Leishmania Promastigotes Lack Phosphatidylserine but Bind Annexin V upon Permeabilization or Miltefosine Treatment

The protozoan parasite Leishmania is an intracellular pathogen infecting and replicating inside vertebrate host macrophages. A recent model suggests that promastigote and amastigote forms of the parasite mimic mammalian apoptotic cells by exposing phosphatidylserine (PS) at the cell surface to trigger their phagocytic uptake into host macrophages. PS presentation at the cell surface is typically analyzed using fluorescence-labeled annexin V. Here we show that Leishmania promastigotes can be stained by fluorescence-labeled annexin V upon permeabilization or miltefosine treatment. However, combined lipid analysis by thin-layer chromatography, mass spectrometry and 31 P nuclear magnetic resonance (NMR) spectroscopy revealed that Leishmania promastigotes lack any detectable amount of PS. Instead, we identified several other phospholipid classes such phosphatidic acid, phosphatidylethanolamine; phosphatidylglycerol and phosphatidylinositol as candidate lipids enabling annexin V staining.

Annexin A1 regulates neutrophil clearance by macrophages in the mouse bone marrow

Under homeostatic conditions, a proportion of senescent CXCR4(hi) neutrophils home from the circulation back to the bone marrow, where they are phagocytosed by bone marrow macrophages. In this study, we have identified an unexpected role for the anti-inflammatory molecule annexin A1 (AnxA1) as a critical regulator of this process. We first observed that AnxA1(-/-) mice have significantly increased neutrophil numbers in their bone marrow while having normal levels of GM and G colony-forming units, monocytes, and macrophages. Although AnxA1(-/-) mice have more neutrophils in the bone marrow, a greater proportion of these cells are senescent, as determined by their higher levels of CXCR4 expression and annexin V binding. Consequently, bone marrow neutrophils from AnxA1(-/-) mice exhibit a reduced migratory capacity in vitro. Studies conducted in vitro also show that expression of AnxA1 is required for bone marrow macrophages, but not peritoneal macrophages, to phagocytose apoptotic neutrophils. Moreover, in vivo experiments indicate a defect in clearance of wild-type neutrophils in the bone marrow of AnxA1(-/-) mice. Thus, we conclude that expression of AnxA1 by resident macrophages is a critical determinant for neutrophil clearance in the bone marrow.-Dalli, J., Jones, C. P., Cavalcanti, D. M., Farsky, S. H., Perretti, M., Rankin, S. M. Annexin A1 regulates neutrophil clearance by macrophages in the mouse bone marrow. FASEB J. 26, 387-396 (2012). www.fasebj.org

Dose-dependent effect of Resveratrol on bladder cancer cells: Chemoprevention and oxidative stress

Background: Over 6 million people die annually in the world because of cancer. Several groups are focused on studying cancer chemoprevention approaches. Resveratrol, a polyphenol, at high dosages, has been reported as antitumor and chemopreventive. However, it has a dose-dependent effect on cell death, even on some cancer cells. Objectives: Our aim was to investigate this dose-dependent effect on human bladder carcinoma ECV304 cells during oxidative stress condition. Methods: For this purpose. ECV304 cells incubated with different Resveratrol concentrations were analyzed as for their metabolic rate, membrane permeability, DNA fragmentation, anti/proapoptotic protein levels and phosphatidylserine exposure after oxidative stress. Results: Resveratrol induced cell death at high concentrations (>20 mu M), but not at low ones (0.1-20 mu M). Pretreatment with 2.5 mu M protected the cells from oxidative damage, whereas 50 mu M intensified the cell death and significantly increased Bad/Bcl-2 ratio (proapoptotic/antiapoptotic proteins). Resveratrol was able to modulate NO and PGE(2) secretion and performed an anti-adhesion activity of neutrophils on PMA-activated ECV304 cells. Conclusions: Resveratrol at high doses induces cell death of ECV304 cells whereas low doses induce protection. Modulation of Bcl-2 protein induced by Resveratrol could be mediating this effect. This information about the role of Resveratrol on cancer alerts us about its dose-dependent effects and could lead the design of future chemoprevention strategies. Published by Elsevier Ireland Ltd.

INFLAMMATORY RESPONSE AND NEUTROPHIL FUNCTIONS IN PLAYERS AFTER A FUTSAL MATCH

de Moura, NR, Cury-Boaventura, MF, Santos, VC, Levada-Pires, AC, Bortolon, JR, Fiamoncini, J, Pithon-Curi, TC, Curi, R, and Hatanaka, E. Inflammatory response and neutrophil functions in players after a futsal match. J Strength Cond Res 26(9): 2507-2514, 2012-Futsal players suffer injuries resulting from muscle fatigue and contact or collision among players. Muscle lesions can be detected by measuring muscle lesion markers such as creatine kinase (CK) and lactate dehydrogenase (LDH) in plasma. After an initial lesion, there is an increase in the plasma levels of C-reactive protein (CRP) and proinflammatory cytokines. These mediators may activate neutrophils and contribute to tissue damage and increase susceptibility to invasive microorganisms. In this study, we investigated the effect of a futsal match on muscle lesion markers, cytokines, and CRP in elite players. The basal and stimulated neutrophil responsiveness after a match was also evaluated based on measurements of neutrophil necrosis, apoptosis, phagocytic capacity, reactive oxygen species (ROS) production, and cytokines (tumor necrosis factor-alpha [TNF-alpha], interleukin [IL]-8, IL-1 beta, IL-10, and IL-1ra) production. Blood samples were taken from 16 players (26.4 +/- 3.2 years, 70.2 +/- 6.9 kg, 59.7 +/- 5.1 ml.kg(-1).min(-1), sports experience of 4.4 +/- 0.9 years) before and immediately after a match. Exercise increased the serum activities of CK (2.5-fold) and LDH (1.3-fold). Playing futsal also increased the serum concentrations of IL-6 (1.6-fold) and CRP (1.6-fold). The TNF-alpha, IL-1 beta, IL-8, IL-1ra, and IL-10 serum levels were not modified in the conditions studied. The futsal match induced neutrophil apoptosis, as indicated by phosphatidylserine externalization (6.0-fold). The exercise induced priming of neutrophils by increasing ROS (1.3-fold), TNF-alpha (5.8-fold), and IL-1 beta (4.8-fold) released in nonstimulated cells. However, in the stimulated condition, the exercise decreased neutrophil function, diminishing the release of ROS by phorbol myristate acetate-stimulated neutrophils (1.5-fold), and the phagocytic capacity (1.6-fold). We concluded that playing futsal induces inflammation, primes and activates neutrophils, and reduces the efficiency of neutrophil phagocytosis immediately after a match.

Quality characteristics and fertilizing ability of ram sperm subpopulations separated by partition in an aqueous two-phase system

Centrifugal countercurrent distribution (CCCD) in an aqueous two-phase system (TPS) is a resolute technique revealing sperm heterogeneity and for the estimation of the fertilizing potential of a given semen sample. However, separated sperm subpopulations have never been tested for their fertilizing ability yet. Here, we have compared sperm quality parameters and the fertilizing ability of sperm subpopulations separated by the CCCD process from ram semen samples maintained at 20 degrees C or cooled down to 5 degrees C. Total and progressive sperm motility was evaluated by computer-assisted analysis using a CASA system and membrane integrity was evaluated by flow cytometry by staining with CFDA/Pl. The capacitation state, staining with chlortetracycline, and apoptosis-related markers, such as phosphatidylserine (PS) translocation detected with Annexin V. and DNA damage detected by the TUNEL assay, were determined by fluorescence microscopy. Additionally, the fertilizing ability of the fractionated subpopulations was comparative assessed by zona binding assay (ZBA). CCCD analysis revealed that the number of spermatozoa displaying membrane and DNA alterations was higher in samples chilled at 5 degrees C than at 20 degrees C. which can be reflected in the displacement to the left of the CCCD profiles. The spermatozoa located in the central and right chambers (more hydrophobic) presented higher values (P<0.01) of membrane integrity, lower PS translocation (P<0.05) and DNA damage (P<0.001) than those in the left part of the profile, where apoptotic markers were significantly increased and the proportion of viable non-capacitated sperm was reduced. We have developed a new protocol to recover spermatozoa from the CCCD fractions and we proved that these differences were related with the fertilizing ability determined by ZBA, because we found that the number of spermatozoa attached per oocyte was significantly higher for spermatozoa recovered from the central and right chambers, in both types of samples. This is the first time, to our knowledge that sperm recovered from a two-phase partition procedure are used for fertilization assays. These results open up new possibilities for using specific subpopulations of sperm for artificial insemination or in vitro fertilization, not only regarding better sperm quality but also certain characteristics such as subpopulations enriched in spermatozoa bearing X or Y chromosome that we have already isolated or any other feature. (C) 2011 Elsevier B.V. All rights reserved.

Influence of the Bilayer Composition on the Binding and Membrane Disrupting Effect of Polybia-MP1, an Antimicrobial Mastoparan Peptide with Leukemic T-Lymphocyte Cell Selectivity

This study shows that MP-1, a peptide from the venom of the Polybia paulista wasp, is more toxic to human leukemic T-lymphocytes than to human primary lymphocytes. By using model membranes and electrophysiology measurements to investigate the molecular mechanisms underlying this selective action, the porelike activity of MP-1 was identified with several bilayer compositions. The highest average conductance was found in bilayers formed by phosphatidylcholine or a mixture of phosphatidylcholine and phosphatidylserine (70:30). The presence of cholesterol or cardiolipin substantially decreases the MP-1 pore activity, suggesting that the membrane fluidity influences the mechanism of selective toxicity. The determination of partition coefficients from the anisotropy of Tip indicated higher coefficients for the anionic bilayers. The partition coefficients were found to be 1 order of magnitude smaller when the bilayers contain cholesterol or a mixture of cholesterol and sphingomyelin. The blue shift fluorescence, anisotropy values, and Stern-Volmer constants are indications of a deeper penetration of MP-1 into anionic bilayers than into zwitterionic bilayers. Our results indicate that MP-1 prefers to target leukemic cell membranes, and its toxicity is probably related to the induction of necrosis and not to DNA fragmentation. This mode of action can be interpreted considering a number of bilayer properties like fluidity, lipid charge, and domain formation. Cholesterol-containing bilayers are less fluid and less charged and have a tendency to form domains. In comparison to healthy cells, leukemic T-lymphocyte membranes are deprived of this lipid, resulting in decreased peptide binding and lower conductance. We showed that the higher content of anionic lipids increases the level of binding of the peptide to bilayers. Additionally, the absence of cholesterol resulted in enhanced pore activity. These findings may drive the selective toxicity of MP-1 to Jurkat cells.

Nuclear Transfer with Apoptotic Bovine Fibroblasts: Can Programmed Cell Death Be Reprogrammed?

Cell death by apoptosis is considered to be irreversible. However, reports have indicated that its reversibility is possible if the cells have not yet reached the "point of no return.'' In order to add new information about this topic, we used cells at different moments of apoptotic process as nuclear donors in somatic cell nuclear transfer (SCNT) in order to test if programmed cell death can be reversed. Adult bovine fibroblasts were treated with 10 mu M of staurosporine (STP) for 3 h and analyzed for phosphatidylserine externalization (Annexin assay) and presence of active caspase-9. Annexin-positive (Anx +) and Caspase-9-positive (Casp-9 +) cells were isolated by FACS and immediately transferred into enucleated in vitro matured bovine oocytes. After STP treatment, 89.9% of cells were Anx + (4.6% in control cells; p < 0.01) and 24.9% were Casp-9 + (2.4% in control cells; p < 0.01). Fusion and cleavage were not affected by the use apoptotic cells (p > 0.05). Also, the use of Anx + cells did not affect blastocyst production compared to control (26.4% vs. 22.9%, respectively; p > 0.05). However, blastocyst formation was affected by the use of Casp-9 + cells (12.3%; p < 0.05). These findings contribute to the idea of that apoptosis is reversible only at early stages. Additionally, we hypothesize that the "point of no return'' for apoptosis may be located around activation of Caspase-9.

Free Rhodium (II) citrate and rhodium (II) citrate magnetic carriers as potential strategies for breast cancer therapy

Abstract Background Rhodium (II) citrate (Rh2(H2cit)4) has significant antitumor, cytotoxic, and cytostatic activity on Ehrlich ascite tumor. Although toxic to normal cells, its lower toxicity when compared to carboxylate analogues of rhodium (II) indicates Rh2(H2cit)4 as a promising agent for chemotherapy. Nevertheless, few studies have been performed to explore this potential. Superparamagnetic particles of iron oxide (SPIOs) represent an attractive platform as carriers in drug delivery systems (DDS) because they can present greater specificity to tumor cells than normal cells. Thus, the association between Rh2(H2cit)4 and SPIOs can represent a strategy to enhance the former's therapeutic action. In this work, we report the cytotoxicity of free rhodium (II) citrate (Rh2(H2cit)4) and rhodium (II) citrate-loaded maghemite nanoparticles or magnetoliposomes, used as drug delivery systems, on both normal and carcinoma breast cell cultures. Results Treatment with free Rh2(H2cit)4 induced cytotoxicity that was dependent on dose, time, and cell line. The IC50 values showed that this effect was more intense on breast normal cells (MCF-10A) than on breast carcinoma cells (MCF-7 and 4T1). However, the treatment with 50 μM Rh2(H2cit)4-loaded maghemite nanoparticles (Magh-Rh2(H2cit)4) and Rh2(H2cit)4-loaded magnetoliposomes (Lip-Magh-Rh2(H2cit)4) induced a higher cytotoxicity on MCF-7 and 4T1 than on MCF-10A (p < 0.05). These treatments enhanced cytotoxicity up to 4.6 times. These cytotoxic effects, induced by free Rh2(H2cit)4, were evidenced by morphological alterations such as nuclear fragmentation, membrane blebbing and phosphatidylserine exposure, reduction of actin filaments, mitochondrial condensation and an increase in number of vacuoles, suggesting that Rh2(H2cit)4 induces cell death by apoptosis. Conclusions The treatment with rhodium (II) citrate-loaded maghemite nanoparticles and magnetoliposomes induced more specific cytotoxicity on breast carcinoma cells than on breast normal cells, which is the opposite of the results observed with free Rh2(H2cit)4 treatment. Thus, magnetic nanoparticles represent an attractive platform as carriers in Rh2(H2cit)4 delivery systems, since they can act preferentially in tumor cells. Therefore, these nanopaticulate systems may be explored as a potential tool for chemotherapy drug development.

Platelet-derived exosomes induce endothelial cell apoptosis through peroxynitrite generation: experimental evidence for a novel mechanism of septic vascular dysfunction

Abstract Introduction Several studies link hematological dysfunction to severity of sepsis. Previously we showed that platelet-derived microparticles from septic patients induce vascular cell apoptosis through the NADPH oxidase-dependent release of superoxide. We sought to further characterize the microparticle-dependent vascular injury pathway. Methods During septic shock there is increased generation of thrombin, TNF-α and nitric oxide (NO). Human platelets were exposed for 1 hour to the NO donor diethylamine-NONOate (0.5 μM), lipopolysaccharide (LPS; 100 ng/ml), TNF-α (40 ng/ml), or thrombin (5 IU/ml). Microparticles were recovered through filtration and ultracentrifugation and analyzed by electron microscopy, flow cytometry or Western blotting for protein identification. Redox activity was characterized by lucigenin (5 μM) or coelenterazine (5 μM) luminescence and by 4,5-diaminofluorescein (10 mM) and 2',7'-dichlorofluorescein (10 mM) fluorescence. Endothelial cell apoptosis was detected by phosphatidylserine exposure and by measurement of caspase-3 activity with an enzyme-linked immunoassay. Results Size, morphology, high exposure of the tetraspanins CD9, CD63, and CD81, together with low phosphatidylserine, showed that platelets exposed to NONOate and LPS, but not to TNF-α or thrombin, generate microparticles similar to those recovered from septic patients, and characterize them as exosomes. Luminescence and fluorescence studies, and the use of specific inhibitors, revealed concomitant superoxide and NO generation. Western blots showed the presence of NO synthase II (but not isoforms I or III) and of the NADPH oxidase subunits p22phox, protein disulfide isomerase and Nox. Endothelial cells exposed to the exosomes underwent apoptosis and caspase-3 activation, which were inhibited by NO synthase inhibitors or by a superoxide dismutase mimetic and totally blocked by urate (1 mM), suggesting a role for the peroxynitrite radical. None of these redox properties and proapoptotic effects was evident in microparticles recovered from platelets exposed to thrombin or TNF-α. Conclusion We showed that, in sepsis, NO and bacterial elements are responsible for type-specific platelet-derived exosome generation. Those exosomes have an active role in vascular signaling as redox-active particles that can induce endothelial cell caspase-3 activation and apoptosis by generating superoxide, NO and peroxynitrite. Thus, exosomes must be considered for further developments in understanding and treating vascular dysfunction in sepsis.