Do synovial fluid acute phase proteins from patients with rheumatoid arthritis originate from serum?

This study was performed in order to gain insight into the occurrence, glycosylation and the possible origin of the acute-phase proteins α1-acid glycoprotein (AGP) and α1-protease inhibitor (PI) in sera and synovial fluid from patients with rheumatoid arthritis (RA). Therefore paired sera and synovial fluid samples from patients with RA, and paired synovial fluid samples from right and left knees of patients with varying degrees of arthritis were studied. Crossed affinity immunoelectrophoresis (CAIE) was used with concanavalin A and Aleuria aurantia lectin for the detection of the degree of branching and fucosylation, respectively, and the monoclonal CSLEX-1 for the detection of Sialyl Lewisx (SLex) groups on AGP. For PI, not only CAIE, but also high-pressure-anion-exchange chromatography with pulsed amperometric detection was used to study the glycosylation. It was established that the concentrations of AGP and PI were increased in the serum of RA patients compared to normal healthy controls, but that the concentration of both proteins, as well as albumin, was significantly lower in synovial fluid than in serum. Furthermore, the type of glycosylation of both AGP and PI found in RA was significantly different from that found in normals, with increased fucosylation, but there were no major differences in the degree of branching of AGP- or PI-glycans in RA, compared to normals. No differences in glycosylation could be established between serum and synovial fluid in RA. For PI an increased fucosylation was found, both in serum and synovial fluid, using both methods of detection, and it could be established that only the α1→3- and not the α1→6-fucosylation of PI was affected by RA. The increased fucosylation of AGP resulted in an increased expression of SLex on AGP-glycans. Since the α1→3- fucosylation of AGP was significantly increased in both serum and synovial fluid from RA patients, and this correlated with systemic but not with local disease parameters, it can be suggested that acute phase proteins in synovial fluid are most probably of hepatic origin. Abbreviations: AGP, α1-acid glycoprotein; AAL, Aleuria Aurantia Lectin; Con A, concanavalin A; PI, α1-protease inhibitor; CAIE, crossed affino-immunoelectrophoresis; SLex, sialyl Lewis X; IL-6, interleukin-6; RA, rheumatoid arthritis; PMN, polymorphonuclear cells; HPAEC, high pressure anion exchange chromatography This revised version was published online in November 2006 with corrections to the Cover Date.     

Loss of nuclear activity of the FBXO7 protein in patients with parkinsonian-pyramidal syndrome (PARK15)

Mutations in the F-box only protein 7 gene (FBXO7) cause PARK15, an autosomal recessive neurodegenerative disease presenting with severe levodopa-responsive parkinsonism and pyramidal disturbances. Understanding the PARK15 pathogenesis might thus provide clues on the mechanisms of maintenance of brain dopaminergic neurons, the same which are lost in Parkinson's disease. The protein(s) encoded by FBXO7 remain very poorly characterized. Here, we show that two protein isoforms are expressed from the FBXO7 gene in normal human cells. The isoform 1 is more abundant, particularly in primary skin fibroblasts. Both isoforms are undetectable in cell lines from the PARK15 patient of an Italian family; the isoform 1 is undetectable and the isoform 2 is severely decreased in the patients from a Dutch PARK15 family. In human cell lines and mouse primary neurons, the endogenous or over-expressed, wild type FBXO7 isoform 1 displays mostly a diffuse nuclear localization. An intact N-terminus is needed for the nuclear FBXO7 localization, as N-terminal modification by PARK15-linked missense mutation, or N-terminus tag leads to cytoplasmic mislocalization. Furthermore, the N-terminus of wild type FBXO7 (but not of mutant FBXO7) is able to confer nuclear localization to profilin (a cytoplasmic protein). Our data also suggest that overexpressed mutant FBXO7 proteins (T22M, R378G and R498X) have decreased stability compared to their wild type counterpart. In human brain, FBXO7 immunoreactivity was highest in the nuclei of neurons throughout the cerebral cortex, intermediate in the globus pallidum and the substantia nigra, and lowest in the hippocampus and cerebellum. In conclusion, the common cellular abnormality found in the PARK15 patients from the Dutch and Italian families is the depletion of the FBXO7 isoform 1, which normally localizes in the cell nucleus. The activity of FBXO7 in the nucleus appears therefore crucial for the maintenance of brain neurons and the pathogenesis of PARK15.     

Current and future management approaches for rheumatoid arthritis

With the introduction of new disease-modifying antirheumatic drugs (DMARDs) and other therapeutic agents, the management of rheumatoid arthritis (RA) has shifted toward earlier, more aggressive therapy. The ultimate goal is to prevent structural joint damage that leads to pain and functional disability. Early diagnosis of RA is therefore essential, and early DMARD treatment combined with nonsteroidal anti-inflammatory drugs is recommended. Combination DMARD regimens and new biologic agents (anti-tumor necrosis factor [TNF] therapies [infliximab, etanercept] and the interleukin [IL]-1 antagonist [anakinra]) have emerged as viable options for early treatment of RA patients. These new biologic agents and future nonbiologic agents that target proteins in signaling cascades are likely to change the landscape of RA treatments.     

The role of carotenoids in preventing oxidative damage in the pigmented yeast, Rhodotorula mucilaginosa

Rhodotorula mucilaginosa is an obligate aerobic yeast which contains a high concentration of carotenoid pigment. To test whether carotenoids are able to protect R. mucilaginosa against oxidative injury, yeast cells in liquid culture were incubated with duroquinone (DQ) (100 microM), a redox-cycling quinone known to generate intracellular O2-. or were grown in a hyperoxic atmosphere (80% O2) under conditions where carotenoid concentrations were altered either intracellularly or extracellularly. Neither of these oxidative challenges affected cell growth unless carotenogenesis was blocked by the addition of diphenylamine (50 microM). In the diphenylamine-treated nonpigmented cells, growth was completely inhibited by DQ and by hyperoxia. In normoxia, however, diphenylamine alone reduced growth by only 30%. The growth inhibition observed in diphenylamine-treated cells exposed to hyperoxia was primarily mycocidal rather than mycostatic since plating of these cells onto solid media revealed that only 25% of the cells were viable after 50 h of incubation when compared to plated control cells. Addition of 10 microM beta-carotene to diphenylamine-treated cells completely prevented the growth inhibition caused by either hyperoxia or DQ. Carotenoids, therefore, are able to prevent oxidant-induced cytotoxicity in R. mucilaginosa. Analysis of the absorption spectra of chloroform extracts of beta-carotene-supplemented cells showed that beta-carotene, not the endogenous carotenoid, torularhodin, was the major carotenoid present in these cells. Superoxide dismutase (SOD) activity in R. mucilaginosa was compared with that of another yeast, Saccharomyces cerevisiae by two methods: (i) activity staining of proteins separated by gel electrophoresis and (ii) measurement of inhibition of ferricytochrome c reduction. By these techniques, the R. mucilaginosa SOD activity had the characteristics of Mn-SOD. No Cu/ZnSOD activity was detected. Thus, the apparent absence of Cu/ZnSOD may make the antioxidant capability of endogenous carotenoids even more critical in preventing oxidative damage in R. mucilaginosa.     

Effect of Phosphate Limitation on Synthesis of Periplasmic Cyclic (beta)-(1,2)-Glucans

Rhizobium meliloti and Agrobacterium tumefaciens synthesize periplasmic cyclic (beta)-(1,2)-glucans during adaptation to hypoosmotic environments. It also appears that these glucans provide important functions during the interactions of these bacteria with plant hosts. A large fraction of these glucans may become modified with anionic substituents such as phosphoglycerol or succinic acid; however, the role(s) of these substituents is unknown. In this study, we show that growth of these bacteria in phosphate-limited media leads to a dramatic reduction in the levels of phosphoglycerol substituents present on the periplasmic cyclic (beta)-(1,2)-glucans. Under these growth conditions, R. meliloti 1021 was found to synthesize anionic cyclic (beta)-(1,2)-glucans containing only succinic acid substituents. Similar results were obtained with R. meliloti 7154 (an exoH mutant which lacks the ability to succinylate its high-molecular-weight exopolysaccharide), revealing that succinylation of the cyclic (beta)-(1,2)-glucans is mediated by an enzyme system distinct from that involved in the succinylation of exopolysaccharide. In contrast, when A. tumefaciens C58 was grown in a phosphate-limited medium, it was found to synthesize only neutral cyclic (beta)-(1,2)-glucans.     

Osmotically-regulated trehalose accumulation and cyclic β-(1,2)-glucan excretion by Rhizobium leguminosarum biovar trifolii TA-1

Rhizobium leguminosarum biovar trifolii TA-1 produced high molecular weight extracellular (EPS) and capsular polysaccharides (CPS) as the main carbohydrate products in a medium (10 g of mannitol and 1 g of glutamic acid per liter) with low osmotic pressure of 0.20 MPa. By increasing the osmotic pressure of the medium with the addition of NaCl or other osmolytes up to 1.44 MPa, the synthesis of EPS and CPS was suppressed. Cyclic -(1,2)-glucans were excreted instead. Concentrations of over 1500 mg of glucans/l medium were produced by a biomass of 520 mg protein at 200 mM NaCl (1.20 MPa). Intracellular cyclic -(1,2)-glucan concentrations remained at 45 to 100 mg/g protein during the stationary phase, independent of the osmotic strength of the medium. Parallel to the increasing osmotic pressure of the medium, the disaccharide trehalose accumulated in the cells as osmo-protectant. Concentrations of up to 130 mg/g protein were reached. Strain TA-1 could tolerate 350 mM NaCl.Abbreviations CPS capsular polysaccharide - EPS extracellular polysaccharide - LM_r low molecular weight - HM_r high molecular weight     

Synthesis of glycerophosphorylated cyclic beta-(1,2)-glucans by Rhizobium meliloti ndv mutants.

The periplasmic cyclic beta-(1,2)-glucans of Rhizobium spp. are believed to provide functions during hypoosmotic adaptation and legume nodulation. In Rhizobium meliloti, cyclic beta-(1,2)-glucans are synthesized at highest levels when cells are grown at low osmolarity, and a considerable fraction (> or = 35%) of these glucans may become substituted with phosphoglycerol moieties. Thus far, two chromosomally encoded proteins, NdvA and NdvB, have been shown to function during cyclic beta-(1,2)-glucan biosynthesis; however, the precise roles for these proteins remain unclear. In the present study, we show that R. meliloti mutants lacking up to one-third of the downstream region of ndvB synthesize cyclic beta-(1,2)-glucans similar to those produced by wild-type cells with respect to size and phosphoglycerol substituent profile. In contrast, no phosphoglycerol substituents were detected on the cyclic beta-(1,2)-glucans synthesized by an R. meliloti ndvA mutant.     

Regulation of cytokines, cytokine inhibitors, and acute-phase proteins following anti-TNF-alpha therapy in rheumatoid arthritis.

Treatment with a chimeric mAb to TNF-alpha has been shown to suppress inflammation and improve patient well-being in rheumatoid arthritis (RA), but the mechanisms of action of such treatment have not been fully explored. Here we show that in vivo administration of anti-TNF-alpha Ab, using a longitudinal analysis, results in the rapid down-regulation of a spectrum of cytokines, cytokine inhibitors, and acute-phase proteins. Marked diurnal variation in the serum levels of some of these were detected. These results were consistent with the concept of a cytokine-dependent cytokine cascade, and the degree of clinical benefit noted after anti-TNF-alpha therapy is probably due to the reduction in many proinflammatory mediators apart from TNF-alpha, such as IL-6, which reached normal levels within 24 h. Serum levels of cytokine inhibitors such as soluble p75 and p55 TNFR were reduced as was IL-1 receptor antagonist. Reductions in acute-phase proteins occurred after serum IL-6 fell and included serum amyloid A, haptoglobin, and fibrinogen. The latter reduction could be of importance, as it is a risk factor for atherosclerosis, which is augmented in RA patients.