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1.
Nat Commun ; 11(1): 4994, 2020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-33020485

RESUMO

Serogroup B meningococcus (MenB) is a leading cause of meningitis and sepsis across the world and vaccination is the most effective way to protect against this disease. 4CMenB is a multi-component vaccine against MenB, which is now licensed for use in subjects >2 months of age in several countries. In this study, we describe the development and use of an ad hoc protein microarray to study the immune response induced by the three major 4CMenB antigenic components (fHbp, NHBA and NadA) in individual sera from vaccinated infants, adolescents and adults. The resulting 4CMenB protein antigen fingerprinting allowed the identification of specific human antibody repertoire correlating with the bactericidal response elicited in each subject. This work represents an example of epitope mapping of the immune response induced by a multicomponent vaccine in different age groups with the identification of protective signatures. It shows the high flexibility of this microarray based methodology in terms of high-throughput information and minimal volume of biological samples needed.


Assuntos
Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Infecções Meningocócicas/imunologia , Vacinas Meningocócicas/imunologia , Neisseria meningitidis Sorogrupo B/imunologia , Adolescente , Adulto , Anticorpos Antibacterianos/sangue , Criança , Pré-Escolar , Mapeamento de Epitopos , Humanos , Lactente , Infecções Meningocócicas/prevenção & controle , Biblioteca de Peptídeos , Análise Serial de Proteínas , Ensaios de Anticorpos Bactericidas Séricos , Adulto Jovem
2.
Sci Rep ; 8(1): 3700, 2018 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-29487324

RESUMO

4CMenB is the first broad coverage vaccine for the prevention of invasive meningococcal disease caused by serogroup B strains. To gain a comprehensive picture of the antibody response induced upon 4CMenB vaccination and to obtain relevant translational information directly from human studies, we have isolated a panel of human monoclonal antibodies from adult vaccinees. Based on the Ig-gene sequence of the variable region, 37 antigen-specific monoclonal antibodies were identified and produced as recombinant Fab fragments, and a subset also produced as full length recombinant IgG1 and functionally characterized. We found that the monoclonal antibodies were cross-reactive against different antigen variants and recognized multiple epitopes on each of the antigens. Interestingly, synergy between antibodies targeting different epitopes enhanced the potency of the bactericidal response. This work represents the first extensive characterization of monoclonal antibodies generated in humans upon 4CMenB immunization and contributes to further unraveling the immunological and functional properties of the vaccine antigens. Moreover, understanding the mechanistic nature of protection induced by vaccination paves the way to more rational vaccine design and implementation.


Assuntos
Infecções Meningocócicas/prevenção & controle , Vacinas Meningocócicas/imunologia , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/imunologia , Células Cultivadas , Reações Cruzadas , Epitopos/imunologia , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Infecções Meningocócicas/imunologia
3.
Physiol Res ; 64(6): 807-19, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26047383

RESUMO

To propose a test to evaluate endothelial function, based on VO(2) on-transition kinetics in sub-anaerobic threshold (AT) constant load exercise, we tested healthy subjects and patients with ischemic-hypertensive cardiopathy by two cardiopulmonary tests on a cycle ergometer endowed with an electric motor to overcome initial inertia: a pre-test and, after at least 24 h, one 6 min constant load exercise at 90 % AT. We measured net phase 3 VO(2)-on kinetics and, by phase 2 time constant (tau), valued endothelial dysfunction. We found shorter tau in repeated tests, shorter time between first and second test, by persisting endothelium-dependent arteriolar vasodilatation and/or several other mechanisms. Reducing load to 80 % and 90 % AT did not produce significant changes in tau of healthy volunteers, while in heart patients an AT load of 70 %, compared to 80 % AT, shortened tau (delta=4.38+/-1.65 s, p=0.013). In heart patients, no correlation was found between NYHA class, ejection fraction (EF), and the two variables derived from incremental cycle cardio-pulmonary exercise, as well as between EF and tau; while NYHA class groups were well correlated with tau duration (r=0.92, p=0.0001). Doxazosin and tadalafil also significantly reduced tau. In conclusion, the O(2) consumption kinetics during the on-transition of constant load exercise below the anaerobic threshold are highly sensitive to endothelial function in muscular microcirculation, and constitute a marker for the evaluation of endothelial dysfunction.


Assuntos
Limiar Anaeróbio , Endotélio Vascular/fisiopatologia , Hipertensão/fisiopatologia , Microcirculação , Isquemia Miocárdica/fisiopatologia , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Exercício Físico/fisiologia , Teste de Esforço , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Músculos/irrigação sanguínea
4.
Physiol Res ; 62(6): 671-9, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23869892

RESUMO

We tested whether the known cytochrome c oxidase (COX) inhibition by nitric oxide (NO) could be quantified by VO(2) kinetics during constant load supra-Anaerobic Threshold (AT) exercises in healthy trained or untrained subjects following aerobic training or nitrate administration. In cycle ergometer constant load exercises supra-AT, identified in previous incremental tests, VO(2) kinetics describe a double exponential curve, one rapid and one appreciably slower, allowing the area between them to be calculate in O(2) l. After training, with increased NO availability, this area decreases in inverse ratio to treatment efficacy. In fact, in 11 healthy subjects after aerobic training for 6-7 weeks, area was decreased on average by 51 %. In 11 untrained subjects, following the assumption of an NO donor, 20 mg isosorbide 5 mononitrate, area was decreased on average by 53 %. In conclusion, supra-AT VO(2) kinetics in constant load exercises permit the quantification of the inhibitory effect NO-dependent on COX after either physical training or nitrate assumption.


Assuntos
Limiar Anaeróbio/fisiologia , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Exercício Físico/fisiologia , Nitratos/farmacologia , Óxido Nítrico/metabolismo , Consumo de Oxigênio/fisiologia , Oxigênio/metabolismo , Adulto , Idoso , Limiar Anaeróbio/efeitos dos fármacos , Feminino , Humanos , Masculino , Oxigênio/análise , Consumo de Oxigênio/efeitos dos fármacos , Valores de Referência
5.
Hum Gene Ther ; 12(8): 871-81, 2001 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-11387053

RESUMO

Baculovirus vectors are efficient tools for gene transfer into mammalian cells in vitro. However, in vivo gene delivery by systemic administration is hindered by the vector inactivation mediated by the complement system. To characterize further the gene transfer efficacy of baculovirus we examined the vector transduction efficiency in skeletal muscle. Vectors expressing vesicular stomatitis virus glycoprotein (VSV-G) in the viral envelope were generated by inserting the VSV-G coding sequence downstream of the polyhedrin promoter. Two viruses were constructed to carry either the Escherichia coli beta-galactosidase (beta-Gal) gene or the mouse erythropoietin (EPO) cDNA cloned downstream of the cytomegalovirus immediate-early promoter and enhancer. The greater gene transduction efficiency of the Bac-G-betaGal vector was confirmed by comparing the beta-Gal expression level in a variety of human and mouse cell lines with that obtained on infection with Bac-betaGal, a vector that lacks VSV-G. Similarly, a 5- to 10-fold increase in beta-Gal expression between Bac-G-betaGal and Bac-betaGal was observed when mouse myoblasts and myotubes were infected. The same increase in beta-Gal expression was detected on injection of the Bac-G-betaGal vector in the quadriceps of BALB/c and C57BL/6 mice. In contrast, a 2-fold difference in transduction was observed between these two vectors in DBA/2J mouse strain. Last, expression of EPO cDNA was detected for at least 178 days in DBA/2J mice on Bac-G-EPO injection into the quadriceps whereas EPO expression declined to normal values by 35 days postinfection in BALB/c and C57BL/6 mice. Thus, these results indicate that baculovirus may be considered a useful vector for gene transfer in mouse skeletal muscle and that persistence of expression may depend on the mouse strain used.


Assuntos
Baculoviridae/genética , Técnicas de Transferência de Genes , Vetores Genéticos , Glicoproteínas de Membrana , Músculo Esquelético/metabolismo , Animais , Western Blotting , Linhagem Celular , DNA Complementar/metabolismo , Elementos Facilitadores Genéticos , Eritropoetina/genética , Escherichia coli/enzimologia , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Músculo Liso/citologia , Músculo Liso/metabolismo , Regiões Promotoras Genéticas , Especificidade da Espécie , Fatores de Tempo , Transdução Genética , Células Tumorais Cultivadas , Proteínas do Envelope Viral/biossíntese , beta-Galactosidase/genética
6.
Proc Natl Acad Sci U S A ; 98(11): 5986-91, 2001 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-11353820

RESUMO

Helper-dependent adenoviral vectors deleted of all viral coding sequences have shown an excellent gene expression profile in a variety of animal models, as well as a reduced toxicity after systemic delivery. What is still unclear is whether long-term expression and therapeutic dosages of these vectors can be obtained also in the presence of a preexisting immunity to adenovirus, a condition found in a high proportion of the adult human population. In this study we performed intramuscular delivery of helper-dependent vectors carrying mouse erythropoietin as a marker transgene. We found that low doses of helper-dependent adenoviral vectors can direct long-lasting gene expression in the muscles of fully immunocompetent mice. The best performance-i.e., 100% of treated animals showing sustained expression after 4 months-was achieved with the latest generation helper-dependent backbones, which replicate and package at high efficiency during vector propagation. Moreover, efficient and prolonged transgene expression after intramuscular injection was observed with limited vector load also in animals previously immunized against the same adenovirus serotype. These data suggest that human gene therapy by intramuscular delivery of helper-dependent adenoviral vectors is feasible.


Assuntos
Adenovírus Humanos/imunologia , Vetores Genéticos/imunologia , Vírus Auxiliares/imunologia , Animais , Eritropoetina/genética , Expressão Gênica , Técnicas de Transferência de Genes , Células HeLa , Humanos , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL
7.
Hum Gene Ther ; 11(13): 1891-900, 2000 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-10986561

RESUMO

We have investigated the efficacy of a gene transfer strategy based on plasmid DNA electroinjection for the correction of anemia associated with renal failure. An expression plasmid encoding the rat erythropoietin (EPO) cDNA under the control of the CMV promoter as constructed and utilized for this work. Electroinjection of pCMV/rEPO in different rat muscles yielded sustained and long-term EPO production and secretion. The muscle-produced EPO corrected the anemia in five of six nephrectomized rats, used as a model of renal failure. The efficiency of muscle transduction was comparable in rats and mice injected with equivalent amounts of DNA per kilogram of body weight. These results demonstrate that gene electrotransfer can be applied to produce therapeutically significant levels of erythropoietin in chronic renal failure.


Assuntos
Anemia/terapia , Eritropoetina/genética , Técnicas de Transferência de Genes , Falência Renal Crônica/complicações , Músculo Esquelético/fisiologia , Anemia/etiologia , Animais , Citomegalovirus/genética , Modelos Animais de Doenças , Eritropoetina/metabolismo , Eritropoetina/farmacologia , Terapia Genética/métodos , Hematócrito , Injeções/métodos , Camundongos , Camundongos Endogâmicos BALB C , Nefrectomia , Plasmídeos/farmacologia , Regiões Promotoras Genéticas , Coelhos , Ratos , Ratos Sprague-Dawley , Transdução Genética
8.
Hum Gene Ther ; 11(6): 859-68, 2000 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-10779163

RESUMO

Helper-dependent (HD) adenoviral (Ad) vectors, in which all viral coding sequences are deleted, have been generated. We show here that intravenous delivery of a mouse EPO (mEPO) expression cassette cloned in an HD vector in immunocompetent mice is effective and long lasting, but not permanent. A precise dose-response relationship between the dose of injected virus and stable EPO serum levels was observed, together with a 100-fold increase in gene expression per infectious particle when compared with a first-generation Ad vector bearing the same cassette. As a direct consequence, therapeutic increases in hematocrit that lasted more than 6 months were achieved with minute amounts of virus, which caused no detectable production of neutralizing antibodies. Intravenous readministration of the HD-mEPO vector in the same mice was as effective as in naive animals without any need for prior immunosuppression. Finally, HD-mEPO injection in subtotally nephrectomized rats improved the anemic status induced by surgery. HD Ad vectors are thus excellent tools for EPO gene therapy.


Assuntos
Adenoviridae/genética , Eritropoetina/genética , Técnicas de Transferência de Genes , Deleção de Sequência , Animais , Formação de Anticorpos , Eritropoetina/imunologia , Eritropoetina/metabolismo , Feminino , Vetores Genéticos , Hematócrito , Injeções Intravenosas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Nefrectomia , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , Fatores de Tempo
9.
Proc Natl Acad Sci U S A ; 97(3): 1002-7, 2000 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-10655474

RESUMO

Helper-dependent (HD) adenoviral vectors devoid of all viral coding sequences provide for safe and highly efficient gene transfer with long-lasting transgene expression. High titer stocks of HD vectors can be generated by using the cre-recombinase system. However, we have encountered difficulties with this system, including rearranged HD vectors and variable efficiency of HD vector rescue. These problems represent a major hindrance, particularly with regard to large-scale production. To overcome these limitations, we have modified the system in two ways: We constructed a new helper virus with a modified packaging signal and enhanced growth characteristics. We also redesigned the vector backbones by including noncoding adenovirus sequences adjacent to the right inverted terminal repeat and by incorporated a number of different segments of noncoding DNA of human origin as "stuffer." Comparison of these vectors showed that the nature of the stuffer sequence affects replication of the HD vector. Optimization of the system resulted in a more robust and consistent production of HD vectors with low helper contamination and high in vivo potency.


Assuntos
Adenoviridae/fisiologia , Vírus Defeituosos/fisiologia , Vetores Genéticos/fisiologia , Vírus Auxiliares/fisiologia , Adenoviridae/genética , Animais , Linhagem Celular , Sequência Consenso , Citomegalovirus/genética , DNA Recombinante/química , DNA Recombinante/genética , Vírus Defeituosos/genética , Eritropoetina/genética , Eritropoetina/metabolismo , Escherichia coli , Genes Reporter , Genes Sintéticos , Vetores Genéticos/genética , Células HeLa , Humanos , Imunocompetência , Luciferases/genética , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes , Recombinação Genética , Segurança , Transfecção , Montagem de Vírus , Replicação Viral
10.
Proc Natl Acad Sci U S A ; 96(11): 6417-22, 1999 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-10339602

RESUMO

We show that an electric treatment in the form of high-frequency, low-voltage electric pulses can increase more than 100-fold the production and secretion of a recombinant protein from mouse skeletal muscle. Therapeutical erythopoietin (EPO) levels were achieved in mice with a single injection of as little as 1 microgram of plasmid DNA, and the increase in hematocrit after EPO production was stable and long-lasting. Pharmacological regulation through a tetracycline-inducible promoter allowed regulation of serum EPO and hematocrit levels. Tissue damage after stimulation was transient. The method described thus provides a potentially safe and low-cost treatment for serum protein deficiencies.


Assuntos
Eritropoetina/genética , Técnicas de Transferência de Genes , Músculo Esquelético/fisiologia , Regiões 5' não Traduzidas/genética , Animais , Citomegalovirus/genética , Estimulação Elétrica , Eletroporação/métodos , Eritropoetina/biossíntese , Eritropoetina/sangue , Feminino , Regulação da Expressão Gênica , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Músculo Esquelético/citologia , Plasmídeos , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Sensibilidade e Especificidade , Fatores de Tempo
11.
EMBO J ; 17(19): 5588-97, 1998 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-9755159

RESUMO

Studies with tumor necrosis factor p55 receptor- and interleukin-6 (IL-6)-deficient mice have shown that IL-6 is required for hepatocyte proliferation and reconstitution of the liver mass after partial hepatectomy. The biological activities of IL-6 are potentiated when this cytokine binds soluble forms of its specific receptor subunit (sIL-6R) and the resulting complex interacts with the transmembrane signaling chain gp130. We show here that double transgenic mice expressing high levels of both human IL-6 and sIL-6R under the control of liver-specific promoters spontaneously develop nodules of hepatocellular hyperplasia around periportal spaces and present signs of sustained hepatocyte proliferation. The resulting picture is identical to that of human nodular regenerative hyperplasia, a condition frequently associated with immunological and myeloproliferative disorders. In high expressors, hyperplastic lesions progress with time into discrete liver adenomas. These data strongly suggest that the IL-6/sIL-6R complex is both a primary stimulus to hepatocyte proliferation and a pathogenic factor of hepatocellular transformation.


Assuntos
Adenoma/patologia , Interleucina-6/biossíntese , Neoplasias Hepáticas/patologia , Fígado/patologia , Receptores de Interleucina-6/biossíntese , Adenoma/metabolismo , Adenoma/mortalidade , Animais , Proteínas de Ligação a DNA/biossíntese , Endotélio Vascular , Regulação da Expressão Gênica , Haptoglobinas/biossíntese , Hiperplasia/mortalidade , Interleucina-6/genética , Fígado/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidade , Regeneração Hepática , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-myc/biossíntese , Receptores de Interleucina-6/genética , Fator de Transcrição STAT3 , Solubilidade , Transativadores/biossíntese
12.
J Autoimmun ; 11(3): 249-53, 1998 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9693973

RESUMO

Treatment of human peripheral blood lymphocytes (PBL) in vitro with the cytokine interleukin-6 (IL-6) induces increased levels of the 90 kDa heat shock protein (hsp90). Hsp90 levels are also elevated in PBLs of human patients with systemic lupus erythematosus (SLE) and in MRL/lpr mice with autoimmune disease. Although IL-6 is elevated in both these situations it has not been shown that it is involved in stimulating elevation of hsp90 levels in vivo. Here we show directly that the elevation of IL-6 in vivo either in mice transgenic for the IL-6 gene or in knock-out mice lacking a functional gene for the transcription factor C/EBP beta (NF-IL-6) does indeed result in elevated hsp90 levels. This overexpression is associated with the specific production of autoantibodies to hsp90 in these mice which is also observed in SLE patients and MRL/1pr mice. Hence IL-6 is likely to play a critical role in the regulation of hsp90 levels both in autoimmune disease states and potentially in normal cells in vivo. In turn the elevated levels of hsp90 produced in autoimmune diseases are likely to be responsible for the observed production of anti-hsp90 autoantibodies.


Assuntos
Autoanticorpos/biossíntese , Proteínas de Choque Térmico HSP90/imunologia , Interleucina-6/imunologia , Animais , Proteínas Estimuladoras de Ligação a CCAAT , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Interleucina-6/biossíntese , Interleucina-6/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Camundongos Transgênicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Recombinantes/biossíntese , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética
13.
Nat Biotechnol ; 15(10): 997-1001, 1997 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9335053

RESUMO

Neutralization of cytokine activity by monoclonal antibodies or receptor antagonists is beneficial in the treatment of immune and neoplastic diseases, but the necessity for continuous parenteral delivery of these anticytokine agents poses considerable practical limitations. A viable alternative is to induce a neutralizing antibody response. Using transgenic mice with high circulating levels of human interleukin-6 (hIL-6), we show that injection of the hIL-6 receptor antagonist Sant1 (an IL-6 variant with seven amino-acid substitutions) induces a strong anti-hIL-6 antibody response. The elicited antibodies bind circulating hIL-6 with very high affinity, totally masking it, and neutralize hIL-6 bioactivity both in vitro and in vivo.


Assuntos
Autoanticorpos/biossíntese , Interleucina-6/imunologia , Receptores de Interleucina-6/antagonistas & inibidores , Hidróxido de Alumínio/química , Animais , Reações Antígeno-Anticorpo , Autoanticorpos/imunologia , Sítios de Ligação , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Regulação da Expressão Gênica/imunologia , Engenharia Genética , Humanos , Injeções Intraperitoneais , Interleucina-6/administração & dosagem , Interleucina-6/genética , Camundongos , Camundongos Transgênicos/imunologia , Proteínas Recombinantes/imunologia , Vacinação
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