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1.
Toxicol Pathol ; 49(2): 315-333, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33167807

RESUMO

Treatment of nonhuman primates and mice with a humanized antigen-binding fragment (Fab) antibody (UCBFab) inhibiting transforming growth factor ß via daily inhalation for up to 13 weeks resulted in low systemic exposure but high local exposure in the lung. Target engagement was demonstrated by reduced levels of signal transducers, phosphoSMAD and plasminogen activator inhibitor-1 in the bronchoalveolar lavage fluid (BALF). Treatment was associated with a high frequency and titer of antidrug antibodies, indicating high local immunogenicity, and local pathology within the lung and draining lymph nodes. Microscopic changes were characterized by perivascular (PV) and peribronchiolar (PB) mononuclear inflammatory cell (MIC) infiltrates that were principally lymphocytic in nature and mixed inflammatory cell infiltrates and/or inflammation within the alveoli. Immunohistochemical investigation revealed a predominantly CD68-positive macrophage and CD3- and CD8>CD4-positive T-cell response in the alveoli, whereas within the airways, there was a variable mixture of CD3-positive T cells, CD20-positive B cells, and CD68-positive macrophages. Increased cellularity of the draining lymph nodes was also noted, indicating the presence of an immune response to the inhaled test article. Morphologic changes did not progress over time, and all changes partially recovered. Increased leukocytes (principally macrophages) in BALF cytology correlated with the changes seen by histopathology.


Assuntos
Anticorpos , Pulmão , Fator de Crescimento Transformador beta , Animais , Anticorpos/toxicidade , Líquido da Lavagem Broncoalveolar , Inflamação , Camundongos , Primatas
2.
Toxicol Pathol ; 49(2): 261-285, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33535023

RESUMO

The inhaled route is still a relatively novel route for delivering biologics and poses additional challenges to those encountered with inhaled small molecules, further complicating the design and interpretation of toxicology studies. A working group formed to summarize the current knowledge of inhaled biologics across industry and to analyze data collated from an anonymized cross-industry survey comprising 12 inhaled biologic case studies (18 individual inhalation toxicity studies on monoclonal antibodies, fragment antibodies, domain antibodies, oligonucleotides, and proteins/peptides). The output of this working group provides valuable insights into the issues faced when conducting toxicology studies with inhaled biologics, including common technical considerations on aerosol generation, use of young and sexually mature nonhuman primates, pharmacokinetic/pharmacodynamic modeling, exposure and immunogenicity assessment, maximum dose setting, and no observed adverse effect levels determination. Although the current data set is too small to allow firm conclusions, testing of novel biologics remains an active area and is likely to remain so for molecules where delivery via the inhaled route is beneficial. In the future, it is hoped others will continue to share their experiences and build on the conclusions of this review to further improve our understanding of these complex issues and, ultimately, facilitate the safe introduction of inhaled biologics into clinical use.


Assuntos
Produtos Biológicos , Administração por Inalação , Aerossóis , Animais , Produtos Biológicos/toxicidade , Testes de Toxicidade
3.
J Chromatogr A ; 1699: 464002, 2023 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-37126878

RESUMO

Determination of the levels of protein cross-linking catalysed by the activity of transglutaminase 2 in various disease states has remained a significant challenge. The ability to quantify the isopeptide ε-(γ-glutamyl) lysine, which can form as a heterogeneous bond within or between proteins has significant analytical and clinical potential as a biomarker in biofluids such as human urine. Increased transglutaminase 2 activity is associated with a number of diseases, such as fibrosis. Previously published methods have been based on classical amino acid analysis, however they require a complex multi-enzyme digestion in order to achieve complete protein digestion, whilst leaving the isopeptide cross link intact. These methods require high levels of enzymes, which contaminate the analysis and alter the dynamics of digestion. The amino acid analysis detection also lacked selectivity, especially where the levels of crosslink are expected to be low relative to the background protein levels. We have systematically addressed these challenges, by optimising the precipitation of the protein in urine, the use of innovative immobilised enzyme technology, which allows for efficient digestion without enzyme contamination and LC-MS/MS detection based on multiple reaction monitoring. This method was validated for its analytical performance characteristics, showing the method has a sensitivity of 0.1 ng/mL of ε-(γ-glutamyl) lysine in human urine with precision of less than 20% CV, and is selective as no interferences were observed that may adversely affect the analysis. As such this approach represents a significant advance in the ability to detect and quantify ε-(γ-glutamyl) lysine.


Assuntos
Lisina , Proteína 2 Glutamina gama-Glutamiltransferase , Humanos , Cromatografia Líquida , Transglutaminases , Espectrometria de Massas em Tandem , Biomarcadores , Dipeptídeos/análise
4.
Drug Discov Today ; 28(2): 103440, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36375739

RESUMO

Harnessing the immune system to kill tumors has been revolutionary and, as a result, has had an enormous benefit for patients in extending life and resulting in effective cures in some. However, activation of the immune system can come at the cost of undesirable adverse events such as cytokine release syndrome, immune-related adverse events, on-target/off-tumor toxicity, neurotoxicity and tumor lysis syndrome, which are safety risks that can be challenging to assess non-clinically. This article provides a review of the biology and mechanisms that can result in immune-mediated adverse effects and describes industry approaches using in vitro and in vivo models to aid in the nonclinical safety risk assessments for immune-oncology modalities. Challenges and limitations of knowledge and models are also discussed.


Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Neoplasias , Humanos , Neoplasias/tratamento farmacológico , Medição de Risco
5.
Drug Discov Today ; 27(6): 1604-1621, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35304340

RESUMO

Many in vitro and in vivo models are used in pharmacological research to evaluate the role of targeted proteins in a disease. Understanding the translational relevance and limitation of these models for analyzing a drug's disposition, pharmacokinetic/pharmacodynamic (PK/PD) profile, mechanism, and efficacy, is essential when selecting the most appropriate model of the disease of interest and predicting clinically efficacious doses of the investigational drug. Selected animal models used in ophthalmology, infectious diseases, oncology, autoimmune diseases, and neuroscience are reviewed here. Each area has specific challenges around translatability and determination of an efficacious dose: new patient-specific dosing methods may help overcome these limitations.


Assuntos
Drogas em Investigação , Oncologia , Animais , Modelos Biológicos
6.
Clin Pharmacol Ther ; 109(6): 1395-1415, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-32757299

RESUMO

Various approaches to first-in-human (FIH) starting dose selection for new molecular entities (NMEs) are designed to minimize risk to trial subjects. One approach uses the minimum anticipated biological effect level (MABEL), which is a conservative method intended to maximize subject safety and designed primarily for NMEs having high perceived safety risks. However, there is concern that the MABEL approach is being inappropriately used for lower risk molecules with negative impacts on drug development and time to patient access. In addition, ambiguity exists in how MABEL is defined and the methods used to determine it. The International Consortium for Innovation and Quality in Pharmaceutical Development convened a working group to understand current use of MABEL and its impact on FIH starting dose selection, and to make recommendations for FIH dose selection going forward. An industry-wide survey suggested the achieved or estimated maximum tolerated dose, efficacious dose, or recommended phase II dose was > 100-fold higher than the MABEL-based starting dose for approximately one third of NMEs, including trials in patients. A decision tree and key risk factor table were developed to provide a consistent, data driven-based, and risk-based approach for selecting FIH starting doses.


Assuntos
Ensaios Clínicos como Assunto/normas , Desenvolvimento de Medicamentos/métodos , Preparações Farmacêuticas/administração & dosagem , Ensaios Clínicos como Assunto/legislação & jurisprudência , Ensaios Clínicos Fase III como Assunto , Desenvolvimento de Medicamentos/legislação & jurisprudência , Indústria Farmacêutica , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Humanos , Dose Máxima Tolerável , Projetos de Pesquisa , Inquéritos e Questionários , Experimentação Humana Terapêutica , Toxicologia
7.
Eur J Immunol ; 39(4): 1025-35, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19266487

RESUMO

Human invariant NKT (iNKT) cells are a unique subset of T cells, which recognize glycolipids presented by the CD1d. Among the iNKT cells, several functionally distinct subsets have been characterized according to CD4 and/or CD8 co-receptor expression. The current study is focussed on the CD4(+) iNKT cell subset and its role in an anti-infectious response. We have examined the role of CD4(+) iNKT cells on the intracellular Brucella suis growth. Our results indicate that CD4(+) iNKT cells impair the intramacrophagic growth of Brucella. This inhibition is due to a combination of soluble and contact-dependent mechanisms: IFN-gamma is weakly involved while cytotoxic activities such as the induction of the Fas pathway and the release of lytic granules are major mechanisms. The impairment of Brucella growth by CD4(+) iNKT cells requires an interaction with CD1d on macrophage surface. Also, we have shown that although CD4 regulates several biological responses of CD4(+) iNKT cells, it is not involved in their antibacterial activity. Here, we have shown for the first time that the CD4(+) iNKT cell population has antibacterial activity and thus, participates directly in the elimination of bacteria and/or in the control of bacterial growth by killing infected cells.


Assuntos
Antígenos CD1d/imunologia , Brucella suis/imunologia , Brucelose/imunologia , Antígenos CD4/imunologia , Macrófagos/imunologia , Células T Matadoras Naturais/imunologia , Degranulação Celular/imunologia , Citotoxicidade Imunológica/imunologia , Proteína Ligante Fas/imunologia , Proteína Ligante Fas/metabolismo , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-4/imunologia , Interleucina-4/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Células T Matadoras Naturais/metabolismo , Células T Matadoras Naturais/microbiologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Receptor fas/imunologia , Receptor fas/metabolismo
8.
Clin Pharmacol Ther ; 107(4): 853-857, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31955414

RESUMO

The availability of multidimensional data together with the development of modern techniques for data analysis represent an exceptional opportunity for clinical pharmacology. Data science-defined in this special issue as the novel approaches to the collection, aggregation, and analysis of data-can significantly contribute to characterize drug-response variability at the individual level, thus enabling clinical pharmacology to become a critical contributor to personalized healthcare through precision dosing. We propose a minireview of methodologies for achieving precision dosing with a focus on an artificial intelligence technique called reinforcement learning, which is currently used for individualizing dosing regimen in patients with life-threatening diseases. We highlight the interplay of such techniques with conventional pharmacokinetic/pharmacodynamic approaches and discuss applicability in drug research and early development.


Assuntos
Inteligência Artificial , Aprendizagem , Modelos Teóricos , Farmacologia Clínica/métodos , Medicina de Precisão/métodos , Reforço Psicológico , Inteligência Artificial/normas , Relação Dose-Resposta a Droga , Humanos , Farmacologia Clínica/normas , Medicina de Precisão/normas
9.
J Leukoc Biol ; 84(1): 224-33, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18456817

RESUMO

NKT cells belong to a conserved T lymphocyte subgroup that has been implicated in the regulation of various immune responses, including responses to viruses, bacteria, and parasites. They express a semi-invariant TCR that recognizes glycolipids presented by the nonpolymorphic MHC class I-like molecule CD1d, and upon activation, they produce various pro- and anti-inflammatory cytokines. Recent studies have shed light on the nature of glycolipids and the environmental signals that may influence the production of cytokines by NKT cells and thus, modulate the immune response. To better understand the regulation mechanisms of NKT cells, we explored their behavior following activation by IL-2 and investigated the signaling pathways and biological responses triggered. We demonstrated that IL-2 activates not only STAT3 and -5 and the PI-3K and ERK-2 pathways as in all IL-2 responder cells but also STAT4 as in NK cells and the p38 MAPK pathway as in alphabeta T cells. We also showed that STAT6 is activated by IL-2 in NKT cells. Moreover, IL-2 induces the production of IFN-gamma and IL-4. The ability of IL-2 to induce pro- and anti-inflammatory cytokine production, in addition to proliferation, could open new therapeutic approaches for use in combination with molecules that activate NKT cells through TCR activation.


Assuntos
Citocinas/biossíntese , Inflamação/imunologia , Interleucina-2/farmacologia , Células Matadoras Naturais/imunologia , Transdução de Sinais/efeitos dos fármacos , DNA/metabolismo , Ativação Enzimática/efeitos dos fármacos , Humanos , Interferon gama/biossíntese , Subunidade alfa de Receptor de Interleucina-2/imunologia , Interleucina-4/biossíntese , Células Matadoras Naturais/enzimologia , Ativação Linfocitária/efeitos dos fármacos , Subpopulações de Linfócitos/efeitos dos fármacos , Subpopulações de Linfócitos/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fenótipo , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Receptores de Antígenos de Linfócitos T/imunologia , Fator de Transcrição STAT4/metabolismo , Fator de Transcrição STAT6/genética , Transcrição Gênica/efeitos dos fármacos
10.
MAbs ; 9(5): 781-791, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28440708

RESUMO

Monoclonal antibodies (mAbs) are a rapidly growing drug class for which great efforts have been made to optimize certain molecular features to achieve the desired pharmacokinetic (PK) properties. One approach is to engineer the interactions of the mAb with the neonatal Fc receptor (FcRn) by introducing specific amino acid sequence mutations, and to assess their effect on the PK profile with in vivo studies. Indeed, FcRn protects mAbs from intracellular degradation, thereby prolongs antibody circulation time in plasma and modulates its systemic clearance. To allow more efficient and focused mAb optimization, in vitro input that helps to identify and quantitatively predict the contribution of different processes driving non-target mediated mAb clearance in vivo and supporting translational PK modeling activities is essential. With this aim, we evaluated the applicability and in vivo-relevance of an in vitro cellular FcRn-mediated transcytosis assay to explain the PK behavior of 25 mAbs in rat or monkey. The assay was able to capture species-specific differences in IgG-FcRn interactions and overall correctly ranked Fc mutants according to their in vivo clearance. However, it could not explain the PK behavior of all tested IgGs, indicating that mAb disposition in vivo is a complex interplay of additional processes besides the FcRn interaction. Overall, the transcytosis assay was considered suitable to rank mAb candidates for their FcRn-mediated clearance component before extensive in vivo testing, and represents a first step toward a multi-factorial in vivo clearance prediction approach based on in vitro data.


Assuntos
Anticorpos Monoclonais Murinos/farmacocinética , Bioensaio/métodos , Antígenos de Histocompatibilidade Classe I/imunologia , Receptores Fc/imunologia , Transcitose/imunologia , Animais , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Monoclonais Murinos/farmacologia , Macaca fascicularis , Camundongos , Ratos , Ratos Wistar
11.
J Leukoc Biol ; 77(5): 652-60, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15668339

RESUMO

Human Vgamma9Vdelta2 T cells play a crucial role in early immune response to intracellular pathogens. In brucellosis infection, this population of cells is drastically increased in the peripheral blood of patients during the acute phase of infection. In vitro, Vgamma9Vdelta2 T cells exhibit strong cytolytic activity against Brucella-infected cells and are able to impair intracellular growth of Brucella suis in autologous macrophages. In this study, we have investigated the relative importance of contact-dependent mechanisms versus soluble factors in the intracellular growth and viability of B. suis. We show that Vgamma9Vdelta2 T cells use contact-dependent mechanisms, such as the release of lytic granules and Fas-mediated signals, to decrease intracellular B. suis through lysis of infected macrophages, but these mechanisms have little impact on Brucella survival. Moreover, we demonstrate that soluble factors secreted by Vgamma9Vdelta2 T cells can directly affect B. suis survival through their potent bactericidal effects. From these results, we conclude that Vgamma9Vdelta2 T cells are able to use a combination of mechanisms that reduce the total numbers of B. suis and thus, may benefit the host by limiting the spread of this intracellular pathogen.


Assuntos
Atividade Bactericida do Sangue/imunologia , Brucella suis/imunologia , Brucelose/imunologia , Comunicação Celular/imunologia , Monócitos/imunologia , Receptores de Antígenos de Linfócitos T gama-delta , Subpopulações de Linfócitos T/imunologia , Sobrevivência Celular/imunologia , Células Cultivadas , Exocitose/imunologia , Proteína Ligante Fas , Humanos , Interferon gama/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Glicoproteínas de Membrana/imunologia , Monócitos/microbiologia , Fator de Necrose Tumoral alfa/imunologia , Receptor fas/imunologia
12.
J Immunother ; 39(7): 279-89, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27404941

RESUMO

CEA TCB is a novel T-cell-bispecific (TCB) antibody targeting the carcinoembryonic antigen (CEA) expressed on tumor cells and the CD3 epsilon chain (CD3e) present on T cells, which is currently in Phase 1 clinical trials (NCT02324257) for the treatment of CEA-positive solid tumors. Because the human CEA (hCEA) binder of CEA TCB does not cross-react with cynomolgus monkey and CEA is absent in rodents, alternative nonclinical safety evaluation approaches were considered. These included the development of a cynomolgus monkey cross-reactive homologous (surrogate) antibody (cyCEA TCB) for its evaluation in cynomolgus monkey and the development of double-transgenic mice, expressing hCEA and human CD3e (hCEA/hCD3e Tg), as a potential alternative species for nonclinical safety studies. However, a battery of nonclinical in vitro/ex vivo experiments demonstrated that neither of the previous approaches provided a suitable and pharmacologically relevant model to assess the safety of CEA TCB. Therefore, an alternative approach, a minimum anticipated biological effect level (MABEL), based on an in vitro tumor lysis assay was used to determine the starting dose for the first-in-human study. Using the most conservative approach to the MABEL assessment, a dose of 52 µg was selected as a safe starting dose for clinical study.


Assuntos
Anticorpos Biespecíficos/metabolismo , Complexo CD3/imunologia , Antígeno Carcinoembrionário/imunologia , Imunoterapia/métodos , Neoplasias/terapia , Animais , Apoptose , Células Cultivadas , Ensaios Clínicos Fase I como Assunto , Reações Cruzadas , Cálculos da Dosagem de Medicamento , Avaliação Pré-Clínica de Medicamentos , Humanos , Macaca fascicularis , Camundongos , Camundongos Transgênicos , Neoplasias/imunologia , Ratos , Homologia Estrutural de Proteína
13.
Clin Cancer Res ; 22(13): 3286-97, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-26861458

RESUMO

PURPOSE: CEA TCB is a novel IgG-based T-cell bispecific (TCB) antibody for the treatment of CEA-expressing solid tumors currently in phase I clinical trials (NCT02324257). Its format incorporates bivalent binding to CEA, a head-to-tail fusion of CEA- and CD3e-binding Fab domains and an engineered Fc region with completely abolished binding to FcγRs and C1q. The study provides novel mechanistic insights into the activity and mode of action of CEA TCB. EXPERIMENTAL DESIGN: CEA TCB activity was characterized on 110 cell lines in vitro and in xenograft tumor models in vivo using NOG mice engrafted with human peripheral blood mononuclear cells. RESULTS: Simultaneous binding of CEA TCB to tumor and T cells leads to formation of immunologic synapses, T-cell activation, secretion of cytotoxic granules, and tumor cell lysis. CEA TCB activity strongly correlates with CEA expression, with higher potency observed in highly CEA-expressing tumor cells and a threshold of approximately 10,000 CEA-binding sites/cell, which allows distinguishing between high- and low-CEA-expressing tumor and primary epithelial cells, respectively. Genetic factors do not affect CEA TCB activity confirming that CEA expression level is the strongest predictor of CEA TCB activity. In vivo, CEA TCB induces regression of CEA-expressing xenograft tumors with variable amounts of immune cell infiltrate, leads to increased frequency of activated T cells, and converts PD-L1 negative into PD-L1-positive tumors. CONCLUSIONS: CEA TCB is a novel generation TCB displaying potent antitumor activity; it is efficacious in poorly infiltrated tumors where it increases T-cell infiltration and generates a highly inflamed tumor microenvironment. Clin Cancer Res; 22(13); 3286-97. ©2016 AACR.


Assuntos
Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Antígeno Carcinoembrionário/imunologia , Imunoterapia/métodos , Neoplasias/terapia , Linfócitos T/imunologia , Animais , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Antineoplásicos/imunologia , Sítios de Ligação/imunologia , Complexo CD3/imunologia , Linhagem Celular Tumoral , Feminino , Humanos , Ativação Linfocitária/imunologia , Camundongos , Receptores Fc/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
15.
AAPS J ; 17(4): 1019-24, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25921938

RESUMO

A novel format was introduced at the recent AAPS NBC Workshop on Method Development, Validation and Troubleshooting in San Diego on 18th May 2014. The workshop format was initiated by Binodh De Silva; Marie Rock and Sherri Dudal joined the initiative to develop and chair the workshop. Questions were solicited by a variety of avenues, including a Linked-In Discussion Group. Once collated and clarified, the topics covered assay development, validation, and analysis of PK, Immunogenicity, and Biomarkers with an additional topic on alternative bioanalytical technologies. A panel of experts (workshop report co-authors) was assigned to each topic to bring forward thought-provoking aspects of each topic. The format of the workshop was developed to target the needs of bioanalytical scientists with intermediate to advanced experience in the field ranging to enable robust discussion and to delve deeper into the current bioanalytical hot topics. While the new format allowed for an interactive session with the topical discussion driven by the audience members, it did not foster equal discussion time for all of the proposed topics, especially Biomarkers and alternative LBA technologies.


Assuntos
Bioensaio/métodos , Biomarcadores/análise , Farmacocinética , Humanos , Ligantes , Estudos de Validação como Assunto
16.
AAPS J ; 17(2): 277-88, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25549614

RESUMO

In September 2013, the FDA released a draft revision of the Bioanalytical Method Validation (BMV) Guidance, which included a number of changes to the expectations for bioanalysis, most notably the inclusion of biomarker assays and data. To provide a forum for an open, inclusive discussion of the revised draft BMV Guidance, the AAPS and FDA once again collaborated to convene a two-and-a-half day workshop during early December 2013 in Baltimore, MD, USA. The resulting format embodied extensive open discussion and each thematic session included only brief, concise descriptions by Agency and industry representatives prior to opening the floor discussion. The Workshop was built around four thematic sessions (Common Topics, Chromatographic, Ligand-Binding Assays, and Biomarkers) and a final session with international regulators, concluding with a review of the outcomes and recommendations from the thematic sessions. This Workshop report summarizes the outcomes and includes topics of agreement, those where the FDA will consider the Industry's perspective, and those where the workshop provided a first open dialogue. This article will be available to the bioanalytical community at http://www.aaps.org/BMV13 .


Assuntos
Bioensaio/métodos , Biomarcadores/análise , Bioensaio/normas , Regulamentação Governamental , Guias como Assunto , Humanos , Estados Unidos , United States Food and Drug Administration , Estudos de Validação como Assunto
17.
Neurobiol Aging ; 25(7): 861-71, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15212840

RESUMO

Alzheimer's disease (AD) is characterized by a progressive cognitive decline leading to dementia and involves the deposition of amyloid-beta (Abeta) peptides into senile plaques. Other neuropathological features that accompany progression of the disease include a decrease in synaptic density, neurofibrillary tangles, dystrophic neurites, inflammation, and neuronal cell loss. In this study, we report the early kinetics of brain amyloid deposition and its associated inflammation in an early onset transgenic mouse model of AD (TgCRND8) harboring the human amyloid precursor protein gene with the Indiana and Swedish mutations. Both diffuse and compact plaques were detected as early as 9-10 weeks of age. Abeta-immunoreactive (Abeta-IR) plaques (4G8-positive) appeared first in the neocortex and amygdala, then in the hippocampal formation, and lastly in the thalamus. Compact plaques (ThioS-positive) with an amyloid core were observed as early as diffuse plaques were detected, but in lower numbers. Amyloid deposition increased progressively with age. The formation of plaques was concurrent with the appearance of activated microglial cells and shortly followed by the clustering of activated astrocytes around plaques at 13-14 weeks of age. This TgCRND8 mouse model allows for a rapid, time-dependent study of the relationship between the fibrillogenic process and the inflammatory response during the brain amyloidogenic process.


Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Inflamação/metabolismo , Placa Amiloide/metabolismo , Fatores Etários , Doença de Alzheimer/genética , Doença de Alzheimer/imunologia , Doença de Alzheimer/patologia , Tonsila do Cerebelo/imunologia , Tonsila do Cerebelo/metabolismo , Tonsila do Cerebelo/patologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/imunologia , Animais , Benzotiazóis , Encéfalo/imunologia , Encéfalo/patologia , Antígeno CD11b/imunologia , Antígeno CD11b/metabolismo , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida/metabolismo , Hipocampo/imunologia , Hipocampo/metabolismo , Hipocampo/patologia , Imuno-Histoquímica , Inflamação/imunologia , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/imunologia , Microglia/metabolismo , Neocórtex/imunologia , Neocórtex/metabolismo , Neocórtex/patologia , Placa Amiloide/genética , Placa Amiloide/imunologia , Placa Amiloide/patologia , Tiazóis/metabolismo
18.
J Neuroimmunol ; 153(1-2): 26-35, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15265660

RESUMO

The microglial inflammatory response to Abeta(1-42) stimulation with or without IFN-gamma priming was investigated in low and high responder strains of mice, A/J and C57BL/6, respectively. A/J microglia showed moderate morphological changes upon stimulation with IFN-gamma alone or with Abeta(1-42). Conversely, C57BL/6 microglia showed major changes in their cellular morphology, which were accompanied by a decrease in NO release and a marked increase in TNF-alpha production. These results indicate that the magnitude of the microglial inflammatory response to Abeta is strongly influenced by genetic factors. Individual differences in the regulation of the microglial response may be a key player in the rate of development of the neuropathology of AD.


Assuntos
Peptídeos beta-Amiloides/toxicidade , Camundongos Endogâmicos C57BL/imunologia , Microglia/efeitos dos fármacos , Fragmentos de Peptídeos/toxicidade , Peptídeos beta-Amiloides/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Células Cultivadas , Sinergismo Farmacológico , Ensaio de Imunoadsorção Enzimática/métodos , Imuno-Histoquímica/métodos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Interferon gama/farmacologia , Camundongos , Microglia/metabolismo , Microglia/patologia , Microscopia Imunoeletrônica/métodos , Nitritos/metabolismo , Fragmentos de Peptídeos/metabolismo , Especificidade da Espécie , Fator de Necrose Tumoral alfa/metabolismo
19.
AAPS J ; 16(2): 194-205, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24343771

RESUMO

As part of the GBC (Global Bioanalysis Consortium), the L3 assay format team has focused on reviewing common platforms used to support ligand binding assays in the detection of biotherapeutics. The following review is an overview of discussions and presentations from around the globe with a group of experts from different companies to allow an international harmonization of common practices and suggestions for different platforms. Some of the major platforms include Gyrolab, Erenna, RIA, AlphaLISA, Delfia, Immuno-PCR, Luminex, BIAcore, and ELISAs. The review is meant to support bioanalysts in taking decisions between different platforms depending on the needs of the analyte with a number of recommendations to help integration of platforms into a GLP environment.


Assuntos
Comportamento Cooperativo , Guias de Prática Clínica como Assunto , Ensaio de Imunoadsorção Enzimática , Ligantes , Reação em Cadeia da Polimerase , Radioimunoensaio
20.
Bioanalysis ; 6(10): 1339-48, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24958118

RESUMO

The bioanalytical scientist plays a key role in the project team for the drug development of biotherapeutics from the discovery to the marketing phase. Information from the project team members is required for assay development and sample analysis during the discovery, preclinical and clinical phases of the project and input is needed from the bioanalytical scientist to help data interpretation. The European Bioanalysis Forum target team 20 discussed many of the gaps in information and communication between the bioanalytical scientist and project team members as a base for providing a perspective on the bioanalytical scientist's role and interactions within the project team.


Assuntos
Preparações Farmacêuticas/análise , Avaliação de Medicamentos/normas , Europa (Continente) , Serviços Terceirizados , Preparações Farmacêuticas/metabolismo , Preparações Farmacêuticas/normas , Controle de Qualidade
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