Whether full-length IL-38 acts as a promoter or inhibitor in activating rheumatoid arthritis fibroblast-like synoviocytes depends on IL-1ß.

AIM: IL-38 is a recently discovered inflammatory factor that belongs to the IL-1 family and has full-length and truncated forms. Clinical findings demonstrated that serum IL-38 levels in people with infectious and autoimmune diseases are significantly different from those in healthy people, but the form remains unclear. We are keenly interested in learning more about the regulatory role of full-length IL-38 in rheumatoid arthritis (RA), a classic autoimmune disease. METHODS: RA-fibroblast-like synoviocytes (RA-FLS) were isolated from six RA patients and stimulated with full-length IL-38 to observe IL-6 and IL-8 secretion. Then, the migration and invasion functions of FLS were assessed. Next, the protein expressions of the MAPK, NF-κB, and JAK pathways were evaluated. In addition, we examined the effect of full-length IL-38 on FLS functions in the presence of IL-1ß. The function of FLS affected by full-length IL-38 was also examined after blocking IL-1 and IL-36 receptors. RESULTS: The functions of FLS were activated after the cells were stimulated with full-length IL-38. IL-6 and IL-8 levels increased with an increase in the full-length IL-38 concentration, and full-length IL-38 induced the acceleration of FLS migration and invasion functions. In addition, the levels of proteins in the MAPK signaling pathway increased after stimulation with full-length IL-38 and depended on its concentration. However, when the FLS were stimulated by IL-38 and IL-1ß simultaneously, all experiments generated opposite results. Full-length IL-38 inhibited FLS function in the presence of IL-1ß. IL-1R and IL-36R blockers terminated all effects of full-length IL-38 on RA-FLS. CONCLUSION: Full-length IL-38 activates FLS functions and acts as a promoter in RA, whereas it inhibits FLS functions and acts as an inhibitor of RA in the presence of IL-1ß. The function of full-length IL-38 can be blocked by IL-1Ra and IL-36Ra.

Response to belimumab among patients with refractory lupus nephritis: a real-world observational retrospective multicenter study.

OBJECTIVES: Belimumab is a biological agent approved for the treatment of active lupus nephritis (LN), but its efficacy on refractory lupus nephritis (LN) is unknown. This study aims to evaluate the efficacy and safety of belimumab in Chinese patients with refractory LN. METHODS: This multicenter, observational, and retrospective study enrolled patients with refractory LN who failed induction therapy with steroids, cyclophosphamide, mycophenolate, and calcineurin inhibitors and received 24-week belimumab treatment before data analysis. Treatment outcomes include the overall clinical response (physician judgment, disease activity, organ damage) and renal response (complete renal response, partial renal response, no renal response). Laboratory indices and adverse events were recorded as well. RESULTS: Of the 45 patients enrolled in the study, 6 (13.3%) achieved complete renal response, 19 (42.2%) achieved partial renal response, and the overall renal response rate was 55.6%. Median rSLEDAI decreased from 12 (IQR 8-12) at baseline to 8 (IQR 4-8) (p < 0.0001), 4 (IQR 4-8) (p < 0.0001) at 12 and 24 weeks. Mean urinary protein decreased more than 50% from 3.2 g/24 h at baseline to 1.0 g/24 h at 24 weeks (p < 0.0001). The conditions of hypoalbuminemia and hypocomplementemia had also gradually improved. The levels of autoantibodies showed a significant downward trend. Additionally, 9 (20.0%) patients successfully reduced the dosage of prednisone to a safe range, and 3 of them achieved their treatment goal of prednisone cessation. The mean prednisone dosage decreased from 32.7 mg/day at baseline to 18.6 mg/day (p < 0.0001), 13.3 mg/day (p < 0.0001) at 12 and 24 weeks. There were 3 adverse events reported, including 2 cases of infection, and 1 case of allergy. No serious events occurred during the follow-up. CONCLUSIONS: Belimumab is effective and safe when used in clinical practice, which can be considered as an add-on therapy for refractory LN. Key Points • A multicenter observational study in the real clinical settings of China. • First revealed the efficacy and safety of belimumab in Chinese patients with refractory LN.

USP1-regulated reciprocal differentiation of Th17 cells and Treg cells by deubiquitinating and stabilizing TAZ.

The balance between inflammatory T helper type 17 (Th17) and immunosuppressive regulatory T (Treg) cells is critical for maintaining immune homeostasis in the human body and is tightly regulated under healthy conditions. An increasing number of studies have reported that deubiquitinases (DUBs) play a vital role in regulating Th17- and Treg-cell differentiation. However, the biological functions of only a small fraction of DUBs in Th17- and Treg-cell differentiation are well defined. In this study, we identified ubiquitin-specific peptidase 1 (USP1) as a vital regulator of CD4+ T-cell differentiation. USP1 promoted Th17-cell differentiation but attenuated Treg-cell differentiation, thereby promoting the development of inflammatory diseases. Mechanistically, USP1 in CD4+ T cells enhanced the activity of RORγt but promoted the proteasomal degradation of Foxp3 through deubiquitination and stabilization of TAZ in vitro and in vivo. Notably, ML323, a specific inhibitor of the USP1/UAF1 deubiquitinase complex, inhibited Th17-cell differentiation and promoted Treg-cell differentiation in vitro and in vivo, indicating that ML323 might be a promising candidate for the treatment of diseases associated with an imbalance between Th17 and Treg cells. Our study highlights the critical role of USP1 in regulating adaptive immune responses and suggests that USP1 might be a drug target for the treatment of diseases associated with an imbalance between Th17 and Treg cells.

[Corrigendum] Role of cysteine­rich angiogenic inducer 61 in fibroblast­like synovial cell proliferation and invasion in rheumatoid arthritis.

Following the publication of the above article, an interested reader drew to the authors' attention that Fig. 4A on p. 921, showing the results from cell migration assay experiments, featured a pair of duplicated data panels. After having consulted their original data, the authors have realized that Fig. 3A on the same page, showing the fluorometric images of apoptotic cells, also contained a pair of duplicated data panels. These errors in the presentation of these figures arose inadvertently as a consequence of selecting the wrong images for the 'RA NC' data panel in Fig. 3A and the NOR-FLS data panel in Fig. 5E. The revised versions of Figs. 3 and 4 are shown on the next two pages. All the authors approve of the publication of this corrigendum, and the authors are grateful to the Editor of Molecular Medicine Reports for granting them the opportunity to publish this. The authors regret their oversight in allowing these errors to be included in the paper, and also apologize to the readership for any inconvenience caused. [Molecular Medicine Reports 11: 917­923, 2015; DOI: 10.3892/mmr.2014.2770].

Antibacterial spirooxindole alkaloids from Penicillium brefeldianum inhibit dimorphism of pathogenic smut fungi.

Three new antibacterial spirooxindole alkaloids, spirobrefeldins A-C (1-3), together with four known analogs, spirotryprostatin M (4), spirotryprostatin G (5), 12ß-hydroxyverruculogen TR-2 (6), and 12α-hydroxyverruculogen TR-2 (7), were isolated from terrestrial fungus Penicillium brefeldianum. All the new compounds were elucidated extensively by the interpretation of their NMR (1D and 2D) spectra and high-resolution mass data, and their absolute configurations were determined by computational chemistry and CD spectra. The absolute configurations of spiro carbon C-2 in spirotryprostatin G (5) and spirotryprostatin C in literature were reported as S, which were revised to R based on experimental and calculated CD spectra. All the compounds were evaluated for their antimicrobial activities toward Pseudomonas aeruginosa PAO1, Dickeya zeae EC1, Staphylococcus epidermidis, Escherichia coli, and Sporisorium scitamineum. Compound 7 displayed moderate inhibitory activity toward dimorphic switch of pathogenic smut fungi Sporisorium scitamineum at 25 µM. Compounds 3 and 6 showed weak antibacterial activities against phytopathogenic bacterial Dickeya zeae EC1 at 100 µM.

Osteoarticular Involvement-Associated Biomarkers and Pathways in Psoriasis: The Shared Pathway With Ankylosing Spondylitis.

Psoriatic arthritis (PsA) is a unique immune-mediated disease with cutaneous and osteoarticular involvement. However, only a few studies have explored the susceptibility of osteoarticular involvement in psoriasis (Ps) at the genetic level. This study investigated the biomarkers associated with osteoarticular participation and potential shared molecular mechanisms for PsA and ankylosing spondylitis (AS). Methods: The RNA-seq data of Ps, PsA, and AS in the Gene Expression Omnibus (GEO) database were obtained. First, we used the limma package and the weighted gene co-expression network analysis (WGCNA) to identify the potential genes related to PsA and AS. Then, the shared genes in PsA and AS were performed using the GO, KEGG, and GSEA analyses. We also used machine learning to screen hub genes. The results were validated using external datasets and native cohorts. Finally, we used the CIBERSORT algorithm to estimate the correlation between hub genes and the abundance of immune cells in tissues. Results: An overlap was observed between the PsA and AS-related modules as 9 genes. For differentially expressed genes in AS and PsA, only one overlapping gene was found (COX7B). Gene enrichment analysis showed that the above 9 genes might be related to the mRNA surveillance pathway. The GSEA analyses showed that COX7B was involved in adaptive immune response, cell activation, etc. The PUM1 and ZFP91, identified from the support vector machine, had preferable values as diagnostic markers for osteoarticular involvement in Ps and AS (AUC > 0.7). Finally, CIBERSORT results showed PUM1 and ZFP91 involvement in changes of the immune microenvironment. Conclusion: For the first time, this study showed that the osteoarticular involvement in psoriasis and AS could be mediated by the mRNA surveillance pathway-mediated abnormal immunologic process. The biological processes may represent the cross talk between PsA and AS. Therefore, PUM1 and ZFP91 could be used as potential biomarkers or therapeutic targets for AS and Ps patients.

Kirenol inhibits RANKL-induced osteoclastogenesis and prevents ovariectomized-induced osteoporosis via suppressing the Ca2+-NFATc1 and Cav-1 signaling pathways.

BACKGROUND: Osteoporosis is a threat to aged people who have excessive osteoclast activation and bone resorption, subsequently causing fracture and even disability. Inhibiting osteoclast differentiation and absorptive functions has become an efficient approach to treat osteoporosis, but osteoclast-targeting inhibitors available clinically remain rare. Kirenol (Kir), a bioactive diterpenoid derived from an antirheumatic Chinese herbal medicine Herba Siegesbeckiae, can treat collagen-induced arthritis in vivo and promote osteoblast differentiation in vitro, while the effects of Kir on osteoclasts are still unclear. PURPOSE: We explore the role of Kir on RANKL-induced osteoclastogenesis in vitro and bone loss in vivo. METHODS: The in vitro effects of Kir on osteoclast differentiation, bone resorption and the underlying mechanisms were evaluated with bone marrow-derived macrophages (BMMs). In vivo experiments were performed using an ovariectomy (OVX)-induced osteoporosis model. RESULTS: We found that Kir remarkably inhibited osteoclast generation and bone resorption in vitro. Mechanistically, Kir significantly inhibited F-actinring formation and repressed RANKL-induced NF-κB p65 activation and p-p38, p-ERK and c-Fos expression. Moreover, Kir inhibited both the expression and nuclear translocation of NFATc1. Ca2+ oscillation and caveolin-1 (Cav-1) were also reduced by Kir during osteoclastogenesis in vitro. Consistent with these findings, 2-10 mg/kg Kir attenuated OVX-induced osteoporosis in vivo as evidenced by decreased osteoclast numbers and downregulated Cav-1 and NFATc1 expression. CONCLUSIONS: Kir suppresses osteoclastogenesis and the Cav-1/NFATc1 signaling pathway both in vitro and in vivo and protects against OVX-induced osteoporosis. Our findings reveal Kir as a potential safe oral treatment for osteoporosis.

Tanshinone IIA Suppresses Proliferation and Inflammatory Cytokine Production of Synovial Fibroblasts from Rheumatoid Arthritis Patients Induced by TNF-α and Attenuates the Inflammatory Response in AIA Mice.

Rheumatoid arthritis (RA) is a chronic and progressive autoimmune disease in which activated RA fibroblast-1ike synoviocytes (RA-FLSs) are one of the main factors responsible for inducing morbidity. Previous reports have shown that RA-FLSs have proliferative features similar to cancer cells, in addition to causing cartilage erosion that eventually causes joint damage. Thus, new therapeutic strategies and drugs that can effectively contain the abnormal hyperplasia of RA-FLSs and restrain RA development are necessary for the treatment of RA. Tanshinone IIA (Tan IIA), one of the main phytochemicals isolated from Salvia miltiorrhiza Bunge, is capable of promoting RA-FLS apoptosis and inhibiting arthritis in an AIA mouse model. In addition, RA patients treated at our clinic with Tan IIA showed significant improvements in their clinical symptoms. However, the details of the molecular mechanism by which Tan IIA effects RA are unknown. To clarify this mechanism, we evaluated the antiproliferative and inhibitory effects of proinflammatory factor production caused by Tan IIA to RA-FLSs. We demonstrated that Tan IIA can restrict the proliferation, migration, and invasion of RA-FLSs in a time- and dose-dependent manner. Moreover, Tan IIA effectively suppressed the increase in mRNA expression of some matrix metalloproteinases and proinflammatory factors induced by TNF-α in RA-FLSs, resulting in inflammatory reactivity inhibition and blocking the destruction of the knee joint. Through the integration of network pharmacology analyses with the experimental data obtained, it is revealed that the effects of Tan IIA on RA can be attributed to its influence on different signaling pathways, including MAPK, AKT/mTOR, HIF-1, and NF-kB. Taken together, these data suggest that the compound Tan IIA has great therapeutic potential for RA treatment.

A Novel Phytochemical, DIM, Inhibits Proliferation, Migration, Invasion and TNF-α Induced Inflammatory Cytokine Production of Synovial Fibroblasts From Rheumatoid Arthritis Patients by Targeting MAPK and AKT/mTOR Signal Pathway.

In rheumatoid arthritis(RA) pathogenesis, activated RA fibroblast-like synoviocytes (RA-FLSs) exhibit similar proliferative features as tumor cells and subsequent erosion to cartilage will eventually lead to joint destruction. Therefore, it is imperative to search for compounds, which can effectively inhibit the abnormal activation of RA-FLSs, and retard RA progression.3'3-Diindolylmethane (DIM), the major product of the acid-catalyzed oligomerization of indole-3-carbinol from cruciferous vegetables, has been reported to be functionally relevant to inhibition of migration, invasion and carcinogenesis in some solid tumors. In this study, we explored the anti-proliferation, anti-metastasis and anti-inflammation effects of DIM on RA-FLSs as well as the underlying molecular mechanisms. To do this, primary RA-FLSs were isolated from RA patients and an animal model. Cell proliferation, migration and invasion were measured using CCK-8, scratch, and Transwell assays, respectively. The effects of DIM on Matrix metalloproteinases (MMPs) and some inflammatory factors mRNA and key molecules such as some inflammatory factors and those involved in aberrantly-activated signaling pathway in response to tumor necrosis factor α(TNF-α), a typical characteristic mediator in RA-FLS, were quantitatively measured by real-time PCR and western blotting. Moreover, the effect of DIM on adjuvant induced arthritis(AIA) models was evaluated with C57BL/6 mice in vivo. The results showed that DIM inhibited proliferation, migration and invasion of RA-FLS in vitro. Meanwhile, DIM dramatically suppressed TNF-α-induced increases in the mRNA levels of MMP-2, MMP-3, MMP-8, and MMP-9; as well as the proinflammatory factors IL-6, IL-8, and IL-1ß. Mechanistic studies revealed that DIM is able to suppress phosphorylated activation not only of p38, JNK in MAPK pathway but of AKT, mTOR and downstream molecules in the AKT/mTOR pathway. Moreover, DIM treatment decreased expression levels of proinflammatory cytokines in the serum and alleviated arthritis severity in the knee joints of AIA mice. Taken together, our findings demonstrate that DIM could inhibit proliferation, migration and invasion of RA-FLSs and reduce proinflammatory factors induced by TNF-α in vitro by blocking MAPK and AKT/mTOR pathway and prevent inflammation and knee joint destruction in vivo, which suggests that DIM might have therapeutic potential for RA.

Efficacy of uric acid-lowering therapy on hypercholesterolemia and hypertriglyceridemia in gouty patients.

AIM: To investigate the effects on hypercholesterolemia and hypertriglyceridemia in gouty patients receiving uric acid-lowering therapy (UALT). METHODS: A retrospective study was performed from January 2015 to December 2017 in gouty patients receiving UALT. A total of 124 gouty patients with hypercholesterolemia or hypertriglyceridemia who were administered UALT were monitored. Of the 124 patients with gout, 52 were treated with febuxostat, 29 were treated with allopurinol, and 43 were treated with benzbromarone. Cholesterol and triglyceride levels were recorded and analyzed following treatment for 8-10 weeks. RESULTS: We compared the efficacy of febuxostat, allopurinol, and benzbromarone. All therapies mildly influenced serum cholesterol and triglyceride levels. Febuxostat significantly decreased cholesterol and triglyceride levels in patients who did not receive lipid-lowering therapy. Allopurinol and benzbromarone modestly decreased triglyceride levels, but cholesterol levels were unaffected. CONCLUSION: Uric acid-lowering therapy benefits hyperlipidemia in gouty patients. Febuxostat effectively improved serum cholesterol and triglyceride levels compared to allopurinol and benzbromarone in patients with gout.

Kirenol Inhibits the Function and Inflammation of Fibroblast-like Synoviocytes in Rheumatoid Arthritis in vitro and in vivo.

Kirenol is a diterpenoid extracted from the Chinese herbal medicine Siegesbeckiae. Siegesbeckiae has been used to treat Rheumatoid arthritis (RA) in China for several centuries. RA is characterized by the proliferation of synoviocytes in inflamed synovia, as well as by their expression of inflammatory cytokines. In the present study, we found that Kirenol inhibited the migration, invasion, and proinflammatory of IL-6 secretion of RA-associated synovial fibroblasts (FLS) at a concentration of 100-200 µg/ml in vitro. Proinflammatory cytokines production and synovium hyperplasia and cartilage erosion were also inhibited in a collagen-induced arthritis (CIA) mouse model upon Kirenol treatment. Together, our results thus confirm that Kirenol has potent therapeutic efficacy in RA owing to its ability to suppress negative FLS activities.

Downregulated microRNA-135a ameliorates rheumatoid arthritis by inactivation of the phosphatidylinositol 3-kinase/AKT signaling pathway via phosphatidylinositol 3-kinase regulatory subunit 2.

Synovial fibroblasts (SFs) of rheumatoid arthritis (RA) are phenotypically aggressive, typically progressing into arthritic cartilage degradation. Throughout our study, we made explorations into the effects of microRNA-135a (miR-135a) on the SFs involved in RA by mediating the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway via regulation of phosphatidylinositol 3-kinase regulatory subunit 2 (PIK3R2). The expression of PI3K was higher, the expression of PIK3R2 was lower, and AKT was phosphorylated in the RA synovial tissues, relative to the levels found in the normal synovial tissues. We predicted miR-135a to be a candidate miR targeting PIK3R2 using an online website, microRNA.org, which was verified with a dual-luciferase reporter gene assay. Subsequently, high miR-135a expression was observed in RA synovial tissues. To study the effect of the interaction between miR-135a and PIK3R2 in RA, the SFs isolated from RA samples were cultured and transfected with mimic, inhibitor, and small interfering RNA. The proliferation, invasion, and apoptosis of the SFs were detected after the transfection. The cells transfected with miR-135a inhibitor showed inhibited cell proliferation, migration, and invasion, while also displaying promoted cell apoptosis, G0/G1 cell ratio, and decreased S cell ratio, through upregulation of PIK3R2 and inactivation of the PI3K/AKT signaling pathway. These findings provided evidence that downregulation of miR-135a inhibits proliferation, migration, and invasion and promotes apoptosis of SFs in RA by upregulating the PIK3R2 coupled with inactivating the PI3K/AKT signaling pathway. The downregulation of miR-135a might be a potential target in the treatment of RA.

A successful treatment of severe systemic lupus erythematosus caused by occult pulmonary infection-associated with hemophagocytic syndrome: A case report.

RATIONALE: A 27-year-old woman with a history of systemic lupus erythaematosus (SLE) developed hemophagocytic syndrome (HPS) secondary due to an unrecognized infection that led to severe SLE with a prolonged recovery. PATIENT CONCERNS: The patient showed a high spiking fever and myalgia. Laboratory data revealed pancytopenia and immunological abnormalities. Pulse methylprednisone plus intravenous immunoglobulin (IVIG) failed to improve the clinical symptoms and laboratory data. DIAGNOSES: As activated macrophages with hemophagocytosis were confirmed in bone marrow histology, the patient was diagnosed as having reactive HPS. INTERVENTIONS AND OUTCOMES: Her reactive HPS was successfully treated with intravenous antibiotics and was followed by oral prednisolone and hydroxychloroquine maintenance therapy. LESSONS: In severe SLE, patients with persistent high fever, cytopenia, and elevated levels of serum ferritin and liver enzymes should be strongly suspected of reactive HPS, and aggressive examination, such as bone marrow biopsy, needs to be considered for early diagnosis and proper treatment.

Transforming Growth Factor Beta is regulated by a Glucocorticoid-Dependent Mechanism in Denervation Mouse Bone.

Bone growth and remodeling is inhibited by denervation in adults and children, resulting in alterations of linear growth and bone mass and increased risk for osteoporosis and pathologic fractures. Transforming growth factor beta (TGF-ß) isoforms are a key group of growth factors that enhance bone formation. To explore the relation between denervation-induced reduction of bone formation and TGF-ß gene expression, we measured mRNA levels of TGF-ß in denervation mouse bone and found decreased mRNA levels of TGF-ß1, TGF-ß2 and TGF-ß3. These changes were accompanied by diminishing weight loss, bone mineral density (BMD), trabecular thickness, trabecular separation and trabecular number of femur and lumbar, serum osteocalcin, total calcium, intact parathyroid hormone, and increased serum C telopeptide. Recombinant human TGF-ß1 (rhTGF-ß1) prevented denervation-induced reduction of BMD further supporting our hypothesis that denervation-induced reduction of bone formation is a result of inhibition of TGF-ß gene expression. In addition, antiprogestins RU 38486 blunted the denervation-induced decrease in mRNA levels of TGF-ß group, while dexamethasone (DEX) decreased TGF-ß group mRNA levels in normal mice. Furthermore, the denervated-mice exhibited a threefold increase in plasma corticosterone. These results suggest that denervation-induced reduction of bone formation may be regulated by glucocorticoids via inhibition of TGF-ß gene expression at least in part.

Role of cysteine­rich angiogenic inducer 61 in fibroblast­like synovial cell proliferation and invasion in rheumatoid arthritis.

Cysteine­rich angiogenic inducer 61 (Cyr61) is a novel molecule that has been shown to be increased in the synovial tissues of patients with rheumatoid arthritis (RA). The present study was conducted in order to investigate the role of Cyr61 in the pathogenesis of RA. A human genome­wide gene assay was used to screen gene expression in synovial tissues obtained from four patients with RA and three patients with osteoarthritis (OA). To examine the role of Cyr61 in the phenotype of RA­fibroblast­like synovial (FLS) cells, Cyr61 expression in RA­FLS cells was knocked down using small interfering RNA (siRNA). Normal FLS cells transduced with lentiviral vectors encoding Cyr61 cDNA were used to further explore the effects of this molecule on FLS cell apoptosis, proliferation and invasion. The study found that the Cyr61 gene was highly expressed in the synovial cells from patients with RA compared with those from patients with OA. Downregulation of Cyr61 by siRNA led to impaired cell proliferation and invasion. Furthermore, it decreased the levels of matrix metalloproteinase (MMP)­3 and MMP­13, and induced apoptosis in RA­FLS cells. Conversely, overexpression of Cyr61 in normal FLS cells led to opposite effects. In conclusion, these results indicate that Cyr61 is capable of promoting RA­FLS cell proliferation and invasion via the suppression of apoptosis and the regulation of MMP expression. Therefore, Cyr61 may be a good target molecule for the treatment and prevention of RA.

Tanshinone IIA induces apoptosis in fibroblast-like synoviocytes in rheumatoid arthritis via blockade of the cell cycle in the G2/M phase and a mitochondrial pathway.

Tanshinone IIA (Tan IIA), a phytochemical derived from the roots of Salvia miltiorrhiza BUNGE, has been documented with anti-tumor, pro-apoptotic, and anti-inflammatory activities. Salvia miltiorrhiza has long been used to treat rheumatoid arthritis (RA). Apoptosis induction of RA-fibroblast-like synoviocytes (FLS) was suggested to be a potential therapeutic approach for RA. The aim of this study was to investigate whether Tan IIA promotes apoptosis in RA-affected FLS. In this study, the viability of an immortalized FLS cell line derived from RA patients was assessed by 3-(4,5-dimethylthiazol-2-yl)-5,3-carboxymethoxyphenyl-2,4-sulfophenyl-2H-tetrazolium (MTS) assay after Tan IIA treatment. Apoptosis was measured by terminal deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) assay and flow cytometry. Cell cycle was evaluated by flow cytometry. The expressions of mitochondrial apoptosis-related molecules, including Bcl-2, Bax, mitochondrial cytochrome c (Cyt-c), cytosolic Cyt-c, apoptotic protease activating factor 1 (Apaf-1), procaspase-9, procaspase-3, caspase-9, and caspase-3 were determined by Western blotting. Our data demonstrate that Tan IIA induced apoptosis of RA-FLS, blocked the cell cycle in the G2/M phase, and regulated the protein expression of Bcl-2, Bax, and Apaf-1, the release of mitochondrial Cyt-c, and the activation of caspase-9 and caspase-3. The results support the conclusion Tan IIA treatment likely induces apoptosis of RA-FLS through blockade of the cell cycle in the G2/M phase and a mitochondrial pathway. These data suggest that Tan IIA may have therapeutic potential for RA.

A monoclonal antibody against a potential cancer biomarker, human ubiquitin-conjugating enzyme E2.

Human ubiquitin-conjugating enzyme E2, also known as UbcH10, is defined as a cyclin-selective ubiquitin carrier protein and is essential for selective degradation of many short-lived proteins in eukaryotic cells. Recently more and more data show that UbcH10 could be a potential cancer biomarker. In this study, we have developed a monoclonal antibody (MAb) against UbcH10 using an expression recombinant protein. Hybridomas F001, F007, and F008 with high affinities belong to IgG1 subclass with κ light and are highly specific for UbcH10. Further experimentation showed that MAbs F001, F007, and F008 are suitable for the development of immunoassay core agents with sufficient sensitivity and specificity in vitro by Western-blot, immunofluorescence, and immunohistochemistry. These MAbs can be used as a tool for further investigation on functions related to the role of UbcH10 in tumorigenesis and development.