The calcineurin-NFATc pathway modulates the lipid mediators in BAL fluid extracellular vesicles, thereby regulating microvascular endothelial cell barrier function.

Extracellular vesicles mediate intercellular communication by transporting biologically active macromolecules. Our prior studies have demonstrated that the nuclear factor of activated T cell cytoplasmic member 3 (NFATc3) is activated in mouse pulmonary macrophages in response to lipopolysaccharide (LPS). Inhibition of NFATc3 activation by a novel cell-permeable calcineurin peptide inhibitor CNI103 mitigated the development of acute lung injury (ALI) in LPS-treated mice. Although pro-inflammatory lipid mediators are known contributors to lung inflammation and injury, it remains unclear whether the calcineurin-NFATc pathway regulates extracellular vesicle (EV) lipid content and if this content contributes to ALI pathogenesis. In this study, EVs from mouse bronchoalveolar lavage fluid (BALF) were analyzed for their lipid mediators by liquid chromatography in conjunction with mass spectrometry (LC-MS/MS). Our data demonstrate that EVs from LPS-treated mice contained significantly higher levels of arachidonic acid (AA) metabolites, which were found in low levels by prior treatment with CNI103. The catalytic activity of lung tissue cytoplasmic phospholipase A2 (cPLA2) increased during ALI, correlating with an increased amount of arachidonic acid (AA) in the EVs. Furthermore, ALI is associated with increased expression of cPLA2, cyclooxygenase 2 (COX2), and lipoxygenases (5-LOX, 12-LOX, and 15-LOX) in lung tissue, and pretreatment with CNI103 inhibited the catalytic activity of cPLA2 and the expression of cPLA2, COX, and LOX transcripts. Furthermore, co-culture of mouse pulmonary microvascular endothelial cell (PMVEC) monolayer and NFAT-luciferase reporter macrophages with BALF EVs from LPS-treated mice increased the pulmonary microvascular endothelial cell (PMVEC) monolayer barrier permeability and luciferase activity in macrophages. However, EVs from CNI103-treated mice had no negative impact on PMVEC monolayer barrier integrity. In summary, BALF EVs from LPS-treated mice carry biologically active NFATc-dependent, AA-derived lipids that play a role in regulating PMVEC monolayer barrier function.

Molecular Regulation of Transforming Growth Factor-ß1-induced Thioredoxin-interacting Protein Ubiquitination and Proteasomal Degradation in Lung Fibroblasts: Implication in Pulmonary Fibrosis.

Thioredoxin-interacting protein (TXNIP) plays a critical role in regulation of cellular redox reactions and inflammatory responses by interacting with thioredoxin (TRX) or the inflammasome. The role of TXNIP in lung fibrosis and molecular regulation of its stability have not been well studied. Therefore, here we investigated the molecular regulation of TXNIP stability and its role in TGF-ß1-mediated signaling in lung fibroblasts. TXNIP protein levels were significantly decreased in lung tissues from bleomycin-challenged mice. Overexpression of TXNIP attenuated transforming growth factor-ß1 (TGF-ß1)-induced phosphorylation of Smad2/3 and fibronectin expression in lung fibroblasts, suggesting that decrease in TXNIP may contribute to the pathogenesis of lung fibrosis. Further, we observed that TGF-ß1 lowered TXNIP protein levels, while TXNIP mRNA levels were unaltered by TGF-ß1 exposure. TGF-ß1 induced TXNIP degradation via the ubiquitin-proteasome system. A serine residue mutant (TNXIP-S308A) was resistant to TGF-ß1-induced degradation. Furthermore, downregulationof ubiquitin-specific protease-13 (USP13) promoted the TGF-ß1-induced TXNIP ubiquitination and degradation. Mechanistic studies revealed that USP13 targeted and deubiquitinated TXNIP. The results of this study revealed that the decrease of TXNIP in lungs apparently contributes to the pathogenesis of pulmonary fibrosis and that USP13 can target TXNP for deubiquitination and regulate its stability.

Troponin I Tyrosine Phosphorylation Beneficially Accelerates Diastolic Function.

BACKGROUND: A healthy heart is able to modify its function and increase relaxation through post-translational modifications of myofilament proteins. While there are known examples of serine/threonine kinases directly phosphorylating myofilament proteins to modify heart function, the roles of tyrosine (Y) phosphorylation to directly modify heart function have not been demonstrated. The myofilament protein TnI (troponin I) is the inhibitory subunit of the troponin complex and is a key regulator of cardiac contraction and relaxation. We previously demonstrated that TnI-Y26 phosphorylation decreases calcium-sensitive force development and accelerates calcium dissociation, suggesting a novel role for tyrosine kinase-mediated TnI-Y26 phosphorylation to regulate cardiac relaxation. Therefore, we hypothesize that increasing TnI-Y26 phosphorylation will increase cardiac relaxation in vivo and be beneficial during pathological diastolic dysfunction. METHODS: The signaling pathway involved in TnI-Y26 phosphorylation was predicted in silico and validated by tyrosine kinase activation and inhibition in primary adult murine cardiomyocytes. To investigate how TnI-Y26 phosphorylation affects cardiac muscle, structure, and function in vivo, we developed a novel TnI-Y26 phosphorylation-mimetic mouse that was subjected to echocardiography, pressure-volume loop hemodynamics, and myofibril mechanical studies. TnI-Y26 phosphorylation-mimetic mice were further subjected to the nephrectomy/DOCA (deoxycorticosterone acetate) model of diastolic dysfunction to investigate the effects of increased TnI-Y26 phosphorylation in disease. RESULTS: Src tyrosine kinase is sufficient to phosphorylate TnI-Y26 in cardiomyocytes. TnI-Y26 phosphorylation accelerates in vivo relaxation without detrimental structural or systolic impairment. In a mouse model of diastolic dysfunction, TnI-Y26 phosphorylation is beneficial and protects against the development of disease. CONCLUSIONS: We have demonstrated that tyrosine kinase phosphorylation of TnI is a novel mechanism to directly and beneficially accelerate myocardial relaxation in vivo.

Non-Lethal Doses of RSL3 Impair Microvascular Endothelial Barrier through Degradation of Sphingosie-1-Phosphate Receptor 1 and Cytoskeletal Arrangement in A Ferroptosis-Independent Manner.

The excess microvascular endothelial permeability is a hallmark of acute inflammatory diseases. Maintenance of microvascular integrity is critical to preventing leakage of vascular components into the surrounding tissues. Sphingosine-1-phosphate (S1P) is an active lysophospholipid that enhances the endothelial cell (EC) barrier via activation of its receptor S1PR1. Here, we delineate the effect of non-lethal doses of RSL3, an inhibitor of glutathione peroxidase 4 (GPX4), on EC barrier function. Low doses of RSL3 (50-100 nM) attenuated S1P-induced human lung microvascular barrier enhancement and the phosphorylation of AKT. To investigate the molecular mechanisms by which RSL3 attenuates S1P's effect, we examined the S1PR1 levels. RSL3 treatment reduced S1PR1 levels in 1 h, whereas the effect was attenuated by the proteasome and lysosome inhibitors as well as a lipid raft inhibitor. Immunofluorescence staining showed that RSL3 induced S1PR1 internalization from the plasma membrane into the cytoplasm. Furthermore, we found that RSL3 (100 and 200 nM) increased EC barrier permeability and cytoskeletal rearrangement without altering cell viability. Taken together, our data delineates that non-lethal doses of RSL3 impair EC barrier function via two mechanisms. RSL3 attenuates S1P1-induced EC barrier enhancement and disrupts EC barrier integrity through the generation of 4-hydroxynonena (4HNE). All these effects are independent of ferroptosis.

Spectrin-Based Regulation of Cardiac Fibroblast Cell-Cell Communication.

Cardiac fibroblasts (CFs) maintain the fibrous extracellular matrix (ECM) that supports proper cardiac function. Cardiac injury induces a transition in the activity of CFs to promote cardiac fibrosis. CFs play a critical role in sensing local injury signals and coordinating the organ level response through paracrine communication to distal cells. However, the mechanisms by which CFs engage cell-cell communication networks in response to stress remain unknown. We tested a role for the action-associated cytoskeletal protein ßIV-spectrin in regulating CF paracrine signaling. Conditioned culture media (CCM) was collected from WT and ßIV-spectrin deficient (qv4J) CFs. WT CFs treated with qv4J CCM showed increased proliferation and collagen gel compaction compared to control. Consistent with the functional measurements, qv4J CCM contained higher levels of pro-inflammatory and pro-fibrotic cytokines and increased concentration of small extracellular vesicles (30-150 nm diameter, exosomes). Treatment of WT CFs with exosomes isolated from qv4J CCM induced a similar phenotypic change as that observed with complete CCM. Treatment of qv4J CFs with an inhibitor of the ßIV-spectrin-associated transcription factor, STAT3, decreased the levels of both cytokines and exosomes in conditioned media. This study expands the role of the ßIV-spectrin/STAT3 complex in stress-induced regulation of CF paracrine signaling.

Classic Phytochemical Antioxidant and Lipoxygenase Inhibitor, Nordihydroguaiaretic Acid, Activates Phospholipase D through Oxidant Signaling and Tyrosine Phosphorylation Leading to Cytotoxicity in Lung Vascular Endothelial Cells.

Nordihydroguaiaretic acid (NDGA), a dicatechol and phytochemical polyphenolic antioxidant and an established inhibitor of human arachidonic acid (AA) 5-lipoxygenase (LOX) and 15-LOX, is widely used to ascertain the role of LOXs in vascular endothelial cell (EC) function. As the modulatory effect of NDGA on phospholipase D (PLD), an important lipid signaling enzyme in ECs, thus far has not been reported, here we have investigated the modulation of PLD activity and its regulation by NDGA in the bovine pulmonary artery ECs (BPAECs). NDGA induced the activation of PLD (phosphatidic acid formation) in cells in a dose- and time-dependent fashion that was significantly attenuated by iron chelator and antioxidants. NDGA induced the formation of reactive oxygen species (ROS) in cells in a dose- and time-dependent manner as evidenced from fluorescence microscopy and fluorimetry of ROS and electron paramagnetic resonance spectroscopy of oxygen radicals. Also, NDGA caused a dose-dependent loss of intracellular glutathione (GSH) in BPAECs. Protein tyrosine kinase (PTyK)-specific inhibitors significantly attenuated NDGA-induced PLD activation in BPAECs. NDGA also induced a dose- and time-dependent phosphorylation of tyrosine in proteins in cells. NDGA caused in situ translocation and relocalization of both PLD1 and PLD2 isoforms, in a time-dependent fashion. Cyclooxygenase (COX) inhibitors were ineffective in attenuating NDGA-induced PLD activation in BPAECs, thus ruling out the activation of COXs by NDGA. NDGA inhibited the AA-LOX activity and leukotriene C4 (LTC4) formation in cells. On the other hand, the 5-LOX-specific inhibitors, 5, 8, 11, 14-eicosatetraynoic acid and kaempferol, were ineffective in activating PLD in BPAECs. Antioxidants and PTyK-specific inhibitors effectively attenuated NDGA cytotoxicity in BPAECs. The PLD-specific inhibitor, 5-fluoro-2-indolyl deschlorohalopemide (FIPI), significantly attenuated and protected against the NDGA-induced PLD activation and cytotoxicity in BPAECs. For the first time, these results demonstrated that NDGA, the classic phytochemical polyphenolic antioxidant and LOX inhibitor, activated PLD causing cytotoxicity in ECs through upstream oxidant signaling and protein tyrosine phosphorylation.

Molecular Regulation of Heme Oxygenase-1 Expression by E2F Transcription Factor 2 in Lung Fibroblast Cells: Relevance to Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal chronic lung disease. Heme oxygenase-1 (HMOX1/HO-1) is an enzyme that catalyzes the degradation of heme. The role of HO-1 in the pathogenesis of IPF has been studied; however, the molecular regulation of HO-1 and its role in IPF are still unclear. In this study, we found that HO-1 protein levels significantly increased in lung myofibroblasts in IPF patients and in lungs in a murine model of bleomycin-induced lung fibrosis. In addition, we observed that administration of a E2F transcription factor inhibitor elevated HO-1 mRNA and protein levels in lung fibroblasts. Downregulation of E2F2 by siRNA transfection increased HO-1 mRNA and protein levels, while overexpression of E2F2 reduced HO-1 levels. However, overexpression of E2F2 did not alter hemin-induced HO-1 protein levels. Furthermore, modulation of HO-1 levels regulated TGF-ß1-induced myofibroblast differentiation without altering the phosphorylation of Smad2/3 in lung fibroblast cells. Moreover, the phosphorylation of protein kinase B (Akt) was significantly upregulated in HO-1-depleted lung fibroblast cells. In summary, this study demonstrated that E2F2 regulates the baseline expression of HO-1, but has no effect on modulating HO-1 expression by hemin. Finally, elevated HO-1 expression contributes to the TGF-ß1-induced lung myofibroblast differentiation through the activation of the serine/threonine kinase AKT pathway. Overall, our findings suggest that targeting E2F2/HO-1 might be a new therapeutic strategy to treat fibrotic diseases such as IPF.

Lipidomic Profiling of Bronchoalveolar Lavage Fluid Extracellular Vesicles Indicates Their Involvement in Lipopolysaccharide-Induced Acute Lung Injury.

Emerging data support the pivotal role of extracellular vesicles (EVs) in normal cellular physiology and disease conditions. However, despite their abundance, there is much less information about the lipid mediators carried in EVs, especially in the context of acute lung injury (ALI). Our data demonstrate that C57BL/6 mice subjected to intranasal Escherichia coli lipopolysaccharide (LPS)-induced ALI release, a higher number of EVs into the alveolar space, compared to saline-treated controls. EVs released during ALI originated from alveolar epithelial cells, macrophages, and neutrophils and carry a diverse array of lipid mediators derived from ω-3 and ω-6 polyunsaturated fatty acids (PUFA). The eicosanoids in EVs correlated with cellular levels of arachidonic acid, expression of cytosolic phospholipase A2, cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome epoxygenase p450 proteins in pulmonary macrophages. Furthermore, EVs from LPS-toll-like receptor 4 knockout (TLR4-/-) mice contained significantly lower amounts of COX and LOX catalyzed eicosanoids and ω-3 PUFA metabolites. More importantly, EVs from LPS-treated wild-type mice increased TNF-α release by macrophages and reduced alveolar epithelial monolayer barrier integrity compared to EVs from LPS-treated TLR4-/- mice. In summary, our study demonstrates for the first time that the EV carried PUFA metabolite profile in part depends on the inflammatory status of the lung macrophages and modulates pulmonary macrophage and alveolar epithelial cell function during LPS-induced ALI.

Pentalinonsterol, a Phytosterol from Pentalinon andrieuxii, is Immunomodulatory through Phospholipase A2 in Macrophages toward its Antileishmanial Action.

Our earlier in vitro and in vivo studies have revealed that the phytosterol, pentalinonsterol (cholest-4,20,24-trien-3-one) (PEN), isolated from the roots of Pentalinon andrieuxii, possesss immunomodulatory properties in macrophages and dendritic cells. Leishmaniasis, caused by the infection of Leishmania spp. (a protozoan parasite), is emerging as the second-leading cause of mortality among the tropical diseases and there is an unmet need for a pharmacological intervention of leishmaniasis. Given the beneficial immunomodulatory actions and lipophilic properties of PEN, the objective of this study was to elucidate the mechanism(s) of action of the immunomodulatory action(s) of PEN in macrophages through the modulation of phospholipase A2 (PLA2) activity that might be crucial in the antileishmanial action of PEN. Therefore, in this study, we investigated whether PEN would modulate the activity of PLA2 in RAW 264.7 macrophages and mouse bone marrow-derived primary macrophages (BMDMs) in vitro and further determined how the upstream PLA2 activation would regulate the downstream cytokine release in the macrophages. Our current results demonstrated that (i) PEN induced PLA2 activation (arachidonic acid release) in a dose- and time-dependent manner that was regulated upstream by the mitogen-activated protein kinases (MAPKs); (ii) the PEN-induced activation of PLA2 was attenuated by the cPLA2-specific pharmacological inhibitors; and (iii) the cPLA2-specific pharmacological inhibitors attenuated the release of inflammatory cytokines from the macrophages. For the first time, our current study demonstrated that PEN exhibited its immunomodulatory actions through the activation of cPLA2 in the macrophages, which potentially could be used in the development of a pharmacological intervention against leishmaniasis.

Lipid Nutrition in Asthma.

Asthma is a heterogeneous pulmonary disease that has constantly increased in prevalence over the past several decades. Primary symptoms include airway constriction, airway hyperresponsiveness, and airway remodeling with additional symptoms such as shortness of breath, wheezing, and difficulty breathing. Allergic asthma involves chronic inflammation of the lungs, and the rise in its yearly diagnosis is potentially associated with the increased global consumption of foods similar to the western diet. Thus, there is growing interest into the link between diet and asthma symptoms, with mounting evidence for an important modulatory role for dietary lipids. Lipids can act as biological mediators in both a proinflammatory and proresolution capacity. Fatty acids play key roles in signaling and in the production of mediators in the allergic and inflammatory pathways. The western diet leads to a disproportionate ω-6:ω-3 ratio, with drastically increased ω-6 levels. To counteract this, consumption of fish and fish oil and the use of dietary oils with anti-inflammatory properties such as olive and sesame oil can increase ω-3 and decrease ω-6 levels. Increasing vitamin intake, lowering LDL cholesterol levels, and limiting consumption of oxidized lipids can help reduce the risk of asthma and the exacerbation of asthmatic symptoms. These dietary changes can be achieved by increasing intake of fruits, vegetables, nuts, oily fish, seeds, animal-related foods (eggs, liver), cheeses, grains, oats, and seeds, and decreasing consumption of fried foods (especially fried in reused oils), fast foods, and heavily processed foods.

NOX4 Mediates Pseudomonas aeruginosa-Induced Nuclear Reactive Oxygen Species Generation and Chromatin Remodeling in Lung Epithelium.

Pseudomonas aeruginosa (PA) infection increases reactive oxygen species (ROS), and earlier, we have shown a role for NADPH oxidase-derived ROS in PA-mediated lung inflammation and injury. Here, we show a role for the lung epithelial cell (LEpC) NOX4 in PA-mediated chromatin remodeling and lung inflammation. Intratracheal administration of PA to Nox4flox/flox mice for 24 h caused lung inflammatory injury; however, epithelial cell-deleted Nox4 mice exhibited reduced lung inflammatory injury, oxidative stress, secretion of pro-inflammatory cytokines, and decreased histone acetylation. In LEpCs, NOX4 was localized both in the cytoplasmic and nuclear fractions, and PA stimulation increased the nuclear NOX4 expression and ROS production. Downregulation or inhibition of NOX4 and PKC δ attenuated the PA-induced nuclear ROS. PA-induced histone acetylation was attenuated by Nox4-specific siRNA, unlike Nox2. PA stimulation increased HDAC1/2 oxidation and reduced HDAC1/2 activity. The PA-induced oxidation of HDAC2 was attenuated by N-acetyl-L-cysteine and siRNA specific for Pkc δ, Sphk2, and Nox4. PA stimulated RAC1 activation in the nucleus and enhanced the association between HDAC2 and RAC1, p-PKC δ, and NOX4 in LEpCs. Our results revealed a critical role for the alveolar epithelial NOX4 in mediating PA-induced lung inflammatory injury via nuclear ROS generation, HDAC1/2 oxidation, and chromatin remodeling.

Defective immunometabolism pathways in cystic fibrosis macrophages.

BACKGROUND: Mitochondria play a key role in immune defense pathways, particularly for macrophages. We and others have previously demonstrated that cystic fibrosis (CF) macrophages exhibit weak autophagy activity and exacerbated inflammatory responses. Previous studies have revealed that mitochondria are defective in CF epithelial cells, but to date, the connection between defective mitochondrial function and CF macrophage immune dysregulation has not been fully elucidated. Here, we present a characterization of mitochondrial dysfunction in CF macrophages. METHODS: Mitochondrial function in wild-type (WT) and CF F508del/F508del murine macrophages was measured using the Seahorse Extracellular Flux analyzer. Mitochondrial morphology was investigated using transmission electron and confocal microscopy. Mitochondrial membrane potential (MMP) as well as mitochondrial reactive oxygen species (mROS) were measured using TMRM and MitoSOX Red fluorescent dyes, respectively. All assays were performed at baseline and following infection by Burkholderia cenocepacia, a multi-drug resistant bacterium that causes detrimental infections in CF patients. RESULTS: We have identified impaired oxygen consumption in CF macrophages without and with B. cenocepacia infection. We also observed increased mitochondrial fragmentation in CF macrophages following infection. Lastly, we observed increased MMP and impaired mROS production in CF macrophages following infection with B. cenocepacia. CONCLUSIONS: The mitochondrial defects identified are key components of the macrophage response to infection. Their presence suggests that mitochondrial dysfunction contributes to impaired bacterial killing in CF macrophages. Our current study will enhance our understanding of the pathobiology of CF and lead to the identification of novel mitochondrial therapeutic targets for CF.

The role of vascular endothelium and exosomes in human protozoan parasitic diseases.

The vascular endothelium is a vital component in maintaining the structure and function of blood vessels. The endothelial cells (ECs) mediate vital regulatory functions such as the proliferation of cells, permeability of various tissue membranes, and exchange of gases, thrombolysis, blood flow, and homeostasis. The vascular endothelium also regulates inflammation and immune cell trafficking, and ECs serve as a replicative niche for many bacterial, viral, and protozoan infectious diseases. Endothelial dysfunction can lead to vasodilation and pro-inflammation, which are the hallmarks of many severe diseases. Exosomes are nanoscale membrane-bound vesicles that emerge from cells and serve as important extracellular components, which facilitate communication between cells and maintain homeostasis during normal and pathophysiological states. Exosomes are also involved in gene transfer, inflammation and antigen presentation, and mediation of the immune response during pathogenic states. Protozoa are a diverse group of unicellular organisms that cause many infectious diseases in humans. In this regard, it is becoming increasingly evident that many protozoan parasites (such as Plasmodium, Trypanosoma, Leishmania, and Toxoplasma) utilize exosomes for the transfer of their virulence factors and effector molecules into the host cells, which manipulate the host gene expression, immune responses, and other biological activities to establish and modulate infection. In this review, we discuss the role of the vascular endothelium and exosomes in and their contribution to pathogenesis in malaria, African sleeping sickness, Chagas disease, and leishmaniasis and toxoplasmosis with an emphasis on their actions on the innate and adaptive immune mechanisms of resistance.

The expression of Mirc1/Mir17-92 cluster in sputum samples correlates with pulmonary exacerbations in cystic fibrosis patients.

INTRODUCTION: Cystic fibrosis (CF) is a multi-organ disorder characterized by chronic sino-pulmonary infections and inflammation. Many patients with CF suffer from repeated pulmonary exacerbations that are predictors of worsened long-term morbidity and mortality. There are no reliable markers that associate with the onset or progression of an exacerbation or pulmonary deterioration. Previously, we found that the Mirc1/Mir17-92a cluster which is comprised of 6 microRNAs (Mirs) is highly expressed in CF mice and negatively regulates autophagy which in turn improves CF transmembrane conductance regulator (CFTR) function. Therefore, here we sought to examine the expression of individual Mirs within the Mirc1/Mir17-92 cluster in human cells and biological fluids and determine their role as biomarkers of pulmonary exacerbations and response to treatment. METHODS: Mirc1/Mir17-92 cluster expression was measured in human CF and non-CF plasma, blood-derived neutrophils, and sputum samples. Values were correlated with pulmonary function, exacerbations and use of CFTR modulators. RESULTS: Mirc1/Mir17-92 cluster expression was not significantly elevated in CF neutrophils nor plasma when compared to the non-CF cohort. Cluster expression in CF sputum was significantly higher than its expression in plasma. Elevated CF sputum Mirc1/Mir17-92 cluster expression positively correlated with pulmonary exacerbations and negatively correlated with lung function. Patients with CF undergoing treatment with the CFTR modulator Ivacaftor/Lumacaftor did not demonstrate significant change in the expression Mirc1/Mir17-92 cluster after six months of treatment. CONCLUSIONS: Mirc1/Mir17-92 cluster expression is a promising biomarker of respiratory status in patients with CF including pulmonary exacerbation.

Pentalinonsterol, a Constituent of Pentalinon andrieuxii, Possesses Potent Immunomodulatory Activity and Primes T Cell Immune Responses.

The use of natural products as adjuvants has emerged as a promising approach for the development of effective vaccine formulations. Pentalinonsterol (PEN) is a recently isolated compound from the roots of Pentalinon andrieuxii and has been shown to possess antileishmanial activity against Leishmania spp. The objective of this study was to examine the immunomodulatory properties of PEN and evaluate its potential as an adjuvant. Macrophages and bone-marrow-derived dendritic cells (BMDCs) were stimulated with PEN and tested for gene expression, cytokine production, and their ability to activate T cells in vitro. PEN was also evaluated for its ability to generate antigen-specific Th1 and Th2 responses in vivo, following ovalbumin (OVA) immunization using PEN as an adjuvant. The results obtained demonstrate that PEN enhances the expression of NF-κB and AP1 transcription factors, promotes gene expression of Tnfα, Il6, Nos2, and Arg1, and upregulates MHCII, CD80, and CD86 in macrophages. PEN also enhanced IL-12 production in BMDCs and promoted BMDC-mediated production of IFN-γ by T cells. Further, mice immunized with OVA and PEN showed enhanced antigen-specific Th1 and Th2 cytokines in their splenocytes and lymph node cells, as well as increased levels of IgG1 and IgG2 in their sera. Taken together, this study demonstrates that PEN is a potent immunomodulatory compound and potentially can be used as an adjuvant for vaccine development against infectious diseases.

EPA + DHA supplementation reduces PMN activation in microenvironment of chronic venous leg ulcers: A randomized, double-blind, controlled study.

Sustained high levels of activated polymorphonuclear leukocytes (PMNs) and PMN-derived proteases in the microenvironment of chronic venous leg ulcers (CVLUs) are linked to chronic inflammation and delayed healing. Uncontrolled PMN activity eventually destroys newly developed tissue and degrades critical growth factors. The bioactive components of fish oil (n-3 eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA]) have strong inflammation-resolving actions and have been shown to assuage PMN activity, but have not been tested in CVLU patients. This randomized controlled study compared the effectiveness of oral EPA + DHA therapy to a placebo for reducing PMN activation in CVLU microenvironments. At Days 0, 28, and 56, markers of PMNs (CD15) and activated PMNs (CD66b), and levels of PMN-derived proteases human neutrophil elastase and matrix metalloproteinase-8 were measured in CVLU fluid from patients receiving standard compression therapy and (1) EPA + DHA therapy (n = 16) or (2) placebo (n = 19). By Day 56, the EPA + DHA Group had a significantly lower percentage of CD66b+ cells in CVLU fluid compared to Day 0 (p = 0.02) and to Day 28 (p = 0.05). Importantly, there were downward trends in levels of both matrix metalloproteinase-8 and human neutrophil elastase over time in the EPA + DHA Group, which also demonstrated greater reductions in wound area by Day 28 (57% reduction) and Day 56 (76% reduction) than the Control Group (35% and 59%, respectively). Moreover, reductions in wound area had significant negative relationships with CD15+ cells in wound fluid at Days 28 (p = 0.008) and 56 (p < 0.001), and CD66b+ cells at Days 28 (p = 0.04) and 56 (p = 0.009). The collective findings provide supplemental evidence that high levels of activated PMNs in CVLU microenvironments inhibit healing, and suggest that EPA + DHA oral therapy may modulate PMN activity and facilitate healing of CVLUs when added to standard care regimens.

Green Synthesized Zinc Oxide (ZnO) Nanoparticles Induce Oxidative Stress and DNA Damage in Lathyrus sativus L. Root Bioassay System.

Zinc oxide nanoparticles (ZnONP-GS) were synthesised from the precursor zinc acetate (Zn(CH3COO)2) through the green route using the milky latex from milk weed (Calotropis gigantea L. R. Br) by alkaline precipitation. Formation of the ZnONP-GS was monitored by UV-visible spectroscopy followed by characterization and confirmation by energy-dispersive X-ray spectroscopy (EDX), transmission electron microscopy (TEM), and X-ray diffraction (XRD). Both the ZnONP-GS and the commercially available ZnONP-S (Sigma-Aldrich) and cationic Zn2+ from Zn(CH3COO)2 were tested in a dose range of 0-100 mg·L-1 for their potency (i) to induce oxidative stress as measured by the generation reactive oxygen species (ROS: O2•-, H2O2 and •OH), cell death, and lipid peroxidation; (ii) to modulate the activities of antioxidant enzymes: catalase (CAT), superoxide dismutase (SOD), guaiacol peroxidase (GPX), and ascorbate peroxidase (APX); and (iii) to cause DNA damage as determined by Comet assay in Lathyrus sativus L. root bioassay system. Antioxidants such as Tiron and dimethylthiourea significantly attenuated the ZnONP-induced oxidative and DNA damage, suggesting the involvement of ROS therein. Our study demonstrated that both ZnONP-GS and ZnONP-S induced oxidative stress and DNA damage to a similar extent but were significantly less potent than Zn2+ alone.