Intra-articular use of radium dichloride ([223Ra] RaCl2) showed relevant anti-inflammatory response on experimental arthritis model.

Rheumatoid arthritis (RA) is an inflammatory chronic autoimmune disease. The treatment of RA is difficult and, in many cases, ineffective, and the arsenal of drugs is limited. Due the longevity of the disease, RA may cause extreme musculoskeletal disorders with a high impact on quality of life. Also, RA is related with severe comorbidities decreasing the life expectancy. Finally, RA has been reported to impact in economy and healthy public. In this direction, the necessity to discover new strategies to efficiently treat RA is immediate. In this direction, we have reported the use of low doses of [223Ra] RaCl2 (radium dichloride) as intra-articular injection to treat RA. Mice were post-treated with [223Ra] RaCl2 (1.48 µCi; i.a.) 24 h after zymosan stimulus. Zymosan-induced arthrithis is responsible for leucocyte recruitment (total leukocytes, neutrophils, and mononuclear cells), which were inhibited by intra-articular injection of [223Ra] RaCl2 (69%, 77%, and 66%, respectively).

Gedunin Binds to Myeloid Differentiation Protein 2 and Impairs Lipopolysaccharide-Induced Toll-Like Receptor 4 Signaling in Macrophages.

Recognition of bacterial lipopolysaccharide (LPS) by innate immune system is mediated by the cluster of differentiation 14/Toll-like receptor 4/myeloid differentiation protein 2 (MD-2) complex. In this study, we investigated the modulatory effect of gedunin, a limonoid from species of the Meliaceae family described as a heat shock protein Hsp90 inhibitor, on LPS-induced response in immortalized murine macrophages. The pretreatment of wild-type (WT) macrophages with gedunin (0.01-100 µM, noncytotoxic concentrations) inhibited LPS (50 ng/ml)-induced calcium influx, tumor necrosis factor-α, and nitric oxide production in a concentration-dependent manner. The selective effect of gedunin on MyD88-adapter-like/myeloid differentiation primary response 88- and TRIF-related adaptor molecule/TIR domain-containing adapter-inducing interferon-ß-dependent signaling pathways was further investigated. The pretreatment of WT, TIR domain-containing adapter-inducing interferon-ß knockout, and MyD88 adapter-like knockout macrophages with gedunin (10 µM) significantly inhibited LPS (50 ng/ml)-induced tumor necrosis factor-α and interleukin-6 production, at 6 hours and 24 hours, suggesting that gedunin modulates a common event between both signaling pathways. Furthermore, gedunin (10 µM) inhibited LPS-induced prostaglandin E2 production, cyclooxygenase-2 expression, and nuclear factor κB translocation into the nucleus of WT macrophages, demonstrating a wide-range effect of this chemical compound. In addition to the ability to inhibit LPS-induced proinflammatory mediators, gedunin also triggered anti-inflammatory factors interleukin-10, heme oxygenase-1, and Hsp70 in macrophages stimulated or not with LPS. In silico modeling studies revealed that gedunin efficiently docked into the MD-2 LPS binding site, a phenomenon further confirmed by surface plasmon resonance. Our results reveal that, in addition to Hsp90 modulation, gedunin acts as a competitive inhibitor of LPS, blocking the formation of the Toll-like receptor 4/MD-2/LPS complex.

Murine IL-17+ Vγ4 T lymphocytes accumulate in the lungs and play a protective role during severe sepsis.

BACKGROUND: Lung inflammation is a major consequence of the systemic inflammatory response caused by severe sepsis. Increased migration of γδ T lymphocytes into the lungs has been previously demonstrated during experimental sepsis; however, the involvement of the γδ T cell subtype Vγ4 has not been previously described. METHODS: Severe sepsis was induced by cecal ligation and puncture (CLP; 9 punctures, 21G needle) in male C57BL/6 mice. γδ and Vγ4 T lymphocyte depletion was performed by 3A10 and UC3-10A6 mAb i.p. administration, respectively. Lung infiltrating T lymphocytes, IL-17 production and mortality rate were evaluated. RESULTS: Severe sepsis induced by CLP in C57BL/6 mice led to an intense lung inflammatory response, marked by the accumulation of γδ T lymphocytes (comprising the Vγ4 subtype). γδ T lymphocytes present in the lungs of CLP mice were likely to be originated from peripheral lymphoid organs and migrated towards CCL2, CCL3 and CCL5, which were highly produced in response to CLP-induced sepsis. Increased expression of CD25 by Vγ4 T lymphocytes was observed in spleen earlier than that by αß T cells, suggesting the early activation of Vγ4 T cells. The Vγ4 T lymphocyte subset predominated among the IL-17(+) cell populations present in the lungs of CLP mice (unlike Vγ1 and αß T lymphocytes) and was strongly biased toward IL-17 rather than toward IFN-γ production. Accordingly, the in vivo administration of anti-Vγ4 mAb abrogated CLP-induced IL-17 production in mouse lungs. Furthermore, anti-Vγ4 mAb treatment accelerated mortality rate in severe septic mice, demonstrating that Vγ4 T lymphocyte play a beneficial role in host defense. CONCLUSIONS: Overall, our findings provide evidence that early-activated Vγ4 T lymphocytes are the main responsible cells for IL-17 production in inflamed lungs during the course of sepsis and delay mortality of septic mice.

Study of the antimalarial properties of hydroxyethylamine derivatives using green fluorescent protein transformed Plasmodium berghei.

A rapid decrease in parasitaemia remains the major goal for new antimalarial drugs and thus, in vivo models must provide precise results concerning parasitaemia modulation. Hydroxyethylamine comprise an important group of alkanolamine compounds that exhibit pharmacological properties as proteases inhibitors that has already been proposed as a new class of antimalarial drugs. Herein, it was tested the antimalarial property of new nine different hydroxyethylamine derivatives using the green fluorescent protein (GFP)-expressing Plasmodium berghei strain. By comparing flow cytometry and microscopic analysis to evaluate parasitaemia recrudescence, it was observed that flow cytometry was a more sensitive methodology. The nine hydroxyethylamine derivatives were obtained by inserting one of the following radical in the para position: H, 4Cl, 4-Br, 4-F, 4-CH3, 4-OCH3, 4-NO2, 4-NH2 and 3-Br. The antimalarial test showed that the compound that received the methyl group (4-CH3) inhibited 70% of parasite growth. Our results suggest that GFP-transfected P. berghei is a useful tool to study the recrudescence of novel antimalarial drugs through parasitaemia examination by flow cytometry. Furthermore, it was demonstrated that the insertion of a methyl group at the para position of the sulfonamide ring appears to be critical for the antimalarial activity of this class of compounds.

Synthesis and trypanocidal activity of novel 2,4,5-triaryl-N-hydroxylimidazole derivatives.

Herein, we report the design, synthesis and trypanocidal activity of some novel trisubstituted imidazole derivatives. These heterocyclic derivatives were structurally planned by exploring the concept of molecular hybridisation between two arylhydrazones derived from megazol, which has potent trypanocidal activity. The trypanocidal activity of these triarylimidazole derivatives was evaluated against infective trypomastigote forms of T. cruzi and the derivative 2'-(4-bromophenyl)-1-methyl-5'-phenyl-1H,3'H-2,4'-biimidazol-3'-ol showed moderate biological activity (IC50 = 23.9 µM) when compared to benznidazole, a standard trypanocidal drug. These compounds did not present cytotoxic effects at concentrations near the trypanocidal IC50, being considered a good starting point for the development of new anti-Chagas drug candidates.

Methyl gallate nanomicelles impairs neutrophil accumulated in zymosan-induced arthritis.

Arthritis is a chronic disease that affects, approximately, 1 % of the total global population. It is characterized by chronic inflammation, accompanied in most of the cases of motor disability and sever pain. The main therapies available have high risk of failure and advanced treatments are scarce and highly cost. In this scenario, search for effective, safe and low-cost treatments is quite desirable. Methyl gallate (MG) is a plant-derived phenolic compound described to present remarkable anti-inflammatory effect in experimental models of arthritis. Thus, in this study we formulated nanomicelles of MG using Pluronic (F-127) as matrix and evaluated in vivo the pharmacokinetic, biodistribution and its effect in the mice model of zymosan-induced arthritis. The nanomicelles were formed with a size 126 nm. The biodistribution showed a ubiquitous tissue deposition with a renal excretion. The pharmacokinetics showed elimination half-life of 1.72 h and a clearance of 0.006 L/h. The oral pretreatment with nanomicelles containing MG (3.5 or 7 mg/kg) demonstrated a reduction in total leukocytes, neutrophils, and mononuclear cells from the inflammation site. The data supports the use of methyl gallate nanomicelles as an alternative drug for arthritis. DATA AVAILABILITY: All the data of this study are transparent.

Mefloquine-oxazolidine derivatives, derived from mefloquine and arenecarbaldehydes: In vitro activity including against the multidrug-resistant tuberculosis strain T113.

Ten new mefloquine-oxazolidine derivatives, 4-[(1S,8aR)-3-(aryl)hexahydro[1,3]oxazolo[3,4-a]pyridin-1-yl]-2,8-bis(trifluoromethyl)quinoline (1: aryl=substituted phenyl) and 4-[(1S,8aR)-3-(heteroaryl)hexahydro[1,3]oxazolo[3,4-a]pyridin-1-yl]-2,8-bis(trifluoromethyl)quinoline [2: heteroaryl=5-nitrothien-2-yl (2a); 5-nitrofuran-2-yl (2b) and 4H-imidazol-2-yl) (2c)], have been synthesized and evaluated against Mycobacterium tuberculosis. Compounds 1f (aryl=3-ethoxyphenyl), 1g (Ar=3,4,5-(MeO)(3)-C(6)H(2)) and 2c were slightly more active than mefloquine (MIC=33µM) with MICs=24.5, 22.5 and 27.4, respectively, whereas compounds 1e (aryl=3,4-(MeO)(2)-C(6)H(3)) and 2a (MICs=11.9 and 12.1µM, respectively) were ca. 2.7 times more active than mefloquine, with a better tuberculostatic activity than the first line tuberculostatic agent ethambutol (MIC=15.9). The compounds were also assayed against the MDR strain T113 and the same MICs were observed. Thus the new derivatives have advantages over such anti-TB drugs as isoniazid, rifampicin, ethambutol and ofloxacin, for which this strain is resistant. The most active compounds were not cytotoxic to Murine Macrophages Cells in a concentration near their MIC values.

Nanoparticle conjugated with aptamer anti-MUC1/Y for inflammatory arthritis.

Aptamers may form well-defined three-dimensional structures binding with high affinity and stability to a specific receptor. The aptamer anti-MUC1 isoform Y is one the most used due the affinity to MUC1, which is overexpressed in several types of cancer and inflammation process. In this study we have developed, characterized, in vitro as in vivo evaluated a nanoaptamer (anti-MUC1/Y) as a nanoagent for rheumatoid arthritis treatment. The results showed that a nanoaptamer with a size range of 241 nm was produced. The entrapment efficacy was 90% with a biodistribution showing a high hepatic uptake (>98%). The results in vivo showed a potent effect in arthritis experimental model, especially in low doses. The results corroborate the applicability of this nanosystem for RA treatment.

Rheumatoid arthritis treatment using hydroxychloroquine and methotrexate co-loaded nanomicelles: In vivo results.

Rheumatoid arthritis (RA) is the most common inflammatory rheumatic disease, affecting almost 1% of the world population. It is a long-lasting autoimmune disease, which mainly affects the joints causing inflammation and swelling of the synovial joint. RA has a significant impact on the ability to perform daily activities including simple work and household chores. Nonetheless, due to the long periods of pain and the continuous use of anti-inflammatory drugs, RA can debilitate the quality of life and increases mortality. Current therapeutic approaches to treat RA aim to achieve prolonged activity and early and persistent remission of the disease, with the gradual adoption of different drugs available. In this study, we developed a novel hydroxychloroquine and methotrexate co-loaded Pluronic® F-127 nanomicelle and evaluated its therapeutic effects against RA. Our results showed that drug-loaded nanomicelles were capable of modulating the inflammatory process of RA and reducing osteoclastogenesis, edema, and cell migration to the joint. Overall, compared to the free drugs, the drug-loaded nanomicelles showed a 2-fold higher therapeutic effect.

Anti-Inflammatory Activity and Chemical Analysis of Different Fractions from Solidago chilensis Inflorescence.

Solidago chilensis Meyen (Compositae) is a species native to South America (Brazil) popularly known as arnica. In Brazilian popular medicine, inflorescences and rhizomes of this plant have been used since the end of the 19th century to replace the exogenous and hepatotoxic Arnica montana L. in the treatment of edema and inflammatory pathologies. Although the anti-inflammatory activity of S. chilensis is evidenced in the literature, there is a lack of studies with enriched fractions or compounds isolated from it. The objective of the current study was to characterize phytochemically and to evaluate the pharmacological action in vivo and in vitro of the crude extract and the different fractions (hexane, dichloromethane, acetal, butanolic, and aqueous) isolated from the inflorescence of S. chilensis. The inflorescence crude extract (ScIE) and fractions were administered by intraperitoneal route to mice at different doses. In an LPS-induced pleurisy model, inhibition of leukocyte influx was observed for the ScIE and all fractions tested, as compared to controls. Dichloromethane (ScDicF), butanolic (ScButF), and aqueous (ScAquF) were selected for further analysis as they showed the best inhibitory effects in leukocyte migration and inflammatory cytokine and chemokine production: TNF-α, CXCL1/KC, CXCL2/MIP-2, and CCL11/eotaxin-1. In LPS-stimulated J774A.1 cell line, ScIE and the ScDicF exhibited an inhibitory effect on nitric oxide (NO) production and downmodulated the COX-2 expression; ScAquF failed to modulate NO production and COX-2 expression. In phytochemical analysis, HPLC-UV-DAD chromatograms of ScDicF and ScAquF showed the main peaks with UV spectrum characteristics of flavonoids; chlorogenic acid and isoquercetin were the most present phytochemicals identified in the ScAquF, and a high number of n-alkanes was found in ScHexF. Our study was the first to address biological effects and correlate them to phytochemically characterized fractions from inflorescences of S. chilensis.

Synthesis and antitubercular activity of heteroaromatic isonicotinoyl and 7-chloro-4-quinolinyl hydrazone derivatives.

Two series of N'-(E)-heteroaromatic-isonicotinohydrazide derivatives (3a-f and 4a-b) and 1-(7-chloroquinolin-4-yl)-2-[(heteroaromatic)methylene]hydrazone derivatives (5a-f and 6a-b) have been synthesized and evaluated for their in vitro antibacterial activity against Mycobacterium tuberculosis H37Rv. Several compounds were noncytotoxic and exhibited significant minimum inhibitory concentration (MIC) activity (3.12, 2.50, 1.25, or 0.60 microg/mL), which can be compared to that of the first-line drugs ethambutol (3.12 microg/mL) and rifampicin (2.0 microg/ml). These results can be considered an important starting point for the rational design of new leads for anti-TB compounds.

IL-4 Receptor α Chain Protects the Kidney Against Tubule-Interstitial Injury Induced by Albumin Overload.

Increasing evidence has highlighted the role of tubule-interstitial injury (TII) as a vital step in the pathogenesis of acute kidney injury (AKI). Incomplete repair of TII during AKI could lead to the development of chronic kidney disease. Changes in albumin endocytosis in proximal tubule epithelial cells (PTECs) is linked to the development of TII. In this context, interleukin (IL)-4 has been shown to be an important factor in modulating recovery of TII. We have studied the possible role of IL-4 in TII induced by albumin overload. A subclinical AKI model characterized by albumin overload in the proximal tubule was used, without changing glomerular function. Four groups were generated: (1) CONT, wild-type mice treated with saline; (2) BSA, wild-type mice treated with 10 g/kg/day bovine serum albumin (BSA); (3) KO, IL4Rα-/- mice treated with saline; and (4) KO + BSA, IL4Rα-/- mice treated with BSA. As reported previously, mice in the BSA group developed TII without changes in glomerular function. The following parameters were increased in the KO + BSA group compared with the BSA group: (1) tubular injury score; (2) urinary γ-glutamyltransferase; (3) CD4+ T cells, dendritic cells, macrophages, and neutrophils are associated with increases in renal IL-6, IL-17, and transforming growth factor ß. A decrease in M2-subtype macrophages associated with a decrease in collagen deposition was observed. Using LLC-PK1 cells, a model of PTECs, we observed that (1) these cells express IL-4 receptor α chain associated with activation of the JAK3/STAT6 pathway; (2) IL-4 alone did not change albumin endocytosis but did reverse the inhibitory effect of higher albumin concentration. This effect was abolished by JAK3 inhibitor. A further increase in urinary protein and creatinine levels was observed in the KO + BSA group compared with the BSA group, but not compared with the CONT group. These observations indicate that IL-4 has a protective role in the development of TII induced by albumin overload that is correlated with modulation of the pro-inflammatory response. We propose that megalin-mediated albumin endocytosis in PTECs could work as a sensor, transducer, and target during the genesis of TII.

Involvement of CC chemokines in gammadelta T lymphocyte trafficking during allergic inflammation: the role of CCL2/CCR2 pathway.

In the present study, we show that the intra-thoracic injection of ovalbumin (OVA, 12.5 microg per cavity) into C57BL/10 mice induced a significant increase in gammadelta T lymphocyte numbers in the pleural cavity, blood and thoracic lymph node of challenged mice. Such increase was significant within 12 h, peaked within 48 h and returned to basal counts within 120 h. Levels of CC chemokine ligand (CCL)-2/monocyte chemotactic protein-1, CCL5/regulated upon activation, normal T cell expressed and secreted, CCL3/macrophage inflammatory protein-1 alpha and CCL25/thymus-expressed chemokine were above control values in pleural washes recovered 24 h after OVA challenge (OPW) and were likely produced by pleural macrophages and mesothelial cells. Antigenic challenge also induced an up-regulation in CC chemokine receptor (CCR)-2, CCR5 and CCR9 on gammadelta T cells from pleural cavities, blood and lymph nodes, suggesting that cells found in mice pleural cavity migrate from secondary lymphoid organs into the inflammatory site via blood stream. The in vitro neutralization of CCL2 (but not of CCL3, CCL5 or CCL25) abrogated OPW-induced gammadelta T lymphocyte transmigration. Confirming such results, the in vivo administration of alpha-CCL2 mAb inhibited gammadelta T lymphocyte accumulation in the pleural cavity of challenged mice, whereas the blockade of CCL3, CCL5 or CCL25 showed no effect on gammadelta T cell mobilization. In addition, OVA challenge failed to induce gammadelta T lymphocyte accumulation in the pleural cavity of C57BL/6 CCR2 knockout mice, which also showed decreased numbers of these cells in blood and lymph nodes when compared with wild-type mice. Overall, such results demonstrate that CCR2/CCL2 pathway is crucial for gammadelta T lymphocyte mobilization during the allergic response.

Synthesis and antitubercular activity of 7-chloro-4-quinolinylhydrazones derivatives.

A series of twenty-one 7-chloro-4-quinolinylhydrazones (3a-u) have been synthesized and evaluated for their in vitro antibacterial activity against Mycobacterium tuberculosis H(37)Rv. The compounds 3f, 3i and 3o were non-cytotoxic and exhibited an important minimum inhibitory concentration (MIC) activity (2.5 microg/mL), which can be compared with that of the first line drugs, ethambutol (3.12 microg/mL) and rifampicin (2.0 microg/mL). These results can be considered an important start point for the rational design of new leads for anti-TB compounds.

Endothelins modulate inflammatory reaction in zymosan-induced arthritis: participation of LTB4, TNF-alpha, and CXCL-1.

Endothelins (ETs) are involved in inflammatory events, including pain, fever, edema, and cell migration. ET-1 levels are increased in plasma and synovial membrane of rheumatoid arthritis (RA) patients, but the evidence that ETs participate in RA physiopathology is limited. The present study investigated the involvement of ETs in neutrophil accumulation and edema formation in the murine model of zymosan-induced arthritis. Intra-articular (i.a.) administration of selective ET(A) or ET(B) receptor antagonists (BQ-123 and BQ-788, respectively; 15 pmol/cavity) prior to i.a. zymosan injection (500 microg/cavity) markedly reduced knee-joint edema formation and neutrophil influx to the synovial cavity 6 h and 24 h after stimulation. Histological analysis showed that ET(A) or ET(B) receptor blockade suppressed zymosan-induced neutrophil accumulation in articular tissue at 6 h. Likewise, dual blockade of ET(A)/ET(B) with bosentan (10 mg/kg, i.v.) also reduced edema formation and neutrophil counts 6 h after zymosan stimulation. Pretreatment with BQ-123 or BQ-788 (i.a.; 15 pmol/cavity) also decreased zymosan-induced TNF-alpha production within 6 h, keratinocyte-derived chemokine/CXCL1 production within 24 h, and leukotriene B(4) at both time-points. Consistent with the demonstration that ET receptor antagonists inhibit zymosan-induced inflammation, i.a. injection of ET-1 (1-30 pmol/cavity) or sarafotoxin S6c (0.1-30 pmol/cavity) also triggered edema formation and neutrophil accumulation within 6 h. Moreover, knee-joint synovial tissue expressed ET(A) and ET(B) receptors. These findings suggest that endogenous ETs contribute to knee-joint inflammation, acting through ET(A) and ET(B) receptors and modulating edema formation, neutrophil recruitment, and production of inflammatory mediators.

Exposure to the UV Filter Octyl Methoxy Cinnamate in the Postnatal Period Induces Thyroid Dysregulation and Perturbs the Immune System of Mice.

Evidence demonstrates the bidirectional communication and regulation between the neuroendocrine and immune systems. Thyroid hormones play key roles in nervous system development and can exert influence on various immune cells contributing to pathophysiological conditions. Octyl methoxycinnamate (OMC) is one of the most commonly used UV filters, and in vitro and in vivo studies have found thyroid disrupting effects. The present study assessed whether OMC administration in mice dams during the lactational period can cause thyroid disruption and generate immunologic alterations in the offspring. Indirect exposure to the OMC (1,000 mg/kg) in the lactational period affected neurodevelopment parameters, such as delayed eye-opening and weight gain in mice of both sexes, and these alterations are corroborated by the decrease in the T4 levels present in the pups' blood. No significant changes were observed in the thymus of these pups, but the number of lymphocytes increased in the spleen of the animals exposed to OMC, similar to the animals treated with propyl-thiouracil (PTU), a well-known thyroid disruptor. OMC modulated the percentage of leukocyte populations in peripheral blood, and the number of circulating polymorphonuclear cells increased two-fold. In vitro, OMC exhibited an inhibitory effect on splenocyte proliferation and IL-2 production induced by anti-CD3 antibody; however, this effect was reversed with the addition of T4 in the cell culture. In summary, the results of the present study demonstrate the influence of OMC on thyroid dysregulation and its impact on the modulation of the immune system in mice pups.

Immunogenicity of synthetic peptide constructs based on PvMSP9E795-A808, a linear B-cell epitope of the P. vivax Merozoite Surface Protein-9.

Plasmodium vivax Merozoite Surface Protein-9 (PvMSP-9) is a malaria vaccine candidate naturally immunogenic in humans and able to induce high antibody titers in animals when delivered as a recombinant protein. Recently, we identified the sequence EAAPENAEPVHENA (PvMSP9E795-A808) as the main linear B-cell epitope in naturally exposed individuals. However, the potential of PvMSP9E795-A808 as an immunogen in experimental animal models remained unexplored. Here we assess the immunogenicity of PvMSP9E795-A808 using synthetic peptides. The peptides tested in BALB/c mice include two repeats of the sequence EAAPENAEPVHENA tested alone (peptide RII), or linked to an autologous (PvMSP9 peptide pL; pLRII) or heterologous (p2 tetanus toxin universal T cell epitope; TTRII) T cell epitope. Immune responses were evaluated by ELISA, FLUOROSPOT, and indirect immunofluorescence. We show that all of the peptide constructs tested were immunogenic eliciting specific IgG antibodies at different levels, with a prevalence of IgG1 and IgG2. Animals immunized with synthetic peptides containing T cell epitopes (pLRII or TTRII) had more efficient antibody responses that resulted in higher antibody titers able to recognize the native protein by immunofluorescence. Relevantly, the frequency of IFN-γ secreting SFC elicited by immunization with TTRII synthetic peptide was comparable to that reported to the PvMSP9-Nt recombinant protein. Taken together, our study indicates that PvMSP9E795-A808 is highly immunogenic in mice and further studies to evaluate its value as promising vaccine target are warranted. Moreover, our study supports the critical role of CD4 T cell epitopes to enhance humoral responses induced by subunit based vaccines.

Activation of human T lymphocytes via integrin signaling induced by RGD-disintegrins.

Adhesive interactions play important roles in coordinating T cell migration and activation, which are mediated by binding of integrins to RGD motif found on extracellular matrix proteins. Disintegrins, isolated from snake venoms, contain the RGD sequence that confers selectivity to integrin interaction. We have investigated the ability of three RGD-disintegrins, ligands of alpha(5)beta(1) and alpha(v)beta(3), Flavoridin (Fl), Kistrin (Kr) and Echistatin (Ech), in modulating the activation of human T lymphocyte. The disintegrins induced T cell proliferation and CD69 expression. This activation parallels with actin cytoskeleton reorganization and tyrosine phosphorylation. Furthermore, the peptides induced focal adhesion kinase (FAK) and phosphoinositide 3-kinase (PI3K) activation. Finally, RGD-disintegrins were capable of driving NF-kappaB nuclear translocation and c-Fos expression, in a PI3K and ERK1/2 activities dependent manner. This report is the first to show that RGD-disintegrins interact with integrins on human T lymphocyte surface, modulating cell proliferation and activation of specific pathways coupled to integrin receptor.

The anti-allergic activity of the acetate fraction of Schinus terebinthifolius leaves in IgE induced mice paw edema and pleurisy.

Schinus is a genus of the Anacardiaceae family and contains Schinus terebinthifolius, the Brazilian pepper tree that is widely used in folk medicine. We investigate the anti-allergic activity of the ethyl acetate fraction of S. terebinthifolius Raddi (ST fraction). HPLC analysis reveled that gallic acid, methyl gallate and 1,2,3,4,6-pentagalloylglucose are the major aromatic components of the fraction. Oral pre-treatment with the ST fraction (100 mg/kg) significantly inhibited paw edema induced by compound 48/80 (100 ng/paw) and to a lesser extent, the allergic paw edema (OVA, 3 microg/paw). The ST fraction (100 and 200 mg/kg) also inhibited the edema induced by histamine (100 microg/paw), preventing mast cell degranulation and, consequently, histamine release in Wistar rat peritoneal mast cells induced by C 48/80 (5 microg/mL). This histamine inhibition was also observed after mast cell pre-treatment with both methyl gallate and 1,2,3,4,6-pentagalloylglucose (100 microg/mL), the isolated compounds from the ethyl acetate fraction. Pre-treatment with the ST fraction (100 mg/kg) significantly inhibited total leukocyte and eosinophil accumulation in pleural cavities 24 h after the intrathoracic injection of OVA (12.5 microg/cavity). This effect was related to the inhibition of CCL11/eotaxin and CCL5/RANTES in pleural lavage fluid. Pre-treatment with this fraction (100 mg/kg) failed to reduce the cell influx that was observed after LPS-injection into pleural cavity (250 ng/cavity). These findings demonstrate the anti-allergic effect of the ST fraction, which includes the inhibition of edema formation and histamine release caused by mast cell degranulation and eosinophil influx into the pleural cavity probably reflected by the decreased levels of chemokines in recovered pleural lavage fluid.

Immunomodulating and antiviral activities of Uncaria tomentosa on human monocytes infected with Dengue Virus-2.

Uncaria tomentosa (Willd.) DC., a large woody vine native to the Amazon and Central American rainforests has been used medicinally by indigenous peoples since ancient times and has scientifically proven immunomodulating, anti-inflammatory, cytotoxic and antioxidant activities. Several inflammatory mediators that are implicated in vascular permeability and shock are produced after Dengue Virus (DENV) infection by monocytes, the primary targets for virus replication. Here we assessed the immunoregulatory and antiviral activities from U. tomentosa-derived samples, which were tested in an in vitro DENV infection model. DENV-2 infected human monocytes were incubated with U. tomentosa hydro-alcoholic extract or either its pentacyclic oxindole alkaloid-enriched or non-alkaloid fractions. The antiviral activity was determined by viral antigen (DENV-Ag) detection in monocytes by flow cytometry. Our results demonstrated an in vitro inhibitory activity by both extract and alkaloidal fraction, reducing DENV-Ag+ cell rates in treated monocytes. A multiple microbead immunoassay was applied for cytokine determination (TNF-alpha, IFN-alpha, IL-6 and IL-10) in infected monocyte culture supernatants. The alkaloidal fraction induced a strong immunomodulation: TNF-alpha and IFN-alpha levels were significantly decreased and there was a tendency towards IL-10 modulation. We conclude that the alkaloidal fraction was the most effective in reducing monocyte infection rates and cytokine levels. The antiviral and immunomodulating in vitro effects from U. tomentosa pentacyclic oxindole alkaloids displayed novel properties regarding therapeutic procedures in Dengue Fever and might be further investigated as a promising candidate for clinical application.