稳定过表达成纤维细胞激活蛋白的口腔鳞状细胞癌细胞株的构建

目的 构建人成纤维细胞激活蛋白（FAP）过表达慢病毒载体,转染口腔鳞状细胞癌（OSCC）细胞株SCC9,构建稳定过表达FAP的OSCC细胞株。方法 采用聚合酶链反应（PCR）技术获得FAP片段,利用基因重组技术构建过表达FAP的慢病毒载体,包埋病毒并收集上清液感染SCC9细胞株,通过流式细胞荧光分选技术（FACS）进行筛选,获得稳定过表达FAP的SCC9细胞株。结果 对阳性克隆进行酶切鉴定和基因测序证明FAP的慢病毒载体构建成功,实时荧光定量PCR和Western blot检测结果证明成功构建稳定过表达FAP的SCC9细胞株。结论 本研究有助于获得纯化FAP蛋白,为进一步研究FAP在OSCC发生发展过程中的机制奠定基础。     

FTY720 ameliorates renal fibrosis by simultaneously affecting leucocyte recruitment and TGF‐β signalling in fibroblasts

Renal fibrosis is the common final manifestation of chronic kidney diseases and usually results in end‐stage renal failure. In this study, we evaluated the effect of fingolimod (FTY720), an analogue of sphingosine 1‐phosphate (S1P), as a treatment for the unilateral ureteral obstruction (UUO)‐induced renal fibrosis animal model. We treated mice with FTY720 at a dosage of 1 mg/kg/day by intragastric administration from day 1 until day 7. The control group received the same amount of saline. FTY720 reduced significantly the urine albumin/creatinine ratio (UACR) in treated UUO mice. FTY720 treatment also caused a significant decrease in interstitial expansion and collagen deposition in the kidney, accompanied by reduced mononuclear cell recruitment and inflammatory cytokine expression. In addition, the expression levels of the endothelial cell adhesion molecules P‐selectin and vascular cell adhesion protein 1 (VCAM‐1) were suppressed in the ligated kidney by FTY720 administration, suggesting reduced renal endothelial cell activation. Furthermore, in renal interstitial fibroblast normal rat kidney (NRK)‐49F cells, FTY720 significantly affected transforming growth factor (TGF)‐β‐induced α‐smooth muscle actin (SMA) expression and collagen synthesis by inhibiting both the Mothers against decapentaplegic homologue (Smad)2/3 and phosphatidylinositol 3‐kinase/protein kinase B/glycogen synthase kinase 3 beta (PI3K/AKT/GSK3β) signalling pathways. S1P1 knock‐down by siRNA reversed this effect significantly in our fibroblast cell culture model. Therefore, FTY720 attenuates renal fibrosis via two different mechanisms: first, FTY720 suppresses the synthesis of extracellular matrix in interstitial fibroblasts by interfering with TGF‐β signalling; and secondly, FTY720 affects endothelial cell activation and chemokine expression, thereby reducing immune cell recruitment into the kidney.     

Diagnostic dilemmas in HLH: Can T‐cell phenotyping help?

The diagnosis of hemophagocytic lymphohistiocytosis (HLH) can be a difficult one, and the distinction between primary versus secondary HLH can be particularly challenging during the early stages of diagnosis. This distinction is important to make as primary HLH requires allogeneic hematopoietic cell transplantation for a definitive cure. Flow cytometric screening tests for many of the genetic forms of HLH are available. However, not all patients with primary HLH are captured by these screening tests, due to the fact that no screening test is 100% sensitive, and additionally, some patients with “primary” forms of HLH may have mutations in genes which are yet to be discovered. In this issue of the European Journal of Immunology, Ammann et al. [Eur. J. Immunol. 2017. 47 : 364–373] compare T‐cell phenotype patterns among patients with primary and secondary HLH, and find that assessment of T‐cell activation and differentiation may assist with the diagnosis of HLH. Furthermore, this phenotypic analysis has the potential to help make the important distinction between primary and secondary HLH.     

Immediate moxifloxacin hypersensitivity: Is there more than currently meets the eye?

Immediate drug hypersensitivity reactions (IDHR) to moxifloxacin constitute a pathomechanistic conundrum and a diagnostic challenge. Our objective was to study whether simultaneous phenotyping and quantification of histamine release might add to our knowledge about the basophil activation properties of moxifloxacin and constitute a reliable diagnostic aid. Fifteen patients with an IDHR to moxifloxacin and nine moxifloxacin challenged controls were selected. All had a basophil activation test (BAT) with moxifloxacin. Flow cytometric analysis of basophil responses implied labeling for CD63, CD203c, and intracellular histamine. Unlike tolerant challenged controls, basophilic upregulation of CD203c in response to moxifloxacin was observed in seven of 15 patients. Only two of these seven patients demonstrated appearance of CD63 and release of histamine. In the remainder eight patients, no basophil responses were demonstrable. In conclusion, immediate hypersensitivity to moxifloxacin might involve mechanisms difficult to capture by traditional CD63‐/CD203c‐based BAT. Deciphering the complexity of quinolone IDHR seems mandatory.     

A good manufacturing practice method to ex vivo expand natural killer cells for clinical use

BackgroundGreat interest has been raised recently by the design of new adoptive immunotherapeutic strategies based on the in vivo infusion of ex vivo-expanded and activated natural killer (NK) cells. The development of good manufacturing practice (GMP) methods for the efficient production of fully functional NK cells is mandatory for clinical application.ResultsNK-cell populations expanded on average 15.7±4.7 fold by day 14, with a viability of 96% ±0.5. At the end of the incubation period, 97% ±1.1 of the expanded population was CD56⁺ NK cells; these effector cells showed significant up-regulation of the activating receptors NKG2D and DNAM-1. Functional tests demonstrated that expanded NK cells are fully functional with no difference whether tested before cryopreservation or after thawing.DiscussionThese data provide the basis for developing new NK-cell-based immunotherapeutic strategies for the treatment of patients with cancer.     

同型半胱氨酸与H型高血压患者血小板活化的相关性及依叶的干预研究

【摘要】 目的 研究H型高血压患者血同型半胱氨酸水平与其体内血小板活化因子（CD41、CD62P）关系,及依那普利叶酸片对H型高血压患者血同型半胱氨酸与血小板活化因子（CD41、CD62P）的影响。方法 选取轻中度H型高血压患者200例,将其随机分为试验组和对照组各100例。试验组给予口服依那普利叶酸片,对照组给予口服依那普利,疗程均为3个月。治疗前后分别测量患者血压、超敏C反应蛋白、同型半胱氨酸、血小板活化因子（CD41、CD62P）。结果 治疗3个月后,与对照组相比,试验组平均动脉压（80.2±6.6 VS 81.6±8.1 mmHg）、超敏C反应蛋白（3.1±1.3 VS 3.7±1.5 mg/L）、血同型半胱氨酸（14.1±3.1 VS 20.2±4.4 umol/L）、血小板活化因子 CD41（47.5±4.1 VS 54.5±3.9%）、CD62P（18.5±3. 1 VS 23.6±3.8%）均下降,差异有统计学意义（P<0.05或P<0.01）。相关分析显示,血同型半胱氨酸水平与H型高血压患体内血小板活化因子呈正相关（CD41 r=0.61,P<005;CD62P r=0.55,P<005）。结论 高同型半胱氨酸可增加H型高血压患者体内血小板活化,依那普利叶酸片在降血压和降血浆同型半胱氨酸浓度的同时还可降低H型高血压患者炎症与血小板活化。与单纯依那普利治疗相比,依那普利叶酸片可更有效减少H型高血压患者心血管发病风险。     

Cyclophosphamide enhances anti-tumor effects of a fibroblast activation protein α-based DNA vaccine in tumor-bearing mice with murine breast carcinoma

Cyclophosphamide (CY) is a DNA alkylating agent, which is widely used with other chemotherapy drugs in the treatment of various types of cancer. It can be used not only as a chemotherapeutic but also as an immunomodulatory agent to inhibit IL-10 expression and T regulatory cells (Tregs). Fibroblast activation protein α (FAPα) is expressed in cancer-associated fibroblasts in the tumor microenvironment. Immunotherapy based on FAPα, as a tumor stromal antigen, typically induces specific immune response targeting the tumor microenvironment. This study evaluated the efficacy of a previously unreported CY combination strategy to enhance the limited anti-tumor effect of a DNA vaccine targeting FAPα. The results suggested CY administration could promote the percentage of splenic CD8^+?T cells and decrease the proportion of CD4?⁺?CD25?⁺?Foxp3^+?Tregs in spleen. In tumor tissues, levels of immunosuppressive cytokines including IL-10 and CXCL-12 were also reduced. Meanwhile, the CY combination did not impair the FAPα-specific immunity induced by the DNA vaccine and further reduced tumor stromal factors. Most importantly, FAP-vaccinated mice also treated with CY chemotherapy showed a marked suppression of tumor growth (inhibition ratio =80%) and a prolongation of survival time. Thus, the combination of FAPα immunotherapy and chemotherapy with CY offers new insights into improving cancer therapies.     

Preparation,Characterization, and Rheological Behaviors of Polysiloxanes Presenting Densely Simple and Well‐Defined Short‐Branched Chains

In this paper, a series of polysiloxanes, presenting different contents of densely simple and well‐defined short‐branched chains (G₁–G₅), are prepared by hydrosilylation of α,ω‐(dimethylvinylsiloxy)‐poly(dimethyl‐methylvinyl)siloxanes (P₁–P₅) with 1,1,1,3,5,5,5‐heptamethyltrisiloxane (MD^HM) and characterized by ¹H and ²⁹Si nuclear magnetic resonance, Fourier transform‐infrared spectroscopy, Abbe refractometry, gel permeation chromatography, and differential scanning calorimetry. The rheological behaviors of the G₁–G₅ products are investigated to study the influences of the short‐branched chains. The rheological parameters including non‐Newtonian index (n ) and the flow activation energy (ΔE_η ) are obtained and discussed. It is observed that the G₁–G₅ polymers belonged to pseudoplastic fluids and the ΔE_η values of the G₁–G₅ products are significantly influenced by the short‐chain branching degree of the polymers.