Michael Bauer

Efficient, biocompatible, and flexible application of CRISPR‐Cas9 remains a major challenge in gene therapy. This study addresses a novel formulation method, focusing on organic solvent‐free lipid nanoparticles for the efficient delivery of CRISPR‐Cas9 components. Therefore, novel stealth lipids comprising the lipid anchor cholesterol hemisuccinate conjugated to poly(2‐ethyl‐2‐oxazoline) (PEtOx) were synthesized and evaluated as a promising in vitro alternative to commercially available poly(ethylene glycol) (PEG)‐based lipids. Our study identified PEtOx‐lipids as markedly more biocompatible compared to PEG‐based analogues (12‐fold reduction in cytotoxicity; 99% reduced hemolytic effect) with high efficiency for mRNA gene delivery. We demonstrate successful CRISPR‐Cas9 activity ex vivo and efficient uptake of PEtOx nanoparticles by macrophages in vivo (66.5% pos. F4/80 ⁺ macrophages). Our data demonstrate that PEtOx‐based stealth lipids are a promising alternative to PEG. The resulting LNPs, optimized for fully aqueous manufacturing, address key limitations of current LNP systems, including immunogenicity and the reliance on ethanol‐based production processes.

ZUSAMMENFASSUNG: Eine effiziente, biokompatible und flexible Anwendung von CRISPR‐Cas9 stellt weiterhin eine wesentliche Herausforderung in der Gentherapie dar. Die vorliegende Studie untersucht eine neuartige Formulierungsmethode mit Fokus auf lösungsmittelfreien Lipidnanopartikeln zur effizienten Verabreichung von CRISPR‐Cas9‐Komponenten. Hierzu wurden neuartige Stealth‐Lipide synthetisiert, die auf dem Lipidanker Cholesterinhemisuccinat basieren und mit Poly(2‐ethyl‐2‐oxazolin) (PEtOx) konjugiert wurden. Diese stellen in vitro eine vielversprechende Alternative zu kommerziell verfügbaren Lipiden auf Basis von Polyethylenglykol (PEG) dar. Unsere Studie identifizierte PEtOx‐Lipide als deutlich biokompatibler im Vergleich zu PEG‐basierten Analoga, mit einer 12‐fachen Reduktion der Zytotoxizität und einer um 99 % reduzierten hämolytischen Wirkung bei gleichzeitig hoher Effizienz für den mRNA Gentransfer. Darüber hinaus konnten wir eine erfolgreiche CRISPR‐Cas9‐Aktivität ex vivo sowie eine effiziente Aufnahme von PEtOx‐Nanopartikeln durch Makrophagen in vivo nachweisen (66,5 % F4/80 ⁺ ‐Makrophagen). Unsere Daten zeigen, dass PEtOx‐basierte Stealth‐Lipide eine vielversprechende Alternative zu PEG darstellen. Die resultierenden LNPs, die für eine vollständig wässrige Herstellung optimiert wurden, adressieren zentrale Limitationen bestehender LNP‐Systeme, darunter die Immunogenität und die Abhängigkeit von ethanolbasierten Herstellungsverfahren.

Zusammenfassung: Vor 90 Jahren erschien in Nature eine bahnbrechende Arbeit des Endokrinologen Hans Selye, in der er eine umfassende Theorie des allgemeinen Anpassungssyndroms („general adaptation syndrome“) von Organismen auf schädigende Reize entwickelte. Selye beobachtete an Ratten, die er unterschiedlichen physikalischen oder chemischen Noxen unterwarf, stereotype hormonelle Reaktionen und führte in späteren Arbeiten den heute ubiquitär verwendeten Begriff „Stress“ hierfür ein. Dabei entlehnte er den Begriff aus der Physik („stress and strain“), wo er die Verformung eines Festkörpers durch einwirkende Kräfte beschreibt. Selye räumte später ein, dass es im biomedizinischen Kontext besser gewesen wäre, statt der auslösenden Faktoren „Stress“ die Belastungs effekte – also „strain“ – zur Beschreibung des allgemeinen Anpassungssyndroms zu nutzen. Analog zum Überdehnen eines Festkörpers bis zum Bruch, wird beim Überschreiten der Reparatur- und Defensivsysteme lebender Organismen ein „point of no return“ erreicht, der durch Organschäden bis hin zum Tod charakterisiert ist. Selyes Forschung hat grundlegend zum Verständnis der Schädigungsreaktionen gerade auch in der Intensivmedizin beigetragen. So lassen sich z. B. traumainduzierte Stresseffekte in chirurgischen Patienten durch Analgesie und Sedierung modulieren. In der vorliegenden Übersicht sollen die Fortschritte in der medizinischen Stressforschung, insbesondere das Konzept der Allostase und der Hormese sowie deren zugrunde liegenden molekularen Mechanismen für das ärztliche Handeln am Beispiel des Organversagens bei Sepsis erläutert werden.

With its high molecular complexity, human breath lends itself as an information-rich sample for non-invasive biomedical diagnostics. Among emerging multi-variate analytical techniques for human breath, laser-based optical spectroscopy constitutes a particularly promising novel combination of short measurement times and high concentration dynamic range for molecular detection. Here, we present an automated system designed for continuous collection of exhaled breath directly from a respirator, and subsequent spectroscopic analysis. By obviating any additional intermediate sample storage or transport, our method minimizes setup complexity and sample contamination, while avoiding interference with the operation of the respirator. In this proof-of-principle study, we present automated and quantitative monitoring of three substances introduced in an artificial test lung ventilated by the respirator. Employing a standard Fourier-transform infrared spectrometer for molecular quantification, individual measurements were performed every 7 min over 24 h. The stability and analytical capability of the breath sampling system presented here, along with improvements in terms of spectral resolution and detection dynamic range obtainable through state-of-the-art laser-based spectroscopy promise fast and quantitative monitoring of the human breath volatilome. Coupled to state-of-the-art spectroscopy this may pave the way to monitor metabolites in real time in critically ill patients over the entire physiologically relevant molecular concentration range.

Background: Functional impairments associated with mental health conditions are on the rise. Predicting functional outcomes may improve the targeting of preventive interventions. While prognostic models have primarily focused on psychosis, early recognition services require a transdiagnostic approach. Objective: This study aimed to predict global functioning within a 2-year follow-up using baseline clinical and structural magnetic resonance imaging (MRI) data in a population-based sample of young, help-seeking individuals presenting with affective and anxiety symptoms as well as attention-deficit hyperactivity disorder. Methods: We classified 357 help-seeking individuals aged 18‐35 years recruited from 9 sites as “impaired” (Global Assessment of Functioning [GAF] ≤60; n=228) or “nonimpaired” (GAF>60; n=129) at year 1 and/or year 2 follow-up. GAF classification group status at follow-up was predicted using linear support vector machine (SVM), decision tree, and large language model (LLM) Llama-3 using clinical assessments and/or structural MRI. Leave-one-site-out (SVM) or external sample (LLM) was used for validation. Results: SVM achieved balanced accuracy of 69.2% using clinical features only. Items related to baseline occupational functioning, interpersonal relationships, cognitive functioning, psychotic and affective symptoms, as well as the presence of anxiety disorder, were most predictive. The decision tree further reduced the feature set to 5 predictive items, achieving balanced accuracy of 76.6%. Although amygdala and hippocampal subregions achieved balanced accuracy of 57.1%, structural MRI did not improve the overall prediction. Llama-3 performed comparably well to SVM (balanced accuracy of 72.6%). Conclusions: Machine learning demonstrated good performance in predicting global functioning. Interestingly, the out-of-the-box LLM performed comparably well without being trained or fine-tuned, highlighting the potential of leveraging free-text data for mental health prognosis.

OBJECTIVE: Bile acids that induce immunosuppression through activation of the Takeda G protein-coupled receptor 5 (TGR5) have recently been identified in the circulation of patients with liver failure and sepsis. These bile acids are associated with increased susceptibility to infection and a high short-term mortality. Furthermore, TGR5 activation by bile acids has been implicated in the development of pruritus. However, therapies targeting TGR5-activating bile acids in patients have not yet been reported. This study investigates the effects of two artificial liver support systems, the Molecular Adsorbent Recirculating System (MARS) and the open albumin dialysis (OPAL). DESIGN: Proof of concept study, multicenter study. SETTING: University Hospital Jena, University Hospital Ulm, and University Hospital Münster, Germany. PATIENTS: Patients with severe liver failure, as defined by the Sequential Organ Failure Assessment hepatic sub-score (i.e., bilirubin > 12 mg/dL). INTERVENTIONS: Patients with severe liver failure were randomized to receive one session of MARS or OPAL for 8 hours, followed by crossover to the other treatment on the next day. MEASUREMENTS AND MAIN RESULTS: Bilirubin, circulating total and individual bile acids, albumin function, and TGR5 activation induced by circulating bile acids were assessed before and after each treatment session. Both OPAL and MARS significantly removed bilirubin, as well as individual and total bile acids from circulation. OPAL was superior to MARS in removing protein-bound lipophilic bile acids and restoring patients’ albumin function (i.e., albumin-binding capacity, detoxification efficiency, and binding efficiency). TGR5 activation induced by circulating bile acids was reduced by 60% with OPAL compared with 39% with MARS (median values, p = 0.051). CONCLUSIONS: Artificial liver support systems effectively remove immunosuppressive bile acids from circulation. The clinical implications of bile acid reduction, however, require further study.

SARS-CoV-2 infection affects multiple immune mechanisms and leads to severe COVID-19 and death, in part related to infection-induced or pre-existing autoantibodies. Here, we describe severe COVID-19 to associate with autoantibodies against interleukin-1 receptor antagonist (IL-1Ra) and progranulin (PGRN), endogenous antagonists of IL-1 and TNF signaling, respectively. These autoantibodies coincide with hyperphosphorylation of IL-1Ra (Thr111) or PGRN (Ser81), form immune complexes independent of phosphorylation, reduce antigen plasma levels, and permit enhanced IL-1 and TNF signaling. Using phage-display selected Fabs specific for hyperphosphorylated isoforms, we track phospho-antigens and autoantibodies in a German national pandemic network cohort. Most seropositive patients show both autoantibodies. Levels peak at baseline and decline over 12 months, with phospho-antigens decreasing before autoantibodies. Seropositivity associates with hyperinflammation and cytokine profiles. Importantly, signaling by key inflammatory cytokines induce IL-1Ra and PGRN hyperphosphorylation in healthy monocytes, but require up to 1000-fold higher doses than in monocytes from previously seropositive severe COVID-19 survivors.

Background: The COVID-19 pandemic has impacted health systems globally, including emergency medical services (EMS). This study evaluates the absorptive and adaptive resilience of EMS in Jena, Germany, by analysing how the system managed significant pandemic-induced shocks to demand and operational workflows. Methods: We conducted an observational, retrospective analysis of EMS missions in Jena from January 2018 to December 2021. Data on mission volumes, patient demographics, health issues, and dispatch and treatment intervals were analysed. Lockdown periods were compared to corresponding pre-pandemic reference periods to determine significant differences. Results: During first and second lockdown EMS mission volume decreased by 16% and 10% respectively, when comparing with reference periods. Patient age increased, with a mean age of 62.2 years during the first lockdown versus 58.9 years in the reference period. There was a notable rise in on-site treatments without hospital transport. Cardiovascular cases decreased during lockdown periods, while occurrence of psychiatric patients increased. Scene times and handover times extended significantly, contributing to longer total mission times. Conclusions: The pandemic led to shift in demand patterns, characterized by reduced overall demand, extended mission times, and a change in treatment focus from cardiovascular to psychiatric and psychosocial issues, reflecting changes in healthcare-seeking behaviour and operational challenges. While regional studies have examined mission volumes, our study provides a higher-resolution analysis of the effects of the COVID-19 pandemic on pre-clinical EMS in Germany and provides important insights to be used for health care planning and policy decision-making.