转Bt玉米暴露对亲代及子代大鼠免疫细胞分型及细胞因子的影响

为研究转苏云金芽胞杆菌(Bacillus thuringiensis,Bt)基因抗虫玉米长期食用对大鼠免疫的影响,以其亲本非转基因玉米饲料及商业饲料为对照,分别采用流式细胞术及液相芯片法检测喂养90 d后亲代及子代刚断乳大鼠外周血、脾脏免疫细胞分型及相关血清细胞因子的表达。结果发现,转Bt基因抗虫玉米饲料组亲代大鼠外周血和脾脏及子代脾脏各淋巴细胞亚群(CD4~+、CD8~+、TCRαβ~+、TCRγδ~+)的细胞数量比例及亲代血清中10种细胞因子表达水平均与非转基因玉米饲料组无显著差异(P0.05);子代外周血B淋巴细胞数量比例显著增高,巨噬细胞炎症蛋白-1α、单核细胞趋化因子-1β、白细胞介素-5表达水平明显降低(P0.05)。结果表明:转Bt基因抗虫玉米暴露90 d对亲代大鼠免疫系统影响不大;对子代刚断乳大鼠的细胞免疫及体液免疫有一定的影响。     

Loss of Interleukin-13-Receptor-Alpha-1 Induces Apoptosis and Promotes EMT in Pancreatic Cancer

In search of new therapies for pancreatic cancer, cytokine pathways have attracted increasing interest in recent years. Cytokines play a vital role in the crosstalk between tumour cells and the tumour microenvironment. The related inflammatory cytokines IL-4 and IL-13 can regularly be detected at increased levels in the microenvironment of pancreatic cancer. They share a receptor heterodimer consisting of IL-4Rα and IL-13Rα1. While IL-4Rα induces a more oncogenic phenotype, the role of IL-13Rα1 was yet to be determined. ShRNA-based knockdown of IL-13Rα1 was performed in Capan-1 and MIA PaCa-2. We assessed cell growth and migratory capacities under the influence of IL-13Rα1. Pathway alterations were detected by immunoblot analysis. We now have demonstrated that the loss of IL-13Rα1 induces apoptosis in pancreatic cancer cells. This was associated with an epithelial-to-mesenchymal transition. Loss of IL-13Rα1 also abolished the effects of exogenous IL-4 and IL-13 stimulation. Interestingly, in wild type cells, cytokine stimulation caused a similar increase in migratory capacities as after IL-13Rα1 knockdown. Overall, our results indicate the vital role of IL-13Rα1 in the progression of pancreatic cancer. The differential expression of IL-4Rα and IL-13Rα1 has to be taken into account when considering a cytokine-targeted therapy in pancreatic cancer.     

Possible Implications of Bacteriospermia on the Sperm Quality,Oxidative Characteristics,and Seminal Cytokine Network in Normozoospermic Men

This study focused on the identification of bacterial profiles of semen in normozoospermic men and their possible involvement in changes to the sperm structural integrity and functional activity. Furthermore, we studied possible fluctuations of selected cytokines, oxidative markers, and antibacterial proteins as a result of bacterial presence in the ejaculate. Sperm motility was assessed with computer-assisted sperm analysis, while sperm apoptosis, necrosis and acrosome integrity were examined with fluorescent methods. Reactive oxygen species (ROS) generation was quantified via luminometry, sperm DNA fragmentation was evaluated using the TUNEL protocol and chromatin-dispersion test, while the JC-1 assay was applied to evaluate the mitochondrial membrane potential. Cytokine levels were quantified with the biochip assay, whilst selected antibacterial proteins were quantified using the ELISA method. The predominant species identified by the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry were Staphylococcus hominis, Staphylococcus capitis and Micrococcus luteus. The results revealed that the sperm quality decreased proportionally to the increasing bacterial load and occurrence of conditionally pathogenic bacteria, including Enterococcus faecalis, Staphylococcus aureus and Escherichia coli. Antimicrobial susceptibility tests revealed a substantial resistance of randomly selected bacterial strains to ampicillin, vancomycin, tobramycin, and tetracycline. Furthermore, an increased bacterial quantity in semen was accompanied by elevated levels of pro-inflammatory cytokines, including interleukin-1, interleukin-2, interleukin-6, tumor necrosis factor alpha as well as ROS overproduction and lipid peroxidation of the sperm membranes. Our results suggest that semen quality may be notably affected by the bacterial quantity as well as quality. It seems that bacteriospermia may be associated with inflammatory processes, oxidative stress, sperm structural deterioration, and a subsequent risk for the development of subfertility, even in normozoospermic males.     

Evaluation of Cell Migration and Cytokines Expression Changes under the Radiofrequency Electromagnetic Field on Wound Healing In Vitro Model

Wound healing (WH) proceeds through four distinct phases: hemostasis, inflammation, proliferation, and remodeling. Impaired WH may be the consequence of the alteration of one of these phases and represents a significant health and economic burden to millions of individuals. Thus, new therapeutic strategies are the topics of intense research worldwide. Although radiofrequency electromagnetic field (RF-EMF) has many medical applications in rehabilitation, pain associated with musculoskeletal disorders, and degenerative joint disorders, its impact on WH is not fully understood. The process of WH begins just after injury and continues during the inflammatory and proliferative phases. A thorough understanding of the mechanisms by which RF-EMF can improve WH is required before it can be used as a non-invasive, inexpensive, and easily self-applicable therapeutic strategy. Thus, the aim of this study is to explore the therapeutic potential of different exposure setups of RF-EMF to drive faster healing, evaluating the keratinocytes migration, cytokines, and matrix metalloproteinases (MMPs) expression. The results showed that RF-EMF treatment promotes keratinocytes’ migration and regulates the expression of genes involved in healing, such as MMPs, tissue inhibitors of metalloproteinases, and pro/anti-inflammatory cytokines, to improve WH.     

利用皮肤模型研究不同分子量透明质酸的抗刺激作用

将十二烷基硫酸钠(SLS)和不同分子量的透明质酸(HA)共同作用于人永生化表皮细胞(HaCaT)和3D皮肤模型,作用24 h后通过MTT比色法检测细胞活性,利用ELISA方法检测培养液中人白细胞介素(IL-1α)的水平,并进行组织学观察细胞形态。研究在不同实验模型中,不同分子量HA影响SLS对细胞的损伤和释放IL-1α的差异。结果表明,皮肤模型可用于HA的体外抗皮肤刺激作用的研究,实验结果更接近人体实际使用情况以及能提供更多的机制信息。     

Tumor Necrosis Factor α and Interleukin-1β Acutely Inhibit AgRP Neurons in the Arcuate Nucleus of the Hypothalamus

Obesity-associated low-grade inflammation favors weight gain, whereas systemic infection frequently leads to anorexia. Thus, inflammatory signals can either induce positive or negative energy balance. In this study, we used whole-cell patch-clamp to investigate the acute effects of three important proinflammatory cytokines, tumor necrosis factor α (TNF-α), interleukin-6, and interleukin-1β (IL-1β) on the membrane excitability of agouti-related peptide (AgRP)- or proopiomelanocortin (POMC)-producing neurons. We found that both TNF-α and IL-1β acutely inhibited the activity of 35–42% of AgRP-producing neurons, whereas very few POMC neurons were depolarized by TNF-α. Interleukin-6 induced no acute changes in the activity of AgRP or POMC neurons. Our findings indicate that the effect of TNF-α and IL-1β, especially on the activity of AgRP-producing neurons, may contribute to inflammation-induced anorexia observed during acute inflammatory conditions.     

Interleukin‐4‐Clicked Surfaces Drive M2 Macrophage Polarization

Driving macrophage (M?) polarization into the M2 phenotype provides potential against inflammatory diseases. Interleukin‐4 (IL‐4) promotes polarization into the M2‐M? phenotype, but its systemic use is constrained by dose‐limiting toxicity. Consequently, we developed IL‐4‐decorated surfaces aiming at sustained and localized activity. IL‐4 muteins were generated by genetic code expansion; Lys42 was replaced by unnatural amino acids (uAAs). Both muteins showed cell‐stimulation ability and binding affinity to IL4Rα similar to those of wt‐IL‐4. Copper‐catalyzed (CuAAC) and copper‐free strain‐promoted (SPAAC) 1,3‐dipolar azide–alkyne cycloadditions were used to site‐selectively anchor IL‐4 to agarose surfaces. These surfaces had sustained IL‐4 activity, as demonstrated by TF‐1 cell proliferation and M2, but not M1, polarization of M‐CSF‐generated human M?. The approach provides a blueprint for the engineering of cytokine‐activated surfaces profiled for sustained and spatially controlled activity.     

Inflammation amplification by versican: the first mediator

The effects of inflammation may not always benefit the individual. Its amplifying nature represents a highly regulated biological program, and the inflammatory microenvironment is its essential component. Growing evidence suggests that the ECM (extracellular matrix) is important for the early steps of inflammation. Versican, a ubiquitous component of the ECM, contributes to the formation of the inflammatory response and is highly regulated by cytokines. Certain cytokines exert their initial effects on versican to alter the homeostasis of the inflammatory milieu, and inappropriate production of versican may promote the next inflammatory response. Therefore, versican could be the first step in the amplification of the inflammatory response, and ongoing research of this molecule may help to explain the pathogenesis of inflammation.     

Synthesis and in vitro evaluation of alpha-GalCer epimers

alpha-GalCer (also known as KRN7000) is an immunomodulatory glycolipid that is known to potently activate invariant natural killer T (NKT) cells upon CD1d-mediated stimulation. Because Th1 and Th2 cytokines, which are released after alpha-GalCer presentation, antagonize each other's effects, alpha-GalCer analogues that induce a biased Th1/Th2 response are highly awaited. In this context, we report the synthesis and in vitro evaluation of alpha-Gal-D-xylo-Cer and two alpha-Gal-L-lyxo-Cer analogues, one with the natural acyl chain, the other with a truncated chain.     

The development of a medium for the in vitro expansion of mammalian neural stem cells

A new medium, PPRF-m2, has been developed for the in vitro expansion of murine neural stem cells. Neural stem cells (NSC) are primitive cells that have the capability to give rise to all of the cells that comprise the central nervous system (CNS). PPRF-m2 was developed in part by examining key aspects of a commercial NSC medium used in our laboratory, and includes the basal media combination RPMI/DMEM/F12 in a 1:1:1 volumetric ratio supplemented with a hormone mixture, sodium bicarbonate (21.4 g/L), Hepes (4.3 g/L), glucose (1.75 g/L), glutamine (0.14 g/L), epidermal growth factor (20 μg/L) and basic fibroblast growth factor (10 μg/L). Cells inoculated into PPRF-m2 effectively doubled 4 times in 5 days to achieve a maximum viable cell density of 1.2 × 10⁶ cells/mL in stationary culture. This was approximately 650% higher than the maximum cell density achieved in the commercial NSC medium. In addition, the PPRF-m2 cultures were observed to contain less cellular debris, and the cells were less granular and exhibited a lower degree of attachment. Immunocytochemistry revealed that cells grown in PPRF-m2 retained the ability to generate neurons, astrocytes, and oligodendrocytes.