Human bone marrow contains high levels of extracellular vesicles with a tissue-specific subtype distribution

Introduction Extracellular vesicles (EV) are shed from a broad variety of cells and play an important role in activation of coagulation, cell to cell interaction and transport of membrane components. They are usually measured as circulating EV in peripheral blood (PB) and other body fluids. However, little is known about the distribution, presence and impact of EV and their sub-populations in bone marrow (BM). In our study, we focused on the analysis of different EV subtypes in human BM as compared to EV subsets in PB. Methods EV in BM and PB from 12 healthy stem cell donors were measured by flow-cytometry using Annexin V and cell-specific antibodies for hematopoietic stem cells, leucocytes, platelets, red blood cells, and endothelial cells. Additionally, concentrations of tissue factor-bearing EV were evaluated. Results High numbers of total EV were present in BM (median value [25-75 percentile]: 14.8 x10(9)/l [8.5-19.3]). Non-significantly lower numbers of total EV were measured in PB (9.2 x10(9)/l [3.8-14.5]). However, distribuation of EV subtypes showed substantial differences between BM and PB: In PB, distribution of EV fractions was similar as previously described. Most EV originated from platelets (93.9%), and only few EV were derived from leucocytes (4.5%), erythrocytes (1.8%), endothelial cells (1.0%), and hematopoietic stem cells (0.7%). In contrast, major fractions of BM-EV were derived from red blood cells or erythropoietic cells (43.2%), followed by megacaryocytes I platelets (27.6%), and by leucocytes as well as their progenitor cells (25,7%);only low EV proportions originated from endothelial cells and hematopoietic stem cells (2.0% and 1.5%, respectively). Similar fractions of tissue factor- bearing EV were found in BM and PB (1.3% and 0.9%). Conculsion Taken together, we describe EV numbers and their subtype distribution in the BM compartment for the first time. The tissue specific EV distribution reflects BM cell composition and favours the idea of a BM-PB barrier existing not only for cells, but also for EV

Allergy / Prevention of allergy by viruslike nanoparticles (VNP) delivering shielded versions of major allergens in a humanized murine allergy model

Background: In highrisk populations, allergenspecific prophylaxis could protect from sensitization and subsequent development of allergic disease. However, such treatment might itself induce sensitization and allergies, thus requiring hypoallergenic vaccine formulations. We here characterized the preventive potential of viruslike nanoparticles (VNP) expressing surfaceexposed or shielded allergens. Methods: Fulllength major mugwort pollen allergen Art v 1 was selectively targeted either to the surface or to the inner side of the lipid bilayer envelope of VNP. Upon biochemical and immunological analysis, their preventive potential was determined in a humanized mouse model of mugwort pollen allergy. Results: Viruslike nanoparticles expressing shielded version of Art v 1, in contrast to those expressing surfaceexposed Art v 1, were hypoallergenic as they hardly induced degranulation of rat basophil leukemia cells sensitized with Art v 1specific mouse or human IgE. Both VNP versions induced proliferation and cytokine production of allergenspecific T cells in vitro. Upon intranasal application in mice, VNP expressing surfaceexposed but not shielded allergen induced allergenspecific antibodies, including IgE. Notably, preventive treatment with VNP expressing shielded allergenprotected mice from subsequent sensitization with mugwort pollen extract. Protection was associated with a Th1/Tregdominated cytokine response, increased Foxp3+ Treg numbers in lungs, and reduced lung resistance when compared to mice treated with empty particles. Conclusion: Viruslike nanoparticles represent a novel and versatile platform for the in vivo delivery of allergens to selectively target T cells and prevent allergies without inducing allergic reactions or allergic sensitization.DKW1248SFB F4605SFB F4609(VLID)313247

The small molecule inhibitor BX-795 uncouples IL-2 production from inhibition of Th2 inflammation and induces CD4+ T cells resembling iTreg

BackgroundTreg cells have been shown to be an important part of immune-homeostasis and IL-2 which is produced upon T cell receptor (TCR)-dependent activation of T lymphocytes has been demonstrated to critically participate in Treg development.ObjectiveTo evaluate small molecule inhibitors (SMI) for the identification of novel IL-2/Treg enhancing compounds.Materials and methodsWe used TCR-dependent and allergen-specific cytokine secretion of human and mouse T cells, next generation messenger ribonucleic acid sequencing (RNA-Seq) and two different models of allergic airway inflammation to examine lead SMI-compounds.ResultsWe show here that the reported 3-phosphoinositide dependent kinase-1 (PDK1) SMI BX-795 increased IL-2 in culture supernatants of Jurkat E6-1 T cells, human peripheral blood mononuclear cells (hPBMC) and allergen-specific mouse T cells upon TCR-dependent and allergen-specific stimulation while concomitantly inhibiting Th2 cytokine secretion. RNA-Seq revealed that the presence of BX-795 during allergen-specific activation of T cells induces a bona fide Treg cell type highly similar to iTreg but lacking Foxp3 expression. When applied in mugwort pollen and house dust mite extract-based models of airway inflammation, BX-795 significantly inhibited Th2 inflammation including expression of Th2 signature transcription factors and cytokines and influx into the lungs of type 2-associated inflammatory cells such as eosinophils.ConclusionsBX-795 potently uncouples IL-2 production from Th2 inflammation and induces Th-IL-2 cells, which highly resemble induced (i)Tregs. Thus, BX-795 may be a useful new compound for the treatment of allergic diseases

ZMYND10 Is Mutated in Primary Ciliary Dyskinesia and Interacts with LRRC6

Defects of motile cilia cause primary ciliary dyskinesia (PCD), characterized by recurrent respiratory infections and male infertility. Using whole-exome resequencing and high-throughput mutation analysis, we identified recessive biallelic mutations in ZMYND10 in 14 families and mutations in the recently identified LRRC6 in 13 families. We show that ZMYND10 and LRRC6 interact and that certain ZMYND10 and LRRC6 mutations abrogate the interaction between the LRRC6 CS domain and the ZMYND10 C-terminal domain. Additionally, ZMYND10 and LRRC6 colocalize with the centriole markers SAS6 and PCM1. Mutations in ZMYND10 result in the absence of the axonemal protein components DNAH5 and DNALI1 from respiratory cilia. Animal models support the association between ZMYND10 and human PCD, given that zmynd10 knockdown in zebrafish caused ciliary paralysis leading to cystic kidneys and otolith defects and that knockdown in Xenopus interfered with ciliogenesis. Our findings suggest that a cytoplasmic protein complex containing ZMYND10 and LRRC6 is necessary for motile ciliary function

Art v 1 IgE epitopes of patients and humanized mice are conformational

Background: Worldwide, pollen of the weed mugwort (Artemisia vulgaris) is a major cause of severe respiratory allergy, with its major allergen, Art v 1, being the key pathogenic molecule for millions of patients. Humanized mice transgenic for a human T-cell receptor specific for the major Art v 1 T-cell epitope and the corresponding HLA have been made. Objective: We sought to characterize IgE epitopes of Art v 1–sensitized patients and humanized mice for molecular immunotherapy of mugwort allergy. Methods: Four overlapping peptides incorporating surface-exposed amino acids representing the full-length Art v 1 sequence were synthesized and used to search for IgE reactivity to sequential epitopes. For indirect mapping, peptide-specific rabbit antibodies were raised to block IgE against surface-exposed epitopes on folded Art v 1. IgE reactivity and basophil activation studies were performed in clinically defined mugwort-allergic patients. Secondary structure of recombinant (r) Art v 1 and peptides was determined by circular dichroism spectroscopy. Results: Mugwort-allergic patients and humanized mice sensitized by allergen inhalation showed IgE reactivity and/or basophil activation mainly to folded, complete Art v 1 but not to unfolded, sequential peptide epitopes. Blocking of allergic patients’ IgE with peptide-specific rabbit antisera identified a hitherto unknown major conformational IgE binding site in the C-terminal Art v 1 domain. Conclusions: Identification of the new major conformational IgE binding site on Art v 1, which can be blocked with IgG raised against non-IgE reactive Art v 1 peptides, is an important basis for the development of a hypoallergenic peptide vaccine for mugwort allergy

Tryptophan immunoadsorption during pregnancy and breastfeeding in patients with acute relapse of multiple sclerosis and neuromyelitis optica

Background: Up to every fourth woman with multiple sclerosis (MS) or neuromyelitis optica spectrum disorder (NMOSD) suffers a clinically relevant relapse during pregnancy. High doses of steroids bear some serious risks, especially within the first trimester of pregnancy. Immunoadsorption (IA) is an effective and more selective treatment option in disabling MS relapse than plasma exchange. Data on the use of IA during pregnancy and breastfeeding are scarce. Methods: In this retrospective multicenter study, we analyzed the safety and efficacy of IA treatment in acute relapses during pregnancy or breastfeeding. The primary outcome parameter - change of acute relapse-related disability after IA - was assessed using Expanded Disability Status Scale (EDSS) and visual acuity (VA) measurements for patients with optic neuritis (ON). Results: A total of 24 patients were analyzed, 23 with relapsing–remitting MS, and 1 with NMOSD. Twenty patients were treated with IA during pregnancy. Four patients received IA postnatally during the breastfeeding period. Treatment was started at a mean 22.5 [standard deviation (SD) 13.9] days after onset of relapse. Patients were treated with a series of 5.8 (mean, SD 0.7) IA treatments within 7–10 days. Sixteen patients received IA because of steroid-refractory relapse, eight were treated without preceding steroid pulse therapy. EDSS improved clinically relevant from 3.5 [median, interquartile range (IQR) 2] before IA to 2.5 (median, IQR 1.1) after IA, p < 0.001. In patients with ON, VA improved in four out of five patients. Altogether, in 83% of patients, a rapid and marked improvement of relapse-related symptoms was observed after IA with either a decrease of ⩾1 EDSS grade or improvement in VA ⩾20%. No clinically relevant side effect was reported in 138 IA treatments. Conclusions: Tryptophan-IA was found to be effective and well tolerated in MS/NMOSD relapses, both as an escalation option after insufficient response to steroid pulse therapy and as first-line relapse treatment during pregnancy and breastfeeding

Microplastic in the water cycle : sampling, sample preparation, analyses, occurrence and assessment

Gedruckt erschienen im Universitätsverlag der TU Berlin, ISBN 978-3-7983-3162-4Das interdisziplinäre Forschungsprojekt MiWa widmete sich grundlegenden Fragestellungen zur Analytik und Wirkung von Mikroplastik-Partikeln im Wasserkreislauf. Es wurden Methoden der Umweltprobennahme, der Probenaufbereitung und verschiedene Detektionsverfahren zur Charakterisierung und Quantifizierung von Mikroplastik intensiv untersucht, miteinander verglichen und weiterentwickelt. Öko- und humantoxikologische Untersuchungen dienten dem Zweck, die potenziell von Mikroplastik ausgehende Gefährdung für die aquatische Umwelt und den Menschen zu analysieren und zu bewerten. Eine Harmonisierung und Standardisierung von Methoden der Probennahme, Probenaufbereitung und Mikroplastik-Detektion sind trotz der erheblichen Fortschritte derzeit nur teilweise möglich. Die ökotoxikologischen Studien zeigen zwar die Aufnahme von Mikroplastik-Partikeln durch einige Organismen, jedoch konnte bisher keine schädigende Wirkung nachgewiesen werden. Dabei wurden für eine Auswahl aquatischer Modellspezies sowohl Szenarien direkter als auch indirekter Exposition innerhalb einer Nahrungskette betrachtet. Interaktionen mit menschlichen Modellzellen wurden bislang nur bei Mikroplastik-Partikeln mit Größen weit unterhalb von 1 µm (also Nanoplastik) beobachtet. Eine umfassende Bewertung ist bislang nicht möglich.The interdisciplinary research project MiWa focused on principle knowledge gaps of analytical detection and effects of microplastic in fresh water cycles. Methods for environmental sampling, sample preparation and different analytical identification and quantification were intensively investigated, compared and further developed. Toxicological studies were conducted to assess potential risks of microplastic particles towards the environment and human health. Harmonization and standardization are still only partially possible despite various improvements. The eco-toxicological experiments confirmed the intake of microplastic particles by different organisms but no hazardous effects could be found. Both direct ingestions and indirect exposition within food webs were tested. Interactions with exemplary human cells were only observed for particle sizes far below 1 µm (thus nanoplastic). An assessment is currently only possible to a limited extent.BMBF, 02WRS1378, Mikroplastik im Wasserkreislauf (MiWa