Mucin 1 (MUC1), a tumor-associated antigen, is a transmembrane glycoprotein expressed by normal epithelial cells and overexpressed by carcinomas of epithelial origin. Autoantibodies against MUC1 are often found in circulation, either free or bound to immune complexes, which might contribute to limit tumor outgrowth and dissemination by antibody-dependent cell-mediated cytotoxicity, and were found favorably predictive of survival in early breast cancer patients. There is no commercial enzyme-linked immunosorbent assay (ELISA) kit for detecting the anti-MUC1 antibodies in human serum thus far. To detect circulating anti-MUC1 antibodies, we established an indirect ELISA (I-ELISA) using a recombinant MUC1 protein containing six tandem repeat sequences of MUC1 after the antigenicity and specificity of the protein were confirmed. The I-ELISA had a sensitivity of 91.3% and a specificity of 94.1% when a competitive I-ELISA was used as a reference test. The results showed that more patients with benign breast tumors (P ‫؍‬ 0.001) and breast cancer patients before primary treatment (P ‫؍‬ 0.010) were found to have anti-MUC1 IgG than healthy women; anti-MUC1 IgG before primary treatment was found more than after primary treatment (P ‫؍‬ 0.016) in breast cancer patients. Interestingly, the anti-MUC1 IgG serum level was reversely correlated to that of CA15-3 antigen in advanced-stage patients (r ‫؍‬ ؊0.4294, P ‫؍‬ 0.046). Our study has demonstrated the suitability of the established I-ELISA for detecting circulating anti-MUC1 antibodies in human serum. Furthermore, we found that circulating anti-MUC1 antibodies may still bind MUC1 shed into blood in stage IV breast cancer, which can support the use of MUC1-target immune therapy strategies.

An oligodeoxynucleotide with CCT repeats (CCT ODN) has been found in our previous study to selectively downregulate Toll-like receptor 7/9 (TLR7/9)-mediated immune responses both in vitro and in vivo. In this study, we unexpectedly found that CCT ODN induced severe patchy hair loss around the mouth in male F1 mice (female Balb/c × male C57BL/6) with lupus-like nephritis induced by injecting allogenic lymphocytes and also in male Balb/c mice, but not in female F1 mice and Balb/c mice and either gender of C57BL/6 mice. Increased infiltration of natural killer group 2, member D (NKG2D+) cells in hair loss skin and upregulated interferon-gamma (IFN-γ) messenger RNA expression in cultured splenocytes were observed in male Balb/c mice. The CCT ODN-conditioned supernatants of cultured mouse splenocytes caused catagen-like changes to hair follicles (HFs). We hypothesized that the CCT ODN could induce patchy hair loss in the male mice with certain genetic traits by mobilizing NKG2D+ cells to HFs and by inducing the production of IFN-γ from immune cells. Taken together these data indicated that a gender and genetic preference of immune-regulatory oligonucleotides is causing unexpected clinical situations such as hair loss.