RESUMO
Enteropathogenic Escherichia coli (EPEC) are important agents of acute diarrhea in children living in developing countries. A severe dysfunction of the intestinal epithelial barrier occurs during EPEC infection, leading to diarrhea and inflammation as consequences. EPEC main virulence factors include the adhesins intimin and bundle-forming pilus (BFP), as well as several effector proteins translocated to the enterocyte by the type-three secretion system. The initial interaction of EPEC with the host cell and the role of effector proteins in this process are well known. However, the role of the EPEC virulence factors in macrophage activation is not fully understood. Hence, we analyzed the ability of intimin and bundle-forming pilus (BfpA) to activate the innate response mediated by macrophages, where the production of the proinflammatory cytokines TNF-α, IL-1, IL-6 and IL-12, as well as the anti-inflammatory cytokine IL-10 and chemokine MCP-1, were evaluated. Our results showed that recombinant intimin and BfpA activate macrophages in a dose-dependent manner, and the stimulated cells produced TNF-α, IL-12, IL-6, IL-10 and MCP-1, but not IL-1β. No synergistic effect was observed in the production of pro-inflammatory cytokines by combining BfpA and intimin, although production of IL-10, an anti-inflammatory mediator, was potentiated at a higher dose. The effect observed was largely attributed to these proteins, as the treatment of proteins with polymyxin B did not alter the production of TNF-α. Thus, herein we showed that intimin and BfpA can activate the innate immune response, inducing the production of pro- and anti-inflammatory cytokines, as well as chemokines, playing additional role as inflammatory molecules in the early steps of EPEC infection.
RESUMO
BACKGROUND: Intimin is an important virulence factor involved in the pathogenesis of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC). Both pathogens are still important causes of diarrhea in children and adults in many developing and industrialized countries. Considering the fact that antibodies are important tools in the detection of various pathogens, an anti-intimin IgG2b monoclonal antibody was previously raised in immunized mice with the conserved sequence of the intimin molecule (int388-667). In immunoblotting assays, this monoclonal antibody showed excellent specificity. Despite good performance, the monoclonal antibody failed to detect some EPEC and EHEC isolates harboring variant amino acids within the 338-667 regions of intimin molecules. Consequently, motivated by its use for diagnosis purposes, in this study we aimed to the cloning and expression of the single-chain variable fragment from this monoclonal antibody (scFv). FINDINGS: Anti-intimin hybridoma mRNA was extracted and reversely transcripted to cDNA, and the light and heavy chains of the variable fragment of the antibody were amplified using commercial primers. The amplified chains were cloned into pGEM-T Easy vector. Specific primers were designed and used in an amplification and chain linkage strategy, obtaining the scFv, which in turn was cloned into pAE vector. E. coli BL21(DE3)pLys strain was transformed with pAE scFv-intimin plasmid and subjected to induction of protein expression. Anti-intimin scFv, expressed as inclusion bodies (insoluble fraction), was denatured, purified and submitted to refolding. The protein yield was 1 mg protein per 100 mL of bacterial culture. To test the functionality of the scFv, ELISA and immunofluorescence assays were performed, showing that 275 ng of scFv reacted with 2 mg of purified intimin, resulting in an absorbance of 0.75 at 492 nm. The immunofluorescence assay showed a strong reactivity with EPEC E2348/69. CONCLUSION: This study demonstrated that the recombinant anti-intimin antibody obtained is able to recognize the conserved region of intimin (Int388-667) in purified form and the EPEC isolate.
RESUMO
Intimin is an important virulence factor involved in the pathogenesis of enteropathogenic Escherichiacoli (EPEC) and enterohemorrhagic Escherichia coli (EHEC). Both pathogens are still important causes of diarrhea inchildren and adults in many developing and industrialized countries. Considering the fact that antibodies areimportant tools in the detection of various pathogens, an anti-intimin IgG2b monoclonal antibody was previously raised in immunized mice with the conserved sequence of the intimin molecule (int388-667). In immunoblotting assays, this monoclonal antibody showed excellent specificity. Despite good performance, the monoclonal antibody failed to detect some EPEC and EHEC isolates harboring variant amino acids within the 338-667 regions of intimin molecules. Consequently, motivated by its use for diagnosis purposes, in this study we aimed to the cloning and expression of the single-chain variable fragment from this monoclonal antibody (scFv).Anti-intimin hybridoma mRNA was extracted and reversely transcripted to cDNA, and the light and heavy chains of the variable fragment of the antibody were amplified using commercial primers. The amplified chains were cloned into pGEM-T Easy vector. Specific primers were designed and used in an amplification and chain linkage strategy, obtaining the scFv, which in turn was cloned into pAE vector. E. coli BL21(DE3)pLys strainwas transformed with pAE scFv-intimin plasmid and subjected to induction of protein expression. Anti-intimin scFv,expressed as inclusion bodies (insoluble fraction), was denatured, purified and submitted to refolding. The proteinyield was 1 mg protein per 100 mL of bacterial culture. To test the functionality of the scFv, ELISA andimmunofluorescence assays were performed, showing that 275 ng of scFv reacted with 2 mg of purified intimin,resulting in an absorbance of 0.75 at 492 nm.
Assuntos
Anticorpos Monoclonais , Escherichia coli Enteropatogênica/imunologia , Escherichia coli Enteropatogênica/patogenicidade , Hibridomas/imunologia , Imunofluorescência/métodosRESUMO
Proteus mirabilis is an important cause of urinary tract infection (UTI) in patients with complicated urinary tracts. Thirty-five strains of P. mirabilis isolated from UTI were examined for the adherence capacity to epithelial cells. All isolates displayed the aggregative adherence (AA) to HEp-2 cells, a phenotype similarly presented in LLC-MK(2) cells. Biofilm formation on polystyrene was also observed in all strains. The mannose-resistant Proteus-like fimbriae (MR/P), Type I fimbriae and AAF/I, II and III fimbriae of enteroaggregative Escherichia coli were searched by the presence of their respective adhesin-encoding genes. Only the MR/P fimbrial subunits encoding genes mrpA and mrpH were detected in all isolates, as well as MR/P expression. A mutation in mrpA demonstrated that MR/P is involved in aggregative adherence to HEp-2 cells, as well as in biofilm formation. However, these phenotypes are multifactorial, because the mrpA mutation reduced but did not abolish both phenotypes. The present results reinforce the importance of MR/P as a virulence factor in P. mirabilis due to its association with AA and biofilm formation, which is an important step for the establishment of UTI in catheterized patients.
Assuntos
Aderência Bacteriana/fisiologia , Células Epiteliais/microbiologia , Proteus mirabilis/fisiologia , Infecções Urinárias/microbiologia , Adesinas Bacterianas/genética , Adesinas de Escherichia coli/genética , Adolescente , Adulto , Idoso , Animais , Aderência Bacteriana/genética , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Linhagem Celular , Criança , Pré-Escolar , Feminino , Proteínas de Fímbrias/genética , Deleção de Genes , Humanos , Lactente , Macaca mulatta , Masculino , Pessoa de Meia-Idade , Poliestirenos , Proteus mirabilis/genética , Proteus mirabilis/isolamento & purificaçãoRESUMO
OBJECTIVE: This article aims at reviewing one of the most important problems faced by pediatricians in the field of child neurology. The paroxystic non-epileptic events are also a frequent reason for pediatric neurology consultations and admission for diagnostic videoelectroencephalogram monitoring. SOURCES: Literature review on the subject was perform on Medline, data were also collected from the main Pediatric Neurology Textbooks, which were found to be an important and unique source of information on the subject. SUMMARY OF THE FINDINGS: Many of the entities discussed in this paper are very common in the pediatric population such as syncope, breath-holding spells and the movement disorders associated with gastroesophageal reflux. Other syndromes are less frequent such as the paroxysmal dystonias and the Segawa Syndrome (dystonia with diurnal variation). CONCLUSIONS: The basic knowledge of these syndromes is very important since it may avoid unnecessary procedures and the wrongful diagnosis of epilepsy. Patients who are mistakenly diagnosed as epileptics are exposed to anticonvulsant medications, which are probably not going to be effective and may expose them to the risk of side effects.