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.

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.

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.

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.

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.

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.

Type I Collagen Suspension Induces Neocollagenesis and Myodifferentiation in Fibroblasts In Vitro.

The ability of a collagen-based matrix to support cell proliferation, migration, and infiltration has been reported; however, the direct effect of an aqueous collagen suspension on cell cultures has not been studied yet. In this work, the effects of a high-concentration aqueous suspension of a micronized type I equine collagen (EC-I) have been evaluated on a normal mouse fibroblast cell line. Immunofluorescence analysis showed the ability of EC-I to induce a significant increase of type I and III collagen levels, parallel with overexpression of crucial proteins in collagen biosynthesis, maturation, and secretion, prolyl 4-hydroxylase (P4H) and heat shock protein 47 (HSP47), as demonstrated by western blot experiments. The treatment led, also, to an increase of α-smooth muscle actin (α-SMA) expression, evaluated through western blot analysis, and cytoskeletal reorganization, as assessed by phalloidin staining. Moreover, scanning electron microscopy analysis highlighted the appearance of plasma membrane extensions and blebbing of extracellular vesicles. Altogether, these results strongly suggest that an aqueous collagen type I suspension is able to induce fibroblast myodifferentiation. Moreover, our findings also support in vitro models as a useful tool to evaluate the effects of a collagen suspension and understand the molecular signaling pathways possibly involved in the effects observed following collagen treatment in vivo.

Soluble Fraction from Lysates of Selected Probiotic Strains Differently Influences Re-Epithelialization of HaCaT Scratched Monolayer through a Mechanism Involving Nitric Oxide Synthase 2.

A growing body of evidence supports the use of probiotics in the treatment of several skin conditions, including wounds. Even if in vitro and in vivo studies have highlighted the pro-healing effects of some probiotic bacteria, the underlying mechanisms are still not fully defined. The current investigation aimed to determine the re-epithelialization potential of the soluble fraction from lysate of seven different probiotic strains belonging to different genera (i.e., Streptococcus, Lactobacillus, and Bifidobacterium) on in vitro physically wounded HaCaT monolayer model. The results suggested that the soluble fraction of S. thermophilus,L. plantarum, and L. acidophilus promoted the re-epithelialization of scratched HaCaT monolayers, whereas those from B. longum,B. infantis, and B. breve significantly inhibited the process. On the other hand, L. bulgaricus showed no significant effect on in vitro wound repair. The mechanisms underlying the pro- or anti-healing properties of selected bacterial strains strictly and positively correlated with their ability to modulate nitric oxide synthase 2 (NOS2) expression and activity. Accordingly, the pre-treatment with aminoguanidine (AG), a specific inhibitor of NOS2 activity, abrogated the pro-healing effects of S. thermophilus, L. plantarum, and L. acidophilus.

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.