RESUMEN
The gastrointestinal tract is a hostile biological environment, yet not all ingested materials are destroyed. The minute differences that determine whether a substance persists or is digested, liberated, adsorbed, excreted, or taken up are still poorly understood. Most attempts to investigate the events occurring during an orogastrointestinal passage rely on simplified in vitro systems where an analyte is exposed to artificial intestinal fluids. To closely mimic the events in the gastrointestinal tract, the exact intestinal fluid composition and the in vivo concentration of its constituents must be known. The widely used lavage procedures, however, dilute the intestinal fluids to an extent that precludes recalculation to the original concentrations. Thus, we developed procedures with which undiluted murine intestinal fluid can be harvested; determined the in vivo concentrations of the digestive enzymes trypsin, chymotrypsin, and elastase and the adsorbents mucin and immunoglobulin A in small intestinal fluid of fasted and unfasted female Balb/c mice; and identified chymotrypsin and immunoglobulin A as valid endogenous dilution markers for the recalculation of aqueous lavages. With these technologies and information at hand, more reliable investigations on the fate of allergens, pathogens, food, and anthropogenic xenobiotics in the gastrointestinal tract will be possible.
Asunto(s)
Bioquímica/métodos , Líquidos Corporales/metabolismo , Intestino Delgado/metabolismo , Proteínas/metabolismo , Animales , Digestión , Ayuno/metabolismo , Femenino , Inmunoglobulina A/metabolismo , Intestino Delgado/citología , Ratones , Ratones Endogámicos BALB C , Mucinas/metabolismo , Péptido Hidrolasas/metabolismo , Irrigación TerapéuticaRESUMEN
Many bioanalytic and diagnostic procedures rely on labels with which the molecule of interest can be tracked in or discriminated from accompanying like substances. Herein, we describe a new labeling and detection system based on derivatives of 2,4-dichlorophenoxyacetic acid (2,4-D) and anti-2,4-D antibodies. The 2,4-D system is highly sensitive with a K(D) of 7 x 10(-11) M for the hapten-antibody pair, can be used on a large variety of biomolecules such as proteins, peptides, carbohydrates, and nucleic acids, is not hampered by endogenous backgrounds because 2,4-D is a xenobiotic, and is robust because 2,4-D is a very stable compound that withstands the conditions of most reactions usually performed on biomolecules. With this unique blend of properties, the 2,4-D system compares favorably with its rivals digoxigenin (DIG)/anti-DIG and biotin/(strept)avidin and provides an interesting and powerful tool in biomolecular labeling.
Asunto(s)
Ácido 2,4-Diclorofenoxiacético/análogos & derivados , Ácido 2,4-Diclorofenoxiacético/metabolismo , Inmunoensayo/métodos , Coloración y Etiquetado/métodos , Ácido 2,4-Diclorofenoxiacético/análisis , Ácido 2,4-Diclorofenoxiacético/inmunología , Aminas/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , ADN/metabolismo , Nucleótidos de Desoxiuracil/química , Nucleótidos de Desoxiuracil/metabolismo , Ambiente , Límite de Detección , Ratones , Polietilenglicoles/metabolismo , Proteínas/química , Proteínas/metabolismoRESUMEN
Vibrio cholerae colonizes the small intestine of adult C57BL/6 mice. In this study, the physical and genetic parameters that facilitate this colonization were investigated. Successful colonization was found to depend upon anesthesia with ketamine-xylazine and neutralization of stomach acid with sodium bicarbonate, but not streptomycin treatment. A variety of common mouse strains were colonized by O1, O139, and non-O1/non-O139 strains. All combinations of mutants in the genes for hemolysin, the multifunctional, autoprocessing RTX toxin (MARTX), and hemagglutinin/protease were assessed, and it was found that hemolysin and MARTX are each sufficient for colonization after a low dose infection. Overall, this study suggests that, after intragastric inoculation, V. cholerae encounters barriers to infection including an acidic environment and an immediate immune response that is circumvented by sodium bicarbonate and the anti-inflammatory effects of ketamine-xylazine. After initial adherence in the small intestine, the bacteria are subjected to additional clearance mechanisms that are evaded by the independent toxic action of hemolysin or MARTX. Once colonization is established, it is suggested that, in humans, these now persisting bacteria initiate synthesis of the major virulence factors to cause cholera disease. This adult mouse model of intestinal V. cholerae infection, now well-characterized and fully optimized, should serve as a valuable tool for studies of pathogenesis and testing vaccine efficacy.
Asunto(s)
Anestesia/métodos , Anestésicos/farmacología , Intestino Delgado/microbiología , Ketamina/farmacología , Vibriosis/metabolismo , Vibrio cholerae/metabolismo , Animales , Eliminación de Gen , Proteínas Hemolisinas/química , Sistema Inmunológico , Intestino Delgado/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Bicarbonato de Sodio/farmacologíaRESUMEN
Cholera epidemics caused by Vibrio cholerae El Tor O1 strains are typified by a large number of asymptomatic carriers who excrete vibrios but do not develop diarrhea. This carriage state was important for the spread of the seventh cholera pandemic as the bacterium was mobilized geographically, allowing the global dispersion of this less virulent strain. Virulence factors associated with the development of the carriage state have not been previously identified. We have developed an animal model of cholera in adult C57BL/6 mice wherein V. cholerae colonizes the mucus layer and forms microcolonies in the crypts of the distal small bowel. Colonization occurred 1 to 3 h after oral inoculation and peaked at 10 to 12 h, when bacterial loads exceeded the inoculum by 10- to 200-fold, indicating bacterial growth within the small intestine. After a clearance phase, the number of bacteria within the small intestine, but not those in the cecum or colon, stabilized and persisted for at least 72 h. The ability of V. cholerae to prevent clearance and establish this prolonged colonization was associated with the accessory toxins hemolysin, the multifunctional autoprocessing RTX toxin, and hemagglutinin/protease and did not require cholera toxin or toxin-coregulated pili. The defect in colonization attributed to the loss of the accessory toxins may be extracellularly complemented by inoculation of the defective strain with an isogenic colonization-proficient V. cholerae strain. This work thus demonstrates that secreted accessory toxins modify the host environment to enable prolonged colonization of the small intestine in the absence of overt disease symptoms and thereby contribute to disease dissemination via asymptomatic carriers.
Asunto(s)
Toxinas Bacterianas/toxicidad , Portador Sano/microbiología , Vibrio cholerae O1/crecimiento & desarrollo , Factores de Virulencia/fisiología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/genética , Cólera/microbiología , Toxina del Cólera/genética , Toxina del Cólera/toxicidad , Recuento de Colonia Microbiana , Femenino , Fimbrias Bacterianas , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/toxicidad , Intestinos/microbiología , Metaloendopeptidasas/genética , Metaloendopeptidasas/toxicidad , Ratones , Ratones Endogámicos C57BL , Vibrio cholerae O1/patogenicidad , Factores de Virulencia/genéticaRESUMEN
The seventh cholera pandemic that started in 1961 was caused by Vibrio cholerae O1 strains of the El Tor biotype. These strains produce the pore-forming toxin hemolysin, a characteristic used clinically to distinguish classical and El Tor biotypes. Even though extensive in vitro data on the cytolytic activities of hemolysin exist, the connection of hemolysin to virulence in vivo is not well characterized. To study the contribution of hemolysin and other accessory toxins to pathogenesis, we utilized the model of intestinal infection in adult mice sensitive to the actions of accessory toxins. In this study, we showed that 4- to 6-week-old streptomycin-fed C57BL/6 mice were susceptible to intestinal infection with El Tor strains, which caused rapid death at high doses. Hemolysin had the predominant role in lethality, with a secondary contribution by the multifunctional autoprocessing RTX (MARTX) toxin. Cholera toxin and hemagglutinin/protease did not contribute to lethality in this model. Rapid death was not caused by increased dissemination due to a damaged epithelium since the numbers of CFU recovered from spleens and livers 6 h after infection did not differ between mice inoculated with hemolysin-expressing strains and those infected with non-hemolysin-expressing strains. Although accessory toxins were linked to virulence, a strain defective in the production of accessory toxins was still immunogenic since mice immunized with a multitoxin-deficient strain were protected from a subsequent lethal challenge with the wild type. These data suggest that hemolysin and MARTX toxin contribute to vaccine reactogenicity but that the genes for these toxins can be deleted from vaccine strains without affecting vaccine efficacy.
Asunto(s)
Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/toxicidad , Cólera/microbiología , Proteínas Hemolisinas/toxicidad , Vibrio cholerae O1/patogenicidad , Factores de Virulencia/toxicidad , Animales , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Vacunas Bacterianas/genética , Vacunas Bacterianas/inmunología , Cólera/inmunología , Cólera/prevención & control , Toxina del Cólera/genética , Toxina del Cólera/toxicidad , Recuento de Colonia Microbiana , Femenino , Proteínas Hemolisinas/genética , Intestino Delgado/patología , Hígado/microbiología , Metaloendopeptidasas/genética , Metaloendopeptidasas/toxicidad , Ratones , Ratones Endogámicos C57BL , Bazo/microbiología , Análisis de Supervivencia , Vibrio cholerae O1/genética , Vibrio cholerae O1/inmunología , Factores de Virulencia/genéticaRESUMEN
Following intranasal inoculation, Vibrio cholerae KFV101 (DeltactxAB DeltahapA DeltahlyA DeltartxA) colonizes and stimulates tumor necrosis factor alpha and interleukin 1beta (IL-1beta) in mice, similar to what occurs with isogenic strain P4 (DeltactxAB), but is less virulent and stimulates reduced levels of IL-6, demonstrating a role for accessory toxins in pathogenesis. Morbidity is enhanced in C3H/HeJ mice, indicating that Toll-like receptor 4 is important for infection containment.
Asunto(s)
Toxina del Cólera/biosíntesis , Toxina del Cólera/genética , Cólera/patología , Receptores Inmunológicos/fisiología , Vibrio cholerae/metabolismo , Animales , Cólera/inmunología , Cólera/metabolismo , Vacunas contra el Cólera/inmunología , Modelos Animales de Enfermedad , Inflamación/inmunología , Inflamación/metabolismo , Interleucina-1/biosíntesis , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/microbiología , Pulmón/patología , Ratones , Ratones Endogámicos C3H , Alveolos Pulmonares/inmunología , Alveolos Pulmonares/microbiología , Alveolos Pulmonares/patología , Receptor Toll-Like 4 , Factor de Necrosis Tumoral alfa/biosíntesis , Vibrio cholerae/inmunologíaRESUMEN
Receptor-mediated drug targeting via nanoengineered particulate delivery systems is an emerging field. However, little is known about how such magic bullets should be assembled to yield optimal targeting efficiency. Here we investigated the influence of targeting ligand flexibility on binding of ligand-coated microparticles to cell surface receptors. Using the ganglioside G(M1)-binding B subunit of cholera toxin as ligand and fluorescent microparticles as a model delivery system, conjugates with different numbers of linkages between ligand and particle were prepared and tested for their efficiency to bind to live fibroblast monolayers. Our results show that multiple bonds between ligand and particle reduce the targeting rate by up to 50% compared to constructs where ligands are attached via single aliphatic chains. Thus, for maximum performance, targeted particulate drug delivery systems should be assembled such that ligands are attached via single sigma bonds only, allowing the ligand molecules to adopt an optimal binding conformation.