Individuals fail to elicit protective antibody after hepatitis B vaccination remain at risk for hepatitis B virus infection. Analysis of the transcriptome of peripheral blood mononuclear cells (PBMCs) is essential to elucidate the characteristics of gene expression in non-responders. In this study, we enrolled seven responders who had received three injections and seven non-responders who had six injections of hepatitis B vaccine before. All the participants were then vaccinated with a three-dose boost regimen. Microarray analysis and Luminex assay were applied to examine mRNA expression and Th1/Th2/Th9/Th17/Th22/Treg cytokine and chemokine profiles in non-responders and responders. Differentially expressed genes in PBMCs of non-responders at 5 time points, i.e. pre-vaccination, 3^rd, 7^th, 28^th day post the first dose vaccination and 7^th day post the second dose vaccination indicated a dense network trend. Compared with responders, nine coding genes (BPI, DEFA1B, DEFA4, CEACAM8, MMP8, FOLR3, LTF, TCN1 and TKTL1) were significantly up-regulated in non-responders at all 5 time points, which could probably be the characteristic genes in hepatitis B vaccine non-responsiveness. Gene ontology analysis revealed that most of the DEGs were related with immune responses. Validation results of these 9 genes using quantitative real-time polymerase chain reaction were mostly consistent with the results of microarray. Cytokine analysis demonstrated that IL-27 and CXCL12 concentrations in responders were significantly higher than non-responders on the 3^rd day after the first dose and 7^th day after the second dose of vaccination, respectively. No significant difference was observed in other cytokine and chemokine signatures between the two groups. In conclusion, our results revealed characteristic transcriptome and cytokine changes in hepatitis B vaccine non-responders after boost immunization.

Mucin 1 (MUC1) is a tumor antigen, and the most important epitopes that can induce cytotoxic T lymphocytes (CTL) reside in the variable‐number tandem repeats (VNTR). Heat shock protein (HSP) complexes isolated from tumors have been shown to induce specific anti‐tumor immunity. HSP alone can also induce nonspecific immunity. To explore the possibility to utilize the specific anti‐tumor immunity induced by MUC1 VNTR and the nonspecific immunity induced by HSP, we constructed a recombinant protein (HSP65‐MUC1) by fusing Bacillus Calmette‐Guérin‐derived HSP65 with the MUC1 VNTR peptide and tested its ability to induce anti‐tumor activities in a tumor challenge model. The growth of MUC1‐expressing tumors was significantly inhibited in mice immunized with HSP65‐MUC1, both before and after tumor challenge. A much larger percentage of immunized mice survived the tumor challenge than non‐immunized mice. Correlating with the anti‐tumor activity, HSP65‐MUC1 was shown to induce MUC1‐specific CTL as well as nonspecific anti‐tumor immunity. In the human system, HSP65‐MUC1‐loaded human DC induced the generation of autologous MUC1‐specific CTL in vitro. These results suggest that exogenously applied HSP65‐MUC1 may be used to treat MUC1 tumors by inducing the epitope‐specific CTL as well as nonspecific anti‐tumor responses mediated by the HSP part of the fusion protein.

A specific oligodeoxynucleotide (ODN), ODN MT01, was found to have positive effects on the proliferation and activation of the osteoblast-like cell line MG 63. In this study, the detailed signaling pathways in which ODN MT01 promoted the differentiation of osteoblasts were systematically examined. ODN MT01 enhanced the expression of osteogenic marker genes, such as osteocalcin and type I collagen. Furthermore, ODN MT01 activated Runx2 phosphorylation via ERK1/2 mitogen-activated protein kinase (MAPK) and p38 MAPK. Consistently, ODN MT01 induced up-regulation of osteocalcin, alkaline phosphatase (ALP) and type I collagen, which was inhibited by pre-treatment with the ERK1/2 inhibitor U0126 and the p38 inhibitor SB203580. These results suggest that the ERK1/2 and p38 MAPK pathways, as well as Runx2 activation, are involved in ODN MT01-induced up-regulation of osteocalcin, type I collagen and the activity of ALP in MG 63 cells.

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.