Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Isotype-Switched Autoantibodies Are Necessary To Facilitate Central Nervous System Autoimmune Disease in Aicda -/- and Ung -/- Mice.

doi: 10.4049/jimmunol.1700729

Figure Lengend Snippet: FIGURE 1. Impaired hMOG- induced EAE in Aicda2/2 mice is restored by a passive transfer of anti-MOG IgG1 Abs. Clinical scores (A–D) and weight loss (C and D) of animals were measured daily fol- lowing immunization until date of harvest. (A) Separately caged WT (n = 25) and Aicda2/2 (n = 26) mice or (B) littermates from Aicda+/2 3 Aicda+/2 parents (WT n = 5; Aicda2/2 n = 5) were immunized with hMOG1–120. (C) Following im- munization, Aicda2/2 mice (n = 5) were treated with 200 mg of anti- MOG IgG1 Ab (clone 818c5) or an isotype control (n = 5) at days 4 and 11 postimmunization. (D) Following immunization, WT mice (n = 5) were treated with 200 mg of anti- MOG IgG1 Ab (clone 818c5) or an isotype control (n = 4) at days 4 and 11 postimmunization. Arrows indi- cate i.v. injections of 818c5 Ab or an isotype control. (A) Represents data pooled over three separate experi- ments. (B–D) Data shown are from a representative experiment from at least two experiments with similar outcomes. Means and SEM are shown. Statistical significance was determined by two-way ANOVA (*p . 0.05, ***p . 0.001).

Article Snippet: DAPI and Alexa Fluor 555 Rat Anti-Mouse IgG1 or IgM (SouthernBiotech) were used to detect nuclei and Abs, respectively.

Techniques: Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Isotype-Switched Autoantibodies Are Necessary To Facilitate Central Nervous System Autoimmune Disease in Aicda -/- and Ung -/- Mice.

doi: 10.4049/jimmunol.1700729

Figure Lengend Snippet: FIGURE 2. WT and Aicda2/2 mice exhibit clinically similar EAE in response to rMOG. (A) Separately caged WT (n = 9) and Aicda2/2 (n = 10) mice were immunized with rMOG1–120, and clinical scores were measured daily until day 26 postimmunization. Data show a representative experiment from two separate experiments with similar outcomes. (B) Titers of anti-rMOG IgM (WT n = 8; Aicda2/2 n = 10) and IgG1 (WT n = 6; Aicda2/2 n = 10) Abs in serum were determined by ELISA. Corresponding dilution curves used to calculate titers can be found in Supplemental Fig. 1A. Coronal sections of formalin-fixed paraffin-embedded thoracic spinal cords from each WT (n = 4) and Aicda2/2 (n = 5) mouse were subjected to H&E staining with a representative example for each genotype shown in (C). Subsequent blinded quantification using one coronal thoracic section for each animal is shown in (D). Coronal sections of formalin-fixed paraffin-embedded thoracic spinal cords from each WT (n = 4) and Aicda2/2 (n = 5) mice were subjected to LFB staining with a representative example for each genotype shown in (E). Subsequent blinded quantification using one coronal thoracic section for each animal is shown in (F). Please refer to Materials and Methods for quantification details. Arrows indicate examples of cellular accumulation (C) or demyelination (E) throughout the white matter area of the spinal cord. Means and SEM (A) or means and SD (B, D, and F) are displayed. Statistical significance was determined by using a two-way ANOVA (A) or a Mann–Whitney U test (B, D, and F) (*p . 0.05, ***p . 0.001).

Article Snippet: DAPI and Alexa Fluor 555 Rat Anti-Mouse IgG1 or IgM (SouthernBiotech) were used to detect nuclei and Abs, respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Isotype-Switched Autoantibodies Are Necessary To Facilitate Central Nervous System Autoimmune Disease in Aicda -/- and Ung -/- Mice.

doi: 10.4049/jimmunol.1700729

Figure Lengend Snippet: FIGURE 5. Ung2/2 mice exhibit an impaired class-switch autoantibody response in response to hMOG. (A) Serum from WT (n = 5; Ung+/2 n = 8) and Ung2/2 (n = 10) mice was diluted to a starting concentration of 0.2 mg/ml anti-MOG IgG1 and added to plates coated with hMOG, followed by washes with increasing concentrations of a stripping buffer (NH4SCN) to generate an affinity index. (B) Serum was measured for relative titers of anti-MOG IgM (WT n = 4; Ung+/2 n = 2; Ung2/2 n = 12) and anti-hMOG IgG1 (WT n = 3; Ung+/2 n = 11; Ung2/2 n = 17). (C) Supernatants from brain homogenates were measured for total IgM (WT n = 5; Ung+/2 n = 8; Ung2/2 n = 10) and anti-hMOG IgG1 (WT n = 6; Ung+/2 n = 8; Ung2/2 n = 10). (D) Supernatants from spinal cord homogenates (WT n = 4; Ung+/2 n = 4; Ung2/2 n = 5) were measured for total IgM and anti-hMOG IgG1. Statistical significance was determined by Mann–Whitney U test (*p . 0.05, **p . 0.01, ***p . 0.001).

Article Snippet: DAPI and Alexa Fluor 555 Rat Anti-Mouse IgG1 or IgM (SouthernBiotech) were used to detect nuclei and Abs, respectively.

Techniques: Concentration Assay, Stripping Membranes, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Isotype-Switched Autoantibodies Are Necessary To Facilitate Central Nervous System Autoimmune Disease in Aicda -/- and Ung -/- Mice.

doi: 10.4049/jimmunol.1700729

Figure Lengend Snippet: FIGURE 6. Class-switched autoantibodies detected in the serum of WTand Ung2/2 mice recognize conformational epitopes of MOG. HEK293 cells transfected with MOG-GFP were overlaid with serum from WT (n = 8), Aicda2/2 (n = 6), or Ung2/2 (n = 11) mice, and IgM (A) or IgG1 (B) Abs were detected by secondary Abs tagged with Alexa Fluor 555 (AF555). DAPI staining for nuclei and merged RGB color images are shown for reference. (C and D) GFP+AF555+DAPI+ cellular events were counted and represented as a ratio of total GFP+DAPI+ cells. Images were captured at original magnification 320. Statistical significance was de- termined by Mann–Whitney U test (**p . 0.01, ***p . 0.001).

Article Snippet: DAPI and Alexa Fluor 555 Rat Anti-Mouse IgG1 or IgM (SouthernBiotech) were used to detect nuclei and Abs, respectively.

Techniques: Transfection, Staining, MANN-WHITNEY