Mirian Farinon

Effect of Aqueous Extract of Giant Horsetail (Equisetum giganteum L.) in Antigen-Induced Arthritis

Equisetum giganteum is a plant used in traditional medicine as diuretic. From our knowledge this is the first time this plant is tested in an in vivo model of acute inflammation. To evaluate the effect of aqueous extract of giant horsetail (AEGH) as immunomodulatory therapy, antigen-induced arthritis (AIA) was generated in mice with methylated bovine serum albumin (mBSA). Inflammation was evaluated by articular nociception, leukocytes migration and lymphocyte proliferation. AEGH reduced nociception at 3, 6 and 24 h (P < 0.01), decreased leukocyte migration (P < 0.015), and inhibited lymphocyte proliferation stimulated with Concanavalin A and Lipopolysaccharide (P < 0.05). In conclusion, AEGH has an anti-inflammatory potential in acute model of inflammation, as well as immunomodulatory effect on both B and T lymphocytes, with an action independent of cytotoxicity.

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.

Intra-articular nonviral gene therapy in mucopolysaccharidosis I mice

ABSTRACT Mucopolysaccharidosis type I (MPS I) is caused by the lysosomal accumulation of glycosaminoglycans (GAGs) due to the deficiency of the enzyme alpha‐L‐iduronidase (IDUA). Currently available treatments may improve several clinical manifestations, but they have limited effects on joint disease, resulting in persistent orthopedic complications and impaired mobility. Thus, this study aimed to perform an intra‐articular administration of cationic nanoemulsions complexed with the plasmid encoding for the IDUA protein (pIDUA) targeting MPS I gene therapy for the synovial joints. Formulations composed of DOPE, DOTAP, MCT (NE), and DSPE‐PEG (NE‐PEG) were prepared by high‐pressure homogenization, and the pIDUA plasmid was associated by adsorption onto the surface of nanoemulsions (pIDUA/NE or pIDUA/NE‐PEG). The physicochemical characterization showed that the presence of DSPE‐PEG in pIDUA/NE‐PEG formulations led to small and highly stable droplets even when incubated with simulated synovial fluid (SSF), when compared to the non‐pegylated complexes (pIDUA/NE). Uptake by fibroblast‐like synoviocytes (FLS) was demonstrated, and high cell viability (70%) in addition with increased IDUA activity (2.5% of normal) were observed after incubation with pIDUA/NE‐PEG. The intra‐articular injection of pIDUA/NE‐PEG complexes in MPS I mice showed that the complexes were localized in the joints, were able to transfect synovial cells, and thus promoted an increase in IDUA activity and expression in the synovial fluid, with no significant activity in other tissues (kidney, liver, lung, and spleen). The overall results demonstrated a contained, safe, tolerable, and effective in situ approach of nonviral intra‐articular gene therapy targeting the reduction or prevention of the debilitating orthopedic complications of MPS I disorder.

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.

Nasal Administration of Cationic Nanoemulsions as Nucleic Acids Delivery Systems Aiming at Mucopolysaccharidosis Type I Gene Therapy

PurposeThis study demonstrates the nasal administration (NA) of nanoemulsions complexed with the plasmid encoding for IDUA protein (pIDUA) as an attempt to reach the brain aiming at MPS I gene therapy.MethodsFormulations composed of DOPE, DOTAP, MCT (NE), and DSPE-PEG (NE-PEG) were prepared by high-pressure homogenization, and assessed in vitro on human fibroblasts from MPS I patients and in vivo on MPS I mice for IDUA production and gene expression.ResultsThe physicochemical results showed that the presence of DSPE-PEG in the formulations led to smaller and more stable droplets even when submitted to dilution in simulated nasal medium (SNM). In vitro assays showed that pIDUA/NE-PEG complexes were internalized by cells, and led to a 5% significant increase in IDUA activity, besides promoting a two-fold increase in IDUA expression. The NA of pIDUA/NE-PEG complexes to MPS I mice demonstrated the ability to reach the brain, promoting increased IDUA activity and expression in this tissue, as well as in kidney and spleen tissues after treatment. An increase in serum IL-6 was observed after treatment, although with no signs of tissue inflammatory infiltrate according to histopathology and CD68 assessments.ConclusionsThese findings demonstrated that pIDUA/NE-PEG complexes could efficiently increase IDUA activity in vitro and in vivo after NA, and represent a potential treatment for the neurological impairment present in MPS I patients.

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

AB0100 Role of gastrin-releasing peptide and the rc-3095, an antagonist of gastrin-releasin peptide receptor, in experimental arthritis synovial fibroblast

Background The gastrin-releasing peptide (GRP) is the mammalian homologue of the bombesin (BN), and its receptor signaling is involved in several functions, including inflammatory response [1]. Both the GRP and its receptor have been found in synovial membrane and fluid of rheumatoid arthritis patients [2]. RC-3095 is an antagonist of the GRP receptor. Objectives To evaluate the role of gatrin-releasing peptide and the RC-3095, a specific antagonist of the gastrin-releasing peptide receptor, in synovial fibroblast proliferation and invasion. Methods Mouse DBA/1J fibroblast-like synoviocyte (FLS) were isolated from the tarsus of the hind paws of collagen-induced arthritis [3]. FLS immunocitochemistry was performed to evaluate the presence of GRP-receptor (GRPR) [4]. FLS (2x104 / 96-wells) viability in 24 h treated with RC-3095 (concentration from 0.05 to 10 mM). FLS proliferation stimulated with Lipopolysaccharide (LPS) (1 and 10 µg/mL) [5] or GRP (0.1, 1 and 10 mM) was performed using the MTT assay in 24 h. The invasion of FLS was assayed in a transwell system using Matrigel-coated inserts from BD (Franklin Lakes, NJ, USA) [6] and treated with GRP (10 mM), RC-3095 (1 mM) and GRP+RC-3095 (GRP 10 mM and after 30 min RC-3095 1 mM) (n= 3 per group). Differences between experimental groups were compared by ANOVA one-way test. Results The immunocitochemistry confirmed the presence of GRPR on FLS. RC-3095 concentrations used were not toxic on FLS, maintaining cellular viability. The dose of 1 μM was defined for other experiments because this was the highest dose with lower cell mortality (p<0.05). The GRP 10 mM increased the fibroblast proliferation in 18%, while LPS 10 mM increased 15% compared to FLS unstipulated (p<0.05). Treatment of highly invasive DBA/1J FLS with RC-3095 (1934 ± 941 cells) significantly decreased the number of cells invading Matrigel over 24 h period in 35.3% (p=0.003) compared to GRP (5371 ± 418.1 cells) and non-different to FLS treated with GRP+RC-3095 (3054 ± 794.5 cells). Conclusions RC-3095 was able to decrease synovial fibroblasts invasion stimulated by GRP. GRP increased FLS proliferation and invasion and could be involved in the development of experimental arthritis through FLS. These findings suggest that interference with the neuropeptide GRP pathway is a potential new strategy for the treatment of arthritis. References – Jensen, R.T. et al. Pharmacol Rev. 2008. 60(1):1-42. – Grimsholm, O. et al. Cells Tissues Organs. 2008.188(3):299-309. – Nishioku, T. et al. J Inflam. 2012. 9:44-47. – Laragione, T. et al. Mol Med. 2010. 16(9-10):352-8 – Miyazawa, S. et al. Rheumatol Int. 2006. 26(8):717-25. – Tolboom, T.C. et al. Arthritis Rheum. 2005. 52:1999-2002. Acknowledgements Finnacial support CAPES, CNPq, FIPE (HCPA) Disclosure of Interest None Declared