First-year cumulative myeloperoxidase-ANCA titres are associated with all-cause mortality in patients with microscopic polyangiitis.

OBJECTIVES: We investigated whether first-year cumulative myeloperoxidase (MPO)-antineutrophil cytoplasmic antibody (ANCA) and proteinase 3 (PR3)-ANCA titres were associated with all-cause mortality and relapse during follow-up in patients with microscopic polyangiitis (MPA) and granMETHODS: Altogether, 74 patients with MPA and 40 with GPA were included in this study. Their clinical data at diagnosis were collected. First-year cumulative ANCA titres were defined as the area under the curve (AUC) of ANCA titres during the first year after MPA or GPA diagnosis, which was obtained using the trapezoidal rule. All-cause mortality and relapse were considered poor outcomes of MPA and GPA. RESULTS: The median ages of patients with MPA and GPA were 65.5 and 60.5 years, respectively. No significant correlation was observed between ANCA titres at diagnosis and concurrent MPA and GPA activity or the inflammatory burden. First-year cumulative MPO-ANCA titres exhibited a significant AUC for all-cause mortality during follow-up in patients with MPA. The optimal cut-off of first-year cumulative MPO-ANCA titres for all-cause mortality was determined as 720.8 IU/mL using receiver operating characteristic curve analysis. MPA patients with first-year cumulative MPO-ANCA titres ≥720.8 IU/mL exhibited a significantly higher risk for all-cause mortality than those without (relative risk 13.250). Additionally, MPA patients with first-year cumulative MPO-ANCA titres ≥720.8 IU/mL exhibited a significantly lower cumulative patients' survival rate than those without. CONCLUSIONS: This is the first study to demonstrate the association between first-year cumulative MPO-ANCA titres and all-cause mortality during follow-up in patients with MPA.

Myeloperoxidase and its derivative hypochlorous acid combined clinical indicators predict new-onset atrial fibrillation in sepsis: a case-control study.

BACKGROUD: New-onset atrial fibrillation (NOAF) is a common complication of sepsis and linked to higher death rates in affected patients. The lack of effective predictive tools hampers early risk assessment for the development of NOAF. This study aims to develop practical and effective predictive tools for identifying the risk of NOAF. METHODS: This case-control study retrospectively analyzed patients with sepsis admitted to the emergency department of Xinhua Hospital, Shanghai Jiao Tong University School of Medicine from September 2017 to January 2023. Based on electrocardiographic reports and electrocardiogram monitoring records, patients were categorized into NOAF and non-NOAF groups. Laboratory tests, including myeloperoxidase (MPO) and hypochlorous acid (HOCl), were collected, along with demographic data and comorbidities. Least absolute shrinkage and selection operator regression and multivariate logistic regression analyses were employed to identify predictors. The area under the curve (AUC) was used to evaluate the predictive model's performance in identifying NOAF. RESULTS: A total of 389 patients with sepsis were included in the study, of which 63 developed NOAF. MPO and HOCl levels were significantly higher in the NOAF group compared to the non-NOAF group. Multivariate logistic regression analysis identified MPO, HOCl, tumor necrosis factor-α (TNF-α), white blood cells (WBC), and the Acute Physiology and Chronic Health Evaluation II (APACHE II) score as independent risk factors for NOAF in sepsis. Additionally, a nomogram model developed using these independent risk factors achieved an AUC of 0.897. CONCLUSION: The combination of MPO and its derivative HOCl with clinical indicators improves the prediction of NOAF in sepsis. The nomogram model can serve as a practical predictive tool for the early identification of NOAF in patients with sepsis.

Assessment of associations between neutrophil extracellular trap biomarkers in blood and thrombi in acute ischemic stroke patients.

Inflammation including immunothrombosis by neutrophil extracellular traps (NETs) has important implications in acute ischemic stroke and can affect reperfusion status, susceptibility to stroke associated infections (SAI) as well as functional clinical outcome. NETs were shown to be prevalent in stroke thrombi and NET associated markers were found in stroke patients' blood. However, little is known whether blood derived NET markers reflect the amount of NETs in thrombi. Conclusions from blood derived markers to thrombus composition might open avenues for novel strategies in diagnostic and therapeutic approaches. We prospectively recruited 166 patients with acute ischemic stroke undergoing mechanical thrombectomy between March 2018 and May 2021. Available thrombi (n = 106) were stained for NET markers DNA-histone-1 complexes and myeloperoxidase (MPO). Cell free DNA (cfDNA), deoxyribonuclease (DNase) activity, MPO-histone complexes and a cytokine-panel were measured before thrombectomy and after seven days. Clinical data, including stroke etiology, reperfusion status, SAI and functional outcome after rehabilitation, were collected of all patients. NET markers were present in all thrombi. At onset the median concentration of cfDNA in blood was 0.19 µg/ml increasing to 0.30 µg/ml at 7 days. Median DNase activity at onset was 4.33 pmol/min/ml increasing to 4.96 pmol/min/ml at 7 days. Within thrombi DNA-histone-1 complexes and MPO correlated with each other (ρ = 0.792; p < 0.001). Moreover, our study provides evidence for an association between the amount of NETs and endogenous DNase activity in blood with amounts of NETs in cerebral thrombi. However, these associations need to be confirmed in larger cohorts, to investigate the potential clinical implications for individualized therapeutic and diagnostic approaches in acute ischemic stroke.

Killer Function of Circulating Neutrophils in Relation to Cytokines in Uterine Myoma and Endometrial Cancer.

The study presents the killer functions of circulating neutrophils: myeloperoxidase activity, the ability to generate ROS, phagocytic activity, receptor status, NETosis, as well as the level of cytokines IL-2, IL-4, IL-6, IL-17A, and IL-18, granulocyte CSF, monocyte chemotactic protein 1, and neutrophil elastase in the serum of patients with uterine myoma and endometrial cancer (FIGO stages I-III). The phagocytic ability of neutrophils in uterine myoma was influenced by serum levels of granulocyte CSF and IL-2 in 54% of the total variance. The degranulation ability of neutrophils in endometrial cancer was determined by circulating IL-18 in 50% of the total variance. In uterine myoma, 66% of the total variance in neutrophil myeloperoxidase activity was explained by a model dependent on blood levels of IL-17A, IL-6, and IL-4. The risk of endometrial cancer increases when elevated levels of monocyte chemotactic protein 1 in circulating neutrophils are associated with reduced ability to capture particles via extracellular traps (96% probability).

[NETosis in lupus nephritis].

AIM: To evaluate the levels of MPO-DNA complex in patients with systemic lupus erythematosus (SLE) and its association with the presence of lupus nephritis (LN). MATERIALS AND METHODS: The study included 77 patients with SLE, of whom 30 had SLE without anti phospholipid syndrome (APS), 47 had SLE with APS, and 20 were healthy individuals serving as the control group. The MPO-DNA complex in the serum was investigated using ELISA. RESULTS: The levels of MPO-DNA complex in serum were significantly higher in patients with SLE compared to healthy controls (p=0.001). Among the patients with SLE, 30 (39%) had elevated levels of MPO-DNA complex. The presence of elevated MPO-DNA complex was significantly associated with the presence of a history of LN (p=0.009). Moreover, among the patients included in the study, 20 had active LN, and patients with elevated MPO-DNA complex levels were more likely to have active LN than patients without elevated MPO-DNA complex concentrations [12 (40%) of 30 vs 8 (17%) of 47, χ2=5.029; p=0.034]. An association was found between elevated levels of MPO-DNA complex and the presence of proteinuria, hematuria, cellular hematic/granular casts and aseptic leukocyturia. A direct correlation of MPO-DNA complex with SLEDAI-R was found in patients with active LN (rs=0.497; p=0.026). CONCLUSION: Elevated levels of MPO-DNA complex were detected in 39% of patients with SLE. These patients had a higher prevalence of LN in their medical history and at the time of inclusion in the study. The correlation between MPO-DNA complex levels and the activity of LN according to SLEDAI-R indicates the potential role of MPO-DNA complex as a biomarker for assessing the activity of renal damage in SLE.

Metabolic reprograming shapes neutrophil functions in severe COVID-19.

To better understand the mechanisms at the basis of neutrophil functions during SARS-CoV-2, we studied patients with severe COVID-19 pneumonia. They had high blood proportion of degranulated neutrophils and elevated plasma levels of myeloperoxidase (MPO), elastase, and MPO-DNA complexes, which are typical markers of neutrophil extracellular traps (NET). Their neutrophils display dysfunctional mitochondria, defective oxidative burst, increased glycolysis, glycogen accumulation in the cytoplasm, and increase glycogenolysis. Hypoxia-inducible factor 1α (ΗΙF-1α) is stabilized in such cells, and it controls the level of glycogen phosphorylase L (PYGL), a key enzyme in glycogenolysis. Inhibiting PYGL abolishes the ability of neutrophils to produce NET. Patients displayed significant increases of plasma levels of molecules involved in the regulation of neutrophils' function including CCL2, CXCL10, CCL20, IL-18, IL-3, IL-6, G-CSF, GM-CSF, IFN-γ. Our data suggest that metabolic remodelling is vital for the formation of NET and for boosting neutrophil inflammatory response, thus, suggesting that modulating ΗΙF-1α or PYGL could represent a novel approach for innovative therapies.

Assessment of serum and synovial fluid MMP-3 and MPO as biomarkers for psoriatic arthritis and their relation to disease activity indices.

Research on biomarkers for the diagnosis and monitoring of psoriatic arthritis (PsA) is ongoing. The purpose of this study was to assess the potential of serum and synovial fluid matrix metalloproteinase-3 (MMP-3) and myeloperoxidase (MPO) as biomarkers for PsA and their relation to disease activity indices. This case-control study involved 156 psoriatic arthritis patients, 50 gonarthrosis patients, and 30 healthy controls. The target parameters were measured with the enzyme-linked immunosorbent assay (ELISA) kits. Serum MMP-3 and MPO levels were elevated in the PsA patients in comparison to the two control groups (p < 0.001) and distinguished PsA from GoA patients and healthy controls with 100% accuracy. Synovial MMP-3 discriminated PsA from GoA patients irrespective of the presence of crystals (AUC = 1.00). PsA patients with crystals in the synovial fluid had elevated synovial MPO (p < 0.001) and were distinguished from PsA patients without crystals with accuracy of 88.50% and from GoA patients with accuracy of 88.30%. Synovial fluid MPO was positively associated with the following indicators of disease activity: VAS (rs = 0.396); DAPSA (rs = 0.365); mCPDAI (rs = 0.323). Synovial MMP-3 showed a weaker positive association with DAPSA (rs = 0.202) and mCPDAI (rs = 0.223). Our results suggest that serum MMP-3 and MPO could serve as biomarkers for PsA. Synovial fluid MMP-3 showed a potential as a biomarker for PsA versus GoA. Synovial MPO could be utilized as a marker for the presence of crystals in PsA patients.

Binding of lactoferrin to the surface of low-density lipoproteins modified by myeloperoxidase prevents intracellular cholesterol accumulation by human blood monocytes.

Myeloperoxidase (MPO) is a unique heme-containing peroxidase that can catalyze the formation of hypochlorous acid (HOCl). The strong interaction of MPO with low-density lipoproteins (LDL) promotes proatherogenic modification of LDL by HOCl. The MPO-modified LDL (Mox-LDL) accumulate in macrophages, resulting in the formation of foam cells, which is the pathognomonic symptom of atherosclerosis. A promising approach to prophylaxis and atherosclerosis therapy is searching for remedies that prevent the modification or accumulation of LDL in macrophages. Lactoferrin (LF) has several application points in obesity pathogenesis. We aimed to study LF binding to Mox-LDL and their accumulation in monocytes transformed into macrophages. Using surface plasmon resonance and ELISA techniques, we observed no LF interaction with intact LDL, whereas Mox-LDL strongly interacted with LF. The affinity of Mox-LDL to LF increased with the degree of oxidative modification of LDL. Moreover, an excess of MPO did not prevent interaction of Mox-LDL with LF. LF inhibits accumulation of cholesterol in macrophages exposed to Mox-LDL. The results obtained reinforce the notion of LF potency as a remedy against atherosclerosis.

Standardised immunophenotypic analysis of myeloperoxidase in acute leukaemia.

Given its myeloid-restricted expression, myeloperoxidase (MPO) is typically used for lineage assignment (myeloid vs. lymphoid) during acute leukaemia (AL) diagnostics. In the present study, a robust flow cytometric definition for MPO positivity was established based on the standardised EuroFlow protocols, the standardised Acute Leukaemia Orientation Tube and 1734 multicentre AL cases (with confirmed assay stability). The best diagnostic performance was achieved by defining MPO positivity as ≥20% of the AL cells exceeding a lymphocyte-based threshold. The methodology employed should be applicable to any form of standardised flow cytometry.

A neutrophil signature is strongly associated with increased cardiovascular risk in gout.

OBJECTIVE: To investigate the association between neutrophil activation and cardiovascular risk in gout patients. We hypothesize that neutrophil activation mediates inflammation and therefore takes part in atherogenesis in gout patients. METHOD: Patient data were collected from 75 consecutive gout patients participating in the Reade gout cohort Amsterdam. Levels of neutrophil extracellular traps (NETs) and neutrophil activation (calprotectin and peroxidase activity) were analysed by ELISA and fluorimetry in plasma and compared with healthy controls. Markers of neutrophil activation were related to clinical markers of cardiovascular risk, including BMI, smoking, blood pressure, lipid profile and 10 year risk of cardiovascular mortality (EU-SCORE). RESULTS: Increased levels of NETs were found in gout patients, although increased levels were not associated with cardiovascular risk. However, markers of neutrophil activation, including peroxidase activity correlated with waist:hip ratio (ß = 0.33, P < 0.001), cholesterol ratio (ß = 0.46, P < 0.005) and triglycerides (ß = 0.60, P < 0.001) as well as the 10 year risk of cardiovascular mortality (ß = 0.44, P = 0.001). Calprotectin levels were elevated in hypertension (P = 0.005) and diabetes (P = 0.02). Finally, gout patients with high levels of both peroxidase and calprotectin ('neutrophil activation signature') had a markedly elevated cardiovascular risk score (P = 0.001), with 68% of the patients having high cardiovascular risk (odds ratio 2.9, P = 0.03). CONCLUSION: We demonstrated elevated levels of neutrophil activation markers, MPO and calprotectin in gout patients as compared with healthy controls. Of note, neutrophil activation markers were associated with several risk factors for cardiovascular disease, including hyperlipidaemia, hypertension and diabetes. Finally, the presence of a neutrophil activation signature was strongly associated with an increased 10 year risk of cardiovascular mortality. Further studies are needed to determine whether gout-specific factors and/or cardiovascular risk factors contribute to the elevated neutrophil activation observed in these patients.

Serum Matrix Metalloproteinase-8 and Myeloperoxidase Predict Survival after Resection of Colorectal Liver Metastases.

INTRODUCTION: Matrix metalloproteinases (MMPs) have been extensively studied in several malignancies, and myeloperoxidase (MPO) is a promising new prognostic biomarker. We investigated the prognostic value of MMP-8, MMP-9, and MPO, as well as carcinoembryonic antigen (CEA), CA19-9, and C-reactive protein (CRP) in colorectal cancer with operable liver metastases. METHODS: This study included 419 patients who underwent liver resection for colorectal metastases at the Helsinki University Hospital between 2000 and 2013. Serum samples were drawn before and 3 months after liver resection. We evaluated associations of MMP-8, MMP-9, MPO, CRP, CEA, and CA19-9 concentrations to disease-free survival (DFS) and overall survival (OS) using the Cox proportional hazards model and Kaplan-Meier log-rank method. RESULTS: In univariate Cox regression analyses, pre- and postoperatively high MMP-8 (HR 1.53, 95% CI: 1.07-2.19, p = 0.021 and HR 1.45, 95% CI: 1.01-2.09, p = 0.044, respectively) associated with worse 10-year OS. Postoperatively high MPO indicated better 5-year DFS (HR 0.70, 95% CI: 0.54-0.90, p = 0.007). Elevated pre- and postoperative CEA and CA19-9 as well as postoperative CRP indicated impaired survival. CONCLUSIONS: Pre- and postoperatively high MMP-8 associates with worse 10-year OS, and postoperatively high MPO associates with better 5-year DFS. CEA, CA19-9, and CRP are also prognostic.

High Expression of MMP-9 in Primary Tumors and High Preoperative MPO in Serum Predict Improved Prognosis in Colorectal Cancer with Operable Liver Metastases.

INTRODUCTION: The liver metastases of colorectal cancer (CRC) can be surgically treated in selected cases, with continuously improving results. Matrix metalloproteinases (MMPs) contribute to cancer invasion by degrading the extracellular matrix, and elevated levels of MMP-2, MMP-8, and MMP-9 have been detected in several malignancies. Myeloperoxidase (MPO) is a mediator of tissue damage that can oxidatively activate latent MMPs. We evaluated the prognostic value of MMP-2, MMP-8, and MMP-9 in tissue samples of primary tumors and liver metastases and the pre- and postoperative serum levels of MMP-8, MMP-9, and MPO in CRC patients undergoing liver resection. METHODS: Tissue and serum samples were obtained from 111 patients who had primary colorectal tumors and their liver metastases surgically treated at the Helsinki University Hospital between 1988 and 2007. Tissue expression of MMP-2, MMP-8, and MMP-9 in primary tumors and liver metastases was evaluated by immunohistochemistry. Pre- and postoperative serum concentrations of MMP-8, MMP-9, and MPO were determined using a time-resolved immunofluorometric assay or commercially available enzyme-linked immunosorbent assay kits. Clinical data were retrieved from patient records and the Central Statistical Office of Finland. Associations with disease-free survival (DFS) and overall survival (OS) were estimated using Cox regression analysis and the Kaplan-Meier method. RESULTS: High expression of MMP-9 in colorectal tumor tissue was associated with better DFS (p = 0.010), and high preoperative MPO in serum with improved DFS and OS (p < 0.001 and p = 0.014, respectively). The prognostic significance varied according to gender, age, and the synchronicity of liver metastases. CONCLUSION: Low preoperative MPO in serum might identify patients at high risk of recurrence and death after resection of colorectal liver metastases. Elevated preoperative MPO and high expression of MMP-9 in colorectal tumor tissue indicate an improved prognosis. The use of these biomarkers should be adjusted according to clinical characteristics.

Myeloperoxidase Targets Apolipoprotein A-I for Site-Specific Tyrosine Chlorination in Atherosclerotic Lesions and Generates Dysfunctional High-Density Lipoprotein.

We previously demonstrated that apolipoprotein A-I (apoA-I), the major protein component of high-density lipoprotein (HDL), is an important target for myeloperoxidase (MPO)-catalyzed tyrosine chlorination in the circulation of subjects with cardiovascular diseases. Oxidation of apoA-I by MPO has been reported to deprive HDL of its protective properties. However, the potential effects of MPO-mediated site-specific tyrosine chlorination of apoA-I on dysfunctional HDL formation and atherosclerosis was unclear. Herein, Tyr192 in apoA-I was found to be the major chlorination site in both lesion and plasma HDL from humans with atherosclerosis, while MPO binding to apoA-I was demonstrated by immunoprecipitation studies in vivo. In vitro, MPO-mediated damage of lipid-free apoA-I impaired its ability to promote cellular cholesterol efflux by the ABCA1 pathway, whereas oxidation to lipid-associated apoA-I inhibited lecithin:cholesterol acyltransferase activation, two key steps in reverse cholesterol transport. Compared with native apoA-I, apoA-I containing a Tyr192 → Phe mutation was moderately resistant to oxidative inactivation by MPO. In high-fat-diet-fed apolipoprotein E-deficient mice, compared with native apoA-I, subcutaneous injection with oxidized apoA-I (MPO treated) failed to mediate the lipid content in aortic plaques while mutant apoA-I (Tyr192 → Phe) showed a slightly stronger ability to reduce the lipid content in vivo. Our observations suggest that oxidative damage of apoA-I and HDL involves MPO-dependent site-specific tyrosine chlorination, raising the feasibility of producing MPO-resistant forms of apoA-I that have stronger antiatherosclerotic activity in vivo.

Taurine supplementation reduces myeloperoxidase and matrix-metalloproteinase-9 levels and improves the effects of exercise in cognition and physical fitness in older women.

Immunosenescence contributes to cognitive impairment and neurodegeneration, and those conditions could be attenuated by non-pharmacological anti-inflammatory strategies, such as exercise and supplementation with the amino acid taurine. Since taurine body content decreases with aging, we investigated the effects of supplementation (alone and combined with exercise) on oxidative stress, extracellular matrix degradation, white blood cells, neurotrophins, cognition and physical fitness of elderly women. Forty-eight women (83.58 ± 6.98 years) were enrolled into exercise training only (EO: n = 13), taurine supplementation (TS: n = 12), exercise training + taurine supplementation (ETTS: n = 11), and control group (CG: n = 12). All interventions lasted 14 weeks. Exercise was applied twice a week, and taurine was given once a day (1.5 g). Data collection occurred before and after interventions with the determination of myeloperoxidase (MPO), matrix metalloproteinase-9 (MMP-9), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) levels, and white blood cell counts (WBC). Montreal cognitive assessment (MoCA) and physical fitness tests were also evaluated. Concentration of MPO and MMP-9 decreased after intervention in TS (p < 0.05). No effect of time or time × group was observed for WBC parameters; however, univariate analysis showed a significant decrease in lymphocytes for TS, while an increase in monocytes occurred in the CG (p < 0.05). MoCA scores decreased over time in the CG (p < 0.05). Improvements in physical fitness occurred in ETTS (better agility and aerobic capacity), mostly likely due to exercise and boosted by taurine supplementation. No changes in BDNF levels were observed (p > 0.05), while NGF concentration were undetectable in almost subjects. Exercise together with taurine supplementation appears to be a valuable strategy to enhance health-related outcomes in older persons.

Immune response and protective efficacy of two new adjuvants, Montanide™ ISA 763B VG and Montanide™ GEL02, administered with a Streptococcus agalactiae ghost vaccine in Nile tilapia (Oreochromis niloticus).

Streptococcus agalactiae is one of the most important pathogens infecting tilapia worldwide and causes meningoencephalitis, septicemia and high mortalities with considerable losses. Various types of vaccines have been developed against S. agalactiae infection, such as inactivated vaccines, live attenuated vaccines and subunit vaccines. Bacterial ghosts (BGs) are nonliving, empty cell envelopes and have been reported as novel vaccine candidates. Therefore, the main aims of this study were to develop an S. agalactiae ghost vaccine (SAGV) and to evaluate the immune response and protective effect of SAGV against S. agalactiae with two novel adjuvants, Montanide™ ISA 763B VG and Montanide™ GEL02. Nile tilapia, mean weight 50 g, were divided into four groups as follows; 1) fish injected with PBS as control, 2) fish injected with the SAGV alone; 3) fish injected with the SAGV+Montanide™ ISA 763B VG; and 4) fish injected with SAGV+Montanide™ GEL02. Following vaccination, innate immunity parameters including serum lysozyme, myeloperoxidase, catalase, and bactericidal activity were all significantly enhanced. Moreover, specific serum IgM antibodies were induced and reached their highest level 2-8 weeks post vaccination. Importantly, the relative percent survival of tilapia vaccinated against the SAGV formulated with both adjuvants was 80-93%. Furthermore, the transcription of immune-related genes (IgM, TCRß, IL-1ß, IL-8 and TNFα) were up-regulated in tilapia after vaccination, indicating that both cellular and humoral immune responses were induced by these adjuvanted vaccines. In summary, Montanide™ ISA 763B VG and Montanide™ GEL02 can enhance immunoprotection induced by the SAGV vaccine against streptococcosis, demonstrating that both have value as potential adjuvants of fish vaccines.

Metformin Is Associated with Reduced Tissue Factor Procoagulant Activity in Patients with Poorly Controlled Diabetes.

PURPOSE: Metformin is the first-line antidiabetic drug and shown to reduce cardiovascular risk independent from its glucose lowering action. Particularly in poorly controlled diabetes, tissue factor (TF) is expressed in the vasculature and accounts for thromboembolic complications. Here, we aimed to assess the effect of metformin on TF activity and markers of vascular inflammation in poorly controlled type 2 diabetes. METHODS: In a cohort of patients with uncontrolled type 2 diabetes (glycosylated hemoglobin 8.39 ± 0.24%, 68.1 ± 2.6 mmol/mol, n = 46) of whom half of the individuals were treated with metformin and the other half did not receive metformin as part of an anti-diabetic combination therapy, we assessed TF activity and markers of vascular inflammation. In vitro, human monocytic cells (THP-1) were exposed to metformin and TF expression measured in the presence and absence of the AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide riboside (AICAR) or the AMPK inhibitor compound C. RESULTS: In the patients, metformin treatment was associated with lower levels of TF protein (241.5 ± 19 vs. 315.4 ± 25 pg/mL, p = 0.03) and reduced TF activity (408.9 ± 49 vs. 643.8 ± 47 U/mL, p = 0.001) compared with controls. Moreover, the patients on metformin showed lower levels of vascular cell adhesion molecule (VCAM)1 (26.6 ± 1.4 vs. 35.03 ± 3.1 ng/mL, p = 0.014) and higher expression of miR-126-3p/U6sno (11.39 ± 2.8 vs. 4.26 ± 0.9, p = 0.006), a known post-transcriptional down regulator of TF and VCAM1. In vitro, metformin dose-dependently reduced lipopolysaccharide (LPS)-induced TF expression in THP-1 cells. The AMPK activator AICAR alone lowered TF expression in THP-1, while the AMPK inhibitor compound C abrogated the metformin-dependent reduction in TF expression. CONCLUSIONS: Our data are the first to report that metformin is associated with reduced plasma TF procoagulant activity possibly explaining-at least in part-the vasculoprotective properties of metformin.

Estimation of numbers of mature and immature neutrophils in blood by a novel, rapid and simple technology.

Counting numbers of blood neutrophils is one of the most common laboratory tests in modern clinical medicine. In this report, we have tested the idea that immunoassay of major constituents of mature neutrophils might serve as proxy of cell counting and allow the development of rapid and simple point-of-care tests. The procedure may also allow for the estimate of the state of maturity of the circulating blood cells. Immunoassays for myeloperoxidase (MPO) and lactoferrin (LF) were used to measure the respective protein in whole blood extracts of 275 unselected hospitalized patient and in 51 healthy controls and leukemia patients of which eight were followed before, during and after remission treatment. MPO was correlated to neutrophil counts in the unselected hospitalized population (r = 0.95, p <.0001). Huge variations were seen in whole blood extracts of patients with AML with very high MPO/LF ratios in half of the AML patients and in all three patients with APL. In extracts from patients with ALL no difference was found in the ratio as compared to healthy persons. The monitoring of AML patients during remission treatment showed intriguing patterns one of which suggested the possibility to monitor the myelopoietic activity in the bone marrow during the recovery phase. We show a novel and easy technology to count mature neutrophils in blood and also to monitor myeloid cell maturity in the blood as well as myelopoietic activity in the bone marrow. The technology lends itself to the development of a rapid and simple point-of-care test.

Antibody subtypes and titers predict clinical outcomes in ANCA-associated vasculitis.

The objective of this study is to evaluate the association between antineutrophil cytoplasmic autoantibody (ANCA) subtype and ANCA titers on clinical outcomes and disease activity among a cohort of patients from Central Appalachia diagnosed with ANCA-associated vasculitis (AAV) over a 3-decade period. This is a retrospective chart review of all patients diagnosed with AAV. ANCA subtypes (myeloperoxidase (MPO) and proteinase 3 (PR3)) and titers at the time of diagnosis and at the time of relapse or last follow-up were evaluated along with patient outcomes. Outcomes of interest included relapse, development of end-stage renal disease (ESRD) and mortality. Sensitivity analysis and multivariable analysis were performed. Of the 202 patients, 111 patients were MPO-ANCA positive and 91 patients were PR3-ANCA positive. Relapse was more frequent among patients with PR3-ANCA compared to MPO-ANCA (35% vs 12%, p < 0.001). In both ANCA subgroups, the strongest predictor of relapse was an increase in titers prior to relapse, HR 8.1 (95% CI 1.6-40), p 0.009. Patients who achieved serological remission had a lower risk of ESRD [sub-HR 0.31 (95% CI 0.11-0.89)] and mortality [HR (95% CI) 0.24 (0.07-0.7)]. PR3-ANCA was associated with higher risk of ESRD [sub-HR 3.1 (95% CI 1.1-8.5)]. There was no difference in mortality between patients with MPO-ANCA and PR3-ANCA. Our study supports the use of both ANCA subtypes and titer levels for predicting clinical outcomes in patients receiving treatment for AAV. Monitoring of ANCA antibody titers may be useful since both serological remission and increase in titers provide prognostic information.

Prognostic value of myeloperoxidase in patients with peripheral artery disease.

OBJECTIVES: The involvement of myeloperoxidase in the production of dysfunctional high-density lipoproteins and oxidised biomolecules leads to oxidative stress in the blood vessel endothelium. This prospective cohort study aimed to examine the prognostic value of myeloperoxidase in patients with peripheral artery disease in relation to major adverse cardiac events (MACEs), target lesion revascularisation, and major adverse limb events (MALEs) and its association with multi-bed vascular disease, which is defined as any combination of the following: peripheral artery disease and coronary artery disease. METHODS: Myeloperoxidase levels were measured in patients with peripheral artery disease and coronary artery disease during angiography. A total of 94 patients were analysed and followed up regarding their MACEs, target lesion revascularisation, and MALEs from August 2016 until February 2019. RESULTS: Among patients with peripheral artery disease, the rates of MACE and mortality were higher in patients with high myeloperoxidase levels than in those with low myeloperoxidase levels; the myeloperoxidase levels were 3.68 times higher in these patients (p < 0.0001). Patients with peripheral artery disease and coronary artery disease (multi-bed vascular disease) had higher myeloperoxidase levels than those with only peripheral artery disease and only coronary artery disease (one-bed vascular disease). Peripheral artery disease patients with higher myeloperoxidase levels had significantly higher rates of limb ischaemia, requiring further revascularisation than those with low myeloperoxidase levels. CONCLUSIONS: High myeloperoxidase levels suggest poor outcomes and are associated with MACE and limb ischaemia. Our findings indicated that myeloperoxidase levels could become a prognostic marker and may be used in conjunction with other methods for risk stratification in patients with peripheral artery disease and multi-bed vascular disease.

Therapeutic Myeloperoxidase Inhibition Attenuates Neutrophil Activation, ANCA-Mediated Endothelial Damage, and Crescentic GN.

BACKGROUND: Myeloperoxidase released after neutrophil and monocyte activation can generate reactive oxygen species, leading to host tissue damage. Extracellular glomerular myeloperoxidase deposition, seen in ANCA-associated vasculitis, may enhance crescentic GN through antigen-specific T and B cell activation. Myeloperoxidase-deficient animals have attenuated GN early on, but augmented T cell responses. We investigated the effect of myeloperoxidase inhibition, using the myeloperoxidase inhibitor AZM198, to understand its potential role in treating crescentic GN. METHODS: We evaluated renal biopsy samples from patients with various forms of crescentic GN for myeloperoxidase and neutrophils, measured serum myeloperoxidase concentration in patients with ANCA-associated vasculitis and controls, and assessed neutrophil extracellular trap formation, reactive oxygen species production, and neutrophil degranulation in ANCA-stimulated neutrophils in the absence and presence of AZM198. We also tested the effect of AZM198 on ANCA-stimulated neutrophil-mediated endothelial cell damage in vitro, as well as on crescentic GN severity and antigen-specific T cell reactivity in the murine model of nephrotoxic nephritis. RESULTS: All biopsy specimens with crescentic GN had extracellular glomerular myeloperoxidase deposition that correlated significantly with eGFR and crescent formation. In vitro, AZM198 led to a significant reduction in neutrophil extracellular trap formation, reactive oxygen species production, and released human neutrophil peptide levels, and attenuated neutrophil-mediated endothelial cell damage. In vivo, delayed AZM198 treatment significantly reduced proteinuria, glomerular thrombosis, serum creatinine, and glomerular macrophage infiltration, without increasing adaptive T cell responses. CONCLUSIONS: Myeloperoxidase inhibition reduced neutrophil degranulation and neutrophil-mediated endothelial cell damage in patients with ANCA-associated vasculitis. In preclinical crescentic GN, delayed myeloperoxidase inhibition suppressed kidney damage without augmenting adaptive immune responses, suggesting it might offer a novel adjunctive therapeutic approach in crescentic GN.