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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).

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Detection by the Raji cell protein synthesis assay of Stx in sera from patients with concomitant fecal Stx during Stx-producing Escherichia coli (STEC)-induced bloody diarrhea. The experiment was performed in duplicate. The presence of the monoclonal antibodies (10 μg) to Stx1a and Stx2a did not affect the controls. *p < 0.05, **p < 0.01 (t-test).
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toxins-07-04564-f004: Detection by the Raji cell protein synthesis assay of Stx in sera from patients with concomitant fecal Stx during Stx-producing Escherichia coli (STEC)-induced bloody diarrhea. The experiment was performed in duplicate. The presence of the monoclonal antibodies (10 μg) to Stx1a and Stx2a did not affect the controls. *p < 0.05, **p < 0.01 (t-test).

Mentions: The results shown in Figure 4 clearly demonstrated the presence of functional Stx1 and Stx2, during the prodromal intestinal phase, detected by the assay described in the present paper. Moreover, data have been validated by the addition of mouse monoclonal IgG to Stx1 (Stx1-13C4) and Stx2 (Stx2-BB12) in order to demonstrate the causal relationship between protein synthesis inhibition and the presence of these specific bacterial toxins in patients’ blood. The simultaneous addition of the two monoclonal antibodies to the same sample was preferred in order to reduce the consumption of the valuable sera, since many other laboratory determinations need to be performed using the same specimen. The characteristics of the three representative patients are shown in Table 2.


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)

Detection by the Raji cell protein synthesis assay of Stx in sera from patients with concomitant fecal Stx during Stx-producing Escherichia coli (STEC)-induced bloody diarrhea. The experiment was performed in duplicate. The presence of the monoclonal antibodies (10 μg) to Stx1a and Stx2a did not affect the controls. *p < 0.05, **p < 0.01 (t-test).
© Copyright Policy
Related In: Results  -  Collection

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

toxins-07-04564-f004: Detection by the Raji cell protein synthesis assay of Stx in sera from patients with concomitant fecal Stx during Stx-producing Escherichia coli (STEC)-induced bloody diarrhea. The experiment was performed in duplicate. The presence of the monoclonal antibodies (10 μg) to Stx1a and Stx2a did not affect the controls. *p < 0.05, **p < 0.01 (t-test).
Mentions: The results shown in Figure 4 clearly demonstrated the presence of functional Stx1 and Stx2, during the prodromal intestinal phase, detected by the assay described in the present paper. Moreover, data have been validated by the addition of mouse monoclonal IgG to Stx1 (Stx1-13C4) and Stx2 (Stx2-BB12) in order to demonstrate the causal relationship between protein synthesis inhibition and the presence of these specific bacterial toxins in patients’ blood. The simultaneous addition of the two monoclonal antibodies to the same sample was preferred in order to reduce the consumption of the valuable sera, since many other laboratory determinations need to be performed using the same specimen. The characteristics of the three representative patients are shown in Table 2.

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