Characterization of novel clonal murine endothelial cell lines with an extended life span

Summary: A murine endothelial cell line was recently established from microvessels that had invaded a subcutaneous sponge implant (Dong, Q. G.; Bernasconi, S.; Lostaglio, S., et al. Arterioscl. Thromb. Vasc. Biol. 17:1599-1604; 1997). From these sponge-induced endothelial (SIE) cells, we have isolated two subpopulations endowed with different phenotypic properties. Clone SIE-F consists of large, highly spread cells that have a relatively slow growth rate, form contact-inhibited monolayers, do not grow under anchorage-independent conditions, express elevated levels of thrombospondin-1 (TSP-1) and are not tumorigenic in vivo. In contrast, clone SIE-S2 consists of small, spindle-shaped cells that have a high proliferation rate, do not show contact-inhibition, grow under anchorage-independent conditions, express very low levels of TSP-1 and are tumorigenic in vivo. Both clones express the endothelial markers vascular endothelial-cadherin and vascular intercellular adhesion molecule-1, but do not express CD31 and E-selectin. In addition, SIE-S2 cells, but not SIE-F cells, express the α-smooth muscle actin isoform. SIE-S2 cells, but not SIE-F cells, are able to form branching tubes in fibrin gels. The SIE-F and SIE-S2 clones, which have properties of nontransformed and transformed cells, respectively, should provide useful tools to investigate physiological and pathological processes involving vascular endotheliu

Pentraxin 3 Plasma Levels and Disease Activity in Systemic Lupus Erythematosus

SLE is an autoimmune disorder that involves polyclonal autoimmunity against multiple autoantigens. PTX3, a marker of the acute-phase inflammatory response, plays an important role in innate immunity and in modulation of the adaptive immune response. Our study tried to resolve some rather controversial aspects of the use of PTX3 as a biomarker of disease activity in SLE patients. We demonstrated that plasma PTX3 concentration of the SLE patients was significantly higher than the healthy control groups and reflected disease activity. ROC curve analysis was used to determine best cut-off point (2.8 ng/mL) with a good sensitivity and specificity. In patients with SLE, PTX3 concentrations were correlated with SLEDAI. Trend to remission (TTR) curve was created by plotting PTX3 levels and SLEDAI and we applied the curve as a model for the analysis of two patients with different follow-up. PTX3 plasma levels declined significantly and this decline occurred parallel to the clinical improvement with a complete remission of disease. In patients who experienced a clinical relapse, an increase in PTX3 levels followed the lupus flare. The proposal of PTX3 cut-off associated with TTR and monitoring of PTX3 plasma levels could be an innovative approach to follow-up of SLE patients

Residual matrix from different separation techniques impacts exosome biological activity

Exosomes are gaining a prominent role in research due to their intriguing biology and several therapeutic opportunities. However, their accurate purification from body fluids and detailed physicochemical characterization remain open issues. We isolated exosomes from serum of patients with Multiple Myeloma by four of the most popular purification methods and assessed the presence of residual contaminants in the preparations through an ad hoc combination of biochemical and biophysical techniques - including Western Blot, colloidal nanoplasmonics, atomic force microscopy (AFM) and scanning helium ion microscopy (HIM). The preparations obtained by iodixanol and sucrose gradients were highly pure. To the contrary, those achieved with limited processing (serial centrifugation or one step precipitation kit) resulted contaminated by a residual matrix, embedding the exosomes. The contaminated preparations showed lower ability to induce NfkB nuclear translocation in endothelial cells with respect to the pure ones, probably because the matrix prevents the interaction and fusion of the exosomes with the cell membrane. These findings suggest that exosome preparation purity must be carefully assessed since it may interfere with exosome biological activity. Contaminants can be reliably probed only by an integrated characterization approach aimed at both the molecular and the colloidal length scales

Association between religiosity or spirituality and internet addiction: A systematic review

Introduction: The literature provides evidence of religiosity being associated with physical and mental health, and also with behavioral addictions. This systematic review examines the data on the link between religiosity or spirituality and the emerging internet addictions. Methods: A systematic literature review was conducted in the PubMed and Scopus databases to identify observational (cross-sectional, cohort, and case-control) studies conducted on adolescents and young adults to investigate the association between religiosity or spirituality and internet addiction. Of the 854 articles identified in the databases, 13 met our inclusion criteria and were included in our systematic review. Results: Eleven of the 13 studies reviewed specifically investigated religiosity and internet addiction: six found an inverse association between religiosity and internet addiction; three found no evidence of any association; and one found a direct association. One study examining both religiosity and spirituality generated mixed results. Only one study investigated spirituality unrelated to religion, and found a direct association with internet addiction. Two of three studies specifically considering internet gaming addiction found it inversely associated with high levels of religiosity, while the third found no association. Conclusion: This review supports a possible role for religiosity as a protective factor, as emerged from the majority of the studies examined. Religiosity also seemed to be associated with lower internet gaming rates among adolescents

Ameloblastomas: diagnosis considerations and therapeutic approaches.

Objetivo: Demostrar as considerações atuais no diagnóstico, mudança na classificação e abordagem terapêuticas, para o tratamento dos ameloblastomas. Método: trata-se de revisão narrativa de literatura aliada a demonstração de casos clínicos originais com o propósito de aproximar o conteúdo teórico revisto com exemplares de aplicação prática da atuação do profissional.  A consulta pela literatura pautou-se em livros de referência mundial como o da Organização Mundial da Saúde associado a busca eletrônica nas bases de dados Medline, Lilacs e SciELO. Resultados: O ameloblastoma é uma neoplasia odontogênica benigna de evolução lenta, assintomática. Em aspecto de exames de imagem as lesões variam entre o radiolúcido unilocular ao multilocular, podendo provocar alterações em dentes próximos. Em aspecto transcirúrgico e histopatológico há diferenças entre ameloblastoma e ameloblastoma cístico para as lesões intraósseas, e o tratamento será influenciado por esse fator, além de idade do paciente, tamanho da lesão e localização. Há ainda na literatura uma certa preferência para as abordagens radicais. As terapias direcionadas contra o gene BRAF parecem um futuro promissor. Conclusão: O conhecimento de variantes do ameloblastoma é primordial e orienta uma reflexão terapêutica do tipo custo/benefício a ser estabelecida em cada caso individualmente a partir de discussão multidisciplinar e escolha do paciente.Objective: To demonstrate the current considerations in the diagnosis, change in the classification and therapeutic approach, for the treatment of ameloblastomas. Method: it is a narrative review of literature combined with the demonstration of original clinical cases to approximate the revised theoretical content with examples of the practical application of the professional's performance. The literature search was based on world reference books such as the World Health Organization's associated electronic search in the Medline, Lilacs and SciELO databases. Results: Ameloblastoma is a slow, asymptomatic benign odontogenic neoplasm. In terms of imaging exams, the lesions vary from unilocular to multilocular radiolucent, which can cause changes in nearby teeth. In trans-surgical and histopathological aspects, there are differences between ameloblastoma and cystic ameloblastoma for intraosseous lesions, and the treatment will be influenced by this factor, in addition to the patient's age, lesion size and location. There is still a certain preference in the literature for radical approaches. Therapies targeting the BRAF gene look like a promising future. Conclusion: The knowledge of ameloblastoma variants is paramount and guides a therapeutic reflection of the cost/benefit type to be established in each case individually based on multidisciplinary discussion and patient choice

NANOSCALE CHARACTERIZATION OF EXTRACELLULAR VESICLE IS MANDATORY TO ASSESS THEIR BIOLOGICAL ACTIVITY AND BIOTECHNOLOGICAL EXPLOITATION

Introduction:Cells release into the extracellular environment diverse types of nanosized extracellularvesicles (EVs) which serve in inter cellular communication by shuttling protein, lipids andsmall nucleic acids (miRNA). It is now becoming clear how they support both physiologicalor pathological mechanisms such as antigen presentation, immunostimulatory or inhibitoryactivities, disease progression.EV properties, including specific targeting, cargo protection, biocompatibility etc. open newexciting perspectives for their application in biotechnology as tools for drug delivery,therapeutic targets or source of new markers.EVs are separated from different biological fluids e.g. blood, cell culture medium, urine anddependingon the applied protocol the resulting sample can be surrounded by differentkind of exogenous nanostructured matrixes. which heavily influence in vitro and in vivoproperties.Here we asses and apply nanodiagnostic techniques used in nanomaterial science tothese new soft natural nanostructures and show that they add important informations ontheir final biological activity.Materials and Methods:Exosomes from serum of Multiple Myeloma (MM) patients were purified following differentpurification protocols: sequential centrifugation steps (P3), discontinuous sugar gradients(sucrose and iodixanol) and a direct vesicle precipitation kit (ExoKit).Samples have been analyzed byWestern Blot, colloidal nanoplasmonics, atomic forcemicroscopy(AFM) and scanning helium ion microscopy (HIM). Resulting data allowed thedetermination of samples purity and their biological activity.Results:Data obtained analyzing samples by Western blot indicate that all the isolation protocolsallowobtaining comparable exosome population expressing characteristic markers(Hsp70, Tsg101, CD63, Annexin V).Colloidal properties and biophysical purity grade has been determined respectively withagarose gel runs and colorimetric nanoplasmonic assay. Results showed that gradientscontain lower quantity of contaminants compared with ExoKit or P3.AFM and HIM imaging highlighted samples microstructure confirming that samples arecomposed of vesicle populations with typical exosome size (50-120 nm) and that residualmatrix are absent in samples isolated by gradients whereas it’s abundant in P3 and ExoKitsamples.To demonstrate if the purity of each preparation can affect their biological effect, humanendothelial cells have been exposed to MM exosome. It is known that EVs cellularinternalization trigger NfkB nuclear translocation, this biological effect has been exploitedto value if surrounding matrix influence samples biological activity. As expected, cellsincubated with exosome obtained from gradients showed a strong NfkB nucleartranslocation signal whereas P3 and ExoKit preparations showed no significant differencescompared to those treated with the control buffer.Discussion:These findings suggest that exosome preparation purity must be carefully assessed since it may interfere with exosome biological activity. Contaminants can be explored only by anintegrated characterization approach aimed at both the molecular and the colloidal lengthscales

Activation of the PI3K/AKT/mTOR Pathway in Cajal–Retzius Cells Leads to Their Survival and Increases Susceptibility to Kainate-Induced Seizures

International audienceCajal–Retzius cells (CRs) are a class of transient neurons in the mammalian cortex that play a critical role in cortical development. Neocortical CRs undergo almost complete elimination in the first two postnatal weeks in rodents and the persistence of CRs during postnatal life has been detected in pathological conditions related to epilepsy. However, it is unclear whether their persistence is a cause or consequence of these diseases. To decipher the molecular mechanisms involved in CR death, we investigated the contribution of the PI3K/AKT/mTOR pathway as it plays a critical role in cell survival. We first showed that this pathway is less active in CRs after birth before massive cell death. We also explored the spatio-temporal activation of both AKT and mTOR pathways and reveal area-specific differences along both the rostro–caudal and medio–lateral axes. Next, using genetic approaches to maintain an active pathway in CRs, we found that the removal of either PTEN or TSC1, two negative regulators of the pathway, lead to differential CR survivals, with a stronger effect in the Pten model. Persistent cells in this latter mutant are still active. They express more Reelin and their persistence is associated with an increase in the duration of kainate-induced seizures in females. Altogether, we show that the decrease in PI3K/AKT/mTOR activity in CRs primes these cells to death by possibly repressing a survival pathway, with the mTORC1 branch contributing less to the phenotype

Aberrant survival of hippocampal Cajal-Retzius cells leads to memory deficits, gamma rhythmopathies and susceptibility to seizures in adult mice

International audienceAbstract Cajal-Retzius cells (CRs) are transient neurons, disappearing almost completely in the postnatal neocortex by programmed cell death (PCD), with a percentage surviving up to adulthood in the hippocampus. Here, we evaluate CR’s role in the establishment of adult neuronal and cognitive function using a mouse model preventing Bax-dependent PCD. CRs abnormal survival resulted in impairment of hippocampus-dependent memory, associated in vivo with attenuated theta oscillations and enhanced gamma activity in the dorsal CA1. At the cellular level, we observed transient changes in the number of NPY + cells and altered CA1 pyramidal cell spine density. At the synaptic level, these changes translated into enhanced inhibitory currents in hippocampal pyramidal cells. Finally, adult mutants displayed an increased susceptibility to lethal tonic-clonic seizures in a kainate model of epilepsy. Our data reveal that aberrant survival of a small proportion of postnatal hippocampal CRs results in cognitive deficits and epilepsy-prone phenotypes in adulthood