Marta Melis

Hydrophobic characterization of intracellular lipids in situ by Nile Red red/yellow emission ratio

Nile Red (9-diethylamino-5H-benzo [alpha] phenoxazine-5-one) is a fluorescent lipophilic dye characterized by a shift of emission from red to yellow according to the degree of hydrophobicity of lipids. Polar lipids (i.e., phospholipids) which are mostly present in membranes, are stained in red whereas neutral lipids (esterified cholesterol and triglycerides) which are present in lipid droplets, are stained in yellow. Besides this marked, qualitative contrast between polar and neutral lipids, small differences of the hydrophobic strength could be assessed by the quantitative ratio of red and yellow emissions, in order to extend the discrimination of lipids within the groups of neutral and polar lipids. On the other hand, ratiometric data of red and yellow emissions have not yet been evaluated in the numerous previous light microscopy investigations which used Nile Red. In this work we show that the Nile Red red/yellow ratio enables discrimination of different lipids (monooleine>oleic acid>phosphatidylcholine>free cholesterol>trioleine>oleyl cholesteryl ester). We also show changes in the Nile Red red/yellow emission ratio of lipid droplets of 3T3 mouse fibroblasts induced by drugs interfering with the cholesterol cycle.

Intracellular cholesterol changes induced by translocator protein (18 kDa) TSPO/PBR ligands

One of the main functions of the translocator protein (18 kDa) or TSPO, previously known as peripheral-type benzodiazepine receptor, is the regulation of cholesterol import into mitochondria for steroid biosynthesis. In this paper we show that TSPO ligands induce changes in the distribution of intracellular cholesterol in astrocytes and fibroblasts. NBD-cholesterol, a fluorescent analog of cholesterol, was rapidly removed from membranes and accumulated into lipid droplets. This change was followed by a block of cholesterol esterification, but not by modification of intracellular cholesterol synthesis. NBD-cholesterol droplets were in part released in the medium, and increased cholesterol efflux was observed in [(3)H]cholesterol-prelabeled cells. TSPO ligands also induced a prominent shrinkage and depolarization of mitochondria and depletion of acidic vesicles with cytoplasmic acidification. Consistent with NBD-cholesterol changes, MTT assay showed enhanced accumulation of formazan into lipid droplets and inhibition of formazan exocytosis after treatment with TSPO ligands. The effects of specific TSPO ligands PK 11195 and Ro5-4864 were reproduced by diazepam, which binds with high affinity both TSPO and central benzodiazepine receptors, but not by clonazepam, which binds exclusively to GABA receptor, and other amphiphilic substances such as DIDS and propranolol. All these effects and the parallel immunocytochemical detection of TSPO in potentially steroidogenic cells (astrocytes) and non-steroidogenic cells (fibroblasts) suggest that TSPO is involved in the regulation and trafficking of intracellular cholesterol by means of mechanisms not necessarily related to steroid biosynthesis.

Influence of treatments in multiple sclerosis disability: A cohort study

Background and objective: A critical aspect of multiple sclerosis (MS) treatments is understanding the effect of disease-modifying drugs (DMDs) on the long-term risk of disability and whether the effect is related to disability at start of treatment. Methods: We performed an observational study on 3060 MS patients. The effect of therapy on progression to Expanded Disability Status Scale (EDSS) 3.0 and 6.0 from onset was analysed in treated vs untreated (UTP) patients using Cox regression analysis adjusted for propensity score and immortal time bias. Results: Compared to UTP, the risks of EDSS 3.0 were 94% and 73% lower in immunomodulant (IMTP-) and immunosuppressant (ISTP-) treated patients, respectively, while the risk of EDSS 6.0 was 86% lower in IMTP. The risk of EDSS 6.0 was, respectively, 91% and 75% lower in 1275 IMTP before and 114 after EDSS 3.0 than in 539 UTP; the risk was higher in IMTP starting therapy after EDSS 3.0 than before (HR = 4.42). Conclusions: DMDs delayed long-term disability in MS patients treated either in the early or, to a lesser extent, in the later phase of the disease. Thus, the window of therapeutic opportunity is relatively extended, assuming that early is better than late treatment, but late is better than never.

Neuroactive steroid levels in plasma and cerebrospinal fluid of male multiple sclerosis patients.

Neuroactive steroid family includes molecules synthesized in peripheral glands (i.e., hormonal steroids) and directly in the nervous system (i.e., neurosteroids) which are key regulators of the nervous function. As already reported in clinical and experimental studies, neurodegenerative diseases affect the levels of neuroactive steroids. However, a careful analysis comparing the levels of these molecules in cerebrospinal fluid (CSF) and in plasma of multiple sclerosis (MS) patients is still missing. To this aim, the levels of neuroactive steroids were evaluated by liquid chromatography‐tandem mass spectrometry in CSF and plasma of male adults affected by Relapsing‐Remitting MS and compared with those collected in control patients. An increase in pregnenolone and isopregnanolone levels associated with a decrease in progesterone metabolites, dihydroprogesterone, and tetrahydroprogesterone was observed in CSF of MS patients. Moreover, an increase of 5α‐androstane‐3α,17β‐diol and of 17β‐estradiol levels associated with a decrease of dihydrotestosterone also occurred. In plasma, an increase in pregnenolone, progesterone, and dihydrotestosterone and a decrease in dihydroprogesterone and tetrahydroprogesterone levels were reported. This study shows for the first time that the levels of several neuroactive steroids, and particularly those of progesterone and testosterone metabolites, are deeply affected in CSF of relapsing‐remitting MS male patients.

Lipid droplet changes in proliferating and quiescent 3T3 fibroblasts

Lipid droplets (LDs) are fat-storing organelles present in virtually all eukaryotic cells and involved in many aspects of cell biology related to lipid metabolism and cholesterol homeostasis. In this study, we investigated the presence of LDs in proliferating and quiescent (contact-inhibited) 3T3 fibroblasts to verify a correlation with cell growth. LDs were characterized by Nile red staining, positivity to adipophilin and negativity to perilipin. LDs were numerous in proliferating cells, but very few in quiescent cells. However, the fraction of quiescent cells, which resumed proliferation after scratch-wound assay, also resumed the formation of LDs. In proliferating cells, the number of LDs correlated with the DNA content, suggesting a continuous accumulation of LDs during cell growth. These findings were supported by biochemical data showing much higher rates of cholesterol esterification and triglyceride synthesis in proliferating cells. Both filipin staining and the fluorescent cholesterol analog dehydroergosterol revealed the presence of an intense traffic of free cholesterol, mediated by acidic vesicles, in proliferating cells. Nile red ratiometric measurements revealed a different lipid composition of LDs in proliferating and quiescent cells. Changes in the number and composition of LDs were also found in growing cells treated with inhibitors of cholesterol esterification (Sandoz 58-035), endosomal cholesterol efflux (U18666A) and V-ATPase (bafilomycin-A1).

What do multiple sclerosis patients and their caregivers perceive as unmet needs

BackgroundMultiple sclerosis (MS) has a major impact on the physical, psychological and social life of patients and their families. The aim of this study was to evaluate the different perceptions of patients and caregivers about management of MS, particularly about the same items, to gather information to ameliorate the care of patients.MethodsWe evaluated what MS patients and caregivers perceive as unmet needs and compared patients’ opinions with caregivers’ opinions using a multidimensional questionnaire. The questionnaire was specifically designed for the study, taking into account different aspects of the global care perceived by patients and care givers, such as information about MS, medical treatment and rehabilitation, patients’ relationships with medical staff and their psychological and social life.ResultsWe administered the questionnaire to 497 patients and 206 caregivers. Results showed that the majority of participants were satisfied with medical staff but expressed a desire that staff be more forthcoming with information about MS. As for medical treatment concerns, more patients found there to be useful a multidisciplinary approach than caregivers did. Both required psychological support for patients but patients felt a greater need for it at the time of diagnosis, whereas caregivers felt it was required post-diagnosis. Both reported significant strains on patient relationships at work but no effect on other social interactions.ConclusionsA better understanding of MS patient needs, starting from the point of view of patients and caregivers, could have a great impact on quality of life and on management of the disease.

BK-virus progressive multifocal leukoencephalitis in a patient with systemic lupus erythematosus

Dear Editor, Progressive multifocal leukoencephalopathy (PML) is a severe demyelinating disease of the central nervous system (CNS), fatal in some cases, and JC virus (JCV) is the usual cause [1]. BK virus (BKV) is a ubiquitous virus and its primary infection is generally asymptomatic. It occurs in childhood [2] and, as well as JCV, belongs to the Polyomaviridae family. Both viruses are associated with a clinical disease in the setting of immunosuppression: JCV commonly in association with PML, while BKV with nephropathy and hemorrhagic cystitis [2]. Here we report a rare case of BKV-associated PML in a patient with systemic lupus erythematosus (SLE).

Percutaneous Endoscopic Transgastric Jejunostomy (PEG-J) Tube Placement for Levodopa-Carbidopa Intrajejunal Gel Therapy in the Interventional Radiology Suite: A Long-term Follow-up

Percutaneous endoscopic gastrojejunostomy (PEG) and radiologically inserted gastrojejunostomy (RIG) are both safe and effective techniques for gastrojejunal tube placement. The authors compared these 2 procedures in patients with advanced Parkinsons disease (PD) who required the continuous intrajejunal delivery of a levodopa/carbidopa gel suspension (LCIG).

Dyskinesia-Hyperpyrexia Syndrome in Parkinson's Disease: A Heat Shock-Related Emergency?: DHS and summer heatwaves

Dyskinesia hyperpyrexia syndrome (DHS) has been reported as a medical emergency in patients with advanced Parkinson’s disease (PD). The few previously published cases have emphasized the role of high dopaminergic daily dose and complex concomitant polytherapy as a risk factor for DHS. We report three patients with advanced PD and Levodopa-Carbidopa intestinal gel (LCIG) who developed DHS during a seasonal heatwave. In this context of climate warming, advanced PD patients, especially when treated with high dopaminergic daily dose (i.e. under LCIG), are a cohort at risk for DHS. In the event of heatwaves, onset of fever and appearance/worsening of severe dyskinesia must be evaluated with the utmost care in order to prevent a DHS emergency in PD. Nonphysiological dopaminergic stimulation can cause Dyskinesia-Hyperpyrexia syndrome (DHS), a rare medical emergency associated with Parkinson’s Disease (PD). DHS is characterized by severe continuous dyskinesia, rhabdomyolysis, and hyperthermia that may progress to mental state alteration, renal and cardiac failure, and death. Here, we report on 3 patients with advanced PD and optimal control of motor fluctuations under levodopa-carbidopa intestinal gel (LCIG) infusion. They all developed DHS during summer heatwaves (Fig. S1).

The long way of a “lost pigtail”: a unique complication of J-tube in Duodopa therapy

An 80-years-old man with a 19-years history of Parkinsons disease (PD) was proposed levodopa/carbidopa intrajejunal gel (LCIG) therapy due to disabling motor fluctuations despite optimized oral levodopa (LD) treatment. In 2015 the patient underwent percutaneous endoscopic gastrojejunostomy (J-PEG) for LCIG infusion with a great benefit on motor symptoms. Two years later, the patient reported the recurrence of fluctuations and dyskinesias, despite increased daily LCIG dosage. Apparently the pump worked normally, without signs of flush hindrance (i.e. blockage or high pressure alarm). Two days later, complete anal exteriorization of the jejunal tube (J-tube) occurred spontaneously, with the classical “pigtail” appearance (Figure) and without further adverse events. In the next days the patient safely returned on LCIG after J-tube replacement. This article is protected by copyright. All rights reserved.