Background. Atypical HUS (aHUS) is thought to be caused by predisposing mutations in genes encoding complement (regulating) proteins, such as Factor H (CFH), Factor I (IF), membrane co-factor protein (MCP) and Factor B (FB), or by auto-antibodies against CFH (αFH) in combination with a homozygous polymorphic deletion of the genes encoding Complement Factor H-related 1 and 3 (ΔCFHR1/3). The clinical impact of this knowledge is high, as it might be a prognostic factor for the outcome of renal transplantations and kidney donations. Methods. Mutational screening, by means of PCR and DNA sequencing, is performed in the above-mentioned genes in a group of 72 aHUS patients. Also, the presence of αFH and ΔCFHR1/3 was tested in patients and controls. Results. In 23 patients, a genetic aberration in at least one gene or the presence of αFH was found. A heterozygous mutation was observed in CFH in nine patients, in IF in seven patients and in MCP in three patients. No mutations were observed in FB. Seven patients presented αFH, of whom five also carried ΔCFHR1/3. Three patients carried a combined mutation (two patients: IF and MCP; one patient: IF, αFH and ΔCFHR1/3). A significant difference between patients and controls was detected for the presence of all three associated polymorphisms in CFH. Conclusions. Genetic abnormalities or the presence of αFH were detected in 31.9% of the aHUS patients. Furthermore, bigenic mutations were present, indicating that routine DNA mutation analysis of all complement factors associated with aHUS is important.
BackgroundIn the majority of pediatric patients, the hemolytic–uremic syndrome (HUS) is caused by an infection with Shiga toxin-producing Escherichia coli (STEC), mostly serotype O157. It is important to discriminate between HUS caused by STEC and complement-mediated HUS (atypical HUS) due to differences in treatment and outcome. As STEC and its toxins can only be detected in the patient’s stool for a short period of time after disease onset, the infectious agent may go undetected using only fecal diagnostic tests. Serum antibodies to lipopolysaccharide (LPS) of STEC persist for several weeks and may therefore be of added value in the diagnosis of STEC.MethodsAll patients with clinical STEC-HUS who were treated at Radboud University Medical Center between 1990 and 2014 were included in this retrospective single-center study. Clinical and diagnostic microbiological data were collected. Immunoglobulin M (IgM) antibodies against LPS of STEC serotype O157 were detected by a serological assay (ELISA).ResultsData from 65 patients weres available for analysis. Fecal diagnostic testing found evidence of an STEC infection in 34/63 patients (54 %). Serological evidence of STEC O157 was obtained in an additional 16 patients. This is an added value of 23 % (p < 0.0001) when the serological antibody assay is used in addition to standard fecal diagnostic tests to confirm the diagnosis STEC-HUS. This added value becomes especially apparent when the tests are performed more than 7 days after the initial manifestation of the gastrointestinal symptoms.ConclusionsThe serological anti-O157 LPS assay clearly makes a positive contribution when used in combination with standard fecal diagnostic tests to diagnose STEC-HUS and should be incorporated in clinical practice.
Cowpea mosaic virus (CPMV) moves from cell to cell by transporting virus particles via tubules formed through plasmodesmata by the movement protein (MP). On the surface of protoplasts, a fusion between the MP and the green fluorescent protein forms similar tubules and peripheral punctate spots. Here it was shown by time-lapse microscopy that tubules can grow out from a subset of these peripheral punctate spots, which are dynamic structures that seem anchored to the plasma membrane. Fluorescence resonance energy transfer experiments showed that MP subunits interacted within the tubule, where they were virtually immobile, confirming that tubules consist of a highly organized MP multimer. Fluorescence recovery after photobleaching experiments with protoplasts, transiently expressing fluorescent plasma membrane-associated proteins of different sizes, indicated that tubules made by CPMV MP do not interact directly with the surrounding plasma membrane. These experiments indicated an indirect interaction between the tubule and the surrounding plasma membrane, possibly via a host plasma membrane protein. INTRODUCTIONFor successful systemic infection, a plant virus must spread throughout the plant, a process that starts with transport from the initially infected cell to neighbouring uninfected cells (cell-to-cell movement) and is followed by transport through the phloem into roots and young developing leaves (systemic movement). Since plant viruses cannot pass through the rigid cell wall, they have evolved ways of exploiting plasmodesmata, the naturally occurring transport channels present between plant cells (Haywood et al., 2002). Normally, only small molecules are able to pass through plasmodesmata, but plant viruses encode one or more proteins, the so-called movement proteins (MPs), that modify the structure of plasmodesmata in such a way that viral transport is enabled. So far, two basic principles for cell-to-cell movement of plant viruses have been described: tubule-guided movement of virus particles and movement as ribonucleoprotein complexes (Lazarowitz & Beachy, 1999).Cowpea mosaic virus (CPMV), a positive-stranded, bipartite RNA virus belonging to the family Comoviridae, moves from cell to cell by transporting virus particles using tubular structures, which connect the infected cell to the neighbouring uninfected cell (Pouwels et al., 2002a). Immunogold labelling has shown that the CPMV MP is present in these tubular structures (van Lent et al., 1990). On the surface of CPMV-infected protoplasts, similar tubular structures are formed, which protrude up to 20 mm into the culture medium, are tightly surrounded by the plasma membrane and have the same ultrastructure as tubules in plant tissue (van Lent et al., 1991). Remarkably, tubules are also formed on protoplasts transiently expressing MP , showing that MP is the only viral protein required for tubule formation. So far, protoplasts have proved extremely useful as a model system for studying targeting and assembly of both wt and mutant MPs (Bertens et al., 20...
Acute renal failure hallmarks the pathogenesis of the epidemic form of hemolytic uremic syndrome (D+HUS), which is caused by E. coli strains that produce Shiga-like toxin (Stx). In this study, we investigated the influence of Stx-1 on nitric oxide (NO) production by human glomerular microvascular endothelial cells (GMVEC) and human mesangial cells. NO synthesis by human mesangial cells is in the micromolar range and that of GMVEC in the picomolar range. Stx-1 reduced NO production in non-stimulated GMVEC (5 nmol/l Stx-1 required) without inhibition of protein synthesis. In non-stimulated and TNFalpha-pretreated mesangial cells, NO production was reduced with a maximal reduction at 10 fmol/l shiga toxin. The cellular iNOS antigen content in mesangial cells was reduced in a concentration-dependent way (10 fmol/l-100 pmol/l), while partial inhibition of protein synthesis required 10 nmol/l Stx-1 in these cells. Our in vitro data suggest that Stx may reduce NO synthesis during the course of HUS development, contributing to the aggravation of the thrombotic microangiopathy and renal failure as observed in HUS.
Shiga-toxin (Stx)-producing Escherichia coli hemolytic-uremic syndrome (STEC-HUS) is one of the most common causes of acute kidney injury in children. Stx-mediated endothelial injury initiates the cascade leading to thrombotic microangiopathy (TMA), still the exact pathogenesis remains elusive. Interestingly, there is wide variability in clinical presentation and outcome. One explanation for this could be the enhancement of TMA through other factors. We hypothesize that heme, as released during extensive hemolysis, contributes to the etiology of TMA. Plasma levels of heme and its scavenger hemopexin and degrading enzyme heme-oxygenase-1 (HO-1) were measured in 48 STEC-HUS patients. Subsequently, the effect of these disease-specific heme concentrations, in combination with Stx, was assessed on primary human glomerular microvascular endothelial cells (HGMVECs). Significantly elevated plasma heme levels up to 21.2 µM were found in STEC-HUS patients compared to controls and were inversely correlated with low or depleted plasma hemopexin levels (R2 −0.74). Plasma levels of HO-1 are significantly elevated compared to controls. Interestingly, especially patients with high heme levels (n = 12, heme levels above 75 quartile range) had high plasma HO-1 levels with median of 332.5 (86–720) ng/ml (p = 0.008). Furthermore, heme is internalized leading to a significant increase in reactive oxygen species production and stimulated both nuclear translocation of NF-κB and increased levels of its target gene (tissue factor). In conclusion, we are the first to show elevated heme levels in patients with STEC-HUS. These increased heme levels mediate endothelial injury by promoting oxidative stress and a pro-inflammatory and pro-thrombotic state. Hence, heme may be a contributing and driving factor in the pathogenesis of STEC-HUS and could potentially amplify the cascade leading to TMA.
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