RESUMO
Vaccine technology is still facing challenges regarding some infectious diseases, which can be addressed by innovative drug delivery systems. In particular, nanoparticle-based vaccines combined with new types of adjuvants are actively explored as a platform for improving the efficacy and durability of immune protection. Here, biodegradable nanoparticles carrying an antigenic model of HIV were formulated with two combinations of poloxamers, 188/407, presenting or not presenting gelling properties, respectively. The study aimed to determine the influence of poloxamers (as a thermosensitive hydrogel or a liquid solution) on the adaptive immune response in mice. The results showed that poloxamer-based formulations were physically stable and did not induce any toxicity using a mouse dendritic cell line. Then, whole-body biodistribution studies using a fluorescent formulation highlighted that the presence of poloxamers influenced positively the dissemination profile by dragging nanoparticles through the lymphatic system until the draining and distant lymph nodes. The strong induction of specific IgG and germinal centers in distant lymph nodes in presence of poloxamers suggested that such adjuvants are promising components in vaccine development.
Assuntos
Poloxâmero , Vacinas , Poloxâmero/metabolismo , Adjuvantes de Vacinas , Distribuição Tecidual , Antígenos , Linfonodos/metabolismo , Adjuvantes Imunológicos/química , Células DendríticasRESUMO
Among mucosal administration routes for vaccines, the sublingual route has been proven capable of inducing a potent systemic and mucosal immune response. However, the absence of a simple and compliant delivery system and the lack of robust mucosal adjuvants impede the development of sublingual vaccines. Here, we describe a mucoadhesive patch made of a layer-by-layer assembly of polysaccharides, chitosan, and hyaluronic acid. The mucoadhesive patch was covered by adjuvanted nanoparticles carrying viral proteins. We showed that the nanoparticles effectively cross the outer layers of the sublingual mucosa to reach the epithelium. Furthermore, the encapsulated adjuvants, 3M-052 and mifamurtide, targeting toll-like receptor (TLR) 7/8 and nucleotide-binding oligomerization domain-2 (NOD2), respectively, remain fully active after encapsulation into nanoparticles and exhibit a cytokine/chemokine signature similar to the mucosal gold-standard adjuvant, the cholera toxin. However, the particulate adjuvants induced more moderate levels of proinflammatory interleukin (IL)-6 and keratinocyte chemoattractant (KC), suggesting a controlled activation of the innate immune response.
Assuntos
Adjuvantes Imunológicos , Imunidade nas Mucosas , Animais , Camundongos , Administração Sublingual , Adjuvantes Imunológicos/farmacologia , Vacinas de Subunidades Antigênicas , Adjuvantes Farmacêuticos , Mucosa , Camundongos Endogâmicos BALB CRESUMO
Combretastatin A-4 and isocombretastatin A-4 derivatives having thiophenes or benzo[b]thiophenes instead of the B ring were prepared and evaluated for their in cellulo tubulin polymerization inhibition (TPI) and antiproliferative activities. The presence of the benzo[b]thiophene ring proved to have a crucial effect as most of the thiophene derivatives, except those having one methoxy group, were inactive to inhibit tubulin polymerization into microtubules. The influence of the attachment position was also studied: benzo[b]thiophenes having iso or cis 3,4,5-trimethoxystyrenes at position 2 were 12-30-fold more active than the 3-regioisomers for the TPI activity. Some of the novel designed compounds exhibited interesting anti-proliferative effects on two different cell lines.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Estilbenos/farmacologia , Tiofenos/farmacologia , Antineoplásicos Fitogênicos/síntese química , Antineoplásicos Fitogênicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Estilbenos/síntese química , Estilbenos/química , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/química , Tubulina (Proteína)/metabolismoRESUMO
Hepatitis B virus remains a major medical burden with more than 250 million chronically infected patients worldwide and 900,000 deaths each year, due to the disease progression towards severe complications (cirrhosis, hepatocellular carcinoma). Despite the availability of a prophylactic vaccine, this infection is still pandemic in Western Pacific and African regions, where around 6% of the adult population is infected. Among novel anti-HBV strategies, innovative drug delivery systems, such as nanoparticle platforms to deliver vaccine antigens or therapeutic molecules have been investigated. Here, we developed polylactic acid-based biodegradable nanoparticles as an innovative and efficient vaccine. They are twice functionalized by (i) the entrapment of Pam3CSK4, an immunomodulator and ligand to Toll-Like-Receptor 1/2, and by (ii) the adsorption/coating of myristoylated (2-48) derived PreS1 from the HBV surface antigen, identified as the major viral attachment site on hepatocytes. We demonstrate that such formulations mimic HBV virion with an efficient peptide recognition by the immune system, and elicit potent and durable antibody responses in naive mice during at least one year. We also show that the most efficient in vitro viral neutralization was observed with NP-Pam3CSK4-dPreS1 sera. The immunogenicity of the derived HBV antigen is modulated by the likely synergistic action of both the dPreS1 coated nanovector and the adjuvant moiety. This formulation represents a promising vaccine alternative to fight HBV infection.
Assuntos
Vírus da Hepatite B , Hepatite B , Camundongos , Animais , Antígenos de Superfície da Hepatite B , Receptor 2 Toll-Like , Vacinas contra Hepatite B , Formação de Anticorpos , Adjuvantes Imunológicos , Hepatite B/tratamento farmacológico , Hepatite B/prevenção & controleRESUMO
BACKGROUND: Hemophilia B (HB) is a bleeding disorder characterized by coagulation factor (F) IX (FIX) deficiency. The current standard-of-care for severe HB is prophylaxis with long-term repetitive intravenous (i.v.) infusions of recombinant FIX (rFIX) with standard half-life or extended half-life. Unmet needs remain regarding the development of non-invasive administration routes for coagulation factors. The aim of this study was to evaluate the effectiveness of intranasal delivery (IND) of rFIX and rFIX fused to Fc fragment (rFIX-Fc) in mice. METHODS: Drops of rFIX and rFIX-Fc were deposited in the nostrils of wild-type, FcRn knock-out, FcRn humanized, and FIX knock-out mice. rFIX mucosal uptake was evaluated by measuring plasma FIX antigen and FIX activity (FIX:C) levels, and by performing histologic analysis of the nasal mucosa following IND. RESULTS: After IND, both rFIX and rFIX-Fc were equally delivered to the blood compartment, irrespective of the mouse strain studied, mostly through a passive mechanism of transportation across the mucosal barrier, independent of FcRn receptor. Both plasma FIX antigen and FIX:C activity levels increased following IND in FIX knock-out mice. CONCLUSION: This proof-of-concept study describes evidence supporting the nasal route as an alternative to FIX i.v. infusion for the treatment of HB.
Assuntos
Hemofilia A , Hemofilia B , Camundongos , Animais , Fator IX/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Hemofilia B/tratamento farmacológico , Hemofilia B/genética , Camundongos Knockout , Hemofilia A/tratamento farmacológico , Proteínas Recombinantes/uso terapêuticoRESUMO
A novel series of combretastatin A-4 heterocyclic analogues was prepared by replacement of the B ring with indole, benzofurane or benzothiophene, attached at the C2 position. These compounds were evaluated for their abilities to inhibit tubulin assembly: derivative cis3b, having a benzothiophene, showed an activity similar to those of colchicine or deoxypodophyllotoxine. The antiproliferative and antimitotic properties of cis3b against keratinocyte cancer cell lines were also evaluated and the intracellular organization of microtubules in the cells after treatment with both stereoisomers of 3b was also determined, using confocal microscopy.
Assuntos
Antimitóticos/síntese química , Compostos Heterocíclicos/química , Estilbenos/química , Antimitóticos/química , Antimitóticos/toxicidade , Benzofuranos/química , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colchicina/farmacologia , Humanos , Indóis/química , Microscopia Confocal , Microtúbulos/química , Microtúbulos/metabolismo , Estereoisomerismo , Estilbenos/síntese química , Estilbenos/toxicidade , Tiofenos/químicaRESUMO
The sublingual mucosa is an appealing route for drug administration. However, in the context of increased use of therapeutic proteins, development of protein delivery systems that will protect the protein bioactivity is needed. As proteins are fragile and complex molecules, current sublingual formulations of proteins are in liquid dosage. Yet, protein dilution and short residence time at the sublingual mucosa are the main barriers for the control of the dose that is delivered. In this work, a simple delivery scaffold based on the assembly of two polysaccharides, chitosan and hyaluronic acid, is presented. The natural polymers were assembled by the Layer-by-Layer methodology to produce a mucoadhesive and oro-dispersible freestanding membrane, shown to be innocuous for epithelial human cells. The functionalization of the membrane with proteins led to the production of a bioactive patch with efficient loading and release of proteins, and suitable mechanical properties for manipulation. Sublingual administration of the patch in mouse evidenced the absence of inflammation and an extended time of contact between the model protein ovalbumin and the mucosa compared to liquid formulation. The delivery of fluorescent ovalbumin in mouse sublingual mucosa demonstrated the penetration of the protein in the epithelium 10 min after the patch administration. Moreover, a migration assay with a chemokine incorporated into the patch showed no decrease in bioactivity of the loaded protein after enzymatic release. This study therefore provides a promising strategy to develop a sublingual protein delivery system. STATEMENT OF SIGNIFICANCE: Although the oral route is largely used for drug delivery, it has limitations for the delivery of proteins that can be degraded by pH or gastric enzymes. The sublingual route therefore appears as an interesting approach for protein administration. In this work, a simple delivery scaffold is presented based on the assembly of two polysaccharides by the Layer-by-Layer methodology to produce a mucoadhesive patch. The produced patch allowed efficient loading and release of proteins, as well as protection of their bioactivity. An extended time of contact between the protein and the mucosa compared to liquid formulation was highlighted in mouse model. This study provides a promising strategy to develop a sublingual protein delivery system.
Assuntos
Mucosa Bucal , Polímeros , Administração Sublingual , Animais , Sistemas de Liberação de Medicamentos , Camundongos , ProteínasRESUMO
The CYP1B1 gene (GenBank: U56438), a member of the cytochrome P450 gene family, has been shown to be mutated in patients with primary congenital glaucoma (PCG), a rare but severely blinding form of glaucoma. Here, we have investigated CYP1B1 mutations in 31 unrelated French PCG patients. Mutations were found in 15 (48%) patients. Six of these mutations were novel. One, g.3979delA, caused a frameshift followed by a stop codon at residue 59. Two mutations, g.4547C>T (p.Q248X) and g.8167C>T (p.R444X), created a stop codon. Three other mutations, g.4499G>C (p.G232R), g.8033T>G (p.I399S), (p.N423Y), induced a significant amino acid change. Seven patients, who were of French descent, were compound heterozygotes. Six patients, whose families came from North Africa or from Portugal, carried a homozygous mutation reflecting their geographic origin. Intriguingly, one mutation, p.E229K, was present in heterozygous state in two unrelated patients. All together, these findings demonstrate the major role and the diversity of CYP1B1 mutations in French PCG patients.
Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Glaucoma/genética , Mutação , Sequência de Aminoácidos , Citocromo P-450 CYP1B1 , DNA/química , DNA/genética , Análise Mutacional de DNA , Mutação da Fase de Leitura , França , Glaucoma/congênito , Glaucoma/enzimologia , Haplótipos , Humanos , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Homologia de Sequência de AminoácidosRESUMO
The migration of DCs is a critical function, enabling information to be carried to where the immunological response occurs. Parasites are known to weaken host immunity by interfering with the functions of DCs and thus, may be a source of molecules with immunomodulatory properties. Here, we demonstrate that the soluble protein, GRA5, specific to Toxoplasma gondii, is able to increase the migration of human CD34-DCs toward CCL19. A synthetic Pep29 derived from the GRA5 hydrophilic NT region (Pep29) was found to be internalized by macropinocytosis and to trigger in vitro migration of CD34-DCs via CCR7 expression without activating DCs. Pep29 also induced a decrease in the number of LCs from human skin epidermis. As local depletion of DCs and migration of immature DCs lead to a disruption of the specific innate response, our results highlight the potential of using pathogen-derived synthetic peptides as novel cell modulators with a therapeutic potential to reduce symptoms in inflammatory disorders.
Assuntos
Antígenos de Protozoários/farmacologia , Movimento Celular/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Peptídeos/farmacologia , Toxoplasma/imunologia , Sequência de Aminoácidos , Antígenos CD34/metabolismo , Antígenos de Protozoários/química , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/metabolismo , Movimento Celular/imunologia , Células Cultivadas , Quimiocina CCL19/imunologia , Quimiocina CCL19/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Imunofenotipagem , Células de Langerhans/imunologia , Células de Langerhans/metabolismo , Sistema de Sinalização das MAP Quinases , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/imunologia , Peptídeos/metabolismo , Pinocitose/imunologia , Domínios e Motivos de Interação entre Proteínas/imunologia , Sinais Direcionadores de Proteínas , Proteínas de Protozoários/química , Proteínas de Protozoários/imunologia , Receptores CCR7/metabolismo , Pele/imunologia , Toxoplasma/químicaRESUMO
Nanoparticles (NPs) have been reported to penetrate into human skin through lesional skin or follicular structures. Therefore, their ability to interact with dendritic cell (DC) was investigated using DCs generated from monocytes (mono-DCs). Hybrid titanium dioxide/para-amino benzoic acid (TiO(2)/PABA) NPs did not induce any cell toxicity. NPs were internalised into DCs through macropinocytosis and not by a receptor-mediated mechanism. Confocal microscopy showed that NPs were not detected in the nucleus. These data are confirmed by electronic microscopy which demonstrated that hybrid NPs were rapidly in contact with cellular membrane and localised into cytoplasmic vesicles without colocalisation with clathrin-coated vesicles. Hybrid NPs did not induce CD86 or HLA-DR overexpression or cytokine secretion (IL-8 and TNF-α) indicating no DC activation. Internalisation of hybrid NPs did not modify DC response towards sensitisers such as nickel and thimerosal or LPS used as positive controls. Moreover, hybrid NPs did not induce any oxidative stress implicated in DC activation process. After mono-DC irradiation by ultraviolet A (UVA), hybrid NP-treated cells did not produce UVA-induced reactive oxygen species (ROS) and exhibited a better cell viability compared with UVA-irradiated control cells, suggesting a protecting effect of hybrid TiO(2)/PABA NPs against UVA-induced ROS.
Assuntos
Ácido 4-Aminobenzoico/toxicidade , Células Dendríticas/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Titânio/toxicidade , Complexo Vitamínico B/toxicidade , Ácido 4-Aminobenzoico/farmacocinética , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Dendríticas/metabolismo , Células Dendríticas/ultraestrutura , Humanos , Microscopia Eletrônica de Transmissão , Pinocitose/efeitos dos fármacos , Pinocitose/fisiologia , Titânio/farmacocinética , Complexo Vitamínico B/farmacocinéticaRESUMO
Odontoblasts, dental pulp fibroblasts and immature dendritic cells (DCs) have been involved in the human dental pulp immune response to oral pathogens that invade dentine during the caries process. How they regulate the inflammatory response to Gram-positive bacteria remains nevertheless largely unknown. In this study we investigated the production of the pro-inflammatory cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-8 (CXCL8) in these three cell types upon stimulation with lipoteichoic acid (LTA), a cell wall component of Gram-positive bacteria that activates the pattern recognition molecule Toll-like receptor 2 (TLR2). We observed that TNF-alpha gene expression was up-regulated in all LTA-stimulated cell types. IL-1beta gene expression was not or barely detectable in odontoblast-like cells and pulp fibroblasts when stimulated or not, but was expressed in immature DCs and increased upon stimulation. TNF-alpha and IL-1beta proteins were detected in DC culture supernatants but not in odontoblast-like cell and pulp fibroblast ones. CXCL8 gene and protein were clearly expressed and increased in the three cell types upon LTA stimulation. These data indicate that LTA-dependent TLR2 activation in odontoblasts and pulp fibroblasts, in contrast to immature DCs, does not lead to significant TNF-alpha and IL-1beta production, but that all three cell types influence the pulp inflammatory/immune response through CXCL8 synthesis and secretion.
Assuntos
Células Dendríticas/metabolismo , Fibroblastos/metabolismo , Bactérias Gram-Positivas/imunologia , Odontoblastos/metabolismo , Receptor 2 Toll-Like/metabolismo , Diferenciação Celular , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/patologia , Polpa Dentária/patologia , Sangue Fetal/citologia , Fibroblastos/imunologia , Fibroblastos/patologia , Perfilação da Expressão Gênica , Infecções por Bactérias Gram-Positivas/imunologia , Humanos , Imunidade Inata , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Interleucina-8/genética , Interleucina-8/imunologia , Interleucina-8/metabolismo , Lipopolissacarídeos/metabolismo , Dente Serotino/patologia , Odontoblastos/imunologia , Odontoblastos/patologia , Ácidos Teicoicos/metabolismo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Gram-positive bacteria entering the dentinal tissue during the carious process are suspected to influence the immune response in human dental pulp. Odontoblasts situated at the pulp/dentin interface are the first cells encountered by these bacteria and therefore could play a crucial role in this response. In the present study, we found that in vitro-differentiated odontoblasts constitutively expressed the pattern recognition receptor TLR1-6 and 9 genes but not TLR7, 8, and 10. Furthermore, lipoteichoic acid (LTA), a wall component of Gram-positive bacteria, triggered the activation of the odontoblasts. LTA up-regulated the expression of its own receptor TLR2, as well as the production of several chemokines. In particular, an increased amount of CCL2 and CXCL10 was detected in supernatants from LTA-stimulated odontoblasts, and those supernatants augmented the migration of immature dendritic cells in vitro compared with controls. Clinical relevance of these observations came from immunohistochemical analysis showing that CCL2 was expressed in vivo by odontoblasts and blood vessels present under active carious lesions but not in healthy dental pulps. In contrast with this inflammatory response, gene expression of major dentin matrix components (type I collagen, dentin sialophosphoprotein) and TGF-beta1 was sharply down-regulated in odontoblasts by LTA. Taken together, these data suggest that odontoblasts activated through TLR2 by Gram-positive bacteria LTA are able to initiate an innate immune response by secreting chemokines that recruit immature dendritic cells while down-regulating their specialized functions of dentin matrix synthesis and mineralization.