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Diagnostic Performance of Non-Invasive Prenatal Testing for the Determination of Fetal Rh Blood Group Genotyping
No full textSeit etwa 25 Jahren besteht die Möglichkeit, zellfreie fetale DNA aus mütterlichem Blut nichtinvasiv auf genetisch determinierte Merkmale zu untersuchen. Damit werden invasive Verfahren wie Amniozentese und Chorionzottenbiopsie, die mit einem niedrigen Abortrisiko verbunden sind, in bestimmten Bereichen ergänzt, etwa bei der pränatalen Erkennung von Aneuploidien und fetalen genetischen Rh-Blutgruppenmerkmalen. Dies ist besonders relevant bei der Risikoevaluierung von Schwangerschaften, in denen die Mutter gegen ein Antigen des Rh-Blutgruppensystems immunisiert ist, da bei einem Fetus mit dem entsprechenden Antigen das Risiko für einen Morbus haemolyticus neonatorum besteht.
Die Einführung der pränatalen und postnatalen Anti-D-Prophylaxe führte zu einem deutlichen Rückgang der Anti-D-Sensibilisierungsrate. In Unkenntnis des pränatalen fetalen RhD-Status erhielt jedoch jede RhD-negative Schwangere eine ungezielte Anti-D-Prophylaxe, obgleich der Fetus in 40% der Fälle das RhD-Antigen nicht exprimierte. Die pränatale Bestimmung des fetalen RhD-Status (NIPT-RhD) ermöglicht den Übergang zu einer gezielten Anti-D-Prophylaxe, welche nur RhD-negative Mütter mit einem RhD-positiven Fetus erhalten.
Durch eine Änderung der Mutterschaftsrichtlinien wurde die pränatale Bestimmung des RhD-Status im Jahr 2021 in den Leistungskatalog der gesetzlichen Krankenkassen aufgenommen. Vor dieser Arbeit existierte kein kommerziell erhältliches Testkit, das der erhöhten Nachfrage an pränatalen RhD-Analysen gerecht werden konnte.
In einem retrospektiven Teil wurden 707 Patientinnen hinsichtlich des postnatalen blutgruppenserologischen Status der Rh-Antigene RhD, C, c und E der Neugeborenen befragt und das Ergebnis mit dem des nichtinvasiven Pränataltests verglichen. In 185 Fällen wurde das pränatale Ergebnis bestätigt, während ein falsch positiver RhD-Status sowie ein falsch negatives Ergebnis für das Rh-Antigen c festgestellt wurden. Letzteres konnte bei einer Wiederholungsuntersuchung zu einem späteren Zeitpunkt während derselben Schwangerschaft nachgewiesen werden, sodass auch diese Schwangerschaft engmaschig mit Doppler-Sonographie der Arteria cerebri media und Kontrollen des Anti-c-Titers überwacht wurde.
In einem prospektiven Teil wurde der fetale RhD-Status durch die Detektion der RHD Exons 5, 7 und 10 mittels des kommerziellen Testkits FetoGnost-Kit-RhD, nach der DNA-Isolation durch den Nukleinsäureextraktor QIAsymphony SP, bestimmt. Die Ergebnisse stimmten in allen 186 Fällen mit einem etablierten Referenztest überein. Im Anschluss wurden die Probandinnen nach dem Ergebnis des blutgruppenserologisch bestimmten postnatalen RhD-Status der Neugeborenen befragt. In einer weiterführenden Untersuchung wurde eine Grundgesamtheit von 1403 Frauen im Alter von 16 bis 47 Jahren (Median: 31 Jahre) betrachtet, die sich zum Zeitpunkt der Blutentnahme zwischen der 9. und 38. Schwangerschaftswoche (Median: 20. Schwangerschaftswoche) befanden. Sie wurden ebenfalls nach dem postnatalen RhD-Phänotyp der Neugeborenen befragt.
Von insgesamt 1696 Fällen lieferte die NIPT-RhD 1589 eindeutige Ergebnisse, die sich in 998 RhD-positive und 591 RhD-negative Befunde unterteilten. Aus 523 Rückmeldungen zum RhD-Status der Neugeborenen ergab sich eine Sensitivität von 99,68% (95%-Konfidenzintervall: 98,21% – 99,94%, n = 313) und eine Spezifität von 99,05% (95%-Konfidenzintervall: 96,59% – 99,74%, n = 210).
Ein falsch negatives Ergebnis ließ sich möglicherweise auf eine lange Transportzeit von sieben Tagen zurückführen, während zwei falsch positive Ergebnisse aus demselben Testlauf stammten, was die Wahrscheinlichkeit einer Kreuzkontamination deutlich erhöht. Insgesamt zeigte sich das FetoGnost-Kit-RhD in Kombination mit dem Nukleinsäureextraktor QIAsymphony SP als präzise und für große Probenserien geeignete Methode zur fetalen RhD-Bestimmung ab der 11+0 Schwangerschaftswoche.
Hinsichtlich der diagnostischen Genauigkeit zeigten sich keine signifikanten Unterschiede zwischen den einzelnen Schwangerschaftstrimestern. Die Methode erwies sich ab der 11+0 Schwangerschaftswoche als zuverlässig, während sich bei Proben vor der 8. Schwangerschaftswoche Limitationen zeigten. Längere Transportzeiten führten zu einer signifikant höheren Hämolyse-Rate, da 42 von 45 Proben (93,3%) mit Hämolyse eine Transportzeit von über fünf Tagen aufwiesen, und möglicherweise in einem Fall zu einem falsch negativen NIPT-RhD-Ergebnis. Daher wird auf Grundlage der gewonnenen Daten und im Kontext der Literatur eine maximale Transportzeit von fünf Tagen empfohlen.
In 1,2% aller Fälle wurden maternale oder fetale RHD Varianten identifiziert. Das Vorhandensein maternaler RHD Varianten wurde zuverlässig erkannt und führte in zwei Dritteln der Fälle zu einem eindeutigen fetalen RhD-Status auf Grundlage der RHD Exons, die in der maternalen DNA fehlten. Spike-Experimente deuteten darauf hin, dass die Multiplex-PCR hierfür ein sicheres Verfahren sein könnte. Ebenso wurden fetale RHD Varianten zuverlässig erkannt, jedoch ermöglichte die verwendete Methode keine eindeutige Aussage zum RhD-Phänotyp und der Immunogenität. Daher wird bei Nachweis einer fetalen RHD Variante eine Anti-D-Prophylaxe empfohlen. Eine Sequenzierung von DNA fetaler RHD Varianten aus maternalem Plasma mittels Next-Generation-Sequencing war nicht erfolgreich.
Eine Lagerung von Plasma von Schwangeren erwies sich bei 4°C für bis zu sieben Tage unproblematisch. Zum Zwecke der internen täglichen Qualitätskontrolle und für Ringversuche kann es bei -20°C mindestens 14 Monate sowie bei -80°C über mindestens 25 Monate gelagert werden.Over the past approximately 25 years, analysis of cell-free fetal DNA (cffDNA) from maternal plasma has enabled the non-invasive assessment of genetically determined fetal characteristics. Non-invasive prenatal testing (NIPT) complements invasive procedures such as amniocentesis and chorionic villus sampling, which are associated with a low but relevant risk of miscarriage, particularly in the prenatal detection of aneuploidies and fetal Rh blood group antigens. This approach is of particular clinical relevance in pregnancies complicated by maternal alloimmunization against Rh antigens, as antigen-positive fetuses are at risk of developing hemolytic disease of the fetus and newborn (HDFN).
The introduction of antenatal and postnatal anti-D immunoprophylaxis has led to a substantial reduction in anti-D alloimmunization rates. However, in the absence of prenatal knowledge of the fetal RhD status, all RhD-negative pregnant women historically received unselective anti-D prophylaxis, despite approximately 40% carrying an RhD-negative fetus. Prenatal determination of the fetal RhD status by NIPT enables a targeted anti-D prophylaxis strategy, limited to RhD-negative women carrying an RhD-positive fetus.
Following an amendment of the German maternity guidelines, prenatal RhD determination was included in the statutory health insurance benefit catalogue in 2021. At that time, no commercially available test kit was capable of meeting the increased demand for prenatal RhD analyses.
In a retrospective cohort, 707 pregnancies were evaluated by comparing NIPT results with postnatal serological typing of neonatal Rh antigens (RhD, C, c, and E). Prenatal results were confirmed in 185 cases; one false-positive RhD result and one false-negative result for the Rhc antigen were identified. The latter was detected upon repeat testing later in pregnancy, enabling close surveillance with middle cerebral artery Doppler ultrasound and anti-c antibody titer monitoring.
In the prospective part of the study, fetal RhD status was determined by detection of RHD exons 5, 7, and 10 using the commercial FetoGnost-Kit-RhD following DNA extraction with the QIAsymphony SP system. Complete concordance with an established reference assay was observed in all 186 cases. In an extended cohort of 1,403 women aged 16–47 years (median: 31 years) sampled between gestational weeks 9 and 38 (median: 20 weeks), postnatal RhD phenotypes of the neonates were obtained by follow-up questionnaires.
Of 1,696 total cases, NIPT-RhD yielded 1,589 conclusive results, comprising 998 RhD-positive and 591 RhD-negative findings. Based on 523 postnatal confirmations, sensitivity was 99.68% (95% CI: 98.21–99.94%, n = 313) and specificity was 99.05% (95% CI: 96.59–99.74%, n = 210). One false-negative result was potentially attributable to an extended sample transport time of seven days, while two false-positive results originated from the same analytical run, suggesting an increased likelihood of cross-contamination.
Overall, the FetoGnost-Kit-RhD in combination with the QIAsymphony SP demonstrated high analytical precision and suitability for high-throughput prenatal RhD determination from gestational week 11+0 onward. No significant differences in diagnostic accuracy were observed across pregnancy trimesters. Samples obtained before gestational week 8 showed methodological limitations. Prolonged transport times were associated with significantly increased hemolysis rates, with 93.3% of hemolytic samples exhibiting transport durations exceeding five days, potentially contributing to false-negative results. A maximum transport time of five days is therefore recommended.
Maternal or fetal RHD variants were identified in 1.2% of cases. Maternal RHD variants were reliably detected and allowed definitive fetal RhD classification in approximately two-thirds of cases based on exons absent in maternal DNA. Spike-in experiments suggested multiplex PCR as a robust approach in this context. Fetal RHD variants were also reliably identified; however, the applied methodology did not allow definitive prediction of RhD phenotype or immunogenicity. Consequently, anti-D prophylaxis is recommended when fetal RHD variants are detected. Attempts to sequence fetal RHD variants from maternal plasma using next-generation sequencing were unsuccessful.
Plasma samples from pregnant women could be stored at 4 °C for up to seven days without adverse effects. For internal quality control and external proficiency testing, storage was feasible for at least 14 months at −20 °C and over 25 months at −80 °C.2026-02-2
Der therapeutische Effekt von Rutin als Antioxidans und Proteindisulfidisomeraseinhibitor auf renale Fibrose
No full textChronic kidney disease (CKD) is a major health condition with a substantial impact on global mortality and morbidity. Pathophysiological, CKD culminates in renal fibrosis due to accumulation of extracellular matrix. Epithelial–mesenchymal transition (EMT), oxidative stress, the protein disulfide isomerase ERp57 and the TGFβ signaling pathway play central roles in this process. Rutin, a flavonoid, has antioxidant properties and the ability to inhibit protein disulfide isomerases such as ERp57. It therefore offers the possibility of being used therapeutically in the treatment of CKD. Under the influence of rutin, renal fibroblasts of the cell line TK173 after treatment with TGFβ, as well as fibrotic murine kidneys following unilateral ureteral obstruction (UUO), were investigated in order to assess the significance of this flavonoid for renal fibrosis in vivo and in vitro. An MTT assay showed that rutin, compared with the control group, had no significant effect on the morphology, cell viability, or metabolic activity of the cell line TK173. Western blot results indicated that rutin altered the proteome of cells under the influence of TGFβ, resulting in a statistically significant reduction in the expression of the examined vimentin (VIM) compared with the control group. Regarding the in vivo experiments, neither the tubular damage score, determined by PAS staining, nor the protein-positive area of Col1, pSmad2, and αSMA, determined by fluorescence microscopy, showed significant changes following treatment with rutin. Considering the results of qRT-PCR as in vivo processes, only gene Col4a1 showed reduced expression under rutin treatment, while transcription of the genes Pdia3, S100a4, and Itgav remained unaffected by the flavonoid. Overall, the results indicated a reduction of profibrotic processes under the influence of rutin only in certain aspects. The study ultimately could not clarify whether the antioxidant effect of rutin or its inhibitory effect on the protein disulfide isomerase ERp57 predominated. The complexity of renal fibrosis, including the TGFβ signaling pathway, requires further investigation and intensive research to identify suitable therapeutic approaches. It may be necessary to target multiple pathways to achieve observable effects. Nevertheless, rutin may still have sufficient potential to be a component of therapeutic strategies.2026-03-1
Effects of pro-inflammatory mediators on contraction in engineered skeletal muscle
No full textIdiopathic inflammatory myopathies, particularly inclusion body myositis, are characterized by chronic muscle inflammation, degenerative changes, and progressive muscle weakness. Pro-inflammatory cytokines are considered as one of the drivers of disease pathology, yet their direct impact on skeletal muscle contractile function remains incompletely understood. In this study, a contractile three-dimensional engineered skeletal muscle model derived from primary human myoblasts was established and compared with conventional two-dimensional myotube cultures. Exposure to interferon-γ and interleukin-1β induced a reproducible inflammatory phenotype in both model systems, reflected by upregulation of MHC-I and CXCL9, while myogenic differentiation and overall tissue morphology of the engineered muscles were largely preserved. Functional assessment of the three-dimensional muscles revealed alterations in contractile force, particularly reduced force at lower stimulation frequencies and after prolonged cytokine exposure, accompanied by changes in muscle fatigability, whereas maximal tetanic force was less markedly affected or even induced. These findings demonstrate that a pro-inflammatory environment can modulate skeletal muscle function and establish engineered skeletal muscle as a suitable platform for studying inflammation-associated muscle dysfunction in myositis.2026-03-1
Reversible cell cycle arrest as a strategy for chemoprotection: Studies in murine intestinal epithelial organoids
No full textDas Wirkprinzip der meisten klassischen Chemotherapeutika in der Krebstherapie, schnell proliferierende Zellen zu treffen, schädigt leider auch physiologisch proliferierendes Normalgewebe wie das Darmepithel. Die Folge sind schwere Nebenwirkungen wie Diarrhoen und Schmerzen. In dieser Arbeit wurde ein Schutzkonzept untersucht, indem die Proliferation des Darmepithels vor der Applikation des Chemotherapeutikums für kurze Zeit medikamentös angehalten wird. Durch die pharmakologische Aktivierung von denjenigen Zellzykluscheckpoints, die im Tumor defekt sind, kann selektiv gesundes Gewebe im Zellzyklus arretiert werden, während die Chemotherapie den weiterproliferierenden Tumor abtötet.
Um diese Strategie zum Schutz des Darmgewebe zu testen, wurde eine Organoidkultur des murinen Dünndarmepithels etabliert. Anschließend wurden Einzel- und Kombinationsbehandlungen mit Zellzyklusinhibitoren (Palbociclib, ALRN-6924) und Zytostatika (Gemcitabin, SN-38, Cisplatin) durchgeführt. Architektur, Viabilität und Wachstum der Organoide wurden mikroskopisch beobachtet und mit mehreren unabhängigen Methoden quantifiziert. Die Auswirkungen der Behandlung auf die Expression verschiedener Zellzyklusgene und Marker für DNA-Schäden und Apoptose wurden, ergänzt durch (immun)histologische Methoden, charakterisiert. Außerdem wurde die Wirkung der Behandlung auf Tumorzelllinien untersucht.
Zunächst wurde durch einen klinisch etablierten Inhibitor der Cyclin-abhängigen Kinase 4/6 (CDK4/6), Palbociclib, die Proliferation der Organoide reversibel angehalten. Nach Entfernung der Behandlung wuchsen die Organoide erneut weiter, wobei Zellzyklusmarker wieder normalisiert wurden. Dieser Zellzyklusarrest schützte die Organoide fast vollständig vor dem Nucleosidanalogon Gemcitabin und vor SN-38, dem Topoisomerase-I-hemmenden Metaboliten von Irinotecan, das sonst besonders schwere intestinale Nebenwirkungen hervorruft. Die Vorbehandlung reduzierte Marker für DNA-Schäden und Apoptose und ermöglichte ein deutlich verbessertes Auswachsen nach Therapieende. Gegen das weniger zellzyklusabhängige Cisplatin war hingegen keine Schutzwirkung nachweisbar. RB1-deletierte Krebszellen wurden durch Palbociclib ebenfalls nicht geschützt. Kurzzeitige Aktivierung von p53 durch den MDM2/MDM4-Inhibitor ALRN-6924, dessen Einsatz bei Patient*innen mit noch häufigeren TP53-mutierten Tumoren denkbar wäre, bewirkte zwar ebenfalls eine reversible Wachstumshemmung, zeigte aber im Gegensatz zu Palbociclib keine protektiven Effekte.
Die Ergebnisse demonstrieren, dass kurzzeitige CDK4/6-Hemmung das Darmepithel effektiv vor Chemotoxizität schützen kann – insbesondere in der Behandlung RB1-defizienter Tumore, die unter CDK4/6-Inhibition weiterproliferieren. Erste klinische Daten zur hämatopoetischen Chemoprotektion durch CDK4/6-Inhibition sind vielversprechend. Unsere Ergebnisse tragen zur Evidenz bei, dass auch gastrointestinale Nebenwirkungen zellzyklusspezifischer Chemotherapie auf diese Weise reduziert werden können.The principle behind most conventional chemotherapeutic agents used in cancer treatment, which is to target rapidly proliferating cells, unfortunately also damages physiologically proliferating normal tissue such as the intestinal epithelium. This results in severe side effects such as diarrhea and pain. This study investigated a protective concept in which the proliferation of the intestinal epithelium is temporarily halted pharmacologically before the application of chemotherapy. Through the pharmacological activation of cell cycle checkpoints that are defective in the tumor, healthy tissue can be selectively arrested in the cell cycle while chemotherapy kills the proliferating tumor.
To test this strategy for protecting intestinal tissue, an organoid culture of the murine small intestinal epithelium was established. Subsequently, single and combination treatments with cell cycle inhibitors (palbociclib, ALRN-6924) and cytostatic drugs (gemcitabine, SN-38, cisplatin) were performed. The architecture, viability, and growth of the organoids were observed microscopically and quantified using several independent methods. The effects of treatment on the expression of various cell cycle genes and markers for DNA damage and apoptosis were characterized using (immuno)histological methods. In addition, the effect of treatment on tumor cell lines was investigated.
First, the proliferation of the organoids was reversibly halted by a clinically established inhibitor of cyclin-dependent kinase 4/6 (CDK4/6), palbociclib. After removal of the treatment, the organoids continued to grow, with cell cycle markers returning to normal. This cell cycle arrest almost completely protected the organoids from the nucleoside analogue gemcitabine and from SN-38, the topoisomerase I-inhibiting metabolite of irinotecan, which otherwise causes particularly severe intestinal side effects. The pretreatment reduced markers for DNA damage and apoptosis and enabled significantly improved growth after the end of therapy. In contrast, no protective effect was detectable against the less cell cycle-dependent cisplatin. RB1-deleted cancer cells were also not protected by palbociclib. Short-term activation of p53 by the MDM2/MDM4 inhibitor ALRN-6924, which could be used in patients with even more common TP53-mutated tumors, also caused reversible growth inhibition, but unlike palbociclib, it showed no protective effects.
The results demonstrate that short-term CDK4/6 inhibition can effectively protect the intestinal epithelium from chemotoxicity, especially for the treatment of RB1-deficient tumors that continue to proliferate under CDK4/6 inhibition. Initial clinical data on hematopoietic chemoprotection through CDK4/6 inhibition look promising. Our results contribute to the evidence that gastrointestinal side effects of cell cycle-specific chemotherapy may also be reduced using this approach.2026-03-2
Roles of Kaposi’s Sarcoma-Associated Herpesvirus Glycoproteins in Viral Entry and Cell Tropism
No full textKaposi’s sarcoma–associated herpesvirus (KSHV) is an oncogenic γ-herpesvirus responsible for conditions such as Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman’s disease (MCD). It is also associated with KSHV inflammatory cytokine syndrome (KICS). The seroprevalence exhibits significant variability, ranging from approximately 20% to 80% in endemic regions such as sub-Saharan Africa, the Mediterranean Basin, and Xinjiang (China), while generally remaining below 10% in most Western nations, reflecting geographic disparities in disease prevalence. Additionally, region-specific indicators (e.g., a purported association with osteosarcoma in Xinjiang) are still exploratory and necessitate further validation and mechanistic investigation.
Despite decades of investigation, two entry checkpoints remained insufficiently characterized. Firstly, the major virion antigen K8.1 is reported to bind to cell surface heparan sulfate and be critical for B-cell infection; however, there is a lack of direct experimental proof regarding whether it mediates attachment, induces or enhances fusion, and whether its role extends to epithelial targets. Secondly, although KSHV mainly enters cells through endocytosis and macropinocytosis, and gB is recognized as the conserved fusogen, there was no definitive evidence showing that low pH directly triggers KSHV gB or that gB itself dictates the endocytosis route. This deficiency is partly attributable to numerous assays utilizing heterologous gB glycoproteins from other γ-herpesviruses, due to the limited fusogenic capability of KSHV gB in standard experimental systems. Clarification of these checkpoints is vital to better understand the molecular mechanisms dictating cell and tissue tropism, as well as to identify potential targets for intervention. This dissertation, therefore, establishes a unifying objective: to define KSHV early entry by exploring (i) a K8.1-dependent attachment checkpoint and (ii) a gB-mediated, low-pH fusion checkpoint, thereby elucidating cell-type tropism and route selection. To achieve this, I employed loss- and gain-of-function approaches, focusing on K8.1 (including a K8.1-knockout virus with genetic complementation). I utilized quantitative attachment and binding assays and conducted fusion tests with gH/gL and gB. Concurrently, we used a dual-fluorescent KSHV to quantify single-particle fusion, engineered a Rhesus monkey rhadinovirus (RRV) chimera carrying KSHV gB, and performed low-pH fusion assays to determine whether gB governs pH-dependent entry.
K8.1 was demonstrated to be critical for the infection of multiple epithelial cell lines (HaCaT, HEK-293T, A549), but it was not necessary in lymphatic and blood endothelial cells. I demonstrated that this phenotype results from K8.1 deletion was associated with the attachment phase (decreased virion attachment), and neither K8.1 nor its RRV homolog R8.1 initiated or amplified gH/gL–gB–mediated fusion. In co-culture experiments with B cells, KSHVΔK8.1 efficiently infected BJAB cells but showed impaired infection in MC116 cells, emphasizing a role beyond attachment. Collectively, these findings designate K8.1 as a cell-type–dependent factor, particularly important for epithelial cell infection. At the fusion checkpoint, single-particle imaging revealed that a low endosomatic pH is required for KSHV particle–cell fusion, with the “time-to-fusion” varying by target cell. Inhibiting vesicle acidification abrogated fusion, and replacing RRV gB with KSHV gB rendered RRV infection of B cells overexpressing a viral fusion receptor, EphB3, acidification-sensitive, demonstrating that KSHV gB dictates a low-pH endocytic route. Thus, KSHV gB is a pH-activated fusogen that commits KSHV to fusion from within acidified endosomes.
Integrating these findings yields a coherent entry model: K8.1 tunes attachment in a cell-type–specific manner upstream of receptor-driven uptake, and low endosomal pH activates gB to execute membrane fusion. This framework reconciles inconsistencies about K8.1’s function and the pH-dependence of KSHV fusion, explains aspects of tropism, and highlights entry-blocking opportunities at attachment (K8.1), uptake, and fusion (endosome acidification/gB triggering), informing future antiviral and vaccine strategies.2026-02-1
Immunocompetent Full-Skin Test System with Primary Immune Cells for Functional and Predictive Characterization of Contact Allergens
No full textThe skin is a complex organ composed of many different cell types that interact with each other, including various types of immune cells. Contact dermatitis is the most common skin disease and can be divided into irritative contact dermatitis (ICD) and allergic contact dermatitis (ACD). In Germany, 12.7 % of the population exhibits clinically relevant contact allergy, while sensitization rates reach 20 %. ACD is a T-cell-mediated, delayed-type hypersensitivity reaction of the skin to an external substance that usually requires repeated exposure to the allergen. This exposure activates epidermal antigen-presenting cells upon barrier penetration.
In light of the EU’s 2013 ban on animal testing for cosmetics and consumer products, full-thickness (FT) skin models that incorporate components of the immune system are a promising alternative. The first part of this thesis focuses on establishing the integration of primary immune cells into an FT skin model, consisting of primary fibroblasts. It describes the development and immunological characterization of such models, utilizing autologous primary keratinocytes and fibroblasts from the same donor. Barrier integrity was confirmed via filaggrin, involucrin, hornerin, and loricrin expression analysis, supported by histological evaluation. This expression was comparable to that of healthy human skin. Elevated expression of cytokeratin-6 and increased secretion of IL-6, CXCL-8, and CCL2 indicate a phenotype resembling early wound healing or inflammation.
A wide range of approaches for incorporating immune cells were assessed, including the addition of predifferentiated Langerhans cells (LCs) and naïve peripheral blood mononuclear cells (PBMCs), which together form a composite immune microenvironment. Flow cytometry and immunohistochemical staining (CD3, CD14, CD20, CD45, CD68) were used to confirm the quantification and localization of immune cells. LC epidermotropism was notably inhibited by the regularly added hydrocortisone in most culture media, a phenomenon that was reversible upon its withdrawal.
In the second phase, immunocompetent skin models were exposed to two important contact allergens, Lyral (a fragrance) and nickel sulfate (a metal), in two different vehicles (PBS, petrolatum). As skin-resident immune cells from allergic patients were not used, the focus was on activating healthy donor cells and evaluating the inflammatory potential of the test substances. Exposure to allergens resulted in the upregulation of T cell-activating cytokines (such as IL-1β, IL-12p70, IL-6), which was modulated by the application vehicle. Nickel exhibited stronger activation of immune cells when applied in PBS than in petrolatum. Significant variations were observed within the treatment groups, suggesting that the activation outcomes may vary due to different immune cells and keratinocyte donors.
Future work will evaluate both sensitization and elicitation phases, including pre-exposure and restimulation protocols. Incorporating patient-derived immune cells will enhance the model’s translational relevance. The established model could serve as a diagnostic platform and a non-animal testing system to evaluate allergenic and immunomodulatory properties of novel compounds in pharmaceutical and cosmetic development. Importantly, personalized medicine applications with patient-derived cells can be used to test for individually tailored therapeutic approaches. 2026-07-0
Magnetic Properties and Electron Transfer Coupled Spin Transitions of Molecular Prussian Blue Analogues
No full textPrussian Blue, discovered in the early 18th century, is renowned for its intense blue color arising from metal-to-metal charge transfer. Replacing one of the Fe centers with other transition metals produces Prussian Blue Analogues (PBAs), a versatile class of compounds with diverse magnetic and electronic properties. Among them, Fe–Co PBAs are particularly interesting because they can undergo electron transfer coupled spin transition (ETCST) processes, typically triggered by external stimuli such as light or temperature.
Despite extensive studies, the exact sequence of electron transfer (ET) and spin transfer (ST) in these systems remains unresolved. Conflicting results have been reported depending on the excitation wavelength and structural type. In particular, tetranuclear PBAs often show selective photoexcitation involving only one Fe–Co pair, raising the question of whether this is a general property or specific to certain systems. This challenge has motivated the exploration of simpler dinuclear PBAs, which may provide clearer insight into the underlying mechanism.
In this thesis, transient absorption spectroscopy and theoretical modeling are applied to study both tetranuclear and dinuclear PBAs. For the tetranuclear systems, cooperative effects in solution were observed for the first time, providing new insight into their dynamic behavior. To reduce the complexity of these architectures, new dinuclear PBAs were also designed using modified Tp- and pentadentate pyridyl-based ligand frameworks. These were developed to achieve transition temperatures closer to room temperature, making them suitable for detailed studies in both solid and solution states.
Overall, this work advances the understanding of ET and ST processes in cyanide-bridged systems and establishes design strategies for future applications in molecular electronics and photo-responsive materials.2026-07-0
Investigations of New Magnetic Resonance Contrast Agents
No full textMagnetic resonance spectroscopy (MRS) and imaging (MRI) are powerful noninvasive techniques for probing biochemical composition and anatomy. However, the inherently low sensitivity of these magnetic resonance techniques confines routine applications to conventional proton (1H) MRI of water and fat, with detection of other biologically relevant molecules hampered by strong background signals. Heteronuclear MRI expands the magnetic resonance toolbox by targeting nuclei beyond 1H that do not suffer from strong endogenous background signals, thereby enabling specific detection. Unlike conventional 1H MRI that mainly reflects total metabolite pools, this approach permits noninvasive tracking of dynamic metabolic fluxes and biodistribution. For 13C , the low natural abundance and sensitivity are overcome using hyperpolarization, which boosts signal intensity by several orders of magnitude and permits real-time in vivo metabolic measurements. Deuterium, while not hyperpolarized, offers short relaxation times for rapid signal averaging and can be referenced to the natural abundance of HDO water, enabling quantitative hotspot mapping. The first part of this thesis focuses on hyperpolarized 13C magnetic resonance. Distinct metabolic conversion patterns between pancreatic and colon tumor xenografts in mice were revealed by localized 13C MRS, which enabled determination of pyruvate to lactate and pyruvate to alanine conversion rate constants using hyperpolarized [1-13C]pyruvate. Building on this, the influence of extracellular acidosis on pancreatic cancer metabolism was
investigated by comparing acid adapted and control Panc02 tumors. Tumors adapted to acidic pH exhibited significantly higher glycolytic activity in vivo, as indicated by increased lactate production measured with hyperpolarized 13C magnetic resonance. Next, the focus shifted from intracellular metabolism to vascular imaging. For this purpose, a novel 13C -labeled angiographic agent, [1-13C]N-acetyl-alanine ethyl ester, was synthesized. As an endogenous metabolic end product, N-acetyl-alanine offers excellent biocompatibility for angiographic imaging. Hyperpolarization and imaging protocols were optimized, and single-shot 13C RARE imaging enabled high-resolution mapping of murine vascular structures with excellent temporal and spatial detail. The second part of this thesis explores deuterium MRI as a complementary modality. Deuterated particle imaging was developed using a new class of 2H-enriched imaging agents: fully acetylated PAMAM generation 5 dendrimers with deuterated methyl groups, yielding nanopolymers with exceptionally high 2H loading. High submillimeter resolution 2H MRI enabled quantitative renal uptake mapping in mice, with endogenous HDO serving as an internal reference. In a separate murine inflammation model, DPI revealed pronounced lymphatic accumulation of the dendrimers in the inflamed leg, demonstrating both anatomical and functional imaging capabilities.
Together, these projects showcase a comprehensive approach to advancing molecular imaging: from real-time metabolic measurements, to angiography, to quantitative anatomical and functional imaging. The strategic combination of molecular design and sequence optimization opens new pathways for metabolic imaging in vivo, establishing a foundation for future clinical translation of heteronuclear MRI.2026-10-1
Immunhistochemical markers for discrimination between pancreatic ductal adenocarcinoma and cholangiocarcinoma
No full textThe distinction between pancreatic ductal adenocarcinoma (PDAC) and cholangiocarcinoma (CCA) is clinically important, as it significantly influences therapeutic decisions, including surgical and adjuvant strategies. Currently, no immunohistochemical biomarkers offer sufficient sensitivity and specificity to reliably differentiate between these tumor entities. Therefore, we conducted an immunohistochemical comparison of PDAC and CCA based on three different biomarkers.
In this study, three proteins—Cathepsin E (CTSE), SH2 Domain Containing 7 (SH2D7), and S100 calcium-binding protein A10 (S100A10) were selected from proteomic data because they were described as moderately to highly expressed in PDAC.
For a comparison of PDAC and CCA, immunohistochemical analysis of tissue microarrays including around 531 PDAC and 70 CCA samples was performed.
CTSE showed positive immunoreactivity in 69.1% of PDAC cases and 25.9% of CCA cases. SH2D7 expression was observed in 18% of PDAC cases, but only in 1.7% of CCA cases. S100A10 was detected in 92.5% of PDAC and 77.8% of CCA samples. Comparative H-score analysis revealed statistically significant differences for CTSE, S100A10 and SH2D7 between PDAC and CCA.
Although H-score analysis revealed statistically significant differences for CTSE and S100A10 between PDAC and CCA, the practical diagnostic value of these markers in routine clinical settings appears to be limited. SH2D7, in particular, demonstrated low sensitivity for PDAC but may still represent a valuable biomarker for differentiating PDAC from CCA due to its high specificity.2026-02-1
Frequency of Intraoperative and Early Postoperative Bleeding Complications in Glaucoma Surgery in Relation to Antithrombotic Medication
No full textWith the increasing age of the population, the number of patients requiring ophthalmologic care continues to rise. Comorbidities and pre-existing conditions often necessitate treatment with antithrombotic medication, which, in the context of ophthalmic surgery, raises concerns regarding perioperative bleeding complications. While guidelines and explicit recommendations have been established in many surgical disciplines, no uniform consensus exists in ophthalmology, particularly in glaucoma surgery, regarding the perioperative management of antithrombotic agents. Consequently, ophthalmologists lack a clear decision-making framework. The present study addresses this clinical uncertainty. Although hemorrhagic events in ophthalmology are rarely hemodynamically relevant, they may nonetheless threaten visual acuity and compromise surgical outcomes. At the same time, discontinuation of antithrombotic therapy must be carefully weighed against the risk of thromboembolic events during and after surgery. To generate robust data, patients who underwent elective glaucoma surgery between 2012 and 2017 were retrospectively analyzed. This observational study evaluated data from 376 patients who underwent trabeculectomy, filtration surgery with drainage implants (Baerveldt or Ahmed), or trabeculotomy. The primary focus was the perioperative management of antithrombotic medication and the occurrence of intraoperative or early postoperative bleeding complications. Additionally, the need for reoperation due to bleeding was assessed. For analytical purposes, patients were categorized into four groups according to their antithrombotic therapy. Overall, 7 intraoperative and 56 early postoperative bleeding complications were observed. Only one bleeding event required reoperation and resulted in permanent visual loss. Patients undergoing angle-based glaucoma surgery exhibited a higher rate of bleeding complications compared to those undergoing filtration procedures. No significant association was found between perioperative bleeding complications and patient sex, age, preoperative intraocular pressure, INR values, or type of anesthesia. However, patients receiving antithrombotic medication demonstrated an increased likelihood of hemorrhagic events. The perioperative discontinuation or continuation of antithrombotic therapy did not reveal a clinically significant difference in bleeding risk. These findings raise the question of whether additional factors not examined in this study, such as vascular status or comorbid conditions, may contribute to bleeding risk. Further studies are required to better elucidate these associations.2026-02-0