Plasma 24S-hydroxycholesterol levels in elderly subjects with late onset Alzheimer's disease or vascular dementia: a case-control study

<p>Abstract</p> <p>Background</p> <p>In central nervous system cholesterol cannot be degraded but is secreted into circulation predominantly in the form of its polar metabolite 24(<it>S</it>)-hydroxycholesterol (24S-OH-Chol). Some studies suggested an association between 24S-OH-Chol metabolism and different neurological diseases including dementia. A possible decrease in 24S-OH-Chol plasma levels has been reported late onset Alzheimer's disease (LOAD) and vascular dementia (VD), but results of previous studies are partially contradictory.</p> <p>Methods</p> <p>By high-speed liquid chromatography/tandem mass spectrometry we evaluated the plasma levels of 24S-OH-Chol in a sample of 160 older individuals: 60 patients with LOAD, 35 patients with VD, 25 subjects affected by cognitive impairment no-dementia (CIND), and 40 (144 for genetics study) cognitively normal Controls. We also investigated the possible association between PPARgamma Pro12Ala polymorphism and dementia or 24S-OH-Chol levels.</p> <p>Results</p> <p>Compared with Controls, plasma 24S-OH-Chol levels were higher in LOAD and lower in VD; a slight not-significant increase in CIND was observed (ANOVA p: 0.001). A positive correlation between 24S-OH-Chol/TC ratio and plasma C reactive protein (CRP) levels was found in the whole sample, independent of possible confounders (multiple regression p: 0.04; r²: 0.10). This correlation was strong in LOAD (r: 0.39), still present in CIND (r: 0.20), but was absent in VD patients (r: 0.08). The PPARgamma Pro12Ala polymorphism was not associated with the diagnosis of LOAD, VD, or CIND; no correlation emerged between the Ala allele and 24S-OH-Chol plasma levels.</p> <p>Conclusions</p> <p>Our results suggest that plasma 24S-OH-Chol levels might be increased in the first stages of LOAD, and this phenomenon might be related with systemic inflammation. The finding of lower 24S-OH-Chol concentrations in VD might be related with a more advanced stage of VD compared with LOAD in our sample, and/or to different pathogenetic mechanisms and evolution of these two forms of dementia.</p

Analisi quantitativa del 24S-idrossicolesterolo come applicazione clinica della LC-MS/MS alle malattie neurologiche: Alzheimer e demenza vascolare

Secondo un recente rapporto dell’Organizzazione Mondiale della Sanità un miliardo di persone, distribuite in tutto il mondo, vive con una malattia neurologica, con 50 milioni di casi di epilessia e 24 milioni di casi di demenza questi ultimi destinati ad aumentare fino a 114 milioni nel 2050, soprattutto per quanto riguarda la malattia di Alzheimer (AD) e la demenza vascolare (VD) che rappresentano le due forme principali, giustificando la prima il 50% e la seconda circa il 20% di tutte le cause di demenza. Nello scenario preoccupante di questa grossa diffusione si inserisce la difficoltà di una diagnosi precoce la quale avrebbe indubbi vantaggi per assicurare interventi tempestivi. Nella fase iniziale è difficile distinguere i sintomi della demenza da un normale invecchiamento, nonché distinguere AD,VD e depressione in quanti i sintomi iniziali sono molto simili. Gli attuali criteri per la diagnosi di AD e VD fanno riferimento ad alcune linee guida e seguono un iter diagnostico basato su test neuropsicologici e test di neuroimaging che però non sono sufficienti per una diagnosi precoce. Si cerca infatti di individuare marker diagnostici correlati al danno cerebrale che avviene in queste patologie, ossia la morte neuronale. Tra i possibili marker, sta suscitando particolare interesse il 24S-idrossicolestrolo, un ossisterolo, principale metabolita del colesterolo a livello del SNC che passando attraverso la barriera ematoencefalica si immette nella circolazione sistemica. Essendo il 24S-idrossicolesterolo di sola origine cerebrale, una sua variazione di concentrazione nel sangue di pazienti patologici rispetto ai normali riflette un’alterata omeostasi del colesterolo a livello del sistema nervoso centrale come conseguenza del danno neurologico. Scopo di questo lavoro è stato quindi quello di sviluppare e validare un metodo analitico in LC-MS/MS per il dosaggio del 24S-idrossi nel siero con successiva applicazione all’analisi di campioni controllo e patologici e precisamente di 20 pazienti affetti da Alzheimer ad insorgenza tardiva (LOAD), 20 pazienti affetti da VD e 20 pazienti con Mild Cognitive Impariment (MCI), uno stadio preclinico di demenza. I risultati mostrano una diminuzione significativa dei valori medi di 24S-idrossicolesterolo nei pazienti con AD e VD rispetto ai controlli, mentre un aumento nei pazienti con MCI. Una differenza poco significativa è stata evidenziata tra pazienti AD e LOAD.According to a recent WHO report, there is 1 billion of people spread all over the world, living with a neurological diseases, in particular 50 million of cases of epilepsy and 24 cases of dementia, that will increase to 114 million within the 2050, especially for Alzheimer’s disease (AD) and vascular dementia (VD), the two main forms of dementia. Alzheimer’s disease represents 50% and vascular dementia 20% of all dementia cases. In the worrying scenario of this wide diffusion there is also a difficulty of early diagnosis that could have got clear advantages to ensure timely intervention. In the early stage is difficult to distinguish symptoms of dementia from a normal ageing as well to distinguish AD from VD and depression. The current standards for dementia diagnosis refer to some guidelines and follow a diagnostic procedure based on neuropsychological and neuroimaging tests that are however not sufficient for early diagnosis. Try to find diagnostic markers linked to brain damage that happens in this kind of pathologies, that is neuronal death. Between the possible markers, it is of great interest 24s-Hydroxycholesterol, oxysterol, the main cholesterol metabolites in the CNS that goes into systemic circulation through the haematoencephalic barrier. The simple variation of concentration of 24S- Hydroxycholesterol, produced only by the brain, in the blood of pathologic patients compared to healthy people entail an altered cholesterol homeostasis in CNS as a consequence of brain damage. The main purpose of this work is to develop and support an analytic method in LC-MS/MS for the serum 24S- Hydroxycholesterol dosage with following application to the analysis of control and pathologic samples (20 persons suffering from late beginning Alzheimer’s disease, 20 persons suffering from VD and 20 persons with MCI, a preclinical dementia stage). The results prove a significant fall in 24S- Hydroxycholesterol average values between people suffering from AD and VD compared to controls, and an increase between sufferers from MCI. A negligible difference has been picked out between AD and LOAD patients

Analisi quantitativa del 24S-idrossicolesterolo come applicazione clinica della LC-MS/MS alle malattie neurologiche: Alzheimer e demenza vascolare

Secondo un recente rapporto dell’Organizzazione Mondiale della Sanità un miliardo di persone, distribuite in tutto il mondo, vive con una malattia neurologica, con 50 milioni di casi di epilessia e 24 milioni di casi di demenza questi ultimi destinati ad aumentare fino a 114 milioni nel 2050, soprattutto per quanto riguarda la malattia di Alzheimer (AD) e la demenza vascolare (VD) che rappresentano le due forme principali, giustificando la prima il 50% e la seconda circa il 20% di tutte le cause di demenza. Nello scenario preoccupante di questa grossa diffusione si inserisce la difficoltà di una diagnosi precoce la quale avrebbe indubbi vantaggi per assicurare interventi tempestivi. Nella fase iniziale è difficile distinguere i sintomi della demenza da un normale invecchiamento, nonché distinguere AD,VD e depressione in quanti i sintomi iniziali sono molto simili. Gli attuali criteri per la diagnosi di AD e VD fanno riferimento ad alcune linee guida e seguono un iter diagnostico basato su test neuropsicologici e test di neuroimaging che però non sono sufficienti per una diagnosi precoce. Si cerca infatti di individuare marker diagnostici correlati al danno cerebrale che avviene in queste patologie, ossia la morte neuronale. Tra i possibili marker, sta suscitando particolare interesse il 24S-idrossicolestrolo, un ossisterolo, principale metabolita del colesterolo a livello del SNC che passando attraverso la barriera ematoencefalica si immette nella circolazione sistemica. Essendo il 24S-idrossicolesterolo di sola origine cerebrale, una sua variazione di concentrazione nel sangue di pazienti patologici rispetto ai normali riflette un’alterata omeostasi del colesterolo a livello del sistema nervoso centrale come conseguenza del danno neurologico. Scopo di questo lavoro è stato quindi quello di sviluppare e validare un metodo analitico in LC-MS/MS per il dosaggio del 24S-idrossi nel siero con successiva applicazione all’analisi di campioni controllo e patologici e precisamente di 20 pazienti affetti da Alzheimer ad insorgenza tardiva (LOAD), 20 pazienti affetti da VD e 20 pazienti con Mild Cognitive Impariment (MCI), uno stadio preclinico di demenza. I risultati mostrano una diminuzione significativa dei valori medi di 24S-idrossicolesterolo nei pazienti con AD e VD rispetto ai controlli, mentre un aumento nei pazienti con MCI. Una differenza poco significativa è stata evidenziata tra pazienti AD e LOAD.According to a recent WHO report, there is 1 billion of people spread all over the world, living with a neurological diseases, in particular 50 million of cases of epilepsy and 24 cases of dementia, that will increase to 114 million within the 2050, especially for Alzheimer’s disease (AD) and vascular dementia (VD), the two main forms of dementia. Alzheimer’s disease represents 50% and vascular dementia 20% of all dementia cases. In the worrying scenario of this wide diffusion there is also a difficulty of early diagnosis that could have got clear advantages to ensure timely intervention. In the early stage is difficult to distinguish symptoms of dementia from a normal ageing as well to distinguish AD from VD and depression. The current standards for dementia diagnosis refer to some guidelines and follow a diagnostic procedure based on neuropsychological and neuroimaging tests that are however not sufficient for early diagnosis. Try to find diagnostic markers linked to brain damage that happens in this kind of pathologies, that is neuronal death. Between the possible markers, it is of great interest 24s-Hydroxycholesterol, oxysterol, the main cholesterol metabolites in the CNS that goes into systemic circulation through the haematoencephalic barrier. The simple variation of concentration of 24S- Hydroxycholesterol, produced only by the brain, in the blood of pathologic patients compared to healthy people entail an altered cholesterol homeostasis in CNS as a consequence of brain damage. The main purpose of this work is to develop and support an analytic method in LC-MS/MS for the serum 24S- Hydroxycholesterol dosage with following application to the analysis of control and pathologic samples (20 persons suffering from late beginning Alzheimer’s disease, 20 persons suffering from VD and 20 persons with MCI, a preclinical dementia stage). The results prove a significant fall in 24S- Hydroxycholesterol average values between people suffering from AD and VD compared to controls, and an increase between sufferers from MCI. A negligible difference has been picked out between AD and LOAD patients

Extracellular Vesicles Tropism: A Comparative Study between Passive Innate Tropism and the Active Engineered Targeting Capability of Lymphocyte-Derived EVs

Cellular communications take place thanks to a well-connected network of chemical–physical signals, biomolecules, growth factors, and vesicular messengers that travel inside or between cells. A deep knowledge of the extracellular vesicle (EV) system allows for a better understanding of the whole series of phenomena responsible for cell proliferation and death. To this purpose, here, a thorough immuno-phenotypic characterization of B-cell EV membranes is presented. Furthermore, the cellular membrane of B lymphocytes, Burkitt lymphoma, and human myeloid leukemic cells were characterized through cytofluorimetry assays and fluorescent microscopy analysis. Through cytotoxicity and internalization tests, the tropism of B lymphocyte-derived EVs was investigated toward the parental cell line and two different cancer cell lines. In this study, an innate capability of passive targeting of the native EVs was distinguished from the active targeting capability of monoclonal antibody-engineered EVs, able to selectively drive the vesicles, enhancing their internalization into the target cancer cells. In particular, the specific targeting ability of anti-CD20 engineered EVs towards Daudi cells, highly expressing CD20 marker on their cell membrane, was proved, while almost no internalization events were observed in HL60 cells, since they did not express an appreciable amount of the CD20 marker on their plasma membranes

Clustering of Major Histocompatibility Complex-Class I Molecules in Healthy and Cancer Colon Cells Revealed from Their Nanomechanical Properties

The activation of the T cell mediated immune response relies on the fine interaction between the T cell receptor on the immune cell and the antigen-presenting major histocompatibility complex (MHC) molecules on the membrane surface of antigen-presenting cells. Both the distribution and quantity of MHC/peptide complexes and their adequate morphological presentation affect the activation of the immune cells. In several types of cancer the immune response is downregulated due to the low expression of MHC-class I (MHC-I) molecules on the cell's surface, and in addition, the mechanical properties of the membrane seem to play a role. Herein, we investigate the distribution of MHC-I molecules and the related nanoscale mechanical environment on the cell surface of two cell lines derived from colon adenocarcinoma and a healthy epithelial colon reference cell line. Atomic force microscopy (AFM) force spectroscopy analysis using an antibody-tagged pyramidal probe specific for MHC-I molecules and a formula that relates the elasticity of the cell to the energy of adhesion revealed the different population distributions of MHC-I molecules in healthy cells compared to cancer cells. We found that MHC-I molecules are significantly less expressed in cancer cells. Moreover, the local elastic modulus is significantly reduced in cancer cells. We speculate that these results might be related to the proven ability of cancer cells to evade the immune system, not only by reducing MHC-I cell surface expression but also by modifying the local mechanical properties affecting the overall morphology of MHC-I synapse presentation to immune cells

Live-cell 3D super-resolution imaging in thick biological samples

We demonstrate three-dimensional (3D) super-resolution live-cell imaging through thick specimens (50-150 \u3bcm), by coupling far-field individual molecule localization with selective plane illumination microscopy (SPIM). The improved signal-to-noise ratio of selective plane illumination allows nanometric localization of single molecules in thick scattering specimens without activating or exciting molecules outside the focal plane. We report 3D super-resolution imaging of cellular spheroids