脊柱关节病患者外周血和关节液单个核细胞基因谱的研究

目的：了解脊柱关节病（SpA）患者外周血和关节液单个核细胞（SFMC）基因的变化及其特点，探讨与SpA关节炎相关的基因及可能的意义。方法：采用含1176个基因的cDNA微阵列，检测6名健康志愿者外周血单个核细胞（PBMC），5例SpA和5例类风湿关节炎（RA）病人PBMC和关节液SFMC的基因表达，挑选SpA SFMC表达异常的9个致炎、抗炎、信号传导基因或受体和粘附分子，扩大病例数以半定量PCR再验证微阵列检测结果。结果：cDNA微阵列和PCR结果显示，1176cDNA微阵列基因图谱和阳性基因数量在RA和SpA病人SFMC组无明显区别，比较SpA或RA病人的SFMC和PBMC，发现SpA和RA的SFMC中的阳性基因均少于各自的PBMC。在RA的PBMC有53个基因明显高于RA SFM（P＜0．05），但在SpA PBMC仅有5个基因明显高于SpA SFMC(P＜0．05）。SpA病人SFMC的IL－1β、TNF-α、TGF-β、TGF-β2、c-jun、JAK-显著高于健康人PBMC（P＜0．05）。结论：SpA患者的SFMC基因表达谱异常，但与RA的SFM未见明显特征型区别，IL－1β、TNF-α、TGF-β、TGF-β2、c-jun、JAK-3与SpA关节炎的发生和发展相关。     

Production of mononuclear cell factor by mononuclear phagocytes from rheumatoid synovial fluid

Collagenase-and PGE1-stimulating activities (mononuclear cell factor or MCF) have been found in culture supernatants from synovial fluid macrophages (SF-M phi) of patients wih rheumatoid arthritis. The apparent molecular weight of the MCF activity present in SF-M phi supernatants was between 15-20,000 daltons, which is similar to interleukin I. All the SF-M phi supernatants from patients tested showed the MCF activity. Our study presents evidence for MCF production by monocyte-macrophages at the local site of the lesion.     

Fibroblast-like synovial cells derived from synovial fluid

OBJECTIVE: To obtain fibroblast-like synovial cells (FLS) from synovial fluid (SF). METHODS: SF aspirated from joints of patients with rheumatoid arthritis (RA), other types of inflammatory arthritis, and osteoarthritis (OA) was centrifuged and the resulting cell pellet resuspended in growth medium. After 2 days, nonadherent cells were removed. FLS were also cultured from surgical specimens of synovial tissue (td-FLS). Phenotype characterization of fluid derived FLS (fd-FLS) was accomplished by flow cytometry and immunohistochemistry staining. Tumor necrosis factor-alpha (TNF-alpha) induced interleukin 6 (IL-6), IL-8, and cyclooxygenase 2 (COX-2) mRNA levels were assessed. RESULTS: Second and later passage fd-FLS exhibited uniform fibroblast-like morphology. Fd-FLS and td-FLS expressed a similar profile of cell surface antigens including the fibroblast marker Thy-1. Less than 2% of either cell type expressed surface markers characteristic of dendritic cells, phagocytic cells, T cells, or leukocytes. Immunohistochemistry staining revealed the presence of fibroblast products prolyl-4 hydroxylase, procollagen I, and procollagen III in both culture types. TNF-a induced increases in IL-6, IL-8, and COX-2 mRNA were suppressed by dexamethasone in both fd-FLS and td-FLS. CONCLUSION: FLS can be cultured from SF. The fibroblast phenotype was confirmed by analysis of surface antigens and intracellular proteins. Inflammatory mediators produced after stimulation of both fd-FLS and td-FLS were suppressed by dexamethasone. In addition to providing a more accessible source of FLS, fd-FLS may also facilitate study of synovial cells in early RA when tissue specimens are not readily available.     

Excitatory amino acid profiles of synovial fluid from patients with arthritis

OBJECTIVE: Previous studies in an experimental synovitis model in rats determined that administration of glutamate and aspartate into the joint produces hyperalgesic responses, while their receptor antagonists provide protection against the development of a hyperalgesic state. We examined concentrations of amino acids in synovial fluid (SF) to determine if increases might be relevant to human joint pathology. METHODS: One hundred forty-four repository SF samples from patients undergoing diagnostic or therapeutic arthrocentesis and 14 SF samples from 7 cadavers were analyzed by high pressure liquid chromatography and compared as arthritic and control cohorts. RESULTS: Compared to the average concentrations from the autopsy cases, the excitatory amino acids (EAA) glutamate and aspartate in SF from patients with synovitis were 54 and 28 times higher, respectively. Increases for all other amino acids ranged from 3 to 18-fold. The values for glutamate and aspartate were significantly higher than the mean increase for other amino acids compared using unpaired t tests (p < 0.0001). The mean ratio of glutamate and aspartate elevations over the mean increase for other amino acids was 4-fold and 2-fold, respectively. The EAA were highest in Reiter's, infectious arthropathies, and systemic lupus erythematosus, but did not appreciably segregate to diagnosis or SF white blood cell count. CONCLUSION: Our data provide evidence of increased glutamate and aspartate in the SF of humans with active arthritis, suggesting that glutamate mediated events may contribute to the pathogenesis of human arthritic conditions.     

Clues to pathogenesis of fibromyalgia in patients with sickle cell disease

OBJECTIVE: To investigate the association between sickle cell disease (SCD) and fibromyalgia (FM). METHODS: Nine patients with SCD for whom a rheumatology consult was requested were assessed for FM by retrospective chart review. Eleven inpatients with other forms of anemia referred for rheumatology consult were also assessed for FM. RESULTS: Eight of 9 patients with SCD fulfilled classification criteria for FM compared to one of 11 patients without SCD (p < 0.001). CONCLUSION: Awareness of the high frequency of FM in SCD can improve treatment of sickle cell crisis. Some pain that is labeled as sickle cell crisis pain may be due to FM, and may improve with tender point injections.     

In vitro migration of mononuclear cells towards synovial fluid and plasma from rheumatoid arthritis patients correlates to RANTES synovial fluid levels and to clinical pain parameters

OBJECTIVE: To evaluate in vitro migration of mononuclear cells towards synovial fluid (SF) and plasma in relation to RANTES synovial fluid levels and clinical disease activity. METHODS: 31 RA patients with synovitis in one knee were included. Modified Boyden chamber technique was used to determine a migratory index defined as: 'In vitro migrating cells towards SF' divided by 'In vitro migrating cells towards plasma'. RANTES was quantified by ELISA. Disease activity was assessed by the swollen joint count, the Ritchie articular index (RAI), global assessment, pain on VAS, HAQ, ESR and CRP. RESULTS: A positive significant correlation was found between the migratory index and the RANTES levels in SF (r=0.48, p=0.006), the RAI (r=0.56, p=0.0001) and pain on VAS (r=0.43, p=0.04). The in vitro migration could be inhibited in 3 of 4 SF samples by neutralising antibodies towards RANTES (12-18%). CONCLUSION: The migratory index correlate to SF levels of RANTES and parameters for joint pain.     

Differential gene expression profiles of peripheral blood mononuclear cells in childhood asthma

Objective: Asthma is a common childhood disease with strong genetic components. This study compared whole-genome expression differences between asthmatic young children and healthy controls to identify gene signatures of childhood asthma. Methods: Total RNA extracted from peripheral blood mononuclear cells (PBMC) was subjected to microarray analysis. QRT-PCR was performed to verify the microarray results. Classification and functional characterization of differential genes were illustrated by hierarchical clustering and gene ontology analysis. Multiple logistic regression (MLR) analysis, receiver operating characteristic (ROC) curve analysis, and discriminate power were used to scan asthma-specific diagnostic markers. Results: For fold-change>2 and p?<?0.05, there were 758 named differential genes. The results of QRT-PCR confirmed successfully the array data. Hierarchical clustering divided 29 highly possible genes into seven categories and the genes in the same cluster were likely to possess similar expression patterns or functions. Gene ontology analysis presented that differential genes primarily enriched in immune response, response to stress or stimulus, and regulation of apoptosis in biological process. MLR and ROC curve analysis revealed that the combination of ADAM33, Smad7, and LIGHT possessed excellent discriminating power. Conclusions: The combination of ADAM33, Smad7, and LIGHT would be a reliable and useful childhood asthma model for prediction and diagnosis.     

Obesity-related glomerulopathy: insights from gene expression profiles of the glomeruli derived from renal biopsy samples

Obesity-related glomerulopathy (ORG) is an important complication of obesity. The pathophysiological mechanism of glomerular injury in ORG is incompletely understood. Gene expression profiles in the glomeruli obtained from renal biopsy samples of patients with ORG were investigated, using a microdissection technique combined with Affymetrix microarray analysis. Six patients presented with obesity, proteinuria, and biopsy-proven ORG were enrolled. Two sex- and age-matched donor kidneys were applied as the controls. Glomeruli were dissected out from renal biopsy samples under microscope, and total RNA was extracted using RNeasy Micro kit. After two rounds of T7 promoter-based RNA amplification, gene expression profiles of the glomeruli samples were detected using Affymetrix U133A gene chips. Bioinformatic tools were applied to analyze the microarray data. Results of candidate ORG-related genes were further confirmed by real-time quantitative PCR and immunohistochemistry staining using renal biopsy samples of a larger pool of 15 ORG patients. Genes related to lipid metabolism, inflammatory cytokines, and insulin resistance were the most highlighted subgroups that significantly changed in the glomerular gene expression profiles of the ORG patients, compared with the controls. The expression levels of several key genes in lipid metabolism were increased over 2-fold, including low-density lipoprotein receptor, fatty acid binding protein 3, and sterol regulatory element binding protein 1. Moreover, some inflammatory cytokines and their downstream molecules were increased as well, including TNF-alpha and its receptors, IL-6 signal transducer, and interferon-gamma. As the indicators of insulin resistance in the local glomerular cells, levels of glucose-transporter 1, leptin receptor, peroxisome proliferator-activated receptor-gamma, and vascular endothelial growth factor increased, too. In addition to lipid dysmetabolism and insulin resistance, the activation of an inflammatory process in the glomeruli might play a unique role in the development of obesity-related glomerulopathy. Our results expand the understanding of obesity-induced glomerular injuries and shed light on new approaches in the treatment of this disease.     

The influence of synovial fluid on adenovirus-mediated gene transfer to the synovial tissue

OBJECTIVE: To determine the effect of synovial fluid (SF) from rheumatoid arthritis (RA) patients on adenovirus type 5 (Ad5)-mediated gene transfer to synoviocytes, and to explore new strategies for vector development based on the neutralization data obtained. METHODS: SF was derived from 63 randomly selected R4 patients. Ten samples were used to study the effect of SF on Ad5-mediated gene transfer in synoviocytes. IgG and <100-kd fractions were purified from these 10 SF, and their effect on gene transfer was determined. Neutralizing activity against wild-type Ad5 (wt-Ad5), wt-Ad26, wt-Ad34, wt-Ad35, and wt-Ad48 was tested in the SF from the remaining 53 patients. RESULTS: Seven of 10 SF samples inhibited Ad5-mediated gene transfer. Purified antibodies exhibited inhibition patterns similar to those seen with unfractionated SF. In 5 of 10 SF samples, low molecular weight fractions inhibited gene transfer at low dilutions. Neutralization of wt-Ad35 by SF from RA patients was less frequent than neutralization of other wt-Ad tested (4% versus 42-72%; n = 53). CONCLUSION: SF from 70% of the RA patients contained neutralizing antibodies that hamper Ad5-mediated gene transfer to synoviocytes. The activity of neutralizing antibodies may be circumvented in the majority of RA patients when vectors based on an Ad35 backbone are used.     

Possible clearance of effete polymorphonuclear leucocytes from synovial fluid by cytophagocytic mononuclear cells: implications for pathogenesis and chronicity in inflammatory arthritis.

A feature common to all forms of chronic inflammatory arthritis, irrespective of the possible underlying cause, is the persistent exudation of large numbers of polymorphonuclear leucocytes (PMNL) into synovial fluid. These cells possess potent degradative enzymes and proinflammatory mediators, and their removal is vital to normal inflammatory resolution. A major route of disposal of extravasated PMNL appears to be programmed cell death (apoptosis), followed by their rapid recognition, and intact phagocytosis, by mature tissue macrophages. Such macrophages, containing PMNL (cytophagocytic mononuclear cells (CPM)), long recognised in synovial fluid as Reiter cells, are commonly found in reactive arthritis, spondyloarthritis, and crystal arthritides, but only rarely in rheumatoid disease. In a retrospective analysis of 187 knee synovial fluid cytospins, the relation between the formation of CPM and the presence of apoptotic (pyknotic) PMNL was investigated. As long as the synovial fluid examined was fresh there was a high correlation between numbers of CPM (as a percentage of macrophages) and pyknotic numbers of PMNL in fluids containing CPM. This suggests that the formation of CPM occurs in vivo and is involved in the disposal of PMNL. Numbers of pyknotic PMNL increased rapidly in stored synovial fluid without a significant change in numbers of CPM, and were highest in synovial fluid which did not contain CPM. The presence or absence of CPM, or their disease associations, could not be explained simply by limiting numbers of macrophages, or apoptotic PMNL in synovial fluid. These findings are consistent with a regulatory role for CPM in synovial fluid, where they may be important in preventing autolysis of PMNL, and thus local tissue damage.     

Synovial fluid mononuclear cell gene expression profiling suggests dysregulation of innate immune genes in enthesitis-related arthritis patients

Objective. Microarray studies have provided insight into the pathogenesis of systemic JIA and have opened new avenues for therapy. Data on the pathogenesis of the enthesitis-related arthritis (ERA) category of JIA are limited, thus we studied the expression profile of ERA patients' peripheral blood and SF mononuclear cells (PBMCs and SFMCs, respectively). PBMCs from healthy subjects were used as controls. Methods. RNA from PBMCs of ERA patients (n?=?17) and healthy controls (n?=?8) and seven ERA SFMCs were converted to labelled cRNA and hybridized to Illumina Human WG-6_v3_BeadChip chips. Expression profiles were analysed using GeneSpring software. Selected genes of interest were validated by real-time PCR. Results. There was no significant difference in PBMC gene expression of ERA and control groups. However, there was a significant difference between expression profiles of SFMCs and PBMCs of patients with ERA, with 131 genes being overexpressed and 216 being underexpressed in SFMCs. Among genes involved with immune function, cluster of differentiation (CD)1b, CD1d, MHC class II alpha and beta chain, and soluble CD163 were overexpressed, whereas genes related to NK cell function, namely, Granzyme H, killer cell lectin-like receptor subfamily F member 1, killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail (KIR3DL3), natural killer group 7 (NKG7) and other genes like CD244, CD248 and Fas apoptotic inhibitory molecule 3 (FAIM3) were underexpressed. Conclusion. ERA SFMCs had a distinct gene expression profile from PBMCs and had higher expression of genes associated with antigen presentation, scavenger function, chemotaxis and proteases, whereas genes involved in NK cell function, cell adhesion and inhibitors of apoptosis were underexpressed.     

Progression of coronary atherosclerosis. Clues to pathogenesis from serial coronary arteriography

Fifty two patients with coronary artery disease underwent repeat coronary arteriographic studies separated by 2-108 (mean 51) months of medical treatment. The results were compared and correlated with symptoms to determine the nature of the progression of coronary atherosclerosis. The condition appeared to progress episodically in the proximal segments of the coronary arteries and in relation to the abrupt development of new symptoms or acute coronary events such as unstable angina or myocardial infarction. Thirty four of 105 (33%) of the pre-existing stenoses showed evidence of progression. Progression to total occlusion was uncommon (13) except for stenoses greater than 90% (six out of 18). New lesions frequently occurred (37) in previously normal segments of the arteries; most of these were stenoses greater than 90% (13) or total occlusions (12). Rapid progression of a mild lesion and new lesions occurred in the form of smooth intimal protrusions into the arterial lumen. Intimal haemorrhages are the likely explanation for these intimal encroachments and also for the episodic nature of the progression of coronary artery disease. Coronary atherosclerosis does not progress gradually in a linear fashion, and local anatomical factors appear to play a dominant role in the natural history.     

Cytophotometric DNA measurements in mononuclear round cells of synovial fluid]

Cytophotometric investigations were performed on synovial fluid mononuclear round cells in patients with various joint diseases. In nearly all instances only round cells with a diploid DNA-content were found. No increased proliferation could be observed neither in patients with rheumatoid arthritis nor in patients with a high relative number of neutrophilic granulocytes in the synovial fluid.