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Abstract Glioblastoma (GBM) represents an aggressive brain tumor, characterized by intra- and inter-tumoral heterogeneity and therapy resistance, leading to unfavourable prognosis. An increasing number of studies pays attention on the regulation of ferroptosis, an iron-dependent cell death, as a strategy to reverse drug resistance in cancer. However, the debate on whether this strategy may have important implications for the treatment of GBM is still ongoing. In the present study, we used ferric ammonium citrate and erastin to evaluate ferroptosis induction effects on two human GBM cell lines, U-251 MG, with proneural characteristics, and T98-G, with a mesenchymal profile. The response to ferroptosis induction was markedly different between cell lines, indeed T98-G cells showed an enhanced antioxidant defence, with increased glutathione levels, as compared to U-251 MG cells. Moreover, using bioinformatic approaches and analysing publicly available datasets from patients’ biopsies, we found that GBM with a mesenchymal phenotype showed an up-regulation of several genes involved in antioxidant mechanisms as compared to proneural subtype. Thus, our results suggest that GBM subtypes differently respond to ferroptosis induction, emphasizing the significance of further molecular studies on GBM to better discriminate between various tumor subtypes and progressively move towards personalized therapy.

Abstract Background The expression of aquaporin 4 (AQP4) and intermediate filament (IF) proteins is altered in malignant glioblastoma (GBM), yet the expression of the major IF-based cytolinker, plectin (PLEC), and its contribution to GBM migration and invasiveness, are unknown. Here, we assessed the contribution of plectin in affecting the distribution of plasmalemmal AQP4 aggregates, migratory properties, and regulation of cell volume in astrocytes. Methods In human GBM, the expression of glial fibrillary acidic protein (GFAP), AQP4 and PLEC transcripts was analyzed using publicly available datasets, and the colocalization of PLEC with AQP4 and with GFAP was determined by immunohistochemistry. We performed experiments on wild-type and plectin-deficient primary and immortalized mouse astrocytes, human astrocytes and permanent cell lines (U-251 MG and T98G) derived from a human malignant GBM. The expression of plectin isoforms in mouse astrocytes was assessed by quantitative real-time PCR. Transfection, immunolabeling and confocal microscopy were used to assess plectin-induced alterations in the distribution of the cytoskeleton, the influence of plectin and its isoforms on the abundance and size of plasmalemmal AQP4 aggregates, and the presence of plectin at the plasma membrane. The release of plectin from cells was measured by ELISA. The migration and dynamics of cell volume regulation of immortalized astrocytes were assessed by the wound-healing assay and calcein labeling, respectively. Results A positive correlation was found between plectin and AQP4 at the level of gene expression and protein localization in tumorous brain samples. Deficiency of plectin led to a decrease in the abundance and size of plasmalemmal AQP4 aggregates and altered distribution and bundling of the cytoskeleton. Astrocytes predominantly expressed P1c, P1e, and P1g plectin isoforms. The predominant plectin isoform associated with plasmalemmal AQP4 aggregates was P1c, which also affected the mobility of astrocytes most prominently. In the absence of plectin, the collective migration of astrocytes was impaired and the dynamics of cytoplasmic volume changes in peripheral cell regions decreased. Plectin’s abundance on the plasma membrane surface and its release from cells were increased in the GBM cell lines. Conclusions Plectin affects cellular properties that contribute to the pathology of GBM. The observed increase in both cell surface and released plectin levels represents a potential biomarker and therapeutic target in the diagnostics and treatment of GBMs.

Abstract Background Non-alcoholic fatty liver disease (NAFLD) is a liver disorder characterized by the ac-cumulation of fat in hepatocytes without alcohol consumption. Mitochondrial dysfunction and endoplasmic reticulum (ER) stress play significant roles in NAFLD pathogenesis. The unfolded protein response in mitochondria (UPRmt) is an adaptive mechanism that aims to restore mitochondrial protein homeostasis and mitigate cellular stress. This study aimed to investigate the effects of ( +)-Lipoic acid (ALA) on UPRmt, inflammation, and oxidative stress in an in vitro model of NAFLD using HepG2 cells treated with palmitic acid and oleic acid to induce steatosis. Results Treatment with palmitic and oleic acids increased UPRmt-related proteins HSP90 and HSP60 (heat shock protein), and decreased CLPP (caseinolytic protease P), indicating ER stress activation. ALA treatment at 1 μM and 5 μM restored UPRmt-related protein levels. PA:OA (palmitic acid:oleic acid)-induced ER stress markers IRE1α (Inositol requiring enzyme-1), CHOP (C/EBP Homologous Protein), BIP (Binding Immunoglobulin Protein), and BAX (Bcl-2-associated X protein) were significantly reduced by ALA treatment. ALA also enhanced ER-mediated protein glycosylation and reduced oxidative stress, as evidenced by decreased GPX1 (Glutathione peroxidase 1), GSTP1 (glutathione S-transferase pi 1), and GSR (glutathione-disulfide reductase) expression and increased GSH (Glutathione) levels, and improved cellular senescence as shown by the markers β-galactosidase, γH2Ax and Klotho-beta. Conclusions In conclusion, ALA ameliorated ER stress, oxidative stress, and inflammation in HepG2 cells treated with palmitic and oleic acids, potentially offering therapeutic benefits for NAFLD providing a possible biochemical mechanism underlying ALA beneficial effects. Graphical Abstract

Abstract Background Follicular thyroid cancer (FTC) is a prevalent form of differentiated thyroid cancer, whereas anaplastic thyroid cancer (ATC) represents a rare, fast-growing, undifferentiated, and highly aggressive tumor, posing significant challenges for eradication. Ferroptosis, an iron-dependent cell death mechanism driven by the excessive production of reactive oxygen species and subsequent lipid peroxidation, emerges as a promising therapeutic strategy for cancer. It has been observed that many cancer cells exhibit sensitivity to ferroptosis, while some other histotypes appear to be resistant, by counteracting the metabolic changes and oxidative stress induced by iron overload. Methods Here we used human biopsies and in vitro approaches to analyse the effects of iron-dependent cell death. We assessed cell proliferation and viability through MTT turnover, clonogenic assays, and cytofluorimetric-assisted analysis. Lipid peroxidation assay and western blot were used to analyse molecular mechanisms underlying ferroptosis modulation. Two distinct thyroid cancer cell lines, FTC-133 (follicular) and 8505C (anaplastic), were utilized. These cell lines were exposed to ferroptosis inducers, Erastin and RSL3, while simulating an iron overload condition using ferric ammonium citrate. Results Our evidence suggests that FTC-133 cell line, exposed to iron overload, reduced their viability and showed increased ferroptosis. In contrast, the 8505C cell line seems to better tolerate ferroptosis, responding by modulating CD71, which is involved in iron internalization and seems to have a role in resistance to iron overload and consequently in maintaining cell viability. Conclusions The differential tolerance to ferroptosis observed in our study may hold clinical implications, particularly in addressing the unmet therapeutic needs associated with ATC treatment, where resistance to ferroptosis appears more pronounced compared to FTC.

Fish oil, renowned for its high content of omega-3 fatty acids, particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), has gained considerable attention for its potential health benefits. EPA and DHA exhibit anti-inflammatory effects by promoting the production of specialized pro-resolving mediators (SPMs), such as resolvins and protectins. Fish oil has been studied for its potential to reduce bronchial inflammation, a key feature of respiratory conditions like asthma and COPD. This study investigates the cellular mechanisms of fish oil in an in vitro model of lung inflammation using lipopolysaccharide (LPS) on a healthy human bronchial epithelium cell line. LPS exposure for 24 h reduced cell viability, elevated reactive oxygen species (ROS), depleted glutathione (GSH), and induced mitochondrial depolarization, indicating oxidative stress and inflammation. Fish oil administration significantly mitigated ROS production, prevented GSH depletion, and reduced mitochondrial depolarization. This was associated with the upregulation of the endogenous antioxidant system, evidenced by restored GSH levels and the increased gene expression of glutathione peroxidase (GPX), catalase (CAT), superoxide dismutase 1 (SOD1), and superoxide dismutase 2 (SOD2). Fish oil also suppressed IL-6 and IL-1β expression and increased anti-inflammatory cytokine IL-10 expression. Furthermore, fish oil upregulated the expression of pro-resolving mediator receptors, suggesting a role in inflammation resolution. These findings highlight the potential of fish oil supplementation as a preventive measure against pulmonary diseases characterized by unresolved inflammation such as lung inflammation.

Background: Nonalcoholic fatty liver disease (NAFLD) is a prevalent condition characterized by hepatic fat accumulation, often progressing to severe liver injury, for which approved treatments are currently lacking. This study explores the potential therapeutic impact of alpha-lipoic acid (ALA), a natural compound crucial in lipid metabolism, on NAFLD using an in vitro model. Methods: HepG2 cells were treated with a palmitic acid:oleic acid (PA:OA) mixture, representing a cellular model of steatosis. Subsequent treatment with ALA at concentrations of 1 µM and 5 µM aimed to evaluate its effects on lipid content and metabolism. Real-time polymerase chain reaction (PCR), BODIPY staining, cytofluorimetric analysis, and lipidomics were used to assess gene expression, lipid droplet accumulation, and fatty acid profiles. Results: Our results showed that ALA significantly reduced lipid droplets in PA:OA-treated HepG2 cells, with a concentration-dependent effect. Analysis of fatty acid profiles demonstrated a decrease in palmitic acid levels with ALA treatment, while oleic acid reduction was observed only at the higher concentration. Moreover, ALA modulated the expression of genes involved in cholesterol biosynthesis and low-density lipoprotein (LDL) metabolism, indicating a potential role in lipid homeostasis. Further insights into molecular mechanisms revealed that ALA modulated peroxisome proliferator activated receptors (PPARs), specifically PPAR-alpha and PPAR-gamma, involved in fatty acid metabolism and insulin sensitivity. Finally, ALA counteracted the overexpression of thermogenic genes induced by exogenous fatty acids, suggesting a regulatory role in energy dissipation pathways. Conclusion: In conclusion, this study highlights ALA as a therapeutic agent in mitigating lipid accumulation and dysregulation in NAFLD.

To date, little is known concerning the circulating levels of biochemically relevant metabolites (antioxidants, oxidative/nitrosative stress biomarkers, purines, and pyrimidines) in patients with primary myelofibrosis (PMF), a rare form of myeloproliferative tumor causing a dramatic decrease in erythropoiesis and angiogenesis. In this study, using a targeted metabolomic approach, serum samples of 22 PMF patients and of 22 control healthy donors were analyzed to quantify the circulating concentrations of hypoxanthine, xanthine, uric acid (as representative purines), uracil, β-pseudouridine, uridine (as representative pyrimidines), reduced glutathione (GSH), ascorbic acid (as two of the main water-soluble antioxidants), malondialdehyde, nitrite, nitrate (as oxidative/nitrosative stress biomarkers) and creatinine, using well-established HPLC method for their determination. Results showed that PMF patients have dramatic depletions of both ascorbic acid and GSH (37.3- and 3.81-times lower circulating concentrations, respectively, than those recorded in healthy controls, p < 0.0001), accompanied by significant increases in malondialdehyde (MDA) and nitrite + nitrate (4.73- and 1.66-times higher circulating concentrations, respectively, than those recorded in healthy controls, p < 0.0001). Additionally, PMF patients have remarkable alterations of circulating purines, pyrimidines, and creatinine, suggesting potential mitochondrial dysfunctions causing energy metabolism imbalance and consequent increases in these cell energy-related compounds. Overall, these results, besides evidencing previously unknown serum metabolic alterations in PMF patients, suggest that the determination of serum levels of the aforementioned compounds may be useful to evaluate PMF patients on hospital admission for adjunctive therapies aimed at recovering their correct antioxidant status, as well as to monitor patients’ status and potential pharmacological treatments.

Abstract Colorectal Cancer (CRC) is one of the most common cancers accounting for 1.8 million new cases worldwide every year. Therefore, the identification of new potential therapeutic targets represents a continuous challenge to improve survival and quality of CRC patient’s life. We performed a microarray analysis dataset consisting of colon biopsies of healthy subjects (HS) and CRC patients. These results were further confirmed in a clinical setting evaluating a series of CRC patients to assess the expression of Resistin-Like Beta (RETNLB) and to correlate it with their clinical data. Our results showed a significant reduction of RETNLB expression in CRC biopsies compared to the HS mucosa. Furthermore, such reduction was significantly associated with the TNM grade and patients’ age. Furthermore, a significantly positive correlation was found within mutated subjects for KRAS, TP53, and BRAF. In particular, patients with poor prognosis at 5 years exhibited RETNLB lower levels. In-silico analysis data were confirmed by histochemical analysis in a series of CRC patients recruited by our group. The results obtained provided that RETNLB low levels are associated with an unfavorable prognosis in CRC patients and its expression is also dependent on adjuvant therapy. Further studies are warranted in order to evaluate the molecular mechanisms underlying the role of RETNLB in CRC progression.

Abstract Since neutrophil extracellular traps formation (NET-osis) can be assessed indirectly by treating healthy neutrophils with blood-derived fluids from patients and then measuring the NETs response, we designed a pilot study to convey high-dimensional cytometry of peripheral blood immune cells and cytokines, combined with clinical features, to understand if NET-osis assessment could be included in the immune risk profiling to early prediction of clinical patterns, disease severity, and viral clearance at 28 days in COVID-19 patients. Immune cells composition of peripheral blood, cytokines concentration and in-vitro NETosis were detected in peripheral blood of 41 consecutive COVID-19 inpatients, including 21 mild breakthrough infections compared to 20 healthy donors, matched for sex and age. Major immune dysregulation in peripheral blood in not-vaccinated COVID-19 patients compared to healthy subjects included: a significant reduction of percentage of unswitched memory B-cells and transitional B-cells; loss of naïve CD3+CD4+CD45RA+ and CD3+CD8+CD45RA+ cells, increase of IL-1β, IL-17A and IFN-γ. Myeloid compartment was affected as well, due to the increase of classical (CD14++CD16−) and intermediate (CD14++CD16+) monocytes, overexpressing the activation marker CD64, negatively associated to the absolute counts of CD8+ CD45R0+ cells, IFN-γ and IL-6, and expansion of monocytic-like myeloid derived suppressor cells. In not-vaccinated patients who achieved viral clearance by 28 days we found at hospital admission lower absolute counts of effector cells, namely CD8+T cells, CD4+ T-cells and CD4+CD45RO+ T cells. Percentage of in-vitro NET-osis induced by patients’ sera and NET-osis density were progressively higher in moderate and severe COVID-19 patients than in mild disease and controls. The percentage of in-vitro induced NET-osis was positively associated to circulating cytokines IL-1β, IFN-γ and IL-6. In breakthrough COVID-19 infections, characterized by mild clinical course, we observed increased percentage of in-vitro NET-osis, higher CD4+ CD45RO+ and CD8+ CD45RO+ T cells healthy or mild-COVID-19 not-vaccinated patients, reduced by 24 h of treatment with ACE inhibitor ramipril. Taken together our data highlight the role of NETs in orchestrating the complex immune response to SARS-COV-2, that should be considered in a multi-target approach for COVID-19 treatment.

In the original publication [...]

Mesenchymal stromal cells (MSCs) are a subset of heterogeneous, non-hematopoietic fibroblast-like cells which play important roles in tissue repair, inflammation, and immune modulation. MSCs residing in the bone marrow microenvironment (BMME) functionally interact with hematopoietic stem progenitor cells regulating hematopoiesis. However, MSCs have also emerged in recent years as key regulators of the tumor microenvironment. Indeed, they are now considered active players in the pathophysiology of hematologic malignancies rather than passive bystanders in the hematopoietic microenvironment. Once a malignant event occurs, the BMME acquires cellular, molecular, and epigenetic abnormalities affecting tumor growth and progression. In this context, MSC behavior is affected by signals coming from cancer cells. Furthermore, it has been shown that stromal cells themselves play a major role in several hematological malignancies’ pathogenesis. This bidirectional crosstalk creates a functional tumor niche unit wherein tumor cells acquire a selective advantage over their normal counterparts and are protected from drug treatment. It is therefore of critical importance to unveil the underlying mechanisms which activate a protumor phenotype of MSCs for defining the unmasked vulnerabilities of hematological cancer cells which could be pharmacologically exploited to disrupt tumor/MSC coupling. The present review focuses on the current knowledge about MSC dysfunction mechanisms in the BMME of hematological cancers, sustaining tumor growth, immune escape, and cancer progression.

Abstract Mesenchymal stromal cells (MSCs) within the protective microenvironment of multiple myeloma (MM) promote tumor growth, confer chemoresistance and support metabolic needs of plasma cells (PCs) even transferring mitochondria. In this scenario, heterocellular communication and dysregulation of critical signaling axes are among the major contributors to progression and treatment failure. Here, we report that myeloma MSCs have decreased reliance on mitochondrial metabolism as compared to healthy MSCs and increased tendency to deliver mitochondria to MM cells, suggesting that this intercellular exchange between PCs and stromal cells can be consider part of MSC pro-tumorigenic phenotype. Interestingly, we also showed that PCs promoted expression of connexin 43 (CX43) in MSCs leading to CXCL12 activation and stimulation of its receptor CXCR4 on MM cells favoring protumor mitochondrial transfer. Consistently, we observed that selective inhibition of CXCR4 by plerixafor resulted in a significant reduction of mitochondria trafficking. Moreover, intracellular expression of CXCR4 in myeloma PCs from BM biopsy specimens demonstrated higher CXCR4 colocalization with CD138+ cells of non-responder patients to bortezomib compared with responder patients, suggesting that CXCR4 mediated chemoresistance in MM. Taken together, our data demonstrated that CXCL12/CXCR4 axis mediates intercellular coupling thus suggesting that the myeloma niche may be exploited as a target to improve and develop therapeutic approaches.

IntroductionPatients with multiple myeloma (MM) frequently reported immune impairment with an increased risk for infection-related mortality. We aimed to evaluate the immune response in MM patients vaccinated for SARS-CoV-2 during active treatment.MethodsWe enrolled 158 patients affected by active MM or smoldering MM (SMM) and 40 healthy subjects. All subjects received 2 or 3 doses of the BNT162b2 (Pfizer/BioNTech) vaccine, and the anti-spike IgG values were evaluated after every dose. We applied the Propensity Score Matching (PSM) as a consequence of the limited sample size and its heterogeneity to adjust for differences in baseline clinical variables between MM patients who achieved or not a vaccine response after 2 or 3 doses.ResultsAt 30 days from the second dose, the median antibodies level in MM was 25.2 AU/mL, lower than in SMM and in the control group. The same results were confirmed after the third dose, with lower median anti-spike IgG levels in MM, compared to SMM and control group. Following PSM, lack of response to SARS-CoV-2 complete vaccination plus boost was associated with age more than 70 years old and use of high-dose of steroids. We failed to identify an association between specific treatment types and reduced vaccine response. The use of prophylaxis with tixagevimab/cilgavimab for 40 non-responder patients after 3 doses of vaccine has proven to be an effective and safe approach in reducing the risk of serious illness in the event of a breakthrough SARS-CoV-2 infection, faced with a mild symptomatic course, and in providing protection instead of long-term humoral immune vaccine responses. Following PSM, only the high-risk cytogenetic abnormalities were associated with an increased risk of developing a breakthrough SARS-CoV-2 infection.ConclusionMonitoring the immune response is fundamental in MM patients that remain highly vulnerable to SARS-CoV-2 despite the vaccine. The use of prophylaxis with tixagevimab/cilgavimab can guarantee better protection from the severe form of the disease.

Chronic myeloproliferative neoplasms encompass the BCR-ABL1-negative neoplasms polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). These are characterized by calreticulin (CALR), myeloproliferative leukemia virus proto-oncogene (MPL) and the tyrosine kinase Janus kinase 2 (JAK2) mutations, eventually establishing a hyperinflammatory tumor microenvironment (TME). Several reports have come to describe how constitutive activation of JAK-STAT and NFκB signaling pathways lead to uncontrolled myeloproliferation and pro-inflammatory cytokines secretion. In such a highly oxidative TME, the balance between Hematopoietic Stem Cells (HSCs) and Mesenchymal Stromal Cells (MSCs) has a crucial role in MPN development. For this reason, we sought to review the current literature concerning the interplay between HSCs and MSCs. The latter have been reported to play an outstanding role in establishing of the typical bone marrow (BM) fibrotic TME as a consequence of the upregulation of different fibrosis-associated genes including PDGF- β upon their exposure to the hyperoxidative TME characterizing MPNs. Therefore, MSCs might turn to be valuable candidates for niche-targeted targeting the synthesis of cytokines and oxidative stress in association with drugs eradicating the hematopoietic clone.

Patients affected by myelofibrosis (MF) or polycythemia vera (PV) and treated with ruxolitinib are at high risk for severe coronavirus disease 2019. Now a vaccine against the virus SARS-CoV-2, which is responsible for this disease, is available. However, sensitivity to vaccines is usually lower in these patients. Moreover, fragile patients were not included in large trials investigating the efficacy of vaccines. Thus, little is known about the efficacy of this approach in this group of patients. In this prospective single-center study, we evaluated 43 patients (30 MF patients and 13 with PV) receiving ruxolitinib as a treatment for their myeloproliferative disease. We measured anti-spike and anti-nucleocapsid IgG against SARS-CoV2 15-30 days after the second and the third BNT162b2 mRNA vaccine booster dose. Patients receiving ruxolitinib showed an impaired antibody response to complete vaccination (2 doses), as 32.5% of patients did not develop any response. After the third booster dose with Comirnaty, results slightly improved, as 80% of these patients produced antibodies above the threshold positivity. However, the quantity of produced antibodies was well below that reached than those reported for healthy individuals. PV patients elicited a better response than patients affected by MF. Thus, different strategies should be considered for this high-risk group of patients.

Accumulating evidence sustains glial cells as critical players during central nervous system (CNS) development, homeostasis and disease. Olfactory ensheathing cells (OECs), a type of specialized glia cells sharing properties with both Schwann cells and astrocytes, are of critical importance in physiological condition during olfactory system development, supporting its regenerative potential throughout the adult life. These characteristics prompted research in the field of cell-based therapy to test OEC grafts in damaged CNS. Neuroprotective mechanisms exerted by OEC grafts are not limited to axonal regeneration and cell differentiation. Indeed, OEC immunomodulatory properties and their phagocytic potential encourage OEC-based approaches for tissue regeneration in case of CNS injury. Herein we reviewed recent advances on the immune role of OECs, their ability to modulate CNS microenvironment via bystander effects and the potential of OECs as a cell-based strategy for tissue regeneration.

Tumor onset and its progression are strictly linked to its metabolic rewiring on the basis of the Warburg effect. In this context, fumarate emerged as a putative oncometabolite mediating cancer progression. Fumarate accumulation is usually driven by fumarate hydratase (FH) loss of function, the enzyme responsible for the reversible conversion of fumarate into malate. Fumarate accumulation acts as a double edge sword: on one hand it takes part in the metabolic rewiring of cancer cells, while on the other it also plays a crucial role in chromatin architecture reorganization. The latter is achieved by competing with a-ketoglutarate-dependent enzymes, eventually altering the cellular methylome profile, which in turn leads to its transcriptome modeling. Furthermore, in recent years, it has emerged that FH has an ability to recruit DNA double strand breaks. The accumulation of fumarate into damaged sites might also determine the DNA repair pathway in charge for the seizure of the lesion, eventually affecting the mutational state of the cells. In this work, we aimed to review the current knowledge on the role of fumarate as an oncometabolite orchestrating the cellular epigenetic landscape and DNA repair machinery.

Abstract Motoneuronal loss is the main feature of amyotrophic lateral sclerosis, although pathogenesis is extremely complex involving both neural and muscle cells. In order to translationally engage the sonic hedgehog pathway, which is a promising target for neural regeneration, recent studies have reported on the neuroprotective effects of clobetasol, an FDA-approved glucocorticoid, able to activate this pathway via smoothened. Herein we sought to examine functional, cellular, and metabolic effects of clobetasol in a neurotoxic mouse model of spinal motoneuronal loss. We found that clobetasol reduces muscle denervation and motor impairments in part by restoring sonic hedgehog signaling and supporting spinal plasticity. These effects were coupled with reduced pro-inflammatory microglia and reactive astrogliosis, reduced muscle atrophy, and support of mitochondrial integrity and metabolism. Our results suggest that clobetasol stimulates a series of compensatory processes and therefore represents a translational approach for intractable denervating and neurodegenerative disorders.

The tumor microenvironment (TME) plays a pivotal role in establishing malignancy, and it is associated with high glycolytic metabolism and lactate release through monocarboxylate transporters (MCTs). Several lines of evidence suggest that lactate also serves as a signaling molecule through its receptor hydroxycarboxylic acid receptor 1 (HCAR1/GPR81), thus functioning as a paracrine and autocrine signaling molecule. The aim of the present study was to investigate the role of lactate in glioblastoma (GBM) progression and metabolic reprogramming in an in vitro and in vivo model. The cell proliferation, migration, and clonogenicity were tested in vitro in three different human GBM cell lines. The expressions of MCT1, MCT4, and HCAR1 were evaluated both in vitro and in a zebrafish GBM model. The results were further validated in patient-derived GBM biopsies. Our results showed that lactate significantly increased the cell proliferation, migration, and colony formation capacity of GBM cells, both in vitro and in vivo. We also showed that lactate increased the expressions of MCT1 and HCAR1. Moreover, lactate modulated the epithelial–mesenchymal transition protein markers E-cadherin and β-catenin. Interestingly, lactate induced mitochondrial mass and the OXPHOS gene, suggesting improved mitochondrial fitness. Similar effects were observed after treatment with 3,5-dihydroxybenzoic acid, a known agonist of HCAR1. Consistently, the GBM zebrafish model exhibited an altered metabolism and increased expressions of MCT1 and HCAR1, leading to high levels of extracellular lactate and, thus, supporting tumor cell proliferation. Our data from human GBM biopsies also showed that, in high proliferative GBM biopsies, Ki67-positive cells expressed significantly higher levels of MCT1 compared to low proliferative GBM cells. In conclusion, our data suggest that lactate and its transporter and receptor play a major role in GBM proliferation and migration, thus representing a potential target for new therapeutic strategies to counteract tumor progression and recurrence.

Abstract In nonsecretory, oligo‐secretory, and light chain multiple myeloma patients, serial sFLC evaluation could precede biochemical and clinical disease progression, even in extramedullary relapse, thus initiating early treatment with novel anti‐MM agents.

Chronic myeloid leukemia (CML), BCR-ABL1-positive, is classified as a myeloproliferative characterized by Philadelphia chromosome/translocation t(9;22) and proliferating granulocytes. Despite the clinical success of tyrosine kinase inhibitors (TKi) agents in the treatment of CML, most patients have minimal residual disease contained in the bone marrow microenvironment, within which stromal cells assume a pro-inflammatory phenotype that determines their transformation in cancer-associated fibroblasts (CAF) which, in turn can play a fundamental role in resistance to therapy. Insulin-like Growth Factor Binding Protein-6 (IGFBP-6) is expressed during tumor development, and is involved in immune-escape and inflammation as well, providing a potential additional target for CML therapy. Here, we aimed at investigating the role of IGFBP-6/SHH/TLR4 axis in TKi response. We used a CML cell line, LAMA84-s, and healthy bone marrow stromal cells, HS-5, in mono- or co-culture. The two cell lines were treated with Dasatinib and/or IGFBP-6, and the expression of inflammatory markers was tested by qRT-PCR; furthermore, expression of IGFBP-6, TLR4 and Gli1 were evaluated by Western blot analysis and immumocytochemistry. The results showed that both co-culture and Dasatinib exposure induce inflammation in stromal and cancer cells so that they modulate the expression of TLR4, and these effects were more marked following IGFBP-6 pre-treatment suggesting that this molecule may confer resistance through the inflammatory processes. This phenomenon was coupled with sonic hedgehog (SHH) signaling. Indeed, our data also demonstrate that HS-5 treatment with PMO (an inducer of SHH) induces significant modulation of TLR4 and overexpression of IGFPB-6 suggesting that the two pathways are interconnected with each other and with the TLR-4 pathway. Finally, we demonstrated that pretreatment with IGFBP-6 and/or PMO restored LAMA-84 cell viability after treatment with Dasatinib, suggesting that both IGFBP-6 and SHH are involved in the resistance mechanisms induced by the modulation of TLR-4, thus indicating that the two pathways may be considered as potential therapeutic targets.

The growing interest in natural bioactive molecules, as an approach to many pathological contexts, is widely justified by the necessity to overcome the disadvantageous benefit–risk ratio related to traditional therapies. Among them, mangiferin (MGF) shows promising beneficial properties such as antioxidant, anti-inflammatory, and immunomodulatory effects. In this study, we aimed to investigate the antioxidant and anti-inflammatory properties of MGF on lipopolysaccharide (LPS)-induced lung NCI-H292 cells, focusing on its role against COVID-19 adsorption. In order to obtain this information, cells treated with LPS, with or without MGF, were analyzed performing wound healing, gene expression of inflammatory cytokines, GSH quantification, and JC-1 staining. Moreover, the inhibition of viral adsorption was evaluated microbiologically and the results were further confirmed by molecular docking analysis. In this regard, MGF downregulates the expression of several inflammatory factors, enhances GSH levels, promotes the wound healing rate, and restores the mitochondrial dysfunction caused by LPS. In addition, MGF significantly inhibits SARS-CoV-2 adsorption as shown by the gene expression of ACE2 and TMPRSS-2, and furtherly confirmed by microbiological and molecular modeling evaluation. Although more investigations are still needed, all data obtained constitute a solid background, demonstrating the cytoprotective role of MGF in inflammatory mechanisms including COVID-19 infection.

Uveal melanoma (UM) is the most common primary intraocular tumor in adults. To date, the main strategies to counteract its progression consist of focal radiation on the tumor site and ocular enucleation. Furthermore, many UM patients develop liver metastasis within 10 years following diagnosis, eventually resulting in a poorer prognosis for those patients. Dissecting the molecular mechanism involved in UM progression may lead to identify novel prognostic markers with significative clinical applications. The aim of the present study was to evaluate the role of Heme Oxygenase 1 (HO-1) in regulating UM progression. UM cell lines (92.1) were treated with Hemin (CONC e time), a strong inducer of HO-1, and VP13/47, a selective inhibitor of its enzymatic activity. Interestingly, our results showed an enhanced 92.1 cellular proliferation and wound healing ability following an HO-1 increase, overall unveiling the role played by this protein in tumor progression. Similar results were obtained following treatment with two different CO releasing molecules (CORM-3 and CORM-A1). These results were further confirmed in a clinical setting using our UM cohort. Our results demonstrated an increased median HO-1 expression in metastasizing UM when compared to nonmetastasizing patients. Overall, our results showed that HO-1 derived CO plays a major role in UM progression and HO-1 protein expression may serve as a potential prognostic and therapeutical factor in UM patients.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting motoneurons (MNs) with a fatal outcome. The typical degeneration of cortico-spinal, spinal, and bulbar MNs, observed in post-mortem biopsies, is associated with the activation of neuroimmune cells. GJA1, a member of the connexins (Cxs) gene family, encodes for connexin 43 (Cx43), a core gap junctions (GJs)- and hemichannels (HCs)-forming protein, involved in cell death, proliferation, and differentiation. Recently, Cx43 expression was found to play a role in ALS pathogenesis. Here, we used microarray and RNA-seq datasets from the NCBI of the spinal cord of control (NDC) and ALS patients, which were stratified according to the GJA1 gene expression. Genes that positively or negatively correlated to GJA1 expression were used to perform a genomic deconvolution analysis (GDA) using neuroimmune signatures. Expression analysis revealed a significantly higher GJA1 expression in the MNs of ALS patients as compared to NDC. Gene deconvolution analysis revealed that positively correlated genes were associated with microglia activation, whereas negatively correlated genes were associated with neuronal activation profiles. Moreover, gene ontology analysis, performed on genes characterizing either microglia or neuronal signature, indicated immune activation or neurogenesis as main biological processes. Finally, using a synthetic analysis of drugs able to revert the GJA1 transcriptomic signatures, we found a specific drug profile for ALS patients with high GJA1 expression levels, composed of amlodipine, sertraline, and prednisolone. In conclusion, our exploratory study suggests GJA1 as a new neuro-immunological gene correlated to microglial cellular profile in the spinal cord of ALS patients. Further studies are warranted to confirm these results and to evaluate the therapeutic potential of drugs able to revert typical GJA1/CX43 signature in ALS patients

Cerebrovascular ischemia is a common clinical disease encompassing a series of complex pathophysiological processes in which oxidative stress plays a major role. The present study aimed to evaluate the effects of Dexmedetomidine, Clonidine, and Propofol in a model of hypoxia/reoxygenation injury. Microglial cells were exposed to 1%hypoxia for 3 h and reoxygenated for 3 h, and oxidative stress was measured by ROS formation and the expression of inflammatory process genes. Mitochondrial dysfunction was assessed by membrane potential maintenance and the levels of various metabolites involved in energetic metabolism. The results showed that Propofol and α2-agonists attenuate the formation of ROS during hypoxia and after reoxygenation. Furthermore, the α2-agonists treatment restored membrane potential to values comparable to the normoxic control and were both more effective than Propofol. At the same time, Propofol, but not α2-agonists, reduces proliferation (Untreated Hypoxia = 1.16 ± 0.2, Untreated 3 h Reoxygenation = 1.28 ± 0.01 vs. Propofol hypoxia = 1.01 ± 0.01 vs. Propofol 3 h Reoxygenation = 1.12 ± 0.03) and microglial migration. Interestingly, all of the treatments reduced inflammatory gene and protein expressions and restored energy metabolism following hypoxia/reoxygenation (ATP content in hypoxia/reoxygenation 3 h: Untreated = 3.11 ± 0.8 vs. Propofol = 7.03 ± 0.4 vs. Dexmedetomidine = 5.44 ± 0.8 vs. Clonidine = 7.70 ± 0.1), showing that the drugs resulted in a different neuroprotective profile. In conclusion, our results may provide clinically relevant insights for neuroprotective strategies in intensive care units.

Hemoglobin and iron overload is considered the major contributor to intracerebral hemorrhage (ICH)-induced brain injury. Accumulation of iron in the brain leads to microglia activation, inflammation and cell loss. Current available treatments for iron overload-mediated disorders are characterized by severe adverse effects, making such conditions an unmet clinical need. We assessed the potential of α-lipoic acid (ALA) as an iron chelator, antioxidant and anti-inflammatory agent in both in vitro and in vivo models of iron overload. ALA was found to revert iron-overload-induced toxicity in HMC3 microglia cell line, preventing cell apoptosis, reactive oxygen species generation and reducing glutathione depletion. Furthermore, ALA regulated gene expression of iron-related markers and inflammatory cytokines, such as IL-6, IL-1β and TNF. Iron toxicity also affects mitochondria fitness and biogenesis, impairments which were prevented by ALA pre-treatment in vitro. Immunocytochemistry assay showed that, although iron treatment caused inflammatory activation of microglia, ALA treatment resulted in increased ARG1 expression, suggesting it promoted an anti-inflammatory phenotype. We also assessed the effects of ALA in an in vivo zebrafish model of iron overload, showing that ALA treatment was able to reduce iron accumulation in the brain and reduced iron-mediated oxidative stress and inflammation. Our data support ALA as a novel approach for iron-overload-induced brain damage.

Relapse in multiple myeloma (MM) decreases therapy efficiency through unclear mechanisms of chemoresistance. Since our group previously demonstrated that heme oxygenase-1 (HO-1) and Toll-like receptor 4 (TLR4) are two signaling pathways protecting MM cells from the proteasome inhibitor bortezomib (BTZ), we here evaluated their cross-regulation by a pharmacological approach. We found that cell toxicity and mitochondrial depolarization by BTZ were increased upon inhibition of HO-1 and TLR4 by using tin protoporphyrin IX (SnPP) and TAK-242, respectively. Furthermore, the combination of TAK-242 and BTZ activated mitophagy and decreased the unfolded protein response (UPR) survival pathway in association with a downregulation in HO-1 expression. Notably, BTZ in combination with SnPP induced effects mirroring the treatment with TAK-242/BTZ, resulting in a blockade of TLR4 upregulation. Interestingly, treatment of cells with either hemin, an HO-1 inducer, or supplementation with carbon monoxide (CO), a by-product of HO-1 enzymatic activity, increased TLR4 expression. In conclusion, we showed that treatment of MM cells with BTZ triggers the TLR4/HO-1/CO axis, serving as a stress-responsive signal that leads to increased cell survival while protecting mitochondria against BTZ and ultimately promoting drug resistance.

The combined derangements in mitochondria network, function and dynamics can affect metabolism and ATP production, redox homeostasis and apoptosis triggering, contributing to cancer development in many different complex ways. In hematological malignancies, there is a strong relationship between cellular metabolism, mitochondrial bioenergetics, interconnections with supportive microenvironment and drug resistance. Lymphoma and chronic lymphocytic leukemia cells, e.g., adapt to intrinsic oxidative stress by increasing mitochondrial biogenesis. In other hematological disorders such as myeloma, on the contrary, bioenergetics changes, associated to increased mitochondrial fitness, derive from the adaptive response to drug-induced stress. In the bone marrow niche, a reverse Warburg effect has been recently described, consisting in metabolic changes occurring in stromal cells in the attempt to metabolically support adjacent cancer cells. Moreover, a physiological dynamic, based on mitochondria transfer, between tumor cells and their supporting stromal microenvironment has been described to sustain oxidative stress associated to proteostasis maintenance in multiple myeloma and leukemia. Increased mitochondrial biogenesis of tumor cells associated to acquisition of new mitochondria transferred by mesenchymal stromal cells results in augmented ATP production through increased oxidative phosphorylation (OX-PHOS), higher drug resistance, and resurgence after treatment. Accordingly, targeting mitochondrial biogenesis, electron transfer, mitochondrial DNA replication, or mitochondrial fatty acid transport increases therapy efficacy. In this review, we summarize selected examples of the mitochondrial derangements in hematological malignancies, which provide metabolic adaptation and apoptosis resistance, also supported by the crosstalk with tumor microenvironment. This field promises a rational design to improve target-therapy including the metabolic phenotype.

Fish skin mucus is an important component of the innate immune mechanism and provides a first barrier against pathogens. Although several studies investigated and demonstrated the antimicrobial properties of skin mucus of fishes, most of them tested antimicrobial properties against marine microbial strains as potential use in aquaculture. Furthermore, most of these studies concerned freshwater species, and far less attention was given to skin mucus of marine fishes for potential application in human health and research toward its antimicrobial properties. This review outlines the importance of marine fishes as an important and effective source of antimicrobial drugs against several human pathogens, thus providing a highly available and low-cost tool to counteract clinical infections.

Iron plays a major role in multiple processes involved in cell homeostasis such as metabolism, respiration and DNA synthesis. Cancer cells exhibit pronounced iron retention as compared to healthy counterpart. This phenomenon also occurs in multiple myeloma (MM), a hematological malignancy characterized by terminally differentiated plasma cells (PCs), in which serum ferritin levels have been reported as a negative prognostic marker. The aim of current study is to evaluate the potential role of iron metabolism in promoting drug resistance in myeloma cancer cells with particular regard to the interactions between PCs and tumor-associated macrophages (TAMs) as a source of iron. Our data showed that myeloma cell lines are able to intake and accumulate iron and thus, increasing their scavenger antioxidant-related genes and mitochondrial mass. We further demonstrated that PCs pre-treated with ferric ammonium citrate (FAC) decreased bortezomib (BTZ)-induced apoptosis in vitro and successfully engrafted in zebrafish larvae treated with BTZ. Treating human macrophages with FAC, we observed a switch toward a M2-like phenotype associated with an increased expression of anti-inflammatory markers such as ARG1, suggesting the establishment of an iron-mediated immune suppressive tumor microenvironment favouring myeloma growth. Using mfap4:tomato mutant zebrafish larvae, we further confirmed the increase of PCs-monocytes interactions after FAC treatment which favour BTZ-resistance. Taken together our data support the hypothesis that targeting iron trafficking in myeloma microenvironment may represent a promising strategy to counteract a tumor-supporting milieu and drug resistance.

In 2021 the World Health Organization published the fifth and latest version of the Central Nervous System tumors classification, which incorporates and summarizes a long list of updates from the Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy work. Among the adult-type diffuse gliomas, glioblastoma represents most primary brain tumors in the neuro-oncology practice of adults. Despite massive efforts in the field of neuro-oncology diagnostics to ensure a proper taxonomy, the identification of glioblastoma-tumor subtypes is not accompanied by personalized therapies, and no improvements in terms of overall survival have been achieved so far, confirming the existence of open and unresolved issues. The aim of this review is to illustrate and elucidate the state of art regarding the foremost biological and molecular mechanisms that guide the beginning and the progression of this cancer, showing the salient features of tumor hallmarks in glioblastoma. Pathophysiology processes are discussed on molecular and cellular levels, highlighting the critical overlaps that are involved into the creation of a complex tumor microenvironment. The description of glioblastoma hallmarks shows how tumoral processes can be linked together, finding their involvement within distinct areas that are engaged for cancer-malignancy establishment and maintenance. The evidence presented provides the promising view that glioblastoma represents interconnected hallmarks that may led to a better understanding of tumor pathophysiology, therefore driving the development of new therapeutic strategies and approaches.

The corneal epithelium, the outermost layer of the cornea, acts as a dynamic barrier preventing access to harmful agents into the intraocular space. It is subjected daily to different insults, and ultraviolet B (UV-B) irradiation represents one of the main causes of injury. In our previous study, we demonstrated the beneficial effects of pituitary adenylate cyclase-activating polypeptide (PACAP) against UV-B radiation damage in the human corneal endothelium. Some of its effects are mediated through the activation of the intracellular factor, known as the activity-dependent protein (ADNP). In the present paper, we have investigated the role of ADNP and the small peptide derived from ADNP, known as NAP, in the corneal epithelium. Here, we have demonstrated, for the first time, ADNP expression in human and rabbit corneal epithelium as well as its protective effect by treating the corneal epithelial cells exposed to UV-B radiations with NAP. Our results showed that NAP treatment prevents ROS formation by reducing UV-B-irradiation-induced apoptotic cell death and JNK signalling pathway activation. Further investigations are needed to deeply investigate the possible therapeutic use of NAP to counteract corneal UV-B damage.

Breast cancer is the most frequent tumor and the leading cause of cancer deaths in women. In recent years, lactate metabolism and, in particular, its receptor GPR81 have been shown to play a vital role in cancer biology. GPR81 is upregulated in breast cancer and promotes tumor growth by tumor cell-derived lactate. Therefore, the search for possible crosstalk and the involvement of new molecules capable of generating this pathology is always in continuous development. In this study, the relationship between GPR81 and IGFBP6 protein in tumor growth and oxidative stress in the human breast cancer cell line MDA-MB-231 was studied. Cells were treated with lactate or the GPR81 receptor agonist and antagonist 3,5-DHBA and 3-OBA, respectively. In addition, oxidative stress and proliferation were also evaluated in cells challenged with the recombinant IGFBP6 protein. Our data showed that lactate induced cell proliferation and wound healing of the MDA-231 breast cancer cell through the overexpression of both the lactate receptor GPR81 and IGFBP6. The increase in IGFBP6 was able, in turn, to improve the mitochondrial fitness and redox state, as suggested by the reduced levels of mitochondrial ROS production after IGFBP6 treatment, presumably mediated by the increase in the ROS detoxifying genes HMOX1, GSTK1 and NQO1. In conclusion, our data highlight a novel axis between GPR81 and IGFBP6 in MDA-231 cells able to modulate lactate metabolism and oxidative stress. This complex signaling may represent a new therapeutic target for breast cancer.

Glioblastoma (GBM) represents the most common primary brain tumor within the adult population. Current therapeutic options are still limited by high rate of recurrences and signalling axes that promote GBM aggressiveness. The contribution of gap junctions (GJs) to tumor growth and progression has been proven by experimental evidence. Concomitantly, tumor microenvironment has received increasing interest as a critical process in dysregulation and homeostatic escape, finding a close link between molecular mechanisms involved in connexin 43 (CX43)-based intercellular communication and tumorigenesis. Moreover, evidence has come to suggest a crucial role of sonic hedgehog (SHH) signalling pathway in GBM proliferation, cell fate and differentiation. Herein, we used two human GBM cell lines, modulating SHH signalling and CX43-based intercellular communication in in vitro models using proliferation and migration assays. Our evidence suggests that modulation of the SHH effector smoothened (SMO), by using a known agonist (i.e., purmorphamine) and a known antagonist (i.e., cyclopamine), affects the CX43 expression levels and therefore the related functions. Moreover, SMO activation also increased cell proliferation and migration. Importantly, inhibition of CX43 channels was able to prevent SMO-induced effects. SHH pathway and CX43 interplay acts inducing tumorigenic program and supporting cell migration, likely representing druggable targets to develop new therapeutic strategies for GBM.

Alzheimer’s disease is a progressive, devastating, and irreversible brain disorder that, day by day, destroys memory skills and social behavior. Despite this, the number of known genes suitable for discriminating between AD patients is insufficient. Among the genes potentially involved in the development of AD, there are the chitinase-like proteins (CLPs) CHI3L1, CHI3L2, and CHID1. The genes of the first two have been extensively investigated while, on the contrary, little information is available on CHID1. In this manuscript, we conducted transcriptome meta-analysis on an extensive sample of brains of healthy control subjects (n = 1849) (NDHC) and brains of AD patients (n = 1170) in order to demonstrate CHID1 involvement. Our analysis revealed an inverse correlation between the brain CHID1 expression levels and the age of NDHC subjects. Significant differences were highlighted comparing CHID1 expression of NDHC subjects and AD patients. Exclusive in AD patients, the CHID1 expression levels were correlated positively to calcium-binding adapter molecule 1 (IBA1) levels. Furthermore, both in NDHC and in AD patient’s brains, the CHID1 expression levels were directly correlated with calbindin 1 (CALB1) and neurogranin (NRGN). According to brain regions, correlation differences were shown between the expression levels of CHID1 in prefrontal, frontal, occipital, cerebellum, temporal, and limbic system. Sex-related differences were only highlighted in NDHC. CHID1 represents a new chitinase potentially involved in the principal processes underlying Alzheimer’s disease.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by hematopoietic stem-cell-derived clonal proliferation, leading to bone marrow (BM) fibrosis. Hematopoiesis alterations are closely associated with modifications of the BM microenvironment, characterized by defective interactions between vascular and endosteal niches. As such, neoangiogenesis, megakaryocytes hyperplasia and extensive bone marrow fibrosis, followed by osteosclerosis and bone damage, are the most relevant consequences of PMF. Moreover, bone tissue deposition, together with progressive fibrosis, represents crucial mechanisms of disabilities in patients. Although the underlying mechanisms of bone damage observed in PMF are still unclear, the involvement of cytokines, growth factors and bone marrow microenvironment resident cells have been linked to disease progression. Herein, we focused on the role of megakaryocytes and their alterations, associated with cytokines and chemokines release, in modulating functions of most of the bone marrow cell populations and in creating a complex network where impaired signaling strongly contributes to progression and disabilities.

The proteasome inhibitor bortezomib (BTZ) has emerged as an effective drug for the treatment of multiple myeloma even though many patients relapse from BTZ therapy. The present study investigated the metabolic pathways underlying the acquisition of bortezomib resistance in multiple myeloma. We used two different clones of multiple myeloma cell lines exhibiting different sensitivities to BTZ (U266 and U266-R) and compared them in terms of metabolic profile, mitochondrial fitness and redox balance homeostasis capacity. Our results showed that the BTZ-resistant clone (U266-R) presented increased glycosylated UDP-derivatives when compared to BTZ-sensitive cells (U266), thus also suggesting higher activities of the hexosamine biosynthetic pathway (HBP), regulating not only protein O- and N-glycosylation but also mitochondrial functions. Notably, U266-R displayed increased mitochondrial biogenesis and mitochondrial dynamics associated with stronger antioxidant defenses. Furthermore, U266-R maintained a significantly higher concentration of substrates for protein glycosylation when compared to U266, particularly for UDP-GlcNac, thus further suggesting the importance of glycosylation in the BTZ pharmacological response. Moreover, BTZ-treated U266-R showed significantly higher ATP/ADP ratios and levels of ECP and also exhibited increased mitochondrial fitness and antioxidant response. In conclusions, our findings suggest that the HBP may play a major role in mitochondrial fitness, driving BTZ resistance in multiple myeloma and thus representing a possible target for new drug development for BTZ-resistant patients.

The use of nanomaterials in several application fields has received in the last decades a great attention due to their peculiar properties, but also raised many doubts about possible toxicity when these materials are used for some specific applications, such as water purification. Indeed a careful investigation is needed in order to exclude possible harmful side effects related to the use of nanotechnology. Nanoparticles effects on the marine organisms may depend on their chemical composition, size, surface structure, solubility, shape and how the individual nanoparticles aggregate together. In order to make the most of their potential, without polluting the environment, many researchers are trying to trap them into some kind of matrix that keeps them active but avoids their dispersion in the environment. In this study we have tested nanocomposite membranes prepared using Nafion polymer combined with various fillers, such as anatase-type TiO2 nanoparticles and graphene oxide. The non-toxicity of these nanocomposites, already shown to be effective for water purification applications in our previous studies, was recognized by testing the effect of the different materials on zebrafish embryos. Zebrafish was considered an excellent model for ecotoxicological studies and for this motivation zebrafish embryos were exposed to different concentrations of free nanoparticles and to the nanocomposite membranes. As biomarkers of exposure, we evaluated the expression of heme-oxygenase 1 and inducible Nitric Oxide Synthases by immunohistochemistry and gene expression. Embryo toxicity test showed that nor sublethal effects neither mortality were caused by the different nanoparticles and nano-systems tested. Only zebrafish larvae exposed to free nanoparticles have shown a different response to antibodies anti-heme-oxygenase 1 and anti- inducible Nitric Oxide Synthases. The immunolocalization analysis in fact has highlighted an increase in the synthesis of these biomarkers.

Nanotechnology is rapidly growing with nanoparticles produced and utilized in a wide range of commercial products worldwide. Among the different types of nanomaterials produced, silver nanoparticles (AgNPs) occupy a predominant position and they are used in electronics, clothing, food industry, cosmetics and medical devices. Nanosilver has also showed excellent performance in antibacterial application. Nowadays, the increasing use of AgNPs has put the evidence on their possible toxicity to the human health and the impact on the environment. This paper focus on adverse effects of AgNPs in adult of Danio rerio. Fishes exposed to increasing concentrations (8, 45, and 70 μg/l) silver nanoparticles (AgNPs, 25 nm in average diameter) and after treatment for 30 days, was quickly euthanized in MS-222. We have evaluated bioaccumulation of AgNPs using ICP-MS and analyzed histological changes, biomarkers of oxidative damage and gene expression in the gut, liver and gills tissues of AgNPs-treated zebrafish. The histological analysis showed lesions of secondary lamellae of the gills with different degrees of toxicity such as hyperplasia, lamellar fusion, subepithelial edema, and even in some cases telangiectasia. Huge necrosis of the intestinal villi was found in the gut. No lesion was detected in the liver. The analysis revealed a high expression of metallothioneins 1 (MTs 1) in animals exposed to AgNPs compared to the control group. The ICP-MS analysis shows that the amount of particles absorbed in all treated samples is almost the same. We can affirm that AgNPs toxicity linked more to their size and state of aggregation than to their concentrations. Silver nanoparticles can damage gills and gut because they are able to pass through the mucosal barrier thanks to their small size. The damage is still reversible because it is not documented injury to the basal membrane.

Azacytidine (5-AZA) is the standard first-choice treatment for high-risk myelodysplasia (MDS) patients. However, the clinical outcome for those patients who interrupt treatment or whose disease failed to respond is very poor. In order to identify the cellular pathways that are modified by long-term exposure to 5-AZA, we evaluated key proteins associated with the autophagy pathway by reverse-phase microarray (RPPA). Comparing bone marrow mononucleated cells (BMMCs) obtained from 20 newly-diagnosed patients and after four 5-AZA cycles we found an increased autophagy signaling. We then evaluated ex-vivo the effect of the combination of 5-AZA with autophagy inhibitors chloroquine (CQ) and leupeptin. Since 5-AZA and CQ showed synergism due to an increase of basal autophagy after 5-AZA exposure, we adopted a sequential treatment treating BMMCs with 5 μM 5-AZA for 72 h followed by 10 μM CQ for 24 h and found increased apoptosis, associated to a reduction of G2M phase and increase in G0-G1 phase. Long-term exposure to 5-AZA induced the reduction of the autophagic marker SQSTM1/p62, reversible by CQ or leupeptin exposure. In conclusion, we identified autophagy as a compensatory pathway occurring in MDS-BM after long-term exposure to 5-AZA and we provided evidences that a sequential treatment of 5-AZA followed by CQ could improve 5-AZA efficacy, providing novel insight for tailored therapy in MDS patients progressing after 5-AZA therapy.

Hypertrophic obesity inhibits activation of peroxisome proliferators-activated receptor gamma (PPARγ), considered the key mediator of the fully differentiated and insulin sensitive adipocyte phenotype. We examined the effects of Caffeic Acid Phenethyl Ester (Cape), isolated from propolis, a honeybee hive product, on Adipose Stem Cells (ASCs) differentiation to the adipocyte lineage. Finally we tested the effects of Cape on insulin-resistant adipocytes. Quantification of Oil Red O-stained cells showed that lipid droplets decreased following Cape treatment as well as radical oxygen species formation. Additionally, exposure of ASC to high glucose levels decreased adiponectin and increased proinflammatory cytokines mRNA levels, which were reversed by Cape-mediated increase of insulin sensitivity. Cape treatment resulted in decreased triglycerides synthesis and increased beta-oxidation. Exposure of ASCs to Lipopolysaccharide (LPS) induced a reduction of PPARγ, an increase of IL-6 levels associated with a well-known stimulation of lipolysis; Cape partially attenuated the LPS-mediated effects. These observations reveal the main role of PPARγ in the adipocyte function and during ASC differentiation. As there is now substantial interest in functional food and nutraceutical products, the observed therapeutic value of Cape in insulin-resistance related diseases should be taken into consideration.

It is well known that mesenchymal stem cells (MSC) have a role in promotion of tumor growth, survival and drug-resistance in chronic myeloid leukemia (CML). Recent reports indicated that a subpopulation of myeloid cells, defined as granulocyte-like myeloid-derived suppressor cells (G-MDSC) is increased in these patients. So far, the role of MSC in MDSC expansion and activation into the BM microenvironment remains unexplored. To address this question, here we use a specific experimental model in vitro, co-culturing MSC with peripheral blood mononucleated cells (PBMC) from normal individuals, in order to generate MSC-educated G-MDSC. Although MSC of healthy donors (HD) and CML patients were able to generate the same amount of MDSC, only CML-MSC-educated G-MDSC exhibited suppressive ability on autologous T lymphocytes. In addition, compared with HD-MSC, CML-MSC over-expressed some immunomodulatory factors including TGFβ, IL6 and IL10, that could be involved in MDSC activation. CML-MSC-educated G-MDSC expressed higher levels of ARG1, TNFα, IL1β, COX2 and IL6 than G-MDSC isolated from co-culture with HD-MSC. Our data provide evidence that CML-MSC may play a critical role in tumor microenvironment by orchestrating G-MDSC activation and regulating T lymphocytes-mediated leukemia surveillance, thus contributing to CML immune escape.

Resistance to chemotherapy occurs in various diseases (i.e., cancer and infection), and for this reason, both are very difficult to treat. Therefore, novel antimicrobial and chemotherapic drugs are needed for effective antibiotic therapy. The aim of the present study was to assess the antimicrobial and anti-proliferative effects of skin mucus derived from Dasyatis pastinaca (Linnaeus, 1758). Our results showed that skin mucus exhibited a significant and specific antibacterial activity against Gram-negative bacteria but not against Gram-positive bacteria. Furthermore, we also observed a significant antifungal activity against some strains of Candida spp. Concerning anti-proliferative activity, we showed that fish mucus was specifically toxic for acute leukemia cells (HL60) with an inhibition of proliferation in a dose dependent manner (about 52% at 1000 μg/mL of fish skin mucous, FSM). Moreover, we did not observe effects in healthy cells, in neuroblastoma cells (SH-SY5Y), and multiple myeloma cell lines (MM1, U266). Finally, it exhibited strong expression and activity of chitinase which may be responsible, at least in part, for the aforementioned results.

Tumor immune tolerance can derive from the recruitment of suppressor cell population, including myeloid derived suppressor cells (MDSCs), able to inhibit T cells activity. We identified a significantly expanded MDSCs population in chronic myeloid leukemia (CML) patients at diagnosis that decreased to normal levels after imatinib therapy. In addition, expression of arginase 1 (Arg1) that depletes microenvironment of arginine, an essential aminoacid for T cell function, resulted in an increase in patients at diagnosis. Purified CML CD11b+CD33+CD14-HLADR- cells markedly suppressed normal donor T cell proliferation in vitro. Comparing CML Gr-MDSCs to autologous polymorphonuclear leukocytes (PMNs) we observed a higher Arg1 expression and activity in PMNs, together with an inhibitory effect on T cells in vitro. Our data indicate that CML cells create an immuno-tolerant environment associated to MDSCs expansion with immunosuppressive capacity mediated by Arg1. In addition, we demonstrated for the first time also an immunosuppressive activity of CML PMNs, suggesting a strong potential immune escape mechanism created by CML cells, which control the anti-tumor reactive T cells. MDSCs should be monitored in imatinib discontinuation trials to understand their importance in relapsing patients.