Certolizumab pegol in combination with dose-optimised methotrexate in DMARD-naïve patients with early, active rheumatoid arthritis with poor prognostic factors: 1-year results from C-EARLY, a randomised, double-blind, placebo-controlled phase III study.

OBJECTIVES: To assess the efficacy and safety of certolizumab pegol (CZP)+dose-optimised methotrexate (MTX) versus placebo (PBO)+dose-optimised MTX in inducing and sustaining clinical remission in DMARD-naïve patients with moderate-to-severe, active, progressive rheumatoid arthritis (RA), with poor prognostic factors over 52 weeks. METHODS: DMARD-naïve patients with ≤1 year of active RA were randomised (3:1) in a double-blind manner to CZP (400 mg Weeks 0, 2, 4, then 200 mg Q2W to Week 52)+MTX or PBO+MTX (the mean optimised-MTX dose=21 and 22 mg/week, respectively). Sustained remission (sREM) and sustained low disease activity (sLDA; DAS28(ESR)<2.6 and DAS28(ESR)≤3.2, respectively, at both Weeks 40 and 52) were the primary and secondary endpoints. RESULTS: Patients were randomised to CZP+MTX (n=660) and PBO+MTX (n=219). At Week 52, significantly more patients assigned to CZP+MTX compared with PBO+MTX achieved sREM (28.9% vs 15.0%, p<0.001) and sLDA (43.8% vs 28.6%, p<0.001). Inhibition of radiographic progression and improvements in physical functioning were significantly greater for CZP+MTX versus PBO+MTX (van der Heijde modified total Sharp score (mTSS) mean absolute change from baseline (CFB): 0.2 vs 1.8, p<0.001, rate of mTSS non-progressors: 70.3% vs 49.7%, p<0.001; least squares (LS) mean CFB in Health Assessment Questionnaire-Disability Index (HAQ-DI): -1.00 vs -0.82, p<0.001). Incidence of adverse events (AEs) and serious AEs was similar between treatment groups. Infection was the most frequent AE, with higher incidence for CZP+MTX (71.8/100 patient-years (PY)) versus PBO+MTX (52.7/100 PY); the rate of serious infection was similar between CZP+MTX (3.3/100 PY) and PBO+MTX (3.7/100 PY). CONCLUSIONS: CZP+dose-optimised MTX treatment of DMARD-naïve early RA resulted in significantly more patients achieving sREM and sLDA, improved physical function and inhibited structural damage compared with PBO+dose-optimised MTX. TRIAL REGISTRATION NUMBER: NCT01519791.

Expression patterns of sarco/endoplasmic reticulum Ca(2+)-ATPase and inositol 1,4,5-trisphosphate receptor isoforms in vascular endothelial cells.

Expression patterns of sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase (SERCA) and inositol 1,4,5-trisphosphate receptor (IP3R) isoforms were studied in endothelial cells at the mRNA level by ratio RT-PCR technique and subsequent restriction-enzyme analysis. Three types of cells have been used in the present study: rat adrenal medulla microvascular endothelial cells (RAMEC), rat aortic endothelial cells (RAEC), and human umbilical vein endothelial cells (HUVEC). Our data show the presence of multiple SERCA and IP3R isoforms in each type of endothelial cells. Freshly isolated HUVEC were an exception in this respect since they contained only SERCA3 without SERCA2b messengers. The expression patterns changed upon cell proliferation: SERCA3 and IP3R-1 messengers decreased, while IP3R-3 increased with culturing. Upon cell differentiation, induced by culturing the cells on Matrigel, the expression pattern of the IP3R changed even further in all endothelial cell types: IP3R-1 was reduced in all three cell kinds, while IP3R-3 raised significantly in RAEC and RAMEC. In HUVEC the expression of SERCA returned, upon differentiation, to the levels observed in the freshly isolated cells. Thus, the plasticity of expression of various SERCA and IP3R isoforms shows that possibly different Ca2+ pools may play distinct roles in cell proliferation and differentiation.

Structure of the human sarco/endoplasmic reticulum Ca2+-ATPase 3 gene. Promoter analysis and alternative splicing of the SERCA3 pre-mRNA.

Human chromosome 17-specific genomic clones extending over 90 kilobases (kb) of DNA and coding for sarco/endoplasmic reticulum Ca2+-ATPase 3 (SERCA3) were isolated. The presence of the D17S1828 genetic marker in the cosmid contig enabled us to map the SERCA3 gene (ATP2A3) 11 centimorgans from the top of the short arm p of chromosome 17, in the vicinity of the cystinosis gene locus. The SERCA3 gene contains 22 exons spread over 50 kb of genomic DNA. The exon/intron boundaries are well conserved between human SERCA3 and SERCA1 genes, except for the junction between exons 8 and 9 which is found in the SERCA1 gene but not in SERCA3 and SERCA2 genes. The transcription start site (+1) is located 152 nucleotides (nt) upstream of the AUG codon. The 5'-flanking region, including exon 1, is embedded in a 1.5-kb CpG island and is characterized by the absence of a TATA box and by the presence of 14 putative Sp1 sites, 11 CACCC boxes, 5 AP-2-binding motifs, 3 GGCTGGGG motifs, 3 CANNTG boxes, a GATA motif, as well as single sites for Ets-1, c-Myc, and TFIIIc. Functional promoter analysis indicated that the GC-rich region (87% G + C) from -135 to -31 is of critical importance in initiating SERCA3 gene transcription in Jurkat cells. Exon 21 (human, 101 base pairs; mouse, 86 base pairs) can be alternatively excluded, partially included, or totally included, thus generating, respectively, SERCA3a (human and mouse, 999 amino acids (aa)), SERCA3b (human, 1043 aa; mouse, 1038 aa), or SERCA3c (human, 1024 aa; mouse, 1021 aa) isoforms with different C termini. Expression of the mouse SERCA3 isoforms in COS-1 cells demonstrated their ability to function as active pumps, although with different apparent affinities for Ca2+.

Isovolumetric regulation in a distal nephron cell line (A6).

The effects of gradually reducing the osmolality of the basolateral solution (pi b) were examined in a renal epithelial cell line (A6). pi b was linearly decreased with time from 260 to 140 mosmol/ kgH2O. Cell volume did not change when pi b was gradually decreased at dilution rates (D(r)) of 1-1.5 mosmol. kgH2O-1. min-1. Increasing D(r) to 3 or 6 mosmol. kgH2O-1. min-1 abolished this isovolumetric regulation (IVR). Replacing Cl- by NO3- or SCN- inhibited IVR markedly, whereas Br- substituted perfectly for Cl-. On the other hand, with all these anions, the regulatory volume decrease (RVD) was completely developed. Ba2+ (30 mM) markedly slowed down RVD but improved IVR. The discrepancies between RVD and IVR suggest that different mechanisms are used to control cell volume during gradual and shockwise hyposmotic perturbations. During gradual and shockwise reductions of pi b, cellular K+ content was reduced to the same extent; 86Rb efflux was only partially inhibited by Ba2+. The amount of intracellular K+ depletion could account for 70% of the cationic osmolyte loss, which suggests that K+ is the major cation excreted during both types of perturbations.

Isovolumetric regulation of C6 rat glioma cells in hyperosmotic media.

Volume regulation of C6 glioma cells was studied in response to a gradual increase of extracellular osmolality from 300 to 440 mosmol/kgH2O at 37 degrees C. Maintenance of cell size depended on the rate of osmolality increase (CR): at CR of 3 mosmol.kg-1.min-1, cell volume was kept constant, whereas it decreased progressively at CR of 6 or 9 mosmol.kg-1.min-1. The ability of C6 cells to maintain their volume is termed isovolumetric regulation (IVR). Reducing temperature to 22 degrees C inhibited IVR significantly. Also, bumetanide and ouabain blocked the regulation, while 5-(N,N-dimethyl)amiloride (DMA) did not affect IVR: Extracellular acidification rate (EAR) was studied by microphysiometry. EAR gradually decreased in the presence and increased in the absence of IVR. Experiments with DMA show that these changes in EAR were related to the activity of the Na+/H+ exchanger. It was stimulated by cell shrinkage but not by hyperosmolality itself. Our data demonstrate that C6 glioma cells are able to prevent volume decrease at a low rate of elevation of external osmolality and at 37 degrees C. This process requires electrolyte uptake by the Na(+)-K(+)-2Cl cotransporter and Na+/K+ pump.

Electrolyte transport mechanisms involved in regulatory volume increase in C6 glioma cells.

Volume regulation of C6 glioma cells was studied with an automatic system for monitoring cell thickness, while increasing bath osmolality from 300 to 440 mosmol/kgH2O. At 37 degrees C, tissues incubated in solutions containing active substances (inositol, D-biotin, hydrocortisone, prostaglandin E1, insulin, transferrin, sodium selenite, and 3,5,3'-triiodothyronine) responded to hyperosmotic challenge with a typical regulatory volume increase (RVI). Lowering temperature or removing the active substances inhibited osmoregulation. Bumetanide, amiloride, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, or ouabain significantly reduced RVI. Ion substitutions of Na+, Cl-, NaCl, or HCO3- also importantly affected the process. Extracellular acidification rate (EAR) was studied by microphysiometry. Hyperosmotic shock induced an increase in EAR with a time course that matched volume recovery. This increase in EAR was prevented by amiloride. The data show that under hyperosmotic conditions C6 cells are able to regulate their volume. Ion substitutions and application of blockers demonstrate that Na+/H+ and Cl-/HCO3- exchangers and Na(+)-K(-)-2Cl- cotransporter are involved in RVI. The rise in EAR is due to the enhanced activity of Na+/H+ antiporter, which seems to be volume dependent but not osmotic dependent.

On the neuro-muscular control of trochlean joints.

This paper presents a simplified dynamical model for the control of one-degree-of-freedom synovial joints considered as pure trochlean joints. This model considers the joint dynamics, the dynamics of the corresponding muscles and their calcium balance dynamics, as well as position and force feedbacks provided by the spindles and the Golgi tendon organs. Delays in the transmission of information are also taken into account as they proved to be of critical importance for the dynamical behavior of the considered systems. The linearized version of this model, which is valid for a rather wide range of movements, also allows us to investigate the stability of the system, as well as its stability robustness with respect to the feedback gains. Further, particular behaviors such as tremor are described.

Volume regulation in rat brain glial cells: lack of a substantial contribution of free amino acids.

Volume regulation of C6 glioma cells was studied while the bath osmolality was reduced from 300 to 150 mosmol/kg. Exposure to a hyposmotic challenge elicited a typical regulatory volume decrease (RVD). No regulatory volume increase was observed upon restoration of isosmotic conditions. During a second subsequent hyposmotic challenge, the cells did not respond with RVD. High extracellular K+ concentration and the K+ channel blockers Ba2+ and quinine inhibited the RVD. RVD was abolished after Cl- was replaced by gluconate and by the Cl- channel blocker 5-nitro-2(3-phenylpropylamino)benzoic acid. Amino acid (AA) concentration in cell and perfusate was determined. In control, cell content was only 26 mmol/l. Hypotonicity increased the efflux of AA from 0.14 to 0.60 mmol/min. During the second hyposmotic challenge, the release was 0.32 mmol/min. The data show that C6 cells adjust their volume under hyposmotic conditions but lose the ability to restore their volume during a subsequent hyposmotic treatment. K+ and Cl- are the main osmolytes involved in volume adjustment through conductive pathways. AA do not contribute substantially to cell volume regulation.

Effect of dexamethasone on sodium channel block and densities in A6 cells.

The association (ON) and dissociation (OFF) rates of either positively charged amiloride or its uncharged analogue, CDPC (6-chloro-3, 5-diaminopyrazine-2-carboxamide), with the apical Na+ channel protein of renal A6 cells were analysed during exposure to the synthetic glucocorticoid, dexamethasone, using noise analysis. These rates were further used to reach specific conclusions about single-channel current, channel density and open probability of the channel in the absence of the blocker. Short-term exposure (3 h) to 10(-7) mol/l dexamethasone at the basolateral side increased the short-circuit current, Isc by 85%, without a change in the ON and OFF rates of the interaction between amiloride and the Na+ channel. A longer incubation (24 h) with dexamethasone tripled the current with a notable increase in the ON rate of the interaction between amiloride and the and channel. The OFF rate remained constant. The effects of dexamethasone on the rate constants of the reaction of amiloride with the channel did not match with the expected changes in membrane potential. On the other hand, ON and OFF rates of the interaction between neutral CDPC and the channel were not influenced by a 24-h incubation with dexamethasone. Further calculations disclosed that the gain in macroscopic current after a 24-h incubation with dexamethasone might be explained by an increase in Na+ channel density, and, to a lesser extent, by a rise in single-channel current. This all occurred without a change in the fraction of time spent by the channel in the conducting state in the absence of the blocker.

Inhibition of endothelium-derived relaxing factor in A6 cells.

The effects of endothelium-derived relaxing factor (EDRF) on Na+ transport in distal renal tubular A6 cells have been studied by inhibition of its synthesis with L-NAME (10(-2) mol/l). Na+ transport was monitored by measuring short-circuit current, cell voltage, transepithelial, apical and basolateral membrane conductances. EDRF production in A6 cells was tested by application of its substrate L-arginine. The blockade of EDRF decreased significantly the Na+ current (11 %), membrane potential (5 mV) and basolateral conductance (33 %), but did not affect the apical membrane conductance. Activation of apical Na+ conductance by dexamethasone incubation (10(-7) mol/l) did not further influence the drop in Na+ current. The involvement of basolateral K+ channels in cell depolarization and in the reduction of basolateral conductance was tested in tissues with elevated basolateral K+/Cl- conductance ratios (by increasing bath osmolarity) and by application of barium (0.5-10(-3) mol/l) a K+ channel blocker. The results showed that the effect of L-NAME on the short-circuit current was more pronounced in A6 cells with increased K+/Cl- conductance ratios, but was almost nullified by barium. Finally, L-arginine fully restored the Na+ current, thus reversing the inhibition induced by L-NAME. We conclude that EDRF is basally released in A6 cells. Inhibition of EDRF by L-NAME directly interferes with Na+ reabsorption. Since apical membrane conductance remains unchanged, the decrease in short-circuit current results from cell depolarization. The latter, together with the drop in basolateral conductance, might reflect inactivation of K+ channels.

Effect of ouabain on membrane conductances and volume in A6 cells.

The present study reports the effect of a reduction in the Na(+)-transport rate on cell volume. A decrease in transport rate was achieved by inhibition of the basolateral Na+/K+ pump with ouabain. Cultured A6 cell monolayers were short-circuited and exposed to ouabain at the basolateral surface. In one series of experiments, cells were impaled with microelectrodes to measure cell voltage, apical fractional resistance and thus derive membrane conductances. Another set, A6, served for cell height measurements. Ouabain decreased short-circuit current (Isc), which is an index of transepithelial Na+ transport: the reduction in transport rate varied from 26 to 79% within 10 min. Equivalent circuit analysis revealed a 20% decrease in apical membrane conductance (ga), whereas basolateral membrane conductance (gb) increased by 66%. A decrease in cell voltage (12 mV) together with drop in ga during ouabain may account for the reduction in Isc. The rise in gb is mainly due to a gain in Cl- conductance which increased from 114 to 613 microS/cm2, compatible with activation of Cl- channels. All of this occurs without a detectable change in cell height. We may conclude from these data that inhibition of Na+ exit by ouabain is quickly compensated by a decrease in apical Na+ entry and an increase in basolateral Cl- conductance. Constant cell volume during ouabain implies that the total cell solute is essentially unchanged.