In-cell ELISA Research Articles

Cell-Based Phenotypic Drug Screening Identifies Luteolin as Candidate Therapeutic for Nephropathic Cystinosis.

Mutations in the gene that encodes the lysosomal cystine transporter cystinosin cause the lysosomal storage disease cystinosis. Defective cystine transport leads to intralysosomal accumulation and crystallization of cystine. The most severe phenotype, nephropathic cystinosis, manifests during the first months of life, as renal Fanconi syndrome. The cystine-depleting agent cysteamine significantly delays symptoms, but it cannot prevent progression to ESKD and does not treat Fanconi syndrome. This suggests the involvement of pathways in nephropathic cystinosis that are unrelated to lysosomal cystine accumulation. Recent data indicate that one such potential pathway, lysosome-mediated degradation of autophagy cargoes, is compromised in cystinosis. To identify drugs that reduce levels of the autophagy-related protein p62/SQSTM1 in cystinotic proximal tubular epithelial cells, we performed a high-throughput screening on the basis of an in-cell ELISA assay. We then tested a promising candidate in cells derived from patients with, and mouse models of, cystinosis, and in preclinical studies in cystinotic zebrafish. Of 46 compounds identified as reducing p62/SQSTM1 levels in cystinotic cells, we selected luteolin on the basis of its efficacy, safety profile, and similarity to genistein, which we previously showed to ameliorate other lysosomal abnormalities of cystinotic cells. Our data show that luteolin improves the autophagy-lysosome degradative pathway, is a powerful antioxidant, and has antiapoptotic properties. Moreover, luteolin stimulates endocytosis and improves the expression of the endocytic receptor megalin. Our data show that luteolin improves defective pathways of cystinosis and has a good safety profile, and thus has potential as a treatment for nephropathic cystinosis and other renal lysosomal storage diseases.

HT-29 and Caco2 Cell Lines Are Suitable Models for Studying the Role of Arachidonic Acid-Metabolizing Enzymes in Intestinal Cell Differentiation

Introduction: Cytochrome (CYP) epoxygenases (CYP2C and CYP2J) and soluble epoxide hydrolase (sEH) participate in the metabolism of arachidonic acid and may also have a potential role in enterocyte differentiation. The first critical step in the study of intestinal cell differentiation is the determination of a suitable in vitro model, which must be as similar as possible to the conditions of a living organism. It is known that HT-29 and Caco2 cell lines derived from human colorectal carcinomas can differentiate into enterocyte-like cells in appropriate culture conditions. Material and Methods: We tested 4 different approaches of enterocyte-like differentiation and determined the most appropriate culture conditions for each model. Subsequently, the changes in the expression of CYP epoxygenases and sEH in undifferentiated and differentiated cells were measured by In-Cell ELISA. These results were compared with immunohistochemical profiles of expression of CYP epoxygenases and sEH in samples of human embryonic and fetal intestines as well as adult duodenum and colon. Results: Our results show that sodium butyrate (NaBt)-differentiated HT-29 cells and spontaneously differentiated Caco2 cells resemble CYP epoxygenases and sEH profiles, corresponding with different types of intestines. Conclusion: Our study revealed that the most suitable models for the study of the role of CYP epoxygenases and sEH expression in differentiation of intestinal epithelium are NaBt-differentiated HT-29 cells and spontaneously differentiated Caco2 cells.

A co-culture model of the bovine alveolus.

The epithelial lining of the lung is often the first point of interaction between the host and inhaled pathogens, allergens and medications. Epithelial cells are therefore the main focus of studies which aim to shed light on host-pathogen interactions, to dissect the mechanisms of local host immunity and study toxicology. If these studies are not to be conducted exclusively in vivo, it is imperative that in vitro models are developed with a high in vitro- in vivo correlation. We describe here a co-culture model of the bovine alveolus, designed to overcome some of the limitations encountered with mono-culture and live animal models. Our system includes bovine pulmonary arterial endothelial cells (BPAECs) seeded onto a permeable membrane in 24 well Transwell format. The BPAECs are overlaid with immortalised bovine alveolar type II epithelial cells and cultured at air-liquid interface for 14 days before use; in our case to study host-mycobacterial interactions. Characterisation of novel cell lines and the co-culture model have provided compelling evidence that immortalised bovine alveolar type II cells are an authentic substitute for primary alveolar type II cells and their co-culture with BPAECs provides a physiologically relevant in vitro model of the bovine alveolus. The co-culture model may be used to study dynamic intracellular and extracellular host-pathogen interactions, using proteomics, genomics, live cell imaging, in-cell ELISA and confocal microscopy. The model presented in this article enables other researchers to establish an in vitro model of the bovine alveolus that is easy to set up, malleable and serves as a comparable alternative to in vivo models, whilst allowing study of early host-pathogen interactions, currently not feasible in vivo. The model therefore achieves one of the 3Rs objectives in that it replaces the use of animals in research of bovine respiratory diseases.

Use of PTC124 for nonsense suppression therapy targeting BMP4 nonsense variants in vitro and the bmp4st72 allele in zebrafish.

Nonsense suppression therapy (NST) utilizes compounds such as PTC124 (Ataluren) to induce translational read-through of stop variants by promoting the insertion of near cognate, aminoacyl tRNAs that yield functional proteins. We used NST with PTC124 to determine if we could successfully rescue nonsense variants in human Bone Morphogenetic Protein 4 (BMP4) in vitro and in a zebrafish bmp4 allele with a stop variant in vivo. We transfected 293T/17 cells with wildtype or mutant human BMP4 cDNA containing p.Arg198* and p.Glu213* and exposed cells to 0–20 μM PTC124. Treatment with 20 μM PTC124 produced a small, non-significant increase in BMP4 when targeting the p.Arg198* allele, but not the p.Glu213* allele, as measured with an In-cell ELISA assay. We then examined the ability of PTC124 to rescue the ventral tail fin defects associated with homozygosity for the p.Glu209* allele of bmp4 (bmp4st72/st72) in Danio rerio. We in-crossed bmp4st72/+ heterozygous fish and found a statistically significant increase in homozygous larvae without tail fin and ventroposterior defects, consistent with phenotypic rescue, after treatment of dechorionated larvae with 0.5 μM PTC124. We conclude that treatment with PTC124 can rescue bmp4 nonsense variants, but that the degree of rescue may depend on sequence specific factors and the amount of RNA transcript available for rescue. Our work also confirms that zebrafish show promise as a useful animal model for assessing the efficacy of PTC124 treatment on nonsense variants.

A bilayer tissue culture model of the bovine alveolus

The epithelial lining of the lung is often the first point of interaction between the host and inhaled pathogens, allergens and medications. Epithelial cells are therefore the main focus of studies which aim to shed light on host-pathogen interactions, to dissect the mechanisms of local host immunity and study toxicology. If these studies are not to be conducted exclusively in vivo, it is imperative that in vitro models are developed with a high in vitro-in vivo correlation. We describe here a co-culture bilayer model of the bovine alveolus, designed to overcome some of the limitations encountered with mono-culture and live animal models. Our system includes bovine pulmonary arterial endothelial cells (BPAECs) seeded onto a permeable membrane in 24 well Transwell format. The BPAECs are overlaid with immortalised bovine alveolar type II epithelial cells and the bilayer cultured at air-liquid interface for 14 days before use; in our case to study host-mycobacterial interactions. Characterisation of novel cell lines and the bilayer model have provided compelling evidence that immortalised bovine alveolar type II cells are an authentic substitute for primary alveolar type II cells and their culture as a bilayer in conjunction with BPAECs provides a physiologically relevant in vitro model of the bovine alveolus. The bilayer model may be used to study dynamic intracellular and extracellular host-pathogen interactions, using proteomics, genomics, live cell imaging, in-cell ELISA and confocal microscopy. The model presented in this article enables other researchers to establish an in vitro model of the bovine alveolus that is easy to set up, malleable and serves as a comparable alternative to in vivo models, whilst allowing study of early host-pathogen interactions, currently not feasible in vivo. The model therefore achieves one of the 3Rs objectives in that it replaces the use of animals in research of bovine respiratory diseases.

Peptide inhibitors of Macrobrachium rosenbergii nodavirus.

Macrobrachium rosenbergii nodavirus (MrNv) causes white tail disease (WTD) in giant freshwater prawns, which leads to devastating economic losses in the aquaculture industry. Despite extensive research on MrNv, there is still no antiviral agent to treat WTD. Thus, the main aim of this study was to identify potential anti-MrNv molecules. A 12-mer phage-displayed peptide library was biopanned against the MrNv virus-like particle (VLP). After four rounds of biopanning, two dominant phages harbouring the amino acid sequences HTKQIPRHIYSA and VSRHQSWHPHDL were selected. An equilibrium binding assay in solution was performed to determine the relative dissociation constant (KDrel) of the interaction between the MrNv VLP and the selected fusion phages. Phage-HTKQIPRHIYSA has a KDrel value of 92.4±22.8 nM, and phage-VSRHQSWHPHDL has a KDrel value of 12.7±3.8 nM. An in-cell elisa was used to determine the inhibitory effect of the synthetic peptides towards the entry of MrNv VLP into Spodoptera frugiperda (Sf9) cells. Peptides HTKQIPRHIYSA and VSRHQSWHPHDL inhibited the entry of the MrNv VLP into Sf9 cells with IC50 values of 30.4±3.6 and 26.5±8.8 µM, respectively. Combination of both peptides showed a significantly higher inhibitory effect with an IC50 of 4.9±0.4 µM. An MTT assay revealed that the viability of MrNv-infected cells increased to about 97 % in the presence of both peptides. A real-time RT-PCR assay showed that simultaneous application of both peptides significantly reduced the number of MrNv per infected cell, from 97±9 to 11±4. These peptides are lead compounds which can be further developed into potent anti-MrNv agents.

Abstract 3828: Pritumumab mAb binds cell surface expressed vimentin on pancreatic cancer cells and inhibits tumor growth

Abstract Monoclonal antibodies (mAbs) as therapeutics for cancer have shown some success in the clinic. These biologics are useful if targeting a cancer specific epitope. Cancer patients can generate tumor-specific B lymphocytes which can be isolated to develop human mAbs against tumor-associated antigens. The best source of anti-tumor antibodies is from sentinel lymph nodes. Pritumumab (also referred to as CLNH-11, CLN-IgG, or ACA-11) is a classic example of a natural human anti-cancer antibody. It is a natural human IgG1 kappa antibody developed by the human hybridoma technology, using B lymphocytes isolated from a regional draining lymph node of a patient with cervical carcinoma. In the original patient from whom the B lymphocyte was isolated, the bioavailability of the target antigen induced a natural immune response resulting in the generation of the pritumumab IgG. The target antigen recognized by pritumumab is cell surface expressed vimentin, also referred to as ecto-domain vimentin (EDV). Vimentin is an intracellular cytoskeletal protein overexpressed during epithelial-to-mesenchymal transition (EMT), a process integral to cancer cell metastasis. Pancreatic cancer is a deadly disease with poor prognosis. No more than 6% of pancreatic cancer patients will survive beyond five years after diagnosis. There is a clear unmet need to help improve outcomes for patients. (A) Purpose of the study: to examine if pritumumab mAb can target pancreatic cancer cells and inhibit pancreatic tumor growth. (B) Experimental Procedures: Immunohistochemical analysis with horseradish peroxidase (HRP)-conjugated pritumumab on pancreatic cancer patient tissue sections along with immunofluorescence, flow cytometry, and In-Cell ELISA to demonstrate antibody binding. Antibody-dependent cell-mediated cytoxicity (ADCC) was performed to determine mechanism of action. In vivo subcutaneous xenograft mouse model treated with pritumumab to assess efficacy of the antibody for tumor inhibition. (C) Results: In this study we demonstrate pritumumab binds to the cell surface of patient pancreatic cancer tissue and binds the surface of established pancreatic cancer cells in culture. We show antibody binding to these cells induces antibody-dependent cell-mediated cytoxicity (ADCC). Furthermore, pritumumab effectively inhibits pancreatic tumor growth in a xenograft mouse model. (D) Statement of Conclusions: overall, these data provide pre-clinical validation of pritumumab mAb as a therapeutic for pancreatic cancer. Citation Format: Ivan Babic, Natsuko Nomura, Eric Glassy, Elmar Nurmemmedov, Venkata Yenugonda, Mark Glassy, Santosh Kesari. Pritumumab mAb binds cell surface expressed vimentin on pancreatic cancer cells and inhibits tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3828.

Controlled Release of Insulin Granules from PLGA Microparticles for Glucose Modulation in Diabetes

Diabetes is a chronic disease deriving from the insufficient production or utilization of insulin. So far, the treatment of diabetes relies on the daily subcutaneous injection of insulin. However, this approach suffers of poor patient compliance and fluctuations in blood glucose levels that increases the risk of hypoglycemia, cardiovascular disease, non-healing wounds and other comorbidities. In this work, a delivery system is proposed for the controlled release of insulin and regulation of glycaemia. Nanoscale granules of insulin were synthesized and encapsulated within the porous matrix of PLGA microparticles (i-μPLs). The granules were obtained with an accurate crystallization process returning spherical nanoparticles with a diameter of 209.9±15.8 nm. μPLs were prepared via a top-down approach resulting in 20 x 5 μm square microparticles, made out of a biodegradable polymer - poly(lactic-co-glycolic acid) (PLGA) - which is already used in clinical applications. In vitro studies were conducted on different cells, including macrophages, fibroblasts and endothelial cells, and confirmed the safety and biological activity of i-μPLs. After insulin stimulation, phosphorylated AKT at Ser473 was assessed in cell lysate using a commercial kit (In-Cell ELISA): i-μPL activity was comparable to that of free, commercial insulin. As preliminary in vivo studies, i-μPLs (0.25 U) or empty μPLs (control) were implanted in the spleen of mice. Twenty-four hours post implantation, fasted mice underwent an oral glucose tolerance test (1 g glucose/Kg body weight). In i-μPL treated mice, the mean blood glucose level was lower as compared to controls at time zero (before gavage) and at 15, 30, 60 and 120 min. Consequently, the glycaemia AUC was reduced in i-μPLs-treated vs. control mice (17828±1527 vs. 20543±413, p=0.06). This preliminary data would suggest that i-μPLs could be effectively used to modulate glucose content in diabetes. Disclosure R. Primavera: None. M. Magnone: None. D. Di Mascolo: None. E. Zocchi: None. A. De Pascale: None. P. Decuzzi: None.

A novel in-cell ELISA method for screening of compounds inhibiting TRKA phosphorylation, using KM12 cell line harboring TRKA rearrangement

Tropomyosin-related kinase A (TRKA) fusion was originally detected in colorectal carcinoma that had resulted in expression of the oncogenic chimeric protein TPM3-TRKA. Lately, many more rearrangements in TRK family of kinases generating oncogenic fusion proteins have been identified. These genetic rearrangements usually result in fusion of cytoplasmic kinase domain of TRK to another gene of interest resulting in constitutive kinase activity. Estimation of TRK inhibitor potency in a cellular context is required for drug discovery programs and is measured by receptor phosphorylation levels upon compound administration. However, since a large chunk of the TRK protein is lost in this rearrangement, it's difficult to set up sandwich ELISA for detection of receptor phosphorylation in any cell assay harboring these fusion proteins. In order to address this issue, we developed a novel and robust in-cell ELISA method which quantifies the phosphorylation of TRK kinase (Tyr 674/675) within the KM12 cells. This cell based method is more versatile & economical than conventional ELISA using engineered overexpressing cell line and/or western blot methods. Performance reliability & robustness for the validated assay were determined by %CV and Z factor in assays with reference molecule larotrectinib. This in-cell ELISA method can be used with any TRKA rearranged oncogenic fusion cell type and can be extended to other TRK isoforms as well. We have used this assay to screen novel molecules in KM12 cells and to study pharmacodynamic properties of compounds in TRKA signaling.

Activation of GPR81 lactate receptors stimulates mitochondrial biogenesis in cerebral microvessel endothelial cells

Introduction. Cerebral endothelial cells express monocarboxylate transporters MCT1 for blood–brain barrier (BBB) transfer of lactate, which are regulated by CD147 activity, as well as lactate receptors GPR81 (HCAR1). Metabolism and intercellular transport of lactate is the crucial mechanism for regulating the function of BBB cells. Objective. To study the effect of activity of GPR81 receptors in cerebral endothelial cells on expression of MCT1, CD147 and the mitochondrial dynamics, which will make it possible to explain the effect of local production of lactate by perivascular astrocytes on angiogenesis in the cerebral tissue. Materials and methods. The culture of cerebral endothelial cells isolated from the brain of 15–17-day-old Wistar rat embryos was used in this study. Mitochondrial biogenesis of cerebral endothelial cells was studied using the standard MitoBiogenesis In-Cell ELISA Kit protocol (Abcam). Chemical hypoxia was induced by incubation in the presence of 50 μM iodoacetate for 30 min. 3Cl-5OH-BA (Calbiochem) at concentrations of 5, 50, and 500 μM was used as an agonist of GPR81 lactate receptors during 24 h. The number of cells expressing GPR81, CD147, and MCT1 molecules was evaluated using indirect double-antibody ELISA. Results. It was found for the first time that prolonged dose-dependent stimulation of GPR81 receptors with 3Cl-5OH-BA intensifies mitochondrial biogenesis (up to 1.5-fold, р<0.05). Meanwhile, a statistically significant (p<0.05) inhibition of expression of monocarboxylate transporters MCT1 (from 81±1.6% to 40.7±4.4%) and the conjugated CD147 protein (from 57.4±3.3% to 48.3±2.9%) in cerebral endothelial cells in the study group compared to the control group. Conclusions. The findings broaden the range of potential applications of GPR81 agonists for modulating intercellular interactions in a neurovascular unit and controlling the functional activity of cerebral microvessel endothelial cells.

Phospho-SMC1 in-Cell ELISA based Detection of Ataxia Telangiectasia

BackgroundAtaxia telangiectasia (A-T) is a common genetically inherited cause of early childhood-onset ataxia. The infrequency of this disease, vast phenotype variation, disorders with features similar to those of A-T, and lack of definite laboratory test, make diagnosis difficult. In addition, there is no rapid reliable laboratory method for identifying A-T heterozygotes, who susceptible to ionizing radiation (IR), atherosclerosis, diabetes, and cancers. We used SMC1pSer966 (pSMC1) in-cell colorimetric ELISA to diagnosis and screen in A-T families.Materials and Methods: With informed consent, 2cc peripheral blood was collected from the 15 A-T patients, their parents, and 24 healthy controls with no family history of malignancy, diabetes, and atherosclerosis. Extracted peripheral blood mononuclear cells (PBMCs) were cultured in poly-L-Lysine treated 96-well plate with density of 70,000 cells per well. SMC1 phosphorylation was evaluated with cell-based ELISA kit 1 hour after 5 Gy IR and the pSMC1data normalized with Glyceraldehyde-3-phosphate dehydrogenase (GAPDH).Results: SMC1 phosphorylation was significantly low in A-T`s PBMC (mean + standard deviation [SD]: 0.075 + 0.034) in comparison to carriers (mean + SD: 0.190 + 0.060) and healthy controls (mean + SD: 0.312 +0.081), but unluckily could only discriminate A-T patients (Area Under the Curve -receiver operating characteristic [AUC-ROC]: 1.00, 1.00-1.00). This method in spite of rapidness and simplicity showed poor imprecision (22.49% coefficient of variation [CV] for intraday imprecision).Conclusion: It seems pSMC1 assessment by in-cell ELISA can be used for detection of A-T patients, but it may not sensitive enough for identification of carriers. This ELISA test is very simple, rapid, and requires less than 2cc blood. Thus it may be proposed for the early differential diagnosis of A-T as an alternative method.

A systematic analysis reveals heterogeneous changes in the endocytic activities of cancer cells.

Metastasis is a multistep process requiring cancer cell signaling, invasion, migration, survival, and proliferation. These processes require dynamic modulation of cell surface proteins by endocytosis. Given this functional connection, it has been suggested that endocytosis is dysregulated in cancer. To test this, we developed In-Cell ELISA assays to measure three different endocytic pathways: clathrin-mediated endocytosis, caveolae-mediated endocytosis, and clathrin-independent endocytosis and compared these activities using two different syngeneic models for normal and oncogene-transformed human lung epithelial cells. We found that all endocytic activities were reduced in the transformed versus normal counterparts. However, when we screened 29 independently isolated non-small cell lung cancer (NSCLC) cell lines to determine whether these changes were systematic, we observed significant heterogeneity. Nonetheless, using hierarchical clustering based on their combined endocytic properties, we identified two phenotypically distinct clusters of NSCLCs. One co-clustered with mutations in KRAS, a mesenchymal phenotype, increased invasion through collagen and decreased growth in soft agar, whereas the second was enriched in cells with an epithelial phenotype. Interestingly, the two clusters also differed significantly in clathrin-independent internalization and surface expression of CD44 and CD59. Taken together, our results suggest that endocytotic alterations in cancer cells that affect cell surface expression of critical molecules have a significant influence on cancer-relevant phenotypes, with potential implications for interventions to control cancer by modulating endocytic dynamics.

Abstract 2511: Therapeutic dendritic cell targeting MIP3α-gp100 DNA vaccination with immunomodulatory αIL-10 and αPD-1 antibodies significantly enhances survival in a mouse melanoma model system

Abstract The current study demonstrates that the combination of a DNA vaccine encoding the chemokine MIP-3α fused to melanoma antigen gp100 with immunomodulatory antibodies prolongs survival in a melanoma mouse model. The current studies utilize the B16F10 mouse spontaneous melanoma syngeneic transplantable mouse model system. Previous published work with this vaccine platform has indicated the MIP-3α component targets nascent peptides to immature dendritic cells leading to processing by class I and class II MHC pathways. It has further been shown in this tumor system that prophylactic vaccination with the DNA construct alone delayed tumor growth. Other studies have provided evidence that αPD-1 or αIL-10 neutralizing antibodies enhance immunological melanoma therapies by modulating the tolerogenic tumor microenvironment. First, this study expanded previous results by finding a significant decrease in tumor burden and increase in survival by prophylactic intramuscular electroporation [IM EP] of the DNA vaccine construct. Adding three therapeutic doses of αIL-10 given subcutaneously [subQ] at the tumor site to the treatment further increased mouse survival. The development of cell-mediated and humoral immune responses were documented for the first time in this system by Elispot and in-cell ELISA assays. Secondly, a novel therapeutic vaccination protocol was developed by initiating vaccination and antibody therapies after tumor challenge. With this therapeutic protocol, we demonstrated for the first time that vaccine significantly delays tumor burden and increases median survival from 17 to 21 days (p = 0.006). Addition of αIL-10 (subQ) and αPD-1 (intra-peritoneally) antibodies led to further highly significant decreases in tumor burden and increased median survival from 17 to 25 days (p = 0.0005). Efficient targeting of antigen to immature dendritic cells with a chemokine fusion vaccine offers a potential alternative approach to the ex vivo dendritic cell antigen loading protocols currently undergoing clinical investigation. Combining this approach with agents able to modulate the tolerogenic tumor microenvironment offers promise as a novel melanoma therapy. Citation Format: James Gordy, Richard B. Markham. Therapeutic dendritic cell targeting MIP3α-gp100 DNA vaccination with immunomodulatory αIL-10 and αPD-1 antibodies significantly enhances survival in a mouse melanoma model system. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2511. doi:10.1158/1538-7445.AM2015-2511

Identification of factors regulating MET receptor endocytosis by high-throughput siRNA screening.

The tyrosine kinase MET, a receptor for hepatocyte growth factor, is a key regulator for normal development and organ renewal via stem cell maintenance. Dysregulated MET signaling contributes to tumor progression and metastasis and is considered a potent therapeutic target for a growing number of malignancies. Toward that goal it is critical to develop high-throughput assays to identify candidate regulators for the termination of MET signaling. We describe here a rapid and efficient method for identifying cellular factors required for MET ubiquitination, which utilizes high-throughput RNA interference screening (HT-siRNA) with a receptor internalization assay and an In-Cell ELISA in a 96-well format. The assay is amenable to a large array of cell surface proteins as well as genome-wide siRNA libraries, with high signal-to-background ratio and low well-to-well variability.

The alpha 7 nicotinic receptor agonist PHA-543613 hydrochloride inhibits Porphyromonas gingivalis-induced expression of interleukin-8 by oral keratinocytes

ObjectiveThe alpha 7 nicotinic receptor (α7nAChR) is expressed by oral keratinocytes. α7nAChR activation mediates anti-inflammatory responses. The objective of this study was to determine if α7nAChR activation inhibited pathogen-induced interleukin-8 (IL-8) expression by oral keratinocytes.Materials and methodsPeriodontal tissue expression of α7nAChR was determined by real-time PCR. OKF6/TERT-2 oral keratinocytes were exposed to Porphyromonas gingivalis in the presence and absence of a α7nAChR agonist (PHA-543613 hydrochloride) alone or after pre-exposure to a specific α7nAChR antagonist (α-bungarotoxin). Interleukin-8 (IL-8) expression was measured by ELISA and real-time PCR. Phosphorylation of the NF-κB p65 subunit was determined using an NF-κB p65 profiler assay and STAT-3 activation by STAT-3 in-cell ELISA. The release of ACh from oral keratinocytes in response to P. gingivalis lipopolysaccharide was determined using a GeneBLAzer M3 CHO-K1-bla cell reporter assay.ResultsExpression of α7nAChR mRNA was elevated in diseased periodontal tissue. PHA-543613 hydrochloride inhibited P. gingivalis-induced expression of IL-8 at the transcriptional level. This effect was abolished when cells were pre-exposed to a specific α7nAChR antagonist, α-bungarotoxin. PHA-543613 hydrochloride downregulated NF-κB signalling through reduced phosphorylation of the NF-κB p65-subunit. In addition, PHA-543613 hydrochloride promoted STAT-3 signalling by maintenance of phosphorylation. Furthermore, oral keratinocytes upregulated ACh release in response to P. gingivalis lipopolysaccharide.ConclusionThese data suggest that α7nAChR plays a role in regulating the innate immune responses of oral keratinocytes.

Dopamine receptor 2 activation inhibits ovarian vascular endothelial growth factor secretion in vitro: implications for treatment of ovarian hyperstimulation syndrome with dopamine receptor 2 agonists

To ascertain whether vascular endothelial growth factor (VEGF) secretion by luteinized granulosa cells (GCs) is modulated by the dopaminergic system in a dose-dependent fashion and how this is related to the differential efficacy of dopamine receptor 2 (D2)-agonists (D2-ag) in preventing ovarian hyperstimulation syndrome (OHSS). The relationship between the dopaminergic system and VEGF secretion in luteinized GCs was evaluated. Archived human ovaries were immunostained to characterize D2 expression. University affiliated infertility center. Premenopausal women and egg donors. Luteinized GCs were cultured with the D2-ag cabergoline. Human ovarian sections were immunostained for D2. The VEGF was measured by ELISA and D2 expression was evaluated by In-Cell ELISA. The D2 expression throughout the luteal phase was characterized by immunohistochemistry. The VEGF secretion was decreased by the D2-ag in a dose-dependent fashion. The efficiency of this process was correlated with the amount of D2 expressed by luteinized GCs. A decrease in D2 expression in ovarian sections was observed during the late luteal phase. The efficacy of D2-ags in preventing OHSS might rely on their capacity to inhibit VEGF secretion by luteinized GCs. Because this capacity is dose-dependent, increasing the intraovarian concentration of D2-ags should be explored as a means of increasing the efficacy of these drugs in preventing OHSS.

Premature Termination Codon Read-Through in the ABCC6 Gene: Potential Treatment for Pseudoxanthoma Elasticum

Pseudoxanthoma elasticum (PXE) is an autosomal recessive disorder manifesting with ectopic connective tissue mineralization, caused by mutations in the ABCC6 gene, ~35% of all mutations being premature termination mutations. In this study, we investigated the therapeutic potential of the nonsense codon read-through-inducing drug, PTC124, in treating PXE. The ability of this drug to facilitate read-through of nonsense mutations was examined in HEK293 cells transfected with human ABCC6 expression constructs harboring seven different PXE associated nonsense mutations, and evaluated by immunofluorescence and In-Cell ELISA. Our data demonstrated that PTC124 did not exhibit cell toxicity in concentrations up to 40 µg/ml, and the facilitated read-through was not only dose dependent but also sequence context dependent. Considering the redundancy of the genetic code, it was postulated that in case of the most common recurrent nonsense mutation, p.R1141X, the read-through may result in substitution of the arginine 1141 by either glycine, tryptophan or cysteine. Their potential pathogenicity was tested in a recently developed zebrafish mRNA rescue assay, and demonstrated that all three mRNA transcripts were able to rescue abcc6a morpholino-induced phenotype of zebrafish. Thus, our results suggest that read-through of nonsense mutations in ABCC6 by PTC124 may have potential for pharmacologic treatment of PXE.

Substance P‐induced increase in Brain Derived Neurotrophic Factor expression and secretion by intestinal smooth muscle is mediated by a rise in intracellular Ca2+

BackgroundWe have previously shown that intestinal longitudinal smooth muscle cells express and secrete brain derived neurotrophic factor (BDNF) and addition of substance P (SP) to cultured smooth muscle cells induces further expression and secretion of BDNF above basal levels. It is not known, however, what cellular signaling pathways mediate SP‐induced expression and secretion of BDNF.AimTo identify the signaling pathways involved in BDNF synthesis and secretion by smooth muscle in response to SP.MethodCultured intestinal smooth muscle cells were treated with 10–100 nM SP for 24 hours in the presence and absence of 1,2 bis (o‐aminophenoxy)ethane‐N,N,N’,N’‐tetraacetic acid (BAPTA) to chelate intracellular Ca2+. BDNF protein expression and secretion were measured by in‐cell western and ELISA, and BDNF mRNA expression was measured by qRT‐PCR.ResultsThe expression of BDNF protein was increased by 10–121% and BDNF secretion was increased by 50% by SP. Consistent with this, BDNF mRNA expression was increased by 161% by SP. Both BDNF protein and mRNA responses to SP were blocked by BAPTA.ConclusionSP stimulation of BDNF expression and secretion requires increased in intracellular Ca2+. The source of the intracellular Ca2+ increased remains to be determined. Supported by grants DK15564 and DK34153 from the National Institutes of Health.

Wound healing properties of jojoba liquid wax: An in vitro study

The wound healing properties of jojoba (Simmondsia chinensis) liquid wax (JLW) were studied in vitro on HaCaT keratinocytes and human dermal fibroblasts, which are involved in wounded skin repair. JLW cytotoxicity was evaluated by the crystal violet staining and the neutral red uptake endpoint. Induction of wound healing by JLW was assessed by scratch wound assay on cell monolayers. The involvement of signaling pathways was evaluated by the use of the Ca(2+) chelator BAPTA and of kinase inhibitors, and by Western blot analysis of cell lysates using anti-phospho antibodies. Collagen and gelatinase secretion by cells were assayed by in-cell ELISA and zymography analysis, respectively. Cytotoxicity assays showed that the toxic effects of JLW to these cells are extremely low. Scratch wound experiments showed that JLW notably accelerates the wound closure of both keratinocytes and fibroblasts. The use of inhibitors and Western blot revealed that the mechanism of action of JLW is strictly Ca(2+) dependent and requires the involvement of the PI3K-Akt-mTOR pathway and of the p38 and ERK1/2 MAPKs. In addition, JLW was found to stimulate collagen I synthesis in fibroblasts, while no effect was detected on the secretion of MMP-2 and MMP-9 gelatinases by HaCaT or fibroblasts. Taken together, data provide a pharmacological characterization of JLW properties on skin cells and suggest that it could be used in the treatment of wounds in clinical settings.

Platelet lysate modulates MMP-2 and MMP-9 expression, matrix deposition and cell-to-matrix adhesion in keratinocytes and fibroblasts

Cell-matrix interactions are an essential element of wound healing, while platelet derivatives are used in clinical settings for the treatment of chronic wounds. We used a platelet lysate (PL), which had been previously shown to accelerate in vitro the wounding of HaCaT keratinocytes and fibroblasts (J Cell Mol Med, 13, 2009, 2030; Br J Dermatol, 159, 2008, 537), to study the modulation of MMP-2 and MMP-9 collagenase expression, collagen type I and III production and syndecan-4 expression and rearrangement in these cells. Zymography and Western blot analyses showed that exposure to 20% (v/v) PL for 24 h induced an apparently ERK1/2- and p38-dependent, NF-kappaB-independent, translational upregulation of MMP-9 in HaCaT, while HaCaT MMP-2 and fibroblast collagenases were almost unaffected. The use of in-cell ELISA showed that PL induced an increase in the collagen III production of fibroblasts. In-cell ELISA and immunofluorescence microscopy revealed an increase in the expression of syndecan-4 and its rearrangement to form focal adhesions in both cell types after PL exposure. Taken together, data indicate that PL promotes keratinocyte epithelialization and regulates fibroblast matrix deposition, thus providing a molecular basis for the ability of this platelet derivative to heal severe and problematic wounds without leading to heavy scarring and keloid formation.