Aquaporins can be involved in the swelling caused by shiga toxin type 2 on hgec and hk-2 cells

Hemolytic uremic syndrome related to Shiga toxin?producing Escherichiacoli (STEC-HUS) is the principal etiology of acute kidney injuryin children in Argentina.Previously, we demonstrated that Shiga toxin type 2 (Stx2) damageshuman glomerular endothelial cells (HGEC) and HK-2 human proximaltubular epithelial cell line by inducing swelling and detachment.In this work, we analyzed cell volume changes of HGEC and HK-2exposed or not to Stx2 or a hypoosmotic (HYPO) medium, in thepresence or not of aquaporins (AQPs) inhibitors (mercuric chloride:HgCl2 and tetraethylammonium: TEA), or an inhibitor of Stx2 receptor(Gb3) synthesis, Eliglustat (EG). For controls, an isosmotic (ISO)medium was used.Cells were grown on 12 well plates and pretreated for 30 minuteswith HgCl2 (10 μM) or TEA (100 μM) or pretreated during 24 h withEG (10 μM). Then, HGEC and HK-2 were incubated with Stx2 (50μM) for an additional 40 minutes. Cell volume was analyzed by lightmicroscopy and measuring cell area by using Image J software.After Stx2 and HYPO medium treatments, a significant increase inthe cell volume of HGEC (Stx2: 42%; HYPO: 36%, n= 3, p<0.05) andHK-2 (Stx2: 70%; HYPO: 55%, n= 3, p<0.05) was detected respectto ISO medium. However, when HGEC and HK-2 were pretreatedwith HgCl2 or TEA a significant swelling prevention was obtained forHGEC (Stx2+HgCl2:100%; Stx2+TEA:86%; HYPO+HgCl2: 42.5%;HYPO+TEA: 83%, n=3, p<0.05) and HK-2 (Stx2+HgCl2: 90%; Stx-2+TEA: 85%; HYPO+HgCl2: 55%; HYPO+TEA: 75%, n=3, p<0.05).In addition, EG also was able to prevent HK-2 swelling in 87 % (n=1)with respect to Stx2 treatment.Results show that AQPs may be involved in the water movement insideHGEC and HK-2 induced by Stx2, since HgCl2 and TEA avoidedthis effect. Furthermore, binding of Stx2 to Gb3 could be the initialstep for the development of cellular mechanisms that possibly triggerthe entry of solutes into the cells and the consequent osmoticgradient responsible for the hypotonic effect.Fil: Gomez, Fernando Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Repetti, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Romero, Romina. Hospital Nacional Profesor Alejandro Posadas.; ArgentinaFil: Sacerdoti, Flavia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Ibarra, Cristina Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Amaral, María Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaLXVI Reunión Anual de la Sociedad Argentina de Investigación Clínica: LXIX Reunión Anual de la Sociedad Argentina de Inmunología; LIII Reunión Anual de la Asociación Argentina de Farmacología Experimental y XI Reunión Anual de la Asociación Argentina de NanomedicinasArgentinaSociedad Argentina de Investigación ClínicaSociedad Argentina de InmunologíaAsociación Argentina de Farmacología ExperimentalAsociación Argentina de Nanomedicina

Therapeutic Antibodies Against Shiga Toxins: Trends and Perspectives

Shiga toxins (Stx) are AB5-type toxins, composed of five B subunits which bind to Gb3 host cell receptors and an active A subunit, whose action on the ribosome leads to protein synthesis suppression. The two Stx types (Stx1 and Stx2) and their subtypes can be produced by Shiga toxin-producing Escherichia coli strains and some Shigella spp. These bacteria colonize the colon and induce diarrhea that may progress to hemorrhagic colitis and in the most severe cases, to hemolytic uremic syndrome, which could lead to death. Since the use of antibiotics in these infections is a topic of great controversy, the treatment remains supportive and there are no specific therapies to ameliorate the course. Therefore, there is an open window for Stx neutralization employing antibodies, which are versatile molecules. Indeed, polyclonal, monoclonal, and recombinant antibodies have been raised and tested in vitro and in vivo assays, showing differences in their neutralizing ability against deleterious effects of Stx. These molecules are in different phases of development for which we decide to present herein an updated report of these antibody molecules, their source, advantages, and disadvantages of the promising ones, as well as the challenges faced until reaching their applicability.Fil: de Macedo Henrique, Izabella. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Sacerdoti, Flavia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Lozzardo Ferreira, Raissa. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Henrique, Camila. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Amaral, María Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Fontes Piazza, Roxane María. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Luz, Daniela. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; Brasi

Detection of serum anti-lipopolysaccharides (lps) antibodies from enterohemorrhagic e. coli (ehec) in asymptomatic kindergarten teachers of buenos aires province

EHEC is responsible for developing typical Hemolytic Uremic Syndrome (HUS) and Argentina has the highest incidence in the world. The endemo-epidemic behavior that HUS presents in our countrywas associated with person-to-person transmission. Since children below 5 years old are the most affected, our objective was to determine the frequency of kindergarten teachers with anti-LPS-EHECantibodies in serum. One hundred and fifty teachers from J.C. Paz district from Buenos Aires Province participated in an informative talk given by LuSUH, a non-governmental association, about good practices to reduce HUS transmission. Then, 63 of them voluntarily gave, under a signedwritten informed consent, a sample of blood obtained by finger puncture. A commercial ELISA (Chemtest, Arg) was used to detect specific IgM and IgG isotypes against 4 types of LPS-EHEC: O157, O145, O121 and O103. These variants represent approximately 87% of the EHEC serotypes associated with HUS in Argentina. The IgM finding was considered as indirect evidence of current infection with the EHEC specific serotype. Forty-two samples were evaluated, 60% (25/42) of them were positive for at least one type of LPS. Fifty-six percent of them (14/25) were IgM positive. Fifty percent of the positive samples were reactive for LPS O157, 26% for LPS O121, 12% for LPS O145 and 2% for LPS O103. Among the samples positive for LPS O157, 5% belonged to IgM isotype, 31% to IgG and 14% had both. The IgG isotype was the unique detected for LPS O145 and O103 (12 and2%, respectively). For LPS O121, the IgM isotype was detected in 2%, IgG in 12% and both isotypes in 12% of the samples. The elevated frequency of LPS-EHEC reactive samples (60%) reflects the high circulation of EHEC strains in our country. This agrees with the prevalence of anti-Stx2 antibodies reported in our population previously. Very strikingly, nearly half of these individuals carry IgM, suggesting that they would be in the active phase of infection.Fil: Fernández Brando, Romina Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Sacerdoti, Flavia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Amaral, María Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Bernal, Alan Mauro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Da Rocha, Marcelo. No especifíca;Fil: Belardo, Marcela. Universidad Nacional de Jose Clemente Paz; ArgentinaFil: Palermo, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Ibarra, Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaLXV Reunión anual de la Sociedad Argentina de Investigación Clínica; LXVIII Reunión anual de la Sociedad Argentina de Inmunología y Reunión anual de la Sociedad Argentina de FisiologíaArgentinaSociedad Argentina de Investigación ClínicaSociedad Argentina de InmunologíaSociedad Argentina De Fisiologí

Development and characterization of soluplus® nanomicelles associated to specific igg as an innovative strategy for the detection and neutralization of shiga toxin type 2

Shiga toxin type 2 (Stx2) is the main virulence factor of Shiga toxinproducing Escherichia coli and is responsible for triggering HemolyticUremic Syndrome (HUS). We aimed to develop and characterizepolymeric nanomicelles (PN) with the amphiphilic polymerSoluplus® coupled to anti-Stx2 IgG in order to introduce innovativeproposals for the detection of Stx2 and treatment of HUS. PN ofSoluplus® were formulated in PBS and coupled with IgG anti Stx2from hyperimmune (PN-IgG-Stx2) or control bovine colostrum (PNIgG-Ctrl). The hydrodynamic size of PN, PN-IgG-Stx2 and PN-IgGCtrlwas evaluated by Dynamic Light Scattering. Morphology of PNor PN-IgG-Stx2 was analyzed by Transmission Electron Microscopy(TEM). The PN toxicity was evaluated on both Vero and Human GlomerularEndothelial cells (HGEC) and cell viability was determinedby neutral red uptake. After coupling PN with IgG, Stx2 neutralizationcapacity of PN-IgG-Stx2 or PN-IgG-Ctrl was evaluated on Vero andHGEC cells and the percentage of cell viability was analyzed. Thehydrodynamic size of the PN of Soluplus® and IgG-Stx2 showed anaverage diameter of 70.2 ± 1.5 nm and 40.9 ± 2 nm, respectively.When both components were coupled, a single peak with a similarhydrodynamic size of the PN was observed (70.4 ± 0.2 nm). TEManalysis revealed circular particles with a diameter corresponding to100 nm either in PN and PN-IgG-Stx2 particles. PN-IgG-Stx2 wereable to neutralize Stx2 on Vero and HGEC cells in a dose dependentmanner. When comparing the neutralization capacity of Stx2 by IgGStx2vs PN-IgG-Stx2 a significant improvement in the cell viabilityof Vero and HGEC was observed with the PN-IgG-Stx2 (p<0.001).The association between anti-Stx2 IgG from bovine colostrum andSoluplus® PN was optimized and characterized. Encouraging resultsof antibody functionality coupled with PN were registered.These results may open the perspective of the design of new nanoplatformsfor neutralization and/or detection of Stx2.Fil: Girón Reyes, Claudio Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Gomez, Fernando Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Amaral, María Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Chiappetta, Diego Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Tecnología Farmacéutica; ArgentinaFil: Moretton, Marcela Analía. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Tecnología Farmacéutica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ibarra, Cristina Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Sacerdoti, Flavia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaLXVI Reunión Anual de la Sociedad Argentina de Investigación Clínica; LXIX Reunión Anual de la Sociedad Argentina de Inmunología; LIII Reunión Anual de la Asociación Argentina de Farmacología Experimental y XI Reunión Anual de la Asociación Argentina de NanomedicinasArgentinaSociedad Argentina de Investigación ClínicaSociedad Argentina de InmunologíaAsociación Argentina de Farmacología ExperimentalAsociación Argentina de Nanomedicina

Combined action of shiga toxin type 2 and subtilase cytotoxin in the pathogenesis of hemolytic uremic syndrome

Shiga toxin-producing E. coli (STEC) produces Stx1 and/or Stx2, and Subtilase cytotoxin (SubAB). Since these toxins may be present simultaneously during STEC infections, the purpose of this work was to study the co-action of Stx2 and SubAB. Stx2 + SubAB was assayed in vitro on monocultures and cocultures of human glomerular endothelial cells (HGEC) with a human proximal tubular epithelial cell line (HK-2) and in vivo in mice after weaning. The effects in vitro of both toxins, co-incubated and individually, were similar, showing that Stx2 and SubAB contribute similarly to renal cell damage. However, in vivo, co-injection of toxins lethal doses reduced the survival time of mice by 24 h and mice also suffered a strong decrease in the body weight associated with a lowered food intake. Co-injected mice also exhibited more severe histological renal alterations and a worsening in renal function that was not as evident in mice treated with each toxin separately. Furthermore, co-treatment induced numerous erythrocyte morphological alterations and an increase of free hemoglobin. This work shows, for the first time, the in vivo effects of Stx2 and SubAB acting together and provides valuable information about their contribution to the damage caused in STEC infections.Fil: Alvarez, Romina Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Gómez, Fernando Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Zotta, Elsa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Paton, Adrienne W.. University of Adelaide; AustraliaFil: Paton, James C.. University of Adelaide; AustraliaFil: Ibarra, Cristina Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Sacerdoti, Flavia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Amaral, María Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentin

Crosstalk between human microvascular endothelial cells and tubular epithelial cells modulates pro-inflammatory responses induced by Shiga toxin type 2 and subtilase cytotoxin

Hemolytic uremic syndrome (HUS) is a consequence of Shiga toxin (Stx)-producingEscherichia coli (STEC) infection and is the most frequent cause of acute renal failure (ARF) in children. Subtilase cytotoxin (SubAB) has also been associated with HUS pathogenesis. We previously reported that Stx2 and SubAB cause different effects on co-cultures of human renalmicrovascular endothelial cells (HGEC) and human proximal tubular epithelial cells (HK-2) relative to HGEC and HK-2 monocultures. In this work we have analyzed the secretion of pro-inflammatory cytokines by co-cultures compared to monocultures exposed or not to Stx2, SubAB, and Stx2+SubAB. Under basal conditions, IL-6, IL-8 and TNF-α secretion was different between monocultures and co-cultures. After toxin treatments, high concentrations of Stx2 and SubAB decreased cytokine secretion by HGEC monocultures, but in contrast, low toxin concentrations increased their release. Toxins did not modulate the cytokine secretion by HK-2 monocultures, but increased their release in the HK-2 co-culture compartment. In addition, HK-2 monocultures were stimulated to release IL-8 after incubation with HGEC conditioned media. Finally, Stx2 and SubAB were detected in HGEC and HK-2 cells from the co-cultures. This work describes, for the first time, the inflammatory responses induced by Stx2 and SubAB, in a crosstalk model of renal endothelial and epithelial cells.Fil: Alvarez, Romina Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Jancic, Carolina Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Garimano, Nicolás Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Sacerdoti, Flavia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Paton, Adrienne W.. University of Adelaide; AustraliaFil: Paton, James C.. University of Adelaide; AustraliaFil: Ibarra, Cristina Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Amaral, María Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentin

Human recombinant fab fragment neutralizes shiga toxin type 2 cytotoxic effects in vitro and in vivo

Shiga toxin (Stx) producing Escherichia coli (STEC) is responsible for causing hemolytic uremic syndrome (HUS), a life-threatening thrombotic microangiopathy characterized by thrombocytopenia, hemolytic anemia, and acute renal failure after bacterially induced hemorrhagic diarrhea. Until now, there has been neither an effective treatment nor method of prevention for the deleterious effects caused by Stx intoxication. Antibodies are well recognized as affinity components of therapeutic drugs; thus, a previously obtained recombinant human FabC11:Stx2 fragment was used to neutralize Stx2 in vitro in a Vero cell viability assay. Herein, we demonstrated that this fragment neutralized, in a dose-dependent manner, the cytotoxic effects of Stx2 on human glomerular endothelial cells, on human proximal tubular epithelial cells, and prevented the morphological alterations induced by Stx2. FabC11:Stx2 protected mice from a lethal dose of Stx2 by toxin-antibody pre-incubation. Altogether, our results show the ability of a new encouraging molecule to prevent Stx-intoxication symptoms during STEC infection.Fil: Luz, Daniela. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Amaral, María Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Sacerdoti, Flavia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Bernal, Alan Mauro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Quintilio, Wagner. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Moro, Ana Maria. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Palermo, Marina Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Ibarra, Cristina Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Piazza, Roxane Maria Fontes. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; Brasi

Detection of Shiga toxin producing Escherichia coli in endocervix of asymptomatic pregnant women: Novel pathogen responsible for adverse pregnancy outcomes?

Brief Introduction: Some studies have demonstrated that vaginal E. coli colonization may produce complicationsduring pregnancy. We have previously reported that Shiga toxin-2 (Stx2) secreted by enterohemorrhagic E. coli canproduce abortion and premature delivery in animals and can impair human trophoblast in vitro.Objective: The aim of this study was to detect virulence factor genes from Shiga toxin producing E. coli (STEC)in the endocervix of asymptomatic pregnant women. Methods: Endocervical swabs were collected from 103 pregnantwomen (12 to 30 weeks of pregnancy) during their antenatal examination. Swab samples were enriched in TrypticSoy Broth overnight at 37 °C and then streaked into selective and differential medium, Sorbitol MacConkey agar. E.coli detection was confirmed by identification of uidA gene by PCR assay. Then, positive samples for E. coli wereanalyzed for STEC virulence factors genes: stx1, stx2, eae, rfbO157, lpfAO113 and hcpA. After that, positive E. colisamples for stx2 gene were grown in Luria-Bertani Broth medium in order to evaluate Stx2 cytotoxic activity. Forthat, the bacterial supernatants were filter-sterilized and cytotoxicity was evaluated on Vero, Swan 71 and HeLa cells.Results: Our results showed that 14.5% of the endocervical samples were positive for E. coli (positive for uidAgene). Furthermore, 9/15 (60%) of the E. coli-positive samples carried the stx2 gene and 6/15 (40%) carried thelpfAO113 and hcpA genes. One bacterial supernatant from an E. coli-positive endocervical sample carrying stx2 andlpfAO113 genes exhibited high cytotoxic activity on the cells evaluated due to Stx2 production confirmed by specificneutralization of Stx2 with an anti-Stx2 antibody.Conclusions: The novelty of this report is the presence of STEC in the endocervix of asymptomatic pregnantwomen. This opens a new perspective with respect to the possible role of this pathogen in woman reproductivehealth care.Fil: Scalise, Maria Lujan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Garimano, Nicolás Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Porporato, Melina Mabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Leonino, Patricia. Hospital Nacional Profesor Alejandro Posadas.; ArgentinaFil: Pereyra, Adriana. Hospital Nacional Profesor Alejandro Posadas.; ArgentinaFil: Ferreiros, José A.. Hospital Nacional Profesor Alejandro Posadas.; ArgentinaFil: Casale, Roberto. Hospital Nacional Profesor Alejandro Posadas.; ArgentinaFil: Amaral, María Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Ibarra, Cristina Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Sacerdoti, Flavia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaInternational Symposium on Reproductive HealthVirtual MeetingSingapurThe Physicologycal SocietyBiochemical Societ

Detection of Shiga Toxin-Producing Escherichia coli (STEC) in the Endocervix of Asymptomatic Pregnant Women. Can STEC Be a Risk Factor for Adverse Pregnancy Outcomes?

The presence of Escherichia coli in the vaginal microbiome has been associated with pregnancy complications. In previous works, we demonstrated that Shiga toxin-producing Escherichia coli (STEC) can produce abortion and premature delivery in rats and that Shiga toxin type 2 (Stx2) can impair human trophoblast cell lines. The hypothesis of this work was that STEC may colonize the lower female reproductive tract and be responsible for adverse pregnancy outcomes. Thus, the aim of this work was to evaluate the presence and prevalence of virulence factor genes from STEC in the endocervix of asymptomatic pregnant women. For that purpose, endocervical swabs were collected from pregnant women during their prenatal examination. Swab samples were enriched in a differential medium to select Enterobacteria. Then, positive samples were analyzed by PCR to detect genes characteristic of Escherichia sp. (such as uidA and yaiO), genes specific for portions of the rfb (O-antigen-encoding) regions of STEC O157 (rfbO157), and STEC virulence factor genes (such as stx1, stx2, eae, lpfAO113, hcpA, iha, sab, subAB). The cytotoxic effects of stx2-positive supernatants from E. coli recovered from the endocervix were evaluated in Vero cells. Our results showed that 11.7% of the endocervical samples were positive for E. coli. Additionally, we found samples positive for stx2 and other virulence factors for STEC. The bacterial supernatant from an isolate identified as E. coli O113:NT, carrying the stx2 gene, exhibited cytotoxic activity in Vero, Swan 71 and Hela cells. Our results open a new perspective regarding the presence of STEC during pregnancy.Fil: Scalise, Maria Lujan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Garimano, Nicolás Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Sanz, Marcelo Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Padola, Nora Lía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Leonino, Patricia. Hospital Nacional Profesor Alejandro Posadas.; ArgentinaFil: Pereyra, Adriana. Hospital Nacional Profesor Alejandro Posadas.; ArgentinaFil: Casale, Roberto. Hospital Nacional Profesor Alejandro Posadas.; ArgentinaFil: Amaral, María Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina. Hospital Nacional Profesor Alejandro Posadas.; ArgentinaFil: Sacerdoti, Flavia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Ibarra, Cristina Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentin

The Deleterious Effects of Shiga Toxin Type 2 Are Neutralized In Vitro by FabF8:Stx2 Recombinant Monoclonal Antibody

Hemolytic Uremic Syndrome (HUS) associated with Shiga-toxigenic Escherichia coli (STEC) infections is the principal cause of acute renal injury in pediatric age groups. Shiga toxin type 2 (Stx2) has in vitro cytotoxic effects on kidney cells, including human glomerular endothelial (HGEC) and Vero cells. Neither a licensed vaccine nor effective therapy for HUS is available for humans. Recombinant antibodies against Stx2, produced in bacteria, appeared as the utmost tool to prevent HUS. Therefore, in this work, a recombinant FabF8:Stx2 was selected from a human Fab antibody library by phage display, characterized, and analyzed for its ability to neutralize the Stx activity from different STEC-Stx2 and Stx1/Stx2 producing strains in a gold standard Vero cell assay, and the Stx2 cytotoxic effects on primary cultures of HGEC. This recombinant Fab showed a dissociation constant of 13.8 nM and a half maximum effective concentration (EC50 ) of 160 ng/mL to Stx2. Additionally, FabF8:Stx2 neutralized, in different percentages, the cytotoxic effects of Stx2 and Stx1/2 from different STEC strains on Vero cells. Moreover, it significantly prevented the deleterious effects of Stx2 in a dose-dependent manner (up to 83%) in HGEC and protected this cell up to 90% from apoptosis and necrosis. Therefore, this novel and simple anti-Stx2 biomolecule will allow further investigation as a new therapeutic option that could improve STEC and HUS patient outcomes.Fil: Luz, Daniela. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Gomez, Fernando Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Ferreira, Raissa L.. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Melo, Bruna S.. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Guth, Beatriz E. C.. Universidade de Sao Paulo; BrasilFil: Quintilio, Wagner. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Moro, Ana Maria. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Presta, Agostina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Sacerdoti, Flavia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Ibarra, Cristina Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Chen, Gang. University of Toronto; CanadáFil: Sidhu, Sachdev S.. University of Toronto; CanadáFil: Amaral, María Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Fontes Piazza, Roxane María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentin