RESUMO
Thirteen human interferon-α (IFNα) subtypes were expressed in Escherichiacoli and purified using an N-terminal affinity tag from the prodomain of subtilisin. IFNα subtypes were expressed in soluble form and purified from cell lysates or refolded and purified from inclusion bodies. Proteins produced by either protocol exhibited biological activities equal to or greater than commercially prepared IFNα preparations. The IFNαs were used to produce an anti-IFNα16 antibody (MAb-1B12) that specifically neutralized the biological activity of IFNα16, but not the 12 other IFNαs. Using MAb-1B12, and a previously generated IFNAR1/IFNAR2-FChk heterodimer, an assay was developed to determine total type I IFN biological activity and IFNα16-derived biological activity in an unknown sample.
Assuntos
Interferon Tipo I/biossíntese , Interferon Tipo I/isolamento & purificação , Interferon-alfa/biossíntese , Interferon-alfa/isolamento & purificação , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/biossíntese , Anticorpos Neutralizantes/imunologia , Escherichia coli , Humanos , Interferon Tipo I/imunologia , Interferon-alfa/imunologiaRESUMO
The nucleotide-binding domain leucine-rich repeat-containing proteins, NLRs, are intracellular sensors of pathogen-associated molecular patterns and damage-associated molecular patterns. A subgroup of NLRs can form inflammasome complexes, which facilitate the maturation of procaspase 1 to caspase 1, leading to IL-1ß and IL-18 cleavage and secretion. NLRC5 is predominantly expressed in hematopoietic cells and has not been studied for inflammasome function. RNA interference-mediated knockdown of NLRC5 nearly eliminated caspase 1, IL-1ß, and IL-18 processing in response to bacterial infection, pathogen-associated molecular patterns, and damage-associated molecular patterns. This was confirmed in primary human monocytic cells. NLRC5, together with procaspase 1, pro-IL-1ß, and the inflammasome adaptor ASC, reconstituted inflammasome activity that showed cooperativity with NLRP3. The range of pathogens that activate NLRC5 inflammasome overlaps with those that activate NLRP3. Furthermore, NLRC5 biochemically associates with NLRP3 in a nucleotide-binding domain-dependent but leucine-rich repeat-inhibitory fashion. These results invoke a model in which NLRC5 interacts with NLRP3 to cooperatively activate the inflammasome.
Assuntos
Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Animais , Proteínas de Transporte/fisiologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Escherichia coli/imunologia , Escherichia coli/patogenicidade , Regulação da Expressão Gênica/imunologia , Humanos , Imunidade Inata , Inflamassomos/biossíntese , Inflamassomos/genética , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucina/metabolismo , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Estrutura Terciária de Proteína/genética , Shigella flexneri/imunologia , Shigella flexneri/patogenicidade , Staphylococcus aureus/imunologia , Staphylococcus aureus/patogenicidadeRESUMO
Heightened DJ-1 (Park7) expression is associated with a reduction in chemotherapeutic-induced cell death and poor prognosis in several cancers, whereas the loss of DJ-1 function is found in a subgroup of Parkinson disease associated with neuronal death. This study describes a novel pathway by which DJ-1 modulates cell survival. Mass spectrometry shows that DJ-1 interacts with BBS1, CLCF1, MTREF, and Cezanne/OTUD7B/Za20d1. Among these, Cezanne is a known deubiquitination enzyme that inhibits NF-κB activity. DJ-1/Cezanne interaction is confirmed by co-immunoprecipitation of overexpressed and endogenous proteins, maps to the amino-terminal 70 residues of DJ-1, and leads to the inhibition of the deubiquitinating activity of Cezanne. Microarray profiling of shRNA-transduced cells shows that DJ-1 and Cezanne regulate IL-8 and ICAM-1 expression in opposing directions. Similarly, DJ-1 enhances NF-κB nuclear translocation and cell survival, whereas Cezanne reduces these outcomes. Analysis of mouse Park7(-/-) primary cells confirms the regulation of ICAM-1 by DJ-1 and Cezanne. As NF-κB is important in cellular survival and transformation, IL-8 functions as an angiogenic factor and pro-survival signal, and ICAM-1 has been implicated in tumor progression, invasion, and metastasis; these data provide an additional modality by which DJ-1 controls cell survival and possibly tumor progression via interaction with Cezanne.
Assuntos
Núcleo Celular/metabolismo , Endopeptidases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , NF-kappa B/metabolismo , Proteínas Oncogênicas/metabolismo , Ubiquitinação , Transporte Ativo do Núcleo Celular/genética , Animais , Núcleo Celular/genética , Sobrevivência Celular/genética , Endopeptidases/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Molécula 1 de Adesão Intercelular/genética , Interleucina-8/biossíntese , Interleucina-8/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Proteínas Associadas aos Microtúbulos , NF-kappa B/genética , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Oncogênicas/genética , Peroxirredoxinas , Proteína Desglicase DJ-1 , Proteínas/genética , Proteínas/metabolismoRESUMO
Dengue viruses (DENVs) are emerging, mosquito-borne flaviviruses which cause dengue fever and dengue hemorrhagic fever. The DENV complex consists of 4 serotypes designated DENV1-DENV4. Following natural infection with DENV, individuals develop serotype specific, neutralizing antibody responses. Monoclonal antibodies (MAbs) have been used to map neutralizing epitopes on dengue and other flaviviruses. Most serotype-specific, neutralizing MAbs bind to the lateral ridge of domain III of E protein (EDIII). It has been widely assumed that the EDIII lateral ridge epitope is conserved within each DENV serotype and a good target for vaccines. Using phylogenetic methods, we compared the amino acid sequence of 175 E proteins representing the different genotypes of DENV3 and identified a panel of surface exposed amino acids, including residues in EDIII, that are highly variant across the four DENV3 genotypes. The variable amino acids include six residues at the lateral ridge of EDIII. We used a panel of DENV3 mouse MAbs to assess the functional significance of naturally occurring amino acid variation. From the panel of antibodies, we identified three neutralizing MAbs that bound to EDIII of DENV3. Recombinant proteins and naturally occurring variant viruses were used to map the binding sites of the three MAbs. The three MAbs bound to overlapping but distinct epitopes on EDIII. Our empirical studies clearly demonstrate that the antibody binding and neutralization capacity of two MAbs was strongly influenced by naturally occurring mutations in DENV3. Our data demonstrate that the lateral ridge "type specific" epitope is not conserved between strains of DENV3. This variability should be considered when designing and evaluating DENV vaccines, especially those targeting EDIII.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Vírus da Dengue/genética , Vírus da Dengue/imunologia , Dengue/imunologia , Dengue/virologia , Aedes/citologia , Animais , Anticorpos Monoclonais/metabolismo , Chlorocebus aethiops , Vírus da Dengue/classificação , Mapeamento de Epitopos , Epitopos/imunologia , Genótipo , Humanos , Camundongos , Mutagênese Sítio-Dirigida , Testes de Neutralização , Ligação Proteica/imunologia , Células U937 , Células Vero , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
PURPOSE: This study aims to determine the effect of loss of breast cancer metastasis suppressor 1 (BRMS1) protein expression on disease-free survival in breast cancer patients stratified by estrogen receptor (ER), progesterone receptor (PR), or HER2 status, and to determine whether loss of BRMS1 protein expression correlated with genomic copy number changes. EXPERIMENTAL DESIGN: A tissue microarray immunohistochemical analysis was done on tumors of 238 newly diagnosed breast cancer patients who underwent surgery at the Cleveland Clinic between January 1, 1995 and December 31, 1996, and a comparison was made with 5-year clinical follow-up data. Genomic copy number changes were determined by array-based comparative genomic hybridization in 47 breast cancer cases from this population and compared with BRMS1 staining. RESULTS: BRMS1 protein expression was lost in nearly 25% of cases. Patients with tumors that were PR negative (P=0.006) or HER2 positive (P=0.039) and <50 years old at diagnosis (P=0.02) were more likely to be BRMS1 negative. No overall correlation between BRMS1 staining and disease-free survival was observed. A significant correlation, however, was seen between loss of BRMS1 protein expression and reduced disease-free survival when stratified by either loss of ER (P=0.008) or PR (P=0.029) or HER2 overexpression (P=0.026). Overall, there was poor correlation between BRMS1 protein staining and copy number status. CONCLUSIONS: These data suggest a mechanistic relationship between BRMS1 expression, hormone receptor status, and HER2 growth factor. BRMS1 staining could potentially be used in patient stratification in conjunction with other prognostic markers. Further, mechanisms other than genomic deletion account for loss of BRMS1 gene expression in breast tumors.
Assuntos
Neoplasias da Mama/diagnóstico , Neoplasias da Mama/metabolismo , Carcinoma/diagnóstico , Carcinoma/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Adulto , Idade de Início , Idoso , Neoplasias da Mama/epidemiologia , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Carcinoma/epidemiologia , Carcinoma/patologia , Carcinoma/terapia , Estudos de Casos e Controles , Cromossomos Humanos Par 11 , Intervalo Livre de Doença , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas dos Microfilamentos/genética , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Prognóstico , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismoRESUMO
Although antibodies are commercially available to allow investigation into the biology of the age-regulating protein Klotho, problems with antibody specificity and application functionality are significant barriers to progress. Chief among these limitations is the inability of current tools to allow in vivo validation of binding partners originally identified through transfection of tagged proteins. To overcome this barrier, we generated a series of hybridoma cell lines by immunizing rats with a GST-KL1 fusion protein. Purified antibodies generated from these cell lines differentially detect human or mouse Klotho protein via Western blot, immunocyto/histochemistry, and immunoprecipitation. Specificity of antibody binding to Klotho was confirmed by mass spectrometry following immunoprecipitation. With this confidence in antibody specificity, co-immunoprecipitation was utilized to validate the interaction of Klotho/FGFR and Klotho/wnt7a in mouse kidney lysates.
Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Glucuronidase/imunologia , Hibridomas/imunologia , Animais , Anticorpos Monoclonais/genética , Western Blotting , Primers do DNA/genética , Células HEK293 , Humanos , Imuno-Histoquímica , Imunoprecipitação , Proteínas Klotho , Espectrometria de Massas , Camundongos , Plasmídeos/genética , RatosRESUMO
Streptococcus pneumoniae causes otitis media, meningitis and pneumonia in patients worldwide; predominantly affecting young children, the elderly, and the immune compromised. Current vaccines against invasive pneumococcal disease are based on the polysaccharide capsules of the most clinically relevant serotypes. Due to serotype replacement, non-vaccine serotypes of S. pneumoniae have become more clinically relevant and as a result pneumococcal vaccines are becoming increasingly complex. These events emphasize the need to evaluate the potential for pneumococcal cross-reactive proteins to contribute to future vaccines. Antibody elicited by the immunization of humans with pneumococcal surface protein A (PspA) can passively protect mice from infection. However, robust in vitro functional assays for antibody to PspA are not available to predict the protective capacity of immune serum. For polysaccharide based vaccines, a standardized opsonophagocytosis killing assay (OPKA) is used. Antibody to PspA, however, does not work well in the standard OPKA. The present studies take advantage of past observations that phagocytosis is more efficient on tissue surfaces than in solution. In a modified surface killing assay (MSKA), monoclonal antibody to PspA, in the presence of complement, opsonized pneumococci for killing by phagocytes on an agar surface. Five monoclonal antibodies to PspA were tested; three demonstrated increased amounts of killing compared to the diluent control and protected mice by passive protection against type 3 pneumococci. The two antibodies that were not functional in the MSKA also failed to protect mice. Thus, an MSKA might be useful as a functional assay for immunity to PspA.
Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Neutralizantes/imunologia , Proteínas de Bactérias/imunologia , Infecções Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/sangue , Humanos , Imunização Passiva , Camundongos , Fagocitose/imunologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/imunologiaRESUMO
Dengue viruses (DENV) are the etiological agents of dengue fever (DF) and dengue hemorrhagic fever (DHF). The DENV complex consists of four closely related viruses designated DENV serotypes 1 through 4. Although infection with one serotype induces cross reactive antibody to all 4 serotypes, the long-term protective antibody response is restricted to the serotype responsible for infection. Cross reactive antibodies appear to enhance infection during a second infection with a different serotype. The goal of the present study was to characterize the binding specificity and functional properties of human DENV immune sera. The study focused on domain III of the viral envelope protein (EDIII), as this region has a well characterized epitope that is recognized by strongly neutralizing serotype-specific mouse monoclonal antibodies (Mabs). Our results demonstrate that EDIII-reactive antibodies are present in primary and secondary DENV immune human sera. Human antibodies bound to a serotype specific epitope on EDIII after primary infection and a serotype cross reactive epitope on EDIII after secondary infection. However, EDIII binding antibodies constituted only a small fraction of the total antibody in immune sera binding to DENV. Studies with complete and EDIII antibody depleted human immune sera demonstrated that EDIII binding antibodies play a minor role in DENV neutralization. We propose that human antibodies directed to other epitopes on the virus are primarily responsible for DENV neutralization. Our results have implications for understanding protective immunity following natural DENV infection and for evaluating DENV vaccines.
Assuntos
Anticorpos Antivirais/sangue , Vírus da Dengue/imunologia , Animais , Antígenos Virais/química , Antígenos Virais/genética , Vírus da Dengue/classificação , Vírus da Dengue/genética , Humanos , Técnicas In Vitro , Camundongos , Testes de Neutralização , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologiaRESUMO
The BRMS1 metastasis suppressor interacts with the protein AT-rich interactive domain 4A (ARID4A, RBBP1) as part of SIN3.histone deacetylase chromatin remodeling complexes. These transcriptional co-repressors regulate diverse cell phenotypes depending upon complex composition. To define BRMS1 complexes and their roles in metastasis suppression, we generated BRMS1 mutants (BRMS1(mut)) and mapped ARID4A interactions. BRMS1(L174D) disrupted direct interaction with ARID4A in yeast two-hybrid genetic screens but retained an indirect association with ARID4A in MDA-MB-231 and -435 human breast cancer cell lines by co-immunoprecipitation. Deletion of the first coiled-coil domain (BRMS1(DeltaCC1)) did not disrupt direct interaction in yeast two-hybrid screens but did prevent association by co-immunoprecipitation. These results suggest altered complex composition with BRMS1(mut). Although basal transcription repression was impaired and the pro-metastatic protein osteopontin was differentially down-regulated by BRMS1(L174D) and BRMS1(DeltaCC1), both down-regulated the epidermal growth factor receptor and suppressed metastasis in MDA-MB-231 and -435 breast cancer xenograft models. We conclude that BRMS1(mut), which modifies the composition of a SIN3.histone deacetylase chromatin remodeling complex, leads to altered gene expression profiles. Because metastasis requires the coordinate expression of multiple genes, down-regulation of at least one important gene, such as the epidermal growth factor receptor, had the ability to suppress metastasis. Understanding which interactions are necessary for particular biochemical/cellular functions may prove important for future strategies targeting metastasis.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Transporte/metabolismo , Montagem e Desmontagem da Cromatina , Regulação Neoplásica da Expressão Gênica , Histona Desacetilases/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Repressoras/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina/genética , Regulação para Baixo/genética , Receptores ErbB/biossíntese , Receptores ErbB/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Histona Desacetilases/genética , Humanos , Camundongos , Mutação , Metástase Neoplásica , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Osteopontina/biossíntese , Osteopontina/genética , Proteínas Repressoras/genética , Proteína 1 de Ligação ao Retinoblastoma , Complexo Correpressor Histona Desacetilase e Sin3 , Transcrição Gênica/genética , Transplante HeterólogoRESUMO
Defective receptor editing or defective B cell checkpoints have been associated with increased frequency of multireactive autoantibodies in autoimmune disease. However, Ig somatic hypermutation and/or class switch recombination may be mechanisms enabling the development of pathogenic multireactive autoantibodies. In this study, we report that, in the BXD2 mouse model of autoimmune disease, elevated expression of activation-induced cytidine deaminase (AID) in recirculating follicular CD86(+) subsets of B cells and increased germinal center B cell activity are associated with the production of pathogenic multireactive autoantibodies. CD4 T cells from BXD2 mice that expressed increased levels of CD28 and an increased proliferative response to anti-CD3 and anti-CD28 stimulation are required for this process. Inhibition of the CD28-CD86 interaction in BXD2 mice with AdCTLA4-Ig resulted in normalization of AID in the B cells and suppression of IgG autoantibodies. This treatment also prevented the development of germinal center autoantibody-producing B cells, suggesting that an optimal microenvironment enabling AID function is important for the formation of pathogenic autoantibodies. Taken together, our data indicate that AID expression in B cells is a promising therapeutic target for the treatment of autoimmune diseases and that suppression of this gene may be a molecular target of CTLA4-Ig therapy.
Assuntos
Autoanticorpos/biossíntese , Doenças Autoimunes/etiologia , Linfócitos B/enzimologia , Linfócitos B/imunologia , Citidina Desaminase/fisiologia , Abatacepte , Adenoviridae/genética , Animais , Antígeno B7-2/análise , Linfócitos T CD4-Positivos/fisiologia , Citidina Desaminase/antagonistas & inibidores , Feminino , Imunoconjugados/genética , Imunoconjugados/uso terapêutico , Switching de Imunoglobulina/genética , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Complemento 3d/análise , Receptores de IgE/análise , Hipermutação Somática de ImunoglobulinaRESUMO
BACKGROUND: The KISS1 protein suppresses metastasis of several tumor models without blocking orthotopic tumor growth, but the mechanism remains elusive. For its role in human sexual maturation, KISS1 protein is secreted and processed to kisspeptins, which bind to the G protein-coupled receptor GPR54. We tested the hypothesis that KISS1 secretion is required for metastasis suppression via GPR54. METHODS: KISS1 containing an internal FLAG epitope with (KFM) or without (KFMdeltaSS) a signal sequence was transfected into C8161.9 human melanoma cells, which do not express endogenous KISS1. Whole-cell lysates and conditioned medium from C8161.9(KFM) and C8161.9(KFMdeltaSS) cells were collected and analyzed for kisspeptins by immunoprecipitation and enzyme-linked immunosorbent assay. GPR54 levels were measured using real-time reverse transcription-polymerase chain reaction. The ability of conditioned medium from C8161.9(KFM) and C8161.9(KFMdeltaSS) cells to stimulate calcium mobilization in GPR54-expressing Chinese hamster ovary cells (CHO-G) and in C8161.9 cells was evaluated. Metastasis was monitored in athymic mice (groups of 10 per experiment) that were injected with C8161.9(KFM) or C8161.9(KFMdeltaSS) cells labeled with enhanced green fluorescent protein. Survival of mice injected with C8161.9 or C8161.9(KFM) cells was analyzed by Kaplan-Meier methods. RESULTS: Full-length KFM and KFMdeltaSS were detected in whole-cell lysates of C8161.9(KFM) and C8161.9(KFMdeltaSS) cells, respectively, but kisspeptins were detected only in conditioned medium of C8161.9(KFM) cells. In vivo, C8161.9(KFM), but not C8161.9(KFMdeltaSS), cells were suppressed for metastasis to lung, eye, kidney, and bone, with corresponding differences in mouse survival (median > 120 versus 42 days). C8161.9(KFM) cells seeded mouse lungs but did not form macroscopic metastases. Conditioned medium from C8161.9(KFM), but not C8161.9(KFMdeltaSS), cells stimulated calcium mobilization in CHO-G cells. GPR54 expression was low in C8161.9 cells, which were not stimulated by conditioned medium from C8161.9(KFM) cells. CONCLUSIONS: KISS1 secretion was required for multiple organ metastasis suppression and for maintenance of disseminated cells in a dormant state. The absence of GPR54 expression in C8161.9 cells (whose metastatic spread was suppressed by KFM) suggests that metastasis suppression is not mediated through this receptor. The results imply the existence of another KISS1 receptor and/or paracrine signaling. The findings raise the possibility that soluble KISS1, kisspeptins, or mimetics could be used to maintain tumor dormancy, rendering treatment of already disseminated tumor cells (i.e., micrometastases) a legitimate target.
Assuntos
Melanoma/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Proteínas de Fluorescência Verde , Humanos , Estimativa de Kaplan-Meier , Kisspeptinas , Camundongos , RNA Mensageiro/análise , Receptores Acoplados a Proteínas G/genética , Receptores de Kisspeptina-1 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Proteínas Supressoras de Tumor/genéticaRESUMO
Although peroxynitrite stimulates apoptosis in many cell types, whether peroxynitrite acts directly as an oxidant or the induction of apoptosis is because of the radicals derived from peroxynitrite decomposition remains unknown. Before undergoing apoptosis because of trophic factor deprivation, primary motor neuron cultures become immunoreactive for nitrotyrosine. We show here using tyrosine-containing peptides that free radical processes mediated by peroxynitrite decomposition products were required for triggering apoptosis in primary motor neurons and in PC12 cells cultures. The same concentrations of tyrosine-containing peptides required to prevent the nitration and apoptosis of motor neurons induced by trophic factor deprivation and of PC12 cells induced by peroxynitrite also prevented peroxynitrite-mediated nitration of motor neurons, brain homogenates, and PC12 cells. The heat shock protein 90 chaperone was nitrated in both trophic factor-deprived motor neurons and PC12 cells incubated with peroxynitrite. Tyrosine-containing peptides did not affect the induction of PC12 cell death by hydrogen peroxide. Tyrosine-containing peptides should protect by scavenging peroxynitrite-derived radicals and not by direct reactions with peroxynitrite as they neither increase the rate of peroxynitrite decomposition nor decrease the bimolecular peroxynitrite-mediated oxidation of thiols. These results reveal an important role for free radical-mediated nitration of tyrosine residues, in apoptosis induced by endogenously produced and exogenously added peroxynitrite; moreover, tyrosine-containing peptides may offer a novel strategy to neutralize the toxic effects of peroxynitrite.
Assuntos
Apoptose/efeitos dos fármacos , Neurônios Motores/patologia , Peptídeos/farmacologia , Ácido Peroxinitroso/farmacologia , Tirosina , Animais , Sequestradores de Radicais Livres/farmacologia , Radicais Livres , Células PC12 , Ratos , Tirosina/análogos & derivadosRESUMO
Parenteral immunization of transgenic mouse models of Alzheimer disease (AD) with synthetic amyloid beta-peptide (Abeta) prevented or reduced Abeta deposits and attenuated their memory and learning deficits. A clinical trial of immunization with synthetic Abeta, however, was halted due to brain inflammation, presumably induced by a toxic Abeta, T-cell- and/or Fc-mediated immune response. Another issue relating to such immunizations is that some AD patients may not be able to raise an adequate immune response to Abeta vaccination due to immunological tolerance or age-associated decline. Because peripheral administration of antibodies against Abeta also induced clearance of amyloid plaques in the model mice, injection of humanized Abeta antibodies has been proposed as a possible therapy for AD. By screening a human single-chain antibody (scFv) library for Abeta immunoreactivity, we have isolated a scFv that specifically reacts with oligomeric Abeta as well as amyloid plaques in the brain. The scFv inhibited Abeta amyloid fibril formation and Abeta-mediated cytotoxicity in vitro. We have tested the efficacy of the human scFv in a mouse model of AD (Tg2576 mice). Relative to control mice, injections of the scFv into the brain of Tg2576 mice reduced Abeta deposits. Because scFvs lack the Fc portion of the immunoglobulin molecule, human scFvs against Abeta may be useful to treat AD patients without eliciting brain inflammation.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/imunologia , Amiloide/antagonistas & inibidores , Região Variável de Imunoglobulina/uso terapêutico , Deficiências da Aprendizagem/tratamento farmacológico , Transtornos da Memória/tratamento farmacológico , Fragmentos de Peptídeos/imunologia , Doença de Alzheimer/imunologia , Doença de Alzheimer/patologia , Amiloide/análise , Animais , Encéfalo/patologia , Química Encefálica , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Músculo Esquelético/química , Músculo Esquelético/patologiaRESUMO
Immunization of mouse models of Alzheimer disease (AD) with amyloid-peptide (Abeta) reduces Abeta deposits and attenuates their memory and learning deficits. Recent clinical trials were halted due to meningoencephalitis, presumably induced by T cell mediated and/or Fc-mediated immune responses. Because injection of anti-Abeta F(ab')(2) antibodies also induces clearance of amyloid plaques in AD mouse models, we have tested a novel gene therapy modality where an adeno-associated virus (AAV) encoding anti-Abeta single-chain antibody (scFv) is injected into the corticohippocampal regions of AD mouse models. One year after injection, expression of scFv was readily detectable in the neurons of the hippocampus without discernible neurotoxicity. AD mouse models subjected to AAV injection had much less amyloid deposits at the injection sites than the mouse models subjected to PBS injection. Because the scFv lacks the Fc portion of the immunoglobulin molecule, this modality may be a feasible solution for AD without eliciting inflammation.