Subtypes Alpha Research Articles

A simple surface plasmon resonance biosensor for detection of PML/RAR\u03b1 based on heterogeneous fusion gene-triggered nonlinear hybridization chain reaction

In this work, a simple and enzyme-free surface plasmon resonance (SPR) biosensing strategy has been developed for highly sensitive detection of two major PML/RARα (promyelocytic leukemia, retinoic acid receptor alpha) subtypes based on the heterogeneous fusion gene-triggered nonlinear hybridization chain reaction (HCR). On the gold chip surface, the cascade self-assembly process is triggered after the introduction of PML/RARα. The different fragments of PML/RARα can specifically hybridize with capture probes (Cp) immobilized on the chip and the hybridization DNA1 (H1). Then, the nonlinear HCR is initiated by the complex of Cp-PML/RARα-H1 with the introduction of two hybridization DNA chains (H2 and H3). As a result, a dendritic nanostructure is achieved on the surface of chip, leading to a significant SPR amplification signal owing to its high molecular weight. The developed method shows good specificity and high sensitivity with detection limit of 0.72 pM for “L” subtype and 0.65 pM for “S” subtype. Moreover, this method has been successfully applied for efficient identification of clinical positive and negative PCR samples of the PML/RARα subtype. Thus, this developed biosensing strategy presents a potential platform for analysis of fusion gene and early diagnosis of clinical disease.

Involvement of estrogen receptor β in androgen receptor-induced growth inhibition in prostate cancer PC-3 cells.

Previous studies have suggested that changes in sex hormone receptor expression may be associated with the initiation and progression of prostate cancer (PCa). Therefore, the present study aimed to investigate the association and possible pathways between two sex hormone receptors and PCa by measuring the expression levels of the androgen receptor (AR) and the estrogen receptor subtypes alpha (ERα) and beta (ERβ) in prostatic cancer PC-3 cell lines. The pcDNA3.1-hERβ plasmid was transfected into PC-3 cell lines. The expression levels of AR, ERα and ERβ were detected at the mRNA level by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR (qPCR). The results demonstrated that the expression levels of AR, ERβ and ERα were downregulated to different degrees: ERβ test group vs. PC-3 cell group (P=0.000; 95% confidence interval: 0.9803–1.6331). ERβ and AR expression was detected continuously in the PC-3 cells, but the expression of ERα was not. AR expression levels exhibited an upward trend whilst the expression of ERβ demonstrated a marked downward trend. There is a correlation between the expression levels of ERβ and the incidence of PCa, and ERβ may inhibit the growth of PC-3 cell lines by regulating the expression levels of AR. ERβ may provide a novel target for PCa therapies.

Transcriptome analysis of pancreatic cells across distant species highlights novel important regulator genes

BackgroundDefining the transcriptome and the genetic pathways of pancreatic cells is of great interest for elucidating the molecular attributes of pancreas disorders such as diabetes and cancer. As the function of the different pancreatic cell types has been maintained during vertebrate evolution, the comparison of their transcriptomes across distant vertebrate species is a means to pinpoint genes under strong evolutionary constraints due to their crucial function, which have therefore preserved their selective expression in these pancreatic cell types.ResultsIn this study, RNA-sequencing was performed on pancreatic alpha, beta, and delta endocrine cells as well as the acinar and ductal exocrine cells isolated from adult zebrafish transgenic lines. Comparison of these transcriptomes identified many novel markers, including transcription factors and signaling pathway components, specific for each cell type. By performing interspecies comparisons, we identified hundreds of genes with conserved enriched expression in endocrine and exocrine cells among human, mouse, and zebrafish. This list includes many genes known as crucial for pancreatic cell formation or function, but also pinpoints many factors whose pancreatic function is still unknown. A large set of endocrine-enriched genes can already be detected at early developmental stages as revealed by the transcriptomic profiling of embryonic endocrine cells, indicating a potential role in cell differentiation. The actual involvement of conserved endocrine genes in pancreatic cell differentiation was demonstrated in zebrafish for myt1b, whose invalidation leads to a reduction of alpha cells, and for cdx4, selectively expressed in endocrine delta cells and crucial for their specification. Intriguingly, comparison of the endocrine alpha and beta cell subtypes from human, mouse, and zebrafish reveals a much lower conservation of the transcriptomic signatures for these two endocrine cell subtypes compared to the signatures of pan-endocrine and exocrine cells. These data suggest that the identity of the alpha and beta cells relies on a few key factors, corroborating numerous examples of inter-conversion between these two endocrine cell subtypes.ConclusionThis study highlights both evolutionary conserved and species-specific features that will help to unveil universal and fundamental regulatory pathways as well as pathways specific to human and laboratory animal models such as mouse and zebrafish.

The G-Protein Coupled Receptors and G-Proteins Alpha Expression in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is a B cell Non-Hodgkin lymphoma comprising approximately 8% of malignant lymphomas. MCL is considered to be an incurable disease even though survival has improved with novel biological therapies that interrupt survival signaling from the lymphoma microenvironment. The signaling pathways that are deregulated and the factors leading to systemic dissemination of the disease are still poorly understood. There is also a clear need to identify additional druggable targets for therapy (Saba and Wiestner, Curr Opin Hematol, 2014). Druggable targets may be G-protein coupled receptors (GPCRs) and G-proteins. GPCRs belong to the family of seven transmembrane receptors, which upon activation by extracellular ligands transfer the signals inside the cell for activation of the specific pathway. The signaling involves G-proteins. G-proteins consist of the subunits: alpha, beta and gamma and their subtypes. During GPCR activation the G alpha subunit is released and the subtype of G-protein subunit determines which downstream signaling pathways (including Akt, PI3K, ERK, JNK, MAPK, NFkappaB and others) is then activated. In the immune cells different subtypes of G-proteins alpha are instrumental for leukocyte migration, architecture of B cells compartments in spleen, thymus, lymph nodes, and gastrointestinal tract (Hwang et al., PlosOne, 2013) and BCR-mediated cell death (Misra et al., J Exp Med, 2010). G alpha13 signaling is involved in development of germinal centre-derived lymphomas (Muppidi et al., Nature, 2014).In order to map the aberrant expression of GPCR and G-proteins in MCL, the expression of genes encoding for GPCRs and G-protein alpha subunits was analyzed by Affymetrix U133A Array of seventeen MCL biopsies and eight non-malignant lymph nodes. Among investigated GPCR and G-protein alpha subunit genes 25/298 were differently expressed in MCL compared to reactive lymph nodes (moderated t-test, adj. p=or< than 0.05). The four most significantly different genes expressed were: LPAR6, GPRC5C, OPN3 and GNAZ (all adj.p<0.003). The latter, GNAZ, encodes for G-protein alpha z. The analysis of GNAZ by qPCR in 77 additional MCL biopsies from a population-based Stockholm MCL cohort showed varied expression of GNAZ. Interestingly, the MCL cases, which expressed the highest levels of GNAZ, had significantly worse survival (Kaplan-Meier, log rank, p<0.003) suggesting that GNAZ has, a yet unknown, role in MCL biology. Further analysis showed that GNAZ expression was correlated to lymphocytosis (Mann-Whitney test, p=0.023) and leukocytosis (Mann-Whitney test, p=0.011) in MCL patients but not to: high tumor cell proliferation, blastoid morphology nor to p53 immunoreactivity. Our results suggest that GNAZ-signaling is of importance for cell-cell interaction in the tumor microenvironment and/or for egress of lymphoma cells to the blood. DisclosuresNo relevant conflicts of interest to declare.

Epigenetic Analysis of Endocrine Cell Subtypes from Human Pancreatic Islets.

Epigenetic mechanisms have been proposed to contribute to multiple complex diseases, including diabetes mellitus. Dissecting the epigenomic landscape of normal and disease states has greatly expanded our understanding of the regulation of key players in disease pathogenesis and is thus opening doors to novel therapeutic avenues. The human pancreatic islet is a pivotal micro-organ that maintains global glucose homeostasis. The heterogeneity of the islet impedes meaningful in-depth epigenetic analysis using whole islet tissue, because important marks in one cell type are masked by signals from other cell types. We describe here a detailed protocol for the isolation of highly purified endocrine cell subtypes (beta, alpha, and delta cells) from human cadaveric islets, to perform cell-type-specific epigenomic analysis of histone modifications as well as global gene expression profiling.

Tumor Necrosis Factor alpha, prognosis and stroke subtype etiology

Abstract Objective To determine the relationship between TNF alpha and the etiology, localization, extension, intima media thickness, carotid atherosclerotic plaque, and outcome after an acute stroke. Methods We randomly selected 75 patients with acute strokes from a total of 253 patients that were admitted prospectively from May, 2008 to December, 2010. We analyzed TNF alpha levels and compared it with demographic data, clinical outcome upon hospital discharge, and at 3 months post discharge with neuroimaging studies. We used the Chi-square test, the U -Mann–Whiney test and the Cox logistic regression adjusted for age, gender and stroke extension. Results We included 47 men and 28 women. The most common etiologies were atherosclerotic (39%) and small vessel disease (27%). TNF alpha levels did not differ between atherosclerotic and cardioembolic stroke etiologies, except for the lacunar infarction, which had the lowest levels ( p = 0. 048), and did not correlate with a worse functional outcome upon hospital discharge ( p = 0. 852) or at 3 months following discharge ( p = 0.194). Additionally, we found a positive relation between intima media thickness >1 mm and TNF alpha ( p = 0. 004). TNF alpha was not associated with the extension of the stroke by an ASPECTS score with CT or MRI ( p = 0.323) or with the arterial territory involved ( p = 0. 289). Conclusions TNF alpha was not globally associated with functional outcome after acute stroke, just in the lacunar infarction, which has the lowest levels. We also found a positive relation between TNF alpha and intima-media thickness.

Evaluation of in vivo estrogenic potency of natural estrogen-active chemical, puerarin, on pituitary function in gonadectomized female rats

AimsPrevious research has revealed that puerarin, the major phytoestrogen in tuberous roots of Pueraria lobata and Pueraria mirifica, acts as a selective estrogen receptor modulator that displays predominantly estrogenic potential for health benefit. However, little is known about the estrogenic potency of puerarin in pituitary, especially in the rat model of postmenopausal females. Main methodsPlasma prolactin and growth hormone levels as well as mRNA expression levels of pituitary estrogen-regulated genes, such as estrogen receptor (ER) subtypes alpha (ERα) and beta (ERβ), truncated ER product-1 (TERP-1) and -2 (TERP-2) and gonadotropin alpha subunit, were examined using radioimmunoassay and TaqMan® real-time PCR, respectively. The effects were compared with the potent ER agonist, 17β-estradiol-3-benzoate (E2B), and both substances were supplemented at low and high doses, i.e., 0.6 or 3g puerarin and 0.0043 or 0.0173g E2B per kilogram of phytoestrogens-free rat chow, and applied to ovariectomized rats (five groups; 11–12 rats per group) for 12weeks. Key findingsPuerarin possessed weak E2B-like activities on pituitary function by acting as ERβ and TERP-1/-2 agonists, which resulted in the downregulation and upregulation of ERβ and TERP-1/-2 mRNA expressions, respectively, and elevation of growth hormone levels. There were trends of decreased levels of alpha subunit mRNA transcripts and increased levels of prolactin in puerarin-treated rats as observed in E2B-treated animals. SignificanceThis is the first report in ovariectomized rats the effects of puerarin on somatotropes and pituitary estrogen-responsive mRNA expressions, which are very weakly estrogenic by acting through ERβ- and TERP-1/-2 mediated pathways.

Interferon Alpha Subtype-Specific Suppression of HIV-1 Infection In Vivo.

Although all 12 subtypes of human interferon alpha (IFN-α) bind the same receptor, recent results have demonstrated that they elicit unique host responses and display distinct efficacies in the control of different viral infections. The IFN-α2 subtype is currently in HIV-1 clinical trials, but it has not consistently reduced viral loads in HIV-1 patients and is not the most effective subtype against HIV-1 in vitro We now demonstrate in humanized mice that, when delivered at the same high clinical dose, the human IFN-α14 subtype has very potent anti-HIV-1 activity whereas IFN-α2 does not. In both postexposure prophylaxis and treatment of acute infections, IFN-α14, but not IFN-α2, significantly suppressed HIV-1 replication and proviral loads. Furthermore, HIV-1-induced immune hyperactivation, which is a prognosticator of disease progression, was reduced by IFN-α14 but not IFN-α2. Whereas ineffective IFN-α2 therapy was associated with CD8(+) T cell activation, successful IFN-α14 therapy was associated with increased intrinsic and innate immunity, including significantly higher induction of tetherin and MX2, increased APOBEC3G signature mutations in HIV-1 proviral DNA, and higher frequencies of TRAIL(+) NK cells. These results identify IFN-α14 as a potent new therapeutic that operates via mechanisms distinct from those of antiretroviral drugs. The ability of IFN-α14 to reduce both viremia and proviral loads in vivo suggests that it has strong potential as a component of a cure strategy for HIV-1 infections. The broad implication of these results is that the antiviral efficacy of each individual IFN-α subtype should be evaluated against the specific virus being treated. The naturally occurring antiviral protein IFN-α2 is used to treat hepatitis viruses but has proven rather ineffective against HIV in comparison to triple therapy with the antiretroviral (ARV) drugs. Although ARVs suppress the replication of HIV, they fail to completely clear infections. Since IFN-α acts by different mechanism than ARVs and has been shown to reduce HIV proviral loads, clinical trials are under way to test whether IFN-α2 combined with ARVs might eradicate HIV-1 infections. IFN-α is actually a family of 12 distinct proteins, and each IFN-α subtype has different efficacies toward different viruses. Here, we use mice that contain a human immune system, so they can be infected with HIV. With this model, we demonstrate that while IFN-α2 is only weakly effective against HIV, IFN-α14 is extremely potent. This discovery identifies IFN-α14 as a more powerful IFN-α subtype for use in combination therapy trials aimed toward an HIV cure.

Characterization of evolutionary trend in squamate estrogen receptor sensitivity

Steroid hormones are a key regulator of reproductive biology in vertebrates, and are largely regulated via nuclear receptor families. Estrogen signaling is regulated by two estrogen receptor (ER) subtypes alpha and beta in the nucleus. In order to understand the role of estrogen in vertebrates, these ER from various species have been isolated and were functionally analyzed using luciferase reporter gene assays. Interestingly, species difference in estrogen sensitivity has been noted in the past, and it was reported that snake ER displayed highest estrogen sensitivity. Here, we isolated additional ER from three lizards: chameleon (Bradypodion pumilum), skink (Plestiodon finitimus), and gecko (Gekko japonicus). We have performed functional characterization of these ERs using reporter gene assay system, and found high estrogen sensitivity in all three species. Furthermore, comparison with results from other tetrapod ER revealed a seemingly uniform gradual pattern of ligand sensitivity evolution. In silico 3D homology modeling of the ligand-binding domain revealed structural variation at three sites, helix 2, and juncture between helices 8 and 9, and caudal region of helix 10/11. Docking simulations indicated that predicted ligand-receptor interaction also correlated with the reporter assay results, and overall squamates displayed highest stabilized interactions. The assay system and homology modeling system provides tool for in-depth comparative analysis of estrogen function, and provides insight toward the evolution of ER among vertebrates.

Variety of Neuronal Pathways to Achieve the Same Hypoxic Preconditioning Effect

The relevance of hypoxic preconditioning is due to its ability to increase the body's resistance to hypoxic/ischemic stress. A single session of moderate hypoxia eliminates the differences in endurance under severe hypoxia in intact rats and those pre-tested under severe hypoxia with high and low innate resistance to it. In these rat groups, the same preconditioning effect is achieved by different synaptic plastic tools (biochemical data). Thus, the synaptic mechanisms of preconditioning are dependent on the prior hypoxic experience. This conclusion confirmed our pharmacological experiments. Antagonists of alpha7 and non-alpha7 subtypes of the nicotinic receptors methyllycaconitine and mecamylamine in single IP injections selectively influenced the resistance to hypoxia of the low-resistant rats and were ineffective against the high-resistant and intact rats. Moreover, in the low-resistant rats, both drugs had ambiguous effects on the resistance to hypoxia after the preconditioning and without it. Based on the data, we substantiated in our review that the following cholinergic neuronal populations and networks were involved in mechanisms of the hypoxic preconditioning: 1) In the high-resistant rats, cholinergic projections from the pedunculopontine and/or laterodorsal tegmental nuclei into the nuclei of the ventrolateral medulla of the medulla oblongata as well as into the subcortical forebrain nuclei and, linked with them, cholinergic projections from these forebrain nuclei into the cortex. 2) In the low-resistant rats, cholinergic C-fibres into the nucleus tractus solitary of the medulla oblongata and influences of unidentified cholinergic neurons through the alpha7 nicotinic receptors in the caudal brainstem areas outside the nucleus tractus solitary. 3) In intact rats, cortical cholinergic interneurons and unidentified cholinergic neurons of the brainstem structures. In conclusion, the variety of neuronal pathways to put off apnea indicates a great adaptive potential of brain, and the specific mechanisms of its realization may be a promising therapeutic targets.

The effect of fenofibrate on expression of genes involved in fatty acids beta-oxidation and associated free-radical processes

Fenofibrate is a synthetic ligand for peroxisome proliferator-activated receptors subtype alpha (PPARa); it is used for the treatment of a wide range of metabolic diseases such as hypertriglyceridemia, dyslipidemia, diabetes and various neurodegenerative diseases. We have studied the effect of fenofibrate on b-oxidation of fatty acids and related free-radical processes. The most effective concentration of fenofibrate (0.3%) added to the chow caused a significant decrease of the body weight of mice. The data obtained by quantitative PCR demonstrated increased hepatic gene expression responsible for b-oxidation of fatty acids in peroxisomes and mitochondria. Enhancement of oxidative processes caused a 2-fold increase in the rate of reactive oxygen species (ROS) production, as evidenced by determination of the level of lipid peroxidation (LPO) products in the liver. Mitochondrial antioxidant systems are more sensitive to elevated ROS production, as they respond by increased expression of SOD2 and PRDX3 genes, than cytoplasmic and peroxisomal antioxidant systems, where expression of CAT1, SOD1, PRDX5 genes remained unaltered.

ID: 41: Treatment with interferon alpha subtype 14 potently suppresses HIV infection in humanized mice

There are twelve human interferon alpha subtypes that differentially trigger intrinsic, innate, and adaptive immune responses to viral infections. IFN α 2 is currently in HIV clinical trials, but the other subtypes have not been tested and may be better HIV therapeutics. We used HIV-1-infected humanized mice to compare the antiviral properties of IFN α 2 with IFN α 14, the subtype we found to induce the most potent anti-HIV effect in vitro. After ten days of post-exposure prophylaxis, IFN α 14 suppressed virus much better than IFN α 2 with no detectable plasma HIV p24 and greatly reduced or undetectable plasma RNA and provirus levels. Furthermore, CXCL10, an inflammatory chemokine indicative of pathological immune activation, was elevated in IFN α 2-treated but not IFN α 14-treated animals. IFN α 14 efficacy was associated with up-regulated BST2 (tetherin) and MX2 transcripts and TRAIL expression on NK cells but no significant activation of adaptive immune responses. Treatment of chronically HIV-1-infected humanized mice with IFN α 14 resulted in significant decrease in plasma HIV p24 and plasma RNA levels. IFN α 14 strongly outperformed IFN α 2 as an HIV anti-viral in humanized mice.

Variation in interferon sensitivity and induction between Usutu and West Nile (lineages 1 and 2) viruses

Given the pivotal role of monocyte-derived dendritic cells (DCs) in determining the magnitude of the antiviral innate immune response, we sought to determine whether Usutu virus (USUV) and West Nile virus (WNV) lineages (L)1 and L2 can infect DCs and affect the rate of type I interferon (IFN) activation. The sensitivity of these viruses to types I and III IFNs was also compared. We found that USUV can infect DCs, induce higher antiviral activities, IFN alpha subtypes and the IFN stimulated gene (ISG)15 pathway, and is more sensitive to types I and III IFNs than WNVs. In contrast, we confirmed that IFN alpha/beta subtypes were more effective against WNV L2 than WNV L1. However, the replication kinetics, induction of IFN alpha subtypes and ISGs in DCs and the sensitivity to IFN lambda 1–3 did not differ between WNV L1 and L2.

Effects of estradiol, estrogen receptor subtype-selective agonists and genistein on glucose metabolism in leptin resistant female Zucker diabetic fatty (ZDF) rats

The leptin resistant Zucker diabetic fatty (ZDF) rats are hyperphagic and become obese, but whereas the males develop type 2 diabetes mellitus (T2DM), the females remain euglycaemic. As estrogen deficiency is known to increase the risk of developing T2DM, we evaluated the role of ER subtypes alpha and beta in the development of glucose tolerance in leptin resistant ovariectomized (OVX) ZDF rats.At least six rats per group were treated with either vehicle (OVX), 17β-estradiol (E2), ER subtype-selective agonists (Alpha and Beta), or genistein (Gen) for 17 weeks. At the end of the treatment period a glucose tolerance assay was performed and the metabolic flux of 13C-glucose for the E2 group was investigated.OVX ZDF rats treated with E2, Alpha, Beta, and Gen tolerated the glucose significantly better than untreated controls. E2 treatment increased absorbance/flux of 13C-glucose to metabolic relevant tissues such liver, adipose tissue, gastrocnemius, and soleus muscle.Moreover, whereas Alpha treatment markedly increased mRNA expression of GLUT4 in gastrocnemius muscle, Beta treatment resulted in the largest fiber sizes of the soleus muscle. Treatment with Gen increased both the mRNA expression of GLUT 4 and the fiber sizes in the skeletal muscle. In addition, E2 and Alpha treatment decreased food intake and body weight gain.In summary, estrogen-improved glucose absorption is mediated via different molecular mechanisms: while activation of ER alpha seems to stimulate muscular GLUT4 functionality, activation of ER beta results in a hypertrophy of muscle fibers. In addition, selective activation of ER alpha decreased food intake and body weight gain. Our data further indicate that ER subtype-selective agonists and genistein improve systemic glucose tolerance also in the absence of a functional leptin signaling pathway.

Prenatal Exposure to Perfluoroalkyl Acids and Serum Testosterone Concentrations at 15 Years of Age in Female ALSPAC Study Participants.

BackgroundExposure to perfluorooctane sulfonic acid (PFOS) or to perfluorooctanoic acid (PFOA) increases mouse and human peroxisome proliferator–activated receptor alpha (PPARα) subtype activity, which influences lipid metabolism. Because cholesterol is the substrate from which testosterone is synthesized, exposure to these substances has the potential to alter testosterone concentrations.ObjectivesWe explored associations of total testosterone and sex hormone–binding globulin (SHBG) concentrations at age 15 years with prenatal exposures to PFOS, PFOA, perfluorohexane sulfonic acid (PFHxS), and perfluoronanoic acid (PFNA) in females.MethodsPrenatal concentrations of the perfluoroalkyl acids (PFAAs) were measured in serum collected from pregnant mothers at enrollment (1991–1992) in the Avon Longitudinal Study of Parents and Children (ALSPAC). The median gestational age when the maternal blood sample was obtained was 16 weeks (interquartile range, 11–28 weeks). Total testosterone and SHBG concentrations were measured in serum obtained from their daughters at 15 years of age. Associations between prenatal PFAAs concentrations and reproductive outcomes were estimated using linear regression models (n = 72).ResultsAdjusted total testosterone concentrations were on average 0.18-nmol/L (95% CI: 0.01, 0.35) higher in daughters with prenatal PFOS in the upper concentration tertile compared with daughters with prenatal PFOS in the lower tertile. Adjusted total testosterone concentrations were also higher in daughters with prenatal concentrations of PFOA (β = 0.24; 95% CI: 0.05, 0.43) and PFHxS (β = 0.18; 95% CI: 0.00, 0.35) in the upper tertile compared with daughters with concentrations in the lower tertile. We did not find evidence of associations between PFNA and total testosterone or between any of the PFAAs and SHBG.ConclusionsOur findings were based on a small study sample and should be interpreted with caution. However, they suggest that prenatal exposure to some PFAAs may alter testosterone concentrations in females.CitationMaisonet M, Calafat AM, Marcus M, Jaakkola JJ, Lashen H. 2015. Prenatal exposure to perfluoroalkyl acids and serum testosterone concentrations at 15 years of age in female ALSPAC study participants. Environ Health Perspect 123:1325–1330; http://dx.doi.org/10.1289/ehp.1408847

Advances in interferon alpha-targeting approaches for systemic lupus erythematosus treatment

Conventional therapies seem to have reached the limit of their ability to treat patients with Systemic Lupus Erythematosus (SLE). To improve the outcome for these patients, new drugs are needed. Several attempts have been made to introduce targeted therapies for this complex disease. One of these targets is Interferon (IFN) alpha, whose production is increased in SLE, contributing to its pathogenesis. In this review we consider some recent advances in IFN alpha targeting-approaches. Three monoclonal antibodies (mAbs) against several IFN alpha subtypes have been tested in phase I and II trials, showing an acceptable safety profile and promising results in terms of reducing the IFN signature and disease activity. A mAb specific for the IFN alpha receptor and active immunization against IFN alpha are also being tested. Further trials will be essential to ascertain the safety and efficacy of all these approaches.

Mechanistic Progress of Estrogen-induced Apoptosis in Estrogen-deprived Breast Cancer Cells

The laboratory discovery of estrogen-induced apoptosis has been translated to treat antihormone resistant patients and to reduce the incidence of breast cancer in postmenopausal hysterectomized women with estrogen replacement therapy (ERT). The key step is the selection pressure exerted by long-term antiestrogen therapy or over 5 years of menopause to specific breast cancer cell populations that will be vulnerable to estrogen-induced apoptosis. However, the mechanisms underlying estrogen-induced apoptosis are currently unclear. At the cellular level, estrogen-induced apoptosis is dependent upon the estrogen receptor (ER), which can be completely blocked by antiestrogen ICI 182,780 or 4-hydroxytamoxifen (4-OHT). Knockdown of ER alpha, but not ER beta, through specific small interfering RNAs effectively blocks estrogen-induced apoptosis, indicating that the ER alpha subtype participates in apoptosis. Further examinations demonstrate that estrogen-induced apoptosis is due to accumulation of endoplasmic reticulum stress, inflammatory responses, and oxidative stress, which, in turn, activate the intrinsic mitochondrial pathway and the extrinsic death receptor pathway to complete the process. This contrasts with paclitaxel, which causes G2 arrest with immediate apoptosis. These stress responses are modulated by glucocorticoids and the c-Src inhibitor to block estrogen-induced apoptosis, but the mechanism for estrogen action is through a genomic pathway rather than a non-genomic pathway. In the nucleus, estrogen activates classic ERE-regulated endogenous genes, but the ERE transcriptional pathway does not directly participate in the estrogen-induced apoptosis in vitro or in vivo. Simultaneously, estrogen activates a non-classic transcriptional pathway involving the interaction of ER with transcription factors such as activator protein-1 (AP-1), which may regulate proliferation, stress responses, or apoptosis. Investigation of how AP-1 modulates the stress responses to trigger estrogen-induced apoptosis will ultimately uncover the mechanisms underlying estrogen-induced apoptosis.

Disruption of EPCR-Dependent Functions By Plasmodium Falciparum Erythrocyte Membrane Protein 1 (PfEMP1) Can be Rescued By a Soluble EPCR Variant

Introduction: The endothelial protein C receptor (EPCR) is essential for the functions of the protein C system. EPCR enhances the activation of protein C and facilitates the activation of protease activated receptors 1 and 3 by activated protein C (APC) that are required for its cytoprotective activities. Recently EPCR was implicated in the pathogenesis of cerebral malaria due to Plasmodium falciparum infection. Cerebral malaria results >500,000 deaths annually, and survivors often suffer neurological impairments. Expression of P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) on the surface of infected erythrocytes (IE) allows for IE sequestration on microvascular endothelial cells in the brain, which causes blood-brain barrier dysfunction, vascular leakage, edema, local thrombosis, and inflammation that ultimately results in coma and death. A subset of PfEMP1 variants, associated with cerebral malaria, was found to contain a Cysteine-rich Inter Domain Region (CIDR) subtype alpha 1 (CIDRα1) that binds with high affinity to EPCR. Here we determined the effects of CIDRα1 on EPCR-dependent functions of the protein C system. Moreover, we demonstrate that disruption of EPCR’s cellular functions by CIDRα1.1 can be effectively rescued by soluble E86A-EPCR that is devoid of (A)PC binding.Materials and Methods: EPCR binding FCR3 IT4VAR20 CIDRα1.1 and non-EPCR binding controls FCR3 IT4VAR15 CIDRα3.5, and Dd2 Dd2VAR01 CIDRa3.1 were produced in insect cells. Protein C and APC were plasma-derived and E86A- and wt-soluble EPCR (sEPCR) were produced in HEK293 cells. Cellular EPCR functions were determined on the EA.hy926 endothelial cells and endothelial permeability was determined by TER using the iCelligence (ACEA). IE adhesion assays were done on HBMECs using the P. falciparum FCR3 strain expressing IT4VAR20 PfEMP1.Results: CIDRα1.1 bound to sEPCR with an apparent Kd of 4.3 nM and was a competitive inhibitor of APC binding to sEPCR. Dose-dependent binding of CIDRα1.1 to EA.hy926 cells but not to EPCR knockdown EA.hy926 cells indicated that EPCR was the main receptor for CIDRα1.1 binding on these cells. CIDRα1.1 inhibited APC binding to EA.hy926 cells with an IC50~35 nM. CIDRα1.1 inhibited protein C activation on EA.hy926 by >75% similar to the anti-EPCR blocking antibody rcr252, whereas control CIDRα3.5 did not affect protein C activation. Furthermore, CIDRα1.1 reduced APC-mediated PAR1 cleavage on EA.hy926 cells >3-fold and accordingly abrogated APC-mediated protection against thrombin-induced barrier disruption. Control CIDRα3.1 did not affect APC-mediated barrier protection.To determine whether CIDRα1.1 inhibited APC binding to EPCR by direct competition or by steric hindrance, binding of CIDRα1.1 to E86A-sEPCR that does not bind APC was determined. Remarkably, CIDRα1.1 bound E86A-sEPCR with similar affinity compared to wt-sEPCR. Therefore, the ability of E86A-sEPCR to compete for CIDRα1.1 binding to cellular EPCR was determined. E86A-sEPCR dose-dependently restored APC binding to wt-sEPCR in the presence of CIDRα1.1. Protein C activation on EA.hy926 cells in the presence of CIDRα1.1 was partially restored at 5 nM and completely restored at 50 nM E86A-sEPCR. Likewise, E86A-sEPCR enhanced PAR1 cleavage by APC in the presence of CIDRα1.1 >4-fold and abrogated inhibition of APC’s barrier protective effects by CIDRα1.1. Finally, cytoadhesion of IE expressing full-length CIDRα1.1-containing PfEMP1 to HBMECs was inhibited ~70% by both E86A- and wt-sEPCR (IC50~30 nM).Conclusions: The binding of CIDRα1.1 to EPCR greatly compromised the ability of EPCR to enhance protein C activation and to facilitate APC-mediated PAR1 cleavage and induction of endothelial barrier protective effects. Based on the well-documented neuroprotective effects of the protein C system in the brain it is likely that the PfEMP1-induced loss of EPCR-dependent functions provide a seminal contribution to the mortality and neurological damages associated with cerebral malaria. Although both E86A- and wt-sEPCR can compete for CIDRα1.1 binding to cellular EPCR, E86A-sEPCR does not bind (A)PC and therefore does not interfere with (A)PC binding to cellular EPCR. In summary, these results provide novel insights into the possible pathogenesis of cerebral malaria and present a proof-of-concept strategy for the development of novel adjunct therapies for cerebral malaria. DisclosuresNo relevant conflicts of interest to declare.

Bisphenol A regulates the estrogen receptor alpha signaling in developing hippocampus of male rats through estrogen receptor.

Bisphenol A (BPA), one of the most common environmental endocrine disruptors, has been recognized to have wide adverse effects on the brain development and behavior. These adversities are related to its ability to bind estrogen receptor (ER) with subsequent alteration of its expression in the target areas. However, very little is known about whether BPA exposure also affects ER phosphorylation and its translocation to nucleus during postnatal development, two critical steps for its function. Here, we found that during development from postnatal day 7 (P7) to P21, the alpha subtype of ER (ERα) in the hippocampus of male rats experienced remarkable alterations in terms of its expression, phosphorylation and translocation to nucleus. Exposure to low level of BPA had bidirectional, development-dependent effects on the expression of ERα mRNA and protein, but decreased ERα phosphorylation and impaired its translocation to nucleus throughout the period investigated. Treatment with low dose of ICI 182,780 (ICI), an ER antagonist to block the binding of ER with BPA, reversed the altered ERα following BPA exposure, highlighting critical involvement of ER. Moreover, ICI treatment rescued the hippocampus-dependent behavioral deficits in the adult rats experiencing early-life BPA exposure. Overall, our results indicate that BPA interferes with the ERα signaling in the developing hippocampus in an ER-dependent manner, which may underlie its adverse behavioral and cognitive outcomes in adult animals.

PPAR-alpha and PPAR-beta expression changes in the hippocampus of rats undergoing global cerebral ischemia/reperfusion due to PPAR-gamma status.

BackgroundPeroxisome proliferator-activated receptors (PPARs, including alpha, beta and gamma subtypes) and their agonists have a protective role in treatment of central nervous system (CNS) diseases. The present study was designed to investigate the expression changes of PPAR-alpha, -beta, -gamma and NF-kappa B in the hippocampus of rats with global cerebral ischemia/reperfusion injury (GCIRI) after treatment with agonists or antagonists of PPAR-gamma.MethodsA rat GCIRI model was established by occlusion of bilateral common carotid arteries and cervical vena retransfusion. GW9662 (5 μg), a selective PPAR- gamma antagonist, was intraventricularly injected at 0.5 h before GCIR; Rosiglitazone (0.8, 2.4 and 7.2 mg/kg), a selective PPAR- gamma agonist, was injected intraperitoneally at 1 h before GCIRI. The expression changes of PPAR-alpha, -beta and -gamma at mRNA and protein levels were detected by RT-PCR and western blotting. The changes of spatial learning and memory (SLM) functions were assessed by using a Morris water maze; the pathohistological changes of hippocampal neurons were evaluated by hematoxylin-eosin (HE) staining; the contents of IL-1, IL-6, IL-10 and TNF-alpha, and the NF- kappa B expression were measured by enzyme-linked immunosorbent assay (ELISA) and immunohistochemical staining. The superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were also detected.ResultsThe SLM function and hippocampal neurons were significantly impaired after the occurrence of GCIRI. The MDA, IL-1, IL-6, IL-10, TNF-alpha content and expression of PPARs increased significantly, but the SOD activity and NF-kappa B expression were weakened in the hippocampus. Rosiglitazone treatment significantly protected rats from SLM function impairment and neuron death, and resulted in higher expressions of SOD activity and NF-kappa B, but lower contents of MDA and inflammatory factors. After treatment with rosiglitazone or GW9662, no significant change in PPAR-alpha or -beta expression was detected.ConclusionsRosiglitazone, a PPAR-gamma agonist, plays a protective role in hippocampal neuron damage of GCIRI rats by inhibiting the oxidative stress response and inflammation. The activation or antagonism of PPAR-gamma did not affect the expression of PPAR-alpha or -beta, indicating that the three subtypes of PPARs act in independent pathways in the CNS.