Bioanalytical chemistry of cytokines – A review

Cytokines are bioactive proteins produced by many different cells of the immune system. Due to their role in different inflammatory disease states and maintaining homeostasis, there is enormous clinical interest in the quantitation of cytokines. The typical standard methods for quantitation of cytokines are immunoassay-based techniques including enzyme-linked immusorbent assays (ELISA) and bead-based immunoassays read by either standard or modified flow cytometers. A review of recent developments in analytical methods for measurements of cytokine proteins is provided. This review briefly covers cytokine biology and the analysis challenges associated with measurement of these biomarker proteins for understanding both health and disease. New techniques applied to immunoassay-based assays are presented along with the uses of aptamers, electrochemistry, mass spectrometry, optical resonator-based methods. Methods used for elucidating the release of cytokines from single cells as well as in vivo collection methods are described.     

脑内免疫炎症的发生机制

中枢神经系统神经元退行性变过程中表达主要组织相容性分子、黏附分子和补体受体,并产生多种细胞因子,从而引发脑内的免疫炎症反应。     

Calotropis procera (Aiton) seeds fixed oil: Physicochemical analysis,GC–MS profiling and evaluation of its in-vivo anti-inflammatory and in-vitro antiparasitic activities

Calotropis procera (Aiton) is a wildly grown shrubbery that has been used by traditional healers in various remedies for centuries. Its different organs are reported to possess a variety of biological activities. However, a few reports chemically investigated the seed fixed oil, but they didn’t prospect its bioactivity. Accordingly, this study aimed to investigate the phytochemical composition, physicochemical properties, and biological activity of the seed oil. As well, exploration of the best extraction method that give the highest oil yield with the best composition. Physicochemical properties, total phenolic content and oxidative stability index of the oil were estimated. GC–MS analysis identified the oil content. Anti- inflammatory activity was examined using a carrageenan-induced rat paw edema assay. Immunomodulatory activity was determined by measuring the pro-inflammatory cytokines levels. Moreover, In-vitro anti-parasitic and antioxidant activities were studied. Soxhlet extraction method gives the highest yield of 40.5 ± 0.76% with no time interval. The oil shows a physicochemical property within the AOCS standards and low phenol content. GC-MS analysis reveals unsaturated fatty acids account for 75.25% of the oil, with an abundance of oleic acid (33.64%) and linoleic acid (31.95%). The oil exhibited potent anti-inflammatory and immunomodulatory activity. At various dose levels, paw edoema volume, pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and myeloperoxidase (MPO) serum levels were reduced. The seed oil is a promising candidate for the treatment of inflammation and/or inflammatory associated diseases (as parasitic infections) besides its immunomodulatory activity.     

肾透明细胞癌组织和细胞系趋化素样因子1的表达及其临床意义

趋化素样因子1(chemokine-like factor 1, CKLF1)在多种恶性肿瘤中发挥了不同的生物学作用,为探讨CKLF1在肾透明细胞癌(clear cell renal cell carcinoma, ccRCC)组织及细胞系中表达水平并分析其临床意义。利用免疫组化法、蛋白质印迹和实时定量PCR法,检测CKLF1在肾透明细胞癌组织和肾癌细胞系中的表达水平,并分析肾透明细胞癌组织中CKLF1表达水平与临床病理Fuhrman分级之间的相关性。免疫组化结果显示:CKLF1在肾透明细胞癌组织和癌旁组织中的表达,差异有显著性(P0.05); CKLF1表达水平的高低在临床病理各分级之间无明显差异(P0.05)。Western blot结果显示:CKLF1蛋白在3种细胞系ACHN、Caki、HKC中的表达差异有统计学意义(P0.05);qRT-PCR结果显示,CKLF1 mRNA在3种细胞系ACHN、Caki、HKC中的表达差异有统计学意义(P0.05)。CKLF1的表达在肾透明细胞癌组织中和肾癌细胞系ACHN、Caki中明显上调,CKLF1是一个潜在的肾透明细胞癌分子标志物。     

浅谈细胞因子网络性

细胞因子是在机体炎症和免疫应答中 ,由免疫细胞产生的一类具有调节与效应功能的小分子多肽或蛋白。它作为细胞间的信号在免疫应答中起作用。细胞因子在非特异性免疫中称单核因子 ,在特异性免疫中又可叫淋巴因子。细胞因子根据其功能的不同可分好多种 ,但不同的细胞因子并不是单一存在 ,它们互相之间相辅相成 ,共同作用 ,使机体处于一个相对稳定的状态。本文即从细胞因子的产生、功能、受体类型等方面论述细胞因子的网络性     

Synthetic Peptide Antibodies as TNF-α Inhibitors: Molecularly Imprinted Polymer Nanogels Neutralize the Inflammatory Activity of TNF-α in THP-1 Derived Macrophages

Tumor Necrosis Factor-α (TNF-α) is a cytokine that is normally produced by immune cells when fighting an infection. But, when too much TNF-α is produced as in autoimmune diseases, this leads to unwanted and persistent inflammation. Anti-TNF-α monoclonal antibodies have revolutionized the therapy of these disorders by blocking TNF-α and preventing its binding to TNF-α receptors, thus suppressing the inflammation. Herein, we propose an alternative in the form of molecularly imprinted polymer nanogels (MIP-NGs). MIP-NGs are synthetic antibodies obtained by nanomoulding the 3-dimensional shape and chemical functionalities of a desired target in a synthetic polymer. Using an in-house developed in silico rational approach, epitope peptides of TNF-α were generated and ‘synthetic peptide antibodies’ were prepared. The resultant MIP-NGs bind the template peptide and recombinant TNF-α with high affinity and selectivity, and can block the binding of TNF-α to its receptor. Consequently they were applied to neutralize pro-inflammatory TNF-α in the supernatant of human THP-1 macrophages, leading to a downregulation of the secretion of pro-inflammatory cytokines. Our results suggest that MIP-NGs, which are thermally and biochemically more stable and easier to manufacture than antibodies, and cost-effective, are very promising as next generation TNF-α inhibitors for the treatment of inflammatory diseases.     

细胞因子在骨质疏松症发病机理中的作用及运动对其的影响

骨质疏松症的发病机理与细胞因子改变有密切的关系。部分细胞因子表达增加或降低会影响骨的微环境中细胞的增殖,分化,使骨形成与骨吸收之间发生紊乱,骨代谢偶联失衡,从而导致骨质疏松症的发生。体育运动可以改变体内细胞因子的表达,从而起到防治骨质疏松症的作用。     

Serum and aqueous humour cytokine response and histopathological alterations during ocular Toxoplasma gondii infection in C57BL/6 mice

Toxoplasma gondii, an obligate intracellular protozoan parasite, infects most species of warm-blooded animals, and in humans it causes toxoplasmosis. Healthy people that become infected rarely present clinical symptoms because the immune system prevents the parasite from causing illness. Congenital toxoplasmosis may result in abortion, hydrocephalus, as well as neurological and ocular disease (most frequently retinochoroiditis) of the newborn. In immunocompromised patients, reactivation of latent disease can cause encephalitis. Cell-mediated immunity to T. gondii antigens involves innate acute inflammatory responses and antigen-specific adaptive immunity. Considering the complexity of the immunological events triggered during toxoplasmosis, systemic and local responses were evaluated by cytokine measurements. Aqueous humour and serum were obtained from non-infected and T. gondii Me-49 strain infected C57BL/6 mice for cytokine quantification. Histopathological analyses were made with eyes enucleated from mice after 30 days of infection. ELISA assays showed an increase of IFN-γ levels both in serum and aqueous humour of infected mice in opposition to a decrease in IL-10 levels. On the other hand, TGF-β was high, whereas IL-12 and TNF-α were present in small levels in both groups. We also detected higher levels of IL-4 and IL-6 in aqueous humour than in serum of infected mice when compared to the control group. MIP-2 presented no significant differences between the two groups. Fas and Fas-L were also present in similar levels in serum of non-infected and infected mice, but both chemokines were increased in the aqueous humour of infected mice. Histopathological analysis of infected mice showed inflammatory infiltrates around blood vessels and alteration of the outer photoreceptor segments, on the external and inner nuclear layer. Parasites were observed in 82% of eyes, inside the blood vessels associated with inflammatory infiltrate. Edema, characterized by the increase of interstitial spaces between the FTR, forming lacunae was also noted. These alterations take the form of projections (retino-vitreal), characteristic of retinochoroiditis. In conclusion, T. gondii infection of C57BL/6 mice revealed that cytokine patterns alone do not assure susceptibility or resistance against infection, thus reinforcing the notion that it is necessary more than cytokine dosage to determine Th1 or Th2 profile in this model.     

Chemical Synthesis of the 12 kDa Human Myokine Irisin by α‐Ketoacid‐Hydroxylamine (KAHA) Ligation

Irisin is a recently discovered protein hormone with a conserved sequence among vertebrates and with putative functions in the regulation of adipose tissue and bone metabolism. We report the first chemical synthesis using two sequential ketoacid‐hydroxylamine (KAHA) ligations to give milligram quantities of unlabeled and fluorescence‐labeled irisin protein. The synthetic proteins were utilized in cell binding assays, which indicated the expected binding characteristics to stromal cells of white adipose tissue. These studies strongly imply the presence of a specific irisin receptor and provide a path to its identification with synthetic irisin.