Induced cytokine response of human PMBC-cultures: correlation of gene expression and secretion profiling and the effect of cryopreservation

The immune system is regulated by the complex interaction of multiple cytokines, which are secreted signaling molecules affecting other cells. In this work, we studied the cytokine response to several well-known stimulants, such as OKT-3, Con A, PWM, and SEB. Healthy donor cells (PBMCs) were cultivated for up to 72 h and the mRNA levels and cytokine release of four key cytokines (IL-2, IL-4, IFN-γ, and TNF-α) were analyzed by RT-PCR and bead-based multiplex analyses. The generated cytokine profiles showed characteristic expression patterns and secretion kinetics for each cytokine and substance. PWM/SEB and OKT-3 led to a very fast and long-lasting immune response, whereas Con A induced the slowest cytokine production. Cytokine concentrations also differed greatly. The highest IFN-γ concentration was 1000 times higher than the respective IL-4 concentration. Gene expression and cytokine concentration profiles were strongly correlated during the time course. The chronological response of the donors' cytokine profiles coincided, but showed individual characteristics regarding the strength of the cytokine release. The comparison of stimulation experiments using freshly isolated and cryopreserved PBMCs showed that, for the observation of an immunological response at early points in time, gene expression experiments are more reliable than the measurement of cytokines in the cell culture supernatant. However, the freezing of cells influences the response significantly. The measurement of secreted proteins is the superior method at later points in time.     

Quantitation of rabbit cytokine mRNA by real-time RT-PCR

Fundamental understanding of rabbit immunology and the use of the rabbit as a disease model have long been hindered by the lack of immunological assays specific to this species. In the present study, we sought to develop a method to quantitate cytokine expression in rabbit cells and tissues. We report the development of a quantitative real-time RT-PCR method for measuring the relative levels of rabbit IFN-gamma, IL-2, IL-4, IL-10 and TNF-alpha mRNA. Quantitation was accomplished by comparison to a standard curve generated using plasmid DNA containing partial sequences of the relevant cytokines. Experimental studies demonstrate applicability of this assay to quantitate cytokine mRNA levels from rabbit spleen cells following mitogen stimulation. We have further utilized this assay to also examine cytokine expression in rabbit tissues during experimental syphilis infection.     

Quantifying Aotus monkey cytokines by real-time quantitative RT-PCR

Aotus spp. monkeys are considered the ideal model for studying the progress of malarial infection and the immune response it elicits. We describe the use of a recently developed technique, real-time quantitative RT-PCR, to quantify several Aotus monkey cytokine mRNAs involved in Th1/Th2 responses (IL-4, IL-10, TNF-beta and IFN-gamma). Specific primers were designed for each cytokine and standard curves were constructed using serial dilutions of pDNA containing each target sequence. Results were normalized to GAPDH housekeeping gene expression levels. Standard curves showed high correlation coefficients and were linear over a wide range of copy numbers. Quantification of Aotus samples showed little intra- and inter-experiment variation, thus, the technique has proven to be highly reproducible and sensitive allowing us to detect as little as 25 copies/microl of target DNA. This technique will allow studying Th1 and Th2 cytokine patterns elicited in response to infection for prospectively evaluating the efficacy of malarial vaccines.     

Vaccination in humans generates broad T cell cytokine responses

In recent years, the quantification of T cell responses to pathogens or immunogens has become a common tool in the evaluation of disease pathogenesis or vaccine immunogenicity. Such measurements are usually limited to enumerating IFN-gamma-producing cells after ex vivo stimulation with Ag, but little is known about the phenotype or complete functional repertoire of the Ag-specific cells. We used 12-color flow cytometry to characterize Ag-specific T cells elicited by vaccines or natural infection to determine lineage and differentiation status as well as the capacity to produce four cytokines (IFN-gamma, TNF-alpha, IL-2, and IL-4) and a chemokine (MIP1beta). As expected, responding cells had a typical memory phenotype; however, the cytokine profiles associated with the responses were highly complex. The pattern of cytokine coexpression in response to specific Ags was a skewed subset of the complete repertoire (revealed by polyclonal stimulation). We found significant differences in the patterns of cytokines elicited by vaccination (where IFN-gamma was by far a subdominant response) vs natural infection; in addition, there was fairly significant intersubject variation. Our findings illustrate the limitation of the evaluation of immune responses using single functional measurements (such as IFN-gamma); in fact, it is likely that sensitive evaluation of Ag-specific T cells will require the coordinate measurement of several cytokines. The presence and variability of these complex response profiles introduce the possibility that selective functional expression patterns may provide correlates for vaccine efficacy or disease progression.     

Characterization of two putative cytokine receptors, gp130 and ciliary neurotrophic factor receptor, from terrestrial salamanders

Cytokines of the gp130 family are fundamental regulators of immune responses and signal through multimeric receptors to initiate intracellular second-messenger cascades. Here, we provide the first characterization of two full-length gp130 cytokine receptors from the cDNA of the red-legged salamander (Plethodon shermani). The first, gp130 (2745 bp), is a common signaling receptor for several multi-functional cytokines in vertebrates. We also isolated the full-length (1104 bp) sequence of the ciliary neurotrophic factor receptor (CNTFR), which forms a heteromeric signaling complex with gp130. The open reading frames of both receptors were predicted to contain many of the conserved features found in mammalian gp130s, such as cytokine binding homology regions and residues known to form disulfide bonds. Finally, we used RT-PCR to show that gp130 and CNTFR were expressed in most P. shermani tissues, including brain, intestine and muscle. The expression profiles, along with the structural predictions, show that gp130, CNTFR, and their cytokine ligands are parts of the immune system of P. shermani and other caudate amphibians.     

Biological functions and therapeutic opportunities of soluble cytokine receptors

Cytokines control the immune system by regulating the proliferation, differentiation and function of immune cells. They activate their target cells through binding to specific receptors, which either are transmembrane proteins or attached to the cell-surface via a GPI-anchor. Different tissues and individual cell types have unique expression profiles of cytokine receptors, and consequently this expression pattern dictates to which cytokines a given cell can respond. Furthermore, soluble variants of several cytokine receptors exist, which are generated by different molecular mechanisms, namely differential mRNA splicing, proteolytic cleavage of the membrane-tethered precursors, and release on extracellular vesicles. These soluble receptors shape the function of cytokines in different ways: they can serve as antagonistic decoy receptors which compete with their membrane-bound counterparts for the ligand, or they can form functional receptor/cytokine complexes which act as agonists and can even activate cells that would usually not respond to the ligand alone. In this review, we focus on the IL-2 and IL-6 families of cytokines and the so-called Th2 cytokines. We summarize for each cytokine which soluble receptors exist, were they originate from, how they are generated, and what their biological functions are. Furthermore, we give an outlook on how these soluble receptors can be exploited for therapeutic purposes.     

Quantification of Leishmania infantum parasites in tissue biopsies by real-time polymerase chain reaction and polymerase chain reaction-enzyme-linked immunosorbent assay

Most of the experimental studies of Leishmania spp. infection require the determination of the parasite load in different tissues. Quantification of parasites by microscopy is not very sensitive and is time consuming, whereas culture microtitrations remain laborious and can be jeopardized by microbial contamination. The aim of this study was to quantify Leishmania infantum parasites by real-time polymerase chain reaction (PCR) using specific DNA TaqMan probes and to compare the efficacy of detection of this technique with a PCR-enzyme-linked immunosorbent assay (ELISA). For this purpose, spleen and liver samples from L. infantum-infected mice were collected during a 3-mo longitudinal study and analyzed by both methods. PCR-ELISA failed to quantify Leishmania spp. DNA in samples with very low or very high numbers of parasites. Real-time PCR was more sensitive than PCR-ELISA, detecting down to a single parasite, and enabled the parasite quantification over a wide, 5-log range. In summary, this study developed a method for absolute quantification of L. infantum parasites in infected organs using real-time TaqMan PCR.     

Species-specific real-time PCR cell number quantification of the bloom-forming cyanobacterium Planktothrix agardhii

A species-specific method to detect and quantify Planktothrix agardhii was developed by combining the SYBR Green I real-time polymerase chain reaction technique with a simplified DNA extraction procedure for standard curve preparation. Newly designed PCR primers were used to amplify a specific fragment within the rpoC1 gene. Since this gene exists in single copy in the genome, it allows the direct achievement of cell concentrations. The cell concentration determined by real-time PCR showed a linear correlation with the cell concentration determined from direct microscopic counts. The detection limit for cell quantification of the method was 8?cells?μL(-1), corresponding to 32 cells per reaction. Furthermore, the real-time qPCR method described in this study allowed a successful quantification of P. agardhii from environmental water samples, showing that this protocol is an accurate and economic tool for a rapid absolute quantification of the potentially toxic cyanobacterium P. agardhii.     

戊型肝炎病毒荧光定量RT-PCR快速检测技术的建立和初步应用

利用DNAMAN软件对GenBank登录的戊型肝炎病毒四个主要基因型代表株的序列进行分析, 选择其高度保守的ORF2区域设计合成引物和探针, 并用包含有扩增区域的核苷酸片段进行体外转录制备标准品cRNA。在对荧光定量RT-PCR的反应条件优化的基础上, 建立了适用于戊型肝炎病毒主要基因型检测的荧光定量RT-PCR检测技术。该检测技术可以有效检测I型和IV型戊型肝炎阳性病料, 而对猪的其它几种疫病阳性病料则为阴性结果, 证实本技术的特异性强、可靠性好。对阳性标准品的检测结果表明, 所建立的TaqMan荧光定量RT-PCR灵敏度可达2.0×101拷贝/反应, 相比于巢式RT-PCR方法, 其灵敏度高10~100倍以上。在对54份临床样品的检测中, 进一步证实了该方法快速、灵敏且重复性好, 可满足戊型肝炎病毒早期快速诊断的需要。