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1.
J Virol ; 95(17): e0186820, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34132574

RESUMO

Pathogenic clade B New World mammarenaviruses (NWM) can cause Argentine, Venezuelan, Brazilian, and Bolivian hemorrhagic fevers. Sequence variability among NWM glycoproteins (GP) poses a challenge to the development of broadly neutralizing therapeutics against the entire clade of viruses. However, blockade of their shared binding site on the apical domain of human transferrin receptor 1 (hTfR1/CD71) presents an opportunity for the development of effective and broadly neutralizing therapeutics. Here, we demonstrate that the murine monoclonal antibody OKT9, which targets the apical domain of hTfR1, can sterically block cellular entry by viral particles presenting clade B NWM glycoproteins (GP1-GP2). OKT9 blockade is also effective against viral particles pseudotyped with glycoproteins of a recently identified pathogenic Sabia-like virus. With nanomolar affinity for hTfR1, the OKT9 antigen binding fragment (OKT9-Fab) sterically blocks clade B NWM-GP1s and reduces infectivity of an attenuated strain of Junin virus. Binding of OKT9 to the hTfR1 ectodomain in its soluble, dimeric state produces stable assemblies that are observable by negative-stain electron microscopy. A model of the OKT9-sTfR1 complex, informed by the known crystallographic structure of sTfR1 and a newly determined structure of the OKT9 antigen binding fragment (Fab), suggests that OKT9 and the Machupo virus GP1 share a binding site on the hTfR1 apical domain. The structural basis for this interaction presents a framework for the design and development of high-affinity, broadly acting agents targeting clade B NWMs. IMPORTANCE Pathogenic clade B NWMs cause grave infectious diseases, the South American hemorrhagic fevers. Their etiological agents are Junin (JUNV), Guanarito (GTOV), Sabiá (SABV), Machupo (MACV), Chapare (CHAV), and a new Sabiá-like (SABV-L) virus recently identified in Brazil. These are priority A pathogens due to their high infectivity and mortality, their potential for person-to-person transmission, and the limited availability of effective therapeutics and vaccines to curb their effects. While low homology between surface glycoproteins of NWMs foils efforts to develop broadly neutralizing therapies targeting NWMs, this work provides structural evidence that OKT9, a monoclonal antibody targeting a single NWM glycoprotein binding site on hTfR1, can efficiently prevent their entry into cells.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Antivirais/administração & dosagem , Arenavirus do Novo Mundo/fisiologia , Glicoproteínas/imunologia , Febre Hemorrágica Americana/prevenção & controle , Receptores da Transferrina/imunologia , Células A549 , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Febre Hemorrágica Americana/imunologia , Febre Hemorrágica Americana/virologia , Humanos , Estrutura Terciária de Proteína , Receptores da Transferrina/química , Receptores da Transferrina/genética
2.
MAbs ; 13(1): 1874121, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33499723

RESUMO

Receptor-mediated transcytosis (RMT) is used to enhance the delivery of monoclonal antibodies (mAb) into the central nervous system (CNS). While the binding to endogenous receptors on the brain capillary endothelial cells (BCECs) may facilitate the uptake of mAbs in the brain, a strong affinity for the receptor may hinder the efficiency of transcytosis. To quantitatively investigate the effect of binding affinity on the pharmacokinetics (PK) of anti-transferrin receptor (TfR) mAbs in different regions of the rat brain, we conducted a microdialysis study to directly measure the concentration of free mAbs at different sites of interest. Our results confirmed that bivalent anti-TfR mAb with an optimal dissociation constant (KD) value (76 nM) among four affinity variants can have up to 10-fold higher transcytosed free mAb exposure in the brain interstitial fluid (bISF) compared to lower and higher affinity mAbs (5 and 174 nM). This bell-shaped relationship between KD values and the increased brain exposure of mAbs was also visible when using whole-brain PK data. However, we found that mAb concentrations in postvascular brain supernatant (obtained after capillary depletion) were almost always higher than the concentrations measured in bISF using microdialysis. We also observed that the increase in mAb area under the concentration curve in CSF compartments was less significant, which highlights the challenge in using CSF measurement as a surrogate for estimating the efficiency of RMT delivery. Our results also suggest that the determination of mAb concentrations in the brain using microdialysis may be necessary to accurately measure the PK of CNS-targeted antibodies at the site-of-actions in the brain.


Assuntos
Anticorpos Monoclonais/farmacocinética , Afinidade de Anticorpos/imunologia , Encéfalo/metabolismo , Microdiálise/métodos , Receptores da Transferrina/imunologia , Animais , Anticorpos Monoclonais/líquido cefalorraquidiano , Anticorpos Monoclonais/imunologia , Antineoplásicos Imunológicos/administração & dosagem , Antineoplásicos Imunológicos/sangue , Área Sob a Curva , Transporte Biológico , Barreira Hematoencefálica/metabolismo , Encéfalo/citologia , Células CHO , Cricetinae , Cricetulus , Células Endoteliais/metabolismo , Humanos , Masculino , Ratos Sprague-Dawley , Transcitose , Trastuzumab/administração & dosagem , Trastuzumab/sangue
3.
FASEB J ; 35(2): e21172, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33241587

RESUMO

Transfer across the blood-brain barrier (BBB) remains a significant hurdle for the development of biopharmaceuticals with therapeutic effects within the central nervous system. We established a functional selection method to identify high affinity single domain antibodies to the transferrin receptor 1 (TfR1) with efficient biotherapeutic delivery across the BBB. A synthetic phage display library based on the variable domain of new antigen receptor (VNAR) was used for in vitro selection against recombinant human TfR1 ectodomain (rh-TfR1-ECD) followed by in vivo selection in mouse for brain parenchyma penetrating antibodies. TXB2 VNAR was identified as a high affinity, species cross-reactive VNAR antibody against TfR1-ECD that does not compete with transferrin or ferritin for receptor binding. IV dosing of TXB2 when fused to human Fc domain (TXB2-hFc) at 25 nmol/kg (1.875 mg/kg) in mice resulted in rapid binding to brain capillaries with subsequent transport into the brain parenchyma and specific uptake into TfR1-positive neurons. Likewise, IV dosing of TXB2-hFc fused with neurotensin (TXB2-hFc-NT) at 25 nmol/kg resulted in a rapid and reversible pharmacological response as measured by body temperature reduction. TXB2-hFc did not elicit any acute adverse reactions, bind, or deplete circulating reticulocytes or reduce BBB-expressed endogenous TfR1 in mice. There was no evidence of target-mediated clearance or accumulation in peripheral organs except lung. In conclusion, TXB2 is a high affinity, species cross-reactive, and brain-selective VNAR antibody to TfR1 that rapidly crosses the BBB and exhibits a favorable pharmacokinetic and safety profile and can be readily adapted to carry a wide variety of biotherapeutics from blood to brain.


Assuntos
Afinidade de Anticorpos , Antígenos CD/imunologia , Transporte Biológico/imunologia , Barreira Hematoencefálica/imunologia , Barreira Hematoencefálica/metabolismo , Receptores da Transferrina/imunologia , Anticorpos de Cadeia Única/imunologia , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Bacteriófagos/imunologia , Transporte Biológico/genética , Reações Cruzadas , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Receptores de Antígenos/imunologia , Receptores de Antígenos/metabolismo , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Anticorpos de Cadeia Única/farmacocinética , Transfecção
4.
Front Immunol ; 11: 597433, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33329589

RESUMO

Newborns are highly susceptible to infectious diseases. The underlying mechanism of neonatal infection susceptibility has generally been related to their under-developed immune system. Nevertheless, this notion has recently been challenged by the discovery of the physiological abundance of immunosuppressive erythroid precursors CD71+ erythroid cells (CECs) in newborn mice and human cord blood. Here, as proof of concept, we show that these cells are also abundant in the peripheral blood of human newborns. Although their frequency appears to be more variable compared to their counterparts in mice, they rapidly decline by 4 weeks of age. However, their proportion remains significantly higher in infants up to six months of age compared to older infants. We found CD45 expressing CECs, as erythroid progenitors, were the prominent source of reactive oxygen species (ROS) production in both humans and mice. Interestingly, a higher proportion of CD45+CECs was observed in the spleen versus bone marrow of neonatal mice, which was associated with a higher ROS production by splenic CECs compared to their siblings in the bone marrow. CECs from human newborns suppressed cytokine production by CD14 monocytes and T cells, which was partially abrogated by apocynin in vitro. Moreover, the depletion of CECs in neonatal mice increased the number of activated effector immune cells in their spleen and liver, which rendered them more resistant to Listeria monocytogenes infection. This was evident by a significant reduction in the bacteria load in the spleen, liver and brain of treated-mice compared to the control group, which enhanced their survival rate. Our finding highlights the immunoregulatory processes mediated by CECs in newborns. Thus, such tightly regulated immune system in newborns/infants may explain one potential mechanism for the asymptomatic or mild COVID-19 infection in this population.


Assuntos
Antígenos CD/imunologia , Células Precursoras Eritroides , Listeria monocytogenes/imunologia , Listeriose , Receptores da Transferrina/imunologia , Animais , Animais Recém-Nascidos , COVID-19/imunologia , COVID-19/patologia , Células Precursoras Eritroides/imunologia , Células Precursoras Eritroides/patologia , Células Precursoras Eritroides/transplante , Feminino , Xenoenxertos , Humanos , Recém-Nascido , Listeriose/imunologia , Listeriose/patologia , Listeriose/terapia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , SARS-CoV-2/imunologia
5.
Fluids Barriers CNS ; 17(1): 62, 2020 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-33054787

RESUMO

BACKGROUND: Preclinical models to determine blood to brain transport ability of therapeutics are often ambiguous. In this study a method is developed that relies on CNS target-engagement and is able to rank brain-penetrating capacities. This method led to the discovery of an anti-transferrin receptor nanobody that is able to deliver a biologically active peptide to the brain via receptor-mediated transcytosis. METHODS: Various nanobodies against the mouse transferrin receptor were fused to neurotensin and injected peripherally in mice. Neurotensin is a neuropeptide that causes hypothermia when present in the brain but is unable to reach the brain from the periphery. Continuous body temperature measurements were used as a readout for brain penetration of nanobody-neurotensin fusions after its peripheral administration. Full temperature curves were analyzed using two-way ANOVA with Dunnett multiple comparisons tests. RESULTS: One anti-transferrin receptor nanobody coupled to neurotensin elicited a drop in body temperature following intravenous injection. Epitope binning indicated that this nanobody bound a distinct transferrin receptor epitope compared to the non-crossing nanobodies. This brain-penetrating nanobody was used to characterize the in vivo hypothermia model. The hypothermic effect caused by neurotensin is dose-dependent and could be used to directly compare peripheral administration routes and various nanobodies in terms of brain exposure. CONCLUSION: This method led to the discovery of an anti-transferrin receptor nanobody that can reach the brain via receptor-mediated transcytosis after peripheral administration. This method could be used to assess novel proteins for brain-penetrating capabilities using a target-engaging readout.


Assuntos
Temperatura Corporal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Neurotensina/farmacologia , Receptores da Transferrina/imunologia , Anticorpos de Domínio Único/farmacologia , Transcitose/fisiologia , Animais , Camelídeos Americanos , Feminino , Masculino , Camundongos , Neurotensina/administração & dosagem , Receptores de Neurotensina/efeitos dos fármacos , Anticorpos de Domínio Único/administração & dosagem
6.
Mol Metab ; 42: 101060, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32763423

RESUMO

OBJECTIVES: The main endocrine cell types in pancreatic islets are alpha, beta, and delta cells. Although these cell types have distinct roles in the regulation of glucose homeostasis, inadequate purification methods preclude the study of cell type-specific effects. We developed a reliable approach that enables simultaneous sorting of live alpha, beta, and delta cells from mouse islets for downstream analyses. METHODS: We developed an antibody panel against cell surface antigens to enable isolation of highly purified endocrine subsets from mouse islets based on the specific differential expression of CD71 on beta cells and CD24 on delta cells. We rigorously demonstrated the reliability and validity of our approach using bulk and single cell qPCR, immunocytochemistry, reporter mice, and transcriptomics. RESULTS: Pancreatic alpha, beta, and delta cells can be separated based on beta cell-specific CD71 surface expression and high expression of CD24 on delta cells. We applied our new sorting strategy to demonstrate that CD71, which is the transferrin receptor mediating the uptake of transferrin-bound iron, is upregulated in beta cells during early postnatal weeks. We found that beta cells express higher levels of several other genes implicated in iron metabolism and iron deprivation significantly impaired beta cell function. In human beta cells, CD71 is similarly required for iron uptake and CD71 surface expression is regulated in a glucose-dependent manner. CONCLUSIONS: This study provides a novel and efficient purification method for murine alpha, beta, and delta cells, identifies for the first time CD71 as a postnatal beta cell-specific marker, and demonstrates a central role of iron metabolism in beta cell function.


Assuntos
Antígenos de Superfície/imunologia , Células Secretoras de Insulina/metabolismo , Ferro/metabolismo , Animais , Antígenos CD/imunologia , Antígenos de Superfície/isolamento & purificação , Antígenos de Superfície/metabolismo , Biomarcadores/metabolismo , Antígeno CD24/imunologia , Linhagem Celular , Feminino , Células Secretoras de Glucagon/imunologia , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Glucagon/fisiologia , Humanos , Imuno-Histoquímica/métodos , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/fisiologia , Ferro/fisiologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pâncreas/metabolismo , Pâncreas/fisiologia , Receptores da Transferrina/imunologia , Reprodutibilidade dos Testes , Células Secretoras de Somatostatina/imunologia , Células Secretoras de Somatostatina/metabolismo , Células Secretoras de Somatostatina/fisiologia
7.
Curr Med Sci ; 40(3): 493-501, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32681254

RESUMO

Transferrin receptor 1 (TfR1), encoded by the TFRC gene, is the gatekeeper of cellular iron uptake for cells. A variety of molecular mechanisms are at work to tightly regulate TfR1 expression, and abnormal TfR1 expression has been associated with various diseases. In the current study, to determine the regulation pattern of TfR1, we cloned and overexpressed the human TFRC gene in HeLa cells. RNA-sequencing (RNA-seq) was used to analyze the global transcript levels in overexpressed (OE) and normal control (NC) samples. A total of 1669 differentially expressed genes (DEGs) were identified between OE and NC. Gene ontology (GO) analysis was carried out to explore the functions of the DEGs. It was found that multiple DEGs were associated with ion transport and immunity. Moreover, the regulatory network was constructed on basis of DEGs associated with ion transport and immunity, highlighting that TFRC was the node gene of the network. These results together suggested that precisely controlled TfR1 expression might be not only essential for iron homeostasis, but also globally important for cell physiology, including ion transport and immunity.


Assuntos
Redes Reguladoras de Genes/genética , Imunidade/genética , Transporte de Íons/genética , Transporte de Íons/imunologia , Ferro/imunologia , Receptores da Transferrina/genética , Receptores da Transferrina/imunologia , Linhagem Celular Tumoral , Redes Reguladoras de Genes/imunologia , Células HeLa , Homeostase/genética , Homeostase/imunologia , Humanos , Imunidade/imunologia
8.
Infect Immun ; 88(9)2020 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-32601108

RESUMO

Chlamydia trachomatis infection of the human fallopian tubes can lead to damaging inflammation and scarring, ultimately resulting in infertility. To study the human cellular responses to chlamydial infection, researchers have frequently used transformed cell lines that can have limited translational relevance. We developed a primary human fallopian tube epithelial cell model based on a method previously established for culture of primary human bronchial epithelial cells. After protease digestion and physical dissociation of excised fallopian tubes, epithelial cell precursors were expanded in growth factor-containing medium. Expanded cells were cryopreserved to generate a biobank of cells from multiple donors and cultured at an air-liquid interface. Culture conditions stimulated cellular differentiation into polarized mucin-secreting and multiciliated cells, recapitulating the architecture of human fallopian tube epithelium. The polarized and differentiated cells were infected with a clinical isolate of C. trachomatis, and inclusions containing chlamydial developmental forms were visualized by fluorescence and electron microscopy. Apical secretions from infected cells contained increased amounts of proteins associated with chlamydial growth and replication, including transferrin receptor protein 1, the amino acid transporters SLC3A2 and SLC1A5, and the T-cell chemoattractants CXCL10, CXCL11, and RANTES. Flow cytometry revealed that chlamydial infection induced cell surface expression of T-cell homing and activation proteins, including ICAM-1, VCAM-1, HLA class I and II, and interferon gamma receptor. This human fallopian tube epithelial cell culture model is an important tool with translational potential for studying cellular responses to Chlamydia and other sexually transmitted pathogens.


Assuntos
Células Epiteliais/imunologia , Regulação da Expressão Gênica/imunologia , Interações entre Hospedeiro e Microrganismos/imunologia , Linfócitos T/imunologia , Adulto , Sistema ASC de Transporte de Aminoácidos/genética , Sistema ASC de Transporte de Aminoácidos/imunologia , Antígenos CD/genética , Antígenos CD/imunologia , Biomarcadores/metabolismo , Quimiocina CCL5/genética , Quimiocina CCL5/imunologia , Quimiocina CXCL10/genética , Quimiocina CXCL10/imunologia , Quimiocina CXCL11/genética , Quimiocina CXCL11/imunologia , Infecções por Chlamydia/genética , Infecções por Chlamydia/imunologia , Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/crescimento & desenvolvimento , Chlamydia trachomatis/imunologia , Células Epiteliais/microbiologia , Tubas Uterinas/citologia , Tubas Uterinas/cirurgia , Feminino , Cadeia Pesada da Proteína-1 Reguladora de Fusão/genética , Cadeia Pesada da Proteína-1 Reguladora de Fusão/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Interações entre Hospedeiro e Microrganismos/genética , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/imunologia , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/imunologia , Modelos Biológicos , Cultura Primária de Células , Receptores de Interferon/genética , Receptores de Interferon/imunologia , Receptores da Transferrina/genética , Receptores da Transferrina/imunologia , Salpingectomia , Linfócitos T/microbiologia , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/imunologia
9.
Nature ; 583(7816): 425-430, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32612231

RESUMO

The vascular interface of the brain, known as the blood-brain barrier (BBB), is understood to maintain brain function in part via its low transcellular permeability1-3. Yet, recent studies have demonstrated that brain ageing is sensitive to circulatory proteins4,5. Thus, it is unclear whether permeability to individually injected exogenous tracers-as is standard in BBB studies-fully represents blood-to-brain transport. Here we label hundreds of proteins constituting the mouse blood plasma proteome, and upon their systemic administration, study the BBB with its physiological ligand. We find that plasma proteins readily permeate the healthy brain parenchyma, with transport maintained by BBB-specific transcriptional programmes. Unlike IgG antibody, plasma protein uptake diminishes in the aged brain, driven by an age-related shift in transport from ligand-specific receptor-mediated to non-specific caveolar transcytosis. This age-related shift occurs alongside a specific loss of pericyte coverage. Pharmacological inhibition of the age-upregulated phosphatase ALPL, a predicted negative regulator of transport, enhances brain uptake of therapeutically relevant transferrin, transferrin receptor antibody and plasma. These findings reveal the extent of physiological protein transcytosis to the healthy brain, a mechanism of widespread BBB dysfunction with age and a strategy for enhanced drug delivery.


Assuntos
Envelhecimento/metabolismo , Envelhecimento/patologia , Barreira Hematoencefálica/metabolismo , Transcitose , Fosfatase Alcalina/metabolismo , Animais , Anticorpos/metabolismo , Transporte Biológico , Proteínas Sanguíneas/administração & dosagem , Proteínas Sanguíneas/metabolismo , Proteínas Sanguíneas/farmacocinética , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Sistemas de Liberação de Medicamentos , Saúde , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasma/metabolismo , Proteoma/administração & dosagem , Proteoma/metabolismo , Proteoma/farmacocinética , Receptores da Transferrina/imunologia , Transcrição Genética , Transferrina/metabolismo
10.
Cell ; 182(2): 267-269, 2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32707092

RESUMO

Brain disorders are at the leading edge of global disease burden worldwide. Effective therapies are lagging behind because most drugs cannot reach their targets in the brain because of the blood-brain barrier (BBB). The new development of a BBB transport vehicle may bring us a step closer to solve this problem.


Assuntos
Barreira Hematoencefálica/metabolismo , Encefalopatias/patologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Barreira Hematoencefálica/efeitos dos fármacos , Encefalopatias/metabolismo , Portadores de Fármacos/química , Humanos , Imunoconjugados/química , Imunoconjugados/imunologia , Imunoconjugados/farmacologia , Receptores da Transferrina/imunologia , Receptores da Transferrina/metabolismo , Transcitose
11.
Immunohorizons ; 4(4): 165-177, 2020 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-32284314

RESUMO

Iron uptake via the transferrin receptor (CD71) is a pivotal mechanism for T cell proliferation. Yet, it is incompletely understood if targeting of CD71 also affects the differentiation and functional polarization of primary human T cells. In this study, we demonstrate that inhibition of iron ingestion with blocking mAbs against CD71 induces nonproliferating T cells, which release high amounts of IL-2. Targeting of CD71 with blocking or nonblocking mAbs did not alter major signaling pathways and the activation of the transcription factors NF-κB, NFAT, or AP-1 as analyzed in Jurkat T cells. Growth arrest in iron-deficient (Fe-def) T cells was prevented upon addition of exogenous iron in the form of ferric ammonium citrate but was not reversible by exogenous IL-2. Surprisingly, protein synthesis was found to be intact in Fe-def T cells as demonstrated by comparable levels of CD69 upregulation and cytokine production with iron-sufficient T cells upon stimulation with CD3 plus CD28 mAbs. Indeed, high amounts of IL-2 were detectable in the supernatant of Fe-def T cells, which was accompanied with a reduced cell surface expression of IL-2R. When we used such Fe-def T cells in allogeneic MLRs, we observed that these cells acquired an accessory cell function and stimulated the proliferation of bystander T cells by providing IL-2. Thus, the results of our study demonstrate that iron deprivation causes nonproliferating, altruistic T cells that can help and stimulate other immune cells by providing cytokines such as IL-2.


Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Proliferação de Células/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Anticorpos Monoclonais Murinos/imunologia , Antígenos CD/imunologia , Doadores de Sangue , Antígenos CD28/antagonistas & inibidores , Antígenos CD28/imunologia , Complexo CD3/antagonistas & inibidores , Complexo CD3/imunologia , Feminino , Compostos Férricos/farmacologia , Sangue Fetal/citologia , Humanos , Interleucina-2/metabolismo , Células Jurkat , Camundongos , Compostos de Amônio Quaternário/farmacologia , Receptores da Transferrina/antagonistas & inibidores , Receptores da Transferrina/imunologia
12.
Fish Shellfish Immunol ; 100: 407-417, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32200071

RESUMO

Transferrin receptors (TfRs) play an essential role in iron-withholding strategy, and are involved in immune response against bacterial infection. In this study, the transferrin receptor 1 (OnTfR1) and transferrin receptor 2 (OnTfR2) genes are identified and characterized in Nile tilapia (Oreochromis niloticus). The open reading frames of OnTfR1 and OnTfR2 are 2220 and 2343 bp of nucleotide sequence, encoding 739 and 780 amino acids, respectively. The deduced proteins of OnTfR1 and OnTfR2 are highly homologous to those of other species, containing three conserved TfR superfamily domains (PA TfR domain, M28 TfR domain and TfR dimer domain). Expression analyses of OnTfRs in the healthy tilapia reveal that the OnTfR1 and OnTfR2 transcripts are the most abundant in the liver. The in vivo studies show that the expressions of OnTfRs are significantly up-regulate in liver and spleen, following infections of Streptococcus agalactiae and Aeromonas hydrophila. In addition, the in vitro studies reveal that the up-regulations of OnTfR expressions are also significant in monocytes/macrophages and hepatocytes upon the stimulations of S. agalactiae and A. hydrophila. Moreover, the iron ion (Fe3+) could significantly increase the expressions of OnTfRs in monocytes/macrophages and hepatocytes. Taken together, the present study indicates that OnTfRs may be involved in host defense against bacterial infection and possess the function of combining or transporting iron ions in Nile tilapia.


Assuntos
Ciclídeos/genética , Resistência à Doença , Proteínas de Peixes/genética , Infecções por Bactérias Gram-Negativas/veterinária , Ferro/metabolismo , Receptores da Transferrina/genética , Animais , Ciclídeos/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/imunologia , Regulação da Expressão Gênica , Bactérias Gram-Negativas/patogenicidade , Infecções por Bactérias Gram-Negativas/imunologia , Interações Hospedeiro-Patógeno , Imunidade Inata , Íons/metabolismo , Fígado/imunologia , Fígado/microbiologia , Macrófagos/imunologia , Receptores da Transferrina/classificação , Receptores da Transferrina/imunologia , Baço/imunologia , Baço/microbiologia
13.
Curr Med Sci ; 40(1): 28-34, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32166662

RESUMO

Selecting an ideal molecular format from diverse structures is a major challenge in developing a bispecific antibody (BsAb). To choose an ideal format of anti-CD3 × anti-transferrin receptor (TfR) bispecific antibodies for clinical application, we constructed TfR bispecific T-cell engager (BiTE) in two extensively applied formats, including single-chain tandem single-chain variable fragments (scFvs) and double-chain diabodies, and evaluated their functional characterizations in vitro. Results demonstrated that TfR-BiTE in both formats directed potent killing of TfR+ HepG2 cells. However, compared to two-chain diabodies, scFvs were more efficient in antigen binding and TfR+ target killing. Furthermore, different domain orders in scFvs would also be evaluated because single-TfR-CD3-His was preferable to single-CD3-TfR-His in immunotherapeutic strategies. Thus, the single-chain tandem TfR-CD3 format was favored for further investigation in cancer therapy.


Assuntos
Anticorpos Biespecíficos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Complexo CD3/imunologia , Receptores da Transferrina/imunologia , Células A549 , Anticorpos Biespecíficos/genética , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células HEK293 , Células Hep G2 , Humanos , Anticorpos de Cadeia Única/imunologia
14.
Mol Immunol ; 121: 1-6, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32135400

RESUMO

The transglutaminase 2 (TG2) is one of the enigmatic enzymes with important functional diversity. It plays an important role in several pathologies such as celiac disease (CD). In patients with active CD, the abnormal retrotranscytosis of IgA/gliadin complexes is mediated by Transferrin Receptor 1 (TfR1). This triad association takes also place in IgA nephropathy (IgA-N). IgA-N is characterized by the formation of nephrotoxic complexes of IgA1 and soluble CD89 (sCD89). These complexes are abnormally deposited in the kidney. Using a humanized mouse model of IgA-N (α1KI-CD89Tg), we showed that IgA1-sCD89 complexes engender mesangial cell activation and proliferation with TfR1 and TG2 up-regulation, associated with IgA-N features. This TG2-TfR1 interaction enhances mesangial IgA1 deposition promoting inflammation. Humanized α1KI-CD89Tg mice deficient for TG2 show a decrease in TfR1 expression in kidney leading to reduced IgA1-sCD89 deposits and an improvement in IgA-N features. Moreover, TG2 is active and overexpressed in the intestine of IgA-N mice and gliadin participates to this renal pathology. In kidney as in intestine, the TG2 has a crucial role in the cooperation between TfR1-IgA and a central role in the pathogenic amplification.


Assuntos
Antígenos CD/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Gliadina/imunologia , Glomerulonefrite por IGA/imunologia , Imunoglobulina A/metabolismo , Receptores da Transferrina/metabolismo , Transglutaminases/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Doença Celíaca/imunologia , Doença Celíaca/patologia , Modelos Animais de Doenças , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/imunologia , Gliadina/metabolismo , Humanos , Imunoglobulina A/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Células Mesangiais/imunologia , Células Mesangiais/patologia , Camundongos , Camundongos Transgênicos , Receptores Fc/genética , Receptores Fc/imunologia , Receptores Fc/metabolismo , Receptores da Transferrina/imunologia , Transglutaminases/genética , Transglutaminases/imunologia , Regulação para Cima/imunologia
15.
Proc Natl Acad Sci U S A ; 117(7): 3405-3414, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32005712

RESUMO

Drug targeting to inflammatory brain pathologies such as stroke and traumatic brain injury remains an elusive goal. Using a mouse model of acute brain inflammation induced by local tumor necrosis factor alpha (TNFα), we found that uptake of intravenously injected antibody to vascular cell adhesion molecule 1 (anti-VCAM) in the inflamed brain is >10-fold greater than antibodies to transferrin receptor-1 and intercellular adhesion molecule 1 (TfR-1 and ICAM-1). Furthermore, uptake of anti-VCAM/liposomes exceeded that of anti-TfR and anti-ICAM counterparts by ∼27- and ∼8-fold, respectively, achieving brain/blood ratio >300-fold higher than that of immunoglobulin G/liposomes. Single-photon emission computed tomography imaging affirmed specific anti-VCAM/liposome targeting to inflamed brain in mice. Intravital microscopy via cranial window and flow cytometry showed that in the inflamed brain anti-VCAM/liposomes bind to endothelium, not to leukocytes. Anti-VCAM/LNP selectively accumulated in the inflamed brain, providing de novo expression of proteins encoded by cargo messenger RNA (mRNA). Anti-VCAM/LNP-mRNA mediated expression of thrombomodulin (a natural endothelial inhibitor of thrombosis, inflammation, and vascular leakage) and alleviated TNFα-induced brain edema. Thus VCAM-directed nanocarriers provide a platform for cerebrovascular targeting to inflamed brain, with the goal of normalizing the integrity of the blood-brain barrier, thus benefiting numerous brain pathologies.


Assuntos
Anticorpos/administração & dosagem , Barreira Hematoencefálica/efeitos dos fármacos , Encefalite/tratamento farmacológico , Endotélio Vascular/efeitos dos fármacos , Nanomedicina/métodos , Animais , Barreira Hematoencefálica/imunologia , Encefalite/genética , Encefalite/imunologia , Endotélio Vascular/imunologia , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/imunologia , Camundongos , Receptores da Transferrina/genética , Receptores da Transferrina/imunologia , Trombomodulina/genética , Trombomodulina/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/imunologia
16.
Nat Commun ; 11(1): 67, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31900422

RESUMO

Certain arenaviruses that circulate in rodent populations can cause life-threatening hemorrhagic fevers when they infect humans. Due to their efficient transmission, arenaviruses pose a severe risk for outbreaks and might be exploited as biological weapons. Effective countermeasures against these viruses are highly desired. Ideally, a single remedy would be effective against many or even all the pathogenic viruses in this family. However, despite the fact that all pathogenic arenaviruses from South America utilize transferrin receptor 1 (TfR1) as a cellular receptor, their viral glycoproteins are highly diversified, impeding efforts to isolate cross-neutralizing antibodies. Here we address this problem using a rational design approach to target TfR1-tropic arenaviruses with high potency and breadth. The pan-reactive molecule is highly effective against all arenaviruses that were tested, offering a universal therapeutic approach. Our design scheme avoids the shortcomings of previous immunoadhesins and can be used to combat other zoonotic pathogens.


Assuntos
Infecções por Arenaviridae/terapia , Arenavirus/imunologia , Imunoterapia , Receptores da Transferrina/química , Receptores da Transferrina/imunologia , Receptores Virais/imunologia , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/química , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Infecções por Arenaviridae/imunologia , Infecções por Arenaviridae/virologia , Arenavirus/química , Arenavirus/genética , Desenho de Fármacos , Humanos , Receptores da Transferrina/genética , Receptores Virais/genética , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/imunologia
17.
J Virol ; 94(4)2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31748396

RESUMO

Several clade B New World arenaviruses (NWAs) can cause severe and often fatal hemorrhagic fever, for which preventive and therapeutic measures are severely limited. These NWAs use human transferrin receptor 1 (hTfR1) as a host cell receptor for virus entry. The most prevalent of the pathogenic NWAs is Junín virus (JUNV), the etiological agent of Argentine hemorrhagic fever. Small animal models of JUNV infection are limited because most laboratory rodent species are refractory to disease. Only guinea pigs are known to develop disease following JUNV infection, but the underlying mechanisms are not well characterized. In the present study, we demonstrate marked susceptibility of Hartley guinea pigs to uniformly lethal disease when challenged with as few as 4 PFU of the Romero strain of JUNV. In vitro, we show that infection of primary guinea pig macrophages results in greater JUNV replication compared to infection of hamster or mouse macrophages. We provide evidence that the guinea pig TfR1 (gpTfR1) is the principal receptor for JUNV, while hamster and mouse orthologs fail to support viral entry/infection of pseudotyped murine leukemia viruses expressing pathogenic NWA glycoproteins or JUNV. Together, our results indicate that gpTfR1 serves as the primary receptor for pathogenic NWAs, enhancing viral infection in guinea pigs.IMPORTANCE JUNV is one of five known NWAs that cause viral hemorrhagic fever in humans. Countermeasures against JUNV infection are limited to immunization with the Candid#1 vaccine and immune plasma, which are available only in Argentina. The gold standard small animal model for JUNV infection is the guinea pig. Here, we demonstrate high sensitivity of this species to severe JUNV infection and identify gpTfR1 as the primary receptor. Use of hTfR1 for host cell entry is a feature shared by pathogenic NWAs. Our results show that expression of gpTfR1 or hTfR1 comparably enhances JUNV virus entry/infectivity. Our findings shed light on JUNV infection in guinea pigs as a model for human disease and suggest that similar pathophysiological mechanisms related to iron sequestration during infection and regulation of TfR1 expression may be shared between humans and guinea pigs. A better understanding of the underlying disease process will guide development of new therapeutic interventions.


Assuntos
Vírus Junin/imunologia , Vírus Junin/patogenicidade , Receptores da Transferrina/metabolismo , Animais , Arenavirus/imunologia , Arenavirus/patogenicidade , Células CHO , Chlorocebus aethiops , Cricetulus , Modelos Animais de Doenças , Feminino , Glicoproteínas/metabolismo , Cobaias/imunologia , Cobaias/metabolismo , Células HEK293 , Febre Hemorrágica Americana/imunologia , Febre Hemorrágica Americana/virologia , Febres Hemorrágicas Virais/imunologia , Febres Hemorrágicas Virais/virologia , Humanos , Vírus Junin/metabolismo , Macrófagos/virologia , Masculino , Receptores da Transferrina/imunologia , Células Vero , Internalização do Vírus , Replicação Viral
18.
J Immunother ; 43(2): 48-52, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31693515

RESUMO

The transferrin receptor 1 (TfR1) is a meaningful target for antibody-based cancer therapy given its overexpression on malignant cells and its central role in cancer pathology. We previously developed a mouse/human chimeric IgG3 targeting human TfR1 (ch128.1), which exhibits significant antitumor activity against multiple myeloma (MM) in xenograft models of SCID-Beige mice bearing disseminated ARH-77 or KMS-11 tumors. This activity is observed in early and late disease stages of disseminated KMS-11 tumors and, in this model, the mechanism of antitumor activity is Fc-mediated, involving macrophages. As human IgG1 is the isotype of choice for therapeutic antibodies targeting malignant cells and has several advantages compared with IgG3, including established manufacturability, we now developed an IgG1 version of ch128.1. A single dose of ch128.1/IgG1 shows significant antitumor activity, not only against early and late stages of disseminated KMS-11 tumors (Asian origin) but also against these stages of disseminated disease following injection of human MM cells MM.1S (African American origin) or its variant that is resistant to dexamethasone MM.1R. Treatment with the Fc mutant version of ch128.1/IgG1 (L234A/L235A/P329S) with impaired effector functions fails to confer protection against MM.1S and MM.1R tumors, indicating a crucial role of the Fc fragment in the antitumor activity, similar to its IgG3 counterpart. In fact, we found that ch128.1/IgG1, but not the mutant, elicits antibody-dependent cell-mediated cytotoxicity and antibody-dependent cell-mediated phagocytosis in the presence of murine bone marrow-derived macrophages. Our results suggest that ch128.1/IgG1 is a promising therapeutic against human B-cell malignancies such as MM.


Assuntos
Imunoglobulina G/imunologia , Mieloma Múltiplo/imunologia , Receptores da Transferrina/imunologia , Animais , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Linfócitos B/imunologia , Feminino , Xenoenxertos/imunologia , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Macrófagos/imunologia , Camundongos , Camundongos SCID , Fagocitose/imunologia
19.
Clin Exp Immunol ; 199(3): 326-336, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31660581

RESUMO

Secretory IgA (SIgA) is a well-known mucosal-surface molecule in first-line defense against extrinsic pathogens and antigens. Its immunomodulatory and pathological roles have also been emphasized, but it is unclear whether it plays a pathological role in lung diseases. In the present study, we aimed to determine the distribution of IgA in idiopathic pulmonary fibrosis (IPF) lungs and whether IgA affects the functions of airway epithelial cells. We performed immunohistochemical analysis of lung sections from patients with IPF and found that mucus accumulated in the airspaces adjacent to the hyperplastic epithelia contained abundant SIgA. This was not true in the lungs of non-IPF subjects. An in-vitro assay revealed that SIgA bound to the surface of A549 cells and significantly promoted production of vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-ß and interleukin (IL)-8, important cytokines in the pathogenesis of IPF. Among the known receptors for IgA, A549 cells expressed high levels of transferrin receptor (TfR)/CD71. Transfection experiments with siRNA targeted against TfR/CD71 followed by stimulation with SIgA suggested that TfR/CD71 may be at least partially involved in the SIgA-induced cytokine production by A549 cells. These phenomena were specific for SIgA, distinct from IgG. SIgA may modulate the progression of IPF by enhancing synthesis of VEGF, TGF-ß and IL-8.


Assuntos
Fibrose Pulmonar Idiopática/imunologia , Imunoglobulina A Secretora/imunologia , Interleucina-8/imunologia , Pulmão/imunologia , Fator de Crescimento Transformador beta/imunologia , Fator A de Crescimento do Endotélio Vascular/imunologia , Células A549 , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos CD/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Imunoglobulina A Secretora/metabolismo , Imunoglobulina A Secretora/farmacologia , Interleucina-8/genética , Interleucina-8/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Pessoa de Meia-Idade , Interferência de RNA , Receptores da Transferrina/genética , Receptores da Transferrina/imunologia , Receptores da Transferrina/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
20.
Analyst ; 145(1): 257-267, 2019 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-31746823

RESUMO

Cancer is a major health problem in the United States with extremely high mortality. The detection and isolation of cancer cells are becoming increasingly important for cancer diagnosis. We describe a microfluidic device modified with silica nanoparticles to enhance the isolation of cancer cells using affinity separation. The isolation of seven different cancer cell lines spiked into liquid biopsies was demonstrated and compared with unmodified separation devices. Cancer cells were isolated using CD71 which has already been demonstrated to be a widespread "net" for capturing cancer cells of any phenotype as the affinity target. The capture efficiency of our nanoparticle (NP)-modified HB chip showed significant differences compared with the normal HB chip, exhibiting an average increase of 16%. The cell enrichment increased by a factor of 2 over unmodified chips. Patient-derived ALL cells, COG-LL-332, were spiked into blood with concentrations ranging from 1% to 20% of total leukocytes, and isolated with the purity of 41%-65%. The results of this study demonstrated that the increase of cell-chip interactions after nanoparticle modification improved capture efficiency and capture purity, and can be applied to a wide range of cell separations.


Assuntos
Separação Celular/métodos , Imunoensaio/métodos , Técnicas Analíticas Microfluídicas/métodos , Nanopartículas/química , Neoplasias/diagnóstico , Anticorpos Imobilizados/imunologia , Antígenos CD/imunologia , Linhagem Celular Tumoral , Humanos , Dispositivos Lab-On-A-Chip , Receptores da Transferrina/imunologia , Dióxido de Silício/química
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