Use of genome sequencing to identify hepatitis C virus transmission in a renal healthcare setting.

BACKGROUND: Hepatitis C virus (HCV) infection is a major health burden worldwide. A patient with no history of HCV infection while on a renal unit was found to seroconvert to HCV. AIM: To report the use of sequencing to postulate how transmission of HCV occurred in a healthcare setting, and how this guided our outbreak investigation. FINDINGS: Based on infection control inspections the transmission event was surmised to be due to ward environmental contamination with blood and subsequent inoculation from intravenous interventions on the patient acquiring HCV. We discuss the interventions put in place in response to the outbreak investigation findings. CONCLUSION: Sequencing of healthcare-acquired HCV infections should be undertaken as routine practice in outbreak investigations.

IL-18 is upregulated in the kidney and primes neutrophil responsiveness in ANCA-associated vasculitis.

In antineutrophil cytoplasm autoantibody (ANCA)-associated systemic vasculitis (ASV), autoantibody-induced neutrophil activation is believed to cause organ damage. In vitro, tumor necrosis factor alpha (TNFalpha) primes neutrophils for ANCA stimulation and TNFalpha blockade has been successfully used to treat ASV. Nonetheless, irreversible organ damage can still occur, suggesting that other cytokines may circumvent TNFalpha blockade. We report that interleukin (IL)-18 deposition, as assessed by immunoperoxidase staining, is increased in renal biopsies from ASV patients. Immunofluorescence microscopy demonstrated that podocytes are the predominant glomerular IL-18-positive cell type, whereas in the interstitium, myofibroblasts, distal tubular epithelium, and infiltrating macrophages stained for IL-18. In vitro, IL-18 primed superoxide production by ANCA-activated neutrophils comparably to TNFalpha. IL-18-primed, ANCA-induced superoxide production was unaffected by anti-TNFalpha antibody, which abrogated TNFalpha priming. Furthermore, TNFalpha and IL-18 phosphorylated neutrophil p38 mitogen-activated protein kinase (MAPK), but IL-18-mediated p38 MAPK phosphorylation was unaffected by anti-TNFalpha antibody. The p38 MAPK inhibitor, SB20358, reduced IL-18-primed, ANCA-induced superoxide production in a concentration-dependent manner. ANCA-induced superoxide release was also sensitive to the Leukotriene B4 (LTB4) inhibitor MK-886. IL-18 priming was not associated with increased ANCA antigen expression on isolated neutrophils. We conclude that IL-18 is likely to be important for neutrophil recruitment and priming in ASV. Therapies targeting single priming agents may have limited efficacy in controlling disease.

Anti-neutrophil cytoplasm antibody IgG subclasses in Wegener's granulomatosis: a possible pathogenic role for the IgG4 subclass.

A characteristic feature of Wegener's granulomatosis is the presence of antineutrophil cytoplasm antibodies (ANCA) to proteinase 3 (PR3). In vitro, ANCA activate neutrophils by co-ligating PR3 and FcgammaRIIa/IIIb receptors. ANCA are predominantly of the IgG isotype, and IgG1, IgG3 and IgG4 subclasses are particularly represented. To address the pathogenic role of individual ANCA-IgG subclass antibodies, patients' sera were screened using indirect immunofluorescence, enzyme-linked immunosorbent assay (ELISA) and subclass PR3-ELISA to identify patients with high titres of PR3-ANCA within the IgG1, IgG3 or IgG4 subclasses. Unfractionated ANCA-IgG and subclass fractions were isolated by affinity chromatography and compared for their capacities to stimulate superoxide production by primed human neutrophils. Donor neutrophils were analysed for constitutive and induced FcgammaRI expression by flow cytometry. The IgG1, IgG3 and IgG4 subclass fractions, isolated from three different ANCA sera, each stimulated superoxide production from neutrophils derived from multiple donors. Subsequently, IgG4 subclass fractions isolated from a further four ANCA positive sera demonstrated varying abilities to stimulate release of superoxide; unrelated to PR3-ANCA titre, neutrophil donor, or neutrophil FcgammaRI expression. The stimulation of superoxide release by IgG1- and IgG3-ANCA subclass fractions is consistent with the proposed mechanism of co-ligation of PR3 antigen and FcgammaRIIa/IIIb receptors. However, the demonstration of similar activity for the IgG4-ANCA subclass fractions isolated from some sera was unexpected. This activity was independent of neutrophil donor and expression of FcgammaRI, suggesting it was capable of activating neutrophils via constitutively expressed FcgammaRIIa/IIIb or co-ligation of other, unidentified, cell surface molecules.

New developments in the pathogenesis of ANCA-associated vasculitis.

In recent years there have been substantial developments in the understanding of the pathogenesis of ANCA-associated vasculitidies. Animal models have now been developed that finally prove a direct pathogenic role for ANCA, a subject fiercely debated since their original identification. We are also closer to understanding how ANCA exert their effects to cause disease. Progress has been made in elucidating how ANCA activate neutrophils, from how they bind antigen and where that antigen is located, to how antigen binding is translated into intracellular activity. The effects of ANCA activation on the effector functions of neutrophils and monocytes are being further dissected and the flow-based assay is allowing interactions with endothelium to be studied in more detail. Knowledge of the role of T cells has been enhanced by examining contributions to disease by differing subsets and their cytokine secretions. Defects in apoptosis playing a role in the initiation of other autoimmune diseases has prompted investigations into whether a similar pathogenesis is relevant in vasculitis, and various genetic polymorphisms have been discovered to be important in determining in whom vasculitis develops. This article reviews how recent research has helped in the understanding of the pathogenesis of small vessel vasculitis.

Antineutrophil cytoplasmic antibodies stabilize adhesion and promote migration of flowing neutrophils on endothelial cells.

OBJECTIVE: Recruitment of neutrophils to sites of inflammation requires coordinated regulation of their capture, activation, and migration on vascular endothelium. This study examines whether exposure of neutrophils to antineutrophil cytoplasmic antibodies (ANCAs) can disrupt this sequence of events. METHODS: Isolated human neutrophils were perfused in the presence or absence of ANCA-positive IgG over endothelial cells that had been activated with either 2 units/ml or 100 units/ml of tumor necrosis factor alpha (TNFalpha) for 4 hours. RESULTS: When endothelial cells were activated with 100 units/ml of TNFalpha, neutrophils were captured from flow, a small proportion of adherent cells rolled, and the majority transmigrated through the endothelial cell monolayer. When neutrophils were treated with ANCA IgG immediately before, 5 minutes before, or 15 minutes before perfusion, none rolled on contact with the endothelium, but the majority still transmigrated. When endothelial cells were activated with 2 units/ml of TNFalpha, the majority of untreated adherent neutrophils rolled, a few transmigrated, and the number that attached decreased with time during washout. In contrast, when neutrophils were pretreated with ANCA IgG just before perfusion, adhesion was stabilized, and the number of neutrophils that transmigrated was increased 10-fold. Priming of the neutrophils with TNFalpha before the addition of ANCA further increased the stability of neutrophil binding, but did not significantly increase transmigration. CONCLUSION: Rather than frustrating the transmigration process, ANCAs promoted the migration of neutrophils through the endothelium. That the effect was evident at a relatively low level of endothelial activation suggests that ANCAs may potentiate the early vasculitic lesion and promote tissue damage and recruitment of other proinflammatory cells.

Is Wegener's granulomatosis an autoimmune disease?

Wegener's granulomatosis is a multisystem disease characterized by granulomata of the respiratory tract and systemic necrotising vasculitis. There is a strong and specific association with autoantibodies directed against proteinase 3, a constituent of neutrophril azurophilic granules. Antibody titers correlate with clinical disease activity and predict relapses. The disease responds favorably to immunosuppressive therapy. The pathogenicity of antineutrophil cytoplasmic antibodies (ANCA), however, remains unproven. In vitro, the expression of proteinase-3 and other ANCA antigens on the surface of neutrophils and monocytes can be induced by priming with proinflammatory cytokines. Antineutrophil cytoplasmic antibodies are then able to activate these leukocytes, stimulating degranulation, the production of reactive oxygen species, and the secretion of further cytokines. Neutrophils activated by ANCA, and possibly ANCA alone, directly damage endothelial cells in vitro. An animal model of proteinase 3-ANCA-induced vasculitis has not been found. Antineutrophil cytoplasmic antibodies directed against another antigen, myeloperoxidase, are not sufficient to cause vasculitis but they promote damage in certain animal models. Thus, a considerable amount of evidence supports the notion that Wegener's granulomatosis is an autoimmune disease.