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1.
J Gen Virol ; 100(3): 431-445, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30714896

RESUMO

A non-replicating form of pseudotyped influenza virus, inactivated by suppression of the haemagglutinin signal sequence (S-FLU), can act as a broadly protective vaccine. S-FLU can infect for a single round only, and induces heterotypic protection predominantly through activation of cross-reactive T cells in the lung. Unlike the licensed live attenuated virus, it cannot reassort a pandemic haemagglutinin (HA) into seasonal influenza. Here we present data on four new forms of S-FLU coated with H7 HAs from either A/Anhui/1/2013, A/Shanghai/1/2013, A/Netherlands/219/2003 or A/New York/107/2003 strains of H7 virus. We show that intranasal vaccination induced a strong local CD8 T cell response and protected against heterosubtypic X31 (H3N2) virus and highly virulent PR8 (H1N1), but not influenza B virus. Intranasal vaccination also induced a strong neutralizing antibody response to the encoded neuraminidase. If given at higher dose in the periphery with intraperitoneal administration, H7 S-FLU induced a specific neutralizing antibody response to H7 HA coating the particle. Polyvalent intraperitoneal vaccination with mixed H7 S-FLU induced a broadly neutralizing antibody response to all four H7 strains. S-FLU is a versatile vaccine candidate that could be rapidly mobilized ahead of a new pandemic threat.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Influenza Humana/prevenção & controle , Neuraminidase/imunologia , Animais , Proteção Cruzada , Glicoproteínas de Hemaglutininação de Vírus da Influenza/administração & dosagem , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/imunologia , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Influenza Humana/virologia , Masculino , Camundongos Endogâmicos C57BL , Neuraminidase/genética , Vacinação
2.
Blood Cells Mol Dis ; 43(2): 180-93, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19487139

RESUMO

Hereditary hemochromatosis is most frequently associated with mutations in HFE, which encodes a class Ib histocompatibility protein. HFE binds to the transferrin receptor-1 (TfR1) in competition with iron-loaded transferrin (Fe-Tf). HFE is released from TfR1 by increasing concentrations of Fe-Tf, and free HFE may then regulate iron homeostasis by binding other ligands. To search for new HFE ligands we expressed recombinant forms of HFE in the human cell line 293T. HFE protein was purified, biotinylated and made into fluorescently labelled tetramers. HFE tetramers bound to TfR1 in competition with Tf, but in addition we detected a binding activity on some cell types that was not blocked by Fe-Tf or by mutations in HFE that prevent binding to TfR1. We identified this second HFE ligand as the cation independent mannose-6-phosphate receptor (CI-MPR, also known as the insulin-like growth factor-2 receptor, IGF2R). HFE:CI-MPR binding was mediated through phosphorylated mannose residues on HFE. Recombinant murine Hfe also bound to CI-MPR. HFE bound to TfR1 was prevented from binding CI-MPR until released by increasing concentrations of Fe-Tf, a feature consistent with an iron sensing mechanism. However, it remains to be determined whether endogenous HFE in vivo also acquires the mannose-6 phosphate modification and binds to CI-MPR.


Assuntos
Antígenos CD/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Manosefosfatos/metabolismo , Proteínas de Membrana/metabolismo , Receptor IGF Tipo 2/metabolismo , Receptores da Transferrina/metabolismo , Transferrina/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proteína da Hemocromatose , Humanos , Ligantes , Camundongos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
3.
Blood Cells Mol Dis ; 40(3): 360-9, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-17977032

RESUMO

Ferroportin is a multi-transmembrane glycoprotein that mediates iron export from cells. Mutations in ferroportin are linked to type IV hemochromatosis, a dominantly inherited disorder of iron metabolism. Multimers of ferroportin, whose existence may relate to the dominant inheritance pattern of disease, have been detected in some studies but not others. We looked for evidence of multimerization in several different types of experiment. We assayed the maturation of mutant and wild-type ferroportin and found that loss-of-function mutants had a reduced half-life but did not alter the stability of coexpressed wild-type. Using bioluminescence resonance energy transfer analysis, we tested how mature wild-type ferroportin behaved in intact live cell membranes. Ferroportin-ferroportin interactions gave the very low acceptor/donor ratio-independent energy transfer levels characteristic of random protein-protein interactions, consistent with ferroportin behaving as a monomer. Consistent with these experiments, we were unable to detect a dominant negative functional effect of mutant ferroportin on wild-type, even when expression of wild-type protein was titrated to low levels. These data suggest that dominantly inherited ferroportin disease does not result from the direct action of a mutated protein inhibiting a wild-type protein within multimers. We propose other possible mechanisms of disease.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Células Epiteliais/metabolismo , Ferritinas/metabolismo , Hemocromatose/metabolismo , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Linhagem Celular , Membrana Celular/metabolismo , Células Epiteliais/citologia , Hemocromatose/genética , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação
4.
Blood ; 106(3): 1092-7, 2005 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-15831700

RESUMO

Ferroportin (FPN) mediates iron export from cells; FPN mutations are associated with the iron overloading disorder hemochromatosis. Previously, we found that the A77D, V162del, and G490D mutations inhibited FPN activity, but that other disease-associated FPN variants retained full iron export capability. The peptide hormone hepcidin inhibits FPN as part of a homeostatic negative feedback loop. We measured surface expression and function of wild-type FPN and fully active FPN mutants in the presence of hepcidin. We found that the Y64N and C326Y mutants of FPN are completely resistant to hepcidin inhibition and that N144D and N144H are partially resistant. Hemochromatosis-associated FPN mutations, therefore, either reduce iron export ability or produce an FPN variant that is insensitive to hepcidin. The former mutation type is associated with Kupffer-cell iron deposition and normal transferrin saturation in vivo, whereas patients with the latter category of FPN mutation have high transferrin saturation and tend to deposit iron throughout the liver parenchyma. FPN-linked hemochromatosis may have a variable pathogenesis depending on the causative FPN mutant.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas de Transporte de Cátions/genética , Resistência a Medicamentos/genética , Hemocromatose/genética , Mutação de Sentido Incorreto , Proteínas de Transporte de Cátions/antagonistas & inibidores , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Hemocromatose/tratamento farmacológico , Hemocromatose/etiologia , Hepcidinas , Humanos , Ferro/metabolismo , Radioisótopos de Ferro/metabolismo , Transferrina/metabolismo
5.
Blood ; 105(10): 4096-102, 2005 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-15692071

RESUMO

Type IV hemochromatosis is associated with dominant mutations in the SLC40A1 gene encoding ferroportin (FPN). Known as the "ferroportin disease," this condition is typically characterized by high serum ferritin, reduced transferrin saturation, and macrophage iron loading. Previously FPN expression in vitro has been shown to cause iron deficiency in human cell lines and mediate iron export from Xenopus oocytes. We confirm these findings by showing that expression of human FPN in a human cell line results in an iron deficiency because of a 3-fold increased export of iron. We show that FPN mutations A77D, V162delta, and G490D that are associated with a typical pattern of disease in vivo cause a loss of iron export function in vitro but do not physically or functionally impede wild-type FPN. These mutants may, therefore, lead to disease by haploinsufficiency. By contrast the variants Y64N, N144D, N144H, Q248H, and C326Y, which can be associated with greater transferrin saturation and more prominent iron deposition in liver parenchyma in vivo, retained iron export function in vitro. Because FPN is a target for negative feedback in iron homeostasis, we postulate that the latter group of mutants may resist inhibition, resulting in a permanently "turned on" iron exporter.


Assuntos
Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Hemocromatose/genética , Mutação/genética , Antígenos CD , Linhagem Celular , Ferritinas/metabolismo , Humanos , Espaço Intracelular/metabolismo , Ferro/metabolismo , Deficiências de Ferro , Fenótipo , Ligação Proteica , Receptores da Transferrina/metabolismo
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