| Objective:1. To evaluate and compare the efficacy of different dosage regime of recombinant human tumor necrosis factor receptor-Fc fusion protein ( rhTNFR-Fc) in Chinese ankylosing spondylitis (AS ) patients.2. To investigate the optimal medication regime of rhTNFR-Fc for Chinese AS.Methods:1. This was a randomized, open-lable trial. Twenty active AS patients were randomly assigned to receive subcutaneous injections of rhTNFR-Fc 25 mg ( 2/w ) or 50 mg ( 1/w ) for 8 times. Clinical assessment was carried out after 0, 1, 4, 8 administration and 20 days after last administration. Laboratory parameters were measured after 0, 4, 8 administration.2 The primary efficacy endpoint was achievement of Assessment in Ankylosing Spondylitis (ASAS) 20%. In addition, the ASAS 50% response rates were also evaluated. The following Indices were also evaluated: the Bath Ankylosing Spondylitis Functional Index (BASFI), a 50% improvement or more of the initial Bath Ankylosing Spondylitis Disease Activity Index (BASDAI 50), as well as improvement in the serum C-reactive protein (CRP) level and Erythrocyte sedimentation rate ( ESR ) during the treatment.3. Data were analyzed using the statistical software CHISS for windows. For comparisons between visits, the paired t-test and the paired Wilcoxon rank sumtest were applied. All comparisons were two-sided. A significance level of 5%was used. No adjustment for repeated significance testing was done.Rusults:There was no significant difference in patients reaching ASAS20, ASAS50 and BASDAI50 after the first, fourth and eighth injection and 20 days after injection completion comparing the patients in the 25 mg group with that in the 50 mg group(P > 0.05). Also, in different medication interval and the same accumulated dose ( 200 mg ), there was no difference in the number of patients who reached ASAS20, ASAS50 and BASDAI50 in both groups, except that BASFI in 50 mg group could reach earlier improvement ( P=0.0289 ). Changes of other indices during injections were not observed.Conclusion:1. Two doses and different medication interval of rhTNFR-Fc may take same curative effect.2. Meanwhile, 50 mg group reached improvement of function earlier.3. 50 mg ( 1/w ) administration group had better convenient and economic.Objective:To study the effect of TNF-α antagonist (rhTNFR-Fc) treatment on the peripheral T cell reactivity (cytokine secretion and proliferation) of ankylosing spondylitis (AS) patients. Methods:1. Peripheral blood mononuclear cells (PBMC) were collected from 10 patients with AS at baseline, after the first, fourth and eighth subcutaneous injections and 20 days after injection completion, and from 13 healthy controls. The number of T cells that secret TNF-α, IL-6, IFN-γ and IL-2 were detected by ELISPOT method. And their correlation with the clinical parameters including BASDAI, BASFI, PGA, night pain, ESR, CRP and PLT was analyzed. And number of T cells level secreting cytokins predicting the clinical response to rhTNFR-Fc in AS were analysed with Logistic regression liklihood ratio tests.2. PBMC were collected from 10 patients with AS at baseline, after the first, fourth and eighth injection and from 13 healthy controls. CD4+ T cell proliferation after stimulation were assayed with WST-1 live cell staining method. And its correlation with the clinical parameters was analyzed.Rusults:1. ELISPOT results(1) TNF-α Compared to the healthy controls, the number of TNF-α producing cells was significant higher in AS patients (P=0.0353) at baseline. A trend of decrease-increase-decrease of TNF was observed after treatment. There was a significant decrease in the number of secreting TNF-α T cell (P=0.0212) 20 days after injection completion of rhTNFR-Fc treatment. Percentage changes ofthe number of TNF-α-secreting cells were all negative value.(2) IL-6: When compared to the healthy controls, the number of IL-6 producing cells was significant higher in AS patients (P=0.0015) at baseline. A trend of decrease-increase-decrease of IL-6 was also observed after treatment. And the number of secreting IL-6 T cell increased significantly (P=0.0253) 20 days after injection completion of rhTNFR-Fc treatment induced. Percentage change of the number of IL-6-secreting cells were all negative value.(3) IL-2: When compared to the healthy controls, the number of IL-2 producing cells was significant higher in AS patients (P value were 0.0004, 0.0371, 0.0221, 0.0027 respectively) at baseline, after the first, fourth and eighth injection and 20 days after injection completion. There was a decrease-increase-decrease trend after treatment but no significant change was found when compared to the baseline. Percentage change of the number of IL-2-secreting cells after the first and injection completion were negative value, and the change after the fourth injection surpassed the baseline.(4) IFN-γ: When compared to the healthy controls, the number of IL-2 producing cells was significant higher in AS patients (P value were respectively 0.0016, 0.0483) at baseline and 20 days after injection completion. A trend of decrease-increase-decrease of TNF was observed after treatment. There was clearly decreasing change after the first injection(P=0.0497 compared to the baseline). Percentage change of the number of IL-6-secreting cells were all negative value.(5) The number of IL-2, IFN-γ-secreting cells were not correlated with the clinical parameters. At the baseline, the number of IL-6-secreting cells were correlated with BASFI. The number of IL-6-secreting cells after the fourth injection were correlated with BASDAI, night pain, global back pain, BASDAI-E. The number of TNF-α-secreting cells after the fourth injection were correlated with BASDAI-E.2. CD4+T cell proliferation rate(1) The proliferation rate of CD4+ T cells at the baseline and after the forth injection was higher than that in the healthy control(P value were respectively 0.0034, 0.0494). Compareing to the baseline there was no significant change.(2) The change of the proliferation rate of CD4+ T cells were all negative value.(3) No correlation was found between the proliferation rate of CD4+ T cells and clinical parameters.3. Cytokine levels and clinical parameters improvement at the evaluation endpointCytokine levels decreased at the evaluation endpoint, and BASDAI, BASFI, PGA, night pain, global back pain, ESR, CRP, PLT decreased either (decreased 63.8%, 50%, 56.7%, 70%, 97.9%, 72.4%, 66.9%, 25.3% respectively).4. Logistic regression analysis showed Cytokine levels at baseline could not predicted ASAS50 response.Conclusion:1. AS patients had a up regulation of the ability of T cells to produce cytokine.2. The treatment with rhTNFR-Fc in AS induced a down regulation of the ability of T cells to produce TNF-α, IL-6, IFN-γ and IL-2.3. The treatment with rhTNFR-Fc in active AS induced a reduction of CD4+T cell proliferation. 4. The number of IL-6-secreting cells had significant correlation with clinical parameters.Objective:(1) To investigate the role of CD4+CD25+ regulatory T cells (Treg) and myeloid dendritic cells (mDC) on the pathogenesis of AS.(2) To study the mechanism of TNF-α blocker on the treatment of AS by counting the number of Treg and mDC before and after the treatment.(3) To investigate the curative effects evaluation of Treg and mDC in AS.(4) To investigate the interactive of Treg, mDC and TNF-α in AS.Methods:1. Peripheral blood from 21 AS patients treated with 50 mg rhTNFR-Fc and 15 AS patients treated with placebo were investigated at week 0, week 2 and week 6 during a randomized, double-blind, placebo-controlled trial. The 19 healthy subjects were mainly heathy blood donors. Three-color flow cytometry analysis were used to investigate the change of the number of Treg and mDC before and after the treatment. And their correlation with the clinical parameters was analyzed. And number of MHC Class II mDC level predicting the clinical response to rhTNFR-Fc in AS were analysed with Logistic regression liklihood ratio tests.Rusults:1. The number of CD4+CD25high regulatory T cells in AS patient was lower than that in healthy controls, but the difference was not statistically significant. CD4+CD25+ regulatory T cell in AS patient was significant lower than in healthy controls(P=0.0013). After 6 weeks of rhTNFR-Fc treatment, there was not a significant increase in Treg levels when compared with week 0 (P>0.05).2. AS patients had less MHC Class II mDC cells than healthycontrols(p=0.0083). A significant increase of MHC Class II mDC cells was observed after 6 weeks of rhTNFR-Fc treatment while no change was recorded in placebo group. Meanwhile, measured by flow cytometry, MHC Class I positive myeloid dendritic cells (Lin-/CD11c+/HLA-ABC+) in AS patients was slightly less than in healthy controls (not statistically significant). No significant change in MHC Class I mDC number was observed after drug treatment. The number of MHC Class II mDC cells were not correlated with clinical parameters.3. Logistic regression analysis showed MHC Class II mDC cells levels at baseline could not predicted ASAS50 response.Conclusion:1. The down regulation of CD4+CD25+T cells may play a role on the pathogenesis of AS.2. The treatment with rhTNFR-Fc in AS induced a significant down regulation of MHC-II DC.
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