Myeloid cells, including parenchymal microglia, perivascular and meningeal macrophages, and dendritic cells (DCs), are present in the central nervous system (CNS) and establish an intricate ...relationship with other cells, playing a crucial role both in health and in neurological diseases. In this context, DCs are critical to orchestrating the immune response linking the innate and adaptive immune systems. Under steady-state conditions, DCs patrol the CNS, sampling their local environment and acting as sentinels. During neuroinflammation, the resulting activation of DCs is a critical step that drives the inflammatory response or the resolution of inflammation with the participation of different cell types of the immune system (macrophages, mast cells, T and B lymphocytes), resident cells of the CNS and soluble factors. Although the importance of DCs is clearly recognized, their exact function in CNS disease is still debated. In this review, we will discuss modern concepts of DC biology in steady-state and during autoimmune neuroinflammation. Here, we will also address some key aspects involving DCs in CNS patrolling, highlighting the neuroprotective nature of DCs and emphasizing their therapeutic potential for the treatment of neurological conditions. Recently, inhibition of the NAD
-dependent deac(et)ylase sirtuin 6 was demonstrated to delay the onset of experimental autoimmune encephalomyelitis, by dampening DC trafficking towards inflamed LNs. Thus, a special focus will be dedicated to sirtuins' role in DCs functions.
Abstract
Background
Experimental autoimmune encephalomyelitis (EAE) is the most common animal model of multiple sclerosis (MS), a neuroinflammatory and demyelinating disease characterized by ...multifocal perivascular infiltrates of immune cells. Although EAE is predominantly considered a T helper 1-driven autoimmune disease, mounting evidence suggests that activated dendritic cells (DC), which are the bridge between innate and adaptive immunity, also contribute to its pathogenesis. Sirtuin 6 (SIRT6), a NAD
+
-dependent deacetylase involved in genome maintenance and in metabolic homeostasis, regulates DC activation, and its pharmacological inhibition could, therefore, play a role in EAE development.
Methods
EAE was induced in female C57bl/6 mice by MOG35-55 injection. The effect of treatment with a small compound SIRT6 inhibitor, administered according to therapeutic and preventive protocols, was assessed by evaluating the clinical EAE score. SIRT6 inhibition was confirmed by Western blot analysis by assessing the acetylation of histone 3 lysine 9, a known SIRT6 substrate. The expression of DC activation and migration markers was evaluated by FACS in mouse lymph nodes. In addition, the expression of inflammatory and anti-inflammatory cytokines in the spinal cord were assessed by qPCR. T cell infiltration in spinal cords was evaluated by immunofluorescence imaging. The effect of Sirt6 inhibition on the migration of resting and activated bone marrow-derived dendritic cells was investigated in in vitro chemotaxis assays.
Results
Preventive pharmacological Sirt6 inhibition effectively delayed EAE disease onset through a novel regulatory mechanism, i.e., by reducing the representation of CXCR4-positive and of CXCR4/CCR7-double-positive DC in lymph nodes. The delay in EAE onset correlated with the early downregulation in the expression of CD40 on activated lymph node DC, with increased level of the anti-inflammatory cytokine IL-10, and with a reduced encephalitogenic T cell infiltration in the central nervous system. Consistent with the in vivo data, in vitro pharmacological Sirt6 inhibition in LPS-stimulated, bone marrow-derived DC reduced CCL19/CCL21- and SDF-1-induced DC migration.
Conclusions
Our findings indicate the ability of Sirt6 inhibition to impair DC migration, to downregulate pathogenic T cell inflammatory responses and to delay EAE onset. Therefore, Sirt6 might represent a valuable target for developing novel therapeutic agents for the treatment of early stages of MS, or of other autoimmune disorders.
Purpose
To present the results of a survival analysis of a series of 147 arthroscopic MAT procedures.
Methods
One-hundred and forty-seven patients (117 males and 30 females) underwent arthroscopic ...MAT without bone plugs (82 medial MAT and 65 lateral MAT) using fresh-frozen, non-irradiated grafts. They were retrospectively reviewed at a mean of 4.0 ± 1.9-year follow-up. Mean age at surgery was 40.9 ± 11.2 (range 16.7–68.8) years; 70 patients (48 %) underwent combined procedures. Clinical evaluation was performed with KOOS, Lysholm and a 0–100 VAS for pain. Survival analysis was performed using two endpoints: surgical failure (revision procedure with direct relation to MAT) and clinical failure (revision procedure or poor Lysholm score, <65).
Results
There was a significant (
p
< 0.05) and clinically relevant decrease in the VAS and increase in KOOS and Lysholm from pre-operative mean score to post-operative mean score. Seven (5 %) patients (two medial and five lateral) experienced surgical failure (five meniscectomies, one lateral graft peripheral suture and one unicompartmental knee arthroplasty). The mean overall survival time was 9.7 years (CI 9.1–10.3). As 16 (11 %) patients presented poor Lysholm score, a total of 23 (16 %) patients were considered clinical failures. The mean overall survival time was 8 years (CI 7.1–8.8). No statistically significant differences in failure and survival rate were present between medial and lateral MAT, isolated or combined MAT, patients >50 or <50 years old and patients with body mass index <25 or >25.
Conclusions
MAT, eventually associated with other needed procedures, was able to significantly relieve pain and improve function of the knee joint at midterm follow-up, with a survival rate from 9.7 to 8.0 years based on failure criteria. Most additional procedures were done in the first 2 post-operative years. MAT eventually associated with other needed procedures could represent an effective treatment for post-meniscectomy syndrome.
Level of evidence
Therapeutic study, retrospective case series, Level IV.
The nicotinamide phosphoribosyltransferase (NAMPT) is considered a very promising therapeutic target because it is overexpressed in pancreatic cancer. Although many inhibitors have been prepared and ...tested, clinical trials have shown that NAMPT inhibition may result in severe haematological toxicity. Therefore, the development of conceptually new inhibitors is an important and challenging task. We synthesized ten β-d-iminoribofuranosides bearing various heterocycle-based chains carbon-linked to the anomeric position starting from non-carbohydrate derivatives. They were then submitted to NAMPT inhibition assays, as well as to pancreatic tumor cells viability and intracellular NAD
depletion evaluation. The biological activity of the compounds was compared to that of the corresponding analogues lacking the carbohydrate unit to assess, for the first time, the contribution of the iminosugar moiety to the properties of these potential antitumor agents.
Sirtuin 6 (SIRT6) is a member of the mammalian NAD
-dependent deac(et)ylase sirtuin family. SIRT6's anti-inflammatory roles are emerging increasingly often in different diseases and cell types, ...including endothelial cells. In this study, the role of SIRT6 in pro-inflammatory conditions was investigated by engineering human umbilical vein endothelial cells to overexpress SIRT6 (SIRT6+ HUVECs). Our results showed that SIRT6 overexpression affected the levels of adhesion molecules and sustained megakaryocyte proliferation and proplatelet formation. Interestingly, the pro-inflammatory activation of the ATP/purinergic axis was reduced in SIRT6+ HUVECs. Specifically, the TNFα-induced release of ATP in the extracellular space and the increase in pannexin-1 hemichannel expression, which mediates ATP efflux, were hampered in SIRT6+ cells. Instead, NAD
release and Connexin43 expression were not modified by SIRT6 levels. Moreover, the Ca
influx in response to ATP and the expression of the purinergic receptor P2X7 were decreased in SIRT6+ HUVECs. Contrary to extracellular ATP, extracellular NAD
did not evoke pro-inflammatory responses in HUVECs. Instead, NAD
administration reduced endothelial cell proliferation and motility and counteracted the TNFα-induced angiogenesis. Altogether, our data reinforce the view of SIRT6 activation as an anti-inflammatory approach in vascular endothelium.
Purpose
The purpose of this systematic review was to summarise and evaluate the clinical outcomes of the collagen meniscus implant (CMI) and its complication and failure rates. These data were then ...used to evaluate the results of the CMI at different follow-up time periods and investigate possible differences in the behaviour of lateral and medial CMI.
Methods
A comprehensive search was performed in PubMed, MEDLINE, CINAHL, Cochrane, EMBASE and Google Scholar databases using various combinations of the following keywords: “collagen meniscus implant” or “collagen meniscal implant”. All studies evaluating medial or lateral CMI using the Lysholm score, visual analogue scale (VAS) for pain, Tegner activity scale and subjective or objective International Knee Documentation Committee (IKDC) scores were included in the systematic review.
Results
Eleven studies were included in the systematic review. The pooled number of patients involved in CMI surgery were 396 (90.2 % medial, 9.8 % lateral), with a mean age at surgery of 37.8 years. Concomitant procedures were present in 48.8 % of patients; most of them were anterior cruciate ligament (ACL) reconstruction, high tibial osteotomy (HTO) and microfractures. The Lysholm score and VAS for pain showed an improvement at six months up to ten years. No noticeable differences were present comparing short-term values of Lysholm score between medial and lateral CMI. The Tegner activity level reached its peak at 12 months after surgery and showed a progressive decrease through five and ten years post CMI implantation, however always remaining above the pre-operative level. Only a few knees were rated as “nearly abnormal” or “abnormal” at IKDC grading at all follow-up evaluations.
Conclusions
The CMI could produce good and stable clinical results, particularly regarding knee function and pain, with low rates of complications and reoperations.
Malignant melanoma is the most lethal form of skin cancer which shows BRAF mutation in 50% of patients. In this context, the identification of BRAF
mutation led to the development of specific ...inhibitors like PLX4032. Nevertheless, although its initial success, its clinical efficacy is reduced after six-months of therapy leading to cancer relapse due to the onset of drug resistance. Therefore, investigating the mechanisms underlying PLX4032 resistance is fundamental to improve therapy efficacy. In this context, several models of PLX4032 resistance have been developed, but the discrepancy between
and
results often limits their clinical translation.
The herein reported model has been realized by treating with PLX4032, for six months, patient-derived BRAF-mutated melanoma cells in order to obtain a reliable model of acquired PLX4032 resistance that could be predictive of patient's treatment responses. Metabolic analyses were performed by evaluating glucose consumption, ATP synthesis, oxygen consumption rate, P/O ratio, ATP/AMP ratio, lactate release, lactate dehydrogenase activity, NAD
/NADH ratio and pyruvate dehydrogenase activity in parental and drug resistant melanoma cells. The intracellular oxidative state was analyzed in terms of reactive oxygen species production, glutathione levels and NADPH/NADP
ratio. In addition, a principal component analysis was conducted in order to identify the variables responsible for the acquisition of targeted therapy resistance.
Collectively, our results demonstrate, for the first time in patient-derived melanoma cells, that the rewiring of oxidative phosphorylation and the maintenance of pyruvate dehydrogenase activity and of high glutathione levels contribute to trigger the onset of PLX4032 resistance.
Therefore, it is possible to hypothesize that inhibitors of glutathione biosynthesis and/or pyruvate dehydrogenase activity could be used in combination with PLX4032 to overcome drug resistance of BRAF-mutated melanoma patients. However, the identification of new adjuvant targets related to drug-induced metabolic reprogramming could be crucial to counteract the failure of targeted therapy in metastatic melanoma.
Limb-girdle muscular dystrophy R3, a rare genetic disorder affecting the limb proximal muscles, is caused by mutations in the α-sarcoglycan gene (Sgca) and aggravated by an immune-mediated damage, ...finely modulated by the extracellular (e)ATP/purinoceptors axis. Currently, no specific drugs are available. The aim of this study was to evaluate the therapeutic effectiveness of a selective P2X7 purinoreceptor antagonist, A438079. Sgca knockout mice were treated with A438079 every two days at 3 mg/Kg for 24 weeks. The P2X7 antagonist improved clinical parameters by ameliorating mice motor function and decreasing serum creatine kinase levels. Histological analysis of muscle morphology indicated a significant reduction of the percentage of central nuclei, of fiber size variability and of the extent of local fibrosis and inflammation. A cytometric characterization of the muscle inflammatory infiltrates showed that A438079 significantly decreased innate immune cells and upregulated the immunosuppressive regulatory T cell subpopulation. In α-sarcoglycan null mice, the selective P2X7 antagonist A438079 has been shown to be effective to counteract the progression of the dystrophic phenotype and to reduce the inflammatory response. P2X7 antagonism via selective inhibitors could be included in the immunosuppressant strategies aimed to dampen the basal immune-mediated damage and to favor a better engraftment of gene-cell therapies.
NAPRT, the rate-limiting enzyme of the Preiss–Handler NAD biosynthetic pathway, has emerged as a key biomarker for the clinical success of NAMPT inhibitors in cancer treatment. Previous studies found ...that high protein levels of NAPRT conferred resistance to NAMPT inhibition in several tumor types whereas the simultaneous blockade of NAMPT and NAPRT results in marked anti-tumor effects. While research has mainly focused on NAMPT inhibitors, the few available NAPRT inhibitors (NAPRTi) have a low affinity for the enzyme and have been scarcely characterized. In this work, a collection of diverse compounds was screened in silico against the NAPRT structure, and the selected hits were tested through cell-based assays in the NAPRT-proficient OVCAR-5 ovarian cell line and on the recombinant hNAPRT. We found different chemotypes that efficiently inhibit the enzyme in the micromolar range concentration and for which direct engagement with the target was verified by differential scanning fluorimetry. Of note, the therapeutic potential of these compounds was evidenced by a synergistic interaction between the NAMPT inhibitor FK866 and the new NAPRTi in terms of decreasing OVCAR-5 intracellular NAD levels and cell viability. For example, compound IM29 can potentiate the effect of FK866 of more than two-fold in reducing intracellular NAD levels. These results pave the way for the development of a new generation of human NAPRTi with anticancer activity.
Nicotinamide adenine dinucleotide (NAD+) is a fundamental molecule in the regulation of energy metabolism, representing both a coenzyme and a substrate for different NAD+ degrading enzymes. Among ...these enzymes, CD38 can be seen under two perspectives: as the enzyme synthesizing Ca2+-mobilizing second messenger, starting from NAD+, and as the major NAD+-consumer, to be inhibited to increase NAD+ levels. Indeed, the regulation of NAD+ availability is a key event during different processes. In this review, we examine the recent studies related to the modulation of CD38 expression and activity, and the consequent changes in NAD(P)(H), in adipose tissue, during inflammation and cold-induced thermogenesis.
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