Protective effects of fatty acid amide hydrolase inhibition in UVB-activated microglia.

Neuroinflammation is a hallmark of several neurodegenerative disorders that has been extensively studied in recent years. Microglia, the primary immune cells of the central nervous system (CNS), are key players in this physiological process, demonstrating a remarkable adaptability in responding to various stimuli in the eye and the brain. Within the complex network of neuroinflammatory signals, the fatty acid N-ethanolamines, in particular N-arachidonylethanolamine (anandamide, AEA), emerged as crucial regulators of microglial activity under both physiological and pathological states. In this study, we interrogated for the first time the impact of the signaling of these bioactive lipids on microglial cell responses to a sub-lethal acute UVB radiation, a physical stressor responsible of microglia reactivity in either the retina or the brain. To this end, we developed an in vitro model using mouse microglial BV-2 cells. Upon 24 h of UVB exposure, BV-2 cells showed elevated oxidative stress markers and, cyclooxygenase (COX-2) expression, enhanced phagocytic and chemotactic activities, along with an altered immune profiling. Notably, UVB exposure led to a selective increase in expression and activity of fatty acid amide hydrolase (FAAH), the main enzyme responsible for degradation of fatty acid ethanolamides. Pharmacological FAAH inhibition via URB597 counteracted the effects of UVB exposure, decreasing tumor necrosis factor α (TNF-α) and nitric oxide (NO) release and reverting reactive oxidative species (ROS), interleukin-1ß (IL-1ß), and interleukin-10 (IL-10) levels to the control levels. Our findings support the potential of enhanced fatty acid amide signaling in mitigating UVB-induced cellular damage, paving the way to further exploration of these lipids in light-induced immune responses.

Efficacy of the Probiotic L. brevis in Counteracting the Demineralizing Process of the Tooth Enamel Surface: Results from an In Vitro Study.

BACKGROUND: Enamel plays an essential role in protecting the underlying layers of the human tooth; therefore, preserving it is vital. This experimental study aimed to evaluate the potential ability of L. brevis to counteract the action of a demineralizing agent on dental enamel morphology and mineral composition in vitro. METHODS: The sample consisted of 12 healthy human posterior teeth. The coronal portion of each tooth was subdivided into two equal parts longitudinally. The specimens were randomly divided into four groups: artificial saliva, L. brevis suspension, demineralizing agent (DA), and DA plus L. brevis. Scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) were used to evaluate the surface micromorphology and the mineral content, respectively. The statistical analysis was conducted using a one-way ANOVA, followed by Tukey's post hoc test. RESULTS: SEM analysis did not highlight significant changes in the enamel microstructure of L. brevis-treated specimens compared to the control. DA-induced damage to the enamel structure was drastically reduced when the specimens were contextually exposed to the probiotic. The treatment with DA substantially reduced the weight % of crucial enamel minerals, i.e., Ca and P. Notably, the probiotic was able to reverse the demineralization process, bringing Ca and P weight % back to basal levels, including the Ca/P ratio. CONCLUSIONS: The findings indicate that L. brevis is able to efficiently protect the dental enamel surface from the damage caused by DA and increase the enamel resistance to demineralization. Overall, L. brevis confirms its efficacy in preventing or counteracting the action of carious lesions through a novel mechanism that protects the tooth surface under a chemical challenge that mimics the caries process.

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.

Evaluation of the Effectiveness of an Innovative Polycomponent Formulation on Adult and Aged Human Dermal Fibroblasts.

Skin aging is a dynamic process that determines structural alterations in ECM and reduction in dermal fibroblasts. The recent availability on the market of an innovative polycomponent formulation (KARISMA Rh Collagen® FACE, K) containing noncrosslinked high-molecular-weight hyaluronic acid (HMW-HA), a human recombinant polypeptide of collagen-1 alpha chain, and carboxymethyl cellulose (CMC), attracted our scientific interest in evaluating its biomolecular effects on human dermal adult and aged fibroblasts. After treatment with increasing K concentrations, cell proliferation, collagen I, prolyl 4-hydroxylase (P4HA1), an essential protein in collagen biosynthesis, and α-SMA levels were assessed. The fibroblast contractility, TGF-ß1 levels, and oxidative stress markers were also evaluated. K formulation exposure led to a significant and dose-dependent increase in the proliferation and migration of adult fibroblasts. Of note, the K exposure counteracted the H2O2-induced aging by promoting cell proliferation, reducing ß-galactosidase activity, and neutralizing the aging-associated oxidative damage. Moreover, an increase in collagen I, P4HA1, α-SMA, TGF-ß1 levels, and improved contractility of adult and aged fibroblasts were observed after treatment. Overall, our results show evidence that the K treatment is efficacious in improving biological functions in adult fibroblasts and suppressing the biomolecular events associated with H2O2-induced cellular aging, thus supporting the regenerative and bio-revitalizing action of the K formulation helpful in preventing or treating skin aging.

Oxidative Stress, Cytotoxic and Inflammatory Effects of Azoles Combinatorial Mixtures in Sertoli TM4 Cells.

Triazole and imidazole fungicides are an emerging class of contaminants with an increasing and ubiquitous presence in the environment. In mammals, their reproductive toxicity has been reported. Concerning male reproduction, a combinatorial activity of tebuconazole (TEB; triazole fungicide) and econazole (ECO; imidazole compound) in inducing mitochondrial impairment, energy depletion, cell cycle arrest, and the sequential activation of autophagy and apoptosis in Sertoli TM4 cells (SCs) has recently been demonstrated. Given the strict relationship between mitochondrial activity and reactive oxygen species (ROS), and the causative role of oxidative stress (OS) in male reproductive dysfunction, the individual and combined potential of TEB and ECO in inducing redox status alterations and OS was investigated. Furthermore, considering the impact of cyclooxygenase (COX)-2 and tumor necrosis factor-alpha (TNF-α) in modulating male fertility, protein expression levels were assessed. In the present study, we demonstrate that azoles-induced cytotoxicity is associated with a significant increase in ROS production, a drastic reduction in superoxide dismutase (SOD) and GSH-S-transferase activity levels, and a marked increase in the levels of oxidized (GSSG) glutathione. Exposure to azoles also induced COX-2 expression and increased TNF-α production. Furthermore, pre-treatment with N-acetylcysteine (NAC) mitigates ROS accumulation, attenuates COX-2 expression and TNF-α production, and rescues SCs from azole-induced apoptosis, suggesting a ROS-dependent molecular mechanism underlying the azole-induced cytotoxicity.

Donkey Colostrum and Milk: How Dietary Probiotics Can Affect Metabolomic Profile, Alkaline Sphingomyelinase and Alkaline Phosphatase Activity.

Positive results on animal health, feed efficiency, and milk's nutritional content have been obtained after oral administration of probiotics. The aim of the present study was therefore to evaluate the effect of dietary supplementation with high numbers of multispecies probiotic formulations on the milk metabolomic profiles of alkaline sphingomyelinase (alk-SMase) and alkaline phosphatase (ALP) in donkeys. Twenty animals were randomly allocated to receive either a normal diet (group B) or a supplemented diet (group A). Colostrum and milk samples were obtained within 48 h, at 15 days (supplementation start), and at 45 days after parturition. Different metabolomic profiles were observed between colostrum and milk, as were the concentrations of 12 metabolites that changed following 30 days of probiotic supplementation. Alk-SMase activity was found to be higher in donkey colostrum (vs. milk at 15 days); this enzyme, together with ALP, increased in milk after 30 days of probiotic supplementation. The results of the present study provide new insight into the complex changes in donkey colostrum and milk composition in the first 45 days of lactation and how the milk metabolome can be modulated by probiotic supplementation.

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.

New Insights into Dose-Dependent Effects of Curcumin on ARPE-19 Cells.

Opposing dose-dependent effects of curcumin (Cur) have been documented in Retinal Pigment Epithelium (RPE); therefore, to shed the light on the mechanisms of action is crucial for ophthalmic applications. On this basis we explored new insights about the dose-dependent mechanisms triggered by Cur in human retinal pigment epithelial cells (ARPE-19). Three concentrations (0.01 mM; 0.05 mM; 0.1 mM) of Cur were tested, followed by morphological, molecular, and functional analysis of the cells. Cur 0.01 mM promotes a significant increase in cell proliferation, not affecting cell cycle progression and apoptosis; by contrast, Cur 0.05 mM and 0.1 mM block cellular proliferation and trigger S-phase cell cycle arrest without inducing apoptosis. The observation of neuronal-like morphological changes in Cur 0.05 mM and 0.1 mM were not associated with neuronal differentiation, as observed by the quantification of Neurofilament-200 and by the analysis of voltage-dependent currents by patch clamp. Evaluation of autophagic markers LC3BII and p62 revealed significant modulations, suggesting an important activation of autophagy in ARPE-19 cells treated with Cur 0.05 mM and Cur 0.1 mM; conversely, Cur 0.01 mM did not affect autophagy. Altogether, our findings show new dose-dependent mechanisms of action of Cur that suggest a wide therapeutic application in ocular diseases with different pathogenesis (i.e., proliferative vitreoretinopathy or Age-Related Macular Degeneration).

Efficacy of probiotic Streptococcus thermophilus in counteracting TGF-ß1-induced fibrotic response in normal human dermal fibroblasts.

BACKGROUND: Abnormal and deregulated skin wound healing associated with prolonged inflammation may result in dermal fibrosis. Since the current therapeutic strategies revealed unsatisfactory, the investigation of alternative approaches such as those based on the use of specific probiotic strains could provide promising therapeutic options. In this study, we aimed to evaluate whether the lysate from S. thermophilus could antagonize the fibrogenic effects of TGF-ß1 in normal human dermal fibroblasts (NHDF). METHODS: NHDF were exposed to TGF-ß1 to establish a fibrotic phenotype. Proliferation rate and cell number were measured using the IncuCyte® Live Cell Imager system and the trypan blue dye exclusion test. Phenoconversion markers (α-SMA and fibronectin) and collagen I levels were assessed by western blot and immunofluorescence. The mRNA levels of TGF-ß1 were evaluated by RT-PCR. The Smad2/3 phosphorylation level as well as ß-catenin and PPARγ expression, were assessed by western blot. The cell contractility function and migration of NHDF were studied using collagen gel retraction assay, and scratch wound healing assay, respectively. The effects of S. thermophilus lysate, alone or combined with TGF-ß1, were evaluated on all of the above-listed parameters and markers associated with TGF-ß1-induced fibrotic phenotype. RESULTS: Exposure to the S. thermophilus lysate significantly reduced the key mediators and events involved in the abnormal activation of myofibroblasts by TGF-ß1 within the fibrotic profile. The S. thermophilus treatment significantly reduced cell proliferation, migration, and myo-differentiation. In addition, the treatment with probiotic lysate reduced the α-SMA, fibronectin, collagen-I expression levels, and affected the collagen contraction ability of activated dermal fibroblasts. Moreover, the probiotic targeted the TGF-ß1 signaling, reducing Smad2/3 activation, TGF-ß1 mRNA level, and ß-catenin expression through the upregulation of PPARγ. CONCLUSION: This is the first report showing that S. thermophilus lysate had a remarkable anti-fibrotic effect in TGF-ß1-activated NHDF by inhibiting Smad signaling. Notably, the probiotic was able to reduce ß-catenin and increase PPARγ levels. The findings support our point that S. thermophilus may help prevent or treat hypertrophic scarring and keloids.

Cyclooxygenase-2 Upregulated by Temozolomide in Glioblastoma Cells Is Shuttled In Extracellular Vesicles Modifying Recipient Cell Phenotype.

Temozolomide (TMZ) resistance is frequent in patients with glioblastoma (GBM), a tumor characterized by a marked inflammatory microenvironment. Recently, we reported that cyclooxygenase-2 (COX-2) is upregulated in TMZ-resistant GBM cells treated with high TMZ concentrations. Moreover, COX-2 activity inhibition significantly counteracted TMZ-resistance of GBM cells. Extracellular vesicles (EV) are considered crucial mediators in orchestrating GBM drug resistance by modulating the tumor microenvironment (TME) and affecting the surrounding recipient cell phenotype and behavior. This work aimed to verify whether TMZ, at low and clinically relevant doses (5-20 µM), could induce COX-2 overexpression in GBM cells (T98G and U87MG) and explore if secreted EV shuttled COX-2 to recipient cells. The effect of COX-2 inhibitors (COXIB), Celecoxib (CXB), or NS398, alone or TMZ-combined, was also investigated. Our results indicated that TMZ at clinically relevant doses upregulated COX-2 in GBM cells. COXIB treatment significantly counteracted TMZ-induced COX-2 expression, confirming the crucial role of the COX-2/PGE2 system in TMZ-resistance. The COXIB specificity was verified on U251MG, COX-2 null GBM cells. Western blotting of GBM-EV cells showed the COX-2 presence, with the same intracellular trend, increasing in EV derived from TMZ-treated cells and decreasing in those derived from COXIB+TMZ-treated cells. We then evaluated the effect of EV secreted by TMZ-treated cells on U937 and U251MG, used as recipient cells. In human macrophage cell line U937, the internalization of EV derived by TMZ-T98G cells led to a shift versus a pro-tumor M2-like phenotype. On the other hand, EV from TMZ-T98G induced a significant decrease in TMZ sensitivity in U251MG cells. Overall, our results, in confirming the crucial role played by COX-2 in TMZ-resistance, provide the first evidence of the presence and effective functional transfer of this enzyme through EV derived from GBM cells, with multiple potential consequences at the level of TME.

Synergistic Activity of Ketoconazole and Miconazole with Prochloraz in Inducing Oxidative Stress, GSH Depletion, Mitochondrial Dysfunction, and Apoptosis in Mouse Sertoli TM4 Cells.

Triazole and imidazole fungicides represent an emerging class of pollutants with endocrine-disrupting properties. Concerning mammalian reproduction, a possible causative role of antifungal compounds in inducing toxicity has been reported, although currently, there is little evidence about potential cooperative toxic effects. Toxicant-induced oxidative stress (OS) may be an important mechanism potentially involved in male reproductive dysfunction. Thus, to clarify the molecular mechanism underlying the effects of azoles on male reproduction, the individual and combined potential of fluconazole (FCZ), prochloraz (PCZ), miconazole (MCZ), and ketoconazole (KCZ) in triggering in vitro toxicity, redox status alterations, and OS in mouse TM4 Sertoli cells (SCs) was investigated. In the present study, we demonstrate that KCZ and MCZ, alone or in synergistic combination with PCZ, strongly impair SC functions, and this event is, at least in part, ascribed to OS. In particular, azoles-induced cytotoxicity is associated with growth inhibitory effects, G0/G1 cell cycle arrest, mitochondrial dysfunction, reactive oxygen species (ROS) generation, imbalance of the superoxide dismutase (SOD) specific activity, glutathione (GSH) depletion, and apoptosis. N-acetylcysteine (NAC) inhibits ROS accumulation and rescues SCs from azole-induced apoptosis. PCZ alone exhibits only cytostatic and pro-oxidant properties, while FCZ, either individually or in combination, shows no cytotoxic effects up to 320 µM.

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.

Soluble Fraction from Lysate of a High Concentration Multi-Strain Probiotic Formulation Inhibits TGF-ß1-Induced Intestinal Fibrosis on CCD-18Co Cells.

Fibrosis is a severe complication of chronic inflammatory disorders, such as inflammatory bowel disease (IBD). Current strategies are not fully effective in treating fibrosis; therefore, innovative anti-fibrotic approaches are urgently needed. TGF-ß1 plays a central role in the fibrotic process by inducing myofibroblast differentiation and excessive extracellular matrix (ECM) protein deposition. Here, we explored the potential anti-fibrotic impact of two high concentration multi-strain probiotic formulations on TGF-ß1-activated human intestinal colonic myofibroblast CCD-18Co. Human colonic fibroblast CCD-18Co cells were cultured in the presence of TGF-ß1 to develop a fibrotic phenotype. Cell viability and growth were measured using the Trypan Blue dye exclusion test. The collagen-I, α-SMA, and pSmad2/3 expression levels were evaluated by Western blot analysis. Fibrosis markers were also analyzed by immunofluorescence and microscopy. The levels of TGF-ß1 in the culture medium were assessed by ELISA. The effects of commercially available probiotic products VSL#3® and Vivomixx® were evaluated as the soluble fraction of bacterial lysates. The results suggested that the soluble fraction of Vivomixx® formulation, but not VSL#3®, was able to antagonize the pro-fibrotic effects of TGF-ß1 on CCD-18Co cells, being able to prevent all of the cellular and molecular parameters that are related to the fibrotic phenotype. The mechanism underlying the observed effect appeared to be associated with inhibition of the TGF-ß1/Smad signaling pathway. To our knowledge, this study provides the first experimental evidence that Vivomixx® could be considered to be a promising candidate against intestinal fibrosis, being able to antagonize TGF-ß1 pro-fibrotic effects. The differences that were observed in our fibrosis model between the two probiotics used could be attributable to the different number of strains in different proportions.

PPARα-Selective Antagonist GW6471 Inhibits Cell Growth in Breast Cancer Stem Cells Inducing Energy Imbalance and Metabolic Stress.

Breast cancer is the most frequent cancer and the second leading cause of death among women. Triple-negative breast cancer is the most aggressive subtype of breast cancer and is characterized by the absence of hormone receptors and human epithelial growth factor receptor 2. Cancer stem cells (CSCs) represent a small population of tumor cells showing a crucial role in tumor progression, metastasis, recurrence, and drug resistance. The presence of CSCs can explain the failure of conventional therapies to completely eradicate cancer. Thus, to overcome this limit, targeting CSCs may constitute a promising approach for breast cancer treatment, especially in the triple-negative form. To this purpose, we isolated and characterized breast cancer stem cells from a triple-negative breast cancer cell line, MDA-MB-231. The obtained mammospheres were then treated with the specific PPARα antagonist GW6471, after which, glucose, lipid metabolism, and invasiveness were analyzed. Notably, GW6471 reduced cancer stem cell viability, proliferation, and spheroid formation, leading to apoptosis and metabolic impairment. Overall, our findings suggest that GW6471 may be used as a potent adjuvant for gold standard therapies for triple-negative breast cancer, opening the possibility for preclinical and clinical trials for this class of compounds.

Olive leaf extract impairs mitochondria by pro-oxidant activity in MDA-MB-231 and OVCAR-3 cancer cells.

Breast and ovarian cancers are the leading and fifth reason for tumor death among females, respectively. Recently, many studies demonstrated antiproliferative activities of natural aliments in cancer. In this study, we investigated the antitumor potential of Olive Leaf Extract (OLE) in triple-negative breast and ovarian cancer cells. A HPLC/DAD analysis on OLE has been performed to assess the total polyphenolics and other secondary metabolites content. HCEpiC, MDA-MB-231, and OVCAR-3 cell lines were used. MTS, Cytofluorimetric, Western Blot analysis were performed to analyze cell viability, cell proliferation, apoptosis, and oxidative stress. Fluorimetric and IncuCyte® analyses were carried out to evaluate apoptosis and mitochondrial function. We confirmed that OLE, containing a quantity of oleuropein of 87 % of the total extract, shows anti-proliferative and pro-apoptotic activity on MDA-MB-231 cells. For the first time, our results indicate that OLE inhibits OVCAR-3 cell viability inducing cell cycle arrest, and it also increases apoptotic cell death up-regulating the protein level of cleaved-PARP and caspase 9. Moreover, our data show that OLE treatment causes a significant decrease in mitochondrial functionality, paralleled by a reduction of mitochondrial membrane potential. Interestingly, OLE increased the level of intracellular and mitochondrial reactive oxygen species (ROS) together with a decreased activity of ROS scavenging enzymes, confirming oxidative stress in both models. Our data demonstrate that mitochondrial ROS generation represented the primary mechanism of OLE antitumor activity, as pretreatment with antioxidant N-acetylcysteine prevented OLE-induced cell cycle arrest and apoptosis.

Antimycotic Activity of Ozonized Oil in Liposome Eye Drops against Candida spp.

Purpose: This study aims to investigate the antifungal activity and mechanism of action of ozonized oil eye drops in liposomes (Ozodrop), commercialized as eye lubricant for the treatment of dry eye syndrome and eye inflammation. The activity was tested against four clinical Candida species: Calbicans,Cglabrata,Ckrusei, and Corthopsilosis. Methods: The antifungal activity of the eye drop solution was ascertained by microdilution method in accordance with EUCAST obtaining the minimum inhibitory concentration for Ozodrop. The mechanism of action was further investigated in Calbicans by measuring cell vitality, intracellular reactive oxygen species production, levels of cellular and mitochondrial (∆Ψm) membrane potential, and the extent of membrane lipid peroxidation. Results: All Candida isolates were susceptible to Ozodrop with minimum inhibitory concentration values ranging from 0.195% (v/v) for Cglabrata to 6.25% (v/v) for Corthopsilosis. After 1 hour of exposure at the minimum inhibitory concentration value about 30% of cells were killed, reaching about 70% at the highest Ozodrop value. After Ozodrop exposure, Calbicans showed cell membrane depolarization, increased levels of lipid peroxidation, depolarized ∆Ψm, and increased reactive oxygen species generation. Conclusions: The significant increases in reactive oxygen species production cause the accumulation of reactive oxygen species-associated damages leading to progressive Candida cell dysfunction. Translational Relevance: The antifungal activity of Ozodrop was demonstrated at concentrations several times lower than the concentration that can be retrieved in ocular surface after its application. The antifungal activity of the eye drops Ozodrop would represent an interesting off-label indication for a product basically conceived as an eye lubricant.

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.

The Effects of Low-Nickel Diet Combined with Oral Administration of Selected Probiotics on Patients with Systemic Nickel Allergy Syndrome (SNAS) and Gut Dysbiosis.

BACKGROUND: Nickel (Ni) oral consumption may elicit systemic reactions in patients affected by systemic nickel allergy syndrome (SNAS), including gastrointestinal symptoms, which in turn are associated with gut dysbiosis. We evaluated the effects of a low-Ni diet alone or in combination with the oral consumption of appropriate probiotics on Ni-sensitivity and urinary dysbiosis markers in SNAS patients. METHODS: n = 51 patients with SNAS and concomitant intestinal dysbiosis were enrolled in the study. According to the urinary indican/skatole levels, quantified through a colorimetric and a high-performance liquid chromatographic method, respectively, patients were assigned to a dysbiosis type/grade and followed a low-Ni diet for three months. Along with the diet, 22 patients also consumed probiotics based on the dysbiosis type. In particular, a Lactobacilli- or Bifidobacteria-containing formulation was administered to patients with fermentative or putrefactive dysbiosis, respectively, while a broad-spectrum probiotic formulation containing both Lactobacilli and Bifidobacteria was administered to patients with mixed dysbiosis. After three months, patients were invited to repeat the Ni-stimulation and the dysbiosis tests. RESULTS: The fermentative dysbiosis group represented the largest group followed by the mixed dysbiosis group, while only two patients had putrefactive dysbiosis. Overall, at three months of treatment in general (diet alone with or without probiotics), the Ni-sensitivity and dysbiosis levels were strongly ameliorated. The association of a low-Ni diet with a specific probiotic oral supplementation was significantly more effective in decreasing dysbiosis levels or reaching eubiosis than with diet alone. CONCLUSION: Our results, while confirming the benefits of a low-Ni diet in SNAS patients, strongly support that appropriate adjuvant treatment with probiotics significantly helps to improve intestinal dysbiosis or restore a healthy microbiota.

Mucoadhesive Buccal Films for Local Delivery of Lactobacillus brevis.

The aim of this work was to prepare mucoadhesive buccal films for local release of Lactobacillus brevis CD2, which shows interesting anti-inflammatory properties due to its high levels of arginine deiminase. Hydroxypropylmethylcellulose-based films were prepared by means of a modified casting method, which allowed L. brevis CD2 loading on one side of the film, before its complete drying. Three batches of films were prepared, stored at +2-8 °C and +23-25 °C for 48 weeks and characterized in terms of physico-chemical and functional properties. For each batch, the L. brevis viable count and arginine deiminase activity were evaluated at different time points in order to assess functional property maintenance over time. Moreover, the mucoadhesive properties and ability of the films to release L. brevis CD2 were evaluated. A good survival of L. brevis CD2 was observed, particularly at the storage temperature of +2-8 °C, while the activity of arginine deiminase was maintained at both temperature values. Films showed good mucoadhesive properties and guaranteed a prolonged release of viable lactobacilli, which can be directed towards the whole buccal cavity or specific mucosa lesions. In conclusion, the proposed preparative method can be successfully employed for the production of buccal films able to release viable L. brevis CD2 cells that maintain the anti-inflammatory enzymatic activity.