Leptin favors imbalance of antigen-specific CD4+ T-cells associated with severity of cat allergy.

Introduction: Obesity can complicate IgE-mediated allergic diseases. In the present study, we aimed to investigate the ability of obesity-related concentrations of leptin to modulate the in vitro effector and regulatory Fel d1-specific CD4+ T-cell subsets in patients allergic to cat, considered the third most common cause of respiratory allergy in humans. Methods: For this study, plasma and peripheral blood mononuclear cells (PBMC) from 30 cat-allergic patients with mild, moderate and severe respiratory symptoms were obtained. The PBMC cultures were stimulated with Fel d1 antigen (10 µg/mL) in the presence or absence of obesity-related leptin dose (50 ηg/mL). After 6 days, the levels of cytokines and IgE in the supernatants were evaluated by multiplex and ELISA, respectively. The frequency of different non-follicular (CXCR5-) and follicular (CXCR5+) Fel d1-specific CD4+ T cell subsets was determined by flow cytometry. The plasma levels of leptin and IgE anti-cat titers were evaluated by ELISA and ImmunoCAP, respectively. Results and conclusions: Fel d1 induced both IgE production and release of cytokines related to Th2, Th9 and Th17 cell phenotypes. Feld1 was more efficient in increasing the frequency of TFHIL-21- cells positive for IL-4, IL-5 and IL-13 than TFHIL-21+ cell subsets. Leptin favored the expansion Th2-like and Th9-like cells and TFHIL-21- cells positive for IL-4, IL-5 and IL-13, but reduced the proportion of conventional (Treg/Tr-1) and follicular (TFR) regulatory CD4+ T-cell subsets expressing or not CD39 marker. Finally, many of the imbalances between Fel d1-specific CD4+ T-cells were also correlated with plasma leptin and anti-Fel d1 IgE titers. In summary, hyperleptinemia should negatively impact on the severity of cat allergies by favoring the expansion of pathogenic Fel d1-specific CD4+ T-cell phenotypes and damaging the functional status of regulatory CD4+ T-cell subsets.

Acute Effects of Different Conditioning Activities on Amateur Soccer Players.

Several warm-up activities are used to prepare soccer players for training and games. However, few studies have focused on comparing different activities (strength vs. stretching) in young and amateur athletes, especially for performance improvement in explosive actions. Thus, in order to compare the effect of four conditioning activities on jump and sprint performances, 12 soccer athletes (age: 19±0.8; weight (kg): 72.8±8.0; height (cm): 180±6.7) performed four warm-up activities: strength exercise (cluster system), combined exercise, plyometric exercise, and static stretching (control). The countermovement jump (CMJ) and 30-meter run (30-m Sprint) were performed 10 min after each experimental condition. A ONE-WAY ANOVA test of repeated measures was conducted with a Tukey's post-hoc test to compare the conditions. The strength conditioning activity protocol (33.68±2.87) showed a significant difference for the CMJ from static stretching (30.96±3.16) (p>0.05). There were significant differences regarding the 30-m Sprint test between strength conditioning (4.72±0.19) and combined activities (4.71±0.21) compared to static stretching (4.84±0.21) (p>0.05). In conclusion, the combined conditioning activity and strength protocols can be chosen in warm-up activities instead of static stretching (control condition) for improved immediate sprint and jump abilities in amateur soccer players.

Elevated proportion of TLR2- and TLR4-expressing Th17-like cells and activated memory B cells was associated with clinical activity of cerebral cavernous malformations.

BACKGROUND: Recent evidences have suggested the involvement of toll-like receptor (TLR)-4 in the pathogenesis of cerebral cavernous malformations (CCM). Elevated frequency of TLR+T-cells has been associated with neurological inflammatory disorders. As T-cells and B-cells are found in CCM lesions, the objective of the present study was to evaluate the cytokine profile of T-cells expressing TLR2 and TLR4, as well as B-cell subsets, in asymptomatic (CCMAsympt) and symptomatic (CCMSympt) patients. METHODS: For our study, the cytokine profile from TLR2+ and TLR4+ T-cell and B-cell subsets in CCMAsympt and CCMSympt patients was investigated using flow cytometry and ELISA. T-cells were stimulated in vitro with anti-CD3/anti-CD28 beads or TLR2 (Pam3C) and TLR4 (LPS) ligands. RESULTS: CCMSymptc patients presented a higher frequency of TLR4+(CD4+ and CD8+) T-cells and greater density of TLR4 expression on these cells. With regard to the cytokine profile, the percentage of TLR2+ and TLR4+ Th17 cells was higher in CCMSympt patients. In addition, an elevated proportion of TLR4+ Tc-1 cells, as well as Tc-17 and Th17.1 cells expressing TLR2 and TLR4, was observed in the symptomatic patients. By contrast, the percentage of TLR4+ IL-10+CD4+ T cells was higher in the CCMAsympt group. Both Pam3C and LPS were more able to elevate the frequency of IL-6+CD4+T cells and Th17.1 cells in CCMSympt cell cultures. Furthermore, in comparison with asymptomatic patients, purified T-cells from the CCMSympt group released higher levels of Th17-related cytokines in response to Pam3C and, mainly, LPS, as well as after activation via TCR/CD28. Concerning the B-cell subsets, a higher frequency of memory and memory activated B-cells was observed in CCMSympt patients. CONCLUSIONS: Our findings reveal an increase in circulating Th17/Tc-17 cell subsets expressing functional TLR2 and, mainly, TLR4 molecules, associated with an increase in memory B-cell subsets in CCM patients with clinical activity of the disease.

Fortification of water kefir with magnetite nanoparticles.

The aim of this work was to evaluate the suitability of incorporating Fe3O4 (magnetite, M) NPs into water kefir (wKef) beverages. Magnetite NPs were synthesized and coated with pectins (cM), and incorporated into wKef beverages obtained by fermentation of a muscovado sugar solution with wKef grains. FeSO4, usually employed as fortifier, was used as a control. Four different beverages were analyzed: wKef, wKef-cM, wKef-M, wKef-FeSO4, indicating wKef beverages fortified with cM, M or FeSO4, respectively. Their stability was assessed by determining the viability of total lactic acid bacteria and yeasts, and the composition of saccharides along storage at 4 °C for up to 30 days. The toxicity of M and cM was evaluated in an in vivo model of Artemia salina. The absorption of iron was quantified by determining ferritin values on intestinal Caco-2/TC7 cells, and its internalization mechanisms, by employing inhibitors of endocytic pathways and quantifying ferritin. M and cM were non-toxic on Artemia salina up to 500 µg/mL, a toxicity even lower than that of FeSO4, which showed a LD50 of 304.08 µg/mL. After 30 days of storage, no significant decrease on yeasts viability was observed, and bacteria viability was above 6 log CFU/mL for the four beverages. In turn, sucrose decreased to undetectable values, concomitantly to an increase in the concentrations of glucose and fructose. Both wKef-M and wKef-cM led to a significant increase in the ferritin values (up to 2 folds) with regard to the basal state. The internalization of M NPs occurred via clathrins and caveolin pathways, whereas that of cM, by macropinocytosis. Safely incorporating M and cM NPs into wKef beverages appear as an innovative strategy for providing bioavailable iron aiming to ameliorate the nutritional status of populations at risk of iron deficiency (e.g., vegans).

Major Depressive Disorder Enhances Th2 and Th17 Cytokines in Patients Suffering from Allergic Rhinitis and Asthma.

INTRODUCTION: Major depressive disorder (MDD) can impact the severity of allergic rhinitis (AR) and asthma (AA). Here, we evaluated the cytokine production by T-cells from AR and AA patients with or without MDD. The effect of serotonin on the in vitro T-cell response was also evaluated. METHODS: The cytokines produced by activated T-cells were measured by Luminex and flow cytometry. In some cell cultures, serotonin was added. RESULTS: MDD not only enhanced the production of Th2- and Th17-related cytokines, but also, the levels of interleukin (IL)-5 and IL-17 were directly correlated with the severity of depression and anxiety symptoms. As compared with AR, the levels of IL-17 were higher and the release of IL-10 was lower in activated T-cell cultures from AA patients, mainly those with MDD. In AA/MDD patients, the severity of anxiety symptoms and lung disease was directly correlated with Th17-like and hybrid Th2/Th17 cells, but inversely correlated with IL-10-secreting CD4+ T-cells. Finally, the addition of serotonin reduced the production of Th2- and Th17-related cytokines, but elevated IL-10 secretion in cell cultures from both AR and AA patients. CONCLUSIONS: Our findings suggest that not only the occurrence of MDD but also the severity of anxiety symptoms, may adversely affect the outcome of allergic reactions by favoring the production of cytokines implicated in the pathogenesis of AR and AA, a phenomenon that was attenuated by serotonin.

Selective serotonin reuptake inhibitor attenuates the hyperresponsiveness of TLR2+ and TLR4+ Th17/Tc17-like cells in multiple sclerosis patients with major depression.

Elevated frequency of Th17-like cells expressing Toll-like receptors (TLRs) has been recently associated with relapsing-remitting multiple sclerosis (MS) pathogenesis, a chronic inflammatory demyelinating autoimmune disease of the central nervous system. We aimed to investigate the impact of current major depressive disorder (MDD) on the behaviour of these cells following in vitro stimulation with TLR2, TLR4, TLR5 and TLR9 agonists. Here, the level of both cell proliferation and cytokine production related to Th17/Tc17 phenotypes in response to TLR2 (Pam3C) and TLR4 (LPS) ligands was significantly higher in CD4+ and CD8+ T-cell cultures from MS/MDD patients when compared to non-depressed patients. These cytokine levels were positively associated with neurological disabilities in patients. No difference for responsiveness to TLR5 (flagellin) and TLR9 (ODN) agonists was observed. LPS, but not Pam3C, induced significant IL-10 release, mainly in patients without MDD. Interestingly, more intense expression of TLR2 and TLR4 on these cells was observed in MDD patients. Finally, in vitro addition of serotonin and treatment of MDD patients with selective serotonin reuptake inhibitors (SSRIs) reduced the production of Th17/Tc17-related cytokines by CD4+ and CD8+ T cells in response to Pam3C and LPS. However, only SSRI therapy diminished the frequency and intensity of TLR2 and TLR4 expression on circulating CD4+ and CD8+ T cells. In summary, although preliminary, our findings suggest that adverse events that elevate circulating levels of TLR2 and TLR4 ligands can affect MS pathogenesis, particularly among depressed patients.

Pregnancy favors circulating IL-21-secreting TFH -like cell recovery in ARV-treated HIV-1-infected women.

PROBLEM: Pregnancy appears to favor maternal antibody production. In contrast, by damaging follicular helper T cells (TFH ), HIV-1 infection compromises protective humoural immune response. Therefore, we aimed to investigate the frequency of different TFH -like cells in HIV-infected pregnant women (PW) before and after antiretroviral (ARV) therapy. METHOD OF STUDY: Peripheral blood mononuclear cells, CD4+ T and B cells, were obtained from asymptomatic HIV-1-infected non-PW and PW just before and after ARV therapy. In some experiments, healthy HIV-1-negative PW were also tested. The frequency of different TFH -like cell subsets was determined by flow cytometry. The plasma titers of IgG anti-tetanus toxoid (TT), anti-HBsAg, and anti-gp41 were determined by ELISA. The in vitro production of total IgG, IL-21, and hormones (estrogen and progesterone) was quantified also by ELISA. RESULTS: Our results demonstrate that antiretroviral (ARV) therapy was more efficient in elevating the percentage of circulating IL-21-secreting TFH cells in HIV-1-infected pregnant women (PW) than in non-pregnant patients (nPW). Moreover, in co-culture systems, CD4+ T cells from ART-treated PW were more efficient in assisting B cells to produce IgG production. The in vivo anti-HBsAg IgG titers after ARV therapy were also significantly higher in PW, and their levels were directly associated with both IL-21+ TFH frequency and plasma concentration of estrogen. CONCLUSION: In summary, our results suggest that pregnancy favors the recovery of TFH -like cells after ARV therapy in HIV-1-infected women, which could help these mothers to protect their newborns from infectious diseases by transferring IgG across the placenta.

Differential activity of lytic α-helical peptides on lactobacilli and lactobacilli-derived liposomes.

Eukaryotic antimicrobial peptides (AMPs) interact with plasma membrane of bacteria, fungi and eukaryotic parasites. Noteworthy, Lactobacillus delbrueckii subsp. lactis (CIDCA 133) and L. delbrueckii subsp. bulgaricus (CIDCA 331) show different susceptibility to human beta-defensins (ß-sheet peptides). In the present work we extended the study to α-helical peptides from anuran amphibian (Aurein 1.2, Citropin 1.1 and Maculatin 1.1). We studied the effect on whole bacteria and liposomes formulated with bacterial lipids through growth kinetics, flow cytometry, leakage of liposome content and studies of peptide insertion in lipid monolayers. Growth of strain CIDCA 331 was dramatically inhibited in the presence of all three peptides and minimal inhibitory concentrations were lower than those for strain CIDCA 133. Flow cytometry revealed that AMPs lead to the permeabilization of bacteria. In addition, CIDCA 331-derived liposomes showed high susceptibility, leading to content leakage and structural disruption. Accordingly, peptide insertion in lipid monolayers demonstrated spontaneous interaction of AMPs with CIDCA 331 lipids. In contrast, lipids monolayers from strain CIDCA 133 were less susceptible. Summarizing we demonstrate that the high resistance of the probiotic strain CIDCA 133 to AMPs extends to α helix peptides Aurein, Citropin and Maculatin. This behavior could be ascribed in part to differences in membrane composition. These findings, along with the previously demonstrated resistance to ß defensins from human origin, suggest that strain CIDCA 133 is well adapted to host innate immune effectors from both mammals and amphibians thus indicating conserved mechanisms of interaction with key components of the innate immune system.

Development and characterization of iron-pectin beads as a novel system for iron delivery to intestinal cells.

Iron deficiency is the most common nutritional deficit worldwide. The goal of this work was to obtain iron-pectin beads by ionic gelation and evaluate their physiological behavior to support their potential application in the food industry. The beads were firstly analyzed by scanning electronic microscopy, and then physical-chemically characterized by performing swelling, thermogravimetric, porosimetry, Mössbauer spectroscopy and X-ray fluorescence analyses, as well as by determining the particle size. Then, physiological assays were carried out by exposing the beads to simulated gastric and intestinal environments, and determining the iron absorption and transepithelial transport into Caco-2/TC7 cells. Iron-pectin beads were spherical (diameter 1-2 mm), with high density (1.29 g/mL) and porosity (93.28%) at low pressure, indicating their high permeability even when exposed to low pressure. Swelling in simulated intestinal medium (pH 8) was higher than in simulated gastric medium. The source of iron [FeSO4 (control) or iron-pectin beads] did not have any significant effect on the mineral absorption. Regarding transport, the iron added to the apical pole of Caco-2/TC7 monolayers was recovered in the basal compartment, and this was proportional with the exposure time. After 4 h of incubation, the transport of iron arising from the beads was significantly higher than that of the iron from the control (FeSO4). For this reason, iron-pectin beads appear as an interesting system to overcome the low efficiency of iron transport, being a potential strategy to enrich food products with iron, without altering the sensory properties.

Efeito do treinamento mental no desempenho de jovens nadadores / Effect of mental training on performance in young swimmers

O objetivo do estudo foi analisar o efeito do treinamento mental sobre o desempenho de jovens nadadores. A amostra foi composta por 35 nadadores com idade entre 15 e 17 anos, divididos aleatoriamente em dois grupos: treinamento mental (GTM, n = 17) e controle (GC, n = 18). O estudo teve duração de 8 semanas. Ambos os grupos fizeram a mesma planificação de treinamento físico/técnico. O GC assistiu vídeos de propagandas, ao passo que o GTM realizou o treinamento mental. Foram realizadas 3 sessões semanais de treinamento mental com intervalo de 48 horas. Utilizou-se o melhor tempo em segundos para determinar o desempenho nas provas de 100 m e 200 m livre. A maturação biológica foi avaliada por intermédio da maturação somática. Conduziu-se a análise multivariada de covariância (MANOVA) de medidas repetidas para comparar o desempenho nas provas de 100 m e 200 m livre entre os grupos (GTM e GC) em função da fase da investigação (pré e pós-intervenção). Os resultados apresentaram efeitos de tempo (F(2, 33) = 32,65, p = 0,01) e grupo (F(2, 33) = 57,31, p = 0,001). Foi identificada diferença significante do desempenho nos 100 m (F(2, 33) = 33,70, p = 0,01, d = 0,5) e 200 m livre (F(2, 33) = 37,09, p = 0,01, d = 0,5) entre GTM e GC após as 8 semanas. Os achados apontaram relação da maturação somática com o desempenho nos 100 m (F(1, 34) = 43,55, p = 0,01) e 200 m livre (F(1, 34) = 34,82, p = 0,01). Concluiu-se que o treinamento mental foi eficiente para maximizar o desempenho nas provas de 100 m e 200 m livre de jovens atletas de natação....(AU)

The objective of this study was to analyze the effect of imaginary training on performance of young swimmers. The sample consisted of 35 swimmers aged between 15 and 17 years, that divided randomly into two groups: mental training (MTG, n = 17) and control (CG, n = 18) group. The study lasted eight weeks. Both groups did the same planning physical/technical training. The CG watched videos of advertisements, while MTG performed the imaginary training. Three weekly sessions of imaginary training interspersed with the period of 48 hours were performed. We used the best time in seconds to determine the performance on 100 m and 200 m freestyle. Biology maturation was evaluated by somatic maturation. We conducted the covariance multivariate measure repeat analysis to compare the 100 m and 200 m performance between the groups (MTG and CG) according to phase investigation. The results presented an time (F(2, 33) = 32,65, p = 0,01) and group (F(2, 33) = 57,31, p = 0,001) effect. Significant difference was identified in 100m (F(2, 33) = 33,70, p = 0,01, d = 0,5) and 200 freestyle (F(2, 33) = 37,09, p = 0,01, d = 0,5) performance between MTG and CG after 8 weeks. The findings appointed relationship between somatic maturation and 100m (F(1, 34) = 43,55, p = 0,01) and 200m (F(1, 34) = 34,82, p = 0,01) performance. It was concluded that imaginary training was efficient to maximize performance on 100m and 200m freestyle in young swimmers. We recommended that the imagery training be included in routine training of young swimmers....(AU)

Efeito de comportamentos de risco para os transtornos alimentares no desempenho nos 50, 100, 200 e 400 m livre em jovens nadadores / Effect of disordered eating behaviors on performance in 50, 100, 200 and 400 m freestyle in young swimmers

O objetivo foi analisar o efeito dos comportamentos de risco para os transtornos alimentares (TA) no desempenho nas provas dos 50, 100, 200 e 400 m livre de nadadores. Participaram 188 atletas de natação do sexo masculino. Utilizou-se o Eating Attitudes Test (EAT-26) para avaliar os comportamentos de risco para TA. Utilizou-se o melhor tempo em segundos para determinar o desempenho nas provas de 50, 100, 200 e 400 m livre. Conduziu-se a análise multivariada de covariância para comparar o desempenho nas provas de 50, 100, 200 e 400 m livre entre nadadores com e sem risco para os TA. Os resultados apontaram que: a) não foi identificada diferença de desempenho na prova dos 50m livre (F(2, 186)=3,40; p=0,18); b) foi encontrada diferença de desempenho nas provas dos 100 m livre (F(2, 186)=16,05; p=0,02), 200 m livre (F(2, 186)=19,61; p=0,01) e 400 m livre (F(2, 186)=23,72; p=0,001) entre os grupos com e sem risco para os TA. Concluiu-se que os nadadores com maior frequência de uso de comportamentos de risco para os TA demonstraram menor desempenho nas provas dos 100, 200 e 400 m livre, fato não replicado para os 50m livre. Do ponto de vista prático, os achados revelaram que a adoção dos comportamentos de risco para os TA pode afetar negativamente o desempenho de nadadores....(AU)

The objective was to analyze the effect of disordered eating (DE) behaviors on 50, 100, 200 and 400 m freestyle performance in swimmers. One hundred and eighty eight male swimmers were recruited. The Eating Attitudes Test (EAT-26) was used to evaluate DE. It was used the best time in seconds to determine the performance in 50, 100, 200 and 400 m freestyle. It was used multivariate analysis of covariance to compare the performance in 50, 100, 200 and 400 m freestyle between swimmers with and without DE. The results showed that: a) there was no difference on 50 m freestyle performance (F(2, 186)=3,40; p=0,18); b) was found difference in performance in 100 (F(2, 186)=16,05; p=0,02), 200m (F(2,186)=19,61; p=0,01) and 400 m freestyle (F(2, 186)=23,72; p=0,001) between groups with and without DE. It was concluded that swimmers with more use DE frequency demonstrated lower performance in 100, 200 and 400 m freestyle, which was not replicated to the 50 m freestyle. From the practical point of view, the findings revealed that the adoption of DE behaviors may negatively affect the performance of swimmers....(AU)

Endocytosis and intracellular traffic of cholesterol-PDMAEMA liposome complexes in human epithelial-like cells.

Liposomes are generally used as delivery systems, as they are capable of encapsulating a wide variety of molecules (i.e. plasmids, recombinant proteins, therapeutic drugs). However, liposomal drug delivery have to fulfill different requirements, such as the effective internalization by the target cells and avoidance of the degradative activity of the intracellular compartments. The use of polymer lipid complexes (PLCs), by including different polymers in the liposome formulation, could improve internalization and intracellular release of drugs. The aim of the present work is to study the mechanisms of cellular uptaking and the intracellular trafficking of PLCs formed with cholesterol-poly(2-(dimethylamino)ethyl methacrylate) CHO-PDMAEMA and lecithin (LC CHO-PD). Calcein-loaded liposomes were used to determine cellular uptake and intracellular localization by flow cytometry and confocal microscopy. Incorporation of CHO-PDMAEMA to lecithin liposomes enhanced the internalization capacity of PLCs. Internalization of PLCs by human epithelial-like cells (HEK-293) diminished at 4°C, suggesting uptake by endocytosis. PLCs showed no co-localization with acidic compartments after internalization. Experiments with endocytosis inhibitors and co-localization of liposomes and albumin, suggested the caveolae endocytic pathway as the most probable route for intracellular trafficking of PLCs. In this work, we demonstrated an efficient uptake of LC CHO-PDs by human epithelial-like cells (HEK-293) through the non-degradative caveolae endocytic pathway. The mode of internalization and the intracellular fate of liposomes under study, suggest a promising use of LC CHO-PDs as drug delivery systems.

O estado de humor e a insatisfação corporal possuem relação com os comportamentos de risco para transtornos alimentares em atletas de esportes coletivos do sexo masculino? / Are mood and body image dissatisfaction related to disordered eating behaviors in male team sports athletes?

Os comportamentos de risco para transtornos alimentares (CRTA) dizem respeito a atitudes patogênicas voltadas para a manutenção ou alteração da massa corporal. As frequentes cobranças as quais os atletas são submetidos para melhorarem seu desempenho competitivo podem levá-los a adotarem essas condutas alimentares, buscando um corpo "ideal e compatível" com a modalidade praticada. Contudo, tais comportamentos podem acarretar desidratação, hiponatremia, e reduções da potência anaeróbia e consumo de oxigênio, considerados efeitos prejudiciais para o rendimento esportivo. Algumas variáveis psicológicas podem estar associadas com os CRTA, dentre elas: o estado de humor e a insatisfação corporal (IC). Assim, o objetivo deste trabalho foi avaliar a relação entre o estado de humor e a IC direcionada à muscularidade com os comportamentos de risco para TA em atletas de esportes coletivos do sexo masculino. A amostra deste estudo constou de atletas de esportes coletivos do sexo masculino [futebol (n = 36), basquete (n = 18) e handebol (n = 16)], que treinavam no estado de Pernambuco. As EAAE, EICCE e BRUMS foram utilizadas para avaliar os CRTA, IC e estado de humor, respectivamente. Conduziu-se o teste Shapiro Wilk para verificar a normalidade dos dados e em seguida, a correlação de Pearson para relacionar as variáveis da pesquisa. Os resultados do presente estudo mostraram que os escores da EICCE e BRUMS não estiveram correlacionados aos da EAAE (p > 0,05), indicando que, em atletas de esportes coletivos do sexo masculino, os CRTA não são influenciados pelo humor e IC...(AU)

The disordered eating (DE) concern pathogenic attitudes aimed at the maintenance or alteration of body mass. The frequent charges that athletes undergo to improve their competitive performance may lead them to adopt these dietary behaviors, seeking an "ideal and compatible" body with the modality practiced. However, such behavior can lead to dehydration, hyponatremia, and reductions in anaerobic potency and oxygen consumption, considered to be detrimental effects on sports performance. Some psychological variables may be associated with DE, among them: mood state and body dissatisfaction (BD). Thus, the objective of this study was to evaluate the relationship between the mood state and the muscularity-directed BD with the DE in male athletes. The sample of this study consisted of male and female athletes [(soccer (n = 36), basketball (n = 18) and handball (n = 16)], who trained in the state of Pernambuco. The DES, EICCE and BRUMS were used to evaluate the DE, BD and mood state, respectively. We conducted the Shapiro Wilk test to verify the normality of the data and then the Pearson correlation to relate the variables of the research. The results of the present study showed that the EICCE and BRUMS scores were not correlated with those of the DES (p> 0.05), indicating that, in male athletes, DE are not influenced by mood and BD...(AU)

Stabilization of polymer lipid complexes prepared with lipids of lactic acid bacteria upon preservation and internalization into eukaryotic cells.

The physicochemical characterization of polymer liposome complexes (PLCs) prepared with lipids of lactic acid bacteria and poly(N,N-dimethylaminoethyl methacrylate) covalently bound to cholesterol (CHO-PDMAEMA) was carried out in an integrated approach, including their stability upon preservation and incorporation into eukaryotic cells. PLCs were prepared with different polymer:lipid molar ratios (0, 0.05 and 0.10). Zeta potential, particle size distribution and polydispersity index were determined. The optimal polymer:lipid ratio and the stability of both bare liposomes and PLCs were evaluated at 37 °C and at different pHs, as well as after storage at 4 °C, -80 °C and freeze-drying in the presence or absence of trehalose 250 mM. Internalization of PLCs by eukaryotic cells was assessed to give a complete picture of the system. Incorporation of CHO-PDMAEMA onto bacterial lipids (ratio 0.05 and 0.10) led to stabilization at 37 °C and pH 7. A slight decrease of pH led to their strong destabilization. Bacteria PLCs showed to be more stable than lecithin (LEC) PLCs (used for comparison) upon preservation at 4 and -80 °C. The harmful nature of the preservation processes led to a strong decrease in the stability of PLCs, bacterial formulations being more stable than LEC PLCs. The addition of trehalose to the suspension of liposomes stabilized LEC PLC and did not have effect on bacterial PLCs. In vitro studies on Raw 264.7 and Caco-2/TC7 cells demonstrated an efficient incorporation of PLCs into the cells. Preparations with higher stability were the ones that showed a better cell-uptake. The nature of the lipid composition is determinant for the stability of PLCs. Lipids from lactic acid bacteria are composed of glycolipids and phospholipids like cardiolipin and phosphatidylglycerol. The presence of negatively charged lipids strongly improves the interaction with the positively charged CHO-PDMAEMA, thus stabilizing liposomes. In addition, glycolipids and phosphatidylglycerol act as intrinsic protectants of PLCs upon preservation. This particular lipid composition of lactic acid bacteria makes them natural formulations potentially useful as drug delivery systems.

Effect of cholesterol-poly(N,N-dimethylaminoethyl methacrylate) on the properties of stimuli-responsive polymer liposome complexes.

The development of new polymer-liposome complexes (PLCs) as delivery systems is the key issue of this work. Three main areas are dealt with: polymer synthesis/characterization, liposome formulation/characterization and evaluation of the PLCs uptake by eukaryotic cells. Poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA) with low molecular weight and narrow polydispersity was synthesized by Atom Transfer Radical Polymerization (ATRP). The polymers were synthesized using two different bromide initiators (cholesteryl-2-bromoisobutyrate and ethyl 2-bromoisobutyrate) as a route to afford PDMAEMA and CHO-PDMAEMA. Both synthesized polymers (PDMAEMA and CHO-PDMAEMA) were incorporated in the preparation of lecithin liposomes (LEC) to obtain PLCs. Three polymer/lipid ratios were investigated: 5, 10 and 20%. Physicochemical characterization of PLCs was carried out by determining the zeta potential, particle size distribution, and the release of fluorescent dyes (carboxyfluorescein CF and calcein) at different temperatures and pHs. The leakage experiments showed that CHO covalently bound to PDMAEMA strongly stabilizes PLCs. The incorporation of 5% CHO-PDMAEMA to LEC (LEC_CHO-PD5) appeared to be the stablest preparation at pH 7.0 and at 37°C. LEC_CHO-PD5 destabilized upon slight changes in pH and temperature, supporting the potential use of CHO-PDMAEMA incorporated to lecithin liposomes (LEC_CHO-PDs) as stimuli-responsive systems. In vitro studies on Raw 264.7 and Caco-2/TC7 cells demonstrated an efficient incorporation of PLCs into the cells. No toxicity of the prepared PLCs was observed according to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. These results substantiate the efficiency of CHO-PDMAEMA incorporated onto LEC to assist for the release of the liposome content in mildly acidic environments, like those found in early endosomes where pH is slightly lower than the physiologic. In summary, the main achievements of this work are: (a) novel synthesis of CHO-PDMAEMA by ATRP, (b) stabilization of LEC by incorporation of CHO-PDMAEMA at neutral pH and destabilization upon slight changes of pH, (c) efficient uptake of LEC_CHO-PDs by phagocytic and non-phagocytic eukaryotic cells.

Effect of human defensins on lactobacilli and liposomes.

AIMS: To study the effect of human ß-defensins (HBD-1 and HBD-2) on lactobacilli membranes as well as on liposomes prepared from purified bacterial lipids. METHODS AND RESULTS: Lactobacillus delbrueckii subsp. bulgaricus CIDCA 331 and Lact. delbrueckii subsp. lactis CIDCA 133 were grown in Man, Rogosa, Sharpe broth for 16 h at 37 °C. After being washed, micro-organisms were treated with 0.1-10 µg ml(-1) of HBD-1 and HBD-2 (30 min, 37 °C). Bacterial damage was determined by flow cytometry after propidium iodide staining. In parallel experiments, release of carboxyfluorescein from liposomes prepared from bacterial lipids was determined fluorometrically (excitation 485/20 nm, emission 528/20 nm) in the presence of HBD-1, HBD-2 or Nisin. Exposure of lactobacilli to HBD-2 resulted in a significant membrane permeabilization being Lact. delbrueckii subsp. bulgaricus CIDCA 331 the most susceptible strain. Liposomes prepared with lipids from strain CIDCA 133 were destabilized neither by HBD-1 nor by HBD-2, whereas liposomes derived from strain CIDCA 331 were susceptible to HBD-2 but not to HBD-1. Effect of defensins was strongly inhibited in the presence of NaCl, and the activity increased in water. CONCLUSIONS: Results reported in the presented work indicate that lipid composition of bacterial membranes lead to a different interaction with cationic peptides such as defensins. SIGNIFICANCE AND IMPACT OF THE STUDY: The results represent an advance in the understanding of the differential effect of HBDs on micro-organisms. Differences in susceptibility to anti-microbial peptides could modify the fate of micro-organisms after the interaction with host's cells.

Lactobacillus delbrueckii subsp lactis (strain CIDCA 133) resists the antimicrobial activity triggered by molecules derived from enterocyte-like Caco-2 cells.

AIMS: The aim of the present study was to assess the ability of a potentially probiotic strain to resist, in vitro, the effect of intestinal antimicrobial molecules. METHODS AND RESULTS: Strain CIDCA 133 of Lactobacillus delbrueckii subsp lactis was studied. Lactobacillus delbrueckii subsp bulgaricus as well as other gram-positive and gram-negative bacteria were used for comparison purposes. The effect of different antimicrobial extracts was determined by diffusion assays, viable counts and growth kinetics. Human-defensins (h beta D1 and h beta D2) were also included in the study. Two types of cellular fractions from Caco-2 cells were tested: (i) cytosolic fractions, obtained by sonication of cultured human enterocytes and (ii) cationic fraction, obtained by batch extraction of the cytosolic fraction with a weak cation exchange resin. In addition, the effect of Caco-2-secreted factors was studied. Strain CIDCA 133 was neither inhibited by Caco-2 secreted, cytosolic nor cationic fractions. Of note, human-defensins were inactive against strain CIDCA 133. In contrast, a related lactobacilli: Lactobacilli delbrueckii subsp bulgaricus (strain CIDCA 331) and other species of gram-positive or gram-negative bacteria were strongly inhibited. CONCLUSIONS: Strain CIDCA 133 is able to survive and grow in the presence of enterocyte-derived antimicrobial molecules. This ability is not a general property of lactobacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: Results could provide a new insight into the mechanisms of the probiotic effect and encourage further studies on this field. Resistance to antimicrobial peptides can be relevant to understand the interaction of potentially probiotic strains with the host's immune system. This ability can be also relevant as a selection criterion for new probiotic strains.

Lactobacilli antagonize biological effects of enterohaemorrhagic Escherichia coli in vitro.

AIMS: To assess the effect of two lactobacilli on the biological activity of enterohaemorrhagic Escherichia coli (EHEC) in vitro. METHODS AND RESULTS: Strains CIDCA 133 (Lactobacillus delbrueckii subsp. lactis) and CIDCA 83114 (Lactobacillus plantarum) were studied. Hep-2 cells were used as an in vitro model to assess the biological effect of a clinical isolate of EHEC. Preincubation of cell monolayers with lactobacilli before EHEC prevented detachment of eukaryotic cells and minimizes both F-actin rearrangements and morphological alterations. Interestingly, the protective effect could not be ascribed to pathogen exclusion. In addition, viability of the lactobacilli was not necessary for protection and other species of the genus Lactobacillus failed to protect eukaryotic cells. CONCLUSIONS: Our results suggest that lactobacilli are antagonizing virulence mechanisms of EHEC either by modification of the microenvironment or by interfering with the signalling cascades triggered by the pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings give a rationale basis for the use of specific probiotic strains for the prophylaxis and prevention of intestinal infections due to EHEC.

Lactobacillus delbrueckii subsp lactis strain CIDCA 133 inhibits nitrate reductase activity of Escherichia coli.

The aim of the present work was to investigate the effect of strain CIDCA 133 on the nitrate reductase activity of a non-pathogenic Escherichia coli strain. Suspensions containing different ratios of the strains under study were coincubated in MRS or MRS without glucose. In some experiments lactobacilli were killed by UV treatment. The nitrate reductase activity was determined by using a diazotization reaction for nitrite. Presence of live lactobacilli leads to a dose-response diminution in the specific nitrate reductase of E. coli even when no acidification occurred. Killing of lactobacilli by UV treatment completely abolished the anti-nitrate reductase effect. In addition, the effect was only partially observed with filtered spent culture supernatants of lactobacilli. Lactobacillus delbrueckii subsp lactis strain CIDCA 133 is able to antagonize the nitrate reductase activity of E. coli. This effect is neither due to a diminution of the viability of E. coli nor is depending on the acidification of the medium by the lactobacilli. Viability is needed for maximal anti-nitrate reductase activity. Modulation of undesirable enzymatic activities of intestinal microorganisms by means of selected microorganisms constitutes a further insight on the mechanisms by which probiotics lead to beneficial effects. Administration of probiotic strains able to modulate microbial intestinal activities could lead to a protection of the host against harmful effects of some members of the intestinal microflora.

[Evaluation of a colorimetric method for studying the interaction between microorganisms and intestinal epithelial cells]. / Evaluación de un método colorimétrico para el estudio de la interacción entre micoorganismos y células de epitelio intestinal.

The aim of the present study was to gain further insight on the reliability of the colorimetric determination of the activity of bacterial nitrate reductase to evaluate bacterial concentrations and interaction between microorganisms and enterocyte-like cells. Nitrite produced after incubation of the samples with a nitrate-formate solution was determined with a diazotization reaction with sulphanilic acid and N-naphthyl-ethylene-diamonium dichloride. Cell association assays were performed with differentiated Caco-2 cells. A biphasic relationship was found between nitrite concentration and bacterial densities. This behavior seems to be due to the sigmoideal character of the kinetics of nitrate reduction. Association to Caco-2 cells was strongly strain dependent being Staphylococcus aureus ATCC 25923 the strain showing the highest values of association. For some strains, percentages of association calculated on the basis of the colorimetric assay were significantly higher than those calculated in terms of viable counts. Bacterial association with enterocyte-like cells can be evaluated by measures of the activity of bacterial nitrate reductase provided that the biphasic relationship between bacterial and nitrite concentrations is taken into account for the calculations. Results presented in this paper show the applicability of the colorimetric method to assess the amount of microorganisms associated to human enterocytes in culture.