Phospholipase A1 Member A Activates Fibroblast-like Synoviocytes through the Autotaxin-Lysophosphatidic Acid Receptor Axis.

Lysophosphatidylserine (lysoPS) is known to regulate immune cell functions. Phospholipase A1 member A (PLA1A) can generate this bioactive lipid through hydrolysis of sn-1 fatty acids on phosphatidylserine (PS). PLA1A has been associated with cancer metastasis, asthma, as well as acute coronary syndrome. However, the functions of PLA1A in the development of systemic autoimmune rheumatic diseases remain elusive. To investigate the possible implication of PLA1A during rheumatic diseases, we monitored PLA1A in synovial fluids from patients with rheumatoid arthritis and plasma of early-diagnosed arthritis (EA) patients and clinically stable systemic lupus erythematosus (SLE) patients. We used human primary fibroblast-like synoviocytes (FLSs) to evaluate the PLA1A-induced biological responses. Our results highlighted that the plasma concentrations of PLA1A in EA and SLE patients were elevated compared to healthy donors. High concentrations of PLA1A were also detected in synovial fluids from rheumatoid arthritis patients compared to those from osteoarthritis (OA) and gout patients. The origin of PLA1A in FLSs and the arthritic joints remained unknown, as healthy human primary FLSs does not express the PLA1A transcript. Besides, the addition of recombinant PLA1A stimulated cultured human primary FLSs to secrete IL-8. Preincubation with heparin, autotaxin (ATX) inhibitor HA130 or lysophosphatidic acid (LPA) receptor antagonist Ki16425 reduced PLA1A-induced-secretion of IL-8. Our data suggested that FLS-associated PLA1A cleaves membrane-exposed PS into lysoPS, which is subsequently converted to LPA by ATX. Since primary FLSs do not express any lysoPS receptors, the data suggested PLA1A-mediated pro-inflammatory responses through the ATX-LPA receptor signaling axis.

From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8.

With ELISAs one detects the ensemble of immunoreactive molecules in biological samples. For biomolecules undergoing proteolysis for activation, potentiation or inhibition, other techniques are necessary to study biology. Here we develop methodology that combines immunosorbent sample preparation and nano-scale liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) for proteoform analysis (ISTAMPA) and apply this to the aglycosyl chemokine CXCL8. CXCL8, the most powerful human chemokine with neutrophil chemotactic and -activating properties, occurs in different NH2-terminal proteoforms due to its susceptibility to site-specific proteolytic modification. Specific proteoforms display up to 30-fold enhanced activity. The immunosorbent ion trap top-down mass spectrometry-based approach for proteoform analysis allows for simultaneous detection and quantification of full-length CXCL8(1-77), elongated CXCL8(-2-77) and all naturally occurring truncated CXCL8 forms in biological samples. For the first time we demonstrate site-specific proteolytic activation of CXCL8 in synovial fluids from patients with chronic joint inflammation and address the importance of sample collection and processing.

Decreased Synovial Fluid Biomarkers Levels Are Associated with Rehabilitation of Function and Pain in Rotator Cuff Tear Patients Following Electroacupuncture Therapy.

BACKGROUND The aim of this study was to assess inflammatory cytokines levels in synovial fluid (SF) before and after electroacupuncture (EA) treatment and to explore whether these biomarkers are associated with function of rotator cuff tear (RCT) patients. MATERIAL AND METHODS We recruited 54 patients with RCT and separated them into an EA group and a control group. The SF biomarker levels were detected at baseline and at 6-week and 6-month follow-up. The symptomatic severity was evaluated by visual analog scale (VAS), Constant-Murley score, and American Shoulder and Elbow Surgeons score (ASES). We also investigated the correlation between symptomatic severity and biomarker levels in SF of the shoulder joint. RESULTS The reductions in VAS and improved functional score (ASES and Constant-Murley score) were significantly different between the 2 groups, and SF biomarker concentrations were significantly lower in the EA group. IL-1ß levels were significantly negatively correlated with Constant-Murley score (r=-0.73, P=0.04) and ASES score (r=-0.59, P<0.001) and positively correlated with VAS scores (r=0.81, P=0.004). IL-6 levels were significantly negatively correlated with Constant-Murley score (r=-0.67, P=0.03) and positively correlated with VAS score (r=0.7, P=0.01). MMP-1 levels were significantly negatively correlated with ASES score (r=-0.57, P<0.001). CONCLUSIONS The biomarkers in SF were directly associated with shoulder pain and shoulder function in rotator cuff tear. EA, as a safe and effective conservative therapy, obviously decreased the level of inflammatory cytokines in RCT patients, accompanied by a reduction in shoulder pain and improved function.

Curcumin reduces inflammation in knee osteoarthritis rats through blocking TLR4 /MyD88/NF-κB signal pathway.

Preclinical Research & Development Curcumin has been shown to possess a series of beneficial effects, such as antiinflammatory, antioxidant, analgesic, and promoting healing. However, the effect and relative mechanism of curcumin on knee osteoarthritis (OA) have not been elucidated. The aim of this study is to explore the protective effect of curcumin on monosodium iodoacetate (MIA)-induced OA. Forty-eight rats were randomized into four experimental groups: control group, OA group, OA + PBS group, and OA + curcumin group, respectively. A single intraarticular injection of MIA was applied to establish the rat model of knee OA. Hematoxylin-eosin staining was used to evaluate histological changes of knee joint. The paw withdrawal threshold was collected and the expression of synovial fluid cytokine levels was measured by ELISA. The protein expression of TRL-4, MyD88, p-IκBα, NF-κB, TNF-α, IL-1ß, and IL6 was measured by western blot. Treating with curcumin can significantly reduce joint diameter and Mankin's score, and increase the paw withdrawal threshold. The expression of synovial fluid inflammatory biomarkers, IL-6, IL-1ß, and TNF-α in the OA + curcumin group were lower than that in OA and OA + PBS group. The protein expression of the TLR4 receptor was increased in the OA, OA + PBS, and OA + curcumin group compared to the control group. However, curcumin treatment can significantly decrease the expression of MyD88, p-IκBα, NF-κB, TNF-α, IL-1ß, and IL6 in OA + curcumin group. These findings may indicate that curcumin could block TLR4/NF-κB signal pathway, and reduce inflammation level to prevent knee wound in OA rats. Curcumin may be a feasible kind of medicament in the treatment of knee OA.

The regulatory role of Dipeptidyl peptidase I on the activation of immune granulocytes.

Dipeptidyl peptidase I (DPPI), a lysosomal cysteine protease, required for activation of serine proteases of granulocytes including mast cells (MCs), neutrophils (NPs) and others, which were found in synovial tissue of patients with rheumatoid arthritis (RA). But, the role of DPPI associated with those cells in RA development is unclear. In this study, the collagen-induced-arthritis (CIA) rat-model was employed to investigate the expression and activity levels of DPPI and its association with RA progress. Primary granulocytes were freshly extracted from bone-marrows of normal or CIA rats, human mast cell line LAD-2 and primary neutrophils, human-recombinant-DPPI, DPPI-inhibitor Gly-Phe-CHN2 , LTB4, anti-IgE antibody, calcium ionophore were used to study the regulatory role of DPPI in cell activations. The increased DPPI activities in synovial fluids, serum, and bone-marrow homogenates of CIA rats associated with RA severities progress were observed after injections. MMP2/9 expressions in SFs and bone-marrow were in different patterns. Regular-Blood-Tests have shown the high leveled DPPI activities associated with granulocytes differentiations in-vivo in blood of CIA rats. In-vitro cell models, DPPI up-regulated the proliferation of primary bone-marrow granulocytes of normal rats, but inhibited that of CIA rats. DPPI up-regulated and Gly-Phe-CHN2 down-regulated MCs intracellular DPPI and chymase activities. Gly-Phe-CHN2 also inhibited the LTB4 -activated-NPs and NP-elastase activities. Following stimulation of calcium ionophore, the net-releases of DPPI and ß-hexosaminidase from MCs were increased over a time-course, while Gly-Phe-CHN2 down-regulated MCs and NPs activation. Our findings demonstrate the role of DPPI in regulating MCs and NPs activation, and modulating proteolysis in the process of RA.

[Anti-inflammatory and synovial-opioid system effects of electroacupuncture intervention on chronic pain in arthritic rats].

OBJECTIVE: To 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 (ß-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). RESULTS: Compared 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 ß-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. CONCLUSION: The 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 ß-END and MOR, KOR, DOR.

Enhanced neutrophil extracellular trap generation in rheumatoid arthritis: analysis of underlying signal transduction pathways and potential diagnostic utility.

INTRODUCTION: Neutrophil extracellular traps (NETs) have recently been implicated in a number of autoimmune conditions, including rheumatoid arthritis (RA). We examined the underlying signaling pathways triggering enhanced NETosis in RA and ascertained whether the products of NETosis had diagnostic implications or usefulness. METHODS: Neutrophils were isolated from RA patients with active disease and from controls. Spontaneous NET formation from RA and control neutrophils was assessed in vitro with microscopy and enzyme-linked immunosorbent assay (ELISA) for NETosis-derived products. The analysis of the signal-transduction cascade included reactive oxygen species (ROS) production, myeloperoxidase (MPO), neutrophil elastase (NE), peptidyl arginine deiminase 4 (PAD4), and citrullinated histone 3 (citH3). NET formation was studied in response to serum and synovial fluid and immunoglobulin G (IgG) depleted and reconstituted serum. Serum was analyzed for NETosis-derived products, for which receiver operator characteristic (ROC) curves were calculated. RESULTS: Neutrophils from RA cases exhibited increased spontaneous NET formation in vitro, associated with elevated ROS production, enhanced NE and MPO expression, nuclear translocation of PAD4, PAD4-mediated citrullination of H3, and altered nuclear morphology. NET formation in both anti-citrullinated peptide antibody (ACPA)-positive and -negative RA was abolished by IgG depletion, but restored only with ACPA-positive IgG. NETosis-derived products in RA serum demonstrated diagnostic potential, the ROC area under the curve for cell-free nucleosomes being >97%, with a sensitivity of 91% and a specificity of 92%. No significant difference was observed between ACPA-positive and -negative cases. CONCLUSIONS: Signaling elements associated with the extrusion of NETs are significantly enhanced to promote NETosis in RA compared with healthy controls. NETosis depended on the presence of ACPA in ACPA-positive RA serum. The quantitation of NETosis-derived products, such as cell-free nucleosomes in serum, may be a useful complementary tool to discriminate between healthy controls and RA cases.

Contribution of mast cell-derived interleukin-1ß to uric acid crystal-induced acute arthritis in mice.

OBJECTIVE: Gouty arthritis is caused by the precipitation of monosodium urate monohydrate (MSU) crystals in the joints. While it has been reported that mast cells (MCs) infiltrate gouty tophi, little is known about the actual roles of MCs during acute attacks of gout. This study was undertaken to assess the role of MCs in a mouse model of MSU crystal-induced acute arthritis. METHODS: We assessed the effects of intraarticular (IA) injection of MSU crystals in various strains of mice with constitutive or inducible MC deficiency or in mice lacking interleukin-1ß (IL-1ß) or other elements of innate immunity. We also assessed the response to IA injection of MSU crystals in genetically MC-deficient mice after IA engraftment of wild-type or IL-1ß(-/-) bone marrow-derived cultured MCs. RESULTS: MCs were found to augment acute tissue swelling following IA injection of MSU crystals in mice. IL-1ß production by MCs contributed importantly to MSU crystal-induced tissue swelling, particularly during its early stages. Selective depletion of synovial MCs was able to diminish MSU crystal-induced acute inflammation in the joints. CONCLUSION: Our findings identify a previously unrecognized role of MCs and MC-derived IL-1ß in the early stages of MSU crystal-induced acute arthritis in mice.

Bacteria and Toll-like receptor and cytokine mRNA expression profiles associated with canine arthritis.

The major forms of inflammatory canine arthritis are immune-mediated arthritis (IMA) and septic arthritis (SA), although some cases of cruciate disease (CD) are associated with significant levels of synovitis. In this study, the bacteria associated with canine arthritis were identified and mRNA expression levels of Toll-like receptors (TLRs) and pro-inflammatory cytokines determined. Of the 40 synovial fluid samples analysed, bacteria were isolated from 12 samples by culture (2 CD, 10 SA) and detected in 4 samples (3 CD, 1 SA) using culture-independent methods. Statistically significant increases in TLR2, tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-12 mRNA expression were seen in all disease groups compared to normal controls. All disease groups had decreased mRNA expression of other TLRs compared to normal controls, but this did not reach statistical significance. Synovial fluid cell counts revealed that the highest number and proportion of mononuclear cells and neutrophils were found in the IMA and SA samples, respectively. Age had an effect on the TLR and cytokine mRNA expression profiles: TNF-α (p=0.043) and IL-12 (p=0.025) mRNA expression was increased and TLR4 mRNA expression was reduced (p=0.033) in dogs up to 4 years of age compared to older animals. In the 10 SA samples from which bacteria were isolated, statistically significant increases in TLR2, TLR7, TNF-α and IL-6 mRNA expression were observed. It is concluded that canine arthritis is associated with increased mRNA levels of pro-inflammatory cytokines, which could in some cases be mediated by bacteria through activation of TLR2.

Anti-inflammatory response of dietary vitamin E and its effects on pain and joint structures during early stages of surgically induced osteoarthritis in dogs.

There is evidence that vitamin E (VE) has anti-inflammatory and analgesic properties in human osteoarthritis (OA). This double-blinded and randomized pilot study used a broad spectrum of clinical and laboratory parameters to investigate whether such beneficial effects could be detected in a canine experimental OA model. Dogs were divided into 2 groups: control (n = 8), which received a placebo, and test group (n = 7), which received 400 IU/animal per day of VE for 55 d, starting the day after transection of the cranial cruciate ligament. Lameness and pain were assessed using a visual analogue scale (VAS), numerical rating scale (NRS), and electrodermal activity (EDA) at day 0, day 28, and day 55. Cartilage and synovial inflammation lesions were assessed. One-side comparison was conducted at an alpha-threshold of 10%. At day 56, dogs were euthanized and concentrations of prostaglandin E2 (PGE2), nitrogen oxides (NOx), and interleukin-1 beta (IL-1ß) were measured in synovial fluid. Concentrations of NOx and PGE2 in synovial fluid were lower in the test group (P < 0.0001 and P = 0.03, respectively). Values of VAS, NRS, and EDA showed a consistent trend to be lower in the test group than in the control, while statistical significance was reached for VAS at day 55 and for EDA at day 28 (adjusted P = 0.07 in both cases). Histological analyses of cartilage showed a significant reduction in the scores of lesions in the test group. This is the first time that a study in dogs with OA using a supplement with a high dose of vitamin E showed a reduction in inflammation joint markers and histological expression, as well as a trend to improving signs of pain.

La vitamine E (VE) est connue par ses propriétés anti-inflammatoires et analgésiques dans le traitement de l'ostéoarthrose (OA) chez l'humain. Dans notre étude pilote nous avons utilisé un ensemble de paramètres cliniques et de laboratoire afin de déterminer si ces effets bénéfiques de la VE pourront être détectés chez le chien arthrosique, dans un modèle expérimental d'OA. Les chiens utilisés ont été divisés en 2 groupes: témoin (n = 8), qui a reçu un placebo et un groupe supplémenté (n = 7), qui a reçu 400 UI de VE/animal/jour pendant 55 jours, la supplémentation orale a commencé un jour après la section du ligament croisé crânial. Avant la chirurgie (J0), J28 et J55 après chirurgie, la boiterie et la douleur ont été évaluées à l'aide d'une échelle visuelle analogique (EVA), d'une échelle d'évaluation numérique (NRS), et par la mesure de l'activité électrodermique (EDA). Les lésions au niveau du cartilage et l'inflammation synoviale ont été évalués. Une seule comparaison statistique a été réalisée avec un seuil alpha à 10 %. Au jour 56, les chiens ont été euthanasiés et les concentrations de prostaglandine E2 (PGE2), d'oxyde d'azote (NOx) et d'interleukine-1 bêta (IL-1ß) ont été mesurées dans le liquide synovial. Les concentrations synoviales de NOx et de PGE2 étaient plus faibles dans le groupe traité (P < 0,0001 et P = 0,03, respectivement). Les valeurs de l'EVA, de NRS et de l'EDA ont montré une tendance constante à être plus faible dans le groupe traité par comparaison au groupe témoin, avec un effet significatif de la VE qui a été observé pour VAS au jour 55 et EDA au jour 28 (P ajustée = 0,07 dans les deux cas). Les analyses histologiques du cartilage ont montré une réduction significative des scores lésionnels chez le groupe traité. Cette étude est la première à démontrer qu'une supplémentation orale avec une dose élevée de VE chez des chiens arthrosiques permet de réduire la libération des marqueurs inflammatoires et les lésions histologiques au niveau du cartilage, ainsi qu'une tendance à améliorer les signes de douleur.(Traduit par Docteur Serge Messier).

[Effects of moxibustion intervention on inflammatory reactions and expression of suppressor of cytokine signaling proteins of synovium cells in rheumatoid arthritis rabbits].

OBJECTIVE: To observe the effect of moxibustion intervention on inflammatory reactions and expression of suppressor of cyfokine signaling 1 (SOCS 1) and SOCS 2 [Which are involved in inhibition of the Janus Kinase-signal transducer and activator of transcrip-tion (JAK/STAT signaling pathway and in sffenuation of cytokine signaling)] in synovium cells of the hind-knee joint in rheumatoid arthritis (RA) rabbits, so as to study its mechanism underlying improvement of RA. METHODS: Forty-two Japanese big-ear white rabbits were randomized into control, model and moxibustion groups respectively, with 14 cases in each group. RA model was established by injection of Freund's Complete Adjuvant (0. 5 mL/kg) into the rabbits' bilateral hind-knee joint cavities. Moxibustion was applied to bilateral "Shenshu" (BL 23) areas, 5 cones every time, once daily for 3 weeks except the Sundays. The perimeters of rabbits' hind legs were measured before and after modeling and after the therapy. The synovial tissue of joint was sampled for analyzing the expression levels of SOCS 1 and SOCS 3 by immunohistochemistry. RESULTS: Before the therapy, the perimeters of bilateral knee joints of the control, model and moxibustion groups were of no statistical significance (P>0. 05). In comparison with the control group, the perimeters of bilateral knee joints were significantly increased on day 1, 7, 14 and 21 in the model group (P<0. 01). Compared with the model group, the perimeters of bilateral knee joints in the moxibustion group were significantly decreased (P<0. 05), suggesting an improvement of the inflammatory reaction after moxibustion intervention. Correspondingly, synovial SOCS 1 and SOCS 3 expression levels were remarkabely higher in the model group than in the control group (P<0. 01), and obviously decreased in the moxibustion group compared with the model group (P<0. 01). CONCLUSIONS: Moxibustion intervention has an anti-inflammatory and detumescent effects in RA rabbits, which may be closely associated with its effects in down-regulating expression of SOCS 1 and SOCS 3 proteins by suppressing negative feedback regulatory JAK/STAT pathway in synovial cells. [KEY WORDS] Moxibustion; Rheumatoid arthritis; Inflammatory reactions; Synovial cells; Suppressor of cytokine signaling proteins; Negative-feedback regulatory factors

Prostaglandin D(2) in inflammatory arthritis and its relation with synovial fluid dendritic cells.

Prostaglandin (PG)D2 has been shown to be an active agent in the resolution of experimentally induced inflammation. This study was undertaken to determine the presence of PGD2 in chronic joint effusions and to explore the potential contributions of dendritic cells (DC) and monocytes to the intra-articular synthesis of PGD2. Synovial fluid (SF) was obtained from patients with inflammatory arthritis and knee effusions. PGD2 and PGE2 were detected in SF by ultrahigh-performance tandem mass spectrometry. Cellular fractions in SF were separated by density-gradient centrifugation and flow cytometry. The expression of hematopoietic prostaglandin D-synthase (hPGDS) and PGE-synthase (PGES) mRNA was determined by RT-PCR. Both PGD2 and PGE2 were detected in blood and SF, with PGD2 being more abundant than PGE2 in SF. mRNA for hPGDS was more abundant in SF mDCs than SF monocytes (P < 0.01) or PB monocytes (P < 0.001). SF mDC expressed significantly more hPGDS than PGES. Expressions of PGD2 and hPGDS were inversely associated with serum C-reactive protein (P < 0.01) and erythrocyte sedimentation rate (P < 0.01). The findings suggest that synovial DCs may be an important source of hPGDS and that systemic disease activity may be influenced by actions of PGD2 in RA and other arthropathies.

TNF-α inhibitory effect of Euphorbia hirta in rats.

CONTEXT: Euphorbia hirta L. (Euphorbiaceae) (E. hirta) is a tree locally used as a traditional medicine in Africa and Australia to treat numerous diseases such as hypertension, respiratory ailments, tumors, and wounds, and it has reported antiallergic, antipyretic, anti-inflammatory activities, etc. OBJECTIVE: This study evaluated the ability of fresh leaves of E. hirta ethanol extract to inhibit the intracellular tumor necrosis factor α (TNF-α) level in the synovial fluid and neutrophils in lipopolysaccharide (LPS)-induced inflamed rat knees. MATERIALS AND METHODS: Female Wister albino rats 140-160 g were used. E. hirta ethanol extract was given orally at 25, 50, 100, and 200 mg/kg, 2 h before an intra-articular (i.a.) injection of LPS. Two and three hours later, synovial fluid and neutrophils levels of intracellular TNF-α production were measured. RESULTS: In the time course of the experiment, E. hirta maximum inhibition at 100 and 200 mg/kg (p.o.) dose showed 16.5 ± 1.34 and 14.4 ± 1.30% of synovial fluid, 4.26 ± 0.36 and 3.78 ± 0.29% of neutrophils levels of intracellular TNF-α productions at 2 h after LPS injection. LPS control displayed 22.97 ± 1.61 and 6.78 ± 0.34% of synovial fluid and neutrophils levels of intracellular TNF-α at 2 h after LPS injection. Intracellular TNF-α was also estimated at 3 h after LPS injection. DISCUSSION AND CONCLUSION: The LPS-injected rat knee model gives a comparative study of acute anti-inflammatory responses. E. hirta inhibition of proinflammatory intracellular cytokine TNF-α production with LPS-induced inflamed rat knee is of great importance in defining the anti-arthritic potential of E. hirta.

The role of human xanthine oxidoreductase (HXOR), anti-HXOR antibodies, and microorganisms in synovial fluid of patients with joint inflammation.

This work is to investigate the levels of human xanthine oxidoreductase (HXOR), its antibodies, and microorganisms in synovial fluid of patients with untreated rheumatoid joint diseases. Synovial fluids were collected from sixty-four patients with rheumatoid joint diseases. Sixty-four age-matched individuals were included as control. Xanthine oxidoreductase (XOR) proteins level and anti-XOR antibodies were determined in the blood and synovial fluid, using human XOR as antigen, by enzyme-linked immunosorbent (ELISA) assay. Synovial fluids were cultured for bacteria and fungi. The titers of XOR protein in the synovial fluid of patients with rheumatoid arthritis were 90.43 ± 23.37 µg/ml (mean ± SD, n = 29) and up to 62.42 ± 8.74 µg/ml (mean ± SD, n = 35) in other joint inflammation. Anti-HXOR antibodies titers in patients were 167.72 ± 23.64 µg/ml, n = 64, which was significantly higher in rheumatoid arthritis patients. The results indicated that anti-HXOR antibodies in synovial fluids have a protective role as high concentrations against XOR were detected in inflammatory arthritis. These antibodies play a role in eliminating XOR from synovial fluids. However, immune complex formation could activate complement and participate in propagating the inflammatory cycle. Synovial aspirate ordinary microbial cultures were negative for any bacteria or fungi, but that does not exclude organisms of special culture requirements.

[Randomized controlled clinical trials of red-hot filiform needle puncturing for knee ostarthritis and inflammatory cytokines in knee articular fluid in senile knee ostarthritis patients].

OBJECTIVE: To observe the effect of red-hot filiform needle puncturing on the pain severity of knee ostarthritis (OA) and the inflammatory cytokine levels in the knee articular cavity (KAC) fluid in senile knee OA patients so as to find a better therapy for knee OA. METHODS: A total of 200 senile knee OA outpatients (who signed an informed consent) were randomized into treatment group and control group (n = 100/group) according to a random number table and their visiting sequence. Futu (LI 18), Neixiyan (EX-LE 4), Yanglingquan (GB 34), Yinlingquan (SP 9), Zusanli (ST 36), etc. on the affected side of the body were punctured with red-hot filiform needles (cauterized on an alcohol burner) or routine filiform needles, with the needles retained for 30 min. The treatment was conducted once every other day, 15 sessions altogether. Before and after the treatment, the patients' KAC fluid was sampled for assaying the contents of IL-1, IL-6 and tumor necrosis factor (TNF)-alpha with enzyme-linked immunosorbent assay (ELISA). The pain severity was tested by using visual analogue scale (VAS) and the comprehensive therapeutic effect was evaluated by using clinical symptoms and signs, functional activity and pain degrees. RESULTS: In comparison with pre-treatment, IL-1, IL-6 and TNF-alpha contents in KAC fluid were decreased significantly in both treatment and control groups (P < 0.01). The IL-1, IL-6 and TNF-alpha levels were considerably lower in the treatment group than in the control group following the treatment (P < 0.01), suggesting a marked relief of inflammation in the affected knee joint. Compared to pre-treatment, the severity scores of illness state of both treatment and control groups were decreased remarkably (P < 0.01), and the VAS score of the treatment group was significantly lower than that of the control group after the treatment (P < 0.05). Of the two 100 knee OA outpatients in the treatment and control groups, 29 (29%) and 21 (21%) were controlled in their symptoms and signs; 46 (46%) and 34 (34%) experienced apparent improvement; 18 (18%) and 29 (29%) were effective; 7 (7%) and 16 (16%) failed in the treatment. The comprehensive therapeutic effect of the treatment group was significantly superior to that of the control group (P < 0.01). CONCLUSION: Red-hot filiform needle puncturing is superior to routine filiform needle puncturing in relieving senile knee OA and in reducing inflammatory cytokine levels in knee OA patients.

Synovial fluid-derived T helper 17 cells correlate with inflammatory activity in arthritis, irrespectively of diagnosis.

We analyzed peripheral blood (PB) and synovial fluid (SF) mononuclear cells from 16 rheumatoid arthritis (RA), 9 spondyloarthritis (SpA), 3 microcrystal arthritis patients, to define the presence of Th17 and Th1 and their relationship with inflammatory activity, and TCR-zeta chain and ZAP-70 levels. Th17 were significantly higher in SF than in PB and more abundant in microcrystal arthritis patients compared to the other groups. Irrespectively of the diagnosis, SF Th17 percentages correlated with joint (SF total leukocyte count, neutrophil percentage) and systemic (C reactive protein [CRP], fibrinogen, erythrocyte sedimentation rate) inflammation markers. SF Th1 percentages directly correlated with inflammation and disease activity (CRP, swollen joint count [SJC]) indices in SpA, but not in RA patients. These observations support the role of Th17 in the pathogenesis of inflammatory arthritides. The TCR-zeta(dim) lymphocytes in SF were found to produce the highest amounts of cytokines including IL-17, whereas no ZAP-70 impairment was associated to Th17.

[Effects of electroacupuncture and simple acupuncture on changes of IL-1, IL-4, IL-6 and IL-10 in peripheral blood and joint fluid in patients with rheumatoid arthritis].

OBJECTIVE: To explore the mechanism of electroacupuncture on rheumatoid arthritis (RA). METHODS: In 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. RESULTS: Both 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). CONCLUSION: Electroacupuncture 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.

Effects of preventive administration of juanbi capsules on TNF-alpha, IL-1 and IL-6 contents of joint fluid in the rabbit with knee osteoarthritis.

OBJECTIVE: To probe into the mechanism of the Chinese herbs with functions of reinforcing kidney and supplementing qi for preventing knee osteoarthritis of the rabbit. METHODS: Totally 72 healthy Japan long-ear white rabbits, aged 4 months, were randomly divided into 6 groups, blank group (A), model group (B), high dose Chinese herb group (C), middle dose Chinese herb group (D), small dose Chinese herb group (E), aminoglucose hydrochloride capsule control group (F), 12 rabbits in each group. All the rabbits in the groups, except the group A, were fixed with plaster cast for six weeks to establish rabbit knee osteoarthritis. At the same time of modeling, the different doses of Juanbi Capsules and aminoglucose hydrochloride capsule were administrated intragastrically in the group C, D, E, F, respectively, for 4 weeks, for preventive treatment. In the group B, the rabbit was administrated intragastrically with equal volume of normal saline to the medication groups, twice each day, in the morning and the evening, and in the group A, nothing was administrated. After modeling for 6 weeks, the joint fluid was taken and TNF-alpha, IL-1 and IL-6 contents were detected with ELISA method, and the articular cartilage was taken for macroscopic and microscopic examinations. RESULTS: In all the preventive treatment groups, the articular cartilage color changed to varying degrees with formation of osteophyte and bone cyst, superficial erosion on the chondral articular surface, and the cartilage defect reached to the mid layer in a part of specimens with cartilage exfoliation, but which in the extent were significantly lower than those in the model group. There were significant differences between the group A and B in TNF-alpha, IL-1 and IL-6 contents in the joint fluid (P < 0.05), indicating that the modeling is successful; and there were significant differences as group B compared with the group C,D, E, F, showing that TNF-alpha , IL-1 and IL-6 contents are decreased in all the medication groups; and significant differences between group C, D, E suggests that the increase of Chinese herb doses strengthened the effect of reducing TNF-alpha, IL-1 and IL-6 contents in joint fluid. CONCLUSION: The Juanbi Capsule prevents osteoarthritis possibly through decreasing serum TNF-alpha, IL-1 and IL-6 contents.

Apolipoprotein A-I and cholesterol in synovial fluid of patients with rheumatoid arthritis, psoriatic arthritis and osteoarthritis.

OBJECTIVE: To investigate lipid and apolipoprotein (Apo) levels in synovial fluid (SF) and serum of patients with rheumatoid arthritis (RA), psoriatic arthritis (PsA) and osteoarthritis (OA). METHODS: SF of 44 patients (14 RA, 14 PsA, 16 OA) was tested for Apo A-I, HDL-C, total cholesterol (TC), IL-1Beta, TNF-alpha, white blood cell count (WBC) and polymorphonucleate (PMN) percentage. Blood samples, collected simultaneously to the SF, were examined for Apo A-I, HDL-C, TC, TNF-alpha, serum amyloid A (SAA) and C-reactive protein (CRP). Thirty-three healthy donors served as a control group. RESULTS: Serum levels of Apo A-I, HDL-C and TC were higher in OA as compared with RA, PsA and the control group. The patients with inflammatory arthritis had lower serum levels of Apo A-I and HDL-C than did the controls. Apo A-I concentrations were higher in SF of RA patients, while PsA showed the highest concentration of TC, though not reaching statistical significance. A negative correlation was found between serum Apo A-I and synovial WBC (r=-0.48 p=0.002) and IL-1Beta (r=-0.42 p=0.016). There was a strong positive correlation between the Apo A-I SF/serum ratio and synovial WBC (r=0.73 p<0.001), IL-1Beta (r=0.68 p<0.001) and a weak, yet significant, correlation with serum CRP (r=0.49 p=0.002) and SAA (r=0.41 p=0.008). CONCLUSION: Our study confirms that in RA Apo A-I and TC levels are decreased in plasma and increased in SF, thus suggesting infiltration of HDL particles in the inflamed joint with inhibition of the local production of proinflammatory cytokines. On the other hand, it can be hypothesized that the sequestration of Apo A-I in the inflamed tissue may, in part, account for the reduction of circulating HDL and the excess cardiovascular risk in RA and PsA patients.

[Study of geniposide-acid on anti-inflammatory action for adjuvant-induced arthritis rats and mechanism of synoviocyte apoptosis in vitro].

OBJECTIVE: To 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. METHOD: Forty-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). RESULT: 200 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). CONCLUSION: GA 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.