Artesunate enhances γδ T-cell-mediated antitumor activity through augmenting γδ T-cell function and reversing immune escape of HepG2 cells.

OBJECTIVE: To explore the effect and mechanism of artesunate on γδ T cell-mediated antitumor immune responses against hepatoma carcinoma cells (HepG2) in vitro. METHODS: Human γδ T cells or HepG2 were respectively treated with artesunate, subjected to co-culture as appropriate, and the following assays were subsequently conducted: CCK8 to examine cell viability; LDH release assay to detect the killing effect of γδ T cells on HepG2 cells; flow cytometry to examine the expression of perforin (PFP) and granzyme B (GraB) of γδ T cells; ELISA to evaluate the levels of TGF-ß1 and IL-10 in the collected supernatant of HepG2 cells pretreated with artesunate; and Western blot analysis to examine Fas, FasL, STAT3, p-STAT3 expression of HepG2 cells induced by artesunate. Results: The results showed that the cytotoxicity effect of γδ T cells pretreated with artesunate on HepG2 cells was augmented via elevating the expression of GraB in γδ T cells. Furthermore, treatment with artesunate reversed the inhibition of HepG2 cells on γδ T cells by reducing the secretion of TGF-ß1 in HepG2 cells supernatant and enhanced the antitumor effect of γδ T cells against HepG2 cells through increasing the expression of Fas on HepG2 cells, which may be attributed to the inhibition of STAT3 signaling protein. CONCLUSION: Artesunate has several mechanisms for augmenting the antitumor immune responses mediated by γδ T cells. These results suggested artesunate may be an efficacious agent in the treatment of hepatocellular carcinoma.

Extracellular Vesicles Derived from Adipose Mesenchymal Stem Cells Regulate the Phenotype of Smooth Muscle Cells to Limit Intimal Hyperplasia.

PURPOSE: Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) play important roles in the reduction of inflammation in multiple disease models. However, their role in vein graft (VG) remodeling is undefined. We aimed to investigate the effect of EVs from adipose MSCs (ADMSC-EVs) on VG intimal hyperplasia and to explore the possible mechanisms. METHODS: After generation and characterization of control-EVs and ADMSC-EVs in vitro, we investigated their effect on the proliferation and migration of vascular smooth muscle cells (VSMCs) in vitro. Next, we established a mouse model of VG transplantation. Mice underwent surgery and received control-EVs or ADMSC-EVs by intraperitoneal injection every other day for 20 days. VG remodeling was evaluated after 4 weeks. We also assessed the effect of ADMSC-EVs on macrophage migration and inflammatory cytokine expression. RESULTS: Significant inhibitory effects of ADMSC-EVs on in vitro VSMC proliferation (p < 0.05) and migration (p < 0.05) were observed compared with control-EVs. The extent of intimal hyperplasia was significantly decreased in ADMSC-EV-treated mice compared with control-EV-treated mice (26 ± 8.4 vs. 45 ± 9.0 µm, p < 0.05). A reduced presence of macrophages was observed in ADMSC-EV-treated mice (p < 0.05). Significantly decreased expression of inflammatory cytokines interleukin (IL)-6 and monocyte chemoattractant protein-1 (MCP-1) was also found in the ADMSC-EV-treated group (both p < 0.05). In addition, phosphorylation of Akt, Erk1/2, and p38 in VGs was decreased in the ADMSC-EV-treated group. CONCLUSIONS: We demonstrated that ADMSC-EVs exert an inhibitory effect on VG neointima formation by regulating VSMC proliferation and migration, macrophage migration, inflammatory cytokine expression, and the related signaling pathways.

Enhancement of CD3AK cell proliferation and killing ability by α-Thujone.

Thujone is a monoterpene ketone natural substance found mainly in wormwood and sage. Previous studies have shown that Thujone has various pharmacological effects, such as anti-tumor, analgesic, and insecticide. The effect of α-Thujone to human immune cells is still unknown. Our study focuses on investigating the effects and mechanism of α-Thujone to CD3AK (anti- CD3 antibody induced activated killer) cells proliferation and cytotoxicity to colon cancer cell lines. With cell proliferation and FCM assay, it is found that α-Thujone could significantly enhance CD3AK cell proliferation and expression of CD107a in a dose-dependent manner. The cytotoxicity to colon cancer cells detected by CCK-8 assay is also improved. The expressions of TNF-α and FasL detected with ELISA assay were not significantly changed. Mechanically, the study shows that α-Thujone could enhance the expression of p-ERK1/2 and p-Akt. In addition, α-Thujone has no cytotoxicity to HCT116 and SW620 cells proliferation. In a word, α-Thujone enhances CD3AK cell proliferation and cytotoxicity via the improvement of expression of CD107a, p-Akt and p-ERK1/2.

[Activation of Wnt/ß-catenin pathway in NK cells by glycogen synthase kinase-3ß inhibitor TWS119 promotes the expression of CD62L].

OBJECTIVE: To investigate the effect of Wnt/ß-catenin pathway activation by glycogen synthase kinase-3ß inhibitor 4,6-disubstituted pyrrolopyrimidine (TWS119) on proliferation and phenotypic characteristics of human nature killer (NK) cells. METHODS: The peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers and added to the complete medium containing recombinant human interleukin-2 (rhIL-2) and human AB serum to isolate NK cells from PBMCs. After co-cultured with 0-8.0 µmol/L TWS119 for 72 hours, growth curve and Wnt/ß-catenin activation of NK cells in each group were determined by CCK-8 and Western blotting. The CD107a and CD62L (L-selectin) expressions in the NK cells were detected using flow cytometry. RESULTS: NK cells were amplified to (61.76 ± 3.74)% after human PBMCs were cultured for 10 days. The 0-2.0 µmol/L TWS119 could promote the growth of NK cells in a dose-dependent manner, and the proliferation rate gradually dropped when TWS119 was more than 2.0 µmol/L. 0-8.0 µmol/L TWS119 could activate Wnt/ß-catenin pathway and up-regulate the expression of CD62L in a dose-dependent manner, but it decreased the expression of CD107a. CONCLUSION: Human NK cells isolated from peripheral blood treated with TWS119 gave rise to early mature CD62L⁺ NK cells.

The ADP antagonist MRS2179 regulates the phenotype of smooth muscle cells to limit intimal hyperplasia.

PURPOSE: ADP plays an important part in platelet aggregation by activating P2Y1 and P2Y12 receptors. The ADP antagonist MRS2179 has been used in thrombosis-related treatments but its effects on vein graft (VG) remodeling is undefined. We examined the effect of MRS2179 on VG intimal hyperplasia and explored the mechanism of action. METHODS: A mouse model of VG transplantation was established. Mice underwent surgery and received MRS2179 by intraperitoneal injection every other day for 3 weeks. VG remodeling was assessed 4-weeks later. Vascular smooth muscle cells (VSMCs) were isolated and treated with MRS2179. The effect of MRS2179 on the proliferation, migration and inflammatory-cytokine expression of VSMCs was also evaluated. RESULTS: MRS2179 significantly inhibited VSMC proliferation compared with the control group. Significant inhibitory effects of MRS2179 on VSMC migration was observed in two-dimensional and three-dimensional models. The extent of intimal hyperplasia was significantly less in MRS2179 treated mice than in controls. Reduced migration of macrophage was found in MRS2179 treated mice. Expression of the inflammatory cytokines IL-1ß and TNF-α was decreased significantly in the MRS2179 treated group. In addition, decreased phosphorylation was found on Akt, Erk1/2 and p38. CONCLUSIONS: These data demonstrate that MRS2179 inhibits neointima formation in VGs by regulating the proliferation, and migration of VSMCs, macrophage migration, inflammatory-cytokine secretion and related signaling pathway. Our study provides novel insights regarding purinergic signaling in SMCs in vivo. The P2Y1 receptor may serve as a therapeutic target in neointima formation.

Enhancement effect of dihydroartemisinin on human γδ T cell proliferation and killing pancreatic cancer cells.

γδ T cells play important roles in innate immunity against tumors and infections. Inhibitory effect of dihydroartemisinin on growth of cancer cells has been found in recent years. In this study, we investigated the effect of dihydroartemisinin on human γδ T cell proliferation by MTT assay and killing activity against pancreatic cancer cells SW1990, BxPC-3 and PANC-1 by LDH release assay in vitro. Intracellular molecule alterations were verified by flow cytometry. The results suggested that appropriate concentration of dihydroartemisinin favored the expansion of γδ T cells and enhanced γδ T cell mediated killing activity against pancreatic cancer cells. Up-regulation of intracellular perforin, granzyme B expression and IFN-γ production may be the important mechanism of dihydroartemisinin on increased antitumor activity of γδ T cells.

In vivo evaluation of stent patency by 64-slice multidetector CT coronary angiography: shall we do it or not?

The diagnostic performance of in-stent restenosis (ISR) by 64-slice multidetector CT coronary angiography (CTCA) has been reported to be influenced by multiple factors. We evaluated individual factors (stent diameter, material and strut thickness) and therefore determined the proper population for follow-up by using this modality. A total of 171 stents were evaluated in 83 consecutive patients with stents imaged with CTCA and conventional coronary angiography. The stent diameter ranged from 2.25 mm to 4.5 mm. 2 models of stainless steel (Taxus Liberte (Boston Scientific, US), 56 stents and Cypher Select (Cordis, US), 34 stents) and 2 models of cobalt alloy (Endeavor (Medtronic, US), 33 stents and Firebird2 (MicroPort, China), 48 stents) were included. By comparing to conventional coronary angiography, the image quality and diagnostic accuracy for ISR were evaluated. The image quality of Taxus, Endeavor and Firebird are markedly better than Cypher in large caliber group (≧3.0 mm) (P < 0.001). Except for Cypher, all other stents with diameter ≧3.0 mm showed excellent diagnostic accuracy (sensitivity 100%, specificity 94.4-96% whereas stents with diameter <3.0 mm had poor diagnostic accuracy (sensitivity 100%, specificity 33.3-70%). Cypher is the stent with thickest strut in our study, and showed reduced image quality and diagnostic accuracy in all stent size, due to large number of unassessable stents. Among 16 binary ISR, 12 lesions were correctly diagnosed by CTCA while the other 4 lesions were unassessable. The main reason for low specificity in small caliber group is the large number of unassessable stents. CTCA has high diagnostic accuracy to identify ISR in selected stents with a diameter of ≧3.0 mm.

Serum levels of osteocalcin are inversely associated with the metabolic syndrome and the severity of coronary artery disease in Chinese men.

OBJECTIVE: Osteocalcin is a bone-derived protein and has been shown to play an important role in regulating glucose and fat metabolism. We therefore investigated the association of serum levels of osteocalcin with the metabolic syndrome (MS) and coronary atherosclerosis in Chinese men. RESEARCH DESIGN AND METHODS: Serum osteocalcin levels were measured by an electrochemiluminescence immunoassay in 181 men who underwent coronary angiography, and their association with the MS and the severity of coronary artery disease (CAD) were studied. RESULTS: Osteocalcin levels in patients with the MS were significantly lower compared with those in non-MS subjects (P < 0·001) and decreased correspondingly with the increasing number of components of the MS (P < 0·001). Multiple logistic regression analysis demonstrated that osteocalcin was independently associated with the MS (OR = 0·060, 95%CI: 0·005-0·651). In multiple stepwise regression analysis, waist circumference (P = 0·001) and fasting plasma glucose (P = 0·002) were independently associated with serum osteocalcin. Subgroup analysis in 60 subjects with normal glucose tolerance showed that serum osteocalcin decreased significantly in patients with CAD compared with those without CAD (P = 0·029) and decreased significantly as the number of stenotic vessels increased (P = 0·033). Furthermore, serum osteocalcin showed an independent correlation with coronary atherosclerosis index (standardized ß = -0·497, P = 0·003). CONCLUSION: Serum osteocalcin is inversely associated with the MS as well as the severity of coronary atherosclerosis in Chinese men, supporting the new concept that bone has the reciprocal regulation with energy metabolism.