Esterified derivatives of DHA and EPA increase bortezomib cytotoxicity in human multiple myeloma cells

BACKGROUND & AIMS: Although the proteasome inhibitor bortezomib has greatly improved the clinical outcome of patients with multiple myeloma (MM), acquired drug resistance remains the greatest obstacle on the road of treating MM. We previously showed that omega-3 polyunsaturated fatty acids (PUFAs), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) with the chemotherapeutic agent bortezomib can overcome its chemoresistance in MM cells. However, most DHA/EPA are esterified shortly after oral administration, which may affect their bioactivity. This study was to evaluate the cytotoxicity of ethyl ester-DHA/EPA in human MM cells. The mechanisms relevant for the cytotoxicity of these esterified-fatty acids were further investigated. METHODS: Human MM cell lines L363, OPM2, U266 were treated with ethyl ester-DHA/EPA with or without bortezomib. The percentage of dead cells and intracellular reactive oxygen species (ROS) levels were analyzed by flow cytometry. RESULTS: Ethyl ester-DHA and -EPA were much more potent than DHA/EPA to induce cytotoxicity in MM cells, even in DHA/EPA-resistant MM cells. Pretreating MM cells with esterified-DHA/EPA before bortezomib potently increased its cytotoxicity. Additionally, intracellular ROS levels were upregulated in MM cells after treatment with ethyl ester-DHA/EPA, which reflected the enhanced oxidative stress in treated cells. CONCLUSIONS: This study provides evidence that ethyl ester-DHA/EPA in combination with bortezomib may improve the overall efficacy in MM cells, similar to DHA/EPA, relieving the concern that esterification of DHA/EPA may affect its bioactivity and further supporting the potential clinical use of fatty acids DHA/EPA for combating drug resistance during MM therapy

До питання про походження імені язичеської богині Мокоша

Приводяться екстралінгвістичні та лінгвістичні докази, що Мокоша пов’язана з водою. Виявлено лексична спорідненість цього ім’я не тільки з індоєвропейськими, але й семітськими мовами. На основі аналізу структурних компонентів слів, які мають спільну сему “вода” та подібність у звучанні, у досліджуваному слові виділено етимологічний корінь о-(а), префікс м- і суфікси -к - і -ош(а). Ключові слова: глубинна етимологія, Мокоша, загальна сема, подібні структурні компоненти, індоєвропейські, семітські мови.Приводятся экстралингвистические и лингвистические доказательства, что Мокоша связана с водой. Обнаружено лексическое родство этого имени не только с индоевропейскими, но и семитскими языками. На основе анализа структурных компонентов слов, которые имеют общую сему “вода” и сходство в звучании, в исследуемом слове выделено этимологический корень о-(а-), префикс м- и суффиксы -к - и -ош(а). Ключевые слова: глубинная этимология, Мокоша, общая сема, похожие структурные компоненты, индоевропейские, семитские языки.Extra-linguistic and linguistic arguments are adduced that Mokosha is connected with water. The lexical affinity of this name was established both with Indo-European and Semitic languages. On the basis of the analysis of structural components of the words which have the common seme “water” and show resemblance in sounding, the etymological root o-(a-), prefix m- and suffixes -k- and -osh(a) were singled out in the investigated word. Keywords: deep etymology, Mokosha, common seme, similar structural components, Indо- European, Semitic languages

Immunoglobulin Free Light Chains Are Increased in Hypersensitivity Pneumonitis and Idiopathic Pulmonary Fibrosis

BACKGROUND: Idiopathic pulmonary fibrosis (IPF), a devastating lung disorder of unknown aetiology, and chronic hypersensitivity pneumonitis (HP), a disease provoked by an immunopathologic reaction to inhaled antigens, are two common interstitial lung diseases with uncertain pathogenic mechanisms. Previously, we have shown in other upper and lower airway diseases that immunoglobulin free light chains (FLCs) are increased and may be involved in initiating a local inflammation. In this study we explored if such a mechanism may also apply to HP and IPF. METHODS: In this study we examined the presence of FLC in serum and BAL fluid from 21 IPF and 22 HP patients and controls. IgG, IgE and tryptase concentrations were measured in BAL fluid only. The presence of FLCs, plasma cells, B cells and mast cells in lung tissue of 3 HP and 3 IPF patients and 1 control was analyzed using immunohistochemistry. RESULTS: FLC concentrations in serum and BAL fluid were increased in IPF and HP patients as compared to control subjects. IgG concentrations were only increased in HP patients, whereas IgE concentrations were comparable to controls in both patient groups. FLC-positive cells, B cells, plasma cells, and large numbers of activated mast cells were all detected in the lungs of HP and IPF patients, not in control lung. CONCLUSION: These results show that FLC concentrations are increased in serum and BAL fluid of IPF and HP patients and that FLCs are present within affected lung tissue. This suggests that FLCs may be involved in mediating pathology in both diseases

Recovery of extracellular vesicles from human breast milk is influenced by sample collection and vesicle isolation procedures

Extracellular vesicles (EV) in breast milk carry immune relevant proteins and could play an important role in the instruction of the neonatal immune system. To further analyze these EV and to elucidate their function it is important that native populations of EV can be recovered from (stored) breast milk samples in a reproducible fashion. However, the impact of isolation and storage procedures on recovery of breast milk EV has remained underexposed. Here, we aimed to define parameters important for EV recovery from fresh and stored breast milk. To compare various protocols across different donors, breast milk was spiked with a well-defined murine EV population. We found that centrifugation of EV down into density gradients largely improved density-based separation and isolation of EV, compared to floatation up into gradients after high-force pelleting of EV. Using cryo-electron microscopy, we identified different subpopulations of human breast milk EV and a not previously described population of lipid tubules. Additionally, the impact of cold storage on breast milk EV was investigated. We determined that storing unprocessed breast milk at −80°C or 4°C caused death of cells present in breast milk, leading to contamination of the breast milk EV population with storage-induced EV. Here, an alternative method is proposed to store breast milk samples for EV analysis at later time points. The proposed adaptations to the breast milk storage and EV isolation procedures can be applied for EV-based biomarker profiling of breast milk and functional analysis of the role of breast milk EV in the development of the neonatal immune system

Predictable Irreversible Switching Between Acute and Chronic Inflammation

Many a disease associates with inflammation. Upon binding of antigen-antibody complexes to immunoglobulin-like receptors, mast cells release tumor necrosis factor-α and proteases, causing fibroblasts to release endogenous antigens that may be cross reactive with exogenous antigens. We made a predictive dynamic map of the corresponding extracellular network. In silico, this map cleared bacterial infections, via acute inflammation, but could also cause chronic inflammation. In the calculations, limited inflammation flipped to strong inflammation when cross-reacting antigen exceeded an “On threshold.” Subsequent reduction of the antigen load to below this “On threshold” did not remove the strong inflammation phenotype unless the antigen load dropped below a much lower and subtler “Off” threshold. In between both thresholds, the network appeared caught either in a “low” or a “high” inflammatory state. This was not simply a matter of bi-stability, however, the transition to the “high” state was temporarily revertible but ultimately irreversible: removing antigen after high exposure reduced the inflammatory phenotype back to “low” levels but if then the antigen dosage was increased only a little, the high inflammation state was already re-attained. This property may explain why the high inflammation state is indeed “chronic,” whereas only the naive low-inflammation state is “acute.” The model demonstrates that therapies of chronic inflammation such as with anti-IgLC should require fibroblast implantation (or corresponding stem cell activation) for permanence in order to redress the irreversible transition

Binding to Iron Quercetin Complexes Increases the Antioxidant Capacity of the Major Birch Pollen Allergen Bet v 1 and Reduces Its Allergenicity

Bet v 1 is the major allergen in birch pollen to which up to 95% of patients sensitized to birch respond. As a member of the pathogenesis-related PR 10 family, its natural function is implicated in plant defense, with a member of the PR10 family being reported to be upregulated under iron deficiency. As such, we assessed the function of Bet v 1 to sequester iron and its immunomodulatory properties on human immune cells. Binding of Bet v 1 to iron quercetin complexes FeQ2 was determined in docking calculations and by spectroscopy. Serum IgE-binding to Bet v 1 with (holoBet v1) and without ligands (apoBet v 1) were assessed by ELISA, blocking experiments and Western Blot. Crosslinking-capacity of apo/holoBet v 1 were assessed on human mast cells and Arylhydrocarbon receptor (AhR) activation with the human reporter cellline AZ-AHR. Human PBMCs were stimulated and assessed for labile iron and phenotypic changes by flow cytometry. Bet v 1 bound to FeQ2 strongly with calculated Kd values of 1 nm surpassing affinities to quercetin alone nearly by a factor of 1000. Binding to FeQ2 masked IgE epitopes and decreased IgE binding up to 80% and impaired degranulation of sensitized human mast cells. Bet v 1 facilitated the shuttling of quercetin, which activated the anti-inflammatory AhR pathway and increased the labile iron pool of human monocytic cells. The increase of labile iron was associated with an anti-inflammatory phenotype in CD14+monocytes and downregulation of HLADR. To summarize, we reveal for the first time that FeQ2 binding reduces the allergenicity of Bet v 1 due to ligand masking, but also actively contributes anti-inflammatory stimuli to human monocytes, thereby fostering tolerance. Nourishing immune cells with complex iron may thus represent a promising antigen-independent immunotherapeutic approach to improve efficacy in allergen immunotherapy

Mast cells disrupt the function of the esophageal epithelial barrier

Mast cells (MCs) accumulate in the epithelium of patients with eosinophilic esophagitis (EoE), an inflammatory disorder characterized by extensive esophageal eosinophilic infiltration. Esophageal barrier dysfunction plays an important role in the pathophysiology of EoE. We hypothesized that MCs contribute to the observed impaired esophageal epithelial barrier. Herein, we demonstrate that coculture of differentiated esophageal epithelial cells with immunoglobulin E-activated MCs significanly decreased epithelial resistance by 30% and increased permeability by 22% compared with non-activated MCs. These changes were associated with decreased messenger RNA expression of barrier proteins filaggrin, desmoglein-1 and involucrin, and antiprotease serine peptidase inhibitor kazal type 7. Using targeted proteomics, we detected various cytokines in coculture supernatants, most notably granulocyte-macrophage colony-stimulating factor and oncostatin M (OSM). OSM expression was increased by 12-fold in active EoE and associated with MC marker genes. Furthermore, OSM receptor-expressing esophageal epithelial cells were found in the esophageal tissue of patients with EoE, suggesting that the epithelial cells may respond to OSM. Stimulation of esophageal epithelial cells with OSM resulted in a dose-dependent decrease in barrier function and expression of filaggrin and desmoglein-1 and an increase in protease calpain-14. Taken together, these data suggest a role for MCs in decreasing esophageal epithelial barrier function in EoE, which may in part be mediated by OSM

Micronutritional supplementation with a holoBLG-based FSMP (food for special medical purposes)-lozenge alleviates allergic symptoms in BALB/c mice: Imitating the protective farm effect

Background: Previously, the protective farm effect was imitated using the whey protein beta-lactoglobulin (BLG) that is spiked with iron-flavonoid complexes. Here, we formulated for clinical translation a lozenge as food for special medical purposes (FSMP) using catechin-iron complexes as ligands for BLG. The lozenge was tested in vitro and in a therapeutical BALB/c mice model. Methods: Binding of iron-catechin into BLG was confirmed by spectroscopy and docking calculations. Serum IgE binding of children allergic or tolerating milk was assessed to loaded (holo-) versus empty (apo-) BLG and for human mast cell degranulation. BLG and Bet v 1 double-sensitized mice were orally treated with the holoBLG or placebo lozenge, and immunologically analysed after systemic allergen challenge. Human PBMCs of pollen allergic subjects were flow cytometrically assessed after stimulation with apoBLG or holoBLG using catechin-iron complexes as ligands. Results: One major IgE and T cell epitope were masked by catechin-iron complexes, which impaired IgE binding of milk-allergic children and degranulation of mast cells. In mice, only supplementation with the holoBLG lozenge reduced clinical reactivity to BLG and Bet v 1, promoted Tregs, and suppressed antigen presentation. In allergic subjects, stimulation of PBMCs with holoBLG led to a significant increase of intracellular iron in circulating CD14+ cells with significantly lower expression of HLADR and CD86 compared to their stimulation with apoBLG. Conclusion: The FSMP lozenge targeted antigen presenting cells and dampened immune activation in human immune cells and allergic mice in an antigen-non-specific manner, thereby conferring immune resilience against allergic symptoms