TNF as Biomarker for Rapid Quantification of Active Staphylococcus Enterotoxin A in Food

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Low levels of aflatoxin B1, ricin, and milk enhance recombinant protein production in mammalian cells.

Gene expression in transduced mammalian cells correlates with virus titer, but high doses of vector for gene therapy leads to toxicity in humans and in animals. Changing the optimal tissue culture medium by adding low levels of environmental stressors, such as 1 µM of the fungal toxin aflatoxin B1 (AFB1), 1 ng of the castor bean protein toxin ricin, or 1% reconstituted milk, enhances transcription and increases production of proteins in transduced mammalian cells as demonstrated by production of the following three recombinant proteins: firefly luciferase, β-galactosidase, and green fluorescent protein (GFP). Higher concentrations of the stress-producing substances damage the cells beyond recovery, resulting in inhibited gene expression and cell death. We also evaluated the effect of the stressor substances on the enhanced infectivity of virus. The presented findings extend methods for large-scale transient recombinant protein production in mammalian cells and suggest that it may be possible to reduce the cytotoxicity of the adenovirus by reducing the virus titer without adversely affecting gene expression levels

Sensitive, Rapid, Quantitative and in Vitro Method for the Detection of Biologically Active Staphylococcal Enterotoxin Type E

Staphylococcus aureus is a major bacterial cause of clinical infections and foodborne illnesses through its production of a group of enterotoxins (SEs) which cause gastroenteritis and also function as superantigens to massively activate T cells. In the present study, we tested Staphylococcal enterotoxin type E (SEE), which was detected in 17 of the 38 suspected staphylococcal food poisoning incidents in a British study and was the causative agent in outbreaks in France, UK and USA. The current method for detection of enterotoxin activity is an in vivo monkey or kitten bioassay; however, this expensive procedure has low sensitivity and poor reproducibility, requires many animals, is impractical to test on a large number of samples, and raises ethical concerns with regard to the use of experimental animals. The purpose of this study is to develop rapid sensitive and quantitative bioassays for detection of active SEE. We apply a genetically engineered T cell-line expressing the luciferase reporter gene under the regulation of nuclear factor of activated T-cells response element (NFAT-RE), combined with a Raji B-cell line that presents the SEE-MHC (major histocompatibility complex) class II to the engineered T cell line. Exposure of the above mixed culture to SEE induces differential expression of the luciferase gene and bioluminescence is read out in a dose dependent manner over a 6-log range. The limit of detection of biologically active SEE is 1 fg/mL which is 109 times more sensitive than the monkey and kitten bioassay

CCD Based Detector for Detection of Abrin Toxin Activity

Abrin is a highly potent and naturally occurring toxin produced in the seeds of Abrus precatorius (Rosary Pea) and is of concern as a potential bioterrorism weapon. There are many rapid and specific assay methods to detect this toxic plant protein, but few are based on detection of toxin activity, critical to discern biologically active toxin that disables ribosomes and thereby inhibits protein synthesis, producing cytotoxic effects in multiple organ systems, from degraded or inactivated toxin which is not a threat. A simple and low-cost CCD detector system was evaluated with colorimetric and fluorometric cell-based assays for abrin activity; in the first instance measuring the abrin suppression of mitochondrial dehydrogenase in Vero cells by the MTT-formazan method and in the second instance measuring the abrin suppression of green fluorescent protein (GFP) expression in transduced Vero and HeLa cells. The limit of detection using the colorimetric assay was 10 pg/mL which was comparable to the fluorometric assay using HeLa cells. However, with GFP transduced Vero cells a hundred-fold improvement in sensitivity was achieved. Results were comparable to those using a more expensive commercial plate reader. Thermal inactivation of abrin was studied in PBS and in milk using the GFP-Vero cell assay. Inactivation at 100 °C for 5 min in both media was complete only at the lowest concentration studied (0.1 ng/mL) while treatment at 63 °C for 30 min was effective in PBS but not milk

T-cell receptor Vβ8 for detection of biologically active streptococcal pyrogenic exotoxin type C

ABSTRACT: Streptococcus pyogenes is an important human pathogen, commonly spread by airborne droplets but also by ingestion of contaminated food. Apart from causing infection, this pathogen produces 13 distinct types of streptococcal pyrogenic exotoxins (SPE). The current method for detection cannot distinguish between the biologically active form of SPE that has been reported to cause foodborne outbreaks and the inactivated toxin that poses no health risk. To measure the biological activity of SPE type C (SPE-C), one such toxin that was linked to foodborne outbreaks associated with milk and milk products, we developed a cell-based assay that can discern between biologically active and inactive SPE-C. To the best of our knowledge, this is the first showing that SPE-C activates T-cells expressing Vβ8. With this finding, we used a T-cell line natively expressing Vβ8 that was genetically engineered to also express the luciferase reporter gene under the regulation of nuclear factor of activated T-cells response element in combination with a B-cell line to present the recombinant SPE-C (rSPE-C) toxin via major histocompatibility complex (MHC) class II to the Vβ8 T-cell receptor (TCR) in an assay to detect and to discern between biologically active and inactive rSPE-C. By using this system, we demonstrated that SPE-C induced significant IL-2 secretion after 72 h and visible light emission after only 5 h, doubling by 24 h. We utilize this finding to assess the specificity of the assay and the effect of pasteurization on SPE-C activity. We observed no cross-reactivity with SPE-B and significant loss of SPE-C biological activity in spiked phosphate-buffered saline while SPE-C spiked into milk is heat stable. Once SPE-C has formed, it is infeasible to eliminate it from milk by thermal treatment

Alternative to Animal Use for Detecting Biologically Active Staphylococcal Enterotoxin Type A

Staphylococcal enterotoxins (SEs) are a food safety concern. Existing methods for biologically active SE detection rely on the emetic response in live kittens or monkeys. This method suffers from low sensitivity, poor reproducibility, and causes ethical concerns regarding the use of experimental animals. The Lautenberg Chemical Safety Act encourages the development and adoption of alternatives to testing on animals for chemical toxicity methodologies. In this study, we utilized the superantigenic effect of SE type A (SEA) and used an ex vivo bioassay as an alternative to live animal testing. We found that interleukin-2 (IL-2) secreted by splenocyte can be utilized for quantifiable detection of SEA in food products. To avoid food matrix interference and attenuation of signal, we separated SEA from spiked food products by employing immunomagnetic beads that were coated with an anti-SEA antibody. This ex vivo method has achieved the detection of 1 ng mL−1 of SEA, which is 107 times more sensitive than the existing live animal testing methods. However, this ex vivo bioassay requires sacrificing of mice. To overcome this limitation, we established a cell based in vitro assay using CCRF-CEM, a human CD4+ T-cell line, for the quantitative detection of SEA. Incubation of SEA with CCRF-CEM human T-cells and Raji cells led to quantifiable and dose dependent secretion of IL-2. This novel cell-based assay is highly specific to biologically active SEA, compared with the related SE toxin subtypes B, D, and E or heat inactivated SEA, which produce no secretion of IL-2. This is the first demonstration of an alternative assay that completely eliminates the use of animals for quantitative detection of active SEA

Non-Linear Relationships between Aflatoxin B1 Levels and the Biological Response of Monkey Kidney Vero Cells

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Plant Compounds Enhance the Assay Sensitivity for Detection of Active Bacillus cereus Toxin

Bacillus cereus is an important food pathogen, producing emetic and diarrheal syndromes, the latter mediated by enterotoxins. The ability to sensitively trace and identify this active toxin is important for food safety. This study evaluated a nonradioactive, sensitive, in vitro cell-based assay, based on B. cereus toxin inhibition of green fluorescent protein (GFP) synthesis in transduced monkey kidney Vero cells, combined with plant extracts or plant compounds that reduce viable count of B. cereus in food. The assay exhibited a dose dependent GFP inhibition response with ~25% inhibition at 50 ng/mL toxin evaluated in culture media or soy milk, rice milk or infant formula, products associated with food poisonings outbreak. The plant extracts of green tea or bitter almond and the plant compounds epicatechin or carvacrol were found to amplify the assay response to ~90% inhibition at the 50 ng/mL toxin concentration greatly increasing the sensitivity of this assay. Additional studies showed that the test formulations also inhibited the growth of the B. cereus bacteria, likely through cell membrane disruption. The results suggest that the improved highly sensitive assay for the toxin and the rapid inactivation of the pathogen producing the toxin have the potential to enhance food safety

Human Leukemia T-Cell Lines as Alternatives to Animal Use for Detecting Biologically Active Staphylococcal Enterotoxin Type B

Staphylococcal enterotoxin type B (SEB) is associated with food poisoning. Current methods for the detection of biologically active SEB rely upon its ability to cause emesis when administered to live kittens or monkeys. This technique suffers from poor reproducibility and low sensitivity and is ethically disfavored over concerns for the welfare of laboratory animals. The data presented here show the first successful implementation of an alternative method to live animal testing that utilizes SEB super-antigenic activity to induce cytokine production for specific novel cell-based assays for quantifiable detection of active SEB. Rather than using or sacrificing live animals, we found that SEB can bind to the major histocompatibility complex (MHC) class II molecules on Raji B-cells. We presented this SEB–MHC class II complex to specific Vβ5.3 regions of the human T-cell line HPB-ALL, which led to a dose-dependent secretion of IL-2 that is capable of being quantified and can further detect 10 pg/mL of SEB. This new assay is 100,000 times more sensitive than the ex vivo murine splenocyte method that achieved a detection limit of 1 µg/mL. The data presented here also demonstrate that SEB induced proliferation in a dose-dependent manner for cells obtained by three different selection methods: by splenocyte cells containing 22% of CD4+ T-cells, by CD4+ T-cells enriched to >90% purity by negative selection methods, and by CD4+ T-cells enriched to >95% purity by positive selection methods. The highly enriched and positively isolated CD4+ T-cells with the lowest concentration of antigen-presenting cells (APC) (below 5%) provided higher cell proliferation than the splenocyte cells containing the highest concentration of APC cells

GFP expression in Vero cells induced by ricin with and without milk.

<p>GFP transduced Vero cells were treated with increasing concentrations of ricin in milk. After incubation of Vero cells with Ad-GFP and ricin for 3 days, GFP expression was quantified fluorometrically. Error bars represent standard errors. (n = 3).</p