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Therapeutic use of pantethine in experimental autoimmune encephalomyelitis
No full textLa pantetina, un tiolo a basso peso molecolare, rappresenta la forma stabile disulfidica della panteteina, il substrato metabolico che costituisce la parte attiva del coenzima A. Grazie alla capacità di abbassare l’indice lipidico senza effetti collaterali documentati, per decenni la pantetina è stata somministrata a pazienti con disfunzioni metaboliche. Studi recenti hanno dimostrato che la somministrazione di pantetina inibisce l’insorgenza della sindrome infiammatoria cerebrale in un modello animale di malaria, determinando la down-regolazione delle principali risposte cellulari pro-infiammatorie. Inoltre, il trattamento con pantetina è in grado di migliorare il decorso clinico in altri modelli animali di malattie neurologiche, tra le quali la malattia di Parkinson. Considerato che l’immunometabolismo è una disciplina che rappresenta una nuova frontiera nell’immunologia, lo scopo di questo studio è quello di studiare l’effetto metabolico associato alla somministrazione di pantetina sulla patogenesi dell’encefalomielite sperimentale autoimmune (EAE), il modello animale della sclerosi multipla (MS) umana.
I nostri esperimenti in vivo hanno dimostrato che il trattamento con pantetina è in grado di interferire con l’evoluzione della forma recidivante - remittente dell’EAE (RR-EAE), ritardando l’esordio della malattia e riducendo la severità del quadro clinico. Inoltre, la somministrazione del farmaco a seguito del manifestarsi dei primi sintomi di malattia, ha determinato un indicativo miglioramento del decorso e della severità della RR-EAE. Come dimostrato da analisi neuropatologiche, questi rilevanti dati clinici sono associati ad una diminuzione degli infiltrati infiammatori e del grado di demielinizzazione a livello del midollo spinale di topi trattati con pantetina. Utilizzando un nuovo sistema di imaging in vivo (IVIS 200; Caliper Life Science), è stato inoltre dimostrato che gli animali trattati con pantetina presentano un ridotto leakage della barriera emato-encefalica (BEE) durante la fase pre-clinica della RR-EAE.
Un ulteriore dato molto importante è la significativa riduzione della capacità proliferativa antigene-specifica delle cellule T isolate dai linfonodi drenanti di animali trattati con pantetina, se paragonato alle cellule isolate da animali non-trattati. I linfociti ottenuti da topi trattati hanno presentato anche una diminuzione nella produzione di citochine pro-infiammatorie durante il picco iniziale di malattia. Inoltre, i studi condotti in vitro suggeriscono che il pre-trattamento con pantetina ha un effetto bloccante sull’adesione integrino-dependente delle cellule T encefalitogeniche. La pantetina ha inoltre un effetto inibitorio anche sull’adesione in vivo delle cellule T attivate, effetto dimostrato tramite l’utilizzo di microscopia intravitale in un modello di infiammazione dei vasi cerebrali. Analisi eseguite tramite ImageStream System hanno evidenziato come l’inibizione sulla capacità adesiva delle cellule T PLP-specifiche non possa essere spiegata tramite cambiamenti di espressione integrinica o della capacità delle integrine di formare cluster indotti tramite attivazione chemochinica. Dato che la funzionalità delle integrine non dipende solo dalla loro avidità per i ligandi, ma anche dalla loro localizzazione all’interno di rafts lipidici ricchi di colesterolo presenti sulla superficie cellulare, si è analizzata l’influenza del trattamento con pantetina sulla conformazione dei rafts lipidici sulle cellule T attivate. Sorprendentemente, gli esperimenti condotti hanno evidenziato che il pre-trattamento con pantetina è in grado di ridurre notevolmente la formazione di rafts lipidici e la loro clusterizzazione sulle cellule T, nonostante le integrine fossero ancora organizzate in grandi caps polarizzati come sulle cellule T non trattate.
Per analizzare più a fondo l’effetto globale della pantetina sulle cellule T attivate, si sono effettuati studi di metabolomica, i quali hanno evidenziato un effetto metabolico molto significativo della pantetina sulle cellule T trattate. In particolare, il trattamento con pantetina ha un effetto molto marcato su cellule T memory, se paragonate a cellule T naïve, modulando almeno 45 diversi pathways metabolici e causando un forte aumento della quantità di coenzima A disponibile, il quale va principalmente ad aumentare l’efficienza del ciclo di Krebs. Questi dati sembrerebbero indicare una capacità da parte della pantetina di regolare molto finemente il metabolismo delle cellule T patogeniche responsabili del danno al sistema nervoso centrale osservato nella MS.
In conclusione i nostri risultati dimostrano che la pantetina ha un effetto immuno-modulatorio sull’EAE attraverso la riduzione dell’attivazione e dell’adesività delle cellule T patogeniche ed il mantenimento dell’integrità’ della BEE. Considerando il basso costo del farmaco, la sua sicura somministrazione in assenza di effetti collaterali e i nostri nuovi risultati, questo tiol a basso peso molecolare potrebbe rappresentare un approccio terapeutico efficace nel trattamento della MS.Pantethine is a low molecular weight thiol and represents the stable disulfate form of pantetheine, the metabolic substrate constituting the active part of coenzyme A (CoA). For decades, pantethine has been administrated to patients with metabolic disorders for its lipid lowering activity, without any side effect reported. Recent data showed that pantethine prevents the occurrence of cerebral malaria, with the down-regulation of key cellular responses to the inflammatory cerebral syndrome. Immunometabolism represents a new frontier in immunology and the aim of this study was to investigate the effect of metabolic intervention with pantethine on experimental autoimmune encephalomyelitis (EAE), the animal model of human multiple sclerosis (MS).
Our in vivo experiments demonstrated that pantethine treatment prevents the development of chronic and relapsing-remitting EAE by delaying the disease onset and reducing the clinical score. Furthermore, pantethine treatment started after disease onset significantly ameliorated disease course and severity. The surprising clinical results were accompanied by a decrease in inflammatory infiltrates and in demyelination in pantethine treated-mice, proved by our neuropathological studies. Moreover, using IVIS 200 system we found that pantethine treated-mice had a reduced BBB leakage during the pre-clinical phase of relapsing-remitting EAE. Importantly, T-cells isolated from draining lymph nodes of mice treated with pantethine showed a significant reduction in antigen-specific proliferation and pro-inflammatory cytokines production at disease peak when compared with untreated-mice.
Furthermore, our data from in vitro experiments demonstrated that pantethine pre-treatment of encephalitogenic T-cells blocked T-cell adhesion on purified integrin ligands. In addition, pantethine inhibited activated T-cell adhesion in vivo using an intravital microscopy model performed in inflamed brain venules. Importantly, pantethine had no effect on chemokine-induced naïve T-cell adhesion, proving that pantethine treatment has a selective effect only on activated T-cells. On the other hand, ImageStream analysis show that the important inhibition of proteolipid-specific T-cells adhesion could not be explain by changes on integrin expression or on their ability to form clusters upon chemokine-induce activation. Integrin functionality does not depend only on their avidity for their ligands but also on its localization into cholesterol-rich membrane rafts on cell surface. Thus, we next investigated the role of pantethine treatment on lipid rafts conformation on activated T-cells. Surprisingly, our in vitro experiments demonstrated that pantethine pre-treatment strongly reduced lipid rafts formation and raft clusters on encephalitogenic T-cells, although integrins were still present in big polarized caps. These results suggest that pantethine dissolve lipid rafts on activated T-cell, possibly interfering with the signal transduction necessary to support integrin-dependent firm adhesion.
To further understand pantethine effect on in T-cell functions, we performed metabolomics analysis on pantethine-treated activated T-cells. Our data suggested a global metabolic effect of pantethine on T-cells. Overall, pantethine treatment significantly and differently affected the metabolism of memory T lymphocytes with respect to naïve T lymphocytes, leading to the modulation of at least 45 metabolic pathways with a huge availability of CoA that greatly enhances the Krebs cycle efficiency. These final consideration made us believe that pantethine could be able to perform a fine metabolic tuning of the pathogenic T-cells involved in demyelinating diseases, as MS.
In conclusion, our results demonstrate that pantethine has an immuno-modulatory effect on EAE by reducing T-cell activation and adhesiveness, and maintaining BBB integrity. As pantethine has a low-cost and has successfully administered in different context to man in the absence of side effects, our results suggest that this low molecular weight thiol may represent a valuable new therapeutic approach in MS
Predictors of Ectopic Fat in Humans
No full textIn the last decade there has been increasing focus on body fat distribution, rather than on the degree of obesity. More recently, great interest has also been dedicated to ectopic fat deposition in overnourished individuals that reflects a failure of the system of intracellular lipid homeostasis, which, in normal conditions, prevents lipotoxicity in the organs, by confining lipid overload to cells specifically designed to store large quantities of surplus calories, the white adipocytes. Consequently, excess body weight leads to fat infiltration of multiple organs including liver, pancreas, skeletal muscle, and heart thus forming "ectopic fat". Although overfeeding is considered the main predictor of ectopic fat deposition, other factors may be also involved. The purpose of this review is to evaluate the current available data on the predictors of ectopic fat deposition in humans
Adipocytes WNT5a mediated dedifferentiation: a possible target in pancreatic cancer microenvironment
Get PDFA significant epidemiological association between obesity and pancreatic ductal adenocarcinoma (PDAC) has previously been described, as well as a correlation between the degree of pancreatic steatosis, PDAC risk and prognosis. The underlying mechanisms are still not completely known.After co-culture of 3T3-L1 adipocytes and MiaPaCa2 with an in vitro transwell system we observed the appearance of fibroblast-like cells, along with a decrease in number and size of remaining adipocytes. RT-PCR analyses of 3T3-L1 adipocytes in co-culture showed a decrease in gene expression of typical markers of mature adipocytes, in parallel with an increased expression of fibroblast-specific and reprogramming genes. We found an increased WNT5a gene and protein expression early in MiaPaCa2 cells in co-culture. Additionally, EMSA of c-Jun and AP1 in 3T3-L1 demonstrated an increased activation in adipocytes after co-culture. Treatment with WNT5a neutralizing antibody completely reverted the activation of c-Jun and AP1 observed in co-cultured adipocytes.Increasing doses of recombinant SFRP-5, a competitive inhibitor for WNT5a receptor, added to the co-culture medium, were able to block the dedifferentiation of adipocytes in co-culture.These data support a WNT5a-mediated dedifferentiation process with adipocytes reprogramming toward fibroblast-like cells that might profoundly influence cancer microenvironment
Vascular inflammation in central nervous system diseases: adhesion receptors controlling leukocyte-endothelial interactions
Get PDFLeukocyte trafficking from the blood into the tissues represents a key process during inflammation and requires multiple steps mediated by adhesion molecules and chemoattractants. Inflammation has a detrimental role in several diseases, and in such cases, the molecular mechanisms controlling leukocyte migration are potential therapeutic targets. Over the past 20 years, leukocyte migration in the CNS has been investigated almost exclusively in the context of stroke and MS. Experimental models of ischemic stroke have led to the characterization of adhesion molecules controlling leukocyte migration during acute inflammation, whereas EAE, the animal model of MS, has provided similar data for chronic inflammation. Such experiments have led to clinical trials of antileukocyte adhesion therapy, with consistently positive outcomes in human subjects with MS, showing that interference with leukocyte adhesion can ameliorate chronic inflammatory CNS diseases. This review summarizes our current understanding of the roles of adhesion molecules controlling leukocyte-endothelial interactions in stroke and MS, focusing on recently discovered, novel migration mechanisms. We also discuss the growing evidence suggesting a role for vascular inflammation and leukocyte trafficking in neurodegenerative diseases such as AD. Moreover, we highlight recent findings suggesting a role for leukocyte-endothelial interactions in the pathogenesis of seizures and epilepsy, thus linking endothelial activation and leukocyte trafficking to neuronal electrical hyperactivity. These emerging roles for leukocytes and leukocyte adhesion mechanisms in CNS diseases provide insight into the mechanisms of brain damage and may contribute to the development of novel therapeutic strategies
Phenotypic Shift of Adipocytes by Cholecalciferol and 1α,25 Dihydroxycholecalciferol in Relation to Inflammatory Status and Calcium Content
No full textRecent experimental data seem to suggest a relevant role for 1,25[OH]2cholecalciferol (1,25[OH]2D3) in adipocyte physiology and pathophysiology, with some studies showing adipogenic and pro-inflammatory properties, and others lipolytic and anti-inflammatory functions. Moreover, to our knowledge, the role of cholecalciferol (D3) in adipocytes function is still not known. Therefore, the aim of this study was to investigate in vitro the effects of 1,25[OH]2D3, as well as of D3, in 3T3-L1 adipocytes in basal and inflammatory conditions, testing the effects of different calcium concentrations in adipocytes culture medium. In 3T3-L1 adipocytes, CYP27A1 and CYP27B1 mRNA were detected in basal conditions and induced after D3 treatment. Pre-treatment of 3T3-L1 adipocytes not only with 1,25[OH]2D3, but also with D3 before inflammatory stimulation, significantly prevented the increase in gene expression and protein secretion of IL-6 and TNF-α, and significantly increased IL-10 mRNA and protein production compared with adipocytes treated only with lipopolysaccharide (LPS). Biological effects of D3 were still present after inhibition of P450 activity with ketokonazole. LPS determined a decrease in cell area compared with controls, paralleled by a significant increase in optical density (OD) of lipid droplets, whereas 1,25[OH]2D3 and D3 alone significantly increased adipocytes area and decreased OD. Pretreatment with both forms of vitamin D preserved cells from the reduction in their area observed after LPS treatment. LPS decreased more the area of cells grown in a high calcium medium than of adipocytes grown in a low calcium medium. In the presence of a high calcium medium, 1,25(OH)2D3 treatment preserved cell area, maintaining its anti-inflammatory and adipogenic properties. In conclusion our results show that D3, besides 1,25[OH]2D3, presents anti-inflammatory effects on 3T3-L1, as well as that adipocytes have the enzymatic pathways necessary to locally regulate the production of active forms of vitamin D, capable of influencing adipocyte phenotype and function
Multidimensional improvements induced by an intensive obesity inpatients rehabilitation programme
Get PDFTo analyse the short-term effectiveness of an intensive multidimensional inpatient programme specifically developed for patients with severe obesity
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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