known to possess essential bioactive properties, notably growth inhibitory effects on tumor cells. In this work, we conducted a series of in vitro studies to examine the influence of FCSPs products from Sargassum henslowianum C. Agardh (FSAR) and Fucus vesiculosus (FVES), respectively, on proliferation of melanoma B16 cells and to investigate the underlying apoptosis promoting mechanisms. Cell viability analysis showed that both FCSPs products, i.e., FSAR and FVES, decreased the proliferation of the melanoma cells in a dose-response fashion, with FSAR being more potent at lower dosages, and FVES being relatively more anti-proliferative than FSAR at higher dosages. Flow cytometric analysis by Annexin V staining of the melanoma cells exposed to the FCSPs products confirmed that both FSAR and FVES induced apoptosis. The FCSPs-induced apoptosis was evidenced by loss of plasma membrane asymmetry and translocation of the cell membrane phospholipids and was accompanied by the activation of caspase-3. The FCSPs bioactivity is proposed to be attributable to distinct structural features of the FCSPs, particularly the presence of sulfated galactofucans (notably in S. henslowianum) and sulfated fucans (notably in F. vesiculosus). This study thus indicates that unfractionated FCSPs may exert bioactive effects on skin cancer cells via induction of apoptosis through cascades of reactions that involve activation of caspase-3

IN THE ABSENCE OF EPSTEIN-BARR VIRUS INFECTION, PHORBOL ESTER MODULATES APOPTOSIS IN CYCLOHEXIMIDE-TREATED BURKITT'S LYMPHOMA (BJA-B) CELLS

We have shown previously that cycloheximide (CHX), a potent protein synthesis inhibitor, induces high levels of apoptosis in Epstein-Barr virus free (EBV(−)) Burkitt’s lymphoma (BJA-B) cells, with comparably reduced levels of apoptosis in the EBV positive (EBV(+)) cells. Modulation of CHX-induced apoptosis in EBV(−) and (+) B cells is reported here using concurrent treatment with phorbol ester (phorbol 12,13-dibutyrate, PdBu). Cells were collected at 0, 3, 6, 12, 24 and 48 hours after treatment with (i) 1 μg/ml CHX, (ii) 0.1 μg/ml PdBu (1 hour pretreatment before 0 h), or (iii) CHX + PdBu (CHX added at 0 h, 1 hour after PdBu). Control cultures were untreated. Apoptotic, necrotic or viable cells were quantified using histological, ultrastructural and biochemical parameters. Protein synthesis was assessed using 35S-methionine incorporation. Intracellular calcium concentrations were measured using flow cytometry. PdBu alone had little effect on cell death. High levels of CHX-induced apoptosis in EBV(−) cells were significantly reduced by concurrent addition of PdBu (P < 0.005). In contrast, low levels of CHX-induced apoptosis in EBV(+) cells were not significantly altered by PdBu treatment. In EBV(−) cells, a negative relationship was observed between levels of apoptosis and calcium concentrations, whereas in EBV(+) cells, there was negligible correlation between these parameters. Thus high levels of CHX-induced apoptosis in EBV(−) cells occur via a PKC-dependent pathway, whereas CHX treatment of EBV(+) cells induces comparatively low levels of apoptosis that occur via a PKC-independent mechanism. The results application in the therapeutic intervention for cancers developing in association with EBV infection

The monitoring possibility of some mammalian cells for zinc concentrations on metallic materials

Zinc plating is widely used to protect steels against corrosion. However, the possibility of a high environmental risk for zinc has been recently discussed among advanced countries and more environmentally-friendly substitutes are required urgently. Therefore, monitoring zinc concentration changes on metallic materials such as steel is very important. We chose to measure zinc concentration changes in some mammalian cells and confirmed that V79 cells were highly sensitive to changes in zinc concentrations. In this study, the following process was applied to the proprietary production for tin-zinc alloy films on steel using V79 cells. Specimens were immersed in PBS to produce extracts. Zinc concentrations in the extracts almost corresponded to zinc concentrations on steel surfaces. When extracts were added to a V79 cell culture, colony formation was inhibited, and inhibition increased with increases in zinc concentrations. Changes in zinc concentrations on steel surfaces with heat treatment could be monitored relatively well by V79 cells, even though the results were still semi-quantitative

Modulation of some cell surface antigens in the monocyte-like cell line U937 in response to β-1,3-1,6 glucan derived from Aureobasidium pullulans

黒酵母(Aureobasidium plulluns)由来のβ-1,3-1,6 glucanによる単球系細胞株(U937)表面上のCD抗原の動態を各種モノクローナル抗体とフローサイトメトリー法を用いて解析した。その結果、β-1,3-1,6 glucanで培養したU937細胞はCD11b (C3R)、CD54(ICAM-1)、CD93(C1qRp)の発現が有意に増強した(P<0.01)。さらに、β-1,3-1,6 glucanで培養したU937細胞はインターロイキン-8(IL-8)およびアポトーシス関連分子である可溶性Fas分子(sFas)の産生も有意に増強した(P<0.01)。以上の結果から、Aureobasidium plulluns由来のβ-1,3-1,6 glucan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 cell surface antigens in a human monocyte-like cell line, U937, cultured with β-1,3-1,6 glucan, produced by the black yeast Aureobasidium pullulans (A. pullulans), using various monoclonal antibodies (mAbs) to human cluster of differentiation (CD) antigens and flow cytometry. The expressions of CD11b (complement component receptor type-3; CR3), CD54 (intercellular adhesion molecule-1; ICAM-1) and CD93 (receptor for complement component 1, subcomponent q phagocytosis; C1qRp) in the U937 cells cultured with β-1,3-1,6 glucan were significantly (P<0.01) enhanced, compared with those in the control cells (cultured without β-1,3-1,6 glucan), at 48 hrs. In addition, the cells cultured with β-1,3-1,6 glucan significantly (P<0.01) enhanced the production of interleukin-8 (IL-8) and an apoptosis-related molecule, soluble Fas (sFas), into the culture supernatants. Together, these findings strongly indicate that the β-1,3-1,6 glucan derived from A. pullulans has some immunopotentiator effects for some human immune responses