The therapeutic effect of mesenchymal stem cell transplantation in experimental autoimmune encephalomyelitis is mediated by peripheral and central mechanisms

Stem cells are currently seen as a treatment for tissue regeneration in neurological diseases such as multiple sclerosis, anticipating that they integrate and differentiate into neural cells. Mesenchymal stem cells (MSCs), a subset of adult progenitor cells, differentiate into cells of the mesodermal lineage but also, under certain experimental circumstances, into cells of the neuronal and glial lineage. Their clinical development, however, has been significantly boosted by the demonstration that MSCs display significant therapeutic plasticity mainly occurring through bystander mechanisms. These features have been exploited in the effective treatment of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis where the inhibition of the autoimmune response resulted in a significant amelioration of disease and decrease of demyelination, immune infiltrates and axonal loss. Surprisingly, these effects do not require MSCs to engraft in the central nervous system but depend on the cells' ability to inhibit pathogenic immune responses both in the periphery and inside the central nervous system and to release neuroprotective and pro-oligodendrogenic molecules favoring tissue repair. These results paved the road for the utilization of MSCs for the treatment of multiple sclerosis

Cost Minimization Analysis of Radiofrequency Compared to Laser Thermal Ablation in Patients with Hepatocellular Carcinoma

BACKGROUND: Over the last decade of years, minimally invasive techniques have been developed for the treatment of hepatocellular carcinoma and liver metastases. We sought to investigate the health costs associated with the management of patients with hepatocellular carcinoma treated with radiofrequency vs laser thermal ablation and their clinical outcomes.METHODS: We performed a retrospective analysis of the ablations performed in two referral centers in southern Italy, from 2009 to 2013. Resource use was valued by year 2017 official prices, in €. Direct healthcare costs (drugs, visits, tests and hospitalizations) of different ablation techniques were compared. Total costs were analyzed from Italian NHS perspective.RESULTS: A total of 140 patients were identified. Baseline demographics and clinical outcomes of interest did not differ between the two groups. Patients treated with laser thermal ablation resulted in an expected annually cost savings of 258.9 € per patient, in one-year follow-up healthcare costs compared with radiofrequency. The largest components of annual medical expenditures were attributable to drugs, regardless of the type of ablative technique.CONCLUSIONS: The ablation using either laser thermal ablation or radiofrequency is equally effective. Laser thermal ablation would carry disposable cost savings as compared to radiofrequency. The costs associated with management of patients with hepatocellular carcinoma, treated with laser thermal ablation were lower than those treated with radiofrequency ablation

BBB-endothelial tight junction response to mesenchymal stem cells in a model of MOG EAE

Experimental autoimmune encephalomyelitis (EAE), an induced autoimmune disease of the central nervous system, simulates the main histopathological and clinical aspects of multiple sclerosis including the impairment of the blood-brain barrier (BBB). In several experimental models of human neurodegenerative diseases, the intravenous (iv) injection of bone marrow-derived mesenchymal stem cells (MSCs) ameliorates clinical symptoms and histopathological features [1,2]. On the basis of these data, we have analyzed the status of BBB tight junctions (TJs) of cerebral cortex microvessels in a model of MOG-EAE with iv injection of MSCs (EAE-MSC). The observations were carried out on EAE-MSC mice sacrificed at 6-24 hrs and 10 days after MSCs iv injection. The expression of endothelial TJ proteins, claudin-5 and occludin, was analyzed in healthy, EAE, and EAE-MSC mice by immunofluorescence confocal microscopy, together with the evaluation of barrier function by FITC-Dextran, as an exogenous permeability tracer. The results demonstrate that unlike EAE animals, characterized by an interrupted junctional staining and a barrier leakage, EAE-MSC mice show together with attenuate disease symptoms, a continuous, control- like claudin-5 and occludin junctional pattern and a functionally recovered barrier efficiency. Overall, these findings suggest that during EAE, the neuroprotective effect of the injected MSCs includes a reparative BBB response that in turn may contribute to the reduction of the inflammatory infiltrates and to the significant amelioration of the disease

Microvascular pericytes involvement in experimental autoimmune encephalomyelitis

In the CNS, pericytes are microvessel wall-encircling cells that, together with endothelial cells, perivascular glial endfeet and basement membrane, form the blood-brain barrier (BBB). Dysfunction of the BBB and migration of autoreactive T lymphocytes into the CNS are histopathological hallmarks of both Multiple Sclerosis (MS), a chronic demyelinating disease, and experimental autoimmune encephalomyelitis (EAE), a widely used MS animal model. The proteoglycan NG2, which has been described to accumulate within MS plaques and at spinal cord (SC) injury sites, is a primary component of pericytes, engaged in pericyte/endothelial cell interaction, proliferation and migration. To explore the role of NG2-expressing pericytes during neuroinflammation and BBB dysfunction, pericyte coverage (pericyte number/vessel length) and density (pericyte number/tissue volume) ratios were studied in brain microvessels by immunohistochemistry and laser confocal microscopy using specific pericyte markers, NG2, RGS5, and CD13. The observations were made in mice affected by MOG-induced chronic EAE with two different genetic C57BL/6 backgrounds: wild type (WT) and homozygous NG2 null (NG2-/-). In literature, NG2-/- mice did not exhibit gross phenotypic or vascular alterations, whereas our results demonstrated an unaltered pericyte density associated with slightly decreased pericyte coverage index and pericyte/endothelial cell ratio. These observations were confirmed in NG2-/- EAE-affected mice, that showed an attenuated disease severity and demyelination, and a milder BBB leakage and leukocyte infiltration, as compared with EAE WT. Taken together these results lend support to the idea of a direct involvement of NG2 proteoglycan in pericyte-endothelial cell interactions essential for the preservation of a proper BBB function

Immunolocalization of CCL2-expressing cells in EAE and EAE-MSC cerebral cortex

The chemokine CCL2 has been considered as a mediator of inflammation in different diseases of the central nervous system, including experimental autoimmune encephalomyelitis (EAE), where the chemokine mediates extravasation of mononuclear leukocytes and loss of microvessel barrier function [1]. Previous studies have demonstrated that cellular sources of CCL2 during both EAE and multiple sclerosis (MS) are astrocytes and microvessel endothelial cells (ECs). Initially, we have demonstrated that in a MOG-induced model of EAE in C57BL/6 mice, 6 hrs after the intravenous treatment with bone marrow derived mesenchymal stem cells (MSCs) [2], the junctional staining patter of blood-brain barrier (BBB) microvessels and their functional effectiveness to permeability tracers seem to be restored. We have subsequently analysed, in the same experimental models, EAE and EAE-MSC mice, expression and immunolocalization of chemokine CCL2 by double immmunolabelling with cell-specific markers: endothelial PECAM-1 (CD31), OPCs (oligodendrocyte precursor cells) proteoglycan NG2, astrocytic GFAP, and Iba1 for microglia cells. Surprisingly, in the adopted model of cerebral cortex EAE, astrocytes and ECs do not show any detectable CCL2 expression, instead a strong staining is observed on activated parenchymal and perivascular microglia. Astrogliosis, microglia activation, and CCL2 overexpression appearing strongly reduced in EAE mice after MSC treatment. These observations identify microglia cells as the major source of CCL2 in EAE mice, whose barrier is damaged, and suggest the downregulation of the chemokine in perivascular microglia as a possible mechanism involved in BBB protection after MSC administration

Tight junction protein changes in experimental autoimmune encephalomyelitis models

Experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis (MS), is characterized by vascular changes, particularly endothelial tight junction (TJ) protein (claudin-5 and occludin) alterations. During blood-brain barrier function, the vascular wall components, endothelial cells, pericytes and perivascular astrocytes, engage in crosstalks through cell-associated molecules and soluble factors. Pericyte-associated NG2 is a large transmembrane proteoglycan participating in these interactions, as well as in the control of pericyte proliferation and migration. We have analyzed the role of NG2 on endothelial TJ arrangement in two groups of mice, wild type (WT) and homozygous NG2 null (NG2-/-), affected by MOG-induced EAE. Expression and distribution of the TJ transmembrane proteins claudin-5 and occludin were analyzed in the cerebral cortex microvessels by immunohistochemistry and laser confocal microscopy. In NG2-/-mice, most cortex vessels showed an altered, chain-like claudin-5 staining pattern with aggregates distributed irregularly along the junctional membranes. Unlike the claudin-5 changes, the occludin staining pattern appeared continuous and linear and only a few cortex microvessels showed protein clustering. These TJ protein expression results in NG2-/- mice affected by EAE were compared with our previous results on WT EAE mice sacrificed at 39 days post immunization. In WT EAE both claudin-5 and occludin appeared severely damaged but occludin changes were related to more severe disease stages. Interestingly, in NG2-/- EAE-affected mice, claudin-5 and occludin formed an apparently unaffected linear and continuous junctional staining, suggesting a compensation of TJ damage, with cerebral cortex microvessels showing a restored claudin-5 and occludin junctional pattern. Overall, these observations suggest that absence of NG2 in the brain microvessels of naïve NG2 null mice may affect the normal arrangement of TJ proteins, whereas under inflammatory stimuli these effects seem to be partly reversed

Sirt6 regulates dendritic cell differentiation, maturation, and function

Dendritic cells (DCs) are antigen-presenting cells that critically influence decisions about immune activation or tolerance. Impaired DC function is at the core of common chronic disorders and contributes to reduce immunocompetence during aging. Knowledge on the mechanisms regulating DC generation and function is necessary to understand the immune system and to prevent disease and immunosenescence. Here we show that the sirtuin Sirt6, which was previously linked to healthspan promotion, stimulates the development of myeloid, conventional DCs (cDCs). Sirt6-knockout (Sirt6KO) mice exhibit low frequencies of bone marrow cDC precursors and low yields of bone marrow-derived cDCs compared to wild-type (WT) animals. Sirt6KO cDCs express lower levels of class II MHC, of costimulatory molecules, and of the chemokine receptor CCR7, and are less immunostimulatory compared to WT cDCs. Similar effects in terms of differentiation and immunostimulatory capacity were observed in human monocyte-derived DCs in response to SIRT6 inhibition. Finally, while Sirt6KO cDCs show an overall reduction in their ability to produce IL-12, TNF-α and IL-6 secretion varies dependent on the stimulus, being reduced in response to CpG, but increased in response to other Toll-like receptor ligands. In conclusion, Sirt6 plays a crucial role in cDC differentiation and function and reduced Sirt6 activity may contribute to immunosenescence

Catastrophic NAD+ Depletion in Activated T Lymphocytes through Nampt Inhibition Reduces Demyelination and Disability in EAE

Nicotinamide phosphoribosyltransferase (Nampt) inhibitors such as FK866 are potent inhibitors of NAD+ synthesis that show promise for the treatment of different forms of cancer. Based on Nampt upregulation in activated T lymphocytes and on preliminary reports of lymphopenia in FK866 treated patients, we have investigated FK866 for its capacity to interfere with T lymphocyte function and survival. Intracellular pyridine nucleotides, ATP, mitochondrial function, viability, proliferation, activation markers and cytokine secretion were assessed in resting and in activated human T lymphocytes. In addition, we used experimental autoimmune encephalomyelitis (EAE) as a model of T-cell mediated autoimmune disease to assess FK866 efficacy in vivo. We show that activated, but not resting, T lymphocytes undergo massive NAD+ depletion upon FK866-mediated Nampt inhibition. As a consequence, impaired proliferation, reduced IFN-γ and TNF-α production, and finally autophagic cell demise result. We demonstrate that upregulation of the NAD+-degrading enzyme poly-(ADP-ribose)-polymerase (PARP) by activated T cells enhances their susceptibility to NAD+ depletion. In addition, we relate defective IFN-γ and TNF-α production in response to FK866 to impaired Sirt6 activity. Finally, we show that FK866 strikingly reduces the neurological damage and the clinical manifestations of EAE. In conclusion, Nampt inhibitors (and possibly Sirt6 inhibitors) could be used to modulate T cell-mediated immune responses and thereby be beneficial in immune-mediated disorders

Performance of the ALICE experiment at the CERN LHC

ALICE is the heavy-ion experiment at the CERN Large Hadron Collider. The experiment continuously took data during the first physics campaign of the machine from fall 2009 until early 2013, using proton and lead-ion beams. In this paper we describe the running environment and the data handling procedures, and discuss the performance of the ALICE detectors and analysis methods for various physics observables