Secreted Alkaline Phosphatase Research Articles

Novel traceable CRISPR-Cas9 engineered human embryonic stem cell line (E1C3 + hSEAP + 2xKO + pCD47), has potential to evade immune detection in pigs

Here we present the generation of a human embryonic stem cell line with the potential to escape immune rejection upon transplantation to an alternate species, in this case sus scrofa. For in vivo detection the cells were modified by CRISPR-Cas9 to express human secreted alkaline phosphatase. To avoid immune recognition and subsequent rejection by host, genes encoding hB2M and hCIITa were knocked out and the porcine gene for CD47 was introduced. Upon editing and subsequent culture, cells maintained molecular and phenotypic pluripotent charactaristica and a normal karyotype supporting viability and functionality of the engineered cell line.

ROLE OF CYTOTOXIC T LYMPHOCYTE IN ADENOVIRUS INFECTION

The successful development of adenovirus vectors for vaccines and gene therapy will require a better understanding of the host immune response. Using the ELISPOT assay to measure IFN-g-secreting CD8þ cells, we identify immunodominant epitopes of the adenovirus hexon and DNA-binding protein in BALB/c and C57BL/6 mice. The T-cell response to the intramuscular administration of adenovirus serotype 5 peaks within a few weeks and gradually declines but is still detectable after 12 weeks. A second administration did not substantially increase the number of reactive T cells. The CD8þ T-cell response was also similar between wild type and E1-deleted adenovirus. When B-cell-deficient mice were injected with adenovirus encoding the gene for secreted alkaline phosphatase, sera phosphatase activity was reduced more quickly in mice pre-exposed to adenovirus. These results add to the evidence that cell-mediated immunity is a substantial barrier to therapeutic adenoviral vectors and provide more quantitative tools to measure cellular immune responses to adenovirus.

Functional Selectivity of Cannabinoid Type 1 G Protein-Coupled Receptor Agonists in Transactivating Glycosylated Receptors on Cancer Cells to Induce Epithelial-Mesenchymal Transition Metastatic Phenotype.

Understanding the role of biased G protein-coupled receptor (GPCR) agonism in receptor signaling may provide novel insights into the opposing effects mediated by cannabinoids, particularly in cancer and cancer metastasis. GPCRs can have more than one active state, a phenomenon called either 'biased agonism', 'functional selectivity', or 'ligand-directed signaling'. However, there are increasing arrays of cannabinoid allosteric ligands with different degrees of modulation, called 'biased modulation', that can vary dramatically in a probe- and pathway-specific manner, not from simple differences in orthosteric ligand efficacy or stimulus-response coupling. Here, emerging evidence proposes the involvement of CB1 GPCRs in a novel biased GPCR signaling paradigm involving the crosstalk between neuraminidase-1 (Neu-1) and matrix metalloproteinase-9 (MMP-9) in the activation of glycosylated receptors through the modification of the receptor glycosylation state. The study findings highlighted the role of CB1 agonists AM-404, Aravnil, and Olvanil in significantly inducing Neu-1 sialidase activity in a dose-dependent fashion in RAW-Blue, PANC-1, and SW-620 cells. This approach was further substantiated by findings that the neuromedin B receptor inhibitor, BIM-23127, MMP-9 inhibitor, MMP9i, and Neu-1 inhibitor, oseltamivir phosphate, could specifically block CB1 agonist-induced Neu-1 sialidase activity. Additionally, we found that CB1 receptors exist in a multimeric receptor complex with Neu-1 in naïve, unstimulated RAW-Blue, PANC-1, and SW-620 cells. This complex implies a molecular link that regulates the interaction and signaling mechanism among these molecules present on the cell surface. Moreover, the study results demonstrate that CB1 agonists induce NFκB-dependent secretory alkaline phosphatase (SEAP) activity in influencing the expression of epithelial-mesenchymal markers, E-cadherin, and vimentin in SW-620 cells, albeit the impact on E-cadherin expression is less pronounced compared to vimentin. In essence, this innovative research begins to elucidate an entirely new molecular mechanism involving a GPCR signaling paradigm in which cannabinoids, as epigenetic stimuli, may traverse to influence gene expression and contribute to cancer and cancer metastasis.

A highly efficient transposon vector system for recombinant protein expression in CHO cells

Chinese hamster ovary (CHO) cells are the most widely used mammalian host cells for the production of recombinant therapeutic proteins. Expression vectors are a key factor in determining the expression level of gene of interest (GOI) in CHO cells. Here, two transposon vector systems were constructed by adding two different pairs of inverted terminal repeats (ITR-1 and ITR-2), and four proteins of interest (POI), including enhanced green fluorescence protein (eGFP), secreted alkaline phosphatase (SEAP), human serum albumin (HSA) and Adalimumab (Adm) were expressed in CHO cells. The stably transfected cell pools and single cell clones were screened, and the expression levels of POIs were detected by qPCR, Western blot and ELISA. The results showed that both transposon vectors could improve the expression levels of POI at both mRNA and protein levels, while the ITR-2 sequence was more effective. Moreover, the positive cell clone rate of transposon system was higher than that of the control. In conclusion, the ITR-2 transposon vector system can be effectively used for the expression of recombinant proteins in CHO cells.

Necrotic Cells from Head and Neck Carcinomas Release Biomolecules That Are Activating Toll-like Receptor 3.

Tumor necrosis is a recurrent characteristic of head and neck squamous cell carcinomas (HNSCCs). There is a need for more investigations on the influence of biomolecules released by these necrotic foci in the HNSCC tumor microenvironment. It is suspected that a fraction of the biomolecules released by necrotic cells are damage-associated molecular patterns (DAMPs), which are known to be natural endogenous ligands of Toll-like receptors (TLRs), including, among others, proteins and nucleic acids. However, there has been no direct demonstration that biomolecules released by HNSCC necrotic cells can activate TLRs. Our aim was to investigate whether some of these molecules could behave as agonists of the TLR3, either in vitro or in vivo. We chose a functional approach based on reporter cell exhibiting artificial TLR3 expression and downstream release of secreted alkaline phosphatase. The production of biomolecules activating TLR3 was first investigated in vitro using three HNSCC cell lines subjected to various pronecrotic stimuli (external irradiation, serum starvation, hypoxia and oxidative stress). TLR3 agonists were also investigated in necrotic tumor fluids from five oral cancer patients and three mouse tumor grafts. The release of biomolecules activating TLR3 was demonstrated for all three HNSCC cell lines. External irradiation was the most consistently efficient stimulus, and corresponding TLR3 agonists were conveyed in extracellular vesicles. TLR3-stimulating activity was detected in the fluids from all five patients and three mouse tumor grafts. In most cases, this activity was greatly reduced by RNAse pretreatment or TLR3 blocking antibodies. Our data indicate that TLR3 agonists are consistently present in necrotic fluids from HNSCC cells and mainly made of dsRNA fragments. These endogenous agonists may induce TLR3, which might lead to a protumorigenic effect. Regarding methodological aspects, our study demonstrates that direct investigations-including functional testing-can be performed on necrotic fluids from patient tumors.

Distribution of Signal Peptides in Microvesicles from Activated Macrophage Cells.

Extracellular vesicles, such as microvesicles (LEV) and exosomes (SEV), play an important role in intercellular signaling by encapsulating functional molecules and delivering them to specific cells. Recent studies showed that signal peptides (SPs), which are derived from sequences at the N-terminal of newly synthesized proteins, exhibited biological activity in the extracellular fluid. We previously reported that SPs were secreted into the extracellular fluid via SEV; however, it remains unclear whether the release of SPs occurs via LEV. In the present study, we demonstrated that SP fragments from human placental secreted alkaline phosphatase (SEAP) were present in LEV as well as SEV released from RAW-Blue cells, which stably express an NF-κB-inducible SEAP reporter. When RAW-Blue cells were treated with LPS at 0-10,000 ng/mL, SEAP SP fragments per particle were more abundant in LEV than in SEV, with fragments in LEV and SEV reaching a maximum at 1000 and 100 ng/mL, respectively. The content of SEAP SP fragments in LEV from IFNγ-stimulated RAW-Blue cells was higher than those from TNFα-stimulated cells, whereas that in SEV from TNFα-stimulated RAW-Blue cells was higher than those from IFNγ-stimulated cells. Moreover, the content of SEAP SP fragments in LEV and SEV decreased in the presence of W13, a calmodulin inhibitor. Collectively, these results indicate that the transportation of SP fragments to extracellular vesicles was changed by cellular activation, and calmodulin was involved in their transportation to LEV and SEV.

Successful AAV8 re-administration: Suppression of capsid-specific neutralizing antibodies by a combination treatment of bortezomib and CD20 mAb in a mouse model of Pompe disease.

A major challenge to AAV mediated gene therapy is the presence of anti-AAV capsid neutralizing antibodies (NAbs), which can block viral vector transduction even at very low titers. Here we examined the ability of a combination immunosuppression (IS) treatment with bortezomib and a mouse-specific CD20 mAb to suppress anti-AAV NAbs and enable re-administration of AAV vectors of the same capsid in mice. An AAV8 vector (AAV8-CB-hGAA) that ubiquitously expresses human α-glucosidase was used for initial gene therapy and a second AAV8 vector (AAV8-LSP-hSEAP) that contains a liver-specific promoter to express human secreted alkaline phosphatase was used for AAV re-administration. Plasma samples were used for determination of anti-AAV8 NAb titers. Cells isolated from whole blood, spleen, and bone marrow were analyzed for B-cell depletion by flow cytometry. The efficiency of AAV re-administration was determined by the secretion of hSEAP in blood. In näive mice, an eight-week IS treatment along with AAV8-CB-hGAA injection effectively depleted CD19+ B220+ B cells from blood, spleen, and bone marrow and prevented the formation of anti-AAV8 NAbs. Following administration of AAV8-LSP-hSEAP, increasing levels of hSEAP were detected in blood for up to 6 weeks, indicating successful AAV re-administration. In mice pre-immunized with AAV8-CB-hGAA, comparison of IS treatment for 8, 12, 16, and 20 weeks revealed that the 16-week IS treatment demonstrated the highest plasma hSEAP level following AAV8-LSP-hSEAP re-administration. Our data suggest that this combination treatment is an effective IS approach that will allow retreatment of patients with AAV mediated gene therapy.

Detection of Nanoparticles' Ability to Stimulate Toll-Like Receptors Using HEK-Blue Reporter Cell Lines.

Nanoparticles can be used to formulate Toll-like receptor (TLR) agonists as vaccine and immunotherapy adjuvants or contain undesirable contaminants (e.g., endotoxin, CpG DNA, flagellin) with TLR-agonist activity. In both scenarios, the activation of the innate immune pattern recognition receptor leads to the inflammatory response that can be beneficial as in the case with vaccines and immunotherapies or adverse as in the case with contaminants. The protocol described herein utilizes commercially available reporter cell lines expressing individual TLRs, which, upon activation with their cognate agonists, stimulate the cells to produce secreted alkaline phosphatase detectable using a plate reader.

The biological activity of serum bacterial lipopolysaccharides associates with disease activity and likelihood of achieving remission in patients with rheumatoid arthritis

BackgroundDysbiotic intestinal and oral microbiota have been implicated in the pathogenesis of rheumatoid arthritis (RA), but the mechanisms how microbiota could impact disease activity have remained elusive. The aim of this study was to assess the association of the biological activity of serum lipopolysaccharides (LPS) with disease activity and likelihood of achieving remission in RA patients.MethodsWe measured Toll-like receptor (TLR) 4-stimulating activity of sera of 58 RA patients with a reporter cell line engineered to produce secreted alkaline phosphatase in response to TLR4 stimulation. Levels of LPS-binding protein, CD14, and CD163 were determined by ELISA assays.ResultsThe patient serum-induced TLR4 activation (biological activity of LPS) was significantly associated with inflammatory parameters and body mass index at baseline and at 12 months and with disease activity (DAS28-CRP, p<0.001) at 12 months. Importantly, baseline LPS bioactivity correlated with disease activity (p=0.031) and, in 28 early RA patients, the likelihood of achieving remission at 12 months (p=0.009). The level of LPS bioactivity was similar at baseline and 12-month visits, suggesting that LPS bioactivity is an independent patient-related factor. Neutralization of LPS in serum by polymyxin B abrogated the TLR4 signaling, suggesting that LPS was the major contributor to TLR4 activation.ConclusionWe describe a novel approach to study the biological activity of serum LPS and their impact in diseases. The results suggest that LPS contribute to the inflammatory burden and disease activity on patients with RA and that serum-induced TLR4 activation assays can serve as an independent prognostic factor.Graphical A graphical summary of the conclusions of the study.

Biochar reduces bioavailability of phosphorus during swine manure composting: Roles of phoD-harboring bacterial community

The bioavailability of phosphorus is a vital index for evaluating the quality of compost products. This study examined the effects of adding wheat straw biochar (WSB) and bamboo charcoal (BC) on the transformation of various phosphorus fractions during composting, as well as analyzing the roles of the phoD-harboring bacterial community in the transformation of phosphorus fractions. Adding WSB and BC reduced the available phosphorus content in the compost products by 35.2 % and 38.5 %, respectively. Redundancy analysis showed that the alkaline phosphatase content and pH were the most important factors that affected the transformation of phosphorus fractions. The addition of biochar resulted in changes in the composition and structures of the phoD-harboring bacteria communities during composting. In addition, the key bacterial genera that secreted alkaline phosphatase and decomposed different forms of phosphorus under WSB and BC were different compared with those under control. Network and correlation analysis demonstrated that the activities of phoD-harboring bacteria could have been enhanced by biochar to accelerate the consumption of available phosphorus, and the activities of key phosphorus-solubilizing bacteria (Lysobacter, Methylobacterium, and Saccharothrix) might be inhibited when the pH increased, thereby increasing the insoluble phosphorus content.

Transcription factor EB protects against endoplasmic reticulum stress in human coronary artery endothelial cells

Oxidative stress and endoplasmic reticulum (ER) stress promote atherogenesis while transcription factor EB (TFEB) inhibits atherosclerosis. Since reducing oxidative stress with antioxidants have failed to reduce atherosclerosis possibly because of aggravation of ER stress, we studied the effect of TFEB on ER stress in human coronary artery endothelial cells. ER stress was measured using the secreted alkaline phosphatase assay. Expression and phosphorylation of key mediators of unfolded protein response (UPR). TFEB, inositol-requiring enzyme 1α (IRE1α), phospho-IRE1α, protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), phospho-PERK, and activating transcription factor 6 (ATF6) expression were measured by Western blot. The effect of TFEB gain- and loss-of-function on ER stress were assessed with a plasmid expressing a constitutively active form of TFEB and via siRNA-mediated silencing, respectively. Treatment with tunicamycin (TM) and thapsigargin (TG) increased TFEB expression by 42.8% and 42.3%, respectively. In HCAEC transfected with the TFEB siRNA, treatment with either TM, TG or high-dextrose increased IRE1α and PERK phosphorylation and ATF6 levels significantly more compared to cells transfected with the control siRNA and treated similarly. Furthermore, transient transfection with a plasmid expressing a constitutively active form of TFEB reduced ER stress. Increased expression of TFEB inhibited ER stress in HCAEC treated with pharmacologic (TM and TG) and physiologic (high-dextrose) ER stress inducers, while TFEB knockout aggravated ER stress caused by these ER stress inducers. TFEB-mediated ER stress reduction may contribute to its anti-atherogenic effects in HCAEC and may be a novel target for drug development.

Broadly Applicable, Virus-Free Dual Reporter Assay to Identify Compounds Interfering with Membrane Fusion: Performance for HSV-1 and SARS-CoV-2.

Membrane fusion constitutes an essential step in the replication cycle of numerous viral pathogens, hence it represents an important druggable target. In the present study, we established a virus-free, stable reporter fusion inhibition assay (SRFIA) specifically designed to identify compounds interfering with virus-induced membrane fusion. The dual reporter assay is based on two stable Vero cell lines harboring the third-generation tetracycline (Tet3G) transactivator and a bicistronic reporter gene cassette under the control of the tetracycline responsive element (TRE3G), respectively. Cell–cell fusion by the transient transfection of viral fusogens in the presence of doxycycline results in the expression of the reporter enzyme secreted alkaline phosphatase (SEAP) and the fluorescent nuclear localization marker EYFPNuc. A constitutively expressed, secreted form of nanoluciferase (secNLuc) functioned as the internal control. The performance of the SRFIA was tested for the quantification of SARS-CoV-2- and HSV-1-induced cell–cell fusion, respectively, showing high sensitivity and specificity, as well as the reliable identification of known fusion inhibitors. Parallel quantification of secNLuc enabled the detection of cytotoxic compounds or insufficient transfection efficacy. In conclusion, the SRFIA reported here is well suited for high-throughput screening for new antiviral agents and essentially will be applicable to all viral fusogens causing cell–cell fusion in Vero cells.

Abstract 385: Fortilin Interacts With Tgf-β1 And Prevents Tgf-β Receptor Activation

Fortilin is a 172-amino acid multifunctional protein present in both intra- and extracellular spaces. Although fortilin binds and regulates various cellular proteins, the biological role of extracellular fortilin remains unknown. Here we report that fortilin specifically interacts with TGF-β1 and prevents it from activating the TGF-β1 signaling pathway. In a standard immunoprecipitation-western blot assay, fortilin co-immunoprecipitates TGF-β1 and its isoforms. The modified ELISA assay shows that TGF-β1 remains complexed with fortilin in human serum. Both bio-layer interferometry and surface plasmon resonance (SPR) reveal that fortilin directly bind TGF-β1. The SPR analysis also reveals that fortilin and the TGF-β receptor II (TGFβRII) compete for TGF-β1. Both luciferase and secreted alkaline phosphatase (SEAP) reporter assays show that fortilin prevents TGF-β1 from activating Smad3 binding to Smad-binding element (Figure). Fortilin inhibits the phosphorylation of Smad3 in both quantitative western blot assays and ELISA. Finally, fortilin inhibits TGFβ-1-induced differentiation of C3H10T1/2 mesenchymal progenitor cells to smooth muscle cells. A computer-assisted virtual docking reveals that fortilin occupies the pocket of TGF-β1 that is normally occupied by TGFβRII and that TGF-β1 can bind either fortilin or TGFβRII at any given time. These data support the role of extracellular fortilin as a negative regulator of the TGF-β1 signaling pathway.

Signal Sequence-Dependent Orientation of Signal Peptide Fragments to Exosomes.

Signal peptides (SPs) not only mediate targeting to the endoplasmic reticulum (ER) but also play important roles as biomarkers and substances with physiological activity in extracellular fluids including blood. SPs are thought to be degraded intracellularly, making it unclear how they are transported from the ER to the extracellular fluid. In a recent study, we showed that a C-terminal fragment of the SP of a type I membrane protein, amyloid precursor protein (APP), was secreted into the extracellular fluid via exosomes using transformed HEK293 cells expressing APP SP flanking a reporter protein. In the present study, we demonstrate that a N-terminal fragment of the SP from a type II membrane protein, human placental secreted alkaline phosphatase (SEAP), is contained in exosomes and secreted into the extracellular fluid using HEK-Blue hTLR3 cells, which express both a human toll-like receptor 3 gene and an inducible SEAP reporter gene. When HEK-Blue hTLR3 cells were stimulated with a TLR3 ligand, a N-terminal fragment of SEAP SP in exosomes was increased in parallel with SEAP secretion in a concentration-dependent manner. These results indicated that SP fragments are exosomal components. In addition, migrating SP fragments were determined by characteristics of the signal–anchor sequence of membrane proteins. Furthermore, we found that SP fragments could bind to calmodulin (CALM), which is a cytosolic protein and also a component of exosomes, suggesting its involvement in the transportation of SP fragments from the endoplasmic reticulum to exosomes.

Fortilin interacts with TGF-\u03b21 and prevents TGF-\u03b2 receptor activation

Fortilin is a 172-amino acid multifunctional protein present in both intra- and extracellular spaces. Although fortilin binds and regulates various cellular proteins, the biological role of extracellular fortilin remains unknown. Here we report that fortilin specifically interacts with TGF-β1 and prevents it from activating the TGF-β1 signaling pathway. In a standard immunoprecipitation-western blot assay, fortilin co-immunoprecipitates TGF-β1 and its isoforms. The modified ELISA assay shows that TGF-β1 remains complexed with fortilin in human serum. Both bio-layer interferometry and surface plasmon resonance (SPR) reveal that fortilin directly bind TGF-β1. The SPR analysis also reveals that fortilin and the TGF-β receptor II (TGFβRII) compete for TGF-β1. Both luciferase and secreted alkaline phosphatase reporter assays show that fortilin prevents TGF-β1 from activating Smad3 binding to Smad-binding element. Fortilin inhibits the phosphorylation of Smad3 in both quantitative western blot assays and ELISA. Finally, fortilin inhibits TGFβ-1-induced differentiation of C3H10T1/2 mesenchymal progenitor cells to smooth muscle cells. A computer-assisted virtual docking reveals that fortilin occupies the pocket of TGF-β1 that is normally occupied by TGFβRII and that TGF-β1 can bind either fortilin or TGFβRII at any given time. These data support the role of extracellular fortilin as a negative regulator of the TGF-β1 signaling pathway.

Synthesis of Glycopolymers Carrying 3′-Sialyllactose for Suppressing Inflammatory Reaction via Siglec-E

Abstract One of the new strategies to treat autoimmune diseases is to target Siglec, a membrane protein receptor with the ability to suppress immune responses. Herein, we synthesized glycopolymers carrying 3′-sialyllactose in various glycounit densities. RAW 264.7 macrophages transfected to express secreted alkaline phosphatase (SEAP) were used to evaluate the immunosuppression ability of the glycopolymers. The inhibition of the signal transmission was dependent on the glycounit densities of the glycopolymers, and was maximized at the moderate density (70%).

Restoration of DNA repair mitigates genome instability and increases productivity of Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are the primary host for manufacturing of therapeutic proteins. However, productivity loss is a major problem and is associated with genome instability, as chromosomal aberrations reduce transgene copy number and decrease protein expression. We analyzed whole-genome sequencing data from 11 CHO cell lines and found deleterious single-nucleotide variants in DNA repair genes. Comparison with primary Chinese hamster cells confirmed DNA repair to be compromised in CHO. Correction of key DNA repair genes by single-nucleotide variant reversal or expression of intact complementary DNAs successfully improved DNA repair and mitigated karyotypic instability. Moreover, overexpression of intact copies of LIG4 and XRCC6 in a CHO cell line expressing secreted alkaline phosphatase mitigated transgene copy loss and improved protein titer retention. These results show that correction of DNA repair genes yields improvements in genome stability in CHO, and provide new opportunities for cell line development for sustainable protein expression.

Novel cell line development strategy for monoclonal antibody manufacturing using translational enhancing technology

Chinese hamster ovary (CHO) cells are widely used for constructing expression systems to produce therapeutic proteins. However, the establishment of high-producer clones remains a laborious and time-consuming process, despite various progresses having been made in cell line development. We previously developed a new strategy for screening high monoclonal antibody (mAb)-producing cells using flow cytometry (FCM). We also reported that p180 and SF3b4 play key roles in active translation on the endoplasmic reticulum, and that the productivity of secreted alkaline phosphatase was enhanced by the overexpression of p180 and SF3b4. Here, we attempted to apply the translational enhancing technology to high mAb-producing cells obtained after high-producer cell sorting. A high mAb-producing CHO clone, L003, which showed an mAb production level of >3g/L in fed-batch culture, was established from a high mAb-producing cell pool fractionated by FCM. Clones generated by the overexpression of p180 and SF3b4 in L003cells were evaluated by fed-batch culture. The specific productivity of clones overexpressing these two factors was ∼3.1-fold higher than that of parental L003cells in the early phase of the culture period. Furthermore, the final mAb concentration was increased to 9.5g/L during 17 days of fed-batch culture after optimizing the medium and culture process. These results indicate that the overexpression of p180 and SF3b4 would be promising for establishing high-producer cell lines applicable to industrial production.

Differential effects of cyclooxygenase-2 (COX-2) inhibitors on endoplasmic reticulum (ER) stress in human coronary artery endothelial cells

Selective cyclooxygenase-2 (COX-2) inhibitor rofecoxib was pulled off the market because of its association with increased risk of adverse cardiovascular effects. The precise underlying mechanism for the differential effects of COX-2 inhibitors on cardiovascular risk is not known. Since endoplasmic reticulum (ER) stress is implicated in atherogenesis, we examined the effects of COX-2 inhibitors on ER stress in primary human coronary artery endothelial cells (HCAEC), human umbilical vein endothelial cells (HUVEC), and human pulmonary artery endothelial cells (HPAEC). ER stress was measured in HCAEC treated with either tunicamycin (TM) or high-concentrations (27.5 mM) of dextrose (HD) using the secreted alkaline phosphatase (ES-TRAP) assay. Markers of the unfolded protein response (UPR) such as activating transcription factor 6 (ATF6), glucose-regulated protein 78 (GRP78), inositol-requiring enzyme 1α (IRE1α), phospho-IRE1α, protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), and phospho-PERK were measured by Western blot. Treatment of HCAEC with TM and HD decreased secreted alkaline phosphatase activity indicating increased ER stress. Treatment of cells exposed to TM or HD with celecoxib, meloxicam, ibuprofen, and acetylsalicylic acid, but not rofecoxib, resulted in a dose-dependent decrease in ER stress. High-dextrose and TM increased IRE1α and PERK phosphorylation and ATF6 and GRP78 expression. Treatment with celecoxib, but not rofecoxib, inhibited these markers of the UPR. Treatment with selective COX-2 inhibitors, with the exception of rofecoxib, suppressed ER stress as measured with both alkaline phosphatase activity assays and markers for the UPR. The inability of rofecoxib to inhibit ER stress, unlike the other cyclooxygenase inhibitors tested, may have contributed to its unfavorable effects on cardiovascular outcomes.

Endpoint and Kinetic Approaches for Assessing Transfection Efficacy in Mammalian Cell Culture.

The efficacy of transfection reagents and nanoparticles is often assessed by measuring levels of expressed reporter protein. Fluorescence and luminescence based assays provide sensitive, quantifiable and repeatable approaches. The genes expressing reporter protein can be integrated into the cells to create stable reporter cell lines or can be expressed from a transfected plasmid. Green fluorescent protein, luciferase, and secreted alkaline phosphatase are well-established reporters with versatile applications. Monitoring changes in live cells during and after transfection offer opportunities to reveal related mechanisms, efficacy, and bottlenecks of transfection.In this chapter, we describe the experimental setup and considerations for in vitro screening of delivery vectors. This can further be extended to measurements in reporter cell lines.