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Introduction Zika virus (ZIKV) is a neurotropic human pathogen that causes neuroinflammation, whose hallmark is elevated translocator protein (TSPO) expression in the brain. This study investigates ZIKV-associated changes in adult brain TSPO expression, evaluates the effectiveness of TSPO radioligands in detecting TSPO expression, and identifies cells that drive brain TSPO expression in a mouse infection model. Methods The interferon-deficient AG129 mouse infected with ZIKV was used as neuroinflammation model. TSPO expression was evaluated by tissue immunostaining. TSPO radioligands, [3H]PK11195 and [18F]FEPPA, were used for in vitro and ex vivo detection of TSPO in infected brains. [18F]FEPPA-PET was used for in vivo detection of TSPO expression. Cell subsets that contribute to TSPO expression were identified by flow cytometry. Results Brain TSPO expression increased with ZIKV disease severity. This increase was contributed by TSPO-positive microglia and infiltrating monocytes; and by influx of TSPO-expressing immune cells into the brain. [3H]PK11195 and [18F]FEPPA distinguish ZIKV-infected brains from normal controls in vitro and ex vivo. [18F]FEPPA brain uptake by PET imaging correlated with disease severity and neuroinflammation. However, TSPO expression by immune cells contributed to significant blood pool [18F]FEPPA activity which could confound [18F]FEPPA-PET imaging results. Conclusions TSPO is a biologically relevant imaging target for ZIKV neuroinflammation. Brain [18F]FEPPA uptake can be a surrogate marker for ZIKV disease and may be a potential PET imaging marker for ZIKV-induced neuroinflammation. Future TSPO-PET/SPECT studies on viral neuroinflammation and related encephalitis should assess the contribution of immune cells on TSPO expression and employ appropriate image correction methods to subtract blood pool activity.

Dengue virus (DENV) non-structural protein 5 (NS5) is critical for viral RNA synthesis within endoplasmic reticulum (ER)-derived replication complexes in the cytoplasm; however a proportion of NS5 is known to be localized to the nucleus of infected cells. The importance of nuclear DENV NS5 on viral replication and pathogenesis is still unclear. We recently discovered a nuclear localization signal (NLS) residing in the C-terminal 18 amino acid (Cter18) region of DENV NS5 and that a single NS5 P884T amino acid substitution adjacent to the NLS is sufficient to relocalize a significant proportion of DENV2 NS5 from the nucleus to the cytoplasm of infected cells. Here, in vitro studies show that the DENV2 NS5 P884T mutant replicates similarly to the parental wild-type infectious clone-derived virus while inducing a greater type I interferon and inflammatory cytokine response, in a manner independent of NS5's ability to degrade STAT2 or regulate SAT1 splicing. In both AG129 mouse and Aedes aegypti mosquito infection models, the P884T virus exhibits lower levels of viral replication only at early timepoints. Intriguingly, there appears to be a tendency for selection pressure to revert to the wild-type proline in P884T-infected Ae. aegypti, in agreement with the high conservation of the proline at this position of NS5 in DENV2, 3, and 4. These results suggest that the predominant nuclear localization of DENV NS5, while not required for viral RNA replication, may play a role in pathogenesis and modulation of the host immune response and contribute to viral fitness in the mosquito host.

This article reviews Brenner’s most recent monograph, New Urban Spaces, in which he demonstrates his systematic and decades-long effort into developing new epistemological perspectives, conceptual proposals, and methodological strategies for urban investigation in our so-called global ‘urban age’. We use images taken in the city of Toronto to interpret and visually explain some of Brenner’s key concepts and arguments. In our critical assessment, we point out that Brenner’s abstract theorizing and overgeneralization may have provoked an ignorance of the local variations in effect globally and a failure to tease out specific testable hypotheses for urban investigation. To conclude, we introduce a newly developed scenic approach, one that extends Brenner’s view on the urban by stressing the importance of local variations and testable hypotheses.

Purpose Zika (ZIKV) is a viral inflammatory disease affecting adults, children, and developing fetuses. It is endemic to tropical and sub-tropical countries, resulting in half the global population at risk of infection. Despite this, there are no approved therapies or vaccines against ZIKV disease. Non-invasive imaging biomarkers are potentially valuable tools for studying viral pathogenesis, prognosticating host response to disease, and evaluating in vivo efficacy of experimental therapeutic interventions. In this study, we evaluated [18F]fluorodeoxyglucose ([18F]FDG)-positron emission tomography (PET) as an imaging biomarker of ZIKV disease in a mouse model and correlated metabolic tracer tissue uptake with real-time biochemical, virological, and inflammatory features of tissue infection. Methods [18F]FDG-PET/CT imaging was performed in an acute, lethal ZIKV mouse infection model, at increasing stages of disease severity. [18F]FDG-PET findings were corroborated with ex vivo wholemount-tissue autoradiography and tracer biodistribution studies. Tracer uptake was also correlated with in situ tissue disease status, including viral burden and inflammatory response. Immune profiling of the spleen by flow cytometry was performed to identify the immune cell subsets driving tissue pathology and enhancing tracer uptake in ZIKV disease. Results Foci of increased [18F]FDG uptake were consistently detected in lymphoid tissues—particularly the spleen—of ZIKV-infected animals. Splenic uptake increased with disease severity, and corroborated findings in tissue pathology. Increased splenic uptake also correlated with increased viral replication and elevated expression of pro-inflammatory cytokines within these tissues. ZIKV-infected spleens were characterized by increased infiltration of myeloid cells, as well as increased proliferation of both myeloid and lymphoid cells. The increased cell proliferation correlated with increased tracer uptake in the spleen. Our findings support the use of [18F]FDG as an imaging biomarker to detect and track ZIKV disease in real time and highlight the dependency of affected tissue on the nature of the viral infection. Conclusion [18F]FDG uptake in the spleen is a useful surrogate for interrogating in situ tissue viral burden and inflammation status in this ZIKV murine model.

Objective JIA is an autoimmune, inflammatory disease with involvement of innate and adaptive immune responses. However, the role of neutrophils in JIA pathogenesis remains unclear. This study aimed to identify and validate neutrophil gene expression signatures in JIA using public microarray datasets and new clinical samples. Methods Three suitable datasets were analysed by significance analysis of microarray and Ingenuity. Neutrophils and peripheral blood mononuclear cells (PBMCs) were isolated from a new cohort of JIA patients and healthy paediatric controls (HCs). Gene expression was validated using quantitative PCR. Serum concentrations of proteins were measured using ELISA. Low-density granulocytes (LDGs) in JIA and HC PBMCs were quantified by flow cytometry using forward/side-scatter properties. Results Ingenuity identified transcriptional regulation (false discovery rate < 0.05) by G-CSF, GM-CSF and IL-8 along with expression of neutrophil granule protein genes including ELANE, MPO, MMP8 and MMP9 in datasets from JIA PBMCs. LDG counts were elevated in JIA compared with HCs (2.5% vs 1.4%; P = 0.007). Transcripts for MMP8 (P = 0.005), MPO (P = 0.0124) and Fcγ Receptor 1B (FCγR1B) (P = 0.0417) were significantly higher in JIA compared with HC neutrophils. MMP9 protein levels were lower in systemic JIA patient sera [355.95 ng/ml (s.d. 250.03)] compared with HCs [675.41 ng/ml (s.d. 181.17); P = 0.007], but levels of elastase, MPO and MMP8 were not significantly different. Conclusion LDGs are elevated in JIA and contribute to the transcriptomic profile of JIA PBMCs. JIA neutrophils express higher levels of MMP8 and FCGR1B, which may be implicated in disease pathology through the release of proteases and reactive oxygen metabolites, causing systemic inflammation and damage to joints.

Development of antiviral therapy against acute viral diseases, such as dengue virus (DENV), suffers from the narrow window of viral load detection in serum during onset and clearance of infection and fever. We explored a biomarker approach using 18F-fluorodeoxyglucose (18F-FDG) PET in established mouse models for primary and antibody-dependent enhancement infection with DENV. 18F-FDG uptake was most prominent in the intestines and correlated with increased virus load and proinflammatory cytokines. Furthermore, a significant temporal trend in 18F-FDG uptake was seen in intestines and selected tissues over the time course of infection. Notably, 18F-FDG uptake and visualization by PET robustly differentiated treatment-naive groups from drug-treated groups as well as nonlethal from lethal infections with a clinical strain of DENV2. Thus, 18F-FDG may serve as a novel DENV infection-associated inflammation biomarker for assessing treatment response during therapeutic intervention trials.

Background T cells are important to systemic lupus erythematosus (SLE) disease progression. This study determined the pro-inflammatory potential of T cells within the rare condition juvenile-onset SLE (JSLE). Method IL-17A and Th1/Th2-related cytokine concentrations were measured in plasma/serum from JSLE patients (n = 19, n = 11) and HC (n = 18, n = 7). IL17A, RORC, IL23 and IL23R mRNA were measured in peripheral blood mononuclear cells (PBMCs) from JSLE and healthy controls (HC) (n = 12). Th17-associated cytokine expression was analysed in the supernatant of CD3/CD28 activated JSLE (n = 7) and HC (n = 6) PBMCs. Results JSLE plasma IL-17A level (21.5 ± 5.2 pg/ml) was higher compared to HC (7.2 ± 2.5 pg/ml, p = 0.028). No differences were found in Th1/Th2 cytokines levels. IL = 17A (p = 0.022), IL-6 (p = 0.028) and IL-21 (p = 0.003) concentrations were increased in supernatants from activated JSLE PBMCs. IL-17 F (p = 0.50) and IL-22 (p = 0.43) were also increased but were not statistically significant. IL17A and IL23 mRNA was significantly higher in JSLE PBMCs (p = 0.018 and p = 0.01). Conclusion JSLE T cells have an increased ability to secrete Th17 associated cytokines once activated, which could contribute to the pro-inflammatory disease phenotype seen in these patients.

Background Neutrophils are one of the first cell types to be recruited to sites of inflammation or infections, yet have been implicated in a wide range of non-infectious inflammatory conditions. Low-density granulocytes (LDGs) are a distinct subset of neutrophils recently documented in a number of systemic autoimmune diseases such as Systemic Lupus Erythematosus (SLE) (1). LDGs show increased ability to undergo neutrophil extracellular trap (NET) formation, leading to potential autoantigen presentation, and subsequent inflammation (2,3). Citrullination of histones in neutrophils, a post-translational histone modification, is a critical step in NET formation (4). Juvenile idiopathic arthritis (JIA) is a heterogeneous autoimmune disease characterized by chronic joint inflammation. We have previously shown increased LDG expression in JSLE patients; however histone citrullination in JSLE and JIA, and LDG populations in JIA has yet to be characterized. Objectives To compare LDG expression in JIA peripheral venous blood to healthy controls. To investigate the presence of histone citrullination in JSLE and JIA patients compared to healthy controls. Methods While mature neutrophils typically sediment with the red-blood cell fraction in a Ficoll-density gradient, LDGs co-purify in the PMBC fraction. LDGs and neutrophils were isolated from whole blood using HetaSep and magnetic bead separation. The percentage populations of LDGs were analysed using flow cytometry in JIA and healthy paediatric controls. Where possible, protein was extracted from the neutrophils of each patient group. Citrullinated histone 3 relative to the expression of histone 3 was measured using Western Blot analysis in JIA and JSLE patients compared to controls. Results are presented as mean ± SEM. Results Preliminary data suggests the expression of LDGs in the peripheral blood of JSLE patients (n=11; 8.31%±2.5) and JIA patients (n=3; 3.6%±2.4) is increased compared to healthy paediatric controls (n=4; 1.62%±0.95). Overall, the ratio of citrullination of histone 3 to pan histone 3 was higher in JSLE neutrophils (0.75±0.15) compared to healthy controls (0.38±0.12; p Conclusions Increased expression of LDGs was observed in JIA and JSLE patients compared to controls. This supports previous research from our group that has shown increased expression of LDGs in JSLE patients correlates with disease activity. LDGs may therefore play an integral role in triggering an autoimmune inflammatory response. Notably, citrullination of histone 3 was increased in JSLE neutrophils compared to controls, but not JIA neutrophils. This may indicate that JSLE neutrophils are intrinsically more primed to undergo NETosis compared to JIA patients. Further investigations are being carried out to increase patient size and to characterize LDG phenotype and function. References Carmona-Rivera C, Kaplan MJ. Semin Immunopathol, 2013. 35(4): p. 455-63 Villanueva E, et al. The Journal of Immunology 2011 July 01;187(1):538-552. Denny MF, et al. The Journal of Immunology 2010 March 15;184(6):3284-3297. Wang, Y, et al. J Cell Biol, 2009. 184(2): p. 205-13 Disclosure of Interest None declared

Background Abnormal cytokine expression is postulated to be a factor in the immunopathology of Systemic Lupus Erythematosus (SLE), leading to inflammation and organ damage. Several studies have shown elevated levels of IL-6, IL-17, IL-21 and TNF-α in adult SLE serum and plasma using ELISAs 1,2 . A spike-and-recovery assessment is used to measure the analyte recovery difference between a natural sample matrix and the standard diluent. Generally 80-120% is considered an acceptable range of recovery. A previous spike-and-recovery study of SLE serum and plasma has shown poor recovery of high mobility group box 1 (HMGB1) protein 3 . Objectives To validate and assess the assay response of juvenile-onset SLE (JSLE) serum and plasma in IL-6, IL-17, IL-21 and TNF-α detection kits. Methods JSLE plasma and serum samples were assayed in spike-and-recovery and linearity-dilution experiments. Three spike-and-recovery ELISA assessments with IL-21 (Biolegend), IL-6 and TNF-α (R&D Systems) were performed, each including 3 JSLE plasma samples and 1 control sample. IL-17 spike-and-recovery ELISA (eBioscience) was executed with 2 JSLE serum and 2 JSLE plasma samples. A spike-and-recovery assessment with IL-17 was performed on 5 JSLE serum samples using a single Bio-Plex Assay (Bio-Rad). Percentage recovery of respective cytokines in spiked and unspiked samples were then calculated and compared against the concentration of spiked sample diluents. Results Recovery percentages in JSLE samples were less than 80% in TNF-α, IL-17 and IL-21 assays (Fig. 1), apart from IL-17 and TNF-α at 1 in 8 dilution which yielded 80% and 113% respectively. Measuring IL-21 in JSLE and control samples showed poor recovery out of acceptable range at all 4 dilutions. IL-6 concentrations fell within the acceptable range and demonstrated good responses in JSLE and control samples. Conclusions Interference was observed in the assay response of JSLE serum and plasma in IL-17, IL-21 and TNF-α assays but not IL-6 ELISA. Recovery increased with sample matrix dilution, indicating that dilution reduced the interference, however this must be balanced against the risk of diluting out the analyte. It can be inferred that constituents in the sample matrix may be affecting the detection of these cytokines. JSLE plasma/serum cytokine concentrations measured by ELISA and single plex assays must be interpreted with caution. Methods such as using specialist buffers or alternative assays should be considered to minimise interference and to increase robustness of future studies. Spike-recovery experiments using a multiplex assay platform are currently underway. References Ohl K et al. J Biomed Biotechnol. 2011;2011. Davas EM et al. Clin Rheumatol. 1999;18(1):17-22. Urbonaviciute V et al. J Leukoc Biol. 2007;81(1):67-74. Disclosure of Interest None Declared