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1.
Toxicol Pathol ; 45(7): 961-976, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28974147

RESUMO

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are neuromuscular disorders that primarily affect boys due to an X-linked mutation in the DMD gene, resulting in reduced to near absence of dystrophin or expression of truncated forms of dystrophin. Some newer therapeutic interventions aim to increase sarcolemmal dystrophin expression, and accurate dystrophin quantification is critical for demonstrating pharmacodynamic relationships in preclinical studies and clinical trials. Current challenges with measuring dystrophin include the variation in protein expression within individual muscle fibers and across whole muscle samples, the presence of preexisting dystrophin-positive revertant fibers, and trace amounts of residual dystrophin. Immunofluorescence quantification of dystrophin can overcome many of these challenges, but manual quantification of protein expression may be complicated by variations in the collection of images, reproducible scoring of fluorescent intensity, and bias introduced by manual scoring of typically only a few high-power fields. This review highlights the pathology of DMD and BMD, discusses animal models of DMD and BMD, and describes dystrophin biomarker quantitation in DMD and BMD, with several image analysis approaches, including a new automated method that evaluates protein expression of individual muscle fibers.


Assuntos
Biomarcadores/metabolismo , Determinação de Ponto Final , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Animais , Modelos Animais de Doenças , Distrofina/deficiência , Regulação da Expressão Gênica , Humanos , Fibras Musculares Esqueléticas/metabolismo , Mutação , Utrofina/genética , Utrofina/metabolismo
2.
J Biol Chem ; 287(53): 44425-34, 2012 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-23148212

RESUMO

Highly specific antibodies to phosphoepitopes are valuable tools to study phosphorylation in disease states, but their discovery is largely empirical, and the molecular mechanisms mediating phosphospecific binding are poorly understood. Here, we report the generation and characterization of extremely specific recombinant chicken antibodies to three phosphoepitopes on the Alzheimer disease-associated protein tau. Each antibody shows full specificity for a single phosphopeptide. The chimeric IgG pT231/pS235_1 exhibits a K(D) of 0.35 nm in 1:1 binding to its cognate phosphopeptide. This IgG is murine ortholog-cross-reactive, specifically recognizing the pathological form of tau in brain samples from Alzheimer patients and a mouse model of tauopathy. To better understand the underlying binding mechanisms allowing such remarkable specificity, we determined the structure of pT231/pS235_1 Fab in complex with its cognate phosphopeptide at 1.9 Å resolution. The Fab fragment exhibits novel complementarity determining region (CDR) structures with a "bowl-like" conformation in CDR-H2 that tightly and specifically interacts with the phospho-Thr-231 phosphate group, as well as a long, disulfide-constrained CDR-H3 that mediates peptide recognition. This binding mechanism differs distinctly from either peptide- or hapten-specific antibodies described to date. Surface plasmon resonance analyses showed that pT231/pS235_1 binds a truly compound epitope, as neither phosphorylated Ser-235 nor free peptide shows any measurable binding affinity.


Assuntos
Doença de Alzheimer/metabolismo , Anticorpos/imunologia , Epitopos/imunologia , Proteínas tau/imunologia , Doença de Alzheimer/genética , Sequência de Aminoácidos , Animais , Anticorpos/química , Anticorpos/genética , Encéfalo/metabolismo , Galinhas , Epitopos/química , Epitopos/genética , Humanos , Imunoglobulina G/química , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Fosforilação , Proteínas tau/química , Proteínas tau/genética , Proteínas tau/metabolismo
3.
Biochem Biophys Res Commun ; 390(3): 372-6, 2009 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-19683511

RESUMO

The HIF (hypoxia inducible factor) hydroxylases EGNL1/PHD2 has been implicated in embryonic development. Here we knocked down EGNL1 in vivo by injecting one-cell murine zygotes with lentivirus-containing RNAi. Progeny with demonstrated EGLN1 inhibition had elevated EPO production and erythropoiesis in vivo. The partial inhibition of EGLN1 in utero is embryonic lethal in some, but not all mice on gestation day 14, and is associated with defects in placental and heart development, similar to those noted in the EGLN1 knockout mouse. Importantly, the in utero inhibition of EGNL1 varied greatly between the embryo proper and the placenta. Using this as a tool we show that the embryopathic effects are associated with knockdown of EGNL1 and the associated induction of Igfbp1 (insulin-like growth factor binding protein-1) mRNA in the placenta, but not the embryo.


Assuntos
Embrião de Mamíferos/anormalidades , Desenvolvimento Embrionário/genética , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/biossíntese , Placenta/metabolismo , Pró-Colágeno-Prolina Dioxigenase/fisiologia , Animais , Embrião de Mamíferos/patologia , Feminino , Coração/embriologia , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/patologia , Prolina Dioxigenases do Fator Induzível por Hipóxia , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Fígado/anormalidades , Fígado/enzimologia , Camundongos , Camundongos Transgênicos , Miocárdio/enzimologia , Miocárdio/patologia , Placenta/anormalidades , Gravidez , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Pró-Colágeno-Prolina Dioxigenase/genética
4.
Arch Pathol Lab Med ; 143(2): 197-205, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30168727

RESUMO

CONTEXT.­: Duchenne muscular dystrophy is a rare, progressive, and fatal neuromuscular disease caused by dystrophin protein loss. Common investigational treatment approaches aim at increasing dystrophin expression in diseased muscle. Some clinical trials include assessments of novel dystrophin production as a surrogate biomarker of efficacy, which may predict a clinical benefit from treatment. OBJECTIVES.­: To establish an immunofluorescent scanning and digital image analysis workflow that provides an objective approach for staining intensity assessment of the immunofluorescence dystrophin labeling and determination of the percentage of biomarker-positive fibers in muscle cryosections. DESIGN.­: Optimal and repeatable digital image capture was achieved by a rigorously qualified fluorescent scanning process. After scanning qualification, the MuscleMap (Flagship Biosciences, Westminster, Colorado) algorithm was validated by comparing high-power microscopic field total and dystrophin-positive fiber counts obtained by trained pathologists to data derived by MuscleMap. Next, the algorithm was tested on whole-slide images of immunofluorescent-labeled muscle sections from Duchenne muscular dystrophy, Becker muscular dystrophy, and control patients. RESULTS.­: When used under the guidance of a trained pathologist, the digital image analysis tool met predefined validation criteria and demonstrated functional and statistical equivalence with manual assessment. This work is the first, to our knowledge, to qualify and validate immunofluorescent scanning and digital tissue image-analysis workflow, respectively, with the rigor required to support the clinical trial environments. CONCLUSIONS.­: MuscleMap enables analysis of all fibers within an entire muscle biopsy section and provides data on a fiber-by-fiber basis. This will allow future clinical trials to objectively investigate myofibers' dystrophin expression at a greater level of consistency and detail.


Assuntos
Distrofina/análise , Interpretação de Imagem Assistida por Computador/métodos , Distrofia Muscular de Duchenne/diagnóstico , Adolescente , Biópsia , Criança , Pré-Escolar , Feminino , Secções Congeladas , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/patologia
5.
Neurology ; 90(24): e2146-e2154, 2018 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-29752304

RESUMO

OBJECTIVE: To describe the quantification of novel dystrophin production in patients with Duchenne muscular dystrophy (DMD) after long-term treatment with eteplirsen. METHODS: Clinical study 202 was an observational, open-label extension of the randomized, controlled study 201 assessing the safety and efficacy of eteplirsen in patients with DMD with a confirmed mutation in the DMD gene amenable to correction by skipping of exon 51. Patients received once-weekly IV doses of eteplirsen 30 or 50 mg/kg. Upper extremity muscle biopsy samples were collected at combined study week 180, blinded, and assessed for dystrophin-related content by Western blot, Bioquant software measurement of dystrophin-associated immunofluorescence intensity, and percent dystrophin-positive fibers (PDPF). Results were contrasted with matched untreated biopsies from patients with DMD. Reverse transcription PCR followed by Sanger sequencing of newly formed slice junctions was used to confirm the mechanism of action of eteplirsen. RESULTS: Reverse transcription PCR analysis and sequencing of the newly formed splice junction confirmed that 100% of treated patients displayed the expected skipped exon 51 sequence. In treated patients vs untreated controls, Western blot analysis of dystrophin content demonstrated an 11.6-fold increase (p = 0.007), and PDPF analysis demonstrated a 7.4-fold increase (p < 0.001). The PDPF findings were confirmed in a re-examination of the sample (15.5-fold increase, p < 0.001). Dystrophin immunofluorescence intensity was 2.4-fold greater in treated patients than in untreated controls (p < 0.001). CONCLUSION: Taken together, the 4 assays, each based on unique evaluation mechanisms, provided evidence of eteplirsen muscle cell penetration, exon skipping, and induction of novel dystrophin expression. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence of the muscle cell penetration, exon skipping, and induction of novel dystrophin expression by eteplirsen, as confirmed by 4 assays.


Assuntos
Distrofina/biossíntese , Éxons/genética , Morfolinos/uso terapêutico , Distrofia Muscular de Duchenne/tratamento farmacológico , Biópsia , Criança , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Resultado do Tratamento
6.
Diagn Microbiol Infect Dis ; 58(1): 59-65, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17300898

RESUMO

The ability of enterococci to acquire resistance to antibiotics and form biofilms in vivo makes these infections, endocarditis in particular, especially difficult to treat. A collection of clinical enterococcal isolates was screened for the presence of various virulence determinants and in an in vitro assay for biofilm formation. Isolates were chosen for the presence or absence of the genes for Esp and gelatinase and different in vitro biofilm phenotypes, and were evaluated in a rat model of endocarditis; all colonized vegetations to similar degrees. Treatment with vancomycin resulted in a 2.7-log reduction in colony-forming unit (CFU) in vegetations for an esp(+)/gel(-) strain, compared with no reduction in CFU for an esp(+)/gel(+) or an esp(-)/gel(-) isolate. These results suggest that although there may not be an absolute role for individual virulence determinants in infectivity, combinations of factors may play a role in allowing a biofilm infection to be more resistant to therapy.


Assuntos
Proteínas de Bactérias/genética , Biofilmes/efeitos dos fármacos , Endocardite Bacteriana/tratamento farmacológico , Enterococcus faecalis/patogenicidade , Resistência a Vancomicina , Animais , Valva Aórtica/microbiologia , Biofilmes/crescimento & desenvolvimento , Contagem de Colônia Microbiana , Endocardite Bacteriana/microbiologia , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/genética , Enterococcus faecalis/crescimento & desenvolvimento , Gelatinases/genética , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Masculino , Proteínas de Membrana/genética , Ratos , Ratos Sprague-Dawley , Vancomicina/farmacologia , Vancomicina/uso terapêutico , Resistência a Vancomicina/genética , Virulência/genética
7.
Toxicol Sci ; 90(1): 149-58, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16357009

RESUMO

Embryonic stem (ES) cells offer unprecedented opportunities for in vitro drug discovery and safety assessment of compounds. Cardiomyocytes derived from ES cells enable development of predictive cardiotoxicity models to increase the safety of novel drugs. Heterogeneity of differentiated ES cells limits the development of reliable in vitro models for compound screening. We report an innovative and robust approach to isolate ES-derived cardiomyocytes using laser microdissection and pressure catapulting (LMPC). LMPC cells were readily applied onto 96-well format in vitro pharmacology assays. The expression of developmental and functional cardiac markers, Nkx 2.5, MLC2V, GATA-4, Connexin 43, Connexin 45, Serca-2a, cardiac alpha actin, and phospholamban, among others, was confirmed in LMPC ES-derived cardiomyocytes. Functional assays exhibited cardiac-like response to increased extracellular calcium (5.4 mM extracellular Ca2+) and L-type calcium channel antagonist (1 microM nifedipine). In conclusion, laser microdissection and pressure catapulting is a robust technology to isolate homogeneous ES-derived cell types from heterogeneous populations applicable to assay development.


Assuntos
Cardiopatias/induzido quimicamente , Microscopia Confocal/métodos , Miócitos Cardíacos/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Xenobióticos/toxicidade , Animais , Bioensaio/métodos , Biomarcadores/metabolismo , Cálcio/metabolismo , Cálcio/farmacologia , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos/métodos , Coração Fetal/citologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Cardiopatias/patologia , Lasers , Camundongos , Camundongos Endogâmicos DBA , Microdissecção/métodos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Nifedipino/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Células-Tronco/metabolismo , Células-Tronco/patologia
8.
Diabetes ; 51(7): 2158-69, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12086946

RESUMO

Type 2 diabetes is characterized by loss of beta-cell mass and concomitant deposition of amyloid derived from islet amyloid polypeptide (IAPP). Previously we have shown that expression of human IAPP (huIAPP) in islets of transgenic mice results in either a rapid onset of hyperglycemia in mice homozygous for the huIAPP transgene on a lean background (FVB/N) or a gradual hyperglycemia in mice hemizygous for the huIAPP transgene on an obese background (A(vy)/A). In both strains, only the males routinely develop diabetes. To investigate this sexual dimorphism, we treated young prediabetic A(vy)/A mice transgenic for huIAPP (huIAPP-A(vy)) with 17beta-estradiol (E2). The treatment completely blocked the progression to hyperglycemia but also prevented the associated weight gain in these mice. Immunohistochemistry of pancreatic sections demonstrated normal islet morphology with no apparent deposition of islet amyloid. E2 treatment of 1-year-old huIAPP-A(vy) diabetic males rapidly reverses obesity and hyperglycemia. To determine the effects of E2 in a nonobese model, we also treated prediabetic, ad libitum-fed and pair-fed Lean-huIAPP transgenic males. E2 completely blocked the progression to hyperglycemia with no significant effect on body weight. Pancreatic insulin content and plasma insulin concentration of Lean-huIAPP transgenic mice increased in a dose-dependent manner. We demonstrated the presence of estrogen receptor (ER)-alpha mRNA in mouse and human islets. By also confirming the presence of ER-alpha protein in islets, we discovered a novel 58-kDa ER-alpha isoform in mice and a 52-kDa isoform in humans, in the absence of the classic 67-kDa protein found in most tissues of both species. The demonstrated presence of ER-alpha in mouse and human islets is consistent with a direct effect on islet function. We conclude that exogenous E2 administered to male mice may block human IAPP-mediated beta-cell loss both by direct action on beta-cells and by decreasing insulin demand through inhibition of weight gain or increasing insulin action.


Assuntos
Amiloide/fisiologia , Diabetes Mellitus/prevenção & controle , Estradiol/uso terapêutico , Obesidade/prevenção & controle , Amiloide/genética , Animais , Implantes de Medicamento , Estradiol/administração & dosagem , Feminino , Homozigoto , Humanos , Hiperglicemia/sangue , Hiperglicemia/genética , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Masculino , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Caracteres Sexuais
9.
Eur J Pharmacol ; 456(1-3): 59-68, 2002 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-12450570

RESUMO

The efficacy of a novel, nonpeptidic, caspase 3/7-selective inhibitor, (S)-(+)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (MMPSI) for reducing ischemic injury in isolated rabbit hearts or cardiomyocytes was evaluated. MMPSI (0.1-10 microM) evoked a concentration-dependent reduction in infarct size (up to 56% vs. control; IC(50)=0.2 microM). Furthermore, apoptosis (DNA laddering, soluble nucleosomes) was reduced in the ischemic area-at-risk. MMPSI inhibited recombinant human caspase-3 with an IC(50)=1.7 microM. Apoptosis in H9c2 cells after 16-h simulated ischemia and 2-h simulated reperfusion was significantly reduced by MMPSI in a concentration-dependent manner (IC(50)=0.5 microM); similar effects were observed in isolated adult rabbit cardiomyocytes (IC(50)=1.5 microM). These data support an important role for caspase-3/7 in mediating myocardial ischemic injury. Furthermore, these data indicate that cardioprotection via caspase-3/7 inhibition is attainable via a small molecule (nonpeptidic) inhibitor, a necessary step in making this approach therapeutically viable.


Assuntos
Inibidores de Caspase , Inibidores de Cisteína Proteinase/farmacologia , Isatina/farmacologia , Isquemia Miocárdica/complicações , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Pirrolidinas/farmacologia , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Caspase 3 , Caspase 7 , Caspases/metabolismo , Linhagem Celular , Células Cultivadas , Circulação Coronária/efeitos dos fármacos , Dipeptídeos/farmacologia , Relação Dose-Resposta a Droga , Frequência Cardíaca/efeitos dos fármacos , Marcação In Situ das Extremidades Cortadas , Isatina/análogos & derivados , Cetonas/farmacologia , Masculino , Microscopia Eletrônica , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/etiologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/enzimologia , Miocárdio/patologia , Miocárdio/ultraestrutura , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Coelhos
10.
PLoS One ; 8(12): e85815, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24392030

RESUMO

Parkinson's disease (PD) is the second most common neurodegenerative disease. A key pathological feature of PD is Lewy bodies, of which the major protein component is α-synuclein (α-syn). Human genetic studies have shown that mutations (A53T, A30P, E46K) and multiplication of the α-syn gene are linked to familial PD. Mice overexpressing the human A53T mutant α-syn gene develop severe movement disorders. However, the molecular mechanisms of α-syn toxicity are not well understood. Recently, mitochondrial dysfunction has been linked with multiple neurodegenerative diseases including Parkinson's disease. Here we investigated whether mitochondrial motility, dynamics and respiratory function are affected in primary neurons from a mouse model expressing the human A53T mutation. We found that mitochondrial motility was selectively inhibited in A53T neurons while transport of other organelles was not affected. In addition, A53T expressing neurons showed impairment in mitochondrial membrane potential and mitochondrial respiratory function. Furthermore, we found that rapamycin, an autophagy inducer, rescued the decreased mitochondrial mobility. Taken together, these data demonstrate that A53T α-syn impairs mitochondrial function and dynamics and the deficit of mitochondrial transport is reversible, providing further understanding of the disease pathogenesis and a potential therapeutic strategy for PD.


Assuntos
Córtex Cerebral/citologia , Mitocôndrias/metabolismo , Mutação , Neurônios/citologia , alfa-Sinucleína/genética , Animais , Transporte Biológico/efeitos dos fármacos , Respiração Celular/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neurônios/efeitos dos fármacos , Fenótipo , Sirolimo/farmacologia
11.
Neurobiol Aging ; 33(7): 1481.e13-23, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22227005

RESUMO

Transgenic mice are used to model increased brain amyloid-ß (Aß) and amyloid plaque formation reflecting Alzheimer's disease pathology. In our study hippocampal network oscillations, population spikes, and long-term potentiation (LTP) were recorded in APPswe/PS1dE9 (APP/PS1) and presenilin1 (PS1) transgenic and wild type mice at 2, 4, and 8 months of age under urethane anesthesia. Hippocampal theta oscillations elicited by brainstem stimulation were similar in wild type and PS1 mice at all age groups. In contrast, APP/PS1 mice showed an age-dependent decrease in hippocampal activity, characterized by a significant decline in elicited theta power and frequency at 4 and 8 months. Magnitudes of population spikes and long-term potentiation in the dentate gyrus were similar across groups at both 4 and 8 months. In APP/PS1 mice, soluble and insoluble Aß, and hippocampal and cortical plaque load increased with age, and the disruption in hippocampal theta oscillation showed a significant correlation with plaque load. Our study shows that, using in vivo electrophysiological methods, early Aß-related functional deficits can be robustly detected in the brainstem-hippocampus multisynaptic network.


Assuntos
Envelhecimento/genética , Envelhecimento/metabolismo , Peptídeos beta-Amiloides/biossíntese , Peptídeos beta-Amiloides/genética , Hipocampo/fisiologia , Ritmo Teta/fisiologia , Precursor de Proteína beta-Amiloide/genética , Animais , Potenciação de Longa Duração/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Presenilina-1/genética
12.
J Neurosci Methods ; 204(1): 179-188, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22019329

RESUMO

Advances in imaging technology have enabled automated approaches for quantitative image analysis. In this study, a high content object based image analysis method was developed for quantification of ß-amyloid (Aß) plaques in postmortem brains of Alzheimer's disease (AD) subjects and in transgenic mice over overexpressing Aß. Digital images acquired from immunohistochemically stained sections of the superior frontal gyrus were analyzed for Aß plaque burden using a Definiens object-based segmentation approach. Blinded evaluation of Aß stained sections from AD and aged matched human subjects accurately identified AD cases with one exception. Brains from transgenic mice overexpressing Aß (PS1APP mice) were also evaluated by our Definiens object based image analysis approach. We observed an age-dependent increase in the amount of Aß plaque load that we quantified in both the hippocampus and cortex. From the contralateral hemisphere, we measured the amount of Aß in brain homogenates biochemically and observed a significant correlation between our biochemical measurements and those that we measured by our object based Definiens system in the hippocampus. Assessment of Aß plaque load in PS1APP mice using a manual segmentation technique (Image-Pro Plus) confirmed the results of our object-based image analysis approach. Image acquisition and analysis of 32 stained human slides and 100 mouse slides were executed in 8 h and 22 h, respectively supporting the relatively high throughput features of the Definiens platform. The data show that digital imaging combined with object based image analysis is a reliable and efficient approach to quantifying Aß plaques in human and mouse brain.


Assuntos
Algoritmos , Encéfalo/patologia , Interpretação de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Microscopia Confocal/métodos , Reconhecimento Automatizado de Padrão/métodos , Placa Amiloide/patologia , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Humanos , Aumento da Imagem/métodos , Masculino , Camundongos , Camundongos Transgênicos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
13.
J Mol Biol ; 421(4-5): 525-36, 2012 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-22197375

RESUMO

Alzheimer's disease, the most common cause of dementia in the elderly and characterized by the deposition and accumulation of plaques, is composed in part of ß-amyloid (Aß) peptides, loss of neurons, and the accumulation of neurofibrillary tangles. Here, we describe ponezumab, a humanized monoclonal antibody, and show how it binds specifically to the carboxyl (C)-terminus of Aß40. Ponezumab can label Aß that is deposited in brain parenchyma found in sections from Alzheimer's disease casualties and in transgenic mouse models that overexpress Aß. Importantly, ponezumab does not label full-length, non-cleaved amyloid precursor protein on the cell surface. The C-terminal epitope of the soluble Aß present in the circulation appears to be available for ponezumab binding because systemic administration of ponezumab greatly elevates plasma Aß40 levels in a dose-dependent fashion after administration to a mouse model that overexpress human Aß. Administration of ponezumab to transgenic mice also led to a dose-dependent reduction in hippocampal amyloid load. To further explore the nature of ponezumab binding to Aß40, we determined the X-ray crystal structure of ponezumab in complex with Aß40 and found that the Aß40 carboxyl moiety makes extensive contacts with ponezumab. Furthermore, the structure-function analysis supported this critical requirement for carboxy group of AßV40 in the Aß-ponezumab interaction. These findings provide novel structural insights into the in vivo conformation of the C-terminus of Aß40 and the brain Aß-lowering efficacy that we observed following administration of ponezumab in transgenic mouse models.


Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/metabolismo , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Sequência de Aminoácidos , Peptídeos beta-Amiloides/sangue , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Encéfalo/patologia , Cristalografia por Raios X , Modelos Animais de Doenças , Humanos , Injeções Intravenosas , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Dados de Sequência Molecular , Fármacos Neuroprotetores/administração & dosagem , Plasma/química , Ligação Proteica , Conformação Proteica
14.
Front Psychiatry ; 2: 14, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21556284

RESUMO

NMDA receptor (NMDAR) antagonists, such as phencyclidine, ketamine, or dizocilpine (MK-801) are commonly used in psychiatric drug discovery in order to model several symptoms of schizophrenia, including psychosis and impairments in working memory. In spite of the widespread use of NMDAR antagonists in preclinical and clinical studies, our understanding of the mode of action of these drugs on brain circuits and neuronal networks is still limited. In the present study spontaneous local field potential (LFP), multi- (MUA) and single-unit activity, and evoked potential, including paired-pulse facilitation (PPF) in response to electrical stimulation of the ipsilateral subiculum were carried out in the medial prefrontal cortex (mPFC) in urethane anesthetized rats. Systemic administration of MK-801 (0.05 mg/kg, i.v.) decreased overall MUA, with a diverse effect on single-unit activity, including increased, decreased, or unchanged firing, and in line with our previous findings shifted delta-frequency power of the LFP and disrupted PPF (Kiss et al., 2011). In order to provide further insight to the mechanisms of action of NMDAR antagonists, MK-801 was administered intracranially into the mPFC and mediodorsal nucleus of the thalamus (MD). Microinjections of MK-801, but not physiological saline, localized into the MD evoked changes in both LFP parameters and PPF similar to the effects of systemically administered MK-801. Local microinjection of MK-801 into the mPFC was without effect on these parameters. Our findings indicate that the primary site of the action of systemic administration of NMDAR antagonists is unlikely to be the cortex. We presume that multiple neuronal networks, involving thalamic nuclei contribute to disrupted behavior and cognition following NMDAR blockade.

15.
Adv Ther ; 27(3): 168-80, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20429046

RESUMO

INTRODUCTION: CD44 is a cell adhesion molecule believed to play a critical role in T cell and monocyte infiltration in the inflammatory process. The reduction of CD44 expression or its ability to properly interact with its key ligand, hyaluronic acid (HA), inhibits migration and subsequent activation of cells within sites of inflammation. CD44-deficient mice exhibit decreased disease in a mouse arthritis model. METHODS: Accordingly, we developed PF-03475952, a fully human IgG2 anti-CD44 monoclonal antibody (mAb). RESULTS: Binding of PF-03475952 to CD44 inhibits binding of HA and induces loss of CD44 from the cell surface. PF-03475952 also passed a series of safety pharmacology assays designed to assess the risk of the mAb to bind Fc gamma receptors, stimulate cytokine release from human whole blood, and stimulate cytokine release from peripheral blood mononuclear cells (PBMC) using plate-bound antibodies. The latter assay was designed specifically to evaluate the risk of cytokine storm that had been observed with TGN1412 (immunostimulatory CD28 superagonist mAb). PF-003475952 exhibits high-affinity binding to both human and cynomolgus monkey CD44, but does not cross-react with rodent CD44. Thus, a rat anti-mouse CD44 mAb was used to demonstrate a dose-dependent decrease of disease in mouse collagen-induced arthritis. Importantly, efficacy was correlated with >50% loss of cell surface CD44 on circulating cells. Loss of CD44 expression on CD3+ lymphocytes was monitored following a single dose of PF-03475952 in cynomolgus monkeys as a pharmacodynamic marker. The recovery of CD44 expression was found to be dose-dependent. PF-03475952 doses of 1, 10, and 100 mg/kg reduced CD44 expression below 50% for 218, 373, and >504 hours, respectively. CONCLUSION: Targeting of CD44 is a unique mechanism of action in the treatment of inflammatory diseases and is expected to reduce joint damage induced by inflammatory mediators, resulting in disease modification in inflammatory diseases such as rheumatoid arthritis.


Assuntos
Anti-Inflamatórios/farmacologia , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Artrite Experimental/tratamento farmacológico , Receptores de Hialuronatos/imunologia , Imunoglobulina G/farmacologia , Animais , Anti-Inflamatórios/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Anticorpos Neutralizantes/uso terapêutico , Artrite Experimental/imunologia , Artrite Experimental/metabolismo , Citocinas/sangue , Ensaio de Imunoadsorção Enzimática , Humanos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , Imunoglobulina G/uso terapêutico , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos DBA , Ativação Plaquetária/efeitos dos fármacos , Ligação Proteica
16.
Arthritis Res Ther ; 10(1): R14, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18234077

RESUMO

INTRODUCTION: CP-690550 is a small molecule inhibitor of Janus kinase 3 (JAK3), a critical enzyme in the signaling pathway of multiple cytokines (interleukin (IL)-2, -7, -15 and -21) that are important in various T cell functions including development, activation and homeostasis. The purpose of this study was to evaluate CP-690550 in murine collagen-induced (CIA) and rat adjuvant-induced (AA) models of rheumatoid arthritis (RA). METHODS: CIA and AA were induced using standard protocols and animals received the JAK3 inhibitor via osmotic mini-pump infusion at doses ranging from 1.5-15 mg/kg/day following disease induction. Arthritis was assessed by clinical scores in the CIA models and paw swelling monitored using a plethysmometer in the AA model until study conclusion, at which time animals were killed and evaluated histologically. RESULTS: CP-690550 dose-dependently decreased endpoints of disease in both RA models with greater than 90% reduction observed at the highest administered dose. An approximate ED50 of approximately 1.5 mg/kg/day was determined for the compound based upon disease endpoints in both RA models examined and corresponds to CP-690550 serum levels of 5.8 ng/ml in mice (day 28) and 24 ng/ml in rats (day 24). The compound also reduced inflammatory cell influx and joint damage as measured histologically. Animals receiving a CP-690550 dose of 15 mg/k/d showed no histological evidence of disease. CONCLUSION: The efficacy observed with CP-690550 in CIA and AA suggests JAK3 inhibition may represent a novel therapeutic target for the treatment of RA.


Assuntos
Artrite Experimental/enzimologia , Artrite Experimental/patologia , Artrite Reumatoide/enzimologia , Artrite Reumatoide/patologia , Cartilagem Articular/enzimologia , Cartilagem Articular/patologia , Janus Quinase 3/antagonistas & inibidores , Adjuvantes Imunológicos , Animais , Artrite Experimental/sangue , Artrite Experimental/imunologia , Artrite Reumatoide/sangue , Artrite Reumatoide/imunologia , Colágeno/imunologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/sangue , Inibidores Enzimáticos/farmacologia , Interleucina-6/sangue , Masculino , Camundongos , Camundongos Endogâmicos DBA , Piperidinas , Pirimidinas/administração & dosagem , Pirimidinas/sangue , Pirimidinas/farmacologia , Pirróis/administração & dosagem , Pirróis/sangue , Pirróis/farmacologia , Ratos , Ratos Endogâmicos Lew
17.
J Lipid Res ; 48(6): 1263-72, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17325387

RESUMO

Cholesteryl ester transfer protein (CETP) inhibitors increase high density lipoprotein-cholesterol (HDL-C) in animals and humans, but whether CETP inhibition will be antiatherogenic is still uncertain. We tested the CETP inhibitor torcetrapib in rabbits fed an atherogenic diet at a dose sufficient to increase HDL-C by at least 3-fold (207 +/- 32 vs. 57 +/- 6 mg/dl in controls at 16 weeks). CETP activity was inhibited by 70-80% throughout the study. Non-HDL-C increased in both groups, but there was no difference apparent by the study's end. At 16 weeks, aortic atherosclerosis was 60% lower in torcetrapib-treated animals (16.4 +/- 3.4% vs. 39.8 +/- 5.4% in controls) and aortic cholesterol content was reduced proportionally. Sera from a separate group of rabbits administered torcetrapib effluxed 48% more cholesterol from Fu5AH cells than did sera from control animals, possibly explaining the reduced aortic cholesterol content. Regression analyses indicated that lesion area in the torcetrapib-treated group was strongly correlated with the ratio of total plasma cholesterol to HDL-C but not with changes in other lipid or lipoprotein levels. CETP inhibition with torcetrapib retards atherosclerosis in rabbits, and the reduced lesion area is associated with increased levels of HDL-C.


Assuntos
Aorta/efeitos dos fármacos , Aterosclerose/prevenção & controle , Proteínas de Transferência de Ésteres de Colesterol/antagonistas & inibidores , Quinolinas/farmacologia , Animais , Anticolesterolemiantes/administração & dosagem , Anticolesterolemiantes/farmacologia , Aorta/metabolismo , Aorta/patologia , Aterosclerose/sangue , Aterosclerose/metabolismo , Transporte Biológico/efeitos dos fármacos , Linhagem Celular Tumoral , Colesterol/sangue , Colesterol/metabolismo , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , HDL-Colesterol/sangue , HDL-Colesterol/metabolismo , Dieta Aterogênica , Suscetibilidade a Doenças/sangue , Suscetibilidade a Doenças/metabolismo , Masculino , Quinolinas/administração & dosagem , Coelhos , Análise de Regressão
18.
Science ; 302(5646): 875-8, 2003 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-14593182

RESUMO

Because of its requirement for signaling by multiple cytokines, Janus kinase 3 (JAK3) is an excellent target for clinical immunosuppression. We report the development of a specific, orally active inhibitor of JAK3, CP-690,550, that significantly prolonged survival in a murine model of heart transplantation and in cynomolgus monkeys receiving kidney transplants. CP-690,550 treatment was not associated with hypertension, hyperlipidemia, or lymphoproliferative disease. On the basis of these preclinical results, we believe JAK3 blockade by CP-690,550 has potential for therapeutically desirable immunosuppression in human organ transplantation and in other clinical settings.


Assuntos
Rejeição de Enxerto/prevenção & controle , Transplante de Coração , Imunossupressores/farmacologia , Transplante de Rim , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Pirróis/farmacologia , Animais , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Inibidores Enzimáticos/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Sobrevivência de Enxerto/efeitos dos fármacos , Humanos , Imunossupressores/administração & dosagem , Imunossupressores/uso terapêutico , Imunossupressores/toxicidade , Interleucina-2/imunologia , Janus Quinase 3 , Ativação Linfocitária/efeitos dos fármacos , Contagem de Linfócitos , Teste de Cultura Mista de Linfócitos , Subpopulações de Linfócitos/efeitos dos fármacos , Macaca fascicularis , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Miocárdio/metabolismo , Piperidinas , Proteínas Tirosina Quinases/metabolismo , Pirimidinas/administração & dosagem , Pirimidinas/uso terapêutico , Pirimidinas/toxicidade , Pirróis/administração & dosagem , Pirróis/uso terapêutico , Pirróis/toxicidade , Transplante Heterotópico , Transplante Homólogo , Células Tumorais Cultivadas
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