RESUMO
T-cell dependent antibody responses to biotherapeutics remain a challenge to the optimal clinical application of biotherapeutics because of their capacity to impair drug efficacy and their potential to cause safety issues. To minimize this clinical immunogenicity risk, preclinical assays measuring the capacity of biotherapeutics to elicit CD4 T cell response in vitro are commonly used. However, there is considerable variability in assay formats and a general poor understanding of their respective predictive value. In this study, we evaluated the performance of three different CD4 T cell proliferation assays in their capacity to predict clinical immunogenicity: a CD8 T cell depleted peripheral blood mononuclear cells (PBMC) assay and two co-culture-based assays between dendritic cells (DCs) and autologous CD4 T cells with or without restimulation with monocytes. A panel of 10 antibodies with a wide range of clinical immunogenicity was selected. The CD8 T cell depleted PBMC assay predicted the clinical immunogenicity in four of the eight highly immunogenic antibodies included in the panel. Similarly, five antibodies with high clinical immunogenicity triggered a response in the DC: CD4 T cell assay but the responses were of lower magnitude than the ones observed in the PBMC assay. Remarkably, three antibodies with high clinical immunogenicity did not trigger any response in either platform. The addition of a monocyte restimulation step to the DC: CD4 T cell assay did not further improve its predictive value. Overall, these results indicate that there are no CD4 T cell assay formats that can predict the clinical immunogenicity of all biotherapeutics and reinforce the need to combine results from various preclinical assays assessing antigen uptake and presentation to fully mitigate the immunogenicity risk of biotherapeutics.
Assuntos
Linfócitos T CD4-Positivos , Células Dendríticas , Humanos , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Medição de Risco , Técnicas de Cocultura , Ativação Linfocitária/imunologia , Leucócitos Mononucleares/imunologia , Proliferação de Células , Linfócitos T CD8-Positivos/imunologia , Avaliação Pré-Clínica de Medicamentos , Produtos Biológicos/imunologia , Produtos Biológicos/efeitos adversos , Anticorpos/imunologia , Células CultivadasRESUMO
1. INTRODUCCIÓNLas inmunodeficiencias primarias son un grupo de más de 400 enfermedades, en las cuales el sistema inmune pierde sus funciones de reconocimiento de patógenos o funciona de forma inapropiada. Algunas de ellas son relativamente comunes; mientras otras son raras. Estas enfermedades son en ocasiones de por vida, debilitantes y costosas1,2.Sin embargo, muchos progresos se han hecho desde su des-cripción original en el año de 1952. Se han dado grandes pasos en cuanto a su entendimiento de las Inmunodeficiencias Pri-marias a nivel genético, de sus características, y tratamiento. Algunos tipos afectan un único tipo de célula; otros afectan más de un componente del sistema inmune2,3.Tomando en cuenta que la aproximación es entre 1-2% de la población, a nivel país se puede decir que un aproximado entre 170 000 a 340 000 pacientes en el país no cuentan con un diagnóstico y muchos mueren por falta de este. El número de afiliados al Instituto Ecuatoriano de Seguridad Social hasta julio de 2021 es de 3 672,611 por lo que se considera que un estimado de 36 726 a 73 452 pacientes podrían presentar este tipo de enfermedades y requerir de atención por infecciones a repetición, enfermedad autoinmune y enfermedades linfopro-liferativas, además de que sin un tratamiento específico po-drían fallecer debido a infecciones graves o tener discapacidad permanente, lo que implica mayor carga para el sistema de Seguridad Social en subsidios y menores ingresos. Ecuador, cuenta con 86 pacientes diagnosticados, según la base de datos de la Sociedad Latino-Americana de Inmunodeficiencias4.Algunas terapias, como la de reemplazo para inmunoglobu-linas, a la que es tributaria más del 60% de estas patologías permite que la esperanza de vida y la morbilidad casi alcancen a aquellos que no presentan la enfermedad57.
1. INTRODUCTIONPrimary immunodeficiencies are a group of more than 400 diseases, in which the immune system loses its pathogen recog-nition functions or functions inappropriately. Some of them are relatively common, while others are rare. These diseases are sometimes lifelong, debilitating, and costly1,2. However, much progress has been made since its original description in 1952. Great strides have been made in understanding Primary Immunodeficiencies at the genetic level, their characteristics, and treatment. Some types affect only one type of cell; others affect more than one component of the immune system2,3. Considering that the approximation is between 1 to 2% of the population, at the country level we could say that approximately between 170 000 to 340 000 patients in the country do not have a diagnosis and many die due to lack of it. The number of social security affiliates until July 2021 is 3 672,611, so we could consider that approximately 36 726 to 73 452 patients could present this type of disease and require care for recurrent infections, autoimmune disease and lymphoproliferative diseases, in addition to the fact that without specific treatment they could die due to serious infections or have permanent disability, which implies a greater burden for the social security system in subsidies and lower income. Currently the country has 86 diagnosed patients, according to the database of the Latin American Society of Immunodeficiencies4. Many of the therapies, such as immunoglobulin replacement therapy, to which more than 60% of these pathologies are de-pendent, allow life expectancy and morbidity to almost reach those who do not have the disease 57.
Assuntos
Humanos , Masculino , Feminino , Imunização Passiva , Doenças da Imunodeficiência Primária , Síndromes de Imunodeficiência , Anticorpos , Anticorpos/imunologia , Células Produtoras de Anticorpos , Terapêutica , Deficiência de IgA , Imunodeficiência de Variável Comum , Técnicas e Procedimentos Diagnósticos , Terapia de Reposição Hormonal , Agamaglobulinemia , Diagnóstico , Equador , Alergia e Imunologia , Síndrome de Imunodeficiência com Hiper-IgM , Formação de AnticorposRESUMO
The objective of this manuscript is to provide the reader with a hypothetical case study to present an immunogenicity risk assessment for a multi-specific therapeutic as part of Investigational New Drug (IND) application. In order to provide context for the bioanalytical strategies used to support the multi-specific therapeutic presented herein, the introduction focuses on known immunogenicity risk factors. The subsequent hypothetical case study applies these principles to a specific example HC-12, based loosely on anti-TNFα and anti-IL-17A bispecific molecules previously in development, structured as an example immunogenicity risk assessment for submission to health authorities. The risk of higher incidence and safety impact of anti-drug antibodies (ADA) due to large protein complexes is explored in the context of multi-specificity and multi-valency of the therapeutic in combination with the oligomeric forms of the targets.
Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos/imunologia , Medição de Risco/métodos , Humanos , Incidência , Interleucina-17/imunologia , Aplicação de Novas Drogas em Teste , Fatores de Risco , Fator de Necrose Tumoral alfa/imunologiaRESUMO
The presence of circulating targets and antidrug antibodies can influence the ability of a bioanalytical method to measure therapeutic protein (TP) concentration relevant to exposure-response evaluations. This project surveyed biosimilar submissions for their bioanalytical methods. Survey results revealed that 97% of pharmacokinetic methods designed to measure theoretically free or partial-free TPs with respect to target indeed measured free or partial-free TPs when considering experimental testing results for target effects. Antidrug antibody effect is less often evaluated. The observed trend of measuring biologically active forms of TP is consistent with the scientific understanding that pharmacokinetics of biologically active forms is more likely to be relevant to the clinical responses and evaluation of clinically meaningful differences to contribute to biosimilarity assessments.
Assuntos
Anticorpos/imunologia , Medicamentos Biossimilares/uso terapêutico , Descoberta de Drogas/métodos , HumanosRESUMO
Cardiovascular disease has remained the world's biggest killer for 30 years. To aid in the diagnosis and prognosis of patients suffering cardiovascular-related disease accurate detection methods are essential. For over 20 years, the cardiac-specific troponins, I (cTnI) and T (cTnT), have acted as sensitive and specific biomarkers to assist in the diagnosis of various types of heart diseases. Various cardiovascular complications were commonly detected in patients with COVID-19, where cTn elevation is detectable, which suggested potential prognostic value of cTn in COVID-19-infected patients. Detection of these biomarkers circulating in the bloodstream is generally facilitated by immunoassays employing cTnI- and/or cTnT-specific antibodies. While several anti-troponin assays are commercially available, there are still obstacles to overcome to achieve optimal troponin detection. Such obstacles include the proteolytic degradation of N and C terminals on cTnI, epitope occlusion of troponin binding-sites by the cTnI/cTnT complex, cross reactivity of antibodies with skeletal troponins or assay interference caused by human anti-species antibodies. Therefore, further research into multi-antibody based platforms, multi-epitope targeting and rigorous validation of immunoassays is required to ensure accurate measurements. Moreover, in combination with various technical advances (e.g. microfluidics), antibody-based troponin detection systems can be more sensitive and rapid for incorporation into portable biosensor systems to be used at point-of care.
Assuntos
Anticorpos/sangue , Biomarcadores/sangue , Doenças Cardiovasculares/sangue , Imunoensaio/métodos , Troponina I/sangue , Troponina T/sangue , Anticorpos/imunologia , COVID-19/complicações , COVID-19/diagnóstico , COVID-19/virologia , Doenças Cardiovasculares/complicações , Doenças Cardiovasculares/diagnóstico , Humanos , Prognóstico , SARS-CoV-2/imunologia , SARS-CoV-2/fisiologia , Troponina I/imunologia , Troponina T/imunologiaRESUMO
Oxytocin is currently of high interest as a biomarker of welfare and stress in humans and animals. The purpose of this study was to validate two new assays (one using a monoclonal antibody and the other using a polyclonal antibody) for the oxytocin measurement in the saliva of dogs. For this purpose, an analytical validation was performed, and these assays were applied in an experimental trial in which dogs were stroked by their owners. In the experimental trial, saliva samples of 17 dogs were collected by the owners at three different times: a basal sample, at the end of 10 min of an affiliative interaction with their owners consisting of stroking and 15 min after the end of the affiliative interaction. The dogs were separated into two groups (group 1, n = 8 and group 2, n = 9) according to the acceptance of the sponge and the response to the stroking. Significant differences in the response of salivary oxytocin after stroking in the two groups were found when the assay with the monoclonal antibody was used. This assay showed a significant increase just after the end of affiliative interaction (P < 0.01) and 15 min after (P < 0.01) in those dogs that had a good acceptance of the sponge and the stroking induced a positive response on them (based in a Likert-type scale from 1 to 10). These data reflect that the assays used in this study can lead to different results when quantifying oxytocin in the saliva of dogs after stroking.
Assuntos
Cães/fisiologia , Ensaio de Imunoadsorção Enzimática/veterinária , Ocitocina/análise , Animais de Estimação/fisiologia , Saliva/química , Animais , Anticorpos/imunologia , Anticorpos Monoclonais/imunologia , Biomarcadores , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , MasculinoRESUMO
The thrombin generation (TG) assay is a well-established tool to capture the clotting potential of any healthy or haemophiliac subject. It measures ex vivo the kinetics of thrombin activation throughout the coagulation. Clinical studies allowed to create two databases gathering the coagulation factor levels and the thrombin generation profile of 40 healthy and 40 haemophiliac A (HA) subjects. Besides, portions of all HA samples were spiked with increasing levels of a TFPI antibody (considered as a possible therapeutic target) and corresponding TG profiles were determined. The non-linear mixed-effect (NLME) modelling aims at describing and explaining the experimentally observed important variability of the TG curves between subjects and the individual effects of spiking with a TFPI antibody. The models consist of an empirical description of the TG kinetics, accounting for an additive residual error and between-subject variability on its parameters. Factor VIII and TFPI were found to significantly explain and reduce the variability of the TG of haemophilia A samples. Besides, the model is shown to correctly reproduce the variability in the response to the ex vivo spiking with the TFPI antibody, by combining the empirical description of TG to a simple Hill equation that accounts for the binding between TFPI and different doses of its antibody. Such models can be useful for clinical practice, with the analysis and comparison of the distributions of TG profiles in healthy and haemophilia populations; and also for research, with the analysis of the effect of TFPI and its neutralization on individual TG profiles.
Assuntos
Hemofilia A/tratamento farmacológico , Lipoproteínas/imunologia , Tempo de Trombina , Trombina/análise , Anticorpos/imunologia , Estudos de Casos e Controles , Hemofilia A/sangue , Humanos , Masculino , Modelos EstatísticosRESUMO
Immunogenicity assays are required to evaluate anti-drug antibody (ADA) responses that can be generated against biotherapeutic modalities. Regulatory guidelines focus on clinical requirements, yet it has become apparent that industry has applied these clinical recommendations for immunogenicity assessment to nonclinical studies in varying degrees. ADAs are an anticipated outcome of dosing a humanized or fully human biotherapeutic into an animal. However, a nonclinical ADA response is rarely predictive of the immunogenic potential in humans. The addendum to ICH S6 recommends that immunogenicity should be explicitly examined where there is: evidence of altered pharmacodynamic activity; unexpected changes in exposure in the absence of a pharmacodynamic marker or evidence of immuno-mediated reactions. The European Bioanalytical Forum has extensively discussed and reached a consensus on a minimal strategic approach of when and what to include for nonclinical immunogenicity assessments. Additionally, this paper recommends a strategy for ADA assay validation and sample analysis for those cases when it is considered necessary to include an immunogenicity assessment in nonclinical toxicology studies.
Assuntos
Anticorpos/análise , Bioensaio , Anticorpos/imunologia , Formação de Anticorpos , Europa (Continente) , HumanosRESUMO
BACKGROUND: Patients can form antibodies to foreign human leukocyte antigen (HLA) Class I antigens after exposure to allogeneic cells. These anti-HLA class I antibodies can bind transfused platelets (PLTs) and mediate their destruction, thus leading to PLT refractoriness. Patients with PLT refractoriness need HLA-matched PLTs, which require expensive HLA typing of donors, antibody analyses of patient sera and/or crossmatching. An alternative approach is to reduce PLT HLA Class I expression using a brief incubation in citric acid on ice at low pH. METHODS AND MATERIALS: Apheresis PLT concentrates were depleted of HLA Class I complexes by 5 minutes incubation in ice-cold citric acid, at pH 3.0. Surface expression of HLA Class I complexes, CD62P, CD63, phosphatidylserine, and complement factor C3c was analyzed by flow cytometry. PLT functionality was tested by thromboelastography (TEG). RESULTS: Acid treatment reduced the expression of HLA Class I complexes by 71% and potential for C3c binding by 11.5-fold compared to untreated PLTs. Acid-treated PLTs were significantly more activated than untreated PLTs, but irrespective of this increase in steady-state activation, CD62P and CD63 were strongly upregulated on both acid-treated and untreated PLTs after stimulation with thrombin receptor agonist peptide. Acid treatment did not induce apoptosis over time. X-ray irradiation did not significantly influence the expression of HLA Class I complexes, CD62P, CD63, and TEG variables on acid treated PLTs. CONCLUSION: The relatively simple acid stripping method can be used with irradiated apheresis PLTs and may prevent transfusion-associated HLA sensitization and overcome PLT refractoriness.
Assuntos
Ácido Cítrico/efeitos adversos , Antígenos de Histocompatibilidade Classe I/efeitos dos fármacos , Transfusão de Plaquetas/métodos , Imunodeficiência Combinada Severa/induzido quimicamente , Anticorpos/imunologia , Tipagem e Reações Cruzadas Sanguíneas/métodos , Plaquetas/efeitos da radiação , Feminino , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe I/efeitos da radiação , Teste de Histocompatibilidade/economia , Teste de Histocompatibilidade/métodos , Humanos , Selectina-P/metabolismo , Transfusão de Plaquetas/efeitos adversos , Plaquetoferese/métodos , Tetraspanina 30/metabolismo , Tromboelastografia/métodos , Trombocitopenia/terapia , Regulação para Cima/genéticaRESUMO
AIM: The antibody levels against a broad spectrum of pathogens were assessed in commercial intravenous immunoglobulin (IVIG) manufactured from pooled plasma obtained from different global regions. METHODS: Twenty-four IVIG commercial lots from eight manufacturers corresponding to 12 brands were analyzed. The plasma was collected in 10 countries/regions. Depending on each pathogen, antibody levels were measured using specific commercial IgG-specific enzyme immunoassay kits or by cell culture neutralization test and guinea pig skin neutralization test. A principal component analysis was performed. RESULTS: For polio and diphtheria (reference markers of the US authorities), all IVIGs had relevant titers in accordance with reference levels. IVIGs from Canada, Australia, and the USA were positive for titers against globally distributed pathogens or those under vaccination programs in the developed world (parainfluenza, Epstein-Barr, varicella-zoster, influenza B, parvovirus B19, and measles viruses). IVIG from Taiwan and Hong Kong showed low antibody titers for these pathogens but high titers for Pseudomonas aeruginosa. IVIG from India had high titers for pathogens frequently found in developing countries (West Nile, dengue, chikungunya, and hepatitis E viruses and Streptococcus pneumoniae). IVIGs from Argentina, Spain, Israel, and Czechia showed intermediate antibody concentrations. CONCLUSION: The antibody profile in IVIG was greatly influenced by regional characteristics including climate, vaccination programs, and the prevalence of pathogens in the different countries and regions.
Assuntos
Imunoglobulinas Intravenosas/imunologia , América , Anticorpos/genética , Anticorpos/imunologia , Ásia , Humanos , Técnicas Imunoenzimáticas , Imunoglobulinas Intravenosas/análise , Imunoglobulinas Intravenosas/economia , Testes de Neutralização , Plasma/química , Plasma/imunologiaRESUMO
Background: Striking similarities have been found between coronavirus disease 2019 (COVID-19) and anti-melanoma differentiation-associated gene 5 (MDA5) antibody (Ab)-related dermatomyositis, implying a shared autoinflammatory aberrance. Herein, we aim to investigate whether the anti-MDA5 Ab is present in COVID-19 and correlates with the severity and adverse outcome of COVID-19 patients. Methods and Findings: We retrospectively recruited 274 adult inpatients with COVID-19 in this study, including 48, 164, and 62 cases of deaths, severe, and non-severe patients respectively. The anti-MDA5 Ab was determined by ELISA and verified by Western Blotting, which indicated that the positive rate of anti-MDA5 Ab in COVID-19 patients was 48.2% (132/274). The clinical and laboratory features, as well as outcomes between patients with positive and negative anti-MDA5 Ab were compared and we found that the anti-MDA5 Ab positive patients tended to represent severe disease (88.6% vs 66.9%, P<0.0001). We also demonstrated that the titer of anti-MDA5 Ab was significantly elevated in the non-survivals (5.95 ± 5.16 vs 8.22 ± 6.64, P=0.030) and the positive rate was also higher than that in the survivals (23.5% vs 12.0%, P=0.012). Regarding severe COVID-19 patients, we found that high titer of anti-MDA5 Ab (≥10.0 U/mL) was more prevalent in the non-survivals (31.2% vs 14.0%, P=0.006). Moreover, a dynamic analysis of anti-MDA5 Ab was conducted at different time-points of COVID-19, which revealed that early profiling of anti-MDA5 Ab could distinguish severe patients from those with non-severe ones. Conclusions: Anti-MDA5 Ab was prevalent in the COVID-19 patients and high titer of this antibody is correlated with severe disease and unfavorable outcomes.
Assuntos
Anticorpos/imunologia , COVID-19/imunologia , Helicase IFIH1 Induzida por Interferon/imunologia , Índice de Gravidade de Doença , Adulto , Idoso , Anticorpos/sangue , COVID-19/epidemiologia , COVID-19/virologia , Progressão da Doença , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Prognóstico , Estudos Retrospectivos , SARS-CoV-2/fisiologiaRESUMO
Insufficient availability of data to evaluate immunogenicity incidence or clinical impact during regulatory review could require further evaluation postapproval. Through a keyword search of all postmarketing requirements and commitments (PMRs/PMCs) associated with products with their original US Food and Drug Administration (FDA) approvals between 2009 and 2018, we identified products that had PMRs/PMCs established to address concerns or uncertainty related to immunogenicity. Of the 113 relevant products, 50% had an immunogenicity-related PMR/PMC; of these, 68% were related to developing immunogenicity assays and 48% requested an assessment of clinical impact. Fifty-five percent of the products with a fulfilled PMR/PMC had a change in the immunogenicity information in their labeling immediately following fulfillment. This work highlights that there are often unknowns associated with immunogenicity incidence and/or impact at the time of approval. Earlier regulatory discussions on immunogenicity assessments in premarket development could improve the understanding and communication of the risk/benefit profile and reduce the need for some immunogenicity PMRs/PMCs.
Assuntos
Anticorpos/imunologia , Aprovação de Drogas , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/imunologia , Testes Imunológicos , Vigilância de Produtos Comercializados , Especificidade de Anticorpos , Qualidade de Produtos para o Consumidor , Reações Cruzadas , Rotulagem de Medicamentos , Epitopos , Humanos , Medição de Risco , Estados Unidos , United States Food and Drug AdministrationRESUMO
With the growth of monoclonal antibodies and other proteins as major modalities in the pharmaceutical industry, there has been an increase in pharmacology and toxicity testing of biotherapeutics in animals. Animals frequently mount an immune response to human therapeutic proteins. This can result in asymptomatic anti-drug antibody formation, immune complexes that affect drug disposition and/or organ function such as kidney, cytokine release responses, fatal hypersensitivity, or a range of reactions in between. In addition, an increasing number of oncology therapeutics are being developed that enhance or directly stimulate immune responses by a variety of mechanisms, which could increase the risk of autoreactivity and an autoimmune-like syndrome in animals and humans. When evaluating the risk of biotherapeutics prior to entering the clinic, the nonclinical safety data may include any of these responses and it is critical to understand whether they represent a safety liability for humans. The DruSafe Leadership group of the IQ Consortium conducted a survey of industry to understand sponsors' experiences with these immune reactions in nonclinical studies related to both immunogenicity and pharmacologically-mediated immune perturbations. The survey covered what pathways were affected, how the immune responses were presented, how the company and health authorities interpreted the data and whether the immune responses were observed in the clinic. Additionally, the survey gathered information on association of these findings with anti-drug antibodies as well as sponsor's use of immunogenicity predictive tools. The data suggests that the ability of a biotherapeutic to activate the immune system, intended or not, plays a significant role on characteristics of the response and whether theys are translatable.
Assuntos
Produtos Biológicos/toxicidade , Sistema Imunitário/efeitos dos fármacos , Animais , Anticorpos/imunologia , Produtos Biológicos/imunologia , Avaliação Pré-Clínica de Medicamentos , Indústria Farmacêutica , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Macaca fascicularis , Camundongos , Ratos , Inquéritos e Questionários , Testes de ToxicidadeRESUMO
OBJECTIVES: To determine if the URO-MCP-1 mouse model for bladder IC/BPS is associated with in vivo bladder hyper-permeability, as measured by contrast-enhanced MRI (CE-MRI), and assess whether molecular-targeted MRI (mt-MRI) can visualize in vivo claudin-2 expression as a result of bladder hyper-permeability. Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic, painful condition of the bladder that affects primarily women. It is known that permeability plays a substantial role in IC/BPS. Claudins are tight junction membrane proteins that are expressed in epithelia and endothelia and form paracellular barriers and pores that determine tight junction permeability. Claudin-2 is a molecular marker that is associated with increased hyperpermeability in the urothelium. MATERIALS AND METHODS: CE-MRI was used to measure bladder hyper-permeability in the URO-MCP-1 mice. A claudin-2-specific mt-MRI probe was used to assess in vivo levels of claudin-2. The mt-MRI probe consists of an antibody against claudin-2 conjugated to albumin that had Gd-DTPA (gadolinium diethylenetriamine pentaacetate) and biotin attached. Verification of the presence of the mt-MRI probe was done by targeting the biotin moiety for the probe with streptavidin-horse radish peroxidase (SA-HRP). Trans-epithelial electrical resistance (TEER) was also used to assess bladder permeability. RESULTS: The URO-MCP-1 mouse model for IC/BPS was found to have a significant increase in bladder permeability, following liposaccharide (LPS) exposure, compared to saline-treated controls. mt-MRI- and histologically-detectable levels of the claudin-2 probe were found to increase with LPS -induced bladder urothelial hyper-permeability in the URO-MCP-1 IC mouse model. Levels of protein expression for claudin-2 were confirmed with immunohistochemistry and immunofluorescence imaging. Claudin-2 was also found to highly co-localize with zonula occlidens-1 (ZO-1), a tight junction protein. CONCLUSION: The combination of CE-MRI and TEER approaches were able to demonstrate hyper-permeability, a known feature associated with some IC/BPS patients, in the LPS-exposed URO-MCP-1 mouse model. This MRI approach could be clinically translated to establish which IC/BPS patients have bladder hyper-permeability and help determine therapeutic options. In addition, the in vivo molecular-targeted imaging approach can provide invaluable information to enhance our understanding associated with bladder urothelium hyper-permeability in IC/BPS patients, and perhaps be used to assist in developing further therapeutic strategies.
Assuntos
Claudina-2/metabolismo , Cistite Intersticial/patologia , Imageamento por Ressonância Magnética/métodos , Sondas Moleculares/química , Bexiga Urinária/fisiopatologia , Animais , Anticorpos/química , Anticorpos/imunologia , Claudina-2/imunologia , Cistite Intersticial/metabolismo , Modelos Animais de Doenças , Gadolínio DTPA/química , Imuno-Histoquímica , Lipopolissacarídeos/toxicidade , Camundongos , Permeabilidade/efeitos dos fármacos , Albumina Sérica/químicaRESUMO
Bisphenol A (BPA) is classified as an endocrine disruptor (ED) and it can interact with variety of hormone receptors leading to hormonal disruption and increased risk of various adverse health effects. Reducing human exposure to BPA is one of the main challenges of public health, as it is constantly present in daily life. A low-cost and commonly applied method to enable determination of BPA in the patient's body has yet to be developed. Currently available techniques are expensive, time-consuming, and require access to highly equipped analytical chemistry laboratories. Here we describe a fast and cheap engineered lateral flow assay of our design, to detect of BPA in urine samples. The technology not only provides an opportunity to perform rapid medical diagnostics without the need for an access to the central laboratory but also a means for self-diagnosis by the patient. The addition of ß-glucuronidase improves the sensitivity of detection as it releases the free BPA from glucuronide complexes in urine. This invention may become a demonstrated analytical means for lowering human exposure to BPA and probably also to other EDs and consequently, may be useful in decrease of the risk for several lifestyle diseases.
Assuntos
Anticorpos/química , Compostos Benzidrílicos/química , Compostos Benzidrílicos/urina , Disruptores Endócrinos/química , Disruptores Endócrinos/urina , Fenóis/química , Fenóis/urina , Adolescente , Adsorção , Adulto , Anticorpos/imunologia , Compostos Benzidrílicos/imunologia , Criança , Pré-Escolar , Cromatografia Líquida , Colódio/química , Disruptores Endócrinos/imunologia , Ensaio de Imunoadsorção Enzimática/economia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Glucuronidase/química , Glucuronídeos/química , Voluntários Saudáveis , Humanos , Masculino , Membranas Artificiais , Fenóis/imunologia , Saúde Pública/métodos , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem , Adulto JovemRESUMO
One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and cytotoxicity, driven by FcγRIIIa (CD16) expressed on natural killer (NK) cells. These observations have led to research focusing on methods to increase Fc receptor-mediated events, which include removal of a fucose moiety found on the Fc portion of the antibody. Further studies have elucidated the mechanistic changes in signaling, cellular processes, and cytotoxic characteristics that increase ADCC activity with afucosylated antibodies. Additionally, other studies have shown the potential benefits of these antibodies in combination with small molecule inhibitors. These experiments demonstrated the molecular and cellular mechanisms underlying the benefits of using afucosylated antibodies in combination settings. Many of these observations were based on an artificial in vitro activation assay in which the FcγRIIIa on human NK cells was activated by therapeutic antibodies. This assay provided the flexibility to study downstream effector NK cell functions, such as cytokine production and degranulation. In addition, this assay has been used to interrogate signaling pathways and identify molecules that can be modulated or used as biomarkers. Finally, other therapeutic molecules (i.e., small molecule inhibitors) have been added to the system to provide insights into the combination of these therapeutics with therapeutic antibodies, which is essential in the current clinical space. This manuscript aims to provide a technical foundation for performing this artificial human NK cell activation assay. The protocol demonstrates key steps for cell activation as well as potential downstream applications that range from functional readouts to more mechanistic observations.
Assuntos
Anticorpos/uso terapêutico , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Receptores de IgG/metabolismo , Anticorpos/imunologia , Genótipo , Humanos , Células Matadoras Naturais/citologia , Ativação Linfocitária , Transdução de SinaisRESUMO
APCs play a key role at initiating adaptive immune responses by presenting antigens to lymphocytes and DCs are professional APCs. It is critical to understand the differential antigen capture and presentation ability of different DC subsets, which is important for DC-targeted immunotherapy. In this section, we give a brief introduction to different antigen presentation pathways and introduce the key concept of cross-presentation, the major antigen presentation pathway used for anti-viral and anti-tumoral immune responses. CD205, a DC restricted receptor, is highly expressed on certain DCs subsets. We find CD205-mediated antigen uptake to be a useful model for studying antigen uptake and defects. These methods provide an introduction to CD205-mediated pre-clinical delivery of antigens to cross-presenting DCs, which can be adapted to the study of targeting to multiple receptors and other C-type lectins. This is a promising strategy to detect the antigen capture capacity and to study the key players orchestrating tolerance and immunity ex vivo.
Assuntos
Anticorpos/imunologia , Apresentação de Antígeno , Células Dendríticas/imunologia , Imunidade Adaptativa , Animais , Antígenos CD/imunologia , Apresentação Cruzada , Endocitose , Humanos , Lectinas Tipo C/imunologia , Camundongos , Antígenos de Histocompatibilidade Menor/imunologia , Receptores de Superfície Celular/imunologiaRESUMO
The circumsporozoite protein (CSP) and thrombospondin-related adhesion protein (TRAP) are major targets for pre-erythrocytic malaria vaccine development. However, the CSP-based vaccine RTS,S provides only marginal protection, highlighting the need for innovative vaccine design and development. Here we design and characterize expression and folding of P. berghei (Pb) and P. falciparum (Pf) TRAP-CSP fusion proteins, and evaluate immunogenicity and sterilizing immunity in mice. TRAP N-terminal domains were fused to the CSP C-terminal αTSR domain with or without the CSP repeat region, expressed in mammalian cells, and evaluated with or without N-glycan shaving. Pb and Pf fusions were each expressed substantially better than the TRAP or CSP components alone; furthermore, the fusions but not the CSP component could be purified to homogeneity and were well folded and monomeric. As yields of TRAP and CSP fragments were insufficient, we immunized BALB/c mice with Pb TRAP-CSP fusions in AddaVax adjuvant and tested the effects of absence or presence of the CSP repeats and absence or presence of high mannose N-glycans on total antibody titer and protection from infection by mosquito bite both 2.5 months and 6 months after the last immunization. Fusions containing the repeats were completely protective against challenge and re-challenge, while those lacking repeats were significantly less effective. These results correlated with higher total antibody titers when repeats were present. Our results show that TRAP-CSP fusions increase protein antigen production, have the potential to yield effective vaccines, and also guide design of effective proteins that can be encoded by nucleic acid-based and virally vectored vaccines.
Assuntos
Vacinas Antimaláricas/farmacologia , Malária/tratamento farmacológico , Proteínas de Protozoários/genética , Animais , Anticorpos/genética , Anticorpos/imunologia , Antígenos/genética , Antígenos/imunologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/imunologia , Humanos , Imunização , Malária/imunologia , Malária/parasitologia , Vacinas Antimaláricas/imunologia , Camundongos , Plasmodium berghei/efeitos dos fármacos , Plasmodium berghei/imunologia , Plasmodium berghei/patogenicidade , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/imunologia , Plasmodium falciparum/patogenicidade , Polissacarídeos/genética , Polissacarídeos/imunologia , Dobramento de Proteína , Proteínas de Protozoários/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Prompt diagnostic evaluation of suspected heparin-induced thrombocytopenia (HIT) is critical for guiding initial patient management. We assessed the performance of 3 immunoassays detecting anti-platelet factor 4 (PF4)/heparin antibodies, derived a diagnostic algorithm with a short analytical turnaround time (TAT), and prospectively validated the algorithm. Plasma samples were analyzed by Zymutest-HIA-IgG, HemosIL-AcuStar-HIT-IgG, and ID-H/PF4-PaGIA in retrospective (n = 221) and prospective (n = 305) derivation cohorts. We calculated likelihood ratios of result intervals and cutoff values with 100% negative (NPV) and positive (PPV) predictive values for a positive gold standard functional assay (heparin-induced platelet activation [HIPA]). A diagnostic algorithm was established based on the Bayesian combination of pretest probability and likelihood ratios of first- and second-line immunoassays. Cutoffs with 100% PPV for positive HIPA were >3.0 U/mL (HemosIL-AcuStar-HIT-IgG) and titer ≥16 (ID-H/PF4-PaGIA); cutoffs with 100% NPV were <0.13 U/mL and ≤1, respectively. During the prospective validation of the derived algorithm (n = 687), HemosIL-AcuStar-HIT-IgG was used as unique testing in 566 (82.4%) of 687 cases (analytical TAT, 30 minutes). In 121 (17.6%) of 687 unresolved cases, ID-H/PF4-PaGIA was used as second-line testing (additional TAT, 30 minutes). The algorithm accurately predicted HIT in 51 (7.4%) of 687 patients and excluded it in 604 (87.9%) of 687 patients, leaving only 20 (2.9%) cases unresolved. We also identified 12 (1.7%) of 687 positive predictions not confirmed by HIPA: 10 patients with probable HIT despite negative HIPA and 2 possible false-positive algorithm predictions. The combination of pretest probability with first- and second-line immunoassays for anti-PF4/heparin antibodies is accurate for ruling in or out HIT in ≥95% of cases within 60 minutes. This diagnostic approach improves initial management of patients with suspected HIT.