Abstract: INTRODUCTION: Pulmonary toxicity of delayed onset is a rare complication of B-lymphocyte depleting antibody therapy and has been almost exclusively reported in older patients with B-cell malignancies. AIMS: To describe a pediatric patient with rituximab-associated lung injury (RALI), to systematically analyze previous reports of pulmonary complications, and to summarize common clinico-pathological features, treatment, and outcome. RESULTS: A teenage boy with focal segmental glomerulosclerosis (FSGS) presented with progressive dyspnea, fever, hypoxemia and fatigue 18 days after the completion of a second course of rituximab infusions for calcineurin inhibitor-dependent nephrotic syndrome. Respiratory symptoms started while he received high-dose prednisone for persistent proteinuria. Bilateral, diffuse ground-glass infiltrates corresponded to the presence of inflammatory cells in the bronchioalveolar lavage fluid. Empiric antibiotic treatment including clarithromycin was given, but the microbiological work-up remained negative. Serum IgE, C3, and C4 concentrations were normal. He recovered within 3 weeks after onset.We systematically reviewed 23 reports describing 30 additional cases of rituximab-associated lung disease. Twenty eight patients had received rituximab for B-cell malignancies, one for graft-versus-host disease and one for immune thrombocytopenia. Median age was 64 years (interquartile range [IQR] 58-69 years). Seventy one percent received concomitant chemotherapy. Time to onset from the last rituximab dose was 14 days (IQR 11-22 days). Eleven of 31 patients required mechanical ventilation, and 9 died (29%). Ventilation was a significant predictor of fatal outcome (odds ratio 46.7; confidence interval 9.5-229.9). High dose glucocorticoid therapy did not improve survival or prevent severe lung disease or death. CONCLUSIONS: With the expanding use of rituximab for novel indications, additional cases of RALI affecting younger age groups are expected to emerge. Mechanical ventilation predicts poor outcome. Glucocorticoids may not be protective.
Abstract: Shiga toxin (Stx)-producing Escherichia coli (STEC)-induced enteropathic HUS (eHUS) is a major cause of acute kidney injury in children and substantial morbidity and mortality in elderly patients. Systemic intestinal absorption of Stx and rapid uptake, through its glycolipid receptor (Gb3), by small vessel endothelial cells, are essential steps in the pathophysiology of STEC disease. HUS is characterized by intravascular hemolytic anemia, thrombocytopenia and acute kidney injury (AKI) that develop abruptly within a week of onset of STEC diarrhea/colitis. Subtle thrombotic changes, attributed to Stx-mediated endothelial injury, may not be limited to HUS. Current treatment of STEC disease targets gastrointestinal, hematological, vascular and renal complications. It includes isotonic volume replacement/expansion, red blood cell and platelet transfusion and, for severe AKI, hemo- or peritoneal dialysis. Plasma exchange is not indicated for eHUS. Novel strategies are being designed for disease prevention or amelioration, including STEC-component vaccines (Stx, protective antigens), toxin neutralizers (Stx-neutralizing monoclonal antibodies [STmAb], Gb3 mimics), and small molecules that block Stx-induced, pathogenic cellular pathways of cell activation/apoptosis. Receptor mimics and STmAb, given parenterally up to 48-72 h after oro-gastric infection, protect experimental animals from otherwise lethal outcomes. Phase II/III mAb studies are planned; however, the narrow, hypothetical therapeutic window makes treatment trials challenging.
Abstract: Shiga toxin (Stx)-producing Escherichia coli (STEC) causes hemorrhagic colitis and hemolytic-uremic syndrome (HUS). The rates of STEC infection and complications, including death, are highest among young children and elderly individuals. There are no causal therapies. Because Stx is the primary pathological agent leading to organ injury in patients with STEC disease, therapeutic antibodies are being developed to neutralize systemically absorbed toxin during the early phase of the infection. Two phase I, single-dose, open-label, nonrandomized studies were conducted to evaluate the safety and pharmacokinetics of the chimeric monoclonal antibodies (antitoxins) against Stx 1 and 2 (calphaStx1 and calphaStx2, respectively). In the first study, 16 volunteers received 1 or 3 mg/kg of body weight of calphaStx1 or calphaStx2 as a single, short (1-h) intravenous infusion (n = 4 per group). In a second study, 10 volunteers received a 1-h infusion of calphaStx1 and calphaStx2 combined at 1 or 3 mg/kg (n = 5 per group). Treatment-emergent adverse events were mild, resolved spontaneously, and were generally unrelated to the antibody infusion. No serious adverse events were observed. Human antichimeric antibodies were detected in a single blood sample collected on day 57. Antibody clearance was slightly greater for calphaStx1 (0.38 +/- 0.16 ml/h/kg [mean +/- standard deviation]) than for calphaStx2 (0.20 +/- 0.07 ml/h/kg) (P = 0.0013, t test). The low clearance is consistent with the long elimination half-lives of calphaStx1 (190.4 +/- 140.2 h) and calphaStx2 (260.6 +/- 112.4 h; P = 0.151). The small volume of distribution (0.08 +/- 0.05 liter/kg, combined data) indicates that the antibodies are retained within the circulation. The conclusion is that calphaStx1 and calphaStx2, given as individual or combined short intravenous infusions, are well tolerated. These results form the basis for future safety and efficacy trials with patients with STEC infections to ameliorate or prevent HUS and other complications.
Abstract: A 13-year-old girl with obesity and hyperinsulinism developed steroid-resistant nephrotic syndrome due to collapsing glomerulopathy with dominant C1q-containing mesangial immune deposits (CG/C1qN). She became overtly diabetic while receiving alternate-day prednisone and tacrolimus, requiring insulin injections. Despite the addition of mycophenolate mofetil to the treatment regimen, renal function subsequently declined. Rituximab (four weekly doses of 375 mg/m2) was tried 6 months after initial presentation and 3 months after weaning all glucocorticoids. Glomerular filtration rate (GFR) and proteinuria improved. Unexpectedly, blood sugar control normalized 6 weeks after antibody infusion. Rituximab was readministered 20 months after the first course because of deteriorating renal function, but the effect on GFR and proteinuria was modest. A retrospective analysis revealed that tubulointerstitial infiltrates present in the biopsies prior to treatment with rituximab contained numerous CD20+ and CD3+ (CD4 > CD8) lymphocyte aggregates. Rebiopsy 10 weeks after repeat rituximab therapy demonstrated the elimination of B-cell infiltrates and the apparent decrease of interstitial T-cell infiltrates, yet persistent, advanced global glomerulosclerosis, interstitial fibrosis and tubular atrophy. In conclusion, CG/C1qN was associated with B- and T-cell-rich tubulointerstitial infiltrates. B-cell-directed therapy delayed clinical progression during early disease but failed to prevent or ameliorate chronic changes, despite effective tissue B-cell clearance. The incidental resolution of diabetes was noted after rituximab treatment.
Abstract: Galloway-Mowat syndrome (GMS) is a rare autosomal recessive disorder characterized by early onset nephrotic syndrome and microcephaly with various anomalies of the central nervous system. GMS likely represents a heterogeneous group of disorders with hitherto unknown genetic etiology. The clinical phenotype to some extent overlaps that of Pierson syndrome (PS), which comprises congenital nephrotic syndrome and distinct ocular abnormalities but which may also include neurodevelopmental deficits and microcephaly. PS is caused by mutations of LAMB2, the gene encoding laminin beta2. We hypothesized that GMS might be allelic to PS or be caused by defects in proteins that interact with laminin beta2. In a cohort of 18 patients with GMS or a GMS-like phenotype we therefore analyzed the genes encoding laminin beta2 (LAMB2), laminin alpha5 (LAMA5), alpha3-integrin (ITGA3), beta1-integrin (ITGB1) and alpha-actinin-4 (ACTN4), but we failed to find causative mutations in these genes. We inferred that LAMA5, ITGA3, ITGB1, and ACTN4 are not directly involved in the pathogenesis of GMS. We excluded LAMB2 as a candidate gene for GMS. Further studies are required, including linkage analysis in families with GMS to identify genes underlying this disease.
Abstract: BACKGROUND: Escherichia coli-derived Shiga toxin (Stx), the cause of the enteropathic hemolytic uremic syndrome, is a potent inducer of apoptotic cell death. The present study was performed to examine the hypothesis that Stx initiates apoptosis by activating the mitochondrial pathway involving mitochondrial-associated, pro-apoptotic Bcl-2 family proteins Bax and Bak. MATERIALS AND METHODS: To determine if Stx2-mediated apoptosis is dependent on Bax or Bak, a gene-silencing approach was employed using sequence-specific small interfering (si)RNA duplexes. Silencing of Bax and Bak protein expression in human renal proximal tubular epithelial (HK-2) cells and its effect on Shiga toxicity was assessed by immunofluorescence microscopy and Western blotting. RESULTS: Transfection of HK-2 cells, shown to be exquisitely sensitive to Stx, with siRNA duplexes successfully diminished Bak, but not Bax protein expression. In order to determine if silencing of pro-apoptotic gene expression affects Stx-induced apoptosis, HK-2 cells were transfected with Bak-specific or control siRNA, exposed to lethal concentrations of Stx2 and assessed for cleavage of poly(ADPribose) polymerase-1 (PARP) as a marker of apoptosis, using Western blot technology. We observed that siRNA-induced reduction of Bak expression levels correlated with decreased PARP cleavage. CONCLUSION: Results suggest that Stx-induced cell death involves pro-apoptotic Bak and that silencing of Bak gene expression affords partial protection against Stx-mediated apoptosis.
Abstract: Neisseria mucosa is part of the normal nasopharyngeal flora and rarely pathogenic in humans. Reports of serious infections associated with this pathogen are very unusual. A 17-year-old boy with end-stage renal disease due to IgA nephropathy presented with acute, spontaneous, symptomatic peritoneal dialysis-associated peritonitis without reported break in sterility or PD catheter exit site infection. beta-lactamase-negative N. mucosa was isolated from the dialysate effluent. Intraperitoneal antibiotic treatment with cephalothin/gentamicin for 5 days and subsequent ceftriaxone led to complete resolution of the infection. This case demonstrates that "non-pathogenic" Neisseria species can cause clinically severe peritonitis with high intraperitoneal neutrophil counts, elevated C-reactive protein levels in the peritoneal effluent (in the presented case, 27,600/mul and 3.6 mg/l, respectively) and impaired peritoneal membrane transport function. To our knowledge, this is the first case of N. mucosa peritonitis complicating chronic peritoneal dialysis in an adolescent patient.
Abstract: BACKGROUND: Nondiarrheal or Streptococcus pneumoniae-related hemolytic uremic syndrome (HUS) represents a heterogeneous group of disorders. This study was performed to: (1) describe the current incidence, causes, demographic features, hospital courses, and short-term outcomes of non-enteropathic HUS; (2) compare findings in patients with non-enteropathic HUS with those obtained from a contemporaneous cohort of children with enteropathic or diarrhea-associated HUS (D+ HUS) diagnosed and treated at the same clinical sites; and (3) identify clinical or laboratory features that differentiate these 2 groups and predict disease severity and the short-term outcome in patients with non-enteropathic HUS. METHODS: Data were collected from patients screened between 1997 and 2001 for enrollment in a multicenter trial of SYNSORB Pk (SYNSORB Biotech Inc, Calgary, Alberta, Canada) in D+ HUS, but who were ineligible because of lack of a diarrhea prodrome. The following features were recorded: age; sex; ethnicity; prodromal symptoms; cause; nadir values for hemoglobin, hematocrit, and platelet count; use of dialysis; and length of hospitalization. RESULTS: Twenty-seven of 247 children with HUS had non-enteropathic HUS (11%). Twenty-four patients (15 boys, 9 girls), whose medical records were complete and available for review, comprise the study cohort. Mean age at onset was 4.2 +/- 0.9 (SE) years. Infection caused by S pneumoniae was diagnosed in 9 patients (38%). Dialysis was performed in 17 patients (71%) for 40 +/- 27 days. Median length of hospitalization was 22 days (range, 2 to 71 days). Children with S pneumoniae-related HUS had a longer hospital stay than those with other causes of non-enteropathic HUS, but all patients with S pneumoniae-related HUS recovered kidney function. Dialysis therapy was required more often (17 of 24 versus 59 of 145 children; P = 0.025) and hospital stays were longer (median, 22 versus 9 days; P = 0.002) in children with non-enteropathic HUS compared with patients with D+ HUS who were enrolled in the SYNSORB Pk clinical trial. CONCLUSION: (1) The incidence of non-enteropathic HUS is approximately one tenth that of D+ HUS; (2) patients with non-enteropathic HUS require dialysis therapy more often and are hospitalized more than twice as long during the acute episode compared with those with D+ HUS; (3) infection caused by S pneumoniae accounts for nearly 40% of cases of non-enteropathic HUS; and (4) although S pneumoniae-related HUS is associated with a less favorable short-term course than other types of non-enteropathic HUS or D+ HUS, the long-term prognosis for recovery of renal function appears to be good in these patients.
Abstract: Escherichia coli-derived verotoxins (VT; Shiga toxins) are causally related to the pathogenesis of enteropathic hemolytic uremic syndrome (HUS). Profound hemolysis is a defining feature of the disease, but it is not known whether the acute intravascular release of heme proteins contributes to HUS pathology. This study examined the biologic effects of hemin and VT by means of tubular epithelial-derived ACHN cells. Hemin at concentrations >/=200 microM caused cell rounding, spike formation, and detachment that was morphologically distinct from verocytotoxicity. VT caused apoptosis at concentrations >100 pM, as demonstrated by nuclear segmentation and poly(ADP-ribose) polymerase cleavage, whereas hemin-mediated injury of ACHN cells grown in serum-containing medium lacked attributes of programmed cell death. Pretreatment of ACHN monolayers with sublethal concentrations (1 to 10 pM) of VT for 12 to 18 h led to superadditive hemin-mediated cytotoxicity. This effect was not limited to ACHN cells, but was similarly noted in microvascular endothelial cells. Heme catabolism is regulated by (inducible) heme oxygenase-1 (HO-1). VT abrogated HO-1 expression in ACHN cells. Stimulation of cells for 6 h with CdCl(2), which markedly increased HO-1 expression before the addition of VT, blunted subsequent hemin injury. In conclusion, VT augments hemin-induced toxicity in renal tubular epithelial cells that can be reversed by prior induction of HO-1. It is proposed that VT subverts the physiologic defense against heme proteins by interfering with the regulated expression of HO-1 and that this mechanism contributes to the renal pathology in patients with Escherichia coli-associated HUS.
Abstract: We describe a 20-year-old patient with cystic fibrosis who developed acute nonoliguric renal failure associated with inhaled tobramycin. Clinical evaluation and renal biopsy findings were consistent with aminoglycoside-induced changes. Renal failure due to inhaled aminoglycosides has not been previously reported. The incidence may rise, however, with the increased use of this treatment modality. Measurable tobramycin levels due to inhalational therapy with conventional dosing in the reported patient indicate that the drug can be systemically absorbed, and renal tubular toxicity may occur.
Abstract: Ninety-five household contacts (aged 2 months to 73 years) of patients with enteropathic hemolytic-uremic syndrome (HUS) were investigated for the presence of immunoglobulin (Ig) G antibodies to Shiga toxins Stx2 and Stx1 by Western blot assay. Thirty-one percent of the household contacts and 19% of 327 controls had anti-Stx2 IgG (heavy and light chain [H + L]), 5 and 8%, respectively, had anti-Stx1 IgG (H + L), and 3 and 2%, respectively, had both anti-Stx2 and anti-Stx1 IgG (H + L). The incidence of infections with Stx-producing Escherichia coli (STEC) was determined based on the following diagnostic criteria: STEC isolation, detection of stx gene sequences, free fecal Stx in stool filtrates, and serum IgM antibodies against E. coli O157 lipopolysaccharide. Evidence of STEC infection was observed in 25 household contacts, of whom 18 (72%) were asymptomatic and represented a potential source of infection. Six of 13 (46%) household contacts with Stx2-producing E. coli O157:H7 in stool culture developed anti-Stx2 IgG (H + L), compared to 71% of Stx2-associated HUS cases. In individuals showing anti-Stx2 IgG (H + L), the antibody response was directed against the B subunit in 69% of household contacts and 71% of controls, in contrast to 28% of HUS patients. In this investigation controls had a significant increase of the median of IgM antibodies to O157 lipopolysaccharide (LPS) with age, up to the fifth decade. The lack of disease in household contacts with B subunit-specific antibodies, as well as the significantly higher median of anti-O157 LPS IgM antibodies in controls beyond 4.9 years of age, suggests a protective role for anti-Stx and anti-O157 LPS antibodies.
Abstract: Saliva antibodies to Escherichia coli O157 were investigated as markers of the immune response in children with enteropathic hemolytic uremic syndrome (HUS). Paired serum and saliva samples were collected from 22 children with HUS during acute disease and convalescence and were tested for E. coli O157 lipopolysaccharide (LPS)-specific IgM and IgA antibodies by ELISA. Serum and saliva samples from 44 age-matched controls were used to establish the cut-off values. Elevated levels of IgM and/or IgA antibodies to O157 LPS were detected in saliva of 13/13 HUS patients with Shiga toxin-producing E. coli (STEC) O157 in stool culture and from 4 of 5 HUS patients in whom STEC were not detected. These results closely mirrored the results obtained with paired serum samples. In contrast, saliva and serum samples from four children with STEC isolates belonging to O-groups O26, O145 (n = 2), and O165 lacked detectable O157 LPS-specific antibodies. The specificity of the ELISA was confirmed by western blotting. In STEC O157 culture-confirmed cases, the sensitivity of the ELISA was 92% for saliva IgM and IgA, based on the first available sample, and 100% and 92%, respectively, when subsequent samples were included. The specificity was 98% for IgM and 100% for IgA. Children with E. coli O157 HUS demonstrate a brisk, easily detectable immune response as reflected by the presence of specific antibodies in their saliva. Saliva-based immunoassays offer a reliable, noninvasive method for the diagnosis of E. coli O157 infection in patients with enteropathic HUS.
Abstract: Escherichia coli O157 lipopolysaccharide (LPS)-specific antibodies were measured in sequential serum samples from 131 children with serologically defined E. coli O157-associated hemolytic-uremic syndrome (HUS), using an enzyme immunoassay. On the basis of evaluation of 66 children with culture-proven E. coli O157 infection and serum samples from 132 age-matched control subjects, the assay showed a sensitivity of 95%, 88%, and 74% and a specificity of 99%, 99%, and 98% for IgM, IgA, and IgG, respectively. Anti-O157 LPS antibodies decreased below the cut-off levels in >50% of the children at 11 (IgM), 5 (IgA), and 11 weeks (IgG) after onset of diarrhea and 10, 4, and 10 weeks, respectively, after the onset of HUS. Children with enteropathic HUS fail to develop a long-lasting humoral immune response to the O157 antigen. Incomplete immunity to E. coli O157 may signal a risk for recurrent infections and has implications for serodiagnostic studies.
Abstract: Fifty-five Shiga toxin (Stx)-producing Escherichia coli (STEC) O26:H11 and O26:H(-) strains isolated from humans between 1965 and 1999 in Germany and the Czech Republic were investigated for their chromosomal and plasmid characteristics. All motile (n = 23) and nonmotile (n = 32) STEC O26 strains were shown to possess the identical flagellin subunit-encoding gene (fliC). We observed a striking recent shift of the stx genotype from stx(1) to stx(2) among the STEC O26 isolates. While stx(1) was the exclusive genotype identified in our collection until 1994, 94% of the isolates obtained after 1997 possessed stx(2) either alone (71%) or together with stx(1) (23%). Plasmid profiling demonstrated a remarkable heterogeneity with respect to plasmid sizes and combinations. Southern blot analysis of plasmid DNA with probes specific to potential accessory virulence genes revealed considerable additional variability in gene composition and arrangement. Pulsed-field gel electrophoresis (PFGE) differentiated 16 subgroups among the 55 STEC O26 strains. Using these techniques we demonstrate the emergence of a new clonal subgroup characterized by PFGE pattern A and a unique combination of virulence markers including stx(2) and a single, approximately 90-kb plasmid harboring the enterhemorrhagic E. coli hlyA and etp genes. The proportion of PFGE subgroup A strains among STEC O26 isolates rose from 30% in 1996 to more than 50% in 1999. Four clusters of infections with the clonal subgroup A were identified. We conclude that the STEC serogroup O26 is diverse and that pathogenic clonal subgroups can rapidly emerge during short intervals. The extensive genetic diversity of STEC O26 provides a basis for molecular subtyping of this important non-O157 STEC serogroup.
Abstract: Shiga toxin-producing Escherichia coli (STEC) have been identified as a worldwide cause of serious human gastrointestinal disease and the life-threatening hemolytic uremic syndrome. The most common serotype implicated is E. coli O157: H7, but infections involving various non-O157 serotypes have been found with increasing frequency in many countries. Food-borne outbreaks caused by STEC can affect large numbers of people and cause serious morbidity, making the bacteria one of the most important emerging pathogens. Because there is no specific treatment of the disease currently available, there is an urgent need for effective preventive measures based on a detailed understanding of the epidemiology of STEC infections. Such measures will also be dependent on the availability of rapid, sensitive, and simple procedures for the detection of the pathogens both in human samples and in samples of nonhuman origin such as food. This review summarizes the current knowledge on the epidemiology of STEC infection and presents a survey of laboratory methods currently available for diagnosis of STEC. Special attention is given to new diagnostic procedures for the less readily detectable non-O157 STEC strains and to simple procedures, usually based on commercially available kits, that can be used in routine clinical microbiological laboratories.
Abstract: Interaction of bipartite Escherichia coli O157-derived verotoxins (VTs) 1 and 2 (Shiga toxin 1 and 2) with vascular endothelium is believed to play a central role in the pathogenesis of the thrombotic microangiopathy and ischemic lesions characteristic of hemolytic uremic syndrome and of E. coli O157-associated hemorrhagic colitis. We defined the effects of VTs on the expression of potent endothelial cell-derived regulators of vascular wall function, namely endothelin-1 (ET-1) and nitric oxide (NO). In quiescent bovine aortic endothelial cells, both VT1 and VT2, but not receptor-binding VT B-subunit which lacks N-glycosidase activity, induced concentration-dependent (0.1-10 nM) increases in steady state preproET-1 mRNA transcript levels, an effect that was maximal at 12-24 h. Metabolic-labeling experiments indicated that VTs increased preproET-1 mRNA transcript levels at concentrations that had trivial effects on nascent DNA, RNA, and protein synthesis. In contrast to preproET-1, endothelin converting enzyme-1 and endothelial constitutive NO synthase mRNA transcript levels remained unchanged. Consistent with these findings, VTs failed to modulate immunoreactive endothelial constitutive NO synthase expression and basal and calcium-dependent L-[14C]arginine to L-[14C]citrulline conversion or the NO chemiluminescence signal. The plant-derived toxin ricin, which shows a similar molecular mechanism of enzymatic ribosomal modification to VTs, caused comparable effects on these endothelial vasomediators and metabolite incorporation, at 3 log orders lower concentrations. Nuclear transcription and actinomycin D chase experiments indicated that VTs stabilize labile preproET-1 mRNA transcripts in endothelial cells. Therefore, VTs potently increase select mRNA transcript levels in endothelial cells at concentrations of toxins that have minimal effects on protein synthesis. Perturbed expression of endothelial-derived vasomediators may play a pathophysiologic role in the microvascular dysfunction that is the hallmark of hemolytic uremic syndrome and hemorrhagic colitis.
Abstract: Helicobacter pylori, the etiologic agent of chronic-active gastritis and duodenal ulcers in humans, and Helicobacter mustelae, a gastric pathogen in ferrets, bind to phosphatidylethanolamine (PE), a constituent of host gastric mucosal cells, and to gangliotetraosylceramide (Gg4) and gangliotriaosylceramide (Gg3). The effect of a bovine colostrum concentrate (BCC) on the interaction of H. pylori and H. mustelae to their lipid receptors was examined. BCC blocked attachment of both species to Gg4, Gg3, and PE. Partial inhibition of binding was observed with native bovine and human colostra. BCC lacked detectable antibodies (by immunoblotting) to H. pylori surface proteins (adhesins). However, colostral lipid extracts contained PE and lyso-PE that bound H. pylori in vitro. These results indicate that colostrum can block the binding of Helicobacter species to select lipids and that binding inhibition is conferred, in part, by colostral PE or PE derivatives. Colostral lipids may modulate the interaction of H. pylori and other adhesin-expressing pathogens with their target tissues.
Abstract: An outbreak of bloody and nonbloody diarrhoea caused by Escherichia coli O157:H7 including one case of haemolytic uraemic syndrome (HUS) and two cases of haemolytic anaemia, in five siblings (aged 2.5 to 11.3 years) and their playmate was investigated. Using sorbitol-MacConkey agar, colony blot hybridisation, and immunomagnetic separation, Shiga toxin 2-producing Escherichia coli O157:H7 was isolated from all children but the HUS patient; however, this patient had high immunoglobulin M antibody titres against Escherichia coli O157 lipopolysaccharide. Escherichia coli O157 isolates from all patients were indistinguishable in serotype, virulence properties, and genomic background, indicating that the same strain caused the infections. These data confirm the importance of person-to-person transmission.
Abstract: The incidence and time course of the immune response to Vero toxin-producing Escherichia coli (VTEC) other than O157 (O26, O55, O111, O128) were determined in 107 children with enteropathic hemolytic uremic syndrome (HUS) by ELISA and compared to antibody profiles of 125 healthy pediatric controls and 100 children with community-acquired uncomplicated diarrhea. Six of 8 HUS patients with non-O157 VTEC isolates exhibited a serologic response to the homologous lipopolysaccharide antigen similar to that of patients infected with E. coli O157. In addition, elevated IgM or IgG antibodies were demonstrated in 7 of 19 culture- and O157 serology-negative HUS patients. Serogroup-specific antibodies decreased below cutoff levels within several months after convalescence. Anti-non-O157 antibodies were found in 14% of the diarrheic controls. Unexpectedly, a high proportion (27/82) of anti-O157 lipopolysaccharide antibody-positive HUS samples reacted with LPS from E. coli O55:B5, suggesting shared epitopes with endemic VTEC O157 strains.
Abstract: Ketoconazole is known to inhibit the metabolism of cyclosporin through inhibition of cytochrome P-450. This pharmacological interaction was used in an 8-year-old renal transplant patient to successfully achieve therapeutic cyclosporin blood concentrations. The addition of ketoconazole to the cyclosporin regimen should be considered when difficulties are encountered in attaining satisfactory cyclosporin levels.
Abstract: The interaction of verotoxins (VTs) with human erythrocytes (RBCs) in vitro was investigated, with particular reference to the role of P blood group glycolipids that are structurally related to the known VT receptors. RBC binding of purified VT1, VT2, VT2c, and VT2e was detected by direct and indirect immunofluorescence. Glycolipids were extracted from defined RBCs, separated by thin-layer chromatography, and assessed for VT binding in an overlay assay by adding toxin and specific antibodies. All VTs bound to P1 phenotype (Pk, P, and P1 antigens) and P2 phenotype (Pk and P antigens) RBCs but not to p phenotype (lacking the Pk, P, and P1 antigens) RBCs. Binding of VT1 and VT2 was approximately 10-fold greater to P1 and the rare Pk2 (Pk antigen but no P1 or P antigen) phenotype cells than to P2 phenotype RBCs, whereas VT2e bound equally well to P1 and P2 phenotype cells. The VT1 and VT2 immunofluorescence results correlated with the detection of P1 and/or increased amounts of Pk (globotriaosylceramide) antigen; VT2e immunofluorescence correlated with the detection of P (globotetraosylceramide) antigen. The Pk band pattern and VT binding observed in the thin-layer chromatogram of human P1 and P phenotype RBC extracts varied from that of human kidney and Pk1 phenotype (Pk and P1 antigens) RBCs. We conclude that each VT binds to human RBCs in vitro by utilizing specific P blood group glycolipids as receptors. On P1 and P phenotype RBCs, the accessibility of the Pk antigen for VTs appeared to be restricted. The occurrence of VT-RBC binding in natural VT-producing Escherichia coli disease and its relevance for the pathophysiology of hemolytic uremic syndrome remain to be established.
Abstract: To assess the importance of infection by Verotoxin (VT) producing Escherichia coli (VTEC) in children with HUS in Central Europe, stool and/or serum samples obtained from 147 patients from 28 paediatric centres were prospectively examined for the presence of VTEC and the kinetics of faecal VT titres (FVT), and for VT neutralization titres and antibodies against E. coli O 157 lipopolysaccharide, respectively. Ninety-two percent of the patients had classic (enteropathic) HUS (E+ HUS). Evidence of VTEC infection was obtained in 86% of them. VTEC/FVT were identified in 55/118 E+ cases (47%). A prominent feature was the frequent isolation of sorbitol-fermenting, VT2-producing E. coli O 157.H-.VT1 (C600/H19) was neutralized by 9%, and VT2 (C600/933W) by 99% of the initial serum samples from E+ patients, compared to 3% (VT1) and 100% (VT2) from age-related controls. Fourfold titre rises against VT1 and/or VT2 were observed in 13/70 (19%), and significantly elevated O 157 LPS IgM and/or IgA antibodies in 106/128 (83%) of the E+ patients. The ubiquitous VT2 neutralizing principle in the serum of HUS patients as of healthy controls warrants further investigations.
Abstract: Intestinal infection by Escherichia coli O157 and other verotoxin (VT) producing E. coli has been increasingly recognized as an important factor for the causation of classic (enteropathic) hemolytic uremic syndrome (HUS) and hemorrhagic colitis (HC). Toxins most frequently involved are VT1 and VT2. As with other toxin-mediated diseases, administration of immunoglobulin (Ig) may be beneficial. However, little is known about the immune response elicited by the toxin(s), and the prevalence of VT neutralizing antibodies in the healthy population. We studied the capacity of seven Igs and a commercial plasma preparation to neutralize four different VTs (VT1, VT2, VT2c and VT2e). The results were compared with the neutralization titers (NT50%) of normal human serum samples from various age groups. Plasma products and normal sera were separated by protein G affinity chromatography to investigate the factor(s) responsible for VT neutralization. All Igs neutralized VT1 (8 to 96 NT50%). None of them inhibited VT2, VT2c or VT2e effectively. In contrast, none of 40 pediatric, and only one of 20 adult control sera (starting dilution 1:4) neutralized VT1 (25 NT50%). All 60 samples as well as the plasma preparation blocked VT2 (22 to 446 NT50%, median 137), but not VT2c and VT2e. The VT1 neutralizing activity was eluted with the IgG fraction. The VT2 neutralizing activity was not bound by protein G, but was recovered in the IgG-free effluent. In conclusion, therapeutic Igs significantly neutralize VT1, but are largely ineffective against other VTs. In contrast, all control sera inhibited VT2, but rarely VT1.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: The paper represents a cross-sectional study from a sample of 1800 out of 3000 school-children from the Braunschweiger Längsschnitt. In this methodical approach we first eliminate approximately the influence of age and stature on the raw data from all body measurements with regression equations. The transformed data were attached to three "types" named 'below normal', 'normal', and 'above normal', in course of which 'normal' means all cases in the range of the standard deviation, whereas the two other "types" are corresponding to the adjacent ranges of values. Subsequently each transformation on the mean of age and stature a discriminant analysis has been performed grouping the cases by the "types" of the width of pelvis, resp. shoulders. There were found great influences of the stature on the chosen measures of width in our investigated class of age. They could be made clear alone by using allometrical methods. Only before correction of the body height the given grouping is supported by other variables, at which different sets of variables dominate the discriminant functions for boys and girls. Out of this new aspects and considerations result for the understanding of the physique and the physique typologies, which would be significant in our opinion for acceleration phenomenon as well as for the comparative examinations on populations.
Abstract: An indirect hemagglutination assay consisting of sheep erythrocytes coated with lipopolysaccharide (LPS) from Shiga-like toxin-producing Escherichia coli O157 was used for the serological diagnosis of E. coli O157 infections in children with classical (enteropathic) hemolytic-uremic syndrome (HUS). One week after the onset of diarrhea (acute phase of the disease), the E. coli O157 antibody titer was greater than or equal to 1:4,096 in 22 of 27 patients with HUS, compared with 4 of 249 controls, the majority of whom had O157 antibody titers of between 1:4 and 1:256. This antibody response was observed in HUS patients with stool cultures positive and negative for E. coli O157. Selective absorption with homologous LPS and heterologous LPS showed that the antibody response was specific for E. coli O157. Because of its simplicity and ease of interpretation, the indirect hemagglutination assay described in this paper is recommended for the serological diagnosis of E. coli O157 infections in patients with HUS.
Abstract: Significant improvements of dialysis techniques and the supportive treatment of the hemolytic uremic syndrome (HUS) during the last three decades do not veil the fact that causal therapy is not yet feasible. This holds true for the classic HUS associated with infections by verotoxin-(VT-)producing E.coli (VTEC) as for the majority of the much less frequent "atypical" forms. The influence of antimicrobial agents on production of verotoxins produced by clinical VTEC isolates was examined. Co-trimoxazole significantly increased the total yield of toxin. In contrast, ciprofloxacin, lincomycin and gentamicin caused significant reduction in toxin yields. The pathogenetic and therapeutic relevance of this finding is not yet known. Analysis of the clinical literature for the postulated relation between antibiotic treatment and development of HUS or its impact on the severity of the disease reveals a controversial picture. Based on the dynamics of the disease, the in vitro findings, and reports on unfavourable outcomes after treatment, it is concluded that antibiotics are not indicated in most cases of the enteropathic HUS or other VTEC infections. Antibiotic prophylaxis has neither been proven to be efficacious nor safe for the prevention of secondary cases during VTEC outbreaks. Apart from the implementations of hygienic measures, prospective studies on the role of antibiotics seem clearly warranted. Possible future strategies may include the design of immunological therapeutic concepts.
Abstract: Shiga-like toxin-producing Escherichia coli strains of serogroup O157 were identified in 26 of 104 patients with hemolytic-uremic syndrome and in 18 of 668 patients with diarrhea. All strains were identified by colony hybridization with DNA probes complementary to Shiga-like toxin I and Shiga-like toxin II gene sequences and characterized by biochemical tests and serotyping. Seventeen of these 44 patients had E. coli O157 strains which were unusual because they fermented sorbitol within 24 h of incubation and were positive for beta-glucuronidase activity. Culture filtrates of these sorbitol-fermenting strains were highly toxic to Vero cells in culture. Serological tests and DNA analysis performed by restriction endonuclease digestion of B-subunit toxin genes revealed that all 17 isolates produced Shiga-like toxin II. Although by using molecular probes we established a high frequency of sorbitol-fermenting E. coli O157 strains in the patients we examined, further studies on the prevalence of such isolates in other areas of endemic disease are clearly warranted.
Abstract: Cell culture tests, DNA colony blot hybridization and polymerase chain reaction were used to examine classical enteropathogenic Escherichia coli (EPEC) for the presence of Shiga-like toxin (SLT). Fifteen of 155 strains from West Germany, originally identified as EPEC on the basis of serotyping, were shown to harbor either SLT-I or SLT-II genes. All strains that hybridized with the 20-base oligonucleotide probes which are complementary to slt-IA or slt-IIA sequences derived from the genomic DNA of enterohemorrhagic E. coli O157:H7 strain 933 produced moderate or high levels of cytotoxin in Vero and HeLa cell assays. Four additional strains of low to moderate cytotoxicity did not hybridize with either probe. Five different serogroups producing SLTs were identified: O26, O55, O111, O119 and O128. All three SLT-positive E. coli O26:H11 and four of five E. coli O111:H- isolates hybridized with a 3.4 kilobase fragment (CVD 419 probe) derived from the 60-megadalton plasmid of EHEC O157:H7. Seven of the 15 SLT-gene positive strains were associated with bloody diarrhea, six isolates were from patients with hemolytic uremic syndrome (HUS). Based on their clinical, epidemiological, pathogenic and genetic features SLT-producing E. coli among classical EPEC mimic enterohemorrhagic E. coli O157:H7 and might be considered as EHEC.
Abstract: Because the classic hemolytic-uremic syndrome has been etiologically linked to intestinal infections by Escherichia coli O157 and other verotoxin-producing E. coli (VTEC), we examined 22 consecutive children with acute hemolytic-uremic syndrome for the presence of VTEC, using microbiologic methods, and for a specific immune response to O157 lipopolysaccharide in acute-phase and follow-up sera, using the indirect hemagglutination assay and the immunoblot procedure. Of 22 children with enteropathic hemolytic-uremic syndrome, 15 (68%) had evidence of VTEC infection by culture of the pathogen or detection of free verotoxin in the feces, or both. Significantly elevated titers of short-lived agglutinins and IgM class antibodies against the O157 lipopolysaccharide were found in 20 (91%) of 22 patients, but not in two of three patients with non-O157 E. coli isolates or in healthy children or children with diarrhea caused by other enteric pathogens (p less than 0.01). The combined microbiologic and serologic procedures provided evidence for VTEC infection in all 22 patients. The high incidence of anti-O157 lipopolysaccharide antibodies in these patients indicates the predominance and the pathogenic potential of this serogroup. Both serologic techniques proved to be valuable tools to further characterize this form of hemolytic-uremic syndrome. Future studies on the induction of protective immunity seem warranted.
Abstract: In 17 of 74 patients, in the 20-40 years of age group, who had undergone an organ transplantation (kidney or liver), the course and outcome of pregnancy were evaluated. In three cases, the pregnancies ended in premature miscarriage and in five cases they were terminated for medical reasons. Nine infants were born alive between the 32nd to the 40th week of gestation, six of them spontaneously, three of them by abdominal Caesarean section. One of these infants born in the 32nd week of gestation with a birth weight of 800 grams died on the second day after birth. One infant born in the 33rd week of gestation showed incidence of a persistent ductus arteriosis Botalli. Four of the nine newborns suffered from intrauterine dystrophy. The birth weight of four further infants corresponded to the 10th to 25th percentile. Neither the incidence of a maternal varicella zoster infection in the early stages of pregnancy nor the reactivation of a herpes simplex (HSV) and cytomegalia virus infection during the pregnancy resulted in any perceptible damage to the infant or transplant. During pregnancy, three of the mothers were treated with immunosuppressants, either with a combination of azathioprine and prednisone (conventional) or cyclosporine (CSA) and prednisone, or with a combination of all three drugs (triple therapy). As opposed to the newborn of those mothers, who had been treated conventionally, the newborn of those treated with CSA showed post partum a tendency towards hypocalcaemia. Two of the mothers gave birth to their infants outside the Federal Republic of Germany.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: Oligonucleotide probes constructed from the sequences published for Shiga-like toxin I (SLT-I) and Shiga-like toxin II (SLT-II) genes and antibody against the purified toxins were used to study the SLT (SLT-IIp) produced by porcine E. coli O138 and O139 strains. By DNA hybridization assays no homology was observed between SLT-I and SLT-IIp. By contrast the oligonucleotide probe derived from the slt-II A gene detected porcine strains of E. coli producing SLT-IIp and E. coli strains associated with human disease producing SLT-II. Homology of nucleotide sequences between SLT-IIp and SLT-II is reflected by serological cross-reactivity as demonstrated by a dot blot ELISA and neutralization of SLT-IIp with anti-SLT-II. The toxins were distinguishable in their ability to kill HeLa S-3 cells. The oligonucleotide probe and anti-SLT-II can facilitate identification of SLT-IIp producing E. coli to further clarify their role in diseased pigs.
Abstract: Cytotoxin production by Escherichia coli O111:H-strain HUS-2 (Hamburg) is associated with a temperate toxin-converting bacteriophage (Tcp-111). E. coli laboratory strain C600 transduced and subsequently lysed by the phage produced and liberated large amounts of cytotoxin (CT111) which was purified by sequential chromatography. When compared with published procedures for toxin release from viable cells, lysis of the C600 culture by the phage was most effective. By SDS-PAGE CT111 as Shiga toxin from Shigella dysenteriae 1 were shown to consist of two polypeptides of MW 31 kd and 4-5 kd. Both toxins share common antigenic epitopes as revealed by immunoblotting and neutralization studies. With rabbit anti-CT111 toxic activity of only 5 out of 8 clinical E. coli O111 isolates was neutralized suggesting the presence of different cytotoxins in E. coli serogroup O111. Taken together, our data established CT111 as a potent cytotoxin with significant enterotoxic and neurotoxic properties similar or identical to Shiga toxin and to Shiga-like toxin I from E. coli O26:H11 and O157:H7 strains.
Abstract: Two immunologically distinct verotoxins purified from Escherichia coli C600, lysogenized with distinct temperate phages from E. coli strain 933 of serotype O157:H7, were compared by SDS-PAGE and different biological assays. The two toxins termed verotoxin 1 (VT1) and verotoxin 2 (VT2) differing in molecular weight exhibited similar biological activities. Both preparations were toxic for HeLa cells and lethal for mice. Epidemiological evidence of verotoxinogenesis in some cases of hemolytic-uremic syndrome (HUS) and the recent observations of inadequate prostacyclin production by endothelial cells associated with HUS prompted us to study the effect of purified verotoxins on prostacyclin synthesis in rat aortic tissue. Our results demonstrate a significant reduction of prostacyclin by both toxins at picomolar levels. The suppression of prostacyclin release by a lower concentration of VT2 as compared with VT1 reflects the relative potencies of these toxins in HeLa cell toxicity and mouse lethality. The results suggest an effect of verotoxins on endothelial cells and support the concept of these toxins as virulence factors in E. coli.
Abstract: The intra-atrial position of Silastic catheters placed in the center of the right atrium was assessed by two-dimensional echocardiography in 25 newborns. In 32% of the infants, the catheter tip was located in the patent foramen ovale, and in another 40% of the infants, the catheter tip was located close to the patent foramen ovale. This placement increases the risk of systemic air and fat embolism. Consequently, the tip of Silastic catheters should be placed just behind the confluence of the superior-inferior vena cava and the right atrium.
Abstract: The microagglutination technique for the detection of antibodies against Y. enterocolitica, serovars 3 and 9 (corresponding to O-groups I and V), was compared with the conventional tube agglutination. An immunoglobulin class specific, indirect ELISA (polyvalent immunoglobulin, IgG, IgM, and IgA) was established employing as antigens formalinized whole bacteria ("OH"-antigens) and LPS preparations (hot phenol-water extraction). ELISA titers and net absorbancy (ELISA-"units") of single serum dilutions were in good agreement; the same was true for ELISA and agglutination results. Specificity (against healthy controls) and sensitivity of both serologic techniques were comparable. Cross-reacting antibodies against serovars 3 and 9 could be identified in the ELISA. Correct serovar-specific diagnosis was possible in 95% with a single assay (polyvalent Ig assay with LPS-antigen). The sensitivity of the LPS-ELISA was superior to the "OH" antigen assay after infections by serovar 3 strains, and antibodies were detected with LPS preparations for a longer period following reconvalescence. Specific IgA, due to its rapid decrease during reconvalescence, on one hand impresses as a valuable marker for the differentiation of recent disease from uncomplicated past infections, while persistence of IgA appears to be associated with Yersinia-induced arthritis. Persisting IgM but rarely IgA titers were characteristically found in patients with prolonged enteric yersiniosis.
Abstract: Two children are described with postnatally acquired acute rubella which induced neurological disease. The first patient with restricted transverse myelitis (Th 11-12) was remarkable for the positive result obtained by the magnetic response (MR) technique of the spinal cord. In the second patient the clinical examination demonstrated a circumscribed, however severe, lasting defect in the extrapyramidal motor system with facial muscle dystonia and complete anarthria; in the latter case the CSF contained rubella specific IgM five days after the onset of exanthema. No abnormalities were noticed by MR five weeks after the clinical onset. The possible significance of MR imaging in virus-induced encephalomyelitis is discussed.
Abstract: A simple and fast isolation procedure in combination with the search for Salmonella and Shigella spp. has been applied for the screening of Yersinia enterocolitica from the faeces. The microorganisms was isolated from 31 out of 4052 patients with gastrointestinal symptoms (0.8%) and from one out of 2295 healthy individuals (0.04%). 42% of the isolates were detected from infants up to the age of 3 years. Y. enterocolitica was excreted on the average at least 22 days after the clinical onset (n = 29). In 17 out of 24 patients (71%) significant agglutinin titers were demonstrated by the first serological examination one to 13 weeks after clinical onset. Acute diarrhoea, the most prominent symptom, lasted for 15 days and was followed in one third of the cases by a protracted course of approximately eight weeks. A reliable diagnosis of "enteric yersiniosis" rests besides the uncharacteristic clinical symptoms on the isolation of the microorganism and/or the demonstration of specific antibodies in significant titers, preferable with fourfold rise or decrease.
