12 research outputs found

    Cloning approach and functional analysis of anti-intimin single-chain variable fragment (scFv)

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    <p>Abstract</p> <p>Background</p> <p>Intimin is an important virulence factor involved in the pathogenesis of enteropathogenic <it>Escherichia coli </it>(EPEC) and enterohemorrhagic <it>Escherichia coli </it>(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 (int<sub>388-667</sub>). 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).</p> <p>Findings</p> <p>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 <it>pGEM-T Easy </it>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. <it>E. coli </it>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.</p> <p>Conclusion</p> <p>This study demonstrated that the recombinant anti-intimin antibody obtained is able to recognize the conserved region of intimin (Int<sub>388-667</sub>) in purified form and the EPEC isolate.</p

    Different Assay Conditions for Detecting the Production and Release of Heat-Labile and Heat-Stable Toxins in Enterotoxigenic Escherichia coli Isolates

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    Enterotoxigenic Escherichia coli (ETEC) produce heat-labile (LT) and/or heat-stable enterotoxins (ST). Despite that, the mechanism of action of both toxins are well known, there is great controversy in the literature concerning the in vitro production and release of LT and, for ST, no major concerns have been discussed. Furthermore, the majority of published papers describe the use of only one or a few ETEC isolates to define the production and release of these toxins, which hinders the detection of ETEC by phenotypic approaches. Thus, the present study was undertaken to obtain a better understanding of ST and LT toxin production and release under laboratory conditions. Accordingly, a collection of 90 LT-, ST-, and ST/LT-producing ETEC isolates was used to determine a protocol for toxin production and release aimed at ETEC detection. for this, we used previously raised anti-LT antibodies and the anti-ST monoclonal and polyclonal antibodies described herein. the presence of bile salts and the use of certain antibiotics improved ETEC toxin production/release. Triton X-100, as chemical treatment, proved to be an alternative method for toxin release. Consequently, a common protocol that can increase the production and release of LT and ST toxins could facilitate and enhance the sensitivity of diagnostic tests for ETEC using the raised and described antibodies in the present work.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Butantan Inst, Bacteriol Lab, BR-05503900 São Paulo, BrazilSão Paulo Trop Med Inst, Seroepidemiol & Immunol Lab, BR-05403000 São Paulo, BrazilFleury Med & Hlth, BR-04344903 São Paulo, BrazilButantan Inst, Immunopathol Lab, BR-05503900 São Paulo, BrazilButantan Inst, Immunochem Lab, BR-05503900 São Paulo, BrazilAdolfo Lutz Inst, Bacteriol Sect, BR-01246000 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol, BR-04923062 São Paulo, SP, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol, BR-04923062 São Paulo, SP, BrazilWeb of Scienc

    Pleiotropic Effect of Hormone Insulin-Like Growth Factor-I in Immune Response and Pathogenesis in Leishmaniases

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    Leishmaniases are diseases caused by several Leishmania species, and many factors contribute to the development of the infection. Because the adaptive immune response does not fully explain the outcome of Leishmania infection and considering that the initial events are crucial in the establishment of the infection, we investigated one of the growth factors, the insulin-like growth factor-I (IGF-I), found in circulation and produced by different cells including macrophages and present in the skin where the parasite is inoculated. Here, we review the role of IGF-I in leishmaniasis experimental models and human patients. IGF-I induces the growth of different Leishmania species in vitro and alters the disease outcome increasing the parasite load and lesion size, especially in L. major- and L. amazonensis-infected mouse leishmaniasis. IGF-I affects the parasite interacting with the IGF-I receptor present on Leishmania. During Leishmania-macrophage interaction, IGF-I acts on the arginine metabolic pathway, resulting in polyamine production both in macrophages and Leishmania. IGF-I and cytokines interact with reciprocal influences on their expression. IL-4 is a hallmark of susceptibility to L. major in murine leishmaniasis, but we observed that IGF-I operates astoundingly as an effector element of the IL-4. Approaching human leishmaniasis, patients with mucosal, disseminated, and visceral diseases presented surprisingly low IGF-I serum levels, suggesting diverse effects than parasite growth. We observed that low IGF-I levels might contribute to the inflammatory response persistence and delayed lesion healing in human cutaneous leishmaniasis and the anemia development in visceral leishmaniasis. We must highlight the complexity of infection revealed depending on the Leishmania species and the parasite’s developmental stages. Because IGF-I exerts pleiotropic effects on the biology of interaction and disease pathogenesis, IGF-I turns up as an attractive tool to explore biological and pathogenic processes underlying infection development. IGF-I pleiotropic effects open further the possibility of approaching IGF-I as a therapeutical target

    Lipopolysaccharide as an Antigen Target for the Formulation of a Universal Vaccine against Escherichia coli O111 Strains â–¿

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    A promising approach to developing a vaccine against O111 strains of diarrheagenic Escherichia coli that exhibit different mechanisms of virulence is to target either the core or the polysaccharide chain (O antigen) of their lipopolysaccharide (LPS). However, due to structural variations found in both these LPS components, to use them as antigen targets for vaccination, it is necessary to formulate a vaccine able to induce a humoral immune response that can recognize all different variants found in E. coli O111 strains. In this study, it was demonstrated that, despite differences in composition of oligosaccharide repeat units between O111ab and O111ac LPS subtypes, antibodies against one O111 subtype can recognize and inhibit the adhesion to human epithelial cells of all categories of O111 E. coli (enteropathogenic E. coli [EPEC], enterohemorrhagic E. coli [EHEC], and enteroaggregative E. coli [EAEC]) strains regardless of the nature of their flagellar antigens, mechanisms of virulence, or O111 polysaccharide subtypes. These antibodies were also able to increase the clearance of different strains of O111 E. coli by macrophages. PCR analyses of the pathways involved in O111 LPS core biosynthesis showed that all EAEC strains have core type R2, whereas typical EPEC and EHEC have core type R3. In contrast, atypical EPEC strains have core types R2 and R3. In summary, the results presented herein indicate that the O111 polysaccharide and LPS core types R2 and R3 are antigen targets for panspecific immunotherapy against all categories of O111 E. coli

    Single Chain Variable Fragments Produced in Escherichia coli against Heat-Labile and Heat-Stable Toxins from Enterotoxigenic E. coli.

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    Diarrhea is a prevalent pathological condition frequently associated to the colonization of the small intestine by enterotoxigenic Escherichia coli (ETEC) strains, known to be endemic in developing countries. These strains can produce two enterotoxins associated with the manifestation of clinical symptoms that can be used to detect these pathogens. Although several detection tests have been developed, minimally equipped laboratories are still in need of simple and cost-effective methods. With the aim to contribute to the development of such diagnostic approaches, we describe here two mouse hybridoma-derived single chain fragment variable (scFv) that were produced in E. coli against enterotoxins of ETEC strains.Recombinant scFv were developed against ETEC heat-labile toxin (LT) and heat-stable toxin (ST), from previously isolated hybridoma clones. This work reports their design, construction, molecular and functional characterization against LT and ST toxins. Both antibody fragments were able to recognize the cell-interacting toxins by immunofluorescence, the purified toxins by ELISA and also LT-, ST- and LT/ST-producing ETEC strains.The developed recombinant scFvs against LT and ST constitute promising starting point for simple and cost-effective ETEC diagnosis

    Construction, expression and purification of scFv-ST.

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    <p>(A) scFv-ST amino acids sequence: V<sub>H</sub>—Heavy chain variable domain, L—Linker and V<sub>L</sub>—Light chain variable domain sequences. CDRs are highlighted in yellow for V<sub>H</sub> and in blue for V<sub>L</sub>. (B) SDS-PAGE analysis of scFv-ST recombinant antibody expression and purification. Lanes: <b>1.</b> BL21(DE3) non-induced fraction; <b>2.</b> BL21(DE3) induced fraction; <b>3.</b> Insoluble fraction; <b>4.</b> Purified scFv-ST.</p

    Reactivity of scFv-ST recombinant antibody against ST toxin.

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    <p>(A) PVDF membranes containing the bacterial lysates fractions after PAGE 10%/tricine gel was subjected to immunoblotting with antibodies. <b>1.</b> 30 μg/mL of scFv-ST. <b>2.</b> 30 μg/mL anti-ST mAb. (B) Immunofluorescence assay after ST interaction with Caco-2 cells. Panel 1. Reactivity with scFv-ST; Panel 2. Reactivity with anti-ST mAb. Panel 3. Reactivity with scFv-ST in the absence of ST toxin. Panel 4. Light microscopy. (C) scFv-ST titration with ST toxin by indirect ELISA. (D) Detection of ST in culture supernatants of ST- and LT/ST-producing ETEC isolates by indirect ELISA.</p

    Reactivity of scFv-LT recombinant antibody against LT toxin.

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    <p>(A) Nitrocellulose membrane containing the LT toxin protein after SDS-PAGE 15% were subjected to immunoblotting with antibodies. Lanes: <b>1.</b> scFv-LT 200 μg/mL, <b>2.</b> 50 μg/mL anti-LT mAb. (B) Immunofluorescence assay after LT interaction with Y-1 cells. Panel 1. Reactivity with scFv-LT; Panel 2. Reactivity with anti-LT mAb. Panel 3. Reactivity with scFv-LT in the absence of LT toxin. Panel 4. Light microscopy. (C) scFv-LT titration with LT toxin by capture ELISA. (D) Detection of LT in culture supernatants of LT- and LT/ST-producing ETEC isolates by capture ELISA.</p

    Construction, expression and purification of scFv-LT.

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    <p>(A) scFv-LT amino acids sequence: V<sub>H</sub>—Heavy chain variable domain, L—Linker and V<sub>L</sub>—Light chain variable domain sequence. CDRs are highlighted in yellow for V<sub>H</sub> and in blue for V<sub>L</sub>. (B) SDS-PAGE analysis of scFv-LT recombinant antibody expression and purification. Lanes: <b>1.</b> C43(DE3) non-induced fraction; <b>2.</b> C43(DE3) induced fraction; <b>3.</b> Insoluble fraction; <b>4.</b> Purified scFv-LT.</p
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