Targeted activation of toll-like receptors: conjugation of a toll-like receptor 7 agonist to a monoclonal antibody maintains antigen binding and specificity

Therapeutic activation of Toll-like receptors (TLR) has potential for cancer immunotherapy, for augmenting the activity of anti-tumor monoclonal antibodies (mAbs), and for improved vaccine adjuvants. A previous attempt to specifically target TLR agonists to dendritic cells (DC) using mAbs failed because conjugation led to non-specific binding and mAbs lost specificity. We demonstrate here for the first time the successful conjugation of a small molecule TLR7 agonist to an anti-tumour mAb (the anti-hCD 20 rituximab) without compromising antigen specificity. The TLR7 agonist UC-1V150 was conjugated to rituximab using two conjugation methods and yield, molecular substitution ratio, retention of TLR7 activity and specificity of antigen binding were compared. Both conjugation methods produced rituximab-UC-1V150 conjugates with UC-1V150 : rituximab ratio ranging from 1:1 to 3:1 with drug loading quantified by UV spectroscopy and drug substitution ratio verified by MALDI TOF mass spectroscopy. The yield of purified conjugates varied with conjugation method, and dropped as low as 31% using a method previously described for conjugating UC-1V150 to proteins, where a bifunctional crosslinker was firstly reacted with rituximab, and secondly to the TLR7 agonist. We therefore developed a direct conjugation method by producing an amine-reactive UV active version of UC-1V150, termed NHS:UC-1V150. Direct conjugation with NHS:UC-1V150 was quick and simple and gave improved conjugate yields of 65-78%. Rituximab-UC-1V150 conjugates had the expected pro-inflammatory activity in vitro (EC50 28-53 nM) with a significantly increased activity over unconjugated UC-1V150 (EC50 547 nM). Antigen binding and specificity of the rituxuimab-UC-1V150 conjugates was retained, and after incubation with human peripheral blood leukocytes, all conjugates bound strongly only to CD20-expressing B cells whilst no non-specific binding to CD20-negative cells was observed. Selective targeting of Toll-like receptor activation directly within tumors or to DC is now feasible.

Impact of chemical structure of flavanol monomers and condensed tannins on in vitro anthelmintic activity against bovine nematodes

Plants containing condensed tannins (CT) may have potential to control gastrointestinal nematodes (GIN) of cattle. The aim was to investigate the anthelmintic activities of four flavan-3-ols, two galloyl derivatives and 14 purified CT fractions, and to define which structural features of CT determine the anti-parasitic effects against the main cattle nematodes. We used in vitro tests targeting L1 larvae (feeding inhibition assay) and adults (motility assay) of Ostertagia ostertagi and Cooperia oncophora. In the larval feeding inhibition assay, O. ostertagi L1 were significantly more susceptible to all CT fractions than C. oncophora L1. The mean degree of polymerization of CT (i.e. average size) was the most important structural parameter: large CT reduced larval feeding more than small CT. The flavan-3-ols of prodelphinidin (PD)-type tannins had a stronger negative influence on parasite activity than the stereochemistry, i.e. cis- vs trans-configurations, or the presence of a gallate group. In contrast, for C. oncophora high reductions in the motility of larvae and adult worms were strongly related with a higher percentage of PDs within the CT fractions while there was no effect of size. Overall, the size and the percentage of PDs within CT seemed to be the most important parameters that influence anti-parasitic activity.

Proanthocyanidins inhibit Ascaris suum glutathione-S-transferase activity and increase susceptibility of larvae to levamisole and ivermectin in vitro

Proanthocyanidins (PAC) are a class of plant secondary metabolites commonly found in the diet that have shown potential to control gastrointestinal nematode infections. The anti-parasitic mechanism(s) of PAC remain obscure, however the protein-binding properties of PAC suggest that disturbance of key enzyme functions may be a potential mode of action. Glutathione-S-transferases (GSTs) are essential for parasite detoxification and have been investigated as drug and vaccine targets. Here, we show that purified PAC strongly inhibit the activity of both recombinant and native GSTs from the parasitic nematode Ascaris suum. As GSTs are involved in detoxifying xenobiotic substances within the parasite, we hypothesised that this inhibition may render parasites hyper-susceptible to anthelmintic drugs. Migration inhibition assays with A. suum larvae demonstrated that the potency of levamisole (LEV) and ivermectin (IVM) were significantly increased in the presence of PAC purified from pine bark (4.6-fold and 3.2-fold reduction in IC50 value for LEV and IVM, respectively). Synergy analysis revealed that the relationship between PAC and LEV appeared to be synergistic in nature, suggesting a specific enhancement of LEV activity, whilst the relationship between PAC and IVM was additive rather than synergistic, suggesting independent actions. Our results demonstrate that these common dietary compounds may increase the efficacy of synthetic anthelmintic drugs in vitro, and also suggest one possible mechanism for their well-known anti-parasitic activity.

Local Corticosterone Infusion Enhances Nocturnal Pineal Melatonin Production In Vivo

Melatonin, an important marker of the endogenous rhythmicity in mammals, also plays a role in the body defence against pathogens and injuries. In vitro experiments have shown that either pro- or anti-inflammatory agents, acting directly in the organ, are able to change noradrenaline-induced pineal indoleamine production. Whereas corticosterone potentiates melatonin production, incubation of the gland with tumour necrosis factor-alpha decreases pineal hormonal production. In the present study, we show that nocturnal melatonin production measured by intra-pineal microdialysis is enhanced in pineals perfused with corticosterone at concentrations similar to those measured in inflamed animals. In vitro experiments suggest that this enhancement may be due to an increase in the activity of the two enzymes that convert serotonin to N-acetylserotonin (NAS) and NAS to melatonin. The present results support the hypothesis that the pineal gland is a sensor of inflammation mediators and that it plays a central role in the control of the inflammatory response.

The Immune-Pineal Axis Stress as a Modulator of Pineal Gland Function

The temporal organization of mammals presents a daily adjustment to the environmental light/dark cycle. The environmental light detected by the retina adjusts the central clock in the suprachiasmatic nuclei, which innervate the pineal gland through a polysynaptic pathway. During the night, this gland produces and releases the nocturnal hormone melatonin, which circulates throughout the whole body and adjusts several bodily functions according to the existence and duration of darkness. We have previously shown that during the time frame of an inflammatory response, pro-inflammatory cytokines, such as tumor necrosis factor-a, inhibit while anti-inflammatory mediators, such as glucocorticoids, enhance the synthesis of melatonin, interfering in the daily adjustment of the light/dark cycle. Therefore, injury disconnects the organism from environmental cycling, while recovery restores the light/dark information to the whole organism. Here, we extend these observations by evaluating the effect of a mild restraint stress, which did not induce macroscopic gastric lesions. After 2 h of restraint, there was an increase in circulating corticosterone, indicating activation of the hypothalamus-pituitary-adrenal (HPA) axis. In parallel, an increase in melatonin production was observed. Taking into account the data obtained with models of inflammation and stress, we reinforce the hypothesis that the activity of the pineal gland is modulated by the state of the immune system and the HPA axis, implicating the darkness hormone melatonin as a modulator of defense responses.

Chronic low frequency/low volume resistance training reduces pro-inflammatory cytokine protein levels and TLR4 mRNA in rat skeletal muscle

Skeletal muscle is the source of pro- and anti-inflammatory cytokines, and recently, it has been recognized as an important source of interleukin 6 (IL-6), a cytokine that exerts inhibitory effects on several pro-inflammatory cytokines. Although dynamic chronic resistance training has been shown to produce the known ""repeated bout effect"", which abolishes the acute muscle damage, performing of high-intensity resistance training has been regarded highly advisable, at least from the hypertrophy perspective. On the other hand, a more therapeutic, ""non-damaging"" resistance training program, mainly composed of concentric forces, low frequency/low volume of training, and the same exercise, could theoretically benefit the muscle when the main issue is to avoid muscle inflammation (as in the treatment of several ""low-grade"" inflammatory diseases) because the acute effect of each resistance exercise session could be diminished/avoided, at the same time that the muscle is still being overloaded in a concentric manner. However, the benefits of such ""less demanding"" resistance training schedule on the muscle inflammatory profile have never been investigated. Therefore, we assessed the protein expression of IL-6, TNF-alpha, IL-10, IL-10/TNF-alpha ratio, and HSP70 levels and mRNA expression of SCF(beta-TrCP), IL-15, and TLR-4 in the skeletal muscle of rats submitted to resistance training. Briefly, animals were randomly assigned to either a control group (S, n = 8) or a resistance-trained group (T, n = 7). Trained rats were exercised over a duration of 12 weeks (two times per day, two times per week). Detection of IL-6, TNF-alpha, IL-10, and HSP70 protein expression was carried out by western blotting and SCF(beta-TrCP) (SKP Cullin F-Box Protein Ligases), a class of enzymes involved in the ubiquitination of protein substrates to proteasomal degradation, IL-15, and TLR-4 by RT-PCR. Our results show a decreased expression of TNF-alpha and TLR4 mRNA (40 and 60%, respectively; p < 0.05) in the plantar muscle from trained, when compared with control rats. In conclusion, exercise training induced decreased TNF-alpha and TLR-4 expressions, resulting in a modified IL-10/TNF-alpha ratio in the skeletal muscle. These data show that, in healthy rats, 12-week resistance training, predominantly composed of concentric stimuli and low frequency/low volume schedule, down regulates skeletal muscle production of cytokines involved in the onset, maintenance, and regulation of inXammation.

Differing effects of exogenous and endogenous hydrogen sulphide in carrageenan-induced knee joint synovitis in the rat

Background and purpose: Recent findings suggest that the noxious gas H(2)S is produced endogenously, and that physiological concentrations of H(2)S are able to modulate pain and inflammation in rodents. This study was undertaken to evaluate the ability of endogenous and exogenous H(2)S to modulate carrageenan-induced synovitis in the rat knee. Experimental approach: Synovitis was induced in Wistar rats by intra-articular injection of carrageenan into the knee joint. Sixty minutes prior to carrageenan injection, the rats were pretreated with indomethacin, an inhibitor of H(2)S formation (dl-propargylglycine) or an H(2)S donor [Lawesson`s reagent (LR)]. Key results: Injection of carrageenan evoked knee inflammation, pain as characterized by impaired gait, secondary tactile allodynia of the ipsilateral hindpaw, joint swelling, histological changes, inflammatory cell infiltration, increased synovial myeloperoxidase, protein nitrotyrosine residues, inducible NOS (iNOS) activity and NO production. Pretreatment with LR or indomethacin significantly attenuated the pain responses, and all the inflammatory and biochemical changes, except for the increased iNOS activity, NO production and 3-NT. Propargylglycine pretreatment potentiated synovial iNOS activity (and NO production), and enhanced macrophage infiltration, but had no effect on other inflammatory parameters. Conclusions and implications: Whereas exogenous H(2)S delivered to the knee joint can produce a significant anti-inflammatory and anti-nociceptive effect, locally produced H(2)S exerts little immunomodulatory effect. These data further support the development and use of H(2)S donors as potential alternatives (or complementary therapies) to the available anti-inflammatory compounds used for treatment of joint inflammation or relief of its symptoms.

Synthesis and in vitro activity of limonene derivatives against Leishmania and Trypanosoma

The synthesis and in vitro activity of R(+)-Limonene derivatives against Leishmania and Trypanosoma cruzi strains are reported. Seven compounds have shown better in vitro activity against Leishmania (V.)braziliensis than the standard drug pentamidine. Additionally, we have identified two promising new anti-T. cruzi limonene derivatives. (C) 2010 Elsevier Masson SAS. All rights reserved.

Low level laser therapy (LLLT) decreases pulmonary microvascular leakage, neutrophil influx and IL-1 beta levels in airway and lung from rat subjected to LPS-induced inflammation

Background and Objective. Low level laser therapy (LLLT) is a known anti-inflammatory therapy. Herein we studied the effect of LLLT on lung permeability and the IL-1 beta level in LPS-induced pulmonary inflammation. Study Design/Methodology. Rats were divided into 12 groups (n = 7 for each group). Lung permeability was measured by quantifying extravasated albumin concentration in lung homogenate, inflammatory cells influx was determined by myeloperoxidase activity, IL-1P in BAL was determined by ELISA and IL-1P mRNA expression in trachea was evaluated by RT-PCR. The rats were irradiated on the skin over the upper bronchus at the site of tracheotomy after LPS. Results. LLLT attenuated lung permeability. In addition, there was reduced neutrophil influx, myeloperoxidase activity and both IL-1 beta in BAL and IL-1 beta mRNA expression in trachea obtained from animals subjected to LPS-induced inflammation. Conclusion. LLLT reduced the lung permeability by a mechanism in which the IL-1 beta seems to have an important role.

Argininosuccinate Synthetase Is a Functional Target for a Snake Venom Anti-hypertensive Peptide ROLE IN ARGININE AND NITRIC OXIDE PRODUCTION

Bj-BPP-10c is a bioactive proline-rich decapeptide, part of the C-type natriuretic peptide precursor, expressed in the brain and in the venom gland of Bothrops jararaca. We recently showed that Bj-BPP-10c displays a strong, sustained anti-hypertensive effect in spontaneous hypertensive rats (SHR), without causing any effect in normotensive rats, by a pharmacological effect independent of angiotensin-converting enzyme inhibition. Therefore, we hypothesized that another mechanism should be involved in the peptide activity. Here we used affinity chromatography to search for kidney cytosolic proteins with affinity for Bj-BPP-10c and demonstrate that argininosuccinate synthetase (AsS) is the major protein binding to the peptide. More importantly, this interaction activates the catalytic activity of AsS in a dose-dependent manner. AsS is recognized as an important player of the citrulline-NO cycle that represents a potential limiting step in NO synthesis. Accordingly, the functional interaction of Bj-BPP-10c and AsS was evidenced by the following effects promoted by the peptide: (i) increase of NO metabolite production in human umbilical vein endothelial cell culture and of arginine in human embryonic kidney cells and (ii) increase of arginine plasma concentration in SHR. Moreover, alpha-methyl-DL-aspartic acid, a specific AsS inhibitor, significantly reduced the anti-hypertensive activity of Bj-BPP-10c in SHR. Taken together, these results suggest that AsS plays a role in the anti-hypertensive action of Bj-BPP-10c. Therefore, we propose the activation of AsS as a new mechanism for the anti-hypertensive effect of Bj-BPP-10c in SHR and AsS as a novel target for the therapy of hypertension-related diseases.

Actions of octocoral and tobacco cembranoids on nicotinic receptors

Nicotinic acetylcholine receptors (AChRs) are pentameric proteins that form agonist-gated cation channels through the plasma membrane. AChR agonists and antagonists are potential candidates for the treatment of neurodegenerative diseases. Cembranoids are naturally occurring diterpenoids that contain a 14-carbon ring. These diterpenoids interact with AChRs in complex ways: as irreversible inhibitors at the agonist sites, as noncompetitive inhibitors, or as positive modulators, but no cembranoid was ever shown to have agonistic activity on AChRs. The cembranoid eupalmerin acetate displays positive modulation of agonist-induced currents in the muscle-type AChR and in the related gamma-aminobutyric acid (GABA) type A receptor. Moreover, cembranoids display important biological effects, many of them mediated by nicotinic receptors. Cembranoids from tobacco are neuroprotective through a nicotinic anti-apoptotic mechanism preventing excitotoxic neuronal death which in part could result from anti-inflammatory properties of cembranoids. Moreover, tobacco cembranoids also have anti-inflammatory properties which could enhance their neuroprotective properties. Cembranoids from tobacco affect nicotine-related behavior: they increase the transient initial ataxia caused by first nicotine injection into naive rats and inhibit the expression of locomotor sensitization to repeated injections of nicotine. In addition, cembranoids are known to act as anti-tumor compounds. In conclusion, cembranoids provide a promising source of lead drugs for many clinical areas, including neuroprotection, smoking-cessation, and anti-cancer therapies. (C) 2009 Elsevier Ltd. All rights reserved.

Volsurf Descriptors to Analyse Anti-HCV and Cytotoxic Activities of Sesquiterpene Lactones from Asteraceae Family

Various significant anti-HCV and cytotoxic sesquiterpene lactones (SLs) have been characterized. In this work, the chemometric tool Principal Component Analysis (PCA) was applied to two sets of SLs and the variance of the biological activity was explored. The first principal component accounts for as much of the variability in the data as possible, and each succeeding component accounts for as much of the remaining variability as possible. The calculations were performed using VolSurf program. For anti-HCV activity, PC1 (First Principal Component) explained 30.3% and PC2 (Second Principal Component) explained 26.5% of matrix total variance, while for cytotoxic activity, PC1 explained 30.9% and PC2 explained 15.6% of the total variance. The formalism employed generated good exploratory and predictive results and we identified some structural features, for both sets, important to the suitable biological activity and pharmacokinetic profile.

Análise botânica, química e biológica comparativa entre flores das espécies sambucus nigra L. e sambucus australis cham. & Schltdt. e avaliação preliminar da estabilidade

Flores das espécies Sambucus nigra, de origem européia, e Sambucus australis, nativa da América do Sul (Caprifoliaceae), denominadas sabugueiro e sabugueirodo- brasil, respectivamente, são utilizadas popularmente sob forma de infusão, como antiinflamatórias, laxativas e para condições febris resultantes de afecções do trato respiratório. Estudos prévios para S. nigra indicam compostos fenólicos como principais constituintes químicos, sendo estes relacionados às principais atividades biológicas avaliadas. Objetivando comparar esta espécie com Sambucus australis, foram realizadas análises botânicas macro e microscópicas das flores, identificando as principais diferenças entre as espécies, tais como o número de lóculos no ovário e presença de idioblastos cristalíferos em algumas estruturas, e observando os possíveis contaminantes (pedicelos). Também foram determinados os parâmetros farmacopéicos: cinzas totais e perda por dessecação. Após a análise química, foi escolhido o flavonóide rutina como marcador das espécies, para realizar análises quantitativas nas 31 amostras adquiridas e / ou coletadas, utilizando método de CLAE previamente validado. Soluções hidroetanólicas apresentaram maior capacidade de extração do produto alvo. Os limites mínimos de rutina observados para ambas as espécies foram de aproximadamente 0,65%. Também foram quantificados, por método espectrofotométrico, os flavonóides totais expressos em quercetina, sendo 0,93% e 1,46% os teores mínimos determinados para S.nigra e S. australis, respectivamente. O estudo da estabilidade acelerada (50°C ± 90% U.R.), avaliando a degradação dos constituintes químicos presentes permitiu sugerir a cinética de degradação de segunda ordem para da rutina nas duas espécies. Comparações de atividades biológicas das espécies foram realizadas pelos ensaios das atividades antiinflamatória (inibição do edema em pata de rato induzido por carragenina) e antioxidante (DPPH). Os resultados para o primeiro ensaio demonstraram ação equivalente em ambas as espécies para extratos hidroetanólicos a 80% (86% de inibição) e aquosos (81%), com atividade semelhante ao padrão indometacina (~83%); para a atividade antioxidante os extratos hidroetanólicos a 80% foram mais ativos (CE50 = 16 μg/ml) que os aquosos (CE50 = 27 μg/ml) em S. australis, e ambos extratos, superiores ao 28 extrato padronizado Gingko biloba (CE50 = 40 μg/ml) e aos extratos de S. nigra (CE50= 50 μg/ml – hidroetanólico e CE50= 32 μg/ml – aquoso).

Potencial terapêutico de um heparinóide isolado do invertebrado marinho Litopenaeus vannamei

The occurrence of bioactive compounds in marine organisms comes awaking the interest of the pharmaceutical industry. Heparin, a sulfated polysaccharide which presence was already identified in several marine invertebrates, is very attractive due its remarkable functional versatility. Besides to intervene in blood coagulation, this molecule has a great anti-inflammatory potential. However, its strong anticoagulant activity difficult the clinical exploitation of its anti-inflammatory properties. Thus, the aims of this work were to evaluate the effect of a heparin-like compound (heparinoid), isolated from the cephalotorax of the Litopenaeus vannamei shrimp, on the inflammatory response, hemostasia and synthesis of antithrombotic heparan sulfate by endothelial cells, besides studying some aspects concerning its structure. The purified heparinoid was structurally characterized following an analytical boarding, involving electrophoresis and chromatography. The structural analysis have shown that this compound possess a high content of glucuronic acid residues and disulfated disaccharide units. In contrast to mammalian heparin, the heparinoid was incapable to stimulate the synthesis of heparan sulfate by endothelial cells in the tested concentrations, beyond to show reduced anticoagulant activity and hemorrhagic effect. In a model of acute inflammation, the compound isolated from the shrimp reduced more than 50% of the cellular infiltration. Besides reduce the activity of MMP-9 and proMMP-2 of the peritoneal lavage of inflamed animals, the heparinoid also reduced the activity of MMP-9 secreted by activated human leukocytes. These results demonstrate the potential of heparinoid from L. vannamei to intervene in the inflammatory response. For possessing reduced anticoagulant activity and hemorrhagic effect, this compound can serve as a structural model to direct the development of more specific therapeutical agents to the treatment of inflammatory diseases

Atividades biológicas de polissacarídeos sulfatados extraídos da alga vermelha gracilaria caudata

In recent years, sulfated polysaccharides (SP) from marine algae have emerged as an important class of natural biopolymers with potential pharmacology applications. Among these, SP isolated from the cell walls of red algae have been study due to their anticoagulant,antithrombotic and anti-inflammatory activities. In the present study, three sulfated polysaccharides fractions denominated F1.5v, F2.0v and F3.0v were obtained from seaweed G. caudate by proteolysis followed to acetone fractionation. Gel electrophoresis using 0.05 M 1,3-diaminopropane-acetate buffer, pH 9,0, stained with 0.1% toluidine blue, showed the presence of SP in all fractions. The chemical analysis demonstrated that all the fractions are composed mainly of galactose. These compounds were evaluated in anticoagulant, antioxidant and antiproliferative activities. In anticoagulant activity evaluated through aPTT and PT tests, no one fractions presented anticoagulant activity at tested concentrations (0.1 mg/mL; 1.0 mg/mL; 2.0 mg/mL).The antioxidant activities of the three fractions were evaluated by the following in vitro systems: Total antioxidant capacity, superoxide and hydroxyl radical scavenging, ferrous chelating activity and reducing power. The fractions were found to have different levels of antioxidant activity in the systems tested. F1.5v shows the highest activity, especially in the ferrous chelating system, with 70% of ferrous inhibiting at 1.0 mg.mL-1. Finally, all the fractions showed dose-dependent antiproliferative activity against HeLa cells. The fractions F1.5v and F2.0v presented the highest antiproliferative activity at 2.0 mg/mL with 42.7% and 37.0% of inhibition, respectively. Ours results suggests that the sulfated polysaccharides from seaweed G. caudata are promising compounds in antioxidant and/or antitumor therapy