Allergen-specific T-cell response in patients with phenytoin hypersensitivity; simultaneous analysis of proliferation and cytokine production by carboxyfluorescein succinimidyl ester (CFSE) dilution assay.
BACKGROUND
Phenytoin can induce diversified adverse reactions including generalized eruptions and the hypersensitivity syndrome. Delayed-type allergic mechanisms have been postulated to underlie these reactions. The tests most widely used to detect T-cell sensitization to drugs are the patch test and the lymphocyte transformation test (LTT), but their sensitivity is not sufficient. Simultaneous assessment of both the frequencies and the cytokine-producing phenotypes of allergen-specific T cells has become possible with the recently introduced carboxyfluorescein succinimidyl ester (CFSE) assay.


METHODS
Seven patients who presented with phenytoin-induced maculopapular exanthema with and without fever were included in this study. Peripheral blood mononuclear cells (PBMCs) were labeled with CFSE and cultured with phenytoin for seven days. The cells were stained with anti-CD4 and cytokine-specific monoclonal antibodies (MoAbs), and analyzed with FACSCalibur.


RESULTS
The phenytoin-specific proliferation of CD4+ cells in patients was significantly higher than in the four controls exposed to phenytoin, and in seven healthy children with no previous phenytoin intake. A significant difference in the percentages of CD4+ IFN-gamma+ cells between patients and the seven healthy children was observed. The sensitivity and specificity of proliferation were 100% and 90.9%, and those of IFN-gamma secretion were 71.4% and 100%, respectively.


CONCLUSIONS
Phenytoin-specific proliferation may be detected with greater sensitivity by the CFSE dilution assay than the conventional LTT. The assay revealed that both CD4+ and CD4- T cells proliferated and produced IFN-gamma and TNF-alpha after stimulation with phenytoin. The CFSE dilution assay might be useful for the diagnosis and understanding of drug hypersensitivity.
Tsuge
Ikuya
I
Department of Pediatrics, School of Medicine, Fujita Health University, Toyoake, Aichi, Japan. itsuge@fujita-hu.ac.jp
Okumura
Akihisa
A
Kondo
Yasuto
Y
Itomi
Seiko
S
Kakami
Michiko
M
Kawamura
Makiko
M
Nakajima
Yoichi
Y
Komatsubara
Ryo
R
Urisu
Atsuo
A
D000293:Adolescent; D000485:Allergens; D000927:Anticonvulsants / Q000009:adverse effects*; D015704:CD4 Antigens / Q000032:analysis; D016022:Case-Control Studies; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D016207:Cytokines / Q000378:metabolism*; D004342:Drug Hypersensitivity / Q000150:complications / Q000175:diagnosis* / Q000276:immunology / Q000378:metabolism; D004395:Dye Dilution Technique; D005076:Exanthema / Q000276:immunology / Q000378:metabolism; D005260:Female; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D007159:Immunologic Tests / Q000379:methods*; D007963:Leukocytes, Mononuclear / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D008213:Lymphocyte Activation / Q000187:drug effects; D008297:Male; D010672:Phenytoin / Q000009:adverse effects*; D011237:Predictive Value of Tests; D015203:Reproducibility of Results; D013388:Succinimides
C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D000485:Allergens; D000927:Anticonvulsants; D015704:CD4 Antigens; D016207:Cytokines; D005452:Fluoresceins; D005456:Fluorescent Dyes; D013388:Succinimides; D010672:Phenytoin