Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 11.627
Filtrar
1.
Sci Transl Med ; 12(564)2020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-32958614

RESUMO

Children and youth infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have milder disease than do adults, and even among those with the recently described multisystem inflammatory syndrome, mortality is rare. The reasons for the differences in clinical manifestations are unknown but suggest that age-dependent factors may modulate the antiviral immune response. We compared cytokine, humoral, and cellular immune responses in pediatric (children and youth, age <24 years) (n = 65) and adult (n = 60) patients with coronavirus disease 2019 (COVID-19) at a metropolitan hospital system in New York City. The pediatric patients had a shorter length of stay, decreased requirement for mechanical ventilation, and lower mortality compared to adults. The serum concentrations of interleukin-17A (IL-17A) and interferon-γ (IFN-γ), but not tumor necrosis factor-α (TNF-α) or IL-6, were inversely related to age. Adults mounted a more robust T cell response to the viral spike protein compared to pediatric patients as evidenced by increased expression of CD25+ on CD4+ T cells and the frequency of IFN-γ+ CD4+ T cells. Moreover, serum neutralizing antibody titers and antibody-dependent cellular phagocytosis were higher in adults compared to pediatric patients with COVID-19. The neutralizing antibody titer correlated positively with age and negatively with IL-17A and IFN-γ serum concentrations. There were no differences in anti-spike protein antibody titers to other human coronaviruses. Together, these findings demonstrate that the poor outcome in hospitalized adults with COVID-19 compared to children may not be attributable to a failure to generate adaptive immune responses.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Hospitalização , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Adolescente , Adulto , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Criança , Infecções por Coronavirus/sangue , Citocinas/sangue , Feminino , Humanos , Imunoglobulina G/metabolismo , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/sangue , Glicoproteína da Espícula de Coronavírus/metabolismo , Resultado do Tratamento
2.
Medicine (Baltimore) ; 99(39): e22341, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32991448

RESUMO

RATIONALE: The Goodpasture syndrome is an extremely rare disease, with renal and pulmonary manifestations, and is mediated by anti-glomerular basement membrane (anti-GBM) antibodies. Renal pathological changes are mainly characterized by glomerular crescent formation and linear immunofluorescent staining for immunoglobulin G on the GBM. There are few reports on the atypical course of the syndrome involving serum-negative anti-GBM antibodies. Therefore, we present a case of Goodpasture syndrome that presented with nephrotic-range proteinuria and was seronegative for anti-GBM antibodies. PATIENT CONCERNS: A 38-year-old Chinese man presented with a lung lesion that was discovered by physical examination a month prior to presentation. The chief concern was occasional hemoptysis without fever, cough, chest pain, and edema. DIAGNOSES: Laboratory testing revealed that the urinary protein level and urine erythrocyte count were 7.4 g/24 hours and 144/high-power field (HPF), respectively. Serological testing for anti-GBM antibodies was negative. Chest computed tomography revealed multiple exudative lesions in both lungs, indicating alveolar infiltration and hemorrhage. Electronic bronchoscopy and pathological examination of the alveolar lavage fluid indicated no abnormalities. However, kidney biopsy suggested cellular crescent formation and segmental necrosis of the globuli, with linear IgG and complement C3 deposition on the GBM. These findings were consistent with the diagnosis of anti-GBM antibody nephritis. INTERVENTIONS: The patient underwent 7 sessions of double filtration plasmapheresis. He was also administered with intravenous methylprednisolone and cyclophosphamide. After renal function stabilization, he was discharged under an immunosuppressive regimen comprising of glucocorticoids and cyclophosphamides. OUTCOMES: Three months later, follow-up examination revealed that the 24-hour urine protein had increased to 13 g. Furthermore, the urine erythrocyte count was 243/HPF. After a 6-month follow-up, the patient achieved partial remission, with a proteinuria level of 3.9 g/24 hours and a urine erythrocyte count of 187/HPF. LESSONS: This extremely rare case of Goodpasture syndrome manifested with seronegativity for anti-GBM antibodies and nephrotic-range proteinuria. Our findings emphasize the importance of renal biopsy for the clinical diagnosis of atypical cases. Furthermore, because renal involvement achieved only partial remission despite therapy, early detection and active treatment of the Goodpasture syndrome is necessary to improve the prognosis of patients.


Assuntos
Doença Antimembrana Basal Glomerular/complicações , Doença Antimembrana Basal Glomerular/imunologia , Autoanticorpos/sangue , Proteinúria/etiologia , Administração Intravenosa , Adulto , Assistência ao Convalescente , Doença Antimembrana Basal Glomerular/diagnóstico , Doença Antimembrana Basal Glomerular/terapia , Grupo com Ancestrais do Continente Asiático/etnologia , Complemento C3/metabolismo , Ciclofosfamida/administração & dosagem , Ciclofosfamida/uso terapêutico , Quimioterapia Combinada , Glucocorticoides/administração & dosagem , Glucocorticoides/uso terapêutico , Hemoptise/diagnóstico , Hemorragia/etiologia , Hemorragia/patologia , Humanos , Imunoglobulina G/metabolismo , Imunossupressores/administração & dosagem , Imunossupressores/uso terapêutico , Rim/patologia , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Pulmão/diagnóstico por imagem , Pulmão/patologia , Masculino , Metilprednisolona/administração & dosagem , Metilprednisolona/uso terapêutico , Nefrite/diagnóstico , Nefrite/imunologia , Plasmaferese/métodos , Tomografia Computadorizada por Raios X/métodos , Resultado do Tratamento
3.
Nat Commun ; 11(1): 4198, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826914

RESUMO

COVID-19 caused by SARS-CoV-2 has become a global pandemic requiring the development of interventions for the prevention or treatment to curtail mortality and morbidity. No vaccine to boost mucosal immunity, or as a therapeutic, has yet been developed to SARS-CoV-2. In this study, we discover and characterize a cross-reactive human IgA monoclonal antibody, MAb362. MAb362 binds to both SARS-CoV and SARS-CoV-2 spike proteins and competitively blocks ACE2 receptor binding, by overlapping the ACE2 structural binding epitope. Furthermore, MAb362 IgA neutralizes both pseudotyped SARS-CoV and SARS-CoV-2 in 293 cells expressing ACE2. When converted to secretory IgA, MAb326 also neutralizes authentic SARS-CoV-2 virus while the IgG isotype shows no neutralization. Our results suggest that SARS-CoV-2 specific IgA antibodies, such as MAb362, may provide effective immunity against SARS-CoV-2 by inducing mucosal immunity within the respiratory system, a potentially critical feature of an effective vaccine.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Betacoronavirus/imunologia , Imunoglobulina A/imunologia , Peptidil Dipeptidase A/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Chlorocebus aethiops , Reações Cruzadas , Epitopos , Células HEK293 , Humanos , Imunoglobulina A/metabolismo , Imunoglobulina A Secretora/imunologia , Imunoglobulina A Secretora/metabolismo , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Vírus da SARS/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero
4.
J Chromatogr A ; 1626: 461319, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32797815

RESUMO

The aim of this study is to model, describe and predict the mass transfer of IgG as a function of the agarose concentration in the protein A stationary phase, taking into account the influence of adsorption on the pore size. Therefore, particle size distribution, bed and bead porosities were examined by light microscopy, pressure-flow behavior and iSEC. Three agarose protein A stationary phases (2 wt%, 4 wt%, 6 wt%) were investigated. The pore size decreased from 116 nm for 2 wt% to 54 nm for 6 wt% and the porosity for the target molecule IgG was reduced by 25%. A shrinking core model approach was used to assess the influence of IgG adsorption on the pore size of the stationary phase and the diffusivity of IgG. Due to IgG adsorption, the pore diameter reduced by 24 nm, which is approximately two times its hydrodynamic diameter. Effective pore diffusivities of IgG were obtained by fitting the general rate model to breakthrough curves. They were in the range between 3.96·10-12m2/s and 6.5·10-12m2/s, decreasing as the agarose concentration increased. The DBC1% has a maximum for the 4 wt% agarose gel, showing optimal tradeoffs between accessibility, specific surface and diffusive mass transfer for IgG. A simple geometrical model was developed to describe the change in pore and filament diameters due to adsorption. The diffusion measured in protein A agarose beads can be described by a modification of the Ogston model. This enables the diffusion measured in protein A agarose networks to be predicted.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Sefarose/química , Adsorção , Cromatografia em Gel , Difusão , Imunoglobulina G/metabolismo , Tamanho da Partícula , Porosidade , Ligação Proteica , Proteína Estafilocócica A/metabolismo
5.
J Chromatogr A ; 1625: 461237, 2020 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-32709313

RESUMO

The quest for ligands alternative to Protein A for the purification of monoclonal antibodies (mAbs) has been pursued for almost three decades. Yet, the IgG-binding peptides known to date still fall short of the host cell protein (HCP) logarithmic removal value (LRV) set by Protein A media (2.5-3.1). In this study, we present an integrated computational-experimental approach leading to the discovery of peptide ligands that provide HCP LRVs on par with Protein A. First, the screening of 60,000 peptide variants was performed using a high-throughput search algorithm to identify sequences that ensure IgG affinity binding. Select sequences WQRHGI, MWRGWQ, RHLGWF, and GWLHQR were then negatively screened in silico against a panel of model HCPs to ensure the selection of peptides with high binding selectivity. Candidate ligands WQRHGI and MWRGWQ were conjugated to chromatographic resins and characterized by isothermal binding and breakthrough assays to quantify static and dynamic binding capacity (Qmax and DBC10%), respectively. The resulting Qmax were 52.6 mg of IgG per mL of adsorbent for WQRHGI and 57.48 mg/mL for MWRGWQ, while the DBC10% (2 minutes residence time) were 30.1 mg/mL for WQRHGI and 36.4 mg/mL for MWRGWQ. Evaluation of the peptides by isothermal titration calorimetry (ITC) confirmed the binding energy predicted in silico, and an amino acid scanning study corroborated the affinity-like binding activity of the peptides. WQRHGI-WorkBeads resin was finally characterized by purification of a monoclonal antibody from a Chinese Hamster Ovary (CHO) cell culture harvest, affording a remarkable HCP LRV of 2.7, and consistent product yield and purity over 100 chromatographic cycles. These results demonstrate the potential of WQRHGI as an effective alternative to Protein A for antibody purification.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Cromatografia de Afinidade/métodos , Peptídeos/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Células CHO , Cricetinae , Cricetulus , Imunoglobulina G/isolamento & purificação , Imunoglobulina G/metabolismo , Ligantes , Peptídeos/síntese química , Peptídeos/metabolismo , Ligação Proteica , Proteína Estafilocócica A/química , Proteína Estafilocócica A/metabolismo
6.
Int J Nanomedicine ; 15: 4079-4090, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32606665

RESUMO

Purpose: The aim of this study is to develop efficient localized therapy of sertaconazole nitrate for the treatment of vaginal candidiasis. Methods: Sertaconazole nitrate-loaded cationic liposomes were prepared by thin-film hydration method and coated with different concentrations of pectin (0.05%, 0.1% and 0.2%) to develop mucoadhesive liposomes. The formulated mucoadhesive vesicles were characterized in terms of morphology, entrapment efficiency, particle size, zeta value, mucoadhesive properties and drug release. The selected formula was incorporated into a gel base and further characterized by an ex vivo permeation study in comparison with conventional sertaconazole gel. Also, the in vivo study was performed to assess the efficacy of sertaconazole mucoadhesive liposomal gel in treating rats with vaginal candidiasis. Results: The mucoadhesive liposomes were spherical. Coating liposomes with pectin results in increased entrapment efficiency and particle size compared with uncoated vesicles. On the contrary, zeta values were reduced upon coating liposomes with pectin indicating efficient coating of liposomes with pectin. Mucoadhesive liposomes showed a more prolonged and sustained drug release compared with uncoated liposomes. Ex vivo study results showed that mucoadhesive liposomal gel increased sertaconazole tissue retention and reduced drug tissue penetration. In the invivo study, the mucoadhesive liposomal gel showed a significant reduction in the microbial count with a subsequent reduction in inflammatory responses with the lowest histopathological change compared with conventional gel. Conclusion: The study confirmed the potentiality of employing mucoadhesive liposomes as a successful carrier for the vaginal delivery of antifungal drugs.


Assuntos
Antifúngicos/uso terapêutico , Candidíase Vulvovaginal/tratamento farmacológico , Imidazóis/uso terapêutico , Muco/química , Tiofenos/uso terapêutico , Adesividade , Animais , Anti-Infecciosos/farmacologia , Biomarcadores/metabolismo , Preparações de Ação Retardada , Liberação Controlada de Fármacos , Feminino , Géis , Humanos , Imidazóis/farmacologia , Imunoglobulina G/metabolismo , Imunoglobulina M/metabolismo , Mediadores da Inflamação/metabolismo , Lipossomos/ultraestrutura , Mucinas/metabolismo , Tamanho da Partícula , Ratos Sprague-Dawley , Ovinos , Eletricidade Estática , Tiofenos/farmacologia , Vagina/patologia , beta-Glucanas/metabolismo
7.
Lancet Infect Dis ; 20(9): e245-e249, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32687805

RESUMO

The collapse of global cooperation and a failure of international solidarity have led to many low-income and middle-income countries being denied access to molecular diagnostics in the COVID-19 pandemic response. Yet the scarcity of knowledge on the dynamics of the immune response to infection has led to hesitation on recommending the use of rapid immunodiagnostic tests, even though rapid serology tests are commercially available and scalable. On the basis of our knowledge and understanding of viral infectivity and host response, we urge countries without the capacity to do molecular testing at scale to research the use of serology tests to triage symptomatic patients in community settings, to test contacts of confirmed cases, and in situational analysis and surveillance. The WHO R&D Blue Print expert group identified eight priorities for research and development, of which the highest is to mobilise research on rapid point-of-care diagnostics for use at the community level. This research should inform control programmes of the required performance and utility of rapid serology tests, which, when applied specifically for appropriate public health measures to then be put in place, can make a huge difference.


Assuntos
Anticorpos Antivirais/metabolismo , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Anticorpos Antivirais/sangue , Betacoronavirus/genética , Betacoronavirus/imunologia , Busca de Comunicante/métodos , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/transmissão , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/metabolismo , Imunoglobulina M/sangue , Imunoglobulina M/metabolismo , Pandemias/prevenção & controle , Pneumonia Viral/imunologia , Pneumonia Viral/prevenção & controle , Pneumonia Viral/transmissão , RNA Viral/análise , Fatores de Tempo , Triagem/métodos , Eliminação de Partículas Virais
8.
Int J Nanomedicine ; 15: 4151-4169, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32606670

RESUMO

Purpose: Focused ultrasound (FUS) is a noninvasive method to produce thermal and mechanical destruction along with an immune-stimulatory effect against cancer. However, FUS ablation alone appears insufficient to generate consistent antitumor immunity. In this study, a multifunctional nanoparticle was designed to boost FUS-induced immune effects and achieve systemic, long-lasting antitumor immunity, along with imaging and thermal enhancement. Materials and Methods: PEGylated PLGA nanoparticles encapsulating astragalus polysaccharides (APS) and gold nanorods (AuNRs) were constructed by a simple double emulsion method, characterized, and tested for cytotoxicity. The abilities of PA imaging and thermal-synergetic ablation efficiency were analyzed in vitro and in vivo. The immune-synergistic effect on dendritic cell (DC) differentiation in vitro and the immune response in vivo were also evaluated. Results: The obtained APS/AuNR/PLGA-PEG nanoparticles have an average diameter of 255.00±0.1717 nm and an APS-loading efficiency of 54.89±2.07%, demonstrating their PA imaging capability and high biocompatibility both in vitro and in vivo. In addition, the as-prepared nanoparticles achieved a higher necrosis cell rate and induced apoptosis rate in an in vitro cell suspension assay, greater necrosis area and decreased energy efficiency factor (EEF) in an in vivo rabbit liver assay, and remarkable thermal-synergic performance. In particular, the nanoparticles upregulated the expression of MHC-II, CD80 and CD86 on cocultured DCs in vitro, followed by declining phagocytic function and enhanced interleukin (IL)-12 and interferon (INF)-γ production. Furthermore, they boosted the production of tumor necrosis factor (TNF)-α, IFN-γ, IL-4, IL-10, and IgG1 (P< 0.001) but not IgG2a. Immune promotion peaked on day 3 after FUS in vivo. Conclusion: The multifunctional APS/AuNR/PLGA-PEG nanoparticles can serve as an excellent synergistic agent for FUS therapy, facilitating real-time imaging, promoting thermal ablation effects, and boosting FUS-induced immune effects, which have the potential to be used for further clinical FUS treatment.


Assuntos
Astrágalo (Planta)/química , Neoplasias da Mama/terapia , Ouro/química , Nanopartículas Multifuncionais/química , Nanotubos/química , Polissacarídeos/química , Terapia por Ultrassom , Animais , Antígenos CD/metabolismo , Apoptose , Morte Celular , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Citocinas/metabolismo , Células Dendríticas/citologia , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Imunoglobulina G/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Camundongos Endogâmicos BALB C , Fagocitose , Técnicas Fotoacústicas , Poliésteres/síntese química , Poliésteres/química , Polietilenoglicóis/síntese química , Polietilenoglicóis/química , Coelhos , Nanomedicina Teranóstica , Fator A de Crescimento do Endotélio Vascular/metabolismo
9.
APMIS ; 128(9): 531-538, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32578252

RESUMO

Despite the interest of researchers in IgG4-related disease (IgG4-RD), many questions still remain unanswered regarding the thyroid gland. We aimed to clarify the relationship between IgG4-positive plasma cells and the histopathological pattern in the Hashimoto thyroiditis (HT) in a Finnish series. HT specimens (n = 280) were retrieved from the Department of Pathology, Fimlab Laboratories. After re-evaluation, 82 (29%) cases (72 females and 10 males, 52 ± 17 years) with significant fibrosis were selected. CD38, IgG and IgG4 positivity in plasma cells was evaluated by immunohistochemistry. Adjusted IgG4-positive plasma cells per HPF > 20 and IgG4- to IgG-positive plasma cell ratio > 30% were adopted as threshold criteria and related to other morphological features. IgG4-positive HT group included 13 cases (15% from fibrotic HT, 4.6% from all HT, 50 ± 15 years, 11 females) with adjusted HPF count 30 ± 5 (23-40) IgG4-positive cells. IgG4-positivity significantly correlated with the presence of lobulation, oncocytic metaplasia and certain type of fibrosis, fibrosis spread outside the gland, lymphocytes/plasma cells epithelial penetration, the predominance of microfollicles and follicular atrophy in the present study. Despite the persisting uncertainty whether HT is IgG4-RD, HT with IgG4-positive plasma cells is histopathologically distinct entity with some geographic variability.


Assuntos
Doença de Hashimoto/imunologia , Imunoglobulina G/metabolismo , Plasmócitos/metabolismo , ADP-Ribosil Ciclase 1/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Feminino , Fibrose , Finlândia , Doença de Hashimoto/patologia , Humanos , Inflamação , Contagem de Linfócitos , Masculino , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Plasmócitos/patologia , Glândula Tireoide/imunologia , Glândula Tireoide/patologia , Adulto Jovem
10.
Proc Natl Acad Sci U S A ; 117(26): 15160-15171, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32541026

RESUMO

IgG antibodies cause inflammation and organ damage in autoimmune diseases such as systemic lupus erythematosus (SLE). We investigated the metabolic profile of macrophages isolated from inflamed tissues in immune complex (IC)-associated diseases, including SLE and rheumatoid arthritis, and following IgG Fcγ receptor cross-linking. We found that human and mouse macrophages undergo a switch to glycolysis in response to IgG IC stimulation, mirroring macrophage metabolic changes in inflamed tissue in vivo. This metabolic reprogramming was required to generate a number of proinflammatory mediators, including IL-1ß, and was dependent on mTOR and hypoxia-inducible factor (HIF)1α. Inhibition of glycolysis, or genetic depletion of HIF1α, attenuated IgG IC-induced activation of macrophages in vitro, including primary human kidney macrophages. In vivo, glycolysis inhibition led to a reduction in kidney macrophage IL-1ß and reduced neutrophil recruitment in a murine model of antibody-mediated nephritis. Together, our data reveal the molecular mechanisms underpinning FcγR-mediated metabolic reprogramming in macrophages and suggest a therapeutic strategy for autoantibody-induced inflammation, including lupus nephritis.


Assuntos
Reprogramação Celular/fisiologia , Nefrite Lúpica/metabolismo , Animais , Células Cultivadas , Dinoprostona/genética , Dinoprostona/metabolismo , Metabolismo Energético , Regulação da Expressão Gênica , Glicólise/fisiologia , Humanos , Imunoglobulina G/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Rim/citologia , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espécies Reativas de Oxigênio , Receptores de IgG/genética , Receptores de IgG/metabolismo
11.
Nat Commun ; 11(1): 3114, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561744

RESUMO

Revealing antibody-antigen interactions at the single-molecule level will deepen our understanding of immunology. However, structural determination under crystal or cryogenic conditions does not provide temporal resolution for resolving transient, physiologically or pathologically relevant functional antibody-antigen complexes. Here, we develop a triangular DNA origami framework with site-specifically anchored and spatially organized artificial epitopes to capture transient conformations of immunoglobulin Gs (IgGs) at room temperature. The DNA origami epitopes (DOEs) allows programmed spatial distribution of epitope spikes, which enables direct imaging of functional complexes with atomic force microscopy (AFM). We establish the critical dependence of the IgG avidity on the lateral distance of epitopes within 3-20 nm at the single-molecule level. High-speed AFM imaging of transient conformations further provides structural and dynamic evidence for the IgG avidity from monovalent to bivalent in a single event, which sheds light on various applications including virus neutralization, diagnostic detection and cancer immunotherapy.


Assuntos
Afinidade de Anticorpos , Epitopos/ultraestrutura , Imunoglobulina G/ultraestrutura , Sondas Moleculares/ultraestrutura , Imagem Individual de Molécula/métodos , Complexo Antígeno-Anticorpo/ultraestrutura , DNA de Cadeia Simples/imunologia , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/ultraestrutura , Epitopos/imunologia , Epitopos/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Microscopia de Força Atômica/métodos , Simulação de Dinâmica Molecular , Sondas Moleculares/imunologia , Sondas Moleculares/metabolismo , Nanotecnologia , Relação Estrutura-Atividade
12.
Int J Mol Sci ; 21(11)2020 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-32503354

RESUMO

Monoclonal antibodies, engineered antibodies, and antibody fragments have become important biological therapeutic platforms. The IgG format with bivalent binding sites has a modular structure with different biological roles, i.e., effector and binding functions, in different domains. We demonstrated the reconstruction of an IgG-like domain structure in vitro by protein ligation using protein trans-splicing. We produced various binding domains to replace the binding domain of IgG from Escherichia coli and the Fc domain of human IgG from Brevibacillus choshinensis as split-intein fusions. We showed that in vitro protein ligation could produce various Fc-fusions at the N-terminus in vitro from the independently produced domains from different organisms. We thus propose an off-the-shelf approach for the combinatorial production of Fc fusions in vitro with several distinct binding domains, particularly from naturally occurring binding domains. Antiviral lectins from algae are known to inhibit virus entry of HIV and SARS coronavirus. We demonstrated that a lectin could be fused with the Fc-domain in vitro by protein ligation, producing an IgG-like molecule as a "lectibody". Such an Fc-fusion could be produced in vitro by this approach, which could be an attractive method for developing potential therapeutic agents against rapidly emerging infectious diseases like SARS coronavirus without any genetic fusion and expression optimization.


Assuntos
Fragmentos Fc das Imunoglobulinas/metabolismo , Lectinas/metabolismo , Trans-Splicing , Brevibacillus/imunologia , Clorófitas/metabolismo , HIV/fisiologia , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Lectinas/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Vírus da SARS/fisiologia , Internalização do Vírus/efeitos dos fármacos
14.
Clin Microbiol Infect ; 26(8): 1082-1087, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32473953

RESUMO

OBJECTIVES: To evaluate the diagnostic performance of seven rapid IgG/IgM tests and the Euroimmun IgA/IgG ELISA for antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in COVID-19 patients. METHODS: Specificity was evaluated in 103 samples collected before January 2020. Sensitivity and time to seropositivity was evaluated in 167 samples from 94 patients with COVID-19 confirmed with RT-PCR on nasopharyngeal swab. RESULTS: Specificity (confidence interval) of lateral flow assays (LFAs) was ≥91.3% (84.0-95.5) for IgM, ≥90.3% (82.9-94.8) for IgG, and ≥85.4% (77.2-91.1) for the combination IgM OR IgG. Specificity of the ELISA was 96.1% (90.1-98.8) for IgG and only 73.8% (64.5-81.4) for IgA. Sensitivity 14-25 days after the onset of symptoms was between ≥92.1% (78.5-98.0) and 100% (95.7-100) for IgG LFA compared to 89.5% (75.3-96.4) for IgG ELISA. Positivity of IgM OR IgG for LFA resulted in a decrease in specificity compared to IgG alone without a gain in diagnostic performance, except for VivaDiag. The results for IgM varied significantly between the LFAs with an average overall agreement of only 70% compared to 89% for IgG. The average dynamic trend to seropositivity for IgM was not shorter than for IgG. At the time of hospital admission the sensitivity of LFA was <60%. CONCLUSIONS: Sensitivity for the detection of IgG antibodies 14-25 days after the onset of symptoms was ≥92.1% for all seven LFAs compared to 89.5% for the IgG ELISA. The results for IgM varied significantly, and including IgM antibodies in addition to IgG for the interpretation of LFAs did not improve the diagnostic performance.


Assuntos
Anticorpos Antivirais/análise , Antígenos Virais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Infecções por Coronavirus/imunologia , Testes Diagnósticos de Rotina , Feminino , Humanos , Imunoglobulina A/metabolismo , Imunoglobulina G/metabolismo , Imunoglobulina M/metabolismo , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/imunologia , Sensibilidade e Especificidade , Fatores de Tempo , Adulto Jovem
15.
PLoS One ; 15(5): e0233047, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32392269

RESUMO

Fruits have been widely considered as the default "health foods" because they contain numerous vitamins and minerals needed to sustain human health. Fermentation strategies have been utilized to enhance the nutritive and flavor features of healthy and readily consumable fruit products while extending their shelf lives. A traditional fermented multi-fruit beverage was made from five fruits including kiwi, guava, papaya, pineapple, and grape fermented by Saccharomyces cerevisiae along with lactic acid bacteria and acetic acid bacteria. The immunomodulatory properties of the fermented multi-fruit beverage, in vivo nonspecific and ovalbumin (OVA)-specific immune response experiments using female BALB/c mice were performed. Administration of the fermented multi-fruit beverage reduced the calorie intake, thus resulting in a less weight gain in mice compared to the water (placebo)-fed mice. In the nonspecific immune study model, the fermented multi-fruit beverage enhanced phagocytosis and T cell proliferation but did not affect B cell proliferation and immunoglobulin G (IgG) production. Analysis of cytokine secretion profile also revealed that the fermented multi-fruit beverage enhanced proinflammatory cytokines interleukin (IL)-6, tumor necrosis factor (TNF)-α, and T helper (Th)1-related cytokine interferon (IFN)-γ production, thus creating an immunostimulatory effect. Nonetheless, in the specific immune study model, the results showed that the fermented multi-fruit beverage decreased the production of proinflammatory cytokines IL-6 and TNF-α production in OVA-immunized mice. Moreover, it also caused a decrease in the production of anti-OVA IgG1, which was accompanied by a decrease in Th2-related cytokines IL-4 and IL-5 production and an increase in Th1-related cytokine IFN-γ production, indicating that it may have the potential to shift the immune system from the allergen-specific Th2 responses toward Th1-type responses. The results indicate that fermented multi-fruit beverage has the potential to modulate immune responses both in a nonspecific and specific manners.


Assuntos
Citocinas/metabolismo , Alimentos e Bebidas Fermentados/microbiologia , Frutas/imunologia , Ovalbumina/administração & dosagem , Linfócitos T/metabolismo , Acetobacteraceae/fisiologia , Administração Oral , Animais , Peso Corporal , Feminino , Imunoglobulina G/metabolismo , Lactobacillales/fisiologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Fagocitose , Saccharomyces cerevisiae/fisiologia , Células Th1/imunologia , Células Th2/imunologia , Vacinação
17.
Transfusion ; 60(5): 1060-1068, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32369193

RESUMO

BACKGROUND: Anti-red blood cell (RBC) alloantibodies consisting of only the immunoglobulin G (IgG) 4 subtype are typically considered clinically insignificant. A US Food and Drug Administration-approved monoclonal anti-human globulin (16H8) is nonreactive with IgG4, which has been considered a benefit to avoid testing interference from IgG4. However, 16H8 also does not recognize two natural IgG3 variants (IgG3-03 and IgG3-13). Thus, 16H8 may miss clinically significant alloantibodies in some settings. STUDY DESIGN AND METHODS: Novel mouse anti-human IgG hybridomas were generated and screened for reactivity with 32 human variants of anti-KEL1 across different IgG subtypes, as well as mutants to allow epitope mapping. Anti-IgG reactivity was determined using KEL1+ RBCs bound by each IgG variant as targets. Binding of anti-IgG was determined by flow cytometry. RESULTS: 16H8 recognized an epitope involving amino acid 419, which is glutamate in IgG4, IgG3-03, and IgG3-13, explaining the lack of 16H8 reactivity with these subtypes/isoallotypes. A new monoclonal antibody (PUMA8) was isolated that, like 16H8, was nonreactive with IgG4 but without blind spots for known variants of IgG1, IgG2, or IgG3. PUMA8 recognized an epitope containing arginine at position 355, which is glutamine in IgG4. However, a recently described new IgG4 variant with an arginine at position 355 results in PUMA8 reactivity. CONCLUSION: PUMA8 represents an alternative to 16H8 that avoids IgG4 but without blind spots for IgG3 variants. However, PUMA8 reacts with one recently described IgG4 variant. In addition to relevance to immunohematology, these studies highlight the importance of patient variation with regards to assay performance in an era of personalized medicine.


Assuntos
Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Imunoglobulina G/imunologia , Imunoglobulinas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Células CHO , Cricetinae , Cricetulus , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Eritrócitos/imunologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Imunoglobulinas/química , Imunoglobulinas/metabolismo , Testes Imunológicos , Isoanticorpos/análise , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ligação Proteica , Análise de Sequência de Proteína
18.
Proc Natl Acad Sci U S A ; 117(23): 12943-12951, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32461366

RESUMO

The IgG Fc domain has the capacity to interact with diverse types of receptors, including the neonatal Fc receptor (FcRn) and Fcγ receptors (FcγRs), which confer pleiotropic biological activities. Whereas FcRn regulates IgG epithelial transport and recycling, Fc effector activities, such as antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis, are mediated by FcγRs, which upon cross-linking transduce signals that modulate the function of effector leukocytes. Despite the well-defined and nonoverlapping functional properties of FcRn and FcγRs, recent studies have suggested that FcγRs mediate transplacental IgG transport, as certain Fc glycoforms were reported to be enriched in fetal circulation. To determine the contribution of FcγRs and FcRn to the maternal-fetal transport of IgG, we characterized the IgG Fc glycosylation in paired maternal-fetal samples from patient cohorts from Uganda and Nicaragua. No differences in IgG1 Fc glycan profiles and minimal differences in IgG2 Fc glycans were noted, whereas the presence or absence of galactose on the Fc glycan of IgG1 did not alter FcγRIIIa or FcRn binding, half-life, or their ability to deplete target cells in FcγR/FcRn humanized mice. Modeling maternal-fetal transport in FcγR/FcRn humanized mice confirmed that only FcRn contributed to transplacental transport of IgG; IgG selectively enhanced for FcRn binding resulted in enhanced accumulation of maternal antibody in the fetus. In contrast, enhancing FcγRIIIa binding did not result in enhanced maternal-fetal transport. These results argue against a role for FcγRs in IgG transplacental transport, suggesting Fc engineering of maternally administered antibody to enhance only FcRn binding as a means to improve maternal-fetal transport of IgG.


Assuntos
Sangue Fetal/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Imunoglobulina G/metabolismo , Troca Materno-Fetal/imunologia , Circulação Placentária/imunologia , Receptores Fc/metabolismo , Animais , Feminino , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Imunoglobulina G/imunologia , Camundongos , Camundongos Transgênicos , Gravidez , Ensaios Clínicos Controlados Aleatórios como Assunto , Receptores Fc/genética , Receptores de IgG/genética , Receptores de IgG/metabolismo
19.
Nat Commun ; 11(1): 2330, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32393818

RESUMO

Recombinant T cell receptors (TCRs) can be used to redirect naïve T cells to eliminate virally infected or cancerous cells; however, they are plagued by low stability and uneven expression. Here, we use molecular modeling to identify mutations in the TCR constant domains (Cα/Cß) that increase the unfolding temperature of Cα/Cß by 20 °C, improve the expression of four separate α/ß TCRs by 3- to 10-fold, and improve the assembly and stability of TCRs with poor intrinsic stability. The stabilizing mutations rescue the expression of TCRs destabilized through variable domain mutation. The improved stability and folding of the TCRs reduces glycosylation, perhaps through conformational stabilization that restricts access to N-linked glycosylation enzymes. The Cα/Cß mutations enables antibody-like expression and assembly of well-behaved bispecific molecules that combine an anti-CD3 antibody with the stabilized TCR. These TCR/CD3 bispecifics can redirect T cells to kill tumor cells with target HLA/peptide on their surfaces in vitro.


Assuntos
Anticorpos Biespecíficos/imunologia , Biologia Computacional/métodos , Receptores de Antígenos de Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Biespecíficos/química , Varredura Diferencial de Calorimetria , Citotoxicidade Imunológica , Imunoglobulina G/metabolismo , Camundongos , Mutação/genética , Polissacarídeos/metabolismo , Desnaturação Proteica , Estabilidade Proteica , Subunidades Proteicas/metabolismo , Receptores de Antígenos de Linfócitos T/química , Proteínas Recombinantes/metabolismo , Solubilidade , Temperatura
20.
Nat Commun ; 11(1): 2070, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: covidwho-116533

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, at the end of 2019, and there are currently no specific antiviral treatments or vaccines available. SARS-CoV-2 has been shown to use the same cell entry receptor as SARS-CoV, angiotensin-converting enzyme 2 (ACE2). In this report, we generate a recombinant protein by connecting the extracellular domain of human ACE2 to the Fc region of the human immunoglobulin IgG1. A fusion protein containing an ACE2 mutant with low catalytic activity is also used in this study. The fusion proteins are then characterized. Both fusion proteins have a high binding affinity for the receptor-binding domains of SARS-CoV and SARS-CoV-2 and exhibit desirable pharmacological properties in mice. Moreover, the fusion proteins neutralize virus pseudotyped with SARS-CoV or SARS-CoV-2 spike proteins in vitro. As these fusion proteins exhibit cross-reactivity against coronaviruses, they have potential applications in the diagnosis, prophylaxis, and treatment of SARS-CoV-2.


Assuntos
Betacoronavirus/efeitos dos fármacos , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Testes de Neutralização , Peptidil Dipeptidase A/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Animais , Betacoronavirus/metabolismo , Ligação Competitiva/efeitos dos fármacos , Reações Cruzadas , Desenho de Fármacos , Humanos , Fragmentos Fc das Imunoglobulinas/metabolismo , Fragmentos Fc das Imunoglobulinas/farmacologia , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Técnicas In Vitro , Concentração Inibidora 50 , Fusão de Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/farmacocinética , Peptidil Dipeptidase A/farmacologia , Domínios Proteicos/genética , Estabilidade Proteica , Receptores Virais/antagonistas & inibidores , Receptores Virais/química , Receptores Virais/genética , Receptores Virais/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacocinética , Vírus da SARS/efeitos dos fármacos , Vírus da SARS/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA