Limits...
A rapid and sensitive method to measure the functional activity of Shiga toxins in human serum.

Arfilli V, Carnicelli D, Ardissino G, Torresani E, Scavia G, Brigotti M - Toxins (Basel) (2015)

Bottom Line: The dramatic effects of these toxins on the microvasculature of different organs, particularly of the kidney, are well known, whereas there is no consensus on the mechanism by which Stx reach the endothelia of target organs and/or indirectly injure these body sites.The assay monitors the translation impairment induced by these powerful inhibitors of protein synthesis, which are identified properly by neutralizing their activity with specific monoclonal antibodies.Recent research has pointed to a dynamic process of Stx-induced renal intoxication in which concurrent and interactive steps are involved.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Sede di Patologia Generale, Università di Bologna, Via San Giacomo 14, 40126 Bologna, Italy. arfilli.v@libero.it.

ABSTRACT
Shiga toxins (Stx) have a definite role in the development of hemolytic uremic syndrome in children with hemorrhagic colitis caused by pathogenic Stx-producing Escherichia coli (STEC) strains. The dramatic effects of these toxins on the microvasculature of different organs, particularly of the kidney, are well known, whereas there is no consensus on the mechanism by which Stx reach the endothelia of target organs and/or indirectly injure these body sites. We hereby describe a quick (4 h), radioactive, Raji cell-based method designed for the detection of Stx in human sera. The assay monitors the translation impairment induced by these powerful inhibitors of protein synthesis, which are identified properly by neutralizing their activity with specific monoclonal antibodies. By this method, we detected for the first time the functional activity of Stx in sera of STEC-infected patients during hemorrhagic colitis. Recent research has pointed to a dynamic process of Stx-induced renal intoxication in which concurrent and interactive steps are involved. Our rapid and specific method could be useful for studying the kinetics of Stx during the natural course of STEC infection and the interplay between Stx activity in serum and Stx presence in different blood fractions (neutrophils, monocytes, platelets, leukocyte-platelet aggregates, microvesicles, lipoproteins).

Show MeSH

Related in: MedlinePlus

Time course of the translation in Raji cells in the presence of human serum added to complete medium (RPMI 1640 containing 10% (v/v) FBS). The experiments were performed according to Scheme 1. The SD values (n = 3) of single points are indicated.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4663520&req=5

toxins-07-04564-f002: Time course of the translation in Raji cells in the presence of human serum added to complete medium (RPMI 1640 containing 10% (v/v) FBS). The experiments were performed according to Scheme 1. The SD values (n = 3) of single points are indicated.

Mentions: The experiment described in Scheme 1 was repeated 10 times in duplicate in the presence of 50 μL human serum from healthy donors added to complete medium (RPMI 1640 containing 10% (v/v) FBS), giving a mean incorporation of 25,449 cpm. The CV(%) (coefficient of variation), calculated as SD/mean × 100, was 7.85%, and the mean percentage of background signal was 2.5%. The limit of detection was determined by the addition of the CV(%) to the mean percentage of background signal. On this basis, inhibitions of protein synthesis above 10% upon challenge of Raji cells with Stx-containing human sera were considered significant. The viability of Raji cells used in the assays (95.8% ± 1.8%; mean ± SD, n = 10) was measured before seeding by the trypan blue dye exclusion test. Moreover, the short incubation time (4 h) strongly reduces further effects on cell viability or proliferation after treatment with toxins rendering the assay quite specific for the determination of protein synthesis inhibition in whole cells. The results obtained in Figure 2 following the final protocol (Scheme 1) also showed that the protein synthesis rate is optimized for efficiency and linearity (r = −0.99) up to 60 min.


A rapid and sensitive method to measure the functional activity of Shiga toxins in human serum.

Arfilli V, Carnicelli D, Ardissino G, Torresani E, Scavia G, Brigotti M - Toxins (Basel) (2015)

Time course of the translation in Raji cells in the presence of human serum added to complete medium (RPMI 1640 containing 10% (v/v) FBS). The experiments were performed according to Scheme 1. The SD values (n = 3) of single points are indicated.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663520&req=5

toxins-07-04564-f002: Time course of the translation in Raji cells in the presence of human serum added to complete medium (RPMI 1640 containing 10% (v/v) FBS). The experiments were performed according to Scheme 1. The SD values (n = 3) of single points are indicated.
Mentions: The experiment described in Scheme 1 was repeated 10 times in duplicate in the presence of 50 μL human serum from healthy donors added to complete medium (RPMI 1640 containing 10% (v/v) FBS), giving a mean incorporation of 25,449 cpm. The CV(%) (coefficient of variation), calculated as SD/mean × 100, was 7.85%, and the mean percentage of background signal was 2.5%. The limit of detection was determined by the addition of the CV(%) to the mean percentage of background signal. On this basis, inhibitions of protein synthesis above 10% upon challenge of Raji cells with Stx-containing human sera were considered significant. The viability of Raji cells used in the assays (95.8% ± 1.8%; mean ± SD, n = 10) was measured before seeding by the trypan blue dye exclusion test. Moreover, the short incubation time (4 h) strongly reduces further effects on cell viability or proliferation after treatment with toxins rendering the assay quite specific for the determination of protein synthesis inhibition in whole cells. The results obtained in Figure 2 following the final protocol (Scheme 1) also showed that the protein synthesis rate is optimized for efficiency and linearity (r = −0.99) up to 60 min.

Bottom Line: The dramatic effects of these toxins on the microvasculature of different organs, particularly of the kidney, are well known, whereas there is no consensus on the mechanism by which Stx reach the endothelia of target organs and/or indirectly injure these body sites.The assay monitors the translation impairment induced by these powerful inhibitors of protein synthesis, which are identified properly by neutralizing their activity with specific monoclonal antibodies.Recent research has pointed to a dynamic process of Stx-induced renal intoxication in which concurrent and interactive steps are involved.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Sede di Patologia Generale, Università di Bologna, Via San Giacomo 14, 40126 Bologna, Italy. arfilli.v@libero.it.

ABSTRACT
Shiga toxins (Stx) have a definite role in the development of hemolytic uremic syndrome in children with hemorrhagic colitis caused by pathogenic Stx-producing Escherichia coli (STEC) strains. The dramatic effects of these toxins on the microvasculature of different organs, particularly of the kidney, are well known, whereas there is no consensus on the mechanism by which Stx reach the endothelia of target organs and/or indirectly injure these body sites. We hereby describe a quick (4 h), radioactive, Raji cell-based method designed for the detection of Stx in human sera. The assay monitors the translation impairment induced by these powerful inhibitors of protein synthesis, which are identified properly by neutralizing their activity with specific monoclonal antibodies. By this method, we detected for the first time the functional activity of Stx in sera of STEC-infected patients during hemorrhagic colitis. Recent research has pointed to a dynamic process of Stx-induced renal intoxication in which concurrent and interactive steps are involved. Our rapid and specific method could be useful for studying the kinetics of Stx during the natural course of STEC infection and the interplay between Stx activity in serum and Stx presence in different blood fractions (neutrophils, monocytes, platelets, leukocyte-platelet aggregates, microvesicles, lipoproteins).

Show MeSH
Related in: MedlinePlus