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Calpains have been implicated in heart diseases. While calpain-1 has been detrimental to the heart, the role of calpain-2 in cardiac pathology remains controversial. In this study we investigated whether sustained over-expression of calpain-2 had any adverse effects on the heart and the underlying mechanisms. Double transgenic mice (Tg-Capn2/tTA) were generated, which express human CAPN2 restricted to cardiomyocytes. The mice were subjected to echocardiography at age 3, 6, 8 and 12 months, and their heart tissues and sera were collected for analyses. We showed that transgenic mice over-expressing calpain-2 restricted to cardiomyocytes had normal heart function with no evidence of cardiac pathological remodeling at age 3 months. However, they exhibited features of dilated cardiomyopathy including increased heart size, enlarged heart chambers and heart dysfunction from age 8 months; histological analysis revealed loss of cardiomyocytes replaced by myocardial fibrosis and cardiomyocyte hypertrophy in transgenic mice from age 8 months. These cardiac alterations closely correlated with aberrant autophagy evidenced by significantly increased LC3BII and p62 protein levels and accumulation of autophagosomes in the hearts of transgenic mice. Notably, injection of 3-methyladenine, a well-established inhibitor of autophagy (30 mg/kg, i.p. once every 3 days starting from age 6 months for 2 months) prevented aberrant autophagy, attenuated myocardial injury and improved heart function in the transgenic mice. In cultured cardiomyocytes, over-expression of calpain-2 blocked autophagic flux by impairing lysosomal function. Furthermore, over-expression of calpain-2 resulted in lower levels of junctophilin-2 protein in the heart of transgenic mice and in cultured cardiomyocytes, which was attenuated by 3-methyladenine. In addition, blockade of autophagic flux by bafilomycin A (100 nM) induced a reduction of junctophilin-2 protein in cardiomyocytes. In summary, transgenic over-expression of calpain-2 induces age-dependent dilated cardiomyopathy in mice, which may be mediated through aberrant autophagy and a reduction of junctophilin-2. Thus, a sustained increase in calpain-2 may be detrimental to the heart.

Gastric cancer tissue-derived mesenchymal stem cells (GC-MSCs) are important resident stromal cells in the tumor microenvironment (TME) and have been shown to play a key role in gastric cancer progression. Whether GC-MSCs exert a tumor-promoting function by affecting anti-tumor immunity is still unclear. In this study, we used GC-MSC conditioned medium (GC-MSC-CM) to pretreat peripheral blood mononuclear cells (PBMCs) from healthy donors. We found that GC-MSC-CM pretreatment markedly reversed the inhibitory effect of PBMCs on gastric cancer growth in vivo, but did not affect functions of PBMCs on gastric cancer cell proliferation, cell cycle and apoptosis in vitro. PBMCs pretreated with GC-MSC-CM significantly promoted gastric cancer migration and epithelial-mesenchymal transition in vitro and liver metastases in vivo. Flow cytometry analysis showed that GC-MSC-CM pretreatment increased the proportion of Treg cells and reduced that of Th17 cells in PBMCs. CFSE labeling and naive CD4+ T cells differentiation analysis revealed that GC-MSC-CM disrupted the Treg/Th17 balance in PBMCs by suppressing Th17 cell proliferation and inducing differentiation of Treg cells. Overall, our collective results indicate that GC-MSCs impair the anti-tumor immune response of PBMCs through disruption of Treg/Th17 balance, thus providing new evidence that gastric cancer tissue-derived MSCs contribute to the immunosuppressive TME.

To investigate the effect of IL-17 on the expression of collagen I/III in cardiac fibroblasts and analyze its molecular mechanism.Cardiac fibroblasts were isolated from 7-14-day-old BALB/c mice and cultured in DMEM with 10% fetal bovine serum (FBS). The cells were collected after IL-17 treatment for 0, 24, 48, 72 h. IL-17 receptors on cardiac fibroblasts were detected by PCR; the collagen I/III expression was analyzed by immunofluorescence; the PKCβ, Erk1/2, NF-κB phosphorylation were investigated by Western blotting.IL-17RA/C was expressed on cardiac fibroblasts; after 24 h of IL-17 stimulation, the collagen I/III expression obviously increased; Western blotting showed that PKCβ, Erk1/2 and NF-κB were phosphorylated on 30, 45, 45 min, respectively.IL-17 could induce the expression of collagen I/III in cardiac fibroblasts, which might be related with PKCβ-ERK1/2-NF-κB phosphorylation.

To construct an enterovirus 71(EV71) multiepitope-mGITRL eukaryotic plasmid and study its immunogenicity in BALB/c mice.We first designed and synthesized VP1' epigene containing two B cells and two T cells epitopes of VP1, and amplified mGITRL gene by PCR. The VP1' epigene and mGITRL gene were then cloned into the expression vector pIRES to construct the recombination plasmid pIRES-VP1'-mGITRL. The recombination plasmid was transfected into COS7 cells by liposome-mediated method. The protein expressions of VP1' and mGITRL were detected by Western blotting. BALB/c mice were immunized with pIRES-VP1'-mGITRL plasmid, and its serum antibody titer was measured by ELISA.The recombination plasmid pIRES-VP1'-mGITRL was successfully constructed as demonstrated by sequencing. Western blot analysis indicated that the VP1'-mGITRL fusion protein was expressed in COS7 cells and muscle cells. After BALB/c mice were immunized with this plasmid, we detected the high titer of anti-VP1 antibody in serum.VP1'-mGITRL fusion protein can be highly expressed in COS7 cells and muscle cells by the construction and transfection of the recombination plasmid pIRES-VP1'-mGITRL, and it could elicit the dramatic immune response in mice.

To investigate whether cardiac fibroblasts (CFs) treated by LPS can actively secrete high-mobility group box protein 1 (HMGB1) and to analyze the correlation between HMGB1 releasing and the accumulation of collagen type I , III .CFs were isolated from the heart of 7-14-day-old BALB/c mice and cultured in DMEM with 10% fetal bovine serum (FBS). We collected the CFs and cell supernatants after treated by LPS for 0, 6, 12, 24, 36, 48 h, respectively. The mRNA and protein expression levels of HMGB1, collagen 1a1 (col1a1) and collagen 3a1 (col3a1) in CFs after LPS stimulation were detected by RT-PCR and Western blotting, respectively. The intracellular localization of HMGB1 in treated CFs was investigated by immunofluorescence.After 0-6 h of LPS stimulation, the mRNA levels of HMGB1, col1a1, col3a1 had no significant changes; but increased obviously at 12, 24, 36, 48 h. HMGB1 was found in the cell supernatant by Western blotting after 24 h LPS stimulation, and its expression decreased following the first rise in CFs. Meanwhile, immunofluorescence showed HMGB1 translocation from nucleus to cytoplasm. The levels of col1a1 and col3a1 were up-regulated in CFs after stimulation.LPS can induce HMGB1 translocation from nucleus to cytoplasm and across cellular membrane to the outside of CFs at a time-dependent manner. Col1a1 and Col3a1, which are closely associated with myocardial fibrosis, were obviously up-regulated by LPS stimulation, which indicates that actively released HMGB1 might contribute to myocardial fibrosis following the endotoxin induced-sepsis.

To construct eukaryotic co-expression vector of Porphyromonas gingivalis outer membrane protein ragB and mouse glucocorticoid-induced tumor necrosis factor receptor ligand (mGITRL) and to analyze its immunogenicity in vivo.The ragB gene was obtained from pMD18-T-ragB, and then cloned into the eukaryotic expression vector pIRES and pIRES-mGITRL, respectively. The eukaryotic expression vectors: pIRES-ragB and pIRES-ragB-mGITRL were identified by double enzyme digestion and DNA sequencing, then transfected into COS7 cells by Lipofectamine(TM);2000. The expressions of ragB or mGITRL in COS7 cells were detected by Western blotting. The mice were immunized with the recombinant pIRES-ragB-mGITRL plasmid. The serum antibody level was determined by ELISA.pIRES-ragB and pIRES-ragB-mGITRL plasmids were successfully constructed. Western blotting showed that the targeted gene was over-expressed in COS7 cells and skeletal muscle cells, respectively. The high titers of antibodies against RagB were detected in mouse serum.The construction of pIRES-ragB-mGITRL co-expression vector provides the experimental basis for Porphyromonas gingivalis vaccine research, prevention and treatment of periodontitis.

To transfect Hlx into mouse dendritic cell line DC2.4 and observe the effect of hlx on function of dendritic cells.The eukaryotic expression vector PIRES2-EGFP/Hlx was transfected into DC2.4 by liposomes. The transfection efficiency was identified through FACS. RT-PCR and Real-time PCR were used to test the transcription level of Hlx in DC2.4. Forty-eight hours after transfection, DC2.4 cells were studied for cytokine production, cell phenotype, phagocytosis, unilateral mixed lymphocyte reaction.The pIRES2-EGFP/Hlx vector was transfected into DC2.4 with the transfection efficiency of up to 60%. Highly expressed Hlx in DC2.4 increased the expression of maturation makers including CD80 and CD86, and major histocompatibility complex-II. Functional assay showed that over-expression of Hlx in DC2.4 increased the interleukin-12 transcription and decreased DC endocytosis. The Hlx modified DC2.4 highly expressed IL-10 and TGF-β at the same time. Furthermore, it was shown that in a unilateral mixed lymphocyte reaction model, Hlx modified DC2.4 inhibited proliferation of lymphocytes.Transient over-expression of Hlx in DC2.4 promotes DC2.4 maturation and up-regulates IL-12, IL-10 and TGF-β expression. However, the Hlx modified DC2.4 cells functionally appear as regulatory dendritic cells.

To express human HMGB1 B box protein and obtain monoclonal antibodies (mAbs) against HMGB1 B box for further study of the function of human HMGB1 protein.pET28-HMGB1 B box plasmid transfected the DH5α, then expressed. And the extracted protein was purified by protein purification system. BALB/c mice were immunized with recombinant human HMGB1 B box protein. Hybridoma cell lines secreting mAb against human HMGB1 B box protein were screened by ELISA and subcloning approach. The characteristics of these mAbs were identified by ELISA and Western blot.Two hybridoma cell lines (1D2F4E3 and 2D4E3A2) stable secreting specific mAbs were successfully obtained.Western blot exhitited the two mAbs binded specifically to human HMGB1 B box protein. The immunoglobulin (Ig) class of two mAbs belonged to IgG, their titers were 1×10(6);, and the A(450); of mAb1D2F4E3, 2D4E3A2 were 0.324±0.093, 0.296±0.085, respectively.Two of high specificity mAbs against human HMGB1 B box protein have been successfully prepared, which laid the foundation for further study of biological function of human HMGB1 protein.