Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Aflatoxin B1 and Epstein-Barr virus-induced CCL22 expression stimulates B cell infection.

doi: 10.1073/pnas.2314426121

Figure Lengend Snippet: Fig. 1. Mycotoxins and EBV exposure increase CCL22 expression. (A) CCL22 relative mRNA expression levels in Louckes and primary B cells exposed to aflatoxin B1 (AFB1) 50 µM and B2 (AFB2) 50 µM. (B) CCL22 protein levels detected in presence and absence of aflatoxin exposure (50 µM) by Western Blot using total cell extract (Left) and immunoprecipitated (IP) CCL22 from cell supernatant (Right). (C and D) RT-qPCR quantification of CCL22 mRNA levels in cells exposed to AFB1 (50 µM), aflatoxicol (AFL) at 25 µM, sterigmatocystin (STC) at 3.13 µM and combination of both (AFL+STC) for 48 h only or infected by EBV 24 h after treatment. (E) The RT-qPCR analyses of CCR4 mRNA levels in Louckes and primary B cells exposed to AFB1 (50 µM) and EBV. DMSO was used as solvent control in all experiments. The histograms represent data mean ± SD and significance level calculated by ANOVA test (P *≤ 0.05, or P** ≤ 0.01, or P*** ≤ 0.001 or P**** ≤ 0.0001), n = 3. (F) Representative images of immunohistochemistry staining for CCL22 performed on EBV-positive (n = 8) and EBV-negative (n = 6) Burkitt Lymphomas.

Article Snippet: Briefly, CCL22 function was neutralized by treating the mice with a neutralizing anti- CCL22 antibody (abCCL22; mouse IgG2B anti human ccl22 R&D system clone 57226 lot BIK0316091) at 20 μg per injection per mouse every week.

Techniques: Expressing, Western Blot, Immunoprecipitation, Quantitative RT-PCR, Infection, Solvent, Control, Immunohistochemistry, Staining

Journal:

Article Title: GM-CSF promoted DC recruitment and survival governs the intestinal mucosal response to enteric attaching-and-effacing bacterial pathogens

doi: 10.1016/j.chom.2010.01.006

Figure Lengend Snippet: (A) mRNA transcripts for the three DC chemoattractant chemokines, CCL22, CCL8 and CCL20, were assayed by real time RT-PCR in the colon of B6 and GM-CSF-/- mice left uninfected or infected with C. rodentium (n = 4-5 mice per group at each time point). (B) CCL22 protein was assayed in colon extracts from B6 and GM-CSF-/- mice. Data are mean ± SEM (n = 5 mice per group at each time point). For (A, B) * p < 0.05 relative to B6 mice at the indicated time points. (C) Colon sections from B6 mice left uninfected and B6 and GM-CSF-/- mice infected for 3 weeks with C. rodentium were stained for CCL22 (red). Original magnification, x 200. (D) Colon sections from B6 mice infected for 3 weeks were stained for CCL22 (red) and β-catenin (green) as an epithelial cell marker. Nuclei were stained with Hoechst 33258 (blue). Specificity controls using control antibody or anti-CCL22 absorbed with CCL22 peptide showed no immunostaining (see Fig. S3). (E) Colon sections from B6 mice infected for 3 weeks were stained for CCL22 (red) and CD11c (green). Original magnification, x 400. (F) WT B6 mice were infected with C. rodentium for 2 weeks. On days 9, 11 and 13 after infection mice were injected i.p. with 20 μg IgG goat anti-CCL22 or control goat IgG.. Colon sections from anti-CCL22 and control goat IgG treated mice were stained for CD11c (red) and F-actin (green). Original magnification, x 200. (G) Cells isolated from colon lamina propria, spleen, and MLN of anti-CCL22 and control goat IgG treated mice were characterized by flow cytometry. Similar results were obtained in 2 independent experiments.

Article Snippet: The following polyclonal antibodies were used: goat anti-mouse and rat CCL22 (IgG) from Santa Cruz for immunostaining experiments, goat anti-mouse CCL22 (IgG) from R&D systems for in vivo neutralization experiments, rabbit anti-FITC (Alexa 488-conjugated, IgG) from Molecular Probes.

Techniques: Quantitative RT-PCR, Infection, Staining, Marker, Control, Immunostaining, Injection, Isolation, Flow Cytometry