Comparison of the concentrations of pentosidine in the synovial fluid, serum and urine of patients with rheumatoid arthritis and osteoarthritis

OBJECTIVE:Pentosidine, an advanced glycation end product (AGE), has recently been observed to be elevated in rheumatoid arthritis (RA). The aim was to elucidate which pentosidine levels, i.e. in serum, synovial fluid or urine, are more related to the disease status of RA. METHODS:We measured levels of pentosidine in serum, synovial fluid or urine in RA compared with osteoarthritis (OA), and examined the relationship between pentosidine and RA disease activity. Subjects were 20 patients with RA and 22 patients with OA. RESULTS:In total RA and OA patients combined, there was a significant correlation between pentosidine in serum, synovial fluid and urine. Pentosidine in serum and synovial fluid was significantly higher in RA than in OA. In RA, there were significant correlations between pentosidine in serum and synovial fluid and C-reactive protein, Lansbury index (LI) and erythrocyte sedimentation rate. CONCLUSIONS:These results demonstrate that pentosidine levels in body fluids correlated with each other, and pentosidine in serum and in synovial fluid is associated with the systemic inflammatory activity of RA. Higher or similar concentrations of pentosidine in serum compared with synovial fluids indicate that the elevated pentosidine levels in serum in RA are not derived from the synovial fluid, but from an increase in the formation of pentosidine in the whole body in RA. Among body fluids, serum pentosidine was the superior indicator for RA disease status.     

Embryonic smooth muscle myosin heavy chain SMemb is expressed in pressure-overloaded cardiac fibroblasts

Left ventricular hypertrophy (LVH) is a secondary adaptation to increased external load. Various qualitative and quantitative changes in myocytes and extracellular components occur during the development of LVH. It has recently been demonstrated that alpha-smooth muscle actin (alpha-SMA)-expressing myofibroblasts appear in the interstitium of the heart subjected to increased workload suggesting that cardiac fibroblasts as well as myocytes alter their phenotype in response to pressure overload. In the present study, to explore the load-induced response and phenotypic modulation of cardiac fibroblasts, the localization of embryonic smooth muscle myosin heavy chain (SMemb) and alpha-SMA in thoracic aorta-constricted rat hearts was investigated by immunohistochemistry, and the morphology of the SMemb-expressing cells was examined by electron microscopy. In addition, to clarify the mechanisms by which SMemb is induced in pressure-overloaded hearts, mRNA expression of SMemb in aorta-constricted rat hearts and in transforming growth factor-beta1 (TGF-beta1)-treated or mechanically-stretched cultured cardiac fibroblasts was investigated. Enhanced staining of SMemb and alpha-SMA was detected in the interstitial spindle-shaped cells in the fibrotic lesions of the pressure-overloaded left ventricles by immunohistochemistry. These cells were demonstrated by electron microscopy to have features specific for activated fibroblasts such as serrated nuclei or prominent rough endoplasmic reticulum. These cells also had characteristic features of myofibroblasts, i.e. irregularly arranged actin filaments and scattered dense bodies. Northern blot analysis revealed increased mRNA levels of SMemb both in aorta-constricted rat hearts and in cultured cardiac fibroblasts stimulated by TGF-beta1 or by mechanical stretch. These results suggest that SMemb may be a molecular marker both for the detection of activated cardiac fibroblasts that may play important roles in the remodeling of pressure-overloaded cardiac interstitium, and for the identification of the regu latory mechanisms that control the phenotypic modulation of cardiac fibroblasts in response to pressure overload.     

Comparison of the activities of multinucleated bone-resorbing giant cells derived from CD14-positive cells in the synovial fluids of rheumatoid arthritis and osteoarthritis patients

OBJECTIVE: To investigate the morphology and function of multinucleated bone-resorbing giant cells derived from CD14-positive cells in the synovial fluids (SF) of patients with rheumatoid arthritis (RA) or osteoarthritis (OA). METHODS: CD14-positive cells were obtained by magnetic-activated cell sorting of primary cultures of mononuclear cells from the SF. Multinucleated bone-resorbing giant cells were induced from the CD14-positive cells in the presence or absence of cytokines. We examined various characteristics, including osteoclast markers, fusion index and bone-resorption activities of the multinucleated giant cells. RESULTS: Multinucleated giant cells were induced from the CD14-positive cells in the SF of the RA and OA patients by the addition of interleukin (IL)-3, IL-5 and IL-7, or granulocyte-macrophage colony-stimulating factor (GM-CSF), respectively. These multinucleated giant cells were positive for tartrate-resistant acid phosphatase (TRAP), carbonic anhydrase II, actin, vitronectin receptor and the calcitonin receptor. However, the average values for the number of nuclei, fusion index and bone-resorption functions of the SF cells from the RA patients were significantly higher than those derived from the OA patients. CONCLUSION: These results suggest that the induction and activities of multinucleated bone-resorbing giant cells may play a pivotal role in bone destruction, and that these processes may be enhanced significantly in RA patients.     

CK2 phosphorylation of eukaryotic translation initiation factor 5 potentiates cell cycle progression

Casein kinase 2 (CK2) is a ubiquitous eukaryotic Ser/Thr protein kinase that plays an important role in cell cycle progression. Although its function in this process remains unclear, it is known to be required for the G(1) and G(2)/M phase transitions in yeast. Here, we show that CK2 activity changes notably during cell cycle progression and is increased within 3 h of serum stimulation of quiescent cells. During the time period in which it exhibits high enzymatic activity, CK2 associates with and phosphorylates a key molecule for translation initiation, eukaryotic translation initiation factor (eIF) 5. Using MS, we show that Ser-389 and -390 of eIF5 are major sites of phosphorylation by CK2. This is confirmed using eIF5 mutants that lack CK2 sites; the phosphorylation levels of mutant eIF5 proteins are significantly reduced, relative to WT eIF5, both in vitro and in vivo. Expression of these mutants reveals that they have a dominant-negative effect on phosphorylation of endogenous eIF5, and that they perturb synchronous progression of cells through S to M phase, resulting in a significant reduction in growth rate. Furthermore, the formation of mature eIF5/eIF2/eIF3 complex is reduced in these cells, and, in fact, restricted diffusional motion of WT eIF5 was almost abolished in a GFP-tagged eIF5 mutant lacking CK2 phosphorylation sites, as measured by fluorescence correlation spectroscopy. These results suggest that CK2 may be involved in the regulation of cell cycle progression by associating with and phosphorylating a key molecule for translation initiation.     

Effect of spironolactone on cardiac sympathetic nerve activity and left ventricular remodeling in patients with dilated cardiomyopathy

OBJECTIVES: We sought to evaluate the effects of spironolactone on cardiac sympathetic nerve activity and left ventricular (LV) remodeling in patients with dilated cardiomyopathy (DCM). BACKGROUND: Aldosterone prevents the uptake of norepinephrine and promotes structural remodeling of the heart. Spironolactone, an aldosterone receptor blocker, improves LV remodeling in patients with DCM, but its influence on cardiac sympathetic nerve activity has not been determined. METHODS: We selected 30 patients with DCM who were treated with an angiotensin-converting enzyme inhibitor and a loop diuretic. Fifteen patients were assigned to receive spironolactone additionally, whereas the remaining 15 patients continued their current regimen. The delayed heart/mediastinum (H/M) count ratio, delayed total defect score (TDS), and washout rate (WR) were determined from iodine-123 ((123)I)-meta-iodobenzylguanidine (MIBG) images before and six months after treatment. The left ventricular end-diastolic volume (LVEDV) and left ventricular ejection fraction (LVEF) were determined by echocardiography, and New York Heart Association (NYHA) functional class was estimated.RESULTS: In the spironolactone group, the TDS decreased from 36 +/- 9 to 24 +/- 13 (p < 0.0001), the H/M ratio increased from 1.64 +/- 0.20 to 1.86 +/- 0.27 (p < 0.0001), and WR decreased from 55 +/- 12% to 41 +/- 15% (p < 0.0005). In addition, the LVEDV decreased from 187 +/- 26 to 154 +/- 41 ml (p < 0.005), and LVEF increased from 33 +/- 6% to 39 +/- 6% (p < 0.005). However, there were no significant changes in these parameters in the control group. There was a significant correlation between changes in the (123)I-MIBG findings and changes in LVEDV with spironolactone treatment (TDS: r = 0.684, p = 0.0038; H/M ratio: r = -0.878, p < 0.0001; and WR: r = 0.737, p = 0.0011). The NYHA functional class improved in both groups but showed a greater improvement in the spironolactone group than in the control group (p < 0.01). CONCLUSIONS: Spironolactone improves cardiac sympathetic nerve activity and LV remodeling in patients with DCM.     

Preferential expression of B7.2 (CD86), but not B7.1 (CD80), on B cells induced by CD40/CD40L interaction is essential for anti-DNA autoantibody production in patients with systemic lupus erythematosus

OBJECTIVE: B7 (CD80/CD86) molecules are over-expressed in patients with SLE. However, it is not clear whether CD80/CD86 molecules are involved in the pathogenic autoantibody production specifically or in the polyclonal antibody production in human SLE. The present study was carried out to characterize B7 molecules on B cells in autoantibody production. METHODS: Expression of costimulatory molecules was analyzed by RT-PCR and two-color immunofluorescence staining. Purified B cells were co-cultured with T cells in the presence of anti-costimulatory molecule antibody. RESULTS: Excessive expression of CD86 and CD80 molecules was evident on freshly isolated B cells in patients with SLE. Normal B cells did not express CD86 molecules spontaneously and expressed it after co-culture with activated T cells. CD86 expression on normal and SLE B cells induced by the activated T cells was inhibited by the addition of anti-CD40L into the cell culture. Furthermore, CD40L expression on T cells upon activation was enhanced in SLE patients. Anti-DNA antibody production by SLE B cells in the presence of activated T cells was markedly inhibited by anti-CD86, but not anti-CD80. Anti-CD86 treatment inhibited polyclonal Ig and anti-SS-A antibody production of SLE B cells, suggesting the preferential involvement of CD86 in polyclonal antibody production. CONCLUSION: SLET T cells express CD40L excessively, and the CD40/CD40L pathway is involved in the CD86 over-expression of SLE B cells; thus T cell abnormality is at least partially involved in B cell hyperactivity. Enhanced CD86 expression of B cells by CD40L is essential for polyclonal antibody production.     

Levels of Rheumatoid Factor Isotypes, Metalloproteinase-3 and Tissue Inhibitor of Metalloproteinase-1 in Synovial Fluid from Various Arthritides

When synovial effusion is the only symptom, it is often difficult to make an exact diagnosis of the arthritic disease. To distinguish various types of arthritis with synovial effusion, we measured the levels of matrix metalloproteinase-3 (MMP-3, Stromelysin), tissue inhibitor of metalloproteinase-1 (TIMP-1) and rheumatoid factor (RF) isotypes in synovial fluid (SF) from patients with rheumatoid arthritis (RA), osteoarthritis (OA), pyogenic arthritis (PA), pseudogouty arthritis (PG), gouty arthritis (GA) and traumatic arthritis (TA). SF was aspirated from the knee joint or the ankle joint. Levels of IgG-, IgM- and IgA-RF isotypes were measured by ELISA. Levels of MMP-3 and TIMP-1 in SF were simultaneously determined by a one-step EIA system. Levels of IgG-RF, IgM-RF and MMP-3 in SF from RA patients were significantly higher than those in OA, PA, PG, GA and TA. However, IgA-RF in SF from RA patients, when compared with PA and GA, did not show a significantly increased level. In addition, TIMP-1 in SF from RA, when compared with PA and TA, also has not shown a significantly increased level. Therefore, in addition to analysing clinical data, measurements of IgG-RF, IgM-RF and MMP-3 in SF may contribute in distinguishing RA from other arthritic diseases. Received: 18 January 1999 / Accepted: 15 June 1999     

Genetic basis for the evolution of vertebrate mineralized tissue

Mineralized tissue is vital to many characteristic adaptive phenotypes in vertebrates. Three primary tissues, enamel (enameloid), dentin, and bone, are found in the body armor of ancient agnathans and mammalian teeth, suggesting that these two organs are homologous. Mammalian enamel forms on enamel-specific proteins such as amelogenin, whereas dentin and bone form on collagen and many acidic proteins, such as SPP1, coordinately regulate their mineralization. We previously reported that genes for three major enamel matrix proteins, five proteins necessary for dentin and bone formation, and milk caseins and salivary proteins arose from a single ancestor by tandem gene duplications and form the secretory calcium-binding phosphoprotein (SCPP) family. Gene structure and protein characteristics show that SCPP genes arose from the 5' region of ancestral sparcl1 (SPARC-like 1). Phylogenetic analysis on SPARC and SPARCL1 suggests that the SCPP genes arose after the divergence of cartilaginous fish and bony fish, implying that early vertebrate mineralization did not use SCPPs and that SPARC may be critical for initial mineralization. Consistent with this inference, we identified SPP1 in a teleost genome but failed to find any genes orthologous to mammalian enamel proteins. Based on these observations, we suggest a scenario for the evolution of vertebrate tissue mineralization, in which body armor initially formed on dermal collagen, which acted as a reinforcement of dermis. We also suggest that mammalian enamel is distinct from fish enameloid. Their similar nature as a hard structural overlay on exoskeleton and teeth is because of convergent evolution.     

Abnormal insulin secretion and glucose metabolism in pancreatic islets from the spontaneously diabetic GK rat

Summary Insulin secretion and islet glucose metabolism were compared in pancreatic islets isolated from GK/Wistar (GK) rats with spontaneous Type 2 (non-insulin-dependent) diabetes mellitus and control Wistar rats. Islet insulin content was 24.5±3.1 U/ng islet DNA in GK rats and 28.8±2.5 U/ng islet DNA in control rats, with a mean (±SEM) islet DNA content of 17.3±1.7 and 26.5±3.4 ng (p < 0.05), respectively. Basal insulin secretion at 3.3 mmol/l glucose was 0.19±0.03 · ng islet DNA^–1· h^–1 in GK rat islets and 0.40±0.07 in control islets. Glucose (16.7 mmol/l) stimulated insulin release in GK rat islets only two-fold while in control islets five-fold. Glucose utilization at 16.7 mmol/l glucose, as measured by the formation of ³H₂O from [5-³ H]glucose, was 2.4 times higher in GK rat islets (3.1±0.7 pmol · ng islet DNA^–1 · h^–1) than in control islets (1.3±0.1 pmol · ng islet DNA^–1 · h^–1; p<0.05). In contrast, glucose oxidation, estimated as the production of ¹⁴CO₂ from [U-¹⁴C]glucose, was similar in both types of islets and corresponded to 15±2 and 30±3 % (p<0.001) of total glucose phosphorylated in GK and control islets, respectively. Glucose cycling, i. e. the rate of dephosphorylation of the total amount of glucose phosphorylated, (determined as production of labelled glucose from islets incubated with ³H₂O) was 16.4±3.4% in GK rat and 6.4±1.0% in control islets, respectively (p<0.01). We conclude that insulin secretion stimulated by glucose is markedly impaired in GK rat islets. Glucose metabolism is also altered in GK rat islets, with diminished ratio between oxidation and utilization of glucose, and increased glucose cycling, suggesting links between impaired glucose-induced insulin release and abnormal glucose metabolism.