Canine glioblastoma-derived extracellular vesicles as precise carriers for glioblastoma imaging: Targeting across the blood-brain barrier.

The treatment of glioblastoma (GBM) faces significant challenges due to the difficulty of delivering drugs through the blood-brain barrier (BBB). Extracellular vesicles (EVs) have emerged as potential carriers for targeted drug delivery to brain tumors. However, their use and distribution in the presence of an intact BBB and their ability to target GBM tissue are still under investigation. This study explored the use of EVs for GBM targeting across the BBB. Canine plasma EVs from healthy dogs and dogs with glioma were isolated, characterized, and loaded with diagnostic agents. Biodistribution studies were conducted in healthy murine models and a novel intranasal model that preserved BBB integrity while initiating early-stage GBM growth. This model assessed EVs' potential for delivering the contrast agent gadoteric acid to intracranial tumors. Imaging techniques, such as bioluminescence and MRI, confirmed EVs' targeting and delivery capabilities thus revealing a selective accumulation of canine glioma-derived EVs in brain tissue under physiological conditions. In the model of brain tumor, MRI experiments demonstrated the ability of EVs to accumulate gadoteric acid within GBM to enhance contrast of the tumoral mass, even when BBB integrity is maintained. This study underscores the potential of EVs derived from glioma for the targeted delivery of drugs to glioblastoma. EVs from dogs with glioma showed capacity to traverse the BBB and selectively accumulate within the brain tumor. Overall, this research represents a foundation for the application of autologous EVs to precision glioblastoma treatment, addressing the challenge of BBB penetration and targeting specificity in brain cancer therapy.

The Potential of JAG Ligands as Therapeutic Targets and Predictive Biomarkers in Multiple Myeloma.

The NOTCH ligands JAG1 and JAG2 have been correlated in vitro with multiple myeloma (MM) cell proliferation, drug resistance, self-renewal and a pathological crosstalk with the tumor microenvironment resulting in angiogenesis and osteoclastogenesis. These findings suggest that a therapeutic approach targeting JAG ligands might be helpful for the care of MM patients and lead us to explore the role of JAG1 and JAG2 in a MM in vivo model and primary patient samples. JAG1 and JAG2 protein expression represents a common feature in MM cell lines; therefore, we assessed their function through JAG1/2 conditional silencing in a MM xenograft model. We observed that JAG1 and JAG2 showed potential as therapeutic targets in MM, as their silencing resulted in a reduction in the tumor burden. Moreover, JAG1 and JAG2 protein expression in MM patients was positively correlated with the presence of MM cells in patients' bone marrow biopsies. Finally, taking advantage of the Multiple Myeloma Research Foundation (MMRF) CoMMpass global dataset, we showed that JAG2 gene expression level was a predictive biomarker associated with patients' overall survival and progression-free survival, independently from other main molecular or clinical features. Overall, these results strengthened the rationale for the development of a JAG1/2-tailored approach and the use of JAG2 as a predictive biomarker in MM.

Ceramide present in cholangiocarcinoma-derived extracellular vesicle induces a pro-inflammatory state in monocytes.

Cholangiocarcinoma (CCA) is a rare cancer characterized by a global increasing incidence. Extracellular vesicles (EV) contribute to many of the hallmarks of cancer through transfer of their cargo molecules. The sphingolipid (SPL) profile of intrahepatic CCA (iCCA)-derived EVs was characterized by liquid chromatography-tandem mass spectrometry analysis. The effect of iCCA-derived EVs as mediators of inflammation was assessed on monocytes by flow cytometry. iCCA-derived EVs showed downregulation of all SPL species. Of note, poorly-differentiated iCCA-derived EVs showed a higher ceramide and dihydroceramide content compared with moderately-differentiated iCCA-derived EVs. Of note, higher dihydroceramide content was associated with vascular invasion. Cancer-derived EVs induced the release of pro-inflammatory cytokines in monocytes. Inhibition of synthesis of ceramide with Myriocin, a specific inhibitor of the serine palmitoyl transferase, reduced the pro-inflammatory activity of iCCA-derived EVs, demonstrating a role for ceramide as mediator of inflammation in iCCA. In conclusion, iCCA-derived EVs may promote iCCA progression by exporting the excess of pro-apoptotic and pro-inflammatory ceramides.

MICA/B-targeted antibody promotes NK cell-driven tumor immunity in patients with intrahepatic cholangiocarcinoma.

The major histocompatibility complex-class I chain related proteins A and B (MICA/B) is upregulated because of cellular stress and MICA/B shedding by cancer cells causes escape from NKG2D recognition favoring the emergence of cancers. Cholangiocarcinoma (CCA) is a relatively rare, though increasingly prevalent, primary liver cancer characterized by a late clinical presentation and a dismal prognosis. We explored the NKG2D-MICA/B axis in NK cells from 41 patients with intrahepatic cholangiocarcinoma (iCCA). The MICA/B-specific 7C6 mAb was used for ex vivo antibody-dependent cytotoxicity (ADCC) experiments using circulating, non tumor liver- and tumor-infiltrating NK cells against the HuCCT-1 cell line and patient-derived primary iCCA cells as targets. MICA/B were more expressed in iCCA than in non-tumoral tissue, MICA transcription being higher in moderately-differentiated compared with poorly-differentiated cancer. Serum MICA was elevated in iCCA patients in line with higher expression of ADAM10 and ADAM17 that are responsible for proteolytic release of MICA/B from tumor. Addition of 7C6 significantly boosted peripheral, liver- and tumor-infiltrating-NK cell degranulation and IFNγ production toward MICA/B-expressing established cell lines and autologous iCCA patient target cells. Our data show that anti-MICA/B drives NK cell anti-tumor activity, and provide preclinical evidence in support of 7C6 as a potential immunotherapeutic tool for iCCA.

Therapeutic Effect of an Antibody-Derived Peptide in a Galleria mellonella Model of Systemic Candidiasis.

The synthetic peptide T11F (TCRVDHRGLTF), with sequence identical to a fragment of the constant region of human IgM, and most of its alanine-substituted derivatives proved to possess a significant candidacidal activity in vitro. In this study, the therapeutic efficacy of T11F, D5A, the derivative most active in vitro, and F11A, characterized by a different conformation, was investigated in Galleria mellonella larvae infected with Candida albicans. A single injection of F11A and D5A derivatives, in contrast with T11F, led to a significant increase in survival of larvae injected with a lethal inoculum of C. albicans cells, in comparison with infected animals treated with saline. Peptide modulation of host immunity upon C. albicans infection was determined by hemocyte analysis and larval histology, highlighting a different immune stimulation by the studied peptides. F11A, particularly, was the most active in eliciting nodule formation, melanization and fat body activation, leading to a better control of yeast infection. Overall, the obtained data suggest a double role for F11A, able to simultaneously target the fungus and the host immune system, resulting in a more efficient pathogen clearance.

CT colonography followed by elective surgery in patients with acute diverticulitis: a radiological-pathological correlation study.

PURPOSE: To perform a radiologic-pathologic correlation analysis of sigmoid colon in patients undergoing pre-operative CT Colonography (CTC) after an episode of acute diverticulitis (AD). METHODS: Fifty-nine consecutive patients (31/28 M/F; 58 ± 13 years) underwent CTC 55 ± 18 days after AD, 8 ± 4 weeks before surgery. Thirty-seven patients (63%) underwent conventional abdominal CT at time of AD. An experienced blinded radiologist retrospectively analyzed all images: disease severity was graded according to the Ambrosetti classification on conventional CT and according to the diverticular disease severity score (DDSS) on CTC. A GI pathologist performed a dedicated analysis, evaluating the presence of acute and chronic inflammation, and fibrosis, using 0-3 point scale for each variable. RESULTS: Of 59 patients, 41 (69%) had at least one previous AD episode; twenty-six patients (44%) had a complicated AD. DDSS was mild-moderate in 34/59 (58%), and severe in 25/59 (42%). All patients had chronic inflammation, while 90% had low-to-severe fibrosis. Patients with moderate/severe fibrosis were older than those with no/mild fibrosis (61 ± 13 versus 54 ± 13). We found a significant correlation between DDSS and chronic inflammation (p = 0.004), as well as DDSS and fibrosis (p = 0.005). Furthermore, fibrosis was correlated with complicated acute diverticulitis (p = 0.0.27), and with age (p = 0.067). At multivariate analysis, complicated diverticulitis was the best predictor of fibrosis (odds ratio 4.4). Patient age and DDSS were other independent predictors. CONCLUSION: DDSS-based assessment on preoperative CTC was a good predictor of chronic colonic inflammation and fibrosis. In addition, the presence of complicated diverticulitis on CT during the acute episode was most predictive of fibrosis.

Unexpected thymoma in a challenging case of hyperparathyroidism.

We report the case of a woman with primary hyperparathyroidism suspected of mediastinal ectopic parathyroid adenoma revealed to be a thymoma. Our aim was to focus on some possible criticisms in distinguishing between ectopic parathyroid and thymus.

Jagged Ligands Enhance the Pro-Angiogenic Activity of Multiple Myeloma Cells.

Multiple myeloma (MM) is an incurable plasma cell malignancy arising primarily within the bone marrow (BM). During MM progression, different modifications occur in the tumor cells and BM microenvironment, including the angiogenic shift characterized by the increased capability of endothelial cells to organize a network, migrate and express angiogenic factors, including vascular endothelial growth factor (VEGF). Here, we studied the functional outcome of the dysregulation of Notch ligands, Jagged1 and Jagged2, occurring during disease progression, on the angiogenic potential of MM cells and BM stromal cells (BMSCs). Jagged1-2 expression was modulated by RNA interference or soluble peptide administration, and the effects on the MM cell lines' ability to induce human pulmonary artery cells (HPAECs) angiogenesis or to indirectly increase the BMSC angiogenic potential was analyzed in vitro; in vivo validation was performed on a zebrafish model and MM patients' BM biopsies. Overall, our results indicate that the MM-derived Jagged ligands (1) increase the tumor cell angiogenic potential by directly triggering Notch activation in the HPAECs or stimulating the release of angiogenic factors, i.e., VEGF; and (2) stimulate the BMSCs to promote angiogenesis through VEGF secretion. The observed pro-angiogenic effect of Notch activation in the BM during MM progression provides further evidence of the potential of a therapy targeting the Jagged ligands.

EPIRETINAL FIBROSIS REMOVAL IN AN ARGUS II-IMPLANTED EYE: Histological Characteristics and Functional Results.

PURPOSE: 1) To investigate morphologic and histochemical characteristics of an epiretinal fibrosis removed in an Argus II-implanted eye; 2) to evaluate the Argus II function before and after the fibrosis removal, and 3) to compare morphologic and functional data. METHODS: Fibrosis, which developed between the Argus II prosthesis and the retina two years after implant, was surgically removed. Its morphologic and histochemical characteristics were evaluated both in light and transmission electron microscopy, with special stains and immunohistochemistry. The Argus II function was evaluated during the follow-up before surgical removal and 1 month later. RESULTS: Fibrosis was successfully removed. It was composed of a fibrotic tissue with spindle cells arranged in nodular aggregates with a symmetric distribution, mixed with an inflammatory infiltrate. Extra- and intracellular, irregular, small iron particles were found and confirmed ultrastructural characterization with degenerative cellular changes. The repositioned Argus II restored, and its function was partially nearly to normal values 1 month after surgery. CONCLUSION: Fibrosis can develop between the Argus II and the retina with increasing reduced function. Morphologic characteristics of the removed fibrosis suggested a pathogenesis based on an inflammatory process involved in a foreign body reaction with progressing connective tissue deposition leading to sclerosis. Adequate clinical follow-up is critical to successful removal of the fibrosis with reactivation of the Argus II function.

Therapeutic Effect of Iron Citrate in Blocking Calcium Deposition in High Pi-Calcified VSMC: Role of Autophagy and Apoptosis.

In chronic kidney disease (CKD), the first cause of mortality is cardiovascular disease induced mainly by vascular calcification (VC). Recently, iron-based phosphate binders have been proposed in advanced CKD to treat hyperphosphatemia. We studied the effect of iron citrate (iron) on the progression of calcification in high-phosphate (Pi) calcified VSMC. Iron arrested further calcification when added on days 7-15 in the presence of high Pi (1.30 ± 0.03 vs 0.61 ± 0.02; OD/mg protein; day 15; Pi vs Pi + Fe, p < 0.01). We next investigated apoptosis and autophagy. Adding iron to high-Pi-treated VSMC, on days 7-11, decreased apoptotic cell number (17.3 ± 2.6 vs 11.6 ± 1.6; Annexin V; % positive cells; day 11; Pi vs Pi + Fe; p < 0.05). The result was confirmed thorough analysis of apoptotic nuclei both in VSMCs and aortic rings treated on days 7-15 (3.8 ± 0.2 vs 2.3 ± 0.3 and 4.0 ± 0.3 vs 2.2 ± 0.2; apoptotic nuclei; arbitrary score; day 15; Pi vs Pi + Fe; VSMCs and aortic rings; p < 0.05). Studying the prosurvival axis GAS6/AXL, we found that iron treatment on days 9-14 counteracted protein high-Pi-stimulated down-regulation and induced its de novo synthesis. Moreover, iron added on days 9-15 potentiated autophagy, as detected by an increased number of autophagosomes with damaged mitochondria and an increase in autophagic flux. Highlighting the effect of iron on apoptosis, we demonstrated its action in blocking the H2O2-induced increase in calcification added both before high Pi treatment and when the calcification was already exacerbated. In conclusion, we demonstrate that iron arrests further high Pi-induced calcium deposition through an anti-apoptotic action and the induction of autophagy on established calcified VSMC.

High-phosphate induced vascular calcification is reduced by iron citrate through inhibition of extracellular matrix osteo-chondrogenic shift in VSMCs.

BACKGROUND: High serum phosphate (Pi) levels strongly associate with cardiovascular morbidity and mortality in chronic kidney disease (CKD) patients with vascular calcification playing a major role in the pathogenesis of related cardiovascular disease. High-Pi challenged vascular smooth muscle cells (VSMCs) undergo simil-osteoblastic transformation and actively deposit calcium-phosphate crystals. Iron-based Pi-binders are used to treat hyperphosphatemia in CKD patients. METHODS: In this study, we investigated the direct effect of iron citrate on extracellular matrix (ECM) modification induced by high-Pi, following either prophylactic or therapeutic approach. RESULTS: Iron prophylactically prevents and therapeutically blocks high-Pi induced calcification. Masson's staining highlights the changes of muscular ECM that after high-Pi stimulation becomes fibrotic and which modifications are prevented or partially reverted by iron. Interestingly, iron preserves glycogen granules and either prevents or partially reverts the formation of non-glycogen granules induced by high-Pi. In parallel, iron addition is able to either prevent or block the high-Pi induced acid mucin deposition. Iron inhibited calcification also by preventing exosome osteo-chondrogenic shift by reducing phosphate load (0,61 ±â€¯0.04vs0,45 ±â€¯0.05, PivsPi + Fe, p < 0,05, nmol Pi/mg protein) and inducing miRNA 30c (0.62 ± 0.05vs3.07 ±â€¯0.62; PivsPi + Fe, p < 0.01, relative expression). Studying aortic rings, we found that iron significantly either prevents or reverts the high-Pi induced collagen deposition and the elastin decrease, preserving elastin structure (0.7 ± 0.1 vs 1.2 ± 0.1; Pi vs Pi + Fe, p < 0.05, elastin mRNA relative expression). CONCLUSIONS: Iron directly either prevents or partially reverts the high-Pi induced osteo-chondrocytic shift of ECM. The protection of muscular nature of VSMC ECM may be one of the mechanisms elucidating the anti-calcific effect of iron.

Inflammatory role of extracellular sphingolipids in Cystic Fibrosis.

Ceramide is emerging as one of the players of inflammation in lung diseases. However, data on its inflammatory role in Cystic Fibrosis (CF) as part of the extracellular machinery driven by lung mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) are missing. We obtained an in vitro model of CF-MSC by treating control human lung MSCs with a specific CFTR inhibitor. We characterized EVs populations derived from MSCs (ctr EVs) and CF-MSCs (CF-EVs) and analyzed their sphingolipid profile by LC-MS/MS. To evaluate their immunomodulatory function, we treated an in vitro human model of CF, with both EVs populations. Our data show that the two EVs populations differ for the average size, amount, and rate of uptake. CF-EVs display higher ceramide and dihydroceramide accumulation as compared to control EVs, suggesting the involvement of the de novo biosynthesis pathway in the parental CF-MSCs. Higher sphingomyelinase activity in CF-MSCs, driven by inflammation-induced ceramide accumulation, sustains the exocytosis of vesicles that export new formed pro-inflammatory ceramide. Our results suggest that CFTR dysfunction associates with an enhanced sphingolipid metabolism leading to the release of EVs that export the excess of pro-inflammatory Cer to the recipient cells, thus contributing to maintain the unresolved inflammatory status of CF.

Lung mesenchymal stem cells-derived extracellular vesicles attenuate the inflammatory profile of Cystic Fibrosis epithelial cells.

BACKGROUND: Mesenchymal stromal/stem cells (MSCs) are multi-potent non-hematopoietic stem cells, residing in most tissues including the lung. MSCs have been used in therapy of chronic inflammatory lung diseases such as Cystic Fibrosis (CF), asthma, and chronic obstructive pulmonary disease (COPD) but the main beneficial effects reside in the anti-inflammatory potential of the released extracellular vesicles (EVs). Recent reports demonstrate that EVs are effective in animal model of asthma, E.coli pneumonia, lung ischemia-reperfusion, and virus airway infection among others. Despite this growing literature, the EVs effects on CF are largely unexplored. METHODS: We treated IB3-1 cells, an in vitro human model of CF, with EVs derived from human lung MSCs under basal and inflammatory conditions (TNFα stimulation). RESULTS: We demonstrated here that treatment of IB3-1 CF cell line with EVs, down-regulates transcription and protein expression of pro-inflammatory cytokines such as IL-1ß, IL-8, IL-6 under TNFα - stimulated conditions. EVs treatment upregulates the mRNA expression of PPARγ, a transcription factor controlling anti-inflammatory and antioxidant mechanisms via NF-kB and HO-1. Accordingly, NF-kB nuclear translocation is reduced resulting in impairment of the downstream inflammation cascade. In addition, the mRNA of HO-1 is enhanced together with the antioxidant defensive response of the cells. CONCLUSIONS: We conclude that the anti-inflammatory and anti-oxidant efficacy of EVs derived from lung MSCs could be mediated by up-regulation of the PPARγ axis, whose down-stream effectors (NF-kB and HO-1) are well-known modulators of these pathways. GENERAL SIGNIFICANCE: EVs could be a novel strategy to control the hyper-inflamed condition in Cystic Fibrosis.

Myriocin treatment of CF lung infection and inflammation: complex analyses for enigmatic lipids.

Our aim was to use quantitative and qualitative analyses to gain further insight into the role of ceramide in cystic fibrosis (CF). Sphingolipid ceramide is a known inflammatory mediator, and its accumulation in inflamed lung has been reported in different types of emphysema, chronic obstructive pulmonary disease and CF. CF is caused by a mutation of the chloride channel and associated with hyperinflammation of the respiratory airways and high susceptibility to ongoing infections. We have previously demonstrated that de novo ceramide synthesis is enhanced in lung inflammation and sustains Pseudomonas aeruginosa pulmonary infection in a CF murine model. We used liquid chromatography and matrix-assisted laser desorption/ionization (MALDI) imaging coupled with mass spectrometry, confocal laser scan microscopy and histology analyses to reveal otherwise undecipherable information. We demonstrated that (i) upregulated ceramide synthesis in the alveoli is strictly related to alveolar infection and inflammation, (ii) alveolar ceramide (C16) can be specifically targeted by nanocarrier delivery of the ceramide synthesis inhibitor myriocin (Myr) and (iii) Myr is able to downmodulate pro-inflammatory lyso-PC, favouring an increase in anti-inflammatory PCs. We concluded that Myr modulates alveolar lipids milieu, reducing hyperinflammation and favouring anti-microbial effective response in CF mouse model.

M1 and M2 macrophages' clinicopathological significance in cutaneous melanoma.

Skin malignant melanoma (MM) is an aggressive cancer with an increasing incidence with limited therapies in advanced stages. Tumor-associated macrophages (TAMs) are the major immune constituent of the MM microenvironment and contribute toward its prognosis. TAMs' characterization and localization in human cancer is important to understand cancer progression and to identify molecular personalized therapies. M2 TAMs in stage I-II MMs are associated with worse prognostic parameters. A comprehensive M1-macrophage and M2-macrophage intratumoral localization and quantification in all stages of skin MMs is documented here with its clinical significance. To highlight immune pathways and possible early indicators of MM progression, we evaluated the number of M1 and M2 TAMs and intratumoral distribution in a large series of skin MMs. CD68 double immunostaining with MRP8-14 or inducible nitric oxide synthase (M1 macrophages) and with CD163 or CD204 (M2 macrophages) was performed in 94 stage I-IV skin MMs with a long duration of follow-up. The accumulation and distribution of M1 and M2 TAMs in intratumoral nests, stroma, and at the invasive front was correlated with clinicopathological variables. Since the early stage of MMs, M1 intratumoral macrophages were fewer than the M2 population; their recruitment was rapidly and progressively overwhelmed by an increase in M2 TAMs during MM progression. Independent of their intratumoral distribution, the accumulation of both M1 and M2 TAMs is associated with poor prognostic indicators and patients' survival. M1-recruited macrophages shift to the M2 phenotype early in MM development, possibly induced by high inducible nitric oxide synthase intratumoral increase peculiarly occurring since the initial MM stages. M2-recruited TAMs overwhelm M1 accumulation in all stages of MM progression, thus favoring neoplastic growth and dissemination. Independent of their intratumoral distribution, the prevalent accumulation of M2 TAMs in MM is statistically confirmed to be a poor indicator of patients' outcome and a potential target of immune therapies.

Overcoming an Extremely Drug Resistant (XDR) Pathogen: Avibactam Restores Susceptibility to Ceftazidime for Burkholderia cepacia Complex Isolates from Cystic Fibrosis Patients.

Burkholderia multivorans is a significant health threat to persons with cystic fibrosis (CF). Infections are difficult to treat as this pathogen is inherently resistant to multiple antibiotics. Susceptibility testing of isolates obtained from CF respiratory cultures revealed that single agents selected from different antibiotic classes were unable to inhibit growth. However, all isolates were found to be susceptible to ceftazidime when combined with the novel non-ß-lactam ß-lactamase inhibitor, avibactam (all minimum inhibitor concentrations (MICs) were ≤8 mg/L of ceftazidime and 4 mg/L of avibactam). Furthermore, a major ß-lactam resistance determinant expressed in B. multivorans, the class A carbapenemase, PenA was readily inhibited by avibactam with a high k2/K of (2 ± 1) × 106 µM-1 s-1 and a slow koff of (2 ± 1) × 10-3 s-1. Mass spectrometry revealed that avibactam formed a stable complex with PenA for up to 24 h and that avibactam recyclized off of PenA, re-forming the active compound. Crystallographic analysis of PenA-avibactam revealed several interactions that stabilized the acyl-enzyme complex. The deacylation water molecule possessed decreased nucleophilicity, preventing decarbamylation. In addition, the hydrogen-bonding interactions with Lys-73 were suggestive of a protonated state. Thus, Lys-73 was unlikely to abstract a proton from Ser-130 to initiate recyclization. Using Galleria mellonella larvae as a model for infection, ceftazidime-avibactam was shown to significantly (p < 0.001) improve survival of larvae infected with B. multivorans. To further support the translational impact, the ceftazidime-avibactam combination was evaluated using susceptibility testing against other strains of Burkholderia spp. that commonly infect individuals with CF, and 90% of the isolates were susceptible to the combination. In summary, ceftazidime-avibactam may serve as a preferred therapy for people that have CF and develop Burkholderia spp. infections and should be considered for clinical trials.

Iron citrate reduces high phosphate-induced vascular calcification by inhibiting apoptosis.

BACKGROUND AND AIMS: High phosphate-induced vascular calcification (VC) and iron deficiency-induced anemia are two major contributors of cardiovascular morbidity and mortality in patients affected by chronic kidney disease (CKD). Since phosphate (Pi) control and iron replacement are common therapies in CKD, the aim of our study was to investigate the effect of iron on high Pi-induced VC in rat vascular smooth muscle cells (VSMCs). METHODS: We treated VSMCs with 5 mM Pi and iron citrate (Fe3+) to evaluate Ca deposition by Alizarin Red destaining, DNA fragmentation by ELISA, gene expression by RT-PCR and protein expression by Western blot. RESULTS: Pretreatment with Fe3+ prevents high Pi-induced calcium (Ca) deposition concentration-dependently, with 90.1% inhibition at 50 µM (0.716 ± 0.04 vs. 0.071 ± 0.01, OD/mg protein; Pi vs. Pi + Fe3+, p < 0.01). We found that 50 µM Fe3+ completely prevents high Pi-induced apoptosis measured as DNA fragmentation (1.51 ± 0.08 vs. 1.03 ± 0.06, Pi vs. Pi + Fe3+; p < 0.01), through the prevention of the downregulation of the pro-survival pathway GAS6/AXL. Moreover, Fe3+ stimulates autophagy, a protective phenomenon in VC, as demonstrated by electron microscopy and by autophagy flux detected by LC3IIß protein expression. Finally, high Pi-induced osteoblastic differentiation is partially affected by Fe3+, since BMP2 increase is prevented and OPN is enhanced, but RUNX2 increase and α-actin and SM22α decrease are not modified. Interestingly, the addition of Fe3+ at different time points after Pi challenge completely blocks the progression of Ca deposition. CONCLUSIONS: In conclusion, iron citrate inhibits high Pi-induced Ca deposition by prevention of apoptosis, induction of autophagy, and partially affecting osteoblastic differentiation.

Multiple myeloma-derived Jagged ligands increases autocrine and paracrine interleukin-6 expression in bone marrow niche.

Multiple myeloma cell growth relies on intrinsic aggressiveness, due to a high karyotypic instability, or on the support from bone marrow (BM) niche.We and other groups have provided evidences that Notch signaling is related to tumor cell growth, pharmacological resistance, localization/recirculation in the BM and bone disease.This study indicates that high gene expression levels of Notch signaling members (JAG1, NOTCH2, HES5 and HES6) correlate with malignant progression or high-risk disease, and Notch signaling may participate in myeloma progression by increasing the BM levels of interleukin-6 (IL-6), a major player in myeloma cell growth and survival. Indeed, in vitro results, confirmed by correlation analysis on gene expression profiles of myeloma patients and immunohistochemical studies, demonstrated that Notch signaling controls IL-6 gene expression in those myeloma cells capable of IL-6 autonomous production as well as in surrounding BM stromal cells. In both cases Notch signaling activation may be triggered by myeloma cell-derived Jagged ligands. The evidence that Notch signaling positively controls IL-6 in the myeloma-associated BM makes this pathway a key mediator of tumor-directed reprogramming of the bone niche.This work strengthens the rationale for a novel Notch-directed therapy in multiple myeloma based on the inhibition of Jagged ligands.

Inhibition of ceramide de novo synthesis by myriocin produces the double effect of reducing pathological inflammation and exerting antifungal activity against A. fumigatus airways infection.

BACKGROUND: Fungal infections develop in pulmonary chronic inflammatory diseases such as asthma, Chronic Obstructive Pulmonary Disease (COPD) and Cystic Fibrosis (CF). The available antifungal drugs may fail to eradicate fungal pathogens, that can invade the lungs and vessels and spread by systemic circulation taking advantage of defective lung immunity. An increased rate of sphingolipid de novo synthesis, leading to ceramide accumulation, was demonstrated in CF and COPD inflamed lungs. The inhibitor of sphingolipid synthesis myriocin reduces inflammation and ameliorates the response against bacterial airway infection in CF mice. Myriocin also inhibits sphingolipid synthesis in fungi and exerts a powerful fungistatic effect. METHODS: We treated Aspergillus fumigatus infected airway epithelial cells with myriocin and we administered myriocin-loaded nanocarriers to A. fumigatus infected mice lung. RESULTS: We demonstrate here that de novo synthesized ceramide mediates the inflammatory response induced by A. fumigatus infection in airway epithelia. CF epithelial cells are chronically inflamed and defective in killing internalized conidia. Myriocin treatment reduced ceramide increase and inflammatory mediator release whereas it upregulated HO1 and NOD2, allowing the recovery of a functional killing of conidia in these cells. Myriocin-loaded nanocarriers, intratracheally administered to mice, significantly reduced both the inflammatory response induced by A. fumigatus pulmonary challenge and fungal lung invasion. CONCLUSIONS: We conclude that inhibition of sphingolipid synthesis can be envisaged as a dual anti-inflammatory and anti-fungal therapy in patients suffering from chronic lung inflammation with compromised immunity. GENERAL SIGNIFICANCE: Myriocin represents a powerful agent for inflammatory diseases and fungal infection.

The sialyl-glycolipid stage-specific embryonic antigen 4 marks a subpopulation of chemotherapy-resistant breast cancer cells with mesenchymal features.

INTRODUCTION: Chemotherapy resistance resulting in incomplete pathologic response is associated with high risk of metastasis and early relapse in breast cancer. The aim of this study was to identify and evaluate biomarkers of treatment-resistant tumor cells. METHODS: We performed a cell surface marker screen in triple-negative breast cancer patient-derived xenograft models treated with standard care genotoxic chemotherapy. Global expression profiling was used to further characterize the identified treatment-resistant subpopulations. RESULTS: High expression of sialyl-glycolipid stage-specific embryonic antigen 4 (SSEA4) was found in residual tumor cells surviving chemotherapy and in samples from metastatic patients who relapsed after neoadjuvant chemotherapy. Gene and microRNA (miRNA) expression profiling linked SSEA4 positivity with a mesenchymal phenotype and a deregulation of drug resistance pathways. Functional assays demonstrated a direct link between epithelial-mesenchymal transition (EMT) and SSEA4 expression. Interestingly, SSEA4 expression, EMT, and drug resistance seemed to be regulated posttranscriptionally. Finally, high expression of CMP-N-acetylneuraminate-ß-galactosamide-α-2,3-sialyltransferase 2 (ST3GAL2), the rate-limiting enzyme of SSEA4 synthesis, was found to be associated with poor clinical outcome in breast and ovarian cancer patients treated with chemotherapy. CONCLUSIONS: In this study, we identified SSEA4 as highly expressed in a subpopulation of tumor cells resistant to multiple commonly used chemotherapy drugs, as well as ST3GAL2, the rate-limiting enzyme of SSEA4 synthesis, as a predictive marker of poor outcome for breast and ovarian cancer patients undergoing chemotherapy. Both biomarkers and additionally identified regulatory miRNAs may be used to further understand chemoresistance, to stratify patient groups in order to avoid ineffective and painful therapies, and to develop alternative treatment regimens for breast cancer patients.