Size-dependent toxicity and cell interaction mechanisms of gold nanoparticles on mouse fibroblasts

Gold nanoparticles (AuNPs) are currently used in several fields including biomedical applications, although no conclusive information on their cytotoxicity is available. For this reason this work has investigated the effects of AuNPs in vitro on Balb/3T3 mouse fibroblasts. Results obtained exposing cells for 72 h to AuNPs 5 and 15 nm citrate stabilized, revealed cytotoxic effects only for AuNPs 5 nm at concentration ≥ 50 μM if measured by colony forming efficiency (CFE). To understand the differences in cytotoxicity observed for the two AuNPs sizes, we investigated the uptake and the intracellular distribution of the nanoparticles. By TEM it was observed that 5 and 15 nm AuNPs are internalized by Balb/3T3 cells and located within intracellular endosomal compartments. Quantification of the uptake by ICP-MS showed that AuNPs internalization enhanced even up to 72 h. Disruption of the actin cytoskeleton was evident, with cell footprints narrow and contracted; effects more remarkable in cells exposed to 5 nm AuNP. The mechanism of NPs cell internalization was investigated using immunocytochemistry and western blot. No significant effect was observed in the expression level of caveolin, while reduction of the expression and degradation of the clathrin heavy chain was observed in cells exposed for 72 h to AuNPs.     

Multidrug resistance protein 4/ ATP binding cassette transporter 4: a new potential therapeutic target for acute myeloid leukemia

Less than a third of adults patients with acute myeloid leukemia (AML) are cured by current treatments, emphasizing the need for new approaches to therapy. We previously demonstrated that besides playing a role in drug-resistant leukemia cell lines, multidrug resistance protein 4 (MRP4/ABCC4) regulates leukemia cell proliferation and differentiation through the endogenous MRP4/ABCC4 substrate, cAMP. Here, we studied the role of MRP4/ABCC4 in tumor progression in a mouse xenograft model and in leukemic stem cells (LSCs) differentiation. We found a decrease in the mitotic index and an increase in the apoptotic index associated with the inhibition of tumor growth when mice were treated with rolipram (PDE4 inhibitor) and/or probenecid (MRPs inhibitor). Genetic silencing and pharmacologic inhibition of MRP4 reduced tumor growth. Furthermore, MRP4 knockdown induced cell cycle arrest and apoptosis in vivo. Interestingly, when LSC population was isolated, we observed that increased cAMP levels and MRP4/ABCC4 blockade resulted in LSCs differentiation. Taken together, our findings show that MRP4/ABCC4 has a relevant role in tumor growth and apoptosis and in the eradication of LSCs, providing the basis for a novel promising target in AML therapy.     

Differential β2-adrenergic receptor expression defines the phenotype of non-tumorigenic and malignant human breast cell lines

Breast cancer is the most frequent malignancy in women. Several reports demonstrated that adrenergic receptors (ARs) are involved in breast cancer. Here we observed that epinephrine (Epi), an endogenous AR agonist, caused opposite effects in non-tumorigenic (MCF-10A and HBL-100) and tumor cells (MCF-7 and MDA-MB-231). Thus, Epi, in non-tumor breast cells, as well as isoproterenol (β-agonist), in all cell lines, maintained a benign phenotype, decreasing cell proliferation and migration, and stimulating cell adhesion. β-AR expression and cAMP levels were higher in MCF-10A than in MCF-7 cells. β₂-AR knock-down caused a significant increase of cell proliferation and migration, and a decrease of cell adhesion both in basal and in Iso-stimulated conditions. Coincidently, β₂-AR over-expression induced a significant decrease of cell proliferation and migration, and an increase of cell adhesion. Therefore, β₂-AR is implied in cell phenotype and its agonists or antagonists could eventually complement cancer therapy.     

The distinctive germinal center phase of IgE+ B lymphocytes limits their contribution to the classical memory response

The mechanisms involved in the maintenance of memory IgE responses are poorly understood, and the role played by germinal center (GC) IgE⁺ cells in memory responses is particularly unclear. IgE⁺ B cell differentiation is characterized by a transient GC phase, a bias toward the plasma cell (PC) fate, and dependence on sequential switching for the production of high-affinity IgE. We show here that IgE⁺ GC B cells are unfit to undergo the conventional GC differentiation program due to impaired B cell receptor function and increased apoptosis. IgE⁺ GC cells fail to populate the GC light zone and are unable to contribute to the memory and long-lived PC compartments. Furthermore, we demonstrate that direct and sequential switching are linked to distinct B cell differentiation fates: direct switching generates IgE⁺ GC cells, whereas sequential switching gives rise to IgE⁺ PCs. We propose a comprehensive model for the generation and memory of IgE responses.IgE antibodies are critical mediators of allergic reactions (Gould and Sutton, 2008). Cross-linking of IgE molecules bound to high affinity FcεRI receptors on mast cells and basophils leads to the rapid release of potent proinflammatory molecules (Kinet, 1999; Galli and Tsai, 2012). In spite of its pathological potential, IgE exhibits the lowest serum concentration and the shortest half-life of all the antibody isotypes (Vieira and Rajewsky, 1988; Gould and Sutton, 2008). The low frequency of IgE-producing cells makes their study particularly challenging. Using mouse models of high IgE responses (Katona et al., 1988; Curotto de Lafaille et al., 2001), we discovered that IgE-producing cells develop via a unique differentiation pathway that occurs during the germinal center (GC) phase of T cell–dependent responses and yet favors the production of plasma cells (PCs; Erazo et al., 2007; Yang et al., 2012). In our early studies a GC IgE⁺ population was not clearly detectable, but the IgE antibodies produced were observed to have undergone affinity maturation, indicating a GC history for IgE⁺ PC. We proposed at the time that high affinity IgE originated from the sequential switching of high affinity IgG1 cells, and hence we speculated that classical IgE⁺ memory cells may be absent in mice (Erazo et al., 2007; Curotto de Lafaille and Lafaille, 2010).Sequential switching of IgG cells to IgE was first discovered by the identification of switch(S)γ region footprints in the Sμ-Sε DNA region of IgE genes (Matsuoka et al., 1990; Yoshida et al., 1990; Jabara et al., 1993; Mandler et al., 1993; Zhang et al., 1994; Baskin et al., 1997), but the biological significance of this finding was at that time unknown. Sequential switching in mice entails two recombination events, Sμ→Sγ1 and SμSγ1→Sε, that may be either continuous or temporally separate events. The latter scenario allows for the existence of an intermediate IgG1 cellular phase in which affinity maturation can occur in GCs. Indeed, stimulation of IgG1 cells in the presence of IL-4 either in vivo or in vitro resulted in the production of IgE antibodies (Erazo et al., 2007; Wesemann et al., 2012). Importantly, mice deficient in class switching to IgG1 due to a mutation in the Iγ1 exon (Lorenz et al., 1995) were unable to produce high affinity IgE antibodies (Xiong et al., 2012a,b), indicating that sequential switching is essential for the formation of high affinity IgE.The recent development of fluorescent reporter mice for IgE has facilitated the identification of IgE GC cells (Talay et al., 2012; Yang et al., 2012). However, the in vivo phenotype and role of IgE GC cells in supporting IgE responses and its relationship with the sequential switching process remain unclear (Lafaille et al., 2012; Xiong et al., 2012a).In the current study, we used a new reporter mouse for class switch recombination (CSR) to IgE, improved methods to functionally study IgE B cells ex vivo and in vivo, and in silico modeling to analyze the origin, functional properties, and population dynamics of IgE GC cells and PC. We show that IgE GC cells are unfit to undergo the conventional GC differentiation program and instead undergo apoptosis at a high rate. This “failure to thrive” of IgE GC cells greatly limits their contribution to the memory pool and high affinity PC compartment. Furthermore, we show that the two types of rearrangement to IgE are associated with distinct B cell differentiation fates. Direct Sμ-Sε rearrangements generate IgE GC cells, whereas sequential switching of IgG1 cells gives rise to IgE PC.     

Decision making in family medicine: Randomized trial of the effects of the InfoClinique and Trip database search engines

Objective

To compare the ability of users of 2 medical search engines, InfoClinique and the Trip database, to provide correct answers to clinical questions and to explore the perceived effects of the tools on the clinical decision-making process.

Design

Randomized trial.

Setting

Three family medicine units of the family medicine program of the Faculty of Medicine at Laval University in Quebec city, Que.

Participants

Fifteen second-year family medicine residents.

Intervention

Residents generated 30 structured questions about therapy or preventive treatment (2 questions per resident) based on clinical encounters. Using an Internet platform designed for the trial, each resident answered 20 of these questions (their own 2, plus 18 of the questions formulated by other residents, selected randomly) before and after searching for information with 1 of the 2 search engines. For each question, 5 residents were randomly assigned to begin their search with InfoClinique and 5 with the Trip database.

Main outcome measures

The ability of residents to provide correct answers to clinical questions using the search engines, as determined by third-party evaluation. After answering each question, participants completed a questionnaire to assess their perception of the engine’s effect on the decision-making process in clinical practice.

Results

Of 300 possible pairs of answers (1 answer before and 1 after the initial search), 254 (85%) were produced by 14 residents. Of these, 132 (52%) and 122 (48%) pairs of answers concerned questions that had been assigned an initial search with InfoClinique and the Trip database, respectively. Both engines produced an important and similar absolute increase in the proportion of correct answers after searching (26% to 62% for InfoClinique, for an increase of 36%; 24% to 63% for the Trip database, for an increase of 39%; P = .68). For all 30 clinical questions, at least 1 resident produced the correct answer after searching with either search engine. The mean (SD) time of the initial search for each question was 23.5 (7.6) minutes with InfoClinique and 22.3 (7.8) minutes with the Trip database (P = .30). Participants’ perceptions of each engine’s effect on the decision-making process were very positive and similar for both search engines.

Conclusion

Family medicine residents’ ability to provide correct answers to clinical questions increased dramatically and similarly with the use of both InfoClinique and the Trip database. These tools have strong potential to increase the quality of medical care.     

The Role of Graduate Medical Education in Global Health: Proceedings From the 2013 Academic Emergency Medicine Consensus Conference

The past 40 years have seen expanded development of emergency medicine (EM) postgraduate residency training programs worldwide. An important part of this educational experience is the ability of resident trainees to participate in experiences abroad. However, little is known about how these experiences shape trainees and the populations they serve. During the 2013 Academic Emergency Medicine consensus conference, a group of educators met to define and outline current trends in graduate medical education (GME) emergency care research. The authors discuss future research questions bridging the gap of GME and global health.     

Energy status and immune system alterations in Elliptio complanata after ingestion of cyanobacteria Anabaena flos-aquae

Cyanobacteria have often been described as nutritionally poor for herbivorous organisms. To gain additional information on the potential impacts of invertebrates feeding on cyanobacteria, we fed Elliptio complanata mussels with two types of algae: Anabaena flos-aquae (cyanobacteria) and Pseudokirchneriella subcapitata (green algae). Physiological parameters were examined at the energy status, immune system and oxidative stress levels. Energy status was examined by following the rate of electron transport activity in mitochondria (a measure of cellular energy expense) and lipid/sugar stores in the visceral mass. The cyanobacteria were not actively producing toxins. Based on the digestive gland index, the mussels fed equally on either regime. However, the energy status in mussels fed A. flos-aquae revealed that the total sugar was lower in the digestive gland, whereas mitochondrial electron transport activity (MET), once corrected against the digestive gland somatic index, showed increased energy expenses. Acetylcholinesterase activity and lipid peroxidation (LPO) were also higher in mussels fed with A. flos-aquae compared with mussels fed with P. subcapitata. LPO was correlated by mitochondrial activity in both the digestive gland and gills, suggesting that oxidative stress resulted from metabolic respiration. Immunocompetence (phagocytic activity, natural killer cell-like activity, haemocyte count and viability) and humoral level of lysozyme were not affected in mussels by the algae or cyanobacteria regime. Moreover, the xenobiotic conjugating enzyme, glutathione S-transferase, hemoprotein oxidase and vitellogenin-like proteins were not affected in mussel organs via ingestion of A. flos-aquae. Our study suggests that ingestion of cyanobacteria leads to increased energy expenses, oxidative stress and increased acetylcholine turnover in mussels.     

Expanding the spectrum of EWSR1‐PATZ1 rearranged CNS tumors: An infantile case with leptomeningeal dissemination

We report on a case of EWSR1‐PATZ1 rearranged brain tumor occurring in a 17 month‐old child, originally interpreted as an infantile glioblastoma. Our case shows important analogies with the 2 previously reported cases, including the intraventricular location, the histologic appearance (pushing borders, oligodendrocyte‐like morphology, rich vascular network) and the glioneural immunophenotype, supporting the role of these features as relevant clues to the diagnosis. On the other hand, our case displays unique characteristics, i.e. the onset in an infant, the presence of a focal high‐grade component and the leptomeningeal dissemination, pointing to the importance of considering this entity in the differential diagnosis of an infantile glial/glioneural tumor.     

Effects of pink grapefruit juice on QT variability in patients with dilated or hypertensive cardiomyopathy and in healthy subjects

Recent evidence shows that pink grapefruit juice, which is a recommended dietary addition that contains high amounts of the antioxidant flavonoid naringenin, prolongs the corrected QT (QT(c)), a noninvasive electrophysiological marker of spatial myocardial repolarization, and does so by inhibiting the rapid component of the delayed rectifier K+ current (I(Kr)). Prompted by the observation that all class III antiarrhythmic drugs inhibit this current, thereby sometimes provoking torsades de pointes, we compared the effects of a liter of freshly squeezed pink grapefruit juice with those of 2 commonly used class III antiarrhythmics amiodarone and sotalol on the major noninvasive markers of temporal variability in myocardial repolarization used to stratify the risk of sudden death from malignant ventricular arrhythmias. In 32 subjects, 10 with postischemic dilated cardiomyopathy, 12 with hypertensive cardiomyopathy, and 10 healthy, we assessed QT(c) and QT variability index (QTVI) after administration of fresh pink grapefruit juice, placebo, amiodarone, or sotalol. After pink grapefruit juice and sotalol, all these indexes increased significantly from values observed after placebo (P<0.05) and from values after amiodarone (P<0.05). Conversely, after amiodarone, QT(c), but not QTVI, increased significantly from values after placebo (P<0.05). Presumably because of its high naringenin glycoside content, pink grapefruit juice prolongs cardiac repolarization and concurrently increases temporal cardiac repolarization dispersion. The potential proarrhythmic actions of pink grapefruit juice might be of concern in patients with major myocardial structural disorders.