Objective To investigate the expressions and significance of interleukin 8 (IL 8) in patients with ankylosing spondylitis (AS). Methods Using microarray and RT PCR to detect the expressions of IL 8 in peripheral blood mononuclear cells (PBMC), synovial fluid mononuclear cells (SFMC) and synovial cells in patients with active AS. Comparing the results with those in PBMC of both healthy volunteers and patients with rheumatoid arthritis (RA), and also with those in synovial cells obtained from patients undergoing knee operations for trauma. Results The expressions of IL 8 in PBMC and SFMC of patients with active AS were significantly higher than those in PBMC of the healthy controls. The expression of IL 8 in PBMC of RA patients was also higher than that of healthy volunteers. Higher expression of IL 8 in the synovial cells were detected in AS than that of patients with knee joint trauma. Conclusions The expression of IL 8 is high in patients with AS. The result may indicate that IL 8 is possibly one of the important mediators of inflammation in AS, and it may mediate the induction and development of inflammation in synovium or other tissues involved in AS

BACKGROUND:MicroRNAs (miRNAs) play an important role in a variety of diseases. Investigation of miRNA expression profile in degenerative lumbar scoliosis is beneficial for understanding its pathogenesis, providing a novel therapeutic target. Therefore, we tested the hypothesis that miRNAs promote intervertebral disc degeneration through the interleukin-10/STAT3 signaling pathway, a potential regulator of intervertebral disc degeneration.
OBJECTIVE:To compare the differentialy expressed miRNAs in the intervertebral disc tissues from patients with degenerative lumbar scoliosis and normal controls and to identify specific miRNAs in degenerative lumbar scoliosis folowed by functional validation.
METHODS: An initial screening of miRNA expression in nucleus pulposus tissues by miRNA Solexa Sequencing was performed in samples from 10 patients with degenerative lumbar scoliosis and 10 controls, respectively. Subsequently, differentialy expressed miRNAs were validated using qRT-PCR. The level of differentialy expressed miRNAs in degenerative nucleus pulposus tissues was investigated. Then, functional analysis of the miRNAs in regulating type II colagen expression was carried out. Western blot and luciferase reporter assay were used to further confirm the target gene.
RESULTS AND CONCLUSION: We identified 30 miRNAs that were differentialy expressed (16 upregulated and 14 downregulated) in patients with degenerative lumbar scoliosis compared with controls. Folowing qRT-PCR confirmation, Has-let-7f was significantly down-regulated in degenerative nucleus pulposus tissues as compared with controls. Moreover, its level was correlated with the severity of disc degeneration. Overexpression of Has-let-7f promoted type II colagen expression in nucleus pulposus cels. Knockout of interleukin-10 induced effects on nucleus pulposus cels similar to Has-let-7f. Bioinformatics target prediction identified interleukin-10 as a putative target of Has-let-7f. Furthermore, luciferase reporter assays demonstrated that Has-let-7f altered the expression of STAT3 and matrix metaloproteinase-2. These findings indicate that the downregulation of Has-let-7f induces type II colagen loss by directly targeting inleukin-10, thereby resulting in intervertebral disc degeneration and degenerative lumbar scoliosis. Has-let-7f is likely to be a novel therapeutic target for degenerative lumbar scoliosis.

BACKGROUND: Neuropeptides, a kind of endogenous active substance in nerve tissues, can modulate physiological functions of multiple body systems.OBJECTIVE: To observe the effects of vascular bundles or sensory nerve tracts implanted into tissue-engineered bone for rabbit large bone defects on the expression levels of calcitonin gene-related peptide (CGRP) and neuropeptide-Y.METHODS: Fifty-four New Zealand rabbits were enrolled to make model of large bone defects, and then, the animal models were randomly divided into three groups, including sensory nerve tract, vascular bundle, and control groups (n=18 per group), followed by implanted with sensory nerve tracts, vascular bundle, and tissue-engineered bone without sensory tracts or vascular bundle, respectively. The defected bone received gross and Masson staining at 4, 8 and 12 weeks after modeling, to compare the expression levels of CGRP and neuropeptide-Y in each group.RESULTS AND CONCLUSION: mRNA expression levels of CGRP and neuropeptide-Y in the sensory nerve tract and vascular bundle groups were significantly higher than those in the control group at different time points after modeling (P < 0.05). mRNA expression levels of CGRP and neuropeptide-Y in the tissue-engineered bone began to be increased and peaked at the 8th week, and then decreased (P < 0.05), which were the lowest at the 4th week (P < 0.05).Immunohistochemical staining results showed that CGRP was mainly found in the bridge, periosteum of newly born bones and around blood vessels; while neuropeptide-Y mainly localized in the medullary cavity and around blood vessels. These results indicate that the implantation of vascular bundle and sensory nerve tracts for bone defects can upregulate the expression levels of CGRP and neuropeptide-Y, and promote bone repair. However, sensory tract implantation may cause sensory impairment; thereafter, vascular bundle implantation is more suitable for ideal tissue-engineered construction to meet physical requirements.

Objective To study T lymphocyte subsets and expression of costimulatory molecule CD154 on T-cells in peripheral blood from patients with ankylosing spondylitis and their changes after treated with Enbrel. Methods Sixty-six patients with AS(39 active and 27 inactive, 35 axial and peripheral joint involvement and 31 axial involvement only), 30 patients with rheumatoid arthritis(RA), 30 healthy volunteers were analyzed. The expression of CD154 on CD3+ T cell as well as T-cells subsets were evaluated using flow cytometry respectively. The changes of the expression of costimulatory molecule CD154 in 39 active AS patients(Enbrel treatment or placebo treatment) were observed in a randomized, double-blind, placebothan that of healthy volunteers, and CD154 expression on CD3+ T cells in peripheral blood in AS patients was on CD3+ T cells in the peripheral blood of active AS or AS patients with peripheral joint involvement were significantly higher than those in inactive or axial involvement only AS patients(P＜0.05), and CD154 on CD3+ placebo, in AS patients group, there was significant reduction in CD154 expression on CD3+ T cells in Enbrel group at week 6(P＜0.05), there was no significant difference between Enbrel group and healthy volunteers at week 6(P＞0.05). Conclusion T lymphocyte subsets are significantly abnormal and CD154 is overexpressed on T-cells in peripheral blood of patients with AS. A six-week course of treatment with Enbrel in active AS can induce a down-regulation of the expression of CD154 on T cells.

BACKGROUND: Human leukocyte antigen (HLA)-B27 is closely connected to the occurrence of some rheumatic diseases such as ankylosing spondylitis and can be used as an important factor for evaluating the diagnosis of ankylosing spondylitis. Immunomagnetic separation and enzymelinked immunosorbent assay (IMS-ELISA) has been applied to the detection of HLA-B27.OBJECTIVE: To explore the accuracy, sensitivity and specificity of IMSELISA for detecting HLA-B27 and its value in the auxiliary diagnosis of ankylosing spondylitis.DESIGN: A clinical trial in comparison with the gold standard.SETTING: Departments of Rheumatology and Clinical Laboratory, Third Affiliated Hospital of Sun Yat-sen University.PARTICIPANTS: Eighty-six patients suffering from low back pain and/or arthritis who were treated for the first time in Department of Rheumatology from December 2002 to April 2003. Inclusion criteria: ① Presence of manifestations of low back pain and/or arthritis; ② Thorough documentation of clinical and other examinations; ③ Informed consent to HLA-B27 examination; ④ Treatment for the first time in the Third Affiliated Hospital of Sun Yet-sen University. Those with other serious diseases or with incomplete record of clinical and/or accessory examinations were excluded. The 86 patients included 56 male and 30 female patients aged from 12 to 65 years.METHODS: Blood sample was detected for HLA-B27 by both IMS-ELISA and microlymphocytotoxicity test, and the latter was selected as the gold standard. The coincidence rate of the results detected by the two methods as well as the sensitivity, specificity, positive and negative predictive values of IMS-ELISA were calculated.MAIN OUTCOME MEASURES: ① The coincidence rate of the results of the two methods. ② The sensitivity and specificity of IMS-ELISA for detecting HLA-B27.RESULTS: None of the patients was lost. For the 33 patients with ankylosing spondylitis, the positivity rate of IMS-ELISA (90.9％) was higher than that of microlymphocytotoxicity test (87.9％), but the difference was not statistically significant (P＞0.05). The total coincidence rate of the two methods was 93.0％ in all the 86 patients. The sensitivity, specificity, positive and negative predictive values of IMS-ELISA were 90.0％, 95.7％,94.7％ and 91.7％ respectively.CONCLUSION: Both IMS-ELISA and microlymphocytotoxicity test are capable of reliable examination of HLA-B27 with high sensitivity and specificity.

BACKGROUND: Spondyloarthropathies(SpA) are a group of interrelated disorders that involve the spine, peripheral joints, and tissues around or outside the joints. SpA are most common in young males, and both the incidence and the mutilation rate are very high. It is important to evaluate the disease activity of patients with SpA since the evaluation can help in therapeutic selection and prognostic prediction of SpA. But up to now there still has not been a widely clinically acceptable criterion to evaluate the disease activity of SpA.OBJECTIVE: To study the worth of color Doppler(CD) in assessing the disease activity of patients with SpA.DESIGN: Non-random and self-control clinical study based on the patients.SETTING: All patients recruited in this study were from department of rheumatology of the Third Affiliated Hospital of Sun Yat-sen University. And all the ultrasonic examinations were conducted in the department of ultrasound of the same hospital.PARTICIPANTS: Thirty(24 males and 6 females) patients with SpA who met the European SpA Study Group criteria for the classification of SpA issued in 1991 and consented to be examined by CD were included.METHODS: CD was used to determine the blood perfusion at 18 major entheses of lower limbs of each patient with a semi-quantitative method. Blood sample was also taken from the patients for the examination of erythrocyte sedimentation rate(ESR) and C reactive protein(CRP) . The disease activity of the patients was also evaluated with the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI).MAIN OUTCOME MEASURES: To observe the inter-correlations between entheseal blood perfusion, ESR, CRP and BASDAI.RESULTS: 80% of the 30 patients were detected to have positive perfusion in at least one enthesis of the 18 examined entheses, and all together 84 entheses were found to have positive perfusion among the 540 entheses being detected(positive rate 15.6% ) . The sum of entheseal perfusion detected in all the 18 sites was statistically correlated with the sum of patients' ESR,CRP, BASDAI respectively with the correlation coefficients of 0. 658, 0. 428and 0. 821 (all P ＜ 0.05) . ESR and CRP were also statistically correlated with BASDAI and their correlation coefficients were 0. 675 and 0. 368(Both P ＜ 0.05) . The sum of perfusion detected in the 18 entheses sites was correlated with BASDAI better than ESR or CRP was.Detecting the.entheseal perfusion by CD can help to determine the disease activity of patients with SpA, and then to guide selecting therapeutic strategy. It is also important for following up and prognosis.

BACKGROUND: Seronegative spondyloarthropathies(SpA) are a group of diseases involving the spine, peripheral joints and structures around the joints that mostly occur in adult males with high morbidity and disability rate. Presently, it is widely believed that enthesis is the initiated site of SpA so that enthesitis is an important pathologic base and one of the characteristic clinical manifestations of SpA, however, there is no specific clinical manifestation enthesitis.OBJECTIVE: To compare the difference on entheseal blood flow between health volunteers and patients with SpA and explore the value of color Doppler(CD) in assessing the pathological change of enthesis.DESIGN: Non-randomized case controlled study to patients SETTING: Department of Rheumatology, Department of Ultrasound of a university, Department of Rheumatology of General Hospital of Chinese PLA.PARTICIPANTS: Totally 30 patients with SpA admitted into Third Affilated Hospital of Sun Yat-sen University during August 2001 to April 2002 and willing to take CD examination were selected as case group. All of them met the classification criteria of SpA set up by the European SpA Study Group in 1991 with 24 males and 6 females. Thirty healthy workers and students working or studying in the same Hospital were enrolled as volunteers for the control group, none of them had any histories of arthropathy or chronic lower back pain, and there were 22 males and 8 females among them.INTERVENTIONS: Color Doppler was used to examine the blood flow of 18entheses in lower limbs of every healthy volunteers and SpA patients and semi-quantitative method was used to classify the blood flow.MAIN OUTCOME MEASURES: Blood flow of enthesesRESULTS: Among 30 healthy volunteers, 5 cases(16.7% ) had been detected blood flow from at least one enthesis. From the 540 entheses being examined, 9entheses were detected blood flow signals of grade Ⅰ, the positive rate was 1.7%. While among 30 SpA patients, 24(80.0%) had at least one enthesis being detected for positive blood flow. Eighty-four of totally 540 exanined entheses were found for positive blood flow signals with a positive rate of 15.6%, and the number of entheses with grade Ⅰ, Ⅱ and Ⅲ of blood flow were 19, 33 and 32 respectively among the 84 positive entheses. There was significantly different in the positive rate of blood flow and classification of entheses between SpA patients and healthy volunteers by x2 test (P＜ 0.05).CONCLUSION: Colour Doppler examination is an effective way to detect the blood flow of enthesis. There were blood flow abnormity in the entheses of lower limbs in patients with SpA.

BACKGROUND: Enthesitis is the most important pathologic change and one of the characteristic manifestations of spondyloarthropathy(SpA). However, its clinical manifestations lack specificity, and medical imaging techniques become an important auxiliary means in its diagnosis. Currently, ultrasound has been applied to examine the entheses of patients with SpA abroad, but so far there is no investigation about the ultrasonic manifestations of entheses in healthy volunteers.OBJECTIVE: To comprehend the acoustic imaging manifestations of entheses in healthy volunteers, and primarily investigate the feasibility of ultrasound in detecting entheses and the possible impact factors of acoustic imaging manifestations.DESIGN: A nonrandomized inter-controlled trail.SETTING and PARTICIPANTS: A total of 30 young healthy workers and medical students who worked or studied in the Third Affiliated Hospital of Sun Yat-sen University were included in our study, including 22 male and 8 female. None of them suffered from any arthropathies or chronic low back pain.INTERVENTION: High frequency and Color Doppler ultrasound were adopted to detect 540 entheses of 30 healthy volunteers, the entheseal thicknesses were compared respectively according to left and right sides, as well as the gender. X-ray was used to inspect if there were any abnormal findings at the attaching bones.MAIN OUTCOME MEASURES: The thickness, the echo inside entheses, the entourage, the attaching bone and the entheseal blood flow were detected.RESULTS: In normal entheses, there was a little lower but equal resonance, in which there was clear funicular fibrin resonance but without blood signal. Most entheses had a little higher membrane resonance, which was clearly divided from entourage. The surface of attaching bone was smooth and continuous. The mean thickness of the lower insertion of rectus femoris tendon, the upper insertion of patellar ligament, the lower insertion of patellar ligament, the lower insertion of tibial collateral ligament, the lower insertion of fibular collateral ligament, the insertion of Achilles tendon and plantar aponeurosis in male volunteers were(0.50 ±0.06), (0. 50 ±0. 07),(0.42±0.04), (0.20±0.03), (0.38±0.04), (0.52±0.07)and (0. 32 ±0.03) cm respectively, while the mean thickness of the corresponding insertions in female volunteers were (0.44 ± 0. 04), (0. 46 ± 0. 03 ),(0.39±0.03), (0.19±0.02), (0.32±0.02), (0.41±0.06)and (0. 28 ± 0.03) cm respectively. All entheses except for the lower insertion of tibial collateral ligament were thicker in male than in female( P ＜ 0. 05 ),but there were no significant differences in the thickness between left and right insertions ( P ＞ 0. 05 ). Calcification, erosion and hyperplasia of the attaching bone or increasing blood flow were found in 11 entheses of 8 volunteers(2.0% of all the 540 entheses detected) . There were 15 items of abnormal signs all together. Some of the abnormal sites had traumatic histories.CONCLUSION: The thicknesses are almost the same in the corresponding insertions between left and right sides, but the thicknesses of most insertions in male are thicker than those in female. Stature, body mass and exercise might impact on entheseal thickness. Trauma can cause abnormal acoustic imaging manifestations of entheses. Ultrasound is an available approach to detecting etheses.

Objective Previous study had shown that signal transduction was changed when Hela cells were transfected with HLA-B27gene.An increase of monocyte chemotactic protein-1(MCP-1)of Hela cells was observed.To determine the role of MCP in the pathogenesis of ankylosing spondylitis(AS),expression of MCP-1,2,3and4in peripheral blood mononuclear cells(PBMC),synovial fluid mononuclear cells(SFMC)and synovial tis-sues of AS patients were tested.Methods Gene expression profiles of PBMC from AS patients and healthy volun-teers were determined by cDNA microarray with1176target gene filter.The differentiated expressed gene MCP-1in PBMC,SFMC and synovial tissue of AS patients were confirmed by semi-quantitative RT-PCR.Results The gene expression profile of SFMC of AS patients was significantly different from those of PBMC from AS and PBMC from healthy volunteers.The MCP-1level was positively correlated with MCP-3(r=0.76,P=0.03).The expressions of MCP-1were higher in synovial tissues of AS than those of healthy volunteers(P=0.0035).MCP-1levels in monocytes of AS patients and control subjects were increased after LPS stimulation for4hours.Conclusions There is increased expression of MCP-1in SFMC and synovial tissue of AS patients.The results indicate that MCP-1may play a potential key role in the homing of cells migrating from blood to joint and in the pathogenesis of joint inflammation in AS patients.

BACKGROUND:MicroRNAs are widely involved in the regulation of protein expression, and play a critical role in many physiological and pathological processes in the body. But microRNA expression profile in degenerative lumbar scoliosis is rarely reported and understood. OBJECTIVE:To compare the microRNA expression profile in the normal intervertebral disc and degenerative lumbar scoliosis and to identify degenerative lumbar scoliosis-specific microRNAs, folowed by functional validation. METHODS: Total RNA samples were extracted from the nucleus pulposus tissues of 57 patients with degenerative lumbar scoliosis as experimental groups and the normal nucleus pulposus tissues of 42 patients with lumbar fractures as control group. An initial screening of differentialy expressed microRNAs in the nucleus pulposus tissues by microRNA microarray was performed in 10 samples from each group. Subsequently, differentialy expressed microRNAs were validated using real-time quantitative RCR. The level of differentialy expressed microRNAs in the degenerative nucleus pulposus tissues was investigated. Then, the functional analysis of microRNAs in regulating colagen II expression was carried out. Western blot and luciferase reporter assay were also used to detect target genes. RESULTS AND CONCLUSION:We identified 22 microRNAs that were differentialy expressed (17 upregulated and 5 downregulated) in degenerative lumbar scoliosis patients compared with the controls. Folowing real-time quantitative RCR confirmation, miR-491-5p was significantly down-regulated in degenerative nucleus pulposus tissues in comparison with the controls. Moreover, its level was closely correlated with the pathological grading of disc degeneration. Overexpression of miR-491-5p promoted type II colagen expression in nucleus pulposus cels. Bioinformatics target prediction identified matrix metaloproteinase-9 as a putative target of miR-491-5p. Furthermore, luciferase reporter assays demonstrated that miR-491-5p directly targeted matrix metaloproteinase-9 and affected its protein expression in nucleus pulposus cels. These results show that the downregulation of miR-491-5p induces type II colagen loss by directly targeting matrix metaloproteinase-9, thereby resulting in degeneration of the intervertebral disc and degenerative lumbar scoliosis. This study also underscores the potential of miR-491-5p as a novel therapeutic target in degenerative lumbar scoliosis.