Synergistic induction of antigen-specific CTL by fusions of TLR-stimulated dendritic cells and heat-stressed tumor cells

Dendritic cell (DC)/tumor cell fusion cells (FCs) can induce potent CTL responses. The therapeutic efficacy of a vaccine requires the improved immunogenicity of both DCs and tumor cells. The DCs stimulated with the TLR agonist penicillin-killed Streptococcus pyogenes (OK-432; OK-DCs) showed higher expression levels of MHC class I and II, CD80, CD86, CD83, IL-12, and heat shock proteins (HSPs) than did immature DCs. Moreover, heat-treated autologous tumor cells displayed a characteristic phenotype with increased expression of HSPs, carcinoembryonic Ag (CEA), MUC1, and MHC class I (HLA-A2 and/or A24). In this study, we have created four types of FC preparation by alternating fusion cell partners: 1) immature DCs fused with unheated tumor cells; 2) immature DCs fused with heat-treated tumor cells; 3) OK-DCs fused with unheated tumor cells; and 4) OK-DCs fused with heat-treated tumor cells. Although OK-DCs fused with unheated tumor cells efficiently enhanced CTL induction, OK-DCs fused with heat-treated tumor cells were most active, as demonstrated by: 1) up-regulation of multiple HSPs, MHC class I and II, CEA, CD80, CD86, CD83, and IL-12; 2) activation of CD4+ and CD8+ T cells able to produce IFN- gamma at higher levels; 3) efficient induction of CTL activity specific for CEA or MUC1 or both against autologous tumor; and 4) superior abilities to induce CD107+ IFN-gamma+ CD8+ T cells and CD154+ IFN-gamma+ CD4+ T cells. These results strongly suggest that synergism between OK-DCs and heat-treated tumor cells enhances the immunogenicity of FCs and provides a promising means of inducing therapeutic antitumor immunity.     

Effect of Alteration of Sterol Composition on the Salt Sensitivity of a Tobacco Cell Suspension Culture

A Nicotiana tabacum cell line, KS-1, which is tolerant to morethan 1% NaCl, was treated with buthiobate. The cells accumulated14a-methylsterols such as obtusifoliol, instead of campesteroland sitosterol. The buthiobate-treated cells lost their salttolerance. These results suggest that the buthiobate-inducedsalt sensitivity is closely associated with changes in the molecularspecies of sterols in the cell membranes. (Received October 16, 1986; Accepted April 13, 1987)     

Accumulation of Rare Phytosterols in Plant Cells on Treatment with Metabolic Inhibitors and Mevalonic Acid

Root application of the metabolic inhibitors D-ethionine (100µM) and L-ethionine (30 µM), which were expectedto inhibit alkylation of the sterol side chains, merely reducedthe sterol content in the roots of Medicago sativa seedlings.The major sterol was stigmasterol. However, when (3RS)-mevalonicacid (2 mM) was applied together with ethionine, cydoartenol(about 50% of the total sterols) accumulated in the roots. Thehypocotyls of the ethionine-treated seedlings accumulated cholesterol(34% of the total sterols), and mevalonic acid showed no additionaleffect in this case. In a suspension culture of Nicotiana tabacum, the most abundantsterol was campesterol. When cells were treated with buthiobate(100 µM), a potent inhibitor of lanosterol 14-demethylationin yeasts and fungi, obtusifoliol accumulated in the treatedcells (50% of the total sterols). 14-Methylfecosterol also accumulatedin the treated cells. The addition of mevalonic acid (1 mM togetherwith buthiobate increased the obtusifoliol content (63% of thetotal sterols). (Received October 16, 1986; Accepted April 2, 1987)     

Variation in the Content and Composition of Sterols in Alfalfa Seedlings Treated with Compactin (ML-236B) and Mevalonic Acid

Compactin (ML-236B), a specific inhibitor of 3-hydroxy-3-methylglutarylCoA reductase, inhibited the elongation of roots and hypocotylsof Medicago sativa seedlings when it was applied to the roots.Addition of mevalonic acid, the direct product of the enzyme,together with compactin relieved the growth inhibition of roots. The contents and compositions of sterols were studied in threeparts of M. sativa seedlings—roots, hypocotyls and cotyledons.Compactin (20 µM) decreased the sterol contents of rootsand hypocotyls by about a half but did not affect that of cotyledons.On the other hand, mevalonic acid (2 mM) increased the sterolcontent of roots more than threefold the nontreated controllevel but not the contents of hypocotyls and cotyledons. Mevalonicacid added in combination with compactin had a similar effecton the sterol content of roots as when it was added alone. The major sterol in all three parts was stigmasterol whetheror not compactin or mevalonic acid was present. However, thevariation of the sterol composition in the roots was distinct;mevalonic acid-treated roots markedly accumulated ⁷-sitosterol,24-methylenecycloartanol and squalene. (Received October 16, 1986; Accepted April 2, 1987)     

Biochemistry of Filamentous Fungi. XIII. Metabolites of Helicobasidium mompa Tanaka

Five crystalline compounds, two colored and three colorless, have been isolated from the mycelium of Helicobasidium mompa Tanaka, a root-infecting plant pathogen. One of the colorless crystals was identified as D-arabitol. Chemical studies of the other substances are going on.     

Streptococcal preparation OK-432 promotes fusion efficiency and enhances induction of antigen-specific CTL by fusions of dendritic cells and colorectal cancer cells

Dendritic/tumor fusion cell (FC) vaccine is an effective approach for various types of cancer but has not yet been standardized. Antitumor activity can be modulated by different mechanisms such as dendritic cell (DC) maturation state. This study addressed optimal strategies for FC preparations to enhance Ag-specific CTL activity. We have created three types of FC preparations by alternating fusion cell partners: 1) immature DCs fused with autologous colorectal carcinoma cells (Imm-FCs); 2) Imm-FCs followed by stimulation with penicillin-inactivated Streptococcus pyogenes (OK-432) (Imm-FCs/OK); and 3) OK-432-stimulated DCs directly fused to autologous colorectal carcinoma cells (OK-FCs). Both OK-FCs and Imm-FCs/OK coexpressed the CEA, MUC1, and significantly higher levels of CD86, CD83, and IL-12 than those obtained with Imm-FCs. Short-term culture of fusion cell preparations promoted the fusion efficiency. Interestingly, OK-FCs were more efficient in stimulating CD4(+) and CD8(+) T cells capable of high levels of IFN-gamma production and cytolysis of autologous tumor or semiallogeneic targets. Moreover, OK-FCs are more effective inducer of CTL activation compared with Imm-FCs/OK on a per fusion cell basis. The pentameric assay confirmed that CEA- and MUC1-specific CTL was induced simultaneously by OK-FCs at high frequency. Furthermore, the cryopreserved OK-FCs retained stimulatory capacity for inducing antitumor immunity. These results suggest that OK-432 promotes fusion efficiency and induction of Ag-specific CTL by fusion cells. We conclude that DCs fused after stimulation by OK-432 may have the potential applicability to the field of antitumor immunotherapy and may provide a platform for adoptive immunotherapy in the clinical setting.     

Observation of water and Na+ in tissues of theBruguiera gymnorrhiza by1H-and23Na-NMR imaging

Distributions of free water, which is called water in this investigation, in mangrove (Bruguiera gymnorrhiza (L.) Lam.) tissues were examined by using¹H-NMR imaging, and accumulation of Na⁺ in hypocotyls was examined by using high resolution²³Na-NMR and²³Na-NMR imaging in relation to their morphology. Water located preferentially in the epidermis and the outer layer of cortex adjacent to the epidermis, and around vascular bundles of a root, a branch stem, and hypocotyls. Amount of water detected in the middle parts of cortex and pith was small unlikeAucuba japonica branch tissue. On the other hand, relatively high concentration of Na⁺ was detected in the pith besides the epidermis and the outer layer of the cortex adjacent to the epidermis, and around vascular bundles of the hypocotyl. The localization of Na⁺ did not correspond to that of water. Concentrations of Na⁺ accumulated (up to 22mM) in the hypocotyl were approximately 10 times higher than those observed in tissues of ordinary plants. The characteristic water distribution and accumulation of Na⁺ in the mangrove are considered to relate to their ecological nature for the adaptation to saline environments.