IL-6 Assay Research Articles

P020 Increased IL-6 signaling in DSA positive renal transplant patients predicts graft failure

Aim IL-6 is associated with chronic inflammation seen in allograft rejection. Clinical trials using anti-IL6 antibodies have found a significant benefit in kidney allograft survival and a reduction in donor-specific anti-HLA antibodies (DSA). We developed a new cell-based IL-6 assay and tested transplant patient sera to determine the temporal relationship between IL-6 and DSA. Methods Serial samples from 45 deceased-donor kidney transplant (Tx) recipients (189 samples total) transplanted between 2011 and 2013 at East Carolina University were tested for both DSA and IL-6. Patients were categorized as DSA-, DSA + stable graft function (DSAstable) or DSA + eventual graft loss (DSAfail). In all patients, samples were tested post-transplant. In the case of the DSA positive cases, the samples were collected pre-DSA appearance and at DSA onset. Similar time points post-transplant in the DSA- patients were used. Results Post-transplant, DSA negative patient samples had significantly lower IL-6-fold-induction (n = 58, mean = 0.83, SE = 0.02) than DSAfail patients (n = 10, mean = 0.98, SE = 0.05; ANOVA, p Conclusions We detected significantly lower levels of IL-6 in DSA negative and DSA stable patients compared to DSA + patients who eventually failed. This testing was done pre-DSA or at the time of DSA onset suggesting IL-6 signaling may be important in the progression to failure in renal transplant patient. Therefore, treating DSA + patients with high IL-6 with an anti-IL6 may prove to be beneficial. Download : Download high-res image (114KB) Download : Download full-size image

Central serotonin attenuates LPS-induced systemic inflammation

Serotonin (5-HT) is a neuromodulator involved in several central-mediated mechanisms, such as endocrine processes, behavior, and sleep. Dysfunction of the serotonergic system is mainly linked to psychiatric disorders, but emerging evidence suggests that immune system activation may also alter brain 5-HT signaling. However, whether central 5-HT modulates systemic inflammation (SI) remains unknown. For this purpose, male Wistar rats (280–350g, 8–9weeks) were submitted to the experimental protocols beginning between 9 and 10AM with the performance of injections. The animals were housed at controlled conditions [temperature (25±1°C), light (06:00–18:00) and humidity (60–65%)]. Thus, we measured 5-HT and its metabolite 5-hydroxyindole-3-acetic acid (5-HIAA) in the anteroventral preoptic region [(AVPO) – the hierarchically most important region for body temperature (Tb) control] during lipopolysaccharide (LPS)-induced SI. We also combined LPS (100μg/kg) treatment with intracerebroventricular (icv) injection of 5-HT (5, 10 and 40μg/μL) and measured Tb (“hallmark” of SI), AVPO prostaglandin E2 [(PGE2) – an essential mediator of fever] and prostaglandin D2 [(PGD2) – a cryogenic mediator], plasma corticosterone [(CORT) – a stress marker with an endogenous anti-inflammatory effect] and interleukin-6 [(IL-6) – an immune mediator] levels. Detection limits of PGE2, PGD2, CORT and IL-6 assays were 39.1–2500pg/mL, 19.5–2500pg/mL, 0.12–2000μg/dL, and 0.125–8ng/mL, respectively. We also assessed tail skin temperature [used to calculate heat loss index (HLI)] to assess a key thermoeffector mechanism. As expected we observed LPS-induced increases in Tb, AVPO PGE2 (whereas PGD2 remained unchanged), plasma CORT and IL-6 levels, as well as a decrease in HLI. These changes were accompanied by reduced levels of AVPO 5-HT and 5-HIAA. Furthermore, we also observed a negative correlation between 5-HT and plasma CORT levels. Moreover, icv 5-HT (5, 10 and 40μg/μL) microinjection caused a U-shaped dose-response curve in LPS fever, in which the intermediate dose reduced the febrile response. Icv 5-HT (10μg/μL) microinjection prevented the LPS-induced increases in AVPO PGE2 (whereas not altering PGD2), plasma CORT and IL-6 levels, as well as preventing reduced HLI. Our data are consistent with the notion that AVPO 5-HT synthesis is down-regulated during SI, favoring AVPO PGE2 synthesis and consequently potentiating the immune response. These results reveal a novel effect of central 5-HT as an anti-inflammatory neuromodulator that may take place during psychiatric disorder treatment with 5-HT reuptake inhibitors as well as suggesting that 5-HT modulation per se is a potential therapeutic approach for inflammatory diseases.

Abstract 5793: Comparison of EMT biomarker expression in 2D monolayer and 3D spheroid cultures in a prostate cancer cell model

Abstract The purpose of this study was to examine the induction of epithelial to mesenchymal transition (EMT) in a prostate cancer cell line by measuring classical biomarker expression in three-dimensional (3D) spheroid cultures compared to traditional 2D monolayers in an effort to develop a more biologically relevant assay. Using Ultra-Low Attachment (ULA) microplates, we grew spheroids from a human prostate cancer cell line (DU 145). Numerous studies have implicated a role for EMT in carcinoma invasion and metastasis. EMT is characterized by rearrangement of the extracellular matrix (ECM) and differential regulation of ECM proteins. We induced EMT using TGF-beta and phorbol-12-myristate-13-acetate (PMA) and compared expression levels of specific biomarkers, such as E-cadherin, fibronectin, and IL-6, using AlphaLISA and LANCE (TR-FRET) assay technologies. We confirmed that treatment of DU 145 cells with TGF-beta is sufficient for inducing changes in both EMT biomarker expression and characteristic cellular morphology in monolayer cultures. However, in 3D spheroid cultures, we observed only a partial EMT response to the same TGF-beta treatment as demonstrated by changes in the expected biomarker expression pattern. Using the small molecule, PMA, we see significant differences in the levels of IL-6 secretion after EMT induction between cells grown in monolayer and those grown in spheroids. Cellular proliferation, growth and vitality were assessed using ATPlite luminescence assays and confocal microscopy of live-stained cells with a high content imaging system. Though we observe increased proliferation in monolayer cultures compared to 3D spheroids, the changes observed in protein expression patterns cannot be sufficiently explained by differences in cell number or viability. These data illustrate the differences in protein expression levels and in cellular tolerance for compound treatment between a human prostate cancer cell line grown in monolayers and those same cells grown in 3D spheroids. Citation Format: Jen Carlstrom, Jeanine M. Hinterneder, Lindsay Nelson, Stephen Hurt. Comparison of EMT biomarker expression in 2D monolayer and 3D spheroid cultures in a prostate cancer cell model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5793. doi:10.1158/1538-7445.AM2017-5793

Abstract 4699: Single domain antibody (sdAb) localizes in cancer cells to inhibit signal transducer and activator of transcription 3 (STAT3) resulting in therapeutic inhibition of multiple cancers

Abstract STAT3 is involved in the pathogenesis of many malignancies, so we developed an anti-STAT3 VHH (variable region of the heavy chain), SBT-100, that internalizes in cancer cells and binds unphosphorylated STAT3 (U-STAT3) and phosphorylated STAT3 (P-STAT3) and results in significant inhibition of multiple cancers. ATCC cell lines for triple negative breast cancers (MDA-MB-231, MDA-MB-468, MDA-MB-453), ER+/PR+ breast cancer (MCF-7), HER2+ breast cancer (BT474), pancreatic cancers (PANC-1, BX-PC3), murine mammary cancer (4T1), STAT3 null cells (PC-3), and castrate-resistant prostate cancer (DU145) were tested. Athymic nude mice were obtained from Envigo. The IL-6 Reporter Cell Assay was obtained from Promega. VEGF inhibition ELISA was done using retinal epithelial cells (ATCC). In vitro growth inhibition was done using a MTT assay. Immunoprecipitation (IP) and western blot studies in MDA-MB-231, PANC-1, HeLa, DU145, 4T1 and PC-3 showed that SBT-100 binds U-STAT3 and P-STAT3. No STAT3 binding was seen in PC-3. Binding to P-STAT3 was seen in lysates from the 4T1 cells, which have constitutively activated STAT3. Since rodent and human STAT3 have a 99% homology, rodents are excellent models for extrapolating to human disease for over production of P-STAT3. Within 24 hrs IL-6 assay showed that SBT-100 blocked the production of IL-6 (p<0.0001) compared to control. The degree of IL-6 suppression was comparable to the negative control, BB1608 (STAT3 inhibitor). VEGF ELISA showed significant (p<0.0001) inhibition of VEGF production within 12 hrs and was maintained for up to 48 hrs. After 3 days with SBT-100 the MTT assay showed growth inhibition (p<0.001) in BT474 (93%), MCF-7 (93%), MDA-MB-231 (77%), MDA-MB-468 (85%), MDA-MB-453 (64%), PANC-1 (79%), BX-PC3 (90%), and DU145 (92%). MDA-MB-231 tumors grown in xenograft athymic mice showed suppression (p<0.001), IHC staining in the cytoplasm, and nucleus after 7 days of SBT-100 treatment. U-STAT3 and P-STAT3 activate genes that promote growth, proliferation, angiogenesis, immune suppression, cancer stem cells, metastasis, and apoptosis inhibition. SBT-100 enters the cancers cells, binds STAT3 and P-STAT3 causing growth inhibition (p<0.001) in MCF-7, BT474, MDA-MB-231, MDA-MB-468, MDA-MB-453, PANC-1, BX-PC3, and DU145. These results suggest that SBT-100 can be developed as a therapeutic for cancers expressing either STAT3 or P-STAT3. Citation Format: Sunanda Singh, Genoveva Murillo, Amanda Rom, Avani Singh, Samara Singh, Meenakshi Parihar, Dong Chen, Rajendra Mehta, Robert Baker, Anjali Singh, Ashutosh S. Parihar. Single domain antibody (sdAb) localizes in cancer cells to inhibit signal transducer and activator of transcription 3 (STAT3) resulting in therapeutic inhibition of multiple cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4699. doi:10.1158/1538-7445.AM2017-4699

Design and synthesis of novel xanthine derivatives as potent and selective A2B adenosine receptor antagonists for the treatment of chronic inflammatory airway diseases

Adenosine induces bronchial hyperresponsiveness and inflammation in asthmatics through activation of A2B adenosine receptor (A2BAdoR). Selective antagonists have been shown to attenuate airway reactivity and improve inflammatory conditions in pre-clinical studies. Hence, the identification of novel, potent and selective A2BAdoR antagonist may be beneficial for the potential treatment of asthma and Chronic Obstructive Pulmonary Disease (COPD). Towards this effort, we explored several prop-2-ynylated C8-aryl or heteroaryl substitutions on xanthine chemotype and found that 1-prop-2-ynyl-1H-pyrazol-4-yl moiety was better tolerated at the C8 position. Compound 59, exhibited binding affinity (Ki) of 62 nM but was non-selective for A2BAdoR over other AdoRs. Incorporation of substituted phenyl on the terminal acetylene increased the binding affinity (Ki) significantly to <10 nM. Various substitutions on terminal phenyl group and different alkyl substitutions on N-1 and N-3 were explored to improve the potency, selectivity for A2BAdoR and the solubility. In general, compounds with meta-substituted phenyl provided better selectivity for A2BAdoR compared to that of para-substituted analogs. Substitutions such as basic amines like pyrrolidine, piperidine, piperazine or cycloalkyls with polar group were tried on terminal acetylene, keeping in mind the poor solubility of xanthine analogs in general. However, these substitutions led to a decrease in affinity compared to compound 59. Subsequent SAR optimization resulted in identification of compound 46 with high human A2BAdoR affinity (Ki = 13 nM), selectivity against other AdoR subtypes and with good pharmacokinetic properties. It was found to be a potent functional A2BAdoR antagonist with a Ki of 8 nM in cAMP assay in hA2B-HEK293 cells and an IC50 of 107 nM in IL6 assay in NIH-3T3 cells. Docking study was performed to rationalize the observed affinity data. Structure-activity relationship (SAR) studies also led to identification of compound 36 as a potent A2BAdoR antagonist with Ki of 1.8 nM in cAMP assay and good aqueous solubility of 529 μM at neutral pH. Compound 46 was further tested for in vivo efficacy and found to be efficacious in ovalbumin-induced allergic asthma model in mice.

Protein Expression Level of Skin Wrinkle-Related Factors in Hairless Mice Fed Hyaluronic Acid.

The aim of this study was to evaluate the wrinkle improving effect of hyaluronic acid intakes. Wrinkles were induced by exposing the skin of hairless mice to ultraviolet B (UVB) irradiation for 14 weeks. Hyaluronic acid was administered to the mice for 14 weeks including 4 weeks before experiments. Skin tissue was assayed by enzyme-linked immunosorbent assay to determine protein expression of wrinkle-related markers. The group supplemented with high concentrations of hyaluronic acid appeared significantly better than control group for collagen, matrix metalloproteinase 1, interleukin (IL)-1β, and IL-6 assay. Transforming growth factor-β1 (TGF-β1) and hyaluronic acid synthase 2 (HAS-2) were not shown to be significantly different. In conclusion, hyaluronic acid administration regulated expression levels of proteins associated with skin integrity, and improved the wrinkle level in skin subjected to UVB irradiation.

Serum interleukin-6 is related to lower cognitive functioning in elderly patients with major depression

ABSTRACTBackground: There is an increased evidence of an association between inflammatory mediators, particularly serum IL-6, depression and cognitive impairment in the elderly. This study aims at exploring the relation of peripheral IL-6 to cognitive functions in elderly patients with major depressive disorder (MDD).Objectives: (1) Assessment of serum IL-6 levels and cognitive functions in elderly patients suffering from major depression and comparing them to healthy age-matched control subjects; (2) correlation between serum IL-6 levels and clinical characteristics of depression and cognitive functions in these patients.Subjects and methods: The study is an observational, case-control study. It consisted of 80 subjects, 40 with the diagnosis of MDD according to the Diagnostic and Statistical Manual of Mental Disorders (DSM IV-TR) with early onset (first episode before the age of 60) and 40 community-dwelling subjects. They were subjected to the Structured Clinical Interview according to DSM-IV, Montreal Cognitive Assessment, Montgomery Asberg Depression Rating Scale, and serum IL-6 assay using ELISA.Results: In the depression group, subjects had lower scores in cognitive testing, than the control group (p = 0.001). Serum IL-6 was found to have a negative correlation with cognitive testing in these patients even after controlling for the severity of depressive status and Body Mass Index (BMI) (p = 0.025).Conclusions: MDD in elderly subjects is associated with decline in cognitive functions that may be related to peripheral IL-6 levels.

Soluble endoglin modulates the pro-inflammatory mediators NF-κB and IL-6 in cultured human endothelial cells

AimsEndoglin is a transmembrane glycoprotein, that plays an important role in regulating endothelium. Proteolytic cleavage of membrane endoglin releases soluble endoglin (sEng), whose increased plasma levels have been detected in diseases related to the cardiovascular system. It was proposed that sEng might damage vascular endothelium, but detailed information about the potential mechanisms involved is not available. Thus, we hypothesized that sEng contributes to endothelial dysfunction, leading to a pro-inflammatory phenotype by a possible modulation of the TGF-β and/or inflammatory pathways. Main methodsHuman umbilical vein endothelial cells (HUVECs) and Human embryonic kidney cell line (HEK293T) were treated with different sEng concentration and time in order to reveal possible effect on biomarkers of inflammation and TGF-β signaling. IL6 and NFκB reporter luciferase assays, quantitative real-time PCR analysis, Western blot analysis and immunofluorescence flow cytometry were used. Key findingssEng treatment results in activation of NF-κB/IL-6 expression, increased expression of membrane endoglin and reduced expression of Id-1. On the other hand, no significant effects on other markers of endothelial dysfunction and inflammation, including eNOS, peNOSS1177, VCAM-1, COX-1, COX-2 and ICAM-1 were detected. SignificanceAs a conclusion, sEng treatment resulted in an activation of NF-κB, IL-6, suggesting activation of pro-inflammatory phenotype in endothelial cells. The precise mechanism of this activation and its consequence remains to be elucidated. A combined treatment of sEng with other cardiovascular risk factors will be necessary in order to reveal whether sEng is not only a biomarker of cardiovascular diseases, but also a protagonist of endothelial dysfunction.

Comparison of rapid MMP-8 and interleukin-6 point-of-care tests to identify intra-amniotic inflammation/infection and impending preterm delivery in patients with preterm labor and intact membranes

Objective: Among patients presenting with preterm labor and intact membranes, those with intra-amniotic inflammation have adverse obstetrical and neonatal outcomes. The diagnosis of intra-amniotic inflammation can easily be made by detecting an elevated concentration of the cytokine interleukin (IL)-6 or the enzyme neutrophil collagenase, also known as matrix metalloproteinase (MMP)-8. The diagnostic performances of MMP-8 and IL-6 enzyme-linked immunosorbent assay tests are similar. Recently, a rapid test has become available for point-of-care determination of either MMP-8 or IL-6. The objectives of this study were to compare the diagnostic indices and predictive values between the rapid MMP-8 and IL-6 tests for the identification of intra-amniotic inflammation in patients with preterm labor and intact membranes.Materials and methods: We performed a retrospective cohort study including 124 women with singleton pregnancies who presented with symptoms of preterm labor and underwent transabdominal amniocentesis for the evaluation of microbial invasion of the amniotic cavity (MIAC). MIAC was defined according to amniotic fluid culture results (aerobic and anaerobic bacteria as well as genital Mycoplasmas). Amniotic fluid white blood cell (WBC) counts were determined using a hemocytometer chamber. An elevated amniotic fluid MMP-8 concentration was assessed using Yoon’s MMP-8 Check® (cutoff: 10 ng/mL). An elevated amniotic fluid IL-6 concentration was scored when there was a positive result for the lateral flow-based immunoassay (cutoff: ≥745 pg/mL and ≥1000 pg/mL). In order to objectively compare rapid MMP-8 and rapid IL-6 tests to identify intra-amniotic inflammation, an amniotic fluid WBC count of ≥50 cells/mm3 was used to define intra-amniotic inflammation.Results: (1) The rapid tests had the same sensitivity for the detection of intra-amniotic inflammation [85.7% (18/21) for all]; (2) the specificity of the rapid MMP-8 test was higher than that of the rapid IL-6 test (cutoff: 745 pg/mL) for the identification of intra-amniotic inflammation [72.8% (75/103) vs. 64.1% (66/103); p < 0.05]; and (3) there were no differences in the sensitivity and specificity between the rapid MMP-8 test and the rapid IL-6 test (cutoff:1000 pg/mL) in the identification of intra-amniotic inflammation. Of 13 patients with discrepant results between the rapid MMP-8 and rapid IL-6 tests, two had a positive MMP-8 but a negative rapid IL-6 test, and both delivered preterm – one within 24 h, and the other within 10 days – and both had acute histologic chorioamnionitis. On the other hand, there were 11 patients with a positive rapid IL-6 but a negative rapid MMP-8 result: 10 delivered preterm, 3 had acute histologic chorioamnionitis and 1 had subacute chorionitis.Conclusion: We conclude that the rapid MMP-8 test has a better specificity than the rapid IL-6 (cutoff: 745 pg/mL) assay for the detection of intra-amniotic infection. Moreover, we observed that among patients who were not identified as having intra-amniotic infection or inflammation by the standard cultivation technique and amniotic fluid WBC count, those who had a positive MMP-8 rapid test delivered preterm and had acute histologic chorioamnionitis.

Micro-bead immunoassays for the detection of IL6 and PDGF-2 proteins on a microfluidic platform, incorporating superhydrophobic passive valves

A microfluidic platform optimized for bead-based immunoassays is presented. It uses a simple design that incorporates passive valves in the inlet and outlet of a microreactor. The superhydrophobic, passive valves are created by selective plasma nanotexturing and deposition of a hydrophobic layer. The anti-sticking operation of the valves simplify the working principle, reduce the reagents needed, and enhance the fluorescence intensity of the assays performed on chip by reducing non-specific binding compared to assays performed on plate. Two different multi-step immunoassays were tested individually and as a mixture to evaluate the platform performance: IL6 and PDGF-2 human protein assays. The results show that the proposed platform offers faster assay kinetics, allows lower reagent use (5μl instead of 50), enhanced fluorescent signals (by a factor of two or more), and reduced incubation time at least by a factor of 2. A possible configuration using the microfluidic platform for a portable biosensing system is also demonstrated.

“In vitro” osteogenic and angiogenic potential evaluation of a coculture of dental pulp stem and endothelial cells grown on the BisGMA/TEGDMA Chitlac coated thermosets

Securing an adequate blood supply for survival of cell transplants is critical for a successful outcome in tissue engineering. Moreover during regeneration of weaken teeth, which is susceptible to reinfection, fracture and loss, the teeth apical canal is open and a limited blood supply is allowed. Thus the interactions between endothelial and dental pulp progenitor stem cells are important for vascularization of regenerating tissue cells. In particular, the interplay of dental pulp stem cells and endothelial cells can enhance “in vitro” osteo/odontogenic and angiogenic potential (Dissanayaka J Endod 2012, 38,454-463) and “in vivo” ensure angiogenesis and pulp regeneration (Dissanayaka Tissue Engin Part A 2015, 3-4, 550-563). Since dental pulp microenvironment supports HUVEC survival and capillary network formation in the absence of scaffolding material and external angiogenic stimulation , “in vitro” osteogenic and angiogenic potential of dental pulp stemcells cocultured with endothelial cells grown on BisGMA/TEGDMA Chitlac coated thermosets was evaluated. Results: DPSCs were grown on BisGMA/TEGDMA Chitlac coated thermosets, a composite material used in dental restoration, in the presence of two different concentrations of endothelial cells (1:1 e 1:5) for 28 days and their metabolic activity and cytotoxic response were evaluated. MTT analysis discloses that cell metabolic activity significantly increases in the presence of endothelial cells, mainly at 21 days of culture, along with cytotoxic response, while at 28 days of culture a light cytotoxic response occurs. An increasing ALP activity is evidenced in the coculture systems up to 28 days, both in the presence and in absence of Chitlac thermosets and this evidence is further supported by Alizarin red staining, which does not detect mineralization in the early stages of differentiation, but is significantly increased at 28 days of culture in both the conditions (1:1 e 1:5). Even though the positive effect on DPSC differentiation, Chitlac thermosets could induce an inflammatory response in the system and thus an ELISA IL6 assay reveals an increased inflammatory response in 1:1 coculture system after 28 days of culture, furtherly increased in 1:5 coculture system. In parallel an increased PGE2 release is evidenced in 1:1 coculture system in the presence of thermosets, reduced in 1:5 coculture system, suggesting the potential occurrence of neoangiogenesis, furtherly supported by a tubular network formation when DPSC are grown on matrigel. These results evidencing that endothelial cells enhance “in vitro” osteo/odontogenic differentiation of DPSCs and angiogenesis, and that this response is revealed in the presence of Chitlac, usually used in dental restorative practice, indicate a coculture of DPSC and endothelial cells as a promising source for regenerative endodontics.

Comparation of Toxic Effect of Silicious Mineral Dusts on Lung Epithelial A549 Cells

Considering the high contents of minerals and the potential health risk of mineral dusts to human and the environment, this paper was aimed to figure out the toxic effect and mechanism of four common mineral particles (quartz, albite, sericite, and montmorillonite). Cytotoxicity assays for cell viability (MTT assay), membrane integrity (LDH assay), oxidative stress (H2O2 assay) and inflammatory cytokines (TNF-α and IL-6 assay) were applied. The results showed the influence of these mineral particles on A549 cell viability followed the order of momtmorillonite > cericite≥quartz > albite. There was no obvious relation between cell viability and the content of SiO2, however, good linear correlation with the content of iron, and the cytotoxicity of mineral dusts was strengthened with increasing iron content. Mineral dusts generated H2O2 in cell or cell-free systems. In particular, H2O2 exhibited a linear correlation with the iron content, which meant that iron in the mineral dusts played an important role in the generation of reactive radical. Among those samples, oxidative stress induced by montmorillonite was distinctly stronger, while there was negligible influence induced by quartz and albite. Besides, all the tested samples induced damage to A549 cell membrane, and triggered the release of TNF-α or IL-6, but differed by the kinds of mineral dusts. In conclusion, composition and structure directly affected, but were not the only factors that contributed to the biological activity of mineral dusts, the evaluation of cell viability, membrane damage, free radicals and inflammatory reaction induced by mineral dusts should take the external morphology, surface active groups, solubility, adsorption and ion exchange properties into consideration.

How to: Measuring blood cytokines in biological psychiatry using commercially available multiplex immunoassays

Cytokines produced by both immune and non-immune cells are likely to play roles in the development and/or progression of psychiatric disorders. Indeed, many investigators have compared the blood cytokine levels in psychiatric patients with those of healthy controls or monitored their levels in patients during disease progression to identify biomarkers. Nevertheless, very few studies have confirmed that such cytokines remain stable in healthy individuals through periods of weeks and months. This is an important issue to consider before using blood cytokine levels as biomarkers of disease traits, disease state, or treatment response. Although multiplex assay technology represents an advance in identifying biomarkers because it allows simultaneous examination of large panels of analytes from a small volume of sample, it is necessary to verify whether these assays yield enough sensitivity and reproducibility when applied to the blood from neuropsychiatric patients. Therefore, we compared two multiplex immunoassays, the bead-based Luminex® (Bio-Rad) and the electro-chemiluminescence-based V-plex® (MesoScaleDiscovery), for the detection and quantification of 31 cytokines, chemokines and growth factors in both the sera and plasma of patients with major depressive episodes (MDE) and age- and sex-matched healthy control subjects during a 30-week period. Although both platforms exhibited low coefficients of variability (CV) between the duplicates in the calibration curves, the linearity was better in general for the V-PLEX® platform. However, neither platform was able to detect the absolute values for all of the tested analytes. Among the 16 analytes that were detected by both assays, the intra-assay reproducibility was in general better with the V-PLEX® platform. Although it is not a general rule that the results from sera and plasma will be correlated, consistent results were more frequent with the V-PLEX® platform. Furthermore, the V-PLEX® results were more consistent with the gold standard ELISA simplex assay for IL-6 in both sera and plasma. The intra-individual variability of the measurements, among the sera and plasma for the 4 samples harvested from each healthy individual, was low for Eotaxin, G-CSF, IL-4, IL-7, IL-9, IL-12p40, IL-12p70, IL-15, MIP-1β, PDGF-BB, TNF, TNF-β and VEGF, but intermediate or high for IFN-γ, IL-6, IL-8, IL-10, and IP10. Together, these data suggest that extreme caution is needed in translating the results of multiplex cytokine profiling into biomarker discovery in psychiatry.

Ezetimibe inhibits platelet activation and uPAR expression on endothelial cells

PurposeLipid lowering therapy constitutes the basis of cardiovascular disease therapy. The purpose of this study was to investigate effects of ezetimibe, a selective inhibitor of intestinal cholesterol absorption, on platelets and endothelial cells in an in vitro endothelial cell model. MethodsAfter a 24h incubation period with ezetimibe (concentrations 1, 50, 100 and 1000ng/ml), human umbilical vein endothelial cells (HUVEC) were stimulated for 1h with lipopolysaccharide (LPS) and were then incubated in direct contact with activated platelets. Following this, the expression of CD40L and CD62P on platelets, and the expression of ICAM-1, VCAM-1, uPAR, and MT1-MMP on endothelial cells were measured by flow cytometry. Supernatants were analysed by enzyme linked immunosorbent assay for soluble MCP-1, IL-6 and MMP-1. ResultsThe increased expression of uPAR on endothelial cells by proinflammatory stimulation with LPS and by direct endothelial contact with activated platelets was significantly reduced through pre-incubation with 100ng/ml and 1000ng/ml ezetimibe (p<0.05). Platelets directly incubated with ezetimibe but without endothelial cell contact showed significantly reduced CD62P and CD40L surface expression (p<0.05). Ezetimibe had no significant effects on HUVEC expression of MT1-MMP, ICAM-1 and VCAM-1 and on CD40L expression on platelets in direct contact with endothelial cells. Levels of soluble IL-6 in HUVEC supernatants were significantly lower after pre-incubation with ezetimibe. ConclusionIn this in vitro analysis, ezetimibe directly attenuates platelet activation and has significant endothelial cell mediated effects on selected markers of atherosclerosis.

Evaluation of highly sensitive immunoassay technologies for quantitative measurements of sub-pg/mL levels of cytokines in human serum

A comprehensive cross-platform and cross-assay evaluation using nine technology platforms and four cytokine immunoassays (IL-6, TNFα, IL-17a, IL-2) was performed by comparing assay precision, sensitivity, parallelism and data correlation between platforms. The precision was acceptable for most evaluated assays. In addition to comparing the analytical assay sensitivity using a spiked recombinant analyte in buffer, forty serum samples from both normal controls and multiple sclerosis patients were used to measure the frequency of endogenous analyte detection (FEAD) as a parameter of each assay's ability to detect the endogenous analyte. The highest FEAD measurements were observed on the Simoa™, Erenna®, Milliplex® and Imperacer® platforms. However, only Simoa and Erenna results showed a high correlation across all evaluated cytokine assays, followed by a more moderate correlation of results across platforms for the V-plex™, high sensitivity ELISA and the Ella™ IL-6 and TNFα assays. In contrast, results from the evaluated cytokine assays on the Milliplex, AMMP™ ViBE® and Imperacer platforms did not correlate to each other nor to other evaluated assays. Acceptable parallelism was observed for the Simoa, Erenna, V-plex and Ella assays but not for the Milliplex, AMMP ViBE and Imperacer assays. In conclusion, the Simoa, Erenna,V-plex and Ella platforms performed well in one or more evaluated cytokine assays. Among those, the Simoa and Erenna assays had the highest sensitivity for detection of cytokines present at sub-pg/mL levels in human serum. In addition, the cross-platform and cross-assay comparisons demonstrated that different immunoassays may yield different results, which underscores the importance of performing such comparative evaluations, especially in the absence of reliable reference standards for the quantitative assessments of biomarkers in immunoassays.

Effect of moderate or high intensity exercise on hypothyroid rats exposed to acute stress.

There are contradictory results about stress response in hypothyroidism and in exercising with variant intensities. We aimed to investigate the potential anxiolytic and protective effects of different intensities of exercise on acute psychological stress in hypothyroidism. Rats (N.=48) were divided as sedentary, moderate intensity (MIE) and high intensity exercise (HIE) groups. Rats were administered intraperitoneally with 6-n-propyl-2-thiouracil (PTU, 10 mg/kg) for 15 days to induce hypothyroidism. Starting by the 3rd week, treadmill exercise was performed moderately (30 min/day) or at high intensity (60 min/day) for 6 weeks, 5 days/week. At the end of the 8th week, exposure to water avoidance stress was used for induction of acute stress. Anxiety-like behavior was determined by holeboard test before and after stress inductions. Serum IL-1β and IL-6 assays, and myeloperoxidase activity (MPO), malondialdehyde (MDA), and glutathione (GSH) measurements, and histological analysis of heart, liver, stomach and small intestine were made. All groups showed increased anxiety-like behavior following acute stress induction. After acute stress induction, increased MPO and MDA levels in heart and elevated MPO activity in liver were inhibited in PTU-treated HIE group. In MIE rats, increased MPO and declined GSH levels of the gastric tissue and small intestine, and elevated MDA levels of gastric tissue were reversed in PTU-treated MIE group. Major histological changes that occurred by both intensities of exercise under stress condition were improved by PTU. Our results indicate that hypothyroid state may be protective against stress- and exhaustive exercise-induced oxidative damage.

Pre-pregnancy maternal plasma cytokine levels and risks of small-for-gestational-age at birth

Objectives: Small-for-gestational-age (SGA) results from abnormalities of the feto-placental-maternal unit. Cytokine profiles during early pregnancy may predict placental changes that lead to SGA, however it is unknown if these altered profiles precede conception. We examined the role of maternal cytokines prior to conception as risk factors for subsequent delivery of an SGA infant. Methods: We included a sample of 80 women and their offspring from a large trial of pre-conceptual multiple micronutrient supplementation. Plasma samples collected before conception were tested with a high sensitivity multiplex assay for IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IFNγ, and TNFα. Results: Pre-conceptual IL-13 and IFNγ were lower in women who gave birth to an SGA child than among control women (0.81 versus 1.14 pg/ml and 7.81 versus 11.01 pg/ml, respectively). Multivariable logistic regression showed that IL-13 (p = 0.029) and IFNγ (p = 0.015) were both inversely associated with SGA. Conclusions: These results suggest maternal immune function prior to conception may indicate an unfavorable immune balance leading to placental abnormalities and high risk of SGA. Preconception assessment of cytokine profiles could potentially contribute to early detection of SGA and to the timely implementation of interventions to prevent it.

α-Mangostin Inhibits α-Synuclein-Induced Microglial Neuroinflammation and Neurotoxicity.

Microglia-mediated neuroinflammation induced by α-synuclein in the substantianigra likely either initiates or aggravates nigral neuro degeneration in Parkinson’s disease (PD). We aimed to explore the effects of α-mangostin (α-M), a polyphenolicxanthone derivative from mangosteen on α-synuclein-stimulated DA neurodegeneration. Primary microglia, mesencephalic neuron, mesencephalic neuron-glianeuronal cultures, and transwell co-cultures were prepared separately. Liquid scintillation counting was used to determine the radioactivity in DA uptake. Enzyme-linked immunosorbent assay (ELISA) was performed in the IL-1β, IL-6, and TNF-α assay. The expression of proteins was analyzed by Western blot. α-M inhibited the increased levels of pro-inflammatory cytokines, NO, and ROS in α-synuclein-stimulated primary microglia. Mechanistic study revealed that α-M functioned by inhibition of nuclear factor kappa B (NF-κB) and NADPH oxidase. Further, α-M protected α-synuclein-induced microglial and direct neurotoxicity. Although detailed mechanisms remain to be defined, our observations suggest a potential compound, which inhibits microglial activation induced by α-synuclein by targeting NADPH oxidase, might be a therapeutic possibility in preventing PD progression.

Effect of β-amyloid peptide 42 on the dynamics of expression and formation of Аβ40, IL -1β, TNF α, IL -6, IL -10 by peripheral blood mononuclear cells in vitro and its correction by curcumin.

The toxic effect of Аβ-oligomers accompanies chronic inflammation, with cytokines as main mediators. Therefore, the cytokine link of inflammation becomes a new target on the way to restrain amyloidosis. The aim of the study was the effect of aggregated Аβ42 on the dynamics of expression and formation of endogenous Аβ40 and cytokines (IL-1β, TNFα, IL-6, IL-10) by peripheral blood mononuclear cells in vitro and its correction by curcumin. A suspension of mononuclear cells isolated ex tempore using ficoll-urografin gradient from venous blood samples of healthy volunteers were used to study the effects of Аβ42 (15 nM), curcumin (54 pM), and their combined action (at similar concentrations) in time dynamics: 0, 1, 3, 6 and 24 h incubation at 37 °C. Polymerase chain reaction with appropriate primers was used to determine the relative expression of mRNA for AβPP, TNFα, IL-1β, IL-6, IL-10 and enzyme-linked immunosorbent assay – to determine the content of Аβ40 and cytokines in mononuclear suspension during all periods of incubation. The individual dynamics AβPP and cytokine expression was shown under the action of the Aβ42, which had influence on the content of Aβ40, TNFα, IL-1β, IL-6 and IL-10 in mononuclear suspension. Curcumin displayed the inhibitory effect on gene expression of AβPP, TNFα and IL6, which resulted in the decrease of the level of these two cytokines and Aβ40. Thus, the dynamics of anti-inflammatory effect of curcumin in vitro for transcriptional and translational levels of cytokine’s formation by mononuclear cells was shown in the work. Direct inhibitory effect of curcumin on the concentration of endogenous Aβ40 during the 24 h incubation in conditions of toxic action of Aβ42 aggregates was established.

Lipids and lipoproteins and inflammatory markers in patients with chronic apical periodontitis.

BackgroundSince chronic apical periodontitis (CAP) appears to be a risk factor for coronary heart disease, the aim of the study was to determine the relationship between the size of CAP lesion and inflammatory markers (hsCRP, IL-6, TNF-α), as well as lipids and lipoproteins (LpPLA2, apoAI, apoB level) in blood serum of patients with CAP.MethodsThe patients studied (n = 43) were divided into groups: patients under 50 and over 50 years of age, and a separate subgroup of the oldest age with the largest size of CAP lesions. Apolipoprotein AI (apoAI) above 150 mg/dL and below 150 mg/dL was used as an important criterion for the division of patients into groups. The CAP lesion size was measured using the Kodak digital imaging system software. The control group consisted of clinically healthy volunteers (n = 20) without CAP. Lipids were measured on a Siemens analyzer (Germany), apoAI, apoB, hsCRP levels were determined by immunonephelometric method, using the Health Care Diagnostic Product (Siemens GmbH, Germany), and IL-6, TNF-α and LpPLAG7 assay kits (ELISA, R&D Systems) were used.ResultsThe findings suggested that in patients with CAP and their age increase, the CAP lesion size, the concentration of inflammatory markers and LpPLA2 mass increased. Correlations between the CAP lesion size and LpPLA2 mass and between the CAP lesion size and TG level in patients with apoAI 150 ≤ mg/dL showed increase TG in atherogenic apoB-containing triglyceride-rich lipoprotein and TC in cholesterol-rich lipoprotein. The patients with a low apoAI and high LpPLA2 level can have a higher risk of odontogenic disease and progression of atherosclerosis and coronary heart disease.ConclusionWe have found a positive correlation between apoAI level and the CAP lesion size and a negative correlation between LpPLA2 level and the CAP lesion size. The results suggest that apoAI and LpPLA2 in HDL particles have antiinflammatory action and together can limit the CAP lesion size in patient with a higher apoAI level. The literature data on the distribution of lipoprotein particles in subjects are still insufficient, so this problem requires further studies.