Characterisation of CD4+ stem cells of the skin and their role in skin homeostasis and tumourigenesis

The skin represents the largest organ of the mammalian body and acts as the primary barrier against environmental stressors. Cellular maintenance and regeneration of the skin is orchestrated by a variety of skin stem cell (SC) populations. Due to their regenerative potential and plasticity, skin SC are an important research focus in regenerative medicine. However, they are also a subject of cancer research since mutations in skin SC can lead to skin tumour formation. The most common neoplasia of the human skin is the basal cell carcinoma (BCC), which most often develops due to Hedgehog (HH) signalling activating mutations in basal cells of the interfollicular epidermis (IFE) and/or from hair follicle (HF) SC. In sporadic BCC, a loss of heterozygosity of the negative HH signalling regulator PATCHED (PTCH) is most commonly observed. Recently our group described the existence of CD4+ non-haematopoietic skin-resident cells, which can give rise to HH signalling-activated BCC upon homozygous Ptch mutation and chemical carcinogenesis. However, the cellular identity and localisation of the CD4+ non-haematopoietic, skin-resident precursors were unclear. Since the knowledge about their occurrence and potential may have critical impact on the understanding of skin cancer development, the main goal of this thesis was to identify and characterise CD4+ non-haematopoietic, skin-resident cells and their descendants. For this purpose, three main strategies were pursued: First, stromal cells, which can give rise to various skin cell types and which can express Cd4/CD4 transcripts, were analysed as possible CD4+ non-haematopoietic ancestors of skin-resident BCC precursors. The respective in vivo and in vitro lineage-tracing analyses revealed that a small population of murine stromal cells of the bone marrow and skin express CD4. However, CD4+ stromal cells could neither be proven nor excluded as the origin of BCC due to missing methods of investigation. Second, epidermal SC were evaluated as the origin of CD4+ non-haematopoietic, skin-resident cells. Using in vivo and in vitro lineage tracing, comparative and single cell transcriptome sequencing and flow cytometric analyses, rare SC-like epidermal cells of the IFE and/or the infundibulum were uncovered as CD4+ non-haematopoietic cells and thus can be considered likely candidates for BCC precursor cells. Moreover, the specific characterisation of CD4+ epidermal cells revealed that their progeny grows permanently and increasingly with age and upon wound healing in adult mice in all IFE layers and as multipotent HF SC of the telogen HF as well as in all compartments of the anagen HF. Thirdly, and highly relevant, the occurrence and characteristics of the previously uncovered CD4+ non-haematopoietic population of human skin was investigated. Flow cytometric and in vitro culture analyses showed that these cells similar to their murine counterparts possess a SC-like character and most likely grow in the IFE and/or the infundibulum of human skin.2022-05-0

Intracellular localization of RORα is isoform and cell line-dependent

AbstractThe retinoid-related orphan receptor α (RORα) belongs to the nuclear receptor superfamily and comprises four isoforms generated by different promotor usage and alternative splicing. To better understand its function, the subcellular distribution of RORα was investigated. We could show that subcellular distribution of RORα is cell line and isoform-dependent. Isoform specific differences were mediated by the A/B domains which with the exception of RORα1 contain a signal that mediates cytoplasmic localization. The lack of this signal in RORα1 results in a complete nuclear localization and prevents cell membrane association observed for RORα2, 3, and 4. The region responsible for membrane association was identified as the C-terminal α-helix 12. Furthermore, the hinge region/ligand binding domain mediates nuclear localization. Our results show that isoform specific activity of RORα is not only regulated by different expression and DNA binding affinities but also by different subcellular distribution. Different access to the nucleus reveals an important mechanism regulating the activity of this constitutively active nuclear receptor

Extracellular signal-regulated kinase-2 phosphorylates RORα4 in vitro

The retinoic acid related orphan receptor RORα activates transcription of genes that play an important role in cerebellar development, the protection against age-related degenerative processes, the regulation of inflammatory responses, and is one of the pivotal participants that control the circadian rhythmicity in the core-clock of mammals. We identified the extracellular signal-regulated kinase 2 (ERK-2) as RORα4 phosphorylating kinase in vitro. The primary sequence of RORα4 contains an ERK-2 recognition motif (P-L-T128-P) within the hinge domain, and mutation of Thr-128 to Ala prevents RORα4 phosphorylation by ERK. The RORα4-T128A mutant exhibits an increased DNA-binding affinity, an increased transcriptional activity and, in the interplay with the opponent RevErbα, acts as a stronger competitor at ROR response elements than RORα4-WT

Spreading of Isolated Ptch Mutant Basal Cell Carcinoma Precursors Is Physiologically Suppressed and Counteracts Tumor Formation in Mice

Basal cell carcinoma (BCC) originate from Hedgehog/Patched signaling-activated epidermal stem cells. However, the chemically induced tumorigenesis of mice with a CD4Cre-mediated biallelic loss of the Hedgehog signaling repressor Patched also induces BCC formation. Here, we identified the cellular origin of CD4Cre-targeted BCC progenitors as rare Keratin 5+ epidermal cells and show that wildtype Patched offspring of these cells spread over the hair follicle/skin complex with increasing mouse age. Intriguingly, Patched mutant counterparts are undetectable in age-matched untreated skin but are getting traceable upon applying the chemical tumorigenesis protocol. Together, our data show that biallelic Patched depletion in rare Keratin 5+ epidermal cells is not sufficient to drive BCC development, because the spread of these cells is physiologically suppressed. However, bypassing the repression of Patched mutant cells, e.g., by exogenous stimuli, leads to an accumulation of BCC precursor cells and, finally, to tumor development

Outpatient parenteral antimicrobial therapy in Germany: a prospective cohort study protocol

Introduction Outpatient parenteral antimicrobial therapy (OPAT) means intravenous administration of antibiotics outside the hospital. The antibiotics are administered at the patient's home. The advantages are the shortening of the inpatient stay, which means that patients can remain in their familiar environment, the reduction of nosocomial infections as well as the reduction of hospital and therapy costs. Nevertheless, OPAT is rarely performed in Germany, despite its international application. Therefore, systematic data on OPAT are not available in Germany. The project objective is to investigate the medical care using OPAT under medical, epidemiological and economic aspects within the framework of the Cologne Network of Infectious Diseases. Methods and analysis Observational study with mixed-methods approach, qualitative analysis to identify physician side factors to assess the attitude of general practitioners in Cologne with regard to possible implementation barriers of an OPAT. Longitudinal analysis of an OPAT patient cohort with respect to clinical and patient-relevant outcomes using descriptive and conclusive statistics. Ethics and dissemination The study has been approved by the Institutional Review Board of the University of Cologne, Germany (19- 1284-1). Written informed consent was obtained from all participants. The results will be submitted for publication in a peer-reviewed journal and presented at one or more scientific conferences

Automated manufacture of ΔNPM1 TCR-engineered T cells for AML therapy

Acute myeloid leukemia (AML) is a heterogeneous malignancy that requires further therapeutic improvement, especially for the elderly and for subgroups with poor prognosis. A recently discovered T cell receptor (TCR) targeting mutant nucleophosmin 1 (ΔNPM1) presents an attractive option for the development of a cancer antigen-targeted cellular therapy. Manufacturing of TCR-modified T cells, however, is still limited by a complex, time-consuming, and laborious procedure. Therefore, this study specifically addressed the requirements for a scaled manufacture of ΔNPM1-specific T cells in an automated, closed, and good manufacturing practice-compliant process. Starting from cryopreserved leukapheresis, 2E8 CD8-positive T cells were enriched, activated, lentivirally transduced, expanded, and finally formulated. By adjusting and optimizing culture conditions, we additionally reduced the manufacturing time from 12 to 8 days while still achieving a clinically relevant yield of up to 5.5E9 ΔNPM1 TCR-engineered T cells. The cellular product mainly consisted of highly viable CD8-positive T cells with an early memory phenotype. ΔNPM1 TCR CD8 T cells manufactured with the optimized process showed specific killing of AML in vitro and in vivo. The process has been implemented in an upcoming phase 1/2 clinical trial for the treatment of NPM1-mutated AML

Globale und regionale Verteilung von Biomassepotenzialen Status-quo und Möglichkeiten der Präzisierung

Diese Veröffentlichung liefert sowohl zu den weltweiten als auch zu den regionalen Biomassepotenzialen in Deutschland Informationen. Dazu wurde der gegenwärtige Stand und (szenarienbasiert) die zukünftige Entwicklung der Biomassepotenziale ermittelt. Im Blick standen hierbei land- und forstwirtschaftliche Biomassepotenziale sowie Reststoffpotenziale. Zu den Reststoffen werden in diesem Kontext u.a. Stroh und Exkremente aus der Nutztierhaltung sowie Bio- und Grünabfälle gezählt. Aufbauend auf den Potenzialberechnungen wurden Auswirkungen des derzeitigen bzw. künftigen Biomasseanbaus auf Aspekte der Raumordnung und Flächennutzung analysiert. In diesem Zusammenhang werden auch die Steuerungsinstrumente der Raumordnung bei Ausbau der Biomassenutzung zu energetischen Zwecken thematisiert. Zusätzlich wird die Eignung eines Modells zur satellitengestützten Fernerkundung von energetischen Biomasseressourcen untersucht