Cytometric Techniques Research Articles

An image cytometric technique is a concise method to detect adenoviruses and host cell proteins and to monitor the infection and cellular responses induced

BackgroundGenetically modified adenoviruses (Ad) with preferential replications in tumor cells have been examined for a possible clinical applicability as an anti-cancer agent. A simple method to detect viral and cellular proteins is valuable to monitor the viral infections and to predict the Ad-mediated cytotoxicity.MethodsWe used type 5 Ad in which the expression of E1A gene was activated by 5′-regulatory sequences of genes that were augmented in the expression in human tumors. The Ad were further modified to have the fiber-knob region replaced with that derived from type 35 Ad. We infected human mesothelioma cells with the fiber-replaced Ad, and sequentially examined cytotoxic processes together with an expression level of the viral E1A, hexon, and cellular cleaved caspase-3 with image cytometric and Western blot analyses.ResultsThe replication-competent Ad produced cytotoxicity on mesothelioma cells. The infected cells expressed E1A and hexon 24 h after the infection and then showed cleavage of caspase-3, all of which were detected with image cytometry and Western blot analysis. Image cytometry furthermore demonstrated that increased Ad doses did not enhance an expression level of E1A and hexon in an individual cell and that caspase-3-cleaved cells were found more frequently in hexon-positive cells than in E1A-positive cells. Image cytometry thus detected these molecular changes in a sensitive manner and at a single cell level. We also showed that an image cytometric technique detected expression changes of other host cell proteins, cyclin-E and phosphorylated histone H3 at a single cell level.ConclusionsImage cytometry is a concise procedure to detect expression changes of Ad and host cell proteins at a single cell level, and is useful to analyze molecular events after the infection.

Application of gene specific mRNA level determinations in individual cells using flow cytometry-based PrimeFlow™ in immunotoxicology

Determining changes in gene expression by measuring mRNA levels is an important capability in biological research. Real-Time Quantitative PCR (RT-qPCR) is the most ubiquitous technique for measuring changes in mRNA transcript levels, but heterogeneity of cell populations and low cell number are serious technical limitations. Recent advances in flow cytometric analytical techniques have enabled the quantification of mRNA levels in individual cells. Here, we present examples demonstrating the strength and challenges of concurrently measuring mRNA using PrimeFlow™ with other endpoints in immunotoxicological studies. Specifically, we demonstrate how measuring gene specific mRNA levels on a per cell basis was used to study: 1) markers of activation and differentiation; 2) cell signaling by measuring intracellular proteins in mature and developing cell types; and 3) a cell type that constitutes a minor population in peripheral blood. We also discuss cell type-specific modifications to the parent technique, which facilitated optimal performance in these cells. While the examples provided are focused on immunotoxicological questions and endpoints, this new strategy can be applied to a wide variety of toxicological research problems.

Isolation of pH-Sensitive Antibody Fragments by Fluorescence-Activated Cell Sorting and Yeast Surface Display.

Fluorescence-activated cell sorting (FACS) in combination with yeast surface display (YSD) has proven to be a valuable tool for the engineering of antibodies. It enables the fast and robust identification and isolation of candidates with prescribed characteristics from combinatorial libraries. A novel application for FACS and YSD that has recently evolved addresses the engineering of antibodies toward pH-switchable antigen binding, aiming at reduced binding at acidic pH, compared to neutral pH. Therefore, we give guidance for the incorporation of such pH switches into antibody variable domains using combinatorial histidine scanning libraries. The protocol describes a flow cytometric sorting technique for the enrichment of antigen-specific molecules. Moreover, we provide information on how to screen the obtained antibody pools from initial sorting to isolate and characterize pH-sensitive variants.

Development of locus specific sub‐clone separation by fluorescence in situ hybridization in suspension in chronic lymphocytic leukemia

Intra-tumor genetic heterogeneity is a hallmark of cancer. The ability to monitor and analyze these sub-clonal cell populations can be considered key to successful treatment, particularly in the modern era of targeted therapies. Although advances in sequencing technologies have significantly improved our ability to analyze the mutational landscape of tumors, this utility is reduced when considering small, but clinically significant sub-clones, that is, those representing <10% of the tumor burden. We have developed a high-throughput method that utilizes a 17-probe labeled bacterial artificial chromosome contig to quantify sub-clonal populations of cells based on deletion of a single locus. Chronic lymphocytic leukemia (CLL) cells harboring deletion of the short arm of chromosome 17 (del17p), an important prognostic marker for CLL were used to demonstrate the technique. Sub-clones of del17p cells were quantified and isolated from heterogeneous CLL populations using fluorescence in situ hybridization in suspension (FISH-IS) and the locus specific probe set. Using the combination of FISH-IS with the locus-specific probe set enables automated analysis of tens of thousands of cells, accurately quantifying and isolating cells carrying a del17p. Based on the fluorescence intensity of 17p probes, 17p (TP53) deleted cells were identified and sorted using flow cytometric techniques, and enrichment was demonstrated using single nucleotide polymorphism analysis. The ability to separate sub-clones of cells based on genetic heterogeneity, independent of the clone size, highlights the potential application of this method not only in the diagnostic and prognostic setting, but also as an unbiased approach to enable further detailed genetic analysis of the sub-clone with deep sequencing approaches. © 2017 International Society for Advancement of Cytometry.

Immune response in Mansonella ozzardi infection modulated by IL-6/IL-10 axis in Amazon region of Brazil

Mansonellosis is an endemic disease in the South and Central America. In Brazil, one of the etiological agents is Mansonella ozzardi. This filarial infection is yet poorly understood, with a controversial morbity, presenting since a oligosymptoms, malaria-like signs or without complaint in humans. The knowledge of the human immune response to microfilariae infection is limited mainly by different evolutionary cycles of the parasite in the host. In addition, the prevalence of this filarial parasite infection is high in several regions of Amazonas State. A cross-sectional study was conducted in an endemic area for microfilariae of M. ozzardi (MF) infection in the Amazonas State, Brazil. Proinflammatory and regulatory cytokines (IL-2, IL-4, IL-6, IL-10, TNF, IFN-gamma, and IL-17A) were measured in cryopreserved serum using the Cytometric Bead Array techniques (CBA) in 54 patients diagnosed with M. ozzardi infection and 55 individuals without the infection were included in the study (Controls). The IL-4, IL-6 and IL-10 level increased in infected patients with MF infection, while IL-17A increased in control only. When we compared controls to patients with high or low parasite load, the increased level of IL-6 and IL-10 were maintained. IL-6 contributes to the proinflammatory activity and IL-10 modulates Th1, Th2 and Th17 immune response. Furthermore, IL-4 was detected as a marker in the MF infection and MF patients with low parasite load, indicating the action of the Th2 cell response. The complex network of cytokines acting during M. ozzardi infection depends on a fine balance to determine a host protective effect or filarial persistence. Therefore, these results suggest that the immune response in MF infection is modulated by IL-6/IL-10 axis.

Knockout of LSD1 Gene by CRISPR/Cas9 System Significantly Inhibited Proliferation and Expression of CD235a in K562 Cells

To study the effect of LSD1 knock-out on human chronic myeloid leukemia cells(K562 cells). The LSD1 gene in K562 cells was knocked-out specifically by using CRISPR/Cas9 system, the single cells were gained by flow cytometric sorting technique, the LSD1+/- and LSD1-/- cell lines were gained after amplificantion and culture, identification of Western blot and sequencing. The MTS assay was used to detect the effect of LSD1 knockout on the proliferation of K562 cells, the flow cytometry was used to examine the expression of K562 cell surface marker after LSD1 knockout. The LSD1 stable knockout cell line of K562 (LSD1+/- and LSD1-/-)were successfully costructed. It was found that knockout of LSD1 significantly inhibited the proliferation of K562 and the expression of CD235a. LSD1 plays a key role in the regulation of K562 cell proliferation and CD235a expression.

T-cell profile and systemic cytokine levels in overweight-obese patients with moderate to very-severe COPD

This study aimed to evaluate the immune profile of lean and overweight-obese COPD patients. Forty patients with moderate to very severe COPD were divided into lean group (n=20; aged 62.00±8.91years; BMI 22.26±1.65kg/m2) or overweight-obese group (n=20; aged 65.40±6.69years; BMI 29.19±3.55kg/m2). The cytokine profile (IL-2, IL-4, IL-6, IL-10, INF-γ, and TNF-α) was evaluated through the Cytometric Bead Array technique, and the expression of CD4, CD8, CD25, CD45ra, CD45ro, CD69, CD195(CCr5) and HLA-DR were evaluated in CD3+ T-cells. Overweight-obese COPD group had lower levels of IL-2 (p=0.01) and higher INF-γ levels (p=0.02) and IL-6 (p=0.003) than lean COPD. Lean COPD patients had higher CD25+ (p=0.01), CCr5 (p=0.04) and HLA-DR (p=0.007) expression on T cell surface compared to overweight-obese COPD participants. These changes are related to immune dysfunction of obesity, and excess of fat mass in COPD can be a key factor to low T-cells activation.

Change in plasma membrane potential of rat thymocytes by tert-butylhydroquinone, a food additive: Possible risk on lymphocytes

Tertiary butylhydroquinone (TBHQ) is a food additive and has various beneficial actions under in vitro and in vivo experimental conditions. Therefore, it is necessary to collect additional data on the toxicity of TBHQ in order to avoid adverse effects during clinical applications. Changes in plasma membrane potential are associated with changes in physiological functions even in non-excitable cells such as lymphocytes. Thus, compounds that affect membrane potential may modify some lymphocytic functions. The effect of TBHQ on plasma membrane potential was examined in rat thymocytes using flow cytometric techniques. Treatment of rat thymocytes with TBHQ caused hyperpolarization and then depolarization. The TBHQ-induced hyperpolarization was due to the activation of Ca2+-dependent K+ channels. TBHQ elevated intracellular Ca2+ levels. The depolarization by TBHQ was caused by a nonspecific increase in membrane ionic permeability. Both the sustained depolarization and elevation of intracellular Ca2+ level by TBHQ are thought to be adverse for thymocytes because such changes disturb membrane and intracellular signaling. The thymus is most active during neonatal and pre-adolescent periods. If TBHQ exerts adverse actions on thymocytes, it may result in an immunotoxic effect in neonates and adolescents.

Redox regulation of leukocyte-derived microparticle release and protein content in response to cold physical plasma-derived oxidants

PurposeRecent results in the field of plasma medicine are increasingly motivating the use of cold physical plasma in clinical settings. Especially two major health issues, non-healing wounds and cancers, are in focus of current research. The outcome of both diseases is highly dependent on inflammation, a process that is priory driven by leukocytes. It is known that these cells are able to tune the behavior and inflammatory status of other cells in a paracrine fashion via release of small particles, so-called microparticles or microvesicles. These phospholipid-bilayers carry a cargo of proteins and RNA and release upon fusion their content into target cells. With different immune cell populations physiologically present in healthy as well as diseased tissue, we investigated their small particle release following plasma exposure in vitro. MethodsFirst, the cytometric analysis technique was established, and analysis of samples revealed a significant modulation of extracellular particle concentrations following exposure to cold plasma. Interestingly, also particle size was affected, indicating alterations in the cellular program of particle generation due to plasma treatment. Proteomic analysis of microparticles using mass spectrometry revealed significant changes in hundreds of proteins regulating, for example, binding, immune processes, transcriptional activity, and redox homeostasis. In the latter, significant modulation of PRX1, PRX2, PRX5, PRX6, TRX, SOD1, SOD2, HSP60, HSP70, and HSP71 protein content of vesicles suggest immune cells to redox-regulate their microenvironment via microparticles in response to plasma. ConclusionThese results further emphasize the potency of cold plasma to not simply damage via oxidation but also to specifically regulate inflammation conceivably of importance in clinical settings.

Interaction between saliva\u2019s adenosine and tick parasitism: effects on feeding and reproduction

BackgroundIt has recently been demonstrated that saliva from Rhipicephalus sanguineus ticks contains adenosine (ADO) and prostaglandin E2 (PGE2), two non-protein molecules that have significant immunomodulatory properties. These molecules can inhibit cytokine production by dendritic cells (DCs), while also reducing the expression of CD40 in these cells. However, more studies are needed for a better understanding of their participation in the feeding of ticks in vivo. This work, therefore, evaluated the importance of ADO during tick infestations. Mice were infested with adult ticks (3 couples/mouse), and their skin was collected at the tick-infested site (3rd and 7th day), and mRNA for receptors of ADO was quantified by real-time PCR.ResultsTick infestation increased by four and two times the expression of the A2b and A3v1 receptors on day 3, respectively, while expression of other ADO receptors was unaltered. In addition, we treated mice (n = 10/group) daily with 8-(p-Sulfophenyl)theophylline, 8-pSPT, 20 mg/kg, i.p.), a non-selective antagonist of ADO receptors, and evaluated the performance of ticks during infestations. Female ticks fed on 8-pSPT-treated mice presented a reduction in their engorgement, weight and hatching rates of egg masses, and survival times of larvae compared to the same parameters presented by ticks in the control group. To investigate if these 8-pSPT-treated mice presented altered immune responses, we performed three tick infestations and collected their lymph node cells to determine the percentages and activation state of DCs and cytokine production by lymphocytes by flow cytometry (Cytometric Bead Array technique, CBA). Our data showed that 8-pSPT-treated mice presented an increase in the percentage of DCs as well as of their stimulatory and co-stimulatory molecules (CD40, CD80 and MHCII). Regarding production of T cell cytokines, we observed a significant increase in the levels of IL-2 and a significant decrease in IL-10, IL-17, TNF-α and IFN-γ cytokines.ConclusionsThese results suggest that ADO produced by ticks helps them feed and reproduce and that this effect may be due to modulation of host DCs and T cells.

Molecular Diagnostic Techniques for Malaria Infection - A Review

Malaria is the most threatening disease protozoal and a major health problem worldwide especially in developing countries. WHO recommended that for every case of suspected malaria, diagnostic test must be done to confirm the disease. A more advanced Diagnostic Techniques were developed to overcome the problem of conventional microscopy. These techniques are known as Molecular diagnostic techniques, and they detect specific sequence in DNA, RNA and proteins to provide clinical information for human pathogens including malaria parasites. There are several techniques involve in molecular diagnostics, some are however discussed in this review. They include Polymerase Chain Reaction (PCR), Loop-mediated isothermal amplification (LAMP), Flow Cytometric technique (FCM), Nucleic acid based sequence amplification (NASBA), and Luminex xMax technology. Among these techniques; LAMP technique is the best techniques that can be deployed in the field settings (clinical and rural settings) because of its simplicity, reliability, stability, detection method as well as point-of- care and confirmatory ability. On the other hand, PCR-based technique is more suitable for research purposes because it can be used to identify drug resistance, follow-up therapeutic response, and detect asymptomatic malaria carriers who may be targeted for treatment. Hence, molecular diagnostic techniques are most innovative science and technical implementations that can be used to diagnose malaria infection and to overcome the limitations.

Prognostic implication of leucocyte subpopulations in diffuse large B-cell lymphoma.

BackgroundRecent studies have suggested that variables related to host adaptive immunity and the tumor microenvironment may predict the outcome in patients with non-Hodgkin's lymphoma. This study was undertaken to determine the prognostic value of peripheral blood leucocyte subpopulations in diffuse large-B-cell lymphoma patients.MethodsWe prospectively analyzed the 16 leukocyte subpopulations using Cytodiff flow cytometric technique in a cohort of 45 diffuse large-B-cell lymphoma patients at a single institution between February and December 2014. The Cox proportional hazards model was used to evaluate prognostic factors for overall survival and progression free survival.ResultsDiffuse large-B-cell lymphoma patients had decreased cytotoxic and non-cytotoxic NK&T cells as well as increased CD16+ monocytes, CD16- monocytes and mature neutrophils. The decreased CD16- monocyte/CD16+ monocyte ratio and increased mature neutrophil/cytotoxic NK&T cell ratio were related to poor progression-free and overall survival outcome in single and multivariate analysis. The co-constructed model using International Prognostic Index and mature neutrophil/cytotoxic NK&T cell ratio can also help discriminate the clinical outcome.ConclusionsThe decreased CD16-monocyte/CD16+monocyte ratio and increased mature neutrophil/cytotoxic NK&T cell ratio predict poor prognosis in diffuse large-B-cell lymphoma patients. This finding provides a strong rationale for the study of cellular immunotherapy in B-cell lymphoma.

A novel method of assessing ASC specks and caspase-1 activity by imaging flow cytometry

Abstract NLRP proteins interact with the adaptor ASC to recruit and activate caspase-1-dependent maturation of IL-1β and IL-18 in a multiprotein ‘inflammasome’ complex. Inflammasome activation occurs in response to structurally diverse agonists triggering NLRP activation leading to rapid localization of NLRP:ASC complexes in a typically singular perinuclear ‘speck’. At present, formation of inflammasome specks can be assayed by microscopy, which is time consuming, or a flow cytometric analysis technique evaluating altered width and height of ASC pulse (TOFIE), which provides no visualization of the speck. We have developed a technique utilizing the advantages of imaging flow cytometry that combines high-throughput sample collection and high magnification (60×) microscopic visualization. This technique not only detects the presence of ASC specks but also determines the distribution of active caspase-1 within the cell using the fluorescent caspase-1 substrate FLICA. This assay allows for high-throughput imaging of ASC-containing specks and characterization of these specks in terms of number and area. Additionally, this technique also determines the location of active caspase-1 and enumerates aggregates of active caspase-1. This method is more sensitive than the present flow cytometric method, TOFIE, which fails to differentiate between active inflammasome specks and false positive signals due to accumulation/aggregation of ASC. The capacity of this method to detect and quantify native as well as reconstituted inflammasome specks with associated caspase-1 activity renders it more powerful for quantifying the impact of mutations on the molecular interaction of NLRP proteins with ASC and accompanying caspase-1 activation.

Physical activity, immune function and inflammation in kidney patients (the PINK study): a feasibility trial protocol

IntroductionPatients with chronic kidney disease (CKD) display increased infection-related mortality and elevated cardiovascular risk only partly attributed to traditional risk factors. Patients with CKD also exhibit a pro-inflammatory environment and...

IL17A Polymorphism Is Not Associated with Human T-Lymphotropic Virus 1-Associated Myelopathy/Tropical Spastic Paraparesis.

The human T-lymphotropic virus 1 (HTLV-1) is the causative agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The present study investigated the association between the rs2275913 polymorphism in the IL17A gene and the development of HAM/TSP. Peripheral blood samples were collected from 116 patients (29 symptomatic patients with HAM/TSP and 87 asymptomatic) with a positive diagnosis of HTLV-1. The single nucleotide polymorphism genotyping was carried out by real time PCR using TaqMan probes. In addition, serum levels of IL-2, IFN-γ, TNF-α, IL-4, IL-6, IL-10, and IL-17 were measured in 64 infected individuals from the study (47 asymptomatic and 17 HAM/TSP), using cytometric bead array technique. No significant differences were found in genotypic and allelic frequencies between the groups. Analysis of cytokine levels showed highest concentrations of IFN-γ and TNF-α in HAM/TSP patients. The results of the present study, therefore, suggest a lack of association between the rs2275913 polymorphism in the IL17A gene and the presence of HAM/TSP.

Zinc-related actions of sublethal levels of benzalkonium chloride: Potentiation of benzalkonium cytotoxicity by zinc

Benzalkonium chloride (BZK) is a common preservative used in pharmaceutical and personal care products. ZnCl2 was recently reported to significantly potentiate the cytotoxicity of some biocidal compounds. In the present study, therefore, we compared the cytotoxic potency of BZK and then further studied the Zn2+-related actions of the most cytotoxic agent among BZK, using flow cytometric techniques with appropriate fluorescent probes in rat thymocytes. Cytotoxicity of benzylcetyldimethylammonium (BZK-C16) was more potent that those of benzyldodecyldimethylammonium and benzyldimethyltetradecylammonium. ZnCl2 (1–10 μM) significantly potentiated the cytotoxicity of BZK-C16 at a sublethal concentration (1 μM). The co-treatment of cells with 3 μM ZnCl2 and 1 μM BZK-C16 increased the population of both living cells with phosphatidylserine exposed on membrane surfaces and dead cells. BZK-C16 at 0.3–1.0 μM elevated intracellular Zn2+ levels by increasing Zn2+ influx, and augmented the cytotoxicity of 100 μM H2O2. Zn2+ is concluded to facilitate the toxicity of BZK. We suggest that the toxicity of BZK is determined after taking extracellular (plasma) and/or environmental Zn2+ levels into account.

Influence of iodine in excess on seminiferous tubular structure and epididymal sperm character in male rats.

Excess iodine induced public health problems are now emerging in many iodine sufficient regions for indiscriminate intake of iodine through various iodized products. It has been reported that excess iodine can disrupt overall male reproductive physiology by generating oxidative stress in the testis. However, information on the possible effect of iodine in excess on spermatozoa found less. In the present investigation flow cytometric techniques and scanning electron microscopy (SEM) have been used to study the spermatozoal functional as well as structural status under the influence of excess iodine; generation of ROS in the spermatozoa as evident by DCFDA, altered acrosomal integrity as observed by fluorescence lectin staining method and depolarized mitochondrial membrane potential (ΔΨm ) noticed by JC-1 staining. Ultrastructure of seminiferous tubule after excess iodine exposure indicated severe deterioration of seminiferous tubular surface architecture. Significant increase in spermatozoal DNA fragmentation and apoptotic sperms were found by acridine orange and Annexin V, respectively, however the plasma membrane integrity/viability was decreased as evident by propidium iodide staining in various incremental doses and durations under iodine excess. The study reveals that excess iodine could cause apoptosis of spermatozoal cells by inducing ROS that ultimately affects male fertility potential.

Hydroxyhydroquinone, a by-product of coffee bean roasting, increases intracellular Ca2+ concentration in rat thymic lymphocytes

Hydroxyhydroquinone (HHQ) is generated during coffee bean roasting. A cup of coffee contains 0.1–1.7 mg of HHQ. The actions of HHQ on mammalian DNA were examined because HHQ is a metabolite of benzene, which causes leukemia. Currently, information on the cellular actions of HHQ is limited. We examined the effects of sublethal levels of HHQ on the concentration of intracellular Ca2+ in rat thymic lymphocytes by using a flow cytometric technique with fluorescent probes. HHQ at 10 μM or more significantly elevated intracellular Ca2+ levels by increasing the membrane permeability of divalent cations, resulting in hyperpolarization via the activation of Ca2+-dependent K+ channels. HHQ-induced changes in the intracellular Ca2+ concentration and membrane potential may affect the cell functions of lymphocytes. HHQ-reduced coffee may be preferable in order to avoid the possible adverse effects of HHQ.

A quantitative real-time approach for discriminating apoptosis and necrosis

Apoptosis and necrosis are the two major forms of cell death mechanisms. Both forms of cell death are involved in several physiological and pathological conditions and also in the elimination of cancer cells following successful chemotherapy. Large number of cellular and biochemical assays have evolved to determine apoptosis or necrosis for qualitative and quantitative purposes. A closer analysis of the assays and their performance reveal the difficulty in using any of these methods as a confirmatory approach, owing to the secondary induction of necrosis in apoptotic cells. This highlights the essential requirement of an approach with a real-time analysis capability for discriminating the two forms of cell death. This paper describes a sensitive live cell-based method for distinguishing apoptosis and necrosis at single-cell level. The method uses cancer cells stably expressing genetically encoded FRET-based active caspase detection probe and DsRed fluorescent protein targeted to mitochondria. Caspase activation is visualized by loss of FRET upon cleavage of the FRET probe, while retention of mitochondrial fluorescence and loss of FRET probe before its cleavage confirms necrosis. The absence of cleavage as well as the retention of mitochondrial fluorescence indicates live cells. The method described here forms an extremely sensitive tool to visualize and quantify apoptosis and necrosis, which is adaptable for diverse microscopic, flow cytometric techniques and high-throughput imaging platforms with potential application in diverse areas of cell biology and oncology drug screening.

Cytokine profile in childhood-onset systemic lupus erythematosus: a cross-sectional and longitudinal study.

Childhood-onset systemic lupus erythematosus (cSLE) exhibits an aggressive clinical phenotype and severe complications. This could be due to a pro-inflammatory cytokine milieu. Therefore, we determined plasma levels of Th1 (IL-2, IFN-γ, TNF), Th2 (IL-4), Th17 (IL-17A, IL-6), and Treg (IL-10) cytokines in a cohort of cSLE patients and healthy controls, and we evaluated the association between these cytokines and disease activity. We conducted a cross-sectional study with 51 cSLE patients from two pediatric rheumatology services. Ten cSLE patients participated in a longitudinal follow-up study. Blood samples were collected from the same patient during active and inactive disease. Disease activity was evaluated according to SLE Disease Activity Index 2000 (SLEDAI-2K). Cytokines levels were measured by cytometric bead array technique. cSLE patients had higher IL-6 (P<0.001) and IL-10 (P<0.001) levels than healthy controls. Patients with active disease had higher IL-6 and IL-10 levels than patients with inactive disease (P=0.001 and P=0.014, respectively) and the control group (both P<0.001). IL-6 (P=0.022), IL-10 (P=0.013), and IL-17A (P=0.041) levels were significantly higher during active than inactive disease. Linear regression analysis revealed IL-6 (P=0.002, 95%CI=0.006-0.025) and IL-10 (P=0.01 95%CI=0.021-0.150) as independent factors for increased SLEDAI-2K. IL-6, IL-10, and IL-17A are candidate biomarkers for disease activity in cSLE patients. This is the first longitudinal study to support their pivotal role in the pathogenesis of the disease.