Conversion of Anergic T Cells Into Foxp3−^- IL-10+^+ Regulatory T Cells by a Second Antigen Stimulus In Vivo

T cell anergy is a common mechanism of T cell tolerance. However, although anergic T cells are retained for longer time periods in their hosts, they remain functionally passive. Here, we describe the induction of anergic CD4$^+$ T cells in vivo by intravenous application of high doses of antigen and their subsequent conversion into suppressive Foxp3$^-$ IL-10$^+$ Tr1 cells but not Foxp3$^+$ Tregs. We describe the kinetics of up-regulation of several memory-, anergy- and suppression-related markers such as CD44, CD73, FR4, CD25, CD28, PD-1, Egr-2, Foxp3 and CTLA-4 in this process. The conversion into suppressive Tr1 cells correlates with the transient intracellular CTLA-4 expression and required the restimulation of anergic cells in a short-term time window. Restimulation after longer time periods, when CTLA-4 is down-regulated again retains the anergic state but does not lead to the induction of suppressor function. Our data require further functional investigations but at this stage may suggest a role for anergic T cells as a circulating pool of passive cells that may be re-activated into Tr1 cells upon short-term restimulation with high and systemic doses of antigen. It is tentative to speculate that such a scenario may represent cases of allergen responses in non-allergic individuals

Klimaschutz in der räumlichen Planung : Gestaltungsmöglichkeiten der Raumordnung und Bauleitplanung ; Kurzdokumentation der Fallstudien

Als Grundlage für die Praxishilfe "Klimaschutz in der räumlichen Planung" wurde im Auftrag des Umweltbundesamtes eine Reihe von Konzepten, Plänen und Programmen auf teil- bzw. gesamtstädtischer, regionaler und Landesebene untersucht, die für die Integration des Klimaschutz in die räumliche Planung beispielhaft sind. Es handelt sich im Schwerpunkt um formelle Instrumente (Bebauungs- und Flächennutzungspläne, Regionalpläne, Landesentwicklungspläne). Komplementär wurden auch informelle Instrumente untersucht, die die fachliche Grundlage für eine integrierte und effektive Bearbeitung des Klimaschutzes in der förmlichen Raumplanung darstellen (v.a. Energieversorgungs- und Klimaschutzkonzepte)

cFLIPL Inhibits Tumor Necrosis Factor-related Apoptosis-inducing Ligand-mediated NF-κB Activation at the Death-inducing Signaling Complex in Human Keratinocytes

Human keratinocytes undergo apoptosis following treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) via surface-expressed TRAIL receptors 1 and 2. In addition, TRAIL triggers nonapoptotic signaling pathways including activation of the transcription factor NF-kappaB, in particular when TRAIL-induced apoptosis is blocked. The intracellular protein cFLIP(L) interferes with TRAIL-induced apoptosis at the death-inducing signaling complex (DISC) in many cell types. To study the role of cFLIP(L) in TRAIL signaling, we established stable HaCaT keratinocyte cell lines expressing varying levels of cFLIP(L). Functional analysis revealed that relative cFLIP(L) levels correlated with apoptosis resistance to TRAIL. Surprisingly, cFLIP(L) specifically blocked TRAIL-induced NF-kappaB activation and TRAIL-dependent induction of the proinflammatory target gene interleukin-8. Biochemical characterization of the signaling pathways involved showed that apoptosis signaling was inhibited at the DISC in cFLIP(L)-overexpressing keratinocytes, although cFLIP(L) did not significantly impair enzymatic activity of the receptor complex. In contrast, recruitment and modification of receptor-interacting protein was blocked in cFLIP(L)-overexpressing cells. Taken together, our data demonstrate that cFLIP(L) is not only a central antiapoptotic modulator of TRAIL-mediated apoptosis but also an inhibitor of TRAIL-induced NF-kappaB activation and subsequent proinflammatory target gene expression. Hence, cFLIP(L) modulation in keratinocytes may not only influence apoptosis sensitivity but may also lead to altered death receptor-dependent skin inflammation

Fluxed M5-instantons in F-theory

We analyse the non-perturbative superpotential due to M5-brane instantons in F-theory compactifications on Calabi-Yau fourfolds. The M5 partition function is obtained via holomorphic factorisation by explicitly performing the sum over chiral 3-form fluxes. Comparison with the partition function of fluxed Euclidean D3-brane instantons in Type IIB orientifolds allows us to fix the spin structure on the intermediate Jacobian of the M5-instanton. We furthermore analyse the contribution of the M5-instanton to the superpotential in the presence of G4 gauge flux, where the superpotential is dressed with matter fields. We explicitly evaluate the pullback of G4 onto the M5-brane as a measure for the presence of charged instanton zero modes. This accounts for the M5 charge both under massless U(1)s, if present, and under what corresponds in Type II language to geometrically massive U(1)s.Comment: 47 pages. v3: refs. added, matches version published in Nuclear Physics

In vivo safety profile and biodistribution of GMP-manufactured human skin-derived ABCB5-positive mesenchymal stromal cells for use in clinical trials

Background aims Human dermal ABCB5-expressing mesenchymal stromal cells (ABCB5+ MSCs) represent a promising candidate for stem cell–based therapy of various currently uncurable diseases in several fields of regenerative medicine. We have developed and validated a method to isolate, from human skin samples, and expand ABCB5+ MSCs that meet the guideline criteria of the International Society for Cellular Therapy. We are able to process these cells into a Good Manufacturing Practice–conforming, MSC-based advanced-therapy medicinal product. Methods To support the development of ABCB5+ MSCs for potential therapeutic topical, intramuscular and intravenous administration, we have tested our product in a series of Good Laboratory Practice–compliant nonclinical in-vivo studies addressing all relevant aspects of biosafety, including potential long-term persistence and proliferation, distribution to nontarget tissues, differentiation into undesired cell types, ectopic tissue formation, tumor formation and local tissue reaction. Results (i) Subcutaneous application of 1 × 107 ABCB5+ MSCs/animal and intravenous application of 2 × 106 ABCB5+ MSCs/animal, respectively, to immunocompromised mice did not result in safety-relevant biodistribution, persistence or proliferation of the cells; (ii) three monthly subcutaneous injections of ABCB5+ MSCs at doses ranging from 1 × 105 to 1 × 107 cells/animal and three biweekly intravenous injections of 2 × 106 ABCB5+ MSCs/animal, respectively, to immunocompromised mice were nontoxic and revealed no tumorigenic potential; and (iii) intramuscular injection of 5 × 106 ABCB5+ MSCs/animal to immunocompromised mice was locally well tolerated. Discussion The present preclinical in vivo data demonstrate the local and systemic safety and tolerability of a novel advanced-therapy medicinal product based on human skin-derived ABCB5+ MSCs

Ex vivo-expanded highly pure ABCB5+ mesenchymal stromal cells as good manufacturing practice-compliant autologous advanced therapy medicinal product for clinical use: Process validation and first in-human data

© 2020 International Society for Cell & Gene Therapy Background aim: Mesenchymal stromal cells (MSCs) hold promise for the treatment of tissue damage and injury. However, MSCs comprise multiple subpopulations with diverse properties, which could explain inconsistent therapeutic outcomes seen among therapeutic attempts. Recently, the adenosine triphosphate-binding cassette transporter ABCB5 has been shown to identify a novel dermal immunomodulatory MSC subpopulation. Methods: The authors have established a validated Good Manufacturing Practice (GMP)-compliant expansion and manufacturing process by which ABCB5+ MSCs can be isolated from skin tissue and processed to generate a highly functional homogeneous cell population manufactured as an advanced therapy medicinal product (ATMP). This product has been approved by the German competent regulatory authority to be tested in a clinical trial to treat therapy-resistant chronic venous ulcers. Results: As of now, 12 wounds in nine patients have been treated with 5 × 105 autologous ABCB5+ MSCs per cm2 wound area, eliciting a median wound size reduction of 63% (range, 32–100%) at 12 weeks and early relief of pain. Conclusions: The authors describe here their GMP- and European Pharmacopoeia-compliant production and quality control process, report on a pre-clinical dose selection study and present the first in-human results. Together, these data substantiate the idea that ABCB5+ MSCs manufactured as ATMPs could deliver a clinically relevant wound closure strategy for patients with chronic therapy-resistant wounds

Rational F-Theory GUTs without exotics

We construct F-theory GUT models without exotic matter, leading to the MSSM matter spectrum with potential singlet extensions. The interplay of engineering explicit geometric setups, absence of four-dimensional anomalies, and realistic phenomenology of the couplings places severe constraints on the allowed local models in a given geometry. In constructions based on the spectral cover we find no model satisfying all these requirements. We then provide a survey of models with additional U(1) symmetries arising from rational sections of the elliptic fibration in toric constructions and obtain phenomenologically appealing models based on SU(5) tops. Furthermore we perform a bottom-up exploration beyond the toric section constructions discussed in the literature so far and identify benchmark models passing all our criteria, which can serve as a guideline for future geometric engineering.Comment: 27 Pages, 1 Figur

Translational development of ABCB5+ dermal mesenchymal stem cells for therapeutic induction of angiogenesis in non-healing diabetic foot ulcers

Background While rapid healing of diabetic foot ulcers (DFUs) is highly desirable to avoid infections, amputations and life-threatening complications, DFUs often respond poorly to standard treatment. GMP-manufactured skin-derived ABCB5+ mesenchymal stem cells (MSCs) might provide a new adjunctive DFU treatment, based on their remarkable skin wound homing and engraftment potential, their ability to adaptively respond to inflammatory signals, and their wound healing-promoting efficacy in mouse wound models and human chronic venous ulcers. Methods The angiogenic potential of ABCB5+ MSCs was characterized with respect to angiogenic factor expression at the mRNA and protein level, in vitro endothelial trans-differentiation and tube formation potential, and perfusion-restoring capacity in a mouse hindlimb ischemia model. Finally, the efficacy and safety of ABCB5+ MSCs for topical adjunctive treatment of chronic, standard therapy-refractory, neuropathic plantar DFUs were assessed in an open-label single-arm clinical trial. Results Hypoxic incubation of ABCB5+ MSCs led to posttranslational stabilization of the hypoxia-inducible transcription factor 1α (HIF-1α) and upregulation of HIF-1α mRNA levels. HIF-1α pathway activation was accompanied by upregulation of vascular endothelial growth factor (VEGF) transcription and increase in VEGF protein secretion. Upon culture in growth factor-supplemented medium, ABCB5+ MSCs expressed the endothelial-lineage marker CD31, and after seeding on gel matrix, ABCB5+ MSCs demonstrated formation of capillary-like structures comparable with human umbilical vein endothelial cells. Intramuscularly injected ABCB5+ MSCs to mice with surgically induced hindlimb ischemia accelerated perfusion recovery as measured by laser Doppler blood perfusion imaging and enhanced capillary proliferation and vascularization in the ischemic muscles. Adjunctive topical application of ABCB5+ MSCs onto therapy-refractory DFUs elicited median wound surface area reductions from baseline of 59 % (full analysis set, n = 23), 64 % (per-protocol set, n = 20) and 67 % (subgroup of responders, n = 17) at week 12, while no treatment-related adverse events were observed. Conclusions The present observations identify GMP-manufactured ABCB5+ dermal MSCs as a potential, safe candidate for adjunctive therapy of otherwise incurable DFUs and justify the conduct of a larger, randomized controlled trial to validate the clinical efficacy. Trial registration ClinicalTrials.gov, NCT03267784, Registered 30 August 2017, https://clinicaltrials.gov/ct2/show/NCT0326778

Untersuchung von spezifischen Protein-Protein-Interaktionen am Beispiel von hGBP1 und strukturelle Charakterisierung von proteinresistenten Peptidthiolen

Am Beispiel der Homodimerisierung von hGBP1 soll ein Einblick in die Möglichkeiten der Biosensorik und die kontrollierte Herstellung von Oberflächen gegeben werden. Der Nachweis von Biomolekülen über maßgeschneiderte Sensoroberflächen ist hierbei ein bedeutender Anwendungsbereich. Gerade aus pharmakologischer Sicht ist die Aufklärung der Struktur und Funktion von Proteinen und die Wechselwirkung mit anderen Proteinen von Bedeutung, um gezieltes $\textit {Drug Design}$ durchzuführen. Bei der Herstellung von Implantaten in der Medizintechnik hat man oft mit dem Problem einer Immunabwehrreaktion des Körpers zu kämpfen. Auf den Implantaten bilden sich unerwünschte Biofilme, die den Einheilungserfolg negativ beeinträchtigen können. Diese Problematik wurde zum Anlass genommen, biomimetische proteinresistente Oberflächen auf der Basis von Peptiden zu schaffen und die proteophoben sowie strukturellen Eigenschaften dieser Peptidthiol-SAMs herauszuarbeiten.Using the protein hGBP1 and its ability to form homodimers this thesis aims at delivering insight into the high potential of biosensory applications and the controlled fabrication of surfaces with desired properties. The specific detection of biomolecules via tailored surfaces is a major application in this context. Especially from pharmacological point of view the elucidation of structure and function of proteins and their interaction with other proteins is of crucial interest for directed drug design. Another aspect of this thesis was the development and structural characterization of protein-resistant surfaces on the basis of peptides. Due to immunoreactions undesirable biofilms are often formed on implants or contact lenses, which hamper osseointegration. This complex of problems prompted the fabrication of biomimetic surfaces on the basis of peptides in order to investigate the proteophobic and structural properties of these peptide thiol SAMs