RESUMO
With the discovery of important biological roles of carbon monoxide (CO), the use of this gas as a therapeutic agent has attracted attention. However, the medical application of this gas has been hampered by the complexity of the administration method. To overcome this problem, several transition-metal carbonyl complexes, such as Ru(CO)(3)Cl(glycinate), [Ru(CO)(3)Cl(2)](2), and Fe(η(4)-2-pyrone)(CO)(3), have been used as CO-releasing molecules both in vitro and in vivo. We sought to develop micellar forms of metal carbonyl complexes that would display slowed diffusion in tissues and thus better ability to target distal tissue drainage sites. Specifically, we aimed to develop a new CO-delivery system using a polymeric micelle having a Ru(CO)(3)Cl(amino acidate) structure as a CO-releasing segment. The CO-releasing micelles were prepared from triblock copolymers composed of a hydrophilic poly(ethylene glycol) block, a poly(ornithine acrylamide) block bearing Ru(CO)(3)Cl(ornithinate) moieties, and a hydrophobic poly(n-butylacrylamide) block. The polymers formed spherical micelles in the range of 30-40 nm in hydrodynamic diameter. Further characterization revealed the high CO-loading capacity of the micelles. CO-release studies showed that the micelles were stable in physiological buffer and serum and released CO in response to thiol-containing compounds such as cysteine. The CO release of the micelles was slower than that of Ru(CO)(3)Cl(glycinate). In addition, the CO-releasing micelles efficiently attenuated the lipopolysaccharide-induced NF-κB activation of human monocytes, while Ru(CO)(3)Cl(glycinate) did not show any beneficial effects. Moreover, cell viability assays revealed that the micelles significantly reduced the cytotoxicity of the Ru(CO)(3)Cl(amino acidate) moiety. This novel CO-delivery system based on CO-releasing micelles may be useful for therapeutic applications of CO.
Assuntos
Monóxido de Carbono/administração & dosagem , Portadores de Fármacos , Imunoterapia/métodos , Micelas , Compostos Organometálicos/química , Rutênio/química , Monóxido de Carbono/química , HumanosRESUMO
Antigen targeting and adjuvancy schemes that respectively facilitate delivery of antigen to dendritic cells and elicit their activation have been explored in vaccine development. Here we investigate whether nanoparticles can be used as a vaccine platform by targeting lymph node-residing dendritic cells via interstitial flow and activating these cells by in situ complement activation. After intradermal injection, interstitial flow transported ultra-small nanoparticles (25 nm) highly efficiently into lymphatic capillaries and their draining lymph nodes, targeting half of the lymph node-residing dendritic cells, whereas 100-nm nanoparticles were only 10% as efficient. The surface chemistry of these nanoparticles activated the complement cascade, generating a danger signal in situ and potently activating dendritic cells. Using nanoparticles conjugated to the model antigen ovalbumin, we demonstrate generation of humoral and cellular immunity in mice in a size- and complement-dependent manner.
Assuntos
Ativação do Complemento , Sistema Linfático/imunologia , Nanopartículas , Vacinas/química , Vacinas/imunologia , Animais , Formação de Anticorpos , Transporte Biológico , Células Cultivadas , Complemento C3/genética , Complemento C3/imunologia , Células Dendríticas/imunologia , Deleção de Genes , Sistema Linfático/citologia , Camundongos , Ovalbumina/imunologiaRESUMO
OBJECTIVE: Interleukin-1 (IL-1) mediates ischemia-reperfusion injury and graft inflammation after heart transplantation. IL-1 affects target cells through two distinct types of transmembrane receptors, type-1 receptor (IL-1R1), which transduces the signal, and the non-signaling type-2 receptor (IL-1R2), which acts as a ligand sink that subtracts IL-1beta from IL-1R1. We analyzed the efficacy of adenovirus (Ad)-mediated gene transfer of a soluble IL-1R2-Ig fusion protein in delaying cardiac allograft rejection and the mechanisms underlying the protective effect. METHODS: IL-1 inhibition by IL-1R2-Ig was tested using an in vitro functional assay whereby endothelial cells preincubated with AdIL-1R2-Ig or control virus were stimulated with recombinant IL-1beta or tumor necrosis factor-alpha (TNF-alpha), and urokinase-type plasminogen activator (u-PA) induction was measured by zymography. AdIL-1R2-Ig was delivered to F344 rat donor hearts ex vivo, which were placed in the abdominal position in LEW hosts. Intragraft inflammatory cell infiltrates and proinflammatory cytokine expression were analyzed by immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. RESULTS: IL-1R2-Ig specifically inhibited IL-1beta-induced u-PA responses in vitro. IL-1R2-Ig gene transfer reduced intragraft monocytes/macrophages and CD4(+) cell infiltrates (p<0.05), TNF-alpha and transforming growth factor-beta (TGF-beta) expression (p<0.05), and prolonged graft survival (15.6+/-5.7 vs 10.3+/-2.5 days with control vector and 10.1+/-2.1 days with buffer alone; p<0.01). AdIL-1R2-Ig combined with a subtherapeutic regimen of cyclosporin A (CsA) was superior to CsA alone (19.4+/-3.0 vs 15.9+/-1.8 days; p<0.05). CONCLUSIONS: Soluble IL-1 type-2 receptor gene transfer attenuates cardiac allograft rejection in a rat model. IL-1 inhibition may be useful as an adjuvant therapy in heart transplantation.
Assuntos
Terapia Genética/métodos , Rejeição de Enxerto/prevenção & controle , Transplante de Coração , Receptores Tipo II de Interleucina-1/genética , Adenoviridae/genética , Animais , Citocinas/biossíntese , Citocinas/genética , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/imunologia , Masculino , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew , Receptores Tipo II de Interleucina-1/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
BACKGROUND: Allograft tolerance might be achieved by expressing immunomodulatory proteins through gene therapy. We have evaluated the possibility of promoting significantly allograft survival in a vascularized cardiac allograft model by performing ex vivo gene transfer. We used a lentiviral vector encoding the chemokine antagonist RANTES 9-68 that is capable of competing with native RANTES. METHODS: The Fisher donor/Lewis recipient rat strain combinations were used and all animals received for the first 5 days posttransplantation a subtherapeutic dose of cyclosporine A (1.5 mg/kg). Ex vivo gene transfer into heart allograft was performed by multiple injections of the SIN.cPPT lentiviral vector, which corresponds to the multiply attenuated, self-inactivating lentivector derived from the human immunodeficiency virus (HIV)-1. RESULTS: About 6% of the cardiac tissue had integrated lentiviral vector, which closely matches the mean in vivo RANTES antagonist expression of 5% obtained by immunohistochemistry. In vivo RANTES 9-68 expression has significantly prolonged graft survival (median [25%-75%]: 20 [17-26] days), compared to the control 15 ([14-15] days; P=0.0007). Furthermore, hearts transduced with RANTES 9-68 showed a significant (P<0.05) reduction in cell infiltration and intragraft expression of TNF-alpha, IFN-gamma, endogenous RANTES, and TGF-beta. CONCLUSION: Lentiviral gene transfer of RANTES 9-68 antagonist attenuates significantly the inflammatory response and delays allograft rejection, despite low levels of transduction. Future improvement of heart transduction by lentiviral vectors, as it has been achieved with other vectors, might become an attractive alternative therapy for treating allografts that require sustained gene expression for better organ preservation.
Assuntos
Quimiocina CCL5/genética , Quimiocina CCL5/uso terapêutico , Sobrevivência de Enxerto , Transplante de Coração , Animais , Sequência de Bases , Linhagem Celular , Quimiocina CCL5/antagonistas & inibidores , DNA Complementar/genética , Técnicas de Transferência de Genes , Terapia Genética , Sobrevivência de Enxerto/imunologia , Proteínas de Fluorescência Verde/genética , Transplante de Coração/imunologia , Humanos , Lentivirus/genética , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapêutico , Transplante HomólogoRESUMO
OBJECTIVE: Interleukin-17 (IL-17), a potent proinflammatory cytokine, has been implicated in allograft rejection. We analyzed the efficacy of an adenoviral vector expressing an IL-17 inhibitor in delaying acute allograft rejection in a rat model of heart transplantation, and the biological mechanisms underlying the protective effect. METHODS: We constructed an adenoviral vector expressing a soluble IL-17 receptor-immunoglobulin (IL-17R-Ig) fusion protein. IL-17R-Ig activity was assessed by inhibition of IL-17-induced IL-6 release in HeLa cells preincubated with the vector. Intracoronary vector administration was performed in F344 donor hearts that were placed as vascularized grafts into Lewis hosts. Inflammatory cells infiltrating the graft were analyzed by immunohistology. Cytokine transcripts in the graft were determined by real-time RT-PCR. RESULTS: IL-17R-Ig gene transfer resulted in prolonged allograft survival (16.1+/-3.1 days vs 10.3+/-2.5 days with control virus and 10.1+/-2.1 days with virus dilution buffer alone; p<0.001). IL-17R-Ig gene transfer reduced inflammatory cell infiltrates, especially monocytes/macrophages and CD4+ T cells (p<0.05). It also reduced intragraft cytokine transcripts for interferon-gamma and transforming growth factor-beta (p<0.05) and, to a lesser extent, IL-1beta and tumor necrosis factor-alpha (p=0.083). CONCLUSIONS: Local expression of soluble IL-17 receptor-immunoglobulin attenuates T helper type 1 (Th1) cytokine responses and leukocyte infiltration in rat cardiac allografts, thereby mediating prolonged graft survival. Intragraft IL-17 inhibition may be useful as an adjuvant therapy to systemic immunosuppression in heart transplantation.
Assuntos
Técnicas de Transferência de Genes , Terapia Genética/métodos , Rejeição de Enxerto/prevenção & controle , Transplante de Coração , Receptores de Interleucina/fisiologia , Adenoviridae/genética , Animais , Linfócitos T CD4-Positivos/imunologia , Citocinas/biossíntese , Citocinas/genética , Vetores Genéticos , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew , Receptores de Interleucina/genética , Receptores de Interleucina-17 , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SolubilidadeRESUMO
Subunit vaccines, employing purified protein antigens rather than intact pathogens, require the addition of adjuvants for enhanced immunogenicity with a correct balance between strong activation of the immune system and low toxicity. Here we show that the endogenous (i.e., autologous) non-toxic TLR4 agonist extra domain A type III repeat of fibronectin (FNIII EDA) can synergize with the exogenous (i.e., bacterial), toxic-at-high-dose, TLR9 agonist CpG to induce efficient cellular immune responses while keeping the dose of CpG low. The efficacy of the combined TLR agonists, even at half-doses, led to stronger dendritic cell activation, enhanced cytotoxic T lymphocyte activation as well as stronger humoral response, compared to the individual agonists given at full doses. Immune cells induced after vaccination with the co-adjuvanted formulation could mediate tumor regression in an E.G7-OVA tumor model, and eradicate circulating hepatitis B virus (HBV) in a transgenic HBV model. Together, these results show that endogenous TLR agonists, such as variants of FNIII EDA, can synergize with exogenous TLR ligands, such as CpG, and strongly enhance cellular immune responses, while improving their safety profile.
Assuntos
Vacinas Anticâncer/imunologia , Fibronectinas/imunologia , Vacinas contra Hepatite B/imunologia , Oligodesoxirribonucleotídeos/imunologia , Linfócitos T Citotóxicos/imunologia , Células Th1/imunologia , Receptor 4 Toll-Like/agonistas , Adjuvantes Imunológicos/administração & dosagem , Animais , Vacinas Anticâncer/administração & dosagem , Células Dendríticas/imunologia , Modelos Animais de Doenças , Fibronectinas/química , Hepatite B/imunologia , Hepatite B/virologia , Vacinas contra Hepatite B/administração & dosagem , Vírus da Hepatite B/imunologia , Imunidade Celular , Imunidade Humoral , Camundongos , Camundongos Transgênicos , Receptores de Reconhecimento de Padrão , Receptor 4 Toll-Like/imunologia , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/imunologia , VacinaçãoRESUMO
BACKGROUND: Among retroviral vectors, lentiviral vectors are unique in that they transduce genes into both dividing and nondividing cells. However, their ability to provide sustained myocardial transgene expression has not been evaluated. METHODS AND RESULTS: Multiply attenuated, self-inactivating lentivectors based on human immunodeficiency virus-1 contained the enhanced green fluorescent protein (EGFP) gene under the transcriptional control of either the cytomegalovirus (CMV) immediate-early enhancer/promoter, the elongation factor-1alpha (EF-1alpha) promoter, or the phosphoglycerate-kinase (PGK) promoter. Lentivectors transduced adult rat cardiomyocytes in a dose-dependent manner (transduction rates, >90%; multiplicity of infection, approximately 5). The CMV promoter achieved higher EGFP expression levels than the EF-1alpha and PGK promoters. Insertion of the central polypurine tract pol sequence improved gene transfer efficiency by approximately 2-fold. In vivo gene transfer kinetics was studied by measuring the copy number of integrated lentivirus DNA and EGFP concentrations in cardiac extracts by real-time polymerase chain reaction and ELISA, respectively. With CMV promoter-containing lentivectors, vector DNA peaked at day 3, declined by approximately 4-fold at day 14, but then remained stable up to week 10. Similarly, EGFP expression peaked at day 7, decreased by approximately 7-fold at day 14, but was essentially stable thereafter. In contrast, vector DNA and EGFP expression declined rapidly with EF-1alpha promoter-containing lentivectors. Peak EGFP expression with titer-matched adenovectors was approximately 35% higher than with CMV lentivectors but was lost rapidly over time. CONCLUSIONS: Lentivectors efficiently transduce and express genes for extended periods of time in cardiomyocytes in vivo. Lentivectors provide a useful tool for studying myocardial biology and a potential system for gene heart therapy.
Assuntos
Vetores Genéticos , Lentivirus/genética , Miócitos Cardíacos/metabolismo , Transdução Genética , Adenoviridae/genética , Animais , Sequência de Bases , Células Cultivadas , DNA Viral/análise , Expressão Gênica , Cinética , Masculino , Miocardite/patologia , Miocardite/virologia , Purinas/química , Ratos , Ratos Wistar , Transgenes , Inativação de VírusRESUMO
BACKGROUND: The inflammation response is modulated by the elaborated chemokine-chemokine receptor system, which also plays an important role in the development of acute rejection (AR). In this study, we hypothesized that functional genetic variants of some of these modulatory proteins might influence the outcome of AR. METHODS: In a retrospective analysis of a cohort of heart transplanted patients (n=158), we examined eight polymorphisms in four genes implicated in this inflammatory process: RANTES, CCR5, CCR2 and CX3CR1. On the basis of timing occurrence, AR episodes (grade>or= 3A) were classified in "early" (0-3 months posttransplantation; EAR) or "late" outcomes (4-12 months posttransplantation; LAR). RESULTS: The incidences of EAR and LAR were 57.6% and 41%, respectively. Number of LAR episodes was significantly higher in subjects that have already experienced one or more EAR episodes, as compared to subjects that had no EAR (median [25%-75%]: 4 () vs. 1 [1-2.5] respectively; P<0.0001). Statistical univariate analysis showed that none of the mentioned polymorphisms were correlated with EAR or LAR. However, allele-allele association analysis showed that subjects carrying both the CX3CR1 249I allele and CCR5 No-E haplotypes were significantly at lower risk of experiencing EAR (OR=0.2 [95%-CI=0.1-0.5], P=0.001). In contrast subjects carrying both the CCR5 E haplotype and the RANTES -403A allele were significantly at higher risk to develop LAR (OR=8.1 [95%-CI=2.3-28.7], P=0.002). CONCLUSIONS: This exploratory study in heart transplantation suggests that the outcomes of EAR and LAR episodes may be influenced by genetic variant interactions such as "CX3CR1 249I*CCR5 No-E" and "CCR5 E*RANTES -403A."
Assuntos
Quimiocina CCL5/genética , Rejeição de Enxerto/genética , Transplante de Coração/imunologia , Proteínas de Membrana/genética , Receptores CCR5/genética , Receptores de Quimiocinas/genética , Doença Aguda , Alelos , Receptor 1 de Quimiocina CX3C , Estudos de Coortes , Haplótipos , Humanos , Polimorfismo Genético , Estudos RetrospectivosRESUMO
Dendritic cell (DC)-derived exosomes (Dexo) contain the machinery necessary to activate potent antigen-specific immune responses. As promising cell-free immunogens, Dexo have been tested in previous clinical trials for cancer vaccine immunotherapy, yet resulted in limited therapeutic benefit. Here, we explore a novel Dexo vaccine formulation composed of Dexo purified from DCs loaded with antigens and matured with either the TLR-3 ligand poly(I:C), the TLR-4 ligand LPS or the TLR-9 ligand CpG-B. When poly(I:C) was used to produce exosomes together with ovalbumin (OVA), the resulting Dexo vaccine strongly stimulated OVA-specific CD8(+) and CD4(+) T cells to proliferate and acquire effector functions. When a B16F10 melanoma cell lysate was used to load DCs with tumor antigens during exosome production together with poly(I:C), we obtained a Dexo vaccine capable of inducing robust activation of melanoma-specific CD8(+) T cells and the recruitment of cytotoxic CD8(+) T cells, NK and NK-T cells to the tumor site, resulting in significantly reduced tumor growth and enhanced survival as compared to a Dexo vaccine formulation similar to the one previously tested on human patients. Our results indicate that poly(I:C) is a particularly favorable TLR agonist for DC maturation during antigen loading and exosome production for cancer immunotherapy.
Assuntos
Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Melanoma Experimental/imunologia , Receptor 3 Toll-Like/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/farmacologia , Células Dendríticas/efeitos dos fármacos , Exossomos/imunologia , Feminino , Imunoterapia/métodos , Células Matadoras Naturais/imunologia , Lipopolissacarídeos/farmacologia , Melanoma Experimental/terapia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovalbumina/imunologia , Ovalbumina/farmacologiaRESUMO
BACKGROUND: The development of nanoparticulate antigen-delivery systems is an important emerging area of vaccinology, being sought to amplify immune responses to recombinant antigens that are poorly immunogenic. Nanoparticle size may play an important role in influencing the activity of such particulate-based adjuvants. METHODS: To explore how the size of nanoparticles that are in the range of many common viruses can modulate the magnitude and quality of mucosal immune responses, the model antigen ovalbumin (OVA) was conjugated to 30 nm or 200 nm polypropylene sulfide nanoparticles (NPs) and administered intranasally to C57BL/6 mice. RESULTS: We show that by increasing the size of the NPs from 30 to 200 nm, OVA was more effectively delivered into both MHC class I and MHC class II-presentation pathways. Intranasal immunization with the 200 nm NPs increased the magnitude of CD4(+) T cell responses in the lungs, as well as systemic and mucosal humoral responses. Most importantly, 200 nm NPs increased the proportion of antigen-specific polyfunctional CD4(+) T cells as compared to 30 nm NPs. CONCLUSIONS: The 200 nm NPs are a very interesting antigen nanocarrier for prophylactic vaccines against mucosal pathogens that require multifunctional CD4(+) T cells for protection. These results contribute to our understanding of how the size of an antigen-conjugated nanoparticle modulates mucosal immune responses to a protein antigen and may be useful to engineer subunit vaccines able to elicit appropriate mucosal immune responses that correlate with protection.
Assuntos
Portadores de Fármacos/administração & dosagem , Imunidade nas Mucosas , Imunização/métodos , Nanopartículas/administração & dosagem , Ovalbumina/imunologia , Vacinas/administração & dosagem , Vacinas/imunologia , Administração Intranasal , Animais , Linfócitos T CD4-Positivos/imunologia , Feminino , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/administração & dosagemRESUMO
Cationic micelles formed from poly(ethylene glycol)-bl-poly(propylene sulfide)-bl-poly(ethylene imine) (PEG-b-PPS-b-PEI) and from mixtures of poly(ethylene glycol)-bl-poly(propylene sulfide) (PEG-b-PPS) with PEG-b-PPS-b-PEI were explored as non-viral vectors for plasmid DNA (pDNA) transfection in a tumor immunotoxicity model. Complexes with pDNA were found to be templated exclusively by the size of the pDNA-free micelles and ranged from 240 nm (for PEG-b-PPS-b-PEI) to 30 nm (for mixed micelles of PEG-b-PPS/PEG-b-PPS-b-PEI). Both formulations transfected melanoma cells well in vitro. As a model with a functional read-out of tumor cell death, one with likely only small bystander effects, tumors were transfected with an antigen transgene, using an antigen to which the recipient animals had been previously vaccinated with a Th1-biasing adjuvant. Reduction in tumor growth, increase in intratumoral infiltration of cytotoxic T lymphocytes and accumulation of Th1-biasing cytokines indicated that both micelle formulations transfected efficiently compared with naked pDNA and with low cytotoxicity.
Assuntos
DNA/metabolismo , Iminas/química , Imunotoxinas/toxicidade , Melanoma Experimental/imunologia , Micelas , Plasmídeos/metabolismo , Polietilenoglicóis/química , Polietilenoimina/análogos & derivados , Polietilenos/química , Sulfetos/química , Animais , Antígenos/imunologia , Proliferação de Células/efeitos dos fármacos , Feminino , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Iminas/síntese química , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Polietilenoglicóis/síntese química , Polietilenoimina/síntese química , Polietilenoimina/química , Polietilenos/síntese química , Sulfetos/síntese química , Testes de Toxicidade , Transfecção , Vírus/efeitos dos fármacosRESUMO
Degradable polymer nanoparticles (NPs, 50 nm) based on polypropylene sulfide (PPS) were conjugated to thiolated antigen and adjuvant proteins by reversible disulfide bonds and evaluated in mucosal vaccination. Ovalbumin was used as a model antigen, and antigen-conjugated NPs were administered intranasally in the mouse. We show penetration of nasal mucosae, transit via M cells, and uptake by antigen-presenting cells in the nasal-associated lymphoid tissue. Ovalbumin-conjugated NPs induced cytotoxic T lymphocytic responses in lung and spleen tissues, as well as humoral response in mucosal airways. Co-conjugation of the TLR5 ligand flagellin further enhanced humoral responses in the airways as well as in the distant vaginal and rectal mucosal compartments and induced cellular immune responses with a Th1 bias, in contrast with free flagellin. The PPS NP platform thus appears interesting as a platform for intranasally-administered mucosal vaccination for inducing broad mucosal immunity.
Assuntos
Portadores de Fármacos/administração & dosagem , Imunidade nas Mucosas , Nanopartículas/administração & dosagem , Vacinação/métodos , Administração Intranasal , Animais , Anticorpos/análise , Células Apresentadoras de Antígenos/imunologia , Feminino , Humanos , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/química , Mucosa Nasal/imunologia , Ovalbumina/imunologia , Polipropilenos/administração & dosagem , Polipropilenos/química , Reto/imunologia , Baço/imunologia , Linfócitos T Citotóxicos/imunologia , Células Th1/imunologia , Vagina/imunologiaRESUMO
Vaccines that drive robust T-cell immunity against Mycobacterium tuberculosis (Mtb) are needed both for prophylactic and therapeutic purposes. We have recently developed a synthetic vaccine delivery platform with Pluronic-stabilized polypropylene sulfide nanoparticles (NPs), which target lymphoid tissues by their small size (â¼ 30 nm) and which activate the complement cascade by their surface chemistry. Here we conjugated the tuberculosis antigen Ag85B to the NPs (NP-Ag85B) and compared their efficacy in eliciting relevant immune responses in mice after intradermal or pulmonary administration. Pulmonary administration of NP-Ag85B with the adjuvant CpG led to enhanced induction of antigen-specific polyfunctional Th1 responses in the spleen, the lung and lung-draining lymph nodes as compared to soluble Ag85B with CpG and to the intradermally-delivered formulations. Mucosal and systemic Th17 responses were also observed with this adjuvanted NP formulation and vaccination route, especially in the lung. We then evaluated protection induced by the adjuvanted NP formulation following a Mtb aerosol challenge and found that vaccination with NP-Ag85B and CpG via the pulmonary route displayed a substantial reduction of the lung bacterial burden, both compared to soluble Ag85B with CpG and to the corresponding intradermally delivered formulations. These findings highlight the potential of administrating NP-based formulations by the pulmonary route for TB vaccination.
Assuntos
Aciltransferases/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Ilhas de CpG/imunologia , Imunoconjugados/imunologia , Nanopartículas/administração & dosagem , Vacinas contra a Tuberculose/administração & dosagem , Vacinas contra a Tuberculose/imunologia , Tuberculose/prevenção & controle , Animais , Antígenos de Bactérias/administração & dosagem , Proteínas do Sistema Complemento/imunologia , Células Dendríticas/imunologia , Imunoconjugados/administração & dosagem , Imunoconjugados/química , Pulmão/citologia , Pulmão/imunologia , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/imunologia , Nanopartículas/química , Tamanho da Partícula , Polipropilenos/química , Baço/imunologia , Células Th1/imunologia , Células Th17/imunologia , Tuberculose/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologiaRESUMO
Interleukin (IL) 18 is a potent pro-inflammatory Th1 cytokine that exerts pleiotropic effector functions in both innate and acquired immune responses. Increased IL-18 production during acute rejection has been reported in experimental heart transplantation models and in kidney transplant recipients. IL-18-binding protein (IL-18BP) binds IL-18 with high affinity and neutralizes its biologic activity. We have analyzed the efficacy of an adenoviral vector expressing an IL-18BP-Ig fusion protein in a rat model of heart transplantation. IL-18BP-Ig gene transfer into Fisher (F344) rat donor hearts resulted in prolonged graft survival in Lewis recipients (15.8 +/- 1.4 days vs. 10.3 +/- 2.5 and 10.1 +/- 2.1 days with control virus and buffer solution alone, respectively; P < 0.001). Immunohistochemical analysis revealed decreased intra-graft infiltrates of monocytes/macrophages, CD4(+), CD8alpha(+) and T-cell receptor alphabeta(+) cells after IL-18BP-Ig versus mock gene transfer (P < 0.05). Real-time reverse transcriptase polymerase chain reaction analysis showed decreased cytokine transcripts for the RANTES chemokine and transforming growth factor-beta after IL-18BP-Ig gene transfer (P < 0.05). IL-18BP-Ig gene transfer attenuates inflammatory cell infiltrates and prolongs cardiac allograft survival in rats. These results suggest a contributory role for IL-18 in acute rejection. Further studies aiming at defining the therapeutic potential of IL-18BP are warranted.
Assuntos
Terapia Genética/métodos , Rejeição de Enxerto/terapia , Sobrevivência de Enxerto , Transplante de Coração/efeitos adversos , Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , Adenoviridae , Animais , Citocinas/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos , Rejeição de Enxerto/metabolismo , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Interleucina-18/metabolismo , Masculino , Miocárdio/metabolismo , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew , Transplantes , Regulação para CimaRESUMO
Endothelial cells (ECs) in normal vessels are poorly transducible by retroviral vectors, which require cell division for gene transduction. Among retroviruses, lentiviruses have the unique ability to integrate their genome into the chromatin of nondividing cells. Here we show that multiply attenuated, self-inactivating, lentiviral vectors transduce both proliferating and growth-arrested human umbilical vein ECs (HUVECs), human coronary artery ECs (HCAECs), and human coronary artery smooth muscle cells (HCASMCs), with high efficacy. Lentiviral vectors containing the enhanced green fluorescence protein (EGFP) transgene driven by either the cytomegalovirus or the elongation factor-1alpha promoter, but not the phosphoglycerate kinase promoter, directed high-level EGFP expression in endothelial and smooth muscle cells. The endothelium-specific Tie2 promoter also directed transgene expression in ECs. Re-insertion of cis-acting sequences from pol of human immunodeficiency virus type 1 (HIV-1) into the vectors improved transgene expression. A lentiviral vector containing the vascular endothelial growth factor transgene promoted EC proliferation and sprouting in vitro. In vivo gene transfer was studied by lumenal infusion of vector containing solutions into rat carotid arteries. Lentivirus-mediated EGFP gene transfer was observed in approximately 5% of ECs. Lentiviral vectors containing the LacZ transgene achieved detectable beta-galactosidase activity in rat arteries, albeit at a lower level compared with adenoviral vectors. This difference was mainly due to the lower concentration of lentiviral vector preparations. Lentivirus-mediated gene transfer was associated with minimal neointimal hyperplasia and scant inflammatory cell infiltrates in the media and adventitia. These observations indicate that lentiviral vectors may be useful for genetic modifications of vascular cells in vitro and in vivo.
Assuntos
Artérias Carótidas/metabolismo , Vasos Coronários/citologia , Vasos Coronários/metabolismo , Vetores Genéticos/genética , Lentivirus/genética , Lentivirus/patogenicidade , Transdução Genética/métodos , Adenoviridae/genética , Animais , Artérias Carótidas/citologia , Proliferação de Células , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Inflamação/genética , Inflamação/virologia , Lentivirus/fisiologia , Masculino , Miócitos de Músculo Liso/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Transdução Genética/instrumentação , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Inativação de VírusRESUMO
BACKGROUND: First-generation, E1-deleted (deltaE1) adenovirus vectors currently used in cardiovascular gene therapy trials are limited by tissue inflammation, mainly due to immune responses to viral gene products. Recently, helper-dependent (HD; also referred to as "gutless") adenovirus vectors devoid of all viral coding sequences have been shown to cause low inflammation when injected intravenously or into skeletal muscles. However, HD vectors have not been evaluated in cardiovascular tissues. METHODS AND RESULTS: HD and deltaE1 vectors containing a cytomegalovirus-driven expression cassette for the green fluorescent protein (GFP) gene were administered intramyocardially to adult rats (n = 54). GFP expression was measured by ELISA at varying time intervals after gene transfer. HD and deltaE1 vectors were equally efficient at transducing the myocardium. Tissue inflammation was assessed by immunostaining for leukocytes and quantitative real-time RT-PCR for cytokine mRNA expression. Monocyte/macrophages, CD4(+) and CD8(+) lymphocytes infiltrating the myocardium were less abundant with HD than deltaE1 vectors. Transcripts levels for pro-inflammatory cytokines such as IL-1beta, tumor necrosis factor-alpha, and RANTES were decreased with HD vectors. However, both vectors were associated with a decline in GFP expression over time, although low-level expression was occasionally detectable 10 weeks after HD vector administration. The two vectors transduced endothelial cells in rat arteries (n = 11) with comparable efficiencies. Vascular GFP expression was not detectable at 10 weeks. CONCLUSIONS: HD vectors are as efficient as deltaE1 vectors at transducing the myocardium and vascular endothelium, while causing less myocardial inflammation. Thus, HD vectors may be superior to earlier-generation adenovirus vectors for cardiovascular gene therapy applications.
Assuntos
Terapia Genética/efeitos adversos , Terapia Genética/métodos , Vetores Genéticos , Miocardite/virologia , Miocárdio/metabolismo , Miocárdio/patologia , Adenoviridae , Infecções por Adenoviridae , Animais , Doenças Cardiovasculares/terapia , Citocinas/metabolismo , Proteínas de Fluorescência Verde/genética , Imuno-Histoquímica , Masculino , Miocardite/metabolismo , Miocardite/patologia , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
AIMS: Polymorphisms in the RANTES (G-403A), monocyte chemoattractant protein-1 (MCP-1; A-2518G), stromal cell-derived factor-1beta (SDF-1beta; G801A), and C-C chemokine receptor-5 (CCR5; Delta32) genes have been associated with functional effects. These chemokines have been implicated in leucocyte recruitment to arterial lesions. In a case-control study, we explored relations between these polymorphisms and coronary artery disease (CAD), with respect to angiographic abnormalities and acute coronary syndromes (ACS). METHODS AND RESULTS: The LUdwigshafen Risk and Cardiovascular health (LURIC) cohort was genotyped by RFLP-PCR. Based on coronary angiography, individuals were sub-divided into CAD cases (n = 2694) and controls (n = 530). RANTES-403 genotype frequencies were significantly different in cases and controls (chi2 = 4.17, p = 0.041), as were A allele carrier frequencies (36.01% vs. 30.19%, OR = 1.30 [95%-CI = 1.06-1.60], p = 0.010). By multivariate analysis, RANTES A-403 retained significant association with CAD (chi2 = 8.40, p = 0.0038). RANTES A-403 was associated with increased ACS prevalence (OR = 1.36 [95%-CI = 1.08-1.71], p = 0.0073). MCP-1 G-2518, SDF-1beta A801, and CCR5 Delta32 were not associated with CAD. CONCLUSIONS: RANTES A-403 was associated with CAD independently from conventional risk factors and CRP or fibrinogen as inflammatory biomarkers. The association was enhanced in smokers and ACS, conditions where platelet activation and inflammation predominate. RANTES A-403 may increase genetic susceptibility to CAD.