Article Figures & Data
Figures
- Figure 1
Characterization of reactive oxygen species (ROS) induced epitopes. A) Carbonyl contents in ROS-modified human-serum-albumin (HSA) (ROS-epitopes-HSA) and unmodified HSA (nHSA). @p=0.0001 versus carbonyl contents present in nHSA. B) ROS induced tryptophan fluorescence alterations in ROS-epitopes-HSA. Fluorescence emission studies of ROS-epitopes-HSA and nHSA. The excitation wavelength was 295 nm. #p=0.0198 versus fluorescence intensity of ROS-epitopes-HSA. C) ROS induced tryptophan-tyrosine fluorescence alterations in ROS-epitopes-HSA. Fluorescence emission studies of ROS-epitopes-HSA and nHSA. The excitation wavelength was 280 nm. $p=0.021 versus fluorescence intensity of ROS-epitopes-HSA. Each whiskers box (min to max) represents the variance in 5 independent assays. The protein was in PBS, pH 7.4, and the concentration of all protein samples was 1 mg/ml. Comparison analysis was performed using the 2-way Analysis of Variance followed by Bonferroni’s post hoc test.
- Figure 2
Direct binding of psoriasis immunoglobulin G to reactive oxygen species (ROS)-epitopes-human-serum-albumin (HSA). A) Levels of anti-ROS-epitopes-HSA-IgGs in psoriasis (PS) patients (n=26) and normal human (NH) controls (n=22). Anti-ROS-epitopes-HSA-PS-immunoglobulin G (IgG) from PS patients’ sera specific to ROS modified HSA (ROS-epitopes-HSA); anti-ROS-epitopes-HSA-NH-IgGs stands for IgG from NH controls’ sera specific to ROS-epitopes-HSA. Black whiskers box (min to max) represents the variance in 26 independent assays, whereas whiskers box (min to max) represents the variance in 22 independent assays. #p=0.021 versus anti-ROS-epitopes-HSA-PS-IgGs. B) Levels of anti-nHSA-IgGs in PS patients (n=26) and NH controls (n=22). Anti-nHSA-PS-IgGs stands for immunoglobulin G from PS patients’ sera specific to native HSA (nHSA); anti-nHSA-NH-IgGs stands for IgG from NH controls’ sera specific to nHSA. Black whiskers box (min to max) represents the variance in 26 independent assays, whereas whiskers box (min to max) represents the variance in 22 independent assays. Anti-nHSA-PS-IgGs versus anti-nHSA-NH-IgGs, p=0.261. C) Levels of anti-ROS-epitopes-HSA-IgGs and anti-nHSA-IgGs in PS patients (n=26). Both whiskers boxes (min to max) represent the variance in 26 independent assays. Microtitre plates were individually coated with ROS-epitopes-HSA or nHSA (10 µg/ml). @p=0.0005 versus ROS-epitopes-HSA. Comparison analysis was performed using the 2-tail test followed by Mann Whitney post hoc analysis.
- Figure 3
Competitive binding assays of psoriasis immunoglobulin G (IgGs) to reactive-oxygen-species (ROS)-epitopes-human-serum-albumin (HSA). A) Inhibition of IgGs from psoriasis patients (PS) and normal human (NH) controls by ROS modified HSA (ROS-epitopes-HSA). #p=0.021 versus PS patients. Black whiskers box (min to max) represents the variance in 26 independent competitive binding assays, whereas whiskers box (min to max) represents the variance in 22 independent competitive binding assays. B) Inhibition of IgGs from PS and NH controls by native HSA (nHSA). Psoriasis patients versus NH controls, p=0.079. C) Inhibition of psoriasis IgGs by ROS-epitopes-HSA and nHSA. Both whiskers boxes (min to max) represent the variance in 26 independent competitive binding assays. Microtitre plates were individually coated with ROS-epitopes-HSA or nHSA (10 µg/ml). @p=0.0005 versus ROS-epitopes-HSA. Comparison analysis was performed using 2-tailed test followed by Mann Whitney post hoc analysis.
- Figure 4
A) Histopathology of thyroid tissue. Hematoxylin and eosin staining of thyroid tissue dissected from rabbit (400X). B) Preparation of thyroglobulin. Gel exclusion chromatography of rabbit thyroid extract on Sephacryl S200 HR column. Indicated fractions in circle were collected and were used as thyroglobulin. SCE - simple cuboidal epithelium, CECs - cuboidal epithelium cells.
- Figure 5
Recognition of thyroid antigens and their oxidized forms by anti-reactive-oxygen-species (ROS)-epitopes-human-serum-albumin (HSA)-immunoglobulin G (IgGs). Binding characteristics of affinity purified anti-ROS-epitopes-HSA-IgGs to thyroid extract, ROS-modified thyroid antigen (ROS-thyroid antigen), thyroglobulin, ROS-modified thyroglobulin (ROS-thyroglobulin), human DNA, ROS-modified human DNA (ROS-human DNA) determined direct binding ELISAs. Reactive-oxygen-species modified HSA (ROS-epitopes-HSA) was used as a positive control, and native HSA (nHSA) was used a negative control for anti-ROS-epitopes-HSA-IgGs. Values above 0.4 (y axis) were considered positive. Each histogram represents as a mean ± SEM. *p=0.000 versus ROS-epitopes-HSA; #p=0.013 versus thyroid antigen; $p=0.008 versus thyroglobulin; @p=0.218 versus human DNA.
- Figure 6
Competitive inhibition of anti-reactive-oxygen-species (ROS)-epitopes-human-serum-albumin (HSA)-immunoglobulin G (IgGs). Inhibition of affinity purified anti-ROS-epitopes-HSA-IgGs by thyroid antigen, ROS-modified thyroid antigen (ROS-thyroid antigen), thyroglobulin, ROS-modified thyroglobulin (ROS-thyroglobulin), human DNA, ROS-modified human DNA (ROS-human DNA) determined by competitive binding assays. Microtitre plates were coated with ROS-epitopes-HSA (10 µg/ml), whereas thyroid extract, ROS-thyroid extract, thyroglobulin, ROS-thyroglobulin, human DNA, ROS-human DNA were used as inhibitors with different concentrations (2.5-10 µg/ml). Each histogram represents a mean ± SEM of 5 independent assays. nHSA - native human-serum-albumin
- Figure 7
Competitive inhibition of psoriasis IgG. Inhibition of affinity purified psoriasis immunoglobulin G (PS-IgG) A) and normal human IgG (normal human [NH]-IgG) B) binding to reactive-oxygen-species (ROS)-epitopes-HSA antigen. Inhibitors used were thyroid extract, ROS-modified thyroid extract (ROS-thyroid extract), thyroglobulin, ROS-modified thyroglobulin (ROS-thyroglobulin), human DNA and ROS-modified human DNA (ROS-human DNA). Inhibitors concentration used in the competitive binding assays was 20 µg/ml. Microtitre plates were coated with ROS-epitopes-HSA (10µg/ml). Each histogram represents a mean ± SEM of four independent assays. *p=0.004 versus thyroid antigen, #p=0.043 versus thyroglobulin, $p=0.845 versus human DNA, **p=0.265 versus thyroid antigen, ##p=0.778 versus thyroglobulin,$$p=1.000 versus human DNA.
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