NET amyloidogenic backbone in human activated neutrophils.

Activated human neutrophils produce a fibrillar DNA network [neutrophil extracellular traps (NETs)] for entrapping and killing bacteria, fungi, protozoa and viruses. Our results suggest that the neutrophil extracellular traps show a resistant amyloidogenic backbone utilized for addressing reputed proteins and DNA against the non-self. The formation of amyloid fibrils in neutrophils is regulated by the imbalance of reactive oxygen species (ROS) in the cytoplasm. The intensity and source of the ROS signal is determinant for promoting stress-associated responses such as amyloidogenesis and closely related events: autophagy, exosome release, activation of the adrenocorticotrophin hormone/α-melanocyte-stimulating hormone (ACTH/α-MSH) loop and synthesis of specific cytokines. These interconnected responses in human activated neutrophils, that have been evaluated from a morphofunctional and quantitative viewpoint, represent primitive, but potent, innate defence mechanisms. In invertebrates, circulating phagocytic immune cells, when activated, show responses similar to those described previously for activated human neutrophils. Invertebrate cells within endoplasmic reticulum cisternae produce a fibrillar material which is then assembled into an amyloidogenic scaffold utilized to convey melanin close to the invader. These findings, in consideration to the critical role played by NET in the development of several pathologies, could explain the structural resistance of these scaffolds and could provide the basis for developing new diagnostic and therapeutic approaches in immunomediated diseases in which the innate branch of the immune system has a pivotal role.

Association between gestational diabetes mellitus and subsequent overactive bladder among premenopausal female twins.

OBJECTIVE: To investigate the association between a history of gestational diabetes mellitus (GDM) and overactive bladder (OAB) in women of premenopausal age. DESIGN: Population-based study. SETTING: The Swedish Twin Register. POPULATION: In 2005, a total of 14 094 female twins born between 1959 and 1985 in the Swedish Twin Registry participated in a comprehensive survey on common exposures and complex diseases. Structured questions provided information on GDM and OAB. The present study was designed as a cross-sectional analysis including all women in the cohort having given birth before 2005 (n = 7855). METHODS: A logistic regression model based on generalised estimating equations was used to derive odds ratios (ORs). MAIN OUTCOME MEASURE: The association between a history of GDM and OAB was estimated using ORs with 95% confidence intervals (CIs). RESULTS: The prevalence of OAB in women with a history of GDM was 19.1% compared with 10.7% in women without GDM. This corresponded to a two-fold increased odds of OAB in women with a history of gestational diabetes (OR 2.13, 95% CI 1.48-3.05). After adjusting the analysis for age, body mass index, parity, smoking, and diabetes mellitus, having had GDM was associated with doubled odds of OAB (OR 1.88, 95% CI 1.26-2.80). CONCLUSIONS: A history of GDM was positively associated with OAB among women of premenopausal age. The association does not seem to be mediated by body mass index or type-I or type-II diabetes mellitus.

No survival benefit from early-start dialysis in a population-based, inception cohort study of Swedish patients with chronic kidney disease.

OBJECTIVE: To investigate how the timing of dialysis initiation is associated with mortality. DESIGN: Population-based, prospective, observational cohort study. SETTING: Clinical laboratories (n = 69) provided information on all patients in Sweden whose serum creatinine level for the first time and exceeded 3.4 mg dL(-1) (men) or 2.8 mg dL(-1) (women) between 20 May 1996 and 31 May 1998. SUBJECTS: All patients (n = 901), aged 18-74 years, in whom the cause of serum creatinine elevation was chronic kidney disease, were included in the study; participants were interviewed and followed for 5-7 years. MAIN OUTCOME MEASURES: Information on date of death was obtained from a national Swedish population register. Early-start dialysis [estimated glomerular filtration rate from serum creatinine (eGFR) ≥7.5 mL min(-1) per 1.73 m(2)] was compared to late start of dialysis (eGFR <7.5 mL min(-1) per 1.73 m(2)), and no dialysis. Relative risk [hazard ratio (HR)] of death was modelled with time-dependent multivariate Cox proportional hazards regression. RESULTS: Mean eGFR was 16.1 mL min(-1) per 1.73 m(2) at inclusion and 7.6 mL min(-1) per 1.73 m(2) at the start of dialysis. Among the 385 patients who started dialysis late, 36% died during follow-up compared to 52% of 323 who started early. The adjusted HR for death was 0.84 [95% confidence interval (CI) 0.64, 1.10] among late versus early starters. The mortality among nondialysed patients increased significantly at eGFR below 7.5 mL min(-1) per 1.73 m(2) (HR 4.65; 95% CI 2.28, 9.49; compared to eGFR 7.5-10 mL min(-1) per 1.73 m(2)). After the start of dialysis, the mortality rate further increased. Compared to nondialysed patients with eGFR ≤15 mL min(-1) per 1.73 m(2), adjusted HR was 2.65 (95% CI 1.80, 3.89) for patients receiving dialysis. CONCLUSION: We found no survival benefit from early initiation of dialysis.

Effects of coffee and tea consumption on urinary incontinence in female twins.

OBJECTIVES: To assess the effect of coffee and tea consumption on symptoms of urinary incontinence. DESIGN: Population-based study. SETTING: The Swedish Twin Register. POPULATION: In 2005, all twins born between 1959 and 1985 in Sweden (n = 42,852) were invited to participate in a web-based survey to screen for common complex diseases and common exposures. The present study was limited to female twins with information about at least one urinary symptoms and coffee and tea consumption (n = 14,031). MAIN OUTCOME MEASURE: The association between coffee and tea consumption and urinary incontinence, as well as nocturia, was estimated as odds ratios (ORs) with 95% confidence intervals. RESULTS: Women with a high coffee intake were at lower risk of any urinary incontinence (OR 0.78, 95% CI 0.64-0.98) compared with women not drinking coffee. Coffee intake and incontinence subtypes showed no significant associations whereas high tea consumption was specifically associated with a risk for overactive bladder (OR 1.34, 95% CI 11.07-1.67) and nocturia (OR 1.18, 95% CI 1.01-1.38). Results from co-twin control analysis suggested that the associations observed in logistic regression were mainly the result of familial effects. CONCLUSIONS: This study suggests that coffee and tea consumption has a limited effect on urinary incontinence symptoms. Familial and genetic effects may have confounded the associations observed in previous studies.

Reversine increases multipotent human mesenchymal cells differentiation potential.

Among different human stem cell sources, adult mesenchymal stem cells from bone marrow (BMSCs), and more recently from adipose tissues (ASCs), have shown their capability to differentiate into a variety of different cell types, including osteoblasts, adipocytes, and muscle cells. However, mesenchymal stem cell differentiation toward certain cell types (including skeletal and cardiac muscle), while shown to be achievable, still suffers of low yields and needs to be greatly improved before any therapeutic application could be foreseen. A possible way of achieving this goal is by using a chemical-pharmacological approach to increase stem cell plasticity. Along this line, we envisioned the possibility of pre-treating BMSCs and ASCs with reversine, a synthetic purine that has been shown to induce adult cells de-differentiation. In the current study we tested reversine effects on both BMSCs and ASCs to increase their differentiation toward osteoblasts, smooth and skeletal muscle cells. Reversine pre-treatment, at very low concentration (50 nM), caused a marked increase in the differentiation yields of both BMSCs and ASCs.

Growth factors and chemokines: a comparative functional approach between invertebrates and vertebrates.

Growth factors and cytokines control and coordinate a broad spectrum of fundamental cellular functions, and are evolutionarily conserved both in vertebrates and invertebrates. In this review, we focus our attention on the functional phylogenetic aspects of growth factors/cytokines like the Transforming Growth Factor-beta (TGF-beta), the Connective Tissue Growth Factor (CTGF), and the Vascular Endothelial Growth Factor (VEGF). We will also delve into the activites of two chemokine families, interleukin (IL)-8 (or CXCL8) and CC chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2). These molecules have been selected for their involvement in immune responses and wound healing processes, where they mediate and finely regulate various regeneration processes like angiogenesis or fibroplasia, not only in vertebrates, but also in invertebrates.

Hematopoietic cell formation in leech wound healing.

The angiogenic process in vertebrates and hirudineans has been compared. The leech Hirudo medicinalis, subjected to an angiogenic stimulus (surgical explant or cytokine treatment) responds, as a vertebrate, with the formation of an extensive network of new vessels accompanied by the production of circulating cells. The reviewed data confirm the surprising similarity between hirudinean and vertebrate processes in wound healing, and suggest that basic common events such as antigenic expressions of endothelial and hemopoietic cells, cytokine secretion and regulation as well as extracellular matrix interactions, are conserved and extended across diverse species, tissues and developmental phases.

Structure and function of the extraembryonic membrane persisting around the larvae of the parasitoid Toxoneuron nigriceps.

The embryo of Toxoneuron nigriceps (Hymenoptera, Braconidae) is surrounded by an extraembryonic membrane, which, at hatching, releases teratocytes and gives rise to a cell layer embedding the body of the 1st instar larva. This cell layer was studied at different developmental times, from soon after hatching up to the first larval moult, in order to elucidate its ultrastructural, immunocytochemical and physiological function. The persisting "larval serosa" shows a striking structural and functional complexity: it is a multifunctional barrier with protective properties, limits the passage of macromolecules and it is actively involved in the enzymatic processing and uptake of nutrients. The reported results emphasizes the important role that the embryo-derived host regulation factors may have in parasitism success in Hymenoptera koinobionts.

GM1 ganglioside-Triton X-100 mixed micelles. Transitions among different micellar species monitored by physicochemical and enzymatic methods.

Aqueous dispersions of two amphiphiles, GM1 ganglioside and Triton X-100, in different proportions, were analysed for some physicochemical properties (surface tension, viscosity, consolution temperature) and for susceptibility to the action of galactose oxidase. By varying the molar ratio between the two components, well defined transitions among different micellar species were recorded by physicochemical measurements. Galactose oxidase was able to recognize the different species of mixed micelles, its kinetics displayed break points which exactly superimposed on those recorded, under the same conditions, by physicochemical methods.

Studies on brain cytosol neuraminadase. I. Isolation and partial characterization of two forms of the enzyme from pig brain.

1. Two forms of cytosol neuraminidase (EC 3.2.1.18) (neuraminidase A and neuraminidase B) were isolated and purified from pig brain homogenate, by proceeding through the following steps: centrifugation of brain homogenate at 105 000 X g (1h); ammonium sulphate fractionation (35-55% saturated fraction); column chromatography on Biogel A 5 m; column chromatography on hydroxy apatite/cellulose gel; affinity chromatography on Affinose-tyrosyl-p-nitrophenyloxamic acid. The separation of the two forms of neuraminidase was provided by chromatography on hydroxylapatite/cellulose gel. Neuraminidase A was purified about 500-fold; neuraminidase B about 400-fold. 2. The pH optima and the maximum activities in various buffers were different for neuraminidase A and B (for instance the pH optimum was in sodium acetate/acetic acid buffer, 4.7 for neuraminidase A and 4.9 for neuraminidase B). Ions affected in a different way the two enzymes: K+ activated neuraminidase A but not neuraminidase B; Na+ and Li+ inhibited neuraminidase A at a higher degree than neuraminidase B. Neuraminidase B seemed to be moderately activated by some bivalent cations (Ca2+; Mg2+; Zn2+); neuraminidase A did not. The Km values for sialyllactose were different: 2.2-10(-3) M for neuramindase A; 0.46-10(-3) M for neuraminidase B.

Optimal conditions for the assay of fibroblast neuraminidase with different natural substrates.

A method for the assay of neuraminidase in human cultured fibroblasts has been worked out. The substrates, all naturally occurring, were: sialyloligosaccharides (alpha(2 lead to 3)sialyllactose, alpha(2 leads to 6)sialyllactose, disialyllactose), sialylglycoplipids (disialogangliosides GD1a and GD1b), sialylglycoproteins and sialylglycopeptides (ovine submaxillary glycoprotein and its pronase-glycopeptides). The method was based on the determination of the enzymically liberated N-acetylneuraminic acid (NeuAc) by a chromatographic-colorimetric microprocedure. The enzyme acted on sialyloligosaccharides and, in the presence of Triton X-100, on gangliosides, while it did not appreciably affect sialylglycoproteins and sialylglycopeptides. The optimum pH was 4.0 for all tested substrates; the Km values were higher for sialyloligosaccharides (about 10(-3) M), lower for gangliosides (about 10(-4) M); the apparent maximum velocity was higher with alpha(2 leads to 3)sialyllactose (400 mU/mg protein); the reaction rate was linear with time for up to 2 h, and with up to 0.6 mg of enzymic protein. The assay method proved to be sufficiently sensitive (3-4 nmol liberated NeuAc), simple, and reproducible (mean activity on pooled fibroblasts with alpha(2 leads to 3)sialyllactose: 400 mU +/- 6 S.E.).

Studies on brain cytosol neuraminidase. II. Extractability, solubility and intraneuronal distribution of the enzyme in pig brain.

The origin and properties of cytosolic neuraminidase (acylneuraminyl hydrolase, EC 3.2.1.18) from pig brain were studied. 1. The brain extracts containing the cytosol derived from neuronal bodies and glial cells carry 0.69 munits neuraminidase/g fresh tissue. The behaviour of neuraminidase during extraction closely paralleled that of authentic cytosolic enzyme, lactate dehydrogenase; whereas, it differed from that of the lysosomal enzymes, beta-hexosaminidase and beta-galactosidase, also found in the extracts. 2. Nerve endings from either crude or purified preparations, when treated by hypoosmotic shock, released neuraminidase activity up to a maximum of 1.25 munits/g fresh tissue. The behaviour of releasable neuraminidase was always identical to that of lactate dehydrogenase and very similar to that of ATPase and acetylcholinesterase. Typical lysosomal enzymes, however, such as beta-galactosidase and beta-hexosaminidase, behaved differently under the same conditions. This neuraminidase activity is thought to be derived from the cytosol of nerve endings. 3. The specific activity of neuraminidase in nerve-ending cytosol is 15--20 times that in neuronal body and glial cell cytosol. Some properties (pH, Km value, V/t relationship) of the cytosolic enzymes of different origin are similar; others (stability on standing at 4 degrees C; resistance to freezing and thawing) are different. Hypoionic solutions caused both cytosolic neuraminidases to slowly precipitate and to assume a stable insoluble form which was still active.

Galactose oxidase action on GM1 ganglioside in micellar and vesicular dispersions.

GM1 ganglioside was dispersed in different membrane-mimicking systems and the effect of dispersion on GM1 oxidation by galactose oxidase was studied. The following membrane-mimicking systems were used: homogeneous micelles of GM1; mixed micelles (at different proportions of constituents) of GM1 with either GD1a ganglioside (which is resistant to the enzyme), or the non-ionic detergent Triton X-100, or bovine serum albumin; small unilamellar vesicles of egg phosphatidylcholine (PC), containing various proportions of GM1. As a reference substrate not involved in membranous systems and freely interacting with the enzyme, the oligosaccharide portion of GM1 (DesGM1) was employed. The apparent Vmax of the enzyme was dramatically dependent on the type of GM1 dispersion. The lowest value was recorded on homogeneous micelles of GM1 and on mixed GM1-GD1a micelles. From this value, the Vmax increased 2-fold with GM1-bovine serum albumin lipoprotein micelles, up to 1400-fold with mixed GM1-Triton X-100 (optimal molar ratio, 1:13.8) micelles, and up to 14000-fold on PC vesicles containing 8 mol% GM1 (this proportion was optimal for enzyme activity on vesicles). The activity developed on these latter vesicles turned out to be still greater (2-fold) than that displayed on DesGM1. The apparent Km had very similar values in all different membrane systems; in contrast, it was markedly greater on DesGM1. Both Triton X-100 micelles and PC vesicles did not appreciably alter the kinetics of galactose oxidase action on pure galactose, indicating that the above effects are dependent on the intrinsic characteristics of the membrane-like systems containing gangliosides.

Interactions of ganglioside GM1 with human and fetal calf sera. Formation of ganglioside-serum albumin complexes.

The interactions of ganglioside GM1 with human and fetal calf sera were studied, the following main results being obtained: (a) GM1, upon incubation with both sera gave origin to two GM1-protein complexes, which also occurred after interaction of GM1 with the albumin fractions prepared from the same sera. Instead no complex formation occurred using the albumin-free fractions. Therefore GM1 appeared to specifically bind serum albumin and to form GM1-albumin complexes. (b) GM1 binding to serum albumin started at ganglioside concentrations surely micellar (above 10(-6) M), was time and concentration dependent, and resulted in a relevant degree of GM1 complexation (up to 80% of total GM1 in human serum and up to 18% in fetal calf serum). (c) the binding kinetics appeared, in both serum and the correspondent albumin fraction, to be biphasic: in the first phase, occurring till about 2 . 10(-4) M GM1, the ratio between bound and total GM1 increased linearly with increasing GM1 concentration; in the second phase, occurring above 2 . 10(-4) M, the ratio remained practically constant. After these findings it should be expected that GM1, when present in serum containing systems, forms complexes with albumin. This should be approximately considered when studying the effects of exogeneous GM1 in in vivo and in vitro (tissue cultures) systems.

Electron paramagnetic resonance studies on the fluidity and surface dynamics of egg phosphatidylcholine vesicles containing gangliosides.

The influence of different gangliosides (GM1, GD1a, GT1b) on the fluidity and surface dynamics of phosphatidylcholine small unilamellar vesicles was studied by electron paramagnetic resonance. 5- and 16-nitroxystearic acid, sounding respectively the region close to the surface and that close to the hydrophobic core of the vesicle, were employed as spin-label probes. The signals released by 5-nitroxystearic acid showed that the presence of gangliosides reduced the mobility of the hydrocarbon chains around the probe. The effect increased by increasing ganglioside concentration, and diminished from GM1 to GD1a and GT1b. The decrease of membrane fluidity was also monitored by the 16-nitroxystearic acid probe. On addition of Ca2+ the fluidity of ganglioside-containing vesicles (as signalled by the 5-nitroxystearic acid probe) promptly decreased, therefore returning slowly to the original value. It is suggested that gangliosides cause strong side-side head group interactions on the bilayer surface--between ganglioside oligosaccharide chains and between ganglioside and phosphatidylcholine polar portions--which lead the lipid chains to assembly in a more rigid fashion. The influence of Ca2+ is interpreted as due to lateral phase separation in the vesicle membrane. This phenomenon can be related to the formation or stabilization of ganglioside clusters on the vesicle surface.

Interactions of pig brain cytosolic sialidase with gangliosides. Formation of catalytically inactive enzyme-ganglioside complexes.

Cytosolic sialidase A was extracted from pig brain and purified about 2000-fold with respect to the starting homogenate (about 550-fold relative to the cytosolic fraction). The enzyme preparation provided a single peak on Ultrogel AcA-34 column chromatography and had an apparent molecular weight of 4 x 10(4). On incubation with micellar ganglioside GT1b, (molecular weight of the micelle, 3.5 x 10(5)) under the conditions used for the enzyme assay, brain cytosolic sialidase A formed two ganglioside-enzyme complexes, I and II, which were isolated and characterized. Complex II had a molecular weight of 4.2 X 10(5), and a ganglioside/protein ratio (w/w) of 4:1. This is consistent with a stoichiometric combination of one ganglioside micelle and two enzyme molecules. Complex I was probably a dimer of complex II. In both complexes I and II cytosolic sialidase was completely inactive. Inactivation of cytosolic sialidase by formation of the corresponding complexes was also obtained with gangliosides GD1a and GD1b, which, like GT1b, are potential substrates for the enzyme and GM1, which is resistant to the enzyme action. Therefore, the enzyme becomes inactive after interacting with ganglioside micelles. GT1b-sialidase complexes acted as excellent substrates for free cytosolic sialidase, as did the complexes with GD1a and GD1b.

Gel phase preference of ganglioside GM1 at low concentration in two-component, two-phase phosphatidylcholine bilayers depends upon the ceramide moiety.

In two-component phosphatidylcholine bilayers with coexisting liquid and P beta' gel phases, the distribution between phases of low concentrations of glycosphingolipids can be determined by freeze-etch electron microscopy after labeling the glycolipid with a suitable protein. We have found that the distribution depends upon the glycosphingolipid species (Rock, P. et al., (1991) Biochemistry 30, 19-25). Using this technique with cholera toxin as the protein label and bilayers formed from dipalmitoyl- and dielaidoylphosphatidylcholine (1:1) containing < 1 mol% GM1, we have studied the distribution of a family of GM1 homologues differing in the acyl chain and sphingoid base moieties. The GM1 preference for the P beta' ripple phase decreases with decreasing acyl chain length and increasing unsaturation. GM1 with either a C18:1 or C20:1 sphingoid base shows similar distributions in liquid and gel phases. When the molecules are preferentially found in the P beta' phase, they are positioned along unique loci in both A and A/2 forms of the ripple structure. This localization and acyl chain dependence reflect the volume, shape and localization of molecular packing defects in the P beta' phase. The ganglioside inclusions stabilize the P beta' phase and form compositional domains of unique topography.

Cytosolic sialidase from pig brain: a 'protein complex' containing catalytic and protective units.

Pig brain cytosolic sialidase purified to homogeneity, showed a single protein band on SDS-PAGE under non-reducing conditions, and three bands using reducing conditions, suggesting a complex of different units. The sialidase complex (molecular mass, M(r), 180 kDa) was resolved into a catalytic unit (M(r) 30 kDa), active but very liable upon storage at 4 degrees C and freezing and thawing, and two protective units (66 kDa and 42 kDa), inactive, but capable to stabilize the catalytic unit. Recombination of the catalytic and protective units (optimal ratio, 1:1, by weight) gave rise to a stable active complex. Using GD1a as substrate, the catalytic unit showed a Michaelis-Menten kinetics, and the complex a sigmoid-shaped kinetics, whereas a Michaelis-Menten kinetics was exhibited with MU-NeuAc in both cases. The apparent Vmax and Km values of the catalytic unit for MU-NeuAc and GD1a were 105.1 and 110.0 mU/mg protein, and 4.2 x 10(-5) and 1.6 x 10(-5) M, respectively. The model we propose for cytosolic sialidase complex is one of each protective units and 2-3 catalytic units. The sialidase complex and protective units did not display any beta-D-galactosidase, beta-D-N- acetylglucosaminidase, alpha-L-fucosidase, alpha-D-glucosidase and carboxypeptidase activities.

Predictors of Ominous Outcome in Infants Undergone to Cardiac Surgery and Cardiopulmonary by-Pass: S100B Protein.

S100B protein has been recently proposed as a consolidated marker of brain damage and death in adult, children and newborn patients. The present study evaluates whether the longitudinal measurement of S100B at different perioperative time-points may be a useful tool to identify the occurrence of perioperative early death in congenital heart disease (CHD) newborns. We conducted a case-control study in 88 CHD infants, without pre-existing neurological disorders or other co-morbidities, of whom 22 were complicated by perioperative death in the first week from surgery. Control group was composed by 66 uncomplicated CHD infants matched for age at surgical procedure. Blood samples were drawn at five predetermined time-points before during and after surgery. In all CHD children, S100B values showed a pattern characterized by a significant increase in protein's concentration from hospital admission up to 24-h after procedure reaching their maximum peak (P<0.01) during cardiopulmonary by-pass and at the end of the surgical procedure. Moreover, S100B concentrations in CHD death group were significantly higher (P<0.01) than controls at all monitoring time-points. The ROC curve analysis showed that S100B measured before surgical procedure was the best predictor of perioperative death, among a series of clinical and laboratory parameters, reaching at a cut-off of 0.1 µg/L a sensitivity of 100% and a specificity of 63.7%. The present data suggest that in CHD infants biochemical monitoring in the perioperative period is becoming possible and S100B can be include among a series of parameters for adverse outcome prediction.

Fluorescence excimer formation imaging: a new technique to investigate association to cells and membrane behavior of glycolipids.

A new fluorescence ratio imaging technique aiming to monitor the lateral distribution of pyrene-labeled lipids in the membranes through visualization of the excimer/monomer (E/M) intensity ratio, has been set up, studying the association of a fluorescent derivative of GM1 ganglioside (pyreneGM1) to rat cerebellar granule cells in culture. The imaging results show that the mean E/M ratio value, under experimental conditions leading to the association of pyreneGM1 with the plasma membrane, is significantly higher in neuritic processes than in cell bodies and, moreover, locally distributed in patches. Fluorescence antisotropy imaging of the fluorescent probe TMA-DPH (trimethylaminodiphenylhexatriene) shows the presence of domains having different fluidity and that the average fluidity is higher in cell bodies than in neuritic processes. Additional experiments using TMA-DPH as a marker of fluid-phase pinocytosis, suggest that the rate of endocytosis is comparable in the two regions of the cell. Taken together, on the one hand these data indicate that the fluorescent ganglioside is present in a more clustered state at the level of neuritic processes than in cell bodies and locally distributed in patches of different size and enrichment. On the other hand, they point out the potentiality of the excimer formation imaging technique to study membrane behavior and dynamics of pyrene-labeled lipids, with a particular insight into their aggregative properties.