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Objective:To investigate the clinical features, risk factors, treatment and prognosis of dermatomyositis (DM) patients with positive anti-melanoma differentiation associated gene 5(MDA5) antibody with rapidly progressive interstitial lung disease (RPILD).Methods:The clinical data of 88 DM patients from June 2019 to June 2020, at the rheumatology department of Guangdong Provincial People's Hospital were collected and retrospectively analyzed. T-test, non-parametric Mann-Whitney U test, Chi-squared test, Fisher exact probability and Logistics regression analysis were used for data analysis. Results:① 37%(36/88) DM patients were positive for anti-MDA5 antibody. The frequency of ulcerative rash, Gottron's sign, arthritis, clinically amyopathic dermatomyositis (CADM), and erythrocyte sedimentation rate (ESR) was significantly higher in patients with anti-MDA5 antibody ( P<0.05). The cell count of white blood cell, neutrophil, lymphocyte, and serum creatine kinase (CK) level were significantly lower in the anti-MDA5 antibody positive group than those in the negative group ( P<0.05). Of anti-MDA5 antibody positive DM patients, 100% developed ILD, 34% (11/32)developed RP-ILD, 16%(5/32) died, which were significantly higher than those of anti-MDA5 antibody negative patients ( P<0.05). ② Of anti-MDA5 antibody positive DM patients, the C reactive protein (CRP) level, positive rate of anti-Ro-52 antibody and mortality rate were significantly higher RPILD group than those in the non-RPILD group [15.70(4.49, 29.00) vs 3.22 (1.66, 7.15), Z=-2.440, P=0.014; 91% vs 43%, P=0.011; 46% vs 0, P=0.002]. Logistics regression analysis indicated that positive anti-Ro-52 antibody [ OR=4.561, 95% CI (1.797, 11.580), P=0.001] might be a risk factor for anti-MDA5 antibody positive DM-RPILD. ③ Among patients with anti-MDA5 antibody with RPILD, serum ferritin and D-dimer level was significantly higher and oxygenation index was significantly lower in the non-survival group than those in the survival group [1 931 (1 377, 7 379) vs 638(196, 876), Z=-2.556, P=0.009; 2 760(1 995, 4 854) vs 985(533, 1 588), Z=-2.379, P=0.017; 230(140, 256) vs 309(262, 382), Z=2.191, P=0.030]. In addition, the delayed intensive treatment time was significantly longer in the non-survival group than those in the survival group [(14.0±2.6) vs (4.5±1.4), t=7.899, P<0.01]. Furthermore, the proportion of combined therapy with two disease modifying antirheumatic drug (DMARDs) was significantly lower in the non-survival group than those in the survival group (0 vs 83%, P=0.015). Conclusion:Anti-MDA5 antibody may be associ-ated with characteristic clinical manifestations of DM, ILD, RPILD and high mortality rate. Positive anti-Ro-52 antibody may be a risk factor for anti-MDA5 antibody positive DM-RPILD. High serum ferritin and D-dimer level and low oxygenation index in RPILD patients may be associated with poor prognosis. Early treatment with two DMARDs may improve the prognosis of RPILD.
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Objective To study the effect of Febuxostat on NALP3 inflammasome in chronic gouty arthri-tis. Methods A total of 89 patients with chronic gouty arthritis and 50 healthy cases were enrolled in this study and 89 patients were divided into Benzbromarone group,Allopurinol group,Febuxostat group and placebo group. The expression of NALP3 inflammasome mRNA and the protein were detected by RT-PCR and Western blot. Re-sults The levels of Uric Acid,NALP3,ASC and caspase-1 mRNA in chronic gouty arthritis patients were higher than those in healthy cases before treatment(#P = 0.000);the level of NALP3 in benzbromarone and allopurinol group had no change after treatment(*P<0.05).The levels of NALP3 mRNA and caspase-1 mRNA in Febuxostat group were lower but the level of ASC mRNA was higher than those in other groups after treatment(*P < 0.05). Conclusions NALP3 inflammatory may be associated with chronic gouty arthritis. Febuxostat can effectively re-duce the level of Uric Acid,and affect the function of NALP3 inflammasome.
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Objective To investigate the effect of glucocorticoid and bisphosphonate on Hedgehog signaling pathway in human bone mesenchymal stem cells (hBMSCs) and bone tissue.Methods ① Bone biopsy test:forty cases of systemic lupus erythematosus (SLE) patients were divided into 2 groups:20 cases in newly diagnosis group,20 cases in the GCs group (the dosage of glucocorticoids was higher than 1 mg·kg-1·d-1).Patients in the GCs group was randomly divided into 2 groups:10 cases in the control group were without anti-osteoporosis treatment,10 cases in treatment group received alendronate 70 mg once a week.All of the patients had bone marrow puncture after24 weeks,and the value of average optical density of Gli1 was tested with immunohistochemistry.② Cell culture:hBMSCs cultured in normal medium were intervened with rh-SHHN and methylprednisolone (10-3 mol/L,10-5 mol/L) after successfully identified.Quantitative polymerase chain reaction (PCR) was used to detectthe mRNA expression of Gli1.The final calcium nodules was detected by Alizarin red staining.hBMSCs cultured in osteogenesis medium were intervened with bisphosphonate and methylprednisolone (10-3 mol/L) after successfully identified.Quantitative PCR was used to detect the expression of Gli1 mRNA.Alizarin red staining was used to detect the final calcium nodules.Comparisons between the two groups were carried out using t-test while the difference analysis of multi-groups were tested by factorial analysis.Results The average optical density of Gli1 in the GCs group (47±7) was less than the newly diagnosed group (61±12) (t=4.442,P＜0.01),and it was less in the control group (51±6) than in the treatment group (42±6) (t=3.701,P=0.002).When normally cultured,high and moderate concentration of methylprednisolone suppressed the mRNA (0.38±0.04; 0.68±0.24) (F=8.748,P＜0.01) expression of Gli1 which was initially stimulated by rh-SHHN (2.01 ±0.38).And the final calcium nodules in groups which contained methylprednisolone were much less than rh-SHHN only group.When hBMSCs were cultured in osteogenesis medium,compared with the methylprednisolone group (0.024±0.011),the expression of Gli1mRNA(0.034-0.006) (t=7.62,P＜0.01) and the final calcium nodules were significantly improved by bisphosphonate.Conclusion High and moderate doses of glucocorticoids inhibit hBMSC osteogenesis by inhibiting Gli1.Low concentration of alendronate can not only stimulate hBMSC osteogenesis differentiation but also can remit the inhibition effects of GCs through the way of Hedgehog.
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Objective To study the anti-fibrotic function and mechanism of peroxisome proliferator activated receptorγ(PPARγ) in connective tissue disease-interstitial lung disease (CTD-ILD).Methods The expression of PPARγin lungs was analyzed in 37 cases with CTD-ILD and 20 control cases by immunohistochemistry.Changes in α-SMA levels were analyzed by Western blotting,and acetylation of Smad3 and Smad3 or PPARγ combined with P300 were analyzed by IP-WB.The data was analyzed by one-way ANOVA,t test or Mann-Whitney test.Results PPARγ' expression in the lung of CTD-ILD was lower than the controls [0.92％(1.44％),3.50％(1.94)％,respectively; Z=-8.924,P＜0.01].Different concentration of PPARγ (0,1,5,10,20,40 pmol/L) ligandinhibited the marked elevation of the protein α-SMA induced by TGF-β1 in a concentration-dependent manner (0.918 ±0.062,0.852±0.042,0.725 ±0.057,0.678 ±0.042,0.418 ±0.022,0.456±0.029; P＜0.05 or P＜0.01).However,this response was blocked by a selective antagonist PPARγ signaling GW9662 (0.946±0.087 vs 0.538±0.120,P＜0.01).Acetylation of Smad3 expression was increased when TGF-β1 was putted into lung fibroblasts after 60,90 and 180 min (0.565±0.047,1.127±0.101,0.873±0.022,0.614±0.407; all P＜0.05).The combination of Smad3 with P300 was also increased (1.46±0.12,0.98±0.09; P＜0.05),compared with the controls.But the ligand of PPARγ could block this effect (0.62±0.10,1.46±0.12; P＜0.05).Meanwhile,the combination of PPARγ and P300 was increased (0.94±0.05,0.76±0.22; P＜0.05).Conclusion PPARγ may play a physiologic role in the regulation of anti-fibrosis response.Its function may be realized by its competition with Smad3 combined with P300.
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Objective To investigate the expression of lipid rafts (LRs) and actin cytoskeleton (F-actin) in the peripheral blood B lymphocytes of patients with systemic lupus erythematosus (SLE).Methods Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Hypaque.B lymphocytes were isolated by positive selection from PBMCs.Membrane staining for LRs was achieved with FITC-conjugated cholera toxin B (CTB).The level and distribution of LRs were studied by flow cytometry and confocal microscopy.Staining for F-actin was carried out with Rhodamine phalloidin.The expression of F-actin was analyzed by confocal microscopy.In an in vitro examination,the effect of Leflunomide on lipid rafts in B lymphocytes from SLE was analyzed.Disease carried out was measured using the SLE disease activity index (SLEDAI).Analysis of the enumerical data was performed using ANOVA or paired-samples t test.Correlation was examined by Pearson's rank correlation test.Results The number of CTB-binding lipid rafts in B cell from active SLE patients or from SLE patients in disease remission.who were treated with immunosuppressive drugs was higher than B cells from healthy controls [(59+4)％,(51±5)％,(33±4)％,F=9.21,P=0.001].Confocal microscopy revealed that in B cell from healthy controls,lipid raft was found to be small and uniformly distributed on the plasma membrane.F-actin was found mainly in the cortical region of the cells.This pattern was different from the pattern seen in B cells from patients with SLE,which presented with stronger staining and irregular large clustering of LRs,with a decrease in F-actin levels.In addition,the number of CTB-binding LRs in B cells from the active SLE patients was correlated significantly with the SLEDAI score (r=0.632,P=0.028).Furthermore,thein vitro results showed that leflunomide treatment reduced the number of CTB-binding LRs in B cell from SLE patients [(48±5)％ vs (39±5)％,t=2.29,P=0.048].Conclusion The altered expression of Lipid raft and F-actin can been seen in B lymphocytes in SLE,and modulation of LRs and F-actin expression may be a potential approach in the treatment of SLE.
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Objective To study the effect of mixed purified autogenic and allogeneic hematopoietic stem cell transplantation for the treatment of systemic lupus erythematosus. Methods Thirty-six MRL/lpr mice were randomly divided into the control group, the study group,the mixed group ( the ratio of autogenic to hematopoietic stem cells, mixed in different proportions were infused intravenously after 60Co irradiation. The study group were treated with daily intraperitoneal infusion of dexamethasone 1 mg·kg-1·d-1, while the control group were treated with intraperitoneal infusion of equivalent volume of saline daily. The changes of serum creatinine level, the urine protein excretion of the mice and blood WBC count were compared. Repeat measures ANOVA was used for data analysis. ELISA was used for anti-nuclear antibody detection Light microscopy, electronic micros-copy, immunofluorescence were applied to detect the pathological changes in renal tissue. Results Serum creatinine and urine protein excretion levels increased with time in the ontrol group, while those of the transplant group and the study group decreased. The reduction in mixed transplantation group and the study group was more evident compared with that of the allogeneic group. The difference was statistically significant (P＜0.05), but there was no significant difference between the mixed transplantation groups and the study group (P＞0.05). The histopathologic damage was most serious in the control group as pathological injury score of most mice were in grade 3 or 4. The majority of the histopathologic damage of the allogeneic group was in grade 2. Most f pathological damage of the study drug group and the mixed transplantation group were grade 1 or 2. Conclusion Mixed hematoopoietic stem cell transplantation for the treatment of murine systemic lupus erythematosus can effectively correct heavy proteinuria in murine systemic lupus erythematosus so improve the renal damage. It is a safe and effectively way to treat murine systemic lupus erythematosus.
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Objective To evaluated intra-articular injection of TNF-α inhibitors into the sacroiliac joint as an effective and viable alternative. Methods Sixteen patients with documented ankylosing spondylitis (AS), without steroids or disease modifying anti-rheumatic drugs (DMARDs) were performed CT-guided intra-articular injections of etanercept (TNF-α antagonist) at week 0, 4 and 8 (25 mg per dose). Similarly, 20 patients with AS in the control group received systemic etanercept therapy at a dose of 50 mg per week for 8 weeks. All patients were followed up clinically and evaluated periodically. Pathological features of sacroiliitis were observed with light microscopy and immunohistochemistry. Expression of cytokines in joint biopsy samples was estimated by RT-PCR. Image changes of sacroiliitis were observed by SPECT/CT and MRI. Ttest, t'tesr and χ2 Fisher's test were selected. Results All the 16 patients who received intra-articular etanercept, the mean value of radiological nuclide decrease of the SIJ ROI (region of interest) in the SPECT improved significantly after 8 weeks treatment [(1.38±0.16 vs 1.45±0.14) P<0.05] . Bone marrow edema and fat deposition in MRI were relieved significantly after 8 weeks (P<0.05). In 8 patients the expression of TNF-α and TGF-β mRNA in joint tissue decreased significantly after 8 weeks [(0.89±0.06, 0.84±0.05) vs (l.08± 0.19, 1.13±0.33) (P<0.05)]. The occurrence of gynonitis, enthesitis, chondritis, subehondral bony plate destruction, bone marrow inflammation and inflammatory cell index also decreased significantly (P<0.05). Participants given intra-articular injection showed significant clinical improvement after 8 weeks and 12 weeks treatment(P<0.01 ) in BASDAI score [(32±13) mm]. Conclusion This study has shown that intra-articular injection of etanercept in SIJ can improve joint function and quality of life. It has a satisfactory safety profile and is cost effective. This mode of treatment is most beneficial in local arthropathy of recent onset and in those patients who do not tolerate systemic etanercept therapy.
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Objective To investigate the effect of leflunomide on the superficial costimulatory molecules expression of T lymphocytes in patients with lupus nephritis ( LN ). Methods The peripheral blood mononuclear cells (PBMCs) of female active LN patients and healthy female were separated by density gradient centrifugation, and cultured by phytohemagglutinin (PHA) or leflunomide active metabolites(A771726).The CD28, CD40L, CTLA-4 and LFA-1a expressions on the peripheral blood T lymphocytes were detected by double-colored flow cytometry. The differences of the means were tested by analysis of variance( ANOVA ) and SNK q test. Results Comparing with healthy controls, there were significantly higher expressions of CD28,CD40L, LFA-1a and CTLA-4 on the peripheral blood T cells in active LN patients (CD28:33.4±6.5 vs14.4±3.2; CD40L: 13.2±3.2 vs 5.4±2.3; LFA-1a: 8.5±2.3 vs2.2±1.1; CTLA-4:4.6±1.5 all P＜0.01) as well as CD28 and CD40L expression on the peripheral blood T cells from healthy controls induced by PHA (CD28:26.8±6.7 vs14.4±3.2; CD40L: 13.9±4.9 vs 5.4±2.3 all P＜0.01 ), but not CTLA-4 and LFA-1a expression.However, CD28, CD40L, LFA-1a and CTLA-4 expressions on T cells stimulated by PHA increased in active LN patients(all P＜0.05 ). A771726 could significantly inhibit over-expression of LFA-1a and CD40L on the T cells from active LN patients (CD40L:8.2±2.0 vs13.3±3.2;LFA-1a:5.1±1.3 vs8.5±2.3 all P＜0.01 ), but not CD28 and CTLA-4 expression. A771726 did not inhibit CD28, CD40L, LFA-1a and CTLA-4 expression on the T cells in healthy controls. Furthermore, A771726 could markedly inhibit the over-expression of all of the above molecules induced by PHA on the T cells of active LN patients (all P＜0.05). Conclusion One of the major mechanisms for LEF treatment of LN is that LEF can down-regulate CD40L and LFA-1a expression but not CD28 and CTLA-4 expression on the peripheral blood T cells in active LN patients.
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Objective To analyze the outcomes and prognostic factors associated with the death of systemic lupus erythematosus (SLE) patients admitted to the intensive care unit (ICU). Methods During June 1996 to June 2007, all SLE patients admitted to the ICU were included. Patients were excluded if the diagnosis of SLE was established at or after ICU admission. A multivariate logistic regression model was applied using variables that were associated with death in the univariate analysis. Results A total of 101 patients meeting the criteria were included. The mortality rate was 48.6%. The most common causes of admission was lung disorder with acute respiratory distress syndrome (ARDS). Multivariate logistic regression analysis suggested that SLICC/ACR DI>7.7 (OR=6.87), APACHE Ⅲ≥21 (OR=29.8), lung disorders with ARDS (OR =55.81 ), septic shock (OR =32.22 ), intracranial haemorrhage (OR =57.35 ), hypocytopenia (OR = 5.89), mean equivalent prednisone dose>25 mg/d (OR=7.65) and prolonged tracheal intubation (OR=5.98) were signi-ficantly associated with death. Whereas sex, age, SLEDAI >27, gastrointestinal bleeding, the cumulative dosage of CTX higher than 1.0 g, pulse intravenous methylprednisolone therapy were not associated with death. Conclusion The mortality rate of critically ill SLE patients in ICU is very high. SLICC/ACR DI> 7.7, APACHE Ⅲ≥21, lung disorders with ARDS, septic shock, intracraniai haemorrhage, average prednisone equivalent dosage higher than 25mg/d and prolonged tracheal intubation (longer than 4 days) are negative prognostic factors in SLE patients admitted to the ICU.
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In order to study the role of the bone marrow-derived mesenchymal stem cells(MSCs)transplanted with or without bone marrow(BM)in the treatment of lupus mice and the effect of MSCs in the onset of systemic lupus erythematosus(SLE).Method Twenty 12-week-old female MRL/lpr mice were randomly divided into four groups,including simple bone marrow transplantation group(SG,BM 1×107),united group-1(UG1,BM 1×107+MSCs 1×104),united group-2(UG2,BM 1×107+MSCs 1×106),the positive control group(PG,no transplantation).BALB/c mice were used as the negative control group(NG,no transplantation).MSCs which were amplified from the bone marrow of male BALB/c mice in vitro were transplanted into the female MRL/lpr mice with or without BM.One month later Y chromosome was detected to confirm if the transplantation was successful or not.The change of weight, white blood cells,urine protein,anti-dsDNA antibody,the pathology and immunofluorescence of renal were observed to evaluate the therapeutic efficacy.Results Y chromosome was detected in all transplanted female mice.Compared with PG,urine protein concentration in SG,UG1 and UG2 significantly decreased 30 days after transplantation(P<0.05).40 days after transplantation,the rite of anti-dsDNA antibodies in sG(0.91±0.27)was still higher than NG,which OD value wag 0.47 s0.10(P<0.05),but there was no statistical difference among UG1(0.76±0.28),uG2(0.73±0.10)and NG(P>0.05).However,50 days after transplantation,there was no marked difference of the tite of anti-dsDNA antibodies in SG(0.55±0.15),UG1(0.57±0.14)and UC2(0.58±0.05)compared with NG(P>0.05).After transplantation there was no vasculitis.no inflammatory cell infiltration in matrix and no obvious intercapillary cells proliferation in the kidney.The immunofluoroscence became negative or weakly positive.Conclusion MSCs transplantation with or without BM Can both improve the pathogenetic condition of MRL/lpr mice.MSCs can accelerate the clearance of anti-dsDNA antibody and promote the restoration of injured organs.We presume that MSCs are important immunological regulation cells in SLE.
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<p><b>OBJECTIVE</b>To investigate the role of IL-17 in the overproduction of autoantibodies and IL-6 overexpression by peripheral blood mononuclear cells (PBMC) of lupus nephritis (LN) patients.</p><p><b>METHODS</b>Fifteen consecutively hospitalized LN patients were selected as subjects and 15 healthy adults as normal controls. PBMC were obtained by Ficoll density gradient centrifugation. IgG, anti-dsDNA antibody and IL-6 protein levels were assessed using enzyme-linked immunosorbent assays (ELISA) on the supernatant of cultured PBMC of LN patients or normal controls. IL-6 mRNA levels in PBMC were measured using reverse transcription-polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>In medium culture, IgG, anti-dsDNA and IL-6 protein levels of the supernatant of PBMC from LN patients were significantly higher than those from normal controls (1492.1 +/- 73.2 ng/ml vs 636.7 +/- 51.9 ng/ml for IgG, 306.6 +/- 53.7 IU/ml vs 95.8 +/- 11.6 IU/ml for anti-dsDNA and 50.92 +/- 15.92 ng/ml vs 1.77 +/- 0.73 ng/ml for IL-6, all P < 0.001). In LN patients, IgG, anti-dsDNA and IL-6 protein levels were higher in the supernatants of PBMC in the IL-17-stimulated culture than the medium culture, but in normal controls, only the IL-6 protein levels were significantly higher. The increase in IgG, anti-dsDNA and IL-6 protein levels induced by IL-17 was dose-dependent and could be completely blocked by IL-17 monoclonal antibody mIgG(28) and partially blocked by dexamethasone. Similarly, IL-6 mRNA overexpression of PBMC in LN patients or normal controls induced by IL-17 was both dose- and time-dependent. During medium culture, IL-6 mRNA levels in LN patients were significantly higher than those in normal controls (1.80 +/- 0.11 vs 0.36 +/- 0.07). During stimulation with IL-17, IL-6 mRNA levels in LN patients were higher than those in normal controls (3.21 +/- 0.24 vs 1.30 +/- 0.14, P < 0.05) and also significantly higher when comparing the stimulated culture with the medium culture either in LN patients or normal control.</p><p><b>CONCLUSIONS</b>IL-17 may play an important role in the pathogenesis of LN through the induction of IgG, anti-dsDNA overproduction and IL-6 overexpression of PBMC in LN patients.</p>