We have used a surface plasmon resonance biosensor (SPR, BIACORE 2000) to detect antibodies against glucose 6-phosphate isomerase (GPI) in synovial fluids of rheumatoid arthritis (RA) and osteoarthritis (OA). Recombinant human GPI proteins fused with or without NusA were expressed in E. coli, purified to homogeneity and immobilized in flow cells of CM5 sensor chips. The flow cells immobilized with NusA protein or bovine serum albumin were used to monitor non-specific binding. Synovial fluid samples from RA patients showed a significantly higher level of binding to recombinant GPI proteins than samples from OA patients. Proteins which bound to the recombinant GPI proteins were confirmed to be immunoglobulin through the administration of anti-human immunoglobulin. NusA fusion protein was excellent for this assay because of a low background binding activity in the SPR analysis and its advantage of increased solubility in recombinant protein production. These results suggested a useful utilization of recombinant NusA-GPI fusion protein for the detection of autoantibodies against GPI in RA patients.
Aged ; Antibodies/*immunology ; Arthritis, Rheumatoid/*immunology ; Female ; Glucose-6-Phosphate Isomerase/genetics/*immunology/metabolism ; Human ; Male ; Middle Aged ; Osteoarthritis/immunology ; Peptide Elongation Factors/genetics/metabolism ; Recombinant Fusion Proteins/genetics/metabolism ; Surface Plasmon Resonance ; Synovial Fluid/*immunology ; Transcription Factors/genetics/metabolism

Nonspecific immune parameters such as natural killer(NK) activity, antibody-dependent cellular cytotoxicity(ADCC), production of leukocyte migration inhibitory factor(LlF) and levels of immune complex(IC) were assessed in 47 patients with rheumatoid arthritis (RA) 20 with degenerative arthritis (DA) and 40 healthy controls. Peripheral blood (PB) as well as synovial fluid (SF) were collected from both RA and DA patients before treatment. Mononuclear cell suspensions and sera were prepared and submitted for the in vitro tests; 4-hr chromium-release assays using human K562 and mouse L1210 cells as targets for NK and ADCC assays respectively, 2-step agarose assay for LIF and platelet aggregation test for IC. Results revealed that 1) LIF activity of PB lymphocytes (PBL) from both RA and DA patients showed a significant (P < 0.05) decrease as compared with that from healthy controls. 2) PB-NK activity from RA patients showed an insignificant decrease as compared with that from DA or healthy controls. However, mononuclear cells isolated from SF (SFL) of RA patients exhibited significantly(P < 0.02) lower NK activity than PBL from the same patients. 3) In ADCC assays with PBL no significant differencies were observed among the 3 groups. 4) Higher titers of IC were detected in both PB and SF from RA patients than DA, and a negative correlation was found between serum IC levels and PB-NK activity. These data are discussed in light of previous reports, and a hypothesis regarding a decreased nonspecific cell-mediated immunity in conjunction with an increased humoral immune response, particularly in local sites, is proposed as one of the mechanisms underlying the pathogenesis of RA.
Adult ; Antibody-Dependent Cell Cytotoxicity ; Antigen-Antibody Complex/immunology ; Arthritis, Rheumatoid/immunology* ; Female ; Human ; Killer Cells, Natural/immunology ; Leukocyte Migration-Inhibitory Factors/biosynthesis ; Male ; Middle Age ; Synovial Fluid/immunology

Effect of interleukin-6 receptor (IL-6R) antibody on polymethyl methacrylate (PMMA) bone cement-mediated expression of osteoprotegerin (OPG) and receptor activator of nuclear factor-kappaB ligand (RANKL) in synovial fibroblasts was investigated. Synovial tissue obtained from total knee arthroplasty was digested and cultured. Inverted microscope was employed to observe the synovial cells and immunocytochemistry (SABC method) staining was used to identify synovial fibroblasts. This experiment was divided into three groups according to different culture media: PMMA group (75 μg/mL PMMA bone cement particles), IL-6R antibody group (10 ng/mL IL-6R antibody+75 μg/mL PMMA bone cement particles), and control group (no IL-6R antibody or PMMA bone cement particles). Influence of IL-6R antibody and PMMA on proliferation of synovial fibroblasts was measured by cell counting kit-8 (CCK-8). ELISA method was used to measure OPG and RANKL levels in culture solution. Fluorescence quantitative real-time PCR (FQ-PCR) was used to detect the expression of OPG and RANKL mRNA. After three consecutive passages, more than 95% of the primary synovial cells became long spindle fibroblast-like cells. SABC staining results showed that the fibroblast-like cells were negative for anti-CD68 antibody and positive for anti-vimentin antibody, with brown madder stained. CCK-8 test demonstrated that the absorbance (A) value at 450 nm was significantly lower in IL-6R antibody group than in PMMA group and control group (P<0.01), but there was no statistically significant difference in A value at 450 nm between the control group and PMMA group (P>0.05). Results of ELISA indicated that the expression of OPG was significantly higher in IL-6R antibody group than in PMMA group and control group (P<0.01). The expression of RANKL was inhibited (P<0.05), and the ratio of OPG/RANKL was significantly increased in IL-6R antibody group as compared with PMMA group and control group. There was no significant difference in the expression of OPG between control group and PMMA group (P>0.05), but the expression of RANKL was higher in PMMA group than in control group (P<0.05), and there was a significant difference in the ratio of OPG/RANKL between them (P<0.05). Results of FQ-PCR revealed the expression of RANKL mRNA was significantly inhibited (P<0.01) and the expression of OPG mRNA was significantly increased (P<0.01) in IL-6R antibody group as compared with PMMA group and control group. The expression of RANKL mRNA was higher in PMMA group than in control group (P<0.05), but the expression of OPG mRNA had no significant difference between them (P>0.05). IL-6R antibody could significantly increase the expression of OPG, but inhibit the expression of RANKL, which might provide a theoretical basis of molecular biology for the prevention and treatment of aseptic loosening of prosthesis.
Antibodies ; administration & dosage ; immunology ; Bone Cements ; Fibroblasts ; immunology ; Gene Expression ; drug effects ; Humans ; Osteoprotegerin ; biosynthesis ; genetics ; Polymethyl Methacrylate ; administration & dosage ; Prostheses and Implants ; RANK Ligand ; biosynthesis ; genetics ; metabolism ; Receptors, Interleukin-6 ; immunology ; metabolism ; Synovial Fluid ; immunology ; metabolism

The natural killer(NK) cell activity of mononuclear cells (MNC) from peripheral blood (PB) and synovial fluid (SF) of 40 rheumatoid arthritis(RA) patients was investigated by employing 51-chromium-(51Cr) release microcytotoxicity and single cell cytotoxicity assays against K562 target cells. It has been revealed that SF-MNC from RA patients showed a significantly lower NK activity than PB-MNC from the same patients and this might be due to an impaired target binding capacity of the effector cells and not due to a deficiency of active NK cells.
Adolescent ; Adult ; Aged ; Arthritis, Rheumatoid/*immunology ; Chromium Radioisotopes/diagnostic use ; Comparative Study ; Cytotoxicity Tests, Immunologic/methods ; *Cytotoxicity, Immunologic ; Female ; Human ; In Vitro ; Killer Cells, Natural/*immunology ; Male ; Middle Age ; Support, Non-U.S. Gov't ; Synovial Fluid/immunology

OBJECTIVETo explore the mechanism of electroacupuncture on rheumatoid arthritis (RA).

METHODSIn a randomized and controlled trial, sixty-three cases with RA were randomly divided into an electroacupuncture group (n = 32) and a simple acupuncture group (n = 31). Baihui (GV 20), Fengchi (GB 20), Quchi (LI 11), Waiguan (TE 5), Guanyuan (CV 4) and Zusanli (ST 36) were selected by coordination method combined whole and local acupoints. The electroacupuncture group was treated with electroacupuncture at the local acupoints near painful joints, continuous wave, retaining needle for 30 minutes, and then electroacupuncture at Back-shu acupoints, retaining needle for 15 minutes, and the simple acupuncture group was treated with the same acupoints selection and acupuncture manipulation without electroacupuncture apparatus. They were all treated once every other day for 20 days as one course. After 3 courses, changes of interleukins in peripheral blood and joint fluid of patients were observed.

RESULTSBoth of electroacupuncture and simple acupuncture had significant effect on IL-1, IL-4, IL-6 and IL-10 in peripheral blood and joint fluid of patients with RA ( P < 0.01, P < 0.05). But after electroacupuncture, the absolute value and improvement value of decreasing IL-1 in peripheral blood and joint fluid were super than those of simple acupuncture (all P < 0.05), and of IL-4 in joint fluid was super than that after simple acupuncture (P < 0.05), and of IL-6 and the absolute value of decreasing IL-10 were almost the same after both treatment (all P > 0.05), and after electroacupuncture, the improvement value of IL-10 in peripheral blood and joint fluid were super than those after simple acupuncture (both P < 0.05).

CONCLUSIONElectroacupuncture can effectively decrease the proinflammatory cytokine of IL-1 and IL-6 and increase the inhibition cytokine of IL-4 and IL-10 and improve the internal environment of occurrence and progression of RA.

Acupuncture Therapy ; Adult ; Aged ; Arthritis, Rheumatoid ; blood ; immunology ; therapy ; Electroacupuncture ; Female ; Humans ; Interleukin-1 ; blood ; Interleukin-10 ; blood ; Interleukin-4 ; blood ; Interleukin-6 ; blood ; Interleukins ; blood ; immunology ; Male ; Middle Aged ; Synovial Fluid ; immunology ; Young Adult

OBJECTIVETo observe the analgesic effect of electroacupuncture (EA) on collagen-induced arthritis (CIA) rats and its regulating effect on inflammation reaction and the endogenous opioid system of synovial tissues. Methods A total of 30 healthy male Wistar rats were randomly divided into a control group, a model group and an EA group, 10 rats in each one. The chronic pain model of CIA rats was made by cattle type-II collagen in the model group and EA group. Rats in the EA group were treated with EA at "Zusanli" (ST 36) and "Kunlun" (BL 60) for 30 min from 16th day after model establishment, once a day for consecutive 10 days. Rats in the control group did not receive any treatment. Rats in the model group were treated with fixation as the EA group. Threshold of pain, arthritis index, paw swelling were measured before model establishment and 16 d, 20 d, 23 d and 25 d after model establishment. The levels of beta-endorphin (β-END), met-enkephalin (met-ENK), dynorphin A (Dyn A) were measured by radioimmunoassay; the mRNA expressions of mu opioid receptor (MOR), kappa opioid receptor (KOR) and delta opioid receptor (DOR) in synovial tissues of CIA rats were detected by I quantitative polymerase chain reaction (qPCR).

RESULTSCompared with the control group, threshold of pain was reduced (all P<0. 01), arthritis index was increased (all P<0. 01) and paw swelling was increased (all P<0. 01) in the model group on the 16th day, 20th day, 23rd day, 25th day after model establishment. Compared with the model group, the threshold of pain was increased in the EA group (all P<0. 01), arthritis index and paw swelling were reduced (all P<0. 01) on the 23rd day and 25th day after model establishment. Compared with the control group, the level of Dyn A in synovial tissues of CIA rats was increased in the model group (P<0. 01); the mRNA expressions of MOR, KOR and DOR were down-regulated lower than 0. 5 fold of normal level. Compared with the model group, the level of β-END in synovial tissues of the knee joint was increased in the EA group (P<0. 05), and the mRNA expressions of MOR, KOR and DOR in synovial tissues of CIA rats were up-regulated more than 2 folds of normal level.

CONCLUSIONThe intervention of EA on chronic pain of CIA rats is superior, which is likely to be related with effects of EA on anti-inflammation and up-regulation of synovial tissue β-END and MOR, KOR, DOR.

Acupuncture Analgesia ; Acupuncture Points ; Analgesics, Opioid ; immunology ; Animals ; Arthritis, Rheumatoid ; immunology ; therapy ; Cattle ; Chronic Pain ; immunology ; therapy ; Dynorphins ; genetics ; immunology ; Electroacupuncture ; Enkephalin, Methionine ; genetics ; immunology ; Humans ; Male ; Rats ; Rats, Wistar ; Receptors, Opioid, mu ; genetics ; immunology ; Synovial Fluid ; immunology ; beta-Endorphin ; genetics ; immunology

INTRODUCTIONOsteoarthritis (OA) is a progressive degenerative disorder of the articular cartilage. Available diagnostic radiography has been poorly associated with the progress and severity of this clinical disease. As osteogenic protein-1 (OP-1) has been identified as a bone morphogenetic protein with a major role in cartilage repair, we aimed to evaluate its potential role in the diagnosis of OA.

METHODSThis was an experimental study conducted at the Department of Biochemistry, Sikkim Manipal Institute of Medical Sciences, India. Polyclonal antibodies (i.e. anti-OP-1[f]) were raised against OP-1 in mice, and subsequently used in a sandwich enzyme-linked immunosorbent assay (ELISA) to detect the presence of OP-1 in the synovial fluids of 75 osteoarthritic patients. For the purpose of correlation, the radiographic assessments of the knees of the 75 patients were graded using the Kellgren-Lawrence scoring system.

RESULTThe polyclonal antibody (i.e. anti-OP-1[f]) raised against OP-1 was able to detect the presence of OP-1 in the synovial fluids of all the osteoarthritic patients via sandwich ELISA. The level of the OP-1 was found to be much higher than the reference range and correlated positively with the severity of OA (r = 0.24; p = 0.04).

CONCLUSIONOur study shows that the polyclonal antibody, anti OP-1(f), could be used for the immunodiagnosis of osteoarthritis via sandwich ELISA.

Adult ; Aged ; Aged, 80 and over ; Animals ; Antibodies ; chemistry ; Bone Morphogenetic Protein 7 ; chemistry ; immunology ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Knee ; physiopathology ; Mice ; Middle Aged ; Osteoarthritis ; diagnosis ; immunology ; Synovial Fluid ; chemistry

OBJECTIVETo detect the disparity of three cytokines interleukin-6 (IL-6), interferon-inducible protein 10 (IP-10) and interleukin-17 (IL-17) in peripheral blood (PB) and synovial fluid (SF) of patients with juvenile idiopathic arthritis (JIA).

METHODSerum concentrations of the three cytokines were measured in 27 patients with 13 systemic-onset JIA (sJIA), 14 polyarticular JIA (pJIA) and 28 healthy controls using enzyme-linked immunosorbent assay (ELISA). Nineteen patients with no marked arthritis symptom or only temporary arthralgia were enrolled in probable sJIA group. SF from 18 patients with 7 sJIA, 11 pJIA were examined for cytokine levels.

RESULT(1) The statistically significant difference in serum IL-6 was detected between sJIA and healthy control group [28.0(4.2-59.2) ng/L vs. 12.3 (2.1-13.8) ng/L, P < 0.05], but no significant difference between probable sJIA and healthy control group [11.8(7.7-39.2) ng/L vs. 12.3 (2.1-13.8) ng/L, P > 0.05] was found. There were statistically significant differences between sJIA group and healthy control group in serum concentrations of IL-17 [14.0(9.8-34.3) ng/L vs. 9.8 (7.9-16.2) ng/L, P < 0.05], yet compared to healthy control group, no significant difference in concentration level of IL-17 was found in pJIA Group [14.2(9.9-16.9) ng/L vs. 9.8(7.9-16.2) ng/L, P > 0.05].(2) In sJIA and pJIA SF, the median IP-10 level was significantly higher compared to respective PB levels [619.7 (160.9, 873.1) ng/L vs. 64.8 (27.4-111.9) ng/L;660.9 (401.9, 1349.8) ng/L vs. 97.4 (41.9-222.1) ng/L, P < 0.01, respectively], but there was only significant difference in IL-17 between pJIA SF and PB [22.9 (17.1, 45.8) ng/L vs. 14.2 (9.9-16.9) ng/L, P < 0.01].

CONCLUSIONIL-6 may play more important role in the pathogenesis of sJIA. Moreover, IL-6 may be the biomarker associated with arthritis in early JIA stage. Both autoinflammation and autoimmune response may be involved in the pathogenesis of sJIA. IL-17 enrichment may only occur in local joint, the levels of IL-17 in PB may not be significantly increased. The prominent expression gradient between SF and PB of IP-10 maybe the basis of performing chemotaxis and further causing joint damage.

Adolescent ; Arthritis, Juvenile ; blood ; immunology ; metabolism ; Case-Control Studies ; Chemokine CXCL10 ; blood ; metabolism ; Child ; Child, Preschool ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Interleukin-17 ; blood ; metabolism ; Interleukin-6 ; blood ; metabolism ; Knee Joint ; metabolism ; Male ; Synovial Fluid ; immunology ; metabolism

Myeloid-related protein (MRP)8/MRP14 is an endogenous Toll-like receptor 4 (TLR4) ligand and is abundant in synovial fluid (SF) of rheumatoid arthritis (RA) patients. Belonging to damage-associated molecular patterns, it amplifies proinflammatory mediators and facilitates a wide range of inflammatory and autoimmune diseases. Interleukin (IL)-17-producing T-helper (Th)17 cells have a crucial role in RA pathogenesis, and IL-6 is the key factor promoting Th17 differentiation. We investigated whether the level of MRP8/MRP14 is positively associated with IL-6 and IL-17 levels in RA SF and found that MRP8/MRP14 level had a significant correlation with IL-6 and IL-17 levels in RA SF. We also observed that MRP8-induced IL-17 production by peripheral blood mononuclear cells but MRP14 did not. Upon stimulation with MRP8, IL-6 production was enhanced by RA fibroblast-like synoviocytes (FLS) and was further elevated by coculturing RA FLS with activated CD4+ T cells. Moreover, we demonstrated that MRP8-activated IL-6 production by RA FLS promoted differentiation of Th17 cells using the coculture system consisting of CD4+ T cells and RA FLS. In addition, IL-6 blockade attenuated Th17 polarization of CD4+ T cells in the cocultures. Inhibitor studies revealed that MRP8 increased IL-6 production in RA FLS via TLR4/phosphoinositide 3-kinase/nuclear factor-kappaB and mitogen-activated protein kinase signaling pathways. Our results show that MRP8 has a crucial role in stimulating IL-6 expression by RA FLS, and subsequently promotes Th17 differentiation in RA, suggesting that neutralizing MRP8 level in RA synovium may be an effective therapeutic strategy in RA treatment.
ATP-Binding Cassette Transporters/*metabolism ; Adult ; Aged ; Arthritis, Rheumatoid/*pathology ; CD4-Positive T-Lymphocytes/metabolism ; Calgranulin B/metabolism ; Cell Differentiation/*immunology ; Fibroblasts/*metabolism/pathology ; Humans ; Inflammation Mediators/metabolism ; Interleukin-17/metabolism ; Interleukin-6/*biosynthesis ; Middle Aged ; Signal Transduction/immunology ; Synovial Fluid/cytology ; Synovial Membrane/metabolism/pathology ; Th17 Cells/*pathology ; Toll-Like Receptor 4/metabolism ; *Up-Regulation

OBJECTIVETo study the effect of geniposide-acid(GA) on the anti-inflammatory action for adjuvant-induced arthritis (AA) rats and the proliferation of synoviocytes in AA rats and the feasible mechanism of apoptosis in vitro.

METHODForty-eight health male Wistar rats were divided randomly into six groups and were administered respectively with 200, 100, 50 mg x kg(-1) GA and 0.75 mg x kg(-1) MTX and normal sodium (normal or model control group) for four weeks when right posterior paw pads of rats excluding normal control group were injected intrademally with complete Freund's adjuvant after 19 days. The left posterior paws swelling degree, swelling inhibition ratio and arthritis index of secondary inflamation were detected. The TNF-alpha and IL-1beta proteins in serum of rats were assayed by enzyme linked immunosorbent assay (ELISA) kits. The synovial fibroblasts of AA rats were exposed to 1-4 micromol x L(-1) GA or 4 micromol x L(-1) MTX. The effect of GA on the proliferation of synoviocytes was detected by MTT assay. The morphologic change of apoptosis cells was observed by Hoechst/PI double stainning and fluorescence microscope. The rate of apoptosis cells was analyzed by flow cytometry. The mRNA expresstion of Bcl-2 and Bax gene was detected by reverse transcription PCR (RT-PCR).

RESULT200 mg kg(-1) or 100 mg kg(-1) GA could decrease significantly the paw swelling degree, arthritis index and the level of TNF-alpha and IL-1beta proteins in serum of AA rats (P < 0.05 or P < 0.01) with 25.4%, 21.37% of the swelling inhibition ratio respectivly, 34.61%, 28% of protein inhibition ratio of TNF-alpha and 29.05%, 21.65% of that of IL-1beta. GA(1-4 micromol x L(-1)) inhibitated significantly the proliferation of synoviocytes culcured for 5 days. Flow cytometry showed that 1, 2, 4 micromol x L(-1) GA increased obviously the rate of apoptosis cells, the apoptosis ratios were 15.8%, 24.3%, 40.7% respectivly (P < 0.01). RT-PCR showed GA could decrease the expression level of Bcl-2 gene but increase that of Bax gene (P < 0.05 or P < 0.01).

CONCLUSIONGA could inhibit the secondary inflamation of AA rats and decrease the level of TNF-alpha and IL-1beta protein in the AA rats serum. GA could inhibit the proliferation of AA rat synoviocytes in vitro and induce apoptosis which mechanism was concerned with down-regulating the mRNA expression of Bcl-2 and up-regulating that of Bax.

Animals ; Anti-Inflammatory Agents ; administration & dosage ; Apoptosis ; drug effects ; Arthritis, Experimental ; chemically induced ; drug therapy ; immunology ; physiopathology ; Cytokines ; immunology ; Disease Models, Animal ; Freund's Adjuvant ; adverse effects ; Glucosides ; administration & dosage ; Humans ; Iridoid Glucosides ; Iridoids ; administration & dosage ; Male ; Random Allocation ; Rats ; Rats, Wistar ; Synovial Fluid ; cytology ; drug effects ; immunology