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Background: Interleukin-6-receptor inhibition with tocilizumab improves myocardial salvage in patients with ST-segment elevation myocardial infarction (STEMI). Reduced levels of neutrophil extracellular traps (NETs), which consist of nuclear material studded with proteins released upon neutrophil activation, might contribute to this effect. Objectives: The purpose of this study was to evaluate the effect of tocilizumab on NETs and investigate the association between NETs and myocardial injury in patients with STEMI. Methods: In the ASSAIL-MI study, 199 patients with STEMI were randomized to tocilizumab or placebo during percutaneous coronary intervention. In this substudy, we analyzed blood levels of the NET markers double-stranded deoxyribonucleic acid (dsDNA), myeloperoxidase-DNA, and citrullinated histone 3 (H3Cit) at admission and after 24 hours and 3 to 7 days. In a subgroup of patients, we assessed regulation of transcripts related to the formation of NETs. We also investigated associations between NET markers and the myocardial salvage index (MSI). Results: All NET markers were lower in the tocilizumab group than in the placebo group at 3 to 7 days (all P

Objectives: This study aimed to explore the role of fungal burden in risk stratification of patients without HIV-negative patients with Pneumocystis pneumonia (PCP). Methods: This was a retrospective analysis of the characteristics associated with 30-day mortality in patients who were positive for P. jirovecii using polymerase chain reaction in bronchoalveolar lavage fluid between 2006 and 2017 in a multicenter cohort from Central Norway. The fungal burden was indicated by the cycle threshold (CT) values from semiquantitative real-time polymerase chain reaction targeting the β-tubulin gene. Results: We included 170 patients with proven or probable PCP. The all-cause 30-day mortality was 18.2%. After adjusting for host characteristics and premorbid corticosteroid use, a higher fungal burden was associated with a higher risk of dying: adjusted odds ratio 1.42 (95% confidence interval 0.48-4.25) for a CT value 31-36, increasing to odds ratio 5.43 (95% confidence interval 1.48-19.9) for a CT value ≤30 compared with patients with a CT value ≥37. The Charlson comorbidity index (CCI) improved the risk stratification: patients with a CT value ≥37 and CCI ≤2 had a 9% mortality risk compared with 70% among those with a CT value ≤30 and CCI ≥6. Comorbid cardiovascular disease, solid tumors, immunological disorders, premorbid corticosteroids, hypoxemia, abnormal leukocyte counts, low serum albumin, and C-reactive protein ≥100 were also independently associated with 30-day mortality. The sensitivity analyses did not suggest selection bias. Conclusion: Fungal burden may improve the risk stratification of patients without HIV-negative patients with PCP.

Antiretroviral treatment (ART) has converted HIV from a lethal disease to a chronic condition, yet co-morbidities persist. Incomplete immune recovery and chronic immune activation, especially in the gut mucosa, contribute to these complications. Inflammasomes, multi-protein complexes activated by innate immune receptors, appear to play a role in these inflammatory responses. In particular, preliminary data indicate the involvement of IFI16 and NLRP3 inflammasomes in chronic HIV infection. This study explores inflammasome function in monocytes from people with HIV (PWH); 22 ART-treated with suppressed viremia and 17 untreated PWH were compared to 33 HIV-negative donors. Monocytes were primed with LPS and inflammasomes activated with ATP in vitro. IFI16 and NLRP3 mRNA expression were examined in a subset of donors. IFI16 and NLRP3 expression in unstimulated monocytes correlated negatively with CD4 T cell counts in untreated PWH. For IFI16, there was also a positive correlation with viral load. Monocytes from untreated PWH exhibit increased release of IL-1α, IL-1β, and TNF compared to treated PWH and HIV-negative donors. However, circulating monocytes in PWH are not pre-primed for inflammasome activation in vivo. The findings suggest a link between IFI16, NLRP3, and HIV progression, emphasizing their potential role in comorbidities such as cardiovascular disease. The study provides insights into inflammasome regulation in HIV pathogenesis and its implications for therapeutic interventions.

Objectives To estimate temporal trends in incidence rate (IR) and case fatality during a 14-year period from 2008 to 2021, and to assess possible shifts in these trends during the COVID-19 pandemic.Setting All Norwegian hospitals 2008–2021.Participants 317 705 patients ≥18 year with a sepsis International Classification of Diseases 10th revision code retrieved from The Norwegian Patient Registry.Primary and secondary measures Annual age-standardised IRs with 95% CIs. Poisson regression was used to estimate changes in IRs across time, and logistic regression was used to estimate ORs for in-hospital death.Results Among 12 619 803 adult hospitalisations, a total of 317 705 (2.5%) hospitalisations in 222 832 (70.0%) unique patients met the sepsis criteria. The overall age-standardised IR of a first sepsis admission was 246/100 000 (95% CI 245 to 247), whereas the age-standardised IR of all sepsis admissions was 352/100 000 (95% CI 351 to 354). In the period 2009–2019, the annual IR for a first sepsis episode was stable (IR ratio (IRR) per year, 0.999; 95% CI 0.994 to 1.004), whereas for recurrent sepsis the IR increased (annual IRR, 1.048; 95% CI 1.037 to 1.059). During the COVID-19 pandemic, the IRR for a first sepsis was 0.877 (95% CI 0.829 to 0.927) in 2020 and 0.929 (95% CI 0.870 to 0.992) in 2021, and for all sepsis it was 0.870 (95% CI 0.810 to 0.935) in 2020 and 0.908 (95% CI 0.840 to 0.980) in 2021, compared with the previous 11-year period. Case fatality among first sepsis admissions declined in the period 2009–2019 (annual OR 0.954 (95% CI 0.950 to 0.958)), whereas case fatality increased during the COVID-19 pandemic in 2020 (OR 1.061 (95% CI 1.001 to 1.124) and in 2021 (OR 1.164 (95% CI 1.098 to 1.233)).Conclusion The overall IR of sepsis increased from 2009 to 2019, due to an increasing IR of recurrent sepsis, and indicates that sepsis awareness with updated guidelines and education must continue.

Abstract Background Community-acquired pneumonia (CAP) is the most frequent infection diagnosis in hospitals. Antimicrobial therapy for CAP is depicted in clinical practice guidelines, but adherence data and effect of antibiotic stewardship measures are lacking. Methods A dedicated antibiotic team pointed out CAP as a potential target for antimicrobial stewardship (AMS) measures at a 1.000-bed, tertiary care, teaching university hospital in Norway from March until May for the years 2016 throughout 2021. The aim of the AMS program was to increase diagnostic and antimicrobial therapy adherence to national clinical practice guideline recommendations through multiple and continuous AMS efforts. Descriptive statistics were retrospectively used to delineate antimicrobial therapy for CAP. The primary outcomes were proportions that received narrow-spectrum beta-lactams, and broad-spectrum antimicrobial therapy. Results 1.112 CAP episodes were identified. The annual proportion that received narrow-spectrum beta-lactams increased from 56.1 to 74.4% (p = 0.045). Correspondingly, the annual proportion that received broad-spectrum antimicrobial therapy decreased from 34.1 to 17.1% (p = 0.002). Trends were affected by the coronavirus pandemic. Mortality and 30-day readmission rates remained unchanged. De-escalation strategies were frequently unutilized, and overall therapy duration exceeded clinical practice guideline recommendations substantially. Microbiologically confirmed CAP episodes increased from 33.7 to 56.2% during the study period. Conclusion CAP is a suitable model condition that is sensitive to AMS measures. A continuous focus on improved microbiological diagnostics and antimicrobial therapy initiation is efficient in increasing adherence to guideline recommendations. There is an unmet need for better antimicrobial de-escalation strategies.

Abstract Observational studies have indicated an association between iron status and risk of sepsis and COVID-19. We estimated the effect of genetically-predicted iron biomarkers on risk of sepsis and risk of being hospitalized with COVID-19, performing a two-sample Mendelian randomization study. For risk of sepsis, one standard deviation increase in genetically-predicted serum iron was associated with odds ratio (OR) of 1.14 (95% confidence interval [CI] 1.01–1.29, P = 0.031). The findings were supported in the analyses for transferrin saturation and total iron binding capacity, while the estimate for ferritin was inconclusive. We found a tendency of higher risk of hospitalization with COVID-19 for serum iron; OR 1.29 (CI 0.97–1.72, P = 0.08), whereas sex-stratified analyses showed OR 1.63 (CI 0.94–2.86, P = 0.09) for women and OR 1.21 (CI 0.92–1.62, P = 0.17) for men. Sensitivity analyses supported the main findings and did not suggest bias due to pleiotropy. Our findings suggest a causal effect of genetically-predicted higher iron status and risk of hospitalization due to sepsis and indications of an increased risk of being hospitalized with COVID-19. These findings warrant further studies to assess iron status in relation to severe infections, including the potential of improved management.

Background Tocilizumab improves myocardial salvage index (MSI) in patients with ST-elevation myocardial infarction (STEMI), but its mechanisms of action are unclear. Here, we explored how cytokines were affected by tocilizumab and their correlations with neutrophils, C-reactive protein (CRP), troponin T, MSI and infarct size.Methods STEMI patients were randomised to receive a single dose of 280 mg tocilizumab (n=101) or placebo (n=98) before percutaneous coronary intervention. Blood samples were collected before infusion of tocilizumab or placebo at baseline, during follow-up at 24–36, 72–168 hours, 3 and 6 months. 27 cytokines were analysed using a multiplex cytokine assay. Cardiac MRI was performed during hospitalisation and 6 months.Results Repeated measures analysis of variance showed significant (p

Abstract Previous studies indicate sex differences in incidence and severity of bloodstream infections (BSI). We examined the effect of sex on risk of BSI, BSI mortality, and BSI caused by the most common infecting bacteria. Using causal mediation analyses, we assessed if this effect is mediated by health behaviours (smoking, alcohol consumption), education, cardiovascular risk factors (systolic blood pressure, non-HDL cholesterol, body mass index) and selected comorbidities. This prospective study included 64,040 participants (46.8% men) in the population-based HUNT2 Survey (1995–1997) linked with hospital records in incident BSI. During median follow-up of 15.2 years, 1840 (2.9%) participants (51.3% men) experienced a BSI and 396 (0.6%) died (56.6% men). Men had 41% higher risk of first-time BSI (95% confidence interval (CI), 28–54%) than women. Together, health behaviours, education, cardiovascular risk factors and comorbidities mediated 34% of the excess risk of BSI observed in men. The HR of BSI mortality was 1.87 (95% CI 1.53–2.28), for BSI due to S. aureus 2.09 (1.28–2.54), S. pneumoniae 1.36 (1.05–1.76), E. coli 0.97 (0.84–1.13) in men vs women. This study shows that men have higher risk of BSI and BSI mortality than women. One-third of this effect was mediated by potential modifiable risk factors for incident BSI.

Abstract Purpose Studies on aetiology of community-acquired pneumonia (CAP) vary in terms of microbial sampling methods, anatomical locations, and laboratory analyses, since no gold standard exists. In this large, multicentre, retrospective, regional study from Norway, our primary objective was to report the results of a strategic diagnostic stewardship intervention, targeting diagnostic yield from lower respiratory tract sampling. The secondary objective was to report hospitalized CAP aetiology and the diagnostic yield of various anatomical sampling locations. Methods Medical records from cases diagnosed with hospitalized CAP were collected retrospectively from March throughout May for three consecutive years at six hospitals. Between year one and two, we launched a diagnostic stewardship intervention at the emergency room level for the university teaching hospital only. The intervention was multifaceted aiming at upscaling specimen collection and enhancing collection techniques. Year one at the interventional hospital and every year at the five other emergency hospitals were used for comparison. Results Of the 1280 included cases of hospitalized CAP, a microbiological diagnosis was established for 29.1% among 1128 blood cultures and 1444 respiratory tract specimens. Blood cultures were positive for a pathogenic respiratory tract microbe in 4.9% of samples, whereas upper and lower respiratory tract samples overall provided a probable microbiological diagnosis in 21.3% and 47.5%, respectively. Expectorated or induced sputum overall provided aetiology in 51.7% of the samples. At the interventional hospital, the number of expectorated or induced sputum samples were significantly increased, and diagnostic yield from expectorated or induced sputum was significantly enhanced from 41.2 to 62.0% after the intervention (p = 0.049). There was an over-representation of samples from the interventional hospital during the study period. Non-typeable Haemophilus influenza and Streptococcus pneumoniae accounted for 25.3% and 24.7% of microbiologically confirmed cases, respectively. Conclusion Expectorated or induced sputum outperformed other sampling methods in providing a reliable microbiological diagnosis for hospitalized CAP. A diagnostic stewardship intervention significantly improved diagnostic yield of lower respiratory tract sampling.

Abstract Background Inflammatory aneurysms and mycotic aneurysms make up a minority of abdominal aortic aneurysms. Mainly autoimmune mechanisms are proposed in the pathogenesis of inflammatory aneurysms, and it is not routine to check for infectious agents as disease culprits. Case presentation A 58-year-old European male with complaints of abdominal and back pain for 8 weeks was admitted after a semi-urgent computed tomography scan revealed an 85 mm inflammatory abdominal aortic aneurysm. The patient had normal vital signs, slightly elevated inflammatory markers, and mild anemia on admission. Clinical examination revealed a tender pulsating mass in his abdomen. His clinical condition was interpreted as impending rupture and urgent repair of the aneurysm was deemed necessary. Due to the patient’s relatively young age and aneurysm neck morphology, open aortic repair was preferred. Preoperatively, the aneurysm appeared inflamed, with fibrous wall thickening and perianeurysmal adhesions. Aneurysm wall biopsies were sent to histopathological and microbiological diagnostics. Routine cultures were negative, but 16S rRNA gene real-time polymerase chain reaction was positive and Borrelia afzelii was identified by DNA sequencing of the polymerase chain reaction product. B. afzelii was also identified by sequencing the polymerase chain reaction product of a Borrelia-specific groEL target. Immunoglobulin G and M anti-Borrelia antibodies were present on serological analysis. Histopathological analysis displayed loss of normal aortic wall structure and diffuse infiltration of lymphocytes and plasma cells. The patient had an uneventful recovery and was discharged after 1 week to a regional rehabilitation facility. Though the patient fares clinically well and inflammatory markers had normalized, antimicrobial treatment with doxycycline continues at 3 months follow-up due to remaining radiologic signs of inflammation. Conclusions Borrelia infection in the setting of acute aortic pathology is a rare entity. To our knowledge, this is the first case report to demonstrate a mycotic abdominal aortic aneurysm as a rare manifestation of Lyme disease. Aortic wall biopsies and real-time polymerase chain reaction analysis of the specimen were essential for accurate diagnosis. This finding may contribute to the understanding of the etiology of inflammatory aneurysmal disease and abdominal aneurysms in general.

Abstract Background Pneumocystis pneumonia (PCP) severely menaces modern chemotherapy and immunosuppression. Detailed description of the epidemiology of Pneumocystis jirovecii today is needed to identify candidates for PCP-prophylaxis. Methods We performed a 12-year retrospective study of patients with P. jirovecii detected by polymerase chain reaction in Central Norway. In total, 297 patients were included. Comprehensive biological, clinical and epidemiological data were abstracted from patients’ medical records. Regional incidence rates and testing trends were also assessed. Results From 2007 to 2017 we found a 3.3-fold increase in testing for P. jirovecii accompanied by a 1.8-fold increase in positive results. Simultaneously, regional incidence rates doubled from 5.0 cases per 100,000 person years to 10.8. A majority of the study population had predisposing conditions other than human immunodeficiency virus (HIV). Hematological (36.0%) and solid cancers (25.3%) dominated. Preceding corticosteroids were a common denominator for 72.1%. Most patients (74.4%) presented with at least two cardinal symptoms; cough, dyspnea or fever. Main clinical findings were hypoxia, cytopenias and radiological features consistent with PCP. A total of 88 (29.6%) patients required intensive care and 121 (40.7%) suffered at least one complication. In-hospital mortality was 21.5%. Three patients (1.0%) had received prophylaxis. Conclusions P. jirovecii is re-emerging; likely due to increasing immunosuppressants use. This opportunistic pathogen threatens the life of heterogenous non-HIV immunosuppressed populations currently at growth. Corticosteroids seem to be a major risk factor. A strategy to increase prophylaxis is called for.

Summary: Background: We recently showed that interleukin (IL)-6 inhibition by tocilizumab improves myocardial salvage in ST-elevation myocardial infarction (STEMI). However, the mechanisms for this effect are not clear. Methods: In this exploratory sub-study of the ASSAIL-MI trial, we examined leukocyte differential counts and their relation to myocardial salvage and peak troponin T (TnT) in STEMI patients randomised to tocilizumab (n = 101) or placebo (n = 98). We performed RNA-sequencing on whole blood (n = 40) and T cells (n = 20). B and T cell subpopulations were examined by flow cytometry (n = 69). Findings: (i) STEMI patients had higher neutrophil counts at hospitalisation compared with stable angina patients. (ii) After percutaneous coronary intervention there was a gradual decline in neutrophils, which was significantly more pronounced in the tocilizumab group. (iii) The decrease in neutrophils in the tocilizumab group was associated with improved myocardial salvage and lower peak TnT. (iv) RNA-sequencing suggested that neutrophil function was also attenuated by tocilizumab. (v) B and T cell sub-populations changed only minimally after STEMI with minor effects of tocilizumab, supported as well by RNA-sequencing analyses of T cells. (vi) However, a low CD8+ count was associated with improved myocardial salvage in patients admitted to the hospital > 3 h after symptom onset. Interpretation: Tocilizumab induced a rapid reduction in neutrophils and seemed to attenuate neutrophil function in STEMI patients potentially related to the beneficial effects of tocilizumab on myocardial salvage. Funding: South-Eastern Norway Regional Health Authority (Nos. 2019067, 2017084), the Central Norway Regional Health Authority and Norwegian Research Council (No. 283867).

Manipulation of Toll-like receptors (TLRs) affects HIV-1 infection and latency reversal. Here, the authors show that HIV-1 is endocytosed and recognized by TLR8 in human primary CD4+T cells and that TLR8 stimulation induces an inflammatory response that promotes HIV-1 replication and reversal of latency.

PurposeSevere bacterial infections are important causes of hospitalization and loss of health worldwide. In this study we aim to characterize the total burden, recurrence and severity of bacterial infections in the general population during a 22-year period.MethodsWe investigated hospitalizations due to bacterial infection from eight different foci in the prospective population-based Trøndelag Health Study (the HUNT Study), where all inhabitants aged ≥ 20 in a Norwegian county were invited to participate. Enrollment was between 1995 and 1997, and between 2006 and 2008, and follow-up ended in February 2017. All hospitalizations, positive blood cultures, emigrations and deaths in the follow-up period were captured through registry linkage.ResultsA total of 79,393 (69.5% and 54.1% of the invited population) people were included, of which 42,237 (53%) were women and mean age was 48.5 years. There were 37,298 hospitalizations due to infection, affecting 15,496 (22% of all included) individuals. The median time of follow-up was 20 years (25th percentile 9.5-75th percentile 20.8). Pneumonia and urinary tract infections were the two dominating foci with incidence rates of 639 and 550 per 100,000 per year, respectively, and with increasing incidence with age. The proportion of recurring admissions ranged from 10.0% (central nervous system) to 30.0% (pneumonia), whilst the proportion with a positive blood culture ranged from 4.7% (skin- and soft tissue infection) to 40.9% (central nervous system). The 30-day mortality varied between 3.2% (skin- and soft tissue infection) and 20.8% (endocarditis).ConclusionsIn this population-based cohort, we observed a great variation in the incidence, positive blood culture rate, recurrence and mortality between common infectious diseases. These results may help guide policy to reduce the infectious disease burden in the population.

ABSTRACT Pneumocystis jirovecii is a threat to iatrogenically immunosuppressed individuals, a heterogeneous population at rapid growth. We assessed the ability of an in-house semiquantitative real-time PCR assay to discriminate Pneumocystis pneumonia (PCP) from colonization and identified risk factors for infection in these patients. Retrospectively, 242 PCR-positive patients were compared according to PCP status, including strata by immunosuppressive conditions, human immunodeficiency virus (HIV) infection excluded. Associations between host characteristics and cycle threshold (CT) values, semiquantitative real-time PCR correlates of fungal loads in lower respiratory tract specimens, were investigated. CT values differed significantly according to PCP status. Overall, a CT value of 36 allowed differentiation between PCP and colonization with sensitivity and specificity of 71.3% and 77.1%, respectively. A CT value of less than 31 confirmed PCP, whereas no CT value permitted exclusion. A considerable diversity was uncovered; solid organ transplant (SOT) recipients had significantly higher fungal loads than patients with hematological malignancies. In SOT recipients, a CT cutoff value of 36 resulted in sensitivity and specificity of 95.0% and 83.3%, respectively. In patients with hematological malignancies, a higher CT cutoff value of 37 improved sensitivity to 88.5% but reduced specificity to 66.7%. For other conditions, assay validity appeared inferior. Corticosteroid usage was an independent predictor of PCP in a multivariable analysis and was associated with higher fungal loads at PCP expression. Semiquantitative real-time PCR improves differentiation between PCP and colonization in immunocompromised HIV-negative individuals with acute respiratory syndromes. However, heterogeneity in disease evolution requires separate cutoff values across intrinsic and iatrogenic predisposition for predicting non-HIV PCP. IMPORTANCE Pneumocystis jirovecii is potentially life threatening to an increasing number of individuals with compromised immune systems. This microorganism can cause severe pneumonia in susceptible hosts, including patients with cancer and autoimmune diseases and people undergoing solid organ transplantation. Together, these patients constitute an ever-diverse population. In this paper, we demonstrate that the heterogeneity herein has important implications for how we diagnose and assess the risk of Pneumocystis pneumonia (PCP). Specifically, low loads of microorganisms are sufficient to cause infection in patients with blood cancer compared to those in solid organ recipients. With this new insight into host versus P. jirovecii biology, clinicians can manage patients at risk of PCP more accurately. As a result, we take a significant step toward offering precision medicine to a vulnerable patient population. One the one hand, these patients have propensity for adverse effects from antimicrobial treatment. On the other hand, this population is susceptible to life-threatening infections, including PCP.

Objective Beyond reducing inflammation and troponin T (TnT) release, the interleukin-6 receptor antagonist tocilizumab reduces neutrophil counts in patients with non-ST segment elevation myocardial infarction (NSTEMI). It is unclear if this is related to formation of neutrophil extracellular traps (NETs), carrying inflammatory and thrombotic properties.Methods In a placebo-controlled trial, 117 patients with NSTEMI were randomised to a single dose of tocilizumab (n=58) or placebo (n=59) before coronary angiography. The NETs related markers double-stranded DNA (dsDNA), myloperoxidase–DNA (MPO–DNA) and citrullinated histone 3 (H3Cit) were measured at five consecutive time points during hospitalisation (days 1–3).Results Our major findings were: (1) H3Cit levels were significantly higher in the tocilizumab compared with the placebo group at all time points (all p

Abstract Background Peripheral intravenous catheters (PIVCs) account for a mean of 38% of catheter associated bloodstream infections (CABSI) with Staphylococcus aureus, which are preventable if deficiencies in best practice are addressed. There exists no feasible and reliable quality surveillance tool assessing all important areas related to PIVC quality. Thus, we aimed to develop and test feasibility and reliability for an efficient quality assessment tool of overall PIVC quality. Methods The Peripheral Intravenous Catheter- mini Questionnaire, PIVC-miniQ, consists of 16 items calculated as a sum score of problems regarding the insertion site, condition of dressing and equipment, documentation, and indication for use. In addition, it contains background variables like PIVC site, size and insertion environment. Two hospitals tested the PIVC-miniQ for feasibility and inter-rater agreement. Each PIVC was assessed twice, 2–5 min apart by two independent raters. We calculated the intraclass correlation coefficient (ICC) for each hospital and overall. For each of the 16 items, we calculated negative agreement, positive agreement, absolute agreement, and Scott’s pi. Results Sixty-three raters evaluated 205 PIVCs in 177 patients, each PIVC was assessed twice by independent raters, in total 410 PIVC observations. ICC between raters was 0.678 for hospital A, 0.577 for hospital B, and 0.604 for the pooled data. Mean time for the bedside assessment of each PIVC was 1.40 (SD 0.0007) minutes. The most frequent insertion site symptom was “pain and tenderness” (14.4%), whereas the most prevalent overall problem was lack of documentation of the PIVC (26.8%). Up to 50% of PIVCs were placed near joints (wrist or antecubital fossae) or were inserted under suboptimal conditions, i.e. emergency department or ambulance. Conclusions Our study highlights the need for PIVC quality surveillance on ward and hospital level and reports the PIVC-miniQ to be a reliable and time efficient tool suitable for frequent point-prevalence audits.

Background: During atherogenesis, cholesterol precipitates into cholesterol crystals (CC) in the vessel wall, which trigger plaque inflammation by activating the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome. We investigated the relationship between CC, complement and NLRP3 in patients with cardiovascular disease. Methods: We analysed plasma, peripheral blood mononuclear cells (PBMC) and carotid plaques from patients with advanced atherosclerosis applying ELISAs, multiplex cytokine assay, qPCR, immunohistochemistry, and gene profiling. Findings: Transcripts of interleukin (IL)-1beta(β) and NLRP3 were increased and correlated in PBMC from patients with acute coronary syndrome (ACS). Priming of these cells with complement factor 5a (C5a) and tumour necrosis factor (TNF) before incubation with CC resulted in increased IL-1β protein when compared to healthy controls. As opposed to healthy controls, systemic complement was significantly increased in patients with stable angina pectoris or ACS. In carotid plaques, complement C1q and C5b-9 complex accumulated around CC-clefts, and complement receptors C5aR1, C5aR2 and C3aR1 were higher in carotid plaques compared to control arteries. Priming human carotid plaques with C5a followed by CC incubation resulted in pronounced release of IL-1β, IL-18 and IL-1α. Additionally, mRNA profiling demonstrated that C5a and TNF priming followed by CC incubation upregulated plaque expression of NLRP3 inflammasome components. Interpretation: We demonstrate that CC are important local- and systemic complement activators, and we reveal that the interaction between CC and complement could exert its effect by activating the NLRP3 inflammasome, thus promoting the progression of atherosclerosis.

Background Interleukin 6 concentration is associated with myocardial injury, heart failure, and mortality after myocardial infarction. In the Norwegian tocilizumab non–ST‐segment–elevation myocardial infarction trial, the first randomized trial of interleukin 6 blockade in myocardial infarction, concentration of both C‐reactive protein and troponin T were reduced in the active treatment arm. In this follow‐up study, an aptamer‐based proteomic approach was employed to discover additional plasma proteins modulated by tocilizumab treatment to gain novel insights into the effects of this therapeutic approach. Methods and Results Plasma from percutaneous coronary intervention–treated patients, 24 in the active intervention and 24 in the placebo‐control arm, drawn 48 hours postrandomization were randomly selected for analysis with the SOMAscan assay. Employing slow off‐rate aptamers, the relative abundance of 1074 circulating proteins was measured. Proteins identified as being significantly different between groups were subsequently measured by enzyme immunoassay in the whole trial cohort (117 patients) at all time points (days 1–3 [7 time points] and 3 and 6 months). Five proteins identified by the SOMAscan assay, and subsequently confirmed by enzyme immunoassay, were significantly altered by tocilizumab administration. The acute‐phase proteins lipopolysaccharide‐binding protein, hepcidin, and insulin‐like growth factor‐binding protein 4 were all reduced during the hospitalization phase, as was the monocyte chemoattractant C‐C motif chemokine ligand 23. Proteinase 3, released primarily from neutrophils, was significantly elevated. Conclusions Employing the SOMAscan aptamer‐based proteomics platform, 5 proteins were newly identified that are modulated by interleukin 6 antagonism and may mediate the therapeutic effects of tocilizumab in non–ST‐segment–elevation myocardial infarction.

Abstract Background Radiation therapy (RT) results in pain relief for about 6 of 10 patients with cancer induced bone pain (CIBP) caused by bone metastases. The high number of non-responders, the long median time from RT to pain response and the risk of adverse effects, makes it important to determine predictors of treatment response. Clinical features such as cancer type, performance status and pain intensity, and biomarkers for osteoclast activity are proposed as predictors of response to RT. However, results are inconsistent and there is a need for better predictors of RT response. A similar argument can be stated for the development of cachexia; there are currently no predictors that can identify patients who will develop cachexia later in the cancer disease trajectory. Experimental and preclinical studies show that pain, depression and cachexia are related to inflammation. However, it is not known if inflammatory biomarkers can predict CIBP, depression or development of cachexia. Methods This multicenter, multinational longitudinal observational study will include 600 adult patients receiving RT for CIBP. Demographic data, clinical variables, osteoclast and inflammatory biomarkers will be assessed before start of RT, and 3, 8, 16, 24 and 52 weeks after last course of RT. The primary aim of the study is to identify potential predictors for pain relief from RT. Secondary aims are to explore potential predictors for development of cachexia, the longitudinal relationship between pain intensity and depression, and if inflammatory biomarkers are associated with changes in pain intensity, cachexia and depression during one-year follow up. Discussion The immediate clinical implication of the PRAIS study is to identify potential predictive factors for a RT response on CIBP, and thereby reduce non-efficacious RT. Patient benefits are fewer hospital visits, reduced risk of adverse effects and more individualized pain treatment. The long-term clinical implication of the PRAIS study is to improve the knowledge about inflammation in relation to CIBP, cachexia and depression and potentially identify associations and mechanisms that can be targeted for treatment. Trial registration ClinicalTrials.gov NCT02107664, date of registration April 8, 2014 (retrospectively registered). Trial sponsor The European Palliative Care Research Centre (PRC), Department of Clinical and Molecular Medicine, NTNU, Faculty of medicine and Health Sciences, Trondheim, N-7491, Norway.

Abstract Background Therapeutic hypothermia is neuroprotective in asphyxiated neonates by counteracting mechanisms contributing to brain injury. Although an initial increased permeability is part of an inflammatory reaction and thereby a natural healing process, an excessive endothelial permeability with edema formation may result in impaired hemodynamics. Reduced permeability may, however, benefit healing. Although plasma and interstitial colloid osmotic pressure are accessible and essential parameters for understanding fluid imbalance, the mechanisms of fluid exchange remain poorly understood. The potential influence of therapeutic hypothermia on plasma and interstitial colloid osmotic pressure, and the relationship between inflammatory markers and colloid osmotic pressure in asphyxiated neonates, was investigated. Methods Seventeen neonates with moderate to severe hypoxic ischemic encephalopathy, born after 35 weeks gestation, received servo-controlled whole body cooling before 6 h of age, followed by gradual rewarming after 72 h. All infants were treated according to a national hypothermia protocol. Interstitial fluid in the skin was collected at 7, 13, 25, 49, and 73 h after birth by subcutaneous implantation of multifilamentous nylon wicks with 60 min of implantation time. Biomarkers of inflammation and colloid osmotic pressure were measured in serum and interstitial fluid. Results A modest decrease in serum and interstitial colloid osmotic pressure was measured, leaving an unaltered difference in colloid osmotic pressure gradient. A decline in mean arterial pressure was observed between 7 and 13 h of life, with a concomitant decrease in positive fluid balance within the same time frame. White blood cell count and leukocyte subclasses dropped significantly throughout treatment, with elevated interstitial interleukin (IL)-1α and decreased serum IL-1RA, IL-6, and IL-10 during treatment time points. Conclusions Colloid osmotic pressures measured in serum and interstitial fluid during asphyxia is lower than previously reported, with small alteration of pressure differences across capillaries, reducing vascular filtration. An inherent local and systemic regulation of inflammation together with changes in colloid osmotic pressure may indicate a possible preventive mechanism of edema generation during neonatal asphyxia and therapeutic hypothermia. Trial registration ClinicalTrials.gov Identifier: NCT01044940. Date of registration: January 8, 2010.

Abstract Background We aimed to evaluate the clinical usefulness of qSOFA as a risk stratification tool for patients admitted with infection compared to traditional SIRS criteria or our triage system; the Rapid Emergency Triage and Treatment System (RETTS). Methods The study was an observational cohort study performed at one Emergency Department (ED) in an urban university teaching hospital in Norway, with approximately 20,000 visits per year. All patients >16 years presenting with symptoms or clinical signs suggesting an infection (n = 1535) were prospectively included in the study from January 1 to December 31, 2012. At arrival in the ED, vital signs were recorded and all patients were triaged according to RETTS vital signs, presenting infection, and sepsis symptoms. These admission data were also used to calculate qSOFA and SIRS. Treatment outcome was later retrieved from the patients’ electronic records (EPR) and mortality data from the Norwegian population registry. Results Of the 1535 admitted patients, 108 (7.0%) fulfilled the Sepsis2 criteria for severe sepsis. The qSOFA score ≥2 identified only 33 (sensitivity 0.32, specificity 0.98) of the patients with severe sepsis, whilst the RETTS-alert ≥ orange identified 92 patients (sensitivity 0.85, specificity 0.55). Twenty-six patients died within 7 days of admission; four (15.4%) of them had a qSOFA ≥2, and 16 (61.5%) had RETTS ≥ orange alert. Of the 68 patients that died within 30 days, only eight (11.9%) scored ≥2 on the qSOFA, and 45 (66.1%) had a RETTS ≥ orange alert. Discussion In order to achieve timely treatment for sepsis, a sensitive screening tool is more important than a specific one. Our study is the fourth study were qSOFA finds few of the sepsis cases in prehospital or at arrival to the ED. We add information on the RETTS triage system, the two highest acuity levels together had a high sensitivity (85%) for identifying sepsis at arrival to the ED - and thus, RETTS should not be replaced by qSOFA as a screening and trigger tool for sepsis at arrival. Conclusion In this observational cohort study, qSOFA failed to identify two thirds of the patients admitted to an ED with severe sepsis. Further, qSOFA failed to be a risk stratification tool as the sensitivity to predict 7-day and 30-day mortality was low. The sensitivity was poorer than the other warning scores already in use at the study site, RETTS-triage and the SIRS criteria.

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TLR8 is the major endosomal sensor of degraded RNA in human monocytes and macrophages. It has been implicated in the sensing of viruses and more recently also bacteria. We previously identified a TLR8-IFN regulatory factor 5 (IRF5) signaling pathway that mediates IFNβ and interleukin-12 (IL-12) induction by Staphylococcus aureus and is antagonized by TLR2. The relative importance of TLR8 for the sensing of various bacterial species is however still unclear. We here compared the role of TLR8 and IRF5 for the sensing of Group B Streptococcus (GBS), S. aureus, and Escherichia coli in human primary monocytes and monocyte-derived macrophages (MDM). GBS induced stronger IFNβ and TNF production as well as IRF5 nuclear translocation compared to S. aureus grown to the stationary phase, while S. aureus in exponential growth appeared similarly potent to GBS. Cytokine induction in primary human monocytes by GBS was not dependent on hemolysins, and induction of IFNβ and IL-12 as well as IRF5 activation were reduced with TLR2 ligand costimulation. Heat inactivation of GBS reduced IRF5 and NF-kB translocation, while only the viable E. coli activated IRF5. The attenuated stimulation correlated with loss of bacterial RNA integrity. The E. coli-induced IRF5 translocation was not inhibited by TLR2 costimulation, suggesting that IRF5 was activated via a TLR8-independent mechanism. Gene silencing of MDM using siRNA revealed that GBS-induced IFNβ, IL-12-p35, and TNF production was dependent on TLR8 and IRF5. In contrast, cytokine induction by E. coli was TLR8 independent but still partly dependent on IRF5. We conclude that TLR8-IRF5 signaling is more important for the sensing of GBS than for stationary grown S. aureus in human primary monocytes and MDM, likely due to reduced resistance of GBS to phagosomal degradation and to a lower production of TLR2 activating lipoproteins. TLR8 does not sense viable E. coli, while IRF5 still contributes to E. coli-induced cytokine production, possibly via a cytosolic nucleic acid sensing mechanism.

The chemokine C-C motif ligand 20 (CCL20) is increased in the colonic mucosa during active inflammatory bowel disease (IBD) and can be found both in the epithelium and immune cells in the lamina propria. The present study investigated CCL20 and C-C motif Chemokine Receptor 6 (CCR6) in peripheral blood mononuclear cells (PBMCs) (n = 40) from IBD patients and healthy controls, to identify inductors of CCL20 release encountered in a local proinflammatory environment. CCL20 release from PBMCs was increased when activating TLR2/1 or NOD2, suggesting that CCL20 is part of a first line response to danger-associated molecular patterns also in immune cells. Overall, ulcerative colitis (UC) had a significantly stronger CCL20 release than Crohn’s disease (CD) (+242%, p < 0.01), indicating that the CCL20-CCR6 axis may be more involved in UC. The CCL20 receptor CCR6 is essential for the chemotactic function of CCL20. UC with active inflammation had significantly decreased CCR6 expression and a reduction in CCR6+ cells in circulation, indicating chemoattraction of CCR6+ cells from circulation towards peripheral tissues. We further examined CCL20 induced release of cytokines from PBMCs. Stimulation with CCL20 combined with TNF increased IL-1β release from PBMCs. By attracting additional immune cells, as well as inducing proinflammatory IL-1β release from immune cells, CCL20 may protract the inflammatory response in ulcerative colitis.

The chemokine CCL20 and its receptor CCR6 are putative drug targets in inflammatory bowel disease, and CCL20 is a novel IBD predilection gene. Previous findings on the CCL20 response in these diseases are divergent. This study was undertaken to examine CCL20 and CCR6 during active and inactive disease, and mechanisms for CCL20 regulation by the innate immune system. As TLR3 has recently emerged as a possible mediator of CCL20 production, we hypothesised that this TLR plays an important role in enterocytic CCL20 production.A large microarray study on colonic pinch biopsies from active and inactive ulcerative colitis and Crohn's disease provided background information. CCL20 and CCR6 were localized and their expression levels assessed in biopsies using in situ hybridization and immunohistochemistry. Regulation of CCL20 was studied in the HT29 cell line using a panel of pattern recognition receptor ligands followed by a TLR3 siRNA assay.CCL20 and CCR6 mRNA abundances were increased during active inflammation (CCL20 5.4-fold in ulcerative colitis and 4.2-fold in Crohn's disease; CCR6 1.8 and 2.0, respectively). CCL20 and CCR6 mRNA positive immune cells in lamina propria were more numerous, and CCL20 immunoreactivity increased massively in the epithelial cells during active inflammation for both diseases. TLR3 stimulation potently induced upregulation and release of CCL20 from HT29 cells, and TLR3 silencing reduced CCL20 mRNA and protein levels.The CCL20-CCR6 axis is involved during active inflammation in both ulcerative colitis and Crohn's disease. The epithelial cells seem particularly involved in the CCL20 response, and results from this study strongly suggest that the innate immune system is important for activation of the epithelium, especially through TLR3.

Interleukin (IL)-10 may have a therapeutic potential in atherosclerosis, but its mechanisms of action have not been clarified. Foam cell formation is a key event in atherogenesis, and apoptosis of these lipid-laden cells may promote plaque destabilization. We sought to explore whether IL-10 could have plaque-stabilizing properties in acute coronary syndromes (ACS). We studied the effect of IL-10 on oxidized low density lipoprotein (oxLDL)-stimulated THP-1 cells and monocyte-derived macrophages from ACS patients and healthy controls using different experimental approaches. Our main findings were: i) IL-10 enhances lipid accumulation in oxLDL-stimulated THP-1 macrophages, at least partly by counteracting oxLDL-induced apoptosis; ii) This antiapoptotic effect of IL-10 involves increased expression of the antiapoptotic genes Bfl-1 and Mcl-1, accompanied by protective effects on mitochondria function; iii) By silencing Bfl-1 and Mcl-1 genes using siRNAs, we were able to abolish this IL-10-mediated effect on lipid accumulation; iv) IL-10 also induced lipid accumulation in oxLDL-stimulated macrophages from patients with ACS, but not in macrophages from healthy controls; v) In ACS patients, this enhancing effect of IL-10 on lipid accumulation was accompanied by enhanced Mcl-1 expression. No such antiapoptotic effect was seen in macrophages from healthy controls.These findings suggest a new mechanism for the effect of IL-10 in atherosclerosis, possibly contributing to plaque stabilization.

BACKGROUND: Scrub typhus, caused by Orientia tsutsugamushi, is endemic in the Asia-Pacific region. Mortality is high if untreated, and even with treatment as high as 10-20%, further knowledge of the immune response during scrub typhus is needed. The current study was aimed at comparing plasma levels of a variety of inflammatory mediators in scrub typhus patients and controls in South India in order to map the broader cytokine profile and their relation to disease severity and clinical outcome. METHODOLOGY/PRINCIPAL FINDINGS: We examined plasma levels of several cytokines in scrub typhus patients (n = 129) compared to healthy controls (n = 31) and infectious disease controls (n = 31), both in the acute phase and after recovery, by multiplex technology and enzyme immunoassays. Scrub typhus patients were characterized by marked changes in the cytokine network during the acute phase, differing not only from healthy controls but also from infectious disease controls. While most of the inflammatory markers were raised in scrub typhus, platelet-derived mediators such as RANTES were markedly decreased, probably reflecting enhanced platelet activation. Some of the inflammatory markers, including various chemokines (e.g., interleukin-8, monocyte chemoattractant peptide-1 and macrophage inflammatory protein-1β) and downstream markers of inflammation (e.g., C-reactive protein and pentraxin-3), were also associated with disease severity and mortality during follow-up, with a particular strong association with interleukin-8. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that scrub typhus is characterized by a certain cytokine profile that includes dysregulated levels of a wide range of mediators, and that this enhanced inflammation could contribute to disease severity and clinical outcome.

BACKGROUND: Biomarkers to differentiate between active tuberculosis (TB) and latent TB infection (LTBI) and to monitor treatment responses are requested to complement TB diagnostics and control, particularly in patients with multi-drug resistant TB. We have studied soluble markers of the Toll-like-receptor 4 (TLR-4) pathway in various stages of TB disease and during anti-TB treatment. METHODS: Plasma samples from patients with culture confirmed drug-sensitive TB (n = 19) were collected before and after 2, 8 and 24 weeks of efficient anti-TB treatment and in a LTBI group (n = 6). Soluble (s) CD14 and myeloid differentiation-2 (MD-2) were analyzed by the Enzyme-linked immunosorbent assay (ELISA). Lipopolysaccharide (LPS) was analyzed by the Limulus Amebocyte Lysate colorimetric assay. Nonparametric statistics were applied. RESULTS: Plasma levels of sCD14 (p

In inflammatory bowel disease (IBD), genetic susceptibility together with environmental factors disturbs gut homeostasis producing chronic inflammation. The two main IBD subtypes are Ulcerative colitis (UC) and Crohn's disease (CD). We present the to-date largest microarray gene expression study on IBD encompassing both inflamed and un-inflamed colonic tissue. A meta-analysis including all available, comparable data was used to explore important aspects of IBD inflammation, thereby validating consistent gene expression patterns.Colon pinch biopsies from IBD patients were analysed using Illumina whole genome gene expression technology. Differential expression (DE) was identified using LIMMA linear model in the R statistical computing environment. Results were enriched for gene ontology (GO) categories. Sets of genes encoding antimicrobial proteins (AMP) and proteins involved in T helper (Th) cell differentiation were used in the interpretation of the results. All available data sets were analysed using the same methods, and results were compared on a global and focused level as t-scores.Gene expression in inflamed mucosa from UC and CD are remarkably similar. The meta-analysis confirmed this. The patterns of AMP and Th cell-related gene expression were also very similar, except for IL23A which was consistently higher expressed in UC than in CD. Un-inflamed tissue from patients demonstrated minimal differences from healthy controls.There is no difference in the Th subgroup involvement between UC and CD. Th1/Th17 related expression, with little Th2 differentiation, dominated both diseases. The different IL23A expression between UC and CD suggests an IBD subtype specific role. AMPs, previously little studied, are strongly overexpressed in IBD. The presented meta-analysis provides a sound background for further research on IBD pathobiology.

The pathophysiological hallmark of spotted fever group rickettsioses comprises vascular inflammation. Based on the emerging importance of the wingless (Wnt) pathways in inflammation and vascular biology, we hypothesized that Dickkopf-1 (DKK-1), as a major modulator of Wnt signaling, could be involved in the pathogenesis in rickettsial infections. Our major findings were: (i) While baseline concentration of DKK-1 in patients with R. conorii infection (n = 32) were not different from levels in controls (n = 24), DKK-1 rose significantly from presentation to first follow-up sample (median 7 days after baseline). (ii) In vitro experiments in human umbilical vein endothelial cells (HUVECs) showed that while heat-inactivated R. conorii enhanced the release of interleukin-6 (IL-6) and IL-8, it down-regulated the release of endothelial-derived DKK-1 in a time- and dose-dependent manner. (iii) Silencing of DKK-1 attenuated the release of IL-6, IL-8 and growth-related oncogene (GRO)α in R. conorii-exposed HUVECs, suggesting inflammatory effects of DKK-1. (iv) Silencing of DKK-1 attenuated the expression of tissue factor and enhanced the expression of thrombomodulin in R. conorii-exposed HUVECs suggesting pro-thrombotic effects of DKK-1. The capacity of R. conorii to down-regulate endothelial-derived DKK-1 and the ability of silencing DKK-1 to attenuate R. conorii-induced inflammation in endothelial cells could potentially reflect a novel mechanism by which R. conorii escapes the immune response at the site of infection.

BACKGROUND: CCL19 and CCL21, acting through CCR7, are termed homeostatic chemokines. Based on their role in concerting immunological responses and their proposed involvement in tissue remodeling, we hypothesized that these chemokines could play a pathogenic role in heart failure (HF). METHODOLOGY/PRINCIPAL FINDINGS: Our main findings were: (i) Serum levels of CCL19 and particularly CCL21 were markedly raised in patients with chronic HF (n = 150) as compared with healthy controls (n = 20). A CCL21 level above median was independently associated with all-cause mortality. (ii) In patients with HF following acute myocardial infarction (MI; n = 232), high versus low CCL21 levels 1 month post-MI were associated with cardiovascular mortality, even after adjustment for established risk factors. (iii). Explanted failing human LV tissue (n = 29) had markedly increased expression of CCL21 as compared with non-failing myocardium (n = 5). (iv) Our studies in CCR7(-/-) mice showed improved survival and attenuated increase in markers of myocardial dysfunction and wall stress in post-MI HF after 1 week, accompanied by increased myocardial expression of markers of regulatory T cells. (v) Six weeks post-MI, there was an increase in markers of myocardial dysfunction and wall stress in CCR7 deficient mice. CONCLUSIONS/SIGNIFICANCE: High serum levels of CCL21 are independently associated with mortality in chronic and acute post-MI HF. Our findings in CCR7 deficient mice may suggest that CCL21 is not only a marker, but also a mediator of myocardial failure. However, while short term inhibition of CCR7 may be beneficial following MI, a total lack of CCR7 during long-term follow-up could be harmful.