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

Reuven Rasooly,Bradley Hernlem,Paula Do,Xiaohua He

doi:10.3390/toxins13050300

Abstract

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.

Highlights

Staphylococcus aureus is a common and widespread bacterial pathogen producing twenty-three different staphylococcal enterotoxins (SEs) that are the cause of some quarter million cases of foodborne illness in the United States per year [1]
We evaluated a specific assay for Staphylococcal enterotoxin type B (SEB) using a cell-based alternative method to the emetic bioassays using monkeys or kittens, which are the currently accepted methods used to measure the biological activity of Staphylococcal enterotoxins
For the first time, successful use of the human T-cell acute lymphoblastic leukemia (HPB-ALL) cell line in combination with a B-cell line, leading to the development of an inexpensive alternative to live animal testing for the detection and quantification of biologically active SEB

Summary

Introduction

Staphylococcus aureus is a common and widespread bacterial pathogen producing twenty-three different staphylococcal enterotoxins (SEs) that are the cause of some quarter million cases of foodborne illness in the United States per year [1]. Class II molecules expressed on the surface of antigen-presenting cells (APC). The other studies using site-directed mutagenesis to create altered SE molecules showed an accompanying reduction in T-cell activation with the loss of emetic responses [8,9]. It was estimated that SEB was responsible for 10% of the staphylococcal enterotoxins in food poisoning outbreak cases [3,10,11]. Among the 71 isolates originating from outbreaks of food poisoning, 54 (76.1%) possessed the SEB, SEA, SEC, or SED genes. Since those strains were capable of producing SEB and

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