RESUMO
We developed and validated a technology platform for designing and testing peptides inhibiting the infectivity of SARS-CoV-2 spike protein-based pseudoviruses. This platform integrates target evaluation, in silico inhibitor design, peptide synthesis, and efficacy screening. We generated a cyclic peptide library derived from the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the angiotensin-converting enzyme 2 (ACE2) receptor. The cell-free validation process by ELISA competition assays and Surface Plasmon Resonance (SPR) studies revealed that the cyclic peptide c9_05, but not its linear form, binds well to ACE2. Moreover, it effectively inhibited the transduction in HEK293, stably expressing the human ACE2 receptor of pseudovirus particles displaying the SARS-CoV-2 spike in the Wuhan or UK variants. However, the inhibitory efficacy of c9_05 was negligible against the Omicron variant, and it failed to impede the entry of pseudoviruses carrying the B.1.351 (South African) spike. These variants contain three or more mutations known to increase affinity to ACE2. This suggests further refinement is needed for potential SARS-CoV-2 inhibition. Our study hints at a promising approach to develop inhibitors targeting viral infectivity receptors, including SARS-CoV-2's. This platform also promises swift identification and evaluation of inhibitors for other emergent viruses.
Assuntos
COVID-19 , Vírus de RNA , Humanos , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2 , Células HEK293 , Peptídeos/farmacologia , Peptídeos Cíclicos , Biblioteca de Peptídeos , Tecnologia , Ligação ProteicaRESUMO
The relevant social and economic costs associated with aging and neurodegenerative diseases, particularly Alzheimer's disease (AD), entail considerable efforts to develop effective preventive and therapeutic strategies. The search for natural compounds, whose intake through diet can help prevent the main biochemical mechanisms responsible for AD onset, led us to screen hops, one of the main ingredients of beer. To explore the chemical variability of hops, we characterized four hop varieties, i.e., Cascade, Saaz, Tettnang, and Summit. We investigated the potential multitarget hop activity, in particular its ability to hinder Aß1-42 peptide aggregation and cytotoxicity, its antioxidant properties, and its ability to enhance autophagy, promoting the clearance of misfolded and aggregated proteins in a human neuroblastoma SH-SY5Y cell line. Moreover, we provided evidence of in vivo hop efficacy using the transgenic CL2006Caenorhabditis elegans strain expressing the Aß3-42 peptide. By combining cell-free and in vitro assays with nuclear magnetic resonance (NMR) and MS-based metabolomics, NMR molecular recognition studies, and atomic force microscopy, we identified feruloyl and p-coumaroylquinic acids flavan-3-ol glycosides and procyanidins as the main anti-Aß components of hop.
Assuntos
Doença de Alzheimer , Humulus , Neuroblastoma , Humanos , Humulus/química , Doença de Alzheimer/prevenção & controle , Cerveja/análise , AntioxidantesRESUMO
The anti-Alzheimer disease (AD) activity reported for an aqueous cinnamon bark extract prompted us to investigate and compare the anti-amyloidogenic properties of cinnamon extracts obtained from both bark and bud, the latter being a very little explored matrix. We prepared the extracts with different procedures (alcoholic, hydroalcoholic, or aqueous extractions). An efficient protocol for the rapid analysis of NMR spectra of cinnamon bud and bark extracts was set up, enabling the automatic identification and quantification of metabolites. Moreover, we exploited preparative reverse-phase (RP) chromatography to prepare fractions enriched in polyphenols, further characterized by UPLC-HR-MS. Then, we combined NMR-based molecular recognition studies, atomic force microscopy, and in vitro biochemical and cellular assays to investigate the anti-amyloidogenic activity of our extracts. Both bud and bark extracts showed a potent anti-amyloidogenic activity. Flavanols, particularly procyanidins, and cinnamaldehydes, are the chemical components of cinnamon hindering Aß peptide on-pathway aggregation and toxicity in a human neuroblastoma SH-SY5Y cell line. Together with the previously reported ability to hinder tau aggregation and filament formation, these data indicate cinnamon polyphenols as natural products possessing multitarget anti-AD activity. Since cinnamon is a spice increasingly present in the human diet, our results support its use to prepare nutraceuticals useful in preventing AD through an active contrast to the biochemical processes that underlie the onset of this disease. Moreover, the structures of cinnamon components responsible for cinnamon anti-AD activities represent molecular templates for designing and synthesizing new anti-amyloidogenic drugs.
RESUMO
The release of neutrophil extracellular traps (NETs), a process termed NETosis, avoids pathogen spread but may cause tissue injury. NETs have been found in severe COVID-19 patients, but their role in disease development is still unknown. The aim of this study is to assess the capacity of NETs to drive epithelial-mesenchymal transition (EMT) of lung epithelial cells and to analyze the involvement of NETs in COVID-19. Bronchoalveolar lavage fluid of severe COVID-19 patients showed high concentration of NETs that correlates with neutrophils count; moreover, the analysis of lung tissues of COVID-19 deceased patients showed a subset of alveolar reactive pneumocytes with a co-expression of epithelial marker and a mesenchymal marker, confirming the induction of EMT mechanism after severe SARS-CoV2 infection. By airway in vitro models, cultivating A549 or 16HBE at air-liquid interface, adding alveolar macrophages (AM), neutrophils and SARS-CoV2, we demonstrated that to trigger a complete EMT expression pattern are necessary the induction of NETosis by SARS-CoV2 and the secretion of AM factors (TGF-ß, IL8 and IL1ß). All our results highlight the possible mechanism that can induce lung fibrosis after SARS-CoV2 infection.
Assuntos
COVID-19/fisiopatologia , Transição Epitelial-Mesenquimal , Armadilhas Extracelulares/metabolismo , Neutrófilos/metabolismo , Adulto , Biópsia , Líquido da Lavagem Broncoalveolar/citologia , COVID-19/complicações , COVID-19/imunologia , Linhagem Celular , Células Epiteliais/patologia , Humanos , Pulmão/patologia , Fibrose Pulmonar/etiologia , Fibrose Pulmonar/metabolismoRESUMO
The metabolic profile of Lavado cocoa was characterized for the first time by NMR spectroscopy, then compared with the profiles of fermented and processed varieties, Natural and commercial cocoa. The significant difference in the contents of theobromine and flavanols prompted us to examine the cocoa varieties to seek correlations between these metabolite concentrations and the anti-amyloidogenic activity reported for cocoa in the literature. We combined NMR spectroscopy, preparative reversed-phase (RP) chromatography, atomic force microscopy, in vitro biochemical and cell assays, to investigate and compare the anti-amyloidogenic properties of extracts and fractions enriched in different metabolite classes. Lavado variety was the most active and the catechins and theobromine were the chemical components of cocoa hindering Aß peptide on-pathway aggregation and toxicity in a human neuroblastoma SH-SY5Y cell line.
Assuntos
Cacau/química , Alimentos Fermentados/análise , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Antioxidantes/química , Cacau/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Flavanonas/análise , Humanos , Espectroscopia de Ressonância Magnética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Agregados Proteicos/efeitos dos fármacos , Teobromina/análiseRESUMO
The growing interest in medicinal plants for the identification of new bioactive compounds and the formulation of new nutraceuticals and drugs prompted us to develop a powerful experimental approach allowing the detailed metabolic profiling of complex plant extracts, the identification of ligands of macromolecular targets of biomedical relevance and a preliminary characterization of their biological activity. To this end, we selected Peucedanum ostruthium, a plant traditionally employed in Austria and Italy for its several potential therapeutic applications, as case study. We combined the use of NMR and UPLC-HR-MS for the identification of the metabolites present in its leaves and rhizome extracts. Due to the significant content of polyphenols, particularly chlorogenic acids, recently identified as anti-amyloidogenic compounds, polyphenols-enriched fractions were prepared and tested for their ability to prevent Aß1-42 peptide aggregation and neurotoxicity in a neuronal human cell line. STD-NMR experiments allowed the detailed identification of Aß oligomers' ligands responsible for the anti-amyloidogenic activity. These data provide experimental protocols and structural information suitable for the development of innovative molecular tools for prevention, therapy and diagnosis of Alzheimer's disease.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Apiaceae/química , Produtos Biológicos/farmacologia , Ressonância Magnética Nuclear Biomolecular , Extratos Vegetais/farmacologia , Peptídeos beta-Amiloides/metabolismo , Produtos Biológicos/análise , Relação Dose-Resposta a Droga , Estrutura Molecular , Extratos Vegetais/análise , Folhas de Planta/química , Relação Estrutura-AtividadeRESUMO
To identify food and beverages that provide the regular intake of natural compounds capable of interfering with toxic amyloidogenic aggregates, we developed an experimental protocol that combines NMR spectroscopy and atomic force microscopy, in vitro biochemical and cell assays to detect anti-Aß molecules in natural edible matrices. We applied this approach to investigate the potential anti-amyloidogenic properties of coffee and its molecular constituents. Our data showed that green and roasted coffee extracts and their main components, 5-O-caffeoylquinic acid and melanoidins, can hinder Aß on-pathway aggregation and toxicity in a human neuroblastoma SH-SY5Y cell line. Coffee extracts and melanoidins also counteract hydrogen peroxide- and rotenone-induced cytotoxicity and modulate some autophagic pathways in the same cell line.
Assuntos
Peptídeos beta-Amiloides/química , Café/química , Manipulação de Alimentos , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Multimerização Proteica/efeitos dos fármacos , Linhagem Celular Tumoral , Cor , Humanos , Espectroscopia de Ressonância MagnéticaRESUMO
Due to their altered metabolism cancer cells are more sensitive to proteasome inhibition or changes of copper levels than normal cells. Thus, the development of copper complexes endowed with proteasome inhibition features has emerged as a promising anticancer strategy. However, limited information is available about the exact mechanism by which copper inhibits proteasome. Here we show that Cu(II) ions simultaneously inhibit the three peptidase activities of isolated 20S proteasomes with potencies (IC50) in the micromolar range. Cu(II) ions, in cell-free conditions, neither catalyze red-ox reactions nor disrupt the assembly of the 20S proteasome but, rather, promote conformational changes associated to impaired channel gating. Notably, HeLa cells grown in a Cu(II)-supplemented medium exhibit decreased proteasome activity. This effect, however, was attenuated in the presence of an antioxidant. Our results suggest that if, on one hand, Cu(II)-inhibited 20S activities may be associated to conformational changes that favor the closed state of the core particle, on the other hand the complex effect induced by Cu(II) ions in cancer cells is the result of several concurring events including ROS-mediated proteasome flooding, and disassembly of the 26S proteasome into its 20S and 19S components.
Assuntos
Cobre/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Células HeLa , Humanos , Concentração Inibidora 50 , Íons , Mutação/genética , Inibidores de Proteassoma/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Espectrometria de Fluorescência , Triptofano/metabolismo , Zinco/farmacologiaRESUMO
A new paradigm for the aetiopathology of phenylketonuria suggests the presence of amyloid-like assemblies in the brains of transgenic mouse models and patients with phenylketonuria, possibly shedding light on the selective cognitive deficit associated with this disease. Paralleling the amyloidogenic route that identifies different stages of peptide aggregation, corresponding to different levels of toxicity, we experimentally address for the first time, the physico-chemical properties of phenylalanine aggregates via Small Angle, Wide Angle X-ray Scattering and Atomic Force Microscopy. Results are consistent with the presence of well-structured, aligned fibres generated by milliMolar concentrations of phenylalanine. Moreover, the amyloid-modulating doxycycline agent affects the local structure of phenylalanine aggregates, preventing the formation of well-ordered crystalline structures. Phenylalanine assemblies prove toxic in vitro to immortalized cell lines and primary neuronal cells. Furthermore, these assemblies also cause dendritic sprouting alterations and synaptic protein impairment in neurons. Doxycycline counteracts these toxic effects, suggesting an approach for the development of future innovative non-dietary preventive therapies.
Assuntos
Amiloide/metabolismo , Doxiciclina/farmacologia , Neurônios/metabolismo , Fenilalanina/metabolismo , Fenilcetonúrias , Sinapses/metabolismo , Animais , Células Hep G2 , Humanos , Camundongos , Neurônios/patologia , Fenilcetonúrias/tratamento farmacológico , Fenilcetonúrias/metabolismo , Fenilcetonúrias/patologia , Sinapses/patologiaRESUMO
The cellular pathways activated by mutant prion protein (PrP) in genetic prion diseases, ultimately leading to neuronal dysfunction and degeneration, are not known. Several mutant PrPs misfold in the early secretory pathway and reside longer in the endoplasmic reticulum (ER) possibly stimulating ER stress-related pathogenic mechanisms. To investigate whether mutant PrP induced maladaptive responses, we checked key elements of the unfolded protein response (UPR) in transgenic mice, primary neurons and transfected cells expressing two different mutant PrPs. Because ER stress favors the formation of untranslocated PrP that might aggregate in the cytosol and impair proteasome function, we also measured the activity of the ubiquitin proteasome system (UPS). Molecular, biochemical and immunohistochemical analyses found no increase in the expression of UPR-regulated genes, such as Grp78/Bip, CHOP/GADD153, or ER stress-dependent splicing of the mRNA encoding the X-box-binding protein 1. No alterations in UPS activity were detected in mutant mouse brains and primary neurons using the Ub(G76V)-GFP reporter and a new fluorogenic peptide for monitoring proteasomal proteolytic activity in vivo. Finally, there was no loss of proteasome function in neurons in which endogenous PrP was forced to accumulate in the cytosol by inhibiting cotranslational translocation. These results indicate that neither ER stress, nor perturbation of proteasome activity plays a major pathogenic role in prion diseases.
Assuntos
Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Mutação , Príons/biossíntese , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Células Cultivadas , Chaperona BiP do Retículo Endoplasmático , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Neurônios/metabolismo , Células PC12 , Peptídeos/química , Príons/genética , Biossíntese de Proteínas , RatosRESUMO
The discovery of methods suitable for the conversion in vitro of native proteins into amyloid fibrils has shed light on the molecular basis of amyloidosis and has provided fundamental tools for drug discovery. We have studied the capacity of a small library of tetracycline analogues to modulate the formation or destructuration of ß2-microglobulin fibrils. The inhibition of fibrillogenesis of the wild type protein was first established in the presence of 20% trifluoroethanol and confirmed under a more physiologic environment including heparin and collagen. The latter conditions were also used to study the highly amyloidogenic variant, P32G. The NMR analysis showed that doxycycline inhibits ß2-microglobulin self-association and stabilizes the native-like species through fast exchange interactions involving specific regions of the protein. Cell viability assays demonstrated that the drug abolishes the natural cytotoxic activity of soluble ß2-microglobulin, further strengthening a possible in vivo therapeutic exploitation of this drug. Doxycycline can disassemble preformed fibrils, but the IC(50) is 5-fold higher than that necessary for the inhibition of fibrillogenesis. Fibril destructuration is a dynamic and time-dependent process characterized by the early formation of cytotoxic protein aggregates that, in a few hours, convert into non-toxic insoluble material. The efficacy of doxycycline as a drug against dialysis-related amyloidosis would benefit from the ability of the drug to accumulate just in the skeletal system where amyloid is formed. In these tissues, the doxycycline concentration reaches values several folds higher than those resulting in inhibition of amyloidogenesis and amyloid destructuration in vitro.
Assuntos
Amiloide/química , Antibacterianos/química , Doxiciclina/química , Microglobulina beta-2/química , Amiloide/metabolismo , Amiloidose/tratamento farmacológico , Amiloidose/metabolismo , Antibacterianos/uso terapêutico , Linhagem Celular Tumoral , Doxiciclina/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Humanos , Ressonância Magnética Nuclear Biomolecular , Trifluoretanol/química , Microglobulina beta-2/metabolismoRESUMO
The ubiquitin-proteasome system plays a critical role in many diseases, making it an attractive biomarker and therapeutic target. However, the impact of results obtained in vitro using purified proteasome particles or whole cell extracts is limited by the lack of efficient methods to assess proteasome activity in living cells. We have engineered an internally quenched fluorogenic peptide with a proteasome-specific cleavage motif fused to TAT and linked to the fluorophores DABCYL and EDANS. This peptide penetrates cell membranes and is rapidly cleaved by the proteasomal chymotrypsin-like activity, generating a quantitative fluorescent reporter of in vivo proteasome activity as assessed by time-lapse or flow cytometry fluorescence analysis. This reporter is an innovative tool for monitoring proteasomal proteolytic activities in physiological and pathological conditions.
Assuntos
Corantes Fluorescentes/síntese química , Peptídeos/síntese química , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Linhagem Celular Tumoral , Citometria de Fluxo , Corantes Fluorescentes/química , Hipocampo/citologia , Humanos , Camundongos , Microscopia de Fluorescência , Modelos Moleculares , Neurônios/enzimologia , Peptídeos/química , Subunidades Proteicas/metabolismoRESUMO
TNF-alpha overexpression may contribute to motor neuron death in amyotrophic lateral sclerosis (ALS). We investigated the intracellular pathway associated with TNF-alpha in the wobbler mouse, a murine model of ALS, at the onset of symptoms. TNF-alpha and TNFR1 overexpression and JNK/p38MAPK phosphorylation occurred in neurons and microglia in early symptomatic mice, suggesting that this activation may contribute to motor neuron damage. The involvement of TNF-alpha was further confirmed by the protective effect of treatment with rhTNF-alpha binding protein (rhTBP-1) from 4 to 9 weeks of age. rhTBP-1 reduced the progression of symptoms, motor neuron loss, gliosis and JNK/p38MAPK phosphorylation in wobbler mice, but did not reduce TNF-alpha and TNFR1 levels. rhTBP-1 might possibly bind TNF-alpha and reduce the downstream phosphorylation of two main effectors of the neuroinflammatory response, p38MAPK and JNK.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/prevenção & controle , Neurônios Motores/patologia , Receptores Tipo I de Fatores de Necrose Tumoral/uso terapêutico , Proteínas Recombinantes/uso terapêutico , Receptores Chamariz do Fator de Necrose Tumoral/uso terapêutico , Fator de Necrose Tumoral alfa/metabolismo , Esclerose Lateral Amiotrófica/genética , Animais , Contagem de Células/métodos , Progressão da Doença , Feminino , Humanos , Masculino , Camundongos , Camundongos Mutantes Neurológicos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/fisiologia , Receptores Tipo I de Fatores de Necrose Tumoral/administração & dosagem , Proteínas Recombinantes/administração & dosagem , Receptores Chamariz do Fator de Necrose Tumoral/administração & dosagem , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
Amyloid fibrils in Gerstmann-Sträussler-Scheinker (GSS) disease are composed of a fragment of the prion protein (PrP), the N and C termini of which correspond to ragged residues 81-90 and 144-153. A synthetic peptide spanning the sequence 82-146 (PrP 82-146) polymerizes into protease-resistant fibrils with the tinctorial properties of amyloid. We investigated the biological activity of PrP 82-146 and of two nonamyloidogenic variants of PrP 82-146 with scrambled amino acid sequence 106-126 or 127-146. Cortical neurons prepared from rat and mouse embryos were chronically exposed to the PrP 82-146 peptides (10-50 microM). PrP 82-146 and the partially scrambled peptides induced neuronal death with a similar dose-response pattern, indicating that neurotoxicity was independent of amyloid fibril formation. Neurotoxicity was significantly reduced by coadministration of an anti-oligomer antibody, suggesting that PrP 82-146 oligomers are primarily responsible for triggering cell death. Neurons from PrP knock-out (Prnp0/0) mice were significantly less sensitive to PrP 82-146 toxicity than neurons expressing PrP. The gliotrophic effect of PrP 82-146 was determined by [methyl-3H]-thymidine incorporation in cultured astrocytes. Treatment with PrP 82-146 stimulated [methyl-3H]-thymidine uptake 3.5-fold. This activity was significantly less when the 106-126 or 127-146 regions were disrupted, indicating that PrP 82-146 amyloid activates the gliotrophic response. Prnp0/0 astrocytes were insensitive to the proliferative stimulus of PrP 82-146. These results underline the role of cerebral accumulation of abnormally folded PrP fragments and indicate that cellular PrP governs the pathogenic process.
Assuntos
Amiloide/química , Astrócitos/efeitos dos fármacos , Doença de Gerstmann-Straussler-Scheinker/metabolismo , Neurônios/efeitos dos fármacos , Proteínas PrPSc/toxicidade , Sequência de Aminoácidos , Amiloide/ultraestrutura , Análise de Variância , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Embrião de Mamíferos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Neuroblastoma , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/toxicidade , Fragmentos de Peptídeos/ultraestrutura , Fosfopiruvato Hidratase/metabolismo , Proteínas PrPSc/genética , Proteínas PrPSc/ultraestrutura , Ratos , Timidina/metabolismo , Fatores de Tempo , Trítio/metabolismoRESUMO
We investigated the effect of long-term, peripheral treatment with enoxaparin, a low molecular weight heparin, in transgenic mice overexpressing human amyloid precursor protein(751). Enoxaparin (6 IU per mouse intraperitoneally, three times a week for 6 months) significantly lowered the number and the area occupied by cortical beta-amyloid deposits and the total beta-amyloid (1-40) cortical concentration. Immunocytochemical analysis of glial fibrillary acid protein-positive cells showed that enoxaparin markedly reduced the number of activated astrocytes surrounding beta-amyloid deposits. In vitro, the drug dose-dependently attenuated the toxic effect of beta-amyloid on neuronal cells. Enoxaparin dose-dependently reduced the ability of beta-amyloid to activate complement and contact systems, two powerful effectors of inflammatory response in AD brain. By reducing the beta-amyloid load and cytotoxicity and proinflammatory activity, enoxaparin offers promise as a tool for slowing the progression of Alzheimer's disease.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Enoxaparina/uso terapêutico , Heparina de Baixo Peso Molecular/uso terapêutico , Placa Amiloide/efeitos dos fármacos , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/efeitos dos fármacos , Peptídeos beta-Amiloides/toxicidade , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Ativação do Complemento/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Proteína Glial Fibrilar Ácida/biossíntese , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Células PC12 , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Placa Amiloide/patologia , RatosRESUMO
To investigate the effect of the terminal complement complex (TCC) on the central nervous system, we injected both the cytolytically active and the inactive complexes into the lateral ventricle of rats. Both complexes promoted accumulation of leukocytes into the cerebrospinal fluid at 4-6 h post-injection. The cells recovered at this time were mostly polymorphonuclear leukocytes (PMN) that were partially replaced by mononuclear cells at 12 h. A direct contribution of the complexes to the in-vivo migration of leukocytes was ruled out by their inability to be chemotactic for rat PMN. Contaminating C5a is unlikely to be responsible for the effect of TCC because it failed to mobilize leukocytes when injected into the lateral ventricle. Histological analysis of rat brains 6 hours after injection of TCC revealed marked leukocyte infiltration of the choroid plexus, increased expression of intercellular adhesion molecule-1 and egression of leukocytes out of the meningeal vessels. The cerebrospinal fluid of rats treated with TCC exhibited chemotactic activity for rat PMN and increased levels of growth related oncogene/cytokine-induced neutrophil chemoattractant-1 and monocyte chemoattractant protein-1 preceding the accumulation of leukocytes. Elevated concentration of IL-1 beta was also found in the cerebrospinal fluid and in periventricular areas of rats treated with TCC.
Assuntos
Encéfalo/patologia , Complexo de Ataque à Membrana do Sistema Complemento/toxicidade , Inflamação/etiologia , Doença Aguda , Animais , Quimiocina CCL2/metabolismo , Complemento C5a/toxicidade , Complexo de Ataque à Membrana do Sistema Complemento/administração & dosagem , Citocinas/biossíntese , Humanos , Injeções Intraventriculares , Leucócitos/fisiologia , Lipopolissacarídeos/toxicidade , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Amyloid-beta protein (Abeta) is implicated in the pathogenesis of Alzheimer's disease because of its neurotoxicity and the ability to trigger local inflammation. Compounds that interact with the amino acids of the N-terminal region or interfere with aggregation can reduce the Abeta biologic activity. We evaluated the effect of heparin on Abeta (1-42) neurotoxicity and on its ability to activate complement and contact system. On differentiated PC12 cells, a reliable model of neuronal cells, heparin at the doses of 10 and 20 microg/ml significantly counteracted Abeta cytotoxicity as assessed by measuring MTT conversion. We then explored the effect of heparin on Abeta (1-42)-induced complement and contact system activation. Abeta (1-42) was incubated with heparin in presence of normal plasma as the source of complement and contact system factors. Heparin reduced, in a dose-dependent manner, complement and contact system activation, assessed by measuring the degree of C4 and high molecular weight kininogen cleavage. The present data show that heparin can attenuate neurotoxic and pro-inflammatory activity of Abeta and suggest that this drug could represent a new strategy to reduce the progressive neurodegeneration in AD.