CpG Oligodeoxynucleotides Prevent the Development of Scleroderma-Like Syndrome in Tight-Skin Mice by Stimulating a Th1 Immune Response Yan Shen, Motohide.

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CpG Oligodeoxynucleotides Prevent the Development of Scleroderma-Like Syndrome in Tight-Skin Mice by Stimulating a Th1 Immune Response Yan Shen, Motohide Ichino, Masatoshi Nakazawa, Mutsuhiko Minami Journal of Investigative Dermatology Volume 124, Issue 6, Pages 1141-1148 (June 2005) DOI: 10.1111/j.0022-202X.2005.23730.x Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Histopathology of the dorsal skin from the treated tight-skin (Tsk/+) mice. Representative paraffin-embedded sections stained with hematoxylin/eosin are shown (× 20) from female (A–D) and male (E–H) mice at 23 wk of age. C57BL/6 pa/pa mice are as control (A, E). Tsk/+ mice were treated seven times with saline (B, F), control oligodeoxynucleotides (ODN (C, G), and CpG ODN (D, H). Scale bar: 100 μm Journal of Investigative Dermatology 2005 124, 1141-1148DOI: (10.1111/j.0022-202X.2005.23730.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Serum levels of autoantibodies from the treated tight-skin (Tsk/+) mice. Tsk/+ mice were treated seven times with saline (Tsk/+ saline), control oligodeoxynucleotides (ODN) (Tsk/+ control), and CpG ODN (Tsk/+ CpG). Sera were collected from the mice at the age of 22 wk (left) and 32 wk (right). (A) ANA titers are evaluated by scoring serum dilutions (from 1:20 to 1:320) and higher titer is defined as detectable in more dilution. Each circle indicates the anti-nuclear antibodies titer of an individual mouse and black circles mean positive response. (B) Serum levels of anti-topoisomerase I antibodies were determined by ELISA. Each circle indicates the antibody index of an individual mouse and horizontal bars represent mean antibody levels. *p<0.05 Journal of Investigative Dermatology 2005 124, 1141-1148DOI: (10.1111/j.0022-202X.2005.23730.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 The number of interferon (IFN)-γ- and interleukin (IL)-4-secreting cells in the spleen of the treated tight-skin (Tsk/+) mice. Tsk/+ mice were treated with saline (Tsk/+ saline), control oligodeoxynucleotides (ODN) (Tsk/+ control), and CpG ODN (Tsk/+ CpG). C57BL/6 pa/pa littermates (C57BL/6 pa/pa) were as control. ELISPOT assays were performed on single cell suspensions of spleen cells from the mice at 32 wk of age with (A) or without (B) boost 1 wk prior to sacrifice. Spleen cells were incubated in the absence (-) or presence of Con A (5 μg per mL). Each bar represents the mean±SD values for three mice. The data is representative of three independent experiments. *p<0.05 as compared with Tsk/+ saline and Tsk/+ control. **p<0.005 as compared with Tsk/+ saline and Tsk/+ control Journal of Investigative Dermatology 2005 124, 1141-1148DOI: (10.1111/j.0022-202X.2005.23730.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 The number of interferon (IFN)-γ- and interleukin (IL)-4-secreting cells in the spleen of tight-skin (Tsk/+) mice receiving single oligodeoxynucleotides (ODN) injection. Naïve 6–8-wk-old Tsk/+ mice were injected once with saline (Tsk/+ saline), control ODN (Tsk/+ control), and CpG ODN (Tsk/+ CpG) 1 wk prior to sacrifice. C57BL/6 pa/pa littermates (C57BL/6 pa/pa) were as control. Spleen cells were incubated in the absence (-) or presence of Con A (5 μg per mL). Each bar represents the mean±SD values for three mice. The data is representative of three independent experiments. *p<0.05 as compared with Tsk/+ saline and Tsk/+ control Journal of Investigative Dermatology 2005 124, 1141-1148DOI: (10.1111/j.0022-202X.2005.23730.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Serum levels of interleukin (IL)-12 in the treated tight-skin (Tsk/+) mice. Tsk/+ mice were treated seven times with saline (Tsk/+ saline), control oligodeoxynucleotides (ODN) (Tsk/+ control), and CpG ODN (Tsk/+ CpG). C57BL/6 pa/pa littermates (C57BL/6 pa/pa) were as control. Serum was collected from mice at 20 wk of age and serum levels of IL-12 were measured by standard ELISA. Each bar represents the mean±SD values for three to nine mice. **p<0.005 as compared with Tsk/+ saline and Tsk/+ control Journal of Investigative Dermatology 2005 124, 1141-1148DOI: (10.1111/j.0022-202X.2005.23730.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions