Antibody Response to Shiga Toxins in Argentinean Children with Enteropathic Hemolytic Uremic Syndrome at Acute and Long-Term Follow-Up Periods

Shiga toxin (Stx)-producing Escherichia coli (STEC) infection is associated with a broad spectrum of clinical manifestations that include diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). Systemic Stx toxemia is considered to be central to the genesis of HUS. Distinct methods have been used to evaluate anti-Stx response for immunodiagnostic or epidemiological analysis of HUS cases. The development of enzyme-linked immunosorbent assay (ELISA) and western blot (WB) assay to detect the presence of specific antibodies to Stx has introduced important advantages for serodiagnosis of HUS. However, application of these methods for seroepidemiological studies in Argentina has been limited. The aim of this work was to develop an ELISA to detect antibodies against the B subunit of Stx2, and a WB to evaluate antibodies against both subunits of Stx2 and Stx1, in order to analyze the pertinence and effectiveness of these techniques in the Argentinean population. We studied 72 normal healthy children (NHC) and 105 HUS patients of the urban pediatric population from the surrounding area of Buenos Aires city. Using the WB method we detected 67% of plasma from NHC reactive for Stx2, but only 8% for Stx1. These results are in agreement with the broad circulation of Stx2-expressing STEC in Argentina and the endemic behavior of HUS in this country. Moreover, the simultaneous evaluation by the two methods allowed us to differentiate acute HUS patients from NHC with a great specificity and accuracy, in order to confirm the HUS etiology when pathogenic bacteria were not isolated from stools

Interactions of Shiga-like toxin with human peripheral blood monocytes

The cytotoxic effect of Shiga-like toxin (Stx; produced by certain Escherichia coli strains) plays a central role in typical hemolytic uremic syndrome (HUS). It damages the renal endothelium by inhibiting the cellular protein synthesis. Also, the monocyte has a specific receptor for Stx but is not sensitive for the cytotoxic effect. In this work, monocytes were studied as a potential transporter for Stx to the renal endothelium. Coincubation of isolated human monocytes loaded with Stx and target cells (vero cells and human umbilical vascular endothelial cells) were performed. Transfer was determined by measuring the protein synthesis of target cells and by flow cytometry. Furthermore, the effect of a temperature shift on loaded monocytes was investigated. Stx-loaded monocytes reduced the protein synthesis of target cells. After adding an antibody against Stx, incomplete recovery occurred. Also, adding only the supernatant of coincubation was followed by protein synthesis inhibition. Stx detached from its receptor on the monocyte after a change in temperature, and no release was detected without this temperature shift. Although the monocyte plays an important role in the pathogenesis of HUS, it has no role in the transfer of Stx

Predicting Hemolytic Uremic Syndrome and Renal Replacement Therapy in Shiga Toxin-producing Escherichia coli-infected Children.

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) infections are leading causes of pediatric acute renal failure. Identifying hemolytic uremic syndrome (HUS) risk factors is needed to guide care. METHODS: We conducted a multicenter, historical cohort study to identify features associated with development of HUS (primary outcome) and need for renal replacement therapy (RRT) (secondary outcome) in STEC-infected children without HUS at initial presentation. Children agedeligible. RESULTS: Of 927 STEC-infected children, 41 (4.4%) had HUS at presentation; of the remaining 886, 126 (14.2%) developed HUS. Predictors (all shown as odds ratio [OR] with 95% confidence interval [CI]) of HUS included younger age (0.77 [.69-.85] per year), leukocyte count ≥13.0 × 103/μL (2.54 [1.42-4.54]), higher hematocrit (1.83 [1.21-2.77] per 5% increase) and serum creatinine (10.82 [1.49-78.69] per 1 mg/dL increase), platelet count \u3c250 \u3e× 103/μL (1.92 [1.02-3.60]), lower serum sodium (1.12 [1.02-1.23 per 1 mmol/L decrease), and intravenous fluid administration initiated ≥4 days following diarrhea onset (2.50 [1.14-5.46]). A longer interval from diarrhea onset to index visit was associated with reduced HUS risk (OR, 0.70 [95% CI, .54-.90]). RRT predictors (all shown as OR [95% CI]) included female sex (2.27 [1.14-4.50]), younger age (0.83 [.74-.92] per year), lower serum sodium (1.15 [1.04-1.27] per mmol/L decrease), higher leukocyte count ≥13.0 × 103/μL (2.35 [1.17-4.72]) and creatinine (7.75 [1.20-50.16] per 1 mg/dL increase) concentrations, and initial intravenous fluid administration ≥4 days following diarrhea onset (2.71 [1.18-6.21]). CONCLUSIONS: The complex nature of STEC infection renders predicting its course a challenge. Risk factors we identified highlight the importance of avoiding dehydration and performing close clinical and laboratory monitoring

Shiga Toxin Binding to Glycolipids and Glycans

Background: Immunologically distinct forms of Shiga toxin (Stx1 and Stx2) display different potencies and disease outcomes, likely due to differences in host cell binding. The glycolipid globotriaosylceramide (Gb3) has been reported to be the receptor for both toxins. While there is considerable data to suggest that Gb3 can bind Stx1, binding of Stx2 to Gb3 is variable. Methodology: We used isothermal titration calorimetry (ITC) and enzyme-linked immunosorbent assay (ELISA) to examine binding of Stx1 and Stx2 to various glycans, glycosphingolipids, and glycosphingolipid mixtures in the presence or absence of membrane components, phosphatidylcholine, and cholesterol. We have also assessed the ability of glycolipids mixtures to neutralize Stx-mediated inhibition of protein synthesis in Vero kidney cells. Results: By ITC, Stx1 bound both Pk (the trisaccharide on Gb3) and P (the tetrasaccharide on globotetraosylceramide, Gb4), while Stx2 did not bind to either glycan. Binding to neutral glycolipids individually and in combination was assessed by ELISA. Stx1 bound to glycolipids Gb3 and Gb4, and Gb3 mixed with other neural glycolipids, while Stx2 only bound to Gb3 mixtures. In the presence of phosphatidylcholine and cholesterol, both Stx1 and Stx2 bound well to Gb3 or Gb4 alone or mixed with other neutral glycolipids. Pre-incubation with Gb3 in the presence of phosphatidylcholine and cholesterol neutralized Stx1, but not Stx2 toxicity to Vero cells

Lipopolysaccharide Renders Transgenic Mice Expressing Human Serum Amyloid P Component Sensitive to Shiga Toxin 2

Transgenic C57BL/6 mice expressing human serum amyloid P component (HuSAP) are resistant to Shiga toxin 2 (Stx2) at dosages that are lethal in HuSAP-negative wild-type mice. However, it is well established that Stx2 initiates extra-intestinal complications such as the haemolytic-uremic syndrome despite the presence of HuSAP in human sera. We now demonstrate that co-administering purified Escherichia coli O55 lipopolysaccharide (LPS), at a dosage of 300 ng/g body weight, to HuSAP-transgenic mice increases their susceptibility to the lethal effects of Stx2. The enhanced susceptibility to Stx2 correlated with an increased expression of genes encoding the pro-inflammatory cytokine TNFα and chemokines of the CXC and CC families in the kidneys of LPS-treated mice, 48 hours after the Stx2/LPS challenge. Co-administering the glucocorticoid dexamethasone, but not the LPS neutralizing cationic peptide LL-37, protected LPS-sensitized HuSAP-transgenic mice from lethal doses of Stx2. Dexamethasone protection was specifically associated with decreased expression of the same inflammatory mediators (CXC and CC-type chemokines and TNFα) linked to enhanced susceptibility caused by LPS. The studies reveal further details about the complex cascade of host-related events that are initiated by Stx2 as well as establish a new animal model system in which to investigate strategies for diminishing serious Stx2-mediated complications in humans infected with enterohemorrhagic E. coli strains

Clinical Use and Therapeutic Potential of IVIG/SCIG, Plasma-Derived IgA or IgM, and Other Alternative Immunoglobulin Preparations

Intravenous and subcutaneous immunoglobulin preparations, consisting of IgG class antibodies, are increasingly used to treat a broad range of pathological conditions, including humoral immune deficiencies, as well as acute and chronic inflammatory or autoimmune disorders. A plethora of Fab- or Fc-mediated immune regulatory mechanisms has been described that might act separately or in concert, depending on pathogenesis or stage of clinical condition. Attempts have been undertaken to improve the efficacy of polyclonal IgG preparations, including the identification of relevant subfractions, mild chemical modification of molecules, or modification of carbohydrate side chains. Furthermore, plasma-derived IgA or IgM preparations may exhibit characteristics that might be exploited therapeutically. The need for improved treatment strategies without increase in plasma demand is a goal and might be achieved by more optimal use of plasma-derived proteins, including the IgA and the IgM fractions. This article provides an overview on the current knowledge and future strategies to improve the efficacy of regular IgG preparations and discusses the potential of human plasma-derived IgA, IgM, and preparations composed of mixtures of IgG, IgA, and IgM

Mutation analysis of 18 nephronophthisis associated ciliopathy disease genes using a DNA pooling and next generation sequencing strategy

Background Nephronophthisis associated ciliopathies (NPHP-AC) comprise a group of autosomal recessive cystic kidney diseases that includes nephronophthisis (NPHP), Senior-Loken syndrome (SLS), Joubert syndrome (JBTS), and Meckel-Gruber syndrome (MKS). To date, causative mutations in NPHP-AC have been described for 18 different genes, rendering mutation analysis tedious and expensive. To overcome the broad genetic locus heterogeneity, a strategy of DNA pooling with consecutive massively parallel resequencing (MPR) was devised.Methods In 120 patients with severe NPHP-AC phenotypes, five pools of genomic DNA with 24 patients each were prepared which were used as templates in order to PCR amplify all 376 exons of 18 NPHP-AC genes (NPHP1, INVS, NPHP3, NPHP4, IQCB1, CEP290, GLIS2, RPGRIP1L, NEK8, TMEM67, INPP5E, TMEM216, AHI1, ARL13B, CC2D2A, TTC21B, MKS1, and XPNPEP3). PCR products were then subjected to MPR on an Illumina Genome-Analyser and mutations were subsequently assigned to their respective mutation carrier via CEL I endonuclease based heteroduplex screening and confirmed by Sanger sequencing.Results For proof of principle, DNA from patients with known mutations was used and detection of 22 out of 24 different alleles (92% sensitivity) was demonstrated. MPR led to the molecular diagnosis in 30/120 patients (25%) and 54 pathogenic mutations (27 novel) were identified in seven different NPHP-AC genes. Additionally, in 24 patients only single heterozygous variants of unknown significance were found.Conclusions The combined approach of DNA pooling followed by MPR strongly facilitates mutation analysis in broadly heterogeneous single gene disorders. The lack of mutations in 75% of patients in this cohort indicates further extensive heterogeneity in NPHP-AC