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Objective:To investigate changes in the peripheral interleukin-35 (IL-35) level in patients with alopecia areata, and to assess its modulatory effect on regulatory T (Treg) cell activities.Methods:Totally, 81 patients with alopecia areata (alopecia areata group) and 27 healthy volunteers (control group) were enrolled from Shanxi Provincial People′s Hospital between December 2019 and January 2021. Sera and peripheral blood mononuclear cells (PBMCs) were isolated. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the serum IL-35 level, real-time fluorescence-based quantitative PCR to determine the mRNA expression of IL-35 subunits EBI3 and IL-12p35, and flow cytometry to determine the proportion of CD4 + CD25 + CD127 dim/- Treg cells. Sorted Treg cells were stimulated by recombinant human IL-35, ELISA was performed to detect levels of perforin and granzyme B in the culture supernatant, and real-time fluorescence-based quantitative PCR to determine the mRNA expression of EBI3, IL-12p35, and immune checkpoint molecules, such as programmed death protein 1 (PD-1) , T cell immunoglobulin and mucin protein-3 (Tim-3) , cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and lymphocyte activation gene-3 (LAG-3) in Treg cells. IL-35-stimulated or unstimulated Treg cells were co-cultured with autologous PBMCs, and cell counting kit-8 (CCK8) assay was conducted to assess cellular proliferative activity. Measurement data were compared between 2 groups by using t test, comparisons among multiple groups were carried out by using one-way analysis of variance, correlation analysis was carried out by using Pearson correlation analysis, and enumeration data were compared by using chi-square test. Results:Compared with the control group, the alopecia areata group showed significantly decreased IL-35 levels (90.10 ± 11.98 ng/L vs. 100.74 ± 28.71 ng/L, t= 2.71, P= 0.008) , mRNA expression of EBI3 and IL-12p35 in PBMCs (EBI3: 1.06 ± 0.15 vs. 1.25 ± 0.11, t= 6.09, P < 0.001; IL-12p35: 1.00 ± 0.15 vs. 1.38 ± 0.22, t= 10.16, P < 0.001) , and proportions of Treg cells (5.91% ± 1.17% vs. 6.85% ± 1.23%, t= 3.54, P= 0.001) . In the alopecia areata group, the proportion of Treg cells was positively correlated with the serum IL-35 level ( r= 0.25, P= 0.026) , and the mRNA expression of EBI3 and IL-12p35 in PBMCs ( r= 0.31, 0.24, P= 0.004, 0.032, respectively) . Compared with the control group, the unstimulated Treg cells from the alopecia areata group showed significantly decreased supernatant levels of perforin and granzyme B, mRNA expression of EBI3, IL-12p35 and immune checkpoint molecules ( P < 0.05 or 0.001) , as well as weakened inhibitory effect on the proliferative activity of PBMCs ( P= 0.013) . There was no significant difference in the level of perforin or granzyme B between the recombinant human IL-35-stimulated and unstimulated Treg cells from the patients with alopecia areata (both P > 0.05) . However, the mRNA expression of EBI3, IL-12p35 and immune checkpoint molecules was significantly higher in the IL-35-stimulated Treg cells than in the unstimulated Treg cells in the alopecia areata group ( P < 0.05 or 0.001) , and the inhibitory effect on the proliferative activity of PBMCs was also significantly enhanced in the IL-35-stimulated Treg cells compared with the unstimulated Treg cells ( P= 0.037) . Conclusion:The peripheral IL-35 level was significantly decreased in the patients with alopecia areata, which was closely associated with reduced activities of Treg cells, and IL-35 may be involved in the occurrence of alopecia areata.
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ObjectiveTo investigate the changes in peripheral blood mononuclear cells ［CD8+ T lymphocytes and natural killer (NK) cells］ and levels of perforin and granzyme B in these cells in children with infectious mononucleosis (IM), as well as their clinical significance in liver injury. MethodsA total of 60 children who met the diagnostic criteria for IM were enrolled, and 30 healthy children were enrolled as control group. With the help of cell surface markers and cytokine staining, flow cytometry was performed to analyze CD8+ T lymphocytes, NK cells, and the expression of perforin and granzyme B. The t-test was used for comparison of continuous data between groups; a one-way analysis of variance was used for comparison between three groups, and the Dunnett-t test was used for further comparison between two groups. ResultsAmong the 60 children with IM, the incidence rate of liver injury was 50% (30/60); 18 (60%) children had an alanine aminotransferase (ALT) level of ＜200 IU/L, 10 (33.3%) had an ALT level of ≥200 IU/L and ＜400 IU/L, and 2 (6.67%) had an ALT level of ≥400 IU/L. All children had normal liver functions after one month of treatment. Compared with the control group, the non-liver injury IM group and the liver injury IM group had significant increases in the expression of perforin and granzyme B in CD8+ T lymphocytes (all P＜0.05). Compared with the non-liver injury subgroup of IM patients, the liver injury subgroup had a significantly higher percentage of CD8+ T lymphocytes and significantly higher expression of perforin and granzyme B in NK cells (all P＜0.05). ConclusionThere is a high incidence rate of liver injury in children with IM, mainly mild or moderate elevation of aminotransferases, which are self-limiting and can be returned to normal. High levels of perforin and granzyme B in NK cells and a high percentage of CD8+ T lymphocytes are the cause of liver injury.
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Objective To investigate the expression of GrB and Bid gene in gastric carcinoma and development of gastric carcinoma,exploring their effect on the mechanism of immune tolerance of tumor.Methods S-P method was used to detect the expression status of GrB and Bid gene in gastric carcinoma and margin tissue.Results The positive expression rate of GrB of gastric carcinoma tissues was 47.62％,the positive rate in the margin tissue was 40.48％,there was no significant difference(P ＞ 0.05) ; The positive expression rate of Bid of gastric carcinoma tissues (26.19％) was significantly lower than that of margin tissue(76.19％) (P ＜ 0.05) ; The expression of GrB had significantly correlation with TNM stage and lymph node metastasis of the tumor (P ＜ 0.05),the expression of Bid in gastric cancer had significantly correlation with differentiation,invasive depth,TNM stage and lymph node metastasis of the tumor (P ＜ 0.05) ; the expression of GrB was negatively correlated with that of Bid in gastric cancaer(rs =-0.311,P ＜ 0.05).Conclusion The expression of GrB and Bid may be involved in development of gastric carcinoma,down-regulated or scarce expression of Bid may be cause cancer cells escaping from endogenous or exogenous GrB-mediated cell apoptosis,and acquire immune tolerance,potential,to escape the immune response.