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1.
Blood ; 121(1): 85-94, 2013 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-23144170

RESUMO

Immunoglobulin class switching from IgM to IgG in response to peptides is generally T cell-dependent and vaccination in T cell-deficient individuals is inefficient. We show that a vaccine consisting of a dense array of peptides on liposomes induced peptide-specific IgG responses totally independent of T-cell help. Independency was confirmed in mice lacking T cells and in mice deficient for MHC class II, CD40L, and CD28. The IgG titers were high, long-lived, and comparable with titers obtained in wild-type animals, and the antibody response was associated with germinal center formation, expression of activation-induced cytidine deaminase, and affinity maturation. The T cell-independent (TI) IgG response was strictly dependent on ligation of TLR4 receptors on B cells, and concomitant TLR4 and cognate B-cell receptor stimulation was required on a single-cell level. Surprisingly, the IgG class switch was mediated by TIR-domain-containing adapter inducing interferon-ß (TRIF), but not by MyD88. This study demonstrates that peptides can induce TI isotype switching when antigen and TLR ligand are assembled and appropriately presented directly to B lymphocytes. A TI vaccine could enable efficient prophylactic and therapeutic vaccination of patients with T-cell deficiencies and find application in diseases where induction of T-cell responses contraindicates vaccination, for example, in Alzheimer disease.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Peptídeos beta-Amiloides/imunologia , Linfócitos B/imunologia , Switching de Imunoglobulina/imunologia , Fragmentos de Peptídeos/imunologia , Receptor 4 Toll-Like/fisiologia , Vacinas de Subunidades Antigênicas/imunologia , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transporte Vesicular/genética , Transferência Adotiva , Sequência de Aminoácidos , Peptídeos beta-Amiloides/administração & dosagem , Animais , Apresentação de Antígeno , Linfócitos B/metabolismo , Antígenos CD28/deficiência , Antígenos CD28/imunologia , Ligante de CD40/deficiência , Ligante de CD40/imunologia , Centro Germinativo/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina M/biossíntese , Receptores de Lipopolissacarídeos/imunologia , Lipossomos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Dados de Sequência Molecular , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Fragmentos de Peptídeos/administração & dosagem , Receptores de Antígenos de Linfócitos B/imunologia , Linfócitos T/imunologia , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genética , Vacinação , Vacinas de Subunidades Antigênicas/administração & dosagem
2.
NPJ Vaccines ; 9(1): 115, 2024 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-38909055

RESUMO

Vaccines generally require T lymphocytes for B-cell activation and immunoglobulin class switching in response to peptide or protein antigens. In the absence of T cells, limited IgG class switch takes place, germinal centers are short-lived, and the B cells lack memory. Here, immunization of mice with liposomes containing 15mer peptides and monophosphoryl lipid A (MPLA) as adjuvant, induced T-cell independent (TI) IgG class switch within three days, as well as germinal center formation. The antibody responses were long-lived, strictly dependent on Toll-like receptor 4 (TLR4) signaling, partly dependent on Bruton's tyrosine kinase (BTK) signal transmission, and independent of signaling through T-cell receptors, MHC class II and inflammasome. The antibody response showed characteristics of both TI type 1 and TI type 2. All IgG subclasses could be boosted months after primary immunization, and the biological function of the secreted antibodies was demonstrated in murine models of allergic anaphylaxis and of bacterial infection. Moreover, antibody responses after immunization with peptide- and MPLA-loaded liposomes could be triggered in neonatal mice and in mice receiving immune-suppressants. This study demonstrates T-cell independent endogenous B-cell memory and recall responses in vivo using a peptide antigen. The stimulation of these antibody responses required a correct and dense assembly and administration of peptide and adjuvant on the surface of liposomes. In the future, TI vaccines may prove beneficial in pathological conditions in which T-cell immunity is compromised through disease or medicines or when rapid, antibody-mediated immune protection is needed.

3.
J Biol Chem ; 287(41): 34786-800, 2012 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-22891248

RESUMO

Increasing evidence implicates Aß peptides self-assembly and fibril formation as crucial events in the pathogenesis of Alzheimer disease. Thus, inhibiting Aß aggregation, among others, has emerged as a potential therapeutic intervention for this disorder. Herein, we employed 3-aminopyrazole as a key fragment in our design of non-dye compounds capable of interacting with Aß42 via a donor-acceptor-donor hydrogen bond pattern complementary to that of the ß-sheet conformation of Aß42. The initial design of the compounds was based on connecting two 3-aminopyrazole moieties via a linker to identify suitable scaffold molecules. Additional aryl substitutions on the two 3-aminopyrazole moieties were also explored to enhance π-π stacking/hydrophobic interactions with amino acids of Aß42. The efficacy of these compounds on inhibiting Aß fibril formation and toxicity in vitro was assessed using a combination of biophysical techniques and viability assays. Using structure activity relationship data from the in vitro assays, we identified compounds capable of preventing pathological self-assembly of Aß42 leading to decreased cell toxicity.


Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Pirazóis/química , Linhagem Celular Tumoral , Citotoxinas/antagonistas & inibidores , Citotoxinas/química , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Estrutura Secundária de Proteína , Relação Estrutura-Atividade
4.
J Biol Chem ; 286(16): 13966-76, 2011 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-21343310

RESUMO

Synthetic peptide immunogens that mimic the conformation of a target epitope of pathological relevance offer the possibility to precisely control the immune response specificity. Here, we performed conformational analyses using a panel of peptides in order to investigate the key parameters controlling their conformation upon integration into liposomal bilayers. These revealed that the peptide lipidation pattern, the lipid anchor chain length, and the liposome surface charge all significantly alter peptide conformation. Peptide aggregation could also be modulated post-liposome assembly by the addition of distinct small molecule ß-sheet breakers. Immunization of both mice and monkeys with a model liposomal vaccine containing ß-sheet aggregated lipopeptide (Palm1-15) induced polyclonal IgG antibodies that specifically recognized ß-sheet multimers over monomer or non-pathological native protein. The rational design of liposome-bound peptide immunogens with defined conformation opens up the possibility to generate vaccines against a range of protein misfolding diseases, such as Alzheimer disease.


Assuntos
Lipossomos/química , Peptídeos/química , Deficiências na Proteostase/metabolismo , Vacinas/química , Doença de Alzheimer/metabolismo , Animais , Benzotiazóis , Dicroísmo Circular , Feminino , Humanos , Imunoglobulina G/química , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Tiazóis/química
6.
Cancer Lett ; 257(2): 165-71, 2007 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-17517470

RESUMO

The clinical use of chemotherapy in cancer treatment is limited by the occurrence of multidrug resistance (MDR) associated with the overexpression of membrane transporters, one of the best known is P-glycoprotein (Pgp), that actively expels drugs out of tumor cells. To overcome Pgp-mediated MDR, synthetic peptides corresponding to fragments from extracellular loops 1, 2 and 4 of the murine Pgp were coupled to polyethylene glycol-distearoylphosphatidylethanolamine and inserted into empty or monophosphoryl lipid A-containing liposomes. This formulation elicited specific antibodies which blocked Pgp-mediated efflux of doxorubicin, resulting in increased intracellular drug accumulation and subsequent potentiation of the cytotoxic effect of doxorubicin on multidrug-resistant P388 (P388R) cells. Previous immunizations with MDR1 peptides improved the efficiency of chemotherapy against P388R cells in vivo, with an increase of 83% of mice survival time. Overall, these results suggest that this approach can modulate Pgp activity by blocking drug efflux and may have clinical relevance as an alternative strategy to toxic chemosensitizers in drug-resistant cancer therapy.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/imunologia , Doxorrubicina/farmacologia , Imunização/métodos , Peptídeos/imunologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Animais , Antibióticos Antineoplásicos/metabolismo , Antibióticos Antineoplásicos/farmacocinética , Antibióticos Antineoplásicos/farmacologia , Anticorpos/administração & dosagem , Anticorpos/imunologia , Anticorpos/farmacologia , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/metabolismo , Doxorrubicina/farmacocinética , Farmacorresistência Bacteriana , Feminino , Citometria de Fluxo , Lipossomos/química , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Neoplasias Experimentais/prevenção & controle , Peptídeos/química , Polietilenoglicóis/química , Análise de Sobrevida , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/química , Vacinas de Subunidades Antigênicas/imunologia
7.
PLoS One ; 11(3): e0152471, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27023444

RESUMO

In Down syndrome (DS) or trisomy of chromosome 21, the ß-amyloid (Aß) peptide product of the amyloid precursor protein (APP) is present in excess. Evidence points to increased APP gene dose and Aß as playing a critical role in cognitive difficulties experienced by people with DS. Particularly, Aß is linked to the late-life emergence of dementia as associated with neuropathological markers of Alzheimer's disease (AD). At present, no treatment targets Aß-related pathogenesis in people with DS. Herein we used a vaccine containing the Aß 1-15 peptide embedded into liposomes together with the adjuvant monophosphoryl lipid A (MPLA). Ts65Dn mice, a model of DS, were immunized with the anti-Aß vaccine at 5 months of age and were examined for cognitive measures at 8 months of age. The status of basal forebrain cholinergic neurons and brain levels of APP and its proteolytic products were measured. Immunization of Ts65Dn mice resulted in robust anti-Aß IgG titers, demonstrating the ability of the vaccine to break self-tolerance. The vaccine-induced antibodies reacted with Aß without detectable binding to either APP or its C-terminal fragments. Vaccination of Ts65Dn mice resulted in a modest, but non-significant reduction in brain Aß levels relative to vehicle-treated Ts65Dn mice, resulting in similar levels of Aß as diploid (2N) mice. Importantly, vaccinated Ts65Dn mice showed resolution of memory deficits in the novel object recognition and contextual fear conditioning tests, as well as reduction of cholinergic neuron atrophy. No treatment adverse effects were observed; vaccine did not result in inflammation, cellular infiltration, or hemorrhage. These data are the first to show that an anti-Aß immunotherapeutic approach may act to target Aß-related pathology in a mouse model of DS.


Assuntos
Peptídeos beta-Amiloides/imunologia , Transtornos Cognitivos/complicações , Transtornos Cognitivos/tratamento farmacológico , Síndrome de Down/complicações , Síndrome de Down/tratamento farmacológico , Vacinas/uso terapêutico , Peptídeos beta-Amiloides/genética , Animais , Animais Recém-Nascidos , Anticorpos/metabolismo , Atrofia , Comportamento Animal , Biomarcadores/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Neurônios Colinérgicos/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Hemorragia/patologia , Inflamação/patologia , Masculino , Memória , Camundongos Transgênicos , Núcleos Septais/patologia , Vacinação
8.
PLoS One ; 9(9): e105641, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25207975

RESUMO

Aggregation of amyloid beta (Aß) into oligomers and fibrils is believed to play an important role in the development of Alzheimer's disease (AD). To gain further insight into the principles of aggregation, we have investigated the induction of ß-sheet secondary conformation from disordered native peptide sequences through lipidation, in 1-2% hexafluoroisopropanol (HFIP) in phosphate buffered saline (PBS). Several parameters, such as type and number of lipid chains, peptide sequence, peptide length and net charge, were explored keeping the ratio peptide/HFIP constant. The resulting lipoconjugates were characterized by several physico-chemical techniques: Circular Dichroism (CD), Attenuated Total Reflection InfraRed (ATR-IR), Thioflavin T (ThT) fluorescence, Dynamic Light Scattering (DLS), solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy and Electron Microscopy (EM). Our data demonstrate the generation of ß-sheet aggregates from numerous unstructured peptides under physiological pH, independent of the amino acid sequence. The amphiphilicity pattern and hydrophobicity of the scaffold were found to be key factors for their assembly into amyloid-like structures.


Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Desenho de Fármacos , Metabolismo dos Lipídeos , Multimerização Proteica , Sequência de Aminoácidos , Sítios de Ligação , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Especificidade por Substrato , Água/química
9.
PLoS One ; 8(8): e72301, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23977276

RESUMO

Progressive aggregation of protein Tau into oligomers and fibrils correlates with cognitive decline and synaptic dysfunction, leading to neurodegeneration in vulnerable brain regions in Alzheimer's disease. The unmet need of effective therapy for Alzheimer's disease, combined with problematic pharmacological approaches, led the field to explore immunotherapy, first against amyloid peptides and recently against protein Tau. Here we adapted the liposome-based amyloid vaccine that proved safe and efficacious, and incorporated a synthetic phosphorylated peptide to mimic the important phospho-epitope of protein Tau at residues pS396/pS404. We demonstrate that the liposome-based vaccine elicited, rapidly and robustly, specific antisera in wild-type mice and in Tau.P301L mice. Long-term vaccination proved to be safe, because it improved the clinical condition and reduced indices of tauopathy in the brain of the Tau.P301L mice, while no signs of neuro-inflammation or other adverse neurological effects were observed. The data corroborate the hypothesis that liposomes carrying phosphorylated peptides of protein Tau have considerable potential as safe and effective treatment against tauopathies, including Alzheimer's disease.


Assuntos
Vacinas contra Alzheimer/imunologia , Anticorpos Neutralizantes/sangue , Peptídeos/imunologia , Fosfoproteínas/imunologia , Tauopatias/tratamento farmacológico , Proteínas tau/imunologia , Vacinas contra Alzheimer/administração & dosagem , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/imunologia , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Humanos , Lipossomos/química , Camundongos , Camundongos Transgênicos , Peptídeos/administração & dosagem , Peptídeos/síntese química , Fosfoproteínas/administração & dosagem , Fosfoproteínas/síntese química , Fosforilação , Desempenho Psicomotor/efeitos dos fármacos , Tauopatias/imunologia , Tauopatias/fisiopatologia , Resultado do Tratamento , Vacinação , Proteínas tau/antagonistas & inibidores , Proteínas tau/genética
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