RESUMO
BACKGROUND: Opsonization, is the molecular mechanism by which target molecules promote interactions with phagocyte cell surface receptors to remove unwanted cells by induced phagocytosis. We designed an in vitro system to demonstrate that this procedure could be driven to eliminate adipocytes, using peptides mimicking regions of the complement protein C3b to promote opsonization and enhance phagocytosis. Two cell lines were used: (1) THP-1 monocytes differentiated to macrophages, expressing the C3b opsonin receptor CR1 in charge of the removal of unwanted coated complexes; (2) 3T3-L1 fibroblasts differentiated to adipocytes, expressing AQP7, to evaluate the potential of peptides to stimulate opsonization. (3) A co-culture of the two cell lines to demonstrate that phagocytosis could be driven to cell withdrawal with high efficiency and specificity. RESULTS: An array of peptides were designed and chemically synthesized p3691 and p3931 joined bound to the CR1 receptor activating phagocytosis (p < 0.033) while p3727 joined the AQP7 protein (p < 0.001) suggesting that opsonization of adipocytes could occur. In the co-culture system p3980 and p3981 increased lipid uptake to 91.2% and 89.0%, respectively, as an indicator of potential adipocyte phagocytosis. CONCLUSIONS: This in vitro model could help understand the receptorligand interaction in the withdrawal of unwanted macromolecules in vivo. The adipocyte-phagocytosis discussed may help to control obesity, since peptides of C3b stimulated the CR1 receptor, promoting opsonisation and phagocytosis of lipidcontaining structures, and recognition of AQP7 in the differentiated adipocytes, favored the phagocytic activity of macrophages, robustly supported by the co-culture strategy.
Assuntos
Fagocitose , Proteínas do Sistema Complemento , Adipócitos , Técnicas In Vitro , Proteínas Opsonizantes , Técnicas de Cocultura , Células Espumosas , Macrófagos , Microscopia de FluorescênciaRESUMO
Freeze-drying of nanoparticle suspensions is capable of generating stable nanoformulations with improved storage times and easier transportation. Nonetheless, nanoparticle aggregation is likely induced during freeze-drying, which reduces its redispersibility upon reconstitution and leads to undesirable effects such as non-specific toxicity and impaired efficacy. In this work, bovine serum albumin (BSA) is described as a suitable protectant for silica nanoparticles (SNPs), which result in solid structures with excellent redispersibility and negligible signs of aggregation even when longer storage times are considered. We experimentally demonstrated that massive system aggregation can be prevented when a saturated BSA corona around the nanoparticle is formed before the lyophilization process. Furthermore, the BSA corona is able to suppress non-specific interactions between these nanoparticles and biological systems, as evidenced by the lack of residual cytotoxicity, hemolytic activity and opsonin adsorption. Hence, BSA can be seriously considered for industry as an additive for nanoparticle freeze-drying since it generates solid and redispersible nanoformulations with improved biocompatibility.
Assuntos
Nanopartículas , Coroa de Proteína , Adsorção , Estabilidade de Medicamentos , Liofilização , Proteínas Opsonizantes , Tamanho da PartículaRESUMO
The pneumococcal whole cell vaccine (PWCV) has been investigated as an alternative to polysaccharide-based vaccines currently in use. It is a non-encapsulated killed vaccine preparation that induces non-capsular antibodies protecting mice against invasive pneumococcal disease (IPD) and reducing nasopharyngeal (NP) carriage via IL-17A activation of mouse phagocytes. Here, we show that PWCV induces antibody and IL-17A production to protect mice against challenge in a fatal aspiration-sepsis model after only one dose. We observed protection even with a boiled preparation, attesting to the stability and robustness of the vaccine. PWCV antibodies were shown to bind to different encapsulated strains, but complement deposition on the pneumococcal surface was observed only on serotype 3 strains; using flow cytometer methodology, variations in PWCV quality, as in the boiled vaccine, were detected. Moreover, anti-PWCV induces phagocytosis of different pneumococcal serotypes by murine peritoneal cells in the presence of complement or IL-17A. These findings suggest that complement and IL-17A may participate in the process of phagocytosis induced by PWCV antibodies. IL-17A can stimulate phagocytic cells to kill pneumococcus and this is enhanced in the presence of PWCV antibodies bound to the bacterial cell surface. Our results provide further support for the PWCV as a broad-range vaccine against all existing serotypes, potentially providing protection for humans against NP colonization and IPD. Additionally, we suggest complement deposition assay as a tool to detect subtle differences between PWCV lots.
Assuntos
Complemento C3/imunologia , Interleucina-17/imunologia , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Animais , Sítios de Ligação de Anticorpos , Citometria de Fluxo , Camundongos , Nasofaringe/microbiologia , Proteínas Opsonizantes/imunologia , Fagocitose , Vacinas Pneumocócicas/administração & dosagem , Sepse/imunologia , Sepse/microbiologia , Sepse/prevenção & controle , Sorogrupo , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologiaRESUMO
Neisseria meningitidis serogroup C (MenC) is the main causative agent of meningitis in Brazil. HIV infection affects the quality of the immune system. HIV+ children have an increased risk of infection to encapsulated bacteria such as N. meningitidis. We evaluated the opsonic antibody (OPA) levels and its correlation with serum bactericidal antibody (SBA) levels induced by one and two doses of a MenC conjugate vaccine in children and adolescents HIV+ and HIV-exposed but uninfected children (HEU) group. Overall the data show the importance of two doses of vaccine for HIV+ individuals. About 79% and 58% of HIV+ patients showed SBA and OPA positive response after two doses of vaccine, respectively. For HEU group, 62% and 41% of patients showed SBA and OPA positive response after one dose of vaccine, respectively. A positive and significant association between SBA and OPA levels was seen after two doses of vaccine in HIV+ patients.
Assuntos
Anticorpos Antibacterianos/sangue , Atividade Bactericida do Sangue , Infecções Meningocócicas/prevenção & controle , Vacinas Meningocócicas/imunologia , Neisseria meningitidis Sorogrupo C/imunologia , Proteínas Opsonizantes/sangue , Adolescente , Brasil , Criança , Pré-Escolar , Feminino , Infecções por HIV/complicações , Humanos , Masculino , Vacinas Meningocócicas/administração & dosagem , Estudos Prospectivos , Vacinas Conjugadas/administração & dosagem , Vacinas Conjugadas/imunologiaRESUMO
BACKGROUND: Plasmodium vivax accounts for the majority of human malaria infections outside Africa and is being increasingly associated in fatal outcomes with anaemia as one of the major complications. One of the causes of malarial anaemia is the augmented removal of circulating non-infected red blood cells (nRBCs), an issue not yet fully understood. High levels of auto-antibodies against RBCs have been associated with severe anaemia and reduced survival of nRBCs in patients with falciparum malaria. Since there are no substantial data about the role of those antibodies in vivax malaria, this study was designed to determine whether or not auto-antibodies against erythrocytes are involved in nRBC clearance. Moreover, the possible immune mechanisms elicited by them that may be associated to induce anaemia in P. vivax infection was investigated. METHODS: Concentrations of total IgG were determined by sandwich ELISA in sera from clinically well-defined groups of P. vivax-infected patients with or without anaemia and in healthy controls never exposed to malaria, whereas the levels of specific IgG to nRBCs were determined by cell-ELISA. Erythrophagocytosis assay was used to investigate the ability of IgGs purified from each studied pooled sera in enhancing nRBC in vitro clearance by THP-1 macrophages. Defocusing microscopy was employed to measure the biomechanical modifications of individual nRBCs opsonized by IgGs purified from each group. RESULTS: Anaemic patients had higher levels of total and specific anti-RBC antibodies in comparison to the non-anaemic ones. Opsonization with purified IgG from anaemic patients significantly enhanced RBCs in vitro phagocytosis by THP-1 macrophages. Auto-antibodies purified from anaemic patients decreased the nRBC dynamic membrane fluctuations suggesting a possible participation of such antibodies in the perturbation of erythrocyte flexibility and morphology integrity maintenance. CONCLUSIONS: These findings revealed that vivax-infected patients with anaemia have increased levels of IgG auto-antibodies against nRBCs and that their deposition on the surface of non-infected erythrocytes decreases their deformability, which, in turn, may enhance nRBC clearance by phagocytes, contributing to the anaemic outcome. These data provide insights into the immune mechanisms associated with vivax malaria anaemia and may be important to the development of new therapy and vaccine strategies.
Assuntos
Anemia/etiologia , Autoanticorpos/sangue , Eritrócitos/imunologia , Malária Vivax/complicações , Proteínas Opsonizantes/sangue , Fagocitose , Adolescente , Adulto , Idoso , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Brucella abortus is an intracellular pathogen of monocytes, macrophages, dendritic cells, and placental trophoblasts. This bacterium causes a chronic disease in bovines and in humans. In these hosts, the bacterium also invades neutrophils; however, it fails to replicate and just resists the killing action of these leukocytes without inducing significant activation or neutrophilia. Moreover, B. abortus causes the premature cell death of human neutrophils. In the murine model, the bacterium is found within macrophages and dendritic cells at early times of infection but seldom in neutrophils. Based on this observation, we explored the interaction of mouse neutrophils with B. abortus In contrast to human, dog, and bovine neutrophils, naive mouse neutrophils fail to recognize smooth B. abortus bacteria at early stages of infection. Murine normal serum components do not opsonize smooth Brucella strains, and neutrophil phagocytosis is achieved only after the appearance of antibodies. Alternatively, mouse normal serum is capable of opsonizing rough Brucella mutants. Despite this, neutrophils still fail to kill Brucella, and the bacterium induces cell death of murine leukocytes. In addition, mouse serum does not opsonize Yersinia enterocolitica O:9, a bacterium displaying the same surface polysaccharide antigen as smooth B. abortus Therefore, the lack of murine serum opsonization and absence of murine neutrophil recognition are specific, and the molecules responsible for the Brucella camouflage are N-formyl-perosamine surface homopolysaccharides. Although the mouse is a valuable model for understanding the immunobiology of brucellosis, direct extrapolation from one animal system to another has to be undertaken with caution.
Assuntos
Brucella abortus/imunologia , Evasão da Resposta Imune , Manose/imunologia , Neutrófilos/microbiologia , Fagocitose , Polissacarídeos Bacterianos/imunologia , Animais , Brucella abortus/crescimento & desenvolvimento , Sequência de Carboidratos , Bovinos , Morte Celular , Cães , Expressão Gênica , Especificidade de Hospedeiro , Humanos , Imunidade Humoral , Imunidade Inata , Manose/análogos & derivados , Camundongos , Neutrófilos/imunologia , Proteínas Opsonizantes/genética , Proteínas Opsonizantes/imunologia , Polissacarídeos Bacterianos/química , Yersinia enterocolitica/crescimento & desenvolvimento , Yersinia enterocolitica/imunologiaRESUMO
BACKGROUND: Widespread use of 7-valent pneumococcal conjugate vaccine (PCV7) in children has led to significant reduction in pneumococcal disease in children and adults. However, diseases caused by serotypes not included in PCV7 have increased. A 15-valent pneumococcal conjugate vaccine (PCV15) containing serotypes in PCV7 and 8 additional serotypes (1, 3, 5, 6A, 7F, 19A, 22F, 33F) was developed and evaluated in toddlers 12 to 15 months of age. METHODS: Ninety toddlers who completed an infant series with PCV7 received a single dose of either aluminum-adjuvanted PCV15, nonadjuvanted PCV15, or PCV7. Injection-site and systemic adverse events (AEs) were collected for 14 days postvaccination and serious AEs (SAEs) were collected for 30 days postvaccination. Solicited AEs included local (pain/tenderness, swelling, nodule and redness) and systemic (fatigue, arthralgia and myalgia) AEs. Serotype-specific immunoglobulin G (IgG) and opsonophagocytic (OPA) responses were measured immediately prior and 30 days postvaccination. RESULTS: Incidences of local and systemic AEs were comparable across vaccine groups. The majority of reported events, regardless of vaccine received, were transient and of mild to moderate intensity. No clinically significant differences were observed when comparing duration and severity of AEs. No vaccine-related SAEs or discontinuations from the study due to AEs were reported. Pneumococcal IgG concentrations and OPA titers increased postvaccination, with appreciable fold rises for all serotypes. Antibody levels were comparable between both PCV15 formulations and generally comparable to PCV7 for the shared serotypes. CONCLUSION: Both formulations of PCV15 display acceptable safety profiles and induce IgG and OPA responses to all vaccine serotypes.
Assuntos
Anticorpos Antibacterianos/sangue , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Proteínas Opsonizantes/sangue , Vacinas Pneumocócicas/efeitos adversos , Vacinas Pneumocócicas/imunologia , Feminino , Humanos , Imunoglobulina G/sangue , Lactente , Masculino , Fagocitose , Vacinas Pneumocócicas/administração & dosagem , Vacinas Conjugadas/administração & dosagem , Vacinas Conjugadas/efeitos adversos , Vacinas Conjugadas/imunologiaRESUMO
This open-label multicenter clinical trial conducted in Mexico assessed the immunogenicity and safety of a 13-valent pneumococcal conjugate vaccine (PCV13) in adults ≥ 50 years of age not previously vaccinated with the 23-valent pneumococcal polysaccharide vaccine (PPSV23). The PCV13 elicited a robust immune response in this study population, as reflected by the magnitude of fold rises in functional antibody levels measured by serotype-specific opsonophagocytic activity (OPA) assays before and 1 month after vaccination. Although the prevaccination OPA geometric mean titers (GMTs) for the majority of the serotypes were significantly lower in the 50- to 64-year age group than those in the ≥ 65-year age group, the postvaccination immune responses were generally similar. The overall immune responses were higher for the majority of the serotypes in the Mexican study population than those in similar adult study populations who received the PCV13 in Europe and the United States. PCV13 was well tolerated, and there were no vaccine-related serious adverse events. In conclusion, PCV13 is safe and immunogenic when administered to adults ≥ 50 years of age in Mexico and has the potential to protect against vaccine-type pneumococcal disease. (This study has been registered at ClinicalTrials.gov under registration no. NCT01432262.).
Assuntos
Anticorpos Antibacterianos/sangue , Vacinas Pneumocócicas/efeitos adversos , Vacinas Pneumocócicas/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Feminino , Humanos , Masculino , México , Pessoa de Meia-Idade , Proteínas Opsonizantes/sangue , Fagocitose , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/administração & dosagemRESUMO
Bordetella parapertussis is a human pathogen that causes whooping cough. The increasing incidence of B. parapertussis has been attributed to the lack of cross protection induced by pertussis vaccines. It was previously shown that B. parapertussis is able to avoid bacterial killing by polymorphonuclear leukocytes (PMN) if specific opsonic antibodies are not present at the site of interaction. Here, we evaluated the outcome of B. parapertussis innate interaction with human macrophages, a less aggressive type of cell and a known reservoir of many persistent pathogens. The results showed that in the absence of opsonins, O antigen allows B. parapertussis to inhibit phagolysosomal fusion and to remain alive inside macrophages. The O antigen targets B. parapertussis to lipid rafts that are retained in the membrane of phagosomes that do not undergo lysosomal maturation. Forty-eight hours after infection, wild-type B. parapertussis bacteria but not the O antigen-deficient mutants were found colocalizing with lipid rafts and alive in nonacidic compartments. Taken together, our data suggest that in the absence of opsonic antibodies, B. parapertussis survives inside macrophages by preventing phagolysosomal maturation in a lipid raft- and O antigen-dependent manner. Two days after infection, about 15% of macrophages were found loaded with live bacteria inside flotillin-enriched phagosomes that had access to nutrients provided by the host cell recycling pathway, suggesting the development of an intracellular infection. IgG opsonization drastically changed this interaction, inducing efficient bacterial killing. These results highlight the need for B. parapertussis opsonic antibodies to induce bacterial clearance and prevent the eventual establishment of cellular reservoirs of this pathogen.