Bacterial Lysate from the Multi-Strain Probiotic SLAB51 Triggers Adaptative Responses to Hypoxia in Human Caco-2 Intestinal Epithelial Cells under Normoxic Conditions and Attenuates LPS-Induced Inflammatory Response.

Hypoxia-inducible factor-1α (HIF-1α), a central player in maintaining gut-microbiota homeostasis, plays a pivotal role in inducing adaptive mechanisms to hypoxia and is negatively regulated by prolyl hydroxylase 2 (PHD2). HIF-1α is stabilized through PI3K/AKT signaling regardless of oxygen levels. Considering the crucial role of the HIF pathway in intestinal mucosal physiology and its relationships with gut microbiota, this study aimed to evaluate the ability of the lysate from the multi-strain probiotic formulation SLAB51 to affect the HIF pathway in a model of in vitro human intestinal epithelium (intestinal epithelial cells, IECs) and to protect from lipopolysaccharide (LPS) challenge. The exposure of IECs to SLAB51 lysate under normoxic conditions led to a dose-dependent increase in HIF-1α protein levels, which was associated with higher glycolytic metabolism and L-lactate production. Probiotic lysate significantly reduced PHD2 levels and HIF-1α hydroxylation, thus leading to HIF-1α stabilization. The ability of SLAB51 lysate to increase HIF-1α levels was also associated with the activation of the PI3K/AKT pathway and with the inhibition of NF-κB, nitric oxide synthase 2 (NOS2), and IL-1ß increase elicited by LPS treatment. Our results suggest that the probiotic treatment, by stabilizing HIF-1α, can protect from an LPS-induced inflammatory response through a mechanism involving PI3K/AKT signaling.

Up-Regulation of Cyclooxygenase-2 (COX-2) Expression by Temozolomide (TMZ) in Human Glioblastoma (GBM) Cell Lines.

TMZ-resistance remains a main limitation in glioblastoma (GBM) treatment. TMZ is an alkylating agent whose cytotoxicity is modulated by O6-methylguanine-DNA methyltransferase (MGMT), whose expression is determined by MGMT gene promoter methylation status. The inflammatory marker COX-2 has been implicated in GBM tumorigenesis, progression, and stemness. COX-2 inhibitors are considered a GBM add-on treatment due to their ability to increase TMZ-sensitivity. We investigated the effect of TMZ on COX-2 expression in GBM cell lines showing different COX-2 levels and TMZ sensitivity (T98G and U251MG). ß-catenin, MGMT, and SOX-2 expression was analyzed. The effects of NS398, COX-2 inhibitor, alone or TMZ-combined, were studied evaluating cell proliferation by the IncuCyte® system, cell cycle/apoptosis, and clonogenic potential. COX-2, ß-catenin, MGMT, and SOX-2 expression was evaluated by RT-PCR, Western blotting, and immunofluorescence and PGE2 by ELISA. Our findings, sustaining the role of COX-2/PGE2 system in TMZ-resistance of GBM, show, for the first time, a relevant, dose-dependent up-regulation of COX-2 expression and activity in TMZ-treated T98G that, in turn, correlated with chemoresistance. Similarly, all the COX-2-dependent signaling pathways involved in TMZ-resistance also resulted in being up-modulated after treatment with TMZ. NS398+TMZ was able to reduce cell proliferation and induce cell cycle arrest and apoptosis. Moreover, NS398+TMZ counteracted the resistance in T98G preventing the TMZ-induced COX-2, ß-catenin, MGMT, and SOX-2 up-regulation.

Distance education during COVID 19: an Italian survey on the university teachers' perspectives and their emotional conditions.

BACKGROUND: Following the COVID-19 pandemic, distance education (DE) replaced traditional "face-to-face" teaching and has become the main method of teaching. The aim of this study was to 1) evaluate the impact of DE by teachers in our department during the second semester of the 2019-20 academic year following the March-May 2020 Italian national lockdown and 2) evaluate the relationship between DE and the emotional well-being of teachers during the period of home confinement. METHODS: Ninety-seven university teachers (51.5% women; most represented age group 60-69 years range, 40.2%) responded to an anonymous online cross-sectional survey between July 15 - September 30, 2020, on the advantages and disadvantages of DE, developed by one online teacher focus group. The emotional conditions were assessed by a short version of the Beck Depression Inventory-II (BDI-II). The internal consistency reliability survey and the 10-item BDI-II were measured by Cronbach's alpha. A correlation analysis (r-Pearson) was conducted between the overall evaluation of the experience of DE and the variables included in the study. RESULTS: Teachers reported difficulties in technical aspects, and in psychological factors, as the discomfort of "speaking in the void" (64.7%). The absence of "face-to-face" eye contact with the students was complained by 81% of teachers. Significant impairments in sleep patterns and loss of energy were reported, with female teachers having greater difficulty concentrating than their male colleagues. A quarter of teachers showed depressive symptoms of varying severity. The most satisfied teachers were those most stimulated by DE (r = 0.752, p < 0.000), who showed a lower impact of depressive symptoms (r = - 0.289, p = 0.005). The teaching load in hours influenced the perception of disadvantages (r = 0.214, p = 0.035) and contributed to a lower appreciation of the challenges of DE. The more significant the manifestation of depressive symptoms during the lockdown was, the greater the subjective recovery of a good emotional condition once the domestic confinement was over (r = 0.344, p = 0.001), despite maintaining DE. CONCLUSIONS: Our study highlights the impact of technical, didactic, and psychological difficulties of DE, reported by our teachers. The appreciation of their new learning promoted by DE seemed related to better emotional well-being of university teachers accepting this "challenge" in their important role in the high-education system, influencing good learning and promoting students' professional success.

Strategic Modification of Gut Microbiota through Oral Bacteriotherapy Influences Hypoxia Inducible Factor-1α: Therapeutic Implication in Alzheimer's Disease.

Dysbiosis contributes to Alzheimer's disease (AD) pathogenesis, and oral bacteriotherapy represents a promising preventative and therapeutic opportunity to remodel gut microbiota and to delay AD onset and progression by reducing neuroinflammation and amyloid and tau proteins aggregation. Specifically, SLAB51 multi-strain probiotic formulation positively influences multiple neuro-chemical pathways, but exact links between probiotics oral consumption and cerebral beneficial effects remain a gap of knowledge. Considering that cerebral blood oxygenation is particularly reduced in AD and that the decreased neurovascular function contributes to AD damages, hypoxia conditioning represents an encouraging strategy to cure diseases of the central nervous system. In this work, 8-week-old 3xTg-AD and wild-type mice were chronically supplemented with SLAB51 to evaluate effects on hypoxia-inducible factor-1α (HIF-1α), a key molecule regulating host-microbial crosstalk and a potential target in neurodegenerative pathologies. We report evidence that chronic supplementation with SLAB51 enhanced cerebral expression of HIF-1α and decreased levels of prolyl hydroxylase 2 (PHD2), an oxygen dependent regulator of HIF-1α degradation; moreover, it successfully counteracted the increase of inducible nitric oxide synthase (iNOS) brain expression and nitric oxide plasma levels in AD mice. Altogether, the results demonstrate an additional mechanism through which SLAB51 exerts neuroprotective and anti-inflammatory effects in this model of AD.

Biological effects of selective COX-2 inhibitor NS398 on human glioblastoma cell lines.

BACKGROUND: Cyclooxygenase-2 (COX-2), an inflammation-associated enzyme, has been implicated in tumorigenesis and progression of glioblastoma (GBM). The poor survival of GBM was mainly associated with the presence of glioma stem cells (GSC) and the markedly inflammatory microenvironment. To further explore the involvement of COX-2 in glioma biology, the effects of NS398, a selective COX-2 inhibitor, were evaluated on GSC derived from COX-2 expressing GBM cell lines, i.e., U87MG and T98G, in terms of neurospheres' growth, autophagy, and extracellular vesicle (EV) release. METHODS: Neurospheres' growth and morphology were evaluated by optical and scanning electron microscopy. Autophagy was measured by staining acidic vesicular organelles. Extracellular vesicles (EV), released from neurospheres, were analyzed by transmission electron microscopy. The autophagic proteins Beclin-1 and LC3B, as well as the EV markers CD63 and CD81, were analyzed by western blotting. The scratch assay test was used to evaluate the NS398 influence on GBM cell migration. RESULTS: Both cell lines were strongly influenced by NS398 exposure, as showed by morphological changes, reduced growth rate, and appearance of autophagy. Furthermore, the inhibitor led to a functional change of EV released by neurospheres. Indeed, EV secreted by NS398-treated GSC, but not those from control cells, were able to significantly inhibit adherent U87MG and T98G cell migration and induced autophagy in recipient cells, thus leading to effects quite similar to those directly caused by NS398 in the same cells. CONCLUSION: Despite the intrinsic diversity and individual genetic features of U87MG and T98G, comparable effects were exerted by the COX-2 inhibitor NS398 on both GBM cell lines. Overall, our findings support the crucial role of the inflammatory-associated COX-2/PGE2 system in glioma and glioma stem cell biology.

Secretome of Adipose Tissue-Derived Stem Cells (ASCs) as a Novel Trend in Chronic Non-Healing Wounds: An Overview of Experimental In Vitro and In Vivo Studies and Methodological Variables.

Wound healing is a complex process with a linear development that involves many actors in a multistep timeline commonly divided into four stages: Hemostasis, inflammation, proliferation, and remodeling. Chronic non-healing wounds fail to progress beyond the inflammatory phase, thus precluding the next steps and, ultimately, wound repair. Many intrinsic or extrinsic factors may contribute to such an occurrence, including patient health conditions, age-related diseases, metabolic deficiencies, advanced age, mechanical pressure, and infections. Great interest is being focused on the adipose tissue-derived stem cell's (ASC) paracrine activity for its potential therapeutic impact on chronic non-healing wounds. In this review, we summarize the results of in vitro and in vivo experimental studies on the pro-wound healing effects of ASC-secretome and/or extracellular vesicles (EVs). To define an overall picture of the available literature data, experimental conditions and applied methodologies are described as well as the in vitro and in vivo models chosen in the reported studies. Even if a comparative analysis of the results obtained by the different groups is challenging due to the large variability of experimental conditions, the available findings are undoubtedly encouraging and fully support the use of cell-free therapies for the treatment of chronic non-healing wounds.

NOS2 inhibitor 1400W Induces Autophagic Flux and Influences Extracellular Vesicle Profile in Human Glioblastoma U87MG Cell Line.

The relevance of nitric oxide synthase 2 (NOS2) as a prognostic factor in Glioblastoma Multiforme (GBM) malignancy is emerging. We analyzed the effect of NOS2 inhibitor 1400W on the autophagic flux and extracellular vesicle (EV) secretion in U87MG glioma cells. The effects of glioma stem cells (GSC)-derived EVs on adherent U87MG were evaluated. Cell proliferation and migration were examined while using Cell Counting Kit-8 assay (CCK-8) and scratch wound healing assay. Cell cycle profile and apoptosis were analyzed by flow cytometry. Autophagy-associated acidic vesicular organelles were detected and quantified by acridine orange staining. The number and size of EVs were assessed by nanoparticle tracking analysis. EV ultrastructure was verified by transmission electron microscopy (TEM). WB was used to analyze protein expression and acid sphingomyelinase was determined through ceramide levels. 1400W induced autophagy and EV secretion in both adherent U87MG and GSCs. EVs secreted by 1400W-treated GSC, but not those from untreated cells, were able to inhibit adherent U87MG cell growth and migration while also inducing a relevant level of autophagy. The hypothesis of NOS2 expression as GBM profile marker or interesting therapeutic target is supported by our findings. Autophagy and EV release following treatment with the NOS2 inhibitor could represent useful elements to better understand the complex biomolecular frame of GBM.

Methods of Isolation, Characterization and Expansion of Human Adipose-Derived Stem Cells (ASCs): An Overview.

Considering the increasing interest in adipose-derived stem cells (ASCs) in regenerative medicine, optimization of methods aimed at isolation, characterization, expansion and evaluation of differentiation potential is critical to ensure (a) the quality of stem cells also in terms of genetic stability; (b) the reproducibility of beneficial effects; and (c) the safety of their use. Numerous studies have been conducted to understand the mechanisms that regulate ASC proliferation, growth and differentiation, however standard protocols about harvesting and processing techniques are not yet defined. It is also important to note that some steps in the procedures of harvesting and/or processing have been reported to affect recovery and/or the physiology of ASCs. Even considering the great opportunity that the ASCs provide for the identification of novel molecular targets for new or old drugs, the definition of homogeneous preparation methods that ensure adequate quality assurance and control, in accordance with current GMPs (good manufacturing practices), is required. Here, we summarize the literature reports to provide a detailed overview of the methodological issues underlying human ASCs isolation, processing, characterization, expansion, differentiation techniques, recalling at the same time their basilar principles, advantages and limits, in particular focusing on how these procedures could affect the ASC quality, functionality and plasticity.

Involvement of NOS2 Activity on Human Glioma Cell Growth, Clonogenic Potential, and Neurosphere Generation.

Aberrant nitric oxide synthase 2 (NOS2) expression has been suggested as an interesting therapeutic target that is being implicated as a component of the molecular profile of several human malignant tumors, including glioblastoma, which is the most aggressive brain tumor with limited therapeutic options and poor prognosis. The aim of the present work was to evaluate the effect of 1400W, a specific NOS2 inhibitor, on human glioma cells in terms of clonogenic potential, proliferation, migration rate, and neurosphere generation ability. NOS2 expression was determined by Western blotting. Nitric oxide (NO) production was measured through nitrite level determination. The trypan blue exclusion test and the plate colony formation assay were performed to evaluate cell proliferation and clonogenic potential. Cell proliferation and migration ability was assessed by the in vitro wound-healing assay. Neurosphere generation in a specific stemcell medium was investigated. NOS2 was confirmed to be expressed in both the glioma cell line and a human glioma primary culture, and overexpressed in relative derived neurospheres. Experiments that aimed to evaluate the influence of 1400W on U-87 MG, T98G (glioblastoma cell lines) and primary glioma cells sustained the crucial role played by NOS2 in proliferation, colony formation, migration, and neurosphere generation, thus supporting the emerging relevance of a NOS2/NO system as a prognostic factor for glioma malignancy and recurrence.

VSL#3 probiotic differently influences IEC-6 intestinal epithelial cell status and function.

The data here reported introduce the wound-healing assay as a tool for testing probiotics aimed at protecting gastrointestinal mucosal surfaces and to verify the consistency of their manufacturing. At the scope, we compared the in vitro effects of two multi-strain high concentration formulations both commercialized under the same brand VSL#3 but sourced from different production sites (USA and Italy) on a non-transformed small-intestinal epithelial cell line, IEC-6. The effects on cellular morphology, viability, migration, and H2 O2 -induced damage, were assessed before and after the treatment with both VSL#3 formulations. While the USA-sourced product ("USA-made") VSL#3 did not affect monolayer morphology and cellular density, the addition of bacteria from the Italy-derived product ("Italy-made") VSL#3 caused clear morphological cell damage and strongly reduced cellularity. The treatment with "USA-made" lysate led to a higher rate of wounded monolayer healing, while the addition of "Italy-made" bacterial lysate did not influence the closure rate as compared to untreated cells. While lysates from "USA-made" VSL#3 clearly enhanced the formation of elongated and aligned stress fibers, "Italy-made" lysates had not similar effect. "USA-made" lysate was able to cause a total inhibition of H2 O2 -induced cytotoxic effect whereas "Italy-made" VSL#3 lysate was unable to protect IEC-6 cells from H2 O2 -induced damage. ROS generation was also differently influenced, thus supporting the hypotesis of a protective action of "USA-made" VSL#3 lysates, as well as the idea that "Italy-made" formulation was unable to prevent significantly the H2 O2 -induced oxidative stress.

PPARα Antagonist AA452 Triggers Metabolic Reprogramming and Increases Sensitivity to Radiation Therapy in Human Glioblastoma Primary Cells.

Glioblastoma (GB) is the most common cancer in the brain and with an increasing incidence. Despite major advances in the field, there is no curative therapy for GB to date. Many solid tumors, including GB, experienced metabolic reprogramming in order to sustain uncontrolled proliferation, hypoxic conditions, and angiogenesis. PPARs, member of the steroid hormone receptor superfamily, are particularly involved in the control of energetic metabolism, particularly lipid metabolism, which has been reported deregulated in gliomas. PPARα was previously indicated by us as a potential therapeutic target for this neoplasm, due to the malignancy grade dependency of its expression, being particularly abundant in GB. In this work, we used a new PPARα antagonist on patient-derived GB primary cells, with particular focus on the effects on lipid metabolism and response to radiotherapy. The results obtained demonstrated that blocking PPARα results in cell death induction, increase of radiosensitivity, and decrease of migration. Therefore, AA452 is proposed as a new adjuvant for the gold standard therapies for GB, opening the possibility for preclinical and clinical trials for this class of compounds. J. Cell. Physiol. 232: 1458-1466, 2017. © 2016 Wiley Periodicals, Inc.

Flavopiridol: An Old Drug With New Perspectives? Implication for Development of New Drugs.

Glioblastoma, the most common brain tumor, is characterized by high proliferation rate, invasion, angiogenesis, and chemo- and radio-resistance. One of most remarkable feature of glioblastoma is the switch toward a glycolytic energetic metabolism that leads to high glucose uptake and consumption and a strong production of lactate. Activation of several oncogene pathways like Akt, c-myc, and ras induces glycolysis and angiogenesis and acts to assure glycolysis prosecution, tumor proliferation, and resistance to therapy. Therefore, the high glycolytic flux depends on the overexpression of glycolysis-related genes resulting in an overproduction of pyruvate and lactate. Metabolism of glioblastoma thus represents a key issue for cancer research. Flavopiridol is a synthetic flavonoid that inhibits a wide range of Cyclin-dependent kinase, that has been demonstrate to inactivate glycogen phosphorylase, decreasing glucose availability for glycolysis. In this work the study of glucose metabolism upon flavopiridol treatment in the two different glioblastoma cell lines. The results obtained point towards an effect of flavopiridol in glycolytic cells, thus suggesting a possible new use of this compound or flavopiridol-derived formulations in combination with anti-proliferative agents in glioblastoma patients. J. Cell. Physiol. 232: 312-322, 2017. © 2016 Wiley Periodicals, Inc.

Increase of Intracellular Cyclic AMP by PDE4 Inhibitors Affects HepG2 Cell Cycle Progression and Survival.

Type 4 cyclic nucleotide phosphodiesterases (PDE4) are major members of a superfamily of enzymes (PDE) involved in modulation of intracellular signaling mediated by cAMP. Broadly expressed in most human tissues and present in large amounts in the liver, PDEs have in the last decade been key therapeutic targets for several inflammatory diseases. Recently, a significant body of work has underscored their involvement in different kinds of cancer, but with no attention paid to liver cancer. The present study investigated the effects of two PDE4 inhibitors, rolipram and DC-TA-46, on the growth of human hepatoma HepG2 cells. Treatment with these inhibitors caused a marked increase of intracellular cAMP level and a dose- and time-dependent effect on cell growth. The concentrations of inhibitors that halved cell proliferation to about 50% were used for cell cycle experiments. Rolipram (10 µM) and DC-TA-46 (0.5 µM) produced a decrease of cyclin expression, in particular of cyclin A, as well as an increase in p21, p27 and p53, as evaluated by Western blot analysis. Changes in the intracellular localization of cyclin D1 were also observed after treatments. In addition, both inhibitors caused apoptosis, as demonstrated by an Annexin-V cytofluorimetric assay and analysis of caspase-3/7 activity. Results demonstrated that treatment with PDE4 inhibitors affected HepG2 cell cycle and survival, suggesting that they might be useful as potential adjuvant, chemotherapeutic or chemopreventive agents in hepatocellular carcinoma. J. Cell. Biochem. 118: 1401-1411, 2017. © 2016 Wiley Periodicals, Inc.

Nitric Oxide Chemical Donor Affects the Early Phases of In Vitro Wound Healing Process.

An artificial wound in a confluent monolayer of human keratinocyte HaCaT cells or mouse embryo fibroblast Swiss NIH 3T3 cells was used to analyze the effects of the nitric oxide (NO) chemical donor, S-nitroso-N-acetylpenicillamine (SNAP). SNAP exposure promoted an enhanced rate of wound closure and accelerated motility of both keratinocytes and fibroblasts compared to control cells. The wounded monolayer cultures of HaCaT and NIH 3T3 cells, treated with or without SNAP, were monitored under a phase contrast microscope. Structural and ultrastructural modifications were analyzed by scanning electron microscopy (SEM). The images were captured by a digital camera at different time points (0-28 h) and the wound area was analyzed through software included in Matlab®. As early as 15 min, SNAP induced significant cytoskeletal remodeling, as shown by immunostaining (phalloidin-labelling), which in turn was associated with increased filopodium number and length rise. NO donor treatment also induced overexpression of Ki-67 protein, a typical marker of cell proliferation, as shown by immunostaining. Both SNAP-induced migration and proliferation were antagonized by the NO-sensitive GC inhibitor 1H-[1,2,4]oxadiazolo[-4,3-a]quinoxalin-1-one (ODQ), which suggests activation of the NO/cGMP signalling cascade in the observed SNAP-induced effects in the early stages of the healing process. Moreover, we provide evidence that PPAR-ß antagonist (GSK0660) may interfere with NO-mediated wound healing process. J. Cell. Physiol. 231: 2185-2195, 2016. © 2016 Wiley Periodicals, Inc.

The PPARß/δ Agonist GW0742 Induces Early Neuronal Maturation of Cortical Post-Mitotic Neurons: Role of PPARß/δ in Neuronal Maturation.

Increasing evidences support that signaling lipids participate in synaptic plasticity and cell survival, and that the lipid signaling is closely associated with neuronal differentiation, learning, and memory and with pathologic events, such as epilepsy and Alzheimer's disease. The Peroxisome Proliferator-Activated Receptors (PPAR) are strongly involved in the fatty acid cell signaling, as many of the natural lypophylic compounds are PPAR ligands. We have previously shown that PPARß/δ is the main isotype present in cortical neuron primary cultures and that during neuronal maturation, PPARß/δ is gradually increased and activated. To get more insight into the molecular mechanism by which PPARß/δ may be involved in neuronal maturation processes, in this work a specific PPARß/δ agonist, GW0742 was used administered alone or in association with a specific PPARß/δ antagonist, the GSK0660, and the parameters involved in neuronal differentiation and maturation were assayed. The data obtained demonstrated the strong involvement of PPARß/δ in neuronal maturation, triggering the agonist an anticipation of neuronal differentiation, and the antagonist abolishing the observed effects. These effects appear to be mediated by the activation of BDNF pathway.

Improving the outcome of fractional CO2 laser resurfacing using a probiotic skin cream: Preliminary clinical evaluation.

As known, fractional CO2 resurfacing treatments are more effective than non-ablative ones against aging signs, but post-operative redness and swelling prolong the overall downtime requiring up to steroid administration in order to reduce these local systems. In the last years, an increasing interest has been focused on the possible use of probiotics for treating inflammatory and allergic conditions suggesting that they can exert profound beneficial effects on skin homeostasis. In this work, the Authors report their experience on fractional CO2 laser resurfacing and provide the results of a new post-operative topical treatment with an experimental cream containing probiotic-derived active principles potentially able to modulate the inflammatory reaction associated to laser-treatment. The cream containing DermaACB (CERABEST™) was administered post-operatively to 42 consecutive patients who were treated with fractional CO2 laser. All patients adopted the cream twice a day for 2 weeks. Grades were given according to outcome scale. The efficacy of the cream containing DermaACB was evaluated comparing the rate of post-operative signs vanishing with a control group of 20 patients topically treated with an antibiotic cream and a hyaluronic acid based cream. Results registered with the experimental treatment were good in 22 patients, moderate in 17, and poor in 3 cases. Patients using the study cream took an average time of 14.3 days for erythema resolution and 9.3 days for swelling vanishing. The post-operative administration of the cream containing DermaACB induces a quicker reduction of post-operative erythema and swelling when compared to a standard treatment.

In vitro evaluation of different methods of handling human liposuction aspirate and their effect on adipocytes and adipose derived stem cells.

Nowadays, fat tissue transplantation is widely used in regenerative and reconstructive surgery. However, a shared method of lipoaspirate handling for ensuring a good quality fat transplant has not yet been established. The study was to identify a method to recover from the lipoaspirate samples the highest number of human viable adipose tissue-derived stem cells (hADSCs) included in stromal vascular fraction (SVF) cells and of adipocytes suitable for transplantation, avoiding an extreme handling. We compared the lipoaspirate spontaneous stratification (10-20-30 min) with the centrifugation technique at different speeds (90-400-1500 × g). After each procedure, lipoaspirate was separated into top oily lipid layer, liquid fraction, "middle layer", and bottom layer. We assessed the number of both adipocytes in the middle layer and SVF cells in all layers. The histology of middle layer and the surface phenotype of SVF cells by stemness markers (CD105+, CD90+, CD45-) was analyzed as well. The results showed a normal architecture in all conditions except for samples centrifuged at 1500 × g. In both methods, the flow cytometry analysis showed that greater number of ADSCs was in middle layer; in the fluid portion and in bottom layer was not revealed significant expression levels of stemness markers. Our findings indicate that spontaneous stratification at 20 min and centrifugation at 400 × g are efficient approaches to obtain highly viable ADSCs cells and adipocytes, ensuring a good thickness of lipoaspirate for autologous fat transfer. Since an important aspect of surgery practice consists of gain time, the 400 × g centrifugation could be the recommended method when the necessary instrumentation is available.

Immunophenotypic characterization of human glioblastoma stem cells: correlation with clinical outcome.

Recently, glioma stem cells have been identified as the main cause of glioma propagation and recurrence and a number of several cell markers have been indicated as putative GSC markers. In the present work, a retrospective study to evaluate the prognostic potential of ability to generate GSCs in our series of 15 glioblastoma patients is described. ß-tubulin III, nestin, CD133, GFAP, and SOX-2 marker expression, both in primary GBM cultures and in respective glioblastoma stem cells (GSCs), was evaluated by flow cytometric analysis. Our results demonstrated various expression levels of these markers in both cell cultures; of note, only those cells expressing SOX-2 at greater than 30% levels were able to produce in vitro neurospheres. Moreover, statistical analysis revealed that the GSCs generation negatively affected overall survival (OS) (P = 0.000) and progression-free survival (PFS) (P = 0.001). In addition, a very poor OS (P = 0.000) and PFS (P = 0.000) were observed among patients whose tumors expressed Ki67, evaluated by immunohistochemistry, and showed the ability to generate in vitro GSCs. Overall, the results suggest that in vitro GSCs generation associated to the expression of Ki67 and SOX-2 may be useful to identify patients at risk of disease progression.

Involvement of Cyclooxygenase-2 in Establishing an Immunosuppressive Microenvironment in Tumorspheres Derived from TMZ-Resistant Glioblastoma Cell Lines and Primary Cultures.

Glioblastoma (GBM) is characterized by an immunosuppressive tumor microenvironment (TME) strictly associated with therapy resistance. Cyclooxygenase-2 (COX-2) fuels GBM proliferation, stemness, and chemoresistance. We previously reported that COX-2 upregulation induced by temozolomide (TMZ) supported chemoresistance. Also, COX-2 transfer by extracellular vesicles released by T98G promoted M2 polarization in macrophages, whereas COX-2 inhibition counteracted these effects. Here, we investigated the COX-2 role in the stemness potential and modulation of the GBM immunosuppressive microenvironment. The presence of macrophages U937 within tumorspheres derived from GBM cell lines and primary cultures exposed to celecoxib (COX-2 inhibitor) with or without TMZ was studied by confocal microscopy. M2 polarization was analyzed by TGFß-1 and CD206 levels. Osteopontin (OPN), a crucial player within the TME by driving the macrophages' infiltration, and CD44 expression was assessed by Western blot. TMZ strongly enhanced tumorsphere size and induced the M2 polarization of infiltrating macrophages. In macrophage-infiltrated tumorspheres, TMZ upregulated OPN and CD44 expression. These TMZ effects were counteracted by the concurrent addition of CXB. Remarkably, exogenous prostaglandin-E2 restored OPN and CD44, highlighting the COX-2 pivotal role in the protumor macrophages' state promotion. COX-2 inhibition interfered with TMZ's ability to induce M2-polarization and counteracted the development of an immunosuppressive TME.

Cognitive behavioral therapy for young people after l'aquila earthquake.

OBJECTIVE: Cognitive behavior therapy (CBT) emerges as the best validated therapeutic approach for children and adolescents who experienced trauma-related symptoms, particularly associated with anxiety or mood disorders. The aim of this study was to evaluate the CBT efficacy among young people exposed to L'Aquila earthquake, in 2009. METHODS: one year after the disaster, 39 young subjects as a case group (CBT treated) and 24 as a comparison group (no CBT treated) were evaluated with the Impact of Event Scale Revised (IES-R), the General Health Questionnaire-12 items (GHQ-12) and the Brief Cope. CBT was conducted in 12 sessions (once per week for 3 months). After CBT intervention, both groups were evaluated again with the same psychometric instruments. RESULTS: our results show a significantly decrease in post traumatic symptoms and psychological distress severity in CBT group. It was attributable to an improvement in each of three PTSD dimensions (intrusion, avoidance, and arousal) and in the total score of IES-R (p< 0.04). Among CBT treated group, subjects that adopted "planning/problem solving" coping strategies (p < .02) and "religiosity" (p < .045) show higher improvement in psychological distress. CONCLUSIONS: our findings show the efficacy of CBT and the influence of individual coping strategies in the improvement of posttraumatic stress symptoms and psychological distress among young people seeking help from an outpatients service for young people with psychiatric problems (the SMILE) after the catastrophic disaster in L'Aquila.