RESUMEN
Biologics have revolutionized disease management in many therapeutic areas by addressing unmet medical needs and overcoming resistance to standard-of-care treatment in numerous patients. However, the development of unwanted immune responses directed against these drugs, humoral and/or cellular, can hinder their efficacy and have safety consequences with various degrees of severity. Health authorities ask that a thorough immunogenicity risk assessment be conducted during drug development to incorporate an appropriate monitoring and mitigation plan in clinical studies. With the rapid diversification and complexification of biologics, which today include modalities such as multi-domain antibodies, cell-based products, AAV delivery vectors, and nucleic acids, developers are faced with the challenge of establishing a risk assessment strategy sometimes in the absence of specific regulatory guidelines. The European Immunogenicity Platform (EIP) Open Symposium on Immunogenicity of Biopharmaceuticals and its one-day training course gives experts and newcomers across academia, industry, and regulatory agencies an opportunity to share experience and knowledge to overcome these challenges. Here, we report the discussions that took place at the EIP's 14th Symposium, held in April 2023. The topics covered included immunogenicity monitoring and clinical relevance, non-clinical immunogenicity risk assessment, regulatory aspects of immunogenicity assessment and reporting, and the challenges associated with new modalities, which were discussed in a dedicated session.
Asunto(s)
Productos Biológicos , Humanos , Anticuerpos , Desarrollo de Medicamentos , Medición de RiesgoRESUMEN
Evolving immunogenicity assay performance expectations and a lack of harmonized neutralizing antibody validation testing and reporting tools have resulted in significant time spent by health authorities and sponsors on resolving filing queries. A team of experts within the American Association of Pharmaceutical Scientists' Therapeutic Product Immunogenicity Community across industry and the Food and Drug Administration addressed challenges unique to cell-based and non-cell-based neutralizing antibody assays. Harmonization of validation expectations and data reporting will facilitate filings to health authorities and are described in this manuscript. This team provides validation testing and reporting strategies and tools for the following assessments: (1) format selection; (2) cut point; (3) assay acceptance criteria; (4) control precision; (5) sensitivity including positive control selection and performance tracking; (6) negative control selection; (7) selectivity/specificity including matrix interference, hemolysis, lipemia, bilirubin, concomitant medications, and structurally similar analytes; (8) drug tolerance; (9) target tolerance; (10) sample stability; and (11) assay robustness.
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Anticuerpos Neutralizantes , Preparaciones Farmacéuticas , Tolerancia a MedicamentosRESUMEN
The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "Context of Use - COU"); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine) and Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) are published in volume 14 of Bioanalysis, issues 9 and 10 (2022), respectively.
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Receptores Quiméricos de Antígenos , Vacunas , Biomarcadores/análisis , Sistemas CRISPR-Cas , Tratamiento Basado en Trasplante de Células y Tejidos , Humanos , Inmunoterapia Activa , Reacción en Cadena de la PolimerasaRESUMEN
Evolving immunogenicity assay performance expectations and a lack of harmonized anti-drug antibody validation testing and reporting tools have resulted in significant time spent by health authorities and sponsors on resolving filing queries. Following debate at the American Association of Pharmaceutical Sciences National Biotechnology Conference, a group was formed to address these gaps. Over the last 3 years, 44 members from 29 organizations (including 5 members from Europe and 10 members from FDA) discussed gaps in understanding immunogenicity assay requirements and have developed harmonization tools for use by industry scientists to facilitate filings to health authorities. Herein, this team provides testing and reporting strategies and tools for the following assessments: (1) pre-study validation cut point; (2) in-study cut points, including procedures for applying cut points to mixed populations; (3) system suitability control criteria for in-study plate acceptance; (4) assay sensitivity, including the selection of an appropriate low positive control; (5) specificity, including drug and target tolerance; (6) sample stability that reflects sample storage and handling conditions; (7) assay selectivity to matrix components, including hemolytic, lipemic, and disease state matrices; (8) domain specificity for multi-domain therapeutics; (9) and minimum required dilution and extraction-based sample processing for titer reporting.
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Anticuerpos , Bioensayo , Europa (Continente) , Estados UnidosRESUMEN
The 14th edition of the Workshop on Recent Issues in Bioanalysis (14th WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14th WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by LCMS were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the Global Bioanalytical Community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity). Part 1 (Innovation in Small Molecules, Hybrid LBA/LCMS & Regulated Bioanalysis), Part 2A (BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation) and Part 2B (Regulatory Input) are published in volume 13 of Bioanalysis, issues 4 and 5 (2020), respectively.
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Tratamiento Basado en Trasplante de Células y Tejidos , Citometría de Flujo , Terapia Genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Vacunas/análisis , Humanos , Control de Calidad , Receptores Quiméricos de Antígenos/análisis , Estados Unidos , United States Food and Drug AdministrationRESUMEN
The 2019 13th Workshop on Recent Issues in Bioanalysis (WRIB) took place in New Orleans, LA on 1-5 April 2019 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers, immunogenicity and gene therapy. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS, LBA cell-based/flow cytometry assays and qPCR approaches. This 2019 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2019 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations on the 2018 FDA BMV guidance, 2019 ICH M10 BMV draft guideline and regulatory agencies' input on bioanalysis, biomarkers, immunogenicity and gene therapy. Part 1 (Innovation in small molecules and oligonucleotides and mass spectrometry method development strategies for large molecules bioanalysis) and Part 3 (New insights in biomarker assay validation, current and effective strategies for critical reagent management, flow cytometry validation in drug discovery and development and CLSI H62, interpretation of the 2019 FDA immunogenicity guidance and gene therapy bioanalytical challenges) are published in volume 10 of Bioanalysis, issues 22 and 24 (2019), respectively.
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Bioensayo/normas , Biomarcadores/análisis , Guías como Asunto , Fenómenos Inmunogenéticos , Informe de Investigación , United States Food and Drug Administration/legislación & jurisprudencia , Humanos , Estados UnidosRESUMEN
The 2018 12th Workshop on Recent Issues in Bioanalysis took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations for PK, PD and ADA assays by hybrid LBA/LCMS and regulatory agencies' input. Part 1 (LCMS for small molecules, peptides, oligonucleotides and small molecule biomarkers) and Part 3 (LBA/cell-based assays: immunogenicity, biomarkers and PK assays) are published in volume 10 of Bioanalysis, issues 22 and 24 (2018), respectively.
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Antígenos/análisis , Bioensayo/normas , Biomarcadores/análisis , Legislación Médica/tendencias , Estados UnidosRESUMEN
Therapeutic proteins provide interventions for some of the most complex and intractable diseases and are an essential part of modern medicine. Immunogenicity is the development of immune responses, usually measured by antibodies, to therapeutic proteins. These responses can adversely affect the safety and efficacy of the therapeutic agent and may have the following consequences: neutralization of a life-saving biotherapeutic, crossreactivity to non-redundant endogenous proteins, and hypersensitivity responses. These concerns have been underscored by the discontinuation of development of several drugs in recent years owing to immunogenicity issues. We review here recent progress in technological approaches that are useful for the clinical and non-clinical risk assessment of immunogenicity as well as mitigation strategies including deimmunizing protein molecules and inducing immune tolerance to the therapeutic protein.
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Productos Biológicos/inmunología , Desarrollo de Medicamentos/métodos , Proteínas/inmunología , Productos Biológicos/administración & dosificación , Productos Biológicos/efectos adversos , Proteínas/administración & dosificación , Proteínas/efectos adversosRESUMEN
European Bioanalysis Forum Workshop, Lisbon, Portugal, September 2016: At the recent European Bioanalysis Forum Focus Workshop, 'current analysis of immunogenicity: best practices and regulatory hurdles', several important challenges facing the bioanalytical community in relation to immunogenicity assays were discussed through a mixture of presentations and panel sessions. The main areas of focus were the evolving regulatory landscape, challenges of assay interferences from either drug or target, cut-point setting and whether alternative assays can be used to replace neutralizing antibody assays. This workshop report captures discussions and potential solutions and/or recommendations made by the speakers and delegates.
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Anticuerpos Neutralizantes/análisis , Productos Biológicos/inmunología , Tolerancia a Medicamentos/inmunología , Inmunoensayo/normas , Productos Biológicos/uso terapéutico , Descubrimiento de Drogas , Guías como Asunto , HumanosRESUMEN
The 2017 11th Workshop on Recent Issues in Bioanalysis (11th WRIB) took place in Los Angeles/Universal City, California on 3-7 April 2017 with participation of close to 750 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event - a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecule analysis involving LCMS, hybrid ligand binding assay (LBA)/LCMS and LBA approaches. This 2017 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2017 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations for biotherapeutics, biomarkers and immunogenicity assays using hybrid LBA/LCMS and regulatory agencies' inputs. Part 1 (LCMS for small molecules, peptides and small molecule biomarkers) and Part 3 (LBA: immunogenicity, biomarkers and pharmacokinetic assays) are published in Volume 9 of Bioanalysis, issues 22 and 24 (2017), respectively.
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Biomarcadores/análisis , Inmunidad Activa , Espectrometría de Masas , Cromatografía Líquida de Alta Presión , Conferencias de Consenso como Asunto , Regulación Gubernamental , LigandosRESUMEN
The 2017 11th Workshop on Recent Issues in Bioanalysis took place in Los Angeles/Universal City, California, on 3-7 April 2017 with participation of close to 750 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule analysis involving LC-MS, hybrid ligand-binding assay (LBA)/LC-MS and LBA approaches. This 2017 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2017 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for large-molecule bioanalysis, biomarkers and immunogenicity using LBA. Part 1 (LC-MS for small molecules, peptides and small molecule biomarkers) and Part 2 (hybrid LBA/LC-MS for biotherapeutics and regulatory agencies' inputs) are published in volume 9 of Bioanalysis, issues 22 and 23 (2017), respectively.
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Biomarcadores/análisis , Inmunidad Activa , Cromatografía Liquida , Conferencias de Consenso como Asunto , Tolerancia a Medicamentos , Guías como Asunto , Ligandos , Espectrometría de Masas , FarmacocinéticaRESUMEN
Neonates are at increased risk of viral encephalopathies that can result in neurological dysfunction, seizures, permanent disability and even death. The neurological damage results from the combined effect of the virus and the immune response it elicits, thus finding tools to facilitate viral clearance from central nervous system (CNS) while minimizing neuron damage remains a critical challenge. Neonatal mice inoculated intraperitoneally with Tacaribe virus (TCRV) develop seizures, hindlimb paralysis and death within 15 days of inoculation. TCRV localizes to the CNS within days of challenge, primarily infecting astrocytes in the cerebellum and brain stem. We show that infection leads to inflammation, T cell and monocyte infiltration into the cerebellar parenchyma, apoptosis of astrocytes, neuronal degeneration and loss of Purkinje cells. Infiltrating antigen-specific T cells fail to clear the virus but drive the disease, as T-cell-deficient CD3É KO mice survive TCRV infection with minimal inflammation or clinical manifestations despite no difference in CNS viral loads in comparison with T-cell sufficient mice. CD8+ T cells drive the pathology, which even in the absence of CD4+ T-cell help, infiltrate the parenchyma and mediate the apoptotic loss of cerebellar astrocytes, neurodegeneration and loss of Purkinje cells resulting in loss of balance, paralysis and death. CD4+ T cells are also pathogenic inducing gliosis and inflammation in the cerebellum and cerebrum that are associated with wasting and death several weeks after CD4+ T-cell transfer. These data demonstrate distinct pathogenic effects of CD4+ and CD8+ T cells and identify them as possible therapeutic targets.
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Infecciones por Arenaviridae/inmunología , Infecciones por Arenaviridae/virología , Arenavirus/fisiología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Meningoencefalitis/inmunología , Meningoencefalitis/virología , Animales , Animales Recién Nacidos , Apoptosis , Infecciones por Arenaviridae/patología , Astrocitos/patología , Sistema Nervioso Central/patología , Sistema Nervioso Central/virología , Gliosis/patología , Meningoencefalitis/patología , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/patología , Degeneración Nerviosa/patología , Neuronas/patología , Células de Purkinje/patología , Linfocitos T/inmunologíaRESUMEN
The 2016 10th Workshop on Recent Issues in Bioanalysis (10th WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event - A Full Immersion Week of Bioanalysis including Biomarkers and Immunogenicity. As usual, it is specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecules involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on biomarkers and immunogenicity. This 2016 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. This White Paper is published in 3 parts due to length. This part (Part 2) discusses the recommendations for Hybrid LBA/LCMS and regulatory inputs from major global health authorities. Parts 1 (small molecule bioanalysis using LCMS) and Part 3 (large molecule bioanalysis using LBA, biomarkers and immunogenicity) have been published in the Bioanalysis journal, issues 22 and 23, respectively.
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Biomarcadores/análisis , Cromatografía Líquida de Alta Presión , Espectrometría de Masas , Anticuerpos Antiidiotipos/análisis , Anticuerpos Antiidiotipos/inmunología , Conferencias de Consenso como Asunto , Agencias Gubernamentales , Humanos , Inmunoensayo , Ligandos , Estudios de Validación como AsuntoRESUMEN
The 2016 10th Workshop on Recent Issues in Bioanalysis (10th WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a weeklong event - A Full Immersion Week of Bioanalysis for PK, Biomarkers and Immunogenicity. As usual, it is specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecules involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on PK, biomarkers and immunogenicity. This 2016 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. This White Paper is published in 3 parts due to length. This part (Part 3) discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Parts 1 (small molecule bioanalysis using LCMS) and Part 2 (Hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in the Bioanalysis journal, issues 22 and 23, respectively.
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Biomarcadores/análisis , Ligandos , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacocinética , Cromatografía Líquida de Alta Presión , Conferencias de Consenso como Asunto , Agencias Gubernamentales , Humanos , Sustancias Macromoleculares/análisis , Sustancias Macromoleculares/inmunología , Sustancias Macromoleculares/farmacocinética , Espectrometría de Masas , Estudios de Validación como AsuntoRESUMEN
Most biotherapeutics can elicit immune responses in dosed recipients generating anti-drug antibodies (ADAs). Neutralizing antibodies (NAbs) are a subpopulation of ADAs that can potentially impact patient safety and directly mediate loss of drug efficacy by blocking the biological activity of a therapeutic product. Therefore, NAb detection is an important aspect of immunogenicity assessment, requiring sensitive and reliable methods reflective of the therapeutic mechanism of action (MoA). Both cell-based and non cell-based assays are viable options for NAb assessment. However, the scientific approach for the selection of a suitable assay format (cell-based or non cell-based) for NAb assessment is not currently well defined. In this manuscript, the authors summarize the design and utility of cell-based and non cell-based NAb assays and recommend a NAb assay format selection approach that relies on a combination of three factors. These include (i) the therapeutic MoA, (ii) the evidence of desirable assay performance characteristics, and (iii) risk of immunogenicity. The utility of correlating NAb response with pharmacodynamic data is also discussed. The aim of this paper is to provide a consistent strategy that will guide the selection of scientifically justified assay formats capable of detecting clinically relevant NAbs for biotherapeutics with varying MoAs and diverse complexity.
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Anticuerpos Neutralizantes/inmunología , Productos Biológicos/inmunología , Animales , HumanosRESUMEN
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.
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Bioensayo/métodos , Biomarcadores/análisis , Farmacocinética , Humanos , Ligandos , Estudios de Validación como AsuntoRESUMEN
Macrophages coexpress both the interleukin (IL)-2Rγ chain (γ(c)) and IL-13Rα1. These receptor chains can heterodimerize with IL-4Rα to form type I or type II IL-4 receptor complexes, respectively. We used macrophages derived from Il2rg and Il13ra1 knockout (KO) mice to evaluate the requirements for these receptor chains for induction of the alternative macrophage activation (AMA) pathway by IL-4 and IL-13. Absence of γ(c) significantly decreased activation of STAT6 by IL-4 but not IL-13. However, although activation of STAT6 by IL-4 was markedly reduced in γ(c) KO macrophages, it was not abolished, indicating that IL-4 can still signal through type II IL-4 receptors via the IL-13Rα1 chain. IL-13 failed to activate STAT6 in macrophages derived from Il13ra1 KO mice; however, these cells remained fully responsive to IL-4. The inability of IL-13 but not IL-4 to signal in Il13ra1(-/-) macrophages correlated with the inability of IL-13 but not IL-4 to induce expression of genes such as Arg1, Retnla and Ccl11 that are characteristically expressed by alternatively activated macrophages. In addition, IL-13 but not IL-4 failed to induce membrane fusion and giant cell formation by Il13ra1 KO macrophages. These findings demonstrate that the IL-13Rα1 chain is essential for induction of the AMA pathway by IL-13 but not IL-4.
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Vía Alternativa del Complemento , Subunidad alfa1 del Receptor de Interleucina-13/metabolismo , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Macrófagos/inmunología , Animales , Arginasa/genética , Arginasa/metabolismo , Antígenos CD11/genética , Antígenos CD11/metabolismo , Células Cultivadas , Vía Alternativa del Complemento/genética , Regulación de la Expresión Génica/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Subunidad gamma Común de Receptores de Interleucina/genética , Interleucina-13/genética , Subunidad alfa1 del Receptor de Interleucina-13/genética , Interleucina-4/genética , Fusión de Membrana/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Transcripción STAT6/metabolismoRESUMEN
Arenaviruses merit significant attention both as causative agents of endemic hemorrhagic fevers and as model systems to study the immune response to acute and persistent viral infections. Development of highly sensitive quantitative screening methods to detect arenavirus is critical for early diagnosis of patients, to screen the rodent population in endemic areas, and as a research tool to confirm effective tissue clearance during the development of anti-viral strategies. This study describes a novel sensitive and reproducible method to quantify prototypic new world arenavirus Tacaribe RNA in cell cultures and tissues using a real-time TaqMan PCR-based detection system. The method has a sensitivity of 100 RNA copies per 200ng of total RNA, making it 2 logs more sensitive than the currently utilized TCID(50) method, and a linear range from 10(2) to 10(9) copies/reaction. The qRT-PCR method is high-throughput and screening can be achieved in <2h allowing for diagnosis of infected patients before the onset of symptoms. This new method is a powerful tool to screen populations for infection and monitor the clearance achieved by available therapies, and serves as a model diagnostic tool for other arenaviruses.
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Arenavirus del Nuevo Mundo/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , ARN Viral/genética , Animales , Chlorocebus aethiops , Ratones , Ratones Endogámicos C57BL , Sensibilidad y Especificidad , Células VeroRESUMEN
Far from being an immune-inert site, the cells of the CNS, including microglia, astrocytes and neurons, express an abundance of innate immune receptors, including toll like receptors (TLR), which recognize conserved structural motifs in pathogens. These TLRs have been implicated in both infectious and non-infectious CNS pathology and appear to play important roles in both tissue surveying and repair. Therapeutics that modulate their activity are being explored for the treatment of infectious and inflammatory diseases.
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Autoinmunidad/inmunología , Enfermedades del Sistema Nervioso Central/tratamiento farmacológico , Enfermedades del Sistema Nervioso Central/inmunología , Inmunidad Innata/inmunología , Receptores Toll-Like/metabolismo , Aminoquinolinas/uso terapéutico , Animales , Humanos , Imiquimod , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Receptores Toll-Like/agonistasRESUMEN
Viral encephalitides are life-threatening diseases in neonates partly due to the irreversible damage inflammation causes to the CNS. This study explored the role of proinflammatory cytokines in the balance between controlling viral replication and eliciting pathologic immune responses in nonlytic viral encephalitis. We show that neonatal mice challenged with arenavirus Tacaribe (TCRV) develop a meningoencephalitis characterized by high IFN-gamma and TNF-alpha levels and mild T cell infiltration. Neutralization of the TNF-alpha using mAb was associated with lower chemokine expression, reduced T cell infiltration, and lower levels of IFN-gamma, and TNF-alpha in the CNS and led to 100% survival. Moreover, treatment with Abs to TNF-alpha improved mobility and increased survival even after the mice developed bilateral hind limb paralysis. Of note, animals treated with anti-TNF-alpha Abs alone did not clear the virus despite generating Abs to TCRV. Direct activation of the innate immune response using CpG oligodeoxynucleotides in combination with anti-TNF-alpha Abs resulted in 100% survival and complete viral clearance. To our knowledge, this is the first demonstration of the use of innate immune modulators plus Abs to TNF-alpha as therapeutics for a lethal neurotropic viral infection.