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Antibodies Anti-CD3 (145-2C11), anti-CD28 (37

Antibodies Anti-CD3 (145-2C11), anti-CD28 (37.51), alexa488-conjugated anti-IL-4 (11B11) and PerCP-Cy5.5-conjugated anti-CD4 (RM4-5) were purchased from BD Biosciences (San Diego, CA, USA). AES16-2M treatment. In addition, quantities of IL-4-, IL-13-, and IL-17-producing CD4 T cells from peripheral lymph nodes and spleens were reduced by injection of AES16-2M. Furthermore, the expression of TSLP was significantly reduced in AES16-2M-treated human keratinocytes. Therefore, these results suggest that AES16-2M can be a novel candidate for AD treatment. (Df) body-induced AD model was established, and 10 mg/kg of AES16-2M was used as the optimal dose determined by preliminary experiments. Surprisingly, AES16-2M administration attenuated AD symptoms and decreased the dermatitis scores significantly in AD mice, which results were comparable to those for the positive control group (dexamethasone) (Figure 1A,B). The histological analysis of dermal tissue sections also revealed that the thickness of the epidermal layer and the dermal infiltration of mononuclear cells following AD induction were improved as well (Figure 1CCE). In addition, the ear thickness of the AES16-2M-treated mice was thinner ML401 than that of vehicle-treated mice (Figure 1F). Since increased IgE and TSLP production is closely related to AD pathology [12], the total IgE levels from sera were determined. As expected, ML401 the serum IgE and TSLP levels were significantly lower in AES16-2M-administrated mice, and these levels were comparable to those in the KCTD18 antibody dexamethasone group (Figure 1G,H). These results demonstrate that AES16-2M ameliorates AD in vivo. Open in a separate window Figure 1 AES16-2M ameliorates (Df)-induced atopic dermatitis (AD). Df-induced AD was established as described in the Materials and Methods section. (A) The skin status was photographed on day 21. (B) The dermatitis score was compared between the AD mice and AES16-2M (10 mg/kg, S.C. injection) treated mice and summarized as mean SD (= 10). Dexamethasone (Dex, 2.5 mg/kg) was used for the positive control group. (C) H&E stain of dermal tissues from day 21 was visualized by microscopy. Arrows reveal the epidermis. (D) The relative epidermal thickness was evaluated via data ML401 derived from (C) (three mice per group in quintuplicate). The thickness was measured using ImageJ and presented as mean relative values to the normal control. (E) Mononuclear cells that had infiltrated into the dermis were counted. (F) Ear thickness was measured using a digital caliper and was expressed as mean SD (= 10). The total IgE (G) and TSLP (H) levels were evaluated from the sera of mice on day 21 (= 5 for IgE, = 3 for TSLP). The animal experiments were performed with a total of 10 mice per group. In the case of ELISA, sera from the dermatitis score-matched mice were used. A non-paired and two-tailed Students 0.001 (vs normal); * 0.05 (vs PBS); ** 0.01 (vs. PBS); *** 0.001 (vs. PBS). 2.2. AES16-2M Reduces the Generation of Th2 and Th17 Cells in the AD Model It is well known that Th2 cells, which secrete IL-4 and IL-13, and IL-17-producing Th17 cells, are involved in AD development [10,21]. For these reasons, the changes in the populations of Th2 and Th17 cells were examined in the spleens and draining lymph nodes of the AD mice. The circulation cytometric results identified that either IL-4-, IL13-, or IL-17-generating CD4 T cells were increased by AD induction, and that these populations were smaller in the spleens and lymph nodes from AES16-2M treated mice (Number 2). These results suggest that AES16-2M efficiently reduces the generation of Th2 and Th17 cells, and attenuates AD pathogenesis. Open in a separate window Number 2 AES16-2M reduces the generation ML401 of either IL-4-, IL-13-, or IL-17-generating CD4 T cells in vivo. (A) Draining lymph nodes and (B) spleens were isolated from mice of dermatitis score-matched five mice per group, and the cells were cultured in the presence of TCR stimuli for 20 h (1 g/mL of CD3and CD28 antibodies). PMA (50 ng/mL), ionomycin (1 g/mL) and monensin (1 g/mL) were added into the cells and ML401 incubated for 4 h. The IL-4-, IL-13-, or IL-17-generating CD4 T cells were evaluated by circulation cytometry (cells gated on CD4+). Dex, dexamethasone..