Quantitative ELISA Assay Research Articles

Application of rapid test kits for the determination of paralytic shellfish poisoning (PSP) toxins in bivalve molluscs from Great Britain

Six different commercial rapid screening assays for Paralytic Shellfish Poisoning toxins were assessed with the analysis of shellfish samples from GB. The performance of each kit was assessed through comparison with the current regulatory HPLC method. Samples assessed consisted of a wide variety of shellfish species of importance to the shellfish industry in GB. These had been sourced over a number of years and with a wide variety of geographical origins. One lateral flow immunoassay was found to provide a quick qualitative assessment of PSP toxicity, with a low proportion of false negative results for PSP-positive samples, but with higher numbers of false positives. The performance of the five quantitative ELISA assays varied considerably, with two demonstrating an inappropriate linear range, with others either over-estimating or under-estimating toxicity. One ELISA from R-Biopharm was found to show a good correlation with the HPLC toxicity results. All ELISAs, however, returned some false negative results, most notably for samples containing high proportions of toxins with low cross reactivity to saxitoxin such as GTX1&4. Whilst the lateral flow assays on the market are of particular use to Food Business Operators for end product testing, further work is required in parallel with instrumental testing methods using a larger number of samples to assess the reliability and accuracy of these kits over the long term.

MHP-1 inhibits cancer metastasis and restores topotecan sensitivity via regulating epithelial-mesenchymal transition and TGF-β signaling in human breast cancer cells

BackgroundCordyceps has long been used to treat cancer. However, its pharmacologically active components as well as the molecular mechanisms underlying its effects are still unclear. PurposeTo investigate the effect of MHP-1, a newly isolated polysaccharide from Mortierella hepialid (the asexual structure of C. sinensis), on breast cancer metastasis. Study designThe effect of MHP-1 on breast cancer cell migration, epithelial-mesenchymal transition (EMT) and TGF-β signaling were investigated in vitro and in vivo. The effect of MHP-1 against topotecan-resistant MCF-7 cells that developed an EMT-like phenotype was also examined. MethodsThe in vitro effect of MHP-1 on breast cancer cell proliferation and migration was evaluated by CCK8 and transwell assay. Morphological changes were observed and EMT markers were detected by western blot. The production of MMPs was measured by quantitative PCR and ELISA assay. To further investigate the mechanism that MHP-1 inhibited breast cancer EMT, western blot, ELISA, luciferase reporter gene assay, siRNA, quantitative PCR, immunohistochemistry, and xenograft tumor model were performed. ResultsMHP-1 inhibited breast cancer cell migration but did not cause any cytotoxicity. MHP-1 significantly surpressed breast cancer EMT, and slightly decreased MMP-9 secretion. TGF-β signaling was selectively inhibited after MHP-1 treatment, and other EMT-related pathways, like Wnt and Notch, were not affected. MHP-1 reduced the secretion of TGF-β1, but rarely affected other EMT-induced cytokines. Dual luciferase assay and Smad2/3 phosphorylation analysis indicated that MHP-1 suppressed TGF-β signaling. We further showed that MHP-1 restored sensitivity in topotecan (TPT)-resistant MCF-7 cells that developed an EMT-like phenotype. Similarly, the effect of TPT on resistant MCF-7 cells was also increased either by ALK5 (TGFβRI) siRNA or by a small molecular inhibitor of ALK5, SB-431542. MHP-1 inhibited breast cancer metastasis in the MDA-MB-231 xenograft model, and the immunohistochemical staining showed dramatically decreased expression of ALK5 and vimentin, and increased expression of E-cadherin. ConclusionMHP-1 significantly inhibited breast cancer metastasis and restored drug sensitivity in TPT-resistant cells via down-regulation of TGF-β signaling and EMT program. The combination of non-toxic agents like MHP-1 and current anti-cancer drugs should be considered in the future treatment of cancer.

Expression level of chromodomain Y (CDY): potential marker for prediction of sperm recovery in non-obstructive azoospermia

The availability of testis specific genes will be of help in choosing the most promising biomarkers for the detection of testicular sperm retrieval in patients with non-obstructive azoospermia (NOA). Testis specific chromodomain protein Y 1 (CDY1) is a histone acetyltransferase which concentrates in the round spermatid nucleus, where histone hyperacetylation occurs and causes the replacement of histones by the sperm-specific DNA packaging proteins, TNPs and PRMs. The aim was to evaluate CDY1 gene as a marker for predicting of successful sperm retrieval in NOA patients. This research was conducted on 29 patients with NOA who had undergone testicular sperm extraction (TESE) procedure. NOA patients were subdivided into patients with successful sperm retrieval (NOA+, n=12) and patients with unsuccessful sperm retrieval (NOA-, n=17). Relative expression of CDY1 gene and chromatin incorporation of CDY1 protein were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and ELISA assay, respectively. Quantification of mRNA relative expression and incorporation of CDY1 protein in chromatin showed significant lower expressions and protein levels of CDY1 in testis tissues of NOA- in comparison to NOA+ group. The findings in this study demonstrated a correlation between the low levels of CDY1 function and unsuccessful sperm recovery in the testicular tissues of NOA- compared to NOA+ patients. Therefore, it can be reasonable to consider CDY1 as a potential biomarker for predicting the presence of spermatozoa, although the claim needs more samples to be confirmed.

416. Enhanced Oncolytic Effect of Tumor Necrosis Factor Alpha Armed Engineered Oncolytic Measles Virus Therapy for Ovarian Carcinoma Cell Line

Introduction. Tumor necrosis factor alpha (TNFa) has been recognized having anticancer properties, and proposed as a cancer treatment early in the 1980s, But its toxicity lead to limited use. Replication competent oncolytic viruses such as live attenuated measles virus (MV), targeting cancer cells selectively, have shown very promising results in both pre and clinical stages. one of its most useful aspects is their ability to produce high amounts of transgene products locally, resulting in high local tumor microenvironment versus systemic concentrations. Therefore, we developed Gene therapy approach to increase anti-tumor activity results from the combination of oncolytic activity, transgene-mediated direct cytotoxic effect such as TNFa-mediated apoptosis. Methods, we created novel oncolytic MV expressing murine TNFa by cloning TNFa gene into MV genome. the gene was amplified by PCR using primers that introduce restriction sites at either end of the coding sequence. The gene cloned into a plasmid encoding the entire measles genome. The insertion site for the TNFa gene was between H and L in the first version. In the second version, transgene was inserted upstream of N. The resulting full-length plasmids [MV-TNFa1 and MV-TNFa2] used to rescue the corresponding recombinant measles virus on 293-3-46 helper cells that was transfected with the MV plasmids. We evaluated the 2 recombinant versions of the MV expressing TNFa with 2 other viruses of MV, one having the sodium iodide symporter (NIS) between H and L, and the second has GFP upstream of N for oncolytic potential against ovarian carcinoma cell line SKOV-3. Infectivity, syncytium formation and cytotoxicity of recombinant MV-TNFs in SKOV-3 cells were evaluated by inverted microscopy and the MTT assay. Transgene expression in SKOV-3 cells after infection was assessed for TNFa functionality and concentration using cells that are genetically engineered for NF-kB Signaling Pathway and TNFa quantitative ELISA assay. Results, we were successful to generate the two versions of the virus with TNFa in different genome locations for each version. MV-TNFa2 shown to produce more TNFa than MV-TNFa1 (Figure-1a), but both viruses were producing functional TNFa by activating NF-kB Signaling Pathway (Figure-1b). Recombinant MV-TNFs viruses were efficiently infected SKOV-3 cell line, resulting in extensive syncytium formation followed by cell death. There was enhanced killing for the MV-TNFa1 infected cells comparing to all other viruses (Figure-1c). In conclusion, engineered MV-TNFs may be a potent and novel cancer gene therapy system. MV expressing TNFa elicited oncolytic effects in ovarian cancer cells, enhancing the killing effect which depend on the insert location.

Circulating MiR-19b-3p, MiR-134-5p and MiR-186-5p are Promising Novel Biomarkers for Early Diagnosis of Acute Myocardial Infarction

Background/Aims: Recent studies have shown that circulating microRNAs (miRNAs) are emerging as promising biomarkers for cardiovascular diseases. This study aimed to determine whether miR-19b-3p, miR-134-5p and miR-186-5p can be used as novel indicators for acute myocardial infarction (AMI). Methods: To investigate the kinetic expression of the three selected miRNAs, we enrolled 18 patients with AMI and 20 matched controls. Plasma samples were collected from each participant, and total RNA was extracted. Quantitative real-time PCR and ELISA assays were used to investigate the expression of circulating miRNAs and cardiac troponin I (cTnI), respectively. Plasma samples from another age- and gender-matched cohort were collected to investigate the impact of medications for AMI on the expression of the selected miRNAs. Results: Levels of plasma miR-19b-3p, miR-134-5p and miR-186-5p were significantly increased in early stage of AMI. Plasma miR-19b-3p and miR-134-5p levels reached peak expression immediately after admission (T0), whereas miR-186-5p achieved peak expression at 4 h after T0. All of these times were earlier than the peak for cTnI (8 h after T0). In addition, all three miRNAs were positively correlated with cTnI. Receiver Operating Characteristic (ROC) analysis indicated that each single miRNA showed considerable diagnostic efficiency for predicting AMI. Furthermore, combining all three miRNAs in a panel increased the efficiency of distinguishing between patients with AMI and controls. Moreover, we found that heparin and medications for AMI did not impact the expression of these circulating miRNAs. Conclusion: Circulating miR-19b-3p, miR-134-5p and miR-186-5p could be considered promising novel diagnostic biomarkers for the early phase of AMI.

Abstract 14697: Novel Assays for Quantification of Lipoprotein-Associated (PCSK9-apoB, PCSK9-Lp(a)) Proprotein Covertase Subtilisin/Kexin Type 9 (PCKS9)

Background: PCSK9 is a major regulator of plasma LDL-C. Monoclonal antibodies to PCSK9 lower LDL-C by 45-65% and Lp(a) by 9-38%. The canonical function of PCSK9 is binding of LDL-receptor (LDLR) via its extracellular EGF-A domain, and subsequently mediating LDLR degradation. However, PCSK9 also weakly associates with plasma lipoproteins, with 20-40% of total plasma PCSK9 found on LDL. However, most LDL particles do not contain PCSK9. Whether PCSK9 also associates with other lipoproteins such as Lp(a) are not well described. Methods: Sensitive and quantitative sandwich-based ELISA assays were developed to measure PCSK9 associated plasma lipoproteins in both mouse and human plasma. For human plasma, commercial rabbit polyclonal antibodies binding to the C-terminal region of PCSK9 (Abgent, ThermoFisher) or REGN727 human monoclonal antibody were bound to microtiter well plates. Plasma was added and monoclonal antibodies MB47 and LPA4, binding to apoB-100 and apo(a) respectively, were used to detect PCSK9-apoB-100 and PCSK9-Lp(a) complexes with a chemiluminescent ELISA. For mouse assays, REGN727 was used as the capture antibody as it detects mouse PCSK9 and monoclonal antibody LF3 was used to detect mouse apoB. Results: PCSK9-apoB and PCSK9-Lp(a) complexes could be detected in both human plasma and in various mouse models expressing apo(a) or Lp(a). The signal to noise ratio was ~20 fold in various clinical samples, including in healthy subjects and in patients with cardiovascular disease. In 536 clinical samples from the Health Professional Follow-Up Study, PCSK9-Lp(a) correlated strongly with Lp(a) (r=0.59, p<0.001, age-adjusted) but not other lipid variables. PCSK9-apoB correlated weakly with PCSK9-Lp(a) (r=0.30, p<0.001, age-adjusted) and LDL-C (r=0.22, p<0.001, age-adjusted). These associations were virtually the same in 526 women in the Nurses’ Health Study. Conclusions: Novel ELISAs were generated to quantitate lipoprotein-associated PCSK9 in transgenic mouse and human plasma, including on apoB and Lp(a). Changes in PCSK9-Lp(a) complexes may provide insights into the Lp(a)-lowering effect of PCSK9 antibodies. Whether these assays will predict CVD outcomes waits to be determined in PCSK9 antibody and epidemiological studies.

Etomidate Mitigates Lipopolysaccharide-Induced CD14 and TREM-1 Expression, NF-κB Activation, and Pro-inflammatory Cytokine Production in Rat Macrophages.

This study was aimed at investigating the effect of etomidate on the viability of rat macrophages and the function of lipopolysaccharide (LPS)-stimulated macrophages as well as the potential mechanisms. Rat macrophages were isolated and treated with different doses of etomidate for 24h, and their viability was determined by the CCK-8 assay. Furthermore, macrophages were treated with, or without, 1μg/ml of LPS, and/or 2.5 or 5μM etomidate in the presence or absence of a TREM-1 inhibitor (LP17, 100ng/ml), and the levels of TNF-α, IL-6, CD14, and TREM-1 in the different groups of cells were determined by quantitative RT-PCR, ELISA, and Western blot assays. The levels of NF-κB activation in the different groups of cells were analyzed by an electrophoretic mobility shift assay (EMSA). Etomidate at 31.25μM or a low dose did not affect the viability of rat macrophages, while etomidate at higher doses reduced the viability of macrophages in vitro. Treatment with 2.5 or 5μM etomidate or with LP17 alone did not affect the levels of TNF-α, IL-6, CD-14, and TREM-1 in macrophages. Treatment with etomidate significantly mitigated LPS-stimulated TNF-α, IL-6, CD-14, and TREM-1 expression (p < 0.05 for all) and inhibited LPS-induced NF-κB activation in macrophages in vitro. However, treatment with both etomidate and LP17 did not enhance the inhibitory effects in macrophages. Hence, etomidate mitigates LPS-up-regulated pro-inflammatory cytokine production and inhibits LPS-enhanced CD14 and TREM-1 expression and NF-κB activation in macrophages.

Oncolytic adenovirus‑mediated mda‑7/IL‑24 expression suppresses osteosarcoma growth and enhances sensitivity to doxorubicin.

Osteosarcoma (OS) is a malignant disease with a high mortality rate and poor response to current chemotherapy. Melanoma differentiation associated gene‑7 (Mda7)/interleukin (IL)‑24 has been demonstrated to suppress the growth of OS. However, the expression level of Mda7/IL‑24 mediated by the current adenoviral vector is limited for effective clinical treatment of OS. In order to solve this issue, an oncolytic adenovirus was employed to express IL‑24 (OA‑IL‑24) in OS cells. Quantitative polymerase chain reaction, immunoblot and ELISA assays verified that OA‑IL‑24 expressed IL‑24 at a higher level than the replication‑deficient adenoviral vector, Ad‑IL24. OA‑IL‑24 infection led to decreased cell viability and increased apoptosis of OS cells, compared with Ad‑IL‑24. Animal studies further confirmed the increased anti‑tumor activity of OA‑IL‑24. Notably, OA‑IL‑24 was also found to sensitize OS cells to doxorubicin. OA‑IL‑24‑induced multiple drug resistance reversion was associated with reduced expression of Pgp and BCRP1, as well as minimized autophagy. Furthermore, restoring Pgp and BCRP1 expression as well as autophagy, was able to rescue the effect of IL‑24 on the cytotoxicity of doxorubicin to OS. In conclusion, a method for inducing a high expression of IL‑24 in OS was provided. In addition, IL‑24 was demonstrated to increase the sensitivity of OS to doxorubicin.

Channeled polymeric scaffolds with polypeptide gel filling for lengthwise guidance of neural cells

CNS damages are often irreversible since neurons of the central nervous system are unable to regenerate after an injury. As a new strategy within the nervous system tissue engineering, multifunctional systems based on two different biomaterials to support axonal guidance in damaged connective tracts have been developed herein. These systems are composed of a channeled scaffold made of ethyl acrylate and hydroxyethyl acrylate copolymer, P(EA-co-HEA), with parallel tubular micropores, combined with an injectable and in situ gelable self-assembling polypeptide (RAD16-I) as pores filler. The polymer scaffold is intended to provide a three-dimensional context for axon growth; subsequently, its morphology and physicochemical parameters have been determined by scanning electron microscopy, density measurements and compression tests. Besides, the hydrogel acts as a cell-friendly nanoenvironment while it creates a gradient of bioactive molecules (nerve growth factor, NGF) along the scaffolds channels; the chemotactic effect of NGF has been evaluated by a quantitative ELISA assay. These multifunctional systems have shown ability to keep circulating NGF, as well as proper short-term in vitro biological response with glial cells and neural progenitors.

Status of soluble ST2 levels in serum of HTLV-1 infected individuals.

ST2 is a member of IL-1 receptor family expressed on Th2 cells and regulates Th2 responces. The gene of ST2 encodes soluble ST2 (sST2) and the transmembrane ST2 (ST2L) isoforms through alternative mRNA splicing. The discovery of IL33/ ST2 signaling pathway, has drawn a great scientific attention to this system. sST2 has been shown to be an indacating factor in various infl ammatory conditions. This study aims to evaluate serum sST2 levels in HTLV-1 infected patients. This study included 49 HTLV-1 seropositive cases of which 14 were sympthomatic. Controls consisted of 30 healthy volunteers. sST2 level was measured using a quantitative ELISA assay and the results of the study groups were compared. Corroborating the previous reports, sST2 was lower in females (P = 0.003). The sST2 levels was slightly increased in HTLV-1 patients, though such increase was not statistically significant (P = 0.91), in addition sST2 level did not correlate significantly to the disease duration (P = 0.78). Despite some other chronic viral infection, HTLV-1 seems not to induce high serum sST2. However owing to relatively high normal variation of sST2 levels and rather small sample size, we stongly recommend further reseach with preferably larger sample size to evalute sST2 in HTLV-1 infected patients.

Transforming growth factor-β, insulin-like growth factor I/insulin-like growth factor I receptor and vascular endothelial growth factor-A: prognostic and predictive markers in triple-negative and non-triple-negative breast cancer.

In the current study, the prognostic and predictive values of serum transforming growth factor-β1 (TGF-β1), insulin-like growth factor I (IGF-I)/IGF-I receptor (IGF-IR) and vascular endothelial growth factor-A (VEGF-A) were evaluated in triple-negative and non-triple-negative breast cancer (TNBC and non-TNBC). The aim was to identify a group of serological biomarkers and to identify possible candidates for targeted therapy in patients with TNBC and non-TNBC. Protein levels of TGF-β1, IGF-I/IGF-IR and VEGF-A in the serum were measured in 43 TNBC, 53 non-TNBC and 20 normal control participants using quantitative ELISA assays. Results were correlated against standard prognostic factors, response to treatment and survival. TNBC was identified to be associated with poor prognosis and serum levels of VEGF-A and IGF/IGF-IR were significantly higher in the TNBC group compared with the non-TNBC group. IGF-IR and VEGF-A overexpression was observed to be correlated with TGF-β1 expression and all of the markers investigated were associated with metastasis and disease progression. In the multivariate analysis, VEGF-A, IGF-I and IGF-IR were observed to be independent predictors for overall survival, whereas TGF-β1 and lymph node status were identified as independent predictors for disease-free survival. The overall response rate was significantly lower in patients with TNBC and those with high levels of TGF-β1, IGF-I/IGF-IR and VEGF-A. In view of the present results, it was concluded that TGF-β1, IGF-I/IGF-IR and VEGF-A overexpression is associated with the presence of aggressive tumors, which exhibit an increased probability of metastasis, a poor response to treatment and reduced survival rate. This indicates that VEGF-A, IGF-IR and IGF-I have the potential to be used as surrogate biomarkers and are promising candidates for targeted therapy, particularly in patients with TNBC.

Tumor suppressive microRNA-200a inhibits renal cell carcinoma development by directly targeting TGFB2.

A large body of evidence indicates that microRNAs play a critical role in tumor initiation and progression by negatively regulating oncogenes or tumor suppressor genes. Here, we report that the expression of miR-200a was notably downregulated in 45 renal cell carcinoma (RCC) samples. Restoration of miR-200a suppressed cell proliferation, migration, and invasion in two RCC cell lines. Furthermore, we used an epithelial-to-mesenchymal transition PCR array to explore the putative target genes of miR-200a. By performing quantitative real-time PCR, ELISA, and luciferase reporter assays, transforming growth factor beta2 (TGFB2) was validated as a direct target gene of miR-200a. Moreover, siRNA-mediated knockdown of TGFB2 partially phenocopied the effect of miR-200a overexpression. These results suggest that miR-200a suppresses RCC development via directly targeting TGFB2, indicating that miR-200a may present a novel target for diagnostic and therapeutic strategies in RCC.

Effects of DENV-2 infection on the expression of IL-29 in primary HUVECs cultured on hydrogel substrates

Objective To analyze the effects of dengue virus 2 (DENV-2) infection on the expression of IL-29 in primary human umbilical vein endothelial cells (HUVECs) cultured on hydrogel substrates. Methods Primary HUVECs were isolated and cultured on hydrogel substrates. DENV-2 stains were used to infect the primary HUVECs at a multiplicity of infection (MOI) of 10. Flow cytometry analysis was performed to detect the apoptosis and infection rate of HUVECs after 48 hours of culturing. The gene chip profiling was performed to analyze mRNA expression. The expression of IL-29 at mRNA and protein levels were measured by real-time fluorescent quantitative PCR analysis and double antibody sandwich ELISA assay, respectively. Results Compared with 96.36% of baby hamster kidney (BHK) cells that were infected with DENV-2 stains, only 4.71% primary HUVECs cultured on hydrogel substrates were infected. The primary HUVECs cultured on hydrogel substrates with or without DENV-2 infection showed no significant differences with the rates of cell apoptosis and infection (P>0.05). A significant difference was observed with the expression of IL-29 at mRNA level between primary HUVECs cultured on hydrogel substrates and the cells cultured in plastic bottles (P<0.05). The results of the real-time quantitative PCR analysis and ELISA assay showed that IL-29 was highly expressed in DENV-2 infected primary HUVECs cultured on hydrogel substrates as compared with those in control groups (P<0.05). Conclusion The expression of IL-29 was detected in DENV-2 infected primary HUVECs cultured on hydrogel substrates, which was significantly different from that in DENV-2 infected primary HUVECs cultured in plastic bottles. The successful establishment of hydrogel substrates as the model of vascular basement membranes might provide a new way for the investigation of the pathogenesis of DENV infection. Key words: Dengue virus; IL-29; Hydrogel; Human umbilical vein endothelial cell

Legionella pneumophila urinary antigen: diagnostic efficiency of a new quantitative Elisa test

Background . To provide a rapid and suitable diagnosis of Legionella pneumophila serogroup1 (LPsg1) infection, a total of 160 urine samples were collected from 56 suspected or diagnosed patients, 8 from the VEQ-NEQAS Program, 96 from healthy blood donors, to detect LPsg1 urinary antigen by a new automated single device quantitative Elisa assay (Urinary Legionella Antigen Chorus, Diesse). Materials and Methods . An immunochromatographic rapid test (V-test Legionella, Coris) was used as a reference. Prevalence of specific IgM and IgG LPsg1 antibodies was assessed on 20 sera collected among the 56 patients with disease. Results . Urinary Ag LPsg1 results of two direct tests from a total of 64 samples (patients and VEQ) showed an overall concordance of 90.6% (58 out of 64); all healthy blood donors resulted as negatives, as well (specificity 100%). Serology positive results (LPsg1 IgM and IgG antibodies) were revealed on 14 out of 20 diagnosed patients sera; instead, both urinary LPsg1 antigen tests resulted as positives in 18 out of 20 urine samples. The combined use of both LPsg1 urinary antigen and specific IgM antibodies improved the diagnostic efficiency than a single test approach, showing 20 out 20 patients with at least one positive LPsg1 specific marker. Conclusions . The new LPsg1 urine antigen quantitative Elisa assay seems to be a useful tool for an early diagnosis of suspected LPsg1 infection and along the follow up of treated patients.

Inhibition of LPS-induced TLR4 signaling products in murine macrophages by phenylmethimazole: an assay methodology for screening potential phenylmethimazole analogs.

Preclinical Research Phenylmethimazole (C10) is an inhibitor of Toll-like receptor (TLR3 and TLR4) expression and signaling. In this study, we carried out a detailed investigation of the effect of C10 on TLR4 and its molecular signaling products in RAW 264.7 macrophages using quantitative real-time polymerase chain reaction (PCR), ELISA and cell toxicity assays, a set of in vitro assays that may be used to screen future C10 analogs. C10 exhibited an inhibitory effect on TLR4 MyD88-dependent and MyD88-independent pathways. Within the TLR4 pathway, C10 inhibited the expression of cytokines, cytokine receptors, kinases, adapter molecules and transcription factors, suggesting a pathway-wide inhibitory effect. We also found that C10 dose-dependently inhibited the expression of TLR4 signaling products, specifically IL-6, inducible nitric oxide (NO) synthase and IFNβ. Additionally, pre-treatment of RAW 264.7 cells with C10 resulted in protection from lipopolysaccharide (LPS) insults, suggesting C10 may be bound to the target thus exhibiting activity during/following LPS stimulation. Also, dimethyl sulfoxide, the solvent for C10 exhibited inhibitory effect on TLR4 signaling products independent from the effects of C10. Combined, this study enhances understanding of the actions of C10 on the TLR4 signaling pathway providing a path for the development of new C10 analogs for inhibiting TLR expression and signaling [corrected].

MicroRNA-495 regulates the proliferation and apoptosis of human umbilical vein endothelial cells by targeting chemokine CCL2

IntroductionEndothelium dysfunction plays a critical role in atherosclerosis. MicroRNAs are endogenous non-coding RNAs that suppress gene expression by binding to the 3’ untranslated regions of target genes. MiR-495 can regulate the proliferation and apoptosis of cancer cells, however, the roles of miR-495 in endothelial cells (ECs) remain unclear. Therefore, this study aims to investigate the roles and mechanisms of miR-495 on ECs proliferation and apoptosis. Materials and MethodsMiR-495 and CCL2 expressions were examined using quantitative RT-PCR, ELISA assay and western blot. Bioinformatics analysis and luciferase reporter assay were used to examine the regulatory relationship between miR-495 and CCL2. CCK8 assay, BrdU incorporation assay and flow cytometry were used to analyze the roles of miR-495 and CCL2 on the proliferation of human umbilical vein endothelial cells (HUVECs). The effects of miR-495 and CCL2 on HUVECs apoptosis were examined by tunnel staining and western blot. ResultsMiR-495 was down-regulated in patients with coronary artery disease compared with healthy controls. CCL2 was a novel target gene of miR-495. MiR-495 significantly promoted HUVECs proliferation by altering cell cycle distribution, and it also inhibited HUVECs apoptosis by affecting the expression of cleaved caspase 3. Effects of miR-495 on HUVECs proliferation and apoptosis were significantly reversed by overexpression of CCL2. ConclusionsMiR-495 could affect HUVECs proliferation and apoptosis by directly targeting CCL2. This is the first report to disclose the roles and mechanisms of miR-495 on HUVECs proliferation and apoptosis, which may provide a theoretical basis for clarifying the mechanisms of atherosclerosis.

Evaluation of qualitative and quantitative immunoassays to detect barley contamination in gluten-free beer with confirmation using LC/MS/MS.

To meet the need for the detection and quantitation of barley gluten in beer, qualitative screening and quantitative immunoassays based on the monoclonal antigluten antibody 401/21 (Skerritt) were validated in a single laboratory. Sample replicates were tested at each stage of beer production using multiple yeast strains and methods of end-stage protein removal. Quantitation was performed using barley-specific standards based on barley flour extracts. Immunoassay results were confirmed using LC/MS/MS for barley-specific peptides. The LOD for the qualitative screening test was 5 mg/L barley gluten. Recovery for the barley-spiked worts ranged from 81 to 128% in the quantitative ELISA assay; the LOD was <1 mg/L, and the LOQ was 5 mg/L. Both screening and confirmation methods were found to be fit for the purposes of detection of low levels of barley gluten in beer.

Abstract 2423: Development of a validated high throughput ELISA assay for gamma H2AX as a pharmacodynamic marker

Abstract Histone H2AX is a 14 kDa ubiquitous member of the H2A histone family that becomes rapidly phosphorylated at Serine 139 by ATM and ATR kinases to yield a form known as γ-H2AX in response to double-strand DNA damage and apoptosis. γ-H2AX is an ideal Pharmacodynamic (PD) surrogate marker to measure molecular responses to a large number of drugs; however, methods such as western blots and immunohistochemistry are widely used but are difficult to validate to regulatory standards, and not suitable for high throughput screening applications. To address this need, we have developed a novel high throughput ELISA assay to measure γ-H2AX levels in cellular extracts and phosphorylation of H2AX in response to therapeutic intervention. This assay documents differences of γ-H2AX levels in PBMC, cultured cells, tissue biopsies, and will be useful in future clinical trials providing one of many needed tools to enable hypothesis-driven preclinical drug design strategies.p Background Histone H2AX is a 14 kDa ubiquitous member of the H2A histone family that contains an evolutionarily conserved SQ motif at the C-terminus in eukaryotes. Serine 139 within this motif becomes rapidly phosphorylated by ATM and ATR kinases to yield a form known as γ-H2AX in response to double-strand DNA damage and apoptosis (1). During the past year investigators have confirmed the value of γ-H2AX as an important Pharmacodynamic (PD) marker (2) and genotoxicity endpoint (3). There are over 21 anticancer drugs that are known to result in γ-H2AX formation. As a result, γ-H2AX is an ideal Pharmacodynamic PD surrogate marker to measure molecular responses to a large number of drugs (4,5,6). While many of these drugs have already garnered regulatory approval, and are currently being used to manage various types of cancers, they are the subject of ongoing clinical studies to evaluate their efficacy when used alone or in combination with molecularly targeted drugs. While methods such as western blots and immunohistochemistry are widely used but are difficult to validate to regulatory standards, the ELISA method is the most quantifiable and easiest to validate, and not suitable for high throughput screening applications. To address this need Trevigen's quantitative pharmacodynamic HT γ-H2AX ELISA assay measures γ-H2AX levels in cellular extracts and phosphorylation of H2AX in response to therapeutic intervention. This assay documents differences of γ-H2AX levels in PBMC, cultured cells, tissue biopsies, and will be useful in future clinical trials providing one of many needed tools to enable hypothesis-driven preclinical drug design strategies. Citation Format: Jay George, Hai Xu. Development of a validated high throughput ELISA assay for gamma H2AX as a pharmacodynamic marker. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2423. doi:10.1158/1538-7445.AM2014-2423

Atherosclerosis-related circulating miRNAs as novel and sensitive predictors for acute myocardial infarction.

BackgroundThe dysregulated expressions of circulating miRNAs have been detected in various cardiovascular diseases. In our previous experiments, the altered expressions of circulating miRNA-21-5p, miRNA-361-5p and miRNA-519e-5p were confirmed in patients with coronary atherosclerosis by miRNA microarrays. However, the expression levels of these circulating miRNAs in the early phase of acute myocardial infarction (AMI) are still unknown. In the present study, our aims were to examine the expressions of circulating miR-21-5p, miR-361-5p and miR-519e-5p in AMI patients, and assess their clinical applications for diagnosing and monitoring AMI.ResultsTwo different cohorts were enrolled in this study. The first cohort included 17 AMI patients and 28 healthy volunteers, and the second cohort included 9 AMI patients, 9 ischemic stroke patients, 8 patients with pulmonary embolism, and 12 healthy volunteers. Quantitative real-time PCR and ELISA assays were preformed to detect the concentrations of plasma miRNAs and cardiac troponin I (cTnI), respectively. The results showed that the plasma levels of miR-21-5p and miR-361-5p were significantly increased in AMI patients, whereas the concentration of circulating miR-519e-5p was reduced. Interestingly, the levels of these circulating miRNAs correlated with the concentrations of plasma cTnI. Receiver operating characteristic (ROC) analysis revealed that these three circulating miRNAs had considerable diagnostic accuracy for AMI with high values of area under ROC curve (AUC). Importantly, combining the three miRNAs significantly increased the diagnostic accuracy. Furthermore, cell experiments demonstrated that these plasma miRNAs may originate from injured cardiomyocytes induced by hypoxia. In addition, the levels of all the three circulating miRNAs in ischemic stroke (IS) and pulmonary embolism (PE) were elevated, whereas the decreased level of plasma miR-519e-5p was only detected in AMI. ROC analysis demonstrated that circulating miR-519e-5p may be a useful biomarker for distinguishing AMI from other ischemic diseases.ConclusionsCirculating miRNAs may be novel and powerful biomarkers for AMI and they could be potential diagnostic tool for AMI.

Complement protein C1q promotes macrophage anti-inflammatory M2-like polarization during the clearance of atherogenic lipoproteins.

Innate immune protein C1q plays a dual role in the chronic inflammatory disease of atherosclerosis. Complement activation via C1q exacerbates pathology in the atherosclerotic lesion in later stages of the disease. However, in early stages of disease C1q is protective. We hypothesize that complement-independent activities of C1q are involved in reprogramming macrophage inflammatory polarization. The influence of C1q on macrophage inflammatory responses during clearance of oxLDL was examined. Changes in cytokines at the gene and protein level were measured by quantitative PCR and ELISA assay. C1q modulated cytokine expression in Raw264.7 macrophages during ingestion of oxLDL. Levels of pro-inflammatory cytokines IL-1β and IL-6 were downregulated by C1q, whereas levels of the anti-inflammatory cytokine IL-10 were increased. In addition, data from an NFκB-luciferase gene reporter assay suggest that C1q suppresses activation of NFκB during lipoprotein clearance in macrophages, providing one mechanism by which C1q downregulates pro-inflammatory cytokine production. C1q-polarization of macrophages toward an anti-inflammatory (M2-like) phenotype may be important in dampening inflammation in the early atherosclerotic lesion. Further investigation of molecular pathways targeted by C1q may provide novel therapeutic targets for this disease.