1. KINETICS OF SHIGA TOXINS (STX) IN THE BLOOD AND FECES OF PATIENTS WITH BLOODY DIARRHEA ASSOCIATED WITH STX-PRODUCING ESCHERICHIA COLI (STEC) Brigotti M. 1, Tazzari P.G. 2, Scavia G. 3, Arfilli V. 1, Carnicelli D. 1, Salardi S. 4, Paglialonga F. 4, Testa S. 4, Ricci F. 2, Pagliaro P. 2, Minelli F. 3, Ardissino G. 4, Caprioli A. 3 1 Dipartimento di Patologia Sperimentale, Università di Bologna; 2 Servizio di Immunoematologia e Trasfusionale, Ospedale S.Orsola-Malpighi, Bologna; 3 Istituto Superiore di Sanità, EU Reference Laboratory for E. coli, Rome; 4 IRCCS Fondazione Cà Granda Ospedale Maggiore Policlinico, Center for HUS Control, Milan, Italy The main steps in the pathogenesis of typical hemolytic uremic syndrome (tHUS) include the colonization of the gut mucosa by STEC, the release of Stx in the intestinal lumen, their passage into the blood and the delivery to the renal and cerebral endothelia harbouring the specific toxin receptors (Gb3Cer). The toxin-induced endothelial injury is considered a main event in the pathogenesis of tHUS and occurs when Stx have reached the kidney or the brain from the gut. To explain the mode of delivery of Stx in the blood stream, two different hypotheses have been proposed: transport of free Stx in the plasma or shuttling by macromolecular or cellular blood components. The first simpler hypothesis is not evidence-based because Stx have never been detected in the plasma of tHUS patients with concomitant detectable fecal toxin. Conversely, the binding of Stx to human polymorphonuclear leukocytes (PMN) has been demonstrated by us and by other groups in those patients. Till now, the studies have been conducted in patients with overt tHUS. In the present investigation, the kinetics of Stx in feces and blood have been studied in patients with STEC-associated bloody diarrhea. Results and Discussion Introduction Eleven children with STEC-induced bloody diarrhea were enrolled and the kinetics of Stx in their feces and blood were studied by a daily sampling to establish possible relationships with the development of tHUS. Although none of the children developed HUS, Stx were detected on PMN as previously described in tHUS patients. Strikingly, the toxins were detected also in patients’ sera in contrast to that observed during overt tHUS. In 4 patients Stx were found only in sera, in 3 patients only on PMN membrane, in 2 patients both in sera and on PMN and in 2 patients the two assays were negative. The panels show three representative patients. Boxes show the time of Stx persistence in blood that was significantly lower in sera with respect to PMN. Conclusions Stx are detectable on PMN membrane and in sera of patients with STEC-induced bloody diarrhea. The presence of Stx in blood, although mandatory for the development of tHUS, is not sufficient in triggering the transition between hemorrhagic colitis and tHUS. Other crucial factors might be involved such as the type of toxin variant, the time-course of toxemia, the amounts of toxins or the presence of concurrent virulence factors such as LPS. Methods Patients presenting with bloody diarrhea and positive for Stx or stx genes at a rapid screening assay (performed by an immunochromatographic commercial test and/or by Reverse Dot blot analysis) were enrolled in the framework of the HUS surveillance network of the Lombardia Region (Italy). This involves 53 pediatric units and aims at the early hospitalization and monitoring of children with STEC-associated bloody diarrhea (potentially in the prodromal phase of tHUS). Further evidence of STEC infection was obtained by: i) detection of stx, eae, and serogroup-associated genes (O157, O26, O103, O111, O145) in enrichment cultures of feces by Real Time PCR; ii) isolation of STEC; iii) detection of serum antibodies against the LPS of serogroups O157, O26, O103, O111, O145 by ELISA. Free fecal Stx was detected and titrated by the Vero cells assay. Detection of free Stx in patients’ sera was performed by a new developed method by using Raji cells (Fig. 1), whereas Stx bound to PMN were detected by indirect flow cytometry analysis (Fig. 2) with mouse monoclonal antibodies to Stx and fluorescent anti-mouse secondary antibodies. PMN carrying Stxs POSITIVE Stx receptor PMN carrying Stx Raji cells are very sensitive to Stx since they express Gb3Cer as TNF-treated human umbilical vein endothelial cells. However, the same IC 50 on protein synthesis (approximately 1 pM Stx, detected by the incorporation of radioactive leucine into proteins) is reached in Raji cells after 3 h challenge, whereas an overnight challenge is required with endothelial cells. This quick response allows us to set up a very sensitive and rapid method for the daily detection of Stx in patients’ sera. Figure 1 Figure 2 Patients with Stx bound to PMN (n=5) Total days with positive PMN 3.8 ± 1.79*(median 4.0) Total days of bloody diarrhea 3.0 ± 2.00 (median 3.0) Total days from the onset of bloody diarrhea to 1 st positive determination 4.0 ± 0.82 (median 4.0) Patients with Stx in sera (n=6) Total days with positive sera 1.83 ± 0.98*(median 1.5) Total days of bloody diarrhea 2.83 ± 1.17 (median 3.0) Total days from the onset of bloody diarrhea to 1 st positive determination 3.83 ± 2.48 (median 4.0) *p<0.05 Student t test