Recovering What Matters: High Protein Recovery after Endotoxin Removal from LPS-Contaminated Formulations Using Novel Anti-Lipid A Antibody Microparticle Conjugates

Bioconjugation; Polystyrene particles; Supramolecular structuresBioconjugación; Partículas de poliestireno; Estructuras supramolecularesBioconjugació; Partícules de poliestirè; Estructures supramolecularsEndotoxins or lipopolysaccharides (LPS), found in the outer membrane of Gram-negative bacterial cell walls, can stimulate the human innate immune system, leading to life-threatening symptoms. Therefore, regulatory limits for endotoxin content apply to injectable pharmaceuticals, and excess LPS must be removed before commercialization. The majority of available endotoxin removal systems are based on the non-specific adsorption of LPS to charged and/or hydrophobic surfaces. Albeit effective to remove endotoxins, the lack of specificity can result in the unwanted loss of essential proteins from the pharmaceutical formulation. In this work, we developed microparticles conjugated to anti-Lipid A antibodies for selective endotoxin removal. Anti-Lipid A particles were characterized using flow cytometry and microscopy techniques. These particles exhibited a depletion capacity > 6 ×103 endotoxin units/mg particles from water, as determined with two independent methods (Limulus Amebocyte Lysate test and nanoparticle tracking analysis). Additionally, we compared these particles with a non-specific endotoxin removal system in a series of formulations of increasing complexity: bovine serum albumin in water < insulin in buffer < birch pollen extracts. We demonstrated that the specific anti-Lipid A particles show a higher protein recovery without compromising their endotoxin removal capacity. Consequently, we believe that the specificity layer integrated by the anti-Lipid A antibody could be advantageous to enhance product yield.The project leading to this application has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 812661, H2020-MSCA-ITN-2018-812661 (ENDONANO)

Behavioral Health Risk Factors and Motivation to Change among Cardiovascular General Hospital Patients Aged 50 to 79 Years

Little is known about the (co-)occurrence of smoking, alcohol at-risk drinking, physical inactivity and overweight, and the motivation to change these behavioral health risk factors (HRFs) in older general hospital patients with cardiovascular disease. Between October and December 2016, all consecutively admitted patients aged 50 to 79 years were proactively recruited on 3 cardiology wards and asked to participate in a survey on HRFs and behavior change motivation. Of the eligible patients, 80.4% participated in the survey (n = 328). The mean age was 66.5 years (standard deviation 9.0), and 65.5% were male. At least 1 HRF was present in 91.8% (n = 280), at least 2 HRFs in 54.4% (n = 166), and 3 or 4 HRFs in 12.1% (n = 37) of participants. The proportion of older adults who contemplated or were changing or planning to change their behavior to meet health behavior recommendations ranged between 66.0% (smoking) and 93.2% (alcohol consumption). The results indicate a notable co-occurrence of behavioral HRFs in older patients with cardiovascular disease. The majority of older adults were at least considering changing the respective behavior. To prevent and treat diseases efficiently, hospitalization may be a suitable moment for systematic multiple HRF screening and intervention.Peer Reviewe

Developmental Stage, Phenotype, and Migration Distinguish Naive- and Effector/Memory-like CD4+ Regulatory T Cells

Regulatory T cells (Tregs) fulfill a central role in immune regulation. We reported previously that the integrin αEβ7 discriminates distinct subsets of murine CD4+ regulatory T cells. Use of this marker has now helped to unravel a fundamental dichotomy among regulatory T cells. αE−CD25+ cells expressed L-selectin and CCR7, enabling recirculation through lymphoid tissues. In contrast, αE-positive subsets (CD25+ and CD25−) displayed an effector/memory phenotype expressing high levels of E/P-selectin–binding ligands, multiple adhesion molecules as well as receptors for inflammatory chemokines, allowing efficient migration into inflamed sites. Accordingly, αE-expressing cells were found to be the most potent suppressors of inflammatory processes in disease models such as antigen-induced arthritis

Curative treatment of oesophageal carcinoma: current options and future developments

Since the 1980s major advances in surgery, radiotherapy and chemotherapy have established multimodal approaches as curative treatment options for oesophageal cancer. In addition the introduction of functional imaging modalities such as PET-CT created new opportunities for a more adequate patient selection and therapy response assessment

Epigenetic Control of the foxp3 Locus in Regulatory T Cells

Compelling evidence suggests that the transcription factor Foxp3 acts as a master switch governing the development and function of CD4(+) regulatory T cells (Tregs). However, whether transcriptional control of Foxp3 expression itself contributes to the development of a stable Treg lineage has thus far not been investigated. We here identified an evolutionarily conserved region within the foxp3 locus upstream of exon-1 possessing transcriptional activity. Bisulphite sequencing and chromatin immunoprecipitation revealed complete demethylation of CpG motifs as well as histone modifications within the conserved region in ex vivo isolated Foxp3(+)CD25(+)CD4(+) Tregs, but not in naïve CD25(−)CD4(+) T cells. Partial DNA demethylation is already found within developing Foxp3(+) thymocytes; however, Tregs induced by TGF-β in vitro display only incomplete demethylation despite high Foxp3 expression. In contrast to natural Tregs, these TGF-β–induced Foxp3(+) Tregs lose both Foxp3 expression and suppressive activity upon restimulation in the absence of TGF-β. Our data suggest that expression of Foxp3 must be stabilized by epigenetic modification to allow the development of a permanent suppressor cell lineage, a finding of significant importance for therapeutic applications involving induction or transfer of Tregs and for the understanding of long-term cell lineage decisions

Development of regulatory T cells and induction of mucosa-specific homing

Bei der Aufrechterhaltung des homeostatischen Gleichgewichts und der peripheren Selbst-Toleranz spielen CD4+CD25+ regulatorische T-Zellen (Tregs) eine wichtige Rolle. In Vorarbeiten wurden Subpopulationen von murinen CD4+ Tregs identifiziert, die sich durch die Expression des Integrins alphaE auszeichnen. Diese alphaE+ Treg Subpopulationen weisen einen Effektor/Memory-ähnlichen Phänotyp auf. In der vorliegenden Dissertation wurde untersucht, welche Bedingungen zur Entwicklung von alphaE+ Effektor/Memory Tregs in vivo führen und aus welchen Vorläuferzellen sie entstehen. Dabei zeigte sich, dass es sich bei den alphaE+ Tregs um Effektor/Memory T-Zellen handelt, die unter physiologischen Bedingungen in vivo ein hohes Maß an Zellteilung aufweisen, welche zum Teil abhängig von der bakteriellen Besiedelung des Darms ist. Darüber hinaus wurde beobachtet, dass alphaE+ Tregs nach oraler, antigen-spezifischer Aktivierung in den darm-assozierten lymphoiden Geweben sowohl aus konventionellen naiven CD4+ T-Zellen, als auch aus thymus-generierten naiven CD4+CD25+ Tregs entstehen können. Zusammenfassend deuten die erzielten Ergebnisse darauf hin, dass das spezifische mukosale Mikroenvironment sowohl die Expansion als auch Konvertierung von Tregs fördert und so eine wichtige Rolle für die Aufrechterhaltung der Homeostase von alphaE+Foxp3+ Tregs spielt. Zudem wurde in dieser Arbeit die Ausbildung von gewebespezifischen Homingrezeptor-Phänotypen von naiven CD4+CD25+ Tregs untersucht. In in vitro Kultur-Systemen zeigte sich, dass selektive Modulation von Tregs, ähnlich wie bei konventionellen T-Zellen, die Induktion von organspezifischen Migrationseigenschaften ermöglicht. So konnte eine effiziente Wanderung von Tregs in den Darm ausgelöst werden. Diese Daten legen den Schluss nahe, dass die Herstellung von Tregs mit spezifischen Wanderungseigenschaften eine Option für therapeutische Anwendungen in der adoptiven T-Zell Therapie sein könnte.Regulatory CD4+CD25+ T cells (Tregs) play an important role in immune homeostasis and in the maintenance of self-tolerance. Previously, subsets of murine CD4+ Tregs characterised by expression of the integrin alphaE had been identified. These alphaE+ Treg subsets display an effector/memory-like phenotype. In the present study the circumstances favouring in vivo generation of effector/memory-like alphaE+ Tregs were analysed. The results presented here show that alphaE+ effector/memory-like Treg subsets contain a large fraction of cycling cells under physiologic conditions in healthy mice. This in vivo proliferation depended, at least in part, on intestinal commensal microflora. Furthermore, it was observed that alphaE+ Tregs not only developed by differentiation of naive-like CD4+CD25+ Tregs, but were also generated de novo from naive CD4+ T cells in the gut-associated lymphoid tissue upon oral antigen delivery. Taken together, these results indicate that the mucosal microenvironment favours both expansion and conversion of Tregs and thereby represents an important mechanism for the homeostatic maintenance of alphaE+Foxp3+ Tregs. In addition, susceptibility of naive CD4+CD25+ Tregs to acquire tissue-specific homing receptor phenotypes was investigated. In vitro culture systems demonstrated that Tregs, similarly to conventional T cells, could be configured with organ-selective homing properties allowing efficient targeting into the gut. These results suggest that generation of Tregs with specific homing properties for therapeutic purposes in adoptive T cell therapy might be a feasible option

The N-terminal amphipathic helix of Pex11p self-interacts to induce membrane remodelling during peroxisome fission

Pex11p plays a crucial role in peroxisome fission. Previously, it was shown that a conserved N-terminal amphipathic helix in Pex11p, termed Pex11-Amph, was necessary for peroxisomal fission in vivo while in vitro studies revealed that this region alone was sufficient to bring about tubulation of liposomes with a lipid consistency resembling the peroxisomal membrane. However, molecular details of how Pex11-Amph remodels the peroxisomal membrane remain unknown. Here we have combined in silico, in vitro and in vivo approaches to gain insights into the molecular mechanisms underlying Pex11-Amph activity. Using molecular dynamics simulations, we observe that Pex11-Amph peptides form linear aggregates on a model membrane. Furthermore, we identify mutations that disrupted this aggregation in silico, which also abolished the peptide's ability to remodel liposomes in vitro, establishing that Pex11p oligomerisation plays a direct role in membrane remodelling. In vivo studies revealed that these mutations resulted in a strong reduction in Pex11 protein levels, indicating that these residues are important for Pex11p function. Taken together, our data demonstrate the power of combining in silico techniques with experimental approaches to investigate the molecular mechanisms underlying Pex11p-dependent membrane remodelling