Vitamin D-Dimer: A Possible Biomolecule Modulator in Cytotoxic and Phagocytosis Processes?

Background: Vitamin D3 complexed to deglycosylated vitamin D binding protein (VitD-dgVDBP) is a water-soluble vitamin D dimeric compound (VitD-dgVDBP). It is not clear how VitD-dgVDBP affects circulating monocytes, macrophages, other immune cell systems, including phagocytosis and apoptosis, and the generation of reactive oxygen species (ROS) compared to dgVDBP. Methods: Flow cytometry was used to measure superoxide anion radical (O2*−) levels and macrophage activity in the presence of VitD-dgVDBP or dgVDBP. VitD-dgVDBP was incubated with normal human lymphocytes (nPBMCs), and several clusters of determination (CDs) were estimated. dgVDBP and VitD-dgVDBP apoptosis was estimated on malignant prostatic cells. Results: The macrophage activity was 2.8-fold higher using VitD-dgVDBP (19.8·106 counts) compared to dgVDBP (7.0·106 counts), but O2*− production was 1.8-fold lower in favor of VitD-dgVDBP (355·103 counts) compared to dgVDBP (630·106 counts). The calculated ratio of the radical/macrophage activity was 5-fold lower compared to that of dgVDBP. Only VitD-dgVDBP activated caspase-3 (8%), caspase-9 (13%), and cytochrome-C (11%) on prostatic cancer cells. PE-Cy7-labeled VitD-dgVDBP was found to bind to cytotoxic suppressor cells, monocytes/macrophages, dendritic and natural killer cells (CD8+), and helper cells (CD4+). After 12 h of co-incubation of nPBMCs with VitD-dgVDBP, significant activation and expression were measured for CD16++/CD16 (0.6 ± 0.1% vs. 0.4 ± 0.1%, p < 0.05), CD45k+ (96.0 ± 6.0% vs. 84.7 ± 9.5%, p < 0.05), CD85k+ (24.3 ± 13.2% vs. 3.8 ± 3.2%, p < 0.05), and CD85k+/CD123+ (46.8 ± 8.1% vs. 3.5 ± 3.7%, p < 0.001) compared to the control experiment. No significant difference was found using CD3+, CD4+, CD8+, CD4/CD8, CD4/CD8, CD16+, CD16++, CD14+, or CD123+. A significant decline in CD14+/CD16+ was obtained in the presence of VitD-dgVDBP (0.7 ± 0.2% vs. 3.1 ± 1.7%; p < 0.01). Conclusion: The newly developed water-soluble VitD3 form VitD-dgVDBP affected cytotoxic suppressor cells by activating the low radical-dependent CD16 pathway and seemed to induce apoptosis in malignant prostatic cells.

The Responsiveness of Bee Venom Phospholipase A2 on Regulatory T Cells Correlates with the CD11c+CD206+Population in Human Peripheral Blood Mononuclear Cells.

Bee venom phospholipase A2 (bvPLA2) has been reported to have therapeutic effects such as neuroprotection, anti-inflammation, anti-nociception, anti-cancer properties, caused by increasing regulatory T cells (Tregs). The mechanism of Tregs modulation by bvPLA2 has been demonstrated by binding with the mannose receptor, CD206 in experimental models of several diseases. However, it remains unknown whether this mechanism can also be applied in human blood. In this study, we collected peripheral blood samples from healthy donors and analyzed the percentages of monocyte-derived dendritic cells with CD206 (CD206+ DCs) before expansion, the proportion of Tregs, and the subpopulations after expansion treated with bvPLA2 or PBS using flow cytometry and the correlations among them. The percentage of Tregs tended to be higher in the bvPLA2 group than in the control group. There were significant positive correlations between the CD206 population in hPBMC and the proportions of Tregs treated with bvPLA2, especially in the Treg fold change comparing the increase ratio of Tregs in bvPLA2 and in PBS. These findings indicate that bvPLA2 increased the proportion of Tregs in healthy human peripheral blood and the number of CD206+ DCs could be a predictor of the bvPLA2 response of different individuals.

Inhibitory effect of porphyran on lipopolysaccharide-induced activation of human immune cells.

In mice, porphyran extracted from Pyropia yezoensis exerts anti-inflammatory effects and suppresses lipopolysaccharide (LPS)-induced immune activation and sepsis. Here, we investigated inhibition of LPS-induced activation of human immune cells by porphyran and the underlying mechanisms. We demonstrated that porphyran may inhibit LPS-induced proinflammatory cytokine production in peripheral blood mononuclear cells, monocyte-derived dendritic cells, and peripheral blood dendritic cells. We also observed that porphyran-mediated LPS-induced activation of DC suppressed syngeneic T cell proliferation, resulting in reduced production of interferon-γ. The mechanism of LPS-induced immune cell activation requires the activation of toll like receptor 4 following binding of LPS to myeloid differentiation factor 2 (MD2). Additionally, we show that porphyran competes with LPS for binding to MD2 and thereby suppresses immune cell activation. Porphyran may, therefore, be a promising therapeutic candidate for the treatment of endotoxin-mediated septic shock and inflammation in humans.

Effect and mechanism of human gingival mesenchymal stem cell on B cells / 器官移植

Objective To investigate the regulating function of human gingival mesenchymal stem cell (GMSC) on the proliferation and differentiation of B cells and its underlying molecular mechanism. Methods GMSC were isolated and B cells were isolated from peripheral blood. GMSC or fibroblasts were co-cultured with B cells in vitro and assigned into the GMSC group and fibroblast group. The proliferation of B cells was detected in two groups. The expression of IgG1 and IgM in the cell supernatants was measured between two groups. The secretion of interleukin (IL)-6, Perforin, interferon (IFN)-γ and tumor necrosis factor (TNF)-α was compared between two groups. The expression levels ofIL-10 and transforming growth factor (TGF)-β in B cells were detected between two groups. The expression of PC-1 in B cells was measured in two groups. The signaling pathway involved with the regulating effect of GMSC on B cell function was investigated. The regulating effect of GMSC on the role of B cells in activating T cell function was assessed. Results Compared with the fibroblast group, the proliferation of B cells was significantly weakened in the GMSC group (P < 0.05). Co-culture of GMSC and B cells significantly inhibited the secretion of IgG1 and IgM from B cells and the secretion ofIL-6, Perforin, IFN-γ and TNF-α (all P < 0.05). Compared with the fibroblast group, the secretion of IL-10 and TGF-βwas significantly higher in the GMSC group (both P < 0.05). The expression level of PC-1 in the GMSC group was significantly down-regulated (P < 0.05). After adding ALK5, an inhibitor of TGF-β receptor, the inhibitory effect of GMSC upon B cells was significantly weakened (P < 0.05). Compared with the fibroblast group, the ability of B cells to activate and proliferate T cells was significantly attenuated in the GMSC group (P < 0.05). Conclusions GMSC can inhibit B cells and their mediated immune responses. The activation of B cells and other related functions can be suppressed through the TGF-β signaling pathway.

Development of a preclinical humanized mouse model to evaluate acute toxicity of an influenza vaccine.

Safety evaluation of a human vaccine is critical for vaccine development and for preventing an unexpected adverse reaction in humans. Nonetheless, to date, very few systems have been described for preclinical studies of human adverse reactions in vivo. Previously, we have identified biomarker genes expressed in the lungs for evaluation of influenza vaccine safety, and their usefulness in rodent models and for adjuvant-containing vaccines has already been reported. Here, our purpose was to develop a novel humanized mouse model retaining human innate-immunity-related cells to assess the safety of influenza vaccines using the previously identified biomarker genes. In the present study, we tested whether the two humanized models, a short-term and long-term reconstitution model of NOD/Shi-scid IL2rγnull mice, are suitable for biomarker gene-based safety evaluation. In the short-term model, human CD14+ cells, plasmacytoid dendritic cells, CD4+ and CD8+ T cells, and B cells were retained in the lungs. Among these cells, human CD14+ cells and plasmacytoid dendritic cells were not detected in the lungs of the long-term model. After the vaccination, the expression levels of human biomarker genes were elevated only in the short-term model when the toxicity reference vaccine was inoculated. This phenomenon was not observed in the long-term model. The levels of human cytokines and chemokines in the lungs increased in response to the toxicity reference vaccine in the short-term mouse model. According to these results, the short-term model provides a better platform for evaluating vaccine safety in terms of human peripheral blood mononuclear cell-mediated initial reactions in vivo.

Increased histone deacetylase activity in peripheral blood mononuclear cells of patients with schizophrenia.

This study investigates the association between histone deacetylase (HDAC) activity in human peripheral blood mononuclear cells (PBMCs) and schizophrenia. Data were derived from a case-control association study of 19 unrelated adult patients with schizophrenia and 21 matched healthy controls. HDAC activity was measured with a HDAC activity colorimetric assay kit. Our findings suggest that HDAC activity in PBMCs is higher in patients with schizophrenia than in healthy people.

In vitro transdifferentiation of human peripheral blood mononuclear cells to photoreceptor-like cells.

Direct reprogramming is a promising, simple and low-cost approach to generate target cells from somatic cells without using induced pluripotent stem cells. Recently, peripheral blood mononuclear cells (PBMCs) have attracted considerable attention as a somatic cell source for reprogramming. As a cell source, PBMCs have an advantage over dermal fibroblasts with respect to the ease of collecting tissues. Based on our studies involving generation of photosensitive photoreceptor cells from human iris cells and human dermal fibroblasts by transduction of photoreceptor-related transcription factors via retrovirus vectors, we transduced these transcription factors into PBMCs via Sendai virus vectors. We found that retinal disease-related genes were efficiently detected in CRX-transduced cells, most of which are crucial to photoreceptor functions. In functional studies, a light-induced inward current was detected in some CRX-transduced cells. Moreover, by modification of the culture conditions including additional transduction of RAX1 and NEUROD1, we found a greater variety of retinal disease-related genes than that observed in CRX-transduced PBMCs. These data suggest that CRX acts as a master control gene for reprogramming PBMCs into photoreceptor-like cells and that our induced photoreceptor-like cells might contribute to individualized drug screening and disease modeling of inherited retinal degeneration.

Cytotoxic and inflammatory responses of TiO2 nanoparticles on human peripheral blood mononuclear cells.

Titanium dioxide nanoparticles (TiO2 -NPs) have been widely used in many applications. Owing to their nanoscale size, interactions between cells and NPs have been expansively investigated. With the health concerns raised regarding the adverse effects of these interactions, closer examination of whether TiO2 -NPs can induce toxicity towards human cells is greatly needed. Therefore, in this study, we investigated the cytotoxicity of TiO2 -NPs towards human blood cells (peripheral blood mononuclear cells [PBMCs]) in serum-free medium, for which there is little information regarding the cytotoxic effects of TiO2 -NPs. Our results provide evidence that PBMCs treated with TiO2 -NPs (at concentrations ≥25 µg ml(-1) ) for 24 h significantly reduced cell viability and significantly increased production of toxic mediators such as reactive oxygen species and inflammatory response cytokines such as interleukin-6 and tumor necrosis factor-α (P < 0.05). Cell apoptosis induction also occurred at these concentrations. Significant expressions of cyclooxygenase-2 and interleukin-1ß were also observed in PBMCs treated with TiO2 -NPs at concentrations ≥125 µg ml(-1) . Our data presented here clearly indicate that the concentration of TiO2 -NPs (at size ~26.4 ± 1.2 nm) applied to human blood cells has a strong impact on cytotoxic induction. Copyright © 2016 John Wiley & Sons, Ltd.

Effects of functionalization of multi-walled carbon nanotubes on human peripheral blood mononuclear cell / 国际生物医学工程杂志

Objective To explore the effects of surface functionalized multi-walled carbon nanotubes (FMWCNTs) on the cytotoxicity of human peripheral blood mononuclear cell (PBMC).Methods Five different types of MWCNTs (hydroxylated,carboxylated,aminated,nickel-plated and pristine MWCNTs (P-MWCNTs)) with the same diameter and length were evaluated the dispersion and characterizations in physiological salt solution by transmission electron microscopy.PBMC were isolated by density gradient centrifugation from human peripheral blood,and 5 types of MWCNTs were ultrasonically dispersed in serum-containing medium respectively.After incubation with PBMC for 12,24,48 or 72 h,cytotoxicity was detected by CCK-8 kits.Results All the MWCNTs had well dispersion,especially the F-MWCNTs.Cytotoxicity results showed that all types of MWCNTs could induced PBMC death,and presented dose-dependence manner and a certain degree of time-dependence manner.Compared with the P-MWCNTs,F-MWCNTs changed cytotoxicity statistically,with the hydroxylated,carboxylated,aminated MWCNTs weakened,aminated MWCNTs significant (P＜0.05),nevertheless the nickel-plated MWCNTs increased.Compared with the P-MWCNTs (25 μg/ml),cell viability of PBMC after 24 and 48 h incubation with the same dose of nickelplated MWCNTs both decreased,and the differences was statistically significant (P＜0.01,P＜0.05).Conclusions The functional group modification affects not only the MWCNTs dispersion in medium,but also the cytotoxicity of the MWCNTs on PBMC.

Targeting Hedgehog signaling pathway and autophagy overcomes drug resistance of BCR-ABL-positive chronic myeloid leukemia.

The frontline tyrosine kinase inhibitor (TKI) imatinib has revolutionized the treatment of patients with chronic myeloid leukemia (CML). However, drug resistance is the major clinical challenge in the treatment of CML. The Hedgehog (Hh) signaling pathway and autophagy are both related to tumorigenesis, cancer therapy, and drug resistance. This study was conducted to explore whether the Hh pathway could regulate autophagy in CML cells and whether simultaneously regulating the Hh pathway and autophagy could induce cell death of drug-sensitive or -resistant BCR-ABL(+) CML cells. Our results indicated that pharmacological or genetic inhibition of Hh pathway could markedly induce autophagy in BCR-ABL(+) CML cells. Autophagic inhibitors or ATG5 and ATG7 silencing could significantly enhance CML cell death induced by Hh pathway suppression. Based on the above findings, our study demonstrated that simultaneously inhibiting the Hh pathway and autophagy could markedly reduce cell viability and induce apoptosis of imatinib-sensitive or -resistant BCR-ABL(+) cells. Moreover, this combination had little cytotoxicity in human peripheral blood mononuclear cells (PBMCs). Furthermore, this combined strategy was related to PARP cleavage, CASP3 and CASP9 cleavage, and inhibition of the BCR-ABL oncoprotein. In conclusion, this study indicated that simultaneously inhibiting the Hh pathway and autophagy could potently kill imatinib-sensitive or -resistant BCR-ABL(+) cells, providing a novel concept that simultaneously inhibiting the Hh pathway and autophagy might be a potent new strategy to overcome CML drug resistance.

Identification of a Bioactive Compound against Adult T-cell Leukaemia from Bitter Gourd Seeds.

In our previous report, an 80% ethanol bitter gourd seed extract (BGSE) was found to suppress proliferation of adult T-cell leukemia (ATL) cell lines. The present study aimed to identify the bioactive compounds from BGSE specific against ATL. From the result of an HPLC-MS analysis, α-eleostearic acid (α-ESA) was present in BGSE at 0.68% ± 0.0022% (±SD, n = 5). In the cell proliferation test, α-ESA potently suppressed proliferation of two ATL cell lines (ED and Su9T01; IC50 = 8.9 and 29.3 µM, respectively) more than several other octadecanoic acids. However, α-ESA moderately inhibited phytohemagglutinin-activated human peripheral blood mononuclear cells (PBMC; IC50 = 31.0 µM). These results suggest that BGSE-derived α-ESA has potential as a functional food constituent because of its activity against ATL, particularly against ED cells. Moreover, α-ESA might be effective for the prevention of moderate adverse effects of ATL on normal T cells.