RESUMO
BACKGROUND: Particular neutralizing mAbs to certain cytokines act as agonists in vivo through protection of the cytokine's active site and prolongation of its half-life. Although this principle might be useful for targeted immunotherapy, its role in the pathogenesis of inflammation and autoimmunity is unclear. OBJECTIVE: We sought to determine whether slight, structurally nonrelevant modifications of the prototypic proinflammatory cytokine IL-1ß during an immune response could elicit polyclonal anti-IL-1ß antibody responses that modulated IL-1ß's in vivo activity. METHODS: We engineered 2 different IL-1ß variants, thereby mimicking the process of cytokine modification occurring during inflammation, and conjugated them to virus-like particles, followed by immunization of mice. The resulting polyclonal anti-IL-1ß antibody responses were assessed by using in vitro and in vivo assays, as well as 2 relevant (auto-) inflammatory murine models. RESULTS: Although antibody responses generated to one variant were potently inhibiting IL-1ß, antibody responses induced by the other variant even potentiated the in vivo effects of IL-1ß; the latter led to enhanced morbidity in 2 different IL-1ß-mediated mouse models, including a model of inflammatory bowel disease and an inflammatory arthritis model. CONCLUSION: These data demonstrate that endogenous polyclonal anti-cytokine antibody responses can enhance the cytokine's activity in inflammatory and autoimmune diseases.
Assuntos
Anticorpos/imunologia , Interleucina-1beta/imunologia , Animais , Células HEK293 , Células HeLa , Humanos , Inflamação/sangue , Inflamação/imunologia , Interleucina-1beta/sangue , Interleucina-1beta/genética , Interleucina-6/sangue , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBARESUMO
Interleukin-1ß (IL-1ß) is a key cytokine involved in inflammatory illnesses including rare hereditary diseases and common chronic inflammatory conditions as gout, rheumatoid arthritis, and type 2 diabetes mellitus, suggesting reduction of IL-1ß activity as new treatment strategy. The objective of our study was to assess safety, antibody response, and preliminary efficacy of a novel vaccine against IL-1ß. The vaccine hIL1bQb consisting of full-length, recombinant IL-1ß coupled to virus-like particles was tested in a preclinical and clinical, randomized, placebo-controlled, double-blind study in patients with type 2 diabetes. The preclinical simian study showed prompt induction of IL-1ß-specific antibodies upon vaccination, while neutralizing antibodies appeared with delay. In the clinical study with 48 type 2 diabetic patients, neutralizing IL-1ß-specific antibody responses were detectable after six injections with doses of 900 µg. The development of neutralizing antibodies was associated with higher number of study drug injections, lower baseline body mass index, improvement of glycemia, and C-reactive protein (CRP). The vaccine hIL1bQb was safe and well-tolerated with no differences regarding adverse events between patients receiving hIL1bQb compared to placebo. This is the first description of a vaccine against IL-1ß and represents a new treatment option for IL-1ß-dependent diseases such as type 2 diabetes mellitus (ClinicalTrials.gov NCT00924105).
Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Diabetes Mellitus Tipo 2/terapia , Interleucina-1beta/imunologia , Vacinas/administração & dosagem , Adulto , Idoso , Animais , Diabetes Mellitus Tipo 2/imunologia , Método Duplo-Cego , Feminino , Humanos , Macaca mulatta , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , Vacinas/imunologiaRESUMO
Interleukin (IL)-1α is a potent proinflammatory cytokine that has been implicated in the development of atherosclerosis. We investigated whether a vaccine inducing IL-1α neutralizing antibodies could interfere with disease progression in a murine model of atherosclerosis. We immunized Apolipoprothin E (ApoE)-deficient mice with a vaccine (IL-1α-C-Qß) consisting of full-length, native IL-1α chemically conjugated to virus-like particles derived from the bacteriophage Qß. ApoE(-/-) mice were administered six injections of IL-1α-C-Qß or nonconjugated Qß over a period of 160 days while being maintained on a western diet. Atherosclerosis was measured in the descending aorta and in cross-sections at the aortic root. Macrophage infiltration in the aorta was measured using CD68. Expression levels of VCAM-1, ICAM-1, and MCP-1 were quantified by RT-PCR. Immunization against IL-1α reduced plaque progression in the descending aorta by 50% and at the aortic root by 37%. Macrophage infiltration in the aorta was reduced by 22%. Inflammation was also reduced in the adventitia, with a decrease of 54% in peri-aortic infiltrate score and reduced expression levels of VCAM-1 and ICAM-1. Active immunization targeting IL-1α reduced both the inflammatory reaction in the plaque as well as plaque progression. In summary, vaccination against IL-1α protected ApoE(-/-) mice against disease, suggesting that this may be a potential treatment option for atherosclerosis.
Assuntos
Aterosclerose/imunologia , Interleucina-1alfa/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Anticorpos/imunologia , Anticorpos Neutralizantes/imunologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/prevenção & controle , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Knockout , Placa Aterosclerótica/genética , Placa Aterosclerótica/imunologia , Placa Aterosclerótica/patologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagemRESUMO
Oxidative stress and inflammation--two components of the natural host response to injury--constitute important etiologic factors in atherogenesis. The pro-inflammatory cytokine interleukin (IL)-1 significantly enhances atherosclerosis, however, the molecular mechanisms of IL-1 induction within the artery wall remain poorly understood. Here we have identified the oxidative stress-responsive transcription factor NF-E2-related 2 (Nrf2) as an essential positive regulator of inflammasome activation and IL-1-mediated vascular inflammation. We show that cholesterol crystals, which accumulate in atherosclerotic plaques, represent an endogenous danger signal that activates Nrf2 and the NLRP3 inflammasome. The resulting vigorous IL-1 response critically depended on expression of Nrf2, and Nrf2-deficient apolipoprotein E (Apoe)-/- mice were highly protected against diet-induced atherogenesis. Importantly, therapeutic neutralization of IL-1α and IL-1ß reduced atherosclerosis in Nrf2+/- Apoe-/- but not in Nrf2-/- Apoe-/- mice, suggesting that the pro-atherogenic effect of Nrf2-signaling was primarily mediated by its permissive role in IL-1 production. Our studies demonstrate a role for Nrf2 in inflammasome activation, and identify cholesterol crystals as disease-relevant triggers of the NLRP3 inflammasome and potent pro-atherogenic cytokine responses. These findings suggest a common pathway through which oxidative stress and metabolic danger signals converge and mutually perpetuate the chronic vascular inflammation that drives atherosclerosis.
Assuntos
Aterosclerose/metabolismo , Aterosclerose/patologia , Colesterol/metabolismo , Inflamassomos/metabolismo , Interleucina-1/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Proteínas de Transporte/metabolismo , Caspase 1/metabolismo , Células Cultivadas , Colesterol/química , Cristalização , Células Dendríticas/metabolismo , Células Espumosas/metabolismo , Interleucina-1/biossíntese , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 2 Relacionado a NF-E2/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR , Estresse Oxidativo , Placa Aterosclerótica/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Since its first appearance in the USA in 1999, West Nile virus (WNV) has spread in the Western hemisphere and continues to represent an important public health concern. In the absence of effective treatment, there is a medical need for the development of a safe and efficient vaccine. Live attenuated WNV vaccines have shown promise in preclinical and clinical studies but might carry inherent risks due to the possibility of reversion to more virulent forms. Subunit vaccines based on the large envelope (E) glycoprotein of WNV have therefore been explored as an alternative approach. Although these vaccines were shown to protect from disease in animal models, multiple injections and/or strong adjuvants were required to reach efficacy, underscoring the need for more immunogenic, yet safe DIII-based vaccines. RESULTS: We produced a conjugate vaccine against WNV consisting of recombinantly expressed domain III (DIII) of the E glycoprotein chemically cross-linked to virus-like particles derived from the recently discovered bacteriophage AP205. In contrast to isolated DIII protein, which required three administrations to induce detectable antibody titers in mice, high titers of DIII-specific antibodies were induced after a single injection of the conjugate vaccine. These antibodies were able to neutralize the virus in vitro and provided partial protection from a challenge with a lethal dose of WNV. Three injections of the vaccine induced high titers of virus-neutralizing antibodies, and completely protected mice from WNV infection. CONCLUSIONS: The immunogenicity of DIII can be strongly enhanced by conjugation to virus-like particles of the bacteriophage AP205. The superior immunogenicity of the conjugate vaccine with respect to other DIII-based subunit vaccines, its anticipated favourable safety profile and low production costs highlight its potential as an efficacious and cost-effective prophylaxis against WNV.
Assuntos
Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Febre do Nilo Ocidental/prevenção & controle , Vacinas contra o Vírus do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental/imunologia , Animais , Bacteriófagos/genética , Bacteriófagos/imunologia , Bacteriófagos/fisiologia , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Terciária de Proteína , Vacinas Conjugadas/administração & dosagem , Vacinas Conjugadas/química , Vacinas Conjugadas/genética , Vacinas Conjugadas/imunologia , Proteínas do Envelope Viral/administração & dosagem , Proteínas do Envelope Viral/genética , Febre do Nilo Ocidental/imunologia , Febre do Nilo Ocidental/mortalidade , Vacinas contra o Vírus do Nilo Ocidental/administração & dosagem , Vacinas contra o Vírus do Nilo Ocidental/química , Vacinas contra o Vírus do Nilo Ocidental/genética , Vírus do Nilo Ocidental/química , Vírus do Nilo Ocidental/genéticaRESUMO
The major chaperone genes of Helicobacter pylori are negatively regulated by HspR, a homologue of the repressor of the dnaK operon of Streptomyces coelicolor. Using an in vitro selection and amplification approach we identified two new chromosomal binding sites of the HspR protein. Both binding sites were characterized by footprinting analysis with purified HspR protein. Intriguingly, these HspR binding sites are located at the 3prime prime or minute ends of two genes coding for predicted proteins with functions unrelated to those of chaperones. This suggests that H. pylori HspR may regulate the expression of genes encoding proteins with diverse functions. Nucleotide sequence alignment of HspR-binding sites highlights conserved nucleotides extending outside the previously proposed consensus binding sequence with structural features predicting geometry of HspR binding as an oligomer.
Assuntos
Proteínas de Bactérias , Genoma Bacteriano , Proteínas de Choque Térmico/genética , Helicobacter pylori/genética , Proteínas Repressoras/genética , Sequência de Bases , Sítios de Ligação/genética , Cromatografia de Afinidade , Clonagem Molecular , Sequência Conservada , Pegada de DNA , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Desoxirribonuclease I/metabolismo , Proteínas de Choque Térmico/metabolismo , Helicobacter pylori/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Proteínas Repressoras/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
Many chronic diseases are caused by non-physiological interactions of certain ligands with their receptors. Conventional treatment of these diseases with synthetic drugs or monoclonal antibodies (mAbs) is efficient, but problematic due to the non-compliance of patients and the risk of adverse side effects. Novel therapeutic approaches are focusing on strategies of active immunisation aimed at the induction of a humoral immune response directed against the deleterious receptor-ligand interaction. Autoantibody production has been achieved by several vaccine formulations, including conjugates of self-antigens to foreign T helper (Th) cell epitopes, virus-like particles coated with self-antigens, and naked DNA vectors. All of these approaches have the potential to be developed for clinical use if important safety issues, related to the possible long-term presence of self-reactive antibodies in the serum of vaccinated individuals and the risk of undesired T cell responses, can be properly addressed.
Assuntos
Anticorpos Bloqueadores/uso terapêutico , Citocinas/antagonistas & inibidores , Receptores de Citocinas/antagonistas & inibidores , Vacinas de DNA/uso terapêutico , Animais , Anticorpos Bloqueadores/efeitos adversos , Citocinas/imunologia , Citocinas/metabolismo , Humanos , Ligantes , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Receptores de Citocinas/imunologia , Receptores de Citocinas/metabolismo , Vacinas de DNA/efeitos adversos , Vacinas de DNA/imunologiaRESUMO
Neutralization of the inflammatory cytokine interleukin-1ß (IL-1ß) is a promising new strategy to prevent the ß-cell destruction, which leads to type 2 diabetes. Here, we describe the preclinical development of a therapeutic vaccine against IL-1ß consisting of a detoxified version of IL-1ß chemically cross-linked to virus-like particles of the bacteriophage Qß. The vaccine was well tolerated and induced robust antibody responses in mice, which neutralized the biological activity of IL-1ß, as shown both in cellular assays and in challenge experiments in vivo. Antibody titers were long lasting but reversible over time and not associated with the development of potentially harmful T cell responses against IL-1ß. Neutralization of IL-1ß by vaccine-induced antibodies had no influence on the immune responses of mice to Listeria monocytogenes and Mycobacterium tuberculosis. In a diet-induced model of type 2 diabetes, immunized mice showed improved glucose tolerance, which was mediated by improved insulin secretion by pancreatic ß-cells. Hence, immunization with IL-1ß conjugated to virus-like particles has the potential to become a safe, efficacious, and cost-effective therapy for the prevention and long-term treatment of type 2 diabetes.
RESUMO
IL-1α and IL-1ß are potent inflammatory cytokines and important mediators of immune responses to intracellular pathogens such as Mycobacterium tuberculosis (Mtb). Here, we investigated the role of IL-1α and IL-1ß during chronic Mtb infection and spontaneous reactivation in mice. For long-term neutralization of IL-1α, IL-1ß or both, mice were immunized with virus-like particles (VLPs) displaying either of the cytokines, inducing strong and long-lasting neutralizing IgG responses. Blocking of IL-1α but not of IL-1ß resulted in increased susceptibility to chronic infection with Mtb. Neutralizing either IL-1α or IL-1ß alone did not lead to increased reactivation of latent tuberculosis. The generation of antibodies neutralizing both IL-1α and IL-1ß simultaneously, did not influence weight gain during Mtb reactivation and the slight increase in pulmonary bacillary counts were not significant when compared to control-immunized group. Thus, the results suggest that IL-1α is the major mediator of the IL-1RI-dependent and protective innate immune responses to Mtb in mice.
Assuntos
Interleucina-1alfa/imunologia , Interleucina-1beta/imunologia , Mycobacterium tuberculosis/imunologia , Tuberculose/imunologia , Animais , Suscetibilidade a Doenças , Feminino , Humanos , Interleucina-1alfa/antagonistas & inibidores , Interleucina-1beta/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/patogenicidade , Tuberculose/microbiologiaRESUMO
West Nile virus (WNV) continues to circulate in the USA and forms a threat to the rest of the Western hemisphere. Since methods for the treatment of WNV infections are not available, there is a need for the development of safe and effective vaccines. Here, we describe the construction of a recombinant influenza virus expressing domain III of the WNV glycoprotein E (Flu-NA-DIII) and its evaluation as a WNV vaccine candidate in a mouse model. FLU-NA-DIII-vaccinated mice were protected from severe body weight loss and mortality caused by WNV infection, whereas control mice succumbed to the infection. In addition, it was shown that one subcutaneous immunization with 10(5) TCID(50) Flu-NA-DIII provided 100% protection against challenge. Adoptive transfer experiments demonstrated that protection was mediated by antibodies and CD4+T cells. Furthermore, mice vaccinated with FLU-NA-DIII developed protective influenza virus-specific antibody titers. It was concluded that this vector system might be an attractive platform for the development of bivalent WNV-influenza vaccines.
Assuntos
Imunidade/imunologia , Vírus da Influenza A/genética , Influenza Humana/imunologia , Recombinação Genética/genética , Proteínas Virais/química , Febre do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular , Cães , Humanos , Imunidade Humoral/imunologia , Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/virologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Estrutura Terciária de Proteína , Especificidade da Espécie , Resultado do Tratamento , Vacinação , Proteínas Virais/imunologia , Redução de Peso/imunologia , Febre do Nilo Ocidental/virologia , Vacinas contra o Vírus do Nilo Ocidental/imunologiaRESUMO
A major challenge for the future is the development of effective vaccines against chronic infections and the application of therapeutic immunization to the treatment of noninfectious diseases, such as cancer, allergy and autoimmune disorders. In recent years, many of the immunological principles governing the immune response to infectious agents have been clarified and can now be exploited for the rational design of new and better vaccines. As an elucidative example, this review will describe the key immunogenic determinants of viruses and discuss how they can be harnessed for the development of tailor-made vaccines against a wide array of human diseases.
Assuntos
Vacinas Virais/imunologia , Vírus/imunologia , Adjuvantes Imunológicos , Antígenos Virais/imunologia , Desenho de Fármacos , Humanos , Imunidade InataRESUMO
IL-1 is an important mediator of inflammation and a major cause of tissue damage in rheumatoid arthritis (RA). Therapeutic administration of recombinant IL-1 receptor antagonist (IL-1Ra) is efficacious in reducing clinical symptoms of disease, but suffers from several drawbacks, including the need for frequent administrations of large amounts. Here, we show that immunization of mice with either IL-1alpha or IL-1beta chemically cross-linked to virus-like particles (VLP) of the bacteriophage Qbeta elicited a rapid and long-lasting autoantibody response. The induced Ab efficiently neutralized the binding of the respective IL-1 molecules to their receptors in vitro and their pro-inflammatory activities in vivo. In the collagen-induced arthritis model, both vaccines strongly protected mice from inflammation and degradation of bone and cartilage. Moreover, immunization with either vaccine showed superior efficacy than daily administrations of high amounts of IL-1Ra. In the T and B cell-independent collagen Ab transfer model, immunization with the IL-1beta vaccine strongly protected from arthritis, whereas immunization with the IL-1alpha vaccine had no effect. Our results suggest that active immunization with IL-1alpha, and especially IL-1beta conjugated to Qbeta VLP, might become an efficacious and cost-effective new treatment option for RA and other systemic IL-1-dependent inflammatory disorders.
Assuntos
Allolevivirus/imunologia , Artrite Experimental/prevenção & controle , Doenças Autoimunes/prevenção & controle , Interleucina-1/uso terapêutico , Vacinas de Subunidades Antigênicas/uso terapêutico , Aminoácidos/sangue , Animais , Formação de Anticorpos/imunologia , Artrite Experimental/induzido quimicamente , Artrite Experimental/imunologia , Autoanticorpos/sangue , Autoanticorpos/imunologia , Doenças Autoimunes/induzido quimicamente , Doenças Autoimunes/imunologia , Colágeno Tipo II/imunologia , Reações Cruzadas/imunologia , Feminino , Proteína Antagonista do Receptor de Interleucina 1/genética , Proteína Antagonista do Receptor de Interleucina 1/imunologia , Interleucina-1/genética , Interleucina-1/imunologia , Interleucina-1alfa/genética , Interleucina-1alfa/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-6/sangue , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Testes de Neutralização , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Receptores Tipo I de Interleucina-1/antagonistas & inibidores , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/uso terapêutico , Vacinação/métodos , Vacinas de Subunidades Antigênicas/síntese química , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
The hrcA and hspR genes of Helicobacter pylori encode two transcriptional repressor proteins that negatively regulate expression of the groES-groEL and hrcA-grpE-dnaK operons. While HspR was previously shown to bind far upstream of the promoters transcribing these operons, the binding sites of HrcA were not identified. Here, we demonstrate by footprinting analysis that HrcA binds to operator elements similar to the so-called CIRCE sequences overlapping both promoters. Binding of HspR and HrcA to their respective operators occurs in an independent manner, but the DNA binding activity of HrcA is increased in the presence of GroESL, suggesting that the GroE chaperonin system corepresses transcription together with HrcA. Comparative transcriptome analysis of the wild-type strain and hspR and hrcA singly and doubly deficient strains revealed that a set of 14 genes is negatively regulated by the action of one or both regulators, while a set of 29 genes is positively regulated. While both positive and negative regulation of transcription by HspR and/or HrcA could be confirmed by RNA primer extension analyses for two representative genes, binding of either regulator to the promoters could not be detected, indicating that transcriptional regulation at these promoters involves indirect mechanisms. Strikingly, 14 of the 29 genes which were found to be positively regulated by HspR or HrcA code for proteins involved in flagellar biosynthesis. Accordingly, loss of motility functions was observed for HspR and HrcA single or double mutants. The possible regulatory intersections of the heat shock response and flagellar assembly are discussed.
Assuntos
Proteínas de Bactérias/fisiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Proteínas de Choque Térmico/fisiologia , Helicobacter pylori/fisiologia , Locomoção/fisiologia , Proteínas Repressoras/fisiologia , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação/genética , Pegada de DNA , DNA Bacteriano/metabolismo , Flagelos/genética , Deleção de Genes , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/genética , Proteínas de Choque Térmico/genética , Helicobacter pylori/genética , Locomoção/genética , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Óperon , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Repressoras/genéticaRESUMO
Helicobacter pylori, a microaerophilic, gram-negative bacterium is a human pathogen that colonizes the gastric niche and is associated with several acute and chronic stomach diseases. In order to survive in the gastric environment and become pathogenic, the bacterium relies on a plethora of virulence factors, which also include heat shock proteins. We previously showed that two out of the three operons encoding the major cellular chaperone machineries are transcriptionally repressed by two regulators, HrcA and HspR. Till now, molecular studies aimed at the understanding of the role of each protein in controlling transcription was hampered by toxicity and insolubility of HrcA in heterologous expression systems. Similar problems were encountered by many other groups studying HrcA from different bacteria. In this study, we analyzed the amino acid sequence of HrcA that predicted association of this protein to the inner membrane, which was experimentally verified. Subsequently, we implemented a dedicated induction protocol which enabled the overexpression of the recombinant His-HrcA protein in the soluble fraction of Escherichia coli cells. Moreover, we developed a purification procedure for His-HrcA that allowed us to obtain highly pure preparation of the protein. The functionality of the purified protein was then confirmed with an in vitro DNA-binding assay.
Assuntos
Helicobacter pylori/química , Proteínas Repressoras/biossíntese , Sequência de Aminoácidos , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Dados de Sequência Molecular , Regiões Promotoras Genéticas/fisiologia , Proteínas Repressoras/isolamento & purificação , Proteínas Repressoras/metabolismoRESUMO
Neutralization of the proinflammatory cytokine TNF-alpha by mAbs or soluble receptors represents an effective treatment for chronic inflammatory disorders such as rheumatoid arthritis, psoriasis, or Crohn's disease. In this study, we describe a novel active immunization approach against TNF-alpha, which results in the induction of high titers of therapeutically active autoantibodies. Immunization of mice with virus-like particles of the bacteriophage Qbeta covalently linked to either the entire soluble TNF-alpha protein (Qbeta-C-TNF(1-156)) or a 20-aa peptide derived from its N terminus (Qbeta-C-TNF(4-23)) yielded specific Abs, which protected from clinical signs of inflammation in a murine model of rheumatoid arthritis. Whereas mice immunized with Qbeta-C-TNF(1-156) showed increased susceptibility to Listeria monocytogenes infection and enhanced reactivation of latent Mycobacterium tuberculosis, mice immunized with Qbeta-C-TNF(4-23) were not immunocompromised with respect to infection with these pathogens. This difference was attributed to recognition of both transmembrane and soluble TNF-alpha by Abs elicited by Qbeta-C-TNF(1-156), and a selective recognition of only soluble TNF-alpha by Abs raised by Qbeta-C-TNF(4-23). Thus, by specifically targeting soluble TNF-alpha, Qbeta-C-TNF(4-23) immunization has the potential to become an effective and safe therapy against inflammatory disorders, which might overcome the risk of opportunistic infections associated with the currently available TNF-alpha antagonists.
Assuntos
Allolevivirus/imunologia , Artrite Experimental/imunologia , Artrite Experimental/prevenção & controle , Tuberculose/imunologia , Fator de Necrose Tumoral alfa/administração & dosagem , Fator de Necrose Tumoral alfa/imunologia , Sequência de Aminoácidos , Animais , Artrite Reumatoide/imunologia , Artrite Reumatoide/prevenção & controle , Feminino , Listeriose/imunologia , Listeriose/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Dados de Sequência Molecular , Tamanho da Partícula , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/efeitos adversos , Fragmentos de Peptídeos/imunologia , Engenharia de Proteínas , Índice de Gravidade de Doença , Solubilidade , Tuberculose/diagnóstico , Tuberculose/prevenção & controle , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/deficiência , Vacinas Conjugadas/administração & dosagem , Vacinas Conjugadas/efeitos adversos , Vacinas Conjugadas/imunologia , Vacinas Virossomais/administração & dosagem , Vacinas Virossomais/efeitos adversos , Vacinas Virossomais/imunologiaRESUMO
The crgA gene of Neisseria meningitidis, which codes for a LysR-type regulator, is divergently oriented with respect to the mdaB gene, which codes for a hypothetical NADPH-quinone oxidoreductase. Transcriptional studies of the intergenic region between crgA and mdaB showed that two overlapping and divergent promoters, P(crgA) and P(mdaB), control transcription of these genes. Deletion of the crgA gene led to a strong increase in transcription from the P(crgA) promoter and a concomitant strong decrease in transcription from the P(mdaB) promoter, indicating that CrgA acts both as an autorepressor of transcription at its own promoter and as an activator of transcription at the mdaB promoter. Addition of alpha-methylene-gamma-butyrolactone (MBL), an inducer of NADPH-quinone oxidoreductase, to wild-type N. meningitidis cells specifically resulted in further activation of transcription of the P(mdaB) promoter and more repression of transcription of the P(crgA) promoter. No such regulation was observed when MBL was added to crgA-deficient cells, indicating that the transcriptional response to MBL is CrgA mediated. Under the same experimental conditions, no regulation of transcription by either CrgA or MBL was detected at the pilus and capsule genes. The role of CrgA in the regulation of gene expression during the infectious cycle of N. meningitidis is discussed.
Assuntos
4-Butirolactona/análogos & derivados , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Neisseria meningitidis/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional/fisiologia , 4-Butirolactona/farmacologia , Cápsulas Bacterianas/genética , Sequência de Bases , Fímbrias Bacterianas/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Teste de Complementação Genética , Dados de Sequência Molecular , NAD(P)H Desidrogenase (Quinona)/genética , Regiões Promotoras Genéticas/fisiologia , Ativação Transcricional/efeitos dos fármacosRESUMO
TNF-related activation-induced cytokine (TRANCE), also known as receptor activator of NF-kappaB ligand (RANKL), is the key molecule responsible for the bone loss observed in osteoporosis. Passive administration of osteoprotegerin, the soluble decoy receptor of TRANCE/RANKL, is efficient in blocking disease progression, but may not find widespread clinical use due to patient compliance problems and the expected high costs. In this study, we describe an efficient, safe, and potentially cost-effective active immunization strategy against TRANCE/RANKL. We show in mice that immunization with TRANCE/RANKL covalently linked to virus-like particles can overcome the natural tolerance of the immune system toward self proteins and produce high levels of specific Abs without the addition of any adjuvant. Serum Abs of immunized mice neutralized TRANCE/RANKL activity in vitro and were highly active in preventing bone loss in a mouse model of osteoporosis. Active immunization against TRANCE/RANKL was essentially reversible and did not produce any measurable immunosuppressive side effects, underscoring its potential as a new therapeutic approach to the treatment of human bone-degenerative disorders.
Assuntos
Proteínas de Transporte/imunologia , Glicoproteínas de Membrana/imunologia , Osteoporose/prevenção & controle , Vacinação , Vírion/imunologia , Animais , Linfócitos B/imunologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Ovariectomia , Ligante RANK , Receptor Ativador de Fator Nuclear kappa-BRESUMO
The major heat shock genes of Helicobacter pylori are regulated by the HspR repressor. In the present study we characterize the transcriptional response of the three known HspR-dependent promoters P(cbp), P(gro), and P(hrc) to different environmental stresses. A temperature shift from 37 to 42 degrees C causes a typical heat shock response at all three promoters characterized by an immediate and strong induction phase of transcription and a subsequent adaptation phase, which is specific for each promoter and whose onset is determined partially by the half-lives of the respective mRNAs. Exposure to high osmolarity induces a similar response on the P(gro) and P(cbp) promoters while no such response is detectable at the P(hrc) promoter. Puromycin treatment induces transcription from all three HspR-dependent promoters, indicating that different environmental stresses are intracellularly sensed by the regulatory machinery through the accumulation of nonnative proteins. The implications of these data for the regulatory network controlling the heat shock response in H. pylori are discussed.
Assuntos
Proteínas de Bactérias/fisiologia , Proteínas de Choque Térmico/fisiologia , Helicobacter pylori/fisiologia , Proteínas Repressoras/fisiologia , Proteínas de Bactérias/genética , Proteínas de Choque Térmico/genética , Helicobacter pylori/genética , Temperatura Alta , Pressão Osmótica , Regiões Promotoras Genéticas , Puromicina/farmacologia , Proteínas Repressoras/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
The Fur protein acts as a regulator of iron-dependent gene transcription in bacteria. In Helicobacter pylori, Fur regulates iron-activated and iron-repressed promoters. It also acts as an autoregulatory rheostat of transcription to fine-tune its own expression in response to iron by binding to three operators at its own promoter Pfur. Using biochemical and genetic analyses, here we show that the distal upstream operator III (centred at -110) is essential for iron regulation of Pfur and functions as an anti-repression site that is bound by the iron-free form of Fur to induce transcription. Furthermore, operator I (centred at -50) may have a dual role both as a high-affinity binding site for Fur and as an UP element. We propose that its role is ensuring that Fur expression is not repressed below a minimum threshold level. Our data supports a novel promoter architecture and mechanism of regulation by Fur.