Characterisation of innate immune dysfunction in fungal sepsis

Sepsis is a complex and severe pathological condition that is characterized by hyper-inflammation followed by leukocyte dysfunction and excessive tissue injury. The mechanisms controlling immune dysregulation remain poorly understood. In this thesis, we show that fungal capture in spleen promotes inflammation and DAMPs-mediated immune dysfunction during systemic candidiasis. We identify the molecules and cellular players and define the order of events that link immune dysfunction between lymphocytes and neutrophils. SIGNR1+ marginal zone macrophages (MZMΦs) capture Candida albicans (C. albicans) and enable fungal colonization of the spleen marginal zone (MZ), triggering aberrant lymphocyte death and release of cell-free chromatin, that synergizes with fungal hyphae to stimulate G-CSF production by CD169+ marginal metallophilic macrophages (MMMΦs). G-CSF and extracellular chromatin selectively reduce the lifespan of mature Ly6Ghigh neutrophils, leading to severe neutropenia. ROS-deficient immature Ly6Glow neutrophils are mobilised and become the predominant peripheral neutrophil population, causing impaired fungal clearance and severe pathology. SIGNR1-blockade effectively limits fungal colonization in the MZ and increases survival. Similarly, T cell- deficiency or neutralization of G-CSF, chromatin or histones consistently reduces inflammation, neutrophil dysfunction and pathology. The release of cell-free actin by dying splenocytes and tissue damage further enhances pathology by interfering with extracellular chromatin clearance. These findings demonstrate that PAMPs and DAMPs mediate inflammation and neutrophil dysregulation, causing a detrimental positive feedback-loop that impairs fungal clearance and increases sepsis pathology

Functional proteomic profiling links deficient DNA clearance with increased mortality in individuals with severe COVID-19 pneumonia

The factors that influence survival during severe infection are unclear. Extracellular chromatin drives pathology, but the mechanisms enabling its accumulation remain elusive. Here, we show that in murine sepsis models, splenocyte death interferes with chromatin clearance through the release of the DNase I inhibitor actin. Actin-mediated inhibition was compensated by upregulation of DNase I or the actin scavenger gelsolin. Splenocyte death and neutrophil extracellular trap (NET) clearance deficiencies were prevalent in individuals with severe COVID-19 pneumonia or microbial sepsis. Activity tracing by plasma proteomic profiling uncovered an association between low NET clearance and increased COVID-19 pathology and mortality. Low NET clearance activity with comparable proteome associations was prevalent in healthy donors with low-grade inflammation, implicating defective chromatin clearance in the development of cardiovascular disease and linking COVID-19 susceptibility to pre-existing conditions. Hence, the combination of aberrant chromatin release with defects in protective clearance mechanisms lead to poor survival outcomes

Enhancement of drug oxidation and conjugation by carcinogens in different rat tissues

OBJECTIVE After endoscopic third ventriculostomy (ETV), some patients develop recurrent symptoms of hydrocephalus. The optimal treatment for these patients is not clear: repeat ETV (re-ETV) or CSF shunting. The goals of the study were to assess the effectiveness of re-ETV relative to initial ETV in pediatric patients and validate the ETV success score (ETVSS) for re-ETV. METHODS Retrospective data of 624 ETV and 93 re-ETV procedures were collected from 6 neurosurgical centers in the Netherlands (1998-2015). Multivariable Cox proportional hazards modeling was used to provide an adjusted estimate of the hazard ratio for re-ETV failure relative to ETV failure. The correlation coefficient between ETVSS and the chance of re-ETV success was calculated using Kendall's tau coefficient. Model discrimination was quantified using the c-statistic. The effects of intraoperative findings and management on re-ETV success were also analyzed. RESULTS The hazard ratio for re-ETV failure relative to ETV failure was 1.23 (95% CI 0.90-1.69; p = 0.20). At 6 months, the success rates for both ETV and re-ETV were 68%. ETVSS was significantly related to the chances of re-ETV success (tau = 0.37; 95% bias corrected and accelerated CI 0.21-0.52; p < 0.001). The c-statistic was 0.74 (95% CI 0.64-0.85). The presence of prepontine arachnoid membranes and use of an external ventricular drain (EVD) were negatively associated with treatment success, with ORs of 4.0 (95% CI 1.5-10.5) and 9.7 (95% CI 3.4-27.8), respectively. CONCLUSIONS Re-ETV seems to be as safe and effective as initial ETV. ETVSS adequately predicts the chance of successful re-ETV. The presence of prepontine arachnoid membranes and the use of EVD negatively influence the chance of success

Microbe capture by splenic macrophages triggers sepsis via T cell-death-dependent neutrophil lifespan shortening

The mechanisms linking systemic infection to hyperinflammation and immune dysfunction in sepsis are poorly understood. Extracellular histones promote sepsis pathology, but their source and mechanism of action remain unclear. Here, we show that by controlling fungi and bacteria captured by splenic macrophages, neutrophil-derived myeloperoxidase attenuates sepsis by suppressing histone release. In systemic candidiasis, microbial capture via the phagocytic receptor SIGNR1 neutralizes myeloperoxidase by facilitating marginal zone infiltration and T cell death-dependent histone release. Histones and hyphae induce cytokines in adjacent CD169 macrophages including G-CSF that selectively depletes mature Ly6G$^{high}$ neutrophils by shortening their lifespan in favour of immature Ly6G$^{low}$ neutrophils with a defective oxidative burst. In sepsis patient plasma, these mediators shorten mature neutrophil lifespan and correlate with neutrophil mortality markers. Consequently, high G-CSF levels and neutrophil lifespan shortening activity are associated with sepsis patient mortality. Hence, by exploiting phagocytic receptors, pathogens degrade innate and adaptive immunity through the detrimental impact of downstream effectors on neutrophil lifespan

Combinatorial multimer staining and spectral flow cytometry facilitate quantification and characterization of polysaccharide-specific B cell immunity

Bacterial capsular polysaccharides are important vaccine immunogens. However, the study of polysaccharide-specific immune responses has been hindered by technical restrictions. Here, we developed and validated a high-throughput method to analyse antigen-specific B cells using combinatorial staining with fluorescently-labelled capsular polysaccharide multimers. Concurrent staining of 25 cellular markers further enables the in-depth characterization of polysaccharide-specific cells. We used this assay to simultaneously analyse 14 Streptococcus pneumoniae or 5 Streptococcus agalactiae serotype-specific B cell populations. The phenotype of polysaccharide-specific B cells was associated with serotype specificity, vaccination history and donor population. For example, we observed a link between non-class switched (IgM+) memory B cells and vaccine-inefficient S. pneumoniae serotypes 1 and 3. Moreover, B cells had increased activation in donors from South Africa, which has high-incidence of S. agalactiae invasive disease, compared to Dutch donors. This assay allows for the characterization of heterogeneity in B cell immunity that may underlie immunization efficacy

HpARI protein secreted by a helminth parasite suppresses interleukin-33

Infection by helminth parasites is associated with amelioration of allergic reactivity, but mechanistic insights into this association are lacking. Products secreted by the mouse parasite Heligmosomoides polygyrus suppress type 2 (allergic) immune responses through interference in the interleukin-33 (IL-33) pathway. Here, we identified H. polygyrus Alarmin Release Inhibitor (HpARI), an IL-33-suppressive 26-kDa protein, containing three predicted complement control protein (CCP) modules. In vivo, recombinant HpARI abrogated IL-33, group 2 innate lymphoid cell (ILC2) and eosinophilic responses to Alternaria allergen administration, and diminished eosinophilic responses to Nippostrongylus brasiliensis, increasing parasite burden. HpARI bound directly to both mouse and human IL-33 (in the cytokine's activated state) and also to nuclear DNA via its N-terminal CCP module pair (CCP1/2), tethering active IL-33 within necrotic cells, preventing its release, and forestalling initiation of type 2 allergic responses. Thus, HpARI employs a novel molecular strategy to suppress type 2 immunity in both infection and allergy. Osbourn et al identified HpARI, a protein secreted by a helminth parasite that is capable of suppressing allergic responses. HpARI binds to IL-33 (a critical inducer of allergy) and nuclear DNA, preventing the release of IL-33 from necrotic epithelial cells

Anti-inflammatory therapy with nebulised dornase alfa in patients with severe COVID-19 pneumonia A Randomised Clinical Trial

SARS-CoV2 infection causes severe, life-threatening pneumonia. Hyper-inflammation, coagulopathy and lymphopenia are associated with pathology and poor outcomes in these patients. Cell-free (cf) DNA is prominent in COVID-19 patients, amplifies inflammation and promotes coagulopathy and immune dysfunction. We hypothesized that cf-DNA clearance by nebulised dornase alfa may reduce inflammation and improve disease outcomes. Here, we evaluated the efficacy of nebulized dornase alfa in patients hospitalised with severe COVID-19 pneumonia. In this randomised controlled single-centre phase 2 proof-of-concept trial, we recruited adult patients admitted to hospital that exhibited stable oxygen saturation (≥94%) on supplementary oxygen and a C-reactive protein (CRP) level ≥30mg/L post dexamethasone treatment. Participants were randomized at a 3:1 ratio to receive twice-daily nebulised dornase alfa in addition to best available care (BAC) or BAC alone for seven days or until hospital discharge. A 2:1 ratio of historical controls to treated individuals (HC, 2:1) were included as the primary endpoint comparators. The primary outcome was a reduction in systemic inflammation measured by blood CRP levels over 7 days post-randomisation, or to discharge if sooner. Secondary and exploratory outcomes included time to discharge, time on oxygen, D-dimer levels, lymphocyte counts and levels of circulating cf-DNA. We screened 75 patients and enrolled 39 participants out of which 30 in dornase alfa arm, and 9 in BAC group. We also matched the recruited patients in the treated group (N=30) to historical controls in the BAC group (N=60). For the the primary outcome, 30 patients in the dornase alfa were compared to 69 patients in the BAC group. Dornase alfa treatment reduced CRP by 33% compared to the BAC group at 7-days (P=0.01). The dornase alfa group least squares mean CRP was 23.23 mg/L (95% CI 17.71 to 30.46) and the BAC group 34.82 mg/L (95% CI 28.55 to 42.47). A significant difference was also observed when only randomised participants were compared. Furthermore, compared to the BAC group, the chance of live discharge was increased by 63% in the dornase alfa group (HR 1.63, 95% CI 1.01 to 2.61, P=0.03), lymphocyte counts were improved (least-square mean: 1.08 vs 0.87, P=0.02) and markers of coagulopathy such as D-dimer were diminished (least-square mean: 570.78 vs 1656.96μg/mL, P=0.004). Moreover, the dornase alfa group exhibited lower circulating cf-DNA levels that correlated with CRP changes over the course of treatment. No differences were recorded in the rates and length of stay in the ICU or the time on oxygen between the groups. Dornase alfa was well-tolerated with no serious adverse events reported. In this proof-of-concept study in patients with severe COVID-19 pneumonia, treatment with nebulised dornase alfa resulted in a significant reduction in inflammation, markers of immune pathology and time to discharge. The effectiveness of dornase alfa in patients with acute respiratory infection and inflammation should be investigated further in larger trials

Exploring consensus on how to measure smoking cessation. A Delphi study

Background Different criteria regarding outcome measures in smoking research are used, which can lead to confusion about study results. Consensus in outcome criteria may enhance the comparability of future studies. This study aims (1) to provide an overview of tobacco researchers’ considered preferences regarding outcome criteria in randomized controlled smoking cessation trials, and (2) to identify the extent to which researchers can reach consensus on the importance of these outcome criteria. Methods A three-round online Delphi study was conducted among smoking cessation experts. In the first round, the most important smoking cessation outcome measures were collected by means of open-ended questions, which were categorized around self-reported and biochemical validation measures. Experts (n = 17) were asked to name the outcome measures (as well as their assessment method and ideal follow-up period) that they thought were important when assessing smoking-related outcomes. In the second (n = 48) and third rounds (n = 37), a list of outcome measures—identified in the first round—was presented to experts. Asking them to rate the importance of each measure on a seven-point scale. Results Experts reached consensus on several items. For self-reports, experts agreed that prolonged abstinence (6 or/and 12 months), point prevalence abstinence (7 days), continuous abstinence (6 months), and the number of cigarettes smoked (7 days) are important outcome measures. Experts reached consensus that biochemical validation methods should not always be used. The preferred biochemical validation methods were carbon monoxide (expired air) and cotinine (saliva). Preferred follow-ups included 6 and/or 12 months, with or without intermediate measurements. Conclusions Findings suggest only partial compliance with the Russell standard and that more outcome measures may be important (including seven-day point-prevalence abstinence, number of cigarettes smoked, and cotinine when using biochemical validation). This study showed where there is and is not consensus, reflecting the need to develop a more comprehensive standard. For these purposes we provided suggestions for the Russell 2.0 standard

Dutch practice nurses' adherence to evidence-based smoking cessation treatment guidelines

Background: Practice nurses in general practice sub-optimally adhere to evidence-based smoking cessation treatment guidelines, but factors explaining their adherence have not yet been investigated. Understanding such factors is important to develop interventions improving practice nurses' smoking cessation guideline adherence and patients' subsequent cessation success. This study explored the association between different socio-cognitive and predisposing factors, and practice nurses' adherence to the Dutch smoking cessation guideline in general (i.e. overall adherence) and to each guideline step individually (i.e. step-based adherence). Methods: A cross-sectional study was conducted among practice nurses (N = 157) in January-March 2015 via web-based questionnaires, assessing constructs from the Integrated Change Model. Spearman's correlations and linear regression analysis were used to identify potential determinants of overall guideline adherence; Mann-Whitney U-tests and logistic regression analyses were used to identify potential determinants of step-based adherence. Results: On average five out of nine steps were completely adhered to by practice nurses; and step-based adherence ranged from 34% to 75%. Overall guideline adherence was associated with high levels of self-efficacy to use a guideline (β = 0.32, P = 0.00), and step-based adherence was additionally associated with spending more time on counselling. Regression results showed positive associations between self-efficacy (8/9 steps) and perceived advantages (7/9 steps) with step-based adherence. Conclusion: This study quantitatively confirmed practice nurses' sub-optimal guideline adherence and found associations between socio-cognitive (self-efficacy and perceived advantages) and predisposing factors (time spent on counselling), and guideline adherence. Detailed insights in these factors offer preliminary directions for intervention development to improve practice nurses' adherence to evidence-based smoking cessation guidelines