Ght/dark), and temperature (2261uC). The air in the facility was passed through a HEPA filter system designed to exclude bacteria and viruses. Animals were fed mouse chow and tap water 10781694 ad libitum. The protocols used in this study were approved by the Animal Care and Use Committee of The Catholic University of Korea.Measurement of Cytokines and IgG TitersThe concentrations of IFN-c and IL-17 in cell culture supernatants and serum were measured using a sandwich ELISA (Duoset; R D Systems, Lille, France). Serum levels of IgG, IgG1 and IgG2a antibodies were measured using a commercially available ELISA kit (Bethyl Laboratories, Montgomery, TX, USA).BMT Model and Histopathology ScoringRecipients (BALB/c) mice were injected intraveneously (i.v.) with 56106 total bone marrow cells from donor mice after lethal irradiation with 800 cGy. To induce acute GVHD, splenocytes were isolated from donor mice. Splenocytes (16107) from MHC major and minor antigen-disparate B6 donors were incubated with 20 nM cucurmin or control vehicle (DMSO) for 1 h at 37uC before adoptive transfer into recipient mice. To create GVHD?negative controls, 56106 T cell epleted (TCD) bone marrow cells were transplanted into irradiated BALB/c recipient mice. All experiments were performed at least three times with six mice per group. Survival after BMT was monitored daily, and the degree of clinical GVHD was assessed using a scoring system that summed changes in five clinical parameters: weight loss, posture, activity, Potassium clavulanate firImmunohistochemistryImmunohistochemistry was performed using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA). Tissues were first incubated with the primary anti-C-jun and anti-C-fos antibodies overnight at 4uC. The primary antibody was detected with a biotinylated secondary linking antibody, followed by K162 incubation with a streptavidin eroxidase complex for 1 h. The final color product was developed using DAB chromogen (DAKO, Carpinteria, CA, USA).Therapeutic Efficacy of Curcumin in Acute GVHDConfocal StainingSpleen tissue was obtained 14 days after BMT and was snapfrozen in liquid nitrogen and stored at 280uC. Tissue cryosections (7-mm thick) were fixed in 4 paraformaldehyde and stained using PE-labeled anti-IFN-c, IL-4, IL-17, or Foxp3 antibody (eBioscience) and FITC-labeled anti-CD4 antibody and APClabeled anti CD25 antibody (Biolegend). After incubation overnight at 4uC, stained sections were analyzed using a confocal microscope (LSM 510 Meta; Zeiss, Gottingen, Germany). CD4+IFN-c+, CD4+IL-4+, CD4+IL-17+, CD4+CD25+Foxp3+ T cells were enumerated visually at higher magnification (projected on a screen) by four individuals.Statistical AnalysisComparison of numerical data between three groups was performed with nonparametric Mann-Whitney tests. Statistical analysis was performed using SPSS 10.0 for Windows (SPSS, Chicago, IL, USA). P values ,0.05 were considered significant. Data are presented as the mean 6 SD.treated splenocytes showed attenuated weight loss, less severe clinical scores of acute GVHD, and significantly delayed acute GVHD lethality in recipient mice, compared to the vehicle-treated group (Fig. 2A). Liver, skin, colon and lung are the target organs of acute GVHD. Mice from each treatment group were sacrificed on day 14 post-BMT after GVHD induction. To determine the protective effects of curcumin on the development of acute GVHD, we evaluated tissue pathology in liver, skin, colon and lung. As shown in Fig. 2B, moderate to seve.Ght/dark), and temperature (2261uC). The air in the facility was passed through a HEPA filter system designed to exclude bacteria and viruses. Animals were fed mouse chow and tap water 10781694 ad libitum. The protocols used in this study were approved by the Animal Care and Use Committee of The Catholic University of Korea.Measurement of Cytokines and IgG TitersThe concentrations of IFN-c and IL-17 in cell culture supernatants and serum were measured using a sandwich ELISA (Duoset; R D Systems, Lille, France). Serum levels of IgG, IgG1 and IgG2a antibodies were measured using a commercially available ELISA kit (Bethyl Laboratories, Montgomery, TX, USA).BMT Model and Histopathology ScoringRecipients (BALB/c) mice were injected intraveneously (i.v.) with 56106 total bone marrow cells from donor mice after lethal irradiation with 800 cGy. To induce acute GVHD, splenocytes were isolated from donor mice. Splenocytes (16107) from MHC major and minor antigen-disparate B6 donors were incubated with 20 nM cucurmin or control vehicle (DMSO) for 1 h at 37uC before adoptive transfer into recipient mice. To create GVHD?negative controls, 56106 T cell epleted (TCD) bone marrow cells were transplanted into irradiated BALB/c recipient mice. All experiments were performed at least three times with six mice per group. Survival after BMT was monitored daily, and the degree of clinical GVHD was assessed using a scoring system that summed changes in five clinical parameters: weight loss, posture, activity, firImmunohistochemistryImmunohistochemistry was performed using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA). Tissues were first incubated with the primary anti-C-jun and anti-C-fos antibodies overnight at 4uC. The primary antibody was detected with a biotinylated secondary linking antibody, followed by incubation with a streptavidin eroxidase complex for 1 h. The final color product was developed using DAB chromogen (DAKO, Carpinteria, CA, USA).Therapeutic Efficacy of Curcumin in Acute GVHDConfocal StainingSpleen tissue was obtained 14 days after BMT and was snapfrozen in liquid nitrogen and stored at 280uC. Tissue cryosections (7-mm thick) were fixed in 4 paraformaldehyde and stained using PE-labeled anti-IFN-c, IL-4, IL-17, or Foxp3 antibody (eBioscience) and FITC-labeled anti-CD4 antibody and APClabeled anti CD25 antibody (Biolegend). After incubation overnight at 4uC, stained sections were analyzed using a confocal microscope (LSM 510 Meta; Zeiss, Gottingen, Germany). CD4+IFN-c+, CD4+IL-4+, CD4+IL-17+, CD4+CD25+Foxp3+ T cells were enumerated visually at higher magnification (projected on a screen) by four individuals.Statistical AnalysisComparison of numerical data between three groups was performed with nonparametric Mann-Whitney tests. Statistical analysis was performed using SPSS 10.0 for Windows (SPSS, Chicago, IL, USA). P values ,0.05 were considered significant. Data are presented as the mean 6 SD.treated splenocytes showed attenuated weight loss, less severe clinical scores of acute GVHD, and significantly delayed acute GVHD lethality in recipient mice, compared to the vehicle-treated group (Fig. 2A). Liver, skin, colon and lung are the target organs of acute GVHD. Mice from each treatment group were sacrificed on day 14 post-BMT after GVHD induction. To determine the protective effects of curcumin on the development of acute GVHD, we evaluated tissue pathology in liver, skin, colon and lung. As shown in Fig. 2B, moderate to seve.
