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Cultured normal adult human astrocytes as a model to investigate patho-physiological mechanisms in chronic inflammatory brain disorders
No full textCome è noto, gli astrociti, unitamente alla microglia, giocano un ruolo importante nei processi infiammatori che si riscontrano in presenza di episodi ischemici acuti e di diverse malattie neurodegenerative croniche (ad es. nel morbo di Alzheimer, morbo di Parkinson, sclerosi laterale amiotrofica, sclerosi multipla, ecc.) del sistema nervoso centrale umano. In presenza di un danno cerebrale si verifica una riattivazione della proliferazione e di diverse attività funzionali degli astrociti, entro i quali si riscontra inoltre un marcato aumento dei gliofilamenti. Gli astrociti, infatti, insieme con la microglia intervengono modulando la risposta immunitaria, in quanto secernono molteplici chemochine, citochine proinfiammatorie ed ossido nitrico (NO).
Da parte mia, lo studio riguardante gli astrociti di Uomo adulto ha avuto inizialmente gli scopi di (1) mettere a punto le condizioni ottimali per l’isolamento e la coltura in vitro di tali cellule e di (2) caratterizzarle da un punto di vista morfologico-biochimico e di (3) analizzare, successivamente, l’eventuale espressione e regolazione delle sintasi dell’ossido nitrico di tipo endoteliale (e-NOS), neuronale (n-NOS) e inducibile (i-NOS) in astrociti in coltura in vitro esposti a stimoli diversi.
I dati ottenuti hanno dimostrato che in vitro gli astrociti umani adulti quiescenti, ossia non attivamente proliferanti e, pertanto, in uno stato paragonabile alla loro condizione fisiologica in vivo, non esprimono livelli determinabili di alcuna delle tre isoforme della sintasi dell’ossido nitrico. Per contro, il trattamento di tali cellule con diverse combinazioni di citochine pro-infiammatorie (come la IL-1ß, il TNF-α e l’INF-γ) ha evidenziato la comparsa della sintasi dell’ossido nitrico di tipo inducibile (i-NOS) sia a livello di espressione dell’mRNA che di proteina. Ciò si è verificato dopo 48-72 ore di trattamento con opportune combinazioni delle sopra menzionate citochine e si è accompagnato con un massiccio accumulo di nitriti/nitrati nel medium di coltura.
Lo studio preliminare di alcune vie segnaletiche coinvolte nell’induzione dell’i-NOS, già descritte in Letteratura per gli astrociti umani fetali o per gli astrociti di altre specie animali, ha inoltre messo in luce che solo 48 ore dopo il trattamento con la combinazioni di citochine pro-infiammatorie (IL-1ß, TNF-α, INF-γ) si riscontra la comparsa dell’mRNA per l’i-NOS, e l’aumento concomitante dell’attività enzimatica delle chinasi MEK1-2/ERK1-2. Più tardivamente (dalle 72 ore dopo il trattamento in poi), si osserva la comparsa della proteina i-NOS che si accompagna con la produzione e il rilascio di NO (dosato come nitriti/nitrati) nel medium. Per chiarire il ruolo delle vie segnaletiche che vedono da una parte il coinvolgimento delle MAP chinasi MEK/ERK, ho utilizzato un inibitore specifico della via MEK/ERK, l’U0126, ed ho osservato che tale composto, una volta somministrato agli astrociti umani adulti in coltura, blocca l’attivazione di MEK1-2 e conseguentemente di ERK1-2, mentre non sopprime la sintesi ex novo della proteina i-NOS. Tuttavia, ho constatato che l’i-NOS sintetizzata in presenza di U0126 non è enzimaticamente attiva, per cui nonostante la sua presenza non ha luogo alcuna produzione di NO. Per tale motivo ho cercato di verificare se la via segnaletica MEK/ERK fosse coinvolta nella sintesi di alcuni cofattori necessari alla dimerizzazione ed all’attivazione funzionale della proteina i-NOS (tra cui rientra la tetraidrobiopterina o BH4) ed ho potuto osservare che l’inibitore di MEK, U0126, è in grado di inibire proprio la sintesi della BH4, bloccando così l’attivazione dell’i-NOS e la produzione di NO.
Da ulteriori approfondimenti, è emerso che la via segnaletica MEK/ERK è coinvolta anche nei segnali trasdotti dai Ca2+-sensing receptors (CaSR) del plasmalemma degli astrociti normali di Uomo adulto. Infatti, ho potuto osservare, che il trattamento degli astrociti in coltura con uno specifico inibitore dei CaSR, l’NPS 89636, previene l’attivazione della via MEK/ERK ed impedisce, di conseguenza, la produzione ed il rilascio di NO. Analogamente a quanto si verifica utilizzando l’inibitore di MEK U0126, la mancata produzione di NO non è dovuta alla soppressione della sintesi della proteina i-NOS, ma al fatto che il trattamento con l’NPS 89636 fa sì che l’i-NOS neosintetizzata non sia enzimaticamente attiva. L’aggiunta di BH4 esogena agli astrociti in coltura consente anche in questo caso di “by-passare” l’inibizione della sintesi della stessa causata dal trattamento con l’NPS 89636 attivando di conseguenza la produzione ed il rilascio di NO.
Da quanto sopra riportato, emerge che l’attivazione di i-NOS, è accompagnata dalla stimolazione della sintesi di BH4 da parte dell’ enzima GTP-cicloidrolasi-I (GTP-CH-I). Attraverso ulteriori studi di immunocitochimica, accompagnati da analisi proteomiche più approfondite, ho dimostrato che l’induzione dell’ enzima GTP-CH-I è parallela a quella di i-NOS ed è mediata da vie segnaletiche che vedono coinvolti sia i segnali mediati dai CaSR che le MAP chinasi.
Infine, mi sono proposta di studiare la regolazione della funzionalità dell’i-NOS negli astrociti normali di Uomo adulto tanto in importanti patologie neurodegenerative quale il morbo di Alzheimer. Infatti è noto che tra gli agenti rilasciati dalla microglia attivata dall’accumulo di peptidi della ß-amiloide Aß(1-40) e Aß(1-42) e dalla loro deposizione in placche fibrillari vi sono le citochine proinfiammatorie, tra cui IL-1ß, TNF-α, INF-γ che inducono, come ho dimostrato negli astrociti, l’espressione e la attivazione di i-NOS con conseguente produzione di NO ed accumulo di perossinitrito, che possono danneggiare in modo irreversibile i neuroni e gli oligodendrociti delle guaine mieliniche, causando la morte di entrambi i tipi cellullari.
Per questo motivo ho cercato di studiare l'effetto diretto dell’Aß (1-40) in forma solubile e della proteina basica della mielina (MBP), che può derivare dal danno subito dalle guaine mieliniche, sugli astrociti normali di Uomo adulto, quiescenti dal punto di vista proliferativo, cioè in condizioni paragonabili a quelle degli astrociti risiedenti nell’encefalo. La scelta di utilizzare questo modello sperimentale è stata dettata anche dal fatto che finora gli studi condotti sugli astrociti, riguardano per lo più astrociti fetali o linee di astrocitoma che ben poco hanno a che fare con la funzionalità di cellule adulte normali e con patologie di tipo cronico degenerativo, quale è il morbo di Alzheimer.When the brain is injured by stroke, head trauma or by the Alzheimer and Parkinson disease processes, astrocytes respond to the build-up of proinflammatory cytokines, such as IL-1ß, TNF-α, and IFN-γ from activated microglia by making among other things the short-lived, weakly oxidizing nitric oxide (NO), which is converted to the long-lived, cytotoxic, strongly oxidizing peroxynitrite radical (ONOO_) that can harm and kill the neurons functionally and physically associated with the astrocytes.
This NO can be made by any one of the three main types of NOS (NO synthase)—neuronal NOS-1 (or nNOS), the inducible and the most productive NOS-2 (or iNOS), and endothelial cell NOS-3 (eNOS or ecNOS).
Since NO injures and kills neurons in variously damaged brains, finding out which nitric oxide synthase induces it and which are the signalling pathways that modulate the enzymatic activity in the human adult astrocytes, should help to design effective therapeutics.
In fact, for example, it has been demonstrated that inhibiting the expression of the NOS-2 gene or the activation of the NOS-2 protein significantly reduces the damage from a head blow or focal ischemia in rat and mouse brains, respectively. For this reason, the aims of my study have been (1) to find out which NOS enzyme is involved in NO-driven killing cascade, and (2) to explicate the mechanisms responsible for the build-up and the regulation of NOS in freshly isolated proliferatively quiescent, phenotypically stable, adult human cerebral astrocytes exposed to the three proinflammatory cytokines (IL-1ß, IFN-γ, TNF-αCM trio) that are most often found in injured or diseased brains.
Finally, I have also investigated (3) the effects of a soluble Aß, the Aß (1-40) peptide driving the development of AD , as well as the myelin basic protein (MBP) that may be produced by neuronal degeneration and oligodendroglial cell damage, on the NO production by the normal adult human astrocytes equivalent to those found in a normal adult brain.
I found that when these astrocytes were exposed to three proinflammatory cytokines (IL-1ß, IFN-γ, TNF-α), they somehow triggered the expression of the NOS-2 gene by MEK–ERK activity-dependent events.
The critical MEK1/MEK2-dependent process had been available for starting NOS-2 activation only between 24 and 24.5 h after CM trio addition because it could not start again and drive NO production for at least 6.5 days after the treatment with the MEK1/MEK2 inhibitor U0126.
The second novel finding was that the U0126-sensitive process in the CM trio-stimulated astrocytes was the synthesis of the NOS-2 cofactor tetrahydrobiopterin (BH4), first because the U0126 prevented the build up of BH4 that accompanies the surge of NO production, and second because adding BH4 to the culture medium enabled the U0126- treated cells to generate nearly normal amounts of NO. The BH4 accumulating along with the new NOS-2 proteins in CM trio-treated cells would have operated by binding with very high affinity to single NOS-2 pre-enzyme proteins and promoting their dimerization into functional enzymes.
I also found that Ca2+-sensing receptors (CASR) signaling was needed to activate the NOS-2 because exposing the cells to NPS 89636 prevented NO production without affecting NOS-2 protein expression. It appeared that NPS 89636 could mimic U0126 by preventing ERK activation, and this inhibition by NPS 89636 could be completely overridden by adding BH4 to the medium.
I then focused my efforts on finding out how CASR signals and MEK-ERK kinases control the production of BH4 by GTP-cyclohydrolase-I (GTP-CH-I) in human astrocytes activated by pro-inflammatory cytokines. In this study I demonstrated that GTP-CH-I is not constitutively expressed and was coinduced in human astrocytes stimulated by CM trio to produce NO. It was my intention to investigate which proteins could be implicated in the activation and post-translational modifications of GTP-CH-I and by mean of proteomic analysis I found that proteins involved probably as adaptors in the signalling transduction of a G-protein-coupled receptor, like CASR were part of the immunocomplexes isolated using an anti-GTP-CH-I antibody from CM trio-treated astrocytes. Some of them could be also involved in the modulation of the intracellular GTP level. GTP is the substrate of GTP-CH-I and modulates GTP-CH-I activity by cooperative binding and thereby changing the enzyme kinetics.
Finally, from my results, I have also learned that a soluble Aß, the Aß (1-40) peptide, as well as the MBP that may be produced by neuronal degeneration and oligodendroglial cell damage, could stimulate non-proliferating phenol typically normal adult human astrocytes equivalent to those found in a normal adult brain to start expressing NOS-2 and manufacturing NO. The mechanism(s) by which Aß (1-40) and MBP might have stimulated NO overproduction in the astrocytes is unknown.
Clearly the next steps will be to find out whether Aßs and MBP induce normal mature adult human astrocytes to express GTP-CH-I and NOS-2 and make NO using the same MEK/ERK-dependent mechanism as the INF-γ+IL-1ß+TNF-α cytokine triad.
Every clarification of the steps leading to the coinduction of GTP-CH-I and NOS-2 activity in the human astrocytes may be an important finding that help the understanding of the molecular events taking place in the neuro inflammation and in the neuronal damage, as manifested in AD brain
Family C G-Protein-coupled receptors in Alzheimer’s disease and therapeutic implications
Full text linkAlzheimer’s disease (AD), particularly its sporadic or late-onset form (SAD/LOAD), is the most prevalent (96–98% of cases) neurodegenerative dementia in aged people. AD’s neuropathology hallmarks are intrabrain accumulation of amyloid-β peptides (Aβs) and of hyperphosphorylated Tau (p-Tau) proteins, diffuse neuroinflammation, and progressive death of neurons and oligodendrocytes. Mounting evidences suggest that family C G-protein-coupled receptors (GPCRs), which include γ-aminobutyric acid B receptors (GABABRs), metabotropic glutamate receptors (mGluR1-8), and the calcium-sensing receptor (CaSR), are involved in many neurotransmitter systems that dysfunction in AD. This review updates the available knowledge about the roles of GPCRs, particularly but not exclusively those expressed by brain astrocytes, in SAD/ LOAD onset and progression, taking stock of their respective mechanisms of action and of their potential as anti-AD therapeutic targets. In particular, GABABRs prevent Aβs synthesis and neuronal hyperexcitability and group I mGluRs play important pathogenetic roles in transgenic AD-model animals. Moreover, the specific binding of Aβs to the CaSRs of human cortical astrocytes and neurons cultured in vitro engenders a pathological signaling that crucially promotes the surplus synthesis and release of Aβs and hyperphosphorylated Tau proteins, and also of nitric oxide, vascular endothelial growth factor-A, and proinflammatory agents. Concurrently, Aβs•CaSR signaling hinders the release of soluble (s)APP-α peptide, a neurotrophic agent and GABABR1a agonist. Altogether these effects progressively kill human cortical neurons in vitro and likely also in vivo. Several CaSR’s negative allosteric modulators suppress all the noxious effects elicited by Aβs•CaSR signaling in human cortical astrocytes and neurons thus safeguarding neurons’ viability in vitro and raising hopes about their potential therapeutic benefits in AD patients. Further basic and clinical investigations on these hot topics are needed taking always heed that activation of the several brain family C GPCRs may elicit divergent upshots according to the models studied
Use of Calcilytic drugs as a pharmacological approach to the treatment and prevention of Alzheimer's Disease, Alzheimer's disease-related disorder, and down's syndrome neuropathies
No full textUse of Calcilytic drugs as a pharmacological approach to the treatment and prevention of Alzheimer's Disease, Alzheimer's disease-related disorder, and down's syndrome neuropathie
Divers roles of protein kinase C-delta in proliferation and apoptosis of rat fibroblasts
No full textIn vitro and in vivo characteristics of frozen/thawed neonatal split-skin strips: A novel biologically active dressing for areas of severe, acute or chronic skin loss.
No full textThe recurrent shortage of human skin autografts or allografts used to close extensive wounds has rekindled the search for feasible alternatives. In the past, adult pig skin was a popular biological dressing, yet doubts regarding its benefits have induced most people to abandon its use. Here we investigated the aptness of neonatal pig split-skin (NPSS) strips to be used as a novel kind of temporary dressing for areas of skin loss. NPSS strips are able to be prepared in bulk amounts, stored at -80 degrees C for up to six months, and recovered by swift thawing at 45 degrees C with no change in histological structure. When set into organ cultures in vitro for up to three weeks, these frozen/thawed NPSS strips exhibited both a skin-typical energy-linked metabolism (i.e., a predominant consumption of L-glutamine instead of glucose), and an enduring ability to secrete cytokines/chemokines such as IL-1alpha, IL-6, GM-CSF, and TNF-alpha; all features alike in quantitative terms to those exhibited by freshly prepared NPSS strips directly set into culture. Moreover, once applied as temporary dressings onto deep burn wounds in vivo, frozen/thawed NPSS strips produced, for at least seven days, porcine IL-1alpha, IL-6, GM-CSF, TNF-alpha, and TGF-beta; the cytokines/chemokines importantly involved in wound healing. Hence, frozen/thawed NPSS strips not only are capable of closing extensive areas of skin loss, but even release several cytokines/chemokines beneficial to tissue regeneration and repair
Modulation of NO synthase isoforms in cultured normal human adult astrocytes
No full textModulation of NO synthase isoforms in cultured normal adult human astrocyte
Danger‐Sensing/Pattern Recognition Receptors and Neuroinflammation in Alzheimer’s Disease
Full text linkFibrillar aggregates and soluble oligomers of both Amyloid‐β peptides (Aβs) and hyperphosphorylated Tau proteins (p‐Tau‐es), as well as a chronic neuroinflammation are the main drivers causing progressive neuronal losses and dementia in Alzheimer’s disease (AD). However, the underlying pathogenetic mechanisms are still much disputed. Several endogenous neurotoxic ligands, including Aβs, and/or p‐Tau‐es activate innate immunity‐related danger‐sensing/pattern recognition receptors (PPRs) thereby advancing AD’s neuroinflammation and progression. The major PRR families involved include scavenger, Toll‐like, NOD‐like, AIM2‐like, RIG‐like, and CLEC‐ 2 receptors, plus the calcium‐sensing receptor (CaSR). This quite intricate picture stresses the need to identify the pathogenetically topmost Aβ‐activated PRR, whose signaling would trigger AD’s three main drivers and their intra‐brain spread. In theory, the candidate might belong to any PRR family. However, results of preclinical studies using in vitro nontumorigenic human cortical neurons and astrocytes and in vivo AD‐model animals have started converging on the CaSR as the pathogenetically upmost PRR candidate. In fact, the CaSR binds both Ca2+ and Aβs and promotes the spread of both Ca2+ dyshomeostasis and AD’s three main drivers, causing a progressive neurons’ death. Since CaSR’s negative allosteric modulators block all these effects, CaSR’s candidacy for topmost pathogenetic PRR has assumed a growing therapeutic potential worth clinical testing
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe 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
Poliuretani/fibroina per applicazioni di ingegneria dei tessuti. II. Interazioni con i fibroblasti umani normali.
No full textSono descritti nuovi scaffold composti da poliuretani e fibroina della seta per impieghi applicativi di ingegneria dei tessuti. Si riportano anche alcune loro interazioni con i fibroblasti umani. normal
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