ABSTRACT
BACKGROUND: Interferon-gamma release assays (IGRAs) are blood tests used to measure the amount of interferon-γ (IFN-γ) released by T lymphocytes after stimulation by antigens specific for the diagnosis of latent tuberculosis infection. A mitogen serves as a positive control to assess the immune function in IGRAs. METHODS: This in vitro study was conducted to evaluate IFN-γ production by human whole blood stimulated with heat-treated and/or cation-supplemented phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM), using QuantiFERON-TB Gold Kit ELISA tests. RESULTS: The optimal concentrations of PWM, Con A and PHA for IGRAs were 2 µg/mL, 5 µg/mL and 10 µg/mL, respectively. The results showed that IFN-γ production in response to PWM was the highest and PHA was the lowest amount. The median values of three mitogens were in the following order: PWM≥Con A≥ positive control>>PHA-P>>negative control. PWM and PHA were heat stable, while Con A was heat sensitive. The mitogen response of lymphocytes to untreated or heat-treated PWM and heat-treated Con A was increased in 1 mM Ca2+-supplemented groups, whereas the response to heat-treated PHA was decreased. Exposure to 1 mM Mg2+ had no effect on untreated or heat-treated PWM, and a concentration of 1 mM Zn2+ inhibited the stimulation of un-treated PWM. We found that calcium supplementation improved the PWM-induced production of IFN-γ. CONCLUSION: Therefore, PWM is an appropriate mitogen for use as a positive control in IGRAs. It is a potential indicator of cytokine production in the diagnostic as well as research settings, and calcium supplementation improved stimulation.
Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Hot Temperature , Interferon-gamma/blood , Lymphocyte Activation/drug effects , Pokeweed Mitogens/pharmacology , T-Lymphocytes/drug effects , Adult , Aged , Cations , Concanavalin A/immunology , Concanavalin A/pharmacology , Female , Humans , Male , Middle Aged , Phytohemagglutinins/immunology , Phytohemagglutinins/pharmacology , Pokeweed Mitogens/immunology , T-Lymphocytes/immunology , Tuberculosis, Pulmonary/blood , Young AdultABSTRACT
While Pichia pastoris has been developed into a versatile recombinant protein expression system, there are only few studies that have investigated the efficacious use of this yeast with human cells. In this study, we demonstrated that P. pastoris can be cultured under mammalian cell culture conditions and co-cultured with human endothelial cells. Co-cultures did not affect endothelial cell morphology or viability. Additionally, P. pastoris was induced to express enhanced green fluorescence protein when co-cultured with human endothelial cell line EA.hy926 under mammalian cell culture conditions. Our study provides data to support the use of P. pastoris as a vehicle for direct delivery of recombinant proteins to mammalian cells during co-culture.