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

Assessing the roles of human serum amyloid p component and neutrophils in shigatoxemia

Bibliography: p. 288-335Some pages are in colour

Recombinant Shiga Toxin B-Subunit-Keyhole Limpet Hemocyanin Conjugate Vaccine Protects Mice from Shigatoxemia

Enterohemorrhagic Escherichia coli (EHEC) causes hemorrhagic colitis in humans and, in a subgroup of infected subjects, a more serious condition called hemolytic-uremic syndrome (HUS). These conditions arise because EHEC produces two antigenically distinct forms of Shiga toxin (Stx), called Stx1 and Stx2. Despite this, the production of Stx2 by virtually all EHEC serotypes and the documented role this toxin plays in HUS make it an attractive vaccine candidate. Previously, we assessed the potential of a purified recombinant Stx2 B-subunit preparation to prevent Shigatoxemia in rabbits. This study revealed that effective immunization could be achieved only if endotoxin was included with the vaccine antigen. Since the presence of endotoxin would be unacceptable in a human vaccine, the object of the studies described herein was to investigate ways to safely augment, in mice, the immunogenicity of the recombinant Stx2 B subunit containing <1 endotoxin unit per ml. The study revealed that sera from mice immunized with such a preparation, conjugated to keyhole limpet hemocyanin and administered with the Ribi adjuvant system, displayed the highest Shiga toxin 2 B-subunit-specific immunoglobulin G1 (IgG1) and IgG2a enzyme-linked immunosorbent assay titers and cytotoxicity-neutralizing activities in Ramos B cells. As well, 100% of the mice vaccinated with this preparation were subsequently protected from a lethal dose of Stx2 holotoxin. These results support further evaluation of a Stx2 B-subunit-based human EHEC vaccine

Sociodemographic correlates of urine culture test utilization in Calgary, Alberta

Abstract Background Many clinical practice guidelines encourage diagnosis and empiric treatment of lower urinary tract infection without laboratory investigation; however, urine culture testing remains one of the largest volume tests in the clinical microbiology laboratory. In this study, we sought to determine if there were specific patient groups to which increased testing was directed. To do so, we combined laboratory data on testing rates with Census Canada sociodemographic data. Methods Urine culture testing data was obtained from the Calgary Laboratory Services information system for 2011. We examined all census dissemination areas within the city of Calgary and, for each area, testing rates were determined for age and gender cohorts. We then compared these testing rates to sociodemographic factors obtained from Census Canada and used Poisson regression and generalized estimating equations to test associations between testing rates and sociodemographic variables. Results Per capita urine culture testing is increasing in Calgary. For 2011, 100,901 individuals (9.2% of all people) received urine cultures and were included in this analysis. The majority of cultures were received from the community (67.9%). Substantial differences in rate of testing were observed across the city. Most notably, urine culture testing was drastically lower in areas of high (≥ $100000) household income (RR = 0.07, p < 0.0001) and higher employment rate (RR = 0.36, p < 0.0001). Aboriginal – First Nations status (RR = 0.29, p = 0.0008) and Chinese visible minority (RR = 0.67, p = 0.0005) were also associated with decreased testing. Recent immigration and visible minority status of South Asian, Filipino or Black were not significant predictors of urine culture testing. Females were more likely to be tested than males (RR = 2.58, p < 0.0001) and individuals aged 15–39 were the most likely to be tested (RR = 1.69, p < 0.0001). Conclusions Considerable differences exist in urine culture testing across Calgary and these are associated with a number of sociodemographic factors. In particular, areas of lower socioeconomic standing had significantly increased rates of testing. These observations highlight specific groups that should be targeted to improve healthcare delivery and, in turn, enhance laboratory utilization

Pulmonary Cryptococcus infections as a manifestation of idiopathic CD4 lymphocytopenia: case report and literature review

Abstract Background Idiopathic CD4 lymphocytopenia (ICL) is a rare clinical disease with relative CD4 deficiency in the absence of HIV infection. The pathogenicity of ICL is poorly understood with an unclear incidence rate in the general population. Sequelae of ICL includes AIDS-defining infections, which most commonly includes Cryptococcus neoformans. Typically, C. neoformans infections present with CNS involvement but rarely with extra-CNS manifestations. Here, we present a rare case of ICL with exclusively primary pulmonary cryptococcus and a review of the literature. Case presentation A 56-year-old female presented to our tertiary care hospital requiring a right hip open reduction intervention. The patient became febrile during admission, prompting a work-up that included a chest X-ray showing a peripheral pulmonary solitary nodule. Transthoracic biopsy revealed encapsulated yeast forms in keeping with C. neoformans. CD4 counts, repeated at least one month apart, were < 200 cells/mm3, with negative HIV testing. Flow cytometry and genetic testing were completed to elucidate the etiology of the immune deficiency, both of which were unremarkable. She was subsequently treated with 12 months of posaconazole with clinical resolution. Conclusions Our patient highlights a rare clinical disease, which a review of literature revealed only five cases in the literature with exclusive pulmonary Cryptococcus in ICL/ This case demonstrates the strong clinical acumen required to properly diagnose and ultimately manage the patient

The Effects of Shiga Toxin 1, 2 and Their Subunits on Cytokine and Chemokine Expression by Human Macrophage-Like THP-1 Cells

Infection by Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC) results in severe diarrhea, hemorrhagic colitis, and, occasionally, hemolytic-uremic syndrome (HUS). HUS is associated with an increase in pro-inflammatory cytokines and chemokines, many of which are produced by macrophages in the kidneys, indicating that localized host innate immunity likely plays a role in renal pathogenesis. EHEC serotypes may express one or two classes of serologically defined but structurally and functionally-related Shiga toxins called Stx1 and Stx2. Of these, Stx2 appears to be linked to higher rates of HUS than Stx1. To investigate a possible reason for this, we exposed human macrophage-like THP-1 cells to Stx1 or Stx2 and then used the Luminex multiplex system to assess cytokine/chemokine concentrations in culture supernatant solutions. This analysis revealed that, relative to Stx1, Stx2 significantly caused increased expression of GRO, G-CSF, IL-1β, IL-8 and TNFα in macrophage-like THP-1 cells. This was determined to not be due to a difference in cytotoxicity since both Stx1 and Stx2 displayed similar cytotoxic activities on macrophage-like THP-1 cells. These observations indicate that, in vitro, Stx2 can provoke a greater pro-inflammatory response than Stx1 in macrophages and provides a possible partial explanation for higher rates of HUS in patients infected with EHEC strains expressing Stx2. To begin to determine a mechanism for Shiga toxin-mediated cytokine production, we exposed macrophage-like THP-1 cells to Stx1 or Stx2 A and B subunits. Luminex analysis of cytokines in cell culture supernatant solutions demonstrated that neither subunit alone induced a cytokine response in THP-1 cells

Impact of the Nature and Size of the Polymeric Backbone on the Ability of Heterobifunctional Ligands to Mediate Shiga Toxin and Serum Amyloid P Component Ternary Complex Formation

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