A laboratory model of toxin-induced hemolytic uremic syndrome.

BACKGROUND: Verocytotoxin-producing (Shiga-like toxin-producing) Escherichia coli infection is the principal cause of hemolytic uremic syndrome (HUS). The pathogenesis is unclear, and there is a need for animal models. These are impeded by the different distribution of verocytotoxin receptors between species. We have circumvented this restriction using ricin, which gains entry into cells via various galactose receptors. Like verocytotoxin, ricin specifically cleaves a single adenine from ribosomal RNA. METHODS: Rats were given ricin at a dose of 6.7 micrograms/100 g body wt, with or without lipopolysaccharide at 10 micrograms/100 g body wt. Lipopolysaccharide alone or saline were used as controls. Changes in glomerular filtration rate, hematological parameters, histology, and plasma cytokine concentrations were measured. RESULTS: Extensive glomerular thrombosis, pyknotic nuclei, and an infiltration of ED1-positive cells into glomeruli were observed eight hours after an injection of ricin. Other vascular beds were unaffected. Histologic changes were preceded by oliguric renal failure, hemolysis, and thrombocytopenia. Ricin produced a rise in plasma concentrations of monocyte chemotactic protein-1, > tumor necrosis factor-alpha, > interleukin-1 beta, > interleukin-6. Interferon-gamma showed a small increase at the end of the experiment. CONCLUSIONS: Ricin induces glomerular thrombotic microangiopathy, closely resembling that which occurs in verocytotoxin-producing E. coli-induced HUS. As in HUS, high concentrations of proinflammatory cytokines are present, which are probably a result of cytokine superinduction by the toxin.

Susceptibility of AML cells to in vitro apoptosis correlates with heat shock protein 70 (hsp 70) expression.

Acute myeloid leukaemia (AML) cells from some individuals rapidly undergo apoptosis during in vitro culture. We have analysed this mode of cell death in AML cells harvested from patients at initial presentation and during subsequent treatment/relapse. Using flow cytometric analysis of propidium iodide-stained cells and quantitation of the subdiploid apoptotic peak, we observed that leukaemic cells from patients with AML displayed a heterogenous susceptibility to apoptosis in terms of the rate of accumulation of apoptotic cells. After 48 h incubation in the absence of added serum or exogenous growth factors the percentage of apoptotic cells ranged from 3% to 99%. This susceptibility to apoptosis correlated significantly with intracellular expression of hsp70 (P = 0.009), but not hsp90, and was also associated with the presence of p53 and low levels of expression of bcl-2.

Analysis of heat-shock protein expression in myeloid leukaemia cells by flow cytometry.

Expression of heat-shock proteins (hsp) was analysed in the leukaemic cells of 12 patients with acute myeloid leukaemia (AML) and nine patients with chronic myeloid leukaemia (CML). Using monoclonal antibodies to hsp70, hsp90 and hsp60 (ML30, a mycobacterial antigen with homology to human hsp60), we measured hsp levels by flow cytometry of permeabilized cells. Mononuclear cells from 10 healthy volunteers were also examined. The results demonstrate that hsp expression is significantly increased (P < 0.01) in the circulating cells of patients with AML compared with cells from CML patients, and compared with normal peripheral blood mononuclear cells. This increased pattern of expression was found for all three heat-shock protein families included in this study. Mononuclear cells from leukaemic patients showed a heterogenous pattern of hsp expression, between different patients, between cells from individual patients, and between the different hsp proteins examined. It is possible that hsp expression relates to the differentiation state or proliferative potential of these leukaemic cells.

Plasminogen activator inhibitor activity in diarrhoea-associated haemolytic uraemic syndrome.

In the haemolytic uraemic syndrome (HUS), platelet microthrombi and deposition of fibrin in glomeruli may contribute to the development of renal failure. The balance of procoagulant and fibrinolytic activities in the renal vasculature may therefore have an important role. We measured plasminogen-activator inhibitor (PAI) activity in the plasma of 81 children with diarrhoea-associated (D+) HUS, and found elevated PAI activity in many patients. When we categorized patients by need for dialysis, only the dialysed group had significantly higher levels of activity, compared with a group of normal controls. We also compared PAI activity with patient outcome after one year, and found that those with a poor outcome had significantly higher PAI activity than those with a good outcome. We suggest that plasma PAI activity may be an acute marker for dialysis requirement in HUS, and may have prognostic value for long-term outcome. The possible role of PAI in the pathogenesis of HUS requires further investigation.

Factor VIII von Willebrand protein in haemolytic uraemic syndrome and systemic vasculitides.

von Willebrand protein (vWF) is reduced by dithiothreitol (DTT) and, as a result, is not detected by enzyme-linked immunosorbent assay (ELISA). Plasma samples from normal subjects, children with haemolytic uraemic syndrome (HUS), and adults with vasculitis and vWF prepared from endothelium were treated with DTT before vWF assay. vWF in HUS and vasculitis resembled the endothelial form in being resistant to reduction. DTT modification of the ELISA assay may be useful as a marker of disease severity in conditions associated with endothelial cell damage.