RESUMO
Membrane expression of fractalkine (CX3CL1)-receptor (CX3CR1) is relevant in monocytes (Mo) because CX3CR1-CX3CL1 interactions might participate on both, homeostatic and pathologic conditions. We have previously demonstrated that CX3CR1 levels are decreased during culture and when Mo are differentiated into dendritic cells, but enhanced when differentiated into macrophages. Regarding soluble factors, lipopolysaccharide (LPS) accelerated the loss of CX3CR1, while interleukin (IL)-10 and Interferon-gamma (IFN-γ) prevented it. However, the comprehensive knowledge about the intracellular pathways that underlay the level of CX3CR1 expression in Mo is still incomplete. In the current work, we studied the effect of anti-inflammatory cytokines (IL-4, IL-13, IL-10), alone or together with IFN- γ on CX3CR1 expression. We found that only IL-10 and IFN-γ separately were able to prevent CX3CR1 down-modulation during culture of human Mo. Besides, Mo incubated with IL-10 plus IFN-γ showed the highest CX3CR1 expression by cell, suggesting cooperation between two different mechanism used by both cytokines. By studying intracellular mechanisms triggered by IL-10 and IFN-γ, we demonstrated that they specifically induced PI3K-dependent serine-phosphorylation of signal transducer and activator of transcription (STAT)3 or STAT1, respectively. Moreover, chemical inhibitors of STAT1 or STAT3 abrogated IFN-γ or IL-10 effects on CX3CR1 expression. Strikingly, only IL-10 increased CX3CR1 mRNA level, as consequence of augmenting mRNA stability. CX3CR1 mRNA increase was PI3K-dependent, supporting the causal link between the action of IL-10 at the CX3CR1 transcript and CX3CR1 protein level on Mo. Thus, both cytokines up-regulate CX3CR1 expression on human Mo by different intracellular mechanisms.
Assuntos
Receptor 1 de Quimiocina CX3C/metabolismo , Interferon gama/metabolismo , Interleucina-10/metabolismo , Monócitos/metabolismo , Regulação para Cima , Receptor 1 de Quimiocina CX3C/genética , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição STAT/metabolismo , Serina/metabolismoRESUMO
Shiga toxin (Stx), produced by Escherichia coli, is the main pathogenic factor of diarrhea-associated hemolytic uremic syndrome (HUS), which is characterized by the obstruction of renal microvasculature by platelet-fibrin thrombi. It is well known that the oxidative imbalance generated by Stx induces platelet activation, contributing to thrombus formation. Moreover, activated platelets release soluble CD40 ligand (sCD40L), which in turn contributes to oxidative imbalance, triggering the release of reactive oxidative species (ROS) on various cellular types. The aim of this work was to determine if the interaction between the oxidative response and platelet-derived sCD40L, as consequence of Stx-induced endothelium damage, participates in the pathogenic mechanism during HUS. Activated human glomerular endothelial cells (HGEC) by Stx2 induced platelets to adhere to them. Although platelet adhesion did not contribute to endothelial damage, high levels of sCD40L were released to the medium. The release of sCD40L by activated platelets was inhibited by antioxidant treatment. Furthermore, we found increased levels of sCD40L in plasma from HUS patients, which were also able to trigger the respiratory burst in monocytes in a sCD40L-dependent manner. Thus, we concluded that platelet-derived sCD40L and the oxidative response are reciprocally stimulated during Stx2-associated HUS. This process may contribute to the evolution of glomerular occlusion and the microangiopathic lesions.
Assuntos
Ligante de CD40/sangue , Células Endoteliais/efeitos dos fármacos , Síndrome Hemolítico-Urêmica/sangue , Toxina Shiga/toxicidade , Células Cultivadas , Criança , Pré-Escolar , Células Endoteliais/patologia , Feminino , Síndrome Hemolítico-Urêmica/induzido quimicamente , Humanos , Lactente , Rim/metabolismo , Rim/patologia , Masculino , Microvasos , Monócitos/metabolismo , Estresse Oxidativo , Ativação Plaquetária/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Objetivo: comparar los resultados motores y sensoriales de los pacientes con esotropía residual tratados con toxina botulínica A con el grupo que recibe cirugía convencional. Métodos: se realizó un estudio experimental (analítico), longitudinal y prospectivo en 27 pacientes que cumplieron con los criterios de inclusión: esotropía residual de 20 a 30 dioptrías independientemente de la edad. Se analizaron las siguientes variables: edad, sexo, diagnóstico previo, tipo de cirugía previa, número de inyección de toxina botulínica A empleada o cirugía realizada en la reintervención, efectos secundarios, complicaciones, ángulo de desviación, fusión y estereopsis preoperatorio y posoperatorio al año. Se consideró éxito quirúrgico la desviación postratamiento igual o menor de 10 dioptrías prismáticas. Resultados: el ángulo de desviación preoperatorio promedio del grupo toxina botulínica A fue de 24,0 ± 1,2 dioptrías y el de cirugía convencional de 25,8 ± 1,1, en oposición con el ángulo de desviación posoperatorio promedio, donde hallamos diferencias significativas (p= 0,003) entre ambos grupos (10,3 ± 1,1 versus 6,0 ± 0,80). El 64,3 por ciento, del grupo de toxina botulínica A y el 92,3 por ciento de cirugía convencional obtuvieron éxito quirúrgico. No se encontraron diferencias significativas ( p= 0,165). Se encontró ptosis palpebral como efecto secundario en el grupo toxina botulínica A y ninguna complicación en ningún grupo. El 44,4 por ciento de los pacientes obtuvo fusión postratamiento pero ninguno alcanzó estereopsis(AU) Conclusiones: en las esotropías residuales de 20 a 30 dioptrías prismáticas tratadas con toxina botulínica se obtienen resultados motores y sensoriales similares al del grupo de cirugía convencional
Objective: to compare the motor and sensory results of patients with residual esotropia, who were treated with botulinum toxin A, and of a group undergoing conventional surgery. Methods: a prospective, longitudinal and experimental (analytical) study was conducted in 27 patients who fulfilled the inclusion criteria: residual esotropia from 20 to 30 dioptries independently of the age. The following variables were analyzed: age, sex, previous diagnosis, type of previous surgery, number of injection of botulinum toxin A applied or type of surgery performed in the reintervención, secondary effects, complications, deviation angle, coalition and preoperative and postoperative estereopsis to the year. It was considered surgical success the deviation same post-treatment or smaller than 10 prismatic dioptries. Results: the angle of preoperative deviation average of the group toxin botulínica A was of 24,0 ± 1,2 dioptres and the one of conventional surgery of 25,8 ± 1,1, in opposition with the angle of posoperative deviation average, where find significant differences ( p= 0,003) between both groups (10,3 ± 1,1 versus 6,0 ± 0,80). The 64,3 percent of the group of toxin botulínica A and the 92,3 percent of conventional surgery obtained surgical success. Significant differences were not find (p= 0,165). Palpebral ptosis was found as secondary effect in the group toxin botulínica A, and any complication in any group. The 44,4 percent of the patients obtained post-treatment fusion but any reached to estereopsis(AU) Conclusions: the residual esotropias of 20 to 30 prismatic dioptres treated with toxin botulínica shows resulted engines and sensory similar to the group of conventional surgery
Assuntos
Humanos , Masculino , Feminino , Criança , Adolescente , Adulto , Blefaroptose , Toxinas Botulínicas Tipo A/uso terapêutico , Esotropia/cirurgia , Estudos Longitudinais , Estudos ProspectivosRESUMO
Chagas disease (Ch) affects 8-10 million people in Latin America, most of them are poor. Sudden death (SD) is the major cause of death in patients with Ch. To the best of our knowledge, the present report covers the largest reported series comparing the SD of Ch versus non-Ch patients Objective: To compare the circadian rhythm of SD in Ch versus non-Ch patients. METHODS: Retrospective analysis of all the cases of SD recorded in our department, including autopsied patients from 1963 until 2011. The pattern of SD of 262 patients (116 Ch and 146 non-Ch), 56.7% men, average age 54, 6 years old, divided into four groups: Group A: Ch with SD (n = 38), Group B: non-Ch with SD (n = 58), Group C. Ch with non SD (n = 89), and Group D: non-Ch with non SD (n = 81). For the statistical analysis, proportion comparison was used. RESULTS: 44.7% (17/38) of SDs in Group A (Ch) occurred between 6 a.m. and 5:59 p.m., while for Group B (not Ch) 70.7% (41 /58) died in that time (p < 0.005). 55.3% (21/38) of the SD in Group A (Ch) occurred between 6 p.m. and 5:59 a.m. compared with 29.3% (17/58) in Group B (p < 0, 005). CONCLUSIONS: Circadian rhythm of SD in patient with Ch differs from those with non-Ch. In Ch patients, SD occurs predominantly during the night compared with non-Ch SD that occurs predominantly during the day especially during the morning.
Assuntos
Doença de Chagas/complicações , Ritmo Circadiano , Morte Súbita Cardíaca/etiologia , Humanos , Estudos RetrospectivosRESUMO
Ruminants are the primary reservoir of Shiga-toxin producing Escherichia coli (STEC) O157:H7 and the main source of infection for humans. The aim of this study was to assess the immunogenic properties of a candidate vaccine consisting on the recombinant proteins of E. coli O157:H7 IntiminC280, the carboxy-terminal fraction of Intimin γ, EspB and the fusion protein between the B subunit of Stx2 and Brucella Lumazine Synthase (BLS)(BLS-Stx2B), in Holstein Fresian calves.To accomplish this goal we vaccinated calves with two doses of different vaccine formulations: 2 antigens (IntiminC280, EspB), 3 antigens (IntiminC280, EspB, BLS-Stx2B), BLS-Stx2B alone and a control non-vaccinated group. All antigens were expressed as recombinant proteins in E. coli. Specific IgG titres increased in vaccinated calves and the inclusion of BLS-Stx2B in the formulation seems to have a stimulatory effect on the humoral response to IntiminC280 and EspB after the booster. The neutralizing activity of antibodies against these two antigens was assessed in Red Blood Cell lysis assays and adherence to Hep-2 cells as a correlate of T3SS activity. Both sera from animals vaccinated with 2 or 3 antigens inhibited both virulence properties. Serological response to Stx2 was observed in animals vaccinated only with BLS-Stx2B and with 3 antigens and neutralization of Stx2 cytotoxicity was also observed in both groups. In conclusion, immunization of calves with BLS-Stx2B, IntiminC280 and EspB elicited a potent humoral response able to neutralize Shiga toxin 2 cytotoxity and the T3SS virulence properties in vitro. These results suggest that this formulation is a good candidate vaccine to reduce STEC shedding in cattle and needs to be further assessed in vivo.
Assuntos
Doenças dos Bovinos/imunologia , Bovinos/imunologia , Infecções por Escherichia coli/veterinária , Vacinas contra Escherichia coli/imunologia , Toxina Shiga II/imunologia , Sistemas de Secreção Tipo III , Adesinas Bacterianas/metabolismo , Animais , Anticorpos Antibacterianos/sangue , Proteínas da Membrana Bacteriana Externa/metabolismo , Derrame de Bactérias , Doenças dos Bovinos/microbiologia , Adesão Celular , Chlorocebus aethiops , Eritrócitos/microbiologia , Infecções por Escherichia coli/imunologia , Escherichia coli O157/patogenicidade , Proteínas de Escherichia coli/metabolismo , Imunidade Humoral , Imunoglobulina G/sangue , Masculino , Proteínas Recombinantes/imunologia , Células Vero , VirulênciaRESUMO
Shiga toxin producing Escherichia coli (STEC) are bacterial pathogens involved in food-borne diseases. Shiga toxin (Stx) is the main virulence factor of STEC and is responsible for systemic complications including Hemolytic Uremic Syndrome (HUS). It has been previously demonstrated that Shiga toxin type 2 (Stx2) induces pregnancy loss in rats in early stage of pregnancy. The main purpose of this study was to determine if an active immunization prevents Stx2 mediated pregnancy loss and confers passive protective immunity to the offspring. For that purpose Sprague Dawley female rats were immunized with the chimera based on the enzyme lumazine synthase from Brucella spp. (BLS) and the B subunit of Shiga toxin 2 (Stx2B) named BLS-Stx2B. After immunization females were mated with males. At day 8 of gestation, dams were challenged intraperitoneally with a sublethal and abortifacient dose of Stx2. The immunization induced high anti-Stx2B-specific antibody titers in sera and most important, prevented pregnancy loss. Pups born and breastfeed by immunized dams had high anti-Stx2B-specific antibody titers in sera. Cross-fostering experiments indicated that passive protective immunity against Stx2 was transmitted through lactation. These results indicate that immunization of adult female rats with BLS-Stx2B prevents Stx2-induced pregnancy loss and confers anti Stx2 protective immunity to the offspring.
Assuntos
Aborto Espontâneo/prevenção & controle , Infecções por Escherichia coli/prevenção & controle , Vacinas contra Escherichia coli/imunologia , Imunidade Materno-Adquirida , Toxina Shiga II/imunologia , Aborto Espontâneo/microbiologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Neutralizantes/sangue , Brucella/enzimologia , Feminino , Doenças Transmitidas por Alimentos/prevenção & controle , Síndrome Hemolítico-Urêmica/prevenção & controle , Masculino , Complexos Multienzimáticos/imunologia , Gravidez , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/imunologia , Escherichia coli Shiga ToxigênicaRESUMO
Shiga toxin (Stx)-producing Escherichia coli (STEC) infections are implicated in the development of the life-threatening Hemolytic Uremic Syndrome (HUS). Despite the magnitude of the social and economic problems caused by STEC infections, no licensed vaccine or effective therapy is presently available for human use. Single chain antibodies (VHH) produced by camelids exhibit several advantages in comparison with conventional antibodies, making them promising tools for diagnosis and therapy. In the present work, the properties of a recently developed immunogen, which induces high affinity and protective antibodies against Stx type 2 (Stx2), were exploited to develop VHHs with therapeutic potential against HUS. We identified a family of VHHs against the B subunit of Stx2 (Stx2B) that neutralize Stx2 in vitro at subnanomolar concentrations. One VHH was selected and was engineered into a trivalent molecule (two copies of anti-Stx2B VHH and one anti-seroalbumin VHH). The resulting molecule presented extended in vivo half-life and high therapeutic activity, as demonstrated in three different mouse models of Stx2-toxicity: a single i.v. lethal dose of Stx2, several i.v. incremental doses of Stx2 and intragastrical STEC infection. This simple antitoxin agent should offer new therapeutic options for treating STEC infections to prevent or ameliorate HUS outcome.
Assuntos
Antitoxinas/isolamento & purificação , Síndrome Hemolítico-Urêmica/terapia , Imunoterapia/métodos , Toxina Shiga II/imunologia , Anticorpos de Cadeia Única/isolamento & purificação , Animais , Antitoxinas/uso terapêutico , Camelus , Modelos Animais de Doenças , Síndrome Hemolítico-Urêmica/diagnóstico , Camundongos , Testes Sorológicos/métodos , Anticorpos de Cadeia Única/uso terapêutico , Terapêutica , Resultado do TratamentoRESUMO
Haemolytic uraemic syndrome (HUS) is the major complication of Escherichia coli gastrointestinal infections that are Shiga toxin (Stx) producing. Monocytes contribute to HUS evolution by producing cytokines that sensitize endothelial cells to Stx action and migration to the injured kidney. As CC chemokine receptors (CCRs) are involved in monocyte recruitment to injured tissue, we analysed the contribution of these receptors to the pathogenesis of HUS. We analysed CCR1, CCR2 and CCR5 expression in peripheral monocytes from HUS patients during the acute period, with healthy children as controls. We observed an increased expression of CCRs per cell in monocytes from HUS patients, accompanied by an increase in the absolute number of monocytes CCR1+, CCR2+ and CCR5+. It is interesting that prospective analysis confirmed that CCR1 expression positively correlated with HUS severity. The evaluation of chemokine levels in plasma showed that regulated on activation of normal T-cell-expressed and -secreted (RANTES) protein was reduced in plasma from patients with severe HUS, and this decrease correlated with thrombocytopenia. Finally, the expression of the higher CCRs was accompanied by a loss of functionality which could be due to a mechanism for desensitization to compensate for altered receptor expression. The increase in CCR expression correlates with HUS severity, suggesting that the dysregulation of these receptors might contribute to an increased risk of renal damage. Activated monocytes could be recruited by chemokines and then receptors could be dysregulated. The dysregulation of CCRs and their ligands observed during the acute period suggests that a chemokine pathway would participate in HUS development.
Assuntos
Quimiocinas/imunologia , Síndrome Hemolítico-Urêmica/metabolismo , Monócitos/metabolismo , Receptores de Quimiocinas/metabolismo , Movimento Celular , Criança , Pré-Escolar , Feminino , Expressão Gênica/fisiologia , Síndrome Hemolítico-Urêmica/imunologia , Humanos , Rim/metabolismo , Masculino , Monócitos/citologia , Estudos ProspectivosRESUMO
Circulating monocytes (Mos) may continuously repopulate macrophage (MAC) or dendritic cell (DC) populations to maintain homeostasis. MACs and DCs are specialized cells that play different and complementary immunological functions. Accordingly, they present distinct migratory properties. Specifically, whereas MACs largely remain in tissues, DCs are capable of migrating from peripheral tissues to lymphoid organs. The aim of this work was to analyze the expression of the fractalkine receptor (CX3CR1) during the monocytic differentiation process. Freshly isolated Mos express high levels of both CX3CR1 mRNA and protein. During the Mo differentiation process, CX3CR1 is downregulated in both DCs and MACs. However, MACs showed significantly higher CX3CR1 expression levels than did DC. We also observed an antagonistic CX3CR1 regulation by interferon (IFN)-γ and interleukin (IL)-4 during MAC activation through the classical and alternative MAC pathways, respectively. IFN-γ inhibited the loss of CX3CR1, but IL-4 induced it. Additionally, we demonstrated an association between CX3CR1 expression and apoptosis prevention by soluble fractalkine (sCX3CL1) in Mos, DCs and MACs. This is the first report demonstrating sequential and differential CX3CR1 modulation during Mo differentiation. Most importantly, we demonstrated a functional link between CX3CR1 expression and cell survival in the presence of sCX3CL1.
Assuntos
Células Dendríticas/citologia , Macrófagos/citologia , Monócitos/citologia , Receptores de Quimiocinas/genética , Apoptose/efeitos dos fármacos , Receptor 1 de Quimiocina CX3C , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Regulação da Expressão Gênica , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interferon gama/farmacologia , Interleucina-4/genética , Interleucina-4/imunologia , Interleucina-4/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Especificidade de Órgãos , Cultura Primária de Células , Receptores de Quimiocinas/imunologia , Transdução de SinaisRESUMO
Hemolytic-uremic syndrome (HUS) is defined as the triad of anemia, thrombocytopenia, and acute kidney injury. Enterohemorrhagic Shiga toxin (Stx)-producing Escherichia coli (EHEC), which causes a prodromal hemorrhagic enteritis, remains the most common etiology of the typical or epidemic form of HUS. Because no licensed vaccine or effective therapy is presently available for human use, we recently developed a novel immunogen based on the B subunit of Shiga toxin 2 (Stx2B) and the enzyme lumazine synthase from Brucella spp. (BLS) (BLS-Stx2B). The aim of this study was to analyze maternal immunization with BLS-Stx2B as a possible approach for transferring anti-Stx2 protection to the offspring. BALB/c female mice were immunized with BLS-Stx2B before mating. Both dams and pups presented comparable titers of anti-Stx2B antibodies in sera and fecal extracts. Moreover, pups were totally protected against a lethal dose of systemic Stx2 injection up to 2 to 3 months postpartum. In addition, pups were resistant to an oral challenge with an Stx2-producing EHEC strain at weaning and did not develop any symptomatology associated with Stx2 toxicity. Fostering experiments demonstrated that anti-Stx2B neutralizing IgG antibodies were transmitted through breast-feeding. Pups that survived the EHEC infection due to maternally transferred immunity prolonged an active and specific immune response that protected them against a subsequent challenge with intravenous Stx2. Our study shows that maternal immunization with BLS-Stx2B was very effective at promoting the transfer of specific antibodies, and suggests that preexposure of adult females to this immunogen could protect their offspring during the early phase of life.
Assuntos
Infecções por Escherichia coli/imunologia , Síndrome Hemolítico-Urêmica/prevenção & controle , Imunidade Materno-Adquirida/imunologia , Imunização/métodos , Toxina Shiga II/imunologia , Vacinas contra Shigella/imunologia , Animais , Anticorpos Antibacterianos/análise , Brucella/imunologia , Modelos Animais de Doenças , Escherichia coli Êntero-Hemorrágica , Feminino , Síndrome Hemolítico-Urêmica/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Complexos Multienzimáticos/imunologia , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Hemolytic uremic syndrome (HUS) is the major complication of gastrointestinal infections with enterohemorrhagic Escherichia coli (EHEC) and is mediated by the production of Shiga toxins (Stx). Although it has been previously reported that not only HUS patients but healthy children have anti-Stx antibodies, very little is known about how these infections impact on mucosal immune system to generate a specific immune response. This work aimed to evaluate the immune responses elicited after a single oral dose of EHEC in a mouse model of HUS at weaning. We found sequential activation of T and B lymphocytes together with an increased percentage of IgA-bearing B cells in Peyer's patches and mesenteric lymph nodes. We also found fecal anti-EHEC IgA and serum anti-Stx2 IgG in EHEC-inoculated mice. Besides, these mice were partially protected against an intravenous challenge with Stx2. These data demonstrate that one episode of EHEC infection is enough to induce activation in the gut-associated lymphoid tissue, especially the B cell compartment, and lead to the production of specific IgA in mucosal tissue and the generation of systemic protection against Stx2 in a percentage of intragastrically inoculated mice. These data also support the epidemiologic observation that a second episode of HUS is very rare.
Assuntos
Sangue/imunologia , Vacinas contra Escherichia coli/administração & dosagem , Vacinas contra Escherichia coli/imunologia , Síndrome Hemolítico-Urêmica/prevenção & controle , Mucosa Intestinal/imunologia , Escherichia coli Shiga Toxigênica/imunologia , Administração Oral , Animais , Anticorpos Antibacterianos/análise , Anticorpos Antibacterianos/sangue , Linfócitos B/imunologia , Modelos Animais de Doenças , Fezes/química , Feminino , Imunoglobulina A/análise , Imunoglobulina G/sangue , Masculino , Camundongos Endogâmicos BALB C , Nódulos Linfáticos Agregados/citologia , Nódulos Linfáticos Agregados/imunologia , Soro/química , Linfócitos T/imunologiaRESUMO
UNLABELLED: Shiga toxins (Stx) are the main agent responsible for the development of hemolytic-uremic syndrome (HUS), the most severe and life-threatening systemic complication of infection with enterohemorrhagic Escherichia coli (EHEC) strains. We previously described Stx2 expression by eukaryotic cells after they were transfected in vitro with the stx2 gene cloned into a prokaryotic plasmid (pStx2). The aim of this study was to evaluate whether mammalian cells were also able to express Stx2 in vivo after pStx2 injection. Mice were inoculated by hydrodynamics-based transfection (HBT) with pStx2. We studied the survival, percentage of polymorphonuclear leukocytes in plasma, plasma urea levels, and histology of the kidneys and the brains of mice. Mice displayed a lethal dose-related response to pStx2. Stx2 mRNA was recovered from the liver, and Stx2 cytotoxic activity was observed in plasma of mice injected with pStx2. Stx2 was detected by immunofluorescence in the brains of mice inoculated with pStx2, and markers of central nervous system (CNS) damage were observed, including increased expression of glial fibrillary acidic protein (GFAP) and fragmentation of NeuN in neurons. Moreover, anti-Stx2B-immunized mice were protected against pStx2 inoculation. Our results show that Stx2 is expressed in vivo from the wild stx2 gene, reproducing pathogenic damage induced by purified Stx2 or secondary to EHEC infection. IMPORTANCE: Enterohemorrhagic Shiga toxin (Stx)-producing Escherichia coli (EHEC) infections are a serious public health problem, and Stx is the main pathogenic agent associated with typical hemolytic-uremic syndrome (HUS). In contrast to the detailed information describing the molecular basis for EHEC adherence to epithelial cells, very little is known about how Stx is released from bacteria in the gut, reaching its target tissues, mainly the kidney and central nervous system (CNS). In order to develop an efficient treatment for EHEC infections, it is necessary to understand the mechanisms involved in Stx expression. In this regard, the present study demonstrates that mammals can synthesize biologically active Stx using the natural promoter associated with the Stx-converting bacteriophage genome. These results could impact the comprehension of EHEC HUS, since local eukaryotic cells transduced and/or infected by bacteriophage encoding Stx2 could be an alternative source of Stx production.
Assuntos
Escherichia coli Êntero-Hemorrágica/metabolismo , Infecções por Escherichia coli/microbiologia , Regiões Promotoras Genéticas , Toxina Shiga II/biossíntese , Toxina Shiga II/genética , Animais , Encéfalo/metabolismo , Encéfalo/microbiologia , Encéfalo/patologia , Escherichia coli Êntero-Hemorrágica/genética , Infecções por Escherichia coli/patologia , Feminino , Humanos , Rim/metabolismo , Rim/microbiologia , Rim/patologia , Fígado/metabolismo , Fígado/microbiologia , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
FUNDAMENTO: A doença de Chagas afeta mais de 15 milhões de pessoas em todo o mundo. Embora a transmissão vetorial tenha diminuído, a transmissão oral tornou-se relevante. Recentemente, nosso grupo publicou as características clínicas e epidemiológicas do maior surto relatado até hoje da doença de Chagas transmitida oralmente. OBJETIVO: Descrever alterações eletrocardiográficas que ocorrem na população de estudo durante o surto causado pela ingestão de suco de goiaba contaminado. MÉTODOS: Avaliamos 103 casos positivos, dos quais 76 (74%) tinham <18 anos de idade (média das idades: 9,1 ± 3,1 anos) e 27 (26%) tinham >18 anos (média das idades: 46 ± 11,8 anos). Todos os pacientes foram submetidos a avaliações clínicas e ECG. Caso os pacientes apresentassem palpitações ou alterações evidentes do ritmo na linha basal, o monitoramento de ECG ambulatorial seria realizado. RESULTADOS: Um total de 68 casos(66%;53 crianças e15 adultos) apresentaram anormalidades no ECG. Além disso, 69,7% (53/76) daqueles com idade < 18anos e 56% (15/27) daqueles com idade >18 anos apresentaram alguma alteração no ECG(p = ns). Anormalidades de ST-T foram observadas em 37,86% casos (39/103) e arritmias foram evidente sem 28,16% casos(29/103). Alterações de ST ocorreram em 72% daqueles com idade < 18 anos em comparação aos de > 18 anos (p < 0,0001). CONCLUSÃO: Este estudo relata o maior número de casos no mesmo surto de doença de Chagas causada por contaminação oral, com ECGs relatados. As alterações no ECG que sugerem miocardite aguda e arritmias foram as anormalidades encontradas com maior frequência.
BACKGROUND: Chagas disease affects more than 15 million people worldwide. Although vector-borne transmission has decreased, oral transmission has become important. Recently, our group published the clinical and epidemiological characteristics of the largest outbreak of orally transmitted Chagas disease reported till date. OBJECTIVE: To describe electrocardiographic changes occurring in the study population during the outbreak caused by ingestion of contaminated guava juice. METHODS: We evaluated 103 positive cases, of which 76 (74%) were aged <18 years (average age: 9.1 ± 3.1 years) and 27 (26%) were aged > 18 years (average age: 46 ± 11.8 years). All patients underwent clinical evaluations and ECG. If the patients had palpitations or evident alterations of rhythm at baseline, ambulatory ECG monitoring was performed. RESULTS: A total of 68 cases (66%; 53 children and 15 adults) had ECG abnormalities. Further, 69.7% (53/76) of those aged < 18 years and 56% (15/27) of those aged >18 years showed some ECG alteration (p = ns). ST-T abnormalities were observed in 37.86% cases (39/103) and arrhythmias were evident in 28.16% cases (29/103). ST alterations occurred in 72% of those aged <18 years compared with 19% of those aged >18 years (p < 0.0001). CONCLUSIONS: This study reports the largest number of cases in the same outbreak of acute Chagas disease caused by oral contamination, with recorded ECGs. ECG changes suggestive of acute myocarditis and arrhythmias were the most frequent abnormalities found.
Assuntos
Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Doença de Chagas/epidemiologia , Doença de Chagas/fisiopatologia , Surtos de Doenças , Fatores Etários , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/fisiopatologia , Bebidas/parasitologia , Doença de Chagas/transmissão , Eletrocardiografia , Contaminação de Alimentos , Trypanosoma cruzi/parasitologia , Venezuela/epidemiologiaRESUMO
The striking feature of enterohemorrhagic Escherichia coli (EHEC) infections is the production of Shiga toxins (Stx) implicated in the development of the life-threatening hemolytic uremic syndrome. Despite the magnitude of the social impact of EHEC infections, no licensed vaccine or effective therapy is available for human use. One of the biggest challenges is to develop an effective and safe immunogen to ensure nontoxicity, as well as a strong input to the immune system to induce long-lasting, high-affinity Abs with anti-Stx-neutralizing capacity. The enzyme lumazine synthase from Brucella spp. (BLS) is a highly stable dimer of pentamers and a scaffold with enormous plasticity on which to display foreign Ags. Taking into account the advantages of BLS and the potential capacity of the B subunit of Stx2 to induce Abs that prevent Stx2 toxicity by blocking its entrance into the host cells, we engineered a new immunogen by inserting the B subunit of Stx2 at the amino termini of BLS. The resulting chimera demonstrated a strong capacity to induce a long-lasting humoral immune response in mice. The chimera induced Abs with high neutralizing capacity for Stx2 and its variants. Moreover, immunized mice were completely protected against i.v. Stx2 challenge, and weaned mice receiving an oral challenge with EHEC were completely protected by the transference of immune sera. We conclude that this novel immunogen represents a promising candidate for vaccine or Ab development with preventive or therapeutic ends, for use in hemolytic uremic syndrome-endemic areas or during future outbreaks caused by pathogenic strains of Stx-producing E. coli.
Assuntos
Síndrome Hemolítico-Urêmica/prevenção & controle , Complexos Multienzimáticos/imunologia , Toxina Shiga II/imunologia , Vacinas contra Shigella/imunologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Neutralizantes/sangue , Brucella , Modelos Animais de Doenças , Escherichia coli Êntero-Hemorrágica , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Complexos Multienzimáticos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Toxina Shiga II/químicaRESUMO
BACKGROUND: Chagas disease affects more than 15 million people worldwide. Although vector-borne transmission has decreased, oral transmission has become important. Recently, our group published the clinical and epidemiological characteristics of the largest outbreak of orally transmitted Chagas disease reported till date. OBJECTIVE: To describe electrocardiographic changes occurring in the study population during the outbreak caused by ingestion of contaminated guava juice. METHODS: We evaluated 103 positive cases, of which 76 (74%) were aged ≤ 18 years (average age: 9.1 ± 3.1 years) and 27 (26%) were aged > 18 years (average age: 46 ± 11.8 years). All patients underwent clinical evaluations and ECG. If the patients had palpitations or evident alterations of rhythm at baseline, ambulatory ECG monitoring was performed. RESULTS: A total of 68 cases (66%; 53 children and 15 adults) had ECG abnormalities. Further, 69.7% (53/76) of those aged ≤ 18 years and 56% (15/27) of those aged >18 years showed some ECG alteration (p = ns). ST-T abnormalities were observed in 37.86% cases (39/103) and arrhythmias were evident in 28.16% cases (29/103). ST alterations occurred in 72% of those aged ≤ 18 years compared with 19% of those aged >18 years (p < 0.0001). CONCLUSIONS: This study reports the largest number of cases in the same outbreak of acute Chagas disease caused by oral contamination, with recorded ECGs. ECG changes suggestive of acute myocarditis and arrhythmias were the most frequent abnormalities found.
Assuntos
Doença de Chagas/epidemiologia , Doença de Chagas/fisiopatologia , Surtos de Doenças , Adolescente , Adulto , Fatores Etários , Idoso , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/fisiopatologia , Bebidas/parasitologia , Doença de Chagas/transmissão , Eletrocardiografia , Feminino , Contaminação de Alimentos , Humanos , Masculino , Pessoa de Meia-Idade , Trypanosoma cruzi/parasitologia , Venezuela/epidemiologia , Adulto JovemRESUMO
Shiga toxins (Stx) are the main virulence factors in enterohemorrhagic Escherichia coli (EHEC) infections, causing diarrhea and hemolytic uremic syndrome (HUS). The genes encoding for Shiga toxin-2 (Stx2) are located in a bacteriophage. The toxin is formed by a single A subunit and five B subunits, each of which has its own promoter sequence. We have previously reported the expression of the B subunit within the eukaryotic environment, probably driven by their own promoter. The aim of this work was to evaluate the ability of the eukaryotic machinery to recognize stx2 sequences as eukaryotic-like promoters. Vero cells were transfected with a plasmid encoding Stx2 under its own promoter. The cytotoxic effect on these cells was similar to that observed upon incubation with purified Stx2. In addition, we showed that Stx2 expression in Stx2-insensitive BHK eukaryotic cells induced drastic morphological and cytoskeletal changes. In order to directly evaluate the capacity of the wild promoter sequences of the A and B subunits to drive protein expression in mammalian cells, GFP was cloned under eukaryotic-like putative promoter sequences. GFP expression was observed in 293T cells transfected with these constructions. These results show a novel and alternative way to synthesize Stx2 that could contribute to the global understanding of EHEC infections with immediate impact on the development of treatments or vaccines against HUS.
Assuntos
Regiões Promotoras Genéticas , Toxina Shiga/genética , Animais , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Homologia de Sequência do Ácido Nucleico , Células VeroRESUMO
Haemolytic uraemic syndrome (HUS) is a rare but life-threatening complication of Shiga toxin (Stx)-producing Escherichia coli (STEC) infections, characterized by acute renal failure, thrombocytopenia and haemolytic anaemia. Although the main infection route is the consumption of contaminated food or water, person-to-person transmission has been suggested in several situations. Moreover, epidemiological data indicate that the horizontal transmission of several pathogens, including STEC, among individuals of the same species requires significantly lower doses than those used in animal models infected with laboratory-cultured bacteria. Thus, the aim of this study was to evaluate whether the passage of a clinically isolated STEC strain through the gastrointestinal tract of mice affects its pathogenicity in mice. To test this, weaned mice were orally inoculated by gavage with either an E. coli O157:H7 isolate from an HUS patient, or the same strain recovered from stools after one or two successive passages through the gastrointestinal tract of the mice. We show that stool-recovered strains are able to induce a more generalized and persistent colonization than the parent strain. Furthermore, a 10(4)-fold-reduced inoculum of the stool-recovered strains still causes gut colonization and mouse mortality, which are not observed with the parent strain. These results indicate an increased pathogenicity in stool-recovered strains that may be associated with an increased ability to colonize the mouse intestine.
Assuntos
Escherichia coli O157/patogenicidade , Trato Gastrointestinal/microbiologia , Animais , Fezes/microbiologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Inoculações Seriadas , Análise de Sobrevida , VirulênciaRESUMO
Muchos pacientes acuden a consulta externa con un conjunto inespecífico de síntomas y signos que sugieren el diagnóstico de astenia, sin encontrarse una causa orgánica que explique dichos síntomas. Se realizó un estudio prospectivo a nivel nacional para diagnosticar y cuantificar la intensidad de la astenia en estos pacientes y medir la eficacia de la sulbutiamina 400 mg/día como tratamiento farmacológicoa corto plazo de primera línea. Se realizó un estudio abierto, prospectivo y multicéntrico,en el cual se ingresaron 341 pacientes, que consultaron ambulatoriamente y que presentaban síntomas de astenia. A través de la escala de Intensidad de Fatiga (por sus siglas en inglés: Fatigue Severity Scale - FSS) se determinó la presencia de astenia en estos pacientes y se evaluó la eficacia de la sulbutiamina 400 mg/día de Laboratorios Leti S.A.V., Venezuela (Tekron®). El estudio tuvo una duración de 15 días, en los cuales el paciente fue evaluado tres veces: día 1 (inicio), día 7 y día 15. Si el paciente presentaba astenia (puntuación ≥ 36 puntos en la FSS), se le indicaba sulbutiamina400 mg/día con el desayuno por 15 días. El 74,7% de los pacientes evaluados fueron del sexo femenino, con una edad media de 43,7 ± 12,5 años y el 25,3% del sexo masculino con una edad media de 41,7 ± 13,5 años. Al inicio del estudio la media de la puntuación de la FSS fue de 49,7 ± 7,3 puntos; a los 7 días de tratamiento con 400 mg/día de sulbutiamina fue de 37,2 ± 8,8 puntos con una disminucióndel 25,2% y al día 15 fue de 28,0 ± 9,8 puntos con una disminución de 43,7% con respecto al inicio del tratamiento, resultando estadísticamente significativo (p< 0,0001 y p< 0,0001 al día 7 y día 15 vs inicio, respectivamente). El 77,7% de los pacientes respondieron al tratamiento al día 15. La sulbutiamina resultó ser un tratamiento muy bien tolerado, se reportaron eventos adversos leves en 132 pacientes (38,7%) al día 7 y en 115 pacientes(33,7%) al día 15...
Many patients attending out patient clinics with a set of nonspecific symptoms and signs that suggest the diagnosis asthenia, without organic cause to explain the symptoms. In order to determine which of was these patients confirmed the diagnosis of functional asthenia, was performed a prospective nation wide, study to diagnose and quantify the intensity of fatigue in these patients and measure the effectiveness of sulbutiamine 400 mg/day treatment short-term drug frontline. Was realized an open, prospective, multicenter study, which entered 341 outpatient patients which showed signs of fati gue. The Fatigue Severity Scale (FSS) was used as a tool for the diagnosis and evaluation of severity of fatigue and was evaluated the effectiveness of sulbutiamine 400 mg/day to Leti Laboratories, S.A.V. (Tekron®), as first line treatment.The duration of the study was 15 days, with 3 evaluations (day 1 or baseline, day 7 and day 15). The score of the FSS had to be ≥ 36 points to be enrolled. 74,7% of patients were females with a mean age of 43,7 ± 12,5 years old and 25,3% were males with a mean age of 41,7 ± 13,5 years old. At baseline, the FSS score was 49,7 ± 7,3 points; at day 7 was 37,2 ± 8,8 points with a decrease of 25,2% and at day 15 was 28,0 ± 9,8 points, with a decrease of 43,7% respective to baseline, being statistically significant (p< 0,0001 and p< 0,0001 at day 7 and day 15, respectively). The percentage of response to treatment was 77,7% at day 15. Sulbutiamine was a very well tolerated treatment, there were reported mild adverse events in 132 patients (38,7%) at day 7 and in 115 patients (33,7%) at day 15. Sulbutiamine 400 mg/day is a secure treatment, it is well tolerated and effective in improving the asthenia symptoms, as demonstrated in this clinical trial by the significant decrease in the FSS mean score and the percentage of patients with asthenia at day 15 of treatment
Assuntos
Adulto Jovem , Astenia/complicações , Astenia/diagnóstico , Fadiga Mental/patologia , Preparações Farmacêuticas/administração & dosagem , Preparações Farmacêuticas , Pesos e MedidasRESUMO
Aun cuando la importancia del tratamiento adecuado y de su cumplimento para alcanzar las metas en salud están claramente demostrados, la falla de adherencia al tratamiento en general supera el 50%. Este es un problema multidimensional donde el paciente es sólo uno de los factores involucrados. Comprender los diferentes factores involucrados así como el impacto de la no adherencia no solamente en la falla de eficacia, sino en el incremento de riesgo y de costos para el sistema de salud, es clave para poder generar acciones que lleven a mejorar la. En Estados Unidos solamente, los costos incrementales relacionados con la no adherencia se calculan por encima de los 300 millardos de dólares.
Although the importance of proper treatment and its compliance to achieve health goals is clearly demonstrated, the failure in treatment adherence generally exceeds 50%. The lack of treatment adherence is a multidimensional problem where the patient is just one of the factors involved. Understanding the different factors in question and the impact of failure in treatment adherence not only in the efficiency, but the increase in risk and cost in the health system, are key for generating actions that lead to improvement. In the U.S. alone incremental costs associated with non-adherence are calculated above 300 billion.
Assuntos
Humanos , Doença Crônica/economia , Custos de Cuidados de Saúde , Adesão à Medicação , Medicina InternaRESUMO
The typical form of hemolytic uremic syndrome (HUS) is the major complication of Shiga toxin-producing Escherichia coli (STEC) infections. HUS is a critical health problem in Argentina since it is the main cause of acute renal failure in children and the second cause of chronic renal failure, giving account for 20% of renal transplants in children and adolescents in our country. In spite of the extensive research in the field, the mainstay of treatment for patients with HUS is supportive therapy, and there are no specific therapies preventing or ameliorating the disease course. In this review, we present the current knowledge about pathogenic mechanisms and discuss traditional and innovative therapeutic approaches, with special focus in national status and contributions made by Argentinean groups.