High-resolution atomic force microscopy as a tool for topographical mapping of surface budding.

Extracellular vesicles (EVs) are membranous nanoparticles secreted by almost all cell types. Reflecting the physiopathological state of the parental cell, EVs circulate in all body fluids, reaching distant cell targets and delivering different bioactive cargoes. As biological carriers, EVs influence their microenvironment altering cellular responses, being considered promising biomarkers for both physiological and pathological conditions. EVs are heterogeneous in terms of size and composition, depending on cell type and exposure to stimuli, and different methods have been developed to characterize their morphological, biophysical, and biochemical features. Among them, electron microscopy (EM) is the main technique used, however, the lack of standardized protocols makes it difficult to characterize EVs with a good reproducibility, thus using multiple approaches may represent a way to obtain more precise information. Furthermore, the relationship between architecture and function, not only in a molecular, but also in a cellular level, is gaining growing emphasis, characterizing morphometric parameters may represent a distinct, but effective approach to study the physiopathological state of the cell. Atomic force microscopy (AFM), may represent a promising method to study in detail EVs dynamics throughout the cell surface and its variations related to the physiological state, overcoming the limits of EM, and providing more reliable information. In this study, human neuroblastoma SH-SY5Y cell line, a cellular model to investigate neurodegeneration and oxidative stress, has been used to perform a comparative morphological and quantitative analysis of membrane budding and isolated large vesicles-enriched (microvesicles-like vesicles; MVs) fraction from control or oxidative stressed cells. Our main goal was to build up a methodology to characterize EVs morphology and spatial distribution over the cell surface in different physiological conditions, and to evaluate the efficacy of AFM against conventional EM. Interestingly, both microscopy techniques were effective for this analysis, but AFM allowed to reveal a differential profiling of plasma membrane budding between the physiological and the stress condition, indicating a potential relationship between mechanical characteristics and functional role. The results obtained may provide interesting perspectives for the use of AFM to study EVs, validating a morphometric approach to understand the pathophysiological state of the cell related to EVs trafficking.

Microvesicles and exosomes in metabolic diseases and inflammation.

Metabolic diseases are based on a dysregulated crosstalk between various cells such as adipocytes, hepatocytes and immune cells. Generally, hormones and metabolites mediate this crosstalk that becomes alterated in metabolic syndrome including obesity and diabetes. Recently, Extracellular Vesicles (EVs) are emerging as a novel way of cell-to-cell communication and represent an attractive strategy to transfer fundamental informations between the cells through the transport of proteins and nucleic acids. EVs, released in the extracellular space, circulate via the various body fluids and modulate the cellular responses following their interaction with the near and far target cells. Clinical and experimental data support their role as biomarkers and bioeffectors in several diseases includimg also the metabolic syndrome. Despite numerous studies on the role of macrophages in the development of metabolic diseases, to date, there are little informations about the influence of metabolic stress on the EVs produced by macrophages and about the role of the released vesicles in the organism. Here, we review current understanding about the role of EVs in metabolic diseases, mainly in inflammation status burst. This knowledge may play a relevant role in health monitoring, medical diagnosis and personalized medicine.

[Primary Prevention in General Medical Practice: A Survey]. / Primärprävention in der Allgemeinarztpraxis: Eine Befragung.

AIM OF THE STUDY: According to the German social insurance code §20 Sec. 1, statutory health insurance companies can reimburse up to 80% of costs incurred by primary prevention programs in physical activity, nutrition, stress management and drug consumption. Whether and how many general practitioners (GPs) provide their patients with information on such programs as part of their own practice is unknown. In this study, we investigate to which primary prevention programs primary care physicians refer their patients and whether they take into account reimbursability of programs. METHODS: Between November 2010 and February 2011, all GPs with a practice in Berlin (n=1 168) received a questionnaire that assessed if patients were referred to prevention programs and the type of programs they were referred to, if they ensured they are reimbursable and if they themselves offered prevention programs. Descriptive statistics and multivariate logistic regression was used for analysis. RESULTS: Of 474 respondents (response rate: 41%), 67% were female. Of the respondents, 22% offered reimbursable prevention programs and 42% at out-of-pocket expense. Patients were referred to reimbursable programs by 63%. GPs younger than 50 were twice as likely to offer reimbursable programs in their practice compared to those older than 50 (OR=1.7; 95% KI 1.1-2,8; p-value 0.025). CONCLUSION: A successful implementation of the new German prevention law needs awareness among GPs about reimbursable prevention programs, which may be lacking in some groups.

Timing the multiple cell death pathways initiated by Rose Bengal acetate photodynamic therapy.

Rose Bengal acetate photodynamic therapy (RBAc-PDT) induced multiple cell death pathways in HeLa cells through ROS and ER stress. Indeed, apoptosis was the first preferred mechanism of death, and it was triggered by at least four different pathways, whose independent temporal activation ensures cell killing when one or several of the pathways are inactivated. Apoptosis occurred as early as 1 h after PDT through activation of intrinsic pathways, followed by activation of extrinsic, caspase-12-dependent and caspase-independent pathways, and by autophagy. The onset of the different apoptotic pathways and autophagy, that in our system had a pro-death role, was timed by determining the levels of caspases 9, 8, 3 and 12; Bcl-2 family; Hsp70; LC3B; GRP78 and phospho-eIF2α proteins. Interestingly, inhibition of one pathway, that is, caspase-9 (Z-LEHD-FMK), caspase-8 (Z-IETD-FMK), pan-caspases (Z-VAD-FMK), autophagy (3-MA) and necrosis (Nec-1), did not impair the activation of the others, suggesting that the independent onset of the different apoptotic pathways and autophagy did not occur in a subordinated manner. Altogether, our data indicate RBAc as a powerful photosensitiser that induces a prolonged cytotoxicity and time-related cell death onset by signals originating from or converging on almost all intracellular organelles. The fact that cancer cells can die through different mechanisms is a relevant clue in the choice and design of anticancer PDT.

Lycopersicon esculentum lectin is a marker of transient amplifying cells in in vitro cultures of isolated limbal stem cells.

The maintenance of a healthy corneal epithelium under both normal and wound healing conditions is achieved by a population of stem cells (SCs) located in the basal epithelium at the corneoscleral limbus. In the light of the development of strategies for reconstruction of the ocular surface in patients with limbal stem cell deficiency, a major challenge in corneal SCs biology remains the ability to identify stem cells in situ and in vitro. To date, not so much markers exist for the identification of different phenotypes. CESCs (corneal epithelial stem cells) isolated from limbal biopsies were maintained in primary culture for 14 days and stained with Hoechst and a panel of FITC-conjugated lectins. All lectins, with the exception of Lycopersicon esculentum, labelled CESCs irrespective of the degree of differentiation. Lycopersicon esculentum, that binds N-acetylglucosamine oligomers, labelled intensely only the surface of TACs (single corneal epithelial stem cells better than colonial cells). These results suggest that Lycopersicon esculentum lectin is a useful and easy-to-use marker for the in vitro identification of TACs (transient amplifying cells) in cultures of isolated CESCs.

Effect of 6mT static magnetic field on the bcl-2, bax, p53 and hsp70 expression in freshly isolated and in vitro aged human lymphocytes.

An increasing number of evidence indicates that static magnetic fields (SMFs) are capable of altering apoptosis, mainly through modulation of Ca(2+) influx. Here we present data that suggest apoptotic-related gene expression as an alternative pathway, through which exposure to 6milliTesla (mT) SMF can interfere with apoptosis. Exposure to 6mT SMF affects the apoptotic rate (spontaneous and drug-induced) and [Ca(2+)](i) in isolated human lymphocytes; the aged cells are more susceptible to exposure than fresh ones. The exposure to 6mT exerted a protective effect on chemical or physical-induced apoptosis, irrespective of the age of the cells. The investigation of the gene expression of bcl-2, bax, p53 and hsp70 in freshly isolated and in culture-aged human lymphocytes indicates that these genes are modulated by SMF exposure in the experimental conditions used, in a gene-, age- and time-dependent manner. The exposure of isolated lymphocytes to SMF for up to 24h modulated increased bax and p53 and decreased hsp70, and bcl-2. The amount of increment and/or decrement of the proteins varied for each gene examined and was independent of the apoptotic inducers. Finally, the same stress applied to freshly isolated or aged lymphocytes resulted in different modulation of bcl-2, bax and hsp70.

Static magnetic field selects undifferentiated myelomonocytes from low-glutamine concentration stimulated U937 cells.

Reduced glutamine (GLN) concentration in the culture medium of a U937 cell line caused them to be differentiated along the monocytic pathway; cells attached to the matrix and to each other by extending pseudopodia and acquired specific functional characteristics, such as the expression of alpha-naphthyl-acetate esterase and the capacity to reduce nitroblue tetrazolium, as well as becoming active phagocytes. When U937 cells were differentiated under continuous exposure to a 6mT static magnetic field (MF) the overall differentiation process was perturbed. Surprisingly, after 5 days' exposure to the static MF, higher cell viability and differentiation were observed in cells cultured in a GLN-deprived medium than in cells grown in the same medium but in the absence of a static MF. The latter cells, particularly those that were still floating in the medium, were stimulated with TPA for a further 3 days. These cells differentiated and attached to the substrate. Conversely, the same treatment applied to cells cultured in GLN-deprived medium in the presence of the static MF resulted in resistance to TPA-induced differentiation. Indeed, these cells exhibited a round shape and in-suspension growth.

Proteinaceous diet inhibits gossypol-induced spermatotoxicity.

The present study was designed to investigate the effect of a proteinaceous dietary supplement, fishmeal, on gossypol-induced spermatotoxicity. Twenty-five adult male Wistar rats, averaging 205 g b.w., were randomly sorted into four experimental groups (I-IV) of 5 animals each, and a control group. Crude cottonseed oil was administered orally to each animal in groups I-IV at a rate that provided 14 mg/kg/d free gossypol; in addition, 3 g/d, 7 g/d, and 10 g/d of fishmeal was provided as meal supplement to each animal in groups I, II and III respectively. The control group received rat pellets and water freely. At the end of the 53-day treatment period, all animals were placed under chloroform anaesthesia; the caudal epididymides were removed, minced and placed in Ham's F10 solution for the evaluation of sperm count and motility. The testes were also processed for histological studies using the eosin and haematoxylin (H & E) method. Our findings revealed a dose-dependent inhibition of gossypol-induced spermatotoxicity by the supplemented fishmeal; this suggests that proteinaceous diets are protective against gossypol-induced male infertility.

Respiratory atopic disease, Ascaris-immunoglobulin E and tuberculin testing in urban South African children.

BACKGROUND: Epidemiological relation of intestinal helminth infection and atopic disease, both associated with a T-helper (Th) 2 immune response, is controversial, as it has been reported that helminth infection may either suppress or pre-dispose to atopic disease. This relation has not been tested in an area with a high burden of Mycobacterium tuberculosis (MTB) infection, a known Th1-stimulating infection. OBJECTIVE: To study the association of intestinal helminth infection and atopic disease in a community where helminth infection is endemic and MTB infection is high. METHODS: Three-hundred and fifty-nine randomly selected children aged 6-14 years from a poor urban suburb were tested with allergy questionnaire, skin prick test (SPT) to common aeroallergens, Ascaris-specific IgE (Ascaris-sIgE), fecal examination for pathogenic intestinal helminths and tuberculin skin testing (TST). Histamine bronchoprovocation was tested in the group of children aged 10 years and older. RESULTS: were corrected for demographic variables, socioeconomic status, parental allergy, environmental tobacco smoke (ETS) exposure in the household, recent anthelminthic treatment and for clustering in the sampling unit. Results Ascaris-sIgE was elevated in 48% of children, Ascaris eggs were found in 15% and TST was positive in 53%. Children with elevated Ascaris-sIgE had significantly increased risk of positive SPT to aeroallergens, particularly house dust mite, atopic asthma (ever and recent), atopic rhinitis (ever and recent) and increased atopy-related bronchial hyper-responsiveness. In children with negative TST (<10 mm), elevated Ascaris-sIgE was associated with significantly increased risk of atopic symptoms (adjusted odds ratio (OR(adj)) 6.5; 95% confidence interval (CI) 1.9-22.4), whereas in those with positive TST (>/=10 mm) this association disappeared (OR(adj) 0.96; 95% CI 0.4-2.8). CONCLUSIONS: These results suggest that immune response to Ascaris (Ascaris-sIgE) may be a risk factor of atopic disease in populations exposed to mild Ascaris infection and that MTB infection may be protective against this risk, probably by stimulation of anti-inflammatory networks.

Neoplasias astrocitárias e correlação com as proteínas p53 mutada e Ki-67 / Astrocytic neoplasms and correlation with mutate p53 and Ki-67 proteins

As neoplasias astrocitárias correspondem a 60 por cento dos tumores do sistema nervoso central, sendo o estudo da biologia molecular um importante passo para a compreensão da gênese e comportamento biológico destas doenças. As proteínas Ki-67, que é um marcador de proliferação celular, e p53, que é o produto do gene supressor de tumor de mesmo nome, são importantes marcadores tumorais. O objetivo deste estudo foi identificar e quantificar as proteínas Ki-67 e produto do gene supressor de tumor TP53 em diferentes graus de malignidade das neoplasias astrocitárias, bem como analisar suas relações com idade e sexo. Foram estudadas por imuno-histoquímica as proteínas Ki-67 e p53 em 47 pacientes com neoplasias astrocitárias ressecadas cirurgicamente, classificadas previamente e revisadas quanto ao grau de malignidade, de acordo com o proposto pela Organização Mundial da Saúde. Os núcleos celulares imunomarcados foram quantificados no programa Imagelab-softium pela razão paramétrica absoluta entre os núcleos de células positivas e o número total de células tumorais, sendo contadas 1000 células. O delineamento utilizado foi transversal não controlado. Para análise estatística as variáveis foram divididas em grupos, que para a Ki-67 foram ausente, <5 por cento e >5 por cento e para a p53 foram ausente (0), <25 por cento (1+), entre 25 e 50 por cento (2+), entre 50 e 75 por cento (3+) e maior que 75 por cento (4+). Ki-67 esteve presente em 37 casos (78,72 por cento) expressando correlação com maior grau de malignidade (p<0,001) . A p53 esteve presente em 14 casos (35,13 por cento) tendo maior correlação com astrocitoma grau IV (p=0,59). Não houve correlação estatisticamente significativa entre p53 e Ki-67, bem com entre estas variáveis, idade e sexo. Concluiu-se que a hipótese de maior presença de Ki-67 e p53 em neoplasias astrocitárias de maior grau de malignidade, com exceção da correlação entre grau III e p53, é corroborada pelos resultados deste estudo.

In vitro study of the interaction of polyalkilimide and polyvinyl alcohol hydrogels with cells.

Hydrogels are a class of polymers that in the last decade have had a great development and application for soft tissue augmentation, due to their similarity to this tissue for their high water content. The in vitro effects of polyalkylmide hydrogel (pAI) and polyvinyl alcohol hydrogel (pVOH) on human lymphocytes and U937 cells viability, apoptosis and cell shape were investigated. Cell viability was always higher than 70%, thus showing the hydrogels were not cytotoxic for both cell lines. Some differences were, however, found. At short time, lymphocytes were very sensitive to the hydrogels incubation, while at long time, U937 cells were the most sensitive cells. Other differences on cell viability were related to the time of incubation, to the type of hydrogel and to the polymers concentration. Cell viability decreased only at the longest time of incubation and with the highest hydrogel concentration. Accordingly, cell death by apoptosis increased; necrosis was never observed in the cultures. Concentration- and hydrogel-dependent modifications of cell shape (bigger cell volume, elongations of cells) were observed in a few percentage of viable cells. In conclusion, the very high in vitro degree of biocompatibility shown by both hydrogels encourages their use as dermal fillers.

Bilateral isodense epidural hematoma: case report

We present a case of a severe head injured 23 year-old male patient. The initial CT scan disclosed bilateral epidural hematoma, isodense with the brain, thus being a pitfall in diagnosis. Brief case report, image and literature review are presented.

Apresentamos o caso de um homem de 23 anos com traumatismo craniano grave. A TC de crânio demonstrou um volumoso hematoma epidural bilateral, isodenso com o cérebro, sendo uma armadilha ao diagnóstico. São apresentados um breve relato, estudo da imagem tomográfica e revisão da literatura.

Differentiation of monocytic U937 cells under static magnetic field exposure.

We present here a morphological, cytochemical and biochemical study of the macrophagic differentiation of human pro-monocytic U937 cells exposed to moderate intensity (6 mT) static magnetic fields (MF). It was found that the following substances induced differentiation in U937 cells to a progressively lower degree: 50 ng/mL 12-0-tetradecanoyl-13-phorbol acetate (TPA), low concentration of glutamine (0,05 mM/L), 10% dimethyl sulfoxide (DMSO) and 100 mM/L Zn++. Differentiated U937 cells shift from a round shape to a macrophage-like morphology, from suspension to adhesion growth and acquire phagocytotic activity, the cytoskeleton adapting accordingly. Exposure to static MF at 6 mT of intensity decreases the degree of differentiation for all differentiating molecules with a consequent fall in cell adhesion and increased polarization of pseudopodia and cytoplasmic protrusions. Differentiation alone, or in combination with exposure to static MFs, affects the distribution and quantity of cell surface sugar residues, the surface expression of markers of macrophage differentiation, and phagocytotic capability. Our results indicate that moderate-intensity static MFs exert a considerable effect on the process of macrophage differentiation of pro-monocytic U937 cells and suggest the need for further studies to investigate the in vivo possible harmful consequences of this.

[Astrocytic neoplasms and correlation with mutate p53 and Ki-67 proteins]. / Neoplasias astrocitárias e correlação com as proteínas p53 mutada e Ki-67.

The astrocytic neoplasms respond by 60% of the central nervous system tumors, being the study of the molecular biology an important step for the understanding of the genesis and biological behavior of these diseases. The Ki-67 proteins, which are markers of the cellular proliferation, and p53, which is the product of the tumor suppressor gene TP53, are both important tumoral markers. This study intends to identify and quantify the Ki-67 and p53 proteins in astrocytic tumors of different grades of malignancy, as well as to analyze their relations with age and gender. Ki-67 and p53 proteins in 47 patients with surgically resected astrocytic neoplasms were studied through immunohistochemistry. They have been previously classified and reviewed concerning their histological grade, as suggested by the World Health Organization. The immunomarked cellular nuclei were quantified by the program Imagelab-softium for the absolute parametric reason between the nuclei of the positive cells and the total amount of tumoral cells, being counted 1000 cells. The lineation used has been transversal not controlled. For the statistical analysis the variables were divided into groups. For the Ki-67 they were absent, <5% and >5% and for p53 they were absent (0), <25% (1+), between 25 and 50% (2+), between 50 and 75% (3+), and higher than 75% (4+). Ki-67 was present in 37 cases (78.72%) evidencing a correlation with a higher malignancy degree (p<0,001). p53 was present in 14 cases (35.13%) with a higher correlation with astrocytoma grade IV (p=0.59). There has not been a statistically significant correlation between p53 and Ki-67, as well as among these variables, age and gender. The hypotheses of a greater presence of Ki-67 and p53 in astrocytic neoplasms with a higher degree of malignancy, except for the correlation between grade III and p53, is corroborated by the results of this study.

In vitro and in vivo biocompatibility evaluation of a polyalkylimide hydrogel for soft tissue augmentation.

Injectable fillers are commonly used in Plastic and Reconstructive Surgery to correct serious and slight aesthetic defects due to their low invasiveness and an easy implant technique procedure. Synthetic hydrogels are proposed as filler materials for their similarity with soft tissue and to avoid many disadvantages of naturally derived materials such as short persistence, allergenicity, and immunogenicity. Our studies are focused on the biocompatibility evaluation of a polyacrylic hydrogel containing alkylimide-amide groups and pyrogen free water (96%) (Bio-Alcamid by means of the in vitro cytotoxicity and mutagenicity assays and the in vivo skin irritation, sensitization test, and subcutaneous implant. All tests conducted on Bio-Alcamid showed no toxicity. It is a substance easy to inject and remove; it does not migrate, and its safety allows it to be a suitable filler for the correction of slight and also very serious aesthetic defects.

Hepatic clearance of apoptotic lymphocytes: simply removal of waste cells?

The in situ liver recognition of apoptotic lymphocytes was studied by using different sources of lymphocytes (i.e. human, rat and mouse) and animal models (i.e. rat and mouse). Lymphocytes were induced to apoptosis using 10(-2)M cycloheximide for up to 24 hours; three types of apoptosing lymphocytes, corresponding to different stages in the apoptotic process, were described: type 1 or early apoptosis, type 2 or mature apoptosis and type 3 or late/necrotic apoptosis. When livers were in situ injected with apoptotic lymphocytes enriched for type 1 (early), 2 (mature) or 3 (late/necrotic) apoptosis, they recognized and internalized apoptosing cells, with an efficiency directly dependent on the stage of the apoptotic process. The highest recognition rate, which was, in all cases, mediated by galactose- and mannose-specific receptors, was obtained with homologous apoptotic cells (i.e. rat lymphocytes and rat liver). Moreover, the drastically reduced efficiency of recognition of human or mouse apoptotic lymphocytes when injected into rat liver, suggested the involvement also of species-specific antigens.

Morphological and ultrastructural analysis of Turritopsis nutricula during life cycle reversal.

The hydrozoa life cycle is characterized, in normal conditions, by the alternation of a post-larval benthic polyp and an adult pelagic medusa; however, some species of Hydrozoa react to environmental stress by reverting their life cycle: i.e. an adult medusa goes back to the juvenile stage of polyp. This very uncommon life cycle could be considered as some sort of inverted metamorphosis. A morphological study of different stages during the reverted life cycle of Turritopsis nutricula led to the characterization of four different stages: healthy medusa, unhealthy medusa, four-leaf clover and cyst. The ultrastructural study of the cellular modifications (during the life cycle reversion of T. nutricula) showed the presence of both degenerative and apoptotic processes. Degeneration was prevalent during the unhealthy medusa and four-leaf clover stages, while the apoptotic rate was higher during the healthy medusa and cyst stages. The significant presence of degenerative and apoptotic processes could be related to the occurrence of a sort of metamorphosis when an adult medusa transforms itself into a polyp.

Lead nitrate and gadolinium chloride administration modify hepatocyte cell surfaces.

Modifications of hepatocyte cell surface were determined after single i.v. injection to rats of Pb(NO(3))(2) (known to induce liver hyperplasia followed by apoptosis) or GdCl(3) (known to induce proliferation of parenchymal cells and Kupffer cell depletion) or administration of GdCl(3) 24 h before Pb(NO(3))(2) injection (known to reduce hyperplasia and apoptosis induced in the parenchymal liver cells by the single Pb(NO(3))(2) injection). Rats were sacrificed at fixed times after the treatments (1, 3 and 5 days) and hepatocytes were isolated by enzymatic liver perfusion. In spite of the intracellular target of the heavy metals, signals leading to liver hyperplasia and apoptosis (with rates different for the different experimental conditions) were generated, which in turn were responsible for cell surface alteration. Increment or decrement of phosphatidylserine (PS) expression, asialoglycoprotein receptors (ASGPRs) and sugar residue expression on hepatocyte surfaces was measured in parallel with apoptosis and proliferation. When GdCl(3) was injected 24 h before Pb(NO(3))(2) injection, liver modifications were significantly reduced, thus suggesting that GdCl(3) could prevent and/or reduce liver damage.

Lymphocytes apoptosis: young versus aged and humans versus rats.

This paper deals with a comparative study of lymphocyte apoptosis in young versus aged and humans versus rats. Apoptotic rate achieved by the use of different apoptogenic inducers, acting at different cellular levels, and cell surface modifications were analyzed. The results showed that aged human lymphocytes and freshly isolated rat lymphocytes were more prone to undergo apoptosis. Therefore, the same apoptotic signal is recognized by human and rat lymphocytes, but the extent of the answer is related to the species, to the intensity of the apoptotic stimulus and to the metabolic/developmental condition of the cells. Surface modifications (lipids and glycans), typical of apoptosis, were observed. Our data showed that cell surface changes are species and age dependent. They are early events, progressively achieved in the course of the apoptotic process involving lateral membrane movements of molecules.