(Homo-)harringtonine prevents endothelial inflammation through IRF-1 dependent downregulation of VCAM1 mRNA expression and inhibition of cell adhesion molecule protein biosynthesis.

The plant alkaloid homoharringtonine (HHT) is a Food and Drug Administration (FDA)-approved drug for the treatment of hematologic malignancies. In addition to its well-established antitumor activity, accumulating evidence attributes anti-inflammatory effects to HHT, which have mainly been studied in leukocytes to date. However, a potential influence of HHT on inflammatory activation processes in endothelial cells, which are a key feature of inflammation and a prerequisite for the leukocyte-endothelial cell interaction and leukocyte extravasation, remains poorly understood. In this study, the anti-inflammatory potential of HHT and its derivative harringtonine (HT) on the TNF-induced leukocyte-endothelial cell interaction was assessed, and the underlying mechanistic basis of these effects was elucidated. HHT affected inflammation in vivo in a murine peritonitis model by reducing leukocyte infiltration and proinflammatory cytokine expression as well as ameliorating abdominal pain behavior. In vitro, HT and HHT impaired the leukocyte-endothelial cell interaction by decreasing the expression of the endothelial cell adhesion molecules intracellular adhesion molecule -1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). This effect was mediated by a bipartite mechanism. While HHT did not affect the prominent TNF-induced pro-inflammatory NF-ĸB signaling cascade, the compound downregulated the VCAM1 mRNA expression in an IRF-1-dependent manner and diminished active ICAM1 mRNA translation as determined by polysome profiling. This study highlights HHT as an anti-inflammatory compound that efficiently hampers the leukocyte-endothelial cell interaction by targeting endothelial activation processes.

Exploring the Impact of French Raw-Milk Cheeses on Oxidative Process Using Caenorhabditis elegans and Human Leukocyte Models.

Fermented foods, including cheeses, have garnered increased interest in recent years for their potential health benefits. This study explores the biological properties of eight French raw-milk cheeses-goat cheese, Saint-Nectaire, Cantal, Bleu d'Auvergne, Roquefort, Comté, Brie de Meaux, and Epoisses-on oxidative processes using both in vivo (Caenorhabditis elegans) and in vitro (human leukocytes) models. A cheese fractionation protocol was adapted to study four fractions for each cheese: a freeze-dried fraction (FDC) corresponding to whole cheese, an apolar (ApE), and two polar extracts (W40 and W70). We showed that all cheese fractions significantly improved Caenorhabditis elegans (C. elegans) survival rates when exposed to oxidative conditions by up to five times compared to the control, regardless of the fractionation protocol and the cheese type. They were also all able to reduce the in vivo accumulation of reactive oxygen species (ROS) by up to 70% under oxidative conditions, thereby safeguarding C. elegans from oxidative damage. These beneficial effects were explained by a reduction in ROS production up to 50% in vitro in human leukocytes and overexpression of antioxidant factor-encoding genes (daf-16, skn-1, ctl-2, and sod-3) in C. elegans.

In Vivo Cytotoxic, Genotoxic and Radiosensitizing Effects of Clofarabine.

BACKGROUND/AIM: ClFdA is a second-generation antineoplastic agent that has demonstrated significant anticancer activity, particularly against acute lymphoblastic leukemia and has been shown to have radiosensitizing activity. The aim of the study was to explore the genotoxic, cytotoxic and radiosensitizing effects of clofarabine (ClFdA) on bone marrow cells (BMCs), normoblasts and leukocytes of mice in vivo. MATERIALS AND METHODS: Cytotoxicity was determined by the reduction in reticulocytes (RET), and genotoxicity was determined by the induction of micronucleated reticulocytes (MN-RET) in the peripheral blood and by DNA break induction in leukocytes determined by single-cell gel electrophoresis (SCGE). The radiosensitizing capacity of ClFdA was determined in leukocytes and BMCs by SCGE. RESULTS: Two mechanisms of MN-RET induction were identified according to the antecedents, that could be due to inhibition of DNA synthesis and demethylation of G-C regions, and subsequent chromosome fragility. ClFdA cytotoxicity causes two contiguous peaks, an early peak that seems to inhibit MN-RET induction and a second peak that seems to be caused by ribonucleotide reductase (RR) and/or DNA synthesis inhibitions. ClFdA induced early DNA damage in noncycling leukocytes, and also radiosensitizes leukocytes immediately after treatment. ClFdA-ionizing radiation (IR) causes two time-dependent episodes of DNA damage, the latest after 80 min triggers a major breakage of DNA. In terms of the number of damaged cells, leukocytes and BMCs are similarly sensitive to ionizing radiation; BMCs are slightly more sensitive than leukocytes to ClFdA, but BMCs are doubly sensitive to combined treatment. CONCLUSION: ClFdA causes early DNA damage and radiosensitivity in non-proliferating leukocytes, which rules out the most favored hypotheses of the participation of RR and DNA polymerase inhibition.

Zerumbone exhibits anti-inflammatory effects by suppressing eicosanoid signaling: Evidence from LPS-induced peripheral blood leukocytes.

Zerumbone, a sesquiterpene isolated from Zingiber zerumbet, has many bioactivities, exhibiting anti-inflammatory properties. However, the effect of zerumbone on the eicosanoid signaling pathway has yet to be examined. Here, we deciphered the anti-eicosanoid properties of zerumbone isolated from ginger. The molecular interaction between zerumbone and eicosanoid metabolizing enzymes (COX-2, 5-LOX, FLAP, and LTA4-hydrolase) and receptors (EP-4, BLT-1, and ICAM-1) along with NOS-2 were assessed using Auto-Dock 4.2 and visualized by chimera and Liggplot+ software. Further, the leukocytes were treated with zerumbone (1-20 µM) and activated using bacterial lipopolysaccharide (LPS-10 nM). The oxidative stress (OS) markers, antioxidant enzymes, and the eicosanoid pathway mediators such as COX-2, 5-LOX, BLT-1, and EP-4 were assessed. The molecular interaction of zerumbone with eicosanoids showed a higher binding affinity with mPGES-1, followed by NOS-2, FLAP, COX-2, LTA-4-hydrolase, and BLT-1. The concentration of 5 µM zerumbone effectively prevented the generation of reactive oxygen species (ROS) and nitric oxide (NO). Likewise, zerumbone significantly (p<0.05) inhibited COX-2, 5-LOX, NOS-2, EP-4, BLT-1, and ICAM-1 expression in LPS-induced peripheral blood leukocytes from rats. Further, the zerumbone treatment on the human PBMCs activated with LPS showed significant inhibition in the expression of ICAM1, COX-2, 5-LOX, and the generation of inflammatory cytokines compared to the control. Overall, the data presented infers that zerumbone positively modulates critical enzymes and receptors of eicosanoids in leukocytes activated with lipopolysaccharides. Thus, zerumbone can be a potential anti-eicosanoid drug in managing inflammation.

Multi-layered effects of Panax notoginseng on immune system.

Recent research has demonstrated the immunomodulatory potential of Panax notoginseng in the treatment of chronic inflammatory diseases and cerebral hemorrhage, suggesting its significance in clinical practice. Nevertheless, the complex immune activity of various components has hindered a comprehensive understanding of the immune-regulating properties of Panax notoginseng, impeding its broader utilization. This review evaluates the effect of Panax notoginseng to various types of white blood cells, elucidates the underlying mechanisms, and compares the immunomodulatory effects of different Panax notoginseng active fractions, aiming to provide the theory basis for future immunomodulatory investigation.

Pyrazoles have a multifaceted anti-inflammatory effect targeting prostaglandin E2, cyclooxygenases and leukocytes' oxidative burst.

Elevated levels of prostaglandin E2 have been implicated in the pathophysiology of various diseases. Anti-inflammatory drugs that act through the inhibition of cyclooxygenase enzymatic activity, thereby leading to the suppression of prostaglandin E2, are often associated with several side effects due to their non-specific inhibition of cyclooxygenase enzymes. Consequently, the targeted suppression of prostaglandin E2 production with innovative molecules and/or mechanisms emerges as a compelling therapeutic strategy for the treatment of inflammatory-related diseases. Therefore, in this study, a systematic analysis of 28 pyrazole derivatives was conducted to explore their potential mechanisms for reducing prostaglandin E2 levels. In this context, the evaluation of these derivatives extended to examining their capacity to reduce prostaglandin E2in vitro in human whole blood, inhibit cyclooxygenase-1 and cyclooxygenase-2 enzymes, modulate cyclooxygenase-2 expression, and suppress oxidative burst in human leukocytes. The results enabled the establishment of significant structure-activity relationships, elucidating key determinants for their activities. In particular, the 4-styryl group on the pyrazole moiety and the presence of chloro substitutions were identified as key determinants. Pyrazole 8 demonstrated the capacity to reduce prostaglandin E2 levels by downregulating cyclooxygenase-2 expression, and pyrazole-1,2,3-triazole 18 emerged as a dual-acting agent, inhibiting human leukocytes' oxidative burst and cyclooxygenase-2 activity. Furthermore, pyrazole 26 demonstrated effective reduction of prostaglandin E2 levels through selective cyclooxygenase-1 inhibition. These results underscore the multifaceted anti-inflammatory potential of pyrazoles, providing new insights into the substitutions and structural frameworks that are beneficial for the studied activity.

The Effects of Fingolimod (FTY720) on Leukocyte Subset Circulation cannot be Behaviourally Conditioned in Rats.

Suppression of immune functions can be elicited by behavioural conditioning using drugs such as cyclosporin A or rapamycin. Nevertheless, little is known about the underlying mechanisms and generalisability of this phenomenon. Against this background, the present study investigated whether the pharmacological properties of fingolimod (FTY720), an immunosuppressive drug widely applied to treat multiple sclerosis, can be conditioned in rats by means of taste-immune associative learning. For this purpose, a conditioned taste avoidance paradigm was used, pairing the presentation of a novel sweet drinking solution (saccharin or sucrose) as conditioned stimulus (CS) with therapeutically effective doses of FTY720 as unconditioned stimulus (US). Subsequent re-exposure to the CS at a later time point revealed that conditioning with FTY720 induced a mild conditioned taste avoidance only when saccharin was employed as CS. However, on an immunological level, neither re-exposure with saccharin nor sucrose altered blood immune cell subsets or splenic cytokine production. Despite the fact that intraperitonally administered FTY720 could be detected in brain regions known to mediate neuro-immune interactions, the present findings show that the physiological action of FTY720 is not inducible by mere taste-immune associative learning. Whether conditioning generalises across all small-molecule drugs with immunosuppressive properties still needs to be investigated with modified paradigms probably using distinct sensory CS. Moreover, these findings emphasize the need to further investigate the underlying mechanisms of conditioned immunomodulation to assess the generalisability and usability of associative learning protocols as supportive therapies in clinical contexts.

Neuroreceptor Inhibition by Clozapine Triggers Mitohormesis and Metabolic Reprogramming in Human Blood Cells.

The antipsychotic drug clozapine demonstrates superior efficacy in treatment-resistant schizophrenia, but its intracellular mode of action is not completely understood. Here, we analysed the effects of clozapine (2.5-20 µM) on metabolic fluxes, cell respiration, and intracellular ATP in human HL60 cells. Some results were confirmed in leukocytes of clozapine-treated patients. Neuroreceptor inhibition under clozapine reduced Akt activation with decreased glucose uptake, thereby inducing ER stress and the unfolded protein response (UPR). Metabolic profiling by liquid-chromatography/mass-spectrometry revealed downregulation of glycolysis and the pentose phosphate pathway, thereby saving glucose to keep the electron transport chain working. Mitochondrial respiration was dampened by upregulation of the F0F1-ATPase inhibitory factor 1 (IF1) leading to 30-40% lower oxygen consumption in HL60 cells. Blocking IF1 expression by cotreatment with epigallocatechin-3-gallate (EGCG) increased apoptosis of HL60 cells. Upregulation of the mitochondrial citrate carrier shifted excess citrate to the cytosol for use in lipogenesis and for storage as triacylglycerol in lipid droplets (LDs). Accordingly, clozapine-treated HL60 cells and leukocytes from clozapine-treated patients contain more LDs than untreated cells. Since mitochondrial disturbances are described in the pathophysiology of schizophrenia, clozapine-induced mitohormesis is an excellent way to escape energy deficits and improve cell survival.

Preclinical evaluation of single domain antibody efficacy in mitigating local tissue damage induced by Bothrops snake envenomation.

Camelid single-domain antibodies (VHH) represent a promising class of immunobiologicals for therapeutic applications due to their remarkable stability, specificity, and therapeutic potential. To enhance the effectiveness of antivenoms for snakebites, various methods have been explored to address limitations associated with serum therapy, particularly focusing on mitigating local damage and ensuring sustainable production. Our study aimed to characterize the pharmacological profile and neutralization capacity of anti-Phospholipase A2 (PLA2) monomeric VHH (Genbank accessions: KC329718). Using a post-envenoming mouse model, we used intravital microscopy to assess leukocyte influx, measured CK and LDH levels, and conducted a histopathology analysis to evaluate VHH KC329718's ability to neutralize myotoxic activity. Our findings demonstrated that VHH KC329718 exhibited heterogeneous distribution in muscle tissue. Treatment with VHH KC329718 reduced leukocyte influx caused by BthTX-I (a Lys-49 PLA2) by 28 %, as observed through intravital microscopy. When administered at a 1:10 ratio [venom or toxin:VHH (w/w)], VHH KC329718 significantly decreased myotoxicity, resulting in a 35-40 % reduction in CK levels from BthTX-I and BthTX-II (an Asp-49 PLA2) and a 60 % decrease in CK levels from B. jararacussu venom. LDH levels also showed reductions of 60%, 80%, and 60% induced by BthTX-I, BthTX-II, and B. jararacussu venom, respectively. Histological analysis confirmed the neutralization potential, displaying a significant reduction in tissue damage and inflammatory cell count in mice treated with VHH KC329718 post B. jararacussu venom inoculation. This study underscores the potential of monomeric anti-PLA2 VHH in mitigating myotoxic effects, suggesting a promising avenue for the development of new generation antivenoms to address current therapeutic limitations.

The proteasome inhibitor carfilzomib exerts anti-inflammatory and antithrombotic effects on the endothelium.

BACKGROUND: Carfilzomib (CFZ) is a second-generation proteasome inhibitor used to treat multiple myeloma. Potent inhibition of the proteasome results in chronic proteotoxic endoplasmic reticulum (ER) stress, leading to apoptosis. While CFZ has improved survival rates in multiple myeloma, it is associated with an increased risk of cardiovascular adverse effects. While this has been putatively linked to cardiotoxicity, CFZ could potentially also exhibit adverse effects on the endothelium. OBJECTIVES: To investigate the effects of CFZ on the endothelium. METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with CFZ, and expression of relevant markers of ER stress, inflammation, and thrombosis was measured and functionally assessed. RESULTS: CFZ failed to induce ER stress in HUVECs but induced the expression of Kruppel-like factor 4, endothelial nitric oxide synthase, tissue plasminogen activator, and thrombomodulin and reduced tumor necrosis factor alpha (TNFα)-mediated intercellular adhesion molecule 1 and tissue factor expression, suggesting a potential protective effect on the endothelium. Consistent with these observations, CFZ reduced leukocyte adhesion under shear stress and reduced factor Xa generation and fibrin clot formation on the endothelium following TNFα treatment and inhibited von Willebrand factor (VWF) and angiopoietin-2 exocytosis from Weibel-Palade bodies. Subsequently, CFZ inhibited the formation of VWF-platelet strings, and moreover, media derived from myeloma cell lines induced VWF release, a process also inhibited by CFZ. CONCLUSION: These data demonstrate that CFZ is unable to induce ER stress in confluent resting endothelial cells and can conversely attenuate the prothrombotic effects of TNFα on the endothelium. This study suggests that CFZ does not negatively alter HUVECs, and proteasome inhibition of the endothelium may offer a potential way to prevent thrombosis.

In-Utero exposure to potential sources of indoor air pollution and umbilical cord blood leukocyte telomere length.

Indoor air pollution (IAP) has been associated with various adverse health effects. However, the evidence regarding such an association with leukocyte telomere length (LTL) in cord blood samples is still scarce. Therefore, the present study aimed to assess the relationship between exposure to indicators of IAP and LTL in umbilical cord blood samples. This cross-sectional study was based on 188 mother-newborn pairs who participated in our study between 2020 and 2022 in Isfahan, Iran. Umbilical LTL was measured by quantitative real-time polymerase chain reaction (qRT-PCR) technique. Linear mixed-effect models were used to assess the relationship between IAP indicators and umbilical LTL, adjusted for relevant covariates. The median (interquartile range (IQR)) of umbilical LTL was 0.92 (0.47). In fully adjusted models, frequency of using degreasing spray during pregnancy (times per month) (ß = -0.047, 95% CI:0.09, -0.05, P-value = 0.02), using air freshener spray during pregnancy (ß = -0.26, 95% CI: -0.5, -0.02, P-value = 0.03) and frequency of using insecticides during pregnancy (times per month) (ß = -0.025, 95% CI: -0.047, -0.003, P-value = 0.02) were significantly associated with shorter umbilical LTL. There was a positive significant relationship between the frequency of using cleaning spray during pregnancy (times per month) with umbilical LTL (ß = 0.019, 95% CI: 0.005, 0.033, P-value = 0.01). Furthermore, the direct connection of the parking with home and the frequency of using barbecue (times per week) were marginally associated with shorter umbilical LTL. For other indicators of IAP, we did not observe any statistically significant associations. Overall, this study suggested a negative association between prenatal exposure to IAP during pregnancy and umbilical LTL.

In vitro effect of diazoxon on cell signaling and second messengers in Nile tilapia (Oreochromis niloticus) leukocytes.

The physiological and molecular responses of leukocytes are altered by organophosphate pesticides. Some reports have shown that diazinon causes immunotoxic effects; diazoxon, the oxon metabolite of diazinon, is attributed to influence the immune response by affecting the leukocyte cholinergic system. In this study, the in vitro effects of diazoxon on molecules involved in cell signaling (cAMP, IP3, DAG, JAK1, and STAT3), which play a crucial role in the activation, differentiation, and survival of leukocytes, were evaluated. Data indicate that diazoxon leads to a decrease in cAMP concentration and an increase in basal IP3 levels. However, diazoxon does not affect basal levels of JAK1 and STAT3 phosphorylation. Instead, diazoxon inhibits leukocyte responsiveness to phorbol myristate acetate and ionomycin, substances that, under normal conditions, enhance JAK/STAT signaling. These findings demonstrate that diazoxon significantly affects key molecular parameters related to cell signaling.

Participation of Hepcidins in the Inflammatory Response Triggered by λ-Carrageenin in Gilthead Seabream (Sparus aurata).

The role of hepcidins, antimicrobial peptides involved in iron metabolism, immunity, and inflammation, is studied. First, gilthead seabream (Sparus aurata L.) head-kidney leucocytes (HKLs) were incubated with λ-carrageenin to study the expression of hepcidin and iron metabolism-related genes. While the expression of most of the genes studied was upregulated, the expression of ferroportin gene (slc40a) was downregulated. In the second part of the study, seabream specimens were injected intramuscularly with λ-carrageenin or buffer (control). The expression of the same genes was evaluated in the head kidney, liver, and skin at different time points after injection. The expression of Hamp1m, ferritin b, and ferroportin genes (hamp1, fthb, and slc40a) was upregulated in the head kidney of fish from the λ-carrageenin-injected group, while the expression of Hamp2C and Hamp2E genes (hamp2.3 and hamp2.7) was downregulated. In the liver, the expression of hamp1, ferritin a (ftha), slc40a, Hamp2J, and Hamp2D (hamp2.5/6) genes was downregulated in the λ-carrageenin-injected group. In the skin, the expression of hamp1 and (Hamp2A Hamp2C) hamp2.1/3/4 genes was upregulated in the λ-carrageenin-injected group. A bioinformatic analysis was performed to predict the presence of transcription factor binding sites in the promoter region of hepcidins. The primary sequence of hepcidin was conserved among the different mature peptides, although changes in specific amino acid residues were identified. These changes affected the charge, hydrophobicity, and probability of hepcidins being antimicrobial peptides. This study sheds light on the poorly understood roles of hepcidins in fish. The results provide insight into the regulatory mechanisms of inflammation in fish and could contribute to the development of new strategies for treat inflammation in farm animals.

The role of leukocyte and platelet-rich fibrin in the prevention of medication-related osteonecrosis of the jaw, in patients requiring dental extractions: an observational study.

PURPOSE: Medication-related osteonecrosis of the jaw (MRONJ) is a significant complication which can present following a dental extraction in patients receiving anti-resorptive and anti-angiogenic medications. The purpose of this study was to investigate the possible beneficial effect of L-PRF in the prevention of MRONJ in patients receiving these medications and requiring dental extractions. METHODS: Thirty-nine patients were included and divided into two groups, depending on whether L-PRF was used after the required dental extraction or not. Subsequently, the patients were categorised into low and high-risk for developing MRONJ, as recommended by the SDCEP guidance. RESULTS: None of the patients in the L-PRF group returned with established MRONJ. Five high-risk patients in the control group presented with established MRONJ in the follow-up appointment. A significant statistical difference (p = 0.04) was observed following a comparison of the high-risk patients of the two groups. CONCLUSION: These encouraging results suggest that L-PRF may be useful in the prevention of MRONJ following a dental extraction especially in patients of the higher risk category. A protocol for the management of this type of patients is also introduced.

Vitamin B12 attenuates leukocyte inflammatory signature in COVID-19 via methyl-dependent changes in epigenetic markings.

COVID-19 induces chromatin remodeling in host immune cells, and it had previously been shown that vitamin B12 downregulates some inflammatory genes via methyl-dependent epigenetic mechanisms. In this work, whole blood cultures from moderate or severe COVID-19 patients were used to assess the potential of B12 as adjuvant drug. The vitamin normalized the expression of a panel of inflammatory genes still dysregulated in the leukocytes despite glucocorticoid therapy during hospitalization. B12 also increased the flux of the sulfur amino acid pathway, that regulates the bioavailability of methyl. Accordingly, B12-induced downregulation of CCL3 strongly and negatively correlated with the hypermethylation of CpGs in its regulatory regions. Transcriptome analysis revealed that B12 attenuates the effects of COVID-19 on most inflammation-related pathways affected by the disease. As far as we are aware, this is the first study to demonstrate that pharmacological modulation of epigenetic markings in leukocytes favorably regulates central components of COVID-19 physiopathology.

Heparin Specifically Interacts with Basic BBXB Motifs of the Chemokine CCL21 to Define CCR7 Signaling.

Chemokines are critically involved in controlling directed leukocyte migration. Spatiotemporal secretion together with local retention processes establish and maintain local chemokine gradients that guide directional cell migration. Extracellular matrix proteins, particularly glycosaminoglycans (GAGs), locally retain chemokines through electrochemical interactions. The two chemokines CCL19 and CCL21 guide CCR7-expressing leukocytes, such as antigen-bearing dendritic cells and T lymphocytes, to draining lymph nodes to initiate adaptive immune responses. CCL21-in contrast to CCL19-is characterized by a unique extended C-terminus composed of highly charged residues to facilitate interactions with GAGs. Notably, both chemokines can trigger common, but also ligand-biased signaling through the same receptor. The underlying molecular mechanism of ligand-biased CCR7 signaling is poorly understood. Using a series of naturally occurring chemokine variants in combination with newly designed site-specific chemokine mutants, we herein assessed CCR7 signaling, as well as GAG interactions. We demonstrate that the charged chemokine C-terminus does not fully confer CCL21-biased CCR7 signaling. Besides the positively charged C-terminus, CCL21 also possesses specific BBXB motifs comprising basic amino acids. We show that CCL21 variants where individual BBXB motifs are mutated retain their capability to trigger G-protein-dependent CCR7 signaling, but lose their ability to interact with heparin. Moreover, we show that heparin specifically interacts with CCL21, but not with CCL19, and thereby competes with ligand-binding to CCR7 and prevents signaling. Hence, we provide evidence that soluble heparin, but not the other GAGs, complexes with CCL21 to define CCR7 signaling in a ligand-dependent manner.

Interleukin-6 inhibition in ST-elevation myocardial infarction: Immune cell profile in the randomised ASSAIL-MI trial.

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).

Methods for Assessing the Effects of Galectins on Leukocyte Trafficking and Clearance.

Numerous protocols exist for investigating leukocyte recruitment and clearance both in vitro and in vivo. Here we describe an in vitro flow chamber assay typically used for studying the mechanisms underpinning leukocyte movement through the endothelium and zymosan-induced peritonitis, an acute in vivo model of inflammation that enables both leukocyte trafficking and clearance to be monitored. Insight is given as to how these models can be used to study the actions of galectins on the inflammatory process.

Susceptibility of Sea Bream (Sparus aurata) to AIP56, an AB-Type Toxin Secreted by Photobacterium damselae subsp. piscicida.

Photobacterium damselae subsp. piscicida (Phdp) is a Gram-negative bacterium that infects a large number of marine fish species in Europe, Asia, and America, both in aquacultures and in the natural environment. Among the affected hosts are economically important cultured fish, such as sea bream (Sparus aurata), sea bass (Dicentrarchus labrax), yellowtail (Seriola quinqueradiata), and cobia (Rachycentron canadum). The best characterized virulence factor of Phdp is the Apoptosis-Inducing Protein of 56 kDa (AIP56), a secreted AB-type toxin that has been shown to induce apoptosis of sea bass phagocytes during infection. AIP56 has an A subunit that displays metalloprotease activity against NF-kB p65 and a B subunit that mediates binding and internalization of the A subunit in susceptible cells. Despite the fact that the aip56 gene is highly prevalent in Phdp isolates from different fish species, the toxicity of AIP56 has only been studied in sea bass. In the present study, the toxicity of AIP56 for sea bream was evaluated. Ex vivo assays showed that sea bream phagocytes are resistant to AIP56 cytotoxicity and that resistance was associated with an inefficient internalization of the toxin by those cells. Accordingly, in vivo intoxication assays revealed that sea bream is much more resistant to AIP56-induced lethality than sea bass. These findings, showing that the effect of AIP56 is different in sea bass and sea bream, set the basis for future studies to characterize the effects of AIP56 and to fully elucidate its virulence role in different Phdp susceptible hosts.

Effects of Formyl Peptide Receptor Agonists Ac9-12 and WKYMV in In Vivo and In Vitro Acute Inflammatory Experimental Models.

Formyl peptide receptors (Fprs) are a G-protein-coupled receptor family mainly expressed on leukocytes. The activation of Fpr1 and Fpr2 triggers a cascade of signaling events, leading to leukocyte migration, cytokine release, and increased phagocytosis. In this study, we evaluate the effects of the Fpr1 and Fpr2 agonists Ac9-12 and WKYMV, respectively, in carrageenan-induced acute peritonitis and LPS-stimulated macrophages. Peritonitis was induced in male C57BL/6 mice through the intraperitoneal injection of 1 mL of 3% carrageenan solution or saline (control). Pre-treatments with Ac9-12 and WKYMV reduced leukocyte influx to the peritoneal cavity, particularly neutrophils and monocytes, and the release of IL-1ß. The addition of the Fpr2 antagonist WRW4 reversed only the anti-inflammatory actions of WKYMV. In vitro, the administration of Boc2 and WRW4 reversed the effects of Ac9-12 and WKYMV, respectively, in the production of IL-6 by LPS-stimulated macrophages. These biological effects of peptides were differently regulated by ERK and p38 signaling pathways. Lipidomic analysis evidenced that Ac9-12 and WKYMV altered the intracellular lipid profile of LPS-stimulated macrophages, revealing an increased concentration of several glycerophospholipids, suggesting regulation of inflammatory pathways triggered by LPS. Overall, our data indicate the therapeutic potential of Ac9-12 and WKYMV via Fpr1 or Fpr2-activation in the inflammatory response and macrophage activation.