Agnese Gugliandolo

Cannabinoid CB2 receptors are involved in the protection of RAW264.7 macrophages against the oxidative stress: an in vitro study

Research in the last decades has widely investigated the anti-oxidant properties of natural products as a therapeutic approach for the prevention and the treatment of oxidative-stress related disorders. In this context, several studies were aimed to evaluate the therapeutic potential of phytocannabinoids, the bioactive compounds of Cannabis sativa. Here, we examined the anti-oxidant ability of Cannabigerol (CBG), a non-psychotropic cannabinoid, still little known, into counteracting the hydrogen peroxide (H2O2)-induced oxidative stress in murine RAW264.7 macrophages. In addition, we tested selective receptor antagonists for cannabinoid receptors and specifically CB1R (SR141716A) and CB2R (AM630) in order to investigate through which CBG may exert its action. Taken together, our in vitro results showed that CBG is able to counteract oxidative stress by activation of CB2 receptors. CB2 antagonist pre-treatment indeed blocked the protective effects of CBG in H2O2 stimulated macrophages, while CB1R was not involved. Specifically, CBG exhibited a potent action in inhibiting oxidative stress, by down-regulation of the main oxidative markers (iNOS, nitrotyrosine and PARP-1), by preventing IκB-α phosphorylation and translocation of the nuclear factor-κB (NF-κB) and also via the modulation of MAP kinases pathway. On the other hand, CBG was found to increase anti-oxidant defense of cells by modulating superoxide dismutase-1 (SOD-1) expression and thus inhibiting cell death (results focused on balance between Bax and Bcl-2). Based on its anti-oxidant activities, CBG may hold great promise as an anti-oxidant agent and therefore used in clinical practice as a new approach in oxidative-stress related disorders.

Transcriptomic analysis of gingival mesenchymal stem cells cultured on 3D bioprinted scaffold: A promising strategy for neuroregeneration

The combined approach of mesenchymal stem cells (MSCs) and scaffolds has been proposed as a potential therapeutic tool for the treatment of neurodegenerative diseases. Indeed, even if MSCs can promote neuronal regeneration, replacing lost neurons or secreting neurotrophic factors, many limitations still exist for their application in regenerative medicine, including the low survival and differentiation rate. The scaffolds, by mimicking the endogenous microenvironment, have shown to promote cell survival, proliferation, and differentiation. In this work, gingival mesenchymal stem cells (GMSCs), isolated from healthy donors, were expanded in vitro, by culturing them adherent in plastic dishes (CTR-GMSCs) or on a poly(lactic acid) scaffold (SC-GMSCs). In order to evaluate the survival and the neurogenic differentiation potential, we performed a comparative transcriptomic analysis between CTR-GMSCs and SC-GMSCs by next generation sequencing. We found that SC-GMSCs showed an increased expression of neurogenic and prosurvival genes. In particular, genes involved in neurotrophin signaling and PI3K/Akt pathways were upregulated. On the contrary, proapoptotic and negative regulator of neuronal growth genes were downregulated. Moreover, nestin and GAP-43 protein levels increased in SC-GMSCs, confirming the neurogenic commitment of these cells. In conclusion, the scaffold, providing a trophic support for MSCs, may promote GMSCs differentiation toward a neuronal phenotype and survival.

Biotherapeutic Effect of Gingival Stem Cells Conditioned Medium in Bone Tissue Restoration

Bone tissue engineering is one of the main branches of regenerative medicine. In this field, the use of a scaffold, which supported bone development, in combination with mesenchymal stem cells (MSCs), has promised better outcomes for bone regeneration. In particular, human gingival mesenchymal stem cells (hGMSCs) may present advantages compared to other MSCs, including the easier isolation. However, MSCs’ secretome has attracted much attention for its potential use in tissue regeneration, such as conditioned medium (CM) that contains different soluble factors proved to be useful for the regenerative purposes. In this study, we evaluated the osteogenic capacity of a poly-(lactide) (3D-PLA) scaffold enriched with hGMSCs and hGMSCs derived CM and its ability to regenerate bone defects in rat calvarias. 3D-PLA alone, 3D-PLA + CM or 3D-PLA + hGMSCs with/without CM were implanted in Wistar male rats subjected to calvarial defects. We observed that 3D-PLA scaffold enriched with hGMSCs and CM showed a better osteogenic capacity, being able to repair the calvarial defect as revealed in vivo by morphological evaluation. Moreover, transcriptomic analysis in vitro revealed the upregulation of genes involved in ossification and regulation of ossification in the 3D-PLA + CM + hGMSCs group. All of these results indicate the great osteogenic ability of 3D-PLA + CM + hGMSCs supporting its use in bone regenerative medicine, in particular in the repair of cranial bone defects. Especially, hGMSCs derived CM played a key role in the induction of the osteogenic process and in bone regeneration.

Successful ceftazidime-avibactam treatment of MDR-KPC-positive Klebsiella pneumoniae infection in a patient with traumatic brain injury: A case report.

Rationale: Carbapenem-resistant Enterobacteriaceae infections are a serious health care problem, because of the high mortality. Carbapenem resistance is mainly caused by carbapenemases production, including Klebsiella pneumoniae carbapenemase (KPC). Ceftazidime-avibactam is a new cephalosporin/&bgr;-lactamase inhibitor combination for the treatment of complicated urinary, intra-abdominal infections, and nosocomial pneumonia caused by gram negative, or other serious gram-negative infections. Patient concerns: We showed the case of a 27-year-old patient, hospitalized for traumatic brain injury and chest trauma, with KPC-producing Klebsiella pneumoniae infection. Diagnoses: Blood and bronchial aspirate culture analysis detected an infection caused by MDR Klebsiella pneumoniae, resistant to meropenem, ertapenem, piperacillin/tazobactam, amoxicillin/clavulanic acid, aztreonam, ceftazidime, cefotaxime, cefepime, amikacin, ciprofloxacin, trimethoprim/sulfamethoxazole, colistin while it showed an intermediate sensitivity to gentamicin and was sensitive to ceftazidime-avibactam. Molecular analyses revealed that the isolate belonged to the epidemic clone sequence type 258 (ST258) carrying blaKPC-3, blaTEM-1, and blaSHV-11genes. Interventions: After various combined antibiotic therapies without improvements, he was treated with ceftazidime-avibactam, on a compassionate-use basis. Outcomes: With ceftazidime-avibactam monotherapy clinical and microbiological clearance was obtained. A week after the end of the therapy microbiological analysis was repeated and a positive rectal swab for KPC-Klebsiella pneumoniae was found, becoming negative after 1 month. Moreover, the patient did not show any relapses for up to 18 weeks. Lessons: This case indicates that ceftazidime-avibactam monotherapy could be efficacious against KPC positive Klebsiella pneumoniae infections.

Role of Vitamin E in the Treatment of Alzheimer’s Disease: Evidence from Animal Models

Alzheimer’s disease (AD) is a neurodegenerative disorder representing the major cause of dementia. It is characterized by memory loss, and cognitive and behavioral decline. In particular, the hallmarks of the pathology are amyloid-β (Aβ) plaques and neurofibrillary tangles (NFTs), formed by aggregated hyperphosphorylated tau protein. Oxidative stress plays a main role in AD, and it is involved in initiation and progression of AD. It is well known that Aβ induced oxidative stress, promoting reactive oxygen species (ROS) production and consequently lipid peroxidation, protein oxidation, tau hyperphosphorylation, results in toxic effects on synapses and neurons. In turn, oxidative stress can increase Aβ production. For these reasons, the administration of an antioxidant therapy in AD patients was suggested. The term vitamin E includes different fat-soluble compounds, divided into tocopherols and tocotrienols, that possess antioxidant action. α-Tocopherol is the most studied, but some studies suggested that tocotrienols may have different health promoting capacities. In this review, we focused our attention on the effects of vitamin E supplementation in AD animal models and AD patients or older population. Experimental models showed that vitamin E supplementation, by decreasing oxidative stress, may be a good strategy to improve cognitive and memory deficits. Furthermore, the combination of vitamin E with other antioxidant or anti-inflammatory compounds may increase its efficacy. However, even if some trials have evidenced some benefits, the effects of vitamin E in AD patients are still under debate.

Three-dimensional printed PLA scaffold and human gingival stem cell-derived extracellular vesicles: a new tool for bone defect repair

BackgroundThe role of bone tissue engineering in the field of regenerative medicine has been a main research topic over the past few years. There has been much interest in the use of three-dimensional (3D) engineered scaffolds (PLA) complexed with human gingival mesenchymal stem cells (hGMSCs) as a new therapeutic strategy to improve bone tissue regeneration. These devices can mimic a more favorable endogenous microenvironment for cells in vivo by providing 3D substrates which are able to support cell survival, proliferation and differentiation. The present study evaluated the in vitro and in vivo capability of bone defect regeneration of 3D PLA, hGMSCs, extracellular vesicles (EVs), or polyethyleneimine (PEI)-engineered EVs (PEI-EVs) in the following experimental groups: 3D-PLA, 3D-PLA + hGMSCs, 3D-PLA + EVs, 3D-PLA + EVs + hGMSCs, 3D-PLA + PEI-EVs, 3D-PLA + PEI-EVs + hGMSCs.MethodsThe structural parameters of the scaffold were evaluated using both scanning electron microscopy and nondestructive microcomputed tomography. Nanotopographic surface features were investigated by means of atomic force microscopy. Scaffolds showed a statistically significant mass loss along the 112-day evaluation.ResultsOur in vitro results revealed that both 3D-PLA + EVs + hGMSCs and 3D-PLA + PEI-EVs + hGMSCs showed no cytotoxicity. However, 3D-PLA + PEI-EVs + hGMSCs exhibited greater osteogenic inductivity as revealed by morphological evaluation and transcriptomic analysis performed by next-generation sequencing (NGS). In addition, in vivo results showed that 3D-PLA + PEI-EVs + hGMSCs and 3D-PLA + PEI-EVs scaffolds implanted in rats subjected to cortical calvaria bone tissue damage were able to improve bone healing by showing better osteogenic properties. These results were supported also by computed tomography evaluation that revealed the repair of bone calvaria damage.ConclusionThe re-establishing of the integrity of the bone lesions could be a promising strategy in the treatment of accidental or surgery trauma, especially for cranial bones.

Moringin Induces Neural Differentiation in the Stem Cell of the Human Periodontal Ligament

The therapeutic strategies for neurodegenerative diseases still represent a vast research field because of the lack of targeted, effective and resolutive treatment for neurodegenerative diseases. The use of stem cell-based therapy is an alternative approach that could lead to the replacement of damaged neuronal tissue. For this purpose, adult mesenchymal stem cells (MSC), including periodontal ligament stem cells (PDLSCs), could be very useful for their differentiation capacity, easy isolation and the ability to perform an autologous implant. The aim of this work was to test whether the Moringin [4-(α-L-rhamnosyloxy) benzyl isothiocyanate; GMG-ITC], an isothiocyanate extracted from Moringa oleifera seeds, was able to induce PDLSCs toward neural progenitor differentiation. Next-generation transcriptomics sequencing showed that moringin treatment increased the expression of genes involved in neuron cortical development and in particular in neuron belonging to upper and deep cortical layers. Moreover, moringin treatment upregulated genes involved in osteogenesis and adipogenesis although with a lower fold change compared to upregulated genes involved in neuronal differentiation. Finally, moringin did not induce the expression of oncogenes resulting in a safe treatment.

Eruca sativa seed extract: A novel natural product able to counteract neuroinflammation

Certain nutrients are able to exert health promoting effects. The consumption of Brassicaceae vegetables has increased given their reported beneficial effects on human health, due to their high content of nutraceutical compounds. The health benefits appear to be associated with the presence of glucosinolates and flavonoids. Certain nutraceutics have been revealed to have anti-inflammatory action. In the present study, the anti-inflammatory properties of Eruca sativa seed extract (ESE) were evaluated in NSC-34 motor neurons exposed to the cell culture medium of lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Treatment with LPS-stimulated RAW 264.7 medium induced apoptosis and the expression of Toll-like receptor 4 (TLR4) and cyclooxygenase 2 (COX2) in NSC-34 motor neurons. Additionally, the stimulation of NSC-34 motor neurons with the medium of LPS-treated macrophages triggered the expression of NLR family pyrin domain containing 3 (NLRP3) inflammasome proteins and the production of pro-inflammatory cytokines. Pre-treatment with ESE counteracted the apoptosis and production of pro-inflammatory cytokines in NSC-34 motor neurons treated with the medium of LPS-treated RAW 264.7. It also eliminated COX2 and TLR4/NLRP3 inflammasome expression. In addition, pre-treatment with ESE was able to restore interleukin 10 expression in NSC-34 cells. These results demonstrate the anti-inflammatory and neuroprotective effects of ESE.

NLRP3 Inflammasome Activation in a Transgenic Amyotrophic Lateral Sclerosis Model

Amyotrophic lateral sclerosis (ALS) is a disabling progressive disease characterized by the degeneration of motor neurons, leading to muscle atrophy and paralysis. The majority of cases are sporadic, but also a familiar form of ALS exists, and some genes causative of the pathology were found. In particular, mutations in superoxide dismutase 1 (SOD1) were found in 20% of familiar cases. It is known that neuroinflammation plays a pivotal role in several neurodegenerative disorders, including ALS. Inflammasomes are protein complexes that induce inflammation in response to various stimuli, involved also in neuroinflammation. The NLRP3 inflammasome, which is the best known, after assembly, induces the activation of caspase 1, which in turn activates interleukin (IL)-1β and IL-18. The aim of this work was the evaluation of inflammasome activation in the brain of SOD1G93A rats, a transgenic model of ALS. We observed the increase in TLR4 and nuclear NF-κB levels in SOD1G93A rats. Their activation is known as priming signal for inflammasome induction. Moreover, NLRP3 protein increased, associated with the presence of active caspase 1, leading to an increase in IL-18 and IL-1β levels. In addition, IL-1β, IL-18, and IFN-γ amount increased in the spleen of SOD1G93A rats, together with an increased expression of CD4, CD8, CD44, and CD68 markers. In conclusion, our results showed the activation of the NLRP3 inflammasome in the brain of SOD1G93A rats, indicating that inflammation plays a main role in ALS.

Reprogramming of Oncogene Expression in Gingival Mesenchymal Stem Cells Following Long-Term Culture In Vitro

Mesenchymal stem cells (MSCs) are a promising resource for stem cell therapy for the treatment of different neurodegenerative disorders. In particular, dental MSCs, given their origin from neural crest and their proneness toward neuronal differentiation, may be more suitable for transplantation. However, if MSCs can undergo spontaneous transformation and give rise to tumor is still debated. Data about transcriptional regulation of oncogenes in MSCs following in vitro expansion are not available. In this work, we compared gene expression levels of oncogenes in gingival-derived MSCs at passage number 10 and 41. We found that the expression of 22 oncogenes was abolished in gingival MSCs at passage number 41 compared to those at passage number 10, and this may indicate a greater safety of high number passage MSCs.