High-content screening of drug combinations of an mPGES-1 inhibitor in multicellular tumor spheroids leads to mechanistic insights into neuroblastoma chemoresistance.

High-throughput drug screening enables the discovery of new anticancer drugs. Although monolayer cell cultures are commonly used for screening, their limited complexity and translational efficiency require alternative models. Three-dimensional cell cultures, such as multicellular tumor spheroids (MCTS), mimic tumor architecture and offer promising opportunities for drug discovery. In this study, we developed a neuroblastoma MCTS model for high-content drug screening. We also aimed to decipher the mechanisms underlying synergistic drug combinations in this disease model. Several agents from different therapeutic categories and with different mechanisms of action were tested alone or in combination with selective inhibition of prostaglandin E2 by pharmacological inhibition of microsomal prostaglandin E synthase-1 (mPGES-1). After a systematic investigation of the sensitivity of individual agents and the effects of pairwise combinations, GFP-transfected MCTS were used in a confirmatory screen to validate the hits. Finally, inhibitory effects on multidrug resistance proteins were examined. In summary, we demonstrate how MCTS-based high-throughput drug screening has the potential to uncover effective drug combinations and provide insights into the mechanism of synergy between an mPGES-1 inhibitor and chemotherapeutic agents.

OTUD1 ameliorates cerebral ischemic injury through inhibiting inflammation by disrupting K63-linked deubiquitination of RIP2.

BACKGROUND: Inflammatory response triggered by innate immunity plays a pivotal element in the progress of ischemic stroke. Receptor-interacting kinase 2 (RIP2) is implicated in maintaining immunity homeostasis and regulating inflammatory response. However, the underlying mechanism of RIP2 in ischemic stroke is still not well understood. Hence, the study investigated the role and the ubiquitination regulatory mechanism of RIP2 in ischemic stroke. METHODS: Focal cerebral ischemia was introduced by middle cerebral artery occlusion (MCAO) in wild-type (WT) and OTUD1-deficient (OTUD1-/-) mice, oxygen glucose deprivation and reoxygenation (OGD/R) models in BV2 cells and primary cultured astrocytes were performed for monitoring of experimental stroke. GSK2983559 (GSK559), a RIP2 inhibitor was intraventricularly administered 30 min before MCAO. Mice brain tissues were collected for TTC staining and histopathology. Protein expression of RIP2, OTUD1, p-NF-κB-p65 and IκBα was determined by western blot. Localization of RIP2 and OTUD1 was examined by immunofluorescence. The change of IL-1ß, IL-6 and TNF-α was detected by ELISA assay and quantitative real-time polymerase chain reaction. Immunoprecipitation and confocal microscopy were used to study the interaction of RIP2 and OTUD1. The activity of NF-κB was examined by dual-luciferase assay. RESULTS: Our results showed upregulated protein levels of RIP2 and OTUD1 in microglia and astrocytes in mice subjected to focal cerebral ischemia. Inhibition of RIP2 by GSK559 ameliorated the cerebral ischemic outcome by repressing the NF-κB activity and the inflammatory response. Mechanistically, OTUD1 interacted with RIP2 and sequentially removed the K63-linked polyubiquitin chains of RIP2, thereby inhibiting NF-κB activation. Furthermore, OTUD1 deficiency exacerbated cerebral ischemic injury in response to inflammation induced by RIP2 ubiquitination. CONCLUSIONS: These findings suggested that RIP2 mediated cerebral ischemic lesion via stimulating inflammatory response, and OTUD1 ameliorated brain injury after ischemia through inhibiting RIP2-induced NF-κB activation by specifically cleaving K63-linked ubiquitination of RIP2.

Identifying novel B-cell targets for chronic inflammatory autoimmune disease by screening of chemical probes in a patient-derived cell assay.

B-cell secretion of autoantibodies drives autoimmune diseases, including systemic lupus erythematosus and idiopathic inflammatory myositis. Few therapies are presently available for treatment of these patients, often resulting in unsatisfactory effects and helping only some of the patients. We developed a screening assay for evaluation of novel targets suspending B-cell maturation into antibody secreting cells, which could contribute to future drug development. The assay was employed for testing 43 high quality chemical probes and compounds inhibiting under-explored protein targets, using primary cells from patients with autoimmune disease. Probes inhibiting bromodomain family proteins and histone methyl transferases demonstrated abrogation of B-cell functions to a degree comparable to a positive control, the JAK inhibitor tofacitinib. Inhibition of each target rendered a specific functional cell and potential disease modifying effect, indicating specific epigenetic protein targets as potential new intervention points for future drug discovery and development efforts.

Anti-Inflammatory Properties of Chemical Probes in Human Whole Blood: Focus on Prostaglandin E2 Production.

We screened 57 chemical probes, high-quality tool compounds, and relevant clinically used drugs to investigate their effect on pro-inflammatory prostaglandin E2 (PGE2) production and interleukin-8 (IL-8) secretion in human whole blood. Freshly drawn blood from healthy volunteers and patients with systemic lupus erythematosus (SLE) or dermatomyositis was incubated with compounds at 0.1 or 1 µM and treated with lipopolysaccharide (LPS, 10 µg/ml) to induce a pro-inflammatory condition. Plasma was collected after 24 h for lipid profiling using liquid chromatography tandem mass spectrometry (LC-MS/MS) and IL-8 quantification using enzyme-linked immunosorbent assay (ELISA). Each compound was tested in at least four donors at one concentration based on prior knowledge of binding affinities and in vitro activity. Our screening suggested that PD0325901 (MEK-1/2 inhibitor), trametinib (MEK-1/2 inhibitor), and selumetinib (MEK-1 inhibitor) decreased while tofacitinib (JAK inhibitor) increased PGE2 production. These findings were validated by concentration-response experiment in two donors. Moreover, the tested MEK inhibitors decreased thromboxane B2 (TXB2) production and IL-8 secretion. We also investigated the lysophophatidylcholine (LPC) profile in plasma from treated whole blood as these lipids are potentially important mediators in inflammation, and we did not observe any changes in LPC profiles. Collectively, we deployed a semi-high throughput and robust methodology to investigate anti-inflammatory properties of new chemical probes.

Exhaustion of CD4+ T-cells mediated by the Kynurenine Pathway in Melanoma.

Kynurenine pathway (KP) activation by the enzymatic activity of indoleamine 2,3-dioxygenase1 (IDO1) and kynurenine (KYN) production represents an attractive target for reducing tumour progression and improving anti-tumour immunity in multiple cancers. However, immunomodulatory properties of other KP metabolites such as 3-hydroxy kynurenine (3-HK) and kynurenic acid (KYNA) are poorly understood. The association of the kynurenine metabolic pathway with T-cell status in the tumour microenvironment were characterized, using gene expression data of 368 cutaneous skin melanoma (SKCM) patients from the TCGA cohort. Based on the identified correlations, we characterized the production of KYN, 3-HK, and KYNA in vitro using melanoma-derived cell lines and primary CD4+ CD25- T-cells. Activation of the CD4+ T-cells produced IFNγ, which yielded increased levels of KYN and KYNA. Concurrently, kynurenine 3-monooxygenase (KMO) expression and proliferation of CD4+ T-cells were reduced, whereas exhaustion markers such as PD-L1, AHR, FOXP3, and CTLA4 were increased. Additionally, an analysis of the correlation network reconstructed using TCGA-SKCM emphasized KMO and KYNU with high variability among BRAF wild-type compared with V600E, which underscored their role in distinct CD4+ T-cell behavior in tumour immunity. Our results suggest that, in addition to IDO1, there is an alternative immune regulatory mechanism associated with the lower KMO expression and the higher KYNA production, which contributes to dysfunctional effector CD4+ T-cell response.

Phosphatase inhibitor PPP1R11 modulates resistance of human T cells toward Treg-mediated suppression of cytokine expression.

Regulatory T cells (Tregs) act as indispensable unit for maintaining peripheral immune tolerance mainly by regulating effector T cells. T cells resistant to suppression by Tregs pose therapeutic challenges in the treatment of autoimmune diseases, while augmenting susceptibility to suppression may be desirable for cancer therapy. To understand the cell intrinsic signals in T cells during suppression by Tregs, we have previously performed a global phosphoproteomic characterization. We revealed altered phosphorylation of protein phosphatase 1 regulatory subunit 11 (PPP1R11; Inhibitor-3) in conventional T cells upon suppression by Tregs. Here, we show that silencing of PPP1R11 renders T cells resistant toward Treg-mediated suppression of TCR-induced cytokine expression. Furthermore, whole-transcriptome sequencing revealed that PPP1R11 differentially regulates not only the expression of specific T cell stimulation-induced cytokines but also other molecules and pathways in T cells. We further confirmed the target of PPP1R11, PP1, to augment TCR-induced cytokine expression. In conclusion, we present PPP1R11 as a novel negative regulator of T cell activation-induced cytokine expression. Targeting PPP1R11 may have therapeutic potential to regulate the T cell activation status including modulating the susceptibility of T cells toward Treg-mediated suppression, specifically altering the stimulation-induced T cell cytokine milieu.

Predictive Systems Toxicology.

In this review we address to what extent computational techniques can augment our ability to predict toxicity. The first section provides a brief history of empirical observations on toxicity dating back to the dawn of Sumerian civilization. Interestingly, the concept of dose emerged very early on, leading up to the modern emphasis on kinetic properties, which in turn encodes the insight that toxicity is not solely a property of a compound but instead depends on the interaction with the host organism. The next logical step is the current conception of evaluating drugs from a personalized medicine point of view. We review recent work on integrating what could be referred to as classical pharmacokinetic analysis with emerging systems biology approaches incorporating multiple omics data. These systems approaches employ advanced statistical analytical data processing complemented with machine learning techniques and use both pharmacokinetic and omics data. We find that such integrated approaches not only provide improved predictions of toxicity but also enable mechanistic interpretations of the molecular mechanisms underpinning toxicity and drug resistance. We conclude the chapter by discussing some of the main challenges, such as how to balance the inherent tension between the predicitive capacity of models, which in practice amounts to constraining the number of features in the models versus allowing for rich mechanistic interpretability, i.e., equipping models with numerous molecular features. This challenge also requires patient-specific predictions on toxicity, which in turn requires proper stratification of patients as regards how they respond, with or without adverse toxic effects. In summary, the transformation of the ancient concept of dose is currently successfully operationalized using rich integrative data encoded in patient-specific models.

Overexpression of endothelin B receptor in glioblastoma: a prognostic marker and therapeutic target?

BACKGROUND: Glioblastoma (GBM) is the most common malignant brain tumor with median survival of 12-15 months. Owing to uncertainty in clinical outcome, additional prognostic marker(s) apart from existing markers are needed. Since overexpression of endothelin B receptor (ETBR) has been demonstrated in gliomas, we aimed to test whether ETBR is a useful prognostic marker in GBM and examine if the clinically available endothelin receptor antagonists (ERA) could be useful in the disease treatment. METHODS: Data from The Cancer Genome Atlas and the Gene Expression Omnibus database were analyzed to assess ETBR expression. For survival analysis, glioblastoma samples from 25 Swedish patients were immunostained for ETBR, and the findings were correlated with clinical history. The druggability of ETBR was assessed by protein-protein interaction network analysis. ERAs were analyzed for toxicity in in vitro assays with GBM and breast cancer cells. RESULTS: By bioinformatics analysis, ETBR was found to be upregulated in glioblastoma patients, and its expression levels were correlated with reduced survival. ETBR interacts with key proteins involved in cancer pathogenesis, suggesting it as a druggable target. In vitro viability assays showed that ERAs may hold promise to treat glioblastoma and breast cancer. CONCLUSIONS: ETBR is overexpressed in glioblastoma and other cancers and may be a prognostic marker in glioblastoma. ERAs may be useful for treating cancer patients.

Poliovirus Receptor-Related 2: A Cholesterol-Responsive Gene Affecting Atherosclerosis Development by Modulating Leukocyte Migration.

OBJECTIVE: Recently, poliovirus receptor-related 2 (Pvrl2) emerged as a top gene in a global gene expression study aiming to detect plasma cholesterol-responsive genes causally related to atherosclerosis regression in hypercholesterolemic mice. PVRL2 is an adherens junction protein implied to play a role in transendothelial migration of leukocytes, a key feature in atherosclerosis development. In this study, we investigated the effect of Pvrl2 deficiency on atherosclerosis development and transendothelial migration of leukocytes activity. APPROACH AND RESULTS: Pvrl2-deficient mice bred onto an atherosclerosis-prone background (Pvrl2-/-Ldlr-/-Apob100/100) had less atherosclerotic lesions and more stable plaques compared with littermate controls (Pvrl2+/+Ldlr-/-Apob100/100). Pvrl2-/-Ldlr-/-Apob100/100 mice also showed a 49% decrease in transendothelial migration of leukocytes activity observed using the in vivo air pouch model. In accordance, augmented arterial wall expression of Pvrl2 during atherosclerosis progression coincided with an increased gene expression of migrating leukocytes into the vessel wall. Both in human and mice, gene and protein expression of PVRL2 was predominantly observed in the vascular endothelium according to the immunohistochemical and gene expression data. In addition, the cholesterol responsiveness of PVRL2 was also observed in humans. CONCLUSIONS: PVRL2 is a plasma cholesterol-responsive gene acting at endothelial sites of vascular inflammation that could potentially be a new therapeutic target for atherosclerosis prevention through its suggested transendothelial migration of leukocytes modulating activity.

Systems Toxicology: Systematic Approach to Predict Toxicity.

Drug discovery is complex and expensive. Numerous drug candidates fail late in clinical trials or even after being released to the market. These failures are not only due to commercial considerations and less optimal drug efficacies but, adverse reactions originating from toxic effects also constitute a major challenge. During the last two decades, significant advances have been made enabling the early prediction of toxic effects using in silico techniques. However, by design, these essentially statistical techniques have not taken the disease driving pathophysiological mechanisms into account. The complexity of such mechanisms in combination with their interactions with drugspecific properties and environmental and life-style related factors renders the task of predicting toxicity on a purely statistical basis which is an insurmountable challenge. In response to this situation, an interdisciplinary field has developed, referred to as systems toxicology, where the notion of a network is used to integrate and model different types of information to better predict drug toxicity. In this study, we briefly review the merits and limitations of such recent promising predictive approaches integrating molecular networks, chemical compound networks, and protein drug association networks.

Human cytomegalovirus may promote tumour progression by upregulating arginase-2.

BACKGROUND: Both arginase (ARG2) and human cytomegalovirus (HCMV) have been implicated in tumorigenesis. However, the role of ARG2 in the pathogenesis of glioblastoma (GBM) and the HCMV effects on ARG2 are unknown. We hypothesize that HCMV may contribute to tumorigenesis by increasing ARG2 expression. RESULTS: ARG2 promotes tumorigenesis by increasing cellular proliferation, migration, invasion and vasculogenic mimicry in GBM cells, at least in part due to overexpression of MMP2/9. The nor-NOHA significantly reduced migration and tube formation of ARG2-overexpressing cells. HCMV immediate-early proteins (IE1/2) or its downstream pathways upregulated the expression of ARG2 in U-251 MG cells. Immunostaining of GBM tissue sections confirmed the overexpression of ARG2, consistent with data from subsets of Gene Expression Omnibus. Moreover, higher levels of ARG2 expression tended to be associated with poorer survival in GBM patient by analyzing data from TCGA. METHODS: The role of ARG2 in tumorigenesis was examined by proliferation-, migration-, invasion-, wound healing- and tube formation assays using an ARG2-overexpressing cell line and ARG inhibitor, N (omega)-hydroxy-nor-L-arginine (nor-NOHA) and siRNA against ARG2 coupled with functional assays measuring MMP2/9 activity, VEGF levels and nitric oxide synthase activity. Association between HCMV and ARG2 were examined in vitro with 3 different GBM cell lines, and ex vivo with immunostaining on GBM tissue sections. The viral mechanism mediating ARG2 induction was examined by siRNA approach. Correlation between ARG2 expression and patient survival was extrapolated from bioinformatics analysis on data from The Cancer Genome Atlas (TCGA). CONCLUSIONS: ARG2 promotes tumorigenesis, and HCMV may contribute to GBM pathogenesis by upregulating ARG2.

Comparative Analysis of Protocols to Induce Human CD4+Foxp3+ Regulatory T Cells by Combinations of IL-2, TGF-beta, Retinoic Acid, Rapamycin and Butyrate.

Regulatory T cells (Tregs) suppress other immune cells and are critical mediators of peripheral tolerance. Therapeutic manipulation of Tregs is subject to numerous clinical investigations including trials for adoptive Treg transfer. Since the number of naturally occurring Tregs (nTregs) is minute, it is highly desirable to develop a complementary approach of inducing Tregs (iTregs) from naïve T cells. Mouse studies exemplify the importance of peripherally induced Tregs as well as the applicability of iTreg transfer in different disease models. Yet, procedures to generate iTregs are currently controversial, particularly for human cells. Here we therefore comprehensively compare different established and define novel protocols of human iTreg generation using TGF-ß in combination with other compounds. We found that human iTregs expressed several Treg signature molecules, such as Foxp3, CTLA-4 and EOS, while exhibiting low expression of the cytokines Interferon-γ, IL-10 and IL-17. Importantly, we identified a novel combination of TGF-ß, retinoic acid and rapamycin as a robust protocol to induce human iTregs with superior suppressive activity in vitro compared to currently established induction protocols. However, iTregs generated by these protocols did not stably retain Foxp3 expression and did not suppress in vivo in a humanized graft-versus-host-disease mouse model, highlighting the need for further research to attain stable, suppressive iTregs. These results advance our understanding of the conditions enabling human iTreg generation and may have important implications for the development of adoptive transfer strategies targeting autoimmune and inflammatory diseases.

Lim domain binding 2: a key driver of transendothelial migration of leukocytes and atherosclerosis.

OBJECTIVE: Using a multi-tissue, genome-wide gene expression approach, we recently identified a gene module linked to the extent of human atherosclerosis. This atherosclerosis module was enriched with inherited risk for coronary and carotid artery disease (CAD) and overlapped with genes in the transendothelial migration of leukocyte (TEML) pathway. Among the atherosclerosis module genes, the transcription cofactor Lim domain binding 2 (LDB2) was the most connected in a CAD vascular wall regulatory gene network. Here, we used human genomics and atherosclerosis-prone mice to evaluate the possible role of LDB2 in TEML and atherosclerosis. APPROACH AND RESULTS: mRNA profiles generated from blood macrophages in patients with CAD were used to infer transcription factor regulatory gene networks; Ldlr(-/-)Apob(100/100) mice were used to study the effects of Ldb2 deficiency on TEML activity and atherogenesis. LDB2 was the most connected gene in a transcription factor regulatory network inferred from TEML and atherosclerosis module genes in CAD macrophages. In Ldlr(-/-)Apob(100/100) mice, loss of Ldb2 increased atherosclerotic lesion size ≈2-fold and decreased plaque stability. The exacerbated atherosclerosis was caused by increased TEML activity, as demonstrated in air-pouch and retinal vasculature models in vivo, by ex vivo perfusion of primary leukocytes, and by leukocyte migration in vitro. In THP1 cells, migration was increased by overexpression and decreased by small interfering RNA inhibition of LDB2. A functional LDB2 variant (rs10939673) was associated with the risk and extent of CAD across several cohorts. CONCLUSIONS: As a key driver of the TEML pathway in CAD macrophages, LDB2 is a novel candidate to target CAD by inhibiting the overall activity of TEML.

Plasma cholesterol-induced lesion networks activated before regression of early, mature, and advanced atherosclerosis.

Plasma cholesterol lowering (PCL) slows and sometimes prevents progression of atherosclerosis and may even lead to regression. Little is known about how molecular processes in the atherosclerotic arterial wall respond to PCL and modify responses to atherosclerosis regression. We studied atherosclerosis regression and global gene expression responses to PCL (≥80%) and to atherosclerosis regression itself in early, mature, and advanced lesions. In atherosclerotic aortic wall from Ldlr(-/-)Apob (100/100) Mttp (flox/flox)Mx1-Cre mice, atherosclerosis regressed after PCL regardless of lesion stage. However, near-complete regression was observed only in mice with early lesions; mice with mature and advanced lesions were left with regression-resistant, relatively unstable plaque remnants. Atherosclerosis genes responding to PCL before regression, unlike those responding to the regression itself, were enriched in inherited risk for coronary artery disease and myocardial infarction, indicating causality. Inference of transcription factor (TF) regulatory networks of these PCL-responsive gene sets revealed largely different networks in early, mature, and advanced lesions. In early lesions, PPARG was identified as a specific master regulator of the PCL-responsive atherosclerosis TF-regulatory network, whereas in mature and advanced lesions, the specific master regulators were MLL5 and SRSF10/XRN2, respectively. In a THP-1 foam cell model of atherosclerosis regression, siRNA targeting of these master regulators activated the time-point-specific TF-regulatory networks and altered the accumulation of cholesterol esters. We conclude that PCL leads to complete atherosclerosis regression only in mice with early lesions. Identified master regulators and related PCL-responsive TF-regulatory networks will be interesting targets to enhance PCL-mediated regression of mature and advanced atherosclerotic lesions.

Multi-organ expression profiling uncovers a gene module in coronary artery disease involving transendothelial migration of leukocytes and LIM domain binding 2: the Stockholm Atherosclerosis Gene Expression (STAGE) study.

Environmental exposures filtered through the genetic make-up of each individual alter the transcriptional repertoire in organs central to metabolic homeostasis, thereby affecting arterial lipid accumulation, inflammation, and the development of coronary artery disease (CAD). The primary aim of the Stockholm Atherosclerosis Gene Expression (STAGE) study was to determine whether there are functionally associated genes (rather than individual genes) important for CAD development. To this end, two-way clustering was used on 278 transcriptional profiles of liver, skeletal muscle, and visceral fat (n = 66/tissue) and atherosclerotic and unaffected arterial wall (n = 40/tissue) isolated from CAD patients during coronary artery bypass surgery. The first step, across all mRNA signals (n = 15,042/12,621 RefSeqs/genes) in each tissue, resulted in a total of 60 tissue clusters (n = 3958 genes). In the second step (performed within tissue clusters), one atherosclerotic lesion (n = 49/48) and one visceral fat (n = 59) cluster segregated the patients into two groups that differed in the extent of coronary stenosis (P = 0.008 and P = 0.00015). The associations of these clusters with coronary atherosclerosis were validated by analyzing carotid atherosclerosis expression profiles. Remarkably, in one cluster (n = 55/54) relating to carotid stenosis (P = 0.04), 27 genes in the two clusters relating to coronary stenosis were confirmed (n = 16/17, P<10(-27 and-30)). Genes in the transendothelial migration of leukocytes (TEML) pathway were overrepresented in all three clusters, referred to as the atherosclerosis module (A-module). In a second validation step, using three independent cohorts, the A-module was found to be genetically enriched with CAD risk by 1.8-fold (P<0.004). The transcription co-factor LIM domain binding 2 (LDB2) was identified as a potential high-hierarchy regulator of the A-module, a notion supported by subnetwork analysis, by cellular and lesion expression of LDB2, and by the expression of 13 TEML genes in Ldb2-deficient arterial wall. Thus, the A-module appears to be important for atherosclerosis development and, together with LDB2, merits further attention in CAD research.

Adenosine A1 and A3 receptors protect astrocytes from hypoxic damage.

Brain levels of adenosine are elevated during hypoxia. Through effects on adenosine receptors (A(1), A(2A), A(2B) and A(3)) on astrocytes, adenosine can influence functions such as glutamate uptake, reactive gliosis, swelling, as well as release of neurotrophic and neurotoxic factors having an impact on the outcome of metabolic stress. We have studied the roles of these receptors in astrocytes by evaluating their susceptibility to damage induced by oxygen deprivation or exposure to the hypoxia mimic cobalt chloride (CoCl(2)). Hypoxia caused ATP breakdown and purine release, whereas CoCl(2) (0.8 mM) mainly reduced ATP by causing cell death in human D384 astrocytoma cells. Further experiments were conducted in primary astrocytes prepared from specific adenosine receptor knock-out (KO) and wild type (WT) mice. In WT cells purine release following CoCl(2) exposure was mainly due to nucleotide release, whereas hypoxia-induced intracellular ATP breakdown followed by nucleoside efflux. N-ethylcarboxamidoadenosine (NECA), an unselective adenosine receptor agonist, protected from cell death following hypoxia. Cytotoxicity was more pronounced in A(1)R KO astrocytes and tended to be higher in WT cells in the presence of the A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). Genetic deletion of A(2A) receptor resulted in less prominent effects. A(3)R KO glial cells were more affected by hypoxia than WT cells. Accordingly, the A(3) receptor agonist 2-chloro-N(6)-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine (CL-IB-MECA) reduced ATP depletion caused by hypoxic conditions. It also reduced apoptosis in human astroglioma D384 cells after oxygen deprivation. In conclusion, the data point to a cytoprotective role of adenosine mediated by both A(1) and A(3) receptors in primary mouse astrocytes.

Quantification of puerarin in plasma by on-line solid-phase extraction column switching liquid chromatography-tandem mass spectrometry and its applications to a pharmacokinetic study.

A highly precise, automatic and rapid method for quantification of puerarin in canine and human plasma using an on-line solid-phase extraction (SPE) column switching procedure combined with liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS) was developed. The eluent of SPE column consisted of acetonitrile/methanol/0.1% formic acid (25/25/50) at a flow rate of 0.2mLmin(-1). Puerarin was analyzed by a linear ion trap mass spectrometer, LTQ-MS, operating in the negative ion and selective reaction monitoring (SRM) acquisition mode. Method validation results demonstrated that the linear calibration curve covered a wide range of 0.39-400.00ngmL(-1), the correlation coefficients (r(2)) were above 0.999. The lower limit of detection (LLOD) with the signal-to-noise (S/N) ratio higher than 12 was 0.39ngmL(-1). The intra- and inter-batch precisions were less than 7.61% and 6.42%, respectively. The accuracy was well within the accept limit. The on-line SPE column switching HPLC-MS system was applied to pharmacokinetic (PK) study of puerarin after a single orally dose in beagles. And the optimum conditions were successfully utilized to quantify puerarin in human plasma, which indicated the feasibility and the reliability of this method for application in preclinical and clinical PK studies of isoflavone drugs.

Structure-efficacy relationships of immunostimulatory activity of CpG-containing oligodeoxynucleotides on mouse spleen cells.

AIM: To study the relationship between primary structures of oligodeoxynucleotides (ODN) containing unmethylated deoxycytidyldeoxyguanosine (CpG) dinucleotide motifs and their immunostimulatory activities in mouse spleen cells. METHODS: A series of CpG ODN with different primary structures were synthesized. Their capabilities to stimulate mouse spleen cell proliferation were determined by [3H]thymidine incorporation assay. Cytokine (interleukin [IL]-6, IL-12, and IFN-alpha) secretion spectra induced by CpG ODN were assessed by ELISA. The ability of CpG ODN to activate natural killer cells was evaluated by standard 4 h (51)Cr-release assay. Flow cytometry was utilized to examine the expressions of various lymphocyte surface molecules on diverse immunocytes. An effective CpG ODN for murine, ODN1826, was set as the template of modification and the positive control. RESULTS: The immunostimulatory activities of CpG ODN with different sequences and compositions varied markedly, both in character and in extent. It was useless for improving the immunostimulatory activity of ODN1826 by simply increasing the functional hexameric CpG motif number, modifying the site of CpG motifs, or changing the distance between multi-CpG motifs. However, an addition of a self-complementary palindrome structure at the 3'-end, but not the 5'-end of CpG ODN, aroused marked improvement in its activity. Several designed ODN had superior comprehensive immunostimulatory properties compared to ODN1826. CONCLUSION: The immunostimulatory activity of a CpG ODN was relevant to its primary structure. It was useless for promoting immunostimulatory activity to simply change CpG motif number, space, or distance. The 3'-end palindrome structure of CpG ODN is associated with enhanced immunostimulatory activity.