Quantitative Analysis of Staphylococcus Enterotoxin A by Differential Expression of IFN-γ in Splenocyte and CD4+ T-Cells

Abstract

Staphylococcus aureus is an important bacterial pathogen that produces a range of Staphylococcal Enterotoxins (SEs) which cause gastroenteritis and superantigen activation of T cells, the mechanism of which is not well understood. The ability to rapidly detect and quantify SEs is very important in order to learn the causes of staphylococcal outbreaks and to stop similar outbreaks in the future. Enzyme-linked immunosorbent assays (ELISAs) have been developed for detection of several SEs. However, these immunological methods cannot distinguish between active and inactive toxin. It is known that interferon-gamma (IFN-γ) expressed in response to stimulation by SEs contributes significantly to the pathogenesis of S. aureus infection. Nonetheless, the cellular source of IFN-γ is still unclear and the contributions of the specific splenocyte types. In our effort to understand the immunologic response to Staphylococcal Enterotoxin A (SEA) exposure, we studied IFN-γ production in mouse splenocytes. We demonstrated that short term ex vivo exposure of splenocytes or primary naïve CD4+ T-cells to biologically active SEA induces differential expression of IFN-γ mRNA in a time and dose dependent manner and the expression levels reflect the levels of IFN-γ secreted protein. Positive isolated CD4+ T-cells accounted for only 10% of IFN-γ production. We also demonstrate that expression of IFN-γ can be used for rapid quantitative analysis of active SEA with a detection limit of 1 ng/mL.

Trace Levels of Staphylococcal Enterotoxin Bioactivity Are Concealed in a Mucosal Niche during Pulmonary Inflammation

“…While there is no doubt that detection is extremely useful in diagnosis and prediction outcomes, there is space for expanding the use of functional bioassays to better understand the effects of a presumed pathogen or factor. For instance, BALF analysis of ICU patients could include functional tests of fluid such as described here and elsewhere [ 32 , 33 ]. An example is the ARDS network established by the NIH which has gathered an impressive collection of BAL and respiratory fluid [ 34 ], among which some could be tested for enterotoxin bioactivity when Staphylococcus aureus is an established risk factor [ 35 ].…”

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

“…The cells were then incubated at 37 • C for up to two days. The supernatants were then harvested after 24…”

T cell Receptor Vβ9 in Method for Rapidly Quantifying Active Staphylococcal Enterotoxin Type-A without Live Animals

“…SEs function as superantigens that activate CD4+ T cells, cause proliferation of T-cells in a dose and time dependent manner [4], and induce differential regulation of CD154 [5] that is responsible for costimulatory signals to B cells. In addition, SEs induce differential expression of interferon-gamma (IFN-γ) [6], tumor necrosis factor (TNF) [7] and cytokine release in a dose-dependent manner [3]. Although superantigenic activity and the gastroenteric syndrome are two separate functions of SEs, there is a high correlation between these activities.…”