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1.
Immunity ; 38(5): 1050-62, 2013 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-23602766

RESUMO

Cord factor, also called trehalose-6,6'-dimycolate (TDM), is a potent mycobacterial adjuvant. We herein report that the C-type lectin MCL (also called Clec4d) is a TDM receptor that is likely to arise from gene duplication of Mincle (also called Clec4e). Mincle is known to be an inducible receptor recognizing TDM, whereas MCL was constitutively expressed in myeloid cells. To examine the contribution of MCL in response to TDM adjuvant, we generated MCL-deficient mice. TDM promoted innate immune responses, such as granuloma formation, which was severely impaired in MCL-deficient mice. TDM-induced acquired immune responses, such as experimental autoimmune encephalomyelitis (EAE), was almost completely dependent on MCL, but not Mincle. Furthermore, by generating Clec4e(gfp) reporter mice, we found that MCL was also crucial for driving Mincle induction upon TDM stimulation. These results suggest that MCL is an FcRγ-coupled activating receptor that mediates the adjuvanticity of TDM.


Assuntos
Fatores Corda/imunologia , Encefalomielite Autoimune Experimental/imunologia , Lectinas Tipo C/imunologia , Proteínas de Membrana/metabolismo , Receptores de IgG/imunologia , Adjuvantes Imunológicos , Animais , Encefalomielite Autoimune Experimental/microbiologia , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Mycobacterium/imunologia , Mycobacterium bovis/imunologia , Mycobacterium tuberculosis/imunologia
2.
Biologicals ; 44(5): 394-402, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27464991

RESUMO

In previous studies, hybridomas producing human immunoglobulin G, the antibodies 5E4 and 5A7 against influenza A and B virus were established using a novel human lymphocyte fusion partner, SPYMEG. In the present study, we succeeded in achieving the recombinant production and secretion of 5E4 and 5A7 in Chinese hamster ovary cells. Our N-glycan analysis by intact-mass detection and liquid chromatography mass spectrometry showed that recombinant 5E4 and 5A7 have one N-glycan and the typical mammalian-type N-glycan structures similar to those in hybridomas. However, the glycan distribution was slightly different among these antibodies. The amount of high-mannose-type structures was under 10% of the total N-glycans of recombinant 5E4 and 5A7, compared to 20% of the 5E4 and 5A7 produced in hybridomas. The amount of galactosylated N-glycans was increased in recombinants. Approximately 80% of the N-glycans of all antibodies was fucosylated, and no sialylated N-glycan was found. Recombinant 5E4 and 5A7 neutralized pandemic influenza A virus specifically, and influenza B virus broadly, quite similar to the 5E4 and 5A7 produced in hybridomas, respectively. Here we demonstrated that recombinants of antibodies identified from hybridomas fused with SPYMEG have normal N-glycans and that their neutralizing activities bear comparison with those of the original antibodies.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Imunoglobulina G/imunologia , Vírus da Influenza A/imunologia , Vírus da Influenza B/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/sangue , Fusão Celular/métodos , Cricetinae , Humanos , Hibridomas/imunologia , Hibridomas/metabolismo , Imunoglobulina G/biossíntese , Linfócitos/imunologia , Linfócitos/metabolismo
3.
J Biol Chem ; 288(7): 4981-90, 2013 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-23264630

RESUMO

The development of vaccination methods that can overcome the emergence of new types of influenza strains caused by escape mutations is desirable to avoid future pandemics. Here, a novel type of immunogen was designed that targeted the conformation of a highly conserved region of influenza A virus hemagglutinin (HA) composed of two separate sequences that associate to form an anti-parallel ß-sheet structure. Our previous study identified this ß-sheet region as the structural core in the epitope of a characteristic antibody (B-1) that strongly neutralizes a wide variety of strains within the H3N2 serotype, and therefore this ß-sheet region was considered a good target to induce broadly reactive immunity against the influenza A virus. To design the immunogen, residues derived from the B-1 epitope were introduced directly onto a part of enhanced green fluorescent protein (EGFP), whose surface is mostly composed of ß-sheets. Through site-directed mutagenesis, several modified EGFPs with an epitope-mimicking structure embedded in their surface were prepared. Two EGFP variants, differing from wild-type (parental) EGFP by only five and nine residues, induced mice to produce antibodies that specifically bind to H3-type HA and neutralize H3N2 virus. Moreover, three of five mice immunized with each of these EGFP variants followed by a booster with equivalent mCherry variants acquired anti-viral immunity against challenge with H3N2 virus at a lethal dosage. In contrast to conventional methods, such as split HA vaccine, preparation of this type of immunogen requires less time and is therefore expected to be quickly responsive to newly emerged influenza viral strains.


Assuntos
Epitopos/química , Proteínas de Fluorescência Verde/metabolismo , Hemaglutininas/química , Animais , Dicroísmo Circular , Escherichia coli/metabolismo , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Hibridomas/metabolismo , Imunização , Vacinas contra Influenza/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese , Testes de Neutralização , Orthomyxoviridae/imunologia , Engenharia de Proteínas/métodos , Estrutura Secundária de Proteína , Proteínas Virais/química
4.
Biochem Biophys Res Commun ; 450(1): 42-8, 2014 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-24858683

RESUMO

Influenza viruses are a continuous threat to human public health because of their ability to evolve rapidly through genetic drift and reassortment. Three human monoclonal antibodies (HuMAbs) were generated in this study, 1H11, 2H5 and 5G2, and they cross-neutralize a diverse range of group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H5N1 and H9N2. The three HuMAbs were prepared by fusing peripheral blood lymphocytes from an H1N1pdm-infected patient with a newly developed fusion partner cell line, SPYMEG. All the HuMAbs had little hemagglutination inhibition activity but had strong membrane-fusion inhibition activity against influenza viruses. A protease digestion assay showed the HuMAbs targeted commonly a short α-helix region in the stalk of the hemagglutinin. Furthermore, Ile45Phe and Glu47Gly double substitutions in the α-helix region made the HA unrecognizable by the HuMAbs. These two amino acid residues are highly conserved in the HAs of H1N1, H5N1 and H9N2 viruses. The HuMAbs reported here may be potential candidates for the development of therapeutic antibodies against group 1 influenza viruses.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Hemaglutininas/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Influenza Humana/virologia , Animais , Anticorpos Antivirais/imunologia , Linhagem Celular , Haplorrinos , Humanos , Rim/imunologia , Rim/virologia , Testes de Neutralização
5.
Biochem Biophys Res Commun ; 452(3): 865-70, 2014 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-25204499

RESUMO

Most neutralizing antibodies elicited during influenza virus infection or by vaccination have a narrow spectrum because they usually target variable epitopes in the globular head region of hemagglutinin (HA). In this study, we describe a human monoclonal antibody (HuMAb), 5D7, that was prepared from the peripheral blood lymphocytes of a vaccinated volunteer using the fusion method. The HuMAb heterosubtypically neutralizes group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H9N2, with a strong hemagglutinin inhibition activity. Selection of an escape mutant showed that the HuMAb targets a novel conformational epitope that is located in the HA head region but is distinct from the receptor binding site. Furthermore, Phe114Ile substitution in the epitope made the HA unrecognizable by the HuMAb. Amino acid residues in the predicted epitope region are also highly conserved in the HAs of H1N1 and H9N2. The HuMAb reported here may be a potential candidate for the development of therapeutic/prophylactic antibodies against H1 and H9 influenza viruses.


Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Proteção Cruzada , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H9N2/imunologia , Influenza Humana/prevenção & controle , Sequência de Aminoácidos , Animais , Antígenos Virais/química , Antígenos Virais/imunologia , Mapeamento de Epitopos , Epitopos/química , Epitopos/imunologia , Testes de Inibição da Hemaglutinação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Hibridomas/imunologia , Vacinas contra Influenza/administração & dosagem , Influenza Humana/imunologia , Influenza Humana/virologia , Masculino , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Vacinação , Adulto Jovem
6.
Artigo em Inglês | MEDLINE | ID: mdl-24437318

RESUMO

We conducted this study to determine the clinical variables associated with the production of human immunodeficiency virus type 1 (HIV-1) circulating recombinant form (CRF) 01_AE neutralizing human monoclonal antibodies (NhMAbs) using a hybridoma technique. This cross sectional study was performed in 20 asymptomatic HIV-1-infected Thais. Peripheral blood mononuclear cells (PBMCs) were obtained from each study participant and fused with SPYMEG cells. Culture supernatant collected from growing hybridomas was tested for neutralizing activity against HIV-1 CRF01_AE Env-recombinant viruses. Fifty hybridomas expressing anti-HIV-1 NhMAbs with strong neutralizing activity against at least 1 CRF01_AE Env-recombinant virus were found. A positive association between the numbers of hybridomas produced and the CD4 counts of study participants (p = 0.019) was observed. NhMAb-producing hybridomas with strong neutralizing activity were mostly found in participans diagnosed with HIV-1 infection within the previous 1 year. The HIV-1 viral load was not significantly correlated with the numbers of either established hybridomas or clones expressing anti-HIV-1 NhMAbs with strong neutralizing activity. To our knowledge, this is the first study of NhMAb-producing hybridomas obtained from HIV-1 CRF01_AE-infected populations identified by antibody binding to HIV-1 V3 loop peptide enzyme-linked immunosorbent assay (ELISA) or TRUGENE HIV-1 Genotyping Assay (HIV-1 pol sequence). It provides important criterion to slect study participants with high CD4 counts who produce large numbers of hybridoma clones. The results are valuable for further studies related to nurtalizing antibodies production and HIV-1 vaccine development.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Adulto , Contagem de Linfócito CD4 , Ensaio de Imunoadsorção Enzimática , Genótipo , Anticorpos Anti-HIV/sangue , Anticorpos Anti-HIV/genética , Infecções por HIV/genética , HIV-1/genética , Humanos , Hibridomas/imunologia , Carga Viral
7.
Sci Rep ; 11(1): 12987, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34155267

RESUMO

Dengue virus (DENV), from the genus flavivirus of the family flaviviridae, causes serious health problems globally. Human monoclonal antibodies (HuMAb) can be used to elucidate the mechanisms of neutralization and antibody-dependent enhancement (ADE) of DENV infections, leading to the development of a vaccine or therapeutic antibodies. Here, we generated eight HuMAb clones from an Indonesian patient infected with DENV. These HuMAbs exhibited the typical characteristics of weak neutralizing antibodies including high cross-reactivity with other flaviviruses and targeting of the fusion loop epitope (FLE). However, one of the HuMAbs, 3G9, exhibited strong neutralization (NT50 < 0.1 µg/ml) and possessed a high somatic hyper-mutation rate of the variable region, indicating affinity-maturation. Administration of this antibody significantly prolonged the survival of interferon-α/ß/γ receptor knockout C57BL/6 mice after a lethal DENV challenge. Additionally, Fc-modified 3G9 that had lost their in vitro ADE activity showed enhanced therapeutic potency in vivo and competed strongly with an ADE-prone antibody in vitro. Taken together, the affinity-matured FLE-targeting antibody 3G9 exhibits promising features for therapeutic application including a low NT50 value, potential for treatment of various kinds of mosquito-borne flavivirus infection, and suppression of ADE. This study demonstrates the therapeutic potency of affinity-matured FLE-targeting antibodies.


Assuntos
Anticorpos Monoclonais/farmacologia , Afinidade de Anticorpos , Antígenos Virais , Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Dengue/virologia , Epitopos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Afinidade de Anticorpos/imunologia , Antígenos Virais/química , Antígenos Virais/imunologia , Antivirais/uso terapêutico , Dengue/tratamento farmacológico , Dengue/imunologia , Vírus da Dengue/imunologia , Mapeamento de Epitopos , Epitopos/química , Epitopos/imunologia , Humanos , Hibridomas , Camundongos , Modelos Moleculares , Testes de Neutralização , Conformação Proteica , Proteínas Recombinantes , Relação Estrutura-Atividade
8.
J Vet Med Sci ; 72(7): 893-901, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20215716

RESUMO

Dogs can be divided into two genetic groups (a minor HK phenotype and a major LK phenotype) based on erythrocyte monovalent cation concentrations, which are controlled by the putative hk and lk allelic genes. HK dogs retain Na,K-ATPase in their erythrocytes due to the high activity of the enzyme in their precursor cells, whereas total loss of reticulocyte Na,K-ATPase occurs in LK dogs. Here, we report that the levels of the lipid raft-associated membrane protein stomatin decrease in parallel with those of Na,K-ATPase during reticulocyte maturation due to its extrusion in exosomes. The stomatin content of HK reticulocytes is higher than that of LK reticulocytes, and remains in the erythrocytes at levels compatible with that in human erythrocytes. However, it is almost absent from LK erythrocytes with the lk/lk genotype; similar to the deficiency seen in human red cells with overhydrated stomatocytosis. LK erythrocytes from hk/lk genotype dogs show reduced, but not negligible, levels of stomatin. These results indicate that the erythrocyte stomatin level is a suitable genotypic marker for the HK/LK red cell phenotype, and suggests a functional association between stomatin and Na,K-ATPase. The absence of morphological abnormalities in the erythrocytes of stomatin-deficient LK dogs also confirms that stomatin deficiency and stomatocytic shape change are independent from each other.


Assuntos
Cães/genética , Potássio/sangue , Sequência de Aminoácidos , Anemia/sangue , Anemia/veterinária , Animais , Anticorpos Monoclonais , Cátions/sangue , Cães/sangue , Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/ultraestrutura , Eritrócitos/metabolismo , Genótipo , Humanos , Proteínas de Membrana/sangue , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Linhagem , Fenótipo , Reticulócitos/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , ATPase Trocadora de Sódio-Potássio/metabolismo
9.
Sci Rep ; 10(1): 21561, 2020 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-33299049

RESUMO

In a secondary dengue virus (DENV) infection, the presence of non-neutralizing antibodies (Abs), developed during a previous infection with a different DENV serotype, is thought to worsen clinical outcomes by enhancing viral production. This phenomenon is called antibody-dependent enhancement (ADE) of infection, and it has delayed the development of therapeutic Abs and vaccines against DENV, as they must be evaluated for the potential to induce ADE. Unfortunately, limited replication of DENV clinical isolates in vitro and in experimental animals hinders this evaluation process. We have, therefore, constructed a recombinant chimeric flavivirus (DV2ChimV), which carries premembrane (prM) and envelope (E) genes of type 2 DENV (DENV-2) R05-624 clinical (Thai) isolate in a backbone of Japanese encephalitis virus (Nakayama strain). DENV E-protein is the most important viral target, not only for neutralizing Abs, but also for infection-enhancing Abs. In contrast to DENV-2 R05-624, DV2ChimV replicated efficiently in cultured mammalian cells and was lethal in interferon-α/ß-γ-receptor double-knockout mice. With DV2ChimV, we were able to perform neutralization assays, in vitro and in vivo ADE assays, and in vivo protection assays. These results suggest that the chimeric virus is a powerful tool for evaluation of Abs against DENV.


Assuntos
Anticorpos Antivirais/imunologia , Vírus da Dengue/metabolismo , Dengue/imunologia , Flavivirus/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Chlorocebus aethiops , Dengue/virologia , Vírus da Dengue/genética , Camundongos , Células Vero , Envelope Viral/imunologia , Proteínas do Envelope Viral/genética
10.
Immunobiology ; 223(3): 319-326, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29107382

RESUMO

Influenza virus causes acute respiratory infection in humans, and is a major public health concern globally. Antibodies play a central role in host protection against influenza virus. We isolated human monoclonal antibodies (hMAb) 206-2-4 and 201-6-8 by a human hybridoma protocol that neutralized various but distinct influenza virus (IFV) A/H1N1 strains, including 2009 pandemic strains. The half-inhibitory concentration of 206-2-4 and 201-6-8 against A/H1N1pdm09 strains was 2-100ng/mL and 5-20µg/mL, respectively. Prophylactic and therapeutic potencies of 206-2-4 were demonstrated in a mouse model of IFV infection at i.p. dosages of 0.25 and 2.5mg/kg, respectively, suggesting that 206-2-4 is one of the most potent hnMAbs against IFV reported thus far. The Ig genes of 206-2-4 and 201-6-8 were originated from distinct germ line repertoires, and accompanied by 63 and 23 somatic hypermutations, respectively. The hemagglutination inhibitory activity indicated that the mechanism of neutralization was to interfere the virus-receptor interaction. The binding epitope of the two antibodies was mapped to hemagglutinin 1 (HA1) amino acid residues 111-120. Additional interaction between the antibody and the HA1 globular head was necessary for neutralization. Such hnMAbs bearing distinct binding epitope have been rarely reported. The potency is likely due to the coverage of a wide surface area of HA protein by these hnMABs. IFV is a highly variable. Our knowledge on the mechanisms by which these cross-reactive hnMAbs function should help design a novel immunogen for the development of a vaccine effective against broader spectrum of IFV strains.


Assuntos
Genes de Imunoglobulinas , Vírus da Influenza A Subtipo H1N1/fisiologia , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Infecções por Orthomyxoviridae/imunologia , Animais , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Células Cultivadas , Reações Cruzadas , Cães , Epitopos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Hibridomas , Influenza Humana/epidemiologia , Japão/epidemiologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Pandemias
11.
J Biosci Bioeng ; 102(4): 297-303, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17116575

RESUMO

The use of transgenic avian species as a transgenic bioreactor for the production of recombinant proteins has been proposed. In recent years, although various procedures for generating transgenic chickens have been reported, the expression of a useful protein at a commercially feasible level has rarely been attained. In this study, we injected a concentrated retroviral vector into quail embryos to generate genetically manipulated quails that produce recombinant proteins. We found that transgene expression in the whole body at a high level was observed for viral injection into the heart of the developing embryos after a 48-h incubation. For the practical production of a useful protein, a retroviral vector encoding an anti-prion scFv-Fc gene under the control of the beta-actin promoter was injected into quail embryos. The quails that hatched stably produced scFv-Fc at a high level in their serum and egg white. The production of scFv-Fc was maintained throughout the breeding period. scFv-Fc purified from the egg white retained the antigen-binding activity. This system exhibited the potential of transgenic quails for the commercial production of recombinant proteins.


Assuntos
Coturnix/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/metabolismo , Transfecção/métodos , Animais , Animais Geneticamente Modificados , Fragmentos Fc das Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Regiões Promotoras Genéticas/genética
12.
J Biosci Bioeng ; 102(6): 518-23, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17270716

RESUMO

We examined the transport of human immunoglobulin G (IgG) subclasses and fusion proteins with the Fc region of human IgG to the egg yolk, after the proteins were injected into a vein of hens. Human IgGs were efficiently transported and accumulated into the yolk, whereas the proteins were not detected in the egg white. Among human IgG subclasses, IgG2 was transported most efficiently. Fc-fusion proteins injected were also transported into the yolk. A fusion protein with the Fc region derived from human IgG2 was more efficiently transported into the yolk than the counterpart fusion with the Fc region from human IgG1. This study shows that the recovery of recombinant antibodies and Fc-fusion proteins from the yolk is an effective method in transgenic chicken bioreactors.


Assuntos
Gema de Ovo/metabolismo , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/metabolismo , Proteínas Recombinantes de Fusão/farmacocinética , Animais , Transporte Biológico Ativo/fisiologia , Embrião de Galinha , Feminino , Humanos , Troca Materno-Fetal , Gravidez
13.
Monoclon Antib Immunodiagn Immunother ; 35(2): 73-82, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26974561

RESUMO

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that bind to and activate the EGF receptor (EGFR/ErbB1) and ErbB4. HB-EGF plays pivotal roles in pathophysiological processes, including cancer. Thus, monoclonal antibodies (mAbs) for HB-EGF detection could be an important tool in the therapeutic diagnosis of HB-EGF-related cancers and other diseases. However, few mAbs, especially those applicable for immunohistochemistry (IHC), have been established to date. In this study, we generated a clone of hybridoma-derived mAb 2-108 by immunizing mice with recombinant human HB-EGF protein expressed by human cells. The mAb 2-108 specifically bound to human HB-EGF but not to mouse HB-EGF and was successful in immunoblotting, even under reducing conditions, immunoprecipitation, and immunofluorescence for unfixed as well as paraformaldehyde-fixed cells. Notably, this mAb was effective in IHC of paraffin-embedded tumor specimens. Epitope mapping analysis showed that mAb 2-108 recognized the N-terminal prodomain in HB-EGF. These results indicate that this new anti-HB-EGF mAb 2-108 would be useful in the diagnosis of HB-EGF-related cancers and would be a strong tool in both basic and clinical research on HB-EGF.


Assuntos
Anticorpos Monoclonais/imunologia , Fator de Crescimento Epidérmico/imunologia , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/imunologia , Neoplasias/imunologia , Animais , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/isolamento & purificação , Mapeamento de Epitopos , Receptores ErbB/imunologia , Receptores ErbB/isolamento & purificação , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/isolamento & purificação , Humanos , Imuno-Histoquímica , Camundongos , Neoplasias/diagnóstico , Parafina
14.
Antiviral Res ; 124: 61-8, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26522769

RESUMO

Antibody-dependent enhancement (ADE) of dengue virus (DENV) infectivity is thought to play a crucial role in severe dengue disease. It occurs when pre-existing sub-neutralizing anti-DENV antibody (Ab) produced from a primary infection encounters a DENV serotype different from that of the initial infection and forms immune complexes, which enable the efficient infection of Fcγ receptor-bearing cells. However, the exact role played by Abs during a secondary infection of patients remains unknown. We previously obtained a broadly cross-reactive neutralizing IgG1 human monoclonal anti-DENV envelope (E) Ab (HuMAb) D23-1G7C2-IgG1 from a DENV-infected patient; however, D23-1G7C2-IgG1 had ADE activity. With the aim of being able to reduce the ADE activity, we exchanged the Fc region of D23-1G7C2 to generate Abs bearing each of the three other IgG subclasses (IgG2-4). In addition, N297A, a mutation known to reduce the affinity of the IgG1 Fc region for Fcγ receptors, was introduced into D23-1G7C2-IgG1. Swapping D23-1G7C2-IgG1 to IgG2 or IgG4 subclasses reduced ADE activity in FcγRI and FcγRII-bearing THP-1 cells. By contrast, in FcγRII-bearing K562 cells, the change to IgG2 increased ADE activity. Introducing the N297A mutation into D23-1G7C2-IgG1 resulted in a marked reduction in ADE activity in both cell types. Compared to D23-1G7C2-IgG1, D23-1G7C2-IgG1-N297A was less protective in IFN-α/ß/γ receptor knockout mice infected with a lethal dose of recombinant chimeric DENV, carrying prME of DENV-2 in Japanese encephalitis virus (80% vs. 40% survival, respectively). These observations provide valuable information regarding the use of recombinant Abs as therapeutics.


Assuntos
Anticorpos Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Dengue/terapia , Dengue/virologia , Receptores de IgG/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/imunologia , Anticorpos Facilitadores/efeitos dos fármacos , Reações Cruzadas , Vírus da Dengue/imunologia , Células HEK293 , Humanos , Imunoglobulina G/imunologia , Células K562 , Camundongos , Mutação , Engenharia de Proteínas , Receptores de IgG/genética , Dengue Grave/imunologia , Células Vero , Proteínas do Envelope Viral/imunologia
15.
J Nucl Med ; 44(11): 1839-44, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14602868

RESUMO

UNLABELLED: We examined whether measurement of the adenosine A(1) receptor (A1-R) with PET can predict the severity of ischemic brain damage using an occlusion and reperfusion model of the cat middle cerebral artery (MCA) and [1-methyl-(11)C]8-dicyclopropylmethyl-1-methyl-3-propylxanthine (MPDX), a positron-emitting radioligand developed at our institution. METHODS: Eighteen adult cats underwent PET measurement of cerebral blood flow (CBF), A1-R, central benzodiazepine receptor (BDZ-R), and glucose metabolism with (15)O-labeled water, MPDX, (11)C-flumazenil (FMZ), and (18)F-FDG, respectively. The right MCAs of 13 cats were transiently occluded via a transorbital approach with microvascular clips. CBF was measured before occlusion of MCA, during occlusion, and immediately after reperfusion. After CBF measurement, A1-R, BDZ-R, and (18)F-FDG uptake were serially measured in the order listed. Two months later, the degree of ischemic damage was evaluated by T2-weighted MR images obtained with an animal MRI system and by analysis of histologic specimens. Five cats that received no operations were used as controls. RESULTS: The cats that underwent occlusion were divided into 3 groups: cats that did not survive the first day because of severe neurologic and systemic conditions (n = 4), cats that survived and had infarcted lesions in both the cortex and the striatum (n = 3), and cats that survived and had infarcted lesions only in the striatum (n = 6). CBF during occlusion of the MCA was significantly lower in all 3 ischemic groups than in the control group, but there was no significant difference among the ischemic groups. Right-to-left ratios of CBF and (18)F-FDG uptake did not significantly differ among the groups. MPDX binding and FMZ binding were significantly lower in the groups with severe ischemic insult than in the groups with little to no insult. CONCLUSION: The degree of decreased MPDX binding to A1-Rs after reperfusion was a sensitive predictor of severe ischemic insult. MPDX PET has good potential to become a suitable in vivo imaging technique for evaluating the function of adenosine and A1-Rs in relation to cerebral ischemia.


Assuntos
Isquemia Encefálica/diagnóstico por imagem , Radioisótopos de Carbono , Receptor A1 de Adenosina/análise , Xantinas/metabolismo , Animais , Gatos , Circulação Cerebrovascular , Imageamento por Ressonância Magnética , Tomografia Computadorizada de Emissão
16.
J Biosci Bioeng ; 95(6): 612-7, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-16233466

RESUMO

Many chemicals that are not structurally related to estrogen have estrogen-like activity. In this study, we tried to apply a quail embryo culture system for assessing the in vivo effects of such chemicals on the development of quail embryos. Beta-estradiol induced feminization of the gonads of genetically male embryos, which was confirmed by the increase in the size of the left gonad and female-specific aromatase expression, while male-specific SOX9 expression was not affected. Nonylphenol, which has a weak estrogenic activity, reduced the viability and body weight of embryos. Simultaneously, several genetically male embryos were feminized in terms of gonadal size and aromatase expression. These results indicate that the avian embryo culture system was useful for evaluating endocrine disrupters.

17.
J Biosci Bioeng ; 95(3): 231-8, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-16233398

RESUMO

We constructed a replication-defective retroviral vector plasmid for the expression of a single-chain antibody fragment (scFv), derived from a chicken anti-human prion protein monoclonal antibody, fused with the Fc region of human IgG1. CHO-K1 and NS-1 cells were transformed with the viral vector pseudotyped with vesicular stomatitis virus G protein (VSV-G), and scFv-Fc producer clones were established. Among the established clones, CHO-2A9 cells produced a large amount of the product with an antibody-like dimerized structure in serum-free culture that facilitated the purification of scFv-Fc. The scFv-Fc specifically recognized the epitope sequence of prion protein in solid-phase enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. The injection test into quails revealed that the scFv became more stable in vivo by fusion with the Fc region. The scFv-Fc will be a useful tool for the detection of mammalian prion proteins.

18.
J Biosci Bioeng ; 98(4): 298-303, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-16233709

RESUMO

Expression vectors for chimeric anti-CD2 antibody were constructed in order to clarify the importance of the expression ratio of heavy (H-) and light (L-) chains of antibody to antibody production in animal cells. The antibody genes were introduced into cells using plasmid DNA vectors or replication-defective retroviral vectors. Productivity was maximal when the expression ratio of H-and L-chains was 1:1, and decreased when the ratio was not equal. We also examined the expression of antibody using one-packed vectors in which the bicistronic expression of H- and L-chain genes was mediated by an internal ribosomal entry site (IRES) sequence derived from encephalomyocarditis virus (EMCV). The translation efficiency was unbalanced between 5'Cap- and IRES-dependent genes. Using the retroviral vectors, it was estimated that the IRES-dependent translation efficiency was 5-fold lower than the 5'Cap-dependent translation efficiency. The cells exhibiting an unbalanced expression of H- and L-chains tended to accumulate H-chain protein.

19.
Biologics ; 7: 175-87, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23983454

RESUMO

BACKGROUND: Hybridomas that produce human monoclonal antibodies (HuMAbs) against Dengue virus (DV) had been prepared previously using peripheral blood lymphocytes from patients with DV during the acute and convalescent phases of a secondary infection. Anti-DV envelope glycoprotein (E) 99 clones, anti-DV premembrane protein (prM) 8 clones, and anti-DV nonstructural protein 1 (NS1) 4 clones were derived from four acute-phase patients, and anti-DV E 2 clones, anti-DV prM 2 clones, and anti-DV NS1 8 clones were derived from five convalescent-phase patients. METHODS AND RESULTS: In the present study, we examined whether these clones cross-reacted with Japanese encephalitis virus (JEV), which belongs to the same virus family. Forty-six of the above-described 99 (46/99) anti-E, 0/8 anti-prM, and 2/4 anti-NS1 HuMAbs from acute-phase, and 0/2 anti-E, 0/2 anti-prM, and 5/8 anti-NS1 HuMAbs from convalescent-phase showed neutralizing activity against JEV. Thus, most of the anti-E and anti-NS1 (but not the anti-prM) antibodies cross-reacted with JEV and neutralized this virus. Interestingly, 3/46 anti-E HuMAbs derived from acute-phase patients and 3/5 anti-NS1 HuMAbs from convalescent-phase patients showed particularly high neutralizing activity against JEV. Consequently, the HuMAbs showing neutralization against JEV mostly consisted of two populations: one was HuMAbs recognizing DV E and showing neutralization activity against all four DV serotypes (complex-type) and the other was HuMAbs recognizing DV NS1 and showing subcomplex-type cross-reaction with DV. CONCLUSION: Anti-DV E from acute phase (46/99) and anti-DV NS1 (7/12) indicate neutralizing activity against JEV. In particular, three of 46 anti-DV E clones from acute phase and three of five anti-NS1 clones from convalescent phase showed strong neutralizing activity against JEV.

20.
Antiviral Res ; 98(3): 423-31, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23545366

RESUMO

Public health concern about dengue diseases, caused by mosquito-borne infections with four serotypes of dengue virus (DENV-1-DENV-4), is escalating in tropical and subtropical countries. Most of the severe dengue cases occur in patients experiencing a secondary infection with a serotype that is different from the first infection. This is believed to be due to antibody-dependent enhancement (ADE), by which one DENV serotype uses pre-existing anti-DENV antibodies elicited in the primary infection to facilitate entry of a different DENV serotype into the Fc receptor-positive macrophages. Recently, we prepared a number of hybridomas producing human monoclonal antibodies (HuMAbs) by using peripheral blood lymphocytes from Thai patients at acute phase of secondary infection with DENV-2. Here, we characterized 17 HuMAbs prepared from two patients with dengue fever (DF) and one patient with dengue hemorrhagic fever (DHF) that were selected as antibodies recognizing viral envelope protein and showing higher neutralization activity to all serotypes. In vivo evaluation using suckling mice revealed near perfect activity to prevent mouse lethality following intracerebral DENV-2 inoculation. In a THP-1 cell assay, these HuMAbs showed ADE activities against DENV-2 at similar levels between HuMAbs derived from DF and DHF patients. However, the F(ab')2 fragment of the HuMAb showed a similar virus neutralization activity as original, with no ADE activity. Thus, these HuMAbs could be one of the therapeutic candidates against DENV infection.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Anticorpos Facilitadores , Vírus da Dengue/imunologia , Dengue/terapia , Adulto , Animais , Anticorpos Monoclonais/uso terapêutico , Antivirais/imunologia , Antivirais/uso terapêutico , Coinfecção/imunologia , Coinfecção/virologia , Dengue/imunologia , Vírus da Dengue/patogenicidade , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Hibridomas/imunologia , Hibridomas/virologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Índice de Gravidade de Doença , Proteínas do Envelope Viral/imunologia , Internalização do Vírus , Adulto Jovem
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