BACKGROUND: The prevalence of diabetes mellitus and its sequelae has been on the rise, and diabetic foot ulcer (DFU) is the leading cause of non-traumatic lower limb amputation globally. The rising occurrence and financial burden associated with DFU necessitate improved clinical assessment and treatment. Diabetes has been found to enhance the formation of neutrophil extracellular traps (NETs) by neutrophils, and excessive NETs have been implicated in tissue damage and impaired wound healing. However, there is as yet insufficient evidence to clarify the value of NETs in assessing and predicting outcomes of DFU.
METHODS: We designed this prospective study with three cohorts formed from type 2 diabetes mellitus (T2DM) patients with DFU (n = 200), newly diagnosed T2DM patients (n = 42), and healthy donors (n = 38). Serum levels of NETs were detected for all groups, and the prognostic value for DFU-related amputation was analyzed.
RESULTS: The results showed that serum NET levels of the DFU group were significantly higher than in the T2DM group (P < 0.05), which also had significantly elevated serum NET levels compared to healthy donors (P < 0.05). Multivariate Cox regression showed that serum NET levels, diabetic foot surgical history, and Wagner grade were the risk factors for amputation (P < 0.05), and these three variables also exhibited the highest coefficient values in additional Lasso Cox regression. For patients with DFU, Kaplan-Meier curves showed that high serum NET levels associated with higher amputation probability (HR = 0.19, P < 0.01) and ROC curve based on NET value showed good validity for amputation (AUC: 0.727, CI 0.651-0.803).
CONCLUSIONS: Elevated serum NET levels serve as an easily accessible serological prognostic marker for assessing the risk of DFU-related amputation, thereby offering evaluation metrics for healthcare providers. Further investigations are necessary to understand the mechanisms driving this relationship.

Neuroendocrine tumors (NETs) are slow-growing tumors that express high levels of somatostatin receptors (SSTRs). Recent studies have shown the superiority of radiolabeled SSTR antagonists in theranostics compared to agonists. In this prospective study, we compared the diagnostic efficacy between [68Ga]Ga-DOTANOC and [68Ga]Ga-DATA5m-LM4 in the detection of primary and metastatic lesions in patients with well differentiated gastroenteropancreatic (GEP) NETs. Histologically proven GEP-NET patients underwent [68Ga]Ga-DOTANOC & [68Ga]Ga-DATA5m-LM4 PET/CT scans, which were analyzed. The qualitative analysis involved the visual judgment of radiotracer uptake validated by the morphological findings using CT, which was considered as the reference standard. Quantitative comparisons were presented as the standardized uptake value (SUV) corrected for lean body mass: SULpeak, SULavg, and tumor-to-background ratios (TBR). In total, 490 lesions were confirmed via diagnostic CT. The lesion-based sensitivity of [68Ga]Ga-DATA5m-LM4 PET/CT was 94.28% (462/490) and 83.46% (409/490) for [68Ga]Ga-DOTANOC PET/CT (p < 0.0001). [68Ga]Ga-DATA5m-LM4 had statistical significance over [68Ga]Ga-DOTANOC in liver metastases [100% vs. 89.4%; p < 0.0001 (292 vs. 253 {283 lesions on CT})] and bone metastases [100% vs. 82.9%; p = 0.005 (45 vs. 34 {41 lesions on CT})]. Statistical significance was also noted for the TBR SULpeak of the primary and liver lesions. [68Ga]Ga-DATA5m-LM4 showed better sensitivity and a higher target-to-background ratio than [68Ga]Ga-DOTANOC PET/CT. [68Ga]Ga-DATA5m-LM4 PET/CT can be used to quantify the extent of skeletal and liver metastases for better planning of SSTR agonist- or antagonist-based therapy.

BACKGROUND: This study assesses the accuracy of neutrophil activation markers, including neutrophil extracellular traps (NETs) and calprotectin, as biomarkers of disease activity in patients with established rheumatoid arthritis (RA). We also analyse the relationship between NETs and various types of therapies as well as their association with autoimmunity.
METHODS: Observational cross-sectional study of patients with RA receiving treatment with biological disease-modifying antirheumatic drugs or Janus kinase inhibitors (JAK-inhibitors) for at least 3 months. Plasma calprotectin levels were measured using an enzyme-linked immunosorbent assay test kit and NETs by measuring their remnants in plasma (neutrophil elastase-DNA and histone-DNA complexes). We also assessed clinical disease activity, joint ultrasound findings and autoantibody status [reumatoid factor (RF), anti-citrullinated peptide/protein antibodies (ACPAs) and anti-carbamylated protein (anti-CarP)]. Associations between neutrophilic biomarkers and clinical or ultrasound scores were sought using correlation analysis. The discriminatory capacity of both neutrophilic biomarkers to detect ultrasound synovitis was analysed through receiver-operating characteristic (ROC) curves.
RESULTS: One hundred fourteen patients were included. Two control groups were included to compare NET levels. The active control group consisted of 15 patients. The second control group consisted of 30 healthy subjects. Plasma NET levels did not correlate with clinical disease status, regardless of the clinic index analysed or the biological therapy administered. No significant correlation was observed between NET remnants and ultrasound synovitis. There was no correlation between plasma NET and autoantibodies. In contrast, plasma calprotectin positively correlated with clinical parameters (swollen joint count [SJC] rho = 0.49; P < 0.001, Clinical Disease Activity Index [CDAI] rho = 0.30; P < 0.001) and ultrasound parameters (rho > 0.50; P < 0.001). Notably, this correlation was stronger than that observed with acute phase reactants.
CONCLUSIONS: While NET formation induced by neutrophils may play a role in RA pathogenesis, our study raises questions about the utility of NET remnants in peripheral circulation as a biomarker for inflammatory activity. In contrast, this study strongly supports the usefulness of calprotectin as a biomarker of inflammatory activity in patients with RA.

OBJECTIVE: The study aimed to evaluate the impact of aging on the formation of neutrophil extracellular traps (NETs). The impaired formation of NETs is the cause of an abnormal innate immune response.
METHODS: The study included a total of 45 healthy male subjects of different age groups. Whole blood was collected from the subjects, and the concentration of myeloperoxidase (MPO), the main biocidal protein in NETs, was determined in serum using ELISA. The serum levels of circulating free DNA (cfDNA), which are the structural basis of NETs, were also measured by fluorescence. In addition, the white blood cell count was determined, whole blood smear was evaluated, and the neutrophillymphocyte ratio was calculated. The variations in the levels of NET biomarkers were analyzed in different age groups.
RESULTS: The low levels of MPO (243.70 ng/ml) and cfDNA (6.24 ng/100 μl) in boys indicated neutrophil insufficiency for NETosis in children. A progressive increase in the levels of MPO and cfDNA with age was observed among adolescents (420.91, p = 0.04; 13.55, p = 0.03, respectively), with the highest level noted in the healthy adult group (466.58, p = 0.01; 14.07, p = 0.01, respectively). The levels of the studied parameters were comparable in adolescents and young adults, which proved that the NETosis process was appropriate and suggested the attainment of neutrophil maturity for the release of NETs in adolescence. The levels of MPO and cfDNA were low in older men (225.46, p < 0.01; 5.19, p < 0.01, respectively) indicating impaired NET formation.
CONCLUSIONS: Data on the generation of NETs in different age groups obtained in this study can allow a better understanding of the ontogenesis of the immune system in terms of the course of NETosis, and also indicate the need to support nonspecific responses in children and adults. Further research should be performed to determine the possibility of regulating the NETosis process. Int J Occup Med Environ Health. 2023;36(3):333-48.

UNASSIGNED: Cumulative research show association of neutrophils and neutrophil extracellular traps (NETs) with poor outcomes in severe COVID-19. However, to date, there is no curative intent therapy able to block neutrophil/NETs-mediated progression of multi-organ dysfunction. Because of emerging neutrophil heterogeneity, the study of subsets of circulating NET-forming neutrophils [NET + Ns] as mediators of multi-organ failure progression among patients with COVID-19 is critical to identification of therapeutic targets.
UNASSIGNED: We conducted a prospective observational study of circulating levels of CD11b + [NET + N] immunotyped for dual endothelin-1/signal peptide receptor (DEspR ±) expression by quantitative immunofluorescence-cytology and causal mediation analysis. In 36 consented adults hospitalized with mod-severe COVID-19, May to September 2020, we measured acute multi-organ failure via SOFA-scores and respiratory failure via SaO2/FiO2 (SF)-ratio at time points t1 (average 5.5 days from ICU/hospital admission) and t2 (the day before ICU-discharge or death), and ICU-free days at day28 (ICUFD). Circulating absolute neutrophil counts (ANC) and [NET + N] subset-specific counts were measured at t1. Spearman correlation and causal mediation analyses were conducted.
UNASSIGNED: Spearman correlation analyses showed correlations of t1-SOFA with t2-SOFA (rho r S  = 0.80) and ICUFD (r S  = -0.76); circulating DEspR + [NET + Ns] with t1-SOFA (r S  = 0.71), t2-SOFA (r S  = 0.62), and ICUFD (r S  = -0.63), and ANC with t1-SOFA (r S  = 0.71), and t2-SOFA (r S  = 0.61).Causal mediation analysis identified DEspR + [NET + Ns] as mediator of 44.1% [95% CI:16.5,110.6] of the causal path between t1-SOFA (exposure) and t2-SOFA (outcome), with 46.9% [15.8,124.6] eliminated when DEspR + [NET + Ns] were theoretically reduced to zero. Concordantly, DEspR + [NET + Ns] mediated 47.1% [22.0,72.3%] of the t1-SOFA to ICUFD causal path, with 51.1% [22.8,80.4%] eliminated if DEspR + [NET + Ns] were reduced to zero. In patients with t1-SOFA > 1, the indirect effect of a hypothetical treatment eliminating DEspR + [NET + Ns] projected a reduction of t2-SOFA by 0.98 [0.29,2.06] points and ICUFD by 3.0 [0.85,7.09] days. In contrast, there was no significant mediation of SF-ratio through DEspR + [NET + Ns], and no significant mediation of SOFA-score through ANC.
UNASSIGNED: Despite equivalent correlations, DEspR + [NET + Ns], but not ANC, mediated progression of multi-organ failure in acute COVID-19, and its hypothetical reduction is projected to improve ICUFD. These translational findings warrant further studies of DEspR + [NET + Ns] as potential patient-stratifier and actionable therapeutic target for multi-organ failure in COVID-19.
UNASSIGNED: The online version contains supplementary material available at 10.1186/s41231-023-00143-x.

UNASSIGNED: The number of patients with non-functional neuroendocrine tumors (NETs) has increased recently, and the rate of liver metastasis of NETs is about 20% in patients at the first diagnosis. Transcatheter arterial embolization (TAE) and everolimus are therapies with reported efficacy, but few reports have described their combined treatment. We therefore aim to evaluate the efficacy and safety of combination therapy with everolimus and TAE in patients with liver metastasis of gastroenteropancreatic neuroendocrine tumors (GEP-NETs) in a prospective study.
UNASSIGNED: We design a single-arm, open-label, prospective study to evaluate the efficacy and safety of combination therapy with everolimus and TAE in patients with liver metastases of GEP-NETs. The study started in June 2021 at Okayama University Hospital and is expected to enroll 18 patients over a 2-year period.
UNASSIGNED: This study is a prospective study investigating a new treatment method for a rare disease called GEP-NETs. We may obtain useful information that contributes to the treatment guidelines in this study. However, NET is a rare disease, and although the number of cases is statistically established, it may not be possible to accurately assess causality.TRIAL REGISTRATION NUMBER: jRCT1061210015.

Among the available neuroendocrine neoplasm (NEN)-specific HR-QoL scales, only the EORTC QLQ-C30 and EORTC QLQ-G.I.NET21 questionnaires have been validated in several languages. We aim to assess patients\' perceptions of these questionnaires. A cross-sectional qualitative pilot study was conducted among 65 adults from four countries with well-differentiated advanced gastro-entero-pancreatic (GEP) or unknown primary NENs. Patients completed the EORTC QLQ-C30 and EORTC QLQ-G.I.NET21 questionnaires and then a survey containing statements concerning the questionnaires. The majority of patients had a small intestine NET (52%). Most tumors were functioning (55%) and grade 2 NET (52%). Almost half of the patients identified limitations in the questionnaires, with nine (14%) patients scoring the questionnaires as poor and 16 (25%) patients as moderate. Overall, 37 (57%) patients were positive towards the questionnaires. Approximately a quarter of patients considered the questionnaires not suitable for all ages, missing some of their complaints, not representative of their overall HR-QoL regarding the treatment of their NET and too superficial. The current validated EORTC QLQ-C30 and EORTC QLQ-G.I.NET21 questionnaires may show some limitations in the design of questions and the patients\' final satisfaction reporting of the questionnaire. Large-scale, high-quality prospective studies are required in HR-QoL assessment regarding NETs.

An early and accurate diagnosis of early onset neonatal sepsis (EONS) and late onset neonatal sepsis (LONS) is essential to improve the outcome of this devastating conditions. Especially, preterm infants are at risk. Reliable biomarkers are rare, clinical decision-making depends on clinical appearance and multiple laboratory findings. Markers of NET formation and NET turnover might improve diagnostic precision. Aim of this study was to evaluate the diagnostic value of NETs in sepsis diagnosis in neonatal preterm infants.
Plasma samples of neonatal preterm infants with suspected sepsis were collected. Blood samples were assayed for markers of NET formation and NET turnover: cfDNA, DNase1, nucleosome, NE, and H3Cit. All clinical findings, values of laboratory markers, and epidemiological characteristics were collected retrospectively. Two subpopulations were created to divide EONS from LONS. EMA sepsis criteria for neonatal sepsis were used to generate a sepsis group (EMA positive) and a control group (EMA negative).
A total of 31 preterm neonates with suspected sepsis were included. Out of these, nine patients met the criteria for sepsis according to EMA. Regarding early onset neonatal sepsis (3 EONS vs. 10 controls), cfDNA, DNase I, nucleosome, and CRP were elevated significantly. H3Cit and NE did not show any significant elevations. In the late onset sepsis collective (6 LONS vs. 12 controls), cfDNA, DNase I, and CRP differed significantly compared to control group.

BACKGROUND: Atrial fibrillation (AF) is the most common age-related cardiac arrhythmia. The etiology underlying AF is still largely unknown. At the intersection of the innate immune system and hemostasis, immunothrombosis may be a possible cause of atrial remodeling, and therefore be an underlying cause of AF.
METHODS: From 1990 to 2014, we followed participants aged 55 and over, free from AF at inclusion. Immunothrombosis factors fibrinogen, von Willebrand factor, ADAMTS13, and neutrophil extracellular traps (NETs) levels were measured at baseline. Participants were followed until either onset of AF, loss-to-follow-up, or reaching the end-date of 01-01-2014. Cox proportional hazard modelling was used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs), adjusted for cardiovascular risk factors.
RESULTS: We followed 6174 participants (mean age 69.1 years, 57% women) for a median follow-up time of 12.8 years. 364 men (13.7%, incidence rate 13.0/1000 person-years) and 365 women (10.4%, incidence rate 8.9/1000 person-years) developed AF. We found no significant association between markers of immunothrombosis and new-onset AF after adjusting for cardiovascular risk factors [HR 1.00 (95% CI 0.93-1.08) for fibrinogen, 1.04 (0.97-1.12) for von Willebrand factor, 1.00 (1.00-1.01) for ADAMTS13, and 1.01 (0.94-1.09) for NETs]. In addition, we found no differences in associations between men and women.
CONCLUSIONS: We found no associations between markers of immunothrombosis and new-onset AF in the general population. Inflammation and immunothrombosis may be associated with AF through other cardiovascular risk factors or predisposing conditions of AF. Our findings challenge the added value of biomarkers in AF risk prediction.

The function of neutrophils in viral infections has long been established and studies have been done to examine the role of neutrophil extracellular traps (NETs). Further study and analysis of NETs in viral infections may reveal a new therapeutic target. Administration of ibuprofen and GS-561937, a fusion protein inhibitor (FPI), have been experimentally shown to decrease the severity of bovine respiratory syncytial virus (BRSV) infection. Our aims were to determine the effect of ibuprofen and FPI on NETs after BRSV infection as a monotherapy or combined therapy.
METHODS: We conducted a randomized placebo-controlled trial of ibuprofen, FPI, or as a dual therapy initiated at 3 or 5 days after experimental infection with BRSV in 36 five to six-week-old Holstein calves (Bos Taurus). Lung tissue samples were collected and stained with antibodies conjugated with fluorescence dyes to visualize and quantify the NETs in situ. We estimated the average NETs in the sample lung tissue slides and compared the areas occupied by NETS within and between the treatment groups.
RESULTS: There were significantly fewer NETs in the lung tissue from calves that were given ibuprofen and both ibuprofen and fusion protein inhibitor from day 3 post infection compared to the placebo group. Calves administered with ibuprofen, fusion protein inhibitor or both from day five had visually fewer NETs than the placebo but the difference was not significant.
CONCLUSIONS: BRSV can induce NET formation in vitro and in vivo. A combination of both drugs (Ibuprofen and FPI) resulted in less NETs observed in lung tissue of BRSV infected calves compared to the placebo or monotherapy groups.