http://www.recercat.cat:80/handle/2072/48817 Tue, 18 Jun 2013 04:52:38 GMT 2013-06-18T04:52:38Z http://www.recercat.cat:80/bitstream/id/34147/ http://www.recercat.cat:80/handle/2072/48817 http://www.recercat.cat:80/handle/2072/212123 Community pharmacist intervention in depressed primary care patients (PRODEFAR study): randomized controlled trial protocol. Rubio Valera, Maria; Serrano Blanco, Antoni; Travé i Mercadé, Pere; Peñarrubia María, María Teresa; Ruiz, Mar; March Pujol, Marian Background: Treatment of depression, the most prevalent and costly mental disorder, needs to be improved. Non-concordance with clinical guidelines and non-adherence can limit the efficacy of pharmacological treatment of depression. Through pharmaceutical care, pharmacists can improve patients' compliance and wellbeing. The aim of this study is to evaluate the effectiveness and costeffectiveness of a community pharmacist intervention developed to improve adherence and outcomes of primary care patients with depression. Methods/design: A randomized controlled trial, with 6-month follow-up, comparing patients receiving a pharmaceutical care support programme in primary care with patients receiving usual care. The total sample comprises 194 patients (aged between 18 and 75) diagnosed with depressive disorder in a primary care health centre in the province of Barcelona (Spain). Subjects will be asked for written informed consent in order to participate in the study. Diagnosis will be confirmed using the SCID-I. The intervention consists of an educational programme focused on improving knowledge about medication, making patients aware of the importance of compliance, reducing stigma, reassuring patients about side-effects and stressing the importance of carrying out general practitioners' advice. Measurements will take place at baseline, and after 3 and 6 months. Main outcome measure is compliance with antidepressants. Secondary outcomes include; clinical severity of depression (PHQ-9), anxiety (STAI-S), health-related quality of life (EuroQol-5D), satisfaction with the treatment received, side-effects, chronic physical conditions and sociodemographics. The use of healthcare and social care services will be assessed with an adapted version of the Client Service Receipt Inventory (CSRI). Discussion: This trial will provide valuable information for health professionals and policy makers on the effectiveness and cost-effectiveness of a pharmaceutical intervention programme in the context of primary care. Trial registration: NCT00794196 http://www.recercat.cat:80/handle/2072/212123 http://www.recercat.cat:80/handle/2072/207492 In vitro antitumor activity of methotrexate via pH-sensitive chitosan nanoparticles Nogueira, Daniele R.; Tavano, Lorena; Mitjans Arnal, Montserrat; Pérez Muñoz, Lourdes; Infante Martínez-Pardo, Ma. Rosa; Vinardell Martínez-Hidalgo, Ma. Pilar Nanoparticles with pH-sensitive behavior may enhance the success of chemotherapy in many cancers by efficient intracellular drug delivery. Here, we investigated the effect of a bioactive surfactant with pH-sensitive properties on the antitumor activity and intracellular behavior of methotrexate-loaded chitosan nanoparticles (MTX-CS-NPs). NPs were prepared using a modified ionotropic complexation process, in which was included the surfactant derived from Nα,Nε-dioctanoyl lysine with an inorganic lithium counterion. The pH-sensitive behavior of NPs allowed accelerated release of MTX in an acidic medium, as well as membrane-lytic pH-dependent activity, which facilitated the cytosolic delivery of endocytosed materials. Moreover, our results clearly proved that MTX-CSNPs were more active against the tumor HeLa and MCF-7 cell lines than the free drug. The feasibilty of using NPs to target acidic tumor extracellular pH was also shown, as cytotoxicity against cancer cells was greater in a mildly acidic environment. Finally, the combined physicochemical and pH-sensitive properties of NPs generally allowed the entrapped drug to induce greater cell cycle arrest and apoptotic effects. Therefore, our overall results suggest that pH-sensitive MTX-CS-NPs could be potentially useful as a carrier system for tumor and intracellular drug delivery in cancer therapy. http://www.recercat.cat:80/handle/2072/207492 http://www.recercat.cat:80/handle/2072/207491 New cationic nanovesicular systems containing lysine-based surfactants for topical administration: Toxicity assessment using representative skin cell lines Nogueira, Daniele R.; Morán Badenas, María del Carmen; Mitjans Arnal, Montserrat; Martínez Ocaña, Verónica; Pérez Muñoz, Lourdes; Vinardell Martínez-Hidalgo, Ma. Pilar Many strategies for treating diseases require the delivery of drugs into the cell cytoplasm following internalization within endosomal vesicles. Thus, compounds triggered by low pH to disrupt membranes and release endosomal contents into the cytosol are of particular interest. Cationic nanovesicles have attracted considerable interest as effective carriers to improve the delivery of biologically active molecules into and through the skin. In this study, lipid-based nanovesicles containing three different cationic lysine-based surfactants were designed for topical administration. We used representative skin cell lines and in vitro assays to assess whether the cationic compounds modulate the toxic responses of these nanocarriers. The nanovesicles were characterized in both water and cell culture medium. In general, significant agglomeration occurred after 24 h incubation under cell culture conditions. We found different cytotoxic responses among the formulations, which depended on the surfactant,cell line (3T3, HaCaT, and THP-1) and endpoint assayed (MTT, NRU, and LDH). Moreover, no potential phototoxicity was detected in fibroblast or keratinocyte cells, whereas only a slight inflammatory response was induced, as detected by IL-1a and IL-8 production in HaCaT and THP-1 cell lines, respectively. A key finding of our research was that the cationic charge position and the alkyl chain length of the surfactants determine the nanovesicles resulting toxicity. The charge on the a-amino group of lysine increased the depletion of cell metabolic activity, as determined by the MTT assay, while a higher hydrophobicity tends to enhance the toxic responses of the nanovesicles. The insights provided here using different cell lines and assays offer a comprehensive toxicological evaluation of this group of new nanomaterials. http://www.recercat.cat:80/handle/2072/207491 http://www.recercat.cat:80/handle/2072/206676 Comparative sensitivity of tumor and non-tumor cell lines as reliable approach for in vitro cytotoxicity screening of lysine-based surfactants with potential pharmaceutical applications Nogueira, Daniele R.; Mitjans Arnal, Montserrat; Infante Martínez-Pardo, Ma. Rosa; Vinardell Martínez-Hidalgo, Ma. Pilar Surfactants are used as additives in topical pharmaceuticals and drug delivery systems. The biocompatibility of amino acid-based surfactants makes them highly suitable for use in these fields, but tests are needed to evaluate their potential toxicity. Here we addressed the sensitivity of tumor (HeLa, MCF-7) and non-tumor (3T3, 3T6, HaCaT, NCTC 2544) cell lines to the toxic effects of lysine-based surfactants by means of two in vitro endpoints (MTT and NRU). This comparative assay may serve as a reliable approach for predictive toxicity screening of chemicals prior to pharmaceutical applications. After 24-h of cell exposure to surfactants, differing toxic responses were observed. NCTC 2544 and 3T6 cell lines were the most sensitive, while both tumor cells and 3T3 fibroblasts were more resistant to the cytotoxic effects of surfactants. IC50-values revealed that cytotoxicity was detected earlier by MTT assay than by NRU assay, regardless of the compound or cell line. The overall results showed that surfactants with organic counterions were less cytotoxic than those with inorganic counterions. Our findings highlight the relevance of the correct choice and combination of cell lines and bioassays in toxicity studies for a safe and reliable screen of chemicals with potential interest in pharmaceutical industry. http://www.recercat.cat:80/handle/2072/206676 http://www.recercat.cat:80/handle/2072/206397 The role of counterions in the membrane-disruptive properties of pH-sensitive lysine-based surfactants Nogueira, Daniele R.; Mitjans Arnal, Montserrat; Infante Martínez-Pardo, Ma. Rosa; Vinardell Martínez-Hidalgo, Ma. Pilar Surfactants are among the most versatile and widely used excipients in pharmaceuticals. This versatility, together with their pH-responsive membrane-disruptive activity and low toxicity, could also enable their potential application in drug delivery systems. Five anionic lysine-based surfactants which differ in the nature of their counterion were studied. Their capacity to disrupt the cell membrane was examined under a range of pH values, concentrations and incubation times, using a standard hemolysis assay as a model for endosomal membranes. The surfactants showed pH-sensitive hemolytic activity and improved kinetics at the endosomal pH range. Low concentrations resulted in negligible hemolysis at physiological pH and high membrane lytic activity at pH 5.4, which is in the range characteristic of late endosomes. With increasing concentration, the surfactants showed an enhanced capacity to lyse cell membranes, and also caused significant membrane disruption at physiological pH. This observation indicates that, at high concentrations, surfactant behavior is independent of pH. The mechanism of surfactant-mediated membrane destabilization was addressed, and scanning electron microscopy studies were also performed to evaluate the effects of the compounds on erythrocyte morphology as a function of pH. The in vitro cytotoxicity of the surfactants was assessed by MTT and NRU assays with the 3T3 cell line. The influence of different types of counterion on hemolytic activity and the potential applications of these surfactants in drug delivery are discussed. The possibility of using pH-sensitive surfactants for endosome disruption could hold great promise for intracellular drug delivery systems in future therapeutic applications. http://www.recercat.cat:80/handle/2072/206397 http://www.recercat.cat:80/handle/2072/204759 Establishment of an In Vitro Photoallergy Test Using NCTC2544 Cells and IL-18 Production Galbiati, Valentina; Martínez Ocaña, Verónica; Bianchi, Sara; Mitjans Arnal, Montserrat; Corsini, Emanuela Differentiation between photoallergenic and phototoxic reactions induced by low molecular weight compounds represents a current problem. The use of eratinocytes as a potential tool for the detection of photoallergens as opposed to photoirritants is considered an interesting strategy for developing in vitro methods. We have previously demonstrated the possibility to use the human keratinocyte cell line NCTC2455 and the production of interleukin-18 (IL-18) to screen low molecular weight sensitizers. The purpose of this work was to explore the possibility to use the NCTC2544 assay to identify photoallergens and discriminate from phototoxic chemicals. First, we identified suitable condition of UV-irradiation (3.5 J/cm2) by investigating the effect of UVAirradiation on intracellular IL-18 on untreated or chloropromazine (a representative phototoxic compound)- treated NCTC2544 cells. Then, the effect of UVA-irradiation over NCTC2544 cells treated with increasing concentrations of 15 compounds including photoallergens (benzophenone, 4-ter-butyl-4-methoxydibenzoylmethane, 2-ethylexyl-p-methoxycinnamate, ketoprofen, 6-methylcumarin); photoirritant and photoallergen (4-aminobenzoic acid, chlorpromazine, promethazine); photoirritants (acridine, ibuprofen, 8-methoxypsoralen, retinoic acid); and negative compounds (lactic acid, SDS and p-phenilendiamine) was investigated. Twenty-four hours after exposure, cytotoxicity was evaluated by the MTT assay or LDH leakage, while ELISA was used to measure the production of IL-18. At the maximal concentration assayed with non-cytotoxic effects (CV80 under irradiated condition), all tested photoallergens induced a significant and a dose-dependent increase of intracellular IL-18 following UVA irratiation, whereas photoirritants failed. We suggest that this system may be useful for the in vitro evaluation of the photoallergic potential of chemicals. http://www.recercat.cat:80/handle/2072/204759 http://www.recercat.cat:80/handle/2072/204585 A diet enriched with cocoa prevents IgE synthesis in a rat allergy model Abril-Gil, Mar; Massot-Cladera, Malén; Pérez-Cano, Francisco J.; Castellote i Bargalló, M. Cristina; Franch i Masferrer, Àngels; Castell, Margarida Previous studies in young rats reported the impact of cocoa intake on healthy immune status and allow suggesting it may have a role in the prevention of some immune-mediated diseases. The aim of this study was to ascertain the effect of a cocoa diet in a model of allergy in young rats. Three-week-old Brown Norway rats were immunized by i.p. injection of ovalbumin (OVA) with alum as adjuvant and Bordetella pertussis toxin. During the next 4 weeks rats received either a cocoa diet (containing 0.2% polyphenols, w/w) or a standard diet. Animals fed a standard diet showed high concentrations of anti-OVA IgG1, IgG2a, IgG2b and high anti-OVA IgE titres, which is the antibody involved in allergic response. In contrast, animals fed a cocoa diet showed significantly lower concentrations of anti-OVA IgG1 and IgG2a antibodies. Interestingly, the cocoa diet prevented anti-OVA IgE synthesis and decreased total serum IgE concentration. Analysis of cytokine production in lymph node cells at the end of the study revealed that, in this compartment, the cocoa diet decreased the tumor necrosis factor (TNF) - alpha and the interleukin (IL) -10 secretion but not IL-4 production. In conclusion, a cocoa-enriched diet in young rats produces an immunomodulatory effect that prevents anti-allergen IgE synthesis, suggesting a potential role for cocoa flavonoids in the prevention or treatment of allergic diseases. http://www.recercat.cat:80/handle/2072/204585 http://www.recercat.cat:80/handle/2072/204436 Effect of a cocoa flavonoid-enriched diet on experimental autoimmune arthritis Ramos-Romero, S.; Pérez-Cano, Francisco J.; Pérez Berezo, Teresa; Castellote i Bargalló, M. Cristina; Franch i Masferrer, Àngels; Castell, Margarida Previously we established that a cocoa-enriched diet in young rats reduces the specific antibody production and the Th lymphocyte proportion in lymphoid tissues. The aim of the present study was to ascertain the modulatory ability of a cocoa flavonoid enriched diet on collagen-induced arthritis (CIA), which is mediated by anti collagen autoantibody response and Th lymphocyte activation. Female LOU rats were fed with a cocoa enriched diet, beginning two weeks before CIA induction. The hind paw swelling and serum cytokine and anti collagen antibody concentrations were determined. Anti collagen antibody-secreting cell counts and lymphocyte subset proportions were established in inguinal lymph nodes. Reactive oxygen species (ROS), NO and TNFα by peritoneal macrophages were determined. Although arthritis cocoa-fed rats showed a similar hind-paw swelling time course as the arthritis animals fed a standard diet, the cocoa intake was able to decrease specific IgG2a, IgG2b, and IgG2c titres. Moreover, cocoa intake in CIA rats reduced ROS production, TNFα and NO release from peritoneal macrophages, and decreased the Th/Tc ratio in inguinal lymph nodes. In conclusion, a cocoa flavonoid-enriched diet in LOU rats with CIA produced no effect on hind-paw swelling but was able to modulate the specific antibody response and also the Th lymphocyte proportions, as well as the synthesis of pro-inflammatory mediators from peritoneal macrophages. Therefore, a cocoa-enriched diet could be a good adjuvant therapy in disorders with oxidative stress or autoimmune pathogenesis. http://www.recercat.cat:80/handle/2072/204436 http://www.recercat.cat:80/handle/2072/204327 Cocoa intake attenuates oxidative stress associated with rat adjuvant arthritis Ramos-Romero, S.; Pérez-Cano, Francisco J.; Ramiro Puig, Emma; Franch i Masferrer, Àngels; Castell, Margarida Cocoa contains flavonoids with antioxidant properties. The aim of this study was to ascertain the effect of cocoa intake on oxidative stress associated with a model of chronic inflammation such as adjuvant arthritis. Female Wistar rats were fed with a 5 or 10% cocoa enriched diet or were given p.o. a quercetin suspension every other day for 10 days. Arthritis was induced by a heat killed Mycobacterium butyricum suspension. Reactive oxygen species (ROS) produced by macrophages, and splenic superoxide dismutase (total, cytoplasmic and mitochondrial) and catalase activities were determined. Clinically, joint swelling in arthritic rats was not reduced by antioxidants; however, the 5% cocoa diet and quercetin administration reduced ROS production. Moreover, the 5% cocoa diet normalized the activities of superoxide dismutase and catalase. In conclusion, a cocoa diet reduces the oxidative stress associated with a chronic inflammatory pathology, although it was not enough to attenuate joint swelling. http://www.recercat.cat:80/handle/2072/204327 http://www.recercat.cat:80/handle/2072/204202 Phospholipid bilayer perturbing-properties underlying lysis induced by pH-sensitive cationic lysine-based surfactants in biomembranes Nogueira, Daniele R.; Mitjans Arnal, Montserrat; Busquets i Viñas Ma. Antonia; Pérez Muñoz, Lourdes; Vinardell Martínez-Hidalgo, Ma. Pilar Many strategies for treating diseases require the delivery of drugs into the cell cytoplasm following internalization within endosomal vesicles. Thus, compounds triggered by low pH to disrupt membranes and release endosomal contents into the cytosol are of particular interest. Here, we report novel cationic lysine-based surfactants (hydrochloride salts of Nε- and Nα-acyl lysine methyl ester) that differ in the position of the positive charge and the length of the alkyl chain. Amino acid-based surfactants could be promising novel biomaterials in drug delivery systems, given their biocompatible properties and low cytotoxic potential. We examined their ability to disrupt the cell membrane in a range of pH values, concentrations and incubation times, using a standard hemolysis assay as a model of endosomal membranes. Furthermore, we addressed the mechanism of surfactant-mediated membrane destabilization, including the effects of each surfactant on erythrocyte morphology as a function of pH. We found that only surfactants with the positive charge on the α-amino group of lysine showed pH-sensitive hemolytic activity and improved kinetics within the endosomal pH range, indicating that the positive charge position is critical for pH-responsive behavior. Moreover, our results showed that an increase in the alkyl chain length from 14 to 16 carbon atoms was associated with a lower ability to disrupt cell membranes. Knowledge on modulating surfactant-lipid bilayer interactions may help us to develop more efficient biocompatible amino acid-based drug delivery devices. http://www.recercat.cat:80/handle/2072/204202 http://www.recercat.cat:80/handle/2072/203740 Immunomodulation by conjugated linoleic acid (CLA) in early life Pérez-Cano, Francisco J.; Ramírez Santana, Carolina; Castell, Margarida; Castellote i Bargalló, M. Cristina; Franch i Masferrer, Àngels Conjugated linoleic acid (CLA) has been reported to exert beneficial physiological effects on body composition and the immune system. However, little information is available on the influence of CLA on immune function during early life periods. The present study evaluates the effect of feeding an 80:20 mixture of cis-9,trans-11- and trans-10,cis-12-CLA isomers duringgestation, suckling and early infancy on the systemic and mucosal immune responses of Wistar rats at three different time points: at the end of the suckling period (21-day-old rats), in early infancy (28-day-old rats), and later in life (adulthood). Cis-9,trans-11- and trans-10,cis-12-CLA isomers were detected in the milk of CLA-fed dams and in the plasma of all CLA-supplemented pups, and the highest content was achieved in those rats supplemented over the longest period. Dietary supplementation with that CLA mix enhances the systemic production of the main in vivo and ex vivo immunoglobulin (Ig) isotypes in 21- and 28-day-old rats. Moreover, CLA supplementation during suckling and early infancy also enhances the humoral immune defenses at intestinal level, by means of mucosal IgA increase, whereas down-regulates thesystemic lymphoproliferative response. Finally, we described herein how feeding a diet enriched with the same isomer mix of cis9,trans11- and trans10,cis12-CLA from gestation to adulthood improves the capacity of adult rats to achieve a specific systemic and mucosal immune responses. All these data support the immunomodulatory effects of dietary supplementation of CLA, particularly of the cis9,trans11-CLA isomer, during early stages of life on immune system development, as well as the long-term effects on the specific immune response in adult age.; Podeu consultar el llibre complet a: http://hdl.handle.net/2445/32393 http://www.recercat.cat:80/handle/2072/203740 http://www.recercat.cat:80/handle/2072/203387 Counter-ion effect on surfactant-DNA gel particles as controlled DNA delivery systems Morán Badenas, María del Carmen; Alonso, Tania; Lima, Filipe S.; Vinardell Martínez-Hidalgo, Ma. Pilar; Miguel, M. Graça; Lindman, Björn, 1942- The ability to entrap drugs within vehicles and subsequently release them has led to new treatments for a number of diseases. Based on an associative phase separation and interfacial diffusion approach, we developed a way to prepare DNA gel particles without adding any kind of cross-linker or organic solvent. Among the various agents studied, cationic surfactants offered particularly efficient control for encapsulation and DNA release from these DNA gel particles. The driving force for this strong association is the electrostatic interaction between the two components, as induced by the entropic increase due to the release of the respective counter-ions. However, little is known about the influence of the respective counter-ions on this surfactant-DNA interaction. Here we examined the effect of different counter-ions on the formation and properties of the DNA gel particles by mixing DNA (either single- (ssDNA) or double-stranded (dsDNA)) with the single chain surfactant dodecyltrimethylammonium (DTA). In particular, we used as counter-ions of this surfactant the hydrogen sulfate and trifluoromethane sulfonate anions and the two halides, chloride and bromide. Effects on the morphology of the particles obtained, the encapsulation of DNA and its release, as well as the haemocompatibility of these particles, are presented, using the counter-ion structure and the DNA conformation as controlling parameters. Analysis of the data indicates that the degree of counter-ion dissociation from the surfactant micelles and the polar/hydrophobic character of the counter-ion are important parameters in the final properties of the particles. The stronger interaction with amphiphiles for ssDNA than for dsDNA suggests the important role of hydrophobic interactions in DNA. http://www.recercat.cat:80/handle/2072/203387 http://www.recercat.cat:80/handle/2072/203386 pH Sensitive surfactants from lysine: assessment of their cytotoxicity and environmental behavior Colomer Flores, Aurora; Pinazo Gassol, Aurora; Garcia Ramón, Maria Teresa; Mitjans Arnal, Montserrat; Vinardell Martínez-Hidalgo, Ma. Pilar; Infante Martínez-Pardo, Ma. Rosa; Martínez Ocaña, Verónica; Pérez Muñoz, Lourdes The toxicity and environmental behavior of new pH-sensitive surfactants from lysine are presented. Three different chemical structures are studied: surfactants with one amino acid and one alkyl chain, surfactants with two amino acids on the polar head and one alkyl chain, and gemini surfactants. The pH sensitivity of these compounds can be tuned by modifying their chemical structures. Cytotoxicity has been evaluated using erythrocytes and fibroblast cells. The toxic effects against these cells depend on the hydrophobicity of the molecules as well as their cationic charge density. The effect of hydrophobicity and cationic charge density on toxicity is different for each type of cells. For erythrocytes, the toxicity increases as hydrophobicity and charge density increases. Nevertheless, for fibroblasts cationic charge density affects cytotoxicity in the opposite way: the higher charge density, the lower the toxicity. The effect of the pH on hemolysis has been evaluated in detail. The aquatic toxicity was established using Daphnia magna. All surfactants yielded EC50 values considerably higher than that reported for cationic surfactants based on quaternary ammonium groups. Finally, their biodegradability was evaluated using the CO2 headspace test (ISO 14593). These lysine derivatives showed high levels of biodegradation under aerobic conditions and can be classified as"readily biodegradable compounds". http://www.recercat.cat:80/handle/2072/203386 http://www.recercat.cat:80/handle/2072/203260 Assessment of the potential skin irritation of lysine-derivative anionic surfactants using mouse fibroblast and human keratinocytes as an alternative to animal testing Sánchez Molina, Lourdes; Mitjans Arnal, Montserrat; Infante Martínez-Pardo, Ma. Rosa; Vinardell Martínez-Hidalgo, Ma. Pilar Purpose. The aim of this study was to identify new surfactants with low skin irritant properties for use in pharmaceutical and cosmetic formulations, employing cell culture as an alternative method to in vivo testing. In addition, we sought to establish whether potential cytotoxic properties were related to the size of the counterions bound to the surfactants. Methods. Cytotoxicity was assessed in the mouse fibroblast cell line 3T6, and the human keratinocyte cell line NCTC 2544, using the MTT assay and uptake of the vital dye neutral red 24 h after dosing (NRU). Results. Lysine-derivative surfactants showed higher IC50s than did commercial anionic irritant compounds such as sodium dodecyl sulphate, proving to be no more harmful than amphoteric betaines. The aggressiveness of the surfactants depended upon the size of their constituent counterions: surfactants associated with lighter counterions showed a proportionally higher aggressivity than those with heavier ones. Conclusions. Synthetic lysine-derivative anionic surfactants are less irritant than commercial surfactants such as sodium dodecyl sulphate and Hexadecyltrimethylammonium bromide and are similar to Betaines. These surfactants may offer promising applications in pharmaceutical and cosmetic preparations, representing a potential alternative to commercial anionic surfactants as a result of their low irritancy potential. http://www.recercat.cat:80/handle/2072/203260 http://www.recercat.cat:80/handle/2072/203259 Photoprotective potential of emulsions formulated with Buriti oil (Mauritia flexuosa) and Vitamin E against UV irradiation on human keratinocytes and fibroblasts cell lines Zanatta, C.F.; Mitjans Arnal, Montserrat; Urgatondo, V.; Rocha-Filho, P.A.; Vinardell Martínez-Hidalgo, Ma. Pilar Considering the belief that natural lipids and edible substances are safer for topical applications and that carotenoids are able to protect cells against photooxidative damage, wea have investigated whether topical creams and lotions, produced with Buriti oil and commercial surfactants, can exert photoprotective effect of against UVA and UVB irradiation. Emulsions and plain Buriti oil were diluted in DMEM medium supplemented with 10% FBS. Cell treatment was divided in two stages, prior and after being exposed to 30 minutes of UVA plus UVB radiation or 60 minutes to UVA radiation. Emulsions prepared with ethoxylated fatty alcohols as surfactants and containing α-tocopherol caused phototoxic damage to the cells, especially when applied prior to UV exposure. Damage reported was due to prooxidant activity and phototoxic effect of the surfactant. Emulsions prepared with Sorbitan Monooleate and PEG-40 castor oil and containing panthenol as active ingredient, were able to reduce the damages caused by radiation when compared to non-treated cells. When the different cells lines used in the study were compared, keratinocytes showed an increase in cell viability higher than fibroblasts. The Buriti oil emulsions can be considered potential vehicles to transport antioxidants precursors and also be used as adjuvant in sun protection. http://www.recercat.cat:80/handle/2072/203259 http://www.recercat.cat:80/handle/2072/202782 Grape epicatechin conjugates prevent erythrocyte membrane protein oxidation Martínez Ocaña, Verónica; Ugartondo Casadevall, Vanessa; Vinardell Martínez-Hidalgo, Ma. Pilar; Torres Simón, Josep Lluís; Mitjans Arnal, Montserrat Epicatechin conjugates obtained from grape have shown antioxidant activity in various systems. However, how these conjugates exert their antioxidant benefits has not been widely studied. We assessed the activity of epicatechin and epicatechin conjugates on the erythrocyte membrane in the presence and absence of a peroxyl radical initiator, to increase our understanding of their mechanisms. Thus, we studied cell membrane fluidity by fluorescence anisotropy measurements, morphology of erythrocytes by scanning electron microscopy, and finally, red cell membrane proteins by sodium dodecyl sulfate&br&polyacrylamide gel electrophoresis. Our data showed that incubation of red cells in the presence of epicatechin derivatives altered membrane fluidity and erythrocyte morphology but not the membrane protein pattern. The presence in the medium of the peroxyl radical initiator 2,2′-azobis(amidinopropane) dihydrochloride (AAPH) resulted in membrane disruptions at all levels analyzed, causing changes in membrane fluidity, cell morphology, and protein degradation. The presence of antioxidants avoided protein oxidation, indicating that the interaction of epicatechin conjugates with the lipid bilayer might reduce the accessibility of AAPH to membranes, which could explain in part the inhibitory ability of these compounds against hemolysis induced by peroxidative insult. http://www.recercat.cat:80/handle/2072/202782 http://www.recercat.cat:80/handle/2072/200208 Mechanisms involved in down-regulation of intestinal IgA in rats by high cocoa intake Pérez Berezo, Teresa; Franch i Masferrer, Àngels; Castellote i Bargalló, M. Cristina; Castell, Margarida; Pérez-Cano, Francisco J. Previous studies have shown that rat intestinal immunoglobulin A (IgA) concentration and lymphocyte composition of the intestinal immune system were influenced by a highly enriched cocoa diet. The aim of this study was to dissect the mechanisms by which a long-term high cocoa intake was capable of modifying gut secretory IgA in Wistar rats. After 7 weeks of nutritional intervention, Peyer's patches, mesenteric lymph nodes and the small intestine were excised for gene expression assessment of IgA, transforming growth factor ß, C-C chemokine receptor-9 (CCR9), interleukin (IL)-6, CD40, retinoic acid receptors (RAR¿ and RARß), C-C chemokine ligand (CCL)-25 and CCL28 chemokines, polymeric immunoglobulin receptor and toll-like receptors (TLR) expression by real-time polymerase chain reaction. As in previous studies, secretory IgA concentration decreased in intestinal wash and fecal samples after cocoa intake. Results from the gene expression showed that cocoa intake reduced IgA and IL¿6 in Peyer's patches and mesenteric lymph nodes, whereas in small intestine, cocoa decreased IgA, CCR9, CCL28, RAR¿ and RARß. Moreover, cocoa-fed animals presented an altered TLR expression pattern in the three compartments studied. In conclusion, a high-cocoa diet down-regulated cytokines such as IL-6, which is required for the activation of B cells to become IgA-secreting cells, chemokines and chemokine receptors, such as CCL28 and CCR9 together with RAR¿ and RARß, which are involved in the gut homing of IgA-secreting cells. Moreover, cocoa modified the cross-talk between microbiota and intestinal cells as was detected by an altered TLR pattern. These overall effects in the intestine may explain the intestinal IgA down-regulatory effect after the consumption of a long-term cocoa-enriched diet. http://www.recercat.cat:80/handle/2072/200208 http://www.recercat.cat:80/handle/2072/200132 Membrane-destabilizing activity of pH-responsive cationic lysine-based surfactants: role of charge position and alkyl chain length Nogueira, Daniele R.; Mitjans Arnal, Montserrat; Morán Badenas, María del Carmen; Pérez Muñoz, Lourdes; Vinardell Martínez-Hidalgo, Ma. Pilar Many strategies for treating diseases require the delivery of drugs into the cell cytoplasm following internalization within endosomal vesicles. Thus, compounds triggered by low pH to disrupt membranes and release endosomal contents into the cytosol are of particular interest. Here, we report novel cationic lysine-based surfactants (hydrochloride salts of N¿- and N¿-acyl lysine methyl ester) that differ in the position of the positive charge and the length of the alkyl chain. Amino acid-based surfactants could be promising novel biomaterials in drug delivery systems, given their biocompatible properties and low cytotoxic potential. We examined their ability to disrupt the cell membrane in a range of pH values, concentrations and incubation times, using a standard hemolysis assay as a model of endosomal membranes. Furthermore, we addressed the mechanism of surfactant-mediated membrane destabilization, including the effects of each surfactant on erythrocyte morphology as a function of pH. We found that only surfactants with the positive charge on the ¿-amino group of lysine showed pH-sensitive hemolytic activity and improved kinetics within the endosomal pH range, indicating that the positive charge position is critical for pH-responsive behavior. Moreover, our results showed that an increase in the alkyl chain length from 14 to 16 carbon atoms was associated with a lower ability to disrupt cell membranes. Knowledge on modulating surfactant-lipid bilayer interactions may help us to develop more efficient biocompatible amino acid-based drug delivery devices. http://www.recercat.cat:80/handle/2072/200132 http://www.recercat.cat:80/handle/2072/199429 CD4 expression decrease by antisense oligonucleotides. Inhibition of rat T CD4+ cell reactivity Rabanal i Tornero, Manel; Franch i Masferrer, Àngels; Noé Mata, Verónica; Pelegrí i Gabaldà, Carme; Ciudad i Gómez, Carlos Julián; Castellote i Bargalló, M. Cristina; Castell, Margarida In previous studies, we have demonstrated the inhibition of CD4 expression in rat lymphocytes treated with phorbol myristate acetate (PMA) by antisense oligonucleotides (AS-ODNs) directed against the AUG start region of the cd4 gene. The aim of the present study was to inhibit CD4 expression in lymphocytes without promoting CD4 synthesis and to determine the effect of this inhibition on CD4+ T cell function. Four 21-mer ODNs against the rat cd4 gene (AS-CD4-1 to AS-CD4-4) were used. Surface CD4 expression was measured by immunofluorescence staining and flow cytometry, and mRNA CD4 expression was measured by RT-PCR. T CD4+ cell function was determined by specific and unspecific proliferative response of rat-primed lymphocytes. After 24 hours of incubation, AS-CD4-2 and AS-CD4-4 reduced lymphocyte surface CD4 expression by 40%. This effect remained for 72 hours and was not observed on other surface molecules, such as CD3, CD5, or CD8. CD4 mRNA expression was reduced up to 40% at 24 hours with AS-CD4-2 and AS-CD4-4. After 48 hours treatment, CD4 mRNA decreased up to 27% and 29% for AS-CD4-2 and AS-CD4-4, respectively. AS-CD4-2 and AS-CD4-4 inhibited T CD4+ cell proliferative response upon antigen-specific and unspecific stimuli. Therefore, AS-ODNs against CD4 molecules inhibited surface and mRNA CD4 expression, under physiologic turnover and, consequently, modulate T CD4+ cell reactivity. http://www.recercat.cat:80/handle/2072/199429 http://www.recercat.cat:80/handle/2072/180191 Blood lymphocyte subsets in rats with adjuvant arthritis Franch i Masferrer, Àngels; Castellote i Bargalló, M. Cristina; Castell, Margarida To determine the phenotype of peripheral blood lymphocytes during the time-course of adjuvant arthritis (AA) to detect alterations that could be involved in the pathogenesis of the arthritic process. METHODS--Phenotype analysis was performed on days 7, 14, 21, 28, 42, 56 and 70 after arthritis induction using monoclonal antibodies to CD5, CD4 and CD8 subsets, and flow cytometry. The proportion of activated lymphocytes and lymphocytes was also assessed with monoclonal antibodies to IL-2R (CD25), to Ia antigen and by polyclonal antibodies to rat Ig. RESULTS--Adjuvant arthritis produced leukocytosis with neutrophilia. Rats with AA showed a marked increase in the number of both CD4+ and CD8+ cells. The ratio CD4/CD8 decreased because the rise in CD8+ cells was more pronounced than the increase in CD4+ cells. Changes in lymphocyte counts showed two well-defined periods: the first, from day 14 to day 28, during which the inflammation of the joints reached a maximum and changes in lymphocyte subsets were more pronounced, that is, there was a threefold increase in CD8+ lymphocytes over normal counts, and the second, from day 42 to day 70, in which modified parameters improved considerably but remained different from controls. CONCLUSION--Alterations were detected in the phenotype of peripheral blood lymphocytes in AA, which provides an additional marker of disease activity. http://www.recercat.cat:80/handle/2072/180191 http://www.recercat.cat:80/handle/2072/167532 Gene expression profiles in rat mesenteric lymph nodes upon supplementation with Conjugated Linoleic Acid during gestation and suckling Selga i Coma, Elisabet; Pérez-Cano, Francisco J.; Franch i Masferrer, Àngels; Ramírez Santana, Carolina; Rivero, Montserrat; Ciudad i Gómez, Carlos Julián; Castellote i Bargalló, M. Cristina; Noé Mata, Verónica Background Diet plays a role on the development of the immune system, and polyunsaturated fatty acids can modulate the expression of a variety of genes. Human milk contains conjugated linoleic acid (CLA), a fatty acid that seems to contribute to immune development. Indeed, recent studies carried out in our group in suckling animals have shown that the immune function is enhanced after feeding them with an 80:20 isomer mix composed of c9,t11 and t10,c12 CLA. However, little work has been done on the effects of CLA on gene expression, and even less regarding immune system development in early life. Results The expression profile of mesenteric lymph nodes from animals supplemented with CLA during gestation and suckling through dam's milk (Group A) or by oral gavage (Group B), supplemented just during suckling (Group C) and control animals (Group D) was determined with the aid of the specific GeneChip® Rat Genome 230 2.0 (Affymettrix). Bioinformatics analyses were performed using the GeneSpring GX software package v10.0.2 and lead to the identification of 89 genes differentially expressed in all three dietary approaches. Generation of a biological association network evidenced several genes, such as connective tissue growth factor (Ctgf), tissue inhibitor of metalloproteinase 1 (Timp1), galanin (Gal), synaptotagmin 1 (Syt1), growth factor receptor bound protein 2 (Grb2), actin gamma 2 (Actg2) and smooth muscle alpha actin (Acta2), as highly interconnected nodes of the resulting network. Gene underexpression was confirmed by Real-Time RT-PCR. Conclusions Ctgf, Timp1, Gal and Syt1, among others, are genes modulated by CLA supplementation that may have a role on mucosal immune responses in early life. http://www.recercat.cat:80/handle/2072/167532 http://www.recercat.cat:80/handle/2072/50623 Muscle protein waste in tumor-bearing rats is effectively antagonized by a beta 2-adrenergic agonist (clenbuterol). Role of the ATP-ubiquitin-dependent proteolytic pathway. Costelli, Paola; García Martínez, Celia; Llovera i Tomàs, Marta; Carbó Carbó, Maria Neus; López-Soriano, Francisco J.; Agell i Jané, Neus; Tessitore, Luciana; Baccino, Francesco M.; Argilés Huguet, Josep Ma. Tissue protein hypercatabolism (TPH) is a most important feature in cancer cachexia, particularly with regard to the skeletal muscle. The rat ascites hepatoma Yoshida AH-130 is a very suitable model system for studying the mechanisms involved in the processes that lead to tissue depletion, since it induces in the host a rapid and progressive muscle waste mainly due to TPH (Tessitore, L., G. Bonelli, and F. M. Baccino. 1987. Biochem. J. 241:153-159). Detectable plasma levels of tumor necrosis factor-alpha associated with marked perturbations in the hormonal homeostasis have been shown to concur in forcing metabolism into a catabolic setting (Tessitore, L., P. Costelli, and F. M. Baccino. 1993. Br. J. Cancer. 67:15-23). The present study was directed to investigate if beta 2-adrenergic agonists, which are known to favor skeletal muscle hypertrophy, could effectively antagonize the enhanced muscle protein breakdown in this cancer cachexia model. One such agent, i.e., clenbuterol, indeed largely prevented skeletal muscle waste in AH-130-bearing rats by restoring protein degradative rates close to control values. This normalization of protein breakdown rates was achieved through a decrease of the hyperactivation of the ATP-ubiquitin-dependent proteolytic pathway, as previously demonstrated in our laboratory (Llovera, M., C. García-Martínez, N. Agell, M. Marzábal, F. J. López-Soriano, and J. M. Argilés. 1994. FEBS (Fed. Eur. Biochem. Soc.) Lett. 338:311-318). By contrast, the drug did not exert any measurable effect on various parenchymal organs, nor did it modify the plasma level of corticosterone and insulin, which were increased and decreased, respectively, in the tumor hosts. The present data give new insights into the mechanisms by which clenbuterol exerts its preventive effect on muscle protein waste and seem to warrant the implementation of experimental protocols involving the use of clenbuterol or alike drugs in the treatment of pathological states involving TPH, particularly in skeletal muscle and heart, such as in the present model of cancer cachexia. http://www.recercat.cat:80/handle/2072/50623