Optimising approaches to active immunotherapy of cancer

The design of therapeutic cancer vaccines is aimed at inducing high numbers and potent T cells that are able to target and eradicate malignant cells. This calls for close collaboration between cells of the innate immune system, in particular dendritic cells (DCs), and cells of the adaptive immune system, notably CD4+ helper T cells and CD8+ cytotoxic T cells. Therapeutic vaccines are aided by adjuvants, which can be, for example, Toll¬like Receptor agonists or agents promoting the cytosolic delivery of antigens, among others. Vaccination with long synthetic peptides (LSPs) is a promising strategy, as the requirement for their intracellular processing will mainly target LSPs to professional antigen presenting cells (APCs), hence avoiding the immune tolerance elicited by the presentation of antigens by non-professional APCs. The unique property of antigen cross-processing and cross-presentation activity by DCs plays an important role in eliciting antitumour immunity given that antigens from engulfed dead tumour cells require this distinct biological process to be processed and presented to CD8+T cells in the context of MHC class I molecules. DCs expressing the XCR1 chemokine receptor are characterised by their superior capability of antigen cross- presentation and priming of highly cytotoxic T lymphocyte (CTL) responses. Recently, XCR1 was found to be also expressed in tissue-residents DCs in humans, with a simitar transcriptional profile to that of cross- presenting murine DCs. This shed light into the value of harnessing this subtype of XCR1+ cross-presenting DCs for therapeutic vaccination of cancer. In this study, we explored ways of adjuvanting and optimising LSP therapeutic vaccinations by the use, in Part I, of the XCLl chemokine that selectively binds to the XCR1 receptor, as a mean to target antigen to the cross-presenting XCR1+ DCs; and in Part II, by the inclusion of Q.S21 in the LSP vaccine formulation, a saponin with adjuvant activity, as well as the ability to promote cytosolic delivery of LSP antigens due to its intrinsic cell membrane insertion activity. In Part I, we designed and produced XCLl-(OVA LSP)-Fc fusion proteins, and showed that their binding to XCR1+ DCs mediate their chemoattraction. In addition, therapeutic vaccinations adjuvanted with XCLl-(OVA LSP)-Fc fusion proteins significantly enhanced the OVA-specific CD8+ T cell response, and led to complete tumour regression in the EL4-OVA model, and significant control of tumour growth in the B16.0VA tumour model. With the aim to optimise the co-delivery of LSP antigen and XCLl to skin-draining lymph nodes we also tested immunisations using nanoparticle (NP)-conjugated OVA LSP in the presence or absence of XCLl chemokine. The NP-mediated delivery of LSP potentiated the CTL response seen in the blood of vaccinated mice, and NP-OVA LSP vaccine in the presence of XCLl led to higher blood frequencies of OVA-specific memory-precursor effector cells. Nevertheless, in these settings, the addition XCLl to NP-OVA LSP vaccine formulation did not increase its antitumour therapeutic effect. In the Part II, we assessed in HLA-A2/DR1 mice the immunogenicity of the Melan-AA27L LSP or the Melan-A26. 35 AA27l short synthetic peptide (SSP) used in conjunction with the saponin adjuvant QS21, aiming to identify a potent adjuvant formulation that elicits a quantitatively and qualitatively strong immune response to tumour antigens. We showed a high CTL immune response elicited by the use of Melan-A LSP or SSP with QS21, which both exerted similar killing capacity upon in vivo transfer of target cells expressing the Melan-A peptide in the context of HLA-A2 molecules. However, the response generated by the LSP immunisation comprised higher percentages of CD8+T cells of the central memory phenotype (CD44hl CD62L+ and CCR7+ CD62L+) than those of SSP immunisation, and most importantly, the strong LSP+QS21 response was strictly CD4+T cell-dependent, as shown upon CD4 T cell depletion. Altogether, these results suggest that both XCLl and QS21 may enhance the ability of LSP to prime CD8 specific T cell responses, and promote a long-term memory response. Therefore, these observations may have important implications for the design of protein or LSP-based cancer vaccines for specific immunotherapy of cancer -- Les vacans thérapeutiques contre le cancer visent à induire une forte et durable réponse immunitaire contre des cellules cancéreuses résiduelles. Cette réponse requiert la collaboration entre le système immunitaire inné, en particulier les cellules dendrites (DCs), et le système immunitaire adaptatif, en l'occurrence les lymphocytes TCD4 hdper et CD8 cytotoxiques. La mise au point d'adjuvants et de molécules mimant un agent pathogène tels les ligands TLRs ou d'autres agents facilitant l'internalisation d'antigènes, est essentielle pour casser la tolérance du système immunitaire contre les cellules cancéreuses afin de générer une réponse effectrice et mémoire contre la tumeur. L'utilisation de longs peptides synthétiques (LSPs) est une approche prometteuse du fait que leur présentation en tant qu'antigénes requiert leur internalisation et leur transformation par les cellules dendrites (DCs, qui sont les mieux à même d'éviter la tolérance immunitaire. Récemment une sous-population de DCs exprimant le récepteur XCR1 a été décrite comme ayant une capacité supérieure dans la cross-présentation d'antigènes, d'où un intérêt à développer des vaccins ciblant les DCs exprimant le XCR1. Durant ma thèse de doctorat, j'ai exploré différentes approches pour optimiser les vaccins avec LSPs. La première partie visait à cibler les XCR1-DCs à l'aide de la chemokine XCL1 spécifique du récepteur XCR1, soit sou s la forme de protéine de fusion XCL1-OVA LSP-Fc, soit associée à des nanoparticules. La deuxième partie a consisté à tester l'association des LSPs avec I adjuvant QS21 dérivant d'une saponine dans le but d'optimiser l'internalisation cytosolique des longs peptides. Les protéines de fusion XCLl-OVA-Fc développées dans la première partie de mon travail, ont démontré leur capacité de liaison spécifique sur les XCRl-DCs associée à leur capacité de chemo-attractio. Lorsque inclues dans une mmunisation de souris porteuse de tumeurs établies, ces protéines de fusion XCL1-0VA LSP-Fc et XCLl-Fc plus OVA LSP ont induites une forte réponse CDS OVA spécifique permettant la complète régression des tumeurs de modèle EL4- 0VA et un retard de croissance significatif de tumeurs de type B16-0VA. Dans le but d'optimiser le drainage des LSPs vers es noyaux lymphatiques, nous avons également testé les LSPs fixés de manière covalente à des nanoparticules co- injectees ou non avec la chemokine XCL1. Cette formulation a également permis une forte réponse CD8 accompagnée d'un effet thérapeutique significatif, mais l'addition de la chemokine XCL1 n'a pas ajouté d'effet anti-tumeur supplémentaire. Dans la deuxième partie de ma thèse, j'ai comparé l'immunogénicité de l'antigène humain Melan A soit sous la forme d un LSP incluant un épitope CD4 et CD8 ou sous la forme d'un peptide ne contenant que l'épitope CD8 (SSP) Les peptides ont été formulés avec l'adjuvant QS21 et testés dans un modèle de souris transgéniques pour les MHC let II humains, respectivement le HLA-A2 et DR1. Les deux peptides LSP et SSP ont généré une forte réponse CD8 similaire assoc.ee a une capacité cytotoxique équivalente lors du transfert in vivo de cellules cibles présentant le peptide SSP' Cependant les souris immunisées avec le Melan A LSP présentaient un pourcentage plus élevé de CD8 ayant un Phénotype «centra, memory» (CD44h' CD62L+ and CCR7+ CD62L+) que les souris immunisées avec le SSP, même dix mois après I'immunisation. Par ailleurs, la réponse CD8 au Melan A LSP était strictement dépendante des lymphocytes CD4, contrairement à l'immunisation par le Melan A SSP qui n'était pas affectée. Dans l'ensemble ces résultats suggèrent que la chemokine XCL1 et l'adjuvant QS21 améliorent la réponse CD8 à un long peptide synthétique, favorisant ainsi le développement d'une réponse anti-tumeur mémoire durable. Ces observations pourraient être utiles au développement de nouveau vaccins thérapeutiques contre les tumeurs.

Triterpenes and new saponins from Ilex chamaedryfolia: chemotaxonomic tool to ilex species differentiation

Three saponins were isolated from leaves of Ilex chamaedryfolia. Their structures were established by spectroscopic and mass spectrometry data as the new saponin 3β-O-β-D-glucopyranosyl-(1-3)-α-L-arabinopyranosyl-20( S)-19α-hydroxyurs-12-en-28-oic acid 28-O-β-D-glucopyranosyl-(1-3)-β-D-glucopyranosyl ester, the new saponin 3β-O-β-D-glucopyranosyl-(1-3)-α-L-arabinopyranosyl-20( S)-19α-hydroxyurs-12-en-28-oic acid 28-O-β-D-glucopyranosyl ester and the known saponin 3β-O-β-D-glucuronopyranosyl-20(R )-19α-hydroxyurs-12-en-28-oic acid 28-O-β-D-glucopyranosyl ester. Ursolic acid and α-amyrin were also isolated.

Otimização das condições de extração de saponinas em Ampelozizyphus amazonicus usando planejamento experimental e metodologia de superfície de resposta

This works describes the use of experimental design and surface response methodology for optimization of saponin extraction from Ampelozizyphus amazonicus. For this purpose, a method employing extraction based on maceration assisted by ultrasound technique was utilized. The following factors were studied: extraction length of time and solvent composition. The total saponin was determined by using a gravimetric method and the results expressed by their relative proportion to total crude extract. For the specific condition, 60% hydro-alcoholic solution and 18 minutes extraction length of time has shown the best results. This method can be useful for extraction of substances with biological importance

Sheep poisoning by Panicum dichotomiflorum in northeastern Brazil

Different species of Panicum, including P. dichotomiflorum,have been reported as a cause of photosensitization in sheep, horses, cattle and goats. An outbreak of hepatogenous photosensitization occurred in 3 flocks of hair sheep in the Brazilian semiarid region. Eighty one out of 365 sheep were affected and 39 died. The main affected animals were nursing lambs and sheep younger than one year old. Donkeys, goats and cattle grazing in the same pasture were not affected. Clinical signs were edema of the head, followed by dermatitis, mainly in the face, ears, and croup, ocular discharge, corneal opacity with blindness, and redness of the coronary band and hoof. At necropsy of one affected lamb the liver was yellowish. Upon histologic examination scattered necrotic hepatocytes were observed in the liver and focal areas of necrosis of myocytes appeared in the heart. Samples of P. dicotomiflorum were analyzed by TLC and those containing saponins were isolated by HPLC using RP-C18 column and eluted with a mixture of MeOH and H2O. The isolated compounds were submitted to ¹H and 13C NMR spectroscopy. Reactions were positive to furostanol saponins with the same Rf of the standard protodioscin (0.21) and methylprotodioscin (0.32). The spectroscopic results indicated a mixture of (25R)- and (25S)-protodioscin isomers in a proportion of 3:1, and methylprotodioscin.

Effect of an isotonic rehydration sports drink and exercise on urolithiasis in rats

The objective of the present study was to evaluate the role of physical exercise as well as the influence of hydration with an isotonic sports drink on renal function in male Wistar rats. Four groups were studied over a period of 42 days: 1) control (N = 9); 2) physical exercise (Exe, N = 7); 3) isotonic drink (Drink, N = 8); 4) physical exercise + isotonic drink (Exe + Drink, N = 8). Physical exercise consisted of running on a motor-driven treadmill for 1 h/day, at 20 m/min, 5 days a week. The isotonic sports drink was a commercial solution used by athletes for rehydration after physical activity, 2 ml administered by gavage twice a day. Urine cultures were performed in all animals. Twenty-four-hour urine samples were collected in metabolic cages at the beginning and at the end of the protocol period. Urinary and plasma parameters (sodium, potassium, urea, creatinine, calcium) did not differ among groups. However, an amorphous material was observed in the bladders of animals in the Exe + Drink and Drink groups. Characterization of the material by Western blot revealed the presence of Tamm-Horsfall protein and angiotensin converting enzyme. Physical exercise and the isotonic drink did not change the plasma or urinary parameters measured. However, the isotonic drink induced the formation of intravesical matrix, suggesting a potential lithogenic risk.

Cholesterol-dependent hemolytic activity of Passiflora quadrangularis leaves

Plants used in traditional medicine are rich sources of hemolysins and cytolysins, which are potential bactericidal and anticancer drugs. The present study demonstrates for the first time the presence of a hemolysin in the leaves of Passiflora quadrangularis L. This hemolysin is heat stable, resistant to trypsin treatment, has the capacity to froth, and acts very rapidly. The hemolysin activity is dose-dependent, with a slope greater than 1 in a double-logarithmic plot. Polyethylene glycols of high molecular weight were able to reduce the rate of hemolysis, while liposomes containing cholesterol completely inhibited it. In contrast, liposomes containing phosphatidylcholine were ineffective. The Passiflora hemolysin markedly increased the conductance of planar lipid bilayers containing cholesterol but was ineffective in cholesterol-free bilayers. Successive extraction of the crude hemolysin with n-hexane, chloroform, ethyl acetate, and n-butanol resulted in a 10-fold purification, with the hemolytic activity being recovered in the n-butanol fraction. The data suggest that membrane cholesterol is the primary target for this hemolysin and that several hemolysin molecules form a large transmembrane water pore. The properties of the Passiflora hemolysin, such as its frothing ability, positive color reaction with vanillin, selective extraction with n-butanol, HPLC profile, cholesterol-dependent membrane susceptibility, formation of a stable complex with cholesterol, and rapid erythrocyte lysis kinetics indicate that it is probably a saponin.

Antinutrients in the cassava (Manihot esculenta Crantz) leaf powder at three ages of the plant

In Brazil, the cassava leaf meal (CLM) has been used to strive against undernourishment because it is a high source of vitamins and minerals. However, the wide variation in the chemical composition of the different cultivars, as well as their antinutritional substances may be a restriction to their uses. The levels of some antinutrients in CLM from five cultivars at three ages of the plant (TAP) were investigated, in order to select the cultivars and plant ages that would be more appropriate for human consumption. The lowest contents of antinutrients were observed in the 12-month old plants, except for nitrate and hemagglutinin from which the lowest contents were found for the 17 month old ones. The cultivar IAC 289-70 had the lowest antinutrient levels, except for saponin and oxalate. Thus, the cultivar IAC 289-70 at 12 months is the most appropriate for human consumption.

Developing novel one-step processes for obtaining food-grade O/W emulsions from pressurized fluid extracts: processes description, state of the art and perspectives

Abstract In this work, a novel on-line process for production of food-grade emulsions containing oily extracts, i.e. oil-in-water (O/W) emulsions, in only one step is presented. This process has been called ESFE, Emulsions from Supercritical Fluid Extraction. With this process, emulsions containing supercritical fluid extracts can be obtained directly from plant materials. The aim in the conception of this process is to propose a new rapid way to obtain emulsions from supercritical fluid extracts. Nowadays the conventional emulsion formulation method is a two-step procedure, i.e. first supercritical fluid extraction for obtaining an extract; secondly emulsion formulation using another device. Other variation of the process was tested and successfully validated originating a new acronymed process: EPFE (Emulsions from Pressurized Fluid Extractions). Both processes exploit the supercritical CO2-essential oils miscibility, in addition, EPFE process exploits the emulsification properties of saponin-rich pressurized aqueous plant extracts. The feasibility of this latter process was demonstrated using Pfaffia glomerata roots as source of saponin-rich extract, water as extracting solvent and clove essential oil, directly extracted using supercritical CO2, as a model dispersed phase. In addition, examples of pressurized fluid-based coupled processes applied for adding value to food bioactive compounds developed in the past five years are reviewed.

Lichens used as monitors of atmospheric pollution around Agadir (Southwestern Morocco) - a case study predating lead-free gasoline

More than 30 epiphytic lichens, collected in Agadir (Morroco) and along a 150-km transect from the Atlantic Ocean eastward, were analyzed for their metal content and lead isotopic composition. This dataset was used to evaluate atmospheric metal contamination and the impact of the city on the surrounding area. The concentrations of Cu, Pb, and Zn (average ± 1 SD) were 20.9 ± 15.2 μg g−1, 13.8 ± 9.0 μg g−1, and 56.6 ± 26.6 μg g−1, respectively, with the highest values observed in lichens collected within the urban area. The 206Pb/207Pb and 208Pb/207Pb ratios in the lichens varied from 1.146 to 1.186 and from 2.423 to 2.460, respectively. Alkyllead-gasoline sold in Morocco by the major petrol companies gave isotopic ratios of 206Pb/207Pb = 1.076–1.081 and 208Pb/207Pb = 2.348–2.360. These new, homogeneous values for gasoline-derived lead improve and update the scarce isotopic database of potential lead sources in Morocco, and may be of great value to future environmental surveys on the presence of lead in natural reservoirs, where it persists over time (e.g., soils and sediments). The interest of normalizing metal concentrations in lichens to concentrations of a lithogenic element is demonstrated by the consistency of the results thus obtained with lead isotopic ratios. Leaded gasoline contributed less than 50% of the total amount of lead accumulated in lichens, even in areas subject to high vehicular traffic. This strongly suggests that the recent banishment of leaded gasoline in Morocco will not trigger a drastic improvement in air quality, at least in Agadir.

Effects of calcium supplementation on body weight reduction in overweight calcium stone formers

A randomized, placebo-controlled trial was conducted in overweight calcium stone-forming (CSF) patients, to evaluate the effect of calcium supplementation associated with a calorie-restricted diet on body weight (BW) and fat reduction and its potential changes upon serum and urinary parameters. Fifteen patients were placed on a hypocaloric diet for 3 months, supplemented with either calcium carbonate (CaCO(3), n = 8) or placebo (n = 7), 500 mg bid. Blood and 24-h urine samples were collected and body composition was assessed at baseline and after the intervention. At the end of the study, final BW was significantly lower vs baseline in both CaCO(3) (74 +/- A 14 vs. 80 +/- A 14 kg, P = 0.01) and placebo groups (80 +/- A 10 vs. 87 +/- A 9 kg, P = 0.02) but the mean percentage of loss of body weight and body fat did not differ between CaCO(3) and placebo (7.0 +/- A 2.0 vs. 8.0 +/- A 3.0%, P = 0.40 and 13.0 +/- A 7.0 vs. 13.0 +/- A 10.0%; P = 0.81, respectively). After CaCO(3) or placebo, no significant differences versus baseline were observed for urinary parameters in both CaCO(3) and placebo, except for a higher mean urinary citrate in placebo group. These data suggest that increasing calcium intake by calcium carbonate supplementation did not contribute to a further reduction of BW and fat in overweight CSF patients submitted to a hypocaloric diet nor altered urinary lithogenic parameters.

Caffeic and chlorogenic acids in Ilex paraguariensis extracts are the main inhibitors of AGE generation by methylglyoxal in model proteins

The present study concentrates on the evaluation of the anti-glycation effect of some bioactive substances present in yerba mate (Ilex paraguariensis): 5-caffeoylquinic acid, caffeic acid and a sapogenin (oleanolic acid). Bovine serum albumin and histones were incubated in the presence of methylglyoxal with or without the addition of 5-caffeoylquinic acid, caffeic acid and oleanolic acid. After the incubation period, advanced glycation end product (AGE) fluorescence spectra were performed and protein structural changes were evaluated by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. Chlorogenic acid, caffeic acid are the main substances responsible for the anti-glycation effect of mate tea. (C) 2009 Elsevier B.V. All rights reserved.

A geochemical and lead isotopic record from a small pond in a remote equatorial island, Fernando de Noronha, Brazil

A 72 cm long core was collected from Lagoa da Viracao (LV), a small poind in the Fernando de Noronha island, northern Brazil. Sediments from the lower section of the core (20-72 cm depth) contain essentially mineral matter, while in the upper section (0-20 cm depth) mineral matter is mixed with organic matter. Lithogenic conservative elements - Si, Al, Fe, Ti, Co, Cr, Cu, Ba, Ga, Hf, Nb, Ni, Y, V, Zn, Zr and REE - exhibit remarkably constant values throughout the core, with concentrations similar to those of the bedrock. The vertical distribution of soluble elements - Ca, Mg, Na, K, P, Mn and Sr - is also homogeneous, but these elements are systematically depleted in relation to the bedrock. LOI, TOC, Br, Se, Hg and Pb, although showing nearly constant values in the lower section of the core, are significantly enriched in the upper section. The concentration profiles of Br and Se suggest that they may be accounted for by natural processes, related to the slight affinity of these elements for organic matter. On the other hand, the elevated levels of Hg and Pb in recent sediments may be explained by their long-range atmospheric transport and deposition. Furthermore, the isotopic composition of Pb clearly indicates that anthropogenic sources contributed to the Pb burden in the uppermost pond sediments.

Use of Saponins as an Effective Surface Modifier in Cellulose Nanocomposites

The present paper deals with the extraction of saponins from the pericarp of Sapindus mukorossi to use as compatibilizer in nanocomposites. The nanofibrils extracted from banana fibres are utilized as reinforcement of nanocomposite. These nanofibers were treated with Saponin, GPS (3-Glycidoxypropyltrimethoxysilane) and APS (3-Aminopropyltriethoxysilane) to compare the effectiveness of surface treatment. The effectiveness of surface modification was reflected on the increase in mechanical (tensile test, flexural modulus, impact test) properties and decrease in the RMS (Roughness Measurement System) roughness investigation by SFM (Scanning force microscopy) analysis.

Effect of the addition of soy lecithin and Yucca schidigera extract on the properties of gelatin and glycerol based biodegradable films

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Porcentagens de saponinas e taninos em vinte e oito cultivares de alfafa (Medicago sativa L.) em duas épocas de corte - Botucatu - SP

O objetivo do estudo foi determinar os conteúdos de saponinas, taninos e a solubilidade da proteína (SP) de 28 cultivares de alfafa: Crioula, Monarca, BR 4, Alto Great, MH 4, SW 9210 A, 5929, BR 1, EL Grande, 5715, MH 15, Valley Plus, BR 2, Rio, SW 8210, Maricopa, ICI 990, 5888, P 30, Alfa-200, WL 516, SW 8112 A, BR 3, Florida 77, Araucana, Falcon, Semit 921 e Sutter. O material analisado foi obtido do 10º e 14º corte, respectivamente; em 08/12/97 e 16/04/98, de um experimento desenvolvido na Faculdade de Medicina Veterinária e Zootecnia, UNESP, Botucatu. Amostras de cada cultivar foram colhidas, pesadas e secas em estufa a 52ºC. As saponinas foram extraídas com solvente hidrofílico e lipofílico e o conteúdo foi calculado pela equação y=0,8121x-1,4759, R² = 1,00. A extração dos taninos totais e condensados foi efetuada por meio de ultra-som (12 min), sendo os taninos totais determinados pela equação: y=44,978 + 0,5644 com R²=0,9977 e os condensados, multiplicando-se a absorbância por 78,26 dividido pelo teor de matéria seca. A SP foi determinada pelo método de KOH, de acordo com a seguinte fórmula: SP (%) = proteína solúvel x 100/proteína bruta da amostra. Os teores de saponinas, taninos totais e condensados e a solubilidade da proteína não diferiram (P>0,05) entre as cultivares. Houve efeito (P<0,05) da época de corte apenas sobre o teor de taninos totais. Os teores médios de saponinas de 1,00% aliados à baixa solubilidade média da proteína bruta (34,11%) não se constituem em fatores limitantes para uso dos cultivares de alfafa estudadas.