Anti-carbamylated Protein Antibody Levels Correlate with Anti-Sa (Citrullinated Vimentin) Antibody Levels in Rheumatoid Arthritis.

OBJECTIVE: The presence of anticitrullinated protein antibodies (ACPA) in rheumatoid arthritis (RA) indicates a breach in immune tolerance. Recent studies indicate that this breach extends to homocitrullination of lysines with the formation of anti-carbamylated protein (anti-CarP) antibodies. We analyzed the clinical and serologic relationships of anti-CarP in 2 RA cohorts. METHODS: Circulating levels of immunoglobulin G anti-CarP antibodies were determined by ELISA in established (Dartmouth-Hitchcock Medical Center) and early (Sherbrooke University Hospital Center) cohorts and evaluated for anticyclic citrullinated peptide antibodies (anti-CCP), specific ACPA, and rheumatoid factor (RF) levels using the Student t test and correlation analysis. RESULTS: We identified elevated anti-CarP antibodies titers in 47.0% of seropositive patients (Dartmouth, n = 164), with relationships to anti-CCP (p < 0.0001) and IgM-RF (p = 0.001). Similarly, 38.2% of seropositive patients from the Sherbrooke cohort (n = 171) had elevated anti-CarP antibodies; titers correlated to anti-CCP (p = 0.01) but not IgM-RF (p = 0.09). A strong correlation with anti-Sa was observed: 47.9% anti-Sa+ patients were anti-CarP antibodies+ versus only 25.4% anti-Sa- in the Sherbrooke cohort (p = 0.0002), and 62.6% anti-Sa+ patients versus 26.9% anti-Sa- were anti-CarP antibodies+ in Dartmouth (p < 0.0001). We found a more variable response for reactivity to citrullinated fibrinogen or to citrullinated peptides from fibrinogen and α enolase. CONCLUSION: In 2 North American RA cohorts, we observed a high prevalence of anti-CarP antibody positivity. We also describe a surprising and unexpected association of anti-CarP with anti-Sa antibodies that could not be explained by cross-reactivity. Further, considerable heterogeneity exists between anti-CarP reactivity and other citrullinated peptide reactivity, raising the question of how the pathogenesis of antibody responses for carbamylated proteins and citrullinated proteins may be linked in vivo.

Evaluation of the antitumoral effect of dihydrocucurbitacin-B in both in vitro and in vivo models.

AIMS: We evaluated both in vitro and in vivo antitumoral properties of an isolated compound from Wilbrandia ebracteata, dihydrocucurbitacin-B (DHCB), using B16F10 cells (murine melanoma). MATERIALS AND METHODS: We made use of MTT and (3)H-Thymidine assays to investigate the cell viability and cell proliferation, flow cytometry analysis to monitor cell cycle and apoptosis, western blot analysis to evaluate the expression of cell cycle proteins, imunofluorescence analysis and in vivo tumor growth and metastasis. RESULTS: Dihydrocucurbitacin-B significantly reduced cell proliferation without important effects on cells viability. DHCB lead cells to accumulate in G2/M phases accompanied by the appearance of polyploid cells, confirmed by fluorescence assays that demonstrated a remarkable alteration in the cell cytoskeleton and formation of binuclear cells. Annexin-V-FITC incorporation demonstrated that DHCB did not induce apoptosis. About 10 microg/mL DHCB was found to decrease cyclin-A, and especially in cyclin-B1. The in vivo experiments showed that DHCB treatment (once a day up to 12 days; p.o.) was able to reduce the tumor growth and lung metastasis up to 83.5 and 50.3%, respectively. CONCLUSIONS: Dihydrocucurbitacin-B reduces cell proliferation due to a decrease in the expression of cyclins, mainly cyclin-B1 and disruption of the actin cytoskeleton, arresting B16F10 cells in G2/M phase. Taken together, the in vitro and in vivo experiments suggest that DHCB was effective against cancer, however, it remains to be proved if DHCB will be a good candidate for drug development.

Anti-inflammatory effects of a triterpenoid isolated from Wilbrandia ebracteata Cogn.

Wilbrandia ebracteata (WE), a Brazilian medicinal plant used in folk medicine for the treatment of rheumatic diseases, displays anti-inflammatory properties and constitutes a rich source of cucurbitacins and cucurbitacin-related compounds. The current study investigated the potential anti-inflammatory properties of Dihydrocucurbitacin B (DHCB), a cucurbitacin-derived compound isolated from roots of WE, in some in vivo and in vitro experimental models. Intraperitoneal treatment of mice with DHCB reduced both carrageenan-induced paw edema (0.3, 1 and 3 mg/kg caused inhibitions of 26, 44 and 56 % at 2 h after stimulation, respectively) and pleurisy (10 mg/kg inhibited leukocyte numbers and LTB(4) levels in the pleural fluid by 51 and 75% at 6 h after cavity challenge, respectively). In vitro, DHCB (up to 10 microg/mL) failed to modify LTB(4) production by human neutrophils or PGE(2) production by COS-7 cells transfected with COX-1, but PGE(2) production by COX-2 transfected COS-7 cells was markedly inhibited (by 72%). The levels of COX-1 or COX-2 proteins in IL-1alpha-stimulated NIH3T3 cells were unaffected by DHCB. The results corroborate the potential anti-inflammatory properties ascribed to W. ebracteata Cogn. in folk medicine and suggest that they might be attributed, at least in part, to the capacity of one of this plants main constituents, DHCB, to inhibit COX-2 activity (but not its expression) during inflammation.

Prostaglandin EP4 receptor agonist protects against acute neurotoxicity.

Under various abnormal physiologic conditions, overactivation of glutamate-gated ion channel receptor family members, including NMDA receptors, causes increase in COX-2 expression and generation of prostaglandins. PGE(2) exerts its physiologic actions mainly through its PGE(2) prostanoid (EP) receptors. In the present study, the role of the EP4 receptor against NMDA-induced excitotoxicity was investigated. Using the EP4 receptor agonist ONO-AE1-329, which has relative selectivity toward murine EP receptors on the order of EP1:EP2:EP3:EP4 of >1000:210:120:1, respectively, we questioned whether activation of the EP4 receptors has the potential to attenuate injury in brain. Mice were pretreated by intracerebroventricular injection with different doses of ONO-AE1-329 (0.1, 1, and 10 nmol; n = 9/group) and, after 20 min, by a single unilateral intrastriatal injection of NMDA (15 nmol, n = 12). NMDA injection produced a significant lesion in the ipsilateral striatum. This lesion volume was significantly reduced in groups that were pretreated with ONO-AE1-329, with maximum protection of more than 32% at 10 nmol. This is the first study revealing the protective effect of ONO-AE1-329 in an acute model of excitotoxicity in brain, and it suggests that preferential stimulation of EP4 receptors attenuates excitotoxic brain injury.

Nitric oxide and cyclooxygenase may participate in the analgesic and anti-inflammatory effect of the cucurbitacins fraction from Wilbrandia ebracteata.

Wilbrandia ebracteata is a medicinal plant from South America used in folk medicine for the treatment of chronic rheumatic diseases. We have shown that the high performance liquid chromatography-characterized (HPLC) dichloromethane fraction isolated from Wilbrandia ebracteata (WEDC) inhibits the parameters observed in experimental models of inflammation in vivo and in vitro. In the present study, we extend our previous observations on the analgesic effects of WEDC by investigating its actions using the hot plate test and zymosan-induced writhing test in mice, as well as zymosan-induced arthritis in rats evaluating articular inflammatory pain, cell migration and determination of NO release into the joint exudate. The effect of WEDC on the activity of COX-1 and COX-2 in vitro and its ulcerogenic capacity in vivo were also investigated. The oral treatment of the animals with WEDC (1-10 mg/kg) produced a significant, dose-dependent reduction of articular incapacitation and abdominal contortions in the writhing test. The same effect was not observed in the hot plate and rota-rod tests. WEDC also reduced nitrite release into the zymosan-inflamed joints. In the evaluation of COX activity, we observed that WEDC was able to selectively inhibit COX-2 but not COX-1 activity in COS-7 cells. Moreover, WEDC treatment did not show gastrointestinal toxicity. Our data confirm the anti-nociceptive activities of the WEDC and indicate that this effect could be associated with inhibition of cyclooxygenase-2 (COX-2) and nitric oxide release. The effects could be attributed to cucurbitacins since several of these were isolated from the WEDC.

Evidence that peroxynitrite affects human osteoblast proliferation and differentiation.

Peroxynitrite (PN), a nitric oxide (NO*)-derived anion, has been associated with NO* damage in various cell types. We examined the effects of adding PN to cultured human osteoblast-like (hOB) cells obtained after hip arthroplasty. Exposure to PN (0.1-0.4 mM) decreased both hOB proliferation and differentiation, measured by [3H]thymidine uptake and alkaline phosphatase production, respectively. Incubation with 3-morpholinosydnonimine (SIN-1; 0.25-1 mM), an NO* and O2- donor that leads to PN release, also reduced both hOB proliferation and differentiation. Coincubation with both superoxide dismutase (SOD; 100 U/ml) and catalase (CAT; 50 U/ml), rendering SIN-1 a pure NO* donor, reversed its effects on hOB proliferation and differentiation. However, SIN-1-induced NO* production, measured by nitrite release to the hOB medium, was not altered by cotreatment with SOD and CAT. Expression of nitrotyrosine by hOB, a marker of PN action, was significantly increased after SIN-1 addition, as compared with untreated cells, as revealed by Western blot analysis. Interleukin-1alpha (IL-1alpha) and interferon gamma (IFN-gamma) but not tumor necrosis factor alpha (TNF-alpha) also significantly increased nitrotyrosine expression in these cells. These data show that PN is at least partially responsible for osteoblast derangement by NO* and that cytokines released during inflammatory arthropathies can induce PN production in hOB cells.

Antigen-induced arthritis in rabbits: a suitable experimental model for the study of arachidonic acid metabolites in articular inflammatory processes

O presente estudo investigou a presença de substâncias de reaçäo lenta da anafilaxia (SRS-A) no líquido sinovial de coelhos com artrite induzida por antígeno utilizando ensaio biológico em ileo isolado de cobaio suspenso em câmara oxigenada contendo atropina, mepiramina e metisergide. Considerou-se SRS-A presente quando a infusäo do líquido sinovial promovia uma contraçäo com pelo menos três minutos de duraçäo que era abolida pela adiçäo de FPL-5512 ao meios. Todos os líquidos sinoviais de joelhos injetados com o antígeno de animais sacrificados até oito horas a partir do início da artrite apresentaram atividade de SRS-A. Nenhum líquido de articulaçöes experimentais com mais de oito horas de artrite, assim como em nenhuma das articulaçöes controles foi detectada a presença dessas substâncias. Esses resultados tornam o modelo experimental em questäo apropriado para o estudo da participaçäo das leucotrienes na fisiopatologia dos processos inflamatórios articulares assim como para o ensaio e desenvolvimento de novas drogas anti-inflamatórias

Artrite induzida por antígeno em coelhos: padronizaçäo dos métodos de induçäo e avaliaçäo do processo inflamatório articular / Antigen-induced sothritis in ralbbit: standardization of the methods of induction and evaluation of the articular inflamatory process

Em 48 coelhos foi induzida artrite por injeçäo intra-articular de soro-albumina bovina, antígeno ao qual os animais haviam sido previamente imunizados de acordo com um protocolo bem definido: os animais foram sacrificados em grupos de três após períodos que variaram de uma hora a oito meses a contar da induçäo da artrite. O estudo do líquido e membranas sinoviais revelou inflamaçäo articular aguda nas primeiras 24 horas após a injeçäo intra-articular do antígeno: em 48 horas as características foram intermediárias entre um processo agudo e crônico, assumindo caráter crônico nos animais sacrificados a partir de entäo. Foram avaliados 11 parâmetros histopatológicos indicadores de sinovite: erosäo da camada de revestimento, proliferaçäo vascular, hemorragia, infiltrado inflamatório, nódulos linfóides, granulomas, dilataçäo e congestäo vascular, hiperplasia e hipertrofia da camada de revestimento, viscosidades, fibrose e vasculite; a presença de três ou mais desses parâmetros forneceu o melhor critério objetivo para o diagnóstico de sinovite. As alteraçöes histopatológicas encontradas foram semelhantes àquelas descritas em membrana sinovial de pacientes com doença reumatóide; a padronizaçäo deste modelo experimental propicia-nos meio de grande utilidade para o estudo dessa patologia humana