Alkaline Phosphatase Reporter Gene Research Articles

305 Usefulness of CHA2DS2-VASc scoring system for predicting risk of embolism in patients with cardiac tumours: a single-centre study

Abstract Aims The link between pro-protein convertase subtilisin/kexin 9 (PCSK9) and inflammatory-related disease has been supported in several experimental and human studies, which show how the protein could acts independent its canonical mechanism, by activation of inflammatory, apoptotic and immune pathways. Among these, TLR4/NFKB signalling pathway has been found to be one of the main pathways mediating the PCSK9-induced increase of pro-inflammatory and pro-thrombotic molecules, such as TNF-alpha, IL-6, IL-1, MCP-1, and TF. To investigate the direct involvement of hTLR4 in recognition of exogenous human recombinant (hr) PCSK9. Methods and results Experiments were carried out in three cell line: (i) HEK293-hTLR4-GFP cell lines obtained by stable co-transfection of HEK293 cells with the pUNO1-hTLR4-GFP, a plasmid expressing the hTLR4 gene fused to a GFP gene; (ii) HEK293-hTLR4+ cells obtained by co-transfection of hTLR4 receptor and its accessory proteins MD-2 and CD14, and an inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene; (iii) HEK293-Null2 cells, a cell line lacking TLR4 expression, used as negative control. NFkB activity were measured by the SEAP reporter gene assay using a fluorescence detection method, while localization of hTLR4 and exogenous hrPCSK9 by confocal microscopy. hrPCSK9 (1 µg/mL) activates NFkB in HEK293-hTLR4+ cells [SEAP activity-OD 620 nm: from a baseline of 0.18 ± 0.06 to 0.68 ± 0.05 (N = 8), P < 0.001] to an extent comparable to lipopolysaccharide, the specific agonist of TLR4. Co-localization of hrPCSK9 and TLR4s was clearly documented by quantitative confocal microscopy in HEK293-hTLR4-GFP cell line, that comprises the analysis of more than 80 fields in ∼2.105 cells/well and showing a percentage of co-localization of ∼4% in membrane spots (P < 0.01). Conclusions Our data support the results reported in previous studies that attribute to PCSK9 a pathophysiological role in development of chronic inflammation and related-diseases such as atherothrombosis. A behavior that extends far beyond LDLR degradation, via a mechanism that might be mediated at least in part by recognition of PCSK9 by the TLRs and later activation of NFkB intracellular pathway. Future studies will be important to better investigate the specific binding site engaged in the PCSK9-TLR interaction and to discriminate the intracellular transduction pathways involved in this response, in order to provide a theoretical basis for the development of innovative therapeutic strategies.

306 Involvement of toll-like receptor 4 in PCSK9-mediated biological effects in cell culture models

Abstract Aims The link between pro-protein convertase subtilisin/kexin 9 (PCSK9) and inflammatory-related disease was supported in several experimental and human studies, which show how the protein could acts independent its canonical mechanism, by activation of inflammatory, apoptotic and immune pathways. Among these, TLR4/NFKB signalling pathway was found to be one of the main pathways mediating the PCSK9-induced increase of pro-inflammatory and pro-thrombotic molecules, such as TNF-alpha, IL-6, IL-1, MCP-1, and TF. To investigate the direct involvement of hTLR4 in recognition of exogenous human recombinant (hr) PCSK9. Methods Experiments were carried out in three cell line: (i) HEK293-hTLR4-GFP cell lines obtained by stable co-transfection of HEK293 cells with the pUNO1-hTLR4-GFP, a plasmid expressing the hTLR4 gene fused to a GFP gene; (ii) HEK293-hTLR4+ cells obtained by co-transfection of hTLR4 receptor and its accessory proteins MD-2 and CD14, and an inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene; (iii) HEK293-Null2 cells, a cell line lacking TLR4 expression, used as negative control. NFkB activity were measured by the SEAP reporter gene assay using a fluorescence detection method, while localization of hTLR4 and exogenous hrPCSK9 by confocal microscopy. Results hrPCSK9 (1 µg/mL) activates NFkB in HEK293-hTLR4+ cells [SEAP activity-OD 620 nm: from a baseline of 0.18 ± 0.06 to 0.68 ± 0.05 (N = 8), P < 0.001] to an extent comparable to Lipopolysaccharide (LPS), the specific agonist of TLR4. Colocalization of hrPCSK9 and TLR4s was clearly documented by quantitative confocal microscopy in HEK293-hTLR4-GFP cell line, that comprises the analysis of more than 80 fields in ∼2.105 cells/well and showing a percentage of co-localization of ∼4% in membrane spots (P < 0.01). Conclusions Our data support the results reported in previous studies that attribute to PCSK9 a pathophysiological role in development of chronic inflammation and related-diseases such as atherothrombosis. A behaviour that extends far beyond LDLR degradation, via a mechanism that might be mediated at least in part by recognition of PCSK9 by the TLRs and later activation of NFKB intracellular pathway. Future studies will be important to better investigate the specific binding site engaged in the PCSK9-TLR interaction and to discriminate the intracellular transduction pathways involved in this response, in order to provide a theoretical basis for the development of innovative therapeutic strategies.

A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay.

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health conditions in both the laboratory and the clinic. However, pre-existing neutralizing antibodies (NAbs) against AAV capsids pose an ongoing challenge for the successful administration of gene therapies in both large animal experimental models and human populations. Preliminary screening for host immunity against AAV is necessary to ensure the efficacy of AAV-based gene therapies as both a research tool and as a clinically viable therapeutic agent. This protocol describes a colorimetric in vitro assay to detect neutralizing factors against AAV serotype 6 (AAV6). The assay utilizes the reaction between an AAV encoding an alkaline phosphatase (AP) reporter gene and its substrate NBT/BCIP, which generates an insoluble quantifiable purple stain upon combination. In this protocol, serum samples are combined with an AAV expressing AP and incubated to permit potential neutralizing activity to occur. Virus serum mixture is subsequently added to cells to allow for viral transduction of any AAVs that have not been neutralized. The NBT/BCIP substrate is added and undergoes a chromogenic reaction, corresponding to viral transduction and neutralizing activity. The proportion of area colored is quantitated using a free software tool to generate neutralizing titers. This assay displays a strong positive correlation between coloration and viral concentration. Assessment of serum samples from sheep before and after administration of a recombinant AAV6 led to a dramatic increase in neutralizing activity (125 to >10,000-fold increase). The assay displayed adequate sensitivity to detect neutralizing activity in >1:32,000 serum dilutions. This assay provides a simple, rapid, and cost-effective method to detect NAbs against AAVs.

Targeting multiple mediators of sepsis using multifunctional tannic acid-Zn2+-gentamicin nanoparticles

Targeting multiple mediators of sepsis using multifunctional tannic acid-Zn2+-gentamicin nanoparticles

Estrogen receptor-dependent and independent roles of benzo[a]pyrene in Ishikawa cells.

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants generated from the incomplete combustion of organic material. PAHs have been studied as genotoxicants, but some also act via non-genotoxic mechanisms in estrogen-dependent malignancies, such as breast cancer. PAHs require metabolic activation to electrophilic metabolites to exert their genotoxicity but non-genotoxic properties may also contribute to their carcinogenicity. The role of PAHs in endometrial cancer, a cancer associated with unopposed estrogen action is unknown. We assessed the metabolism of the representative PAH, benzo[a]pyrene (B[a]P), to estrogenic compounds in Ishikawa human endometrial cells in the presence and absence of cytochrome P450 induction. Using stable-isotope dilution high-performance liquid chromatography and APCI tandem mass spectrometry in the selected reaction monitoring mode, we analyzed B[a]P metabolism in Ishikawa cells. Estrogenic activity of B[a]P metabolites was determined by the endogenous estrogen inducible alkaline phosphatase reporter gene and an exogenous estrogen response element (ERE) luciferase reporter gene construct. We also assessed whether PAHs can induce a proliferative phenotype via estrogen receptor (ER)- and non-ER-regulated pathways. We demonstrate that B[a]P can be metabolized in human endometrial cells into 3-OH-B[a]P and B[a]P-7,8-dione in sufficient amounts to activate ERs. We also show that only B[a]P-7,8-dione induces endometrial cell proliferation at concentrations lower than required to activate the ER; instead non-genomic signaling by the EGF receptor (EGFR) and activation of the mitogen-activated protein kinase (MAPK) pathway was responsible. This work indicates that human endometrial cells can metabolize PAHs into estrogenic metabolites, which may induce cell proliferation through non-ER-regulated pathways.

Identification and characterization of Fbxl22, a novel skeletal muscle atrophy-promoting E3 ubiquitin ligase.

Muscle-specific E3 ubiquitin ligases have been identified in muscle atrophy-inducing conditions. The purpose of the current study was to explore the functional role of F-box and leucine-rich protein 22 (Fbxl22), and a newly identified splice variant (Fbxl22-193), in skeletal muscle homeostasis and neurogenic muscle atrophy. In mouse C2C12 muscle cells, promoter fragments of the Fbxl22 gene were cloned and fused with the secreted alkaline phosphatase reporter gene to assess the transcriptional regulation of Fbxl22. The tibialis anterior muscles of male C57/BL6 mice (12-16 wk old) were electroporated with expression plasmids containing the cDNA of two Fbxl22 splice variants and tissues collected after 7, 14, and 28 days. Gastrocnemius muscles of wild-type and muscle-specific RING finger 1 knockout (MuRF1 KO) mice were electroporated with an Fbxl22 RNAi or empty plasmid and denervated 3 days posttransfection, and tissues were collected 7 days postdenervation. The full-length gene and novel splice variant are transcriptionally induced early (after 3 days) during neurogenic muscle atrophy. In vivo overexpression of Fbxl22 isoforms in mouse skeletal muscle leads to evidence of myopathy/atrophy, suggesting that both are involved in the process of neurogenic muscle atrophy. Knockdown of Fbxl22 in the muscles of MuRF1 KO mice resulted in significant additive muscle sparing 7 days after denervation. Targeting two E3 ubiquitin ligases appears to have a strong additive effect on protecting muscle mass loss with denervation, and these findings have important implications in the development of therapeutic strategies to treat muscle atrophy.

Isorhamnetin Has Potential for the Treatment of Escherichia coli-Induced Sepsis.

Isorhamnetin is a flavonoid that is abundant in the fruit of Hippophae rhamnoides L. It is widely studied for its ability to modulate inflammatory responses. In this study, we evaluated the potential of isorhamnetin to prevent gram-negative sepsis. We investigated its efficacy using an Escherichia coli-induced sepsis model. Our study reveals that isorhamnetin treatment significantly enhances survival and reduces proinflammatory cytokine levels in the serum and lung tissue of E. coli-infected mice. Further, isorhamnetin treatment also significantly reduces the levels of aspartate aminotransferase, alanine amino transferase and blood urea nitrogen, suggesting that it can improve liver and kidney function in infected mice. Docking studies reveal that isorhamnetin binds deep in the hydrophobic binding pocket of MD-2 via extensive hydrophobic interactions and hydrogen bonding with Tyr102, preventing TLR4/MD-2 dimerization. Notably, binding and secreted alkaline phosphatase reporter gene assays show that isorhamnetin can interact directly with the TLR4/MD-2 complex, thus inhibiting the TLR4 cascade, which eventually causes systemic inflammation, resulting in death due to cytokine storms. We therefore presume that isorhamnetin could be a suitable therapeutic candidate to treat bacterial sepsis.

Antiseptic Effect of Ps-K18: Mechanism of Its Antibacterial and Anti-Inflammatory Activities.

Recently, bioactive peptides have attracted attention for their therapeutic applications in the pharmaceutical industry. Among them, antimicrobial peptides are candidates for new antibiotic drugs. Since pseudin-2 (Ps), isolated from the skin of the paradoxical frog Pseudis paradoxa, shows broad-spectrum antibacterial activity with high cytotoxicity, we previously designed Ps-K18 with a Lys substitution for Leu18 in Ps, which showed high antibacterial activity and low toxicity. Here, we examined the potency of Ps-K18, aiming to develop antibiotics derived from bioactive peptides for the treatment of Gram-negative sepsis. We first investigated the antibacterial mechanism of Ps-K18 based on confocal micrographs and field emission scanning electron microscopy, confirming that Ps-K18 targets the bacterial membrane. Anti-inflammatory mechanism of Ps-K18 was investigated by secreted alkaline phosphatase reporter gene assays and RT-PCR, which revealed that Ps-K18 activates innate defense via Toll-like receptor 4-mediated nuclear factor-kappa B signaling pathways. Moreover, we investigated the antiseptic effect of Ps-K18 using a lipopolysaccharide or Escherichia coli K1-induced septic shock mouse model. Ps-K18 significantly reduced bacterial growth and inflammatory responses in the septic shock model. Ps-K18 showed low renal and liver toxicity and attenuated lung damage effectively. This study suggests that Ps-K18 is a potent peptide antibiotic that could be applied therapeutically to Gram-negative sepsis.

Ttc39c is upregulated during skeletal muscle atrophy and modulates ERK1/2 MAP kinase and hedgehog signaling.

Tetratricopeptide repeat domain containing 39c (Ttc39c) is expressed in skeletal muscle and is transcriptionally activated in response to neurogenic atrophy in mice. Expression analysis using quantitative polymerase chain reaction and Western blots revealed that Ttc39c is expressed in both proliferating and differentiated muscle cells, peaking during early differentiation and then decreasing as cells progress further through the differentiation process. To further analyze the transcriptional regulation of Ttc39c, promoter fragments of the gene were cloned and fused with the secreted alkaline phosphatase reporter gene. The Ttc39c reporter plasmids were then transfected into cultured mouse muscle cells and found to have transcriptional activity. Furthermore, overexpression of MyoD and myogenin resulted in significant transcriptional repression of the Ttc39c reporter genes. To determine subcellular localization, an expression plasmid with the Ttc39c complementary DNA fused with green fluorescent protein was transfected into muscle cells and analyzed by confocal fluorescent microscopy showing that Tct39c localizes exclusively to the cytoplasm of cultured cells. To assess potential function in muscle, Ttc39c was overexpressed leading to vitiated muscle cell differentiation, impaired ERK1/2 MAP Kinase and Hedgehog signaling, and increased expression of IFT144 and IFT43, which are part of the IFT-A complex involved in retrograde transport in primary cilia. Interestingly, Ttc39c knockdown also resulted in inhibition of muscle cell differentiation and impaired Hedgehog and MAP Kinase signaling but did not affect IFT144 or IFT433 expression. The results of this study demonstrate that muscle cell differentiation is sensitive to abnormal Ttc39c expression and that normal Ttc39c expression appears to be necessary for proper MAP Kinase and Hedgehog signal transduction in developing muscle cells.

Development of a novel short 12-meric papiliocin-derived peptide that is effective against Gram-negative sepsis

The development of novel peptide antibiotics with potent activity against multidrug-resistant Gram-negative bacteria and anti-septic activity is urgently needed. In this study, we designed short, 12-meric antimicrobial peptides by substituting amino acids from the N-terminal 12 residues of the papiliocin (Pap12-1) peptide to alter cationicity and amphipathicity and improve antibacterial activity and bacterial membrane interactions. Pap12-6, with an amphipathic α-helical structure and Trp12 at the C-terminus, showed broad-spectrum antibacterial activity, especially against multidrug-resistant Gram-negative bacteria. Dye leakage, membrane depolarization, and electron microscopy data proved that Pap12-6 kills bacteria by permeabilizing the bacterial membrane. Additionally, Pap12-6 significantly reduced the secretion of NO, TNF-α, and IL-6 and secreted alkaline phosphatase reporter gene activity confirmed that Pap12-6 shows anti-inflammatory activity via a TLR4-mediated NF-κB signaling pathway. In a mouse sepsis model, Pap12-6 significantly improved survival, reduced bacterial growth in organs, and reduced LPS and inflammatory cytokine levels in the serum and organs. Pap12-6 showed minimal cytotoxicity towards mammalian cells and controlled liver and kidney damage, proving its high bacterial selectivity. Our results suggest that Pap12-6 is a promising peptide antibiotic for the therapeutic treatment of Gram-negative sepsis via dual bactericidal and immunomodulatory effects on the host.

Effect of Robinia pseudoacacia Leaf Extract on Interleukin-1β-mediated Tumor Angiogenesis.

Interleukin (IL)-1β is a pro-inflammatory cytokine that has recently been established as a stimulator of angiogenesis via regulation of proangiogenic factor expression in the tumor microenvironment. This study aimed to demonstrate the inhibitory effects of Robinia pseudoacacia leaf extract (RP) on IL-1β-mediated tumor angiogenesis. Secreted embryonic alkaline phosphatase (SEAP) reporter gene assay, ex vivo and in vitro tube formation assay, western blot, and quantitative PCR were used to analyze the inhibitory effect of RP on IL-1β-mediated angiogenesis. RP inhibited secretion of SEAP, blocked IL-1β signaling, and inhibited IL-1β-mediated angiogenesis in ex vivo and in vitro assays. RP inhibited nuclear translocation of NF-ĸB by suppressing phosphorylation of IL-1β signaling protein kinases and inhibited mRNA expression of IL-1β-induced pro-angiogenic factors including VEGFA, FGF2, ICAM1, CXCL8, and IL6. RP suppressed IL-1β-mediated angiogenesis and, thus, could be a promising agent in anticancer therapy.

High-throughput screening for small molecule inhibitors of the type-I interferon signaling pathway

Interferons (IFNs) are cytokines with fundamental roles in resistance to infections, cancer and other diseases. Type-I IFNs, interferon α (IFN-α) and interferon β (IFN-β), act through a shared receptor complex (IFNAR) comprised of IFNAR1 and IFNAR2 subunits. Binding of type-I IFN to IFNAR1 will robustly activate Janus activated kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway. Aberrant activation of the type-I IFN response results in a spectrum of disorders called interferonopathies. The purpose of this research is to develop an assay for high-throughput screening (HTS) of small molecule inhibitors of the type-I IFN signaling pathway. Inhibition of type-I IFN signaling can be beneficial in terms of therapeutic use and understanding the underlying mechanism of action. We report here a HTS campaign with the secreted embryonic alkaline phosphatase (SEAP) reporter gene assay against 32,000 compounds which yielded 25 confirmed hits. These compounds were subsequently characterized for their cytotoxicity, effects on STAT phosphorylation and activities in IFN regulatory factor (IRF) transcription.

Broad Antagonistic Activity of a Fatty Acid Binding Protein of Fasciola hepatica (Fh12) in the Presence of Whole Bacteria Extract Prelude a Broad Spectrum of Action

Inflammation process could be initiated by activation of single or multiple toll‐like receptors on antigen presenting cells via molecular pattern recognition of molecules of foreign pathogens like Gram positive and negative bacteria. The exacerbated immune response to these molecules can lead to septic shock syndrome that could be fatal. The identification of TLR‐antagonists that could block or diminish the activation of these receptors and suppress the inflammatory mediators has become in an excellent therapeutic alternative against sepsis. In the last decade, scientists have shown interest in the strong anti‐inflammatory role that some helminth parasites exhibit. Helminths use several immunomodulatory strategies to modify immune responses in order to survive into the mammalian host. Fasciola hepatica is able to induce long‐lasting disease in the host associated to Th2 responses, which is linked to an anti‐inflammatory phenotype of immune responses. This parasite secretes and expresses proteins on their surfaces that are essential either, its metabolism or immunomodulating the host's immune response. One of these proteins is a 12kDa fatty acid binding protein (Fh12). We had previously demonstrated that Fh12 could suppress the TLR4 activation. The current study aimed to ascertain whether Fh12 could also suppress other TLRs. THP1‐Blue CD14 cells is a cell line of human monocytes that express NF‐κB/AP‐1 inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene. Cells were cultured for 18h, 37°C, 5% CO2 with 10μg Fh12 in the presence or absence of specific ligands for each TLR as well as with or without heat attenuated whole extracts of (1 × 108 cells) Escherichia coli, Klebsiella pneumonia or Enterococcus faecalis. Cells cultured only with RPMI‐medium, TLR‐ligands, Fh12 or whole bacteria extracts alone served as controls. Results demonstrated that Fh12 was capable to significantly suppress the activation of NF‐κB to Gram negative and Gram‐positive bacteria by 65% and 90%, respectively. In consistency with this finding Fh12 also suppressed the NF‐κB activation induced by specific ligands for TLR2, TLR4, TLR5, TLR6 and TLR8 by 80% to 89% respectively. FACS analysis also revealed that Fh12 also induced significant reduction in the expression of co‐stimulatory molecules like CD40, CD80 and CD86, which play crucial role in the initiation and maintenance of inflammatory immune response. Our results suggest that Fh12 not only impacts TLR4, but also could impact multiple TLRs and which prelude a broader anti‐inflammatory effect for Fh12. Further studies directed to explore the potential of Fh12 as a new class of drug against septic shock caused by bacteria and also others inflammatory diseases are under study.Support or Funding InformationThis study was supported by MBRS‐RISE R25GM061838‐13, and NIH grants G12MD007600 and 2P40OD012217.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fasciola hepatica Glutathione S‐Transferase (FhGST) conformational studies and its effect on the suppression of toll‐like receptors stimulation

Fasciola hepatica is a parasite well known as a master of immunomodulation because of its ability to suppress the Th1 immune responses and elicit a strong Th2/Treg response. This occurs when the parasite releases excretory‐secretory products (ESPs), which are comprised of a myriad of glycoproteins that are believed to play an important role in the immune response. F. hepatica Glutathione S‐Transferase (FhGST) is one of the ESP components recognized for its anti‐oxidant properties and its capacity to provide part of the parasite's defense by detoxifying the secondary products of lipid peroxidation that are produced due to the immune free‐radical attack on the host or the parasite membrane. Our preliminary studies demonstrate that FhGST exerts a suppressive effect on the activation of TLR4 in macrophages which is evidenced by significant suppression of NF‐kB activation and down‐regulation of TNFα and IL1‐β in response to LPS‐stimuli. We speculated that the integrity of the tertiary structure, as well as the glycans that decorate the protein moiety of FhGST, could be essential for its capacity to suppress the NF‐kB activation. To test our hypothesis, we first subjected FhGST to a thermal denaturing process by boiling it at 90°C for 10 min followed by a treatment with PNGase F. Circular dichroism analysis (CD) comparing the FhGST spectra before and after heat denaturing served to confirm that FhGST was full and irreversibly denatured. Next, denatured or native FhGST were added alone or in the presence of the whole extract of 1 x 108 cells Klebsiella pneumonia (Kp) to a culture of human monocytes (THP1‐CD14) that express most TLRs as well as an NF‐kb inducible secreted embryonic alkaline phosphatase reporter gene. Cells stimulated with an attenuated whole extract of 1 × 108 cells Klebsiella pneumonia (Kp) were used as activation control. Our results demonstrated that native FhGST suppresses NF‐kB activation by 83%, whereas the heat‐denatured protein suppresses NF‐kB activation by 84%. These suppressions suggest that in contrast to expected, the capacity of FhGST to suppress the NF‐κB activation in THP1‐CD14 cells is independent of the integrity of its secondary/tertiary structure and is not associated to the glycans. Docking analysis in which the FhGST‐protein moiety was docked with the CD14 and MD2 co‐receptor structures suggested that FhGST could target these co‐receptors as part of its mechanisms of action.Support or Funding InformationThis study was supported by MBRS‐RISE R25GM061838‐13, 5R25GM061151‐16 and NIH grants G12MD007600 and 2P40OD012217This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The role of unfolded protein response and ER-phagy in quantum dots-induced nephrotoxicity: an in vitro and in vivo study.

Unfolded protein response (UPR) and endoplasmic reticulum (ER)-phagy are essential for cell homeostasis. Quantum dots (QDs), which have been widely used for biomedical applications, can accumulate in the kidney tissues and may cause renal dysfunction. However, the molecular mechanism of QDs-induced nephrotoxicity is still obscure. The present study was aimed to elucidate the role and mechanism of UPR and ER-phagy in QDs-induced nephrotoxicity. Herein, human embyronic kidney (HEK) cells were exposed to 15, 30, 45, and 60nM cadmium telluride (CdTe)-QDs for 12 and 24h. And CdTe-QDs (30-60nM) inhibited the HEK cell viability. The clathrin-dependent endocytosis was determined as the main pathway of CdTe-QDs cellular uptake. Within cells, CdTe-QDs disrupted ER ultrastructure and induced UPR and FAM134B-dependent ER-phagy. Blocking UPR with inhibitors or siRNA rescued the FAM134B-dependent ER-phagy, which was triggered by CdTe-QDs. Moreover, suppression of UPR or FAM134B-dependent ER-phagy restored the cell vability. In vivo, mice were intravenously injected with 8 and 16nmol/kg body weight CdTe-QDs for 24h. Kidney was shown as one of highest distributed organs of CdTe-QDs, resulting in renal dysfunction, as well as UPR and FAM134B-dependent ER-phagy in it. Thus, for the first time, we demonstrated that ER-phagy can be triggered by nanomaterials both in vitro and in vivo. In addition, blocking of UPR and ER-phagy showed protective effects against CdTe-QDs-induced toxicity in kideny cells. Notably, a secreted alkaline phosphatase reporter gene system has been developed as a sensitive and rapid method for evaluating the ER quality under the exposure of nanomaterials.

Candida albicans-derived mannoproteins activate NF-κB in reporter cells expressing TLR4, MD2 and CD14.

The ability of soluble C. albicans 20A (serotype A) mannoprotein (CMP) to serve as a ligand for toll-like receptor 4 (TLR4) and its co-receptors was examined using commercially available and stably-transfected HEK293 cells that express human TLR4, MD2 and CD14, but not MR. These TLR4 reporter cells also express an NF-κB-dependent, secreted embryonic alkaline phosphatase (SEAP) reporter gene. TLR4-reporter cells exhibited a dose-dependent SEAP response to both LPS and CMP, wherein peak activation was achieved after stimulation with 40–50 μg/mL of CMP. Incubation on polymyxin B resin had no effect on CMP’s ligand activity, but neutralized LPS-spiked controls. HEK293 Null cells lacking TLR4 and possessing the same SEAP reporter failed to respond to LPS or CMP, but produced SEAP when activated with TNFα. Reporter cell NF-κB responses were accompanied by transcription of IL-8, TNFα, and COX-2 genes. Celecoxib inhibited LPS-, CMP-, and TNFα-dependent NF-κB responses; whereas, indomethacin had limited effect on LPS and CMP responses. SEAP production in response to C. albicans A9 mnn4Δ mutant CMP, lacking phosphomannosylations on N-linked glycans, was significantly greater (p ≤ 0.005) than SEAP responses to CMP derived from parental A9 (both serotype B). These data confirm that engineered human cells expressing TLR4, MD2 and CD14 can respond to CMP with NF-κB activation and the response can be influenced by variations in CMP-mannosylation. Future characterizations of CMPs from other sources and their application in this model may provide further insight into variations observed with TLR4 dependent innate immune responses targeting different C. albicans strains.

Screening for Novel Endogenous Inflammatory Stimuli Using the Secreted Embryonic Alkaline Phosphatase NF-κB Reporter Assay.

An immune response can be activated by pathogenic stimuli, as well as endogenous danger signals, triggering the activation of pattern recognition receptors and initiating signalling cascades that lead to inflammation. This method uses THP1-Blue™ cells, a human monocytic cell line which contains an embryonic alkaline phosphatase reporter gene allowing the detection of NF-κB-induced transcriptional activation. We validated this protocol by assessing NF-κB activation after stimulation of toll-like receptor 4 (TLR4) by two different agonists: lipopolysaccharide (LPS), derived from the cell wall of Gram negative bacteria, and tenascin-C, an extracellular matrix protein whose expression is induced upon tissue injury. We then used this protocol to screen for potential new endogenous TLR4 agonists, but this method can also be used as a quick, economical and reliable means to assay the activity of other inflammatory stimuli resulting in TLR-dependent NF-κB activation.

Hydrophobic and electrostatic interactions between cell penetrating peptides and plasmid DNA are important for stable non-covalent complexation and intracellular delivery.

Cell penetrating peptides are useful tools for intracellular delivery of nucleic acids. Delivery of plasmid DNA, a large nucleic acid, poses a challenge for peptide mediated transport. The paper investigates and compares efficacy of five novel peptide designs for complexation of plasmid DNA and subsequent delivery into cells. The peptides were designed to contain reported DNA condensing agents and basic cell penetrating sequences, octa-arginine (R8 ) and CHK6 HC coupled to cell penetration accelerating peptides such as Bax inhibitory mutant peptide (KLPVM) and a peptide derived from the Kaposi fibroblast growth factor (kFGF) membrane translocating sequence. A tryptophan rich peptide, an analogue of Pep-3, flanked with CH3 on either ends was also a part of the study. The peptides were analysed for plasmid DNA complexation, protection of peptide-plasmid DNA complexes against DNase I, serum components and competitive ligands by simple agarose gel electrophoresis techniques. Hemolysis of rat red blood corpuscles (RBCs) in the presence of the peptides was used as a measure of peptide cytotoxicity. Plasmid DNA delivery through the designed peptides was evaluated in two cell lines, human cervical cancer cell line (HeLa) and (NIH/3 T3) mouse embryonic fibroblasts via expression of the secreted alkaline phosphatase (SEAP) reporter gene. The importance of hydrophobic sequences in addition to cationic sequences in peptides for non-covalent plasmid DNA complexation and delivery has been illustrated. An alternative to the employment of fatty acid moieties for enhanced gene transfer has been proposed. Comparison of peptides for plasmid DNA complexation and delivery of peptide-plasmid DNA complexes to cells estimated by expression of a reporter gene, SEAP. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Abstract 2732: Myelosuppression in patients treated with the telomerase inhibitor imetelstat is not mediated through activation of toll-like receptors

Abstract Imetelstat is a covalently lipidated 13-mer N3′-P5′ thio-phosphoramidate (NPS) oligonucleotide (5′-TAGGGTTAGACAA-3′) that is complementary to the RNA template region (hTR; 3′-AUCCCAACCUGUU-5′) of telomerase. Imetelstat is believed to act as a direct competitive inhibitor of telomerase enzyme activity and to not have an antisense mechanism of action (MOA) typical of other oligonucleotides. The purpose of this study was to assess whether the thrombocytopenia observed in some patients with myeloproliferative neoplasm treated with imetelstat can be attributed to activation of toll-like receptors (TLRs) by imetelstat. Imetelstat and related control oligonucleotides (Mis-match and Sense) were tested using 7 different HEK-Blue™ TLR cell lines, each stably coexpressing a human TLR gene (TLR2, 3, 4, 5, 7, 8, and 9) and a NF-κB-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene. After 20 hours’ incubation, the cells were measured for absorbance at 650 nm for SEAP activity. Each TLR experiment was performed twice, with 3 replicates per experiment. The results showed that the positive control for each individual TLR was recognized by the corresponding TLR, leading to a strong SEAP activity, whereas imetelstat at a clinically relevant concentration range (10 μM-60 μM) and the control oligonucleotides related to imetelstat yielded no induction of SEAP activity (similar level to untreated). These data demonstrate that imetelstat has no stimulatory effect on the majority of the tested TLRs. Imetelstat yielded slightly higher induction on TLR8 than the untreated control, but significantly lower than the positive control. Although thrombocytopenia has been observed in patients with myeloproliferative neoplasms treated with imetelstat, these data show that it cannot result from imetelstat acting as a ligand for TLRs. TLR9, associated with thrombocytopenia, was not activated by imetelstat and the slight activity of imetelstat on TLR8 is likely irrelevant as no link between TLR8 activation and thrombocytopenia has been reported. These findings are consistent with 13-mer sequence of imetelstat, differing from oligonucleotides known to stimulate TLR9, which comprise 2 CpG islands separated by 6-10 nucleotides and are longer than 21 nucleotides. Ex vivo studies have provided evidence that imetelstat may induce thrombocytopenia in patients with myeloproliferative neoplasms by blocking the terminal maturation of normal megakaryocyte precursors: megakaryocytic differentiation requires upregulation of telomerase activity which is inhibited by imetelstat. Correct understanding of the MOA of imetelstat is vital to guide its clinical use and strategies to mitigate thrombocytopenia in patients with myeloproliferative neoplasms. Citation Format: Gabriela M. Baerlocher, Joshua Rusbuldt, Fei Huang, Jacqueline Bussolari. Myelosuppression in patients treated with the telomerase inhibitor imetelstat is not mediated through activation of toll-like receptors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2732.

181. Optimization of In Vivo Delivery of Baculovirus Gene Therapy Vectors for the Treatment of Alpha-1 Antitrypsin Deficiency

Alpha-1 antitrypsin deficiency (AATD) is a single-gene disorder commonly associated with adult onset lung diseases, including emphysema, chronic obstructive pulmonary disease and airway inflammation. Alpha-1 antitrypsin (AAT) is produced primarily in hepatocytes and is subsequently secreted into the bloodstream and travels to the lung where it inhibits neutrophil elastase. Unregulated neutrophil elastase activity in individuals with AATD causes degradation of various components of the extracellular matrix producing damage to alveolar and airway epithelial cells. Furthermore, mutated AAT folds incorrectly causing intracellular aggregation within hepatocytes, which can result in liver damage in addition to pulmonary symptoms. The development of gene therapy strategies has been a popular approach for the treatment of AATD due to monogenic nature of this genetic disease. Recently, baculovirus based vectors have garnered attention for possible application in gene therapy due to multiple factors. Baculoviruses can tolerate large gene insertions (>38kb), are capable of transducing mammalian cells but not replicating or integrating into host chromosomes and finally, humans lack pre-existing immunity to these insect viruses. Although there are many characteristics that make baculovirus vectors attractive for use in gene therapy, there is little in vivo data on bio-distribution and tissue tropism in mammalian models. As AATD is a disease affecting both the liver and the lung, we examined several delivery routes of a baculovirus vector expressing the human placental alkaline phosphatase reporter gene in C57BL/6 mice. To target the lung we investigated intranasal and intratracheal administration and for liver delivery intravenous, intrahepatic and intraportal vein injections were utilized. Lung delivery of wild type baculovirus yielded little transduction, however intrahepatic injection resulted in moderate liver transduction as well as unexpected spread to other tissues. Different formulations of the baculovirus vector were examined for pulmonary delivery including a variety of viscoeleastic gels. Additionally, pharmacological agents such as clodronate liposomes to deplete macrophages and cobra venom factor to ablate complement activity were examined in an effort to bolster transduction.