Resveratrol inhibits early T cell activation and co-stimulatory molecule expression in APCs <i>in vitro</i>.

<p>DO11.10 mice-derived splenocytes were un-stimulated (no treat) or stimulated with 300 µg/ml OVA plus 12 pM CT and were cultured in the presence or absence of 10 or 30 µM resveratrol for 1 day. Then the analysis described below was performed. A. After 1 day-culture, whole splenocytes were stained with PE-conjugated anti-KJ1–26 and APC-conjugated anti-CD25 antibody. Representative FACS plots are shown. Numbers indicate the proportion of the KJ1–26⁺ CD25⁺ cells. B. Quantitative analysis of A (n = 3 per group). C. After 1 day-culture, the supernatants were collected and IL-2 concentrations were measured by ELISA (n = 9 per group). D. After 1 day-culture, RNA samples were extracted from the splenocytes and the cDNA samples were synthesized using reverse transcriptase system. Quantitative real-time PCR analysis was then performed to assess CD80 and CD86 mRNA levels. Relative expression levels are shown (n = 3 per group). Values represent the mean ± SD. *P<0.05 in comparison to the corresponding controls. (ND: non-detected).</p

Resveratrol inhibits OVA plus CT-induced Th1/2 cell differentiation <i>in vitro</i>.

<p>DO11.10 mice-derived splenocytes were un-stimulated or stimulated with 300 µg/ml OVA plus 12 pM CT and were cultured in the presence or absence of 10 or 30 µM resveratrol for the indicated times. Then the analysis described below was performed. A. After 3 day-culture, RNA samples were extracted from the cells and the cDNA samples were synthesized using reverse transcriptase system. Quantitative real-time PCR analysis was then done for T-bet, GATA3, and Foxp3 mRNAs. Relative expression levels are shown (n = 3 per group). B. After 3 day-culture, the supernatants were collected and IFN-ã, IL-4, and IL-13 concentrations were measured by ELISA (n = 9 per group). C. After 3 day-culture, the cells were subjected to a WST assay for evaluation of the cell viability (n = 4 per group). Values represent the mean ± SD. *P<0.05 in comparison to the corresponding controls. (ND: non-detected).</p

Dietary resveratrol protects mice against a lethal dose of CT.

<p>Survival of 8-week-old C57BL/6 mice orally challenged with CT at a dose of 150 µg monitored until day 10 after challenge (n = 5 per group). Representative results from 2 independent experiments with similar results are shown. (S: standard diet-fed mice, S/R: standard diet plus resveratrol-fed mice).</p

Resveratrol inhibits CT-driven mucosal sensitization to OVA in mice.

<p>Mice were fed the standard diet or standard diet plus resveratrol (22.4 mg/kg diet, 0.01% resveratrol) for 5 weeks (day 0–35). The mice were orally administered 50 mg of OVA with 10 µg of CT 1 week after the start of resveratrol feeding and 4 times a week for 4 weeks and sacrificed at the 5 weeks (on day 35) for the analysis below. A. Experimental protocol. B. Serum samples were collected on day 35 from the standard diet-fed and standard diet plus resveratrol-fed mice with or without OVA plus CT sensitization. OVA-specific serum IgE concentrations were measured by ELISA (n = 7 per group). C. The standard diet-fed and standard diet plus resveratrol-fed mice sensitized with OVA plus CT were challenged intraperitoneally with OVA on day 35 and rectal temperatures were measured with a digital thermometer at 0, 5, 30, and 60 minutes after the challenge (n = 10 per group). D. Splenocytes (SPL) and mesenteric lymph node (MLN) cells were obtained on day 35 from the standard diet-fed and standard diet plus resveratrol-fed mice with or without OVA plus CT sensitization and the cells were re-stimulated <i>in vitro</i> with OVA for 3 days (n = 5 per group). The culture supernatants were then collected and IL-13 and IFN-ã concentrations were measured by ELISA. (S: standard diet-fed mice, S/R: standard diet plus resveratrol-fed mice) Values represent the mean ± SD. *P<0.05 in comparison to the corresponding controls. Representative results from 2 independent experiments with same results are shown.</p

Resveratrol inhibits CT-induced cAMP elevation in BMDCs.

<p>A. Cultured BMDCs were un-stimulated or stimulated with 12 pM CT in the presence or absence of 10 or 30 µM resveratrol for 1 hour or 10 nM PGE2 (as a positive control) and intracellular cAMP levels were measured using a cAMP EIA kit. Relative changes in intracellular cAMP levels are indicated. Values represent the mean ± SD (n = 3 per group). B. Cultured BMDCs were un-stimulated (no treat) or stimulated with 300 µg/ml OVA plus 12 pM CT and were cultured in the presence or absence of 10 or 30 µM resveratrol for 3 days. The cells were then stained with APC-conjugated anti-CD11c and FITC-conjugated anti-CD80 or CD86 antibody and were subjected to FACS analysis. A representative histogram regarding surface CD80 and CD86 expression levels on CD11c gated cells is shown. Numbers indicate the mean fluorescence intensity (MFI) of CD80 and CD86-stained cells. The filled histogram indicates un-stimulated (no treat) BMDCs for comparison. Representative results from 3 independent experiments with similar results are shown.</p

Mechanism of mAb 4713-induced cell death involving the cytoskeleton.

<p>(A) Impact of chemical inhibitors on the cytotoxic activity of mAb 4713 against L428 and Raji cells. The inhibitors were added 1 or 2 h before the cytotoxicity assay. The percentage of dead cells was determined by trypan blue dye exclusion. **p = 0.01 versus none. ***p = 0.001 versus none. The agents were caspase inhibitors (z-VAD-FMK and z-Asp-DCB), PI-3 kinase inhibitors (wortmannin and LY294002), cytoskeletal inhibitors (cytochalasin D and latrunculin B), necroptosis inhibitor (Necrostatin-1), necrosis inhibitor (IM-54), a cathepsin inhibitor (Cath inib III), and reactive oxygen species scavenger (Tiron). (B) Limiting dilution experiments in which L428 cells were seeded (0.3 cells/well; 96 wells) with 3 μg/ml mAb 4713 or control IgG. After 2 h, the number of live cells per well was counted. **p = 0.01 versus isotype control. ***p = 0.001 versus control IgG. Each value represents the mean ± SD.</p

Reactivity and cytotoxic activity of mAb 4713 against lymphoma cell lines.

<p>(A) Flow cytometry analysis of mAb 4713 reactivity analyzed by incubating different lymphoma cell lines with mAb 4713 (4°C; 2 h), followed by Alexa 488-conjugated rat anti-mouse Igs (green histogram). Cytotoxicity was analyzed based on propidium iodide (PI) staining of dead cells (red histogram). (B) Cytotoxic effects of mAb 4713 on IL-2-dependent or IL-2-independent ATL cell lines analyzed by PI staining after incubation with mAb 4713 (37°C; 1 or 2 h). Similar data were obtained in two independent experiments. (C) Kaplan–Meier survival analysis of mAb 4713-treated C.B-17/ICR-SCID mice bearing Raji lymphoma xenografts. The Raji-injected SCID mice (n = 5) were treated with mAb 4713 once (red line) or twice (brown line), or with control IgG once (green line). **p = 0.01 versus isotype control.</p

Cytotoxic effects of mAb4713 on lymphoma/leukemia cell lines.

<p>Cytotoxic effects of mAb4713 on lymphoma/leukemia cell lines.</p

Microscopy analysis of mAb 4713-induced single cell death shows giant pores.

<p>(A) Light microscopy image showing that the incubation of L428 cells with mAb 4713 caused rapid aggregation and death of single cells after 30 min, as shown by trypan blue exclusion staining. ←, single cell death; ∆, aggregation of cells. Bar: 50 μm (B) Scanning electron microscopy image showing the formation of giant pores on L428 Hodgkin lymphoma cells after a 15 or 30 min incubation with mAb 4713.</p