ROCK2 interacts with p22phox to phosphorylate p47phox and to control NADPH oxidase activation in human monocytes.

Monocytes play a key role in innate immunity by eliminating pathogens, releasing high levels of cytokines, and differentiating into several cell types, including macrophages and dendritic cells. Similar to other phagocytes, monocytes produce superoxide anions through the NADPH oxidase complex, which is composed of two membrane proteins (p22phox and gp91phox/NOX2) and four cytosolic proteins (p47phox, p67phox, p40phox and Rac1). The pathways involved in NADPH oxidase activation in monocytes are less known than those in neutrophils. Here, we show that p22phox is associated with Rho-associated coiled-coil kinase 2 (ROCK2) in human monocytes but not neutrophils. This interaction occurs between the cytosolic region of p22phox (amino acids 132 to 195) and the coiled-coil region of ROCK2 (amino acids 400 to 967). Interestingly, ROCK2 does not phosphorylate p22phox, p40phox, p67phox, or gp91phox in vitro but phosphorylates p47phox on Ser304, Ser315, Ser320 and Ser328. Furthermore, KD025, a selective inhibitor of ROCK2, inhibited reactive oxygen species (ROS) production and p47phox phosphorylation in monocytes. Specific inhibition of ROCK2 expression in THP1-monocytic cell line by siRNA inhibited ROS production. These data show that ROCK2 interacts with p22phox and phosphorylates p47phox, and suggest that p22phox could be a shuttle for ROCK2 to allow p47phox phosphorylation and NADPH oxidase activation in human monocytes.

epiTCR: a highly sensitive predictor for TCR-peptide binding.

MOTIVATION: Predicting the binding between T-cell receptor (TCR) and peptide presented by human leucocyte antigen molecule is a highly challenging task and a key bottleneck in the development of immunotherapy. Existing prediction tools, despite exhibiting good performance on the datasets they were built with, suffer from low true positive rates when used to predict epitopes capable of eliciting T-cell responses in patients. Therefore, an improved tool for TCR-peptide prediction built upon a large dataset combining existing publicly available data is still needed. RESULTS: We collected data from five public databases (IEDB, TBAdb, VDJdb, McPAS-TCR, and 10X) to form a dataset of >3 million TCR-peptide pairs, 3.27% of which were binding interactions. We proposed epiTCR, a Random Forest-based method dedicated to predicting the TCR-peptide interactions. epiTCR used simple input of TCR CDR3ß sequences and antigen sequences, which are encoded by flattened BLOSUM62. epiTCR performed with area under the curve (0.98) and higher sensitivity (0.94) than other existing tools (NetTCR, Imrex, ATM-TCR, and pMTnet), while maintaining comparable prediction specificity (0.9). We identified seven epitopes that contributed to 98.67% of false positives predicted by epiTCR and exerted similar effects on other tools. We also demonstrated a considerable influence of peptide sequences on prediction, highlighting the need for more diverse peptides in a more balanced dataset. In conclusion, epiTCR is among the most well-performing tools, thanks to the use of combined data from public sources and its use will contribute to the quest in identifying neoantigens for precision cancer immunotherapy. AVAILABILITY AND IMPLEMENTATION: epiTCR is available on GitHub (https://github.com/ddiem-ri-4D/epiTCR).

Impaired p47phox phosphorylation in neutrophils from patients with p67phox-deficient chronic granulomatous disease.

Superoxide production by the phagocyte reduced NAD phosphate (NADPH) oxidase is essential for innate immunity as shown in chronic granulomatous disease (CGD), an immunodeficiency disease resulting from mutations in 1 of its genes. The NADPH oxidase is composed of 2 membrane proteins (gp91phox/NOX2 and p22phox) and 4 cytosolic proteins (p47phox, p67phox, p40phox, and Rac1/2). The phosphorylation of p47phox is required for NADPH oxidase activation in cells. As p47phox and p67phox can form a tight complex in cells, we hypothesized that p67phox could regulate p47phox phosphorylation. To investigate this hypothesis, we used phospho-specific antibodies against 5 major p47phox-phosphorylated sites (Ser304, Ser315, Ser320, Ser328, and Ser345) and neutrophils from healthy donors and from p67phox-/- CGD patients. Results showed that formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate induced a time- and a concentration-dependent phosphorylation of p47phox on Ser304, Ser315, Ser320, and Ser328 in healthy human neutrophils. Interestingly, in neutrophils and Epstein-Barr virus-transformed B lymphocytes from p67phox-/- CGD patients, phosphorylation of p47phox on serine residues was dramatically reduced. In COSphox cells, the presence of p67phox led to increased phosphorylation of p47phox. In vitro studies showed that recombinant p47phox was phosphorylated on Ser304, Ser315, Ser320, and Ser328 by different PKC isoforms and the addition of recombinant p67phox alone or in combination with p40phox potentiated this process. Thus, p67phox and p40phox are required for optimal p47phox phosphorylation on Ser304, Ser315, Ser320, and Ser328 in intact cells. Therefore, p67phox and p40phox are novel regulators of p47phox-phosphorylation.

Application of the Hollow-Fiber Infection Model to Personalized Precision Dosing of Isoniazid in a Clinical Setting.

BACKGROUND: The hollow-fiber infection model (HFIM) is a valuable tool for evaluating pharmacokinetics/pharmacodynamics relationships and determining the optimal antibiotic dose in monotherapy or combination therapy, but the application for personalized precision medicine in tuberculosis treatment remains limited. This study aimed to evaluate the efficacy of adjusted antibiotic doses for a tuberculosis patient using HFIM. METHODS: Model-based Bayesian forecasting was utilized to assess the proposed reduction of the isoniazid dose from 300 mg daily to 150 mg daily in a patient with an ultra-slow-acetylation phenotype. The efficacy of the adjusted 150-mg dose was evaluated in a time-to-kill assay performed using the bacterial isolate Mycobacterium tuberculosis (Mtb) H37Ra in a HFIM that mimicked the individual pharmacokinetic profile of the patient. RESULTS: The isoniazid concentration observed in the HFIM adequately reflected the target drug exposures simulated by the model. After 7 days of repeated dose administration, isoniazid killed 4 log10 Mtb CFU/mL in the treatment arm, while the control arm without isoniazid increased 1.6 log10 CFU/mL. CONCLUSION: Our results provide an example of the utility of the HFIM for predicting the efficacy of specific recommended doses of anti-tuberculosis drugs in real clinical setting.

Covid-19 during Pregnancy - Histopathological Lesions of the Placenta.

INTRODUCTION: Pregnant women and their offspring represented a vulnerable patient collective during the Covid-19 pandemic. Beyond the direct effect of SARS-CoV-2 via vertical transmission, an indirect impact on the fetus can occur through placental lesions deteriorating placental villous function. We performed a histopathological analysis of placentas of parturients with SARS-CoV-2 compared to healthy controls. METHODS AND MATERIALS: Between February 2022 and July 2022 we conducted a prospective case-control study analyzing placental specimens of parturients with SARS-CoV-2 infection compared to specimens of placentas of healthy controls. Patient history, Covid-19-specific symptoms, and obstetric outcomes were recorded. Statistical analysis was performed. RESULTS: During the observation period 71 patients were included with a gestational age 37 1/7-41 5/7 weeks. Thirty-six patients presented with SARS-CoV-2 infection. The control group consisted of 35 patients and showed no placental abnormalities. Among SARS-CoV-2-positive parturients, 66.7% of placentas of the case group showed histopathological abnormalities classified as vascular or inflammatory abnormalities. 22.2% of placentas showed acute ischemic infarction areas. 8.3% of placentas showed subchorionic layered thrombi. There was one case of severe acute subchorionitis. SARS-CoV-2 increased the risk of placental lesions significantly (OR 3.000, CI 1.890-4.762, p=0.0001). Placental lesions had no significant impact on perinatal acidosis (OR 0.455, CI 0.044-4.667, p=0.498) or number of cesarean sections (OR 2.314, CI 0.717-7.473, p=0.156). CONCLUSION: SARS-CoV-2 infection during labor and delivery increased the risk of adverse outcomes. Histopathological analysis indicated that the placenta as a maternal-fetal interface was affected by SARS-CoV-2, leading to systemic vasculopathy and inflammation.

Evaluation of Vectra® XT 3D Surface Imaging Technology in Measuring Breast Symmetry and Breast Volume.

BACKGROUND: Breast symmetry is an essential component of breast cosmesis. The Harvard Cosmesis scale is the most widely adopted method of breast symmetry assessment. However, this scale lacks reproducibility and reliability, limiting its application in clinical practice. The VECTRA® XT 3D (VECTRA®) is a novel breast surface imaging system that, when combined with breast contour measuring software (Mirror®), aims to produce a more accurate and reproducible measurement of breast contour to aid operative planning in breast surgery. OBJECTIVES: This study aims to compare the reliability and reproducibility of subjective (Harvard Cosmesis scale) with objective (VECTRA®) symmetry assessment on the same cohort of patients. METHODS: Patients at a tertiary institution had 2D and 3D photographs of their breasts. Seven assessors scored the 2D photographs using the Harvard Cosmesis scale. Two independent assessors used Mirror® software to objectively calculate breast symmetry by analysing 3D images of the breasts. RESULTS: Intra-observer agreement ranged from none to moderate (kappa - 0.005-0.7) amongst the assessors using the Harvard Cosmesis scale. Inter-observer agreement was weak (kappa 0.078-0.454) amongst Harvard scores compared to VECTRA® measurements. Kappa values ranged 0.537-0.674 for intra-observer agreement (p < 0.001) with Root Mean Square (RMS) scores. RMS had a moderate correlation with the Harvard Cosmesis scale (rs = 0.613). Furthermore, absolute volume difference between breasts had poor correlation with RMS (R2 = 0.133). CONCLUSION: VECTRA® and Mirror® software have potential in clinical practice as objectifying breast symmetry, but in the current form, it is not an ideal test. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Caspase Inhibition Modulates Monocyte-Derived Macrophage Polarization in Damaged Tissues.

Circulating monocytes are recruited in damaged tissues to generate macrophages that modulate disease progression. Colony-stimulating factor-1 (CSF-1) promotes the generation of monocyte-derived macrophages, which involves caspase activation. Here, we demonstrate that activated caspase-3 and caspase-7 are located to the vicinity of the mitochondria in CSF1-treated human monocytes. Active caspase-7 cleaves p47PHOX at aspartate 34, which promotes the formation of the NADPH (nicotinamide adenine dinucleotide phosphate) oxidase complex NOX2 and the production of cytosolic superoxide anions. Monocyte response to CSF-1 is altered in patients with a chronic granulomatous disease, which are constitutively defective in NOX2. Both caspase-7 down-regulation and radical oxygen species scavenging decrease the migration of CSF-1-induced macrophages. Inhibition or deletion of caspases prevents the development of lung fibrosis in mice exposed to bleomycin. Altogether, a non-conventional pathway that involves caspases and activates NOX2 is involved in CSF1-driven monocyte differentiation and could be therapeutically targeted to modulate macrophage polarization in damaged tissues.

Computational Investigation of the Formation of Substituted Isoindole N-Oxides through the Photo-oxidative Cyclization of 2'-Alkynylacetophenone Oximes.

Our recently published joint experiment-theory study of the photo-oxidative intramolecular cyclization of 2'-alkynylacetophenone oximes, performed in collaboration with the de Lijser group, presented the first reported formation of isoindole N-oxides. That study focused on determining a mechanistic explanation for the unexpected chemistry observed when three 2'-alkynylacetophenone oximes were photo-oxidized with 9,10-dicyanoanthracene (DCA), specifically the derivatives with a phenyl, isopropyl, or n-butyl substituent at the alkynyl group. Here, we use density functional theory to develop a broader understanding of the scope of this chemistry. In particular, we demonstrate that substituents on the alkynyl group and on the central benzene ring can significantly modulate the thermodynamic driving force for oxime radical cation generation when DCA is used as the photosensitizer. In contrast, substituents are shown to have a small impact on the chemical reactivity of the radical cation intermediates. In particular, 5-exo radical cation cyclization, which ultimately results in an isoindole N-oxide product, is always kinetically and sometimes also thermodynamically preferred over 6-endo radical cation cyclization, which would produce an isoquinoline N-oxide product. Overall, this study provides mechanistic insights into the diversity of isoindole N-oxides that can be produced through the photo-oxidative cyclization of 2'-alkynylacetophenone oximes.

The Kinesin Light Chain-Related Protein PAT1 Promotes Superoxide Anion Production in Human Phagocytes.

Superoxide anion production by the phagocyte NADPH oxidase plays a crucial role in host defenses and inflammatory reaction. The phagocyte NADPH oxidase is composed of cytosolic components (p40phox, p47phox, p67phox, and Rac1/2) and the membrane flavocytochrome b558, which is composed of two proteins: p22phox and gp91phox/NOX2. p22phox plays a crucial role in the stabilization of gp91phox in phagocytes and is also a docking site for p47phox during activation. In the current study, we have used a yeast two-hybrid approach to identify unknown partners of p22phox. Using the cytosolic C-terminal region of p22phox as bait to screen a human spleen cDNA library, we identified the protein interacting with amyloid precursor protein tail 1 (PAT1) as a potential partner of p22phox. The interaction between p22phox and PAT1 was further confirmed by in vitro GST pulldown and overlay assays and in intact neutrophils and COSphox cells by coimmunoprecipitation. We demonstrated that PAT1 is expressed in human neutrophils and monocytes and colocalizes with p22phox, as shown by confocal microscopy. Overexpression of PAT1 in human monocytes and in COSphox cells increased superoxide anion production and depletion of PAT1 by specific small interfering RNA inhibited this process. These data clearly identify PAT1 as a novel regulator of NADPH oxidase activation and superoxide anion production, a key phagocyte function.

Whole genome sequencing and genetic variations in several dengue virus type 1 strains from unusual dengue epidemic of 2017 in Vietnam.

BACKGROUND: Dengue hemorrhagic fever is an acute viral infection transmitted by mosquitoes. In the 2017, a dengue epidemic occurred in Hanoi in a short time interval and many cases were serious with associated mortality. This was the largest and unusual dengue fever outbreak in the North of Vietnam over the past 20 years. The objective of the present study was to understand the genetic characteristics of the DENV-1 strain in the 2017 epidemic and its relationship with previous viruses in Vietnam and the rest of the world. METHODS: Complete genomes of 72 DENV-1 from patients in the 2017 epidemic were sequenced using NGS. The full genome sequences were then analyzed to find out the genetic variants in the groups of 72 strains, followed by their comparison with other strains that caused disease in Vietnam previously and several other regions of the world, revealing a genetic relationship between them. RESULTS: The complete genome sequence of 72 DENV-1 strains comprised 10,697 nucleotides with an open reading frame coding for 3392 amino acids. The genomic analysis revealed different amino acid substitutions in all genes, especially varying at position S75 (Capsid), M125 (PrM), D54 (E), T147, V180 (NS1), G45, Y126, I154 (NS2A), A94 (NS2B), M298 (NS3), K47, V68 (NS4A), I29 (NS4B), and R166, E536, G614, T821 (NS5). The genetic analysis suggested that the viruses were most closely related to the causative virus of the dengue outbreak in Vietnam and Cambodia from 2006 to 2008. These results indicated that DENV-1 from the dengue epidemic 2017 in Northern Vietnam originated from the virus that caused the dengue outbreak during the 2007 to 2008 period in Vietnam. CONCLUSION: The present study is the first of its kind to describe complete genome sequence as well as genetic variants and phylogenetic analysis of DENV-1 associated with the unusual dengue epidemic of 2017 in northern Vietnam. These results provide detailed evidence to elucidate the origin, circulation, and genetic evolution of DENV in Vietnam.

Gold nanorod/molybdenum sulfide core-shell nanostructures synthesized by a photo-induced reduction process.

Coupling of plasmonic nanostructures and semiconductors gives promising hybrid nanostructures that can be used in different applications such as photosensing and energy conversion. In this report, we describe an approach for fabricating a new hybrid material by coupling a gold nanorod (Au NR) core and amorphous molybdenum sulfide (MoSx) shell. The Au NR/MoSx core-shell structure is achieved by exploiting the hot electrons generated in the plasmonic excitation of Au NRs to drive the reduction of [MoS4]2-, which is pre-adsorbed on the Au NR surface, producing a thin MoSx layer. This approach allows us to control the thickness of the MoSx coating layer on the Au NR surface. The resultant Au NR/MoSx hybrid is characterized by absorption spectroscopy, scanning electron microscopy, transmission electron microscopy, energy-dispersive x-ray spectroscopy elemental mapping, x-ray diffraction and x-ray photoelectron spectroscopy.

Activation of the phagocyte NADPH oxidase/NOX2 and myeloperoxidase in the mouse brain during pilocarpine-induced temporal lobe epilepsy and inhibition by ketamine.

Excessive reactive oxygen species (ROS) production can induce tissue injury involved in a variety of neurodegenerative disorders such as neurodegeneration observed in pilocarpine-induced temporal lobe epilepsy. Ketamine, a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist has beneficial effects in pilocarpine-induced temporal lobe epilepsy, when administered within minutes of seizure to avoid the harmful neurological lesions induced by pilocarpine. However, the enzymes involved in ROS productions and the effect of ketamine on this process remain less documented. Here we show that during pilocarpine-induced epilepsy in mice, the expression of the phagocyte NADPH oxidase NOX2 subunits (NOX2/gp91phox, p22phox, and p47phox) and the expression of myeloperoxidase (MPO) were dramatically increased in mice brain treated with pilocarpine. Interestingly, treatment of mice with ketamine before or after pilocarpine administration decreased this process, mainly when injected before pilocarpine. Finally, our results showed that pilocarpine induced p47phox phosphorylation and H2O2 production in mice brain and ketamine was able to inhibit these processes. Our results show that pilocarpine induced NOX2 activation to produce ROS in mice brain and that administration of ketamine before or after the induction of temporal lobe epilepsy by pilocarpine inhibited this activation in mice brain. These results suggest a key role of the phagocyte NADPH oxidase NOX2 and MPO in epilepsy and identify a novel effect of ketamine.

Priming of the neutrophil respiratory burst: role in host defense and inflammation.

Neutrophils are the major circulating white blood cells in humans. They play an essential role in host defense against pathogens. In healthy individuals, circulating neutrophils are in a dormant state with very low efficiency of capture and arrest on the quiescent endothelium. Upon infection and subsequent release of pro-inflammatory mediators, the vascular endothelium signals to circulating neutrophils to roll, adhere, and cross the endothelial barrier. Neutrophils migrate toward the infection site along a gradient of chemo-attractants, then recognize and engulf the pathogen. To kill this pathogen entrapped inside the vacuole, neutrophils produce and release high quantities of antibacterial peptides, proteases, and reactive oxygen species (ROS). The robust ROS production is also called 'the respiratory burst', and the NADPH oxidase or NOX2 is the enzyme responsible for the production of superoxide anion, leading to other ROS. In vitro, several soluble and particulate agonists induce neutrophil ROS production. This process can be enhanced by prior neutrophil treatment with 'priming' agents, which alone do not induce a respiratory burst. In this review, we will describe the priming process and discuss the beneficial role of controlled neutrophil priming in host defense and the detrimental effect of excessive neutrophil priming in inflammatory diseases.

NOX5 and p22phox are 2 novel regulators of human monocytic differentiation into dendritic cells.

Dendritic cells (DCs) are a heterogeneous population of professional antigen-presenting cells and are key cells of the immune system, acquiring different phenotypes in accordance with their localization during the immune response. A subset of inflammatory DCs is derived from circulating monocytes (Mo) and has a key role in inflammation and infection. The pathways controlling Mo-DC differentiation are not fully understood. Our objective was to investigate the possible role of nicotinamide adenine dinucleotide phosphate reduced form oxidases (NOXs) in Mo-DC differentiation. In this study, we revealed that Mo-DC differentiation was inhibited by NOX inhibitors and reactive oxygen species scavengers. We show that the Mo-DC differentiation was dependent on p22phox, and not on gp91phox/NOX2, as shown by the reduced Mo-DC differentiation observed in chronic granulomatous disease patients lacking p22phox. Moreover, we revealed that NOX5 expression was strongly increased during Mo-DC differentiation, but not during Mo-macrophage differentiation. NOX5 was expressed in circulating myeloid DC, and at a lower level in plasmacytoid DC. Interestingly, NOX5 was localized at the outer membrane of the mitochondria and interacted with p22phox in Mo-DC. Selective inhibitors and small interfering RNAs for NOX5 indicated that NOX5 controlled Mo-DC differentiation by regulating the JAK/STAT/MAPK and NFκB pathways. These data demonstrate that the NOX5-p22phox complex drives Mo-DC differentiation, and thus could be critical for immunity and inflammation.

Case Report: A Case of Chromoblastomycosis Caused by Fonsecaea pedrosoi in Vietnam.

BACKGROUND: Chromoblastomycosis is a chronic fungal infection of the skin and subcutaneous tissues caused by different melanized fungi. The disease occurs worldwide, particularly in tropical and subtropical regions but not reported in Vietnam. A 47-year-old women was admitted to hospital 103, Hanoi, Vietnam, with a 10-year lasting lesion on backside of her right shank. Diagnosis of chromoblastomycosis was made after discovery of a muriform cell in histopathological examination. A black, slow-growth fungus was isolated and identified as Fonsecaea pedrosoi after molecular analysis. After 1-month treatment with itraconazole, the lesion has significant improvement. CONCLUSION: This is the first case of chromoblastomycosis caused by Fonsecaea pedrosoi reported in Vietnam.

NADPH oxidase activation in neutrophils: Role of the phosphorylation of its subunits.

Neutrophils are key cells of innate immunity and during inflammation. Upon activation, they produce large amounts of superoxide anion (O2 -. ) and ensuing reactive oxygen species (ROS) to kill phagocytized microbes. The enzyme responsible for O2 -. production is called the phagocyte NADPH oxidase. This is a multicomponent enzyme system that becomes active after assembly of four cytosolic proteins (p47phox , p67phox , p40phox and Rac2) with the transmembrane proteins (p22phox and gp91phox , which form the cytochrome b558 ). gp91phox represents the catalytic subunit of the NADPH oxidase and is also called NOX2. NADPH oxidase-derived ROS are essential for microbial killing and innate immunity; however, excessive ROS production induces tissue injury and prolonged inflammatory reactions that contribute to inflammatory diseases. Thus, NADPH oxidase activation must be tightly regulated in time and space to limit ROS production. NADPH oxidase activation is regulated by several processes such as phosphorylation of its components, exchange of GDP/GTP on Rac2 and binding of p47phox and p40phox to phospholipids. This review aims to provide new insights into the role of the phosphorylation of the NADPH oxidase components, that is gp91phox , p22phox , p47phox , p67phox and p40phox , in the activation of this enzyme.

Density functional theory study of mechanism of epoxy-carboxylic acid curing reaction.

A comprehensive picture on the mechanism of the epoxy-carboxylic acid curing reactions is presented using the density functional theory B3LYP/6-31G(d,p) and simplified physical molecular models to examine all possible reaction pathways. Carboxylic acid can act as its own promoter by using the OH group of an additional acid molecule to stabilize the transition states, and thus lower the rate-limiting barriers by 45 kJ/mol. For comparison, in the uncatalyzed reaction, an epoxy ring is opened by a phenol with an apparent barrier of about 107 kJ/mol. In catalyzed reaction, catalysts facilitate the epoxy ring opening prior to curing that lowers the apparent barriers by 35 kJ/mol. However, this can be competed in highly basic catalysts such as amine-based catalysts, where catalysts can enhance the nucleophilicity of the acid by forming hydrogen-bonded complex with it. Our theoretical results predict the activation energy in the range of 71 to 94 kJ/mol, which agrees well with the reported experimental range for catalyzed reactions. © 2017 Wiley Periodicals, Inc.

Oleuropein and hydroxytyrosol inhibit the N-formyl-methionyl-leucyl-phenylalanine-induced neutrophil degranulation and chemotaxis via AKT, p38, and ERK1/2 MAP-Kinase inhibition.

PURPOSE: Oleuropein and hydroxytyrosol are polyphenols that are extracted from olives and are major biological active components of olives and olive oil. Oleuropein and hydroxytyrosol exhibit interesting pharmacological effects on cells, and have been shown to have many health benefits such as anti-inflammatory effects. These effects were mainly attributed to their ability to scavenge the reactive oxygen species (ROS) produced by phagocytes such as neutrophils. The aim of this study was to investigate the effect of oleuropein and hydroxytyrosol on other neutrophil functions. METHODS: Human neutrophils were isolated from healthy donors. ROS production was measured by luminol-amplified chemiluminescence. Degranulation was assessed by measuring myeloperoxidase activity and Western blots. Chemotaxis was assessed by the under-agarose chemotaxis assay. Phosphorylated proteins were assessed by gel electrophoresis and Western blots. RESULTS: We show that in addition to their ROS scavenging effect, oleuropein and hydroxytyrosol significantly inhibited the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLF)-induced degranulation of azurophilic and specific granules as measured by myeloperoxidase and lactoferrin release, respectively. We also show that oleuropein and hydroxytyrosol reduced fMLF-induced neutrophil chemotaxis. Interestingly, both agents impaired the fMLF-induced AKT, p38MAPKinase, and ERK1/2 phosphorylation, signaling molecules that are involved in pathways regulating neutrophil functions. CONCLUSION: Our data suggest that the anti-inflammatory properties of oleuropein and hydroxytyrosol are not only restricted to their ROS scavenging effect, but also involve the inhibition of two other major pro-inflammatory neutrophil functions.