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Results: 6

1.
Fig. 6

Fig. 6. From: Structure-based selection of small molecules to alter allele-specific MHC class II antigen presentation.

Correlation between IL-10 Elispots with and without glyphosine in the presence of B:9–23 peptide. Mean IL-10 Elispots from (A) DQ8 Individuals with type 1 diabetes (n=14) and (B) non-diabetic DQ8 controls (n=8). There is a statistically significant correlation between the insulin B:9–23 spot number and spots in the presence of peptide and glyphosine for individuals with type 1 diabetes (p=0.002, r2=0.56). There is no correlation with the DQ8 controls (p=0.30, r2=0.17). T1D = type 1 diabetes.

Aaron W Michels, et al. J Immunol. ;187(11):5921-5930.
2.
Fig. 1

Fig. 1. From: Structure-based selection of small molecules to alter allele-specific MHC class II antigen presentation.

Small molecule inhibitors used in B:9–23 activation of T cells. (A) Diagram of the structural pockets along the I-Ag7 binding groove with the proposed binding sites for the compound structures based upon in silico molecular modeling. (B–F) Inhibition curves of compounds that block T cell responses to the B:9–23 peptide. Three TCRs are depicted; ● 8-1.1α1 and ○ BDC 12-4.1 recognize insulin B:9–23 peptide while ■ BDC 2.5 responds to a chromogranin mimotope. Percentage of inhibition was calculated from the stimulation of peptide alone for each TCR. Data points represent triplicate wells for each concentration of small molecule with peptide. Inhibition curves are representative of at least 3 independent experiments.

Aaron W Michels, et al. J Immunol. ;187(11):5921-5930.
3.
Fig. 5

Fig. 5. From: Structure-based selection of small molecules to alter allele-specific MHC class II antigen presentation.

IL-10 and IFN-γ Elispot assays from DQ8 type 1 diabetic individuals and controls. (A) IL-10 Elispot in type 1 diabetic subjects (n=14) showing an increase in IL-10 spots with glyphosine and B:9–23 peptide compared to B:9–23 peptide alone. (B) IFN-γ Elispot showing no change in number when glyphosine is added to culture with or without B:9–23 peptide. (C) IL-10 and (D) IFN-γ Elispots from nondiabetic DQ8 controls (n=8) showing no change in spot number with glyphosine. All patients produced IFN-γ in response to a tetanus toxin peptide (used as a positive control) which did not change in response to glyphosine. Each data point represents the mean spot number from triplicate wells for an individual. Wilcoxon matched pairs test was used to compare means between groups and p values are labeled on graphs for ease of comparison.

Aaron W Michels, et al. J Immunol. ;187(11):5921-5930.
4.
Fig. 3

Fig. 3. From: Structure-based selection of small molecules to alter allele-specific MHC class II antigen presentation.

Glyphosine specifically enhances the T cell response to insulin B:9–23 presented by I-Ag7. (A) Glyphosine enhanced stimulation of the T cell hybridoma 8-1.1α1 only when insulin B:9–23 peptide is present. Glyphosine concentrations are indicated and the reaction mixture contained B:9–23 peptide unless otherwise noted. The glyphosine concentration used in subsequent experiments was 500 nM. TT = tetanus toxin (negative control peptide). *p=0.011, **p=0.049 (unpaired Student’s t-test). (B) The glyphosine response in the presence of B:9–23 is blocked by a monoclonal antibody to I-Ag7 and unchanged with an isotype control antibody. *p<0.001. (C–D) Glyphosine does not alter TCR reactivity to a chromogranin peptide presented by I-Ag7 to the BDC 2.5 T cell hybridoma or to ovalbumin presented by I-Ad to the DO11.10 T cell hybridoma. Results are given as a stimulation index (SI) from triplicate cultures ± SEM. Data are representative of at least three independent experiments.

Aaron W Michels, et al. J Immunol. ;187(11):5921-5930.
5.
Fig. 2

Fig. 2. From: Structure-based selection of small molecules to alter allele-specific MHC class II antigen presentation.

Small molecule inhibitors block presentation of endogenously processed insulin by NOD splenocytes. (A) Inhibition curves for p1 molecules and (B) p6 molecules. Dotted line represents IL-2 production by the 4–8 TCR to insulin in the absence of a small molecule. (C) IL-2 production from a T cell hybridoma cultured with whole islets used as antigen presenting cells. p6:4 molecule inhibits IL-2 production when islets isolated from an adult NOD mouse without diabetes are used for antigen presentation to the 4–8 TCR. *p=0.004, **p=0.002, ***p<0.001 (D) p6:4 inhibits B:9–23 peptide binding to I-Ag7, as determined using a soluble binding assay with purified I-Ag7 protein and biotinylated insulin B:9–23 peptide. Negative control is uncut I-Ag7 (thrombin was not used to cleave the flexible linker and linked peptide) with biotinylated B:9–23 peptide added to the assay. *p=0.023, **p<0.001. (E) p6:4 inhibits a human DQ8 restricted TCR to insulin B:9–23. TT = tetanus toxin (negative control peptide). *p=0.004, **p<0.001.

Aaron W Michels, et al. J Immunol. ;187(11):5921-5930.
6.
Fig. 4

Fig. 4. From: Structure-based selection of small molecules to alter allele-specific MHC class II antigen presentation.

Glyphosine stimulates IL-10 production from NOD splenocytes. (A) IL-10 Elispot with 500 nm glyphosine. Number of spots for each well is shown in the upper left hand corner. (B–C) Ex vivo IL-10 and IFN-γ Elispot assays from 10 week old female NOD mice treated with 80 mg/kg/day of glyphosine by intraperitoneal administration for 5 days. IL-10 is increased with glyphosine while IFN-γ spot number remains unchanged. *p=0.014. (D) IL-10 Elispot assays from mice with differing I-A molecules. *p=0.011, **p=0.008. Each bar represents the mean spot number of triplicates ± SEM. from at least four mice per group. (E) Glyphosine treatment prevents diabetes development. Survival curve of a diabetes prevention study in 4 week old NOD mice obtained from Jackson Laboratories. The glyphosine treated mice are statistically different (p<0.001) compared to controls receiving phosphate buffered saline.

Aaron W Michels, et al. J Immunol. ;187(11):5921-5930.

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