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The transport and inactivation kinetics of bacterial lipopolysaccharide influence its immunological potency in vivo.

ABSTRACT: The extraordinary potency and pathological relevance of gram-negative bacterial LPSs have made them very popular experimental agonists, yet little is known about what happens to these stimulatory molecules within animal tissues. We tracked fluorescent and radiolabeled LPS from a s.c. inoculation site to its draining lymph nodes (DLN), blood, and liver. Although we found FITC-labeled LPS in DLN within minutes of injection, drainage of radiolabeled LPS continued for >6 wk. Within the DLN, most of the LPS was found in the subcapsular sinus or medulla, near or within lymphatic endothelial cells and CD169(+) macrophages. Whereas most of the LPS seemed to pass through the DLN without entering B cell follicles, by 24 h after injection a small amount of LPS was found in the paracortex. In wild-type mice, ?70% of the injected radiolabeled LPS underwent inactivation by deacylation before it left the footpad; in animals that lacked acyloxyacyl hydrolase, the LPS-deacylating enzyme, prolonged drainage of fully acylated (active) LPS boosted polyclonal IgM and IgG3 Ab titers. LPS egress from a s.c. injection site thus occurred during many weeks and was mainly via lymphatic channels. Its immunological potency, as measured by its ability to stimulate polyclonal Ab production, was greatly influenced by the kinetics of both lymphatic drainage and enzymatic inactivation.

SUBMITTER: Lu M

PROVIDER: S-EPMC3169744 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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The transport and inactivation kinetics of bacterial lipopolysaccharide influence its immunological potency in vivo.

Lu Mingfang M Munford Robert S RS

Journal of immunology (Baltimore, Md. : 1950) 20110817 6

The extraordinary potency and pathological relevance of gram-negative bacterial LPSs have made them very popular experimental agonists, yet little is known about what happens to these stimulatory molecules within animal tissues. We tracked fluorescent and radiolabeled LPS from a s.c. inoculation site to its draining lymph nodes (DLN), blood, and liver. Although we found FITC-labeled LPS in DLN within minutes of injection, drainage of radiolabeled LPS continued for >6 wk. Within the DLN, most of ...[more]

PMID: 21849675

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Project description:BackgroundThe inhibitory effect of andrographolide sodium bisulphite (ASB) on jack bean urease (JBU) and Helicobacter pylori urease (HPU) was performed to elucidate the inhibitory potency, kinetics and mechanism of inhibition in 20 mM phosphate buffer, pH 7.0, 2 mM EDTA, 25 °C.MethodsThe ammonia formations, indicator of urease activity, were examined using modified spectrophotometric Berthelot (phenol-hypochlorite) method. The inhibitory effect of ASB was characterized with IC50 values. Lineweaver-Burk and Dixon plots for JBU inhibition of ASB was constructed from the kinetic data. SH-blocking reagents and competitive active site Ni2+ binding inhibitors were employed for mechanism study. Molecular docking technique was used to provide some information on binding conformations as well as confirm the inhibition mode.ResultsThe IC50 of ASB against JBU and HPU was 3.28±0.13 mM and 3.17±0.34 mM, respectively. The inhibition proved to be competitive and concentration- dependent in a slow-binding progress. The rapid formation of initial ASB-JBU complex with an inhibition constant of Ki=2.86×10(-3) mM was followed by a slow isomerization into the final complex with an overall inhibition constant of Ki*=1.33×10(-4) mM. The protective experiment proved that the urease active site is involved in the binding of ASB. Thiol reagents (L-cysteine and dithiothreithol) strongly protect the enzyme from the loss of enzymatic activity, while boric acid and fluoride show weaker protection, indicating that the active-site sulfhydryl group of JBU was potentially involved in the blocking process. Moreover, inhibition of ASB proved to be reversible since ASB-inactivated JBU could be reactivated by dithiothreitol application. Molecular docking assay suggested that ASB made contacts with the important sulfhydryl group Cys-592 residue and restricted the mobility of the active-site flap.ConclusionsASB was a competitive inhibitor targeting thiol groups of urease in a slow-binding manner both reversibly and concentration-dependently, serving as a promising urease inhibitor for the treatment of urease-related diseases.

Project description:Low-temperature atmospheric-pressure plasma has been studied for disinfection purposes. When plasma is exposed to water, reactive oxygen and nitrogen species are generated and preserved in the water fraction (plasma-treated water [PTW]), which consequently exhibits bactericidal activity. At low temperatures, one of the bactericidal components of PTW is peroxynitric acid (PNA). Importantly, PNA can also be synthesized by chemical reaction, without exposure to plasma. In this study, we evaluated the bactericidal properties of PNA based on reaction kinetics in comparison with other disinfectants. The analysis, based on dose-dependent effects, showed that PNA exhibited about 1 and 10 times the bactericidal activity of hypochlorous acid (HOCl) and peracetic acid, respectively. In addition, we evaluated the influence of organic contaminants on the bactericidal effects of PNA and HOCl. The bactericidal potential of both disinfectants was reduced by bovine serum albumin (BSA); however, PNA showed about 30-times-higher resistance against BSA inhibition than HOCl. Analysis of the dose-dependent effects of PNA revealed that the inhibition of bactericidal effect was caused by its consumption. Further experiments using model substrates containing particular amino acid residues (Met, Cys, Lys, and Leu) suggested that the bacterial inactivation by PNA is less affected by BSA due to the low reactivity and narrow reactivity spectrum of PNA for amino acid residues. Overall, our results suggest that PNA has a great disinfection potential, especially in the presence of organic contaminants (e.g., on the surface of the human body and on medical instruments contaminated with biological fluids).IMPORTANCE A good disinfectant for the human body should have various properties, such as strong bactericidal activity, harmlessness to living tissues, and resistance against biological fluids (or other organic contaminants). Peroxynitric acid (PNA) showed a bactericidal effect that was several tens up to several hundred times higher per unit of molarity than that of sodium hypochlorite and peracetic acid, which are used as general disinfectants for medical equipment. Moreover, the high resistance of PNA to organic load was confirmed, indicating that PNA will inactivate bacteria effectively even on contaminated surfaces, such as used medical devices or the human body surface. Therefore, we propose that PNA can be used as a strong disinfectant for the human body.

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The transport and inactivation kinetics of bacterial lipopolysaccharide influence its immunological potency in vivo.

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The transport and inactivation kinetics of bacterial lipopolysaccharide influence its immunological potency in vivo.

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