Are rheumatologists adhering to the concepts window of opportunity and treat-to-target? Earlier and more intense disease-modifying anti-rheumatic drug treatment over time in patients with early arthritis in the PEARL study.

OBJECTIVES: To analyse changes over time in the treatment with disease modifying anti-rheumatic drugs and biological therapies prescribed to patients from an early arthritis register and whether these changes had an impact on their outcome. METHODS: This was a longitudinal retrospective 2-year study based on data collected in the PEARL study. The population was clustered in three groups depending on year of symptoms onset (2000-2004, 2005-2009, 2010-2014). Intensity of disease-modifying anti-rheumatic drug treatment was calculated and the percentage of patients receiving biological therapy during the first 2-year follow-up was collected. Disease activity and remission at the end of follow-up, as well as radiological progression were the outcomes analysed. Multivariable analyses were fitted to determine which variables including the three period times were associated with the outcomes. RESULTS: A significant increase in treatment intensity was observed in patients with undifferentiated arthritis, getting closer to that prescribed to patients fulfilling the 1987 RA criteria at the last period studied (2010-2014). This finding was associated with a significantly higher percentage of patients in remission and lower progression of the erosion component of the Sharp van der Heijde score. CONCLUSIONS: During the last 15 years, the treatment of patients with early arthritis in our hospital has been progressively increased and it has been associated with significantly better outcomes.

Anti-CCR7 therapy exerts a potent anti-tumor activity in a xenograft model of human mantle cell lymphoma.

BACKGROUND: The chemokine receptor CCR7 mediates lymphoid dissemination of many cancers, including lymphomas and epithelial carcinomas, thus representing an attractive therapeutic target. Previous results have highlighted the potential of the anti-CCR7 monoclonal antibodies to inhibit migration in transwell assays. The present study aimed to evaluate the in vivo therapeutic efficacy of an anti-CCR7 antibody in a xenografted human mantle cell lymphoma model. METHODS: NOD/SCID mice were either subcutaneously or intravenously inoculated with Granta-519 cells, a human cell line derived from a leukemic mantle cell lymphoma. The anti-CCR7 mAb treatment (3 × 200 µg) was started on day 2 or 7 to target lymphoma cells in either a peri-implantation or a post-implantation stage, respectively. RESULTS: The anti-CCR7 therapy significantly delayed the tumor appearance and also reduced the volumes of tumors in the subcutaneous model. Moreover, an increased number of apoptotic tumor cells was detected in mice treated with the anti-CCR7 mAb compared to the untreated animals. In addition, significantly reduced number of Granta-519 cells migrated from subcutaneous tumors to distant lymphoid organs, such as bone marrow and spleen in the anti-CCR7 treated mice. In the intravenous models, the anti-CCR7 mAb drastically increased survival of the mice. Accordingly, dissemination and infiltration of tumor cells in lymphoid and non-lymphoid organs, including lungs and central nervous system, was almost abrogated. CONCLUSIONS: The anti-CCR7 mAb exerts a potent anti-tumor activity and might represent an interesting therapeutic alternative to conventional therapies.

Angiotensin II differentially modulates cyclooxygenase-2, microsomal prostaglandin E2 synthase-1 and prostaglandin I2 synthase expression in adventitial fibroblasts exposed to inflammatory stimuli.

AIMS: To assess whether angiotensin II (Ang II) modulates key enzymes of the cyclooxygenase (COX)-2/prostanoid pathway, including prostaglandin E synthase-1 (mPGES-1) and prostacyclin synthase (PGIS) in rat aortic adventitial fibroblasts in the presence or absence of an inflammatory stimulus [interleukin (IL)-1ß]. METHODS AND RESULTS: Fibroblasts stimulated with IL-1ß (10 ng/ml, 24 h) and/or Ang II (0.1 µmol/l, 24 h) were used. IL-1ß up-regulated COX-2 and mPGES-1 (protein and mRNA) and increased PGI2 and PGE2 release, without altering PGIS protein expression. Ang II did modify neither COX-2 and mPGES-1 expression nor prostanoid levels, but it induced PGIS expression. Interestingly, Ang II further enhanced IL-1ß-induced COX-2 expression and PGI2 release and concomitantly reduced IL-1ß-induced mPGES-1 expression. The AT1 receptor antagonist losartan prevented the effects of Ang II on IL-1ß-induced COX-2 or mPGES-1 expression. IL-1ß activated p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK)1/2 pathways, and coincubation with Ang II resulted in a higher and more sustained phosphorylation of both MAPK. Inhibition of either p38 MAPK (SB203580) or ERK1/2 (PD98059) reduced COX-2 and mPGES-1 expression in cells treated with IL-1ß or the combination of IL-1ß and Ang II. Ang II did not modify COX-2 transcriptional activity but increased COX-2 mRNA stability in IL-1ß-treated cells; by contrast, it increased PGIS mRNA levels through a transcriptional mechanism. CONCLUSION: Ang II differentially modulates key enzymes involved in prostanoid biosynthesis thereby altering the balance between PGI2/PGE2 in vascular cells exposed to inflammatory stimuli.

Analysis of migratory and prosurvival pathways induced by the homeostatic chemokines CCL19 and CCL21 in B-cell chronic lymphocytic leukemia.

OBJECTIVE: The CCR7 chemokine receptor has been reported to promote homing of B-cell chronic lymphocytic leukemia (CLL) cells into lymph nodes and support their survival, but the mechanisms mediating these effects are largely unknown. We investigated the role of different signaling pathways triggered by CCR7 engagement by its ligands, the chemokines CCL19 and CCL21, in the control of CLL migration and survival. MATERIALS AND METHODS: Chemotaxis and apoptosis assays were performed in the presence of pharmacologic inhibitors and genetic mutants of the phosphatidylinositol-3-OH kinase (PI3K), Rho guanosine triphosphatase, and mitogen-activated protein kinase (MAPK) signaling cascades to assess the role of these pathways on primary CLL migration and survival in response to CCR7 activation. Kinase activation was determined by immunoblotting and pull-down experiments. RESULTS: CLL chemotactic activity induced by CCL19 or CCL21 was markedly reduced by inhibitors of PI3K and the Rho effector molecule Rho-associated coiled-coil forming protein kinases (ROCK), and also by the expression of dominant negative forms of PI3K and RhoA, whereas constitutively activated PI3K and RhoA mutants strongly promoted CLL migration. In contrast, MAPKs were not significantly involved in CLL migration to CCL19/CCL21. Conversely, extracellular signal-regulated kinase and c-Jun-N-terminal kinase, along with PI3K, had a role in CCR7-mediated CLL cell survival. Biochemical experiments confirmed that CCL19/21 induced PI3K-dependent phosphorylation of Akt/protein kinase B, activation of the Rho/Rho-associated coiled-coil forming protein kinases/myosin light chain pathway and MAPKs phosphorylation. CONCLUSIONS: The role of PI3K, Rho guanosine triphosphatases, and MAPKs in CCR7-mediated CLL cells migration and survival suggests that these signal transduction pathways could represent promising targets for CLL therapy.

p38 MAPK contributes to angiotensin II-induced COX-2 expression in aortic fibroblasts from normotensive and hypertensive rats.

OBJECTIVE: To investigate the effect of angiotensin II on cyclooxygenase-2 (COX-2) expression in aortic adventitial fibroblasts from normotensive [Wistar-Kyoto (WKY)] rats and spontaneously hypertensive rats (SHRs). METHODS: Protein expression was determined by western blot, mRNA levels by real-time PCR, transcriptional activity by luciferase assays, superoxide anion (O2*-) production by dihydroethidine fluorescence and prostaglandin E2 by enzyme immunoassay. RESULTS: Angiotensin II (0.1 micromol/l, 0.5-6 h) time dependently induced COX-2 protein expression, this effect being transient in fibroblasts from WKY rats and maintained over time in SHRs. Angiotensin II effect was abolished by valsartan (1 micromol/l), an angiotensin II type 1 receptor antagonist. Angiotensin II-induced prostaglandin E2 production was reduced by valsartan and the COX-2 inhibitor NS398 (1 micromol/l). Angiotensin II increased O2*- production more in SHR than WKY rats. This increase was reduced by apocynin (30 micromol/l) and allopurinol (10 micromol/l), respective nicotinamide adenine dinucleotide phosphate (NADPH) and xanthine oxidase inhibitors. However, angiotensin II-induced COX-2 expression was unaffected by apocynin, allopurinol, tempol (1 mmol/l) or catalase (1000 U/ml). Angiotensin II (2-30 min) induced p38 mitogen-activated protein kinase (MAPK) phosphorylation, transiently in WKY rats but sustained in SHRs. The p38 inhibitor SB203580 (10 micromol/l) reduced angiotensin II-induced COX-2 protein and mRNA levels. The angiotensin II effect was not prevented by inhibition of mRNA synthesis, and angiotensin II was unable to modulate COX-2 transcriptional activity. CONCLUSIONS: Angiotensin II increases COX-2 expression in aortic fibroblasts through mechanisms including p38 MAPK pathway, independent of reactive oxygen species production and nonmediated by COX-2 transcriptional activity modulation. The sustained angiotensin-induced p38 MAPK activation in SHR cells might be related to the maintained COX-2 expression in this strain.

Hypertension increases contractile responses to hydrogen peroxide in resistance arteries through increased thromboxane A2, Ca2+, and superoxide anion levels.

This study investigated the mechanisms underlying the response to hydrogen peroxide (H(2)O(2)) in mesenteric resistance arteries from spontaneously hypertensive rats (SHRs) and normotensive Wistar Kyoto (WKY) rats. Arteries were mounted in microvascular myographs for isometric tension recording and for simultaneous measurements of intracellular Ca(2+) concentration ([Ca(2+)](i)), superoxide anion (O(2)(.)) production was evaluated by dihydroethidium fluorescence and confocal microscopy, and thromboxane A(2) (TXA(2)) production was evaluated by enzyme immunoassay. H(2)O(2) (1-100 microM) induced biphasic responses characterized by a transient endothelium-dependent contraction followed by relaxation. Simultaneous measurements of tension and Ca(2+) showed a greater effect of H(2)O(2) in arteries from hypertensive than normotensive rats. The cyclooxygenase (cox) inhibitor, indomethacin [1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1-H-indole-3-acetic acid] (1 microM); the COX-1 inhibitor, SC-58560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethyl pyrazole] (1 microM); the thromboxane (TXA(2)) synthase inhibitor, furegrelate [5-(3-pyridinylmethyl)-2-benzofurancarboxylic acid, sodium salt] (10 microM); and the TXA(2)/prostaglandin H(2) receptor antagonist, SQ 29,548 ([1S-[1.alpha.,2.alpha.(Z),3.alpha.,4.alpha.]]-7-[3-[[2-[(phenylamino) carbonyl] hydrazino] methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid)) (1 microM) abolished H(2)O(2) contraction in arteries from WKY rats but only reduced it in SHRs. The O(2)(.) scavenger, tiron (4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt) (1 mM), and the NADPH oxidase inhibitor, apocynin (4'-hydroxy-3'-methoxyacetophenone) (0.3 mM), decreased H(2)O(2) contraction in arteries from SHRs but not in WKY rats. H(2)O(2) induced TXA(2) and O(2)(.) production that was greater in SHRs than in WKY rats. The TXA(2) analog, U46619 [9,11-di-deoxy-11 alpha,9 alpha-epoxymethano prostaglandin F(2 alpha) (0.1 nM-1 microM)], also increased O(2)(.) production in SHR vessels. H(2)O(2)-induced TXA(2) production was decreased by SC-58560. H(2)O(2)-induced O(2)(.) production was decreased by tiron, apocynin, and SQ 29,548. In conclusion, the enhanced H(2)O(2) contraction in resistance arteries from SHRs seems to be mediated by increased TXA(2) release from COX-1 followed by elevations in vascular smooth muscle [Ca(2+)](i) levels and O(2)(.) production. This reveals a new mechanism of oxidative stress-induced vascular damage in hypertension.

Ouabain treatment increases nitric oxide bioavailability and decreases superoxide anion production in cerebral vessels.

OBJECTIVE: Chronic administration of ouabain induces hypertension and increases the contribution of nitric oxide to vasoconstrictor responses in peripheral arteries. The aim of this study was to analyse whether ouabain treatment alters the nitric oxide bioavailability in cerebral arteries. METHODS: Basilar arteries from control and ouabain-treated rats ( approximately 8.0 microg/day, 5 weeks) were used. Vascular reactivity was analysed by isometric tension recording, protein expression by western blot, nitric oxide levels by diaminofluorescein-induced fluorescence, superoxide anion (O2) production by ethidium fluorescence and lucigenin chemiluminescence and plasma total antioxidant status by a commercial kit. RESULTS: The relaxations induced by bradykinin (1 nmol/l-10 micromol/l) and L-arginine (0.01-300 micromol/l) and the contractile responses induced by both N-nitro-L-arginine methyl ester (0.1-100 micromol/l) and oxyhaemoglobin (0.01-10 micromol/l) were greater in arteries from ouabain-treated than control rats. However, the relaxation to diethylamine NONOate-nitric oxide (0.1 nmol/l-10 micromol/l) and the contractions to KCl (7.5-120 mmol/l) and 5-hydroxytryptamine (0.01-10 micromol/l) were similar in arteries from both groups. Ouabain treatment increased basal nitric oxide levels but did not modify endothelial and neuronal nitric oxide synthase protein expression. O2 production was lower in cerebral arteries from ouabain-treated rats; however, plasma total antioxidant status and vascular protein expression of Cu/Zn-superoxide dismutase, Mn-superoxide dismutase and extracellular superoxide dismutase were similar in both groups. CONCLUSION: Chronic ouabain treatment increased nitric oxide basal levels in basilar arteries probably due to the decreased O2 levels. This might be an adaptive mechanism of the cerebral vasculature to the increase in blood pressure.

Losartan reduces the increased participation of cyclooxygenase-2-derived products in vascular responses of hypertensive rats.

This study analyzes the role of angiotensin II (Ang II), via AT1) receptors, in the involvement of cyclooxygenase (COX)-2-derived prostanoids in phenylephrine responses in normotensive rats (Wistar Kyoto; WKY) and spontaneously hypertensive rats (SHR). Aorta from rats untreated or treated for 12 weeks with losartan (15 mg/kg . day) or hydralazine plus hydrochlorothiazide (44 and 9.4 mg/kg . day, respectively) and vascular smooth muscle cells (VSMC) from SHR were used. Vascular reactivity was analyzed by isometric recording; COX-2 expression by Western blot and reverse transcription-polymerase chain reaction; prostaglandin (PG)I2, PGF(2alpha), 8-isoprostane, and total antioxidant status (TAS) by commercial kits; superoxide anion (O2*-) by lucigenin chemiluminescence; and plasmatic malondialdehyde (MDA) by thiobarbituric acid assay. The COX-2 inhibitor N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methane sulfonamide (NS-398) at 1 microM reduced phenylephrine responses more in SHR than in WKY rats. COX-2 protein and mRNA expressions, PGF(2alpha), PGI2, 8-isoprostane, and O2*- production, and MDA levels were higher in SHR, but TAS was similar in both strains. Losartan, but not hydralazine-hydrochlorothiazide treatment, reduced COX-2 expression and the effect of NS-398 on phenylephrine responses in SHR. Losartan also increased TAS and reduced PGF(2alpha), PGI2, 8-isoprostane, and O2*- production and MDA levels in SHR. Ang II (0.1 microM) induced COX-2 expression in VSMC from SHR that was reduced by 30 microM apocynin and 100 microM allopurinol, NADPH oxidase, and xanthine oxidase inhibitors, respectively. In conclusion, AT1 receptor activation by Ang II could be involved in the increased participation of COX-2-derived contractile prostanoids in vasoconstriction to phenylephrine with hypertension, probably through COX-2 expression regulation. The increased oxidative stress seems to be one of the mechanisms involved.

High glucose enhances inducible nitric oxide synthase expression. Role of protein kinase C-betaII.

The aim was to determine whether high glucose levels interfere with nitric oxide (NO) production and inducible NO synthase (iNOS) protein expression in interleukin-1beta-stimulated vascular smooth muscle cells from normotensive Wistar Kyoto and spontaneously hypertensive rats. Cells were incubated with either normal (5.5 mM) or high (22 mM) d-glucose for 72 h and with interleukin-1beta (10 ng/ml) for the last 24 h. High glucose increased nitrite levels, iNOS expression and protein kinase C activity in cells from normotensive rats and had no effect in cells from hypertensive rats. High glucose effects on nitrite production and iNOS expression was abolished by the selective inhibitor for the protein kinase C-betaII, 5,21:12,17-dimetheno-18H-dibenzo[i,o]pyrrolo[3,4-1] [1,8]diacyclohexadecine-18,20 (19H)-dione, 8-[(dimethylamino) methyl]-6,7,8,9,10,11-hexahydro-monomethanesulfonate (LY379196, 30 nM). Calphostin C (1 microM) and LY379196 (10 microM) reduced nitrite levels and iNOS expression only in cells from normotensive rats treated with both media. These results suggest that high glucose increases inducible nitric oxide synthase induction and subsequent NO production by activating the protein kinase C-betaII; this mechanism seems to be altered in hypertension.

Vascular effects of the Mangifera indica L. extract (Vimang).

The effects of the Mangiferia indica L. (Vimang) extract, and mangiferin (a C-glucosylxanthone of Vimang) on the inducible isoforms of cyclooxygenase (cyclooxygenase-2) and nitric oxide synthase (iNOS) expression and on vasoconstrictor responses were investigated in vascular smooth muscle cells and mesenteric resistance arteries, respectively, from Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. Vimang (0.5-0.1 mg/ml) and mangiferin (0.025 mg/ml) inhibited the interleukin-1beta (1 ng/ml)-induced iNOS expression more in SHR than in WKY, and cyclooxygenase-2 expression more in WKY than in SHR. Vimang (0.25-1 mg/ml) reduced noradrenaline (0.1-30 microM)- and U46619 (1 nM-30 microM)- but not KCl (15-70 mM)-induced contractions. Mangiferin (0.05 mg/ml) did not affect noradrenaline-induced contraction. In conclusion, the antiinflammatory action of Vimang would be related with the inhibition of iNOS and cyclooxygenase-2 expression, but not with its effect on vasoconstrictor responses. Alterations in the regulation of both enzymes in hypertension would explain the differences observed in the Vimang effect.

Heparin and low molecular weight heparin decrease nitric oxide production by human polymorphonuclear cells

Background. Heparin and heparin derivatives with low anticoagulant activity exhibit a wide spectrum of biological functions affecting adhesion, activation and trafficking of luekocytes. Methods. We investigated the in vitro effect of heparin and low molecular weight heparin derivative (LMWH) on nitric oxide (NO) production by human polymorphonuclear leukocytes (PMN). Results. N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated NO production was significantly decreased by heparin at doses of 0.5 and 5 µg/mL, while LMWH was only effective at doses of 50 and 200 µg/mL by means of a mechanism not related to No synthase (NOS) activity. Conclusions. These results support the hypothesis that heparin and LMWH derivatives may offet therapeutic benefit for inflammatory diseases where No plays a protagonic role