| Objective:To evaluate the relationship between the plasma TPO level and the outcome of treatment of steroids and correlation of abnormalities of T, B cell function with the activities of the illness in the adult ITP patients.Methods:1,Collected clinical data and blood samples of the adult ITP patients in Peking Union Medical College Hospital from July 2008 to July 2009.2,Three groups haved been enrolled to the study:primary immune thrombocytopenia (PITP),ITP secondary to Systemic lupus erythematosus (SLE-ITP) and normal control.Plasma thrombopoietin (TPO) levels were measured using Enzyme-linked immunosorbent assay (Elisa) and compared among the three groups.3,Cytometric bead array (CBA) was used to measure the plasma concentration of seven Thl/Th2/Thl7-associated cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, Tumor necrosis factor (TNF), interferon-gamma (IFN-γ)).4,Anti-GPⅡb/Ⅲa and anti-GPIb/IX autoantibodis were detected by modified monoclonal antibody immobilization of platelet antigens assay (MAIPA).5,B cell enzyme-linked immunosorbent spot (ELISPOT) assay were used to detect human effective and memory B cells producing anti-GPⅡb/Ⅲa antibodies in human peripheral blood mononuclear cells(PBMCs).6,Concentrations of Th1/Th2/Th17-associated cytokines in the supernatant of mononuclear cells culture were determined by Cytometric bead array (CBA).Results:1.252 thrombocytopenic patients, including 132 PITP and 54 secondary ITP, were enrolled into the study. Among 132 case of PITP, female-to-male ratio was 81:51, with a median age of 38 years old, median platelet count of 25 x 109/L, median megakaryocyte number in bone marrow was 186 per smear. Out of 132 cases,33 (25%) were ANA positive,4 cases LA positive,3 casesβ2GP1 positive.88 patients who were treated by steroids have received telephone inquisition in April 2010. Among them,53 (60%) achieved complete remission (CR),14 cases (16%) had no response. In comparison with the ANA negative patients, ANA positive patients had more steroid non-responders and higher recrudescence (P<0.05)2. PITP group (256.20±846.20pg/ml) and SLE-ITP group (195.69±383.15 pg/ml) had markedly elevated TPO levels in comparison with the control (22.83±32.46 pg/ml) (P< 0.01).There was a significant negative correlation between their plasma TPO concentrations and platelet counts (r=-0.488, P<0.01), no significant correlation was found between plasma TPO concentrations and megakaryocyte count (r=-0.160, P=0.116).There was no significant difference in TPO levels between steroid CR and steroid non-responders group.3. The levels of IL-2, IL-4, IL-6, IL-10, TNF, IFN-y and IL-17A had no significant difference between PITP and normal control. The IL-10 level in SLE-ITP was significant higher than that in PITP. There was a weakly positive correlation between platelet counts and the level of IL-17A (r=0.149, P=0.038),and also between patelet and IFN-y (r=0.142, P=0.046). The level of IL-17A was positively correlative to level of IL-2,IL-4,IL-6,IL-10,TNF or IFN-y (r=0.647,0.892,0.879,0.644,0.253,0.921,P<0.01)4. The positive rates of antibodies against platelet GPIb and GPIIb in PITP group were 22.6% and 23.7%, whereas they were 46.2% and 57.7% in SLE-ITP group, respecitively. In 8 PITP-CR patients, one patient had positive MAIPA-GpIIb only. In comparison with patients with negative MAIPA detection, those who had positive detection had higher levels of IL-6 (P<0.05) and IL-10 (P<0.01)5. The frequencies of GPIIb-IIIa-reactive B cells determined by ELI SPOT were 8.18±27.22/106PBMC in PITP group (n=70),7.90±20.81/106PBMC in SLE-ITP group (n-16) and 3.50±4.47/106 PBMC in PITP—CR group, respectively. There were no significant differences among three groups. The frequency in PITP-active group was significantly higher than that in control group (1±1.13 per 106PBMC,n=10), P value was 0.03. The total reactive B cell frequencies in PWM+SAC stimulated group was significantly higher than no stimulate group (288.26±355.43/105 PBMC vs 28.53±54.82/105 PBMC).The actual gpIIb-IIIa-reactive memory B cells frequencies in PITP-active group (27.35±30.13%) were significantly higher than SLE-ITP group (11.49±16.19%), but no significantly difference between PITP-active and PITP-CR groups.6. The levels of IL-4,IL-6,IL10,TNF and IL-17A in cell culture supernatant in PWM+SAC stimulated group were significantly higher than those in no stimulate group. There was a significant positive correlation between level of IL-4,IL-6,IL10,TNF or IL-17A in cell culture supernatant in PWM+SAC stimulated group and the number of total reactive B cells (P<0.01), also a significant positive correlation between levels of IL-4,IL-6,IL10 and actual gpIIb-IIIa-reactive memory B cells frequencies.Conclusions:1. Most cases of thrombocytopenia were primary ITP. Secondary ITP comprised heterogeneous disorders which were usually associated with systemic autoimmune diseases, especially SLE. In some PITP patients, ANA were positive, which might predict steroid refractory.2. The level of TPO in adult chronic PITP patients was significantly higher than that in healthy controls, and lower platelet count was associated with higher TPO level. The relationship between megakaryocyte hyperplasia, outcome of steroid therapy and TPO levels were not clear.3. No significant differences in the concentration of Th1/Th2/Th17 cytokines were observed between patients with PITP and the control.Th2 cells may play an important role in the production of platelet autoantibody, Th17 may be associated with the production of antibodies against platelet GPIb.4. The frequencies of GPIIb-IIIa-reactive B cell in peripheral blood from PITP and SLE-ITP were significantly higher than that from normal control. Detection of GPIIb-IIIa-reactive B cell by ELISPOT was not affected by steroid treatment, but may not be helpful in distinguishing PITP and SLE-ITP.5. Th2 cells played a key role in the activation of ITP memory B cells in vitro, steroid therapy could not clear the memory B cells out of peripheral blood of PITP patients. 6. The actual gpIIb-IIIa-reactive memory B cells frequencies in PITP-active group were significantly higher than SLE-ITP group, the actual gpIIb-IIIa-reactive memory B cells frequencies may be helpful in distinguishing PITP and SLE-ITP.
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