Myriocin modulates the altered lipid metabolism and storage in cystic fibrosis.

Cystic fibrosis (CF) is a hereditary disease mostly related to ΔF508 CFTR mutation causing a proteinopathy that is characterized by multiple organ dysfunction, primarily lungs chronic inflammation, and infection. Defective autophagy and accumulation of the inflammatory lipid ceramide have been proposed as therapeutic targets. Accumulation of lipids and cholesterol was reported in the airways of CF patients, together with altered triglycerides and cholesterol levels in plasma, thus suggesting a disease-related dyslipidemia. Myriocin, an inhibitor of sphingolipids synthesis, significantly reduces inflammation and activates TFEB-induced response to stress, enhancing fatty acids oxidation and promoting autophagy. Myriocin ameliorates the response against microbial infection in CF models and patients' monocytes. Here we show that CF broncho-epithelial cells exhibit an altered distribution of intracellular lipids. We demonstrated that lipid accumulation is supported by an enhanced synthesis of fatty acids containing molecules and that Myriocin is able to reduce such accumulation. Moreover, Myriocin modulated the transcriptional profile of CF cells in order to restore autophagy, activate an anti-oxidative response, stimulate lipid metabolism and reduce lipid peroxidation. Moreover, lipid storage may be altered in CF cells, since we observed a reduced expression of lipid droplets related proteins named perilipin 3 and 5 and seipin. To note, Myriocin up-regulates the expression of genes that are involved in lipid droplets biosynthesis and maturation. We suggest that targeting sphingolipids de novo synthesis may counteract lipids accumulation by modulating CF altered transcriptional profile, thus restoring autophagy and lipid metabolism homeostasis

Complementary Use of Carbohydrate Antigens Lewis a, Lewis b, and Sialyl-Lewis a (CA19.9 Epitope) in Gastrointestinal Cancers: Biological Rationale towards a Personalized Clinical Application

Carbohydrate antigen 19.9 (CA19.9) is used as a tumor marker for clinical and research purposes assuming that it is abundantly produced by gastrointestinal cancer cells due to a cancer-associated aberrant glycosylation favoring its synthesis. Recent data has instead suggested a different picture, where immunodetection on tissue sections matches biochemical and molecular data. In addition to CA19.9, structurally related carbohydrate antigens Lewis a and Lewis b are, in fact, undetectable in colon cancer, due to the down-regulation of a galactosyltransferase necessary for their synthesis. In the pancreas, no differential expression of CA19.9 or cognate glycosyltransferases occurs in cancer. Ductal cells only express such Lewis antigens in a pattern affected by the relative levels of each glycosyltransferase, which are genetically and epigenetically determined. The elevation of circulating antigens seems to depend on the obstruction of neoplastic ducts and loss of polarity occurring in malignant ductal cells. Circulating Lewis a and Lewis b are indeed promising candidates for monitoring pancreatic cancer patients that are negative for CA19.9, but not for improving the low diagnostic performance of such an antigen. Insufficient biological data are available for gastric and bile duct cancer. Studying each patient in a personalized manner determining all Lewis antigens in the surgical specimens and in the blood, together with the status of the tissue-specific glycosylation machinery, promises fruitful advances in translational research and clinical practice

2-Acetyl-5-tetrahydroxybutyl imidazole (THI) protects 661W cells against oxidative stress

Retinal degeneration and in particular retinitis pigmentosa (RP) is associated to ceramide (Cer) accumulation and cell death induction. Cer and sphingosine-1-phosphate (S1P) belong to the sphingolipids class and exert a pro-apoptotic and pro-survival activity, respectively. Our aim is to target sphingolipid metabolism by inhibiting S1P lyase that regulates one of the S1P degradation pathways, to reduce retinal photoreceptor damage. The murine 661W cone-like cell line was pretreated with THI, an inhibitor of S1P lyase and exposed to H2O2-induced oxidative stress. 661W cell viability and apoptosis were evaluated by Trypan Blue and TUNEL assay, respectively. Protein expression of mediators of the survival/death pathway (ERK1/2, Akt, Bcl-2, Bax) was analyzed by Western blotting. RT-PCR was performed to establish HO-1 transcript changes and LC-MS analysis to measure Cer intracellular content. THI rescues inhibitory H2O2-effect on 661W cell viability and impairs H2O2-induced apoptosis by increasing Bcl-2/Bax ratio. THI administration counteracts the oxidative stress effects of H2O2 on 661W cells by activating the Nrf2/HO-1 pathway, regulating ERK and Akt phosphorylation levels, and decreasing Cer intracellular content. We conclude that sphingolipid metabolism manipulation can be considered a therapeutic target to promote photoreceptor survival. © 2017 Springer-Verlag Berlin HeidelbergPeer reviewe

Endocarditis infecciosa. Análisis retrospectivo de tres años (1995-1997)

A retrospective analysis of the medical histories of 24 patients suffering from infective endocarditis from January 1995 to December 1997 was made, with a view to finding out the characteristics of this group of patients. Half of the patients had permanent pacemakers and one third were carriers of native valve cardiopathies. Access route for the disease was detected in 45.8 % of them whereas pacemaker pocket sepsis was the origin of the infection in almost two-thirds of them. Microorganisms causing the sepsis were determined in 79.2 % of cases and the most commom were positive staphylococcus coagulase, negative staphylococcus coagulase and streptococcus viridans. 14 patients were treated with surgical therapy and 2 with medical treatment. Eight patients died from heart septic complications. We concluded that a strict antibiotic prophylaxis should be followed in patients having heart valve protheses or permanent pacemakers in the face of any surgical intervention because of the risks posed by infective endocarditis.Fueron analizadas retrospectivamente las historias clínicas de 24 pacientes con endocarditis infecciosa en el período comprendido entre enero de 1995 y diciembre de 1997, con el objetivo de conocer las características de este grupo. La mitad de los pacientes tenían implantados marcapasos permanentes y la tercera parte eran portadores de cardiopatías valvulares primitivas. La puerta de entrada se identificó en el 45,8 % de los pacientes y en casi las dos terceras partes de ellos fue la sepsis del bolsillo del marcapasos el inicio de la infección. Los microorganismos causales se determinaron en el 79,2 % y los gérmenes más frecuentes fueron el estafilococo coagulasa positivo, el estafilococo coagulasa negativo y el estreptococo viridans. La forma de resolución fue quirúrgica en 14 enfermos y médica en 2. Fallecieron 8 pacientes como consecuencia de complicaciones cardíacas y sépticas. Concluimos que debe realizarse una rigurosa profilaxis antibiótica a los pacientes con prótesis valvular cardíaca o con marcapasos permanentes ante cualquier instrumentación quirúrgica por el alto riesgo de que se presente la endocarditis infecciosa

Endocarditis infecciosa. Análisis retrospectivo de tres años (1995-1997)

A retrospective analysis of the medical histories of 24 patients suffering from infective endocarditis from January 1995 to December 1997 was made, with a view to finding out the characteristics of this group of patients. Half of the patients had permanent pacemakers and one third were carriers of native valve cardiopathies. Access route for the disease was detected in 45.8 % of them whereas pacemaker pocket sepsis was the origin of the infection in almost two-thirds of them. Microorganisms causing the sepsis were determined in 79.2 % of cases and the most commom were positive staphylococcus coagulase, negative staphylococcus coagulase and streptococcus viridans. 14 patients were treated with surgical therapy and 2 with medical treatment. Eight patients died from heart septic complications. We concluded that a strict antibiotic prophylaxis should be followed in patients having heart valve protheses or permanent pacemakers in the face of any surgical intervention because of the risks posed by infective endocarditis.Fueron analizadas retrospectivamente las historias clínicas de 24 pacientes con endocarditis infecciosa en el período comprendido entre enero de 1995 y diciembre de 1997, con el objetivo de conocer las características de este grupo. La mitad de los pacientes tenían implantados marcapasos permanentes y la tercera parte eran portadores de cardiopatías valvulares primitivas. La puerta de entrada se identificó en el 45,8 % de los pacientes y en casi las dos terceras partes de ellos fue la sepsis del bolsillo del marcapasos el inicio de la infección. Los microorganismos causales se determinaron en el 79,2 % y los gérmenes más frecuentes fueron el estafilococo coagulasa positivo, el estafilococo coagulasa negativo y el estreptococo viridans. La forma de resolución fue quirúrgica en 14 enfermos y médica en 2. Fallecieron 8 pacientes como consecuencia de complicaciones cardíacas y sépticas. Concluimos que debe realizarse una rigurosa profilaxis antibiótica a los pacientes con prótesis valvular cardíaca o con marcapasos permanentes ante cualquier instrumentación quirúrgica por el alto riesgo de que se presente la endocarditis infecciosa

An Update on Sphingolipidomics: Is Something Still Missing? Some Considerations on the Analysis of Complex Sphingolipids and Free-Sphingoid Bases in Plasma and Red Blood Cells

The main concerns in targeted “sphingolipidomics” are the extraction and proper handling of biological samples to avoid interferences and achieve a quantitative yield well representing all the sphingolipids in the matrix. Our work aimed to compare different pre-analytical procedures and to evaluate a derivatization step for sphingoid bases quantification, to avoid interferences and improve sensitivity. We tested four protocols for the extraction of sphingolipids from human plasma, at different temperatures and durations, and two derivatization procedures for the conversion of sphingoid bases into phenylthiourea derivatives. Different columns and LC-MS/MS chromatographic conditions were also tested. The protocol that worked better for sphingolipids analysis involved a single-phase extraction in methanol/chloroform mixture (2:1, v/v) for 1 h at 38 °C, followed by a 2 h alkaline methanolysis at 38 °C, for the suppression of phospholipids signals. The derivatization of sphingoid bases promotes the sensibility of non-phosphorylated species but we proved that it is not superior to a careful choice of the appropriate column and a full-length elution gradient. Our procedure was eventually validated by analyzing plasma and erythrocyte samples of 20 volunteers. While both extraction and methanolysis are pivotal steps, our final consideration is to analyze sphingolipids and sphingoid bases under different chromatographic conditions, minding the interferences

Inhibitors of ceramide de novo biosynthesis rescue damages induced by cigarette smoke in airways epithelia

Exposure to cigarette smoke represents the most important risk factor for the development of chronic obstructive pulmonary disease (COPD). COPD is characterized by chronic inflammation of the airways, imbalance of proteolytic activity resulting in the destruction of lung parenchyma, alveolar hypoxia, oxidative stress, and apoptosis. Sphingolipids are structural membrane components whose metabolism is altered during stress. Known as apoptosis and inflammation inducer, the sphingolipid ceramide was found to accumulate in COPD airways and its plasma concentration increased as well. The present study investigates the role of sphingolipids in the cigarette smoke-induced damage of human airway epithelial cells. Lung epithelial cells were pre-treated with sphingolipid synthesis inhibitors (myriocin or XM462) and then exposed to a mixture of nicotine, acrolein, formaldehyde, and acetaldehyde, the major toxic cigarette smoke components. The inflammatory and proteolytic responses were investigated by analysis of the mRNA expression (RT-PCR) of cytokines IL-1β and IL-8, and matrix metalloproteinase-9 and of the protein expression (ELISA) of IL-8. Ceramide intracellular amounts were measured by LC-MS technique. Ferric-reducing antioxidant power test and superoxide anion radical scavenging activity assay were used to assess the antioxidant power of the inhibitors of ceramide synthesis. We here show that ceramide synthesis is enhanced under treatment with a cigarette smoke mixture correlating with increased expression of inflammatory cytokines and matrix metalloproteinase 9. The use of inhibitors of ceramide synthesis protected from smoke induced damages such as inflammation, oxidative stress, and proteolytic imbalance in airways epithelia. © 2017, Springer-Verlag Berlin Heidelberg.Financial support from the Italian Cystic Fibrosis Research Foundation (Grant no. FFC20-2013) is acknowledged. We thank the Health Sciences Department, University of Milan, for the Post-Doctoral fellowship to support A. Zulueta and the PhD program in Molecular and Translational Medicine to support G.M. Campisi.Peer reviewe