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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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IC50 of Stx2a on Raji cells’ protein synthesis in the absence and in the presence of human serum from healthy donor 2 (see Table 1) added to complete medium (RPMI 1640 containing 10% (v/v) FBS).
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toxins-07-04564-f003: IC50 of Stx2a on Raji cells’ protein synthesis in the absence and in the presence of human serum from healthy donor 2 (see Table 1) added to complete medium (RPMI 1640 containing 10% (v/v) FBS).

Mentions: The addition of different concentrations of both toxins to culture media allowed the calculation of similar IC50 on Raji translation for Stx1a (0.8 pM; 54.4 pg/mL; r = −0.97) and Stx2a (2.2 pM; 149.6 pg/mL; r = −0.99). The values are quite similar to those obtained by Quinones and colleagues [41] by using a very sensitive method based on a Vero cell line harboring a destabilized variant (t1/2 = 2 h) of the enhanced green fluorescent protein (d2EGFP) to monitor the Stx-induced inhibition of protein synthesis. Conversely, the presence of human sera from healthy donors did not significantly change the IC50 of Stx1a (0.3 pM; 20.4 pg/mL; r = −0.92), whereas, as expected, that of Stx2a showed a marked increase (IC50 ranging between 40 and 75 pM; 2.7–5.1 ng/mL) caused by the known Stx2 inhibitor present in human sera (HuSAP) and not in FBS [25], as depicted in a representative experiment (Figure 3) and summarized in Table 1. Although its concentration in serum is relatively stable, circulating HuSAP has been reported to be lower in females and infants, and slight variations exist among different subjects [27,42]. This would have an impact on the sensitivity of the assay, hence explaining the differences in Stx2 activity observed in Table 1. On the other hand, HuSAP is not an acute phase protein, thus differences between donors or patients in Stx2 neutralizing activities of their sera reflect the individual behaviors measured by the proposed assay. In conclusion, the limit of detection (concentration of toxins giving 10% inhibition of protein synthesis) of Stx in the presence of human sera, calculated by the reported plots (see above and Table 1), was ~2 pg/mL (~0.03 pM) for Stx1a and ~100 pg/mL for Stx2a (~1.5 pM).


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)

IC50 of Stx2a on Raji cells’ protein synthesis in the absence and in the presence of human serum from healthy donor 2 (see Table 1) added to complete medium (RPMI 1640 containing 10% (v/v) FBS).
© Copyright Policy
Related In: Results  -  Collection

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

toxins-07-04564-f003: IC50 of Stx2a on Raji cells’ protein synthesis in the absence and in the presence of human serum from healthy donor 2 (see Table 1) added to complete medium (RPMI 1640 containing 10% (v/v) FBS).
Mentions: The addition of different concentrations of both toxins to culture media allowed the calculation of similar IC50 on Raji translation for Stx1a (0.8 pM; 54.4 pg/mL; r = −0.97) and Stx2a (2.2 pM; 149.6 pg/mL; r = −0.99). The values are quite similar to those obtained by Quinones and colleagues [41] by using a very sensitive method based on a Vero cell line harboring a destabilized variant (t1/2 = 2 h) of the enhanced green fluorescent protein (d2EGFP) to monitor the Stx-induced inhibition of protein synthesis. Conversely, the presence of human sera from healthy donors did not significantly change the IC50 of Stx1a (0.3 pM; 20.4 pg/mL; r = −0.92), whereas, as expected, that of Stx2a showed a marked increase (IC50 ranging between 40 and 75 pM; 2.7–5.1 ng/mL) caused by the known Stx2 inhibitor present in human sera (HuSAP) and not in FBS [25], as depicted in a representative experiment (Figure 3) and summarized in Table 1. Although its concentration in serum is relatively stable, circulating HuSAP has been reported to be lower in females and infants, and slight variations exist among different subjects [27,42]. This would have an impact on the sensitivity of the assay, hence explaining the differences in Stx2 activity observed in Table 1. On the other hand, HuSAP is not an acute phase protein, thus differences between donors or patients in Stx2 neutralizing activities of their sera reflect the individual behaviors measured by the proposed assay. In conclusion, the limit of detection (concentration of toxins giving 10% inhibition of protein synthesis) of Stx in the presence of human sera, calculated by the reported plots (see above and Table 1), was ~2 pg/mL (~0.03 pM) for Stx1a and ~100 pg/mL for Stx2a (~1.5 pM).

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