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Investigations into Alzheimer's disease pathogenetic mechanisms using normal adult human astrocytes in culture
No full textLa malattia di Alzheimer è una delle principali patologie neurodegenerative legate alla terza età: i casi infatti che si sviluppano in età precoce rappresentano meno dell’1% e sono dovuti ad alterazioni geniche specifiche. L’eziologia di questa malattia resta ancora sconosciuta, sebbene la presenza di placche senili, composte principalmente di Aβ1-40 e Aβ1-42, o aggregati fibrillari intraneuronali della proteina tau, siano caratteristiche ormai note del suo stadio avanzato. Diversi processi patologici sono stati messi in relazione con il danno neuronale e il declino cognitivo tipici della malattia. Tra questi uno dei più studiati è il processo infiammatorio indotto da aggregati di amiloide negli spazi perineuronali e mediato principalmente da astrociti e cellule della microglia. Recentemente anche alterazioni dei meccanismi neurovascolari e della struttura della barriera emato-encefalica sono stati correlati con la progressione del processo patologico e con l’induzione locale di angiogenesi: la presenza di VEGF, una delle principali citochine ad azione angiogenica, è stata dimostrata infatti in corrispondenza di cluster di astrociti reattivi in pazienti AD. Per chiarire il possibile legame tra la neoangiogenesi, la presenza di aggregati di amiloide e la secrezione di mediatori infiammatori, si è deciso di analizzare la modulazione dell’espressione del complesso trascrizionale HIF-1α•HIF-1β in astrociti umani normali adulti (NAHA) a seguito del trattamento con la triade di citochine proinfiammatorie (CM-trio, IL-1β + IFN-γ + TNF-α) e con Aβ25-35, il frammento attivo dell’Aβ1-42. Il legame di HIF-1α•HIF-1β alla sequenza HRE presente nella regione promotoriale di geni correlati alla malattia, quali VEGF e BACE-1, è responsabile della loro induzione in risposta a stimoli quali stress ipossico. Entrambi i trattamenti, pur non alterando in nessun modo la trascrizione delle subunità HIF-1α e HIF-1β, inducono sia una maggiore stabilità della frazione proteica di entrambi i fattori, sia un’aumentata traslocazione del complesso a livello nucleare con conseguente legame alla sequenza HRE. Il trattamento invece con CM-trio determina una significativa riduzione dell’espressione di un altro fattore implicato nell’induzione della trascrizione del VEGF-A, PGC-1α, dimostrando che HIF-1α•HIF-1β è il solo complesso coinvolto nella modulazione del fattore angiogenico. La traslocazione nucleare dell’eterodimero HIF-1α•HIF-1β e il suo legame alla sequenza promotoriale HRE corrisponde ad un aumento dell’espressione di tre varianti di splicing del VEGF-A (121, 165 e 189), ma non all’ accumulo della corrispondente frazione proteica a livello intracellulare. Analisi mediante ELISA hanno dimostrato un aumento del rilascio del VEGF-A nel medium di coltura di cellule trattate con Aβ25-35, con la coppia IFN-γ + TNF-α, ma soprattutto con CM-trio. L’insieme di Aβ25-35 + CM-trio non è in grado di esercitare un’azione sinergica sul rilascio, dimostrando che la triade di citochine proinfiammatorie è il principale induttore della secrezione. Il complesso HIF-1α•HIF-1β si è rivelato inoltre implicato nella regolazione della trascrizione di BACE-1, una delle secretasi coinvolte nella produzione di Aβ. Il trattamento con Aβ25-35 stimola infatti la maggiore espressione dell’mRNA e della proteina BACE-1, inducendo inoltre un aumento della sua attività e di quella della γ-secretasi, altro enzima responsabile del processamento dell’APP. La produzione di peptidi Aβ in NAHA trattati con Aβ25-35 è stata dimostrata tramite immunoblotting e immunofluorescenza; quest’ultima tecnica, in particolare, ha permesso di evidenziare la presenza di aggregati di Aβ25-35 nel citoplasma di astrociti trattati, delineando un loro possibile ruolo nella rimozione di aggregati di amiloide potenzialmente patologici.Alterations in neurovascular mechanisms and blood-brain barrier (BBB) disorders have been related to late-onset Alzheimer’s disease (AD). An increased expression of vascular endhotelial growth factor (VEGF) has seen in cluster of reactive astrocytes in AD brains and into rat hippocampus Aβ1-42 injected. We found that the Aβ25-35, an amyloid β1-42 active fragment, and proinflammatory cytokines (CM-trio, IL-1β + IFN-γ + TNF-α) treatments has induced an increased protein steady state levels of the hypoxia-inducible factor transcriptional complex (HIF-1α•HIF-1β) in normal adult human astrocytes (NAHAs). This transcription factor is the main regulator of the VEGF expression, but also the PGC-1α cofactor seems to be related to its upregulation in AD. We analyzed the mRNA expression of both these factors, but only the complex HIF-1α•HIF-1β was implicated in the VEGF-A expression. Indeed, the enhanced HIF-1α•HIF-1β nuclear translocation and its binding at the hypoxia response element (HRE) sequence in the VEGF-A promoter, found after both treatments, were correlated with the increased VEGF-A mRNA splice variants (121, 165, and 189) expression. The CM-trio was the most powerful stimulus inducing the VEGF-A secretion in NAHAs culture medium, also induced, less than CM-trio, by the paired cytokines IFN-γ + TNF-α, Aβ25-35 and CM-trio + Aβ25-35 treatments. Hence the astrocytes in an AD brain can produce a raised amount of VEGF-A in response to Aβ peptide and the proinflammatory cytokines promoting the neoangiogenic process. We found that the HIF-1α•HIF-1β complex was furthermore responsible for the β-secretase (BACE-1) mRNA and protein up-regulation in our experimental model treated with Aβ25-35. After the same treatment we found moreover an enhancement of the β and γ-secretase activities, the two secretases involved in the amyloid precursor protein (APP) processing. The immunofluorescence and immunoblotting assays confirmed the Aβ production in NAHAs, suggesting the existence of a positive feed-back loop for the Aβ synthesis in astrocytes. This findings support the theory that astrocytes, as well as neurones, are involved in the AD decline of cognitive functions. Moreover, the immunofluorescence assay shows the Aβ25-35 uptake, defining a possible role of astocytes as Aβ peptides scavengers
Arnica Montana effects on gene expression in a human macrophage cell line. Evaluation by quantitative Real-Time PCR
Full text linkPremesse:
l'Arnica Montana è un preparato tradizionale popolare molto usato in medicina complementare, anche per le sue proprietà curative.
Nonostante la sua azione riconosciuta in ambito clinico nelle diverse dosi, gli aspetti molecolari che favoriscono la guarigione delle ferite
devono essere ancora chiariti. Per cercare di ovviare a questa mancanza, abbiamo valutato l'estratto della pianta, in un ampio spettro di
diluizioni, nelle cellule umane THP-1, differenziate tra macrofagi completamente sviluppati e macrofagi aVENTl fenotipo alternativo IL-4, coinvolto nella ricomposizione e nella guarigione dei tessuti.
Metodi:
L'analisi PCR (Polymerase Chain Reaction) è stata applicata per analizzare i cambi di espressione di un pannello di "geni chiave", soprattutto citochine. recettori e fattori di trascrizione.
Risultati:
Sui macrofagi sviluppati verso il fenotipo che favorisce la guarigione, l'Arnica Montana influenza l'espressione di svariati geni. In particolare il gene CXCL1 ha mostrato l'incremento di espressione più evidente, mentre il gene CXCL2, il gene IL8 e quello della proteina BMP2 sono risultati modificati verso l'alto, suggerendo la possibilità di un effetto positivo dell'Arnica Montana sulla selezione dei neutrofili e sull'angiogenesi. A sua volta, il gene MMP1, esprimente una metalloproteinasi in grado di suddividere i substrati extra-cellulari, risulta regolata verso il basso.
La maggior parte dei risultati ottenuti mostra un andamento non lineare della relazione dose-effetto.
Conclusioni:
Questo studio esplorativo fornisce una nuova visuale nello studio del meccanismo di azione di Arnica Montana come rimedio per favorire la guarigione, poiché alcuni dei geni da essa modificati sono geni regolatori della ricomposizione dei tessuti, del grado di infiammazione e della chemiotassi.Background:
Arnica montana is a popular traditional remedy widely used in complementary medicine, also for its
wound healing properties. Despite its acknowledged action in clinical settings at various doses, the
molecular aspects relating to how Arnica montana promotes wound healing remain to be elucidated.
To fill this gap, we evaluated the whole plant extract, in a wide range of dilutions, in THP-1 human
cells, differentiated into mature macrophages and into an alternative IL-4-activated phenotype
involved in tissue remodelling and healing.
Methods:
Real-time quantitative Reverse Transcription Polymerase Chain Reaction (PCR) analysis was used
To study the changes in the expression of a customized panel of key genes, mainly cytokines,
receptors and transcription factors.
Results:
On macrophages differentiated towards the wound healing phenotype, Arnica montana affected the
expression of several genes. In particular CXC chemokine ligand 1 (CXCL1), coding for a chief chemo-
kine, exhibited the most consistent increase of expression, while also CXC chemokine ligand 2 (CXCL2),
Interleukin8 (IL8) and bone morphogenetic protein (BMP2) were slightly up-regulated, suggesting a
positive influence of Arnica montana on neutrophil recruitment and on angiogenesis. MMP1, coding for
a metalloproteinase capable of cleaving extracellular matrix substrates, was down-regulated.
Most results showed non-linearity of the dose-effect relationship.
Conclusions:
This exploratory study provides new insights into the cellular and molecular mechanisms of action of
Arnica montana as a promoter of healing, since some of the genes it modifies are key regulators of
tissue remodelling, inflammation and chemotaxis
Biological activity of interferon gamma and lipopolysaccharide on the nitric oxide production in C6 astroglioma cells and some unexpected effects of potentization
No full textBackground: The search for new therapeutic approaches with fewer side effects and better treatment efficacy to the Chagas Disease has been a major challenge. Aim: To evaluate the effects of Kalium causticum, Conium maculatum, and Lycopodium clavatum 13 cH in mice inoculated with the Y strain of Trypanosoma cruzi. Materials and methods: In a blind, controlled, randomized study, 102 male Swiss mice, eight weeks old, were inoculated with 1,400 trypomastigotes of the Y strain of T. cruzi and distributed into the following groups: CI (treated with 7% hydroalcoholic solution), Ca (treated with Kalium causticum 13cH), Co (treated with Conium maculatum 13cH), and Ly (treated with Lycopodium clavatum 13cH). The medicines were selected by three homeopaths using Lince Expert System Software (Albuquerque, NM, USA), considering the behavioral characteristics of the mice. The treatments were performed 48 hours before and 48, 96, and 144 hours after infection [1]. The following parameters were evaluated: infectivity, prepatent period, parasitemia peak, total parasitemia, tissue tropism, inflammatory infiltrate, and survival. Results: The prepatent period was greater in the Ly group than in the CI group (p = 0.02). The number of trypomastigotes on the 8th day after infection was lower in the Ca group than in the CI group (p < 0.05). Total parasitemia was significantly lower in the Ca, Co, and Ly groups than in the CI group. On the 12th day after infection, the Ca, Co, and Ly groups had fewer nests of amastigotes and amastigotes/nest in the heart than the CI group (p < 0.05) (Figure-I). A decrease in the number of nests and amastigotes in the intestine were observed in the Ly group compared with the CI group (p < 0.05). In the liver (day 12), Ly significantly prevented the formation of inflammatory foci compared with the other groups. In muscle, Co and Ly decreased the formation of inflammatory foci compared with CI (p < 0.05). Ly afforded greater animal survival compared with CI, Ca, and Co (p < 0.05). The animals in the Co group died prematurely compared with the CI group (p = 0.031). (Figure-II) Conclusion: All of the experimental homeopathic medications with 13cH dynamization studied herein reduced the parasite peak and total parasitemia. Ly had significantly more benefits in the treatment of mice infected with T. cruzi, reducing the number of blood parasites, amastigotes nests in tissue and the number of amastigotes per nest, resulting in the increasing animal survival. The data may contribute to changes in management strategies in individuals with Chagas disease
Angiogenic factor induction in and secretion by cultured phenotypically normal adult human astrocytes (NAHA) exposed to beta-amyloid peptides and proinflammatory cytokines
No full textThe induction and secretion of VEGF-A by cultured phenotypically normal adult human astrocytes (NAHAs) exposed to beta-amyloid peptides and proinflammatory cytokines is reported for the first time
Cell sensitivity, non-linearity and inverse effects
No full textIt has been claimed that the homeopathic principle of ‘similarity’ (or ‘similia’) and the use of individualized remedies in extremely low doses conflicts with scientific laws, but this opinion can be disputed on the basis of recent scientific advances. Several mechanisms to explain the responsiveness of cells to ultra-low doses and the similarity as inversion of drug effects, have again been suggested in the framework of hormesis and modern paradoxical pharmacology. Low doses or high dilutions of a drug interact only with the enhanced sensitivities of regulatory systems, functioning as minute harmful stimuli to trigger specific compensatory healing reactions. Here we review hypotheses about homeopathic drug action at cellular and molecular levels, and present a new conceptual model of the principle of similarity based on allosteric drug action. While many common drugs act through orthostatic chemical interactions aimed at blocking undesired activities of enzymes or receptors, allosteric interactions are associated with dynamic conformational changes and functional transitions in target proteins, which enhance or inhibit specific cellular actions in normal or disease states. The concept of allostery and the way it controls physiological activities can be broadened to include diluted/dynamized compounds, and may constitute a working hypothesis for the study of molecular mechanisms underlying the inversion of drug effects
Proinflammatory cytokines and Amyloid β-(25-35) respectively stimulate VEGF-A production and secretion by cultured phenotypically normal adult human cerebral astrocytes
No full textProinflammatory cytokines and Amyloid β-(25-35) stimulate VEGF-A production and secretion by cultured phenotypically normal adult human cerebral astrocytes in cultur
Amyloid-beta peptide induces HIF transcription factor and VEGF expression in cultured normal adult human astrocytes.
No full textAmyloid-beta peptide induces HIF transcription factor and VEGF expression in cultured normal adult human astrocytes
Detection of nanostructures in solutions of Zincum metallicum and the vehicle lactose
No full textBackground: Nanoparticles (NP), because of their size (< 1μm) and high relative surface area, are highly reactive forms of their source material with biological, chemical, optical, electromagnetic, and thermal properties different from larger bulk forms of the same material. It has been speculated that NPs can occur in homeopathic products as a function of trituration and shaking into glass containers. Moreover, the presence of sugars additives like lactose and of silicates leaked from the glassware are reported to stabilize the nanoparticles.1 Up to now some authors observed nanoparticles in highly diluted samples2, 3, but further studies are needed to know the nature of the NPs. Actually, nanostructures in the solutions may derive from the source materials but also from stuff contaminations and also may be constituted by nano-bubbles.4, 5 Moreover, the mechanism by which the nanostructures can be formed and the effect of serial dilution/succussions of the NPs suspension should be studied.6 Many tools are available to analyze the nanostructures both in solutions or in dried samples and these give complementary information about the concentration, stability, structure and chemical nature of the NPs. In the present report we describe preliminary observations obtained by the nanoparticle tracking analysis (NTA) in Zincum metallicum (Zinc met.) solutions at low-grade dilution/dynamization and their lactose controls. This study is part of a formal Brazilian-Italian inter-university collaboration
Abeta42 induction by its proxy, Abeta25-35, in cerebral normal human astrocytes requires calcium-sensing receptor (CaSR) signaling and nuclear translocation of HIF-1alpha/HIF-1beta complexes
No full textIntroduction: Astrocytes in Abeta42-hoarding human brains with Alzheimer’s disease (AD) upregulate vascular endothelial growth factor (VEGF)-A synthesis and accumulate Abeta42 intracellularly. Previously, we showed that fibrillary Abeta25-35 (a surrogate of Abeta42) induces VEGF-A synthesis and secretion and Abeta42 intracellular buildup in cultured early passage crebral normal adult astrocytes (NAHAs), both effects being mediated via the stabilization and nuclear translocation of HIF-1alpha/HIF-1beta complexes.
Aims. Here, we investigated whether calcium-sensing receptor (CaSR) signaling plays any role in such Abeta25-35-elicited effects.
Methods: NAHA cultures were treated with Abeta25-35 plus/minus the CaSR inhibitor NPS-89696 (a gift of Dr. E.F. Nemeth) and the Abeta42 contents of their protein extracts and HRE-DNA- HIF-1alpha/HIF-1beta complexes were analyzed via immunoblotting and EMSA, respectively.
Results: trating NAHAs with Abeta25-35+NPS-89696 significantly reduced the de novo synthesis and accumulation of Abeta42 induced by Abeta25-35 alone. Exposure to Abeta25-35+NPS-89696 also hindered the stabilization and nuclear translocation of the HIF-1alpha/HIF-1beta complexes then elicited by Abeta25-35 itself.
Conclusions: Our preliminary findings show that caSR signaling is importantly involved in Abeta42-production by Abeta25-35-exposed NAHAs. Previously, we showed that CaSR signaling is required also for nitric oxide synthase (NOS)-2 induction and NO hyperproduction by NAHAs treated with Abeta25-35 and/or combined proinflammatory cytokines. Therefore, the present results emphasize the notion that CaSR signaling plays a central role in the several trophic, proinflammatory and perhaps cytotoxic (release of produced Abeta42?) responses of the activated astrocytes in AD brains. Further, our findings suggest that administration CaSR inhibitors may be potentially beneficial in AD
Amyloid-β25-35, an Amyloid-β1-42 Surrogate, and Proinflammatory Cytokines Stimulate VEGF-A Secretion by Cultured, Early Passage, Normoxic Adult Human Cerebral Astrocytes
Full text linkCerebrovascular angiopathy affects late-onset Alzheimer's disease (LOAD) brains by possibly increasing vascular endothelial growth factor (VEGF). A expression, thereby stimulating endothelial cell proliferation and migration. Indeed, VEGF-A gene upregulation, with increased VEGF-A protein content of reactive astrocytes and microglia, occurs in LOAD brains, and neovascularization was observed one week after injecting amyloid-β (Aβ)1-42 into rat hippocampus. We have now found, with cultured 'normoxic' normal adult human astrocytes (NAHAs), that fibrillar Aβ25-35 (an active Aβ1-42 fragment) or a cytokine mixture (the (CM)-trio (interleukin [IL]-1β+interferon [IFN]-γ+tumor necrosis factor [TNF]-α), or pair (IFN-γ+TNF-α) like those produced in LOAD brains) stimulates the nuclear translocation of stabilized hypoxia-inducible factor (HIF)-1α protein and its binding to VEGF-A hypoxia-response elements; the mRNA synthesis for three VEGF-A splice variants (121, 165, 189); and the secretion of VEGF-A165. The CM-trio was the most powerful stimulus, IFN-γ+TNF-α was less potent, and other cytokine pairs or single cytokines or Aβ35-25 were ineffective. While Aβ25-35 did not change HIF-1β protein levels, the CM-trio increased both HIF-1α and HIF-1β protein levels, thereby giving an earlier and stronger stimulus to VEGF-A secretion by NAHAs. Thus, increased VEGF-A secretion from astrocytes stimulated by Aβ1-42 and by microglia-released cytokines might restore angiogenesis and Aβ1-42 vascular clearance
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