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Objective To investigate the significant of peripheral CD4+CD69+T lymphocytes in patients with autoimmune hemolytic anemia (AIHA)/Evans syndrome (ES).Methods In this study peripheral blood samples from 32 patients with AIHA/ES (15 hemolytic episode patients,17 remission patients) and 13 healthy controls were collected.Patients with AIHA/ES were recruited in Tianjin Medical University General Hospital from October 2015 to May 2016.The percentages of CD69+ T lymphocytes were analyzed by flow cytometry.The expression of CD69 mRNA in CD4+T lymphocytes which was sorted from peripheral blood by magnetic activated cell sorting (MACS) was detected using real-time PCR.Soluable CD69 was measured by ELISA.Results In hemolytic episode patients,the ratio of CD3+CD69+/CD3+T lymphocytes [(3.08 ± 1.48)％] was significantly higher than that in healthy controls [(1.28 ± 0.83)％,P＜0.01] and in remission group[(1.96± 1.33)％,P＜0.05].The absolute count of CD3+CD69+T lymphocytes in hemolytic episode group [(2.94± 1.81)× 107/L] was higher than that in healthy controls [(1.48± 1.42)× 107/L,P＜0.05].The ratio of CD3+CD4+CD69+/CD3+CD4+T cells in hemolytic episode group [(2.16± 1.56)％] was significantly higher than that in remission group [(1.16±0.62)％,P＜0.05] and healthy controls[(0.94±0.78)％,P＜0.05].The quantity of CD3+CD4+CD69+T lymphocytes in hemolytic episode group[(1.04±0.98)× 107/L] was higher than in healthy controls [(0.44± 0.38) × 107 / L,P＜0.05].The ratio of CD3+CD8+CD69+/CD3+ CD8+T lymphocyte in hemolytic episode group [(4.87±2.56)％] was significantly higher than that in healthy controls[(1.83± 1.27)％,P＜0.01].The quantity of CD3+CDs+CD69+T lymphocytes in three groups did not show significant difference.The ratio of CD3+CD4+CD69+/CD3+CD4+T lymphocytes in hemolytic episode group was negatively correlated with hemoglobin (Hb) (P＜0.01),positively correlated with the percentage of reticulocytes (Ret％)(P=0.01)total bilirubin(TBil),indirect bilirubin(IBil) (P＜0.01) and not correlated with absolute reticulocytes count,lactic dehydrogenase (LDH),complement 3(C3),complement 4 (C4).The ratio of CD3+CD4+CD69+/CD3+CD4+T lymphocytes in remission group was negatively correlated with Hb (P＜0.05).In hemolytic episode patients CD69 mRNA (32.26±35.11) was significantly higher than that in remission group(6.05±5.87)(P＜0.05)and healthy controls (1.76± 1.85)(P＜0.01).CD69 mRNA in remission group was significantly higher than healthy controls (P＜0.05).Serum CD69 in hemolytic episode patients [(494.21 ± 16.06) ng/L] was significantly higher than that in healthy controls [(441.39± 104.6) ng/L,P＜0.05].Conclusion Our findings suggest that the proportion of CD4+CD69+ T lymphocytes increase in AIHA/ES patients,which is correlated with the severity of disease.
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Objective To investigate changes in expressions of activation antigens CD69 and HLA-DR in CD3+T lymphocytes in peripheral blood and skin lesions in patients with psoriasis vulgaris. Methods Peripheral blood samples were obtained from 20 patients with psoriasis vulgaris and 20 healthy controls, and skin specimens from the lesions of 15 out of the 20 patients and 10 healthy controls. Flow cytometry was performed to quantify the expressions of CD69 and HLA-DR in peripheral blood CD3+T cells, and an immunohistochemical study to measure the expression of HLA-DR in skin specimens. Statistical analysis was carried out by a two-sample t-test and Pearson correlation analysis with the SPSS 19.0 software. Results Compared with the healthy controls, the patients with psoriasis vulgaris showed increased expression rates of CD69 (4.70%± 1.90%vs. 1.56%± 0.95%, t=6.629, P<0.01)and HLA-DR (8.97%± 1.79% vs. 3.02% ± 1.15%, t= 6.204, P< 0.01)in peripheral blood. Pearson correlation analysis revealed that the percentage of CD3+HLA-DR+cells in peripheral blood was positively correlated with the psoriasis area and severity index (PASI)score (r=0.5626, P<0.05). The expression rate of HLA-DR was significantly higher in the dermis (64.87%± 17.31%vs. 19.80%± 5.69%, t=7.916, P<0.01), but lower in the epidermis(11.80%± 5.55%vs. 27.40%± 8.61%, t=5.479, P<0.01)in the psoriatic specimens compared with the control specimens. Immunohistochemically, HLA-DR was widely expressed in the dermis of psoriatic lesions, but mainly distributed around blood vessels in the control skin. Conclusions There is an aberrant activation of CD3+T cells in peripheral blood and inflammatory cells in skin lesions in patients with psoriasis vulgaris, and the percentage of CD3 +HLA-DR+ cells in peripheral blood is correlated with the severity of psoriasis vulagaris.
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Objective To investigate the effects of psoriatic keratinocytes on the expression of CD25 and CD69 in T lymphocytes. Methods Keratinocytes were isolated from the biopsy samples resected from the lesions and adjacent non-lesional area of 10 patients with psoriasis, and cultured in 5% CO2 at 37 ℃ in 24-well plates. Density gradient centrifugalization and glass adherence method were applied to detach peripheral blood mononuclear cells (PBMC) and peripheral blood T lymphocytes (PBTL) from anticoagulant blood samples of the same 10 psoriatic patients and 10 normal controls. PBMCs of 1×105/well were added to the wells containing cultured keratinocytcs of 1×105/well, then gamma rays were used to inactivate these cells. Following that, PBTLs of 1×106/well were inoculated into the 24-well plate containing inactivated keratinocytes and PBMCs, and cultured in 5% CO2 at 37 ℃. Those PBTLs cultured without the presence of keratinocytes or PBMCs served as the natural growth control. Three days later, flow cytometry was performed to detect the expression of CD25 and CD69 in PBTLs. Results There was a significant increase in the expression of CD25 and CD69 in psoriatic PBTLs cocultured with lesional kcratinocytes compared with those cocultured with non-lesional keratinocytes and natural psoriatic controls. Also, the expression of CD25 and CD69 was increased in normal PBTLs cocultured with lesional or non-lesional keratinocytes of psoriatc patients than those in the natural normal controls. No significant differenco was observed in the expression of CD25 or CD69 between psoriatic PBTLs cocultured with non-lesional keratinocytes and natural psoriatic PBTLs, or between the normal PBTLs cocultrred with lesional keratinocytes and those with non-lesienal keratinocytes (P>0.05). Conclusions Psoriatic keratinocytes may act as an autoantigen to trigger autoimmune response and eventually lead to a chronic local inflammation in patients with psoriasis.
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0.05).Expression of CD25and CD69on CD8 + T cells was significantly higher in patients(16.30%?5.97%and32.61%?6.36%)than that in control group(9.21%?2.14%and10.03%?2.35%)(P
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AIM: To study cellular and molecular mechanism involved in increasing susceptibility of infection in psychological stress persons. METHODS: Comparative studies were performed with double staining and flow cytometry analysis on immunophenotyping and in vitro expression of early activating surface molecule CD69 in response to mitogens on T cells from peripheral blood of 20 healthy college student volunteers before and after psychological stress. A series of term final examinations was defined as psychological stress. RESULTS: Immunophenotyping analysis showed no statistically significant difference in the percentage of CD2, CD3, CD4, CD8, CD19, CD20, CD16 and CD56 positive lymphocyte populations before and after psychological stress. There was a statistically significant decrease in the in vitro expression of CD69 in response to polyclonal stimulators on the T cells from persons after psychological stress than those before psychological stress. The percentage of CD69 expression (CD69+CD3+/CD3+%) in response to PHA and PDB in the whole blood culture for 72 hours decreased respectively from 28.1?4.1 and 80.7?6.8 on the T cells obtained before psychological stress to 17.6?3.8 and 65.8?7.9 on those obtained after psychological stress, while there was no statistically significant difference between the CD69 expression rates without stimulators on the T cells obtained before and after psychological stress. CONCLUSIONS: The effects of psychological stress to immune system is not on the level of changing proportions of the sub-populations within peripheral blood lymphocytes. Psychological stress can decrease the activating response of T cells in healthy persons, which may be responsible for the increase of susceptibility to infection in the psychological stress persons.