RESUMO
Shiga toxin (Stx)-producing Escherichia coli (STEC) cause post-diarrhea Hemolytic Uremic Syndrome (HUS), which is the most common cause of acute renal failure in children in many parts of the world. Several non-O157 STEC strains also produce Subtilase cytotoxin (SubAB) that may contribute to HUS pathogenesis. The aim of the present work was to examine the cytotoxic effects of SubAB on primary cultures of human cortical renal tubular epithelial cells (HRTEC) and compare its effects with those produced by Shiga toxin type 2 (Stx2), in order to evaluate their contribution to renal injury in HUS. For this purpose, cell viability, proliferation rate, and apoptosis were assayed on HRTEC incubated with SubAB and/or Stx2 toxins. SubAB significantly reduced cell viability and cell proliferation rate, as well as stimulating cell apoptosis in HRTEC cultures in a time dependent manner. However, HRTEC cultures were significantly more sensitive to the cytotoxic effects of Stx2 than those produced by SubAB. No synergism was observed when HRTEC were co-incubated with both SubAB and Stx2. When HRTEC were incubated with the inactive SubAA272B toxin, results were similar to those in untreated control cells. Similar stimulation of apoptosis was observed in Vero cells incubated with SubAB or/and Stx2, compared to HRTEC. In conclusion, primary cultures of HRTEC are significantly sensitive to the cytotoxic effects of SubAB, although, in a lesser extent compared to Stx2.
Assuntos
Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Proteínas de Escherichia coli/efeitos adversos , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/microbiologia , Toxina Shiga II/efeitos adversos , Subtilisinas/efeitos adversos , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Humanos , Células Vero/efeitos dos fármacos , Células Vero/microbiologiaRESUMO
The dengue virus non-structural 1 (NS1) protein contributes to evasion of host immune defenses and represents a target for immune responses. Evidences generated in experimental models, as well as the immune responses elicited by infected individuals, showed that induction of anti-NS1 immunity correlates with protective immunity but may also result in the generation of cross-reactive antibodies that recognize platelets and proteins involved in the coagulation cascade. In the present work, we evaluated the immune responses, protection to type 2 dengue virus (DENV2) challenges and safety parameters in BALB/c mice vaccinated with a recombinant NS1 protein in combination with three different adjuvants: aluminum hydroxide (alum), Freund's adjuvant (FA) or a genetically detoxified derivative of the heat-labile toxin (LT(G33D)), originally produced by some enterotoxigenic Escherichia coli (ETEC) strains. Mice were subcutaneously (s.c.) immunized with different vaccine formulations and the induced NS1-specific responses, including serum antibodies and T cell responses, were measured. Mice were also subjected to lethal challenges with the DENV2 NGC strain. The results showed that maximal protective immunity (50%) was achieved in mice vaccinated with NS1 in combination with LT(G33D). Analyses of the NS1-specific immune responses showed that the anti-virus protection correlated mainly with the serum anti-NS1 antibody responses including higher avidity to the target antigen. Mice immunized with LT(G33D) elicited a prevailing IgG2a subclass response and generated antibodies with stronger affinity to the antigen than those generated in mice immunized with the other vaccine formulations. The vaccine formulations were also evaluated regarding induction of deleterious side effects and, in contrast to mice immunized with the FA-adjuvanted vaccine, no significant hepatic damage or enhanced C-reactive protein levels were detected in mice immunized with NS1 and LT(G33D.) Similarly, no detectable alterations in bleeding time and hematological parameters were detected in mice vaccinated with NS1 and LT(G33D). Altogether, these results indicate that the combination of a purified recombinant NS1 and a nontoxic LT derivative is a promising alternative for the generation of safe and effective protein-based anti-dengue vaccine.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Toxinas Bacterianas/administração & dosagem , Vacinas contra Dengue/imunologia , Vírus da Dengue/imunologia , Enterotoxinas/administração & dosagem , Proteínas de Escherichia coli/administração & dosagem , Toxoides/administração & dosagem , Proteínas não Estruturais Virais/imunologia , Adjuvantes Imunológicos/efeitos adversos , Adjuvantes Imunológicos/genética , Hidróxido de Alumínio/administração & dosagem , Animais , Anticorpos Antivirais/sangue , Toxinas Bacterianas/efeitos adversos , Toxinas Bacterianas/genética , Dengue/mortalidade , Dengue/patologia , Vacinas contra Dengue/administração & dosagem , Vacinas contra Dengue/efeitos adversos , Vírus da Dengue/genética , Enterotoxinas/efeitos adversos , Enterotoxinas/genética , Proteínas de Escherichia coli/efeitos adversos , Proteínas de Escherichia coli/genética , Adjuvante de Freund/administração & dosagem , Humanos , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Análise de Sobrevida , Linfócitos T/imunologia , Toxoides/efeitos adversos , Toxoides/genética , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/efeitos adversos , Vacinas Sintéticas/imunologia , Proteínas não Estruturais Virais/genéticaRESUMO
A leptospirose é uma zoonose causada por bactérias patogênicas do gênero Leptospira. O sequenciamento do genoma completo de L. interrogans sorovar Copenhageni tem permitido a obtenção e caracterização de proteínas potencialmente envolvidas na patogênese desta bactéria, como lipoproteínas e proteínas de membrana externa. Neste trabalho, foram estudados três genes, OmpL1, LIC10731 e LIC10645, dos quais o gene OmpL1 foi o mais frequente em diferentes espécies de Leptospira. As proteínas recombinantes foram purificadas por cromatografia de afinidade ao metal. As três proteínas recombinantes promoveram resposta humoral e celular após imunização em camundongos. Ensaios de adesão mostraram que as proteínas se ligam à laminina e plasminogênio, e adicionalmente a proteína OmpL1 se liga ao fibrinogênio e fibronectina plasmática. A proteína OmpL1 foi bastante reativa com soro de pacientes de leptospirose. Os resultados sugerem que as proteínas referentes aos genes estudados podem desempenhar um papel na patogênese da bactéria.
Leptospirosis is a zoonosis caused by pathogenic bacteria of genus Leptospira. Annotation of the genome sequences of L. interrogans serovar Copenhageni allows the identification and characterization of proteins potentially involved in the pathogenesis of this bacterium, such as lipoproteins and outer-membrane proteins. The present study characterized three genes, OmpL1, LIC10731 and LIC10645, and one of them, OmpL1, was the most frequent among different species of Leptospira. The recombinant proteins were purified by metal-chelating chromatography. All three recombinant proteins promoted humoral and cellular response after immunization in mice. Binding assays showed that all proteins interact to laminin and plasminogen, and additionally protein OmpL1 binds to fibrinogen and plasma fibronectin. OmpL1 was highly reactive with positive-leptospirosis human sera. The results suggest that the proteins encoded by these genes may play a role in the bacterium pathogenesis.
Assuntos
Humanos , Leptospira interrogans , Leptospirose , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/efeitos adversos , Proteínas de Escherichia coli/imunologia , Vacinas/imunologiaRESUMO
The colonization factors (CF) of enterotoxigenic Escherichia coli (ETEC) are being targeted for inclusion in a multi-subunit ETEC vaccine. This study was designed to examine the preclinical safety and immunogenicity of CF CS6, encapsulated in a biodegradable poly(DL-lactide-co-glycolide) (meCS6), and administered in the presence or absence of a mutated heat-labile enterotoxin, LT(R192G), in the non-human primate, Aotus nancymae. A. nancymae were inoculated intranasally (IN) with meCS6 (200 microg; positive control), or intragastrically (IG) with meCS6 (200 or 1000 microg) with or without 2 microg LT(R192G) in three doses given at 2-week intervals. In a second experiment, A. nancymae were inoculated IG with 950 microg of meCS6 with or without 2 microg LT(R192G) in four doses given every 48 h. Blood was collected to assess anti-CS6 and -LT serum immunoglobulin G (IgG) and IgA responses and safety variables (complete blood count and chemistry). Safety parameters were unchanged from baseline following all vaccinations. In Experiment 1, a dose-related serologic response to CS6 was observed; 78.6 and 57.1% of monkeys given 1000 microg meCS6 (n = 14) had a serum IgG and IgA response, respectively, compared to only 28.6% of monkeys given 200 microg meCS6 (n = 14) with a serum IgG and IgA response. No significant effect on the number of responders or the magnitude of responses was observed with the addition of LT(R192G). The three-dose, 2-week regimen with 1000 microg meCS6 was more effective at eliciting an immune response than the four-dose, 48-h regimen with 950 microg meCS6. Results from this study indicate that A. nancymae provide a useful ETEC preclinical safety and immunogenicity model.