Flow cytometry detection of Shiga toxins in the blood from children with hemolytic uremic syndrome

Abstract

Results: The method was quick (3 h), sensitive (femtomoles), and capable of detecting both Stxs. The presence of Stxs was detected on PMNs from six patients with HUS: four patients had serologic or microbiological evidence of STEC infection, whereas the other two patients had no evidence of STEC infection when employing the standard diagnostic methods. Conclusions: The method described is rapid, simple, and based on commercially available reagents, and it might be more sensitive than the standard methods for diagnosis of STEC infection. It also allows the detection of Stxs in blood, a key step to monitor the pathogenesis of HUS.

The Antibiotic Polymyxin B Impairs the Interactions between Shiga Toxins and Human Neutrophils

“…Briefly, the seminal paper demonstrating for the first time such an interaction (18) was followed by a second paper (20) in which some of the authors reversed their position. However, we and other groups confirmed these findings in vitro and in patients (16,(21)(22)(23)(24)(25).…”

“…Stx1 or Stx2 bound on neutrophils were detected by flow cytometry as previously described (25). Neutrophils carrying Stx1 or Stx2 (see above) were incubated with a mouse mAb against Stx1 or Stx2 in the presence of human serum to saturate FcRs on polymorphonuclear leukocytes (PMN).…”

“…Neutrophils were checked by staining with mAb to Ags associated to granulocytes (FITC-CD16 and FITC-CD65, Beckman Coulter, Miami, FL). The mean channel value of fluorescence (MCV) of the single histogram was chosen to measure the extent of binding of Stx to PMN (25). The single values were calculated by subtracting the control MCV (range, 0.4-0.6), that is, the MCV of neutrophils from the same donor incubated with primary and secondary Abs in the absence of the toxin.…”

“…The same values (MCV = 0.4-0.6) were obtained when anti-Stx mouse mAbs were omitted in the assay in the presence of toxins and secondary Abs. The assay has been validated previously by comparing control subjects and HUS patients in a double-blind fashion (25) and by challenging Stx + PMN with a negative control Ab (35).…”

Change in Conformation with Reduction of α-Helix Content Causes Loss of Neutrophil Binding Activity in Fully Cytotoxic Shiga Toxin 1

“…The cells were spun down at 200 ϫ g for 5 min and washed three times with 100 l of the same buffer containing BSA at 37°C. Native Stx1 bound to PMN was detected by flow cytometry as previously described (10,14). Briefly, PMN were incubated with appropriately diluted mouse monoclonal antibody against Stx1 in the presence of human serum to saturate Fc receptors.…”