Risk factors of significant relapse and appropriate maintenance therapy strategy in SLE-associated immune thrombocytopenia.

Background: Systemic lupus erythematosus-associated immune thrombocytopenia (SLE-ITP) is characterized by relapse. The risk factors of relapse and appropriate maintenance therapy strategy deserve further exploration. Objectives: To determine the risk factors for relapse and appropriate maintenance therapy in significant SLE-ITP patients (a platelet count ⩽30 × 109/l) after the first complete response. Design: Retrospective cohort study using the medical records of 105 patients diagnosed as significant SLE-ITP in Fujian Medical University Union Hospital during December 2012 to March 2021. Patients were followed through a call for observations in January 2022. Methods: Data including demographics, initial clinical feature, induction and maintenance therapy, and outcome at the end of follow-up were analyzed. Risk factors for significant relapse were analyzed using multivariate logistic regression models. The cumulative hazard of significant relapse and the duration of response were estimated, and the differences in outcome between groups were compared using the Cox regression analysis. Results: A total of 65 significant SLE-ITP patients were eligible for the final analysis. Median [interquartile range (IQR)] follow-up duration and median [IQR] duration of response were 62.2 [41.0-79.6] months and 43.4 [20.3-68.7] months, respectively. After the first complete response, 19/65 (29.2%) had a significant relapse. Compared with sustained clinical remission (SCR) + sustained response (SR) group, significant relapse group had a higher proportion of discontinued patients (47.4% versus 8.7%, p = 0.001). Among the 13 discontinued patients, the duration of maintenance therapy of the patients in significant relapse group was significantly shorter than that of the patients in SCR + SR group (months, median [IQR], 43.1 [32.0-62.4] versus 12.0 [5.1-22.0], p = 0.009). Multivariate logistic regression analysis showed that drug withdrawal was an independent risk factor for significant relapse [odds ratio (OR) = 10.4, confidence interval (CI) 95% 2.2-47.8, p = 0.003]. There was no significant difference between glucocorticoids (GCs) + hydroxychloroquine (HCQ) group and GCs + HCQ + immunosuppressive agents (ISAs) group in significant relapse rate (26.7% versus 22.2%, p > 0.05). The two SR curves of GCs + HCQ and GCs + HCQ+ ISA group basically coincided by the Cox regression analysis, demonstrating comparable long-term outcomes (p > 0.05). Conclusion: Drug withdrawal, especially abrupt withdrawal with insufficient duration of maintenance therapy, is an independent risk factor for significant relapse of SLE-ITP. HCQ combined with GCs is expected to be the first choice of the maintenance therapy for SLE-ITP patients.

Paeonol inhibited TNFα-induced GM-CSF expression in fibroblast-like synoviocytes.

OBJECTIVES: Granulocyte macrophage colony-stimulating factor (GM-CSF) has been proved to be among the most important chemokines, playing a key role in rheumatoid arthritis (RA). However, the mechanism underlying the regulation of GM-CSF has not been established clearly yet. The aim of this study was to investigate the influence of paeonol in the expression of GM-CSF in fibroblast-like synoviocytes (FLS). METHODS: The expression of GM-CSF was detected both at protein and mRNA levels in FLS after the stimulation of TNF-α at diverse concentrations and times. And then GM-CSF was detected again after pre-treatment with paeonol. Phosphorylation of PI3K/Akt and expression of NF-κB and p-I-κB-αwere detected with western blot. Meanwhile, inhibitors of the pathways were used to investigate the mechanism of regulation of GM-CSF. RESULTS: Recombinant TNF-α up-regulated GM-CSF in a concentration- and time-dependent manner in FLS, which was significantly suppressed by paeonol. Paeonol also exerted its ability to suppress the promoting effects of TNF-α on the phosphorylation of PI3K/Akt and activation of NF-κB pathway. Administration of the inhibitors LY294002, perifosine, BAY11-7082, and SC-514 confirmed the roles of PI3K/Akt and NF-κB on the production of GM-CSF. Furthermore, TNF-α induced proliferation, while paeonol suppressed proliferation of FLS. CONCLUSION: These results demonstrate that paeonol suppressed TNF-α-induced GM-CSF production via the PI3K/Akt/NF-κB pathway.

[The effect of culture supernatant from MSCs on the proliferation and expression of COX-2, MMP-2 in rat synovial cell line].

OBJECTIVE: To investigate the effect of culture supernatant form mesenchymal stem cells (MSCs) on the proliferation of rat fibroblast-like synovial cell line RSC-364 and the expression of COX-2, MMP-2. METHODS: After isolation and identification of MSCs, the effect of MSCs supernatant liquid (MSCs-SL) on the proliferation of RSC-364 and the expression of COX-2, MMP-2 were detected by MTT assay and RT-PCR. RESULTS: MSCs-SL enhanced the proliferation of synoviocyte and elevated the expression of COX-2 and MMP-2 in synoviocytes (P < 0.05). CONCLUSION: MSCs may influence the proliferation of synoviocytes by secreting soluble cytokines and altered the expression level of some enzymes in synoviocytes.

[Effect of HepG2 cells modified with CD80-IgG1 Fc fusion protein on anti-tumor immune response by lymphocytes].

AIM: To investigate the anti-tumor immune response of lymphocytes elicited by HepG2 cells modified with CD80-IgG1 Fc fragment fusion protein (CD80-Fc). METHODS: HepG2 cells were modified with CD80-Fc, then the expression of CD80 on the cell surface was analyzed by flow cytometry (FCM). After mixed lymphocyte-tumour cell reaction (MLTR) of the modified HepG2 cells and peripheral lymphocytes of healthy volunteers, the proliferation and cytotoxicity of the lymphocytes were tested by MTT colorimetry and LDH release assay,respectively. RESULTS: CD80-Fc could be efficiently bound on HepG2 cells. HepG2 cells modified with CD80-Fc fusion protein dramatically elicited proliferation and cytotoxicity of normal lymphocytes. CONCLUSION: CD80 may play an important role in anti-tumour immune response. HepG2 cells modified with CD80-Fc fusion protein can elicited potent anti-tumor immune response. This fusion protein provides a convenient means for further potential use in immunotherapy of tumor.

[Construction of eukaryotic expression vector of CD80-IgG1 Fc fragment fusion protein and its expression in CHO cells].

AIM: To construct an eukaryotic expression vector of CD80-IgG1 Fc, and to express the fusion protein in CHO cells. METHODS: The gene encoding the CD80-IgG1 Fc fusion protein were constructed in eukaryotic expression vector pcDNA3.1(+) by means of T-A cloning and subcloning techniques, then was transfected into CHO cells for stable expression. The expression of the fusion protein was detected by Western blot and ELISA. RESULTS: DNA sequencing and restriction endonuclease digestion analysis indicated that the eukaryotic expression vector CD80-IgG1 Fc/pcDNA3.1(+) was successfully constructed. After the recombinant plasmid was transfected into CHO cells, the stable expression of the fusion protein was demonstrated by Western blot and ELISA. CONCLUSION: The eukaryotic expression vector of CD80-IgG1 Fc/pcDNA3.1(+) was successfully constructed and stably expressed in CHO cells, providing basis for anti-tumor study.

[Inhibition of CD40 expression on CA46 cell lines by hairpin siRNA eukaryotic expression vectors].

AIM: To construct human CD40 hairpin siRNA eukaryotic expression vectors and investigate their effects on CD40 expression, proliferation and apoptosis of CA46 cells. METHODS: Two DNA oligonucleotides encoding CD40 hairpin siRNAs were synthesized, and cloned into pSilenCircle to construct recombinant plasmid siCD40/pSilenCircle that expressed hairpin siRNAs under the control of pol III U6 promoter. At the same time, antiCD40/pSilenCircle encoding the antisense CD40 RNA and siFly/pSilenCircle encoding siRNA against luciferase were constructed as control vectors. The recombinants were identified by restriction enzyme digestion analysis and sequencing. The recombinant plasmids were transfected into CA46 cells. CD40 expression on CA46 cells and apoptosis were detected by flow cytometry. The proliferation of transfected CA46 cells was detected by MTS colorimetry. RESULTS: (1)siCD40/pSilenCircle, antiCD40/pSilenCircle and siFly/pSilenCircle had been constructed successfully. (2) CD40 expressions on siCD40/pSilenCircle- and antiCD40/pSilenCircle-transfected CA46 cells were significantly reduced, as compared with that on siFly/pSilenCircle-transfected CA46 cells (P<0.01 and P<0.05, respectively). Both hairpin siRNAs and antisense RNA had no influence on the proliferation and apoptosis of CA46 cells. CONCLUSION: Constructed siCD40/pSilenCircle and antiCD40/pSilenCircle can significantly inhibit CD40 expression, but have no significant influence on the proliferation and apoptosis of CA46 cells.

[Effect of antisense CD40 RNA on CD40 expression and proliferation of EB virus-transformed B lymphocytes].

AIM: To investigate the changes of CD40 expression and proliferation pattern of EB virus-transformed B lymphocytes that had been transfected by antisense CD40 RNA. METHODS: Eukaryotic expression vector pcDNA3/CD40 of human CD40antisense RNA was constructed by means of T-A cloning and subcloning, and then was transfected into EB virus-transformed B lymphocytes. Changes of CD40 expression on the B cells and their proliferation were tested by flow cytometry and MTT colorimetry assay, respectively. RESULTS: CD40 expression and proliferation of the B cells transfected with antisense CD40 RNA were significantly lower than those of the cells transfected with pcDNA3 alone (P < 0. 01). CONCLUSION: Antisense CD40 RNA may be served as an effective means of regulating immune function, and the CD40 gene itself also play an important role in cell proliferation.