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Mitogen-Induced Interferon Gamma Production in Human Whole Blood: The Effect of Heat and Cations

[ Vol. 20 , Issue. 7 ]

Author(s):

Ji-Hyun Nam, Bomi Cha, Jun-Young Park, Fukushi Abekura, Cheorl-Ho Kim* and Jeong-Ran Kim*   Pages 562 - 572 ( 11 )

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.

Keywords:

Pokeweed mitogen, phytohemagglutinin, concanavalin A, interferon gamma, heat stability, calcium, magnesium, zinc.

Affiliation:

Department of Research and Development, The Korean Institute of Tuberculosis, 168-5 Osongsaengmyeong4-ro, Heungdeok-gu, Cheongju-si, Chungcheongbuk-do 28158, Department of Research and Development, The Korean Institute of Tuberculosis, 168-5 Osongsaengmyeong4-ro, Heungdeok-gu, Cheongju-si, Chungcheongbuk-do 28158, Molecular and Cellular Glycobiology Unit, Department of Biological Sciences, SungKyunKwan University, Seoburo, Jangan-Gu, Kyunggi-Do 16419, Molecular and Cellular Glycobiology Unit, Department of Biological Sciences, SungKyunKwan University, Seoburo, Jangan-Gu, Kyunggi-Do 16419, Molecular and Cellular Glycobiology Unit, Department of Biological Sciences, SungKyunKwan University, Seoburo, Jangan-Gu, Kyunggi-Do 16419, Department of Research and Development, The Korean Institute of Tuberculosis, 168-5 Osongsaengmyeong4-ro, Heungdeok-gu, Cheongju-si, Chungcheongbuk-do 28158

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