Hsa_circ_0044235 and hsa_circ_0001947 as novel biomarkers in plasma of patients with new-onset systemic lupus erythematosus.

Circular RNA (circRNA) are novel types of non-coding RNA that may be used as non-invasive noninvasive biomarkers in clinical plasma samples. However, the role of circRNA in plasma samples from patients with new-onset systemic lupus erythematosus (SLE) has not been extensively investigated. In the present study, reverse transcription-quantitative PCR was used to screen differentially-expressed circRNA (hsa_circ_0000175, hsa_circ_0044235, hsa_circ_0068367, hsa_circ_0002316, hsa_circ_0104871, hsa_circ_0001947, hsa_circ_0001481, hsa_circ_0008675, hsa_circ_0082689 and hsa_circ_0082688) in plasma samples isolated from 22 patients with new-onset SLE and 22 healthy control (HC). The results indicated hsa_circ_0000175, hsa_circ_ 0044235, hsa_circ_0068367, and hsa_circ_0001947 expression levels were significantly lower in plasma samples from new-onset SLE patients compared with corresponding levels in HC subjects and patients with new-onset rheumatoid arthritis (RA). Multivariate analysis indicated expression levels of hsa_circ_0044235 and hsa_circ_0001947 in plasma were independent risk factors for SLE. ROC curve analysis suggested that the combination of hsa_circ_0044235 and hsa_circ_0001947 indicated significant value in discriminating new-onset SLE from HC subjects and patients with RA. Moreover, the levels of hsa_circ_0044235 in plasma samples from patients with new-onset SLE were associated with platelet count, platelet-crit, and platelet distribution width; the expression of hsa_circ_0001947 in plasma from patients with SLE was associated with treatment. Thus, the present study demonstrated a promise for the combination of plasma hsa_circ_0044235 and hsa_circ_0001947 expression as potential diagnostic and prognostic biomarkers in patients with new-onset SLE.

Increased TIGIT+PD­1+CXCR5­CD4+T cells are associated with disease activity in rheumatoid arthritis.

It is well established that increased programmed cell death protein 1 (PD-1)+C-X-C chemokine receptor type 5 (CXCR5)- CD4+T cells are found in patients with rheumatoid arthritis (RA). T cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) is a co-inhibitory receptor that is expressed on T cells. However, the expression patterns and immunomodulatory roles of TIGIT on PD1+CXCR5-CD4+T cells in RA are poorly understood. Patients with RA were recruited and their clinical characteristics were recorded. The expression level of TIGIT on PD-1+CXCR5-CD4+T cells was examined using flow cytometry. Subsequently, the correlation between various parameters of TIGIT+PD-1+CXCR5-CD4+T cells [percentage of the cells, mean fluorescence intensity (MFI) of PD-1 and TIGIT in the cells] in patients with RA and clinical data, including autoantibodies, inflammatory indicators and RA activity, were analyzed. In addition, the risk factors of RA were assessed using univariate and multivariate regression analyses. The percentages of TIGIT+CXCR5-CD4+T, PD-1+CXCR5-CD4+T, TIGIT+PD-1+CXCR5-CD4+T, TIGIT-PD-1+CXCR5-CD4+T cells, the MFI of PD-1 in these cells and the MFI of TIGIT in TIGIT+PD-1+CXCR5-CD4+T cells were revealed to be significantly increased in patients with RA compared with those in healthy individuals. The parameters of TIGIT+PD-1+CXCR5-CD4+T cells (percentage and/or MFI of PD-1) in patients with RA were found to be associated with the levels of anti-cyclic citrullinated peptide antibodies, rheumatoid factor and inflammatory markers, such as lymphocyte count, lymphocyte percentage, neutrophil percentage, lymphocyte-to-monocyte ratio, neutrophil-to-lymphocyte ratio, systemic immune inflammation index, derived neutrophil-to-lymphocyte ratio, erythrocyte sedimentation rate and C-reactive protein. In addition, the MFI of PD-1 in TIGIT+PD-1+CXCR5-CD4+T cells was associated with a disease activity score of 28 and the patient visual analogue scale. Multivariate logistic regression analysis demonstrated that the percentage of TIGIT+PD-1+CXCR5-CD4+T cells and the MFI of PD-1 in TIGIT+PD-1+CXCR5-CD4+T cells were risk factors for RA. The present study suggests that the increase in TIGIT+PD-1+CXCR5-CD4+T cells is associated with the production of autoantibodies and RA activity and may serve a role in RA pathogenesis.

Increased TIM-3+PD-1+ NK cells are associated with the disease activity and severity of systemic lupus erythematosus.

It is well established that natural killer (NK) cells are dysregulated in systemic lupus erythematosus (SLE) patients. However, the functions of NK cells and the mechanisms regulated by them in SLE remain incompletely understood. Patients with SLE were recruited from The First Affiliated Hospital of Nanchang University, and their clinical characteristics and treatments were recorded. The expression levels of T cell immunoglobulin mucin-3 (TIM-3) and programmed cell death protein 1 (PD-1) on NK cells were examined using flow cytometry. The correlations between the increase in TIM-3+PD-1+ NK cells in the SLE patients and clinical traits, including inflammatory markers, auto-antibodies, disease activity and severity of SLE, were examined. The TIM-3+NK cells, PD-1+NK cells and TIM-3+PD-1+ NK cells were significantly increased in the SLE patients. The increase in TIM-3+PD-1+ NK cells in the patients with SLE was associated with erythrocyte sedimentation rate, C-reactive protein, anti-double stranded DNA, anti-ribosomal P, SLE disease activity index and clinical features. The frequency of TIM-3+PD-1+NK cells in SLE patients with a cardiovascular disease (CVD) was significantly lower than that in SLE patients without a CVD. Moreover, the increased TIM-3+PD-1+ NK cells were significantly decreased in SLE patients following treatment. The present study suggested that the increased TIM-3+PD-1+ NK cells were associated with the disease activity and severity of SLE and may play a role in SLE pathogenesis.

Circular RNAs hsa-circ0000175 and hsa-circ0044235 in plasma are novel biomarkers for new-onset rheumatoid arthritis.

Circular RNAs (circRNAs) are a class of non-coding RNAs that could serve as potential molecular markers for disease diagnosis. However, the role of circRNAs in plasma from new-onset rheumatoid arthritis (RA) has not been extensively investigated. In this study, the expression of hsa-circ0000175 and hsa-circ0044235 in plasma from RA patients, healthy controls (HCs), systemic lupus erythematosus (SLE) patients, osteoarthritis (OA), and undiagnosed arthritis (UA) patients were determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Correlation analysis was used to assess the correlation of the two circRNAs and clinical variables of RA. The receiver operating characteristic (ROC) curves were created to evaluate the diagnostic value and multivariate analysis (logistic regression) was performed to analyse the risk factors. We confirmed that hsa-circ0000175 was significantly elevated in plasma from patients with new-onset RA compared with HC and patients with new-onset SLE, but significantly was reduced when compared with OA + UA patients. Hsa-circ0044235 was found to be significantly decreased in plasma from patients with new-onset RA compared with HC and OA + UA patients, but was significantly increased compared with SLE patients. The expression of plasma hsa-circ0000175 in new-onset RA patients was associated with platelet count (PLT), plateletcrit (PCT), and platelet large cell ratio (PLR), the expression of plasma hsa-circ0044235 new-onset RA patients was associated with swollen joint count (SJC), painful joint count (PJC), and disease activity score 28 (DAS28). ROC curve analysis suggested that the combination of hsa-circ0000175 and hsa-circ0044235 has some value in the diagnosis of new-onset RA from HC, patients with SLE and patients with OA + UA. The logistic regression analysis revealed that the expression of hsa-circ0000175 and hsa-circ0044235 in plasma were risk factors for RA. This study suggests that the combination of plasma hsa-circ0000175 and hsa-circ0044235 improves the diagnostic accuracy for new-onset RA. Moreover, the expression levels of plasma hsa-circ0000175 and hsa-circ0044235 were associated with disease activity and severity of RA.

Interleukin-17A-Induced Epithelial-Mesenchymal Transition of Human Intrahepatic Biliary Epithelial Cells: Implications for Primary Biliary Cirrhosis.

Primary biliary cirrhosis (PBC) is an autoimmune chronic liver disease with worldwide increasing morbidity. However, the etiology of PBC is still unclear. Recently, the epithelial-mesenchymal transition (EMT) and interleukin-17A (IL-17A), a pro-inflammatory cytokine, were proposed to be involved in the pathogenesis of PBC. Therefore, in this study, we aimed to clarify the roles of IL-17A and/or EMT in the onset of PBC. The results showed that the median serum IL-17A level was significantly higher in 29 PBC patients (average course of 40.69 months) than that of 11 healthy controls. The intrahepatic biliary epithelial cells (IBECs), the major target of destruction in PBC, underwent EMT in PBC patients. The immunohistochemical analysis revealed that the protein levels of IL-17A receptor were increased in IBECs and the IL-17A protein was accumulated around the IBECs in the PBC patients. These results imply that the IL-17A-mediated signaling and EMT of intrahepatic biliary epithelial cells (IBEC-EMT) are key pathogenic processes of PBC. To study the association between IL-17A and IBECs-EMT, we then examined if IL-17A induced EMT using a human cell line of IBECs (HIBECs). After the treatment with IL-17A for 48 h, HIBECs changed into bipolar cells with a fibroblastic morphology. Additionally, the results of real-time PCR and Western blot analyses demonstrated that IL-17A up-regulated the expression of a mesenchymal marker vimentin and down-regulated the expression of an epithelial marker E-cadherin in HIBECs in the dose- and time-dependent manners. These results suggest that IL-17A may play an important role in the IBECs-EMT.

Long non-coding RNA RP5-833A20.1 inhibits proliferation, metastasis and cell cycle progression by suppressing the expression of NFIA in U251 cells.

Early reports suggest that nuclear factor IA (NFIA) is important in the pathogenesis of glioma. Our previous study demonstrated that the long non­coding RNA (lncRNA), RP5­833A20.1, suppressed the expression of NFIA in THP­1 macrophage-derived foam cells. However, the effect and possible mechanism of RP5­833A20.1 on glioma remains to be fully elucidated, and whether the NFIA-dependent pathway is involved in its progression has not been investigated. In the present study, the mechanisms by which RP5­833A20.1 regulates the expression of NFIA in glioma were investigated. The expression levels of RP5­833A20.1 and NFIA were determined in U251 cells and clinical samples using reverse transcription­quantitative polymerase chain reaction (PCR) analysis. The effects of RP5­833A20.1 on cell proliferation, invasion, cell cycle and apoptosis were evaluated using in vitro assays. The potential changes in protein expression were investigated using western blot analysis. The methylation status of the CpG island in the NFIA promoter was determined using bisulfite PCR (BSP) sequencing. It was found that the expression of RP5­833A20.1 was downregulated, whereas the expression of NFIA was upregulated in glioma tissues, compared with corresponding adjacent nontumor tissues from 20 patients with glioma. The overexpression of RP5­833A20.1 inhibited proliferation and cell cycle progression, and induced apoptosis in the U251 cells. The mRNA and protein levels of NFIA were markedly inhibited by overexpression of RP5­833A20.1 in the U251 cells. The overexpression of RP5­833A20.1 increased the expression of microRNA­382­5p in the U251 cells. The BSP assay revealed that the overexpression of RP5­833A20.1 enhanced the methylation level of the NFIA promoter. These results demonstrated that RP5­833A20.1 inhibited tumor cell proliferation, induced apoptosis and inhibited cell­cycle progression by suppressing the expression of NFIA in U251 cells. Collectively, these results indicated RP5­833A20.1 as a novel therapeutic target for glioma.

PD-L1-expressing neutrophils as a novel indicator to assess disease activity and severity of systemic lupus erythematosus.

BACKGROUND: It is well-known that increased frequency of neutrophils was found in patients with systemic lupus erythematosus (SLE). However, the immunomodulatory roles and mechanisms of neutrophils in SLE are poorly understood. METHODS: Patients with SLE were recruited from the First Affiliated Hospital of Nanchang University. The medical history, clinical manifestations, physical examination, laboratory measurements, therapeutic regimen and treatment response were recorded. The expression of costimulatory molecules including programmed death 1 (PD-1), programmed death ligand 1 (PD-L1), T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3), CD40, T cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory domains (TIGIT), CD80 and CD86 on neutrophils were determined by flow cytometry. The frequencies of PD-L1-expressing neutrophils in patients with SLE were further analyzed for their correlation with markers of autoimmune response, inflammation, disease activity and severity of SLE. RESULTS: The frequency of PD-L1-expressing neutrophils was significantly elevated in SLE patients compared to the healthy controls (P < 0.0001). The frequency of PD-L1-expressing neutrophils in patients with SLE was increased significantly in subjects with high ANA titre, high anti-nRNP/Sm, high levels of inflammatory markers and high SLE Disease Activity Index (SLEDAI) score. Furthermore, the percentages of PD-L1-expressing neutrophils were significantly decreased in SLE patients that received a 15-day regular treatment with corticosteroids and immunosuppressive drugs (P = 0.0075). CONCLUSION: The frequency of PD-L1-expressing neutrophils is elevates in patients with SLE, correlates with the disease activity and severity of SLE, and may serves as a negative feedback mechanism preventing potential tissue damage caused by excessive autoimmune responses in patients with SLE.

[Role of glycogen synthase kinase 3ß in maturation and function of murine myeloid dendritic cells in vitro].

OBJECTIVE: To investigate the role of glycogen synthase kinase 3ß (GSK-3ß) in the maturation and function of murine bone marrow-derived dendritic cells (BMDCs). METHODS: Mature DCs (mDCs) induced by LPS were examined for GSK-3ß phosphorylation level with Western blotting before and after LPS exposure. To explore the role of GSK-3ß in maturation and function of DCs, we added SB216763, a selective inhibitor of GSK-3ß, in the cell culture of immature DCs (iDCs), and examined CD40 and CD86 expressions in the cells by flow cytometry and the expression of IL-6, IL-12 and IL-10 mRNA by real-time PCR; the changes of the immunogenicity of the cells was evaluated by mixed lymphocyte reaction. The expression of GSK-3ß and RelB was examined by Western blotting in DC2.4 cells transfected with a lentiviral vector over-expressing murine GSK-3ß gene. RESULTS: LPS exposure significantly lowered GSK-3ß activity in iDCs as demonstrated by increased Ser9 phosphorylation and reduced Tyr216 phosphorylation. GSK-3ß inhibition induced DC maturation by increasing the expression of surface costimulatory molecules CD40 and CD86, lowered the expressions of IL-6 and IL-12 while enhanced the expression of IL-10 in iDCs, and impaired mixed lymphocyte reaction of the cells. In DC2.4 cells, lentivirus-mediated over-expression of GSK-3ß obviously down-regulated the expression of RelB. CONCLUSIONS: GSK-3ß is a crucial enzyme involved in the differentiation and maintenance of an immature phenotype of DCs. GSK-3ß is constitutively active in iDCs to inhibit their spontaneous maturation. DCs become phenotypically mature after inhibition of GSK-3ß, which also executes a proinflammatory task in DC activation. The reduction of RelB protein levels as a result of GSK-3ß overexpression supports GSK-3ß as a new target for inducing tolerogenic DCs.

Effect of WFDC 2 silencing on the proliferation, motility and invasion of human serous ovarian cancer cells in vitro.

OBJECTIVE: To investigate effect and possible mechanisms of silencing human WFDC2 (HE4) gene on biological behavior changes as cell proliferation, apoptosis, movement and invasion of human serous ovarian cancer cell line SKOV3. METHODS: Lentiviral WFDC2 gene sequence of small interfering siRNA was stablely transfected into SKOV3 identified by Q-PCR and western-blot. Obtained SKOV3 stable strains with silenced HE4 were measured by proliferation, apoptosis, migration, and invasion. RESULTS: Gene sequencing showed that the oligonucleotides were successfully inserted into the expected site. After silencing HE4 in the SKOV3, proliferation was significantly inhibited (P<0.05). G(0)/G(1) phase was arrested by the cell cycle (P<0.01) and capacity of the migration and invasion decreased significantly (P<0.01). Slight early apoptosis ratio and no change of late apoptosis were found without change of Caspase-3 or Bcl-2 protein. Proteins involved in ERK pathway as phosphorylated protein as p-EGFR, p-ERK decreased and protease protein involved in tissue remodeling as matrix metalloproteinases MMP-9, MMP-2 and cathepsin B decreased compared with control group. CONCLUSIONS: HE4 gene plays an important role in regulating proliferation, apoptosis, migration, invasion of serous ovarian cancer cells by ERK pathway and protease system. Its role in apoptosis needs to be further explored, and it may be a potential target for serous ovarian cancer.

Expression of serum human epididymal secretory protein E4 at low grade and high grade serous carcinomas.

OBJECTIVE: To investigate the value of serum human epididymis protein 4 (HE4) in differential diagnosis of patients with low-grade serous (LGSC) and high-grade serous carcinoma (HGSC) serous ovarian cancer. METHODS: LGSC and HGSC serous ovarian cancer were diagnosed by the two-tier grade system, serum levels of HE4 and carbohydrate antigen 125 (CA125) were measured by ELISA and radioisotope method, respectively in 60 serous ovarian cancer patients. HE4 and TP53 protein in cancer tissue were measured by immunohistochemical method. RESULTS: The difference in density of HE4 and TP53 protein was significant between LGSC and HGSC tissue, while serum CA125 did not show significant difference between different serum samples. There was significant difference in serum HE4 levels between LGSC and HGSC, and the result was different within FIGO (I+II) stage, suggesting HE4 was not a reliable biomarker for the discrimination between LGSC and HGSC. HE4 had potential as a biomarker for the discrimination between LGSC and HGSC but the role in early diagnosis was limited. CONCLUSIONS: HE4 may be a reliable marker for differential diagnosis of LGSC and HGSC. But its role in early diagnosis of LGSC and HGSC need to be confirmed from the perspective of two-tier grade system.

[Diagnostic value of anti-alpha-fodrin antibody testing for Sjögren's syndrome: a meta-analysis].

OBJECTIVE: To evaluate the diagnostic accuracy of anti-alpha-fodrin antibody for Sjögren's syndrome (SS). METHODS: Qualified literatures on evaluation of anti-alpha-fodrin antibody in diagnosis of SS in English and Chinese published between January 1997 and December 2007 were retrieved from the Cochrane Library, Medline, Embase, and China National Knowledge Infrastructure (CNKI) databases, etc. Two reviewers independently assessed the methodological quality of each study with the tool QUADAS (quality assessment of diagnostic accuracy studies). Statistical analysis was performed by employing MATLAB, Review Manager 4.2 and Meta-Disc1.4. A meta-analysis of the reported sensitivity and specificity of each study and Summary Receiver Operating Characteristic (SROC) curve was performed. RESULTS: Eighteen literatures were included at last. After testing the heterogeneity of the included articles, proper effect model was selected to calculate the pooled weighted sensitivity and specificity with 95% confidence interval: for anti-alpha-fodrin antibody IgG, the sensitivity was 0.40 [95%CI (0.37-0.43)] and the specificity was 0.82 [95%CI (0.79-0.84)], the area under the curve (AUC) of SROC was 0.8029 (SE=0.0580). Eight studies tested anti-alpha-fodrin antibody IgA, the pooled weighted sensitivity and specificity with 95% confidence interval were 0.34 [95%CI (0.30-0.38)] and 0.83 [95%CI (0.79-0.86)] respectively, the AUC of SROC was 0.6374 (SE=0.1841), the synthesis data all showed heterogeneity. The subgroups were analyzed to identify the sources of heterogeneity according to age, race, assay method, agent source, diagnostic criteria, and country. There was homogeneity among the 4 studies from China, and the 6 studies from Japan, the AUC of SROC were 0.7343 (SE=0.0448) and 0.9273 (SE=0.0394), respectively. CONCLUSION: Diagnostic criteria, agent source, assay method, age, race, and country are the important sources of heterogeneity. Anti-alpha-fodrin antibodies IgG and IgA have relatively low pooled sensitivity and relatively high pooled specificity. Negative anti-alpha-fodrin antibody has not important value in excluding SS, but positive anti-alpha-fodrin antibody may be a useful parameter in clinical diagnosis of SS.

[Diagnostic value of anti-cyclic citrullinated peptide antibody for rheumatoid arthritis: a meta-analysis].

OBJECTIVE: To evaluate the diagnostic value of anti-cyclic citrullinated peptide (CCP) antibody for rheumatoid arthritis. METHODS: Data about RA from January 2000 to December 2005 were retrieved through Cochrane Library, Pubmed database, Excerpta Medica Database (EMBASE), OVID database, and China National Knowledge Infrastructure (CNKI), especially through the Annul of the Rheumatic Disease and relevant gray literatures, by entering the words "cyclic citrullinated peptides", "rheumatoid arthritis", "sensitivity", and "specificity". The inclusion of qualified literatures was based on the criteria for diagnostic research recommended by the Cochrane Methods Group on Screening and Diagnostic Test. Statistical analysis wes performed by employing the softwares of MATLAB and Review Manager 4, 2, and summary receiver operation characteristic (SROC) curve method. RESULTS: Twenty-two articles, 15 in English and 7 in Chinese, were extracted. The reported sensitivity of anti-CCP for the diagnosis of RA ranged from 39.2% to 84.6%, and the reported specificity ranged from 90% to 97.9%. The heterogeneity of the included articles was tested, a proper effect model was selected to calculate the pooled weighted sensitivity and specificity with 95% confidence interval for anti-CCP antibody as 77.3% (63.1%, 89.2%) and 93.85% (85.5%, 98.1%), and the positive and negative likelihood ratios as 12.0 and 0.24 respectively. The area under the curve of SROC was 0.8976, and the Q value was 0.87. The sensitivity of the patients with the duration of illness < 1 year was 43%, significantly lower than that of the patients with a duration of illness > 1 year (70.2%, P < 0.01); and the specificity of the patients with the duration of illness < 1 year was 94.2%, not significantly different from that of the patients with the duration of illness > 1 year (95.2%, P = 0.94). CONCLUSION: With relatively high sensitivity and specificity, anti-CCP antibody may be a useful parameter in the clinical diagnosis pf RA.