Application of lymphocytes test in peripheral blood of patients with systemic sclerosis during the treatment / 北京大学学报(医学版)

OBJECTIVE@#To explore the significance of lymphocytes in systemic sclerosis (SSc), by detecting the levels of T lymphocytes, B lymphocytes and natural killer (NK) cells, and analyzing the correlation between the lymphocytes and clinical laboratory indexes.@*METHODS@#The numbers and proportion of T, CD4+T, CD8+T, B, and NK cells were detected by flow cytometry in peripheral blood of 32 SSc patients who had taken immunosuppressive drugs and 30 healthy controls (HC). The comparison of the lymphocyte subsets in SSc with them in the HC groups, and the correlation between the lymphocytes and other clinical and laboratory indicators were analyzed by the relevant statistical analysis.@*RESULTS@#Compared with the HC group, the numbers of T, CD4+T, CD8+T, and NK cells in peripheral blood of SSc group, who had taken immunosuppressive drugs, were significantly decreased (P < 0.05). More-over, the proportion of NK cells in peripheral blood of the SSc group was also significantly lower than that in the HC group (P=0.004). In addition, all the lymphocyte subsets were decreased in peripheral blood of more than 65% of the SSc patients who had taken immunosuppressive drugs. Compared with CD4+T normal group, the positivity of Raynaud's phenomenon, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) was significantly increased in CD4+T reduction group, respectively (P＝0.024, P < 0.001, P=0.018). ESR was higher in CD8+T reduction group than CD8+T normal group (P＝0.022). The prevalence of fingertip ulcer was significantly increased in B cell decrease group (P=0.019). Compared with NK cell normal group, the prevalence of fingertip ulcer was significantly increased in NK cell lower group (P=0.033), IgM was remarkablely decreased yet (P=0.049). The correlation analysis showed that ESR was negatively correlated with the counts of T lymphocytes (r＝－0.455, P＝0.009), CD4+T lymphocytes (r＝－0.416, P＝0.018), CD8+T lymphocytes (r＝－0.430, P=0.014), B cells (r＝－0.366, P＝0.039).@*CONCLUSION@#The number of CD4+T, CD8+T, B, and NK cells significantly decreased in peripheral blood of SSc patients who had used immunosuppressive drugs, some lymphocyte subsets might be related with Raynaud's phenomenon and fingertip ulcer, and reflected the disease activity by negatively correlated with ESR and CRP; the numbers of lymphocyte subsets in peripheral blood should be detected regularly in SSc patients who had taken immunosuppressive drugs.

Significance of anti-tubulin-α-1C autoantibody in systemic sclerosis / 北京大学学报(医学版)

OBJECTIVE@#To detect the serum level of a novel autoantibody, anti-tubulin-α-1C, in patients with systemic sclerosis (SSc) and to investigate its clinical significance.@*METHODS@#Anti-tubulin-α-1C antibody levels were determined by enzyme-linked immunosorbent assay (ELISA) in 62 patients with SSc, 38 systemic lupus erythematosus (SLE), 24 primary Sjögren's syndrome (pSS) patients, and 30 healthy controls (HCs). Erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), immunoglobulin A(IgA), immunoglobulin M (IgM), immunoglobulin G (IgG), C3, C4, rheumatoid factor (RF), antinuclear antibody(ANA), anti-centromere antibodies(ACA), anticardiolipin (aCL), anti-dsDNA antibody, anti-Sm antibody, anti-RNP antibody, anti-Scl-70 antibody, anti-Ro52 antibody, anti-SSA antibody, anti-SSB antibody, centromere protein A(CENP-A), centromere protein B (CENP-B) were measured by standard laboratory techniques. Raynaud's phenomenon and modified Rodnan skin score(MRSS) were recorded to evaluate the disease status of SSc. Independent sample t test, Chi square test, Mann-Whitney U test, Spearman rank correlation were used for statistical analyses.@*RESULTS@#The serum anti-tubulin-α-1C antibody concentration in SSc group was 81.24±34.38, the serum anti-tubulin-α-1C antibody concentration in SLE group was 87.84±38.52, the serum anti-tubulin-α-1C antibody concentration in pSS group was 59.79±25.24, and the serum anti-tubulin-α-1C antibody concentration in healthy group was 39.37±18.7. Multivariate analysis revealed that anti-tubulin-α-1C antibody levels were significantly increased in the SSc and SLE patients. The expression level of anti-tubulin-α-1C antibody in SSc was higher compared with the pSS group and the health control group (P < 0.01). Further analysis demonstrated that the elevated anti-tubulin-α-1C antibody were correlated with the SSc inflammation and disease activity markers ESR(r=0.313, P=0.019), The levels of anti-tubulin-α-1C antibody were also significantly correlated with MRSS(r=0.636, P < 0.01). The best cut-off value for the diagnose of SSc was 76.77 as mean+2SD value. The proportion of Raynaud's phenomenon was higher in the group of anti-tubulin-α-1C autoantibody-postive SSc patients than that in anti-tubulin-α-1C autoantibody negative group(71.4% vs. 37.5%, P=0.039). The proportions of anti-Scl-70 antibody, anti-CENP antibody and anti-cardiolipin antibody were higher in the group of anti-tubulin-α-1C autoantibody-postive SSc patients than in the anti-tubulin-α-1C autoantibody negative group (37.9% vs. 15.2%, 34.5% vs. 12.1%, 13.8 vs. 0, respectively, all P < 0.05).@*CONCLUSION@#Based on this explorative stu-dy, the level of anti-tubulin-α-1C antibody increased in the serum of the patients with SSc. There were correlations between anti-tubulin-α-1C autoantibody and clinical and laboratory indicators of the SSc patients. It may become a novel biomarker indicative of active SSc and could be applied in future clinical practice.

Significance of anti-carbamylated fibrinogen antibodies in systemic lupus erythematosus / 北京大学学报(医学版)

OBJECTIVE@#Antibodies against carbamylated protein (anti-CarP) were found to be a promising marker to evaluate joint damage and disease activity in patients with rheumatoid arthritis (RA). However, whether anti-CarP antibodies were present in systemic lupus erythematosus (SLE) remained ambiguity. We have therefore undertaken this study to assess the levels of serum anti-CarP antibodies and to evaluate their clinical value in SLE.@*METHODS@#Serum levels of antibodies against carbamylatedfibrinogen (anti-CarP) were measured by enzyme-linked immunosorbent assay (ELISA) in 105 SLE patients and 73 healthy controls. Other clinical and laboratory measurements of the SLE patients were collected from medical records. Data analyses between anti-CarP antibodies and other laboratory measurements were performed using SPSS software for Windows 24.0.@*RESULTS@#The levels of serum anti-CarP antibodies in the patients with SLE were significantly higher than those in the healthy controls (P<0.05). There were significant differences between the anti-CarP-positive group and anti-CarP-negative group in many clinical features. The disease duration, values of ESR, CRP, RF, anti-cardiolipin, anti-dsDNA, D-dipolymer, IgA and IgG were significantly higher in the anti-CarP-positive group compared with the negative group (P<0.05). Conversely, the values of complement 3, complement 4, peripheral blood RBC, and hemoglobin were significantly lower in anti-CarP-positive group than in the negative group(P<0.05). Moreover, the incidence of increase of erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), rheumatoid factor (RF), D-dipolymer, decrease of peripheral blood RBC, hemoglobin, complement 3, complement 4, and positive rate of anti-dsDNA were significant different between the two groups(P<0.05). The positive rate of anti-CarP (21.9%) was higher than that of anti-Sm (15.24%), and close to anti-ribosomal P protein (22.86%) in our SLE patients. In addition, anti-CarP antibody was present in the SLE patients lacking the disease specific antibodies, including anti-Sm (anti-CarP positive rate 20.2%, 18/89), anti-dsDNA (anti-CarP positive rate 9.3%, 4/43), anti-nucleosome (anti-CarP positive rate 12.5%, 6/48), and anti-ribosomal P protein antibody (anti-CarP positive rate 20.9%, 17/81). Moreover, the high levels of anti-CarP antibodies were correlated with short disease duration, low C3, C4, RBC, and hemoglobin (P<0.05), high ESR, CRP, IgA, IgG, RF, anti-cardiolipin, anti-dsDNA, and D-dipolymer (P<0.05).@*CONCLUSION@#The level of anti-CarP antibody was increased in the serum of patients with SLE. There were correlations between anti-CarP antibodies and clinical and laboratory indicators of SLE patients.

Effects of integrin metalloproteinases on osteogenic differentiation / 北京大学学报(医学版)

OBJECTIVE@#To study the effects of disintegrin and metalloproteinase (ADAM) 9, 15 and 17 on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs).@*METHODS@#BMMSCs of ADAM9, ADAM15, ADAM17 conditional knockout mice and wild type mice (WT) were induced and cultured. Alkaline phosphatase (ALP) activity was measured by colorimetry, early osteogenic transcription factors Runx and Osterix were detected by Real-time PCR, and mineral formation was analyzed by alizarin red staining.@*RESULTS@#ALP activity was lower in ADAM9 group (8.08±0.34), ADAM15 group (6.46±3.40), ADAM17 group (9.30±2.30) than that in WT group (9.44±2.50), but there was no significant difference (P>0.05). Stimulated with bone morphogenetic protein 2(BMP2),there was significant difference (P<0.05) between ADAM9 group (14.22±3.25), ADAM15 group (10.14±2.40) and WT group (20.89±3.40), and ADAM 17 group (23.56±2.50) was higher than WT group (20.89±3.40), but no significant difference (P>0.05). Similarly, cultured by osteogenic induction medium (OST), compared with WT group (12.97±1.30), ADAM9 group (9.63±1.00) and ADAM15 group (7.75±1.30) were lower, ADAM17 group (20.09±1.68) was higher, and the difference was statistically significant (P<0.05). Using stimulated culture by BMP2 and OST combined, ADAM9 group (15.75±1.30), ADAM 15 group (12.43±1.30) were less than WT group (26.15 ±1.50), while ADAM17 group (29.55±2.10) was higher than WT group were statistically significant (P<0.05). The expression of Runx2 in ADAM9 group (2.02±0.24), ADAM15 group (3.09±0.19), ADAM17 group (3.89±0.91) had no significant difference compared with WT (2.02±0.21) group (P>0.05). ADAM9 group stimulated by BMP2 (7.00±0.23), ADAM15 group (6.04±0.23) were lower than WT group (12.6±0.23), ADAM17 group (18.52±1.39) was higher than WT group (12.6±0.23), and the difference was statistically significant (P<0.05). In non-stimulating culture, there was no significant difference in Osterix expression between ADAM9 group (9.60±3.87), ADAM17 group (12.40±3.00) and WT group (10.9±1.10, P>0.05), but in ADAM15 group (6.50±1.51) it was slightly lower than that in WT group (P<0.05). After BMP2 stimulation, ADAM9 group (39.20±3.23) and ADAM15 group (20.50±4.80) were less than WT group (60.30±5.93), while ADAM17 group (80.20±3.30) was higher than WT group (P<0.05). Alizarin red staining showed no obvious orange-red mass in the non-induction group. Local calcified nodules could be seen in the BMP2, OST, OST + BMP2 induction culture conditions in all the experimental groups, but there was no significant difference in quantitative analysis (P>0.05).@*CONCLUSION@#ADAM9, 15, 17 took part in the osteogenic differentiation of BMMSCs, and provided new targets for its regulation.

Elevated Levels of Soluble ST2 were Associated with Rheumatoid Arthritis Disease Activity and Ameliorated Inflammation in Synovial Fibroblasts / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Much evidence has demonstrated that interleukin (IL)-33 plays an important role in rheumatoid arthritis (RA). However, there have been limited studies about soluble ST2, a receptor for IL-33, in RA. The aims of this study were to detect the levels of ST2 in the serum and synovial fluid of RA patients and to reveal the association of these levels with disease activity and the function of ST2 in RA.</p><p><b>METHODS</b>A total of 56 RA patients and 38 age-matched healthy controls were enrolled in this study. Synovial fluid samples were collected from another 30 RA patients and 20 osteoarthritis patients. Serum and synovial fluid levels of ST2 were measured by ELISA. In addition, the levels of ST2 in the serum of RA patients before and after therapy were detected. The function of ST2 in RA was revealed by the results of an in vitro cell assay, where recombinant ST2 proteins were used to treat peripheral blood mononuclear cells (PBMCs) and RA synovial fibroblasts (RASFs).</p><p><b>RESULTS</b>Serum-soluble ST2 levels were significantly higher in RA patients (127.14 ± 61.43 pg/ml) than those in healthy controls (78.37 ± 41.93 pg/ml, P < 0.01). Synovial fluid-soluble ST2 levels (41.90 ± 33.58 pg/ml) were much higher in RA patients than those in osteoarthritis patients (19.71 ± 16.72 pg/ml, P < 0.05). RA patients who received effective therapy for 6 months showed decreased serum-soluble ST2 levels (113.01 ± 53.90 pg/ml) compared to baseline (139.59 ± 68.36 pg/ml) (P = 0.01). RA patients with high disease activity had higher serum-soluble ST2 levels (162.02 ± 56.78 pg/ml) than those with low disease activity (94.67 ± 40.27 pg/ml, P = 0.001). Soluble ST2 did not affect IL-1β, IL-6, IL-8, or tumor necrosis factor-α (TNF-α) expression in PBMCs from RA patients. However, soluble ST2 ameliorated the expressions of IL-33 and IL-1β but not that of IL-6, IL-8, or TNF-α in resting RASFs. Interestingly, in the RASFs stimulated by TNF-α plus IL-1β, soluble ST2 showed extensive suppressive effects on the expression of IL-6, IL-8, and TNF-α.</p><p><b>CONCLUSION</b>Elevated levels of ST2 in the serum and synovial fluid were associated with disease activity and ameliorated IL-33 expression and IL-33-induced inflammation in RASFs, suggesting that soluble ST2 might be a potential therapeutic candidate for RA.</p>

Serum IgA against type 3 muscarinic acetylcholine receptor is a novel marker in diagnosis of Sjögren's syndrome / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Antibodies against type 3 muscarinic acetylcholine receptor (M3R) are involved in the pathogenesis of Sjögren's syndrome (SS), but the clinical value of them in SS patients has been controversial. The aims of this study were to: (1) establish an improved enzyme-linked immunosorbent assay (ELISA) to detect IgA antibodies against M3R; (2) evaluate the value of IgA antibodies against the second extracellular loop of M3R205-220 (c2M3RP) in diagnosis of SS.</p><p><b>METHODS</b>To increase the ELISA sensitivity, c2M3RP was coupled to bovine serum albumin (BSA) by the glutaraldehyde method and a 96-well microplate was treated by ultraviolet rays before coated. Concentrations of anti-c2M3RP, anti-SSA, and anti-SSB were measured in the sera of 240 individuals: 91 patients with primary SS and 149 controls (16 secondary SS, 27 systemic lupus erythematosus, 40 rheumatoid arthritis and 66 healthy controls). Diagnostic properties of anti-c2M3RP were determined by receiver-operating characteristic curve analysis.</p><p><b>RESULTS</b>The prevalence of serum IgA anti-c2M3RP antibodies in patients with pSS (46%, 42/91) was significantly higher than that in patients with systemic lupus erythematosus (19%, 5/27), in rheumatoid arthritis (15%, 6/40) and in healthy controls (5%, 3/66). However, there was no significant difference between the two SS groups (P = 0.727). The diagnostic performance of IgA anti-M3RP antibodies was similar to anti-SSA assay, but had 22% higher sensitivity than anti-SSB. By analyzing of IgA anti-c2M3RP antibodies, combination of anti-SSA and anti-SSB resulted in increased sensitivity, whereas their specificity was not significantly changed.</p><p><b>CONCLUSIONS</b>The improved anti-c2M3RP ELISA is a novel, sensitive, and specific serological test for the diagnosis of SS. The combined application of anti-c2M3RP, anti-SSA and anti-SSB tests can improve the laboratory diagnosis of SS. The IgA anti-c2M3RP antibodies may serve as a novel diagnostic marker for SS.</p>