A Requirement for Neutrophil Glycosaminoglycans in Chemokine:Receptor Interactions Is Revealed by the Streptococcal Protease SpyCEP.

To evade the immune system, the lethal human pathogen Streptococcus pyogenes produces SpyCEP, an enzyme that cleaves the C-terminal α-helix of CXCL8, resulting in markedly impaired recruitment of neutrophils to sites of invasive infection. The basis for chemokine inactivation by SpyCEP is, however, poorly understood, as the core domain of CXCL8 known to interact with CXCL8 receptors is unaffected by enzymatic cleavage. We examined the in vitro migration of human neutrophils and observed that their ability to efficiently navigate a CXCL8 gradient was compromised following CXCL8 cleavage by SpyCEP. SpyCEP-mediated cleavage of CXCL8 also impaired CXCL8-induced migration of transfectants expressing the human chemokine receptors CXCR1 or CXCR2. Despite possessing an intact N terminus and preserved disulfide bonds, SpyCEP-cleaved CXCL8 had impaired binding to both CXCR1 and CXCR2, pointing to a requirement for the C-terminal α-helix. SpyCEP-cleaved CXCL8 had similarly impaired binding to the glycosaminoglycan heparin. Enzymatic removal of neutrophil glycosaminoglycans was observed to ablate neutrophil navigation of a CXCL8 gradient, whereas navigation of an fMLF gradient remained largely intact. We conclude, therefore, that SpyCEP cleavage of CXCL8 results in chemokine inactivation because of a requirement for glycosaminoglycan binding in productive chemokine:receptor interactions. This may inform strategies to inhibit the activity of SpyCEP, but may also influence future approaches to inhibit unwanted chemokine-induced inflammation.

Azithromycin susceptibility testing for Salmonella enterica isolates: discordances in results using MIC gradient strips.

BACKGROUND: Azithromycin resistance is emerging in typhoidal Salmonella. Confirmation of azithromycin MIC is the most frequent antibiotic susceptibility request made to the Gastrointestinal Bacteria Reference Unit (GBRU) laboratory in England by local diagnostic laboratories. OBJECTIVES: (i) Determine concordance between local diagnostic and reference laboratory estimations of azithromycin MIC by gradient strip in Salmonella enterica serovars Typhi and Paratyphi. (ii) Consider causes of variation. METHODS: Isolates from patients with enteric fever attending a central London hospital between May 2011 and April 2019 were tested for azithromycin susceptibility using gradient strips, according to EUCAST methodology. Matched local diagnostic and reference laboratory estimations of azithromycin and ciprofloxacin (as a comparator) MICs were included; concordance in estimations was examined. RESULTS: Local diagnostic laboratory readings overestimated azithromycin MIC values compared with the reference laboratory, resulting in poor concordance in susceptibility/resistance attribution (concordant susceptibility interpretation in 8/19, κ = 0). In contrast, ciprofloxacin MIC estimation demonstrated superior concordance (concordant susceptibility interpretation in 16/17, κ = 0.85). None of the isolates was resistant to azithromycin at the reference laboratory and no known genes associated with azithromycin resistance were detected in any isolate using WGS. CONCLUSIONS: Overestimation of azithromycin resistance is likely to be due to difficulty in interpreting the point of intersection of the 'trailing edge' with the gradient strip, used to determine MIC. We advise local diagnostic laboratories to review their experience and consider adopting a 'second reader' system to mitigate this.

The true impact of fatigue in primary biliary cirrhosis: a population study.

BACKGROUND & AIMS: Patient surveys suggest that fatigue is a common problem in primary biliary cirrhosis (PBC). The actual extent of the problems caused by fatigue in PBC has yet to be determined as previous studies addressing this question have tended to use selected patient subgroups and subjective or non-quantitative fatigue assessment tools. Here, we have attempted to more accurately assess the extent of fatigue in PBC, and the specificity of the symptom for this disease, by the application of an objective measure of fatigue impact (the fatigue impact score [FIS]) to a geographically based patient cohort, age- and sex-matched normal controls, and chronic liver disease controls. METHODS: Postal completion of the FIS and linked symptom assessment tools. RESULTS: Median FIS was significantly higher in patients (n = 136) than community controls (40 [0-138] vs. 28 [0-156]; P < 0.0001) and chronic liver disease controls (n = 38) (20.5 [0-145]; P < 0.05). Fatigue scores in the 11 patients who had undergone liver transplantation (median 3.5 years previously) were the same as those in non-transplanted patients with advanced disease. CONCLUSIONS: Fatigue is a significant and specific problem in PBC. It is not, however, universal and affects fewer patients than has previously been thought to be the case based on data from selected patient cohorts. This definition of the "normal range" for fatigue in PBC will assist in future studies of etiology and therapy.