Expansion of exhausted CD8+ T cells associates with increased pulmonary fungal infection risk in anti-melanoma differentiation associated gene 5 dermatomyositis.

OBJECTIVES: Anti-MDA5+ dermatomyositis was associated with poor prognosis due to the high incidence of rapid progressive interstitial lung disease, pulmonary infection. The aim of this study is to investigate the abundance and clinical relevance of exhaustion markers on peripheral CD8 T cells from patients with idiopathic inflammatory myopathy (IIM). METHODS: Twenty-nine healthy controls (HCs) and 71 patients with IIM were enrolled, including 42 with anti-MDA5+ and 18 with anti-MDA5- dermatomyositis (DM) and 11 with anti-synthetase syndrome (ASS). Flow cytometry was applied to detect PD-1, TIM-3 and LAG-3 in CD8 T cells. The clinical associations of the CD8 T cell exhaustion phenotype in patients with anti-MDA5+ DM were analysed. RESULTS: CD8 T cells from patients with anti-MDA5+ DM showed significantly increased LAG-3, TIM-3 and PD-1 compared to those from patients with anti-MDA5- IIM (18 with anti-MDA5- DM and 11 with ASS) or HCs (adjusted p all < 0.05). CD8 T cells with distinct exhaustion levels were all significantly increased in anti-MDA5+ DM patients compared with HCs (p all < 0.05). Patients with high level of PD-1+ TIM-3+LAG-3+ CD8+ T cells had a significant higher incidence of pulmonary fungal infections but lower counts of CD4+ and CD8+ T cells. ROC analysis revealed that the frequency of PD-1+TIM-3+LAG-3+CD8+ T cell significantly predicted pulmonary fungal infections (area under the curve: 0.828). CONCLUSIONS: CD8 T cells from patients with anti-MDA5+ DM show significant exhausted phenotype, and increased exhausted CD8 T cells were associated with high risk of pulmonary fungal infection.

Evaluation and validation of the prognostic value of anti-MDA5 IgG subclasses in dermatomyositis-associated interstitial lung disease.

OBJECTIVE: To investigate the association between the anti-melanoma differentiation associated gene 5 (MDA5) IgG subclasses and prognosis of patients with dermatomyositis (DM)-associated interstitial lung disease (ILD). METHODS: This retrospective study included 122 anti-MDA5 positive DM-ILD patients admitted from October 2017 to October 2020 as training cohort, and additional 68 patients from August 2014 to September 2017 as validation cohort. The levels of anti-MDA5 total IgG and IgG subclasses were measured using in-house enzyme-linked immunosorbent assays, and analysed in association with the patient prognosis. RESULTS: In the training cohort, the concentrations of anti-MDA5 IgG1 and IgG3 in non-survivors were significantly higher than in survivors (P < 0.05), whereas there were no significant differences in the IgG2 and IgG4 levels. Kaplan-Meier survival analysis revealed that the levels of anti-MDA5 total IgG, IgG1 and IgG3 were associated with mortality (P < 0.05). Multivariate analysis revealed anti-MDA5 IgG1 >13 U/ml and anti-MDA5 IgG3 >11 U/ml were independent risk factors for death of DM-ILD patients (P < 0.05). Anti-MDA5 IgG1 was confirmed as an independent risk factor in the validation cohort, while anti-MDA5 IgG3 was not. Anti-MDA5 IgG1 showed greater discriminable power for patient prognosis (Youden index 0.494) than anti-MDA5 total IgG, IgG3, or the combination of IgG1 and IgG3 (Youden index 0.356, 0.32 and 0.447, respectively). CONCLUSION: Anti-MDA5 IgG1 and IgG3 are significantly associated with poor prognosis in DM-ILD patients, and anti-MDA5 IgG1 is more efficient as a prognostic biomarker in DM-ILD patients.

Contribution of Necroptosis to Myofiber Death in Idiopathic Inflammatory Myopathies.

OBJECTIVE: Myofiber necrosis is a significant pathologic characteristic of idiopathic inflammatory myopathies (IIMs), and its molecular mechanism is largely unknown. Necroptosis is a recently identified form of regulated necrotic cell death, and its activation might have crucial biologic consequences. The aim of the present study was to investigate the role of necroptosis in IIM muscle damage. METHODS: Western blot and immunohistochemistry analyses were performed to examine the expression of receptor-interacting protein 3 (RIP-3) and mixed-lineage kinase domain-like (MLKL) proteins in 26 IIM patients and 4 healthy controls, as well as necroptosis-related damage-associated molecular pattern molecules. Tumor necrosis factor (TNF) was used to stimulate cultured C2C12 myoblasts, and the involvement of necroptosis in cell death of C2C12 cells was studied in vitro. RESULTS: The expression of RIP-3 and MLKL proteins and their phosphorylated forms was significantly increased in the muscle tissue of IIM patients compared to that of healthy controls. The expression levels of RIP-3 and MLKL proteins were associated with the severity of muscle damage in patients with IIM. Significant colocalization of MLKL with high mobility group box chromosomal protein 1 in necrotizing myofibers was observed in muscle biopsy tissue from patients with IIM. Stimulation of C2C12 myoblasts with TNF and a pan-caspase inhibitor, Z-VAD, resulted in the overactivation of necroptosis and significantly increased necrotic cell death. Strategies involving either inhibition of necroptosis with necrostatin-1 or knockdown of MLKL expression successfully prevented necroptosis-induced cell death of C2C12 cells. CONCLUSION: These findings demonstrate that overactivated necroptosis contributes to muscle damage in IIMs and suggest that necroptosis inhibitors could represent a new therapeutic target in the treatment of IIMs.

Aberrantly Expressed Galectin-9 Is Involved in the Immunopathogenesis of Anti-MDA5-Positive Dermatomyositis-Associated Interstitial Lung Disease.

BACKGROUND: Dermatomyositis (DM) associated rapidly progressive interstitial lung disease (RP-ILD) has high mortality rate and poor prognosis. Galectin-9 (Gal-9) plays multiple functions in immune regulation. We investigated Gal-9 expression in DM patients and its association with DM-ILD. METHODS: A total of 154 idiopathic inflammatory myopathy patients and 30 healthy controls were enrolled in the study. Cross-sectional and longitudinal studies were used to analyze the association between serum Gal-9 levels and clinical features. Enzyme-linked immunosorbent assay and qRT-PCR were used to examine Gal-9 expression in the sera and isolated peripheral blood mononuclear cells (PBMCs) from DM patients. Immunohistochemistry was performed to analyze the expression of Gal-9 and its ligand (T-cell immunoglobulin mucin (Tim)-3 and CD44) in lung tissues from anti-melanoma differentiation-associated gene 5 (MDA5)-positive patients. The effect of Gal-9 on human lung fibroblasts (MRC-5) was investigated in vitro. RESULTS: Serum Gal-9 levels were significantly higher in DM patients than in immune-mediated necrotizing myopathy patients and healthy controls (all p < 0.001). Higher serum Gal-9 levels were observed in anti-MDA5-positive DM patients than in anti-MDA5-negative DM patients [33.8 (21.9-44.7) vs. 16.2 (10.0-26.9) ng/mL, p < 0.001]. Among the anti-MDA5-positive DM patients, serum Gal-9 levels were associated with RP-ILD severity. Serum Gal-9 levels were significantly correlated with disease activity in anti-MDA5-positive DM patients in both cross-sectional and longitudinal studies. PBMCs isolated from anti-MDA5-positive DM patients (3.7 ± 2.3 ng/mL) produced higher levels of Gal-9 than those from immune-mediated necrotizing myopathy patients (1.1 ± 0.3 ng/mL, p = 0.022) and healthy controls (1.4 ± 1.2 ng/mL, p = 0.045). The mRNA levels of Gal-9 were positively correlated with the levels of type-I interferon-inducible genes MX1 (r = 0.659, p = 0.020) and IFIH1 (r = 0.787, p = 0.002) in PBMCs from anti-MDA5-positive DM patients. Immunohistochemistry revealed increased Gal-9 and Tim-3 expression in the lung tissues of patients with DM and RP-ILD. In vitro stimulation with Gal-9 protein increased CCL2 mRNA expression in MRC-5 fibroblasts. CONCLUSIONS: Among anti-MDA5-positive DM patients, Gal-9 could be a promising biomarker for monitoring disease activity, particularly for RP-ILD severity. Aberrant expression of the Gal-9/Tim-3 axis may be involved in the immunopathogenesis of DM-ILD.

Increased Levels of Soluble CD206 Associated with Rapidly Progressive Interstitial Lung Disease in Patients with Dermatomyositis.

OBJECTIVE: Soluble CD206 (sCD206) is considered a macrophage activation marker, and a previous study proved it as a potential biomarker to predict the severity of anti-melanoma differentiation-associated gene 5- (anti-MDA-5-) positive dermatomyositis- (DM-) associated interstitial lung disease (ILD). To investigate the role of sCD206 in various subtypes of DM, we evaluated the serum level of sCD206 in patients with different myositis-specific autoantibodies besides anti-MDA-5 and clarified its clinical significance. METHODS: Commercial enzyme-linked immunosorbent assay kits were used to detect serum concentrations of sCD206 in 150 patients with DM and 52 healthy controls (HCs). Correlations between sCD206 levels and clinical features, laboratory examinations, and pulmonary function test parameters were analysed. RESULTS: The median concentrations of serum sCD206 in DM patients were significantly higher than those in HCs (p < 0.0001). Furthermore, median sCD206 levels were elevated in patients with ILD (p = 0.001), especially in those with rapidly progressive ILD (RP-ILD) (p < 0.0001). In addition, sCD206 levels were negatively correlated with the pulmonary function test results, including the percent predicted forced vital capacity (r = -0.234, p = 0.023), percent predicted forced expiratory volume in one second (r = -0.225, p = 0.030), and percent predicted carbon monoxide diffusion capacity (r = -0.261, p = 0.014). Age- and gender-adjusted multivariable analysis showed that sCD206 was an independent prognostic factor for RP-ILD in patients with DM. A longitudinal study showed that sCD206 levels were positively correlated with the physician global assessment visual analog scale scores (ß = 54.201, p = 0.001). CONCLUSION: Serum sCD206 levels were significantly increased in patients with DM and significantly associated with RP-ILD, suggesting that sCD206 is an important biological predictor of RP-ILD in patients with DM.

A novel autoantibody targeting calreticulin is associated with cancer in patients with idiopathic inflammatory myopathies.

OBJECTIVES: To investigate the prevalence and clinical significance of anti-calreticulin autoantibodies (anti-CRT Ab) in a large cohort of idiopathic inflammatory myopathy (IIM) patients. METHODS: Sera from 469 patients with IIM, 196 patients with other connective tissue diseases, 28 patients with solid tumors and 81 healthy controls were screened for anti-CRT Ab by enzyme-linked immunosorbent assay using human recombinant CRT protein. Sera from 35 IIM patients were tested using an immunoprecipitation assay to confirm the presence of anti-CRT Ab. Subsequently, IIM-cancer patients were identified and divided into new-onset, remission and recurrent groups based on their cancer status. The relationships between anti-CRT Ab levels and IIM disease activity were also investigated. RESULTS: Serum anti-CRT Ab was detected positive in 81 of the 469 (17.3%) IIM patients. Immunoprecipitated bands were observed at a molecular weight of 60 kDa corresponding to the CRT protein. The IIM patients with anti-CRT Ab more frequently had cancers compared to the patients without anti-CRT Ab. Moreover, the prevalence of anti-CRT Ab differed according to the cancer status. The IIM patients with recurrent cancers had a much higher prevalence of anti-CRT Ab than those with cancers in remission. Also, serum anti-CRT Ab levels positively correlated with disease activity at baseline and at follow-up visits. CONCLUSION: We report the existence of serum anti-CRT Ab in IIM patients and demonstrate the possible association of anti-CRT Ab with malignancy in IIM patients. Serum anti-CRT Ab could serve as a novel candidate marker of cancer in IIM patients.

Anti-Mi-2 antibodies characterize a distinct clinical subset of dermatomyositis with favourable prognosis.

BACKGROUND: Anti-Mi-2 antibody is a type of myositis-specific autoantibody found in idiopathic inflammatory myopathy patients. OBJECTIVES: To investigate the clinical features and long-term outcomes in anti-Mi-2-positive dermatomyositis (DM) patients. MATERIALS AND METHODS: Serum anti-Mi-2ß antibodies were detected in 357 DM patients by enzyme-linked immunosorbent assays, and possible associated clinical features were investigated based on cross-sectional and longitudinal studies. RESULTS: Of the DM patients, 40/357 (11.2%) were positive for anti-Mi-2ß antibodies and found to have a significantly higher frequency of V sign (72.5% vs 45.7%; p = 0.001), shawl sign (60.0% vs 35.6%; p = 0.003), and muscle weakness (77.5% vs 57.1%; p = 0.013), but a lower incidence of interstitial lung disease (ILD) (37.5% vs 60.9%; p = 0.005) and malignancy (0% vs 12.0%; p = 0.041) than anti-Mi-2ß-negative patients. Anti-Mi-2ß antibody levels positively correlated with disease activity. After a median follow-up period of 44 months, 97.0% of patients showed clinical remission. Twenty-six anti-Mi-2ß-positive patients had a disease course longer than two years, and 16/26 (61.5%) were monocyclic without relapse. Moreover, five patients (15.1%) were drug-free with complete remission for more than three months. Kaplan-Meier survival curves showed that DM patients with positive anti-Mi-2ß had a significantly lower mortality rate compared to anti-Mi-2ß-negative patients (log-rank; p = 0.035). Interestingly, anti-Mi-2ß antibodies did not disappear in all patients over time. CONCLUSION: Anti-Mi-2ß antibodies were associated with a subgroup of DM with a low frequency of ILD and malignancy, good treatment response, and favourable outcome. Moreover, anti-Mi-2ß levels correlated with disease activity.

Identification of a novel autoantibody against heat shock factor 1 in idiopathic inflammatory myopathy.

OBJECTIVES: Myositis autoantibodies show great utility in the diagnosis and clinico-serological phenotyping of idiopathic inflammatory myopathy (IIM). We identified a novel autoantibody against heat shock factor 1 (HSF1) and further evaluated its disease specificity and clinical significance in IIM patients. METHODS: A human protein microarray was used to identify autoantibodies in myositis sera. ELISA, immunoblot and dot blot assays were applied to examine anti-HSF1 autoantibodies in IIM patients and controls. Immunofluorescence was used to detect HSF1 expression in muscle tissues. RESULTS: Anti-HSF1 was identified as a novel autoantibody by protein microarray and the seroreactivity was confirmed by immunoprecipitation, ELISA, immunoblot and dot blot assays. Anti-HSF1 autoantibodies were present in 64/581 (11.0%) IIM, 4/37 (10.8%) rheumatoid arthritis, 5/40 (12.5%) primary Sjögren's syndrome, 2/40 (5%) systemic lupus erythematosus, while largely negative in healthy controls. Anti-HSF1 autoantibodies were significantly associated with pruritus, hypergammaglobulinaemia, and elevated erythrocyte sedimentation rate in IIM patients. Anti-HSF1 autoantibodies were more prevalent in cancer-associated myositis (CAM) compared to non-CAM patients (17.2% vs. 7.5%, p=0.009), nevertheless were undetectable in cancer controls. Meanwhile, cross-sectional and longitudinal analyses revealed positive correlations between anti-HSF1 levels and disease activity in IIM patients without cancer. Additionally, increased expression of HSF1 was found in regenerating muscle cells of myositis muscle tissues. CONCLUSIONS: These data reveal anti-HSF1 as a new autoantibody associated with CAM in IIM. The autoimmunity against HSF1 may be involved in the immunopathogenesis of myositis.

Targeted capture sequencing identifies novel genetic variations in Chinese patients with idiopathic inflammatory myopathies.

OBJECTIVES: Previous association studies have identified genetic variants in the human leukocyte antigen (HLA) complex as substantial risk factors for idiopathic inflammatory myopathies (IIMs). However, a great number of genes are located in the HLA region, and thus fine mapping is quite necessary. METHODS: Targeted capture sequencing were performed on the whole HLA region in 42 IIM patients and 24 healthy controls. A microarray analysis was applied to analyze gene expression profiles in additional 20 newly diagnosed IIM and five healthy controls. RESULTS: The HLA region was confirmed to be associated with IIMs in Chinese patients. By gene expression profiling and pathway analysis, several genes were identified as candidates for IIM risk factors, including HLA-A, HLA-B, HLA-DRB5, HLA-DRB1, HLA-DQA1, HLA-DQB1 and HLA-DQB2. Interestingly, p.Y107V of the HLA-DRB1 was predicted to be a potential causal non-synonymous variation for IIMs that may affect the antigen-binding groove of the HLA-II molecule. CONCLUSIONS: Our data have revealed novel genetic variations in the HLA region of IIM patients and provide new insight into the pathogenesis and diagnosis of IIMs.

The prevalence and clinical significance of anti-PUF60 antibodies in patients with idiopathic inflammatory myopathy.

Autoantibodies against poly-U-binding factor 60 kDa protein (PUF60) have been reported in Caucasian dermatomyositis (DM) patients. However, their clinical significance in idiopathic inflammatory myopathy (IIM) remains to be fully clarified. Our objective was to analyze the prevalence and clinical significance of anti-PUF60 antibodies in a large cohort of Chinese IIM patients. In our study, 388 IIM patients, 301 disease controls, and 167 healthy controls (HCs) were involved. An enzyme-linked immunosorbent assay (ELISA) was developed to detect serum anti-PUF60 levels and was validated using immunoblotting methods. Unpaired Mann-Whitney U test and Spearman correlation analysis were used when appropriate. Anti-PUF60 antibodies were observed in IIM patients at a frequency of 10.6% (41/388). Subgrouping analysis revealed that the prevalence of anti-PUF60 antibodies was 10% in DM, 5.5% in polymyositis (PM), 10% in immune-mediated necrotizing myositis (IMNM), and 26.5% in myositis-overlap syndrome. Anti-PUF60 antibodies were also observed in systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and Sjögren's syndrome (SS) patients at a positive rate of 17.3, 14.5, and 10.1% respectively. Intriguingly, anti-PUF60 antibodies were frequently observed in clinically amyopathic dermatomyositis (CADM) patients and DM patients without currently known myositis autoantibodies. Furthermore, DM patients with anti-PUF60 antibodies had higher prevalence of skin ulcerations. Moreover, longitudinal investigation in eight DM patients with anti-PUF60 antibodies revealed that the antibodies levels decreased with disease remission. Anti-PUF60 antibodies were nonspecific for myositis, since they could be detected in other rheumatic diseases. Further investigation of anti-PUF60 antibodies may reveal shared pathogenic pathways in systemic autoimmune disorders.

Transcriptomic profiling of long non-coding RNAs in dermatomyositis by microarray analysis.

Long non-coding RNAs (lncRNAs) are prevalently transcribed in the genome and have been found to be of functional importance. However, the potential roles of lncRNAs in dermatomyositis (DM) remain unknown. In this study, a lncRNA + mRNA microarray analysis was performed to profile lncRNAs and mRNAs from 15 treatment-naive DM patients and 5 healthy controls. We revealed a total of 1198 lncRNAs (322 up-regulated and 876 down-regulated) and 1213 mRNAs (665 up-regulated and 548 down-regulated) were significantly differentially expressed in DM patients compared with the healthy controls (fold change>2, P < 0.05). Subgrouping DM patients according to the presence of interstitial lung disease and anti-Jo-1 antibody revealed different expression patterns of the lncRNAs. Pathway and gene ontology analysis for the differentially expressed mRNAs confirmed that type 1 interferon signaling was the most significantly dysregulated pathway in all DM subgroups. In addition, distinct pathways that uniquely associated with DM subgroup were also identified. Bioinformatics prediction suggested that linc-DGCR6-1 may be a lncRNA that regulates type 1 interferon-inducible gene USP18, which was found highly expressed in the perifascicular areas of the muscle fibers of DM patients. Our findings provide an overview of aberrantly expressed lncRNAs in DM muscle and further broaden the understanding of DM pathogenesis.

Elevated Serum Levels of Soluble CD163 in Polymyositis and Dermatomyositis: Associated with Macrophage Infiltration in Muscle Tissue.

OBJECTIVE: To investigate serum levels of soluble CD163 (sCD163) in patients with polymyositis (PM) and dermatomyositis (DM), and to correlate these to clinical manifestations and laboratory data. METHODS: Serum levels of sCD163 were detected in 24 patients with PM, 84 patients with DM, and 46 healthy controls by using the ELISA method. Immunohistochemistry staining of macrophage infiltration in muscle tissue using anti-CD163 monoclonal antibody was conducted on muscle biopsy specimens from 13 patients with PM and 17 with DM. RESULTS: Serum levels of sCD163 were significantly increased in patients compared with healthy controls (p < 0.001). Patients with interstitial lung disease (ILD) had statistically higher sCD163 levels than patients without ILD (p < 0.001). High serum sCD163 levels were associated with increased incidence of antinuclear antibody (p < 0.05), higher serum levels of immunoglobulin G (p < 0.01) and immunoglobulin A (p < 0.05), and increased erythrocyte sedimentation rates (p < 0.01). Serum sCD163 levels were inversely correlated with CD3+ T cell counts in peripheral blood of patients (r = -0.306, p < 0.01). Cross-sectional assessment and longitudinal study revealed a significant correlation between serum sCD163 levels and disease activity. Patients with high serum sCD163 levels showed a higher incidence of CD163+ macrophage infiltration in muscle tissue than patients with normal sCD163 levels (chi-square value = 10.804, p < 0.01). CONCLUSION: Serum levels of sCD163 were significantly elevated and correlated with disease severity in patients with PM/DM, suggesting serum sCD163 as a promising biomarker in the disease evaluation of PM/DM. Our finding of elevated serum sCD163 levels associated with muscle macrophage infiltration highlights the role activated macrophage plays in the pathogenesis of PM/DM.

B-cell activating factor as a serological biomarker for polymyositis and dermatomyositis.

AIM: To investigate serum levels of B-cell activating factor (BAFF) in the patients with polymyositis (PM) and dermatomyositis (DM), and to systematically examine the association between serum BAFF levels and disease activity in PM/DM patients. PATIENTS & METHODS: A cross-sectional analysis included 92 PM/DM patients and 25 healthy control subjects. A longitudinal study followed 24 patients. Serum BAFF concentrations were detected by the ELISA method. RESULTS: Serum BAFF levels in PM/DM patients were significantly higher than those in healthy controls. A cross-sectional assessment revealed a modest correlation between serum BAFF levels and global disease activity and a mild correlation between serum BAFF levels and muscle disease activity. The longitudinal study showed that serum BAFF levels modestly correlated with global disease activity and muscle disease activity. CONCLUSION: Resulting data showed high serum BAFF levels in PM/DM patients and suggested BAFF as a serological biomarker for PM/DM disease activity.

Expression of tumor necrosis factor-like weak inducer of apoptosis and fibroblast growth factor-inducible 14 in patients with polymyositis and dermatomyositis.

INTRODUCTION: The aim of this study was to investigate the expression of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) in patients with polymyositis (PM) and dermatomyositis (DM), and their relation to clinical manifestations. METHODS: Serum levels of TWEAK were detected in 98 PM/DM patients and 37 healthy controls by using the ELISA method. Total RNA isolated from fresh-frozen muscle tissue samples of 36 PM/DM patients and 10 healthy controls were used for analyzing the mRNA levels of TWEAK and Fn14 by quantitative reverse transcription polymerase chain reaction (RT-PCR). Immunofluorescence staining of TWEAK and Fn14 was conducted on muscle biopsy specimens from 23 PM/DM patients and seven healthy controls. RESULTS: Serum levels of TWEAK were significantly decreased in the PM/DM patients compared to those in the healthy controls (P < 0.001), and serum TWEAK levels negatively correlated with serum CD163 levels in PM/DM patients (r = -0.49, P < 0.001). The expression of Fn14 mRNA was significantly increased in the muscle tissue of PM/DM patients than in the muscle tissue of healthy controls (P < 0.01), whereas the expression of TWEAK mRNA in PM/DM patients was not statistically different from that of the healthy controls (P > 0.05). Fn14 mRNA levels in muscle tissue positively correlated with muscle disease activity (r = 0.512, P < 0.01). Patients with oropharyngeal dysphagia had significantly higher Fn14 mRNA levels than patients without oropharyngeal dysphagia (P < 0.05). The results of immunofluorescence staining showed that 19 out of 23 PM/DM patients were TWEAK-positive, and 20 out of 23 PM/DM patients were Fn14-positive. No detectable expressions of TWEAK or Fn14 were observed in the healthy controls. CONCLUSIONS: TWEAK-Fn14 axis may be involved in the pathogenesis of PM/DM. Further understanding of TWEAK-Fn14 function in PM/DM may help to define therapeutic targets for PM/DM.

[The levels and clinical significance of serum B cell activating factor in Chinese patients with polymyositis or dermatomyositis].

OBJECTIVES: To investigate serum levels of B cell activating factor (BAFF) in Chinese patients with polymyositis (PM) or dermatomyositis (DM), and analyze the correlation of BAFF with autoantibodies and clinical phenotypes. METHODS: Serum BAFF levels of 28 PM patients and 30 DM patients (study group), and 25 matched healthy controls (control group) were measured by ELISA. Serum anti-Jo-1 antibody levels were also measured by ELISA in all the subjects. The results of the two groups were compared by unpaired t test and the relevance was analyzed by Pearson's correlation analysis. RESULTS: Serum levels of BAFF in PM/DM patients were significantly higher compared to healthy controls (P = 0.000), but there was no statistically significant difference between the PM and DM patients (P > 0.05). Patients with interstitial lung disease (ILD) had significantly higher serum BAFF level than the patients without ILD (P = 0.000) or the controls (P = 0.000). Serum BAFF levels of patients with positive anti-nuclear antibody (ANA) were significantly higher than those with negative ANA (P = 0.003). For patients with anti-Jo-1 antibodies, the serum BAFF levels were correlated with the serum concentration of anti-Jo-1 antibodies (r = 0.799, P = 0.006). CONCLUSIONS: Serum levels of BAFF are increased in Chinese PM/DM patients. These findings indicate that BAFF may be possibly enrolled in the pathogenesis of PM/DM. Detecting serum BAFF levels could have some implication for the diagnosis and treatment of PM/DM.

[Distribution and species of mercury in water and sediments from Xiangjiang River section flowing through Zhuzhou, Xiangtan, Changsha].

Total mercury content in water in dry season is (1.881 +/- 1.854) microg x L(-1) in Xiangjiang River section flowing through Zhuzhou, Xiangtan, Changsha. Mercury in water is mainly in the form of particle mercury. The maxima of total mercury and soluble mercury content discovered in Xiawan attenuating rapidly in section of Xiawan-Xiangtan but slowly in section of Xiangtan-Lake Dongting. The average content of total mercury in sediment is 0.846 mg x kg(-1) up to the highest level in rivers of China. The highest content of total mercury discovered in Xiawan is 3.268 mg x kg(-1) which far more exceeds III-level soil pollution norms of China. In the section of Xiawan-Majiahe mercury mainly exists in sediment and average content total mercury (in sediments) is 2.218 mg x kg(-1). With coming down by a big margin total mercury content is 0.442 mg x kg(-1) in section of Yijiawan-Juzizhou. In the downstream of Changsha total mercury attenuating slowly. The mercury average content in pore-water of sediments is 0.035 microg x L(-1). There is no significant correlation between pore-water mercury and total mercury. Mercury (in sediments) is mainly in the form of residue, soluble plus exchangeable ions,and organic matter bound that will become active and pollution again. From upstream to downstream the fraction bound to organic matter decreases, but residue mercury increases to a small extent that indicates the change of mercury form from the organic matter bounded to the form of residue. In the section analysis of mercury (in sediments), Hg reaches 65 cm deep. As the locations close to sediments top, total mercury content increases until the distance to sediment top is 30-25 cm.

[Linkage of modified human papillomavirus type 16 E7 to CD40 ligand enhances specific CD8+ T-lymphocyte induction and anti-tumour activity of DNA vaccine].

OBJECTIVE: To develop human papillomavirus (HPV) 16 DNA vaccine for the treatment of HPV16 infection and its related tumors. METHODS: HPV16 oncogene E7 was modified by combined approaches including insertion and replication of specific region of E7 gene, murine codon optimization, and point-mutation at transforming regions of the E7 protein. The resulting artificial gene, named as mE7, was obtained by gene synthesis. The mE7 gene was then genetically fused to murine CD40 ligand (CD40L) by overlapping PCR to form the mE7/CD40L fusion gene. The mE7/CD40L gene was inserted into pVR1012 plasmid and then immunized C57/BL6 mice intramuscularly. The E7-specific IFN-gamma-secreting CD8+ T cells were analyzed with EIISPOT, and E7-specific antibody was measured by indirect ELISA. FACS assays were performed to analyze the activation of E7-specific Th cells. Mice were vaccinated, followed by tumor challenged or challenged before immunization. Tumor growth was observed. RESULTS: The mE7 DNA vaccine elicited an increased E7-specific antibody level (P < 0.01), E7-specific IFN-gamma-secreting CD8+ T (P < 0.01), and CD4+ T cells number (P < 0.05), compared with those of mice immunized with wE7 gene. Furthermore, the mE7/CD40L DNA vaccine elicited an increased number of E7-specific IFN-gamma secreting CD8+ T cell compared with that of mice immunized with mE7 gene (P < 0.01); however, no significant differences were found between mice immunized with the mE7 gene and mE7/CD40L fusion gene in the E7-specific antibody production and Th cell activation. In the preventive experiment, all mice received the mE7 or mE7/CD40L remained tumor-free 7 weeks after challenges with TC-1 tumor cells, while the wE7 group exhibited tumor growth within 2 weeks. In the therapeutic experiment, all the mice in the wE7 group exhibited tumor growth within 8 days, while among mice receiving the mE7 and mE7/CD40L, 30% and 45% of mice remained tumor-free after TC-1 challenge, respectively. HE staining of tumor tissues showed copious lymphocytes infiltration around tumor cells in mE7 and mE7/CD40L mice with regression of tumor growth. CONCLUSIONS: The mE7 DNA vaccine increases the E7-specific humoral and cellular immune responses, and the fusion of CD40L to mE7 gene enhances the specific immune responses and anti-tumor effects against HPV16 E7-expressing murine tumors. mE7/CD40L may therefore be a suitable and promising target for HPV16 therapeutic vaccine.