| Co-Investigator(Kenkyū-buntansha) |
YAMAMOTO Kazuhiko Medical Institute of Bioregulation Kyushu Univ.Professor & Chairman, 臨床免疫学, 教授 (80191394)
MAEDA Toshiro Dept.of Dermatol, St.Marianna Univ.Assistant, 医学部, 助手 (80278027)
TAKAHAMA Hideto Dept.of Dermatol, St.Marianna Univ.Assistant, 医学部, 助手 (90267633)
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| Budget Amount *help |
¥7,100,000 (Direct Cost: ¥7,100,000)
Fiscal Year 1996: ¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 1995: ¥4,000,000 (Direct Cost: ¥4,000,000)
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| Research Abstract |
T cells may play an important role in developing atopic dermatitis (AD) lesions. We investigated whether T cells clonally expand in the lesions of AD,using a novel system, reverse transcriptase-polymerase chain reaction single-strand conformation polymorphism (RT-PCR/SSCP), which allows us to detect clonal accumulation of T cells.
We examined patients with AD and obtained two different biopsy samples from uninvolved lesions of those patients and normal skin samples of healthy volunteers were also examined. After isolating total RNA and converting it to cDNA,we performed PCR using a set of BV subfamily-specific primers and a BC primer. Thereafter we denatured the amplified DNA and analyzed it in non-denaturing polyacrylamide gel electrophoresis (SSCP) to discriminate the differences within the complementarity-determining region (CDR3) of T cell receptor beta chain, which is supposed to be crucial for antigen recognition. In all the samples of AD lesions, we found distinct band (s) by SSC
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P analysis, indicating that antigen-specific T cell clonotypes were oligoclonally accumulated in those lesions. In addition, by the simultaneous electrophoresis of two samples obtained from the different AD lesions of the same patient, we noticed the identical T cell clonotypes, which may recognize the same antigens. To clarify whether the Dermatophagoldes pteronyssinus (Dp) specific T cell clones accumulated in the involved skin of AD patients, we obtained PBMC from patients and established antigen specific T cell clones by DP,which was kindly provided by Dr.Ohta (Nagoya city Univ). These PCR products from established antigen specific T cell clones by Dp and those from AD lesions were electrophoresed in the same gels. In all patients with AD,we found certain distinct bands, which migrated to the same position, in the lanes of AD involved skin and the established antigen specific T clone by Dp. DNA sequence analysis previously confirmed that the presence of bands migrating to the same position denote identical T cell clones with a similar nucleotide sequence in CDR3 of TCR BV chain, Therefore, we next performed DNA sequence analysis of the several identically-migrating bands of AD lesions and T cell clones, which revealed that they have the same nucleotide sequence.
From those results, we speculate that oligoclonally accumulated T cell clonotypes, one of which is Dp specific, exist in the AD lesions. Less
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