Vanessa Schuck Clarimundo

N-acetylcysteine prevents behavioral and biochemical changes induced by alcohol cessation in rats

N-acetylcysteine (NAC), a glutamate-modulating agent with antioxidant and anti-inflammatory properties, has been considered as a potential anti-addictive drug. Beneficial effects were reported for cocaine, cannabis, and tobacco addicts, but the effect of NAC in alcoholics or in alcohol animal models is unknown. The aggravation of alcohol withdrawal symptoms, such as anxiety, has been associated with increased levels of serum corticosterone and leptin. Thus, the aim of this study was to assess the effects of NAC on anxiety, as well as corticosterone and leptin serum levels, after cessation of chronic alcohol treatment in rats. Male Wistar rats were treated with 2 g/kg ethanol, twice daily, by gavage for 30 days; control animals received an appropriate dose of glucose to balance caloric intake. Rats were treated for 4 days with NAC (60 and 90 mg/kg, intra-peritoneally [i.p.]) or saline after alcohol cessation. Twenty-four hours after the last treatment, rats were exposed to a 5-min session in the open-field test (OF). Corticosterone and leptin serum levels were determined by ELISA in samples collected within 30 min after the OF. Results showed that rats were hypoactive (decreased rearing, peripheral, and total crossings), and that corticosterone and leptin levels were increased 5 days after alcohol cessation. Four days of NAC prevented the behavioral and biochemical changes brought about by alcohol cessation. We suggest that, in addition to the anti-addictive properties reported for other drugs of abuse, NAC is potentially useful in the management of alcohol withdrawal.

Characterization of Ectonucleotidases in Human Medulloblastoma Cell Lines: ecto-5′NT/CD73 in Metastasis as Potential Prognostic Factor

Medulloblastoma (MB) is the most common malignant brain tumor in children and occurs mainly in the cerebellum. Important intracellular signaling molecules, such those present in the Sonic Hedgehog and Wnt pathways, are involved in its development and can also be employed to determine tumor grade and prognosis. Ectonucleotidases, particularly ecto-5′NT/CD73, are important enzymes in the malignant process of different tumor types regulating extracellular ATP and adenosine levels. Here, we investigated the activity of ectonucleotidases in three malignant human cell lines: Daoy and ONS76, being representative of primary MB, and the D283 cell line, derived from a metastatic MB. All cell lines secreted ATP into the extracellular medium while hydrolyze poorly this nucleotide, which is in agreement with the low expression and activity of pyrophosphate/phosphodiesterase, NTPDases and alkaline phosphatase. The analysis of AMP hydrolysis showed that Daoy and ONS76 completely hydrolyzed AMP, with parallel adenosine production (Daoy) and inosine accumulation (ONS76). On the other hand, D283 cell line did not hydrolyze AMP. Moreover, primary MB tumor cells, Daoy and ONS76 express the ecto-5′NT/CD73 while D283 representative of a metastatic tumor, revealed poor expression of this enzyme, while the ecto-adenosine deaminase showed higher expression in D283 compared to Daoy and ONS76 cells. Nuclear beta-catenin has been suggested as a marker for MB prognosis. Further it can promotes expression of ecto-5′NT/CD73 and suppression of adenosine deaminase. It was observed that Daoy and ONS76 showed greater nuclear beta-catenin immunoreactivity than D283, which presented mainly cytoplasmic immunoreactivity. In summary, the absence of ecto-5′NT/CD73 in the D283 cell line, a metastatic MB phenotype, suggests that high expression levels of this ectonucleotidase could be correlated with a poor prognosis in patients with MB.

Disease modifying anti-rheumatic activity of the alkaloid montanine on experimental arthritis and fibroblast-like synoviocytes

Abstract Montanine is an alkaloid isolated from Rhodophiala bifida bulb with potential anti‐arthritic activity. In this context, we evaluated whether montanine has a disease modifying anti‐rheumatic activity in two arthritis models and its effect in vitro on lymphocyte proliferation and on invasiveness of fibroblast‐like synoviocytes (FLS). Antigen‐induced arthritis (AIA) was performed in Balb/C mice with methylated bovine serum albumin, and nociception and leukocytes migration into the knee joint were evaluated. Collagen‐induced arthritis (CIA) was performed in DBA/1 J mice, and arthritis development and severity were assessed by clinical and histological scoring and articular nociception. Montanine was administered intraperitoneally twice a day. Lymphocyte proliferation stimulated by concanavalin A in 48 h was performed with MTT assay, while FLS invasion in 24 h was assayed in a Matrigel‐coated transwell system. Administration of montanine decreased nociception (P<0.001) and leukocyte articular migration (P<0.001) in mice with AIA. In mice with CIA, treatment with montanine reduced severity of arthritis and joint damage assessed by clinical (P<0.001) and histological (P<0.05) scores and ameliorated articular nociception (P<0.05). In vitro, montanine inhibited lymphocyte proliferation stimulated with ConA (P<0.001) and decreased FLS invasion (P<0.05) by 54%, with an action independent of cytotoxicity. Our findings suggest that montanine can be further explored as an innovative pharmacological approach for autoimmune diseases such as arthritis. Graphical abstract Figure. No caption available.

Gastrin-releasing peptide and its receptor increase arthritis fibroblast-like synoviocytes invasiveness through activating the PI3K/AKT pathway

&NA; Rheumatoid arthritis (RA) is an autoimmune disease that leads to joint destruction. The fibroblast‐like synoviocytes (FLS) has a central role on the disease pathophysiology. The present study aimed to examine the role of gastrin‐releasing peptide (GRP) and its receptor (GRPR) on invasive behavior of mice fibroblast‐like synoviocytes (FLS), as well as to evaluate GRP‐induced signaling on PI3K/AKT pathway. The expression of GRPR in FLS was investigated by immunocytochemistry, western blot (WB) and qRT‐PCR. The proliferation and invasion were assessed by SRB and matrigel‐transwell assay after treatment with GRP and/or RC‐3095 (GRPR antagonist), and/or Ly294002 (inhibitor of PI3K/AKT pathway). Finally, AKT phosphorylation was assessed by WB. GRPR protein was detected in FLS and the exposure to GRP increased FLS invasion by nearly two‐fold, compared with untreated cells (p < 0.05), while RC‐3095 reversed that effect (p < 0.001). GRP also increased phosphorylated AKT expression in FLS. When Ly294002 was added with GRP, it prevented the GRP‐induced increased cell invasiveness (p < 0.001). These data suggest that GRPR expression in FLS and that exogenous GRP are able to activate FLS invasion. This effect occurs at least in part through the AKT activation. Therefore, understanding of the GRP/GRPR pathway could be relevant in the development of FLS‐targeted therapy for RA. HighlightsFibroblast‐like synoviocytes express gastrin‐releasing receptor protein.GRP increased FLS invasiveness and RC‐3095 reverse that effect.The effect of GRP could be through activation of PI3K/AKT pathway.GRP/GRPR signaling could be a new target for the treatment of arthritis.

THU0072 Gastrin-Releasing Peptide (GRP) and RC-3095, a GRP Receptor Antagonist, Regulates Arthritic Mice Synovial Fibroblasts

Background Rheumatoid Arthritis (RA) is characterized by invasion of fibroblast-like synoviocytes (FLS) into de articular cartilage and bone erosion leading to progressive joint destruction. FLS invasiveness correlates with articular damage in RA, yet little is known about this regulation. Gastrin-releasing peptide (GRP) is a functional homologue of bombesin, and its receptor signaling is involved in several functions, including the inflammatory response. GRP and its receptor (GRPR) have been found in synovial membrane and fluid of RA patients. RC-3095 is an antagonist of GRPR. Objectives To evaluate the presence and role of gastrin-releasing peptide and its receptor on FLS from arthritic mice. Methods FLS were isolated from the ankle joints of DBA/1J mice with collagen-induced arthritis. Expression of GRPR protein in FLS was analyzed by immunocitochemistry and western blot (WB), and qPCR was used to measure expression levels of GRPR and GRP. Viability of FLS treated with GRP (0.1, 1 and 10 μM) or RC-3095 (0.05 to 10μM) was assessed by MTT assay in 24h and 48h. FLS (n=6) invasion was measured using a Matrigel-coated transwell invasion system in the presence of GRP (10 μM), RC-3095 (1μM) or GRP+RC-3095 (GRP 10 μM and RC 1μM) over 24 hours, in duplicate. Cytosol and culture supernatant GRP was measured by ELISA after treatment with GRP (10 μM), RC-3095 (1 μM) or GRP+RC-3095 (GRP 10 μM and RC-3095 1μM). Results GRPR protein and mRNA was present in FLS. GRP mRNA was also detected. RC-3095 and GRP were not cytotoxic for FLS in the used concentrations. GRP increased FLS invasion by nearly two-fold (5356±767) compared with untreated FLS (2888±386) (p<0,02), whereas RC-3095 reversed that effect (1722±271) (p<0,005). GRP+RC-3095 (2670±499) decreased FLS invasion compared with GRP (p<0,0001). RC-3095 (206±10,55) increased GRP levels in supernatant comparing with GRP (334±61,26) (p<0,02) and with GRP+RC-3095 (211±12,39) (p<0,03). In cytosol no statistical difference was observed. Conclusions This is the first study to identify the presence and activity of the GRP-GRPR pathway in FLS. These cells expressed the GRPR receptor and secreted GRP. RC-3095 was able to decrease FLS invasion while GRP increased. These findings suggest that interference with the GRP pathway is a potential therapeutic target on FLS for treatment to rheumatoid arthritis using RC-3095 for future clinical trials. References Grimsholm O, et al. Neuropeptides. 2008. Laragione T, Gulko PS. Mol Med. 2012. Oliveira PG, et al. Arthritis Rheum. 2011. Acknowledgements FIPE, CNPq, CAPES Disclosure of Interest None declared

AB0129 Montanine: An Alkaloid Isolated from Rhodophiala Bifida with Anti-Inflammatory and Immunomodulatory Properties

Background Montanine is an alkaloid isolated from Rhodophiala bifida, a plant used in folk medicine (1) and never before tested in inflammatory diseases. Objectives To evaluate the effect of montanine as an in vivo anti-inflammatory therapy in Antigen-induced Arthritis (AIA) and Collagen-induced Arthritis (CIA) models, and to characterize its effects on immune and hematologic phenotypes in vivo and in vitro, including fibroblast-like synoviocytes (FLS) invasiveness. Methods AIA was induced in 24 Male BALB/c mice with methylated bovine serum albumin (mBSA) and mice divided into 4 groups: vehicle (saline) or montanine at 0.3, 1 or 3mg/kg, intraperitoneal twice a day. Treatment started one day before intra-articular injection of mBSA. Paw nociception at 0, 3, 5 and 24 hours and leukocytes migration into the knee joint at 24 hours were evaluated. DBA/1J mice were induced with CIA and divided into a preventive (n=24; 16 days of treatment at doses 0.05; 0.25 and 0.5 mg/kg) or therapeutic group (n=23; 10 days of treatment at doses 0.5 and 1.5 mg/kg starting after the onset of arthritis). BALB/c controls (n=36) were treated for 14 days at doses 0.05, 0.5 and 1.5 mg/kg and hematology, cytokine and leukocyte subpopulation and histophatology of lymphoid organs were evaluated. Montanine 1mM was used for lymphocyte proliferation assay with ConA (n=7). FLS invasion over 24 hours was assayed in a transwell Matrigel-coated chamber in the presence or absence of Montanine 1mM (n=5). Statistical analysis: ANOVA or t-test. Results In AIA Montanine decreased leukocyte articular migration (figure 1A) in a dose-dependent manner by as much as 90% (3mg/kg: 4.15±1.46x104 leukocytes/cavity) compared with vehicle (43.5±9.73x104 leukocytes/cavity) (p<0.001) and reduced nociception in all doses at 5 and 24h (p<0.01), compared with vehicle. In the CIA preventive treatment (figure 1B), montanine 0.25 and 0.5 mg/kg reduced articular score that could be detected as early as day 8 and persisted throughout the observation period (p<0.05). Montanine 0.5mg/kg also significantly reduced CIA joint severity score by as much as 39,52% compared to vehicle in the therapeutic arm (figure 1C), an effect that was noticed as early as day 3 and persisted throughout the observation period (p<0.01), with reduced histology damage (p<0.03). Nociception was significantly reduced at days 2 and 10 (p<0.05) compared with vehicle. Montanine inhibited lymphocyte proliferation stimulated with ConA by 54.78% (p<0.01). Montanine 1mM decreased FLS invasion by 54% (p=0.031) (figure 1D). Montanine did not have any significant effect on the hematologic and immunological parameters analyzed in BLAB/c controls.Figure 1 Conclusions Montanine significantly improved experimental arthritis, reduced joint damage and nociception in both acute and chronic models. Moreover, montanine reduced lymphocyte proliferation and FLS invasion, two processes implicated in RA pathogenesis and did not have any obvious toxicity. These results suggest that montanine has the potential to become a new class of drugs to treat inflammatory and autoimmune diseases such as arthritis. References Louw CAM et al. J. Ethnopharmacol. 2002:82: 147-154. Acknowledgements FIPE-HCPA, CAPES, CNPq Disclosure of Interest None declared

AB0130 Immunomodulatory and Antiinflamatory Properties of Antigen B, A Lipoprotein Secreted on Hydatic Cyst of Echinococcus Granulosus, in Experimental Arthritis

Background Antigen B (AgB) is a lipoprotein secreted in hydatic cyst by Echinococcus granulosus larval stage (1) and seems to be responsible to regulate immune balance via Th2 response to promotes survival of the parasite (2). A Th2 response can suppress the pro-inflammatory Th1 response generated in several immunopathologies. Objectives To evaluate the effect of AgB in three animal models of arthritis. Methods In all models, mice were divided into three groups: vehicle (saline) or AgB (2 and 10μg – once a day, intraperitoneal). BALB/c mice (n=21) were treated and then injected with zymosan into knee joint for development of zymosan Induced-arthritis (ZYA). Nociception in 0, 4 and 6 hours and leukocytes articular migration 6 hours after intra-articular (ia) injection were assessed. Antigen Induced-arthritis (AIA) was induced in BALB/c (n=36) with methylated bovine serum albumin (mBSA) and treatment started one day before ia injection of mBSA. Paw nociception in 0, 3, 5, 7 and 24h and neutrophils migration into knee joints 24h after ia injection of mBSA were evaluated. DBA/1J mice had Collagen Induced-arthritis (CIA) and were divided into preventive (n=25, 18 days of treatment) or therapeutic groups (n=27, 10 days of treatment starting after the onset of arthritis). In the preventive group, articular score, nociception, paw edema and body weight were evaluated. In the therapeutic group, articular score and nociception were evaluated and knee joints were collected to analyses cytokine th1/th2/th17 profile. Statistical analysis: ANOVA or Kruskal-Wallis. Results In ZYA, both treatment reduced nociception in 4 and 6h (p<0.001) and inhibited leukocytes migration to inflammatory site (39.67±8.57, 55±13.71x104 leukocytes/cavity, respectively) compared with vehicle (159.7±39.32x104 leukocytes/cavity) (p<0.05). In AIA, both doses of AgB treatment reduced nociception at 3, 5, 7 and 24h compared with vehicle (p<0.01) and inhibited neutrophils migration (7.75±2,58, 8.99±2.18x104 neutrophils/cavity, respectively) compared with vehicle (55.93±9.79x104 neutrophils/cavity) (p<0.001). In CIA, preventive treatment improved nociception at the 2μg dose at days 14 and 18 (p<0.05) but did not improved articular score, paw edema or body weight. Therapeutic treatment did not improved articular score or reduced the nociception however the 2μg dose was able to reduce IL-6 (p<0.05) and TNF-a (p<0.05) levels, comparing with vehicle. Conclusions Treatment with AgB significantly improved acute experimental arthritis, attenuated nociception and immune cells articular migration. On the other hand, AgB had no effect in chronic experimental arthritis, although therapeutic treatment reduced Il-6 and TNF-a levels, two important pro-inflammatory cytokines and prophylactic treatment improved nociception. We believe that the adjuvant use of AgB with a DMARD can lead to an additive effect on amelioration of immune-mediated arthritis short-term symptoms. References Oriol R. et al. Am J Trop Med Hyg. 1971; 20(4):569. Siracusano A. et al. Exp Parasitol. 2008; 119(4):483-9. Acknowledgements FIPE-HCPA, CAPES, CNPq Disclosure of Interest None declared

AB0135 Immunomodulatory and Antiinflammatory Properties of Antigen B, A Protein Secreted by Echinococcus Granulosus Larval Stage, in Experimental Arthritis

Background Antigen B (AgB) is a protein secreted by Echinococcus granulosus larval stage (1) and seems to be responsible for immuneregulation via Th2 response (2). response to promotes survival of the parasite (2,3). A Th2 response can suppress the pro-inflammatory Th1 response generated in several immunopathology (2), providing therapeutic potential in autoimmune diseases. Objectives To evaluate the effect of AgB in antigen-induced arthritis (AIA), zymosan-induced arthritis (ZYA) and collagen-induced arthritis (CIA) models. Methods In all models, mice were divided intro three groups: vehicle (saline) and AgB (2 and 10mg – once a day). BALB/c mice (n=21) were treated with AgB and then injected with zymosan into intraarticular knee joint for ZYA. Nociception was assessed in 0, 4 and 6 h and leukocytes migration analyzed 6h after intraarticular injection. AIA was induced in BALB/c mice (n=36) with methylated bovine serum albumin (mBSA) and AgB treatment started one day before intraarticular mBSA injection. Nociception was evaluated in 0, 3, 5, 7 and 24h and neutrophils migration into knee joints was evaluated 24h after intraarticular mBSA injection CIA was induced in DBA/1J mice (n=27) with bovine collagen type II and were monitored daily for arthritis, AgB treatment starting after onset of disease and lasting for 10 days. Articular score and nociception were evaluated and knee joints were collected to analyze the cytokine (CBA th1/th2/th17). Statistical analysis was assessed with ANOVA. Results In ZYA, both dose treatment reduced nociception in 4 and 6 h (p<0.001) and inhibited leukocytes migration (39.67±8.57x104 and 55±13.71x104 leukocytes/cavity, respectively) compared with vehicle (159.7±39.32x104 leukocytes/cavity) (p<0.05). In AIA, both doses of AgB reduced nociception at 3, 5, 7 and 24h compared with vehicle (p<0.01) and inhibited neutrophils migration (7.75±2,58x104 and 8.99±2.18x104 neutrophils/cavity, respectively) compared with vehicle (55.93±9.79x104 neutrophils/cavity) (p<0.001). AgB treatment in CIA did not improved clinical score or reduced nociception, but the 2mg dose was able to reduce IL-6 (p<0.05) and TNF-a (p<0.05) levels. Conclusions Treatment with AgB improved acute experimental arthritis attenuating nociception and cells migration to the joint. Although AgB had no effect on nociception and clinical score in chronic arthritis, it was able to reduce IL-6 and TNF-a levels, two important pro-inflammatory cytokines. We believe that a prophylactic intervention or the adjuvant use of AgB can attenuate joint damage and nociception also in chronic arthritis. References Oriol R, Williams JF, Esandi MVP, Oriol C. Purification of lipoprotein antigens of Echinococcus granulosus from sheep hydatid fluid. American Journal of Tropical Medicine and Hygiene. 1971;20(4):569; Siracusano A, Rigano R, Ortona E, Profumo E, Margutti P, Buttari B, et al. Immunomodulatory mechanisms during Echinococcus granulosus infection. Experimental Parasitology. 2008;119(4):483-9. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3897