Recombinant PilS: Cloning, Expression and Biochemical Characterization of a Pil-Fimbriae Subunit.

Pil-fimbriae is a type IV pili member, which is a remarkably versatile component with a wide variety of functions, including motility, attachment to different surfaces, electrical conductance, DNA acquisition, and secretion of a broad range of structurally distinct protein substrates. Despite the previous functional characterization of Pil, more studies are required to understand the regulation of Pil expression and production, since the exact mechanisms involved in these steps are still unknown. Therefore it is extremely important to have a protein with the correct secondary and tertiary structure that will enable an accurate characterization and a specific antisera generation. For this reason, the aim of this work was to generate potential tools for further investigations to comprehend the mechanisms involved in Pil regulation and its role in pathogenic E. coli infections with the obtaining of a precise native-like recombinant PilS and the corresponding antisera. The pilS gene was successfully cloned into an expression vector, and recombinant PilS (rPilS) was efficiently solubilized and purified by metal affinity chromatography. Protein characterization analyses indicated that rPilS presented native-like secondary and tertiary structures after the refolding process. The generated anti-rPilS sera efficiently recognized recombinant and native proteins from atypical enteropathogenic E. coli strains.

Recombinant PilS: cloning, expression and biochemical characterization of a Pil-Fimbriae subunit

Pil-fimbriae is a type IV pili member, which is a remarkably versatile component with a wide variety of functions, including motility, attachment to different surfaces, electrical conductance, DNA acquisition, and secretion of a broad range of structurally distinct protein substrates. Despite the previous functional characterization of Pil, more studies are required to understand the regulation of Pil expression and production, since the exact mechanisms involved in these steps are still unknown. Therefore it is extremely important to have a protein with the correct secondary and tertiary structure that will enable an accurate characterization and a specific antisera generation. For this reason, the aim of this work was to generate potential tools for further investigations to comprehend the mechanisms involved in Pil regulation and its role in pathogenic E. coli infections with the obtaining of a precise native-like recombinant PilS and the corresponding antisera. The pilS gene was successfully cloned into an expression vector, and recombinant PilS (rPilS) was efficiently solubilized and purified by metal affinity chromatography. Protein characterization analyses indicated that rPilS presented native-like secondary and tertiary structures after the refolding process. The generated anti-rPilS sera efficiently recognized recombinant and native proteins from atypical enteropathogenic E. coli strains.

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

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.

[Vaginal delivery of bi-scarred uterus]. / L'accouchement par voie basse sur utérus bi-cicatriciel.

Despite awareness of obstetricians to the constant increase in the number of caesarean sections in recent years, certain dogmas concerning uterine scar still persist in our practices and influence clinical decisions. Fear of a uterine scar rupture, a major obstetric complication, is always in mind. As for bi-uterine scar, it was considered, until recently in Belgium, as a full and definitive indication against an attempted vaginal delivery. However, several previous clinical studies clearly showed that, under certain conditions, vaginal birth after two caesarean sections was usually successful with very good results in terms of maternal and fetal morbidities. Even if such a clinical situation is not common, this article aims to sensitize obstetricians to the lack of objective clinical arguments to reject a vaginal delivery in a patient having a previous history of two caesarean sections. Such a patient must be motivated and followed up within a specific framework. Moreover, this type of delivery should receive optimal monitoring.

Résumé : Malgré la sensibilisation des obstétriciens à l'augmentation constante du nombre de césariennes ces dernières années, certains dogmes concernant l'utérus cicatriciel persistent encore dans nos pratiques et influencent les décisions du praticien. La peur d'une rupture de cicatrice utérine, complication obstétricale majeure, reste toujours présente à l'esprit. Quant à l'utérus bi-cicatriciel, ce dernier était considéré, jusque il y a peu dans notre pays, comme une contre-indication totale et définitive à une tentative d'accouchement par voie basse. La littérature nous apprend pourtant que plusieurs études observationnelles ont montré que, sous certaines conditions, une tentative d'accouchement par voie basse après deux césariennes était le plus souvent couronnée de succès, avec de très bons résultats en ce qui concerne la morbidité maternelle et foetale. Même si pareille situation clinique n'est pas fréquente, le but de cet article est de sensibiliser les obstétriciens au manque d'arguments cliniques objectifs pour refuser un accouchement par voie basse chez une patiente ayant deux antécédents de césarienne, à condition que la demande soit motivée et rentre dans un cadre bien précis. Ce type d'accouchement doit bénéficier d'une surveillance optimale.

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

BACKGROUND: 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. METHODS AND FINDINGS: 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. CONCLUSION: The developed recombinant scFvs against LT and ST constitute promising starting point for simple and cost-effective ETEC diagnosis.

[Involvement of matrix metalloproteinases in obstetrics: from implantation to delivery]. / Implication des métalloprotéases matricielles en obstétrique: de la nidation à l'accouchement.

Matrix metalloproteinases, which remodel the extracellular matrix, are involved in all physiological and pathophysiological processes. In particular, they contribute to the success of a pregnancy: from embryo implantation in the endometrium to uterine cervical ripening and uterine involution. A misregulation of their expression and/or of their activity is observed in two major diseases in pregnancy such as spontaneous abortion and preeclampsia.

Different assay conditions for detecting the production and release of heat-labile and heat-stable toxins in enterotoxigenic Escherichia coli isolates.

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.

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

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.

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

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.

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

A promising approach to developing a vaccine against O111 strains of diarrheagenic Escherichia coli thatexhibit 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 humoralimmune 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 havecore type R3. In contrast, atypical EPEC strains have core types R2 and R3. In summary, the results presentedherein 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.

Lipopolysaccharide as an antigen target for the formulation of a universal vaccine against Escherichia coli O111 strains.

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.

Effects of perilipin (PLIN) gene variation on metabolic syndrome risk and weight loss in obese children and adolescents.

CONTEXT: Genetic polymorphisms at the perilipin (PLIN) locus have been investigated for their potential utility as markers for obesity and metabolic syndrome (MS). We examined in obese children and adolescents (OCA) aged 7-14 yr the association of single-nucleotide polymorphisms (SNP) at the PLIN locus with anthropometric, metabolic traits, and weight loss after 20-wk multidisciplinary behavioral and nutritional treatment without medication. DESIGN: A total of 234 OCA [body mass index (BMI = 30.4 +/- 4.4 kg/m(2); BMI Z-score = 2.31 +/- 0.4) were evaluated at baseline and after intervention. We genotyped four SNPs (PLIN1 6209T-->C, PLIN4 11482G-->A, PLIN5 13041A-->G, and PLIN6 14995A-->T). RESULTS: Allele frequencies were similar to other populations, PLIN1 and PLIN4 were in linkage disequilibrium (D' = 0.999; P < 0.001). At baseline, no anthropometric differences were observed, but minor allele A at PLIN4 was associated with higher triglycerides (111 +/- 49 vs. 94 +/- 42 mg/dl; P = 0.003), lower high-density lipoprotein cholesterol (40 +/- 9 vs. 44 +/- 10 mg/dl; P = 0.003) and higher homeostasis model assessment for insulin resistance (4.0 +/- 2.3 vs. 3.5 +/- 2.1; P = 0.015). Minor allele A at PLIN4 was associated with MS risk (age and sex adjusted) hazard ratio 2.4 (95% confidence interval = 1.1-4.9) for genotype GA and 3.5 (95% confidence interval = 1.2-9.9) for AA. After intervention, subjects carrying minor allele T at PLIN6 had increased weight loss (3.3 +/- 3.7 vs. 1.9 +/- 3.4 kg; P = 0.002) and increased loss of the BMI Z-score (0.23 +/- 0.18 vs. 0.18 +/- 0.15; P = 0.003). Due to group size, risk of by-chance findings cannot be excluded. CONCLUSION: The minor A allele at PLIN4 was associated with higher risk of MS at baseline, whereas the PLIN6 SNP was associated with better weight loss, suggesting that these polymorphisms may predict outcome strategies based on multidisciplinary treatment for OCA.

Binge eating symptoms, diet composition and metabolic characteristics of obese children and adolescents.

This study aimed to determine the occurrence of symptoms of binge eating (BE) among children and adolescents seeking treatment for their obesity, as well as to evaluate their diet composition and metabolic characteristics. The Binge Eating Scale (BES) was answered by 128 children and adolescents (10.77+/-2.04 years, BMI 29.15+/-4.98 kg/m2, BMI Z score 2.28+/-0.46, 53.91% pubescent), who were classified into two subgroups--binge eaters (score greater than or equal to 18 points) and non-binge eaters (score lower than 18 points). Anthropometric data, body composition and Tanner stages were collected and dietary evaluation conducted. Blood pressure was determined, and glucose, lipid profile and insulin assays were performed. Insulin resistance was determined using HOMA-IR. BE symptoms were present in 39.06% of patients. Carbohydrate intake in diet composition was significantly higher among binge eaters. Children with BE did not demonstrate significant dissimilar metabolic characteristics when compared to their counterparts without BE. Therefore, BE seems to be a prevalent problem among children and adolescents seeking help for their obesity. When associated with obesity, this eating behaviour can influence macronutrient consumption through increased carbohydrate intake. Further research would be valuable to verify the reproducibility of these findings.

Galactose alpha 1-3 galactose and anti-alpha galactose antibody in normal and pathological pregnancies.

The galactose alpha 1-3 galactose (Gal alpha 1-3 Gal) residue is a carbohydrate widely distributed in many non-human mammals. Since Gal alpha 1-3 Gal residues are described on the cell surface of tumor cells, we have examined the possibility of their expression on human trophoblastic cells at different stages of placental implantation and in various pregnancy-associated conditions. Using immunohistochemical methods, Gal alpha 1-3 Gal was demonstrated on interstitial and vascular trophoblast during pregnancy. For villous trophoblast, the staining disappeared in second trimester pregnancies. The density of staining for Gal alpha 1-3 Gal was increased in highly invasive trophoblast (mole and choriocarcinoma) and decreased in poorly invasive specimens (spontaneous abortion, XO monosomia). No cells displaying Gal alpha 1-3 Gal at their surface were identified in some segments of spiral arteries from pre-eclamptic women. The anti-Gal antibody titer increased in the first trimester of pregnancy and in the sera of pre-eclamptic and eclamptic patients. These findings suggest that Gal alpha 1-3 Gal residues could be considered as markers for trophoblast invasive capacity and that the binding of maternal anti-Gal antibodies to the trophoblast could contribute to limit trophoblastic invasion and thus participate to the immunological control of implantation.

Reconstituted basement membrane matrix stimulates interstitial procollagenase synthesis by human fibroblasts in culture.

Synthesis of interstitial collagenase by human fibroblasts was compared when cultured on plastic, in the presence or absence of soluble laminin, on a type I collagen gel and on a gel of basement membrane components (matrigel). Fibroblasts cultured on matrigel or on type I collagen gel displayed an increase in the steady-state levels of mRNA for interstitial procollagenase that was proportional to its enzymatic activity. Laminin, the main component of matrigel, had no effect on the interstitial collagenase synthesis by fibroblasts. We suggest that matrigel, which stimulates the interstitial collagenase production at a transcriptional step, could regulate the catabolic potential of fibroblasts.

Type IV and interstitial collagenolytic activities in normal and malignant trophoblast cells are specifically regulated by the extracellular matrix.

Type IV and interstitial collagenolytic activities were compared in human malignant and normal trophoblast cells cultured on plastic, in presence or absence of laminin in solution, on matrigel (a gel of basement membrane components) and on type I collagen gel. Laminin highly stimulated the type IV collagenolytic activity but not the interstitial collagenolytic activity, in malignant trophoblast cells. This glycoprotein had no effect on the interstitial collagenolytic activity and doubled the type IV collagenolytic activity in normal trophoblast cells. Thus malignant trophoblast cells produce preferentially the enzyme able to degrade basement membrane when in contact with laminin, and the enzyme able to degrade interstitial collagen fibers when cultured on type I collagen. On the contrary, type I collagen gel and matrigel equally increased both type IV and interstitial collagenolytic activities by normal trophoblast cells. Interactions of tumor trophoblast or normal trophoblast cells with the extracellular matrix result thus in distinct stimulations of collagenolytic activities. Increased production of type IV collagenolytic activity upon exposure to laminin appears to be specific of the metastatic phenotype.

Diagnóstico endoscópico da hemorragia digestiva alta: estudo retrospectivo em hospital universitario / Endoscopia diagnosis of upper gastrointestinal bleeding: retrospective study in university Hospital

Hemorragia digestiva alta (HDA)constitui emergência médica freqüente. Apesar das numerosas e variadas publicaçöes similares, foram estudadas as peculiaridade do problema no nosso meio. Desde janeiro de 1982 até junho de 1987 foram atendidas no HC-UNICAMP, 1095 pacientes portadores de HDA. Todos foram submetidos a esofagogastroduodenoscopia até 24 horas após admissäo. Representaram 12% do total de 9.100 exames realizados no Serviço de Endoscopia Digestiva no mesmo período, sendo 772 (70,5%) do sexo masculino. A faixa etária predominante foi dos 20 aos 30 anos (20%)

Laminin and type III procollagen peptide in human preovulatory follicular fluid.

The levels of laminin P1 fragment, a marker of basement membrane, and of the aminoterminal sequence of type III procollagen, a marker of interstitial connective tissue, were measured in human preovulatory follicular fluids. The concentrations of these peptides correlated with progesterone levels but not with those of estradiol or testosterone. Immunocytochemical studies confirmed the remodeling of the perifollicular basement membrane and interstitial matrix during oocyte maturation. The studies suggest that monitoring of the ovarian connective tissue macromolecules could be useful for estimating follicular maturation.

Analysis of tridimensional mixed cultures of mouse B16 melanoma cells and 3T3 fibroblasts.

The interactions between invasive malignant cells and normal fibroblastic cells were studied in a cellular spheroid model in vitro. Murine B16 melanoma cells (previously cultured in monolayer for a short period) and 3T3 mouse fibroblasts (greater than or equal to 130 passages in monolayers) were cultured under tridimensional conditions (pure or mixed spheroids). As compared to pure 3T3 or mixed spheroids, B16 spheroids were smaller and characterized by a higher proliferation rate, a lower degree of necrosis, and a less abundant extracellular matrix. Disintegration was observed in some pure 3T3 or mixed spheroids. Melanogenesis progressively increased inside B16 or mixed spheroids. By immunohistochemical methods and electron microscopy, laminin, fibronectin and collagen I, III and IV in extracellular matrix were studied in the three types of spheroids.