Murine peritoneal macrophages were preincubated with amphotericin B (AMPH) and were then stimulated with bacterial lipopolysaccharide or streptococcal preparation (OK432). These macrophages produced a large amount of tumor necrosis factor. When administered to mice, the priming activity of amphotericin B for tumor necrosis factor production in vivo was also observed.Amphotericin B (AMPH) is known to activate macrophage functions in terms of oxidative burst (17) and fungicidal activity (10). It was also reported to induce the production of a marginal level of tumor necrosis factor-a (TNF) in murine macrophages in vitro (2, 3). There is much evidence that TNF exhibits many types of immunological properties (1,4,14). This suggests that some part of the immunological or pharmacological actions of AMPH may be mediated by TNF. Efficient induction of TNF by macrophages is known to require at least two types of stimulation (7). Here, we report that AMPH induces a relatively large amount of TNF in vitro and in vivo when combined with other macrophage stimulators.C3H/HeN mice were obtained from Shizuoka Experimental Animal Corporation, Hamamatsu, Japan. Deoxycholatefree AMPH supplied from Bristol-Myers Squibb K. K., Tokyo, Japan, was used unless indicated otherwise. AMPH was dissolved in dimethyl sulfoxide (DMSO), and final concentrations of DMSO in culture media were less than 0.05%. Endotoxin contamination of all tested AMPH preparations except AMPH as Fungizone (Bristol-Myers Squibb Co.) was estimated to be less than 15 pg/i ,ug of AMPH by a Limulus assay (Endospecy; Seikagaku Industrial Co., Tokyo, Japan). Bacterial lipopolysaccharide (LPS) from Escherichia coli 0127:B8 was purchased from Difco Laboratories. A streptococcal preparation, OK432, was kindly supplied by Chugai Chemical Co., Tokyo, Japan. Antitumor mannoglucan polyalcohol (MGA) was supplied by Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan. Fetal calf serum, in which contaminating endotoxin was present at less than 3 pg/ml, was purchased from Hyclone Co., Logan, Utah.Male C3H/HeN mice (6 weeks old) were injected intraperitoneally with 3 ml of 3% thioglycolate. Four days later, peritoneal cells were collected, washed twice with RPMI 1640 medium containing 5% fetal calf serum, and adjusted to 1.0 x 106 cells per ml in the same medium. They were seeded into flat-bottom microtiter plates (2 x 105 cells per well) and incubated for 2 h in a 5% CO2 incubator at 37°C, and nonadherent cells were removed. The monolayers, which consisted of macrophages (>95%) (9), were incubated in the presence of several concentrations of AMPH for 1 h and were then restimulated by the addition of * Corresponding author. LPS or OK432 for an additional 2 h. The viabilities of the macrophages, which were checked by the trypan blue dye exclusion test, were always greater than 95% before stimulation, and AMPH at a concentration of less than 5 ,ug/ml did not affected viability. The culture supernatants of each well were collected and were used immediately for the TNF assay. The TNF activi...

It has been reported that epigallocatechin-3-O-(3″-O-methyl)-gallate (EGCG3″Me) and the EGCG3″Me-rich green tea ( Camellia sinensis L.) cultivar 'Benifuuki' exhibit antiallergic effects. The objective of this study was to investigate the effect of various tea leaf catechins on histamine release from murine bone marrow mast cells (BMMC). At a dose of 50 μg/mL, the rank order of histamine release inhibition was observed to be epicatechin-3-O-(3″-O-methyl)-gallate (ECG3″Me) > gallocatechin-3-O-(3″-O-methyl)-gallate (GCG3″Me) > EGCG3″Me > gallocatechin-gallate (GCG) > catechin-gallate (CG) > EGCG > epicatechin-gallate (ECG) > epigallocatechin (EGC) > gallocatechin (GC). Of the various tea cultivars analyzed by HPLC, the greatest content of ECG3″Me was found in the third crop of 'Benifuuki' (1.05% dry weight). Moreover, ECG3″Me content was positively correlated with EGCG3″Me content in 'Benifuuki' tea leaves. In an assay of mixtures of ECG3″Me and EGCG3″Me, inhibitory activity (50 μg/mL in total) was increased as the content of ECG3″Me increased. This suggests that ECG3″Me might link to the antiallergic effect of 'Benifuuki' tea, as has been reported for EGCG3″Me.

A staining method with crystal violet (CV) was demonstrated to be useful for a simple, quick and objective assessment of in vitro growth inhibitory activity of leukocytes against Candida albicans cells. Candida cells incubated with murine neutrophils or macrophages for 14 hr in microwells were stained with CV and, after washing with 0.25% sodium dodecyl sulfate (SDS), treated with isopropanol containing HC1 (0.04 N) to extract Candida cell-bound CV. Then the absorbance at 590 nm of the isopropanol extract was photometrically measured. The results showed that the photometrical absorbance was proportional to the amount of 3H-glucose taken up by C. albicans cells, which reflected the number of viable Candida cells.

1. The immunomodulating activity of antifungal drugs was reviewed. Although results are conflicting, all azole drugs tend to be immunosuppressive, except for fluconazole, which has no immunologic effect. In contrast, the polyene antibiotic amphotericin B (AMPH) is immunostimulatory. 2. AMPH induced host resistance to Pseudomonas aeruginosa infection in mice, whereas no azole drugs did so. 3. Polymorphonuclear leukocytes are activated by AMPH, but not by any azole drugs, in terms of the level of their adherence. 4. No azole drugs induce in vitro tumor necrosis factor alpha (TNF) production by macrophages, whereas AMPH slightly but substantially does so. 5. AMPH potently primes macrophages in vitro and in vivo so that they produce large amounts of TNF after the secondary stimulation (triggering) by bacterial lipopolysaccharides or a streptococcal preparation used for antitumor immunotherapy, OK432. 6. Viable or heat-killed Candida albicans cells are capable of inducing in vitro TNF production by macrophages. This activity of the fungal cells is enhanced by AMPH.

These results suggested that 'Sunrouge' might protect humans from humans from AChE-related diseases by suppressing AChE activity. To obtain sufficient amounts of anthocyanins, catechins and/or caffeine for a functional food material, 'Sunrouge' from the third crop season should be used.

Tea polyphenols, e.g., (-)-epigallocatechin-3-O-(3-O-methyl gallate (EGCG3"Me), (-)-epigallocatechin-3-O-gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin-3-O-gallate (ECG), and (-)-epicatechin (EC), are believed to be responsible for the beneficial effects of tea. 'Benifuuki', a tea (Camellia sinensis L.) cultivar grown in Japan, is rich in the anti-allergic molecule epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3"Me). Pulverized Benifuuki green tea powder (BGP) is more widely distributed than leaf tea in Japan. Japanese people mix their pulverized tea with water directly, whereas it is common to drink leaf tea after extraction. However, few studies of the effects of BGP particle size on polyphenol bioavailability have been performed. This study was conducted to investigate the absorption of catechins in rats after the intragastric administration of Benifuuki green tea. Therefore, we assessed the plasma concentrations of catechins following the ingestion of BGP with different mean particle sizes (2.86, 18.6, and 76.1 μm) or Benifuuki green tea infusion (BGI) as a control in rats. The bioavailabilities of EGCG3"Me, EGCG, ECG, EGC, and EC were analyzed after the oral administration of a single dose of Benifuuki green tea (125 mg/rat) to rats. The plasma concentrations of tea catechins were determined by HPLC analysis combined with of electrochemical detection (ECD) using a coulometric array. The AUC (area under the drug concentration versus time curve; min μg/mL) of ester-type catechins (EGCG3"Me, EGCG, and ECG) for the BGP 2.86 μm were significantly higher than those in the infusion and 18.6 and 76.1 μm BGP groups, but the AUC of free-type catechins (EGC and EC) showed no differences between these groups. Regarding the peak plasma level of EGCG3"Me adjusted for intake, BGP 2.86 μm and BGI showed higher values than the BGP 18.6 and 76.1 μm groups, and the peak plasma levels of the other catechins displayed the same tendency. The present study demonstrates that the bioavailability of ester-type catechins (EGCG and ECG) can be improved by reducing the particle size of green tea, but the plasma level of EGCG3"Me in the BGI group was similar to that in the BGP 2.86 μm group. This result suggests that drinking Benifuuki green tea with a particle size of around 2 μm would deliver the anti-allergic EGCG3"Me and the anti-oxidant EGCG efficiently.

We investigated the phagocytosis-enhancing activity of green tea polyphenols, such as epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), epicatechin (EC) catechin (+C) and strictinin, using VD3-differentiated HL60 cells. EGCG, EGC, ECG and strictinin, but not EC and +C, increased the phagocytic activity of macrophage-like cells, and a caspase inhibitor significantly inhibited phagocytic activities. These results suggest that the pyrogallol-type structure in green tea polyphenols may be important for enhancement of the phagocytic activity through caspase signaling pathways.

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