The aim of this study is to demonstrate the effect of extracellular calcium ion (Ca2+) and inorganic phosphate (Pi) concentrations on the growth and differentiation of bone-marrow-derived mesenchymal stem cells (MSCs), which is essential to understand the interaction between calcium phosphate ceramic (CPC) scaffolds and seeded cells during the construction of tissue-engineered bones. MSCs were separated from rabbits and cultured in media with different concentrations of Ca2+ and Pi supplements. Their proliferation, apoptosis, mineralization and osteogenic differentiation were determined by the MTT assay, TUNEL assay, Vonkossa stain and RT-PCR examination. A two-way ANOVA calculation with comparisons of estimated marginal means by LSD was used for statistical analysis. Results showed that the optimal extracellular Ca2+ and Pi concentrations for the cells to proliferate and differentiate were 1.8 mM and 0.09 mM, respectively, which are the concentrations supplied in many commonly used culture media such as DMEM and alpha-MEM. Cell proliferation and differentiation decreased significantly with greater or lower concentrations of the Pi supplement. Greater Pi concentrations also led to significant cell apoptosis. Greater Ca2+ concentrations did not change cell proliferation but significantly inhibited cell differentiation. In addition, greater Ca2+ concentrations could significantly enhance cell mineralization. In conclusion, extracellular Ca2+ and Pi significantly influence the growth and osteogenic differentiation of MSCs. It is important to take the cellular effect of Ca2+ and Pi into consideration when designing or constructing scaffolds for bone tissue engineering with CPC.

Umbilical cord mesenchymal stem cells (UC-MSCs) have been suggested as a candidate for various clinical applications, however, major limitations include the lack of organ-specific accumulation and low survival rates of transplanted cells. In the present study, it was hypothesized that the paracrine effects of UC-MSCs may enhance stem cell-based tissue repair and regeneration by promoting the specific homing of stem/progenitor cells and the overall ability to drive them to the damaged area. UC-MSCs-derived conditioned medium (UC-CM) was analyzed using liquid chip and ELISA techniques. In vitro tube formation assays of human umbilical vein endothelial cells (HUVECs) and UC-MSCs were then performed to assess the angiogenic properties of UC-CM. Subsequently, UC-MSCs, HUVECs and fibroblasts were labeled with PKH26 for an in vivo cell migration assay. The expression levels of C-X-C chemokine receptor 4 (CXCR4), C-C chemokine receptor 2 (CCR2) and c-met were determined in the UC-MSCs, HUVECs and fibroblasts using reverse transcription-quantitative polymerase chain reaction and flow cytometry. UC-CM was incubated with or without antibodies, and the contribution of stromal cell-derived factor 1 (SDF-1), monocyte chemotactic protein 1 (MCP-1) and hepatocyte growth factor (HGF) on the migration of cells was investigated in vitro. The results demonstrated that UC-MSCs secreted different cytokines and chemokines, including increased quantities of SDF-1, MCP-1 and HGF, in addition to the angiogenic factors, vascular cell adhesion protein-1, interleukin-8, insulin-like growth factor-1 and vascular endothelial growth factor. The total lengths of the tubes were significantly increased in the UC-MSCs and HUVECs incubated in UC-CM compared with those incubated in Dulbecco’s modified Eagle’s medium. In vivo cell migration assays demonstrated that UC-CM was a chemotactic stimulus for the UC-MSCs and HUVECs. In vitro Matrigel migration and scratch healing assays demonstrated that UC-CM increased the migration of CXCR4-postive or/and CCR2-positive cells in a dose-dependent manner. In addition, different molecules were screened under antibody-based blocking migration conditions. The data revealed that the SDF-1/CXCR4 and MCP-1/CCR2 axes were involved in the chemoattractive activity of UC-CM and suggested that the effective paracrine factor of UC-CM is a large complex rather than a single factor. The results of the present study supported the hypothesis that UC-MSCs release soluble factors, which may extend the therapeutic applicability of stem cells.

No abstract

γδ-T cells play an indispensable role in host defense against different viruses, including influenza A virus. However, whether these cells have cytotoxic activity against influenza virus-infected lung alveolar epithelial cells and subsequently contribute to virus clearance remains unknown. Using influenza virus-infected A549 cells, human lung alveolar epithelial cells, we investigated the cytotoxic activity of aminobisphosphonate pamidronate (PAM)-expanded human Vγ9Vδ2-T cells and their underlying mechanisms. We found that PAM could selectively activate and expand human Vγ9Vδ2-T cells. PAM-expanded human Vγ9Vδ2-T cells efficiently killed influenza virus-infected lung alveolar epithelial cells and inhibited virus replication. The cytotoxic activity of PAM-expanded Vγ9Vδ2-T cells was dependent on cell-to-cell contact and required NKG2D activation. Perforin-granzyme B, tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas-Fas ligand (FasL) pathways were involved in their cytotoxicity. Our study suggests that targeting γδ-T cells by PAM can potentially offer an alternative option for the treatment of influenza virus.

The human zona pellucida (ZP) is a highly organized glycoprotein matrix that encircles oocytes and plays an essential role in successful reproduction. Previous studies have reported that mutations in human ZP1, ZP2 and ZP3 influence their functions and result in a lack of ZP or in an abnormal oocytes and empty follicle syndrome, which leads to female infertility. Here, we performed whole‐exome sequencing in two probands with primary infertility whose oocytes lacked a ZP, and we identified a heterozygous mutation in ZP1 (NM_207341:c.326G>A p.Arg109His), which is situated in the N‐terminus, and a heterozygous mutation in ZP3 (NM_001110354:c.400G>A p.Ala134Thr), which is situated in the ZP domain. The effects of the mutations were investigated through structure prediction and in vitro studies in HeLa cells. The results, which were in line with the phenotype, suggested that these mutations might impede the function of cross‐linking and secretion of ZP proteins. Our study showed that the two mutations in ZP1 and ZP3 influenced the formation of the ZP, causing female infertility. Meanwhile, these data highlight the importance of the ZP1 N‐terminus in addition to the conserved domains for ZP1 function and ZP formation. Additionally, the patient with the ZP1 mutation delivered a baby following intracytoplasmic sperm injection (ICSI); thus, we suggest the targeted genetic diagnosis of ZP genes to choose appropriate fertilization methods and improve the success rate of assisted reproductive technology (ART) treatments.

Various proteins are expressed during different stages of schistosome development that are essential for cercarial penetration of vertebrate skin and evasion of host immune response. CD4(+)CD25(+) regulatory T cells are important in modulating immune responses towards helminth infections. Schistosoma japonicum protein Sj16 present in the secretions of schistosomula has been shown to have anti-inflammatory effects; however, it is uncertain whether Sj16 can induce CD4(+)CD25(+) regulatory T cells to participate in the regulation of early infection. In this study, we demonstrate a relationship between recombinant Sj16 (rSj16) and the induction of CD4(+)CD25(+) Foxp3(+) regulatory T cells. An increase in CD4(+)CD25(+) T cells was observed both in splenic cells from mice injected with rSj16 and the cells pretreated with rSj16, respectively. The induced CD4(+)CD25(+) T cells suppressed CD4(+)CD25(-) T-cell proliferation; furthermore, IFN-γ and IL-10 released from rSj16-stimulated cells contribute to this suppression. Additionally, rSj16-treated bone marrow dendritic cells (BMDCs) demonstrate an immature phenotype and play a role in the conversion of CD4(+)CD25(-) T cells into suppressive CD4(+)CD25(+) regulatory T cells. Our study identified a new CD4(+)CD25(+) T-cell population that induced by rSj16 and suggests that an IFN-γ-biased microenvironment during early infection of schistosome may favour the establishment of infection.

Hepatic stellate cells (HSCs) play a key role in the development of liver fibrosis caused by schistosomiasis. Chemokines were widely expressed and involved in cellular activation, proliferation and migration in inflammatory and infectious diseases. However, little is known about the expressions of chemokines on HSCs in the schistosoma infection. In addition, the roles of chemokines in pathogenesis of liver fibrosis are not totally clear. In our study, we used microarray to analyze the temporal gene expressions of primary HSCs isolated from mice with both acute and chronic schistosomiasis. Our microarray data showed that most of the chemokines expressed on HSCs were upregulated at 3 weeks post-infection (p.i) when the egg granulomatous response was not obviously evoked in the liver. However, some of them like CXCL9, CXCL10 and CXCL11 were subsequently decreased at 6 weeks p.i when the granulomatous response reached the peak. In the chronic stage, most of the differentially expressed chemokines maintained persistent high-abundances. Furthermore, several chemokines including CCR2, CCR5, CCR7, CXCR3, CXCR4, CCL2, CCL5, CCL21, CXCL9 and CXCL10 were expressed by HCSs and the abundances of them were changed following the praziquantel treatment in the chronic stage, indicating that chemokines were possibly necessary for the persistence of the chronic stage. In vitro experiments, hepatic non-parenchymal cells, primary HSCs and human HSCs line LX-2 were stimulated by chemokines. The results showed that CXCL9 and CXCL10, but not CXCL11 or CXCL4, significantly inhibited the gene expressions of Col1α1, Col3α1 and α-SMA, indicating the potential anti-fibrosis effect of CXCL9 and CXCL10 in schistosomiasis. More interestingly, soluble egg antigen (SEA) of Schistosoma japonicum was able to inhibit transcriptional expressions of some chemokines by LX-2 cells, suggesting that SEA was capable of regulating the expression pattern of chemokine family and modulating the hepatic immune microenvironment in schistosomiasis.

Human induced pluripotent stem cells (hiPSCs) have potential applications in cell replacement therapy and regenerative medicine. However, limited information is available regarding the immunologic features of iPSCs. In this study, expression of MHC and T cell co-stimulatory molecules in hiPSCs, and the effects on activation, proliferation and cytokine production in allogeneic human peripheral blood mononuclear cells were examined. We found that no-integrate hiPSCs had no MHC-II and T cell co-stimulatory molecules expressions but had moderate level of MHC-I and HLA-G expressions. In contrast to human skin fibroblasts (HSFs) which significantly induced allogeneic T cell activation and proliferation, hiPSCs failed to induce allogeneic CD45+ lymphocyte and CD8+ T cell activation and proliferation but could induce a low level of allogeneic CD4+ T cell proliferation. Unlike HSFs which induced allogeneic lymphocytes to produce high levels of IFN-γ, TNF-α and IL-17, hiPSCs only induced allogeneic lymphocytes to produce IL-2 and IL-10, and promote IL-10-secreting regulatory T cell (Treg) generation. Our study suggests that the integration-free hiPSCs had low or negligible immunogenicity, which may result from their induction of IL-10-secreting Treg.