Investigation of the role of KATP channels in the cytotoxic effect of cypermethrin on rat-derived aortic smooth muscle cells.

We investigate role of ATP sensitive potassium (KATP) channel in cytotoxic effect of cypermethrin on rat aortic smooth muscle cells. Cytotoxicity analysis was performed at 0, 0.1, 0.5, 10, 50, and 100 µM concentrations of cypermethrin and the cell index (CI) was calculated. KATP currents were recorded using patch clamp technique for 50 and 100 µM concentrations and channel conductivity was determined by obtaining current-voltage characteristics. No cytotoxic effect was observed in the first 72 hours. At the 96th hour, only at 100 µM concentration, the CI value decreased significantly compared to control group and at 120 and 144th hours, it was observed that the CI value decreased significantly at all concentrations. Currents and conductivities were significantly decreased at 50 and 100 µM concentrations. Results gave clues that cypermethrin causes a cytotoxic effect on vascular smooth muscles and that KATP channels may have a role in the emergence of this effect.

Modulation of Bronchial Epithelial Barrier Integrity by Low Molecular Weight Components from Birch Pollen.

Pollen, in addition to allergens, comprise low molecular weight components (LMC) smaller than 3 kDa. Emerging evidence indicates the relevance of LMC in allergic immune responses. However, the interaction of birch pollen (BP)-derived LMC and epithelial cells has not been extensively studied. We investigated epithelial barrier modifications induced by exposure to BP LMC, using the human bronchial epithelial cell line 16HBE14o-. Epithelial cell monolayers were apically exposed to the major BP allergen Bet v 1, aqueous BP extract or BP-derived LMC. Barrier integrity after the treatments was monitored by measuring transepithelial electrical resistance at regular intervals and by using the xCELLigence Real-Time Cell Analysis system. The polarized release of cytokines 24 h following treatment was measured using a multiplex immunoassay. Epithelial barrier integrity was significantly enhanced upon exposure to BP LMC. Moreover, BP LMC induced the repair of papain-mediated epithelial barrier damage. The apical release of CCL5 and TNF-α was significantly reduced after exposure to BP LMC, while the basolateral release of IL-6 significantly increased. In conclusion, the results of our study demonstrate that BP-derived LMC modify the physical and immunological properties of bronchial epithelial cells and thus regulate airway epithelial barrier responses.

Growth rates of human induced pluripotent stem cells and neural stem cells from attention-deficit hyperactivity disorder patients: a preliminary study.

Attention-deficit hyperactivity disorder (ADHD) is a neurodevelopmental polygenic disorder that affects more than 5% of children and adolescents around the world. Genetic and environmental factors play important roles in ADHD etiology, which leads to a wide range of clinical outcomes and biological phenotypes across the population. Brain maturation delays of a 4-year lag are commonly found in patients, when compared to controls of the same age. Possible differences in cellular growth rates might reflect the clinical observations in ADHD patients. However, the cellular mechanisms are still not elucidated. To test this hypothesis, we analysed the proliferation of induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs) derived from male children and adolescents diagnosed with ADHD and with genetic predisposition to it (assessed using polygenic risk scores), as well as their respective matched controls. In the current pilot study, it was noticeable that NSCs from the ADHD group proliferate less than controls, while no differences were seen at the iPSC developmental stage. Our results from two distinct proliferation methods indicate that the functional and structural delays found in patients might be associated with these in vitro phenotypic differences, but start at a distinct neurodevelopmental stage. These findings are the first ones in the field of disease modelling of ADHD and might be crucial to better understand the pathophysiology of this disorder.

Establishment of an in vitro model from the vulnerable fish species Coregonus maraena (maraena whitefish): Optimization of growth conditions and characterization of the cell line.

In this study, a cell line of the fish species Coregonus maraena was produced for the first time. C. maraena is an endangered species, and studies indicate that this fish species will be affected by further population declines due to climate change. This cell line, designated CMAfin1, has been maintained in Leibovitz L-15 supplemented with 10% fetal bovine serum over 3 years. Both subculturing and storage (short-term storage at -80°C and long-term storage in liquid nitrogen) was successful. Cell morphology and growth rate were consistent from passage 10 onwards. Immunocytochemical examination of cellular proteins and matrix components confirmed the mechanical stability of the cells. Actin, fibronectin, vinculin, vimentin, and tubulin are present in the cells and form a network. In addition, the transport of molecules is ensured by the necessary proteins. Gene expression analysis showed a shift in the expressions of stem cell markers between younger and higher passages. While SOX2 and IGF1 were more highly expressed in the seventh passage, SOX9 and IGF2 expressions were significantly increased in higher passages. Therefore, the stable cell culture CMAfin1 can be used for applied analysis to further understand the cell physiology of C. maranea.

Biochemical characterization and detection of antitumor activity of l-asparaginase from thermophilic Geobacillus kaustophilus DSM 7263T.

L-asparaginases, which are oncolytic enzymes, have been used in clinical applications for many years. These enzymes are also important in food processing industry due to their potential in acrylamide-mitigation. In this study, the gene for l-asparaginase (GkASN) from a thermophilic bacterium, Geobacillus kaustophilus, was cloned and expressed in E. coli Rosetta™2 (DE3) cells utilizing the pET-22b(+) vector. The 6xHis-tag attached enzyme was purified and analyzed both biochemically and structurally. The molecular mass of GkASN was determined as ∼36 kDa by SDS-PAGE, Western Blotting, and MALDI-TOF MS analyses. Optimum temperature and pH for the enzyme was determined as 55 °C and 8.5, respectively. The enzyme retained 89% of its thermal stability at 37 °C and 75% at 55 °C after 6 h of incubation. The enzyme activity was inhibited in the presence of Cu2+, Fe3+, Zn2+, and EDTA, while the activity was enhanced in the presence of Mn2+, Mg2+, and thiol group protective agents such as 2-mercaptoethanol and DTT. The structural modeling analysis demonstrated that the catalytic residues of the enzyme were partially similar to other asparaginases. The therapeutic potential of GkASN was tested on hepatocellular carcinoma cells, a solid cancer type with high mortality rate and rapidly increasing incidence in recent years. We showed that the GkASN-induced asparagine deficiency effectively reduced the metastatic synergy in HCC SNU387 cells on a xCELLigence system with differentiated epithelial Hep3B and poorly differentiated metastatic mesenchymal HCC SNU387 cells.

MPP+-Induced Changes in Cellular Impedance as a Measure for Organic Cation Transporter (SLC22A1-3) Activity and Inhibition.

The organic cation transporters OCT1-3 (SLC22A1-3) facilitate the transport of cationic endo- and xenobiotics and are important mediators of drug distribution and elimination. Their polyspecific nature makes OCTs highly susceptible to drug-drug interactions (DDIs). Currently, screening of OCT inhibitors depends on uptake assays that require labeled substrates to detect transport activity. However, these uptake assays have several limitations. Hence, there is a need to develop novel assays to study OCT activity in a physiological relevant environment without the need to label the substrate. Here, a label-free impedance-based transport assay is established that detects OCT-mediated transport activity and inhibition utilizing the neurotoxin MPP+. Uptake of MPP+ by OCTs induced concentration-dependent changes in cellular impedance that were inhibited by decynium-22, corticosterone, and Tyrosine Kinase inhibitors. OCT-mediated MPP+ transport activity and inhibition were quantified on both OCT1-3 overexpressing cells and HeLa cells endogenously expressing OCT3. Moreover, the method presented here is a valuable tool to identify novel inhibitors and potential DDI partners for MPP+ transporting solute carrier proteins (SLCs) in general.

In Vitro Evaluation of the Cytotoxic Effect of Streptococcus pyogenes Strains, Protegrin PG-1, Cathelicidin LL-37, Nerve Growth Factor and Chemotherapy on the C6 Glioma Cell Line.

Brain cancer treatment, where glioblastoma represents up to 50% of all CNS malignancies, is one of the most challenging calls for neurooncologists. The major driver of this study was a search for new approaches for the treatment of glioblastoma. We tested live S. pyogenes, cathelicidin family peptides and NGF, assessing the oncolytic activity of these compounds as monotherapy or in combination with chemotherapeutics. For cytotoxicity evaluation, we used the MTT assay, trypan blue assay and the xCELLigence system. To evaluate the safety of the studied therapeutic approaches, we performed experiments on normal human fibroblasts. Streptococci and peptides demonstrated high antitumor efficiency against glioma C6 cells in all assays applied, surpassing the effect of chemotherapeutics (doxorubicin, carboplatin, cisplatin, etoposide). A real-time cytotoxicity analysis showed that the cell viability index dropped to 21% 2-5 h after S. pyogenes strain exposure. It was shown that LL-37, PG-1 and NGF also exhibited strong antitumor effects on C6 glioma cells when applied at less than 10-4 M. Synergistic effects for combinations of PG-1 with carboplatin and LL-37 with etoposide were shown. Combinations of S. pyogenes strain #7 with NGF or LL-37 demonstrated a cytotoxic effect (56.7% and 57.3%, accordingly) on C6 glioma cells after 3 h of exposure.

Effect of Finishing-Polishing Procedures on Cytotoxicity of Resin-Based Restorative Materials via Real-Time Cell Analysis.

OBJECTIVE: The aim of this study was to evaluate the effect of finishing and polishing procedures of compomer and bulk-fill composite resins on cytotoxicity against human gingival fibroblasts by xCELLigence analysis. STUDY DESIGN: Filtek™ Bulk Fill composite and Dyract XP compomer were used. After curing, the specimens were randomly divided into two groups and finishing-polishing procedures were applied to one group; no finishing-polishing procedures were applied to the other group. For the first time in this study, pure gold samples were prepared with the same weight and base area as the test specimens and the wells containing the pure gold samples were determined as the control group. xCELLigence system was used to assess the response of the human gingival fibroblasts after exposure to test specimens. Measurements were recorded for 72 hours after adding specimens. RESULTS: Finishing and polishing procedures caused a significant increase in cell viability of Dyract XP compomer samples at all time periods; the percentage of cell viability reached above 70% after finishing and polishing procedures. However, significant effects were not observed in Filtek™ Bulk Fill composite samples at any time period. CONCLUSION: Finishing and polishing procedures play an essential role in increasing the biocompatibility of Dyract XP compomer. It is recommended to apply finishing and polishing procedures even though a smooth surface may be obtained in restorations with matrix strips.

A novel cell-based assay for dynamically detecting neutrophil extracellular traps-induced lung epithelial injuries.

Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS) are common lung disorders characterized by alveolar-capillary barrier disruption and dyspnea, which can cause substantial morbidity and mortality. Currently, a cluster of acute respiratory illnesses, known as novel coronavirus (2019-nCoV)-infected pneumonia (NCIP), which allegedly originally occurred in Wuhan, China, has increased rapidly worldwide. The critically ill patients with ARDS have high mortality in subjects with comorbidities. Previously, the excessive recruitment and activation of neutrophils (polymorphonuclear leukocytes [PMNs]), accompanied by neutrophil extracellular traps (NETs) formation were reported being implicated in the pathogenesis of ALI/ARDS. However, the direct visualization of lung epithelial injuries caused by NETs, and the qualitative and quantitative evaluations of this damage are still lacking. Additionally, those already reported methods are limited for their neglect of the pathological role exerted by NETs and focusing only on the morphological features of NETosis. Therefore, we established a cell-based assay for detecting NETs during lung epithelial cells-neutrophils co-culture using the xCELLigence system, a recognized real-time, dynamic, label-free, sensitive, and high-throughput apparatus. Our results demonstrated that lung epithelial injuries, reflected by declines in cell index (CI) values, could be induced by lipopolysaccharide (LPS)-activated PMNs, or NETs in a time and dose-dependent manner. NETs generation was verified to be the major contributor to the cytotoxicity of activated PMNs; protein components of NETs were the prevailing cytotoxic mediators. Moreover, this cell-based assay identified that PMNs from severe pneumonia patients had a high NETs formative potential. Additionally, acetylsalicylic acid (ASA) and acetaminophen (APAP) were discovered alleviating NETs formation. Thus, this study not only presents a new methodology for detecting the pathophysiologic role of NETs but also lays down a foundation for exploring therapeutic interventions in an effort to cure ALI/ARDS in the clinical setting of severe pneumonia, including the emerging of NCIP.

Long-term exposure to triclosan increases migration and invasion of human breast epithelial cells in vitro.

Extensive use of triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether) as an antimicrobial agent in household and personal care products has resulted in global exposure of the human population. Its presence in human tissues, including milk, and its oestrogen-disrupting properties raise concerns for an involvement in breast cancer. Because metastatic tumour spread is the main cause of breast cancer mortality, we have investigated the effects of triclosan on cell migration and invasion using three human breast epithelial cell lines and using concentrations comparable with those in human tissues. Long-term exposure to 10-7 M of triclosan resulted in increased migration and invasion as measured by xCELLigence technology for all three cell lines, for the immortalized but nontransformed MCF-10F breast epithelial cells (after 28 weeks), the oestrogen-responsive MCF-7 breast cancer cells (after 17 weeks) and the oestrogen-unresponsive MDA-MB-231 breast cancer cells (after 20 weeks). The effects were therefore not limited to cancerous cells or to oestrogen-responsive cells. This was paralleled in the MCF-10F and MCF-7 (but not MDA-MB-231) cells by a reduction in levels of E-cadherin mRNA as measured by reverse transcription-polymerase chain reaction (RT-PCR) and of E-cadherin protein as measured by western immunoblotting, suggesting a mechanism involving epithelial-to-mesenchymal transition. This adds triclosan to the increasing list of ingredients of personal care products that can not only enter human breast tissue and increase cell proliferation but also influence cell motility. If mixtures of components in household and personal care products contribute to increasing cell migration and invasion, then reduction in exposure could offer a strategy for reducing breast cancer spread.

Lactobacilli spp.: real-time evaluation of biofilm growth.

BACKGROUND: Biofilm is a fundamental bacterial survival mode which proceeds through three main generalized phases: adhesion, maturation, and dispersion. Lactobacilli spp. (LB) are critical components of gut and reproductive health and are widely used probiotics. Evaluation of time-dependent mechanisms of biofilm formation is important for understanding of host-microbial interaction and development of therapeutic interventions. Time-dependent LB biofilm growth was studied in two systems: large biofilm output in continuous flow system (microfermenter (M), Institute Pasteur, France) and electrical impedance-based real time label-free cell analyzer (C) (xCELLigence, ACEA Bioscience Inc., San Diego, CA). L. plantarum biofilm growth in M system was video-recorded, followed by analyses using IMARIS software (Bitplane, Oxford Instrument Company, Concord, MA, USA). Additionally, whole genome expression and analyses of attached (A) and dispersed (D) biofilm phases at 24 and 48 h were performed. RESULTS: The dynamic of biofilm growth of L. plantarum was similar in both systems except for D phases. Comparison of the transcriptome of A and D phases revealed, that 121 transcripts differ between two phases at 24 h. and 35 transcripts - at 48 h. of M growth. The main pathways, down-regulated in A compared to D phases after 24 h. were transcriptional regulation, purine nucleotide biosynthesis, and L-aspartate biosynthesis, and the upregulated pathways were fatty acid and phospholipid metabolism as well as ABC transporters and purine nucleotide biosynthesis. Four LB species differed in the duration and amplitude of attachment phases, while growth phases were similar. CONCLUSION: LB spp. biofilm growth and propagation area dynamic, time-dependent processes with species-specific and time specific characteristics. The dynamic of LB biofilm growth agrees with published pathophysiological data and points out that real time evaluation is an important tool in understanding growth of microbial communities.

Enhanced activation of human NK cells by drug-exposed hepatocytes.

Drug-induced liver injury (DILI) represents one of the major causes why drugs have to be withdrawn from the market. In this study, we describe a new interaction between drug-exposed hepatocytes and natural killer (NK) cells. In a previous genome-wide expression analysis of primary human hepatocytes that had been exposed to clinically relevant concentrations of 148 drugs, we found that several activating ligands for NK cell receptors were regulated by various drugs (e.g., valproic acid, ketoconazole, promethazine, isoniazid). Especially expression of the activating NKG2D ligands (MICA, MICB and ULBPs) and the NKp30 ligand B7-H6 were upregulated in primary human hepatocytes upon exposure to many different drugs. Using the human hepatocyte cell lines Huh7 and HepG2, we confirmed that protein levels of activating NK cell ligands were elevated after drug exposure. Hepatocyte cell lines or primary human hepatocytes co-cultivated with NK cells caused enhanced NK cell activation after pretreatment with drugs at in vivo relevant concentrations compared to solvent controls. Enhanced NK cell activation was evident by increased cytotoxicity against hepatocytes and interferon (IFN)-γ production. NK cell activation could be blocked by specific antibodies against activating NK cell receptors. These data support the hypothesis that NK cells can modulate drug-induced liver injury by direct interaction with hepatocytes resulting in cytotoxicity and IFN-γ production.

Analysis of Cytotoxicity of Selected Asteraceae Plant Extracts in Real Time, Their Antioxidant Properties and Polyphenolic Profile.

Plants from Asteraceae family are widely used for their therapeutic effects in the treatment of gastrointestinal diseases, but the consequences of excessive intake still need to be studied. The aims of this study were the evaluation of cytotoxicity, measurement of antioxidant properties and determination of polyphenolic profile of Tanacetum vulgare L. (tansy), Achillea millefolium L. (yarrow) and Solidago gigantea Ait. (goldenrod) ethanolic extracts. The cytotoxicity of extracts was monitored by xCELLigence system in real time by using porcine intestinal epithelial cell line (IPEC-1) and by measurement of changes in metabolic activity ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay). The antioxidant properties were measured by spectrophotometric methods and polyphenolic profiles were determined by HPLC-DAD for 50% ethanol extracts (10% w/v). Strong cytotoxic effect was recorded for tansy and yarrow extracts (125-1000 µg/mL) by xCELLigence system and MTS assay. Conversely, a supportive effect on cell proliferation was recorded for goldenrod extracts (125 µg/mL) by the same methods (p < 0.001). The antioxidant activity was in good correlation with total polyphenolic content, and the highest value was recorded for goldenrod leaves, followed by tansy leaves, goldenrod flowers and yarrow leaf extracts. The goldenrod extracts were abundant with flavonoids, whereas phenolic acid derivatives predominated in the polyphenolic profile of tansy and yarrow.

Hypercapnia and Hypoxia Stimulate Proliferation of Astrocytes and Neurons In Vitro.

We compared proliferative activity and hypoxic tolerance in a co-culture of neurons and astrocytes in vitro after preliminary exposure to normobaric hypoxia and/or permissive hypercapnia in vivo. Preliminary hypoxic exposure increased the cell index throughout the 72-h period of observation, the effect of hypercapnia was observed on days 1 and 3 of the experiment, and the effect of hypercapnic hypoxia was noted only on day 1. Preliminary hypoxic exposure has a protective effect on nerve cells under conditions of chemical hypoxia. This suggests that hypercapnia and hypoxia activate proliferative activity of nerve cells, which can be viewed as a mechanism of their neuroprotective effectiveness.

Comparative detection method of early onset cytokine-induced stress in ß-cells (INS-1E).

ß-Cells contain a prominent endoplasmic reticulum (ER), disrupting ER homeostasis and function, activating the unfolded protein response (UPR). Currently, no direct protocols measure the UPR initiation. Current methods to measure ER stress include the quantification of nitric oxide (NO) (indirect method), Western blotting, and qRT-PCR of downstream components. However, these methods do not account for the overlap with mitochondrial dysfunction. In this study, INS-1E cells were exposed to proinflammatory cytokines to induce ER stress, as determined using NO, thioflavin T (ThT) binding, and ß-cell functionality (insulin production). ER stress was confirmed through the upregulation of CHOP. Cell viability was monitored using MTT, sulforhodamine B, and the xCELLigence system. Morphological changes were monitored using electron microscopy. IL-1ß exposure-induced ß-cell stress after 4 H, decreased insulin levels, and increased thioflavin binding were noted. Increased NO production was only detected after 10 H, highlighting its lack of sensitivity, and the need for a continuous, selective, rapid, convenient, and economical detection method for early onset of ER stress. Standard methods (MTT and NO) failed to detect early ER stress. The xCELLigence coupled with a functional assay such as the detection of insulin levels or ThT are better predictors of ER stress in INS-1E cells.

Polyphenols Content, Antioxidant Activity, and Cytotoxicity Assessment of Taraxacum officinale Extracts Prepared through the Micelle-Mediated Extraction Method.

This experiment was conducted with extracts prepared from dandelion (Taraxacum officinale F. H. Wigg) leaves and flowers, using the micelle-mediated extraction method, with the surface active compound Triton X-100 and water⁻acetone as the extraction solvents. Extracts were, first, examined for the content of total phenols and the antioxidant capacity. All extracts showed good anti-radical properties, especially for leaves, in comparison to the flower samples. Flavonoids (mainly luteolin derivatives) and phenolic acids, predominated among the determined polyphenols. Quantitative analyses indicated acetone extract to be the richest in phenols (up to 0.535 mg/mL), in the case of dandelion leaves, and Triton X-100 extract in the case of flowers (0.385 mg/mL). Extracts were also evaluated for cytotoxicity to the model cell line (epithelial rabbit kidney cells RK13), using the colorimetric 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) test and the real-time cell analysis method ((RTCA); xCELLigence system). The obtained results indicated that surfactants, especially non-ionic ones, can be effectively used as modifiers in the aqueous extraction of phenolic compounds from plant materials. An advantage over the traditional organic solvents is their non-flammability. Furthermore, surfactants might also be used at low concentrations. Studies on cell lines, however, indicated the cytotoxic effect of this type of compound, even in the trace amounts present in the extracts.

Functional analysis of ESM1 by siRNA knockdown in primary and metastatic head and neck cancer cells.

BACKGROUND: Genetic factors play a large role in cancer, and thus, there is a great desire to understand the effects of different genes in cancer and to also develop gene therapy for better treatments. Therefore, the development of alternative diagnosis and therapy modalities is of utmost importance. The aim of our study was to illuminate the role of ESM1 (endothelial cell-specific molecule-1, also known as Endocan) in proliferation and migration of head and neck cancer, thus helping to pave the way for new treatment modalities and predictive biomarkers. METHODS: ESM1 expression was shown with immunofluorescence assay using confocal laser scanning microscope in primary and metastatic head and neck cancer cells. ESM1 expression was knocked down by RNA interference in head and neck cancer cells. Knockdown efficiency was evaluated by quantitative real-time RT-PCR and Western blot. Cell proliferation and migration assays were performed by xCELLigence real-time cell analysis system. RESULTS: Immunofluorescence assay showed nuclear localization and high expression of ESM1 in primary and metastatic head and neck cancer cells. ESM1 mRNA and protein levels were significantly decreased in ESM1-knockdown cells compared to control. ESM1-knockdown cells showed reduced proliferation and migration activity when compared to control cells. CONCLUSION: These findings suggest that ESM1 has roles on proliferation and migration of head and neck cancer cells.

Study on the Quality Evaluation of Compound Danshen Preparations Based on the xCELLigence Real-Time Cell-Based Assay and Pharmacodynamic Authentication.

Objective: To perform a preliminary study on the quality evaluation of compound Danshen preparations based on the xCELLigence Real-Time Cell-based Assay (RTCA) system and make a pharmacodynamics verification. Methods: The compound Danshen was discussed as a methodological example, and the bioactivity of the compound Danshen preparations were evaluated by real-time cell electronic analysis technology. Meanwhile, an in vivo experiment on an acute blood stasis rat model was performed in order to verify this novel evaluation through the curative effect of dissipating blood stasis. Results: We determined the cell index (CI) and IC50 of the compound Danshen preparations and produced time/dose-dependent cell response profiles (TCRPs). The quality of the three kinds of compound Danshen preparations was evaluated through the RTCA data. The trend of CI and TCRPs reflected the effect of drugs on the cell (promoting or inhibiting), and it was verified that the results correlated with the biological activity of the drugs using a pharmacodynamics experiment. Conclusion: The RTCA system can be used to evaluate the quality of compound Danshen Preparations, and it can provide a new idea and new method for quantitatively characterizing the biological activity of traditional Chinese medicines (TCMs).

A novel release kinetics evaluation of Chinese compound medicine: Application of the xCELLigence RTCA system to determine the release characteristics of Sedum sarmentosum compound sustained-release pellets.

Purpose: To establish a novel release kinetics evaluation method of Chinese compound medicine (Sedum Sarmentosum compound) with xCELLigence Real-Time Cell-based Assay (RTCA) system. Methods: Cell lines sensitive to Sedum Sarmentosum compound are screened, and cell index-time (CI-T) graphs and cell index release kinetics models are established based on the cell index (CI) monitored. The methodological studies of precision and repeatability were processed by the cell monitors system. The release profiles of the sustained-release Sedum Sarmentosum compound were determined. Consequently, the sustained-release property was characterized by the kinetic parameters based on the cell-index. Results: The accumulative release rate based on cell index of Sedum sarmentosum compound sustained-release preparation was determined and it had a good correlation with time, fitting better with First-order model, Higuchi model and Ritger-Peppas model, and fitting best with Weibull model. It indicated that the release rate is proportional with the diffusion coefficient. Conclusion: The new method of cell-index release kinetics may provide a quantitative description for the release of the multi active agents from Traditional Chinese Medicines. The application of xCELLigence RTCA system for evaluating the release kinetics of Chinese compound medicine is feasible.

Avidity characterization of genetically engineered T-cells with novel and established approaches.

BACKGROUND: Adoptive transfer of genetically engineered autologous T-cells is becoming a successful therapy for cancer. The avidity of the engineered T-cells is of crucial importance for therapy success. We have in the past cloned a T-cell receptor (TCR) that recognizes an HLA-A2 (MHC class I)-restricted peptide from the prostate and breast cancer- associated antigen TARP. Herein we perform a side-by-side comparison of the TARP-specific TCR (TARP-TCR) with a newly cloned TCR specific for an HLA-A2-restricted peptide from the cytomegalovirus (CMV) pp65 antigen. RESULTS: Both CD8(+) T-cells and CD4(+) T-cells transduced with the HLA-A2-restricted TARP-TCR could readily be detected by multimer analysis, indicating that the binding is rather strong, since binding occured also without the CD8 co-receptor of HLA-A2. Not surprisingly, the TARP-TCR, which is directed against a self-antigen, had weaker binding to the HLA-A2/peptide complex than the CMV pp65-specific TCR (pp65-TCR), which is directed against a viral epitope. Higher peptide concentrations were needed to achieve efficient cytokine release and killing of target cells when the TARP-TCR was used. We further introduce the LigandTracer technology to study cell-cell interactions in real time by evaluating the interaction between TCR-engineered T-cells and peptide-pulsed cancer cells. We were able to successfully detect TCR-engineered T-cell binding kinetics to the target cells. We also used the xCELLigence technology to analyzed cell growth of target cells to assess the killing potency of the TCR-engineered T-cells. T-cells transduced with the pp65 - TCR exhibited more pronounced cytotoxicity, being able to kill their targets at both lower effector to target ratios and lower peptide concentrations. CONCLUSION: The combination of binding assay with functional assays yields data suggesting that TARP-TCR-engineered T-cells bind to their target, but need more antigen stimulation compared to the pp65-TCR to achieve full effector response. Nonetheless, we believe that the TARP-TCR is an attractive candidate for immunotherapy development for prostate and/or breast cancer.