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Shiga toxin type 2 (Stx2) produced by Escherichia coli O:157H7 can cause hemolytic-uremic syndrome in children, a disease for which there is neither a vaccine nor an effective treatment. This toxin consists of an enzymatically active A subunit and a pentameric B subunit responsible for the toxin binding to host cells, and also found to be immunogenic in rabbits. In this study we developed eukaryotic plasmids expressing the B subunit gene of Stx2 (pStx2B) and the B subunit plus the gene coding for the A subunit with an active-site deletion (pStx2 Delta A). Transfection of eukaryotic cells with these plasmids produced proteins of the expected molecular weight which reacted with specific monoclonal antibodies. Newborn and adult BALB/c mice immunized with two intramuscular injections of each plasmid, either alone or together with the same vector expressing the granulocyte and monocyte colony-stimulating factor (pGM-CSF), elicited a specific Th1-biased humoral response. The effect of pGM-CSF as an adjuvant plasmid was particularly notable in newborn mice and in pStx2B-vaccinated adult mice. Stx2-neutralizing activity, evaluated in vitro on VERO cell monolayers, correlated with in vivo protection. This is the first report using plasmids to induce a neutralizing humoral immune response against the Stx2.

The concept that during an immune challenge the release of glucocorticoids (GC) provides feedback inhibition on evolving immune responses has been drawn primarily from studies of autoimmune and/or inflammatory processes in animal models. The epidemic form of haemolytic uraemic syndrome (HUS) occurs secondary to infection with Gram-negative bacteria that produce Shiga toxin (Stx). Although Stx binding to the specific receptors present on renal tissue is the primary pathogenic mechanism, inflammatory or immune interactions are necessary for the development of the complete form of HUS. The aim of this study was to investigate the influence of endogenous GC on Stx-toxicity in a mouse model. Stx2 was injected into GC-deprived mice and survival rate, renal damage and serum urea levels were evaluated. Plasma corticosterone and cytosolic GC receptor (GR) concentration were also determined at multiple intervals post-Stx2 treatment. Higher sensitivity to Stx2 was observed in mice lacking endogenous GC, evidenced by an increase in mortality rates, circulating urea levels and renal histological damage. Moreover, Stx2 injection was associated with a transient but significant rise in corticosterone secretion. Interestingly, 24 h after Stx inoculation significant increases in total GR were detected in circulating neutrophils. These results indicate that interactions between the neuroendocrine and immune systems can modulate the level of damage significantly during a bacterial infection.

The hemolytic uremic syndrome (HUS) is the most-common cause of acute renal failure in children. Several researchers have reported the presence of neutrophil (PMN) activating cytokines, such as interleukin-8 and tumor necrosis factor-alpha, in the sera of HUS patients. Moreover, PMN-derived products, such as elastase, were increased. These observations have lead to the hypothesis that activated PMN could act as mediators of endothelial damage. The objective of this investigation was to directly evaluate the activation status of peripheral PMN from children with HUS. For this purpose, 12 children with typical HUS were bled during the acute period, before dialysis and/or transfusion, and 8 of them were also bled after 1 month follow-up. Additionally, blood samples from healthy control children admitted for routine surgical procedures, chronic uremic children, and neutrophilic children with acute infections not related to HUS were collected and processed in an identical manner. The function and membrane activation markers of PMN from these groups were evaluated. We found that during the acute period of HUS, PMN had reduced expression of FcgammaRIII (CD16) and CD11b, were degranulated, and exhibited an impaired antibody-dependent cellular cytotoxicity. These parameters returned to normal after clinical recuperation. We conclude that PMN activation in HUS patients is a very early and transient event, and upon hospitalization before dialysis PMN show a phenotype and functional pattern of partial deactivation.

It has been demonstrated that infections due to Shiga toxins (Stx) producing Escherichia coli are the main cause of the hemolytic uremic syndrome (HUS). Although it is recognized that Stx damage the glomerular endothelium, clinical and experimental evidence suggests that the inflammatory response is able to potentiate Stx toxicity. Lipopolysaccharides (LPS) and neutrophils (PMN) represent two central components of inflammation during a gram-negative infection. In this regard, patients with high peripheral PMN counts at presentation have a poor prognosis. Since the murine model has been used to study LPS-Stx interactions, we analyzed the effects of Stx alone or in combination with LPS on the kinetics of neutrophil production and activation and their participation in renal damage. We observed a sustained neutrophilia after Stx2 injection. Moreover, these neutrophils showed increased expression of CD11b, enhanced cytotoxic capacity, and greater adhesive properties. Regarding the cooperative effects of LPS on Stx2 action, we demonstrated potentiation of neutrophilia and CD11b induction at early times by pretreatment with LPS. Finally, a positive correlation between neutrophil percentage and renal damage (assayed as plasmatic urea) firmly suggests a role for PMN in the pathogenesis of HUS.

Haemolytic uraemic syndrome (HUS) has been closely associated with infection with a group of Shiga toxin-producing enterohaemorrhagic Eschericchia coli in young children. Shiga toxins (Stx) have been implicated as pathogenic agents of HUS by binding to the surface receptor of endothelial cells. LPS is a central product of the Gram-negative bacteria and several reports have documented that both LPS and Stx are important for disease development. In this study the reciprocal interactions between LPS and Stx2 are analysed in a mouse model. The results demonstrated that LPS was able to reduce or enhance Stx2 toxicity, depending on the dose and the timing of the injection. The involvement of the main early cytokines induced by LPS, tumour necrosis factor alpha (TNF-alpha) and IL-1beta, in those LPS opposite effects on Stx2 toxicity was evaluated. Stx2 toxicity was enhanced by in vivo injection of murine TNF-alpha and low doses of murine IL-1beta. However, at higher doses of IL-1beta which induced corticosteroid increase in serum, Stx2 lethality was decreased. Considering that dexamethasone and IL-1beta reproduce the LPS protective effects, it is suggested that endogenous corticosteroids secondary to the inflammatory response induced by LPS, mediate the protection against Stx2. It can be concluded that the fine equilibrium between proinflammatory and anti-inflammatory activities strongly influences Stx2 toxicity.