miRNA-21 inhibition enhances RANTES and IP-10 release in MCF-7 via PIAS3 and STAT3 signalling and causes increased lymphocyte migration

MicroRNAs (miRNAs) are a class of small endogenous gene regulators that have been implicated in various developmental and pathological processes. However, the precise identities and functions of miRNAs involved in antitumor immunity are not yet well understood. miRNA-21 is an oncogenic miRNA that can be detected in various tumours. In this study, we report that a miRNA-21 inhibitor enhances the release of chemoattractants RANTES and IP-10 in the MCF-7 breast cancer cell line and results in increased lymphocyte migration. Thus, miRNA-21 is a potential therapeutic target for cancer immunotherapy. We further demonstrated that PIAS3, a protein inhibitor of activated STAT3, is a target of miRNA-21 in MCF-7. Thus, miRNA-21 is a novel miRNA regulating immune cell recruitment, which acts at least in part via its inhibition of PIAS3 expression and oncogenic STAT3 signalling in tumour cells.     

The discovery of BMS-457, a potent and selective CCR1 antagonist

A series of compounds which exhibited good human CCR1 binding and functional potency was modified resulting in the discovery of a novel series of high affinity, functionally potent antagonists of the CCR1 receptor. Issues of PXR activity, ion-channel potency, and poor metabolic stability were addressed by the addition of a hydroxyl group to an otherwise lipophilic area in the molecule resulting in the discovery of preclinical candidate BMS-457 for the treatment of rheumatoid arthritis.     

CXCR6 protects from inflammation and fibrosis in NEMOLPC-KO mice

Chronic inflammation in the liver provokes fibrosis and, on long-term, carcinogenesis. This sequence is prototypically recapitulated in mice with hepatocyte-specific knock-out of the NF-κB essential modulator (NEMO), termed NEMO^LPC-KO mice, in which increased hepatocyte apoptosis and compensatory regeneration cause steatosis, inflammation and fibrosis. Natural killer T (NKT) cells carrying the chemokine receptor CXCR6 participate in liver inflammation and injury responses. Here, we investigated the role of CXCR6 in the NEMO^LPC-KO mouse model. Unexpectedly, genetic deletion of CXCR6 enhanced hepatocyte death, inflammation and fibrosis in NEMO^LPC-KO mice. Although CXCR6 expression is restricted to immune cells in the liver, the adoptive transfer of CXCR6⁺ cells did not protect NEMO^LPC-KOCxcr6^?/? mice from hepatic injury. Gene array analyses revealed up-regulated stress response and metabolism pathways in hepatocytes from NEMO^LPC-KOCxcr6^?/? mice, functionally corresponding to an increased susceptibility of these hepatocytes to TNFα-induced cell death in vitro. These data revealed a novel CXCR6-dependent mechanism of suppressing inflammatory hepatocytic responses to cellular stress.     

趋化因子受体CX3CR1调控人主动脉瓣膜间质细胞成骨分化的作用研究

目的:探究趋化因子受体CX3CR1调控人主动脉瓣膜间质细胞成骨分化的作用和机制,为钙化性主动脉瓣膜疾病的早期干预和治疗提供新思路。方法:取非钙化主动脉瓣(3例)和钙化主动脉瓣(5例),免疫组织化学染色检测成骨相关转录因子Runx2、骨桥蛋白OPN和骨钙蛋白OCN的表达;取3例非钙化的主动脉瓣,采用胶原酶连续消化法分离人主动脉瓣膜间质细胞,观察细胞形态及生长状态,并采用细胞免疫荧光进行表型鉴定。对成骨诱导培养的人主动脉瓣膜间质细胞分别过表达和干扰趋化因子受体CX3CR1,平行设置CM组、OM组和negative control+OM组,采用qPCR和Western blot检测Runx2、OPN和OCN的表达,Western blot检测AKT和p-AKT的表达。茜素红S染色评价晚期钙结节形成情况。结果:临床标本显示钙化的主动脉瓣较非钙化的主动脉瓣高表达CX3CR1(P 0. 05);成功分离人主动脉瓣膜间质细胞,α-SMA和Vimentin阳性,vWF阴性。与CM、OM、negative control组比较,CX3CR1+OM组Runx2、OPN和p-AKT表达上调(P 0. 05),且茜素红S染色可见明显钙结节;与CM、OM、negative siRNA control+OM组比较,si CX3CR1+OM组Runx2、OPN和p-AKT表达下调(P 0. 05),且茜素红S染色可见钙结节减少。结论:趋化因子受体CX3CR1可能通过AKT信号通路促进人主动脉瓣膜间质细胞成骨分化。     

慢性阻塞性肺疾病患者中血清亲环素A、趋化因子CX3CL1表达水平及临床意义

摘要 目的：探讨慢性阻塞性肺疾病（COPD）患者中血清亲环素A（CyPA）、趋化因子CX3CL1以及其他炎性指标的表达水平及其临床意义。方法：选取2019年1月-2021年6月本院收治的COPD患者120例作为研究对象,其中68例患者为急性加重期组,52例患者为稳定期组;另选取体检健康者45例作为对照组。收集所有受试者的临床资料,检测其血清中CyPA、CX3CL1、C反应蛋白（CRP）、白细胞介素-6（IL-6）以及基质金属蛋白酶-9（MMP-9）水平,比较3组患者各参数的差异,并与肺功能进行相关性分析,比较急性加重期患者治疗前后各指标的差异。结果：急性加重期组患者的血清 CyPA、CX3CL1、CRP、IL-6、MMP-9水平均明显高于稳定期组和对照组患者,稳定期组患者的血清 CyPA、CX3CL1、CRP、IL-6、MMP-9水平均明显高于对照组患者（均P＜0.05）;而急性加重期组患者的第1s用力呼气量（FEV₁）、用力肺活量（FVC）、第1s用力呼气量（FEV₁）与用力肺活量（FVC）的比值（FEV₁%）、最大呼气峰流速（PEF）以及最大呼气中期流速（MMEF）明显低于稳定期组和对照组患者,稳定期组患者的FEV₁、FVC、FEV₁/FVC、PEF、MMEF明显低于对照组患者（均P＜0.05）。COPD患者的血清CyPA、CX3CL1、CRP、IL-6、MMP-9水平与FEV₁、FVC、FEV₁/FVC、PEF、MMEF呈负相关（P<0.05）。与治疗前相比较,急性加重期组患者在治疗后血清CyPA、CX3CL1、CRP、IL-6、MMP-9水平明显下降,而FEV₁、FVC、FEV₁/FVC、PEF、MMEF明显上升（均P＜0.05）。结论：COPD患者的血清CyPA、CX3CL1、CRP、IL-6、MMP-9水平可在一定程度上预测患者的严重程度,同时也可以作为急性加重期治疗后效果的评价指标。     

口腔扁平苔藓患者血清白介素-17、P物质、趋化素水平与疾病活动和免疫功能的关系研究

摘要 目的：研究口腔扁平苔藓（OLP）患者血清白介素-17（IL-17）、P物质（SP）、趋化素（Chemerin）水平与疾病活动和免疫功能的关系。方法：选取我院2019年3月~2021年5月期间收治的117例OLP患者作为观察组,其中非糜烂型69例,糜烂型48例。另选取同期来我院体检的健康志愿者60例为对照组。对比对照组及不同分型患者血清SP、IL-17、Chemerin水平,对比不同分型患者的疾病活动相关评分和免疫功能指标,应用Pearson相关分析血清SP、IL-17、Chemerin水平与疾病活动相关评分和免疫功能指标的相关性。结果：不同分型OLP患者血清SP、IL-17、Chemerin水平均高于对照组（P<0.05）,且糜烂型OLP患者的血清SP、IL-17、Chemerin水平高于非糜烂型OLP患者（P<0.05）。糜烂型OLP患者的CD3⁺、CD4⁺低于非糜烂型OLP患者（P<0.05）,糜烂型OLP患者的CD8⁺、免疫球蛋白M（IgM）水平、OLP网纹-糜烂-溃疡疾病活动积分（REU）总分高于非糜烂型OLP患者（P<0.05）。糜烂型与非糜烂型OLP患者间免疫球蛋白A（IgA）、免疫球蛋白G（IgG）水平对比差异无统计学意义（P>0.05）。Pearson相关性分析结果显示,血清SP、IL-17、Chemerin水平与IgM水平、REU总分均呈正相关（P<0.05）。结论：OLP患者血清IL-17、SP、Chemerin水平异常升高,三者共同参与了OLP患者的病情进展及免疫功能调节。     

Clusterin induces CXCR4 expression and migration of cardiac progenitor cells

Clusterin (CST) is a stress-responding protein with multiple biological functions, including the inhibition of apoptosis and inflammation and transport of lipids. It may also participate in cell traffic and migration. In the process of post-infarct cardiac tissue repair, stem cells migrate into the damaged myocardium under the influence of chemoattractive substances such as stromal cell-derived factor (SDF). This study aimed at testing whether CST enhances expression of stem cell homing receptor and migration of cardiac progenitor cells (CPCs). CPCs isolated from fetal canine hearts transduced by CST cDNA expressed high levels of CXCR4, a receptor for SDF-1. The transfected cells also showed an increased migratory response to SDF-1 stimulation. The SDF-1-mediated migration of the CST-expressing CPCs was attenuated by PI3 kinase inhibitor LY294002 but not by mitogen-activated protein/ERK kinase inhibitor PD98059. Analysis of cell cycle by flow cytometry revealed no significant difference in cell cycle between the transduced and control CPCs. Thus, CST expression may increase CPCs migration via increasing CXCR4 expression and SDF-1/chemokine receptor signaling in a PI3/Akt-dependent manner.     

Genetic variation at chemokine receptor CCR5 in leporids: alteration at the 2nd extracellular domain by gene conversion with CCR2 in Oryctolagus, but not in Sylvilagus and Lepus species

Whereas in its natural host (Sylvilagus sps.) the effects of myxoma virus infections are benign, in European rabbit (Oryctolagus cuniculus), it causes a highly infectious disease with very high mortality rate, known as myxomatosis. There is evidence that, as with HIV-1 virus in human, myxoma virus may use chemokine receptors such as CCR5 of the host target cell for entry and activation of pathways of immune avoidance. We have characterized and compared CCR5 genes of leporid species with different susceptibility levels to myxomatosis. The CCR5 protein of O. cuniculus differs markedly from all those known from other species. The most striking was the replacement of a specific peptide motif of the second extracellular loop (ECL2) by a motif, which in other species characterizes the CCR2 molecules. While absent in Sylvilagus and Lepus species, this CCR2 imposed CCR5–ECL2 alteration was observed in all genomes of 25 European rabbits, representing the subspecies O. cuniculus algirus and O. cuniculus cuniculus. Allelic variation at the rabbit CCR5 locus confirmed that the gene conversion predates the subspecies split (1–2 Ma).     

Development of surface-based assays for transmembrane proteins: selective immobilization of functional CCR5, a G protein-coupled receptor

A general method to develop surface-based assays for transmembrane (TM) receptor function(s) without the need to isolate, purify, and reconstitute the proteins is presented. Based on the formation of an active surface that selectively immobilizes membrane vesicles, the method is illustrated using the chemokine receptor CCR5, a member of the largest family of cell surface eukaryotic TM proteins, the G protein-coupled receptors (GPCRs). The method begins with a protein-resistant surface containing a low percentage (1-5%) of surface-bound biotin on gold as the initial template. Surface plasmon resonance (SPR) data show specific immobilization of functional CCR5 after the initial template is activated by immobilization of rho 1D4 antibody, an anti-rhodopsin monoclonal antibody specific for the carboxyl terminal nine amino acids on bovine rhodopsin that had been engineered into the carboxyl terminus of CCR5, and exposure to vesicles obtained from mammalian cells transfected with a synthetic human CCR5 gene. Activation of the initial template is effected by sequential immobilization of avidin, which binds to the biotin in the initial template, a biotinylated goat anti-mouse immunoglobulin G (Bt-IgG), which binds to the avidin binding sites distal to the surface and the F(c) portion of the rho 1D4 antibody through its F(ab) region(s) and finally rho 1D4. This approach establishes a broad outline for the development and application of various assays for CCR5 functions. SPR data also showed that vesicle immobilization could be achieved through an integrin-integrin antibody interaction after activation of the initial template with a goat anti-human integrin beta1 antibody. These results suggest that the generic nature of the initial platform and flexibility of the subsequent surface activation for specific immobilization of membrane vesicles can be applied to the development of assays for other GPCRs or TM receptors for which antibodies are available or can be engineered to contain a particular antibody epitope.