Invited review: Role of bacterial endotoxins in the etiopathogenesis of periparturient diseases of transition dairy cows

Shiga toxin-producing Escherichia coli is an important cause of foodborne illness, with cases attributable to beef, fresh produce and other sources. Many serotypes of the pathogen cause disease, and differentiating one serotype from another requires specific identification of the O antigen located on the lipopolysaccharide (LPS) molecule. The amphiphilic structure of LPS poses a challenge when using classical detection methods, which do not take into account its lipoglycan biochemistry. Typically, detection of LPS requires heat or chemical treatment of samples and relies on bioactivity assays for the conserved lipid A portion of the molecule. Our goal was to develop assays to facilitate the direct and discriminative detection of the entire LPS molecule and its O antigen in complex matrices using minimal sample processing. To perform serogroup identification of LPS, we used a method called membrane insertion on a waveguide biosensor, and tested three serogroups of LPS. The membrane insertion technique allows for the hydrophobic association of LPS with a lipid bilayer, where the exposed O antigen can be targeted for specific detection. Samples of beef lysate were spiked with LPS to perform O antigen specific detection of LPS from E. coli O157. To validate assay performance, we evaluated the biophysical interactions of LPS with lipid bilayers both in- and outside of a flow cell using fluorescence microscopy and fluorescently doped lipids. Our results indicate that membrane insertion allows for the qualitative and reliable identification of amphiphilic LPS in complex samples like beef homogenates. We also demonstrated that LPS-induced hole formation does not occur under the conditions of the membrane insertion assays. Together, these findings describe for the first time the serogroup-specific detection of amphiphilic LPS in complex samples using a membrane insertion assay, and highlight the importance of LPS molecular conformations in detection architectures.

/pdf/membrane-insertion-for-the-detection-of-lipopolysaccharides-302ksdi1hs.pdf

Membrane Insertion for the Detection of Lipopolysaccharides: Exploring the Dynamics of Amphiphile-in-Lipid Assays

This dissertation describes the development of new tailored methods for the discriminative detection of amphiphilic lipopolysaccharide (LPS) antigens, so as to improve screening methodologies for food-safety applications, and detection of amphiphiles in general. LPS is associated with the outer membrane of Gramnegative bacteria, and is a primary virulence biomarker of several pathogens. Direct detection of amphiphilic LPS in the aqueous matrices of the host/sample requires an appreciation of the complex biochemistry of the molecule, and forms the basis for this research. The unique structure of this molecule can be used for identification of both the serogroup and strain of pathogen. However, current detection methods lack sensitivity, and are also not serogroup specific. To achieve discriminative detection, we have first created a unique repertoire of associated reagents by isolating amphiphilic LPS from seven strains of Shiga toxin-producing Escherichia coli, and developing highly specific monoclonal antibodies against the O antigen regions of the same. We demonstrate the use of a targeted detection technique, called membrane insertion, which facilitates the physiological presentation of LPS by inserting the hydrophobic lipid A portion of the molecule into a lipid bilayer, leaving the O antigen exposed. This method is advantageous because it minimizes exposure of the highly conserved lipid A epitopes, and maximizes exposure of the serogroup specific O antigens. In addition, we present the first comprehensive biophysical analysis of the interaction of LPS with supported lipid bilayer

Differential Interactions of Lipopolysaccharides with Lipid Bilayers: Applications for Pathogen Detection

Peritonitis is one of the most severe complications of various diseases and injuries of the abdominal organs. The diagnosis of peritonitis in a general sense implies any form and severity of inflammation of the peritoneum. Currently, the problem of peritonitis remains actual, despite the achievements of scientific and technological progress. So, despite the successes of modern surgery, the achievements of asepsis and antiseptics, the rather wide possibilities of antibacterial, infusion and detoxification therapy, the incidence of peritonitis and mortality from it remain at a high level. The aim of the study was to elucidate the significance of the activity of liver arginase and Kupffer cells in the development of secondary atherogenic dyslipidemia and the formation of thyroid status in rats with experimental peritonitis. It has been established that under conditions of experimental peritonitis in rats, the activity of liver arginase decreases, the content of NO3 – /NO2 – increases and the level of iodine-containing thyroid hormones in the blood decreases, secondary atherogenic dyslipoproteinemia develops. Liver arginase and Kupffer cells are involved in changes in the content of total cholesterol in the liver, total cholesterol, lipoprotein cholesterol, the level of iodine-containing hormones in the blood and body temperature in peritonitis. A decrease in the activity of Kupffer cells in peritonitis is accompanied by an increase in the level of triiodothyronine in the blood, a less pronounced decrease in the activity of liver arginase and a weakening of the development of characteristic changes in the content of total cholesterol in the liver, lipoprotein cholesterol in the blood and prevents the development of secondary dyslipoproteinemia. Depression of liver arginase in conditions of peritonitis aggravates changes in the content of total cholesterol in blood and liver lipoproteins, triiodothyronine in the blood and contributes to the development of secondary dyslipoproteinemia. 

/pdf/on-the-significance-of-the-activity-of-liver-arginase-and-139u5lz7.pdf

On the significance of the activity of liver arginase and Kupffer cells in the development of secondary atherogenic dyslipidemia and the formation of thyroid status in rats with experimental peritonitis

Objective: To characterise the lipid profiles in dogs with parvoviral enteritis.



Methods: Blood was collected before treatment from 30 dogs that fulfilled the criteria for severe sepsis including hypo- or hyperthermia, hypotension, leucopenia, thrombocytopenia and evidences of organ dysfunction. Canine parvovirus was detected by haemagglutination and indirect fluorescence antibody tests in the faeces. Twenty control dogs were also enrolled on the basis of normal physical examination results, complete blood count and serum biochemistry profiles.



Results: Tachycardia, tachypnoea, hypotension, leucopenia, thrombocytopenia and increased serum markers of tissue injury (alanine aminotransferase, creatinine kinase myocardial isoenzyme [CK-MB], blood urea nitrogen and creatinine) were observed in dogs with parvoviral enteritis. Serum total cholesterol, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol levels were lower, but serum triglyceride level was higher in dogs with parvoviral enteritis than those in control dogs (P<0·001). Circulating tumour necrosis factor α correlated negatively with total cholesterol (r=−0·979; P<0·001) but positively with triglyceride (r=0·953; P<0·001) in dogs with parvoviral enteritis. Serum total cholesterol and high-density lipoprotein cholesterol levels were lower in non-survival (n=9) dogs than in survival dogs (n=21, P<0·001).



Clinical Significance: Serum total cholesterol and high-density lipoprotein cholesterol levels decreased, but serum triglyceride level increased in dogs with parvoviral enteritis. Low serum total cholesterol and high-density lipoprotein cholesterol levels may be used as an index of the severity of parvoviral enteritis.

Characterisation of lipid profiles in dogs with parvoviral enteritis

Binding of [3H]-lipopolysaccharide toxin (LPS) and complexes of LPS with serum [125I]-labeled low density lipoproteins (LDL) to primary culture of rat liver macrophages (Kupffer cells) has been studied Total, specific and nonspecific binding was determined The receptor interaction was shown to dominate for both LPS and LDL-LPS complexes, representing 70–77% and 80–85%, respectively The Scatchard plot was essentially non-linear for LPS binding but linear for the LDL-LPS complexes At the Scatchard graph of LPS binding, however, two regions approximately fitting the linear regression could be identified These regions correspond to two different types of specific binding sites: the first is for lower toxin concentrations of 025–050 μg/ml with K
 d
 = 075 μg/ml; while the second is for higher LPS concentrations of 75–15 μg/ml with K
 d
 = 539 μg/ml For LDL-LPS complexes only K
 d
 of 280 μg/ml was obtained The LDL-LPS complexes significantly blocked the LPS binding (−40%) while acetylated or oxidized LDLs exerted a less pronounced effect LPS inhibited binding of LDL-LPS complexes (−60%), while acetylated or oxidized LDLs suppressed interaction of LDL-LPS complexes with Kupffer cells insignificantly It is suggested that, while binding to the Kupffer cell surface, a substantial portion of both LPS and LDL-LPS complexes share the same scavenger receptors with which, however, modified LDLs interact weakly The LDL-LPS complexes can interact, apart from receptors common with LPS, with other receptors exhibiting similar binding parameters, with the apo-B/E receptors playing an inessential role

Binding of lipopolysaccharide and complexes of lipopolysaccharide to serum low density lipoproteins to liver macrophages

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Binding of lipopolysaccharide and complexes of lipopolysaccharide to serum low density lipoproteins to liver macrophages