Multiple myeloma-derived exosomes are enriched of amphiregulin (AREG) and activate the epidermal growth factor pathway in the bone microenvironment leading to osteoclastogenesis

Background: Multiple myeloma (MM) is a clonal plasma cell malignancy associated with osteolytic bone disease. Recently, the role of MM-derived exosomes in the osteoclastogenesis has been demonstrated although the underlying mechanism is still unknown. Since exosomes-derived epidermal growth factor receptor ligands (EGFR) are involved in tumor-associated osteolysis, we hypothesize that the EGFR ligand amphiregulin (AREG) can be delivered by MM-derived exosomes and participate in MM-induced osteoclastogenesis. Methods: Exosomes were isolated from the conditioned medium of MM1.S cell line and from bone marrow (BM) plasma samples of MM patients. The murine cell line RAW264.7 and primary human CD14 + cells were used as osteoclast (OC) sources. Results: We found that AREG was specifically enriched in exosomes from MM samples and that exosomes-derived AREG led to the activation of EGFR in pre-OC, as showed by the increase of mRNA expression of its downstream SNAIL in both RAW264.7 and CD14 + cells. The presence of neutralizing anti-AREG monoclonal antibody (mAb) reverted this effect. Consequently, we showed that the effect of MM-derived exosomes on osteoclast differentiation was inhibited by the pre-treatment of exosomes with anti-AREG mAb. In addition, we demonstrated the ability of MM-derived AREG-enriched exosomes to be internalized into human mesenchymal stromal cells (MSCs) blocking osteoblast (OB) differentiation, increasing MM cell adhesion and the release of the pro-osteoclastogenic cytokine interleukin-8 (IL8). Accordingly, anti-AREG mAb inhibited the release of IL8 by MSCs suggesting that both direct and indirect effects are responsible for AREG-enriched exosomes involvement on MM-induced osteoclastogenesis. Conclusions: In conclusion, our data indicate that AREG is packed into MM-derived exosomes and implicated in OC differentiation through an indirect mechanism mediated by OBs

Tumour cell-derived small extracellular vesicles modulate macrophage immunosuppressive phenotype associated with PD-L1 expression

Introduction: Tumour-associated macrophages (TAMs) play a key role in promoting tumour progression, by exerting an immunosuppressive phenotype associated with M2 polarization and with the expression of CD204 and programmed cell death ligand 1 (PD-L1). It is well known that tumour-derived extracellular vesicles (TEVs) play a pivotal role in the tumour microenvironment, influencing TAM behaviour. The study was aimed to examine the effect of TEVs derived from colon cancer and multiple myeloma cells on macrophage functions. Methods: Non-polarized macrophages (M0) differentiated from THP-1 cells were co-cultured, for 3 up to 48 hours, with TEVs derived from a colon cancer cell line, SW480, and multiple myeloma cell line, MM1.S. The expression of M2 and TAM markers (respectively CD163 and CD204) as well as of PD-L1 and Interleukin 6 (IL6) were evaluated at mRNA and protein level. The apoptotic rate of CD3+ T cells cocultured with TEV-treated M0 macrophages was analysed by FACS. Results: Our results indicate that TEVs can significantly upregulate the expression of surface markers of M2-like phenotype (CD163) and TAM (CD204) as well as of PD-L1, inducing macrophages to acquire an immunosuppressive phenotype. In parallel, we found that TEVs were also able to induce a significant increase of IL6 expression at both mRNA and protein levels and to activate the STAT3 signalling pathway. Since PD-1/PD-L1 axis is involved in the inhibition of T cells, we assessed the ability of macrophages treated with TEVs to affect T cell viability. We found that CD3+ T cells co-cultured with TEVs-treated M0 showed an increase of their apoptotic rate in comparison to CD3 + T cells grown in the presence of untreated macrophages. Summary/Conclusion: Cumulatively, these preliminary data suggest that TEVs contribute to the immunosuppressive status of TAMs, promoting tumour growth and progression. Funding: Grant from the Fondazione AIRC per la Ricerca sul Cancro to Riccardo Alessandro (grant n° 18783)

Citral-Enriched Fraction of Lemon Essential Oil Mitigates LPS-Induced Hepatocyte Injuries

Simple Summary To date, essential oil fractions are emerging as functional compounds of interest for the food and perfume industries. The aim of this study is to evaluate the ability of citral-enriched fractions obtained from lemon essential oil (Cfr-LEO) to counteract, in healthy human hepatocytes, the activity of lipopolysaccharide (LPS), a trigger of inflammation, oxidative stress, and epithelial-mesenchymal transition. In our paper, we report that the pretreatment of hepatocytes with Cfr-LEO counteracts the effects induced by LPS. The data obtained lay the basis for the development of commercial products such as food and drink aimed at preventing or alleviating chronic conditions associated with liver dysfunction.Abstract Lemon essential oil (LEO) is known for its aromatic and healthy properties; however, less consideration is given to the biological properties of the fractions obtained from LEO. This study aims to evaluate the ability of a citral-enriched fraction obtained from LEO (Cfr-LEO) to counteract lipopolysaccharide (LPS)-mediated inflammation, oxidative stress, and epithelial-mesenchymal transition (EMT) in healthy human hepatocytes. Human immortalized hepatocytes (THLE-2 cell line) were pretreated with Cfr-LEO and subsequently exposed to LPS at various time points. We report that the pretreatment with Cfr-LEO counteracts LPS-mediated effects by inhibiting inflammation, oxidative stress, and epithelial-mesenchymal transition in THLE-2. In particular, we found that pretreatment with Cfr-LEO reduced NF-kappa B activation and the subsequent proinflammatory cytokines release, ROS production, and NRF2 and p53 expression. Furthermore, the pretreatment with Cfr-LEO showed its beneficial effect in counteracting LPS-induced EMT. Taken together, these results support Cfr-LEO application in the nutraceutical research field not only for its organoleptic properties, conferred by citral enrichment, but also for its biological activity. Our study could lay the basis for the development of foods/drinks enriched with Cfr-LEO, aimed at preventing or alleviating chronic conditions associated with liver dysfunction

Curcumin and Andrographolide Co-Administration Safely Prevent Steatosis Induction and ROS Production in HepG2 Cell Line

Non-alcoholic fatty liver disease (NAFLD) is an emerging chronic liver disease worldwide. Curcumin and andrographolide are famous for improving hepatic functions, being able to reverse oxidative stress and release pro-inflammatory cytokines, and they are implicated in hepatic stellate cell activation and in liver fibrosis development. Thus, we tested curcumin and andrographolide separately and in combination to determine their effect on triglyceride accumulation and ROS production, identifying the differential expression of genes involved in fatty liver and oxidative stress development. In vitro steatosis was induced in HepG2 cells and the protective effect of curcumin, andrographolide, and their combination was observed evaluating cell viability, lipid and triglyceride content, ROS levels, and microarray differential gene expression. Curcumin, andrographolide, and their association were effective in reducing steatosis, triglyceride content, and ROS stress, downregulating the genes involved in lipid accumulation. Moreover, the treatments were able to protect the cytotoxic effect of steatosis, promoting the expression of survival and anti-inflammatory genes. The present study showed that the association of curcumin and andrographolide could be used as a therapeutic approach to counter high lipid content and ROS levels in steatosis liver, avoiding the possible hepatotoxic effect of curcumin. Furthermore, this study improved our understanding of the antisteatosis and hepatoprotective properties of a curcumin and andrographolide combination

Accuracy of ChatGPT-Generated Information on Head and Neck and Oromaxillofacial Surgery: A Multicenter Collaborative Analysis

Objective: To investigate the accuracy of Chat-Based Generative Pre-trained Transformer (ChatGPT) in answering questions and solving clinical scenarios of head and neck surgery. Study design: Observational and valuative study. Setting: Eighteen surgeons from 14 Italian head and neck surgery units. Methods: A total of 144 clinical questions encompassing different subspecialities of head and neck surgery and 15 comprehensive clinical scenarios were developed. Questions and scenarios were inputted into ChatGPT4, and the resulting answers were evaluated by the researchers using accuracy (range 1-6), completeness (range 1-3), and references' quality Likert scales. Results: The overall median score of open-ended questions was 6 (interquartile range[IQR]: 5-6) for accuracy and 3 (IQR: 2-3) for completeness. Overall, the reviewers rated the answer as entirely or nearly entirely correct in 87.2% of cases and as comprehensive and covering all aspects of the question in 73% of cases. The artificial intelligence (AI) model achieved a correct response in 84.7% of the closed-ended questions (11 wrong answers). As for the clinical scenarios, ChatGPT provided a fully or nearly fully correct diagnosis in 81.7% of cases. The proposed diagnostic or therapeutic procedure was judged to be complete in 56.7% of cases. The overall quality of the bibliographic references was poor, and sources were nonexistent in 46.4% of the cases. Conclusion: The results generally demonstrate a good level of accuracy in the AI's answers. The AI's ability to resolve complex clinical scenarios is promising, but it still falls short of being considered a reliable support for the decision-making process of specialists in head-neck surgery

Exosomes analysis in Non Small Cell Lung Cancer: from in vitro models to preclinical application

Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related deaths worldwide. The majority of patients are diagnosed in advanced disease stage. Bone metastasis is the most frequent complication in NSCLC resulting in osteolytic lesions. The perfect balance between bone-resorbing osteoclasts and bone-forming osteoblasts activity is lost in bone metastasis, inducing osteoclastogenesis. In NSCLC, the epidermal growth factor receptor (EGFR) pathway is constitutively activated. EGFR binds Amphiregulin (AREG) that is overexpressed in several cancers such as colon, breast and lung. Its levels in plasma of NSCLC patients correlate with poor prognosis and AREG was recently found as a signaling molecule in exosomes derived from cancer cell lines. Exosomes have a key role in the cell-cell communication and they were recently indicated as important actors in metastatic niche preparation. In the present work, we hypothesize a role of AREG carried by exosomes derived from NSCLC in bone metastasis induction. We observed that NSCLC-exosomes, containing AREG, induce EGFR pathway activation in pre-osteoclasts that in turn causes an increased expression of RANKL. RANKL is able to induce the expression of proteolytic enzymes, well-known markers of osteoclastogenesis, triggering a vicious cycle in osteolytic bone metastasis

Comprendere il dolore oro-facciale

La diagnosi differenziale del dolore oro-facciale cronico rappresenta una vera sfida clinica. Nella maggior parte dei casi il dolore oro-facciale proviene dalla cavità orale e dalle strutture circostanti, tuttavia l’origine non dentale è frequente. Al fine di evitare terapie dentali inadeguate è necessaria la corretta diagnosi di condizioni quali dolore facciale idiopatico cronico, disordini temporomandibolari, sindrome della bocca bruciante e nevralgia trigeminale. L’odontoiatra deve tenere in considerazione che il dolore oro-facciale di origine non dentale può essere influenzato da fattori cranio-facciali, muscoloscheletrici, stomatognatici, neurologici, vascolari e/o psicologici che si modificano in modo dinamico durante la vita e che l’eziologia può essere multifattoriale. Le sindromi dolorose croniche del distretto oro-facciale possono essere associate a diversi sintomi che sorgono da una causa contingente, come il dolore post operatorio o conseguente a una patologia maligna, ma possono anche essere condizioni in cui il dolore costituisce il problema primario, come le cefalee o il dolore neuropatico. La diagnosi delle sindromi dolorose si avvale dell’interpretazione dei dati anamnestici, di laboratorio, radiologici ed elettrodiagnostici, della valutazione del comportamento sociale, professionale e dell’esame obiettivo

Extracellular Vesicles and Tumor-Immune Escape: Biological Functions and Clinical Perspectives

The modulation of the immune system is one of the hallmarks of cancer. It is now widely described that cancer cells are able to evade the immune response and thus establish immune tolerance. The exploration of the mechanisms underlying this ability of cancer cells has always attracted the scientific community and is the basis for the development of new promising cancer therapies. Recent evidence has highlighted how extracellular vesicles (EVs) represent a mechanism by which cancer cells promote immune escape by inducing phenotypic changes on different immune cell populations. In this review, we will discuss the recent findings on the role of tumor-derived extracellular vesicles (TEVs) in regulating immune checkpoints, focusing on the PD-L1/PD-1 axis

Prevalence of primary headache related to work activity in a group of temporomandibular disorder patients

Since headache is one of the most frequent complaints in temporomandibular disorder (TMD) patient, its burden in terms of the both social cost and individual sufferance is considerable. Headache is a frequent cause of absence from work and decreased productivity. This study was aimed at identifying specific occupational factors able to modify the prevalence of headache in a group of TMD patients

Effects of exosomes released by NSCLC cells on osteoclasts differentiation.

Effects of exosomes released by NSCLC cells on osteoclasts differentiation Pucci M., Taverna S., Corrado C., Giallombardo M., Rolfo C. and Alessandro R. Non-small cell lung cancer (NSCLC) has a poorly 5-year survival rate, as a consequence of the delay in the detection of the disease. The majority of patients are diagnosed in an advanced disease stage. Bone metastasis is the most frequent complication in NSCLC resulting in osteolytic lesions. The perfect balance between bone-resorbing osteoclasts (OCs) and bone-forming osteoblasts (OBs) activity is lost in bone metastasis, inducing osteoclastogenesis. Most of the patients with lung cancer are treated with EGFR inhibitors (TKIs). Numerous studies show in patients with NSCLC the presence of somatic mutations at the level of the exons coding for tyrosine kinase domain of the EGFR. The cell line CRL 2868 presents the “activating” mutation of exon 19, that confers sensitivity to treatment with TKIs. Recent studies demonstrated that exosomes, nano-size vesicles that shuttle proteins, lipids and nucleic acids, have a key role in tumor progression and premetastatic niche formation. Exosomes are involved in modulation of metastasis formation; we investigated the role of NSCLC cell-derived exosomes in OCs differentiation. We show that CRL 2868 cells release exosomes which are actively internalized by RAW 264.7 cell line, a cellular model of preosteoclasts. CRL 2868 cell-derived exosomes positively modulate pre-osteoclast migration and play a significant pro-differentiative role of RAW 264.7 cells, inducing the expression of osteoclast markers such as Cathepsin K (CTSK), Matrix Metalloproteinases 9 (MMP9) and Tartrate-resistant Acid Phosphatase (TRAP). Our data indicate that CRL2868-exosomes modulate OCs function and differentiation. Further studies will be carried out to investigate the role of TKI in OCs differentiation