RESUMEN
Background and Purpose- Mechanisms contributing to acute hematoma growth in intracerebral hemorrhage are not well understood. Neuropathological studies suggest that the initial hematoma may create mass effect that can tear vessels in the vicinity by shearing, causing further bleeding and hematoma growth. Methods- To test this in mice, we simulated initial intracerebral hemorrhage by intrastriatal injection of a liquid polymer that coagulates upon contact with tissue and measured the presence and volume of bleeding secondary to the mass effect using Hemoglobin ELISA 15 minutes after injection. Results- Secondary hemorrhage occurred in a volume-dependent (4, 7.5, or 15 µL of polymer) and rate-dependent (0.05, 0.5, or 5 µL/s) manner. Anticoagulation (warfarin or dabigatran) exacerbated the secondary hemorrhage volume. In a second model of hematoma expansion, we confirmed that intrastriatal whole blood injection (15 µL, 0.5 µL/s) also caused secondary bleeding, using acute Evans blue extravasation as a surrogate. Anticoagulation once again exacerbated secondary hemorrhage after intrastriatal whole blood injection. Secondary hemorrhage directly and significantly correlated with arterial blood pressures in both nonanticoagulated and anticoagulated mice, when modulated by phenylephrine or labetalol. Conclusions- Our study provides the first proof of concept for secondary vessel rupture and bleeding as a potential mechanism for intracerebral hematoma growth.
Asunto(s)
Hemorragia Cerebral/complicaciones , Hemorragia Cerebral/patología , Hemorragia/etiología , Hemorragia/patología , Enfermedad Aguda , Animales , Anticoagulantes/administración & dosificación , Hemorragia Cerebral/tratamiento farmacológico , Dabigatrán/administración & dosificación , Hemorragia/tratamiento farmacológico , Masculino , Ratones , Distribución Aleatoria , Warfarina/administración & dosificaciónRESUMEN
One of the key pathological features of Alzheimer's disease (AD) is the existence of extracellular deposition of amyloid plaques formed with misfolded amyloid-ß (Aß). The conformational change of proteins leads to enriched contents of ß sheets, resulting in remarkable changes of vibrational spectra, especially the spectral shifts of the amide I mode. Here, we applied stimulated Raman scattering (SRS) microscopy to image amyloid plaques in the brain tissue of an AD mouse model. We have demonstrated the capability of SRS microscopy as a rapid, label-free imaging modality to differentiate misfolded from normal proteins based on the blue shift (~10 cm-1) of amide I SRS spectra. Furthermore, SRS imaging of Aß plaques was verified by antibody staining of frozen thin sections and fluorescence imaging of fresh tissues. Our method may provide a new approach for studies of AD pathology, as well as other neurodegenerative diseases associated with protein misfolding.
Asunto(s)
Enfermedad de Alzheimer/patología , Modelos Animales de Enfermedad , Microscopía Óptica no Lineal/métodos , Placa Amiloide/patología , Enfermedad de Alzheimer/diagnóstico por imagen , Precursor de Proteína beta-Amiloide/genética , Animales , Humanos , Ratones , Ratones Transgénicos , Placa Amiloide/diagnóstico por imagen , Presenilinas/genéticaRESUMEN
One of the main neuropathological lesions observed at brain autopsy of Alzheimer's disease (AD) patients are the extracellular senile plaques mainly composed of amyloid-beta (Abeta) peptides. Abeta is generated by proteolytic processing of amyloid precursor protein (APP) via beta and gamma-secretases. The beta-secretase APP cleaving enzyme 1 (BACE1) has become a target of intense research aimed at blocking the enzyme activity. Recent studies showed that BACE1 is involved in processing other non-APP substrates, and that other proteases are involved in APP processing. We have recently established a novel approach to inhibit Abeta production via antibodies against the beta-secretase cleavage site of APP. These antibodies bind wild type and Swedish mutated APP expressed in transgenic mice brain tissues. The isolated antibodies do not bind any form of Abeta peptides. Antibody up-take experiments, using Chinese hamster ovary cells expressing wild-type APP, suggest that antibody internalization and trafficking are mediated via the endocytic pathway. Administration of antibodies to the cells growing media resulted in a considerable decrease in intracellular Abeta levels, as well as in the levels of the corresponding C-terminal fragment (C99). The relevance of intra-neuronal accumulation of mainly Abeta42 as an early event in AD pathogenesis suggests that this approach may be applicable as a novel therapeutic strategy in AD treatment.
Asunto(s)
Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/terapia , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Precursor de Proteína beta-Amiloide/efectos de los fármacos , Anticuerpos/farmacología , Inmunoterapia/métodos , Secretasas de la Proteína Precursora del Amiloide/inmunología , Precursor de Proteína beta-Amiloide/metabolismo , Animales , HumanosRESUMEN
We present here the development of antibodies against prion protein in BALB/C mice using as antigen human helix 1 of PrP. This sequence is suggested to be involved in protein pathological conformational changes, and is distinguished from that of mice by one amino acid. The immune tolerance to an 'almost-self' epitope and the poor immunogenicity of short peptides was overcome by using Multiple Antigen Peptide displaying eight copies of helix 1. The generated antibodies recognize the whole prion protein with a high binding constant and the established protocol may lead to an active immunization towards therapeutics of prion disease.
Asunto(s)
Anticuerpos Heterófilos/biosíntesis , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/inmunología , Proteínas PrPC/inmunología , Animales , Anticuerpos Heterófilos/metabolismo , Anticuerpos Monoclonales/metabolismo , Antígenos Heterófilos/inmunología , Antígenos Heterófilos/metabolismo , Sitios de Unión de Anticuerpos , Bovinos , Línea Celular Tumoral , Epítopos/inmunología , Epítopos/metabolismo , Humanos , Sueros Inmunes/metabolismo , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/metabolismo , Proteínas PrPC/metabolismo , Estructura Secundaria de Proteína , Conejos , Ovinos , Vacunación/métodosRESUMEN
Although extracellular Abeta plaques are a hallmark of Alzheimer's disease, it remains to be determined whether extracellular or intracellular Abeta accumulation initiates the disease process. A recent paper from Gunnar Gouras' group showed that Abeta antibodies lead to reduced intracellular Abeta levels in neurons via antibody internalization after binding to the Abeta domain of amyloid precursor protein (APP) at the plasma membrane. This work suggests novel avenues for the immunotherapy of Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/terapia , Péptidos beta-Amiloides/inmunología , Inmunoterapia , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Anticuerpos Monoclonales/uso terapéutico , HumanosRESUMEN
BACKGROUND/OBJECTIVES: Active and passive immunization methodologies against amyloid-beta (Abeta) are employed to clear and reduce cerebral Abetatowards treatment of Alzheimer's disease (AD) patients. The therapeutic potential of these antibodies in AD patients is limited because of adverse inflammatory reactions and cerebral hemorrhage, which are associated with the treatment. We propose a novel approach to inhibit Abeta production via antibodies against the beta-secretase cleavage site of the amyloid precursor protein (APP). Such an approach limits APP processing by beta-secretase, mainly through the endocytic pathway, and overcomes some of the limitations of BACE inhibition. Anti-APP beta-site antibodies, tested in a cellular model expressing wild-type APP, were found to bind full-length APP, internalize into the cells and interfere with BACE activity, inhibiting both intra- and extracellular Abeta peptide formation. METHODS: We investigated the effect of anti-beta-site antibodies in an AD animal model regarding antibody efficacy, as well as possible adverse effects in the brain and periphery that may result from antibody treatment. RESULTS/CONCLUSIONS: Here, we show that long-term systemic administration of anti-APP beta-site antibodies to Tg2576 transgenic mice improved mouse cognitive functions associated with a reduction in both brain inflammation and the incidence of microhemorrhage. Furthermore, antibody treatment did not induce any peripheral autoimmunity responses. In spite of the beneficial effects observed in antibody-treated mice, brain Abeta levels were not altered as a result of antibody treatment.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/antagonistas & inhibidores , Precursor de Proteína beta-Amiloide/antagonistas & inhibidores , Anticuerpos/farmacología , Encefalitis/tratamiento farmacológico , Inmunoterapia/métodos , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/biosíntesis , Precursor de Proteína beta-Amiloide/inmunología , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Anticuerpos/inmunología , Anticuerpos/uso terapéutico , Autoinmunidad/efectos de los fármacos , Autoinmunidad/inmunología , Cognición/efectos de los fármacos , Cognición/fisiología , Trastornos del Conocimiento/tratamiento farmacológico , Trastornos del Conocimiento/inmunología , Trastornos del Conocimiento/fisiopatología , Modelos Animales de Enfermedad , Esquema de Medicación , Encefalitis/inmunología , Encefalitis/fisiopatología , Femenino , Humanos , Hemorragias Intracraneales/tratamiento farmacológico , Hemorragias Intracraneales/inmunología , Hemorragias Intracraneales/fisiopatología , Memoria/efectos de los fármacos , Memoria/fisiología , Ratones , Ratones Transgénicos , Estructura Terciaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/genética , Tiempo , Resultado del TratamientoRESUMEN
Amyloid-beta peptide (AbetaP) that accumulates in the Alzheimer's diseased brain is derived from proteolytic processing of the amyloid precursor protein (APP) by means of beta- and gamma-secretases. The beta-secretase APP cleaving enzyme (BACE), which generates the N terminus of AbetaP, has become a target of intense research aimed at blocking the enzyme activity, thus reducing AbetaP and, subsequently, plaque formation. The search for specific inhibitors of beta-secretase activity as a possible treatment for Alzheimer's disease intensified with the discovery that BACE may be involved in processing other non-APP substrates. The presence of the APP-BACE complex in early endosomes highlights the cell surface as a potential therapeutic target, suggesting that interference in APP-BACE interaction at the cell surface may affect amyloid-beta production. We present here a unique approach to inhibit AbetaP production by means of antibodies against the beta-secretase cleavage site of APP. These antibodies were found to bind human APP overexpressed by CHO cells, and the formed immunocomplex was visualized in the early endosomes. Indeed, blocking of the beta-secretase site by these antibodies interfered with BACE activity and inhibited both intracellular and extracellular AbetaP formation in these cells.