Isolation of anti-extra-cellular vesicle single-domain antibodies by direct panning on vesicle-enriched fractions.

BACKGROUND: The thorough understanding of the physiological and pathological processes mediated by extracellular vesicles (EVs) is challenged by purification methods which are cumbersome, not reproducible, or insufficient to yield homogeneous material. Chromatography based on both ion-exchange and immune-capture can represent an effective method to improve EV purification and successive analysis. METHODS: Cell culture supernatant was used as a model sample for assessing the capacity of anion-exchange chromatography to separate distinct EV fractions and to isolate nanobodies by direct panning on whole EVs to recover binders specific for the native conformation of EV-surface epitopes and suitable to develop EV immune-capture reagents. RESULTS: Anion-exchange chromatography of cell culture supernatant separated distinct protein-containing fractions and all of them were positive for CD9, a biomarker associated to some EVs. This suggested the existence of several EV fractions but did not help in separating EVs from other contaminants. We further isolated several nanobodies instrumental for implementing immune-affinity protocols. These were able to immobilize EVs from both cell culture supernatant and biological samples, to be used in ELISA, flow-cytometry, and immune-purification. CONCLUSIONS: Here we report the first successful isolation of anti-EV nanobodies for the use in immunoaffinity-based EV capture by panning a phage library directly on partially purified EVs. This achievement paves the way for the application of direct EV panning for the discovery of novel antibody-vesicle surface biomarker pairs and represents the preliminary requirement for the development of selective immune-capture that, in combination with anion-exchange chromatography, can simplify the systematic stratification of EV sub-populations and their individual characterization.

Characterization of the Proinflammatory Profile of Synovial Fluid-Derived Exosomes of Patients with Osteoarthritis.

The purpose of this study is to characterize synovial fluid- (SF-) derived exosomes of patients with gonarthrosis comparing two methods of isolation and to investigate their immune regulatory properties. Extracellular vesicles (EVs) have been isolated from inflamed SF by polymer precipitation method and quantified by Exocet kit and by nanoparticle tracking analysis. Vesicles expressed all the specific exosomal markers by immunoblot and FACS. After isolation with Exoquick, a relevant contamination by immune complexes was detected, which required further magnetic bead-based purification to remove. SF-derived exosomes significantly stimulated the release of several inflammatory cytokines and chemokines and metalloproteinases by M1 macrophages but did not influence the expression of CD80 and CD86 costimulatory molecules. In conclusion, we characterized purified exosomes isolated from inflamed SF and demonstrate that purified exosomes are functionally active in their ability to stimulate the release of proinflammatory factors from M1 macrophages. Our data indicate that SF-derived exosomes from gonarthrosis patients play a role in disease progression.

In patients with metastatic breast cancer the identification of circulating tumor cells in epithelial-to-mesenchymal transition is associated with a poor prognosis.

BACKGROUND: Although recent models suggest that the detection of Circulating Tumor Cells (CTC) in epithelial-to-mesenchymal transition (EM CTC) might be related to disease progression in metastatic breast cancer (MBC) patients, current detection methods are not efficient in identifying this subpopulation of cells. Furthermore, the possible association of EM CTC with both clinicopathological features and prognosis of MBC patients has still to be demonstrated. Aims of this study were: first, to optimize a DEPArray-based protocol meant to identify, quantify and sort single, viable EM CTC and, subsequently, to test the association of EM CTC frequency with clinical data. METHODS: This prospective observational study enrolled 56 MBC patients regardless of the line of treatment. Blood samples, depleted of CD45(pos) leukocytes, were stained with an antibody cocktail recognizing both epithelial and mesenchymal markers. Four CD45(neg) cell subpopulations were identified: cells expressing only epithelial markers (E CTC), cells co-expressing epithelial and mesenchymal markers (EM CTC), cells expressing only mesenchymal markers (MES) and cells negative for every tested marker (NEG). CTC subpopulations were quantified as both absolute cell count and relative frequency. The association of CTC subpopulations with clinicopathological features, progression free survival (PFS), and overall survival (OS) was explored by Wilcoxon-Mann-Whitney test and Univariate Cox Regression Analysis, respectively. RESULTS: By employing the DEPArray-based strategy, we were able to assess the presence of cells pertaining to the above-described classes in every MBC patient. We observed a significant association between specific CD45(neg) subpopulations and tumor subtypes (e.g. NEG and triple negative), proliferation (NEG and Ki67 expression) and sites of metastatic spread (e.g. E CTC and bone; NEG and brain). Importantly, the fraction of CD45(neg) cells co-expressing epithelial and mesenchymal markers (EM CTC) was significantly associated with poorer PFS and OS, computed, this latter, both from the diagnosis of a stage IV disease and from the initial CTC assessment. CONCLUSION: This study suggests the importance of dissecting the heterogeneity of CTC in MBC. Precise characterization of CTC could help in estimating both metastatization pattern and outcome, driving clinical decision-making and surveillance strategies.

Mast cells control the expansion and differentiation of IL-10-competent B cells.

The discovery of B cell subsets with regulatory properties, dependent on IL-10 production, has expanded our view on the mechanisms that control inflammation. Regulatory B cells acquire the ability to produce IL-10 in a stepwise process: first, they become IL-10 competent, a poised state in which B cells are sensitive to trigger signals but do not actually express the Il-10 gene; then, when exposed to appropriate stimuli, they start producing IL-10. Even if the existence of IL-10-competent B cells is now well established, it is not yet known how different immune cell types cross talk with B cells and affect IL-10-competent B cell differentiation and expansion. Mast cells (MCs) contribute to the differentiation and influence the effector functions of various immune cells, including B lymphocytes. In this study, we explored whether MCs could play a role in the expansion of IL-10-competent B cells and addressed the in vivo relevance of MC deficiency on the generation of these cells. We show that MCs can expand IL-10-competent B cells, but they do not directly induce IL-10 production; moreover, the absence of MCs negatively affects IL-10-competent B cell differentiation. Noteworthy, our findings reveal that the CD40L/CD40 axis plays a significant role in MC-driven expansion of IL-10-competent B cells in vitro and highlight the importance of MC CD40L signaling in the colon.

Ex vivo molecular rejuvenation improves the therapeutic activity of senescent human cardiac stem cells in a mouse model of myocardial infarction.

Cardiac stem cells (CSC) from explanted decompensated hearts (E-CSC) are, with respect to those obtained from healthy donors (D-CSC), senescent and functionally impaired. We aimed to identify alterations in signaling pathways that are associated with CSC senescence. Additionally, we investigated if pharmacological modulation of altered pathways can reduce CSC senescence in vitro and enhance their reparative ability in vivo. Measurement of secreted factors showed that E-CSC release larger amounts of proinflammatory cytokine IL1ß compared with D-CSC. Using blocking antibodies, we verified that IL1ß hampers the paracrine protective action of E-CSC on cardiomyocyte viability. IL1ß acts intracranially inducing IKKß signaling, a mechanism that via nuclear factor-κB upregulates the expression of IL1ß itself. Moreover, E-CSC show reduced levels of AMP protein kinase (AMPK) activating phosphorylation. This latter event, together with enhanced IKKß signaling, increases TORC1 activity, thereby impairing the autophagic flux and inhibiting the phosphorylation of Akt and cAMP response element-binding protein. The combined use of rapamycin and resveratrol enhanced AMPK, thereby restoring downstream signaling and reducing IL1ß secretion. These molecular corrections reduced E-CSC senescence, re-establishing their protective activity on cardiomyocytes. Moreover ex vivo treatment with rapamycin and resveratrol improved E-CSC capacity to induce cardiac repair upon injection in the mouse infarcted heart, leading to reduced cardiomyocyte senescence and apoptosis and increased abundance of endogenous c-Kit(+) CSC in the peri-infarct area. Molecular rejuvenation of patient-derived CSC by short pharmacologic conditioning boosts their in vivo reparative abilities. This approach might prove useful for refinement of CSC-based therapies.

Glioma-associated stem cells: a novel class of tumor-supporting cells able to predict prognosis of human low-grade gliomas.

BACKGROUND: Translational medicine aims at transferring advances in basic science research into new approaches for diagnosis and treatment of diseases. Low-grade gliomas (LGG) have a heterogeneous clinical behavior that can be only partially predicted employing current state-of-the-art markers, hindering the decision-making process. To deepen our comprehension on tumor heterogeneity, we dissected the mechanism of interaction between tumor cells and relevant components of the neoplastic environment, isolating, from LGG and high-grade gliomas (HGG), proliferating stem cell lines from both the glioma stroma and, where possible, the neoplasm. METHODS AND FINDINGS: We isolated glioma-associated stem cells (GASC) from LGG (n=40) and HGG (n=73). GASC showed stem cell features, anchorage-independent growth, and supported the malignant properties of both A172 cells and human glioma-stem cells, mainly through the release of exosomes. Finally, starting from GASC obtained from HGG (n=13) and LGG (n=12) we defined a score, based on the expression of 9 GASC surface markers, whose prognostic value was assayed on 40 subsequent LGG-patients. At the multivariate Cox analysis, the GASC-based score was the only independent predictor of overall survival and malignant progression free-survival. CONCLUSIONS: The microenvironment of both LGG and HGG hosts non-tumorigenic multipotent stem cells that can increase in vitro the biological aggressiveness of glioma-initiating cells through the release of exosomes. The clinical importance of this finding is supported by the strong prognostic value associated with the characteristics of GASC. This patient-based approach can provide a groundbreaking method to predict prognosis and to exploit novel strategies that target the tumor stroma.

Serological and cellular response to mRNA-SARS-CoV2 vaccine in patients with hematological lymphoid malignancies: Results of the study "Cervax".

messenger RNA (mRNA)-Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) vaccines such as BNT162b2 became available in late 2020, but hematological malignancy patients (HM pts) were not evaluated in initial registration trials. We hereby report the results of a prospective, unicentric, observational study Response to COVID-19 Vaccination in hEmatological malignancies (CERVAX) developed to assess the postvaccine serological and T-cell-mediated response in a cohort of SARS-CoV2-negative HM pts vaccinated with BNT162b2. Patients with lymphomas [non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL)], chronic lymphocytic leukemia (CLL), and multiple myeloma (MM); off-therapy for at least 3 months; in a watch-and-wait program; or in treatment with ibrutinib, venetoclax, and lenalidomide were included. Different time points were considered to assess the serological response to the vaccine: before the second dose (T1), at 3-6-12 months after the first dose (T2-3-4, respectively). Since March 2021, 39 pts have been enrolled: 15 (38%) NHL, 12 (31%) CLL, and 12 (31%) MM. There were 13 of the 39 pts (33%) seroconverted at T1; an increase of the serological response was registered after the second dose (T2) (22/39 pts, 56%) and maintained after 6 months (22/39 pts, 56%) and 12 months (24/39 pts, 61%) from the first dose (T3-T4, respectively). Non-serological responders at T4 were 7/39 (18%): 0/15 NHL, 1/12 MM (8%), and 6/12 CLL (50%). All of them were on therapy (one lenalidomide, three ibrutinib, and three venetoclax). SARS-CoV2-reactive T-cell analysis (interferon gamma release assays) was available since June 2022 and was evaluated at 12 months (T4) from the first dose of vaccine in 31/39 pts (79%). T-cell-mediated-responders were 17/31 (55%): most of them were NHL and MM (47%, 41% and 12% for NHL, MM, and CLL, respectively). Both serological and T-cell non-responders were represented by pts on active therapy (venetoclax/ibrutinib). During the period of observation, eight (20.5%) pts developed mild SARS-CoV2 infection; no coronavirus disease 19 (COVID-19)-related deaths or hospitalizations were registered. In conclusion, in our cohort of lymphoproliferative pts receiving BNT162b2, CLL diagnosis and venetoclax/ibrutinib seem to be related with a lower humoral or T-mediated response. Nevertheless, the efficacy of mRNA vaccine in HM pts and the importance to continue the vaccine program even in non-responders after the first dose are supported in our study by demonstrating that a humoral and T-cell-mediated seroconversion should be observed even in the subsets of heavily immunocompromised pts.

Effects of age and heart failure on human cardiac stem cell function.

Currently, it is unknown whether defects in stem cell growth and differentiation contribute to myocardial aging and chronic heart failure (CHF), and whether a compartment of functional human cardiac stem cells (hCSCs) persists in the decompensated heart. To determine whether aging and CHF are critical determinants of the loss in growth reserve of the heart, the properties of hCSCs were evaluated in 18 control and 23 explanted hearts. Age and CHF showed a progressive decrease in functionally competent hCSCs. Chronological age was a major predictor of five biomarkers of hCSC senescence: telomeric shortening, attenuated telomerase activity, telomere dysfunction-induced foci, and p21(Cip1) and p16(INK4a) expression. CHF had similar consequences for hCSCs, suggesting that defects in the balance between cardiomyocyte mass and the pool of nonsenescent hCSCs may condition the evolution of the decompensated myopathy. A correlation was found previously between telomere length in circulating bone marrow cells and cardiovascular diseases, but that analysis was restricted to average telomere length in a cell population, neglecting the fact that telomere attrition does not occur uniformly in all cells. The present study provides the first demonstration that dysfunctional telomeres in hCSCs are biomarkers of aging and heart failure. The biomarkers of cellular senescence identified here can be used to define the birth date of hCSCs and to sort young cells with potential therapeutic efficacy.

Circulating stem cell vary with NYHA stage in heart failure patients.

We have investigated the blood levels of sub-classes of stem cells (SCs) [mesenchymal stem cells (MSCs), haematopoietic stem cells (HSCs), endothelial progenitor cells/circulating endothelial cells (EPCs/CECs) and tissue-committed stem cells (TCSCs)] in heart failure (HF) patients at different stage of pathology and correlated it with plasmatic levels of proangiogenic cytokines. Peripheral blood level of SCs were analysed in 97 HF patients (24 in NYHA class I, 41 in class II, 17 in class III and 15 in class IV) and in 23 healthy controls. Plasmatic levels of PDGF-BB, bFGF, HGF, vascular endothelial growth factor (VEGF), SDF-1α, TNF-α and NTproBNP were also measured. Compared with healthy individuals, MSC, and in particular the sub-classes CD45(-) CD34(-) CD90(+) , CD45(-) CD34(-) CD105(+) and CD45(-) CD34(-) CXCR4(+) were significantly enhanced in NYHA class IV patients (16.8-, 6.4- and 2.7-fold, respectively). Level of CD45(-) CD34(-) CD90(+) CXCR4(+) cells progressively increased from class II to class IV (fold increases compared with controls: 8.5, 12 and 21.5, respectively). A significant involvement of CXCR4(+) subpopulation of HSC (CD45(+) CD34(+) CD90(+) CXCR4(+) , 1.4 versus 13.3 cells/µl in controls and NYHA class III patients, respectively) and TCSC (CD45(-) CD34(+) CXCR4(+) , 1.5 cells/ µl in controls versus 12.4 and 28.6 cells/µl in NYHA classes II and IV, respectively) were also observed. All tested cytokines were enhanced in HF patients. In particular, for PDGF-BB and SDF-1α we studied specific ligand/receptors pairs. Interestingly, the first one positively correlated with TCSCs expressing PDGFR (r = 0.52, P = 0.001), whereas the second one correlated with TCSCs (r = 0.34, P = 0.005) and with MSCs CD90(+) expressing CXCR4 (r = 0.39, P = 0.001). HF is characterized by the increase in the circulating levels of different MSC, HSC, EPC and TCSC subsets. Both the entity and kinetic of this process varied in distinct cell subsets. Specifically, differently from HSCs and EPCs/CECs, MSCs and TCSCs significantly increased with the progression of the disease, suggesting a possible distinct role of these cells in the pathophysiology of HF.

Multipotent progenitor cells are present in human peripheral blood.

To determine whether the peripheral blood in humans contains a population of multipotent progenitor cells (MPCs), products of leukapheresis were obtained from healthy donor volunteers following the administration of granulocyte colony-stimulating factor. Small clusters of adherent proliferating cells were collected, and these cells continued to divide up to 40 population doublings without reaching replicative senescence and growth arrest. MPCs were positive for the transcription factors Nanog, Oct3/4, Sox2, c-Myc, and Klf4 and expressed several antigens characteristic of mesenchymal stem cells. However, they were negative for markers of hematopoietic stem/progenitor cells and bone marrow cell lineages. MPCs had a cloning efficiency of approximately 3%, and following their expansion, retained a highly immature phenotype. Under permissive culture conditions, MPCs differentiated into neurons, glial cells, hepatocytes, cardiomyocytes, endothelial cells, and osteoblasts. Moreover, the gene expression profile of MPCs partially overlapped with that of neural and embryonic stem cells, further demonstrating their primitive, uncommitted phenotype. Following subcutaneous transplantation in nonimmunosuppressed mice, MPCs migrated to distant organs and integrated structurally and functionally within the new tissue, acquiring the identity of resident parenchymal cells. In conclusion, undifferentiated cells with properties of embryonic stem cells can be isolated and expanded from human peripheral blood after granulocyte colony-stimulating factor administration. This cell pool may constitute a unique source of autologous cells with critical clinical import.

Biologically driven cut-off definition of lymphocyte ratios in metastatic breast cancer and association with exosomal subpopulations and prognosis.

High neutrophil to lymphocyte ratio (NLR) and monocyte to lymphocyte ratio (MLR) are respectively associated with systemic inflammation and immune suppression and have been associated with a poor outcome. Plasmatic exosomes are extracellular vesicles involved in the intercellular communication system that can exert an immunosuppressive function. Aim of this study was to investigate the interplay between the immune system and circulating exosomes in metastatic breast cancer (MBC). A threshold capable to classify patients according to MLR, NLR and PLR, was computed through a receiving operator curve analysis after propensity score matching with a series of female blood donors. Exosomes were isolated from plasma by ExoQuick solution and characterized by flow-cytometry. NLR, MLR, PLR and exosomal subpopulations potentially involved in the pre-metastatic niche were significantly different in MBC patients with respect to controls. MLR was significantly associated with number of sites at the onset of metastatic disease, while high levels of MLR and NLR were found to be associated with poor prognosis. Furthermore, exosomal subpopulations varied according to NLR, MLR, PLR and both were associated with different breast cancer subtypes and sites of distant involvement. This study highlights the nuanced role of immunity in MBC spread, progression and outcome. Moreover, they suggest potential interaction mechanisms between immunity, MBC and the metastatic niche.

An NF-κB signature predicts low-grade glioma prognosis: a precision medicine approach based on patient-derived stem cells.

Background: While recent genome-wide association studies have suggested novel low-grade glioma (LGG) stratification models based on a molecular classification, we explored the potential clinical utility of patient-derived cells. Specifically, we assayed glioma-associated stem cells (GASC) that are patient-derived and representative of the glioma microenvironment. Methods: By next-generation sequencing, we analyzed the transcriptional profile of GASC derived from patients who underwent anaplastic transformation either within 48 months (GASC-BAD) or ≥7 years (GASC-GOOD) after surgery. Gene set enrichment and pathway enrichment analyses were applied. The prognostic role of a nuclear factor-kappaB (NF-κB) signature derived from GASC-BAD was tested in 530 newly diagnosed diffuse LGG patients comprised within The Cancer Genome Atlas (TCGA) database. The prognostic value of the GASC upstream regulator p65 NF-κB was assessed, by univariate and multivariate Cox analyses, in a single center case study, including 146 grade II LGGs. Results: The key elements differentiating the transcriptome of GASC isolated from LGG with different prognoses were mostly related to hallmarks of cancer (eg, inflammatory/immune process, NF-κB activation). Consistently, the NF-κB signature extrapolated from the GASC study was prognostic in the dataset of TCGA. Finally, the nuclear expression of the NF-kB-p65 protein, assessed using an inexpensive immunohistochemical method, was an independent predictor of both overall survival and malignant progression-free survival in 146 grade II LGGs. Conclusion: This study demonstrates for the first time the independent prognostic role of NF-kB activation in LGG and outlines the role of patient-based stem cell models as a tool for precision medicine approaches.

Systemic T Cells Immunosuppression of Glioma Stem Cell-Derived Exosomes Is Mediated by Monocytic Myeloid-Derived Suppressor Cells.

A major contributing factor to glioma development and progression is its ability to evade the immune system. Nano-meter sized vesicles, exosomes, secreted by glioma-stem cells (GSC) can act as mediators of intercellular communication to promote tumor immune escape. Here, we investigated the immunomodulatory properties of GCS-derived exosomes on different peripheral immune cell populations. Healthy donor peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3, anti-CD28 and IL-2, were treated with GSC-derived exosomes. Phenotypic characterization, cell proliferation, Th1/Th2 cytokine secretion and intracellular cytokine production were analysed by distinguishing among effector T cells, regulatory T cells and monocytes. In unfractionated PBMCs, GSC-derived exosomes inhibited T cell activation (CD25 and CD69 expression), proliferation and Th1 cytokine production, and did not affect cell viability or regulatory T-cell suppression ability. Furthermore, exosomes were able to enhance proliferation of purified CD4+ T cells. In PBMCs culture, glioma-derived exosomes directly promoted IL-10 and arginase-1 production and downregulation of HLA-DR by unstimulated CD14+ monocytic cells, that displayed an immunophenotype resembling that of monocytic myeloid-derived suppressor cells (Mo-MDSCs). Importantly, the removal of CD14+ monocytic cell fraction from PBMCs restored T-cell proliferation. The same results were observed with exosomes purified from plasma of glioblastoma patients. Our results indicate that glioma-derived exosomes suppress T-cell immune response by acting on monocyte maturation rather than on direct interaction with T cells. Selective targeting of Mo-MDSC to treat glioma should be considered with regard to how immune cells allow the acquirement of effector functions and therefore counteracting tumor progression.

Immunoglobulin K light chain deficiency: A rare, but probably underestimated, humoral immune defect.

Human immunoglobulin molecules are generated by a pair of identical heavy chains, which identify the immunoglobulin class, and a pair of identical light chains, Kappa or Lambda alternatively, which characterize the immunoglobulin type. In normal conditions, Kappa light chains represent approximately 2/3 of the light chains of total immunoglobulins, both circulating and lymphocyte surface bound. Very few cases of immunoglobulin Kappa or Lambda light chain defects have been reported. Furthermore, the genetic basis of this defect has been extensively explored only in a single case. We report a case of a patient suffering of serious recurrent bacterial infections, which was caused by a very rare form of immunoglobulin disorder, consisting of a pure defect of Kappa light chain. We evaluated major serum immunoglobulin concentrations, as well as total and free Kappa and Lambda light chain concentrations. Lymphocyte phenotyping was also performed and finally we tested the Kappa chain VJ rearrangement as well as the constant Kappa region sequence. Studies performed on VJ rearrangement showed a polyclonal genetic arrangement, whereas the gene sequencing for the constant region of Kappa chain showed a homozygous T to G substitution at the position 1288 (rs200765148). This mutation causes a substitution from Cys to Gly in the protein sequence and, therefore, determines the abnormal folding of the constant region of Kappa chain. We suggest that this defect could lead to an effective reduction of the variability of total antibody repertoire and a consequent defect of an apparently normal immunoglobulin response to common antigens.

Identification of tumorigenesis-related mRNAs associated with RNA-binding protein HuR in thyroid cancer cells.

RNA binding proteins (RBPs) play a central role in cell physiology and pathology. Among them, HuR is a nuclear RBP, which shuttles to the cytoplasm to allow its RNA targets processing. HuR over-expression and delocalization are often associated to cell transformation. Numerous cancers display increased HuR protein levels and its high cytoplasmic levels has been associated with a worse prognosis.In our study, we first evaluated HuR expression in normal and cancer thyroid tissues and then evaluated its function in thyroid cell lines. HuR is over-expressed in all thyroid tumor tissues; high cytoplasmic levels are detected in all thyroid carcinomas. HuR silencing decreased cell viability and determined apoptotic cell death, in a non-tumorigenic (Nthy-ori-3.1) and a tumorigenic (BCPAP) thyroid cell line. Global transcriptome analysis indicated that HuR silencing, though having similar biological effects, induces distinct gene expression modifications in the two cell lines. By using the RIP-seq approach, the HuR-bound RNA profiles of different thyroid cell lines were evaluated. We show that in distinct cell lines HuR-bound RNA profiles are different. A set of 114 HuR-bound RNAs distinguishing tumorigenic cell lines from the non-tumorigenic one was identified.Altogether, our data indicate that HuR plays a role in thyroid tumorigenesis. Moreover, our findings are a proof of concept that RBP targets differ between cells with the same origin but with distinct biological behavior.

Critical role of lysosomes in the dysfunction of human Cardiac Stem Cells obtained from failing hearts.

UNLABELLED: The in vivo reparative potential of Cardiac Stem Cells (CSC), cultured from explanted failing hearts (E-), is impaired by cellular senescence. Moreover, E-CSC are characterized, with respect to CSC obtained from healthy donors (D-), by an arrest in the autophagic degradation. Although the lysosome plays a pivotal role in cellular homeostasis and defects of this organelle may be associated with aging and heart failure, the lysosomal function of CSC has never been investigated. The aim of this work was to focus on the Lysosomal Compartment (LC) of E-CSC, evaluating elements that could jeopardize lysosome functionality. METHODS AND RESULTS: Bioinformatics analysis conducted on genes differentially expressed between D- and E-CSC identified lysosomal-related gene sets as significantly enriched. Moreover, 29 differentially expressed genes were part of CLEAR (Coordinated Lysosomal Expression and Regulation) gene network, by which Transcription Factor EB (TFEB) regulates cellular clearance. Consistently, live cell imaging and flow cytometry analyses showed that the lysosomes of E-CSC are less acidic than the D-CSC ones. Furthermore, confocal microscopy showed in E-CSC: an accumulation of intralysosomal lipofuscins, a reduction of cathepsin B activity, evidence of lysosome membrane permeabilization, and the reduction of the nuclear active TFEB. The use of Rapamycin (TORC1 inhibitor) was able on one hand to increase TFEB activation and, on the other hand, to reduce lipofuscin mass, potentiating the lysosomal functionality. CONCLUSIONS: This study demonstrated for the first time that E-CSC are characterized by a blunted activation of TFEB and an altered proteostasis. TORC1 hyperactivation plays a central role in this phenomenon.

MCM5 as a target of BET inhibitors in thyroid cancer cells.

Anaplastic thyroid carcinoma (ATC) is an extremely aggressive thyroid cancer subtype, refractory to the current medical treatment. Among various epigenetic anticancer drugs, bromodomain and extra-terminal inhibitors (BETis) are considered to be an appealing novel class of compounds. BETi target the bromodomain and extra-terminal of BET proteins that act as regulators of gene transcription, interacting with histone acetyl groups. The goal of this study is to delineate which pathway underlies the biological effects derived from BET inhibition, in order to find new potential therapeutic targets in ATC. We investigated the effects of BET inhibition on two human anaplastic thyroid cancer-derived cell lines (FRO and SW1736). The treatment with two BETis, JQ1 and I-BET762, decreased cell viability, reduced cell cycle S-phase, and determined cell death. In order to find BETi effectors, FRO and SW1736 were subjected to a global transcriptome analysis after JQ1 treatment. A significant portion of deregulated genes belongs to cell cycle regulators. Among them, MCM5 was decreased at both mRNA and protein levels in both tested cell lines. Chromatin immunoprecipitation (ChIP) experiments indicate that MCM5 is directly bound by the BET protein BRD4. MCM5 silencing reduced cell proliferation, thus underlining its involvement in the block of proliferation induced by BETis. Furthermore, MCM5 immunohistochemical evaluation in human thyroid tumor tissues demonstrated its overexpression in several papillary thyroid carcinomas and in all ATCs. MCM5 was also overexpressed in a murine model of ATC, and JQ1 treatment reduced Mcm5 mRNA expression in two murine ATC cell lines. Thus, MCM5 could represent a new target in the therapeutic approach against ATC.

Adipose tissue derived stem cells: in vitro and in vivo analysis of a standard and three commercially available cell-assisted lipotransfer techniques.

INTRODUCTION: Autologous fat grafting is commonly used to correct soft-tissue contour deformities. However, results are impaired by a variable and unpredictable resorption rate. Autologous adipose-derived stromal cells in combination with lipoinjection (cell-assisted lipotransfer) seem to favor a long-term persistence of fat grafts, thus fostering the development of devices to be used in the operating room at the point of care, to isolate the stromal vascular fraction (SVF) and produce SVF-enhanced fat grafts with safe and standardized protocols. Focusing on patients undergoing breast reconstruction by lipostructure, we analyzed a standard technique, a modification of the Coleman's procedure, and three different commercially available devices (Lipokit, Cytori, Fastem), in terms of 1) ability to enrich fat grafts in stem cells and 2) clinical outcome at 6 and 12 months. METHODS: To evaluate the ability to enrich stem cells, we compared, for each patient (n=20), the standard lipoaspirate with the respective stem cell-enriched one, analyzing yield, immunophenotype and colony-forming capacity of the SVF cells as well as immunophenotype, clonogenicity and multipotency of the obtained adipose stem cells (ASCs). Regarding the clinical outcome, we compared, by ultrasonography imaging, changes at 6 and 12 months in the subcutaneous thickness of patients treated with stem-cell enriched (n=14) and standard lipoaspirates (n=16). RESULTS: Both methods relying on the enzymatic isolation of primitive cells led to significant increase in the frequency, in the fat grafts, of SVF cells as well as of clonogenic and multipotent ASCs, while the enrichment was less prominent for the device based on the mechanical isolation of the SVF. From a clinical point of view, patients treated with SVF-enhanced fat grafts demonstrated, at six months, a significant superior gain of thickness of both the central and superior-medial quadrants with respect to patients treated with standard lipotransfer. In the median-median quadrant the effect was still persistent at 12 months, confirming an advantage of lipotransfer technique in enriching improving long-term fat grafts. CONCLUSIONS: This comparative study, based on reproducible biological and clinical parameters and endpoints, showed an advantage of lipotransfer technique in enriching fat grafts in stem cells and in favoring, clinically, long-term fat grafts.

IL-10 production by B cells is differentially regulated by immune-mediated and infectious stimuli and requires p38 activation.

IL-10 is an immune suppressive cytokine with pleiotropic effects on B cell biology. IL-10 production has a pivotal role for the regulatory suppressive functions that B cells exert in many physiological and pathological settings. Several exogenous stimuli and endogenous immune mediators can trigger IL-10-producing B cell maturation. To clarify and gain a better insight into the mechanisms of IL-10 production by B cells, we first compared the effects of LPS, CpG, agonistic CD40 mAb and BAFF on IL-10 production, and then we investigated the signal transduction mechanisms responsible for these responses. While infectious/danger stimuli determine the rapid production and release of IL-10 by B cells, a limited subset of CD40-poised, IL-10-competent B cells produce IL-10 in response to a later antigenic or infectious signal. Although BAFF is able to induce a similar subset of IL-10-competent B cells, these cells do not similarly respond to the same antigenic or infectious signals. Importantly, by using specific inhibitors of the MAP kinase pathways, we found that while il-10 gene expression triggered by the TLR agonists LPS and CpG is strongly dependent on p38 activity, the induction of IL-10 competence in CD40-activated B cells does not depend on ERK1/2, p38 or JNK pathways.

Role of tumor associated fibroblasts in human liver regeneration, cirrhosis, and cancer.

Tumor associated fibroblasts (TAFs) are considered a microenvironmental element critical for tumor growth and progression. Experimental studies suggest that their origin could be from mesenchymal stem cells (MSCs) derived from the bone marrow. However, the role played by TAFs in cirrhosis, hepatocellular carcinoma development, and progression is largely unknown, and in vitro human models are missing. This paper for the first time demonstrates that (1) human neoplastic livers possess a population of multipotent adult stem cells (MASCs) with properties of TAFs; (2) a population of MASC-derived TAFs is already present in cirrhotic, not yet neoplastic, livers; (3) MASCs isolated from nonneoplastic and noncirrhotic liver scan acquire a TAF phenotype when grown in a medium conditioned by tumor cell lines, supporting the notion that TAF could originate from resident primitive cells (MASCs), possibly through a paracrine mechanism.