University Hospital Carl Gustav Carus

The skin is a diverse reservoir of immune cells with strong potential for immunotherapeutic delivery. Langerhans cells in the epidermis are antigen-presenting cells, accessible for vaccination using carbohydrate-conjugated therapeutics targeting their endocytic lectin receptor, Langerin. As carbohydrate-lectin binding is highly dependent on valency, scaffolds that enable control over ligand spacing and stoichiometry are instrumental in enhancing receptor binding and selectivity. Here we utilized a self-assembled nucleic acid-based Holliday Junction scaffold, fully modified for nuclease protection and with a well-defined carbohydrate arrangement to optimize drug delivery to Langerhans cells. In vitro screening with Langerin-expressing cells revealed that mannosylated Holliday Junctions showed the strongest binding. This was confirmed in human epidermal cell suspensions, demonstrating specificity and valency-driven interactions. Topical administration of mannosylated scaffolds on skin explants enabled effective targeting of epidermal Langerhans cells. Finally, in an antigen-presentation assay, in vitro differentiated Langerhans cells loaded with mannosylated and peptide-conjugated scaffolds significantly enhanced T cell activation. Overall, our study presents a promising nucleic acid-based platform for precise Langerhans cell targeting and drug delivery, with broad potential for skin-directed immunotherapies.

Machine learning (ML) accelerates progress in many areas, including biomedical and clinical research. ML algorithms provide powerful options for efficiently analyzing multivariate data sets. We developed and validated an ML pipeline to detect myelodysplastic syndrome (MDS)-associated pathological alterations of extracellular matrices (ECMs) by time-of-flight secondary ion mass spectrometry (ToF-SIMS). A Bayesian-optimized neural network (NN) was trained and applied to classify ToF-SIMS spectra of ECM secreted by mesenchymal stromal cells (MSCs) derived from MDS patients and healthy reference donors. Validated by principal component analysis, the explainer tool SHapley Additive exPlanations (known as SHAP) was integrated into the analysis pipeline to unravel characteristic compositional and structural differences of the ECM variants. Our results demonstrate the potential of ToF-SIMS-ML for the label-free investigation of pathogenic alterations of the ECM. Integrated into the multiscale ECM analysis of cell and organoid-based disease models, the introduced methodology may facilitate advances in the development of novel diagnostic and therapeutic strategies.

Handheld ultrasound (HHUS) devices are increasingly used in clinical practice due to their portability and cost-effectiveness. However, HHUS image quality remains variable, and a standardized expert-based evaluation protocol is lacking. This study compares the image quality of B-mode recordings of five HHUS models with a high-end reference system (HEUS) using a blinded assessment to identify differences, covariates, offering a standardized evaluation framework.In this blinded, multi-center study, video sequences of the liver, pancreas, and sigmoid colon were recorded in ten volunteers using 5 HHUS devices - Vscan Air, Butterfly iQ+, Philips Lumify, Clarius C3, and Clarius C3 HD3 - and one HEUS (GE Logiq E10). Twenty-two physicians with many years of abdominal ultrasound experience rated blinded clips for 5 image quality parameters.The HEUS consistently received the highest ratings. Image quality differed significantly between HHUS devices. Clarius systems showed the smallest deviation from the reference, followed by Vscan Air and Philips Lumify. All image parameters significantly correlated with the overall impression, without a single dominant factor. The patients' BMI had no relevant influence. Examiner experience was less impactful than device familiarity and personal preference.The image quality of HEUS remained superior to all tested HHUS devices. Further subdivision of image quality aspects did not provide additional information. Furthermore, we could not identify significant covariates in our setup. These findings could justify simplified setups with reduced questionnaires and requirements for examiners.

Monitoring of unfractionated heparin (UFH) in critically ill patients is challenging due to disease-related alterations of the coagulation system, directly affecting conventional laboratory tests, including activated partial thromboplastin time (aPTT). Measurement of anti-factor Xa activity (Anti-Xa) as an alternative to aPTT often lacks 24/7 availability. Whole blood viscoelastic testing (VET) may provide a feasible point-of-care alternative to guide UFH dosing in this setting. Additionally, VET is readily available in the environment found in our clinic, The aim of this study was to evaluate the diagnostic performance of viscoelastic testing compared with activated partial thromboplastin time for monitoring unfractionated heparin in critically ill patients and to define clinically applicable viscoelastic thresholds using anti-factor Xa activity as the reference standard. In this prospective observational study for UFH monitoring Anti-Xa, aPTT, and the VET clotting time IN/HI ratio (ClotPro®; Haemonetics, Boston, MA, USA) were determined in simultaneously taken blood samples. Anti-Xa results were used as the reference standard to categorize ongoing UFH anticoagulation intensity into four categories (prophylactic, sub-therapeutic, therapeutic, and overdosed). Diagnostic performance of aPTT and CT IN/HI ratio were evaluated against Anti-Xa using ROC analysis and Cohen’s Kappa. Test performance was evaluated using ROC analysis, multiclass classification, and weighted Cohen’s kappa. Between September 2020 and July 2022, 466 datasets from 75 ICU patients receiving intravenous UFH were analyzed. Compared to aPTT, AUC values for CT IN/HI were higher for detecting prophylactic (0.817 vs. 0.726), therapeutic (0.690 vs. 0.494) and overdosed (0.845 vs. 0.708) UFH levels. Thresholds of > 1.4, > 1.9, and > 2.3 for the CT IN/HI ratio corresponded to sub-therapeutic, therapeutic, and overdosed ranges, respectively. The CT IN/HI ratio showed moderate agreement with Anti-Xa classification (κ = 0.450), while aPTT agreement was insufficient (κ = 0.220). The CT IN/HI ratio offers a reliable point-of-care alternative to standard UFH monitoring, in the case it is readily available. It provides better alignment with Anti-Xa activity than aPTT and may improve anticoagulation management in critically ill patients, especially when Anti-Xa testing is delayed or unavailable. DRKS-ID DRKS00028689 (https://drks.de/search/de), PI: Lars Heubner, retrospectively registered 30.03.2022.

Desmoid-type fibromatosis (DT) is a rare, locally aggressive soft tissue tumor with a frequently chronic course and substantial impact on health-related quality of life (HRQoL). While international studies have demonstrated considerable symptom burden and psychosocial impairment, data from Germany are lacking. This study aimed to assess HRQoL in German patients and to identify factors associated with HRQoL. In this cross-sectional analysis, adult patients with histologically confirmed DT completed the EORTC QLQ-C30 and the disease-specific Desmoid-Type Fibromatosis Quality of Life Questionnaire (DTF-QoL). HRQoL scores were compared with reference data from the German general population, German sarcoma patients, and international DT cohorts. Sociodemographic, disease-, tumor-, and treatment-related factors associated with HRQoL were examined using multivariate linear regression analyses. A total of 155 patients were included (69.7% female; mean age 45.0 years). Compared with the German general population, DT patients reported clinically relevant impairments in role, social, and emotional functioning and higher symptom burden, particularly pain, fatigue, and insomnia. Compared with sarcoma patients, DT patients showed better physical, role, and social functioning, while emotional functioning and symptom burden were largely comparable. German DT patients reported consistently worse HRQoL than Dutch/UK cohorts and moderately worse outcomes than Indian cohorts. Female gender, unemployment or disability pension, intensive multimodal treatment, multiple lines of systemic therapy, and tumor location in the lower extremities were independently associated with poorer HRQoL. DT is associated with sustained and clinically meaningful HRQoL impairment. HRQoL is driven primarily by psychosocial and treatment-related factors rather than disease duration, supporting the concept of DT as a chronic condition requiring long-term, multidisciplinary supportive care.

Positron emission tomography (PET) of the brain using [18F]fluorodeoxyglucose (FDG) is becoming increasingly important for the diagnosis and differential diagnosis of atypical parkinsonian syndrome such as multiple system atrophy (MSA), which is characterized by hypometabolism of the putamen, pons, and cerebellum. We report on a patient with clinically established MSA based on a rapidly progressive, poorly levodopa-responsive parkinsonian syndrome, multidomain autonomic failure, and imaging findings where hereditary spastic paraplegia was discussed as a differential diagnosis. PET images revealed a well-preserved glucose metabolism in the striatum, specifically in the putamen, while metabolism in the cerebellum was significantly reduced. This pattern of glucose metabolism might indicate a distinct subtype of synucleinopathy as proven by seed-amplification assay and should be taken into account when diagnosing patients with MSA.

Membrane protection against oxidative insults is achieved by the concerted action of glutathione peroxidase 4 (GPX4) and endogenous lipophilic antioxidants such as ubiquinone and vitamin E. More recently, ferroptosis suppressor protein 1 (FSP1) was identified as a critical ferroptosis inhibitor, acting via the regeneration of membrane-embedded antioxidants. Yet, regulators of FSP1 are largely uncharacterized, and their identification is essential for understanding the mechanisms buffering phospholipid peroxidation and ferroptosis. Here we report a focused CRISPR-Cas9 screen to uncover factors influencing FSP1 function, identifying riboflavin (vitamin B2) as a modulator of ferroptosis sensitivity. We demonstrate that riboflavin supports FSP1 stability and the recycling of lipid-soluble antioxidants, thereby mitigating phospholipid peroxidation. Furthermore, we show that the riboflavin antimetabolite roseoflavin markedly impairs FSP1 function and sensitizes cancer cells to ferroptosis. Our findings provide a rational strategy to modulate the FSP1-antioxidant recycling pathway and underscore the therapeutic potential of targeting riboflavin metabolism, with implications for understanding the interaction of nutrients, as well as their contributions to a cell's antioxidant capacity.

Metabolic reprogramming is known to critically contribute to therapy response in cancer, including the resistance of triple-negative breast cancer (TNBC) patients to conventional radio/(chemo)therapy. Our study, therefore, characterized the metabolic response of TNBC cell models to irradiation using untargeted metabolomics. We identified radiation-induced metabolic changes by harnessing a metabolite-gene interaction network comprising 44 genes. Nine genes with significant radiosensitizing potential in TNBC cells were identified by RNAi-screening. Among the druggable genes, high expression of pyruvate kinase M (PKM) was associated with worse overall survival in the METABRIC-TNBC cohort. Pharmacological PKM inhibition led to radiosensitization in TNBC cell models, which was attributed to S-phase related metabolic changes. Selective disruption of the S-phase by PKM inhibition led to both reduced DNA synthesis and increased replication stress, as evidenced by increased DNA damage at active replication forks. This resulted in prolonged cell cycle arrest after irradiation. Metabolic profiling upon PKM depletion and irradiation revealed two positively PKM-associated gene clusters, predominantly involved in the glycolysis pathway. Each cluster was functionally distinct, one shared the PKM-associated effects in survival reduction, while the other correlated with its radiosensitization. High expression of the first cluster was significantly correlated with worse overall survival, whereas high expression of the second cluster, consisting of PKM, ENO1, GAPDH and GPI, predicted poor response to radiotherapy in the METABRIC-TNBC cohort. Thus, our study suggests that glycolytic-associated metabolic reprogramming upon irradiation plays a major role in radioresistance of TNBC. Analysis of our identified glycolysis-related gene cluster in patients could predict the response to radiotherapy, and targeting PKM and the related glycolytic pathway holds promise for radiosensitization in TNBC patients.

Health services research faces the challenge of providing sound recommendations for action for the further development of health systems and care. The application of causal inference methods offers health services researchers an excellent opportunity to identify causal relationships under everyday conditions. The role of clinical trials with a classic randomised controlled trial (RCT) design is recognised as suitable for gaining insights that help establish causal inference, but other methodological approaches to generating evidence also play an important role in health services research.The discussion paper presents key concepts and assumptions of causal inference and highlights their relevance for health services research. The paper makes it clear that in order to fulfil the assumptions, it is necessary to integrate theory, contextual knowledge, understanding of mechanisms and formal concepts, such as directed acyclic graphs (DAGs), into a suitable empirical study design. To this end, RCTs, quasi-experimental methods, causal machine learning, target trial emulation, in silico trials and the mixed-methods approach of integrated inference are presented and discussed in terms of their applicability in health services research and their internal and external validity.All of the approaches presented here can contribute to the estimation of causal effects when used in a targeted manner and in accordance with the central assumptions. Their suitability depends largely on the research question, data quality, theoretical modelling and contextual knowledge. The combination of complementary designs and high quality data sources can increase the robustness of causal conclusions.Causal inference in health services research is not only a methodological procedure, but an integrative process that systematically combines theory, methodology and contextual knowledge. By consistently linking these aspects, health services research can generate differentiated and actionable insights that go beyond correlative analyses and enable an understanding of the mechanisms of causal processes. This can lead, for example, to evidence-supported recommendations that critically examine the often non-evidence-based status quo and reliably evaluate the benefits of new models.