OBJECTIVE: This study aimed to compare the mean percentages and absolute counts of CD4+ memory T cell subsets between: (i) non-allergic controls and AR patients; (ii) mild AR patients and moderate-severe AR patients.
METHODS: Sensitization to Dermatophagoides farinae and Dermatophagoides pteronyssinus were determined in 33 non -allergic controls, 28 mild AR and 29 moderate-severe AR patients. Flow cytometry was used to determine the percentage of CD4+ na?ve (TN; CD45RA+CCR7+), central memory (TCM; CD45RA-CCR7+), effector memory (TEM; CD45RA-CCR7-) and TEMRA (CD45RA+CCR7-) T cells from the peripheral blood. The absolute counts of CD4+ T cell subsets were obtained by dual platform method from flow cytometer and hematology analyzer.
RESULTS: There were no significant differences in the mean percentages and absolute counts of CD4+ T cell subsets between non-allergic controls and AR patients sensitized to HDMs. However, there were significant reduction in the mean percentage (p=0.0307) and absolute count (p=0.0309) of CD4+ TEMRA cells in moderate-severe AR patients compared to mild AR patients sensitized to HDMs and 13/24 (54.2%) moderate-severe AR patients sensitized to HDMs had persistent symptoms.
CONCLUSION: Reduction in the mean percentage and absolute count of CD4+CD45RA+CCR7- TEMRA cells were observed in moderate-severe AR patients compared to mild AR patients in our population of AR patients sensitized to HDMs.
MATERIALS AND METHODS: The differentiation of fibroblast-like cells from SHED was carried out by using specific human recombinant connective tissue growth factor (CTGF). To characterize fibroblastic differentiation, the induced cells were subjected to morphological changes, proliferation rate, gene expression analysis using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), flow cytometry, and immunofluorescence staining. The commercial primary human gingival fibroblasts served as positive control in this study.
RESULTS: The results from characterization analysis were compared with that of commercial cells to ensure that the cells differentiated from SHED were fibroblast-like cells. The results showed the inductive effect of CTGF for fibroblastic differentiation in SHED. SHED-derived fibroblasts were successfully characterized despite having similar morphological appearance, i.e., (i) significant proliferation rate between fibroblast-like cells and SHED, (ii) high expression of fibroblast-associated markers in qRT-PCR analysis, and (iii) positive staining against collagen type 1, fibroblast-specific protein 1, and human thymic fibroblasts in flow cytometry analysis and immunofluorescence staining. The same expression patterns were found in primary human gingival fibroblasts, respectively. SHED as negative control showed lower expression or no signal, thus confirming the cells differentiated from SHED were fibroblast-like cells.
CONCLUSIONS: Taken together, the protocol adopted in this study suggests CTGF to be an appropriate inducer in the differentiation of SHED into fibroblast-like cells.
CLINICAL RELEVANCE: The fibroblast-like cells differentiated from SHED could be used in future in vitro and in vivo dental tissue regeneration studies as well as in clinical applications where these cells are needed.