Antigens of Mycobacterium tuberculosis Recognized by Antibodies during Incipient, Subclinical Tuberculosis

Serum samples obtained from human immunodeficiency virus (HIV)-infected tuberculosis (TB) patients months prior to clinical TB were used to delineate the profile of Mycobacterium tuberculosis culture filtrate proteins recognized during subclinical TB. A subset of ~12 antigens was recognized by antibodies in these serum samples. Antibodies to two of these antigens (81 [88]-kDa malate synthase [GlcB] and MPT51) were present in serum samples obtained during incipient subclinical TB in 19 (~90%) of the 21 HIV-infected TB patients tested. These antigens will be useful for devising diagnostic tests that can identify HIV-positive individuals who are at a high risk for developing clinical TB.     

p53 Contributes to Quercetin-Induced Apoptosis in Human Rheumatoid Arthritis Fibroblast-like Synoviocytes

In the present study, we sought to explore the mechanism of quercetin-induced apoptosis in rheumatoid arthritis fibroblast-like synoviocytes (RAFLSs). DNA fragmentation assay was used to detect quercetin-induced apoptosis in RAFLSs. The cleavages of caspase-3 and caspase-9 and the accumulation of cytosolic cytochrome C were measured by western blot in quercetin-treated RAFLSs. Mitochondrial membrane potential was tested by flow cytometry. Small interfering RNAs were used to knock down the expression of protein 53 (p53) and analyze the role of p53 in quercetin-induced apoptosis in RAFLSs. DNA fragmentation assay showed that quercetin dose-dependently elevated the apoptosis of RAFLSs, accompanying with enhanced caspase-3 and caspase-9 cleavages. Moreover, quercetin caused a concentration-dependent loss of mitochondrial membrane potential and cytochrome c release to cytosol and also decreased Bcl-2/Bax ratio, indicating that quercetin-induced apoptosis is through mitochondrial pathway. Quercetin also elevated p53 phosphorylation at ser15. Pretreatment with pifithrin-α, a p53 inhibitor, significantly diminished p53 phosphorylation at the concentration of 30 μM and abrogated quercetin-induced apoptosis in a dose-dependent manner. Quercetin-induced apoptosis was also significantly blocked by p53 silencing, further suggesting the involvement of p53 in quercetin-induced apoptosis in RAFLSs. Our study indicated that quercetin-induced apoptosis of RAFLSs is through mitochondrial pathway, in which p53 plays an important role.     

The Presence of Anti-Protein A Antibodies in Patients with Rheumatoid Arthritis

Sixteen patients with rheumatoid arthritis (RA) were examined for the presence of anti-protein A antibodies. The F(ab')2 preparations from five RA patients showed significant binding to IgG-free protein A on ELISA. The protein A binding was further examined by immunoblotting. The F(ab')2 preparations of high protein A-binding protein gave a specific reaction with IgG-free protein A on nitrocellulose paper. This demonstrates the presence of anti-protein A antibodies in patients with RA. Those RA patients with anti-protein A antibodies had more active disease as judged by the Lansbury's activity index. The level of serum rheumatoid factor (RAHA) was significantly higher in patients with anti-protein A antibodies than in those without anti-protein A antibodies.     

Effects of Resveratrol on the Proliferation and Apoptosis in Synoviocytes of Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic systemic inflamma-tory disorder of unknown etiologic cause, and characterizedby symmetric involvement of the peripheral joints with thetypical histopathological changes, showing hyperplasia ofthe synovium, and often …     

Mechanical Stretch Regulates the Expression of Matrix Metalloproteinase in Rheumatoid Arthritis Fibroblast-Like Synoviocytes

Mechanical stretch plays a crucial role in articular joints. In rheumatoid arthritis (RA), it is well known that fibroblast-like synoviocytes (FLS) produce matrix metalloproteinases (MMPs), resulting in local invasion into and degradation of bone and cartilage. We sought to examine whether mechanical stretch regulates the expression and underlying signal pathways of MMP secretion (MMP-1, -3, -13) in RA-FLS. FLS were grown on elastic silicone membrane in an equibiaxial strain apparatus and were exposed to 6% mechanical stretch (equivalent to gentle stretch exercise) for 15 min and 75 min, respectively. Semiquantitative PCR and real-time PCR were used to measure and analyze gene expression. Protein levels were determined by Western blotting. The results showed that 15 min of mechanical stretch inhibited MMP-1 and MMP-13 mRNA and protein level. However, the degree of inhibition by 75 min of stretch in expression of MMP-1 and MMP-13 was lower compared with 15 min stretch groups. The mRNA expression of ERK-1, ets-1 and citied-2 were increased by 6% mechanical stretch under both time points, however c-jun and c-fos mRNA level were affected differently after 15 min and 75 min mechanical stretch compared to control group. There were no significant changes on MMP-3 and ets-2 mRNA level under both 6% mechanical stretch time points. In the presence of pro-inflammatory cytokines (IL-1β and TNF-α), the stretch also reduced the mRNA expression of MMP-1 and MMP-13. In short, our results showed that gentle mechanical strain affects MMP-1 and MMP-13 expression, potentially through the ERK-1-ets-1-cited-2-c-jun signaling pathway.     

SUMO-Conjugating Enzyme UBC9 Promotes Proliferation and Migration of Fibroblast-like Synoviocytes in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease with high morbidity and mortality. Fibroblast-like synoviocytes (FLS) in the synovial tissues play critical roles in joint destruction. Recent studies implicate the sumoylation in the regulation of the inflammation and arthritis. Thus, we explored whether SUMO-conjugating enzyme UBC9 is involved in the progression of RA using a mouse collagen-induced arthritis (CIA) model. The effects of UBC9 siRNA on cell invasion and migration in human RA-FLS were also assessed in vitro. Treatment with siRNA against UBC9 for 3 weeks reduced the arthritis score and joint destruction. The expression of SUMO-1 and UBC9 protein in CIA joints was inhibited by UBC9 knockdown. Serum levels of anti-collagen (CII) antibodies, vascular endothelial growth factor A (VEGF-A), matrix metalloproteinases (MMP)-3, and MMP-9 were also decreased in CIA mice. In vitro, UBC9 silencing inhibited the secretion of VEGF-A, MMP-3, and MMP-9 from TNF-α-stimulated human RA-FLS. TNF-α-induced RA-FLS proliferation and migration were significantly attenuated by UBC9 knockdown. These findings indicate that SUMO-conjugating enzyme UBC9 promotes proliferation and migration of fibroblast-like synoviocytes in rheumatoid arthritis. Inhibition of UBC9 activity may be a viable therapeutic target in amelioration of disease progression in RA by attenuating FLS proliferation, migration, and invasion.     

IgA Rheumatoid Factor and IgG Dietary Protein Antibodies are Associated in Rheumatoid Arthritis

This study sought to determine whether patients with rheumatoid arthritis (RA) were immunologically sensitised to dietary protein (DP). Using an enzyme linked immunosorbent assay (ELISA), antibodies to milk and wheat proteins were measured in 93 unselected out-patients with classical or definite RA. Of these 93, 53 had raised levels of IgG antibodies to one or both dietary proteins (DP). In the DP antibody positive group, 48 patients (90%) also had raised levels of IgA rheumatoid factor (measured by ELISA) while only 7 (17%) of the 40 DP antibody negative patients had detectable IgA RF; P<0.02. There was no association between IgM rheumatoid factor and dietary protein antibodies. These results demonstrate that in RA, raised levels of IgA RF are associated with an increased IgG response to antigens which enter the body through the gastrointestinal tract. A breakdown in gastrointestinal tolerance to dietary antigens may play a role in the immunopathogenesis of RA in these patients who might therefore benefit from dietary manipulation.     

Expression of Activation Antigens on T Cells in Rheumatoid Arthritis Patients

The aim of our study was to identify differences in cell surface marker expression between T cells taken from the peripheral blood (PB) of healthy individuals and T cells recovered from inflamed joints of rheumatoid arthritis (RA) patients. Out of 118 monoclonal antibodies (MoAbs) directed against activation antigens on haematopoietic cells, 12 MoAbs recognizing nine distinct surface molecules were selected after a screening procedure to study the expression of the corresponding antigens on T cells from the PB, synovial fluid and synovial tissue of RA patients, and also on T cells from PB and spleens of controls. Using two-colour flow cytometry and immunohistology we found the molecules B-C5, CD39, CD40, CD45 R0, CD54, CD76 and potentially 1D11 to be substantially up-regulated on T cells from various body compartments in RA patients. We thus could determine that the cell surface of T cells in RA patients not only differs in MHC class II expression, but also in a number of other activation-associated cell surface molecules from T cells in healthy individuals.     

Isoelectrophoretic Characterization of Protein Antigens Present in Mycobacterial Culture Filtrates and Recognized by Monoclonal Antibodies Directed Against the Mycobacterium bovis BCG Antigen 85 Complex

Nine monoclonal antibodies (MoAbs) were produced against Mycobacterium bovis BCG antigen 85 complex. Using isoelectric focusing combined with Western (immunoblot) blot analysis, antigenically related proteins could be identified in culture filtrates from M. tuberculosis, M. bovis, M. Kansasii, M. avium, M. xenopi, M. gordonae, M. fortuitum, M. phlei and M. smegmatis. Most of the MoAbs were found to be broadly cross-reactive between the various mycobacterial species, albeit some minor differences were observed. These MoAbs reacted generally, in each species, with different components. One MoAb (VID1-14) was found to be specifically directed only against antigen 85B from M. bovis, M. tuberculosis and M. kansasii.     

The Role of Anti-Cyclic Cytrullinate Antibodies Testing in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic, progressive inflammatory disease, which leads to joint destruction and deformity and is often accompanied by systemic complications. It is generally considered an autoimmune disease characterized by several autoantibodies. The impressive advances made in understanding the biological mechanisms of RA have led to more focused, directed therapies that have joined, and in many cases overcome, more traditional treatments. Along the last decade, the so-called biological anti-TNF-alpha agents have been shown to reduce disease activity, to slow disease progression and to improve patients’ quality of life. The clear evidence that an early therapeutic intervention improves the overall outcome of the disease supports the importance of an early diagnosis. In the last years, several studies showed that anti-cyclic citrullinated peptide antibodies (anti-CCP) represent a sensitive and specific serologic marker for RA. Moreover, a large body of evidence has shown that anti-CCP may also serve as an early diagnostic and prognostic marker in RA. The aim of this article is to provide an overview of the current state of knowledge regarding anti-CCP focusing in particular on their clinical specificity and prognostic value in RA.     

Autoantibodies from Rheumatoid Arthritis Patients Recognize Antigens on the Synoviocyte Surface

We have found autoantibodies in the sera from rheumatoid arthritis (RA) patients which recognize two cell surface antigens of approximately 70 kDa and 28 kDa from synoviocyte extracts as detected by immunoprecipitation analysis. These polypeptides were immuno-precipitated from extracts containing mainly macrophage-like synoviocytes (type A) but not from extracts of homogeneous fibroblast-like synoviocytes (type B). These autoantigens are not selectively expressed by RA synoviocytes, since both RA and non-rheumatoid synovia were reactive for RA sera. From the panel of different RA sera tested, 64% immunoprecipitated the 70 kDa band, and 27% recognized the 28 kDa polypeptide. These differences in the specificity of the sera seemed to be related to the clinical state of the donor. The sera from patients suffering from other autoimmune diseases such as autoimmune thyroiditis and systemic lupus erythematosus (SLE) do not appear to be reactive for these specificities, but sera from patients with Sjögren's syndrome, psoriatic arthritis, and Crohn's disease showed a weak cross-reactivity with the 70 kDa polypeptide. This autoreactivity against synovial cells in RA supports the idea that these cells participate in the initial immune response of the disease.     

Intrinsic Cell Membrane Antigens Recognized by Antichromatin Autoantibodies

The main object of this study was to see whether or not chromatin constituents are present in cell membranes. The binding of antinuclear antibodies (ANA) to plasma membranes of leucocytes was studied by using autoantibodies and induced antibodies to different histones and histone peptides, and autoantibodies to double-stranded DNA (dsDNA) in adsorption-elution experiments. Eluates were subsequently tested for antinuclear antibodies by a solid-phase ELISA. No ANA activities in the eluates were observed, except when true cross-reacting ANA were employed in the study. Furthermore, no binding of these antibodies to plasma membranes could be visualized by indirect membrane fluorescence tests. The conclusion of these studies was that freshly isolated viable leucocytes did not carry detectable amounts of ectopic chromatin components at the level of plasma membranes. Thus, it seems fairly unlikely that chromatin autoantibodies in general exert some tissue damage by binding to homologous nuclear antigens associated with plasma membranes in vivo.     

miR-483-3p Promotes IL-33 Production from Fibroblast-Like Synoviocytes by Regulating ERK Signaling in Rheumatoid Arthritis

Inflammation - Our previous studies have identified miR-483-3p to be highly expressed in synoviocytes from patients with rheumatoid arhtirits (RA); however, its effects on inflammation of RA...     

Genistein Inhibit Cytokines or Growth Factor-Induced Proliferation and Transformation Phenotype in Fibroblast-Like Synoviocytes of Rheumatoid Arthritis

The purpose of this research is to study the effect of genistein on cytokines or growth factor-induced proliferation and transformation phenotype of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). RA-FLS were primarily cultured. With respective stimulation of IL-1β, TNF-α, and EGF, genistein was applied to elucidate its effect on synoviocytes’ growth number, cell proliferation assay, cell cycle using cell counts, ³H-TdR incorporation and flow cytometry, the colony numbers under anchorage-independent condition, and the expression of MMP-2 and MMP-9 in synovial fibroblasts. EGF, IL-1β, and TNF-α increased ³H incorporation in RA-FLS, respectively. EGF augmented clone numbers of RA-FLS under anchorage-independent condition and not IL-1β and TNF-α. Genistein had an inhibitory role on cell number and ³H-TdR incorporation of RA-FLS stimulated with IL-1β, TNF-α and EGF; genistein arrested the cell cycle at G₁ restriction point; genistein decreased colony numbers under anchorage-independent condition stimulated by EGF in serum condition. IL-1β or TNF-α increased expression of MMP-9 and MMP-2 in rheumatoid synoviocytes; EGF stimulated expression of MMP-9 but not of MMP-2; genistein suppressed production of MMP-9 more than MMP-2 induced by IL-1β or TNF-α; rMMP-9, rMMP-2, or their inhibitors had no effect on the ³H-TdR incorporation of synovial cells. Erk1/2 inhibitor (PD098 059) had obvious inhibitory effect on the ³H incorporation induced by TNF-α or IL-1β; inhibitors of JNK (SP600 125) had no significant effect on the ³H incorporation. While pretreatment with PD098059 had no marked inhibitory effect on MMP-9 expression induced by TNF-α or IL-1β, SP600125 decreased significantly the MMP-9 expression induced by TNF-α or IL-1β. Neither PD098059 nor SP600 125 could inhibit the MMP-2 expression induced by TNF-α or IL-1β. Genistein inhibited IL-1β, TNF-α or EGF-induced proliferation and MMP-9 expression in fibroblast-like synoviocytes of rheumatoid arthritis; the proliferation of RA-FLS was mediated by Erk1/2 but not JNK activation, while JNK activation was involved in the signal transduction pathway leading to MMP-9 expression in rheumatoid synoviocytes.     

IL-18 Upregulates the Production of Key Regulators of Osteoclastogenesis from Fibroblast-Like Synoviocytes in Rheumatoid Arthritis

Recent data have demonstrated the importance of IL-18 in the induction and perpetuation of chronic inflammation in experimental arthritis. The aim of the present study was to elucidate whether IL-18 has any indirect effects on osteoclastogenesis by regulating the production of molecules from fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA). Human FLS were isolated from RA synovial tissue and cultured in vitro for three to five passages. The expression of IL-18 receptor was determined by RT-PCR. The levels of soluble receptor activator of nuclear factor κB ligand (RANKL), osteoprotegerin (OPG), macrophage colony-stimulating factor (M-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) in culture supernatants were determined by ELISA. Membrane-bound RANKL expression was analyzed by flow cytometry. Both α and β chains of IL-18 receptor were confirmed in cultured FLS. IL-18 upregulated membrane-bound RANKL expression and soluble RANKL production by FLS in both time- and dose-dependent manners. In addition, IL-18 enhanced production of M-CSF, GM-CSF, and OPG from cultured FLS in a dose-dependent manner. IL-18 also increased the ratio of RANKL/OPG, suggesting that the net effect of IL-18 on FLS favors for the induction of osteoclast formation and bone resorption. In conclusion, IL-18 upregulates the production of key regulators of osteoclastogenesis from FLS in RA.