[Value of serum YKL-40 in the diagnosis of anti-MDA5-positive patients with dermatomyositis complicated with severe pulmonary injury].

OBJECTIVE: To investigate the value of serum and bronchoalveolar lavage fluid (BALF) chitinase-3-like-1 protein (YKL-40) in the diagnosis of anti-melanoma differentiation-associated gene 5 (MDA5)-positive dermatomyositis (DM) patients complicated with serious pulmonary injury, including rapidly progressive interstitial lung disease (RP-ILD) and pulmonary infection. METHODS: Anti-MDA5 antibodies positive patients with DM who were hospitalized in the Department of Rheumatology of China-Japan Friendship Hospital from 2013 to 2018 were involved in this study. Demographic information, clinical, laboratory and imaging data were retrospectively collected. ELISA was used to detect the serum and BALF levels of YKL-40. The receiver operating characteristic (ROC) curve was drawn, and the area under ROC curve (AUC) was used to evaluate the diagnostic value of serum YKL-40 for pulmonary injury.Interstitial lung disease (ILD) was confirmed by chest high-resolution CT (HRCT). RP-ILD was defined as progressive respiratory symptoms such as dyspnea and hypoxemia within 3 months, and/or deterioration of interstitial changes or appearace of new pulmonary interstitial lesions on chest HRCT. Pulmonary infection was considered as positive pathogens detected in qualified sputum, blood, bronchoalveolar lavage fluid or lung biopsy specimens. RESULTS: A total of 168 anti-MDA5-positive DM patients including 108 females and 60 males were enrolled in the study. Of these patients, 154 had ILD, and 66(39.3%) of them presented RP-ILD. Seventy patients with pulmonary infection were confirmed by etiology. In the patients with RP-ILD, 39 (59.1%) of them were complicated with pulmonary infection. While only 31 cases(30.4%) had pulmonary infection in the non-RP-ILD patients. The incidence of pulmonary infection in the patients with RP-ILD was significantly higher than that of those with non-RP-ILD (P < 0.001). The serum YKL-40 levels in the RP-ILD patients with pulmonary infection were the highest compared with RP-ILD without pulmonary infection, non-RP-ILD with pulmonary infection and non-RP-ILD without pulmonary infection groups among all the patients [83 (42-142) vs. 42 (21-91) vs. 43 (24-79) vs. 38 (22-69), P < 0.01].The sensitivity, specificity and AUC of serum YKL-40 in the diagnosis of RP-ILD complicated with pulmonary infection were 75%, 67%, and 0.72, respectively. The AUC of diagnosed of anti-MDA5 positive DM patients complicated with RP-ILD and pulmonary infection was higher than that of patients complicated with only RP-ILD and only pulmonary infection (0.72 vs. 0.54 and 0.55, Z=2.10 and 2.11, P < 0.05). CONCLUSION: The prognosis of anti-MDA5-positive DM patients with RP-ILD and pulmonary infection were poor. Serum YKL-40 level can be used as a helpful tool for the diagnosis of coexistence of these conditions in the patients.

[GPCR48 promotes invasion and metastasis by inducing epithelial-mesenchymal transition in hepatocellular carcinoma cells].

Objective: To observe the G protein-coupled receptor 48 (GPCR48) expression in hepatocellular carcinoma (HCC) cell lines with different metastatic potential and its characteristics effect on the invasion and metastasis of Huh7 hepatoma cells via epithelial-mesenchymal transition (EMT). Methods: Western blot was used to detect the protein expression level of GPCR48 in HCC cells with different metastatic potential. The lentivirus vector expressing GPCR48 gene was constructed. GPCR48 was overexpressed in Huh7 hepatoma cells. The GPCR48 overexpression level was detected by real-time PCR and Western blot. Transwell invasion and migration assay was used to detect the Huh7 hepatoma cells invasion and migration ability in the Control, Mock and GPCR48 overexpression group. Real-time PCR and Western blot were used to detect Huh7 hepatoma cells mRNA and protein expression levels of the EMT related markers (E-cadherin, N-cadherin, vimentin, and γ catenin) in the Control, Mock and GPCR48 overexpression groups, respectively. Analysis of variance was used to compare the differences between data sets. Results: GPCR48 protein expression level in metastatic HCC cell lines was significantly higher than non-metastatic HCC cell lines (P < 0.05). The lentivirus vector expressing the GPCR48 gene had effectively transfected the Huh7 hepatoma cells and stably expressed the GPCR48mRNA and protein. Compared with the Mock and the Control group, Huh7 hepatoma cells invasion and migration ability in the GPCR48 overexpression group was significantly enhanced (F≥5.54, P < 0.05), and the mRNA and protein expression levels of epithelial phenotypic markers E-cadherin and γ-catenin were decreased (P < 0.05). The mRNA and protein expression levels of the mesenchymal phenotypic markers N-cadherin and Vimentin were increased (P < 0.05), indicating that EMT changes occurred in Huh7 hepatoma cells had overexpressed GPCR48. Conclusion: GPCR48 expression level is positively correlated with the metastatic potential of HCC cells. GPCR48 overexpression can down-regulate the expression of epithelial phenotypic markers and up-regulate the expression of mesenchymal phenotypic markers, and induce EMT changes in HCC cells, thus promoting HCC cells invasion and migration.

A high level of serum neopterin is associated with rapidly progressive interstitial lung disease and reduced survival in dermatomyositis.

Neopterin is primarily synthesized and released by activated macrophages/monocytes upon stimulation with interferon-γ and is considered as a marker for macrophage activation. This study aimed to analyze the serum levels of neopterin in patients with dermatomyositis (DM) in association with clinical manifestations, laboratory data and patient prognosis. One hundred and eighty-two consecutive DM patients and 30 healthy controls were retrospectively enrolled into the study. Serum levels of neopterin were significantly increased in DM patients compared to healthy controls (P < 0·001). High serum neopterin levels were associated with anti-melanoma differentiation-associated gene (MDA5) antibody, rapidly progressive interstitial lung disease (RP-ILD) and characteristic DM cutaneous involvement. Longitudinal assessment of serum samples revealed that the serum neopterin levels were closely correlated with disease severity (ß = 30·24, P < 0·001). In addition, a significant increase in serum neopterin concentration of non-survivors was observed when compared to that of survivors (P < 0·001). Receiver operator characteristic curves showed that serum neopterin could distinguish non-survivors and survivors at an optimal cut-off level of 22·1 nmol/l with a sensitivity and specificity of 0·804 and 0·625, respectively (P < 0·001). Kaplan-Meier survival curves revealed that DM patients with serum neopterin > 22·1 nmol/l had a significantly higher mortality compared to the patient group with serum neopterin < 22·1 nmol/l (log-rank P < 0·001). Multivariate regression analysis identified high serum neopterin concentration to be an independent risk factor for poor prognosis in DM (adjusted hazard ratio = 4·619, 95% confidence interval = 2·092-10·195, P < 0·001). In conclusion, increased serum levels of neopterin were significantly associated with RP-ILD and reduced survival in DM patients, suggesting it as a promising biomarker in disease evaluation of DM.

[Clinical and pathological characteristics of immune mediated necrotizing myopathy].

OBJECTIVE: To investigate the clinical and pathological features of immune-mediated necrotic myopathies (IMNM) with different myositis-specific antibodies (MSAs). METHODS: In the study, 104 IMNM patients who met any of the following three criteria were selected from idiopathic inflammatory myopathy patients who had MSAs results and underwent muscle biopsy from 2008 to 2018 in China-Japan Friendship Hospital: (1) Anti-signal recognition particle (SRP) antibody positive; (2) Anti-3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) antibody positive; (3) MSAs negative and consistent with the pathological diagnostic criteria of IMNM defined by the European Neuromuscular Centre in 2004. The clinical, laboratory and muscle pathological information of the IMNM patients were retrospectively collected and compared in anti-SRP, anti-HMGCR and MSAs negative groups. RESULTS: Of 104 IMNM patients, 47 patients (45.2%) were positive for anti-SRP antibody, 23 (22.1%) were positive for anti-HMGCR antibody, and 34 (32.7%) were negative for MSAs. The common symptoms of IMNM patients were muscle weakness (92.3%), elevated serum creatine kinase level (92.3%), dysphagia (33.7%) and interstitial lung diseases (ILD) (49.5%). The anti-HMGCR-positive patients were more frequent to have "V" sign (30.4% vs. 4.3% and 5.9%, P<0.01) as compared with the anti-SRP-positive and MSAs-negative patients. The incidence of ILD in the anti-SRP-positive patients was higher than that in the anti-HMGCR-positive and MSAs negative patients (64.4% vs. 34.8% and 29.0%, P<0.01). The prevalence of the patients combined with other connective tissue diseases in MSAs-negative IMNM was higher than that in the other two groups (32.4% vs. 8.5% and 4.3%, P<0.01). 93.3% of the anti-SRP-positive patients were found with antinuclear antibody positivity, higher than those of the anti-HMGCR-positive and MSAs-negative patients (93.3% vs. 36.4% and 58.8%, P<0.001). The common pathological features of IMNM were muscle fibre necrosis (94.2%), regeneration (67.3%) and phagocytosis (65.4%), overexpression of major histocompatibility complex1 on sarcolemma (78.8%), infiltration of CD4+ T cells (81.7%) and CD68+ macrophage (79.8%) and expression of membrane attack complex (MAC) (77.8%). The endomysial infiltration of CD4+ T cells and CD68+ macrophage and MAC expression on sarcolemma in the MSAs-negative group were more common than that in the anti-SRP and anti-HMGCR groups (88.2% vs. 57.4% and 60.9%, 91.2% vs. 59.1% and 38.1%, 76.5% vs. 45.5% and 42.9%, respectively, P<0.01). CONCLUSION: There is heterogeneity in anti-SRP-positive, anti-HMGCR-positive or MSAs-negative patients. The detection of MSAs and performing of muscle biopsy are useful for distinguishing different types of IMNM.

[Effect of cyclophosphamide on ovarian function in patients with systemic lupus erythematosus in childbearing age].

Objective: Serum anti Müllerian hormone (AMH) was used to evaluate the effect of cyclophosphamide (CTX) on ovarian function in female patients with systemic lupus erythematosus (SLE). Methods: A total of 121 female patients who were 18-50 years old with normal menstruation were selected. Among them, 54 patients were treated with CTX as the study group and the remaining 67 cases as the control group. Before and after treatment for 6 months, the clinical characteristics, menstruation and AMH level of all patients were recorded and detected. At the same time, the method of using CTX and the cumulative measurement are recorded. Results: (1) Before treatment, there was no significant difference in AMH and mean age, duration of disease and SLEDAI score between the CTX treatment group and the control group. The renal injury in the CTX treatment group (44.4%) was higher than that of the control group (34.3%), and the difference was statistically significant (P<0.05). (2) After 6 months of treatment, the AMH of group CTX decreased from (2.39±1.58) µg/L to (1.56±1.42) µg/L, and the difference was statistically significant (P<0.01). But there was no significant change in the control group. In 54 cases of CTX treatment group, 23 cases (42.6%) had different degree of menstrual abnormalities, while 67 cases had only 8 cases (11.9%) in the control group. Moreover, the AMH level of 31 cases with abnormal menstruation was (0.95±0.59) µg/L, which was significantly lower than that of the other 90 normal cases (2.36±1.58) µg/L. (3) In 54 cases of CTX treatment group, the cumulative dose of CTX was less than 3 g in 14 cases, 33 cases of 3-6 g, 7 cases greater than 6 g. AMH was all were lower than those before treatment. But there was a statistical difference between the 3 g group and 3-6 g group before treatment, and there were statistical differences between the groups. Conclusion: CTX can damage ovarian function in women of childbearing age SLE. Low dose intravenous CTX may have less damage. Serum AMH can be used to monitor ovarian function in patients with SLE and to guide individualized treatment.

[Effects of integrin metalloproteinases on osteogenic differentiation].

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.

Genetic association of HLA-DRB1 multiple polymorphisms with dermatomyositis in Chinese population.

Genetic variation in HLA plays an important role in the pathogenesis of dermatomyositis (DM). The aim of this study was to investigate the association of HLA class II with DM in China. Two hundred and twenty-four DM patients and 300 healthy controls were randomly enrolled at China-Japan Friendship Hospital. High-resolution typing of HLA-DRB1 alleles was performed by sequencing based typing. The HLA-DQA1 and HLA-DQB1 alleles were determined by polymerase chain reaction sequence-specific primers. The frequencies of HLA-DRB1*09:01 (28.6% vs 11.3%, P < .0001, odds ratio, OR = 3.14, 95% confidence interval, CI = 2.47-3.99) and HLA-DRB1*12:01 (29.0% vs 11.0%, P < .0001, OR = 3.30, 95% CI = 2.59-4.20) in DM patients were significantly higher than that in healthy controls. No significant difference was found in HLA-DQA1 or DQB1 alleles between DM patients and healthy controls. Furthermore, DM patients with anti-melanoma differentiation-associated gene 5 antibody (anti-MDA5) had a significantly higher frequency of HLA-DRB1*12:01 compared to that for patients without anti-MDA5 (P < .0001, OR = 4.77, 95% CI: 2.29-9.93). Multivariate binary logistic regression analysis was performed to identify the risk factors for interstitial lung disease. The HLA-DRB1*09:01 allele was a poor prognostic factor (P = .01, OR = 9.21, 95% CI: 1.47-57.50) for DM patients with anti-MDA5 autoantibody. In summary, our findings indicate that HLA-DRB1*09:01 and HLA-DRB1*12:01 alleles may contribute to susceptibility of adult DM in Han Chinese population. In addition, the DRB1*12:01 genotype is significantly associated with the presence of anti-MDA5 antibody in DM patients.

The g.243A>G mutation in intron 17 of MUC4 is significantly associated with susceptibility/resistance to ETEC F4ab/ac infection in pigs.

Using a porcine radiation hybrid panel, we assigned the mucin 4 (MUC4) gene to SSC13q41, which harbours the enterotoxigenic Escherichia coli (ETEC) F4ab/ac receptor locus. In addition, we identified two SNPs in intron 17 of MUC4 (DQ124298:g.243A>G and DQ124298:g.334A>G) in the parental population of a White Duroc x Erhualian cross. Association analysis showed that the MUC4 g.243A>G mutation was strongly associated with ETEC F4ab/ac, and especially with F4ac adhesion phenotypes in the White Duroc x Erhualian resource population, indicating that this polymorphism was in a significant linkage disequlibrium with the ETEC F4ab/ac receptor locus. Because of different linkage disequlibrium values between the ETEC F4ab and F4ac adhesion phenotypes and the MUC4 g.243A>G mutation, we argue that the inheritance of F4ab and F4ac receptors might be under the control of two closely linked loci.

Pycnogenol protects neurons from amyloid-beta peptide-induced apoptosis.

Neuronal apoptosis is one of the pathological features of Alzheimer's disease (AD). Morphological pathology reveals that neuronal apoptosis is associated with senile plaques containing amyloid-beta peptide (Abeta) in AD brains. Reactive oxygen species (ROS) has been proposed to be involved in the apoptotic mechanism of Abeta-mediated neurotoxicity. In the present study, using a rat pheochromocytoma (PC12) cell line, we investigated the effect of Pycnogenol (PYC), a potent antioxidant and ROS scavenger, on Abeta(25-35)-induced apoptosis and ROS generation. We used vitamin E, a known antioxidant agent, to verify the effect of PYC. Abeta(25-35)-induced apoptosis in PC12 cells was demonstrated by: (1) a dose-dependent loss of cell viability; (2) a time- and dose-dependent increase in the apoptotic cells; (3) an induction of DNA fragmentation; and (4) an increase in caspase-3 activity and cleavage of poly (ADP-ribose) polymerase (PARP). Our data showed that a significant increase in ROS formation preceded apoptotic events after PC12 cells were exposed to Abeta(25-35). We further found that PYC not only suppressed the generation of ROS but also attenuated caspase-3 activation, DNA fragmentation, PARP cleavage, and eventually protected against Abeta-induced apoptosis. Vitamin E also suppressed cell death and caspase-3 activation induced by Abeta(25-35). Taken together, these results suggest that ROS may be involved in Abeta-induced apoptosis in PC12 cells. They further suggest that PYC can reduce apoptosis, possibly by decreasing free radical generation in PC12 cells.

Pycnogenol enhances endothelial cell antioxidant defenses.

Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), superoxide anion (O2(-)), and hydroxyl radical (OH(•)) have been implicated in mediating various pathological events such as cancer, atherosclerosis, diabetes, ischemia, inflammatory diseases, and the aging process. The glutathione (GSH) redox cycle and antioxidant enzymes-superoxide dismutase (SOD) and catalase (CAT)-play an important role in scavenging ROS and preventing cell injury. Pycnogenol has been shown to protect endothelial cells against oxidant-induced injury. The present study determined the effects of pycnogenol on cellular metabolism of H2O2 and O2(-) and on glutathione-dependent and -independent antioxidant enzymes in bovine pulmonary artery endothelial cells (PAEC). Confluent monolayers of PAEC were incubated with pycnogenol, and oxidative stress was triggered by hypoxanthine and xanthine oxidase or H2O2. Pycnogenol caused a concentration-dependent enhancement of H2O2 and O2(-) clearance. It increased the intracellular GSH content and the activities of GSH peroxidase and GSH disulfide reductase. It also increased the activities of SOD and CAT. The results suggest that pycnogenol promotes a protective antioxidant state by upregulating important enzymatic and nonenzymatic oxidant scavenging systems.