Skeletal muscle of young females under resistance exercise exhibits a unique innate immune cell infiltration profile compared to males and elderly individuals.

Muscle damage resulting from physical activities such as exercise triggers an immune response crucial for tissue repair and recovery. This study investigates the immune cell profiles in muscle biopsies of individuals engaged in resistance exercise (RE) and explores the impact of age and sex on the immune response following exercise-induced muscle damage. Microarray datasets from muscle biopsies of young and old subjects were analyzed, focusing on the gene expression patterns associated with immune cell activation. Genes were compared with immune cell signatures to reveal the cellular landscape during exercise. Results show that the most significant modulated gene after RE was Folliculin Interacting Protein 2 (FNIP2) a crucial regulator in cellular homeostasis. Moreover, the transcriptome was stratified based on the expression of FNIP2 and the 203 genes common to the groups obtained based on sex and age. Gene ontology analysis highlighted the FLCN-FNIP1-FNIP2 complex, which exerts as a negative feedback loop to Pi3k-Akt-mTORC1 pathway. Furthermore, we highlighted that the young females exhibit a distinct innate immune cell activation signature compared to males after a RE session. Specifically, young females demonstrate a notable overlap with dendritic cells (DCs), M1 macrophages, M2 macrophages, and neutrophils, while young males overlap with M1 macrophages, M2 macrophages, and motor neurons. Interestingly, in elderly subjects, both sexes display M1 macrophage activation signatures. Comparison of young and elderly signatures reveals an increased M1 macrophage percentage in young subjects. Additionally, common genes were identified in both sexes across different age groups, elucidating biological functions related to cell remodeling and immune activation. This study underscores the intricate interplay between sex, age, and the immune response in muscle tissue following RE, offering potential directions for future research. Nevertheless, there is a need for further studies to delve deeper and confirm the dynamics of immune cells in response to exercise-induced muscle damage.

Microglia and glioblastoma heterocellular interplay sustains tumour growth and proliferation as an off-target effect of radiotherapy.

Glioblastoma (GBM), a WHO grade IV glioma, is a malignant primary brain tumour for which combination of surgery, chemotherapy and radiotherapy is the first-line approach despite adverse effects. Tumour microenvironment (TME) is characterized by an interplay of cells and soluble factors holding a critical role in neoplastic development. Significant pathophysiological changes have been found in GBM TME, such as glia activation and oxidative stress. Microglia play a crucial role in favouring GBM growth, representing target cells of immune escape mechanisms. Our study aims at analysing radiation-induced effects in modulating intercellular communication and identifying the basis of protective mechanisms in radiation-naïve GBM cells. Tumour cells were treated with conditioned media (CM) derived from 0, 2 or 15 Gy irradiated GBM cells or 0, 2 or 15 Gy irradiated human microglia. We demonstrated that irradiated microglia promote an increase of GBM cell lines proliferation through paracrine signalling. On the contrary, irradiated GBM-derived CM affect viability, triggering cell death mechanisms. In addition, we investigated whether these processes involve mitochondrial mass, fitness and oxidative phosphorylation and how GBM cells respond at these induced alterations. Our study suggests that off-target radiotherapy modulates microglia to support GBM proliferation and induce metabolic modifications.

(+)-Lipoic acid reduces mitochondrial unfolded protein response and attenuates oxidative stress and aging in an in vitro model of non-alcoholic fatty liver disease.

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 ß-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.

Correction: Carota et al. Neuroprotective Role of α-Lipoic Acid in Iron-Overload-Mediated Toxicity and Inflammation in In Vitro and In Vivo Models. Antioxidants 2022, 11, 1596.

In the original publication [...].

The Gestational Pathologies Effect on the Human Milk Redox Homeostasis: A First Step towards Its Definition.

BACKGROUND: Human Milk (HM) is a dynamic nourishment; its composition is influenced by several conditions such as gestational age, maternal diet and ethnicity. It appears important to evaluate the impact that gestational pathologies have on HM components and if their presence, as a source of oxidative stress in the mother, influence milk's redox homeostasis. To assess the effect of Preeclampsia (PE) and Gestational Diabetes Mellitus (GDM) on some aspects of human milk redox homeostasis, we chose to investigate both oxidative and antioxidant aspects, with, respectively, Lipid hydroperoxides (LOOHs) and Glutathione (GSH). METHODS: Women with PE, GDM and who were healthy were recruited for this study. Colostrum, transitional and mature milk samples were collected. GSH and LOOHs levels were measured using a spectrophotometric test. To investigate the effect of pathology on redox homeostasis, a mixed linear model with unistructural covariance structure was performed. RESULTS: A total of 120 mothers were recruited. The GSH concentration results were significantly lower in GDM women than in healthy women only in colostrum (p < 0.01). No other differences emerged. LOOHs was not detectable in almost all the samples. DISCUSSION: Our study is the first to extensively evaluate these components in the HM of women with these gestational pathologies. The main observation is that GDM can alter the GSH level of HM, mainly in colostrum.

Sigma-1 Receptor Inhibition Reduces Mechanical Allodynia and Modulate Neuroinflammation in Chronic Neuropathic Pain.

Neuropathic pain is one of the most debilitating forms of chronic pain, resulting from an injury or disease of the somatosensory nervous system, which induces abnormal painful sensations including allodynia and hyperalgesia. Available treatments are limited by severe side-effects and reduced efficacy in the chronic phase of the disease. Sigma-1 receptor (σ1R) has been identified as a chaperone protein, which modulate opioid receptors activities and the functioning of several ion channels, exerting a role in pain transmission. As such, it represents a druggable target to treat neuropathic pain. This study aims at investigating the therapeutic potential of the novel compound (+)-2R/S-LP2, a σ1R antagonist, in reducing painful behaviour and modulating the neuroinflammatory environment. We showed that repeated administration of the compound significantly inhibited mechanical allodynia in neuropathic rats, increasing the withdrawal threshold as compared to CCI-vehicle rats. Moreover, we found that (+)-2R/S-LP2-mediated effects resolve the neuroinflammatory microenvironment by reducing central gliosis and pro-inflammatory cytokines expression levels. This effect was coupled with a significant reduction of connexin 43 (Cx43) expression levels and gap junctions/hemichannels mediated microglia-to-astrocyte communication. These results suggest that inhibition of σ1R significantly attenuates neuropathic pain chronicization, thus representing a viable effective strategy.

Anaplastic thyroid cancer cells reduce CD71 levels to increase iron overload tolerance.

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.

Cytotoxicity, mutagenicity and genotoxicity of electronic cigarettes emission aerosols compared to cigarette smoke: the REPLICA project.

Concerns have recently increased that the integrity of some scientific research is questionable due to the inability to reproduce the claimed results of some experiments and thereby confirm that the original researcher's conclusions were justified. This phenomenon has been described as 'reproducibility crisis' and affects various fields from medicine to basic applied sciences. In this context, the REPLICA project aims to replicate previously conducted in vitro studies on the toxicity of cigarette smoke and e-cigarette aerosol, sometimes adding experiments or conditions where necessary, in order to verify the robustness and replicability of the data. In this work the REPLICA Team replicated biological and toxicological assessment published by Rudd and colleagues in 2020. As in the original paper, we performed Neutral Red Uptake (NRU) assay for the evaluation of cytotoxicity, Ames test for the evaluation of mutagenesis and In Vitro Micronuclei (IVMN) assay for the evaluation of genotoxicity on cells treated with cigarette smoke or e-cigarette aerosol. The results showed high cytotoxicity, mutagenicity and genotoxicity induced by cigarette smoke, but slight or no cytotoxic, mutagenic and genotoxic effects induced by the e-cigarette aerosol. Although the two studies presented some methodological differences, the findings supported those previously presented by Rudd and colleagues.

Analysis of oral lichen planus severity on micro-RNA linked with malignant transformation risks.

OBJECTIVE: The present study evaluated the oral tissue expression of micro-RNA (miRNAs) linked to the potential malignant evolution of oral lichen planus (OLP). Furthermore, the correlation between OLP severity and miRNAs expression was assessed, and possible predictors of miRNAs in OLP patients were identified. METHODS: The present study enrolled 41 patients with OLP (median age 58 years) and 42 healthy controls (median age 59 years). In each patient, miRNA levels (miR-7a-3p,-7a2-3p,-7a-5p,-21-3p,-21-5p,-100-3p,-100-5p,-125b-2-3p,-125b-5p,-200b-3p,-200b-5p) were assessed and analyzed through reverse transcription polymerase chain reaction. Clinical parameters and the eventual presence of OLP symptoms, signs, and disease severity scores in each patient were reported using an anamnestic questionnaire. RESULTS: In comparison with healthy controls, OLP patients showed significantly higher miR-7a-3p,-7a-2-3p,-21-3p, miR-21-5p and miR-100-5p levels (p < 0.05) and significantly lower miR-125b-2-3p,-125b-5p,-200b-3p, and -200b-5p levels (p < 0.05). Furthermore, OLP symptoms and signs and disease severity scores were significantly correlated and were also predictors of all analyzed miRNAs (p < 0.05). CONCLUSIONS: In comparison with healthy subjects, OLP patients exhibited unbalanced oral miRNAs expression linked to the risk of potential malignant evolution of OLP. Furthermore, some miRNAs were correlated with OLP extent and were significant predictors of OLP symptoms, signs, and disease severity scores.

The Pleiotropic Effects of Fumarate: From Mitochondrial Respiration to Epigenetic Rewiring and DNA Repair Mechanisms.

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.

Soluble peptidoglycan fragments produced by Limosilactobacillus fermentum with antiproliferative activity are suitable for potential therapeutic development: A preliminary report.

Currently, the use of probiotic strains and their products represents a promising innovative approach as an antagonist treatment against many human diseases. Previous studies showed that a strain of Limosilactobacillus fermentum (LAC92), previously defined as Lactobacillus fermentum, exhibited a suitable amensalistic property. The present study aimed to purify the active components from LAC92 to evaluate the biological properties of soluble peptidoglycan fragments (SPFs). The cell-free supernatant (CFS) and bacterial cells were separated after 48 h of growth in MRS medium broth and treated for isolation of SPFs. Antimicrobial activity and proliferation analysis on the human cell line HTC116 were performed using technologies such as xCELLigence, count and viability, and clonogenic analysis. MALDI-MS investigation and docking analysis were performed to determine the molecular structure and hypothetical mode of action, respectively. Our results showed that the antimicrobial activity was mainly due to SPFs. Moreover, the results obtained when investigating the SPF effect on the cell line HCT116 showed substantial preliminary evidence, suggesting their significant cytostatic and quite antiproliferative properties. Although MALDI was unable to identify the molecular structure, it was subsequently revealed by analysis of the bacterial genome. The amino acid structure is called peptide 92. Furthermore, we confirmed by molecular docking studies the interaction of peptide 92 with MDM2 protein, the negative regulator of p53. This study showed that SPFs from the LAC92 strain exerted anticancer effects on the human colon cancer HCT116 cell line via antiproliferation and inducing apoptosis. These findings indicated that this probiotic strain might be a potential candidate for applications in functional products in the future. Further examination is needed to understand the specific advantages of this probiotic strain and improve its functional features to confirm these data. Moreover, deeper research on peptide 92 could increase our knowledge and help us understand if it will be possible to apply to specific diseases such as CRC.

Mesenchymal stromal cells in tumor microenvironment remodeling of BCR-ABL negative myeloproliferative diseases.

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- ß 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.

Lactate trafficking inhibition restores sensitivity to proteasome inhibitors and orchestrates immuno-microenvironment in multiple myeloma.

Metabolic changes of malignant plasma cells (PCs) and adaptation to tumour microenvironment represent one of the hallmarks of multiple myeloma (MM). We previously showed that MM mesenchymal stromal cells are more glycolytic and produce more lactate than healthy counterpart. Hence, we aimed to explore the impact of high lactate concentration on metabolism of tumour PCs and its impact on the efficacy of proteasome inhibitors (PIs). Lactate concentration was performed by colorimetric assay on MM patient's sera. The metabolism of MM cell treated with lactate was assessed by seahorse and real time Polymerase Chain Reaction (PCR). Cytometry was used to evaluate mitochondrial reactive oxygen species (mROS), apoptosis and mitochondrial depolarization. Lactate concentration resulted increased in MM patient's sera. Therefore, PCs were treated with lactate and we observed an increase of oxidative phosphorylation-related genes, mROS and oxygen consumption rate. Lactate supplementation exhibited a significant reduction in cell proliferation and less responsive to PIs. These data were confirmed by pharmacological inhibition of monocarboxylate transporter 1 (MCT1) by AZD3965 which was able to overcame metabolic protective effect of lactate against PIs. Consistently, high levels of circulating lactate caused expansion of Treg and monocytic myeloid derived suppressor cells and such effect was significantly reduced by AZD3965. Overall, these findings showed that targeting lactate trafficking in TME inhibits metabolic rewiring of tumour PCs, lactate-dependent immune evasion and thus improving therapy efficacy.

Resistin-like beta reduction is associated to low survival rate and is downregulated by adjuvant therapy in colorectal cancer patients.

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.

Impact of periodontitis on gingival crevicular fluid miRNAs profiles associated with cardiovascular disease risk.

BACKGROUND AND OBJECTIVE: Recent emerging evidence has shown that microRNA (miRNAs) is involved in several epigenetic processes linked with periodontitis, increased oxidative stress and cardiovascular disease (CVD). The present study aimed to assess the impact of periodontitis on gingival crevicular fluid (GCF) miRNAs expression associated with CVD risk and to evaluate possible confounders that influenced this association. MATERIALS AND METHODS: For the present study, healthy controls (n = 28) and subjects with CVD (n = 28), periodontitis (n = 30) and periodontitis + CVD (n = 29) were enrolled. All subjects underwent regular periodontal examinations and blood sampling. In addition, GCF sampling was performed, and miRNAs 7a-5p, 21-3p, 21-5p, 100-5p, 125-5p, 200b-3p, and 200b-5p expression was analyzed using a real-time quantitative polymerase chain reaction (RT-PCR). RESULTS: The results showed that periodontitis and periodontitis + CVD subjects presented significantly different GCF miRNAs expression compared to healthy controls and CVD subjects. More specifically, compared to healthy controls and CVD, subjects with periodontitis and periodontitis + CVD showed higher GCF miRNA 7a-5p, miRNA 21-3p, miRNA 21-5p, miRNA 200b-3p, and miRNA 200b-5p (p < .05) and lower miRNA 100-5p, miRNA 125-5p levels (p < .05). Furthermore, the multivariate regression analysis evidenced that periodontitis (miRNA 21-3p, 100-5p) and periodontal inflamed surface area (PISA) (miRNA 7a-5p, 21-3p, 21-5p, 100-5p, 125-5p, 200b-3p) were significant predictors of GCF miRNAs concentration (p < .05). CONCLUSION: The results of the study highlighted that the periodontitis and periodontitis + CVD group showed higher GCF miRNAs expression than healthy controls and CVD subjects. Furthermore, periodontitis and its extent (PISA) were revealed as significant predictors of GCF miRNAs associated with CVD risk.

Comparative assessment of electronic nicotine delivery systems aerosol and cigarette smoke on endothelial cell migration: The Replica Project.

Cigarette smoking is associated with impairment of repair mechanisms necessary for vascular endothelium homeostasis. Reducing the exposure to smoke toxicants may result in the mitigation of the harmful effect on the endothelium and cardiovascular disease development. Previous investigations evaluated in vitro the effect of electronic cigarette (EC) compared with cigarette smoke demonstrating a significant reduction in human umbilical vein endothelial cells (HUVECs) migration inhibition following EC aerosol exposure. In the present study, we replicated one of these studies, evaluating the effects of cigarette smoke on endothelial cell migration compared with aerosol from EC and heated tobacco products (HTPs). We performed an in vitro scratch wound assay on endothelial cells with a multi-center approach (ring-study) to verify the robustness and reliability of the results obtained in the replicated study, also testing the effect of aerosol from two HTPs on endothelial cells. Consistently with the original study, we observed a substantial reduction of the effects of aerosol from EC and HTPs on endothelial cell migration compared with cigarette smoke. While cigarette smoke reduced endothelial wound healing ability already at low concentrations (12.5%) and in a concentration-dependent manner, EC and HTPs aerosol showed no effect on endothelial cells until 80%-100% concentrations. In conclusion, our study further confirms the importance of EC and tobacco heated products as a possible harm reduction strategy for cardiovascular diseases development in smokers.

In vitro cytoxicity profile of e-cigarette liquid samples on primary human bronchial epithelial cells.

Cigarette smoke is associated to severe chronic diseases. The most harmful components of cigarette smoke derive from the combustion process, which are significantly reduced in the electronic cigarette aerosol, thus providing a valid option in harm reduction strategies. To develop safer products, it is therefore necessary to screen electronic cigarette liquids (e-liquids) to meet high safety standards defined by government regulations. The aim of the present study was to evaluate the presence of metal- and plastic-derived contaminants in four different commercial e-liquids with high concentration of nicotine and their cytotoxic effect in normal human bronchial epithelial cells by a number of in vitro assays, in comparison with the 1R6F reference cigarette, using an air-liquid interface (ALI) exposure system. Moreover, we evaluated the effect of aerosol exposure on oxidative stress by measuring the production of reactive oxygen species and mitochondrial potential. Our results showed no contaminants in all e-liquids and a significantly reduced cytotoxic effect of e-liquid aerosol compared to cigarette smoke as well as a maintained mitochondria integrity. Moreover, no production of reactive oxygen species was detected with e-cigarette aerosol. In conclusion, these results support the reduced toxicity potential of e-cigs compared to tobacco cigarettes in an in vitro model resembling real life smoke exposure.