Effects of Human RelA Transgene on Murine Macrophage Inflammatory Responses.

The NFκB transcription factors are major regulators of innate immune responses, and NFκB signal pathway dysregulation is linked to inflammatory disease. Here, we utilised bone marrow-derived macrophages from the p65-DsRedxp/IκBα-eGFP transgenic strain to study the functional implication of xenogeneic (human) RelA(p65) protein introduced into the mouse genome. Confocal imaging showed that human RelA is expressed in the cells and can translocate to the nucleus following activation of Toll-like receptor 4. RNA sequencing of lipid A-stimulated macrophages, revealed that human RelA impacts on murine gene transcription, affecting both non-NFκB and NFκB target genes, including immediate-early and late response genes, e.g., Fos and Cxcl10. Validation experiments on NFκB targets revealed markedly reduced mRNA levels, but similar kinetic profiles in transgenic cells compared to wild-type. Enrichment pathway analysis of differentially expressed genes revealed interferon and cytokine signaling were affected. These immune response pathways were also affected in macrophages treated with tumor necrosis factor. Data suggests that the presence of xenogeneic RelA protein likely has inhibitory activity, altering specific transcriptional profiles of key molecules involved in immune responses. It is therefore essential that this information be taken into consideration when designing and interpreting future experiments using this transgenic strain.

Novel Methods in the Surveillance of Influenza-Like Illness in Germany Using Data From a Symptom Assessment App (Ada): Observational Case Study.

BACKGROUND: Participatory epidemiology is an emerging field harnessing consumer data entries of symptoms. The free app Ada allows users to enter the symptoms they are experiencing and applies a probabilistic reasoning model to provide a list of possible causes for these symptoms. OBJECTIVE: The objective of our study is to explore the potential contribution of Ada data to syndromic surveillance by comparing symptoms of influenza-like illness (ILI) entered by Ada users in Germany with data from a national population-based reporting system called GrippeWeb. METHODS: We extracted data for all assessments performed by Ada users in Germany over 3 seasons (2017/18, 2018/19, and 2019/20) and identified those with ILI (report of fever with cough or sore throat). The weekly proportion of assessments in which ILI was reported was calculated (overall and stratified by age group), standardized for the German population, and compared with trends in ILI rates reported by GrippeWeb using time series graphs, scatterplots, and Pearson correlation coefficient. RESULTS: In total, 2.1 million Ada assessments (for any symptoms) were included. Within seasons and across age groups, the Ada data broadly replicated trends in estimated weekly ILI rates when compared with GrippeWeb data (Pearson correlation-2017-18: r=0.86, 95% CI 0.76-0.92; P<.001; 2018-19: r=0.90, 95% CI 0.84-0.94; P<.001; 2019-20: r=0.64, 95% CI 0.44-0.78; P<.001). However, there were differences in the exact timing and nature of the epidemic curves between years. CONCLUSIONS: With careful interpretation, Ada data could contribute to identifying broad ILI trends in countries without existing population-based monitoring systems or to the syndromic surveillance of symptoms not covered by existing systems.

Impact of Interleukin 10 Deficiency on Intestinal Epithelium Responses to Inflammatory Signals.

Interleukin 10 (IL-10) is a pleiotropic, anti-inflammatory cytokine that has a major protective role in the intestine. Although its production by cells of the innate and adaptive immune system has been extensively studied, its intrinsic role in intestinal epithelial cells is poorly understood. In this study, we utilised both ATAC sequencing and RNA sequencing to define the transcriptional response of murine enteroids to tumour necrosis factor (TNF). We identified that the key early phase drivers of the transcriptional response to TNF within intestinal epithelium were NFκB transcription factor dependent. Using wild-type and Il10-/- enteroid cultures, we showed an intrinsic, intestinal epithelium specific effect of IL-10 deficiency on TNF-induced gene transcription, with significant downregulation of identified NFκB target genes Tnf, Ccl20, and Cxcl10, and delayed overexpression of NFκB inhibitor encoding genes, Nfkbia and Tnfaip3. IL-10 deficiency, or immunoblockade of IL-10 receptor, impacted on TNF-induced endogenous NFκB activity and downstream NFκB target gene transcription. Intestinal epithelium-derived IL-10 appears to play a crucial role as a positive regulator of the canonical NFκB pathway, contributing to maintenance of intestinal homeostasis. This is particularly important in the context of an inflammatory environment and highlights the potential for future tissue-targeted IL-10 therapeutic intervention.

Risk Factors and Predictors of Mortality in Streptococcal Necrotizing Soft-tissue Infections: A Multicenter Prospective Study.

BACKGROUND: Necrotizing soft-tissue infections (NSTI) are life-threatening conditions often caused by ß-hemolytic streptococci, group A Streptococcus (GAS) in particular. Optimal treatment is contentious. The INFECT cohort includes the largest set of prospectively enrolled streptococcal NSTI cases to date. METHODS: From the INFECT cohort of 409 adults admitted with NSTI to 5 clinical centers in Scandinavia, patients culture-positive for GAS or Streptococcus dysgalactiae (SD) were selected. Risk factors were identified by comparison with a cohort of nonnecrotizing streptococcal cellulitis. The impact of baseline factors and treatment on 90-day mortality was explored using Lasso regression. Whole-genome sequencing of bacterial isolates was used for emm typing and virulence gene profiling. RESULTS: The 126 GAS NSTI cases and 27 cases caused by SD constituted 31% and 7% of the whole NSTI cohort, respectively. When comparing to nonnecrotizing streptococcal cellulitis, streptococcal NSTI was associated to blunt trauma, absence of preexisting skin lesions, and a lower body mass index. Septic shock was significantly more frequent in GAS (65%) compared to SD (41%) and polymicrobial, nonstreptococcal NSTI (46%). Age, male sex, septic shock, and no administration of intravenous immunoglobulin (IVIG) were among factors associated with 90-day mortality. Predominant emm types were emm1, emm3, and emm28 in GAS and stG62647 in SD. CONCLUSIONS: Streptococcal NSTI was associated with several risk factors, including blunt trauma. Septic shock was more frequent in NSTI caused by GAS than in cases due to SD. Factors associated with mortality in GAS NSTI included age, septic shock, and no administration of IVIG.

Disease Activity Patterns of Crohn's Disease in the First Ten Years After Diagnosis in the Population-based IBD South Limburg Cohort.

BACKGROUND AND AIMS: Real-life data on long-term disease activity in Crohn's disease [CD] are scarce. Most studies describe disease course by using proxies, such as drug exposure, need for surgery or hospitalisations, and disease progression. We aimed to describe disease course by long-term disease activity and to identify distinctive disease activity patterns in the population-based IBD South Limburg cohort [IBDSL]. METHODS: All CD patients in IBDSL with ≥10 years follow-up [n = 432] were included. Disease activity was defined for each yearly quarter by mucosal inflammation on endoscopy or imaging, hospitalisation, surgery, or treatment adjustment for increased symptoms. Six distinct disease activity clusters were defined. Subsequently, the associations between clinical characteristics and the patterns were assessed using multivariable logistic regression models. RESULTS: On average, patients experienced 5.44 (standard deviation [SD] 3.96) quarters of disease activity during the first 10 years after diagnosis. Notably, 28.2% of the patients were classified to a quiescent pattern [≤2 active quarters in 10 years], and 89.8% of those never received immunomodulators nor biologics. Surgery at diagnosis (odds ratio [OR] 2.99; 95% confidence interval [CI] 1.07-8.34) and higher age [OR 1.03; 95% CI 1.01-1.06] were positively associated with the quiescent pattern, whereas inverse associations were observed for ileocolonic location [OR 0.44; 95% CI 0.19-1.00], smoking [OR 0.43; 95% CI 0.24-0.76] and need for steroids <6 months [OR 0.24; 95% CI 0.11-0.52]. CONCLUSIONS: Considering long-term disease activity, 28.2% of CD patients were classified to a quiescent cluster. Given the complex risk-benefit balance of immunosuppressive drugs, our findings underline the importance of identifying better predictive markers to prevent both over-treatment and under-treatment.

Syndromic Surveillance Insights from a Symptom Assessment App Before and During COVID-19 Measures in Germany and the United Kingdom: Results From Repeated Cross-Sectional Analyses.

BACKGROUND: Unprecedented lockdown measures have been introduced in countries worldwide to mitigate the spread and consequences of COVID-19. Although attention has been focused on the effects of these measures on epidemiological indicators relating directly to the infection, there is increased recognition of their broader health implications. However, assessing these implications in real time is a challenge, due to the limitations of existing syndromic surveillance data and tools. OBJECTIVE: The aim of this study is to explore the added value of mobile phone app-based symptom assessment tools as real-time health insight providers to inform public health policy makers. METHODS: A comparative and descriptive analysis of the proportion of all self-reported symptoms entered by users during an assessment within the Ada app in Germany and the United Kingdom was conducted between two periods, namely before and after the implementation of "Phase One" COVID-19 measures. Additional analyses were performed to explore the association between symptom trends and seasonality, and symptom trends and weather. Differences in the proportion of unique symptoms between the periods were analyzed using a Pearson chi-square test and reported as log2 fold changes. RESULTS: Overall, 48,300-54,900 symptomatic users reported 140,500-170,400 symptoms during the Baseline and Measures periods in Germany. Overall, 34,200-37,400 symptomatic users in the United Kingdom reported 112,100-131,900 symptoms during the Baseline and Measures periods. The majority of symptomatic users were female (Germany: 68,600/103,200, 66.52%; United Kingdom: 51,200/71,600, 72.74%). The majority were aged 10-29 years (Germany: 68,500/100,000, 68.45%; United Kingdom: 50,900/68,800, 73.91%), and about one-quarter were aged 30-59 years (Germany: 26,200/100,000, 26.15%; United Kingdom: 14,900/68,800, 21.65%). Overall, 103 symptoms were reported either more or less frequently (with statistically significant differences) during the Measures period as compared to the Baseline period, and 34 of these were reported in both countries. The following mental health symptoms (log2 fold change, P value) were reported less often during the Measures period: inability to manage constant stress and demands at work (-1.07, P<.001), memory difficulty (-0.56, P<.001), depressed mood (-0.42, P<.001), and impaired concentration (-0.46, P<.001). Diminished sense of taste (2.26, P<.001) and hyposmia (2.20, P<.001) were reported more frequently during the Measures period. None of the 34 symptoms were found to be different between the same dates in 2019. In total, 14 of the 34 symptoms had statistically significant associations with weather variables. CONCLUSIONS: Symptom assessment apps have an important role to play in facilitating improved understanding of the implications of public health policies such as COVID-19 lockdown measures. Not only do they provide the means to complement and cross-validate hypotheses based on data collected through more traditional channels, they can also generate novel insights through a real-time syndromic surveillance system.

Using systems medicine to identify a therapeutic agent with potential for repurposing in inflammatory bowel disease.

Inflammatory bowel diseases (IBDs) cause significant morbidity and mortality. Aberrant NF-κB signalling is strongly associated with these conditions, and several established drugs influence the NF-κB signalling network to exert their effect. This study aimed to identify drugs that alter NF-κB signalling and could be repositioned for use in IBD. The SysmedIBD Consortium established a novel drug-repurposing pipeline based on a combination of in silico drug discovery and biological assays targeted at demonstrating an impact on NF-κB signalling, and a murine model of IBD. The drug discovery algorithm identified several drugs already established in IBD, including corticosteroids. The highest-ranked drug was the macrolide antibiotic clarithromycin, which has previously been reported to have anti-inflammatory effects in aseptic conditions. The effects of clarithromycin effects were validated in several experiments: it influenced NF-κB-mediated transcription in murine peritoneal macrophages and intestinal enteroids; it suppressed NF-κB protein shuttling in murine reporter enteroids; it suppressed NF-κB (p65) DNA binding in the small intestine of mice exposed to lipopolysaccharide; and it reduced the severity of dextran sulphate sodium-induced colitis in C57BL/6 mice. Clarithromycin also suppressed NF-κB (p65) nuclear translocation in human intestinal enteroids. These findings demonstrate that in silico drug repositioning algorithms can viably be allied to laboratory validation assays in the context of IBD, and that further clinical assessment of clarithromycin in the management of IBD is required.This article has an associated First Person interview with the joint first authors of the paper.

Correlation Between Immunoglobulin Dose Administered and Plasma Neutralization of Streptococcal Superantigens in Patients With Necrotizing Soft Tissue Infections.

Analyses of plasma collected pre- and postadministration of intravenous immunoglobulin (IVIG) from patients with group A Streptococcus necrotizing soft tissue infections demonstrated a negative correlation between IVIG dose and toxin-triggered T-cell proliferation (r = -.67, P < .0001). One 25-g IVIG dose was sufficient to yield plasma-neutralizing activity against streptococcal superantigens. Clinical Trials Registration. NCT01790698 and NCT02111161.

Macrophage-Specific NF-κB Activation Dynamics Can Segregate Inflammatory Bowel Disease Patients.

The heterogeneous nature of inflammatory bowel disease (IBD) presents challenges, particularly when choosing therapy. Activation of the NF-κB transcription factor is a highly regulated, dynamic event in IBD pathogenesis. Using a lentivirus approach, NF-κB-regulated luciferase was expressed in patient macrophages, isolated from frozen peripheral blood mononuclear cell samples. Following activation, samples could be segregated into three clusters based on the NF-κB-regulated luciferase response. The ulcerative colitis (UC) samples appeared only in the hypo-responsive Cluster 1, and in Cluster 2. Conversely, Crohn's disease (CD) patients appeared in all Clusters with their percentage being higher in the hyper-responsive Cluster 3. A positive correlation was seen between NF-κB-induced luciferase activity and the concentrations of cytokines released into medium from stimulated macrophages, but not with serum or biopsy cytokine levels. Confocal imaging of lentivirally-expressed p65 activation revealed that a higher proportion of macrophages from CD patients responded to endotoxin lipid A compared to controls. In contrast, cells from UC patients exhibited a shorter duration of NF-κB p65 subunit nuclear localization compared to healthy controls, and CD donors. Analysis of macrophage cytokine responses and patient metadata revealed a strong correlation between CD patients who smoked and hyper-activation of p65. These in vitro dynamic assays of NF-κB activation in blood-derived macrophages have the potential to segregate IBD patients into groups with different phenotypes and may therefore help determine response to therapy.

Patient's characteristics and outcomes in necrotising soft-tissue infections: results from a Scandinavian, multicentre, prospective cohort study.

PURPOSE: Necrotising soft-tissue infections (NSTI) are characterised by necrosis, fast progression, and high rates of morbidity and mortality, but our knowledge is primarily derived from small prospective studies and retrospective studies. METHODS: We performed an international, multicentre, prospective cohort study of adults with NSTI describing patient's characteristics and associations between baseline variables and microbiological findings, amputation, and 90-day mortality. RESULTS: We included 409 patients with NSTI; 402 were admitted to the ICU. Cardiovascular disease [169 patients (41%)] and diabetes [98 (24%)] were the most common comorbidities; 122 patients (30%) had no comorbidity. Before surgery, bruising of the skin [210 patients (51%)] and pain requiring opioids [172 (42%)] were common. The sites most commonly affected were the abdomen/ano-genital area [140 patients (34%)] and lower extremities [126 (31%)]. Monomicrobial infection was seen in 179 patients (44%). NSTI of the upper or lower extremities was associated with monomicrobial group A streptococcus (GAS) infection, and NSTI located to the abdomen/ano-genital area was associated with polymicrobial infection. Septic shock [202 patients (50%)] and acute kidney injury [82 (20%)] were common. Amputation occurred in 22% of patients with NSTI of an extremity and was associated with higher lactate level. All-cause 90-day mortality was 18% (95% CI 14-22); age and higher lactate levels were associated with increased mortality and GAS aetiology with decreased mortality. CONCLUSIONS: Patients with NSTI were heterogeneous regarding co-morbidities, initial symptoms, infectious localisation, and microbiological findings. Higher age and lactate levels were associated with increased mortality, and GAS infection with decreased mortality.