Orbital fibrosis in a mouse model of Graves' disease induced by genetic immunization of thyrotropin receptor cDNA

The TSH receptor (TSHR) is the critical target for antibody production in Graves' disease (GD). Insulin-like growth factor 1 receptor (IGF1R) has been proposed as a second autoantigen in complications of GD such as orbitopathy. We attempted to induce orbital tissue remodeling in mice undergoing immunizations with plasmids encoding TSHR and IGF1R delivered by in vivo skeletal muscle electroporation, a procedure known to give a sustained, long-term antibody response. Female BALB/c mice were challenged with TSHR A-subunit or IGF1Rα subunit plasmid by injection and electroporation. Mice challenged with TSHR A-subunit plasmid resulted in high frequency (75%) of hyperthyroidism and thyroid-stimulating antibodies. But strikingly, immunization with TSHR A-subunit plasmid also elicited antibody to IGF1Rα subunit. Mice challenged in the same manner with IGF1Rα subunit plasmid produced strong antibody responses to IGF1R, but did not undergo any changes in phenotype. Simultaneous challenge by double antigen immunization with the two plasmids in distant anatomical sites reduced the incidence of hyperthyroidism, potentially as a consequence of antigenic competition. Thyroid glands from the TSHR A-subunit plasmid-challenged group were enlarged with patchy microscopic infiltrates. Histological analysis of the orbital tissues demonstrated moderate connective tissue fibrosis and deposition of Masson's trichrome staining material. Our findings imply that immunization with TSHR A-subunit plasmid leads to generation of IGF1R antibodies, which together with thyroid-stimulating antibodies may precipitate remodeling of orbital tissue, raising our understanding of its close association with GD

Immunomodulation by imiquimod in patients with high-risk primary melanoma.

Imiquimod is a synthetic Toll-like receptor 7 (TLR7) agonist approved for the topical treatment of actinic keratoses, superficial basal cell carcinoma, and genital warts. Imiquimod leads to an 80-100% cure rate of lentigo maligna; however, studies of invasive melanoma are lacking. We conducted a pilot study to characterize the local, regional, and systemic immune responses induced by imiquimod in patients with high-risk melanoma. After treatment of the primary melanoma biopsy site with placebo or imiquimod cream, we measured immune responses in the treated skin, sentinel lymph nodes (SLNs), and peripheral blood. Treatment of primary melanomas with 5% imiquimod cream was associated with an increase in both CD4+ and CD8+ T cells in the skin, and CD4+ T cells in the SLN. Most of the CD8+ T cells in the skin were CD25 negative. We could not detect any increases in CD8+ T cells specifically recognizing HLA-A(*)0201-restricted melanoma epitopes in the peripheral blood. The findings from this small pilot study demonstrate that topical imiquimod treatment results in enhanced local and regional T-cell numbers in both the skin and SLN. Further research into TLR7 immunomodulating pathways as a basis for effective immunotherapy against melanoma in conjunction with surgery is warranted

Nuclear Targeting of IGF-1 Receptor in Orbital Fibroblasts from Graves' Disease: Apparent Role of ADAM17

Insulin-like growth factor-1 receptor (IGF-1R) comprises two subunits, including a ligand binding domain on extra- cellular IGF-1Rα and a tyrosine phosphorylation site located on IGF-1Rβ. IGF-1R is over-expressed by orbital fibroblasts in the autoimmune syndrome, Graves' disease (GD). When activated by IGF-1 or GD-derived IgG (GD-IgG), these fibroblasts produce RANTES and IL-16, while those from healthy donors do not. We now report that IGF-1 and GD-IgG provoke IGF-1R accumulation in the cell nucleus of GD fibroblasts where it co-localizes with chromatin. Nuclear IGF-1R is detected with anti-IGF-1Rα-specific mAb and migrates to approximately 110 kDa, consistent with its identity as an IGF-1R fragment. Nuclear IGF-1R migrating as a 200 kDa protein and consistent with an intact receptor was undetectable when probed with either anti-IGF-1Rα or anti-IGF-1Rβ mAbs. Nuclear redistribution of IGF-1R is absent in control orbital fibroblasts. In GD fibroblasts, it can be abolished by an IGF-1R-blocking mAb, 1H7 and by physiological concentrations of glucocorticoids. When cell-surface IGF-1R is cross-linked with 125I IGF-1, 125I-IGF-1/IGF-1R complexes accumulate in the nuclei of GD fibroblasts. This requires active ADAM17, a membrane associated metalloproteinase, and the phosphorylation of IGF-1R. In contrast, virally encoded IGF-1Rα/GFP fusion protein localizes equivalently in nuclei in both control and GD fibroblasts. This result suggests that generation of IGF-1R fragments may limit the accumulation of nuclear IGF-1R. We thus identify a heretofore-unrecognized behavior of IGF-1R that appears limited to GD-derived fibroblasts. Nuclear IGF-1R may play a role in disease pathogenesis

Effect of CTCF binding activity on interactions among <i>PAX6</i>, <i>RCN1</i> and <i>ADAM17</i> gene promoters.

<p>(<b><i>A</i></b>) Detection of CTCF binding activities at sites 2 and 3 in Pax6 promoter region by ChIP based PCRs in both lentivirus-infected Lv-control and CTCF-knocked down CTCF-shRNA cells during differentiation. (<b><i>B</i></b>) Detection of decreased CTCF-binding on site 2 of <i>RCN1</i> gene promoter during differentiation of both Lv-control and CTCF-shRNA cells. (<b><i>C</i></b>) Detection of decreased CTCF-binding on site 2 of <i>ADAM17</i> gene in differentiated Lv-control and CTCF -shRNA cells. (<b><i>D</i></b>) Statistic analysis of the significant decreases in CTCF binding in promoter regions of <i>PAX6</i>, <i>RCN1</i> and <i>ADAM17</i> genes in differentiated Lv-control and CTCF-shRNA cells, respectively. ChIP-based PCR was performed to amplify the selected CTCF bound DNA fragments in <i>PAX6</i>, <i>RCN1</i> and <i>ADAM17</i> promoter regions, respectively. Input and CTCF AB^- experiments were performed as controls with non-immunoprecipitated chromatins and in the absence of CTCF-specific antibody, respectively. Data were obtained from six independent ChIP and PCR experiments. Symbols “*” and “**” indicate significant differences between control and differentiated cells, control and CTCF-shRNA cells and differentiated control and CTCF-shRNA cells, respectively (<i>p</i><0.05, n = 6).</p

Characterization of differentiation-induced corneal epithelial cells.

<p>(<b><i>A</i></b>) Morphological changes in culture during differentiation of HTCE cells. (<b><i>B</i></b>) AE3 and AE1 keratin panel staining in HTCE cell differentiation. (<b><i>C</i></b>) Time course of K12 mRNA expression following induced differentiation of HTCE cells. Differentiation of HTCE cells was induced by adding 1.2 mM calcium and 5% FBS in the normal culture condition up to 120 h. (<b><i>D</i></b>) Detection of K12 and p63 expression in HTCE cell differentiation by Western blot up to 120 h. (<b><i>E</i></b>) Expression of K12 and p63 mRNAs and proteins in human limbal stem/progenitor (HLS/P) and corneal epithelial (HCE) cells. Cells were grown and induced to differentiation in chamber slides and photos were taken by light microscopy. These cells were then washed in PBS and fixed with 95% ethanol before staining. Staining protocols were used as suggested by manufactures. Furthermore, RNAs were extracted from HTCE, HLS/P and HCE cells, and reverse-transcribed into cDNAs before real-time qPCR analysis was performed using K12-specific primers as indicated in Materials and Methods. Symbol “*” indicates significant differences after 72 h induction (<i>p</i><0.05, n = 6). Photos were taken by a Zeiss AXIO microscope (x20).</p

Effect of altered CTCF levels on expressions of <i>PAX6</i>, <i>RCN1</i> and <i>ADAM17</i>.

<p>(<b><i>A</i></b>) Expression patterns of CTCF, PAX6, ADAM17 and RCN1 proteins in Lv-control and CTCF mRNA knocked down CTCF-shRNA cells following a differentiation time course. (<b><i>B</i></b>) Statistical analysis of the effect of knocking down <i>CTCF</i> mRNA by <i>CTCF</i>-specific shRNA on expressions of CTCF, PAX6, ADAM17 and RCN1 proteins during HTCE cell differentiation at 48 h. (<b><i>C</i></b>) Comparison of expression in RNA levels of <i>K12</i>, <i>PAX6</i>, <i>ADAM17</i>, and <i>RCN1</i> in lentivirus-infected control and CTCF-shRNA cells following differentiation time courses. (<b><i>D</i></b>) Significant alteration of G₀/G₁ and S phases in cell cycle distribution of differentiation-induced CTCF-shRNA cells. Western blots and RT-qPCR were described in details in materials and methods. Treatments are as indicated. Cell cycle analysis was also described previously. Symbols “*” and “**” indicate the statistical significance between Lv-control and CTCF-shRNA cells before and after differentiation, respectively. Significant differences among groups were determined by One-way ANOVA and Tukey’s tests, and then Student’s <i>t</i> test was used to determine the significant difference between two samples at <i>P</i><0.05 (n = 4 to 6).</p

Correlation of CTCF and PAX6 expression during corneal epithelial cell differentiation.

<p>(<b><i>A</i></b>) Cell cycle analysis by flow cytometry revealed significantly increased and decreased cell populations in G₀/G₁ and S phases, respectively. (<b><i>B</i></b>) Time courses of CTCF and <i>PAX6</i> mRNA expressions in HTCE cell differentiation. (<b><i>C</i></b>) Detection and analysis of CTCF and <i>PAX6</i> mRNA expressions in HLS/P and HCE cells. (<b><i>D</i></b>) Western analysis demonstrated that there is an opposite expression pattern between CTCF and Pax6 following a time course of HTCE cell differentiation (Diff). Flow cytometric analysis of HTCE cells with or without differentiation was performed as described in materials and methods section. Expressions of CTCF and Pax6 in both their protein and RNA levels were detected by Western blots and quantitative real-time RT-PCR during HTCE cell differentiation for as long as 96 h after induction. Symbol “*” indicates significant differences (<i>p</i><0.05, n = 4).</p