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Objective:To investigate changes of natural killer (NK) cell subsets and interleukin-18 (IL-18) level in peripheral blood of patients with alopecia areata, and to assess the regulatory effect of IL-18 on NK cell activity.Methods:A total of 67 patients with alopecia areata (alopecia areata group) and 25 healthy volunteers (control group) were collected from Shanxi Provincial People′s Hospital between December 2019 and January 2021. Peripheral blood mononuclear cells (PBMCs) and plasma were isolated. The percentage of NK cell subsets was investigated by flow cytometry, and plasma IL-18 level was measured by enzyme-linked immunosorbent assay. PBMCs were stimulated with recombinant human IL-18, and co-culture systems of PBMCs with 721.221 cells, K562 cells and P815-Ab cells were established separately. NK cell function was assessed by determining the percentage of CD107a-expressing NK cells and fluorescence intensity of CD16 + NK cells. Comparisons between groups were performed using t test or paired t test. Results:Compared with the control group, the alopecia areata group showed significantly decreased percentage of CD56 +CD16 - NK cells (8.12% ± 3.14% vs. 10.78% ± 4.08%, t = 3.33, P = 0.001) , but significantly increased percentage of CD56 +CD16 + NK cells (46.08% ± 15.21% vs. 32.14% ± 10.45%, t = 4.22, P < 0.001) , and there was no significant difference in the percentage of CD56 -CD16 + NK cells between the alopecia areata group and control group (28.81% ± 8.65% vs. 27.09% ± 7.62%, t = 0.88, P = 0.383) . The plasma IL-18 level was significantly higher in the alopecia areata group than in the control group (112.0 ± 23.72 pg/ml vs. 99.34 ± 15.15 pg/ml, t = 2.48, P = 0.015) . After co-culture with 721.221 cells, K562 cells and P815-Ab cells, the percentage of CD107a-expressing NK cells was significantly higher in NK cells from the alopecia areata group (9.53% ± 1.70%, 5.15% ± 1.35%, 6.50% ± 1.64%, respectively) than in those from the control group (5.00% ± 1.17%, 4.40% ± 1.09%, 5.13% ± 1.36%, respectively, all P < 0.05) . After the stimulation with P815-Ab cells, the alopecia areata group showed significantly decreased fluorescence intensity of CD16 + NK cells (151.10% ± 59.30%) compared with the control group (221.90% ± 93.56%, t = 4.31, P < 0.001) . After IL-18 stimulation, the percentage of CD107a-expressing NK cells significantly increased in the co-culture system of NK cells with 721.221 cells compared with the unstimulated co-culture system (14.47% ± 2.67% vs. 9.93% ± 1.94%, t = 6.00, P < 0.001) , while there was no significant difference between the IL-8-stimulated co-culture system of NK cells with K562 cells or P815-Ab cells and the unstimulated co-culture systems (both P > 0.05) . Conclusion:IL-18 could enhance NK cell activity in patients with alopecia areata, likely by promoting natural cytotoxicity receptor-mediated cytotoxicity.
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Objective@#To investigate the role of lysosomes in manganese-induced toxicity in human neuroblastoma SK-N-SH cells.@*Methods@#SK-N-SH cells were treated with MnCl2 at doses of 0.062 5, 0.125, 0.25, 0.5, 1.0, 2.0 and 4.0 mmol/L for 24 h, and the cell viability was detected by MTT assay. Cells were treated with MnCl2 at doses of 0.125, 0.25, 0.5 and 1.0mmol/L for 24 h, and lysosomes labeled with lysotracker red were observed by laser confocal microscopy, the expression levels of LAMP1 and CTSD were detected by western blot, and CTSD activity was detected by Cathepsin D Activity Fluorometric Assay Kit.@*Results@#Compared with the control group, the survival rates of SK-N-SH cells were decreased significantly in the 0.5-4.0 mmol/L MnCl2 treatment groups (P<0.01) , the relative fluorescence intensities of 0.5 and 1.0 mmol/L MnCl2 treatment groups were increased (P<0.01) . Compared with the control group, the 0.125-0.5 mmol/L MnCl2 treatment groups had significant increase in the the expression of LAMP1 (P<0.01) . Compared with the control group, the expression of m-CTSD was significantly increased at the does of 0.125-0.25 mmol/L MnCl2, while it was decreased at the does of 1.0 mmol/L (P<0.01) . Otherwise, it wasn’t observed significant difference of the activity of CTSD between different MnCl2 treatment groups.@*Conclusion@#MnCl2 could cause cytotoxicity in SK-N-SH cells. Lysosomes may play a normal function at low doses of manganese, but they may be damaged at high doses of manganese. As an organelle that can degradate substrates in autophagy, lysosomes participate in the neurotoxic mechanism of manganese.
ABSTRACT
It is a challenge in diagnosis of hemophagocytic lymphohistocytosis (HLH).Rapid diagnosis is the key to make decisions in clinical management of HLH.The timely and reliable diagnosis of HLH can be assisted by laboratory tests including nature killer (NK) cell activity, sCD25, hemophagocytosis, ferritin, surface CD107a, and genetic testing.Here is introduced lab assays and evaluate the clinical applications in HLH.The aim is to raise awareness of the values of laboratory evaluations in diagnosing HLH.
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Objective To investigate the effects of high density lipoprotein (HDL) on thrombin-activated platelet al-pha-granulemembrane protein (CD62P) and lysosome intact membrane protein (CD63) expressions in vitro. Methods The equivalent volume of washed platelets prepared by hand was preincubated with HDL (1 g/L) in 37℃water for 15 minutes, which was then stimulated with different concentrations of thrombin (0.5 U/mL, 1 U/mL and 10 U/mL) for 10 minutes in wa-ter of 37℃. Meanwhile another three groups of washed platelets were incubated with thrombin (0.5 U/mL, 1 U/mL and 10 U/mL) for 10 minutes, respectively. The CD62P and CD63 from each sample were analyzed by flow cytometry (FCM). Results The CD62P positive rates of HDL-preincubated groups were significantly lower than those of different concentrations of thrombin groups (0.5 U/mL,1 U/mL and 10 U/mL) in the absence of HDL (11.55%± 1.34% vs 18.14%± 1.50%, 17.19%± 0.17% vs 26.24%± 0.77% and 19.79%± 0.32% vs 80.38%± 5.66%,P ＜ 0.01). Meanwhile, The CD63 positive rates of HDL-preincubated groups were also significantly lower than those of thrombin-treated (0.5 U/mL, 1 U/mL and 10 U/mL) groups without HDL, namely,2.92%±0.22%vs 8.09%±0.48%(P＜0.001), 4.20%±0.98%vs 14.15%±1.39%(P＜0.001) and 5.12%± 0.09% vs 24.48%± 1.71%(P ＜ 0.01). Conclusion HDL inhibits the expression of CD62P and CD63 on throm-bin-stimulated platelets in vitro.