The clinical significance of cytoplasmic inclusions(CPI) in synovial fluid(SF) examination was evaluated. We examined SF specimens collected from major rheumatology clinics in the Philadelphia area during the period of January to December 1995. Among 759 patients in the initial study group, 419 cases with established diagnoses and full synovial analyses were included. Their diagnoses and SF analysis results including leukocyte counts, differential counts and wet preparations were collected and analysed. Ninety seven of the 419 SF specimens were found to have CPI. CPI were found in SF from almost all rheumatic diseases. They were most likely to be found in inflammatory arthropathy including rheumatoid arthritis(RA, 46%), juvenile rheumatoid arthritis(JRA, 78%) and psoriatic arthritis(55%). On the contrary, CPI were least common in crystal-induced arthropathy among the inflammatory arthropathy. CPI were found 8 out of 98 gout cases(8%) and 2 among 53 calcium pyrophosphate dihydrate(CPPD) deposition disease(4%). In noninflammatory arthropathy, CPI were found in only 6 cases(6%) out of the 103 osteoarthritis(OA). In RA cases with non-inflammatory SF, 4 of the 20 SF(20%) had CPI while only 6% of OA SF had CPI. OA SF with CPI were all noninflammatory SF. In summary, CPI were a common finding on SF examination. CPI were more likely to be found in inflammatory arthropathy than noninflammatory. Among inflammatory arthropathy, CPI can favor non-crystal arthropathy than crystal arthropathy. Awareness of the presence of CPI is suggested as an addendum to routine SF analysis. Renewed investigation of the several types of CPI may add further to the understanding of joint disease.
Inclusion Bodies/*pathology ; Rheumatic Diseases/*physiopathology ; Synovial Fluid/*cytology

This study was purposed to investigate the influence of inflammatory microenvironment on mesenchymal stem cells (MSCs) and regulatory effect of MSCs on osteoblast formation. The MSCs were isolated from synovial fluid of patients with rheumatoid arthritis (RASF-MSCs) and were cultured, the immunotypes of RASF-MSCs were detected by flow cytometry, the ability to differentiate RASF-MSCs into osteoblasts and adipocytes was determined by means of osteogenic and adipogenic induction, the regulatory effect of RASF-MSCs on osteoblast formation was assayed by co-culturing RASF-MSCs whth CD14(+) monocytes and in situ tartrate-resistant acid phosphatase staining. The results showed that RASF-MSCs highly expressed CD105, CD73, CD29, CD44, CD166 and HLA-ABC. Meanwhile, they lowly expressed CD34, CD45, CD31, HLA-DR, CD80 and CD86. However, RASF-MSCs decreased multi-differentiation capability as compared with BM-MSCs. More interestingly, RASF-MSC significantly promoted osteoclasts formation (p < 0.05) when co-cultured with monocytes. It is concluded that MSCs from rheumatoid arthritis synovial fluid exert typical MSC phenotypes but displayed decline of multi-differentiation capability. RASF-MSCs especially show promoting effect on osteoclastogenesis. The findings of this study may contribute to the understanding biological behavior of MSCs in pathological microenvironment.
Arthritis, Rheumatoid ; Bone Marrow Cells ; cytology ; Cell Differentiation ; Cells, Cultured ; Humans ; Mesenchymal Stromal Cells ; cytology ; Osteoblasts ; cytology ; Synovial Fluid ; cytology

OBJECTIVETo investigate the role of natural killer-22 (NK-22) cells in the synovial fluid in the proliferation of fibroblast-like synoviocytes (FLS) in patients with rheumatoid arthritis (RA) and explore the possible signal pathway involved.

METHODSNK-22 cells in the SF of RA patients were sorted by flow cytometry. NK-22 cells were cultured for two weeks and the purity was detected by flow cytometry before stimulation with 20 ng/ml phorbol 12-myristate 13-acetate and 0.5 µmol/L ionomycin for 4 h. The level of interleukin-22 (IL-22) in the culture medium supernatant was then measured with ELISA. The proliferation of FLS in the presence of the culture supernatant of NK-22 cells was assessed with MTT assay at 24, 48 and 72 h, and the effect of IL-22 antibody on FLS proliferation was also observed. Real-time PCR and Western blotting were used to detect Stat3 mRNA and p-Stat3 protein levels, respectively, in the FLS exposed to rhIL-22 and AG490.

RESULTSNK-22 cells were successfully sorted by flow cytometry with a purity exceeding 90%. The levels of IL-22 in the supernatant of NKp44(+)NK cell culture averaged 1273.42∓254.48 pg/ml. The FLS proliferated rapidly 24, 48, and 72 h after the addition of culture supernatant of NK-22 cells (P<0.05). IL-22 antibody obviously inhibited the proliferation of FLS induced by NK-22 cell culture supernatant (P<0.05). Exposure of the FLS to rhIL-22 obviously increased cellular Stat3 expression levels, which were significantly lowered by the addition of AG490 (P<0.05).

CONCLUSIONNK-22 cells in the SF of RA patients can produce high concentrations of IL-22 to promote the proliferation of FLS through the STAT3 signal pathway.

Arthritis, Rheumatoid ; metabolism ; Cell Proliferation ; Cells, Cultured ; Fibroblasts ; cytology ; Humans ; Interleukins ; metabolism ; Killer Cells, Natural ; cytology ; metabolism ; STAT3 Transcription Factor ; metabolism ; Signal Transduction ; Synovial Fluid ; cytology ; Synovial Membrane ; cytology

OBJECTIVETo investigate the pathological expression and significance of VEGF in patients with active ankylosing spondylitis.

METHODSThe expression of VEGF in the synovial tissues of cacroiliac joint of patients with active AS was detected by using in situ hybridization and the results were compared with those in the patients with pelvic fracture using image analysis system.

RESULTSThe positive expressions of VEGF in the synovial tissues of cacroiliac joint of patients with active AS were stronger than those in the control group (P<0.01).

CONCLUSIONVEGF are important factors in patients with active AS. They are tightly correlated with the process of osteoclasia and pathological new bone formation in the cacroiliac joint of patients with active AS. If we can reduce the expressions of VEGF in the patients with active AS, the process of osteoclasia and pathological new bone formation will be interrupted and this provides a new strategy for the treatment of ankylosing spondylitis.

Adult ; Case-Control Studies ; Female ; Gene Expression Regulation ; Humans ; Male ; RNA, Messenger ; genetics ; metabolism ; Spondylitis, Ankylosing ; genetics ; Synovial Fluid ; cytology ; metabolism ; Vascular Endothelial Growth Factor A ; genetics

OBJECTIVETo investigate the effect of NK-22 cells isolated from the synovial fluid (SF) of patients with rheumatoid arthritis (RA) on the proliferation of fibroblast-like synoviocytes and explore its possible mechanism.

METHODSThe proportions of NK-22 cells in the peripheral blood (PB) and the SF of 20 RA patients and 20 healthy individuals were determined by flow cytometry. NK-22 cells in the SF sorted by flow cytometry were cultured for two weeks followed by a 4-h stimulation with 20 ng/ml phorbol 12-myristate 13-acetate and 0.5 µ mol/L ionomycin. The culture supernatant of NK-22 cells was then harvested, in which the levels of IL-22 and TNF-α were measured by ELISA. The fibroblast-like synoviocytes were exposed to the culture supernatant for 24, 48, 72, and 96 h, and the changes in the cell proliferation were detected by MTT assay.

RESULTSRA patients showed a significantly greater proportion of NK-22 cells in both the SF and PB than the normal control subjects (P<0.05). NK-22 cells sorted by flow cytometry reached a purity exceeding 90%, and the levels of IL-22 and TNF-α in the culture supernatant of NK-22 cells cultured for two weeks were 941.16 pg/ml and 368.1 pg/ml, respectively. The culture supernatant of NK-22 cells caused a rapid proliferation of the fibroblast-like synoviocytes at 24, 48, 72, and 96 h after the exposure.

CONCLUSIONNK-22 cells in the SF of RA patients can promote the proliferation of fibroblast-like synoviocytes possibly due to the capacity of NK-22 cells to produce IL-22 and TNF-α.

Adult ; Arthritis, Rheumatoid ; pathology ; Case-Control Studies ; Cell Proliferation ; Cells, Cultured ; Female ; Fibroblasts ; cytology ; Flow Cytometry ; Humans ; Interleukins ; metabolism ; Killer Cells, Natural ; cytology ; Male ; Middle Aged ; Synovial Fluid ; cytology ; Tumor Necrosis Factor-alpha ; metabolism

Rheumatoid arthritis (RA) is an autoimmune disease involving the synovial membrane of peripheral joints. T cells specific for self antigens may play a critical role. Identification of T cell receptors (TCR) of such specific T cell clones is very important for treatment, prevention and identification of relevant autoantigens. To identify specific T cells, TCR V beta family repertoire and the clonal expansion of T cells were analyzed in this study. The percentage of V beta 5+ or V beta 8+ cells in the synovial fluid mononuclear cells (SFMCs) was similar to that in the peripheral blood mononuclear cells (PBMCs). However, the percentage of DR+ T cells in the SFMCs was higher (p< 0.01). Analyzing the clonality of T cells in 8 V beta families (V beta 1, V beta 5, V beta 8, V beta 14, V beta 16, V beta 17, V beta 18, V beta 20), clonal expansions in CD8+ T cells from the SFMCs were found more frequently than in the PBMCs. The patterns of clonal expansions were discrepant between the SFMCs and the PBMCs even in the same patient, which suggests several inflamed tissue specific T cell clonal expansions in the SFMCs. These T cell clones might be activated by autoantigens which are not identified yet and responsible for the RA pathogenesis.
Arthritis, Rheumatoid/*metabolism/pathology ; Base Sequence ; Blood Cells/*metabolism ; Clone Cells ; Female ; Human ; Male ; Molecular Probes ; Molecular Sequence Data ; Polymerase Chain Reaction ; Receptors, Antigen, T-Cell/genetics/*metabolism ; Support, Non-U.S. Gov't ; Synovial Fluid/cytology/*metabolism ; T-Lymphocytes/*metabolism

OBJECTIVETo evaluate the effects of Artesunate on tumor necrosis factor alpha (TNF-alpha), monocyte chemoattractant protein (MCP-1), and on reduced activation normal T cell expressed and secreted (RANTES) in the serum and the synoviocyte culture supernate of collagen-induced arthritis (CIA) rats.

METHODSEighty male Wistar rats were selected to establish the CIA rat model. On the 6th day after modeling, 60 rats with the sum of arthritis index of right metapedes and two propodium > or = 6 were selected, and randomly divided into 6 groups (n = 10), i.e., the blank control group, the CIA model control group (treated with normal saline, abbreviated as the CIA group), the MTX positive control group (abbreviated as the MTX group), the large dose Artesunate group (at the daily dose of 20 mg/kg), the moderate dose Artesunate group (at the daily dose of 10 mg/ kg), and the small dose of Artesunate group (at the daily dose of 2.5 mg/kg). Mice were sacrificed 7 days of immune injection and their venous blood was collected to obtain the serum. Meanwhile, the synovial tissues of the knee joint were taken by aseptic techniques and primary cultured for 48 h. The supernate was collected by centrifuge. The changes of MCP-1, RANTES, and TNF-alpha in the serum and the synoviocyte culture supernate were observed in each group before and after treatment using ELISA.

RESULTSArtesunate significantly decreased the expressions of TNF-alpha in the serum and the synoviocyte culture supernate, showing significant difference when compared with the model control groups (P < 0.05). There was no statistical difference in the large dose Artesunate group and the moderate dose Artesunate group when compared with the MTX group (P > 0.05). But statistical difference existed in the large dose Artesunate group, the moderate dose Artesunate group, and the MTX group when compared with the small dose Artesunate group (P < 0.05). Artesunate could significantly decrease the expressions of MCP-1 and RANTES in the serum and the synoviocyte culture supernate, showing statistical difference when compared with the model control group (P < 0.05). But no statistical difference existed when compared with the MTX group (P > 0.05).

CONCLUSIONThe mechanism of anti-inflammatory action and immune regulation of Artesunate might be correlated with the inhibition of inflammatory factor TNF-alpha and chemotactic factors MCP-1 and RANTES.

Animals ; Artemisinins ; pharmacology ; Arthritis, Experimental ; blood ; metabolism ; Cells, Cultured ; Chemokine CCL2 ; blood ; metabolism ; Chemokine CCL5 ; blood ; metabolism ; Disease Models, Animal ; Male ; Rats ; Rats, Wistar ; Synovial Fluid ; cytology ; metabolism ; Tumor Necrosis Factor-alpha ; blood ; metabolism

Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disorder that causes the immune system to attack the joints. Transforming growth factor-beta1 (TGF-beta1) is a secreted protein that promotes differentiation of synovial fibroblasts to alpha-smooth muscle actin (alpha-SMA)-positive myofibroblasts to repair the damaged joints. Synovial fluid from patients with RA (RA-SF) induced expression of alpha-SMA in human adipose tissue-derived mesenchymal stem cells (hASCs). RA-SF-induced alpha-SMA expression was abrogated by immunodepletion of TGF-beta1 from RA-SF with anti-TGF-beta1 antibody. Furthermore, pretreatment of hASCs with the TGF-beta type I receptor inhibitor SB431542 or lentiviral small hairpin RNA-mediated silencing of TGF-beta type I receptor expression in hASCs blocked RA-SF-induced alpha-SMA expression. Small interfering RNA-mediated silencing of Smad2 or adenoviral overexpression of Smad7 (an inhibitory Smad isoform) completely inhibited RA-SF-stimulated alpha-SMA expression. These results suggest that TGF-beta1 plays a pivotal role in RA-SF-induced differentiation of hASCs to alpha-SMA-positive cells.
Actins/*metabolism ; Adipose Tissue/*cytology ; Arthritis, Rheumatoid/*metabolism ; Humans ; Mesenchymal Stem Cells/*metabolism ; Receptors, Lysophosphatidic Acid/metabolism ; Signal Transduction ; Smad2 Protein/metabolism ; Stress Fibers/metabolism ; Synovial Fluid/*metabolism ; Transforming Growth Factor beta1/*metabolism

OBJECTIVETo compare and estimate the diagnostic value and characteristic of different diagnostic methods (blood laboratory test, histological analysis, synovial fluid cytological test and microbiological examination) in detecting the presence of periprosthetic joint infection.

METHODSData of 52 patients underwent hip or knee joint revision in Peking University People's Hospital Arthritis Clinic and Research Center between July 2013 and March 2015 were analyzed retrospectively. For each patient, results of blood laboratory tests(peripheral-blood white blood cell, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), interleukin-6 (IL-6) and high-sensitivity C-reactive protein (Hs-CRP)), histological analysis, synovial fluid white cell count (SWCC), microbiological examinations (synovial fluid, tissue and prosthetic joint sonication fluid) were collected. Data were analyzed by t-test, independent sample median test or χ(2) test, respectively. Sensitivity, specificity, positive predictive value, negative predictive value and accuracy for each method were calculated and compared by receiver operating characteristic curve.

RESULTSThere were 30 female and 22 male patients. Twenty-one patients (40.4%) were diagnosed as PJI. The levels of CRP, ESR, IL-6 and Hs-CRP in patients with PJI were higher than that in aseptic failure patients (Z=23.084, 13.499, 5.796, 17.045, all P<0.05). The sensitivities of CRP, ESR, IL-6 and Hs-CRP were 90.5%, 81.0%, 95.0% and 90.0%. The sensitivities of histological analysis and SWCC were 55.0% and 70.6%, while they had high specificity as 89.7% and 85.7%. The sensitivity of sonication fluid culture was 90.0%, which was higher than that of tissue culture (71.4%) and synovial fluid culture (65.0%) (χ(2) = 5.333, 6.400, all P<0.05).

CONCLUSIONSThe tests of CRP, ESR, IL-6 and Hs-CRP have good value in detecting PJI preoperatively. Histological analysis and SWCC have high specificity, which could help to exclude PJI. Sonication fluid culture has a higher sensitivity than tissue culture and synovial fluid culture.

Arthroplasty, Replacement, Hip ; Arthroplasty, Replacement, Knee ; Blood Sedimentation ; C-Reactive Protein ; metabolism ; Female ; Humans ; Interleukin-6 ; blood ; Knee Joint ; Male ; Prosthesis-Related Infections ; diagnosis ; ROC Curve ; Retrospective Studies ; Sensitivity and Specificity ; Synovial Fluid ; cytology

OBJECTIVETo observe beta-catenin expression of Wnt signaling pathway in rats with knee osteoarthritis, and influence of Bushen Huoxue decoction on beta-catenin and MMP-7 expression of synoviocytes in rats with knee osteoarthritis (OA).

METHODSRats model with knee osteoarthritis were established by Hulth method. Primary synoviocytes and OA synoviocytes were cultured with collagenase digestion method. The cultured synoviocytes were divided into normal group, OA model group and Bushen Huoxue decoction group. Western blotting method was used to detect beta-catenin, MMP-7 protein expression of synoviocytes after acting by Bushen Huoxue decoction for 48 h; ELISA method was used to detect MMP-7 expression of synovial supernatant.

RESULTSOA synoviocytes were cultured successfully. Western blotting showed that beta-catenin, MMP-7 expression in OA synoviocytes was significantly higher than normal group, Bushen Huoxue decoction could significantly reduce beta-catenin, MMP-7 expression; ELISA results showed that MMP-7 expression of OA synovial supernatant was significantly higher than normal synoviocytes supernatant, Bushen Huoxue decoction significantly regulated the level MMP-7 down.

CONCLUSION(1) High expression of beta-catenin in OA synoviocytes indicates that Wnt classical signal pathway is activated in rat with knee osteoarthritis; (2) High expression of MMP-7 expression in OA synoviocytes confirms the MMP-7 is downstream genes of Wnt/beta-catenin signal pathway; (3) Activation of Wnt signal pathway and increase of MMP-7 may cause degradation of articular cartilage, and promote the formation of osteoarthritis; (4) Bushen Huoxue decoction can reduce expression of MMP-7, and promote cartilage repair, which may be one of mechanisms of osteoarthritis.

Animals ; Blotting, Western ; Drugs, Chinese Herbal ; pharmacology ; Enzyme-Linked Immunosorbent Assay ; Male ; Matrix Metalloproteinase 7 ; analysis ; Osteoarthritis, Knee ; drug therapy ; metabolism ; Rats ; Rats, Wistar ; Synovial Fluid ; chemistry ; cytology ; beta Catenin ; analysis