Polyamine Oxidation Is Indispensable for Wheat (Triticum aestivum L.) Oxidative Response and Necrotic Reactions during Leaf Rust (Puccinia triticina Eriks.) Infection

Hydrogen peroxide is a signal and effector molecule in the plant response to pathogen infection. Wheat resistance to Puccinia triticina Eriks. is associated with necrosis triggered by oxidative burst. We investigated which enzyme system dominated in host oxidative reaction to P. triticina infection. The susceptible Thatcher cultivar and isogenic lines with defined resistance genes were inoculated with P. triticina spores. Using diamine oxidase (DAO) and polyamine oxidase (PAO) inhibitors, accumulation of H2O2 was analyzed in the infection sites. Both enzymes participated in the oxidative burst during compatible and incompatible interactions. Accumulation of H2O2 in guard cells, i.e., the first phase of the response, depended on DAO and the role of PAO was negligible. During the second phase, the patterns of H2O2 accumulation in the infection sites were more complex. Accumulation of H2O2 during compatible interaction (Thatcher and TcLr34 line) moderately depended on DAO and the reaction of TcLr34 was stronger than that of Thatcher. Accumulation of H2O2 during incompatible interaction of moderately resistant plants (TcLr24, TcLr25 and TcLr29) was DAO-dependent in TcLr29, while the changes in the remaining lines were not statistically significant. A strong oxidative burst in resistant plants (TcLr9, TcLr19, TcLr26) was associated with both enzymes’ activities in TcLr9 and only with DAO in TcLr19 and TcLr26. The results are discussed in relation to other host oxidative systems, necrosis, and resistance level.

Impairment of Neutrophil Functions and Homeostasis in COVID-19 Patients: Association With Disease Severity

Background Emerging data based on analyses of peripheral and pulmonary immune responses to SARS-CoV-2 increasingly suggest that a dysregulated immune response underpins the development of severe disease in COVID-19 patients. Neutrophils are key components of early innate immunity that, if not tightly regulated, contribute to uncontrolled systemic inflammation. We sought to decipher the role of neutrophil phenotypes, functions, and homeostasis in COVID-19 disease severity and outcome. Methods This longitudinal study compares study compares peripheral whole-blood neutrophils from 90 COVID-19 ICU patients with those of 22 SARS-CoV-2 – patients hospitalized for severe community-acquired pneumonia (CAP) and 38 healthy controls. We also assessed correlations between these phenotypic and functional indicators and markers of endothelial damage as well as disease severity. Results At ICU admission, the circulating neutrophils of the COVID-19 patients showed continuous basal hyperactivation not seen in CAP patients, associated with higher circulating levels of soluble E- and P-selectin, which reflect platelet and endothelial activation. Furthermore, COVID-19 patients had expanded aged-angiogenic and reverse transmigrated neutrophil subsets — both involved in endothelial dysfunction and vascular inflammation. Simultaneously, COVID-19 patients had significantly lower levels of neutrophil oxidative burst in response to bacterial formyl peptide, an abnormality that was greater in superinfected than non-superinfected COVID-19 patients. Moreover, patients dying of COVID-19 had significantly higher expansion of aged-angiogenic neutrophil subset and greater impairment of oxidative burst response than survivors. Conclusions These data suggest that neutrophil exhaustion may play a central role in the pathogenesis of severe COVID-19 and identify angiogenic neutrophils as a potentially harmful subset involved in fatal outcome.

H2O2-Elicitation of Black Carrot Hairy Roots Induces a Controlled Oxidative Burst Leading to Increased Anthocyanin Production

Hairy roots (HRs) grown in vitro are a powerful platform for plant biotechnological advances and for the bio-based production of metabolites of interest. In this work, black carrot HRs able to accumulate anthocyanin as major secondary metabolite were used. Biomass and anthocyanin accumulation were improved by modulating growth medium composition—different Murashige & Skoog (MS)-based media—and H2O2-elicitation, and the level of the main antioxidant enzymes on elicited HRs was measured. Higher growth was obtained on liquid 1/2 MS medium supplemented with 60 g/L sucrose for HRs grown over 20 days. In this medium, 200 µM H2O2 applied on day 12 induced anthocyanin accumulation by 20%. The activity of superoxide dismutase (SOD)—which generates H2O2 from O2•−—increased by over 50%, whereas the activity of H2O2-scavenging enzymes was not enhanced. Elicitation in the HRs can result in a controlled oxidative burst, in which SOD activity increased H2O2 levels, whereas anthocyanins, as effective reactive oxygen species scavengers, could be induced to modulate the oxidative burst generated. Moreover, given the proven stability of the HR lines used and their remarkable productivity, this system appears as suitable for elucidating the interplay between antioxidant and secondary metabolism.

Fungal oxysterol-binding protein-related proteins promote pathogen virulence and activate plant immunity

Oxysterol-binding protein-related proteins (ORPs) are a conserved class of lipid transfer proteins that are closely involved in multiple cellular processes in eukaryotes but their roles in plant-pathogen interactions are mostly unknown. We showed that transient expression of ORPs of Magnaporthe oryzae (MoORPs) in Nicotiana benthamina plants triggered oxidative burst and cell death; treatment of tobacco Bright Yellow-2 suspension cells with recombinant MoORPs elicited the production of reactive oxygen species. Despite that ORPs are normally described as intracellular proteins, we detected MoORPs in fungal cultural filtrates and intercellular fluids from barley plants infected with the fungus. More importantly, infiltration of Arabidopsis plants with recombinant Arabidopsis or fungal ORPs activated oxidative burst, callose deposition, PR1 gene expression, and enhanced plant disease resistance, implying that ORPs may function as endogenous and exogenous danger signals triggering plant innate immunity. Extracellular application of fungal ORPs exerted an opposite impact on salicylic acid and jasmonic acid/ethylene signaling pathways. The Brassinosteroid Insensitive 1-associated Kinase 1 was dispensable for the ORP-activated defense. Besides, simultaneous knockout of MoORP1 and MoORP3 abolished fungal colony radial growth and conidiation, whereas double knockout of MoORP1 and MoORP2 compromised fungal virulence on barley and rice plants. These observations collectively highlight the multifaceted role of MoORPs in the modulation of plant innate immunity and promotion of fungal development and virulence in M. oryzae.

A pilot study on the effect of a novel feed additive containing exogenous enzymes, acidifiers, sodium butyrate and silicon dioxide nanoparticles on selected cellular immune indices and body weight gains of calves

Introduction The rearing of calves is a difficult period for farmers due to health problems to which the animals are prone this time. Since the use of antibiotics as growth promoters has been forbidden, various innovative feed additives have been tested in many countries around the world. Material and Methods In this study, experimental (E) calves were supplemented with a novel feed additive consisting of the pancreatic-like enzymes protease and lipase, a fat-coated mixture of organic fumaric, malic, citric and sorbic acids, sodium butyrate and silicon dioxide nanoparticles. Control (C) calves received feed without additive. During the supplementation, white blood cell (WBC) counts with leukocyte differentiation, percentages of B lymphocytes and T lymphocytes and their subpopulations, phagocytic activity and oxidative burst of circulating monocytes and granulocytes were examined. Body weight (b.w.) gains of the calves were also monitored. Results The WBC counts in the E and C calves were within the reference ranges throughout the study. In the analysis of the percentages of the lymphocyte subpopulations, phagocytic activity and oxidative burst, no statistically significant differences were reported between the E and C groups. However, higher average daily body weight gains were obtained for the E calves. Conclusion The study revealed that the examined feed additive did not modulate the immune response of the calves significantly. The tendency to higher daily average b.w. gains in the E calves than in the C calves suggests a beneficial effect of this feed additive.

Maternal and umbilical cord blood polymorphonuclear leukocytes showed moderate oxidative burst at phagocytosis of Gardnerella vaginalis

Objective Pregnant women with bacterial vaginosis due to Gardnerella vaginalis (GV) infection presents with a wide-ranging disease symptomatology. We speculate this may be due to interaction that varies between host immune response and the pathogen. We studied the oxidative burst in polymorphonuclear leukocytes (PMNL)s from maternal blood (MB) and cord blood (CB) upon phagocytosis of GV and compared against E. coli and Group B Streptococcus (GBS). Results The PHAGOBURST™ assay detects fluorescence from oxidized dihydrorhodamine during oxidative burst. The average percentage of PMNL showing oxidative burst was almost two-fold greater with GBS (99.5%) and E. coli (98.2%) than GV (56.9%) (p < 0.01) in MB, but a similar proportion of PMNL with burst activity was seen in CB (84.7%). The mean fluorescence intensity (MFI) of oxidative burst in MB PMNL with GV was lower compared to E. coli but comparable to GBS. The MFI of CB PMNL (1580 ± 245.8) was significantly higher than MB PMNL (1198 ± 262.1) with GV, p = 0.031. The live-cell imaging showed neutrophil oxidative burst upon phagocytosis of GV produces hypochlorous acid (HOCl). Overall, the HOCL-mediated microbicidal activity against GV is more variable and less robust than E. coli and GBS, especially in maternal than CB PMNL.

Dysregulated Neutrophil Iron Homeostasis in β-Thalassemia Impairs Phagocytosis and Reactive Oxygen Species Production

Introduction Beta thalassemia is an inherited disorder characterised by ineffective erythropoiesis leading to anemia and secondary iron overload. Neutrophils are the first line of innate immune defence against infection is dysfunctional in thalassemia patients. Iron is required for the oxidative response of neutrophils to allow the production of reactive oxygen species (ROS). However, the role of iron contributing to the neutrophil dysfunction is unclear. This is the first study to characterise the neutrophil iron metabolism in β-thalassemia and its association with oxidative burst capacity, phagocytosis, and systemic iron homeostasis. Method Sixteen thalassemia patients and fourteen healthy individuals were recruited in the department of Haematology, Christian Medical College, Vellore, India. Neutrophils were purified from human whole blood collected in EDTA tube, using neutrophil magnetic isolation kit (Miltenyi Biotec). Purified population (&gt;95%) was confirmed by the presence of the surface marker CD62L evaluated using flow cytometry. Haematological parameters were analysed according to standard methods. Serum ferritin, iron, soluble transferrin receptor were measured using immunoassay. Serum hepcidin was measured using ELISA. Neutrophil RNA was isolated using trizol method and was reverse transcribed into complementary DNA using QIAGEN kit. The relative quantification of iron related genes were measured using real-time PCR. In oxidative burst assay, neutrophils were incubated with dihydrorhodamine 123 (DHR) and stimulated with Phorbol 12-Myristate 13-Acetate (PMA). Respiratory burst of the cell was analysed by flow cytometry. Phagocytosis and acidification capacity of human neutrophils were quantified using the pHrodo Green Staphylococcus aureus BioParticles kit (Thermo Fisher). Acquisition was performed using the Beckman Coulter(Navios) flow cytometer and analysed using kaluza software. Statistical analysis was performed using SPSS software. Results We investigated a cohort of β thalassemia Major (n=5), intermedia (n=6) and sickle beta thalassemia (n=5) patients who were on regular iron chelation therapy. The demographic and biochemical parameters are tabulated in Table 1. Serum iron, ferritin levels and transferrin saturation were significantly increased in thalassemia cohort as compared to healthy donors (Fig1a). There was no significant association between ferritin and hepcidin levels. The percentage of neutrophils in thalassemia was significantly reduced incomparision to healthy donors (p=0.032). Oxidative burst capacity of neutrophils from thalassemia major and intermedia patients were significantly decreased (p=0.002) compared to healthy donors upon stimulation with PMA (Fig1b). Neutrophil phagocytosis capacity indicated by the relative amounts of phagocytized fluorescein S.aureus particles were significantly lower in thalassemia patients compared with controls, after 15min/30min incubation (Fig1c). The recognition capacity of neutrophils towards bioparticles significantly decreased at 45-minute incubation in patients (p=0.017) (Fig1d). Serum iron overload and transferrin saturation had negative association with neutrophil phagocytosis capacity (r=-0.714; p=0.045 & r=-0.857; p=0.014), respectively. Neutrophil iron related gene expression was analysed and found significantly lower expression of FPN 1A (FPN1 containing iron regulatory element (IRE)), DMT1B without IRE region and IRP2 respectively (p=0.029, p=0.029 & p=0.016) (Fig1e). FPN1B without IRE region was upregulated in thalassemia patients (p=0.016). Serum ferritin had positive correlation with FPN1B (r=0.786; p=0.036). Soluble transferrin receptor had negative association with DMT1A (containing IRE region) and IRP2 respectively (r=-0.900; p=0.037 and r=-0.750; p=0.052). Aberrant neutrophil function was found in all thalassemia patients. Although oxidative burst activity was decreased in thalassemia major and intermedia patients, sickle β-thalassemia patients had normal burst activity. Systemic iron status had inverse correlation with phagocytosis capacity of neutrophils. Dysregulation of iron transporters in neutrophils was indicated by decreased expression of DMT1 and augmented FPN1B expression, despite systemic iron overload. These findings have to be explored in a larger cohort to elucidate the clinical significance. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Anti-inflammatory and Antioxidant Properties of Finger Millet (Eleusine coracana (L.) Gaertn.) Varieties Cultivated in Sri Lanka

The prevalence of inflammatory-mediated and oxidative stress-associated diseases is increasing worldwide, creating an increasing demand for novel sources of anti-inflammatory agents and antioxidants. This study was focused on determining the in vitro arachidonate 5-lipoxygenase (A5-LOX), xanthine oxidase (XO), hyaluronidase and oxidative burst inhibitory activities, and antioxidant properties of Ravi, Rawana, and Oshadha finger millet varieties using ethanolic and methanolic extracts. Among all extracts, the methanolic extract of Oshadha exhibited the highest A5-LOX (IC50 value: 484.42 μg/ml) and XO (IC50 value: 764.34 μg/ml) inhibitory activities. All extracts showed less than 50% hyaluronidase inhibitory activity at 1 mg/ml concentration. Methanolic extracts showed moderate inhibitory potential on reactive oxygen species (ROS) generated from whole blood phagocytes, with IC50 values ranging between 26.9 and 27.7 μg/ml, when compared to ibuprofen (IC50 value: 11.18 μg/ml). All extracts showed potent inhibition of ROS produced from polymorphonuclear neutrophils isolated from human blood when compared to ibuprofen (IC50 value: 2.47 μg/ml) and IC50 values of methanolic and ethanolic extracts ranged from 0.29 to 0.47 μg/ml and 1.35 to 1.70 μg/ml, respectively. All extracts had significantly high amounts of phenolic compounds including flavonoids and the potential to scavenge 2,2 ′ -azino-bis (3-ethylbenzothiazoline-6-sulfonic) acid (ABTS) cation, 2,2-diphenyl-1-picryl-hydrazyl (DPPH), and oxygen radicals. Besides, they were able to reduce metal ions and chelate metal ions terminating radical generating reactions. This is the first report of A5-LOX, XO, hyaluronidase, and oxidative burst inhibitory properties of any extract of any finger millet variety cultivated in Sri Lanka. The findings revealed the potential of using these finger millet extracts as natural sources of anti-inflammatory drug candidates. Additionally, the findings indicated that Ravi, Rawana, and Oshadha varieties are good sources of antioxidants. Therefore, consumption of these finger millet varieties on a regular basis may play an important role in the prevention and dietary management of oxidative stress-associated diseases.