scholarly journals A Novel Highly Sensitive Method for Measuring Inflammatory Neural-Derived APC Activity in Glial Cell Lines, Mouse Brain and Human CSF

2020 ◽  
Vol 21 (7) ◽  
pp. 2422
Author(s):  
Valery Golderman ◽  
Shany G. Gofrit ◽  
Nicola Maggio ◽  
Orna Gera ◽  
Alexandra Gerasimov ◽  
...  
Keyword(s):  
Cell Lines ◽  
Neuroprotective Effect ◽  
Activated Protein C ◽  
Brain Slices ◽  
Fluorogenic Substrate ◽  
Low Concentrations ◽  
Highly Sensitive ◽  
Novel Method ◽  
Mice Brain ◽  
Low Levels

Background: Neural inflammation is linked to coagulation. Low levels of thrombin have a neuroprotective effect, mediated by activated protein C (APC). We describe a sensitive novel method for the measurement of APC activity at the low concentrations found in neural tissue. Methods: APC activity was measured using a fluorogenic substrate, Pyr-Pro-Arg-AMC, cleaved preferentially by APC. Selectivity was assessed using specific inhibitors and activators. APC levels were measured in human plasma, in glia cell lines, in mice brain slices following mild traumatic brain injury (mTBI) and systemic lipopolysaccharide (LPS) injection, and in cerebrospinal fluid (CSF) taken from viral meningoencephalitis patients and controls. Results: Selectivity required apixaban and alpha-naphthylsulphonylglycyl-4-amidinophenylalanine piperidine (NAPAP). APC levels were easily measurable in plasma and were significantly increased by Protac and CaCl2. APC activity was significantly higher in the microglial compared to astrocytic cell line and specifically lowered by LPS. Brain APC levels were higher in posterior regions and increased by mTBI and LPS. Highly elevated APC activity was measured in viral meningoencephalitis patients CSF. Conclusions: This method is selective and sensitive for the measurement of APC activity that significantly changes during inflammation in cell lines, animal models and human CSF.

Glycosaminoglycans from two human malignant mesothelioma cell lines: determination, distribution, and effect of platelet-derived growth factor on their synthesis

1995 ◽  
Vol 73 (1-2) ◽  
pp. 59-66 ◽  
Author(s):  
George N. Tzanakakis ◽  
Nikos K. Karamanos ◽  
Julius Klominek ◽  
Anders Hjerpe
Keyword(s):  
Growth Factor ◽  
Cell Lines ◽  
Malignant Mesothelioma ◽  
Dermatan Sulfate ◽  
Platelet Derived Growth Factor ◽  
Agarose Gel Electrophoresis ◽  
Differentiated Cells ◽  
Low Concentrations ◽  
Highly Sensitive ◽  
Mesothelioma Cell

The synthesis and distribution of glycosaminoglycans (GAGs) were studied in two human malignant mesothelioma cell lines: one with fibroblast-like morphology and the other with epithelial differentiation. Analyses using highly sensitive high-pressure liquid chromatography techniques and agarose gel electrophoresis showed that these cells produce not only hyaluronan (HA) but also galactosaminoglycans (GalAGs, chondroitin sulfate and (or) dermatan sulfate) and heparan sulfate (HS). In both cell lines most of the HA (87–90%) and GalAGs (57–66%) are secreted into the extracellular matrix. Although HS is mainly bound to the cell surface in fibroblast-differentiated cells (75%), in epithelial type cells only 40% occurs in the cell-associated fraction. The amounts of secreted GAGs are 6- to 8-fold higher in epithelial than in fibroblast-like mesothelioma cultures. In cells with the fibroblast phenotype, the β-homodimer of platelet-derived growth factor (PDGF) in a concentration of 1.5 ng/mL stimulates HA and GalAG synthesis 5-fold and that of HS 10-fold, whereas higher concentrations suppress this stimulatory effect. The stimulatory effect, observed at low concentrations of this growth factor, was completely blocked by the addition of antibodies against this factor. In epithelially differentiated cells, the production of all GAGs was suppressed after addition of this factor, even at low concentrations. We therefore suggest that mesothelioma cells can produce GAGs, the synthesis of which is dependent on the presence and concentration of PDGF β-homodimer. The differences between the two cell lines regarding the effect of this growth factor on GAG synthesis indicates that the regulation of this synthesis is complex, other factors also being important.Key words: mesothelioma, differentiation, glycosaminoglycans, synthesis, growth factors.

Differential regulation of c-fos gene expression by two types of human endothelin receptor in Chinese hamster ovary cells

1994 ◽  
Vol 12 (2) ◽  
pp. 173-180 ◽  
Author(s):  
H Tabuchi ◽  
Y Furuichi ◽  
C Miyamoto
Keyword(s):  
Signal Transduction ◽  
Cell Lines ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Physiological Concentration ◽  
Ovary Cells ◽  
Colorimetric Measurement ◽  
Low Concentrations ◽  
Highly Sensitive

ABSTRACT To investigate the nuclear signalling pathway induced by endothelin (ET) isopeptides, we have established permanent Chinese hamster ovary (CHO) cell lines, CHO-ETA/fos-lacZ and CHO-ETB/fos-lacZ, that produce both a c-fos-β-galactosidase fusion protein and either the type A or the type B human ET receptor. These cell lines permitted a colorimetric measurement of c-fos expression, which was induced by the signal transduction system with ET receptors and ET isopeptides. We found that the ET-1-dependent c-fos expression was so efficient that it could respond to low concentrations (even a physiological concentration) of ET-1. For example, CHO-ETA/fos-lacZ and CHO-ETB/fos-lacZ responded to ET concentrations of 5×10−9m and 5×10−13m respectively. Using this highly sensitive system, the H-7 sensitive protein kinase was found to be involved in signal transduction mediated by ETA, and also partly in the ETB-mediated pathway. These lines of evidence suggest that c-fos expression occurs through at least two different pathways, depending on the concentration of ET in plasma.

Arsenic removal by MF membrane with chemical sludge adsorption and NF membrane equipped with vibratory shear enhanced process

2003 ◽  
Vol 3 (5-6) ◽  
pp. 303-310 ◽  
Author(s):  
S.-H. Yi ◽  
S. Ahmed ◽  
Y. Watanabe ◽  
K. Watari
Keyword(s):  
Arsenic Removal ◽  
Membrane Surface ◽  
Membrane Process ◽  
Low Concentrations ◽  
Strong Shear ◽  
Low Levels ◽  
Removal Processes ◽  
Removal Of Arsenic ◽  
Concentration Polarization Layer ◽  
Chemical Sludge

Conventional arsenic removal processes have difficulty removing low concentrations of arsenic ion from water. Therefore, it is very hard to comply with stringent low levels of arsenic, such as below 10 μg/L. So, we have developed two arsenic removal processes which are able to comply with more stringent arsenic regulations. They are the MF membrane process combined with chemical sludge adsorption and NF membrane process equipped with the vibratory shear enhanced process (VSEP). In this paper, we examine the performance of these new processes for the removal of arsenic ion of a low concentration from water. We found that chemical sludge produced in the conventional rapid sand filtration plants can effectively remove As (V) ions of H2AsO4- and HAsO42- through anion exchange reaction. The removal efficiency of MF membrane process combined with chemical sludge adsorption increased to about 36%, compared to MF membrane alone. The strong shear force on the NF membrane surface produced by vibration on the VSEP causes the concentration polarization layer to thin through increased back transport velocity of particles. So, it can remove even dissolved constituents effectively. Therefore, As (V) ions such as H2AsO4- and HAsO42- can be removed. The concentration of As (V) ions decreased from 50 μg/L to below 10 μg/L and condensation factor in recirculating water increased up to 7 times by using NF membrane equipped with VSEP.

scholarly journals Co-Occurrence of Regulated and Emerging Mycotoxins in Corn Silage: Relationships with Fermentation Quality and Bacterial Communities

Toxins ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 232
Author(s):  
Antonio Gallo ◽  
Francesca Ghilardelli ◽  
Alberto Stanislao Atzori ◽  
Severino Zara ◽  
Barbara Novak ◽  
...  
Keyword(s):  
Bacterial Community ◽  
Bacterial Communities ◽  
Dry Matter ◽  
Kojic Acid ◽  
Corn Silage ◽  
Fungal Metabolites ◽  
Fusarium Mycotoxins ◽  
Low Concentrations ◽  
Fermentation Quality ◽  
Low Levels

Sixty-four corn silages were characterized for chemicals, bacterial community, and concentrations of several fungal metabolites. Silages were grouped in five clusters, based on detected mycotoxins, and they were characterized for being contaminated by (1) low levels of Aspergillus- and Penicillium-mycotoxins; (2) low levels of fumonisins and other Fusarium-mycotoxins; (3) high levels of Aspergillus-mycotoxins; (4) high levels of non-regulated Fusarium-mycotoxins; (5) high levels of fumonisins and their metabolites. Altersetin was detected in clusters 1, 3, and 5. Rugulusovin or brevianamide F were detected in several samples, with the highest concentration in cluster 3. Emodin was detected in more than 50.0% of samples of clusters 1, 3 and 5, respectively. Kojic acid occurred mainly in clusters 1 and 2 at very low concentrations. Regarding Fusarium mycotoxins, high occurrences were observed for FB3, FB4, FA1, whereas the average concentrations of FB6 and FA2 were lower than 12.4 µg/kg dry matter. Emerging Fusarium-produced mycotoxins, such as siccanol, moniliformin, equisetin, epiequisetin and bikaverin were detected in the majority of analyzed corn silages. Pestalotin, oxaline, phenopirrozin and questiomycin A were detected at high incidences. Concluding, this work highlighted that corn silages could be contaminated by a high number of regulated and emerging mycotoxins.

scholarly journals Bioanalytical screening of low levels of dioxins and dioxin-like PCBs in pig meat (pork) for checking compliance with EU maximum and action levels using highly sensitive “third generation” recombinant H4L7.5c2 rat hepatoma cells

2021 ◽  
Vol 33 (1) ◽  
Author(s):  
Johannes Haedrich ◽  
Claudia Stumpf ◽  
Michael S. Denison
Keyword(s):  
European Union ◽  
Hepatoma Cell ◽  
Animal Origin ◽  
The European Union ◽  
Highly Sensitive ◽  
Commission Regulation ◽  
Pig Meat ◽  
Low Levels ◽  
Action Levels ◽  
Rat Hepatoma

Abstract Background Low maximum and action levels set by the European Union for polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (DL-PCBs) in pig meat (pork) have led to a demand for reliable and cost-effective bioanalytical screening methods implemented upstream of gas chromatography/high-resolution mass spectrometry confirmatory technology, that can detect low levels of contamination in EU-regulated foods with quick turn-around times. Results Based on the Chemically Activated LUciferase gene eXpression (CALUX) bioassay, extraction and clean-up steps were optimized for recovery and reproducibility within working ranges significantly lower than in current bioassays. A highly sensitive “3rd generation” recombinant rat hepatoma cell line (H4L7.5c2) containing 20 dioxin responsive elements was exposed to pork sample extracts, and their PCDD/Fs and DL-PCBs levels were evaluated by measuring luciferase activity. The method was validated according to the provisions of Commission Regulation (EU) 2017/644 of 5 April 2017 with spiking experiments performed selectively for PCDD/Fs and DL-PCBs and individual calibration for PCDD/Fs, DL-PCBs and the calculated sum of PCDD/Fs and DL-PCBs. The resulting performance parameters met all legal specifications as confirmed by re-calibration using authentic samples. Cut-off concentrations for assessing compliance with low maximum levels and action levels set for PCDD/Fs and DL-PCBs within a range of 0.50–1.25 pg WHO-TEQ/g fat were derived, ensuring low rates of false-compliant results (ß-error < 1%) and keeping the rate of false-noncompliant results well under control (α-error < 12%). Conclusions We present a fast and efficient bioanalytical routine method validated according to the European Union’s legal requirements on the basis of authentic samples, allowing the analyst to reliably identify pork samples and any other EU-regulated foods of animal origin suspected to be noncompliant with a high level of performance and turn-around times of 52 h. This was facilitated in particular by a quick and efficient extraction step followed by selective clean-up, use of a highly sensitive “3rd generation” H4L7.5c2 recombinant rat hepatoma cell CALUX bioassay, and optimized assay performance with improved calibrator precision and reduced lack-of-fit errors. New restrictions are proposed for the calibrator bias and the unspecific background contribution to reportable results. The procedure can utilize comparably small sample amounts and allows an annual throughput of 840–1000 samples per lab technician. The described bioanalytical method contributes to the European Commission's objective of generating accurate and reproducible analytical results according to Commission Regulation (EU) 2017/644 across the European Union.

Hepatoma Cell Cultures as In Vitro Models for the Hepatotoxicity of Xenobiotics

1988 ◽  
Vol 16 (1) ◽  
pp. 32-37
Author(s):  
Margherita Ferro ◽  
Anna Maria Bassi ◽  
Giorgio Nanni
Keyword(s):  
Cell Lines ◽  
Cell Cultures ◽  
Membrane Lipids ◽  
Hepatoma Cell ◽  
Positive Control ◽  
In Vitro Models ◽  
Low Concentrations ◽  
Formation Efficiency ◽  
Dose Dependent

Two hepatoma cell cultures were examined as in vitro models to be used in genotoxicity and cytotoxicity tests without the addition of bioactivating enzymes. The MH1C1, and HTC hepatoma lines were used in this study to establish their sensitivity to a number of xenobiotics, namely, cyclophosphamide (CP), the classical positive control in bioactivation tests; benzaldehyde (BA), a short-chain aldehyde; and 4-hydroxynonenal (HNE), a major toxic end-product of the peroxidative degradation of cell membrane lipids. As a first approach, we compared the following cytotoxicity tests: release of lactate dehydrogenase (LDH), and colony formation efficiency (CF). Colony-forming cells were exposed to the drugs according to different procedures, before or after the anchorage phase. The leakage of LDH into the medium following exposure of both cell lines to HNE, CP and BA for up to 24 hours was found not to be a good index of cytotoxicity. A better indicator of cytotoxicity was CF, as evaluated by exposure of the cells 24 hours after seeding. The effects were detectable at very low concentrations, corresponding to 10, 90 and 100μM for HNE, CP and BA, respectively. The impairment of CF efficiency was dose-dependent and time-dependent, and several differences between the two cell lines were observed.

scholarly journals AB0050 A NOVEL METHOD FOR ISOLATION OF EXOSOMES FROM SYNOVIAL FLUID

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 1057.2-1057
Author(s):  
Y. Liu ◽  
Y. Huang ◽  
Q. Huang ◽  
S. Sun ◽  
Z. Ji ◽  
...  
Keyword(s):  
Synovial Fluid ◽  
Western Blot ◽  
Biological Fluid ◽  
High Sensitivity ◽  
Protein Distribution ◽  
Sds Page ◽  
Close Relationship ◽  
Novel Method ◽  
Low Levels

Background:Exosomes in synovial fluid (SF) has a close relationship with the pathogenesis of rheumatiod arthritis. As a complex biological fluid, SF presents challenges for exosomes isolation using standard methods, such as ExoquickTM kit and ultracentrifugation.Objectives:The study aims to compared the quality of exosomes separated by ExoquickTM kit (TM), ExoquickTM kit+ExoquickTC kit (TM-TC), ultracentrifugation (UC) and TM-TC+UC(TM-TC-UC) from SF.Methods:Exosomes was separated by TM, TM-TC, UC and TM-TC-UC respectively. The size and concentrations of exosomes were detected by high sensitivity flow cytometry for nanoparticle analysis. Total protein and RNA were extracted from exosomes. SDS-PAGE was used to detect the protein distribution of exosomes. Western blot was used to examine the level of albumin and exosomes marker (TSG101 and CD81).Results:There was no statistic difference in the diameters of exosomes separated by the four methods. The concentrations of exosomes in TM, TM-TC, TM-TC-UC and UC were (5.65±0.93), (3.02±1.19), (1.67±0.25) and (4.61±0.73) *109Particles/mL. The protein concentrations of exosomes separated by the four methods were consistent with the concentrations of exosomes. SDS-PAGE showed that the protein distribution of exosomes separated by the four methods were different. Low levels of albumin were detected in TM-TC and TM-TC-UC, while high levels of albumin in TM and UC. Total RNA concentrations from exosomes in TM-TC was higher than other groups.Conclusion:TM-TC can be used to obtain higher quality exosomes from SF for the study of exosome-enriched components.References:[1]Helwa I, et al, A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents. PloS one, 2017. 12(1): p. e0170628-e0170628.Figure 1.A: SDS-PAGE showed the protein distribution of exosomes; B: the detection of albumin, TSG101 and CD81 by western blot.Disclosure of Interests:None declared

scholarly journals Generation of Genetically RGD σ1-Modified Oncolytic Reovirus That Enhances JAM-A-Independent Infection of Tumor Cells

2020 ◽  
Vol 94 (23) ◽  
Author(s):  
Takahiro Kawagishi ◽  
Yuta Kanai ◽  
Ryotaro Nouda ◽  
Ichika Fukui ◽  
Jeffery A. Nurdin ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Cell Attachment ◽  
Genetically Modified ◽  
Cancer Cell Lines ◽  
Oncolytic Viruses ◽  
Peptide Sequence ◽  
Attachment Protein ◽  
Low Levels ◽  
Entry Receptor

ABSTRACT Mammalian reovirus (MRV) strain type 3 Dearing (T3D) is a naturally occurring oncolytic virus that has been developed as a potential cancer therapeutic. However, MRV treatment cannot be applied to cancer cells expressing low levels of junctional adhesion molecule A (JAM-A), which is the entry receptor of MRV. In this study, we developed a reverse genetics system for MRV strain T3D-L, which showed high oncolytic potency. To modify the cell tropism of MRV, an arginine–glycine–aspartic acid (RGD) peptide with an affinity to integrin was inserted at the C terminus or loop structures of the viral cell attachment protein σ1. The recombinant RGD σ1-modified viruses induced remarkable cell lysis in human cancer cell lines with marginal JAM-A expression and in JAM-A knockout cancer cell lines generated by a CRISPR/Cas9 system. Pretreatment of cells with anti-integrin antibody decreased cell death caused by the RGD σ1-modified virus, suggesting the infection to the cells was via a specific interaction with integrin αV. By using mouse models, we assessed virulence of the RGD σ1-modified viruses in vivo. This system will open new avenues for the use of genetically modified oncolytic MRV for use as a cancer therapy. IMPORTANCE Oncolytic viruses kill tumors without affecting normal cells. A variety of oncolytic viruses are used as cancer therapeutics. Mammalian reovirus (MRV), which belongs to the genus Orthoreovirus, family Reoviridae, is one such natural oncolytic virus. The anticancer effects of MRV are being evaluated in clinical trials. Unlike other oncolytic viruses, MRV has not been genetically modified for use as a cancer therapeutic in clinical trials. Here, we used a reverse genetic approach to introduce an integrin-affinity peptide sequence into the MRV cell attachment protein σ1 to alter the natural tropism of the virus. The recombinant viruses were able to infect cancer cell lines expressing very low levels of the MRV entry receptor, junctional adhesion molecule A (JAM-A), and cause tumor cell death while maintaining its original tropism via JAM-A. This is a novel report of a genetically modified oncolytic MRV by introducing a peptide sequence into σ1.

Invasion of human glioma biopsy specimens in cultures of rodent brain slices: a quantitative analysis

2002 ◽  
Vol 97 (1) ◽  
pp. 169-176 ◽  
Author(s):  
Sophie de Boüard ◽  
Christo Christov ◽  
Jean-Sébastien Guillamo ◽  
Lina Kassar-Duchossoy ◽  
Stéphane Palfi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Brain Slices ◽  
Lower Grade ◽  
Human Glioma ◽  
Newborn Mice ◽  
Content Type ◽  
Biopsy Specimens ◽  
Rodent Brain ◽  
Fine Print

Object. The reliable assessment of the invasiveness of gliomas in vitro has proved elusive, because most invasion assays inadequately model in vivo invasion in its complexity. Recently, organotypical brain cultures were successfully used in short-term invasion studies on glioma cell lines. In this paper the authors report that the invasiveness of human glioma biopsy specimens directly implanted into rodent brain slices by using the intraslice implantation system (ISIS) can be quantified with precision. The model was first validated by the demonstration that, in long-term studies, established glioma cells survive in the ISIS and follow pathways of invasion similar to those in vivo. Methods. Brain slices (400 µm thick) from newborn mice were maintained on millicell membranes for 15 days. Cells from two human and one rodent glioblastoma multiforme (GBM) cell lines injected into the ISIS were detected by immunohistochemistry or after transfection with green fluorescent protein—containing vectors. Preferential migration along blood vessels was identified using confocal and fluorescent microscopy. Freshly isolated (≤ 24 hours after removal) 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate—prelabeled human glioma biopsy specimens were successfully implanted in 19 (83%) of 23 cases, including 12 GBMs and seven lower grade gliomas (LGGs). Morphometric quantification of distance and density of tumor cell invasion showed that the GBMs were two to four times more invasive than the LGGs. Heterogeneity of invasion was also observed among GBMs and LGGs. Directly implanted glioma fragments were more invasive than spheroids derived from the same biopsy specimen. Conclusions. The ISIS combines a high success rate, technical simplicity, and detailed quantitative measurements and may, therefore, be used to study the invasiveness of biopsy specimens of gliomas of different grades.

TMOD-31. AN ORGANOTYPIC TISSUE PLATFORM TO BRIDGE IN VITRO AND IN VIVO ASSAYS FOR BRAIN CANCER TREATMENT

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi222-vi222
Author(s):  
Breanna Mann ◽  
Noah Bell ◽  
Denise Dunn ◽  
Scott Floyd ◽  
Shawn Hingtgen ◽  
...  
Keyword(s):  
Cell Lines ◽  
Brain Cancer ◽  
Brain Slice ◽  
Brain Slices ◽  
In Vitro Assays ◽  
Preclinical Model ◽  
Short Period ◽  
The Brain

Abstract Brain cancers remain one of the greatest medical challenges. The lack of experimentally tractable models that recapitulate brain structure/function represents a major impediment. Platforms that enable functional testing in high-fidelity models are urgently needed to accelerate the identification and translation of therapies to improve outcomes for patients suffering from brain cancer. In vitro assays are often too simple and artificial while in vivo studies can be time-intensive and complicated. Our live, organotypic brain slice platform can be used to seed and grow brain cancer cell lines, allowing us to bridge the existing gap in models. These tumors can rapidly establish within the brain slice microenvironment, and morphologic features of the tumor can be seen within a short period of time. The growth, migration, and treatment dynamics of tumors seen on the slices recapitulate what is observed in vivo yet is missed by in vitro models. Additionally, the brain slice platform allows for the dual seeding of different cell lines to simulate characteristics of heterogeneous tumors. Furthermore, live brain slices with embedded tumor can be generated from tumor-bearing mice. This method allows us to quantify tumor burden more effectively and allows for treatment and retreatment of the slices to understand treatment response and resistance that may occur in vivo. This brain slice platform lays the groundwork for a new clinically relevant preclinical model which provides physiologically relevant answers in a short amount of time leading to an acceleration of therapeutic translation.

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