James Brunton

Interferon-Mediated Immunopathological Events Are Associated with Atypical Innate and Adaptive Immune Responses in Patients with Severe Acute Respiratory Syndrome

ABSTRACT It is not understood how immune inflammation influences the pathogenesis of severe acute respiratory syndrome (SARS). One area of strong controversy is the role of interferon (IFN) responses in the natural history of SARS. The fact that the majority of SARS patients recover after relatively moderate illness suggests that the prevailing notion of deficient type I IFN-mediated immunity, with hypercytokinemia driving a poor clinical course, is oversimplified. We used proteomic and genomic technology to systematically analyze host innate and adaptive immune responses of 40 clinically well-described patients with SARS during discrete phases of illness from the onset of symptoms to discharge or a fatal outcome. A novel signature of high IFN-α, IFN-γ, and IFN-stimulated chemokine levels, plus robust antiviral IFN-stimulated gene (ISG) expression, accompanied early SARS sequelae. As acute illness progressed, SARS patients entered a crisis phase linked to oxygen saturation profiles. The majority of SARS patients resolved IFN responses at crisis and expressed adaptive immune genes. In contrast, patients with poor outcomes showed deviated ISG and immunoglobulin gene expression levels, persistent chemokine levels, and deficient anti-SARS spike antibody production. We contend that unregulated IFN responses during acute-phase SARS may culminate in a malfunction of the switch from innate immunity to adaptive immunity. The potential for the use of the gene signatures we describe in this study to better assess the immunopathology and clinical management of severe viral infections, such as SARS and avian influenza (H5N1), is therefore worth careful examination.

Cloning and nucleotide sequence of the eae gene homologue from enterohemorrhagic Escherichia coli serotype O157:H7

The eae gene has recently been shown to be necessary for the attaching and effacing (AE) activity of enteropathogenic Escherichia coli (EPEC) on intestinal epithelial cells. In this paper we report the cloning and nucleotide sequence of a similar gene from a strain of enterohemorrhagic E. coli (EHEC) serotype O157:H7. An EHEC eae sequence was identified which was 97% homologous to the EPEC eae gene for the first 2200 bp and 59% homologous over the last 800 bp. Both eae sequences show 50% homology to the central region of the Yersinia pseudotuberculosis inv gene. The receptor-binding domain of the inv gene product lies near the carboxyl terminus. This suggests that the predicted amino acid sequence divergence in the carboxyl termini of the eae gene products might result in different antigenic and receptor specificity of these putative adhesins.

Is Surveillance for Multidrug-Resistant Enterobacteriaceae an Effective Infection Control Strategy in the Absence of an Outbreak?

Multidrug-resistant enterobacteriaceae (MDRE) are an important cause of nosocomial infections. The effectiveness of screening for MDRE in the nonoutbreak setting in an attempt to prevent transmission is unknown. Patients admitted for new organ transplantation were screened for MDRE colonization. Prospective clinical data were collected, and pulsed-field gel electrophoresis and plasmid and integron analysis of isolates were performed. Colonized patients were not isolated except when required by standard precautions. Of the 287 patients, 69 (24%) were colonized, and 6 (9%) of the 69 developed clinical infections. Most colonizing isolates (66/69) were unique. No clinical infections resulted from patient-to-patient transmission. Analysis of clinical isolates from nonstudy patients demonstrated no evidence of transmission leading to clinical disease. The annual cost of a surveillance program was calculated at Canadian

The CXCR4/CXCR7/SDF-1 pathway contributes to the pathogenesis of Shiga toxin–associated hemolytic uremic syndrome in humans and mice

1,130,184.44. Thus, the routine and costly use of MDRE surveillance and isolation precautions are not warranted in the absence of a clonal outbreak in this population.

The identification of three biologically relevant globotriaosyl ceramide receptor binding sites on the Verotoxin 1 B subunit

Hemolytic uremic syndrome (HUS) is a potentially life-threatening condition. It often occurs after gastrointestinal infection with E. coli O157:H7, which produces Shiga toxins (Stx) that cause hemolytic anemia, thrombocytopenia, and renal injury. Stx-mediated changes in endothelial phenotype have been linked to the pathogenesis of HUS. Here we report our studies investigating Stx-induced changes in gene expression and their contribution to the pathogenesis of HUS. Stx function by inactivating host ribosomes but can also alter gene expression at concentrations that minimally affect global protein synthesis. Gene expression profiling of human microvascular endothelium treated with Stx implicated a role for activation of CXCR4 and CXCR7 by their shared cognate chemokine ligand (stromal cell-derived factor-1 [SDF-1]) in Stx-mediated pathophysiology. The changes in gene expression required a catalytically active Stx A subunit and were mediated by enhanced transcription and mRNA stability. Stx also enhanced the association of CXCR4, CXCR7, and SDF1 mRNAs with ribosomes. In a mouse model of Stx-mediated pathology, we noted changes in plasma and tissue content of CXCR4, CXCR7, and SDF-1 after Stx exposure. Furthermore, inhibition of the CXCR4/SDF-1 interaction decreased endothelial activation and organ injury and improved animal survival. Finally, in children infected with E. coli O157:H7, plasma SDF-1 levels were elevated in individuals who progressed to HUS. Collectively, these data implicate the CXCR4/CXCR7/SDF-1 pathway in Stx-mediated pathogenesis and suggest novel therapeutic strategies for prevention and/or treatment of complications associated with E. coli O157:H7 infection.

Neurological manifestations of West Nile virus infection.

The Verotoxin 1 (VT1) B subunit binds to the glycosphingolipid receptor globotriaosylceramide (Gb3). Receptor‐binding specificity is associated with the terminally linked Galα(1–4) Galβ disaccharide sequence of the receptor. Recently, three globotriose (Galα[1–4] Galβ [1–4] Glcβ) binding sites per B‐subunit monomer were identified by crystallography. Two of these sites (sites I and II) are located adjacent to phenylalanine‐30. Site I was originally predicted as a potential Gb3 binding site on the basis of sequence conservation, and site II was additionally predicted based on computer modelling and receptor docking. The third (site III) was also identified by crystallography and is located at the N‐terminal end of the α‐helix. To determine the biological significance of sites II and III, and to support our previous findings of the significance of site I, we examined the binding properties and cytotoxicity of VT1 mutants designed to block Gb3 binding at each site selectively. The Scatchard analysis of saturation‐binding data for each mutant revealed that only the amino acid substitutions predicted to affect site I (D‐17E) or site II (G‐62T) caused reductions in the binding affinity and capacity of VT1 for Gb3. Similarly, those mutations at sites I and II also caused significant reductions in both Vero and MRC‐5 cell cytotoxicity (by seven and five logs, respectively, for G‐62T and by four and two logs, respectively, for D‐17E). In contrast, the substitution of alanine for W‐34 at site III did not reduce the high‐affinity binding of the B subunit, despite causing a fourfold reduction in the receptor‐binding capacity. The corresponding mutant W‐34A holotoxin had a two‐log reduction in cytotoxicity on Vero cells and no statistically significant reduction on MRC‐5 cells. We conclude that the high‐affinity receptor binding most relevant for cell cytotoxicity occurs at sites I and II. In contrast, site III appears to mediate the recognition of additional Gb3 receptor epitopes but with lower affinity. Our results support the significance of the indole ring of W‐34 for binding at this site.

Foscarnet therapy of cytomegalovirus retinitis in AIDS

BACKGROUND Over the past four years, West Nile virus (WNV) has become a significant health issue in North America. In 2002, WNV infection made its first appearance in the human population in Canada. METHODS Patients who presented to the University Health Network and Mount Sinai Hospital in Toronto with neurological disease attributed to WNV infection were identified and followed by the neurology service. Clinical features and results of laboratory, electrodiagnostic, radiological and pathological studies are presented. RESULTS In August and September 2002, 26 patients were admitted with WNV infection; 14 presented with neurological illness. Encephalitis was the most common presentation (11 patients). Eleven patients developed neuromuscular disease; two at presentation and nine after encephalitis. While the majority had a motor process that localized to the anterior horn cell and/or motor neuron, two patients had evidence of a demyelinating neuropathy and one a sensorimotor axonal neuropathy. Less common manifestations included rhombencephalitis, ataxia, myelopathy and parkinsonism. Death occurred in four patients; two > 75 years of age, and two who were immunocompromised. CONCLUSIONS The most common neurological manifestation of WNV infection was encephalitis with subsequent neuromuscular involvement. The diversity of clinical and pathological findings, however, suggests widespread involvement of the central and peripheral nervous system. A poorer prognosis for neurological recovery and overall survival was seen in older and immunocompromised patients.

Lack of Efficacy of Oral Bacitracin Plus Doxycycline for the Eradication of Stool Colonization with Vancomycin-Resistant Enterococcus faecium

Cytomegalovirus (CMV) retinitis is the most common cause of blindness in AIDS. Twenty patients were treated with a 21-day course of foscarnet therapy by continuous infusion. Response to therapy was good in eight (47%) of 17 evaluable patients; partial arrest of progression was observed in eight (47%); and no response was obtained in one (6%). Foscarnet therapy did not lead to suppression of urinary excretion of CMV in four of 12 patients who nonetheless had improvement in retinal lesions. Toxic effects, especially reversible renal failure, were common, with blood creatinine increase in 50% and dialysis in two patients. Renal toxicity occurred primarily during the third week of therapy. Anemia (hemoglobin <80 g/L) occurred in 10 patients after a mean of 14.5 + 5.1 days of therapy and required transfusion. Review of this study and of data from a previous case series, however, was inconclusive regarding the additional benefit of a third week of therapy. Maintenance therapy was given to seven patients. Four had recurrence of CMV retinitis at a mean interval of 62 + 52 days. Only one patient has maintained prolonged remission on maintenance (>24 weeks). Toxicity on the maintenance protocol included anemia (two of seven patients) and increased creatinine blood levels (one of seven patients). Zidovudine therapy in six patients did not contribute to increased toxicity of induction or maintenance therapy. Drug levels during continuous infusion were stable for individual patients but showed wide inter-patient variability. Peak levels of post-maintenance infusion varied both within and between patients.

A blinded comparison of three laboratory protocols for the identification of patients colonized with methicillin-resistant Staphylococcus aureus.

In a prospective observational cohort study designed to assess the role of oral bacitracin solution plus doxycycline in the eradication of intestinal carriage of vancomycin-resistant Enterococcus faecium (VREF) in patients on a renal ward, rectal swab specimens were obtained from 15 treated and 24 control patients. Cultures of the rectal swabs were negative for 15 (100%) of the antibiotic-treated vs. eight (33.3%) of the untreated patients (P < .001) on day 14. However, follow-up for a mean of 127 and 130 days revealed 9 of 15 (60%) and 15 of 24 (62.5%) in the treated and untreated cohorts (P = .86), respectively, carried VREF intermittently or persistently. Quantitative VREF stool cultures in the treated cohort revealed an initial 3.1-log10/g decrease, but there was an increase to pretreatment levels at 2-4 and 5-7 weeks post-treatment (7.8 and 7.4 log10/g). Oral bacitracin and doxycycline were not efficacious in reducing the carriage of VREF beyond the 2-week interval during which they were given.

A multicenter randomized double-blind placebo-controlled trial of adjunctive corticosteroids in the treatment of Pneumocystis carinii pneumonia complicating the acquired immune deficiency syndrome.

OBJECTIVE To compare three laboratory screening protocols for the detection of methicillin-resistant Staphylococcus aureus (MRSA) from surveillance specimens (mannitol-salt agar containing 2 microg/mL of oxacillin [MSA-2], mannitol-salt agar containing 4 microg/mL of oxacillin [MSA-4], and a broth-containing protocol as recommended by the American Society for Microbiology [M-ASM]). DESIGN Blinded comparative laboratory study and cost analysis. SETTING University-affiliated microbiology laboratory. METHODS Outcome measurements included rate of detection of MRSA-positive specimens and patients, turnaround time, and media and technologist costs. All MRSA culture swabs obtained from any patient site from November 1998 to April 1999 were included. RESULTS The M-ASM protocol detected between 19.1% and 32.0% more MRSA-positive specimens and between 13.3% and 23.3% more MRSA-positive patients per surveillance event than the MSA-4 and MSA-2 protocols, respectively. There was no difference in positive-culture reporting time between the M-ASM and MSA4 protocols. The broth-containing protocol was 2- to 2.5-fold more expensive than the simpler protocols, taking into account media and laboratory personnel costs. CONCLUSIONS It remains to be determined whether it is cost beneficial for a hospital to adopt the M-ASM, as the potential cost of MRSA transmission from unidentified MRSA-colonized patients is unknown. A broth-containing protocol should be considered the gold standard in future studies examining newer MRSA screening protocols