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Scientific Unit
Background: Intima proliferation and in-stent restenosis is a challenging situation in interventional treatment of small vessel obstruction. Al/Al2O3 nanowires have been shown to accelerate vascular endothelial cell proliferation and migration in vitro, while suppressing vascular smooth muscle cell growth. Moreover, surface modification of Al/Al2O3 nanowires with poly[bis(2,2,2-trifluoromethoxy)phosphazene (PTFEP) coating enables further advantages such as reduced platelet adhesion. Therefore, the study's goal was to compare the biocompatibility of novel Al/Al2O3 + PTFEP coated nanowire bare-metal stents to uncoated control stents in vivo using optical coherence tomography (OCT), quantitative angiography and histomorphometric assessment. Methods: 15 Al/Al2O3 + PTFEP coated and 19 control stents were implanted in the cervical arteries of 9 Aachen minipigs. After 90 days, in-stent stenosis, thrombogenicity, and inflammatory response were assessed. Scanning electron microscopy was used to analyse the stent surface. Results: OCT analysis revealed that neointimal proliferation in Al/Al2O3 + PTFEP coated stents was significantly reduced compared to control stents. The neointimal area was 1.16 ± 0.77 mm2 in Al/Al2O3 + PTFEP coated stents vs. 1.98 ± 1.04 mm2 in control stents (p = 0.004), and the neointimal thickness was 0.28 ± 0.20 vs. 0.47 ± 0.10 (p = 0.003). Quantitative angiography showed a tendency to less neointimal growth in coated stents. Histomorphometry showed no significant difference between the two groups and revealed an apparent inflammatory reaction surrounding the stent struts. Conclusions: At long-term follow-up, Al/Al2O3 + PTFEP coated stents placed in peripheral arteries demonstrated good tolerance with no treatment-associated vascular obstruction and reduced in-stent restenosis in OCT. These preliminary in vivo findings indicate that Al/Al2O3 + PTFEP coated nanowire stents may have translational potential to be used for the prevention of in-stent restenosis.
Solution structure and synaptic analyses reveal determinants of bispecific T cell engager potency
(2025)
Bispecific T cell engagers (TcEs) link T cell receptors to tumor-associated antigens on cancer cells, forming cytotoxic immunological synapses (IS). Close membrane-to-membrane contact (≤13 nm) has been proposed as a key mechanism of TcE function. To investigate this and identify potential additional mechanisms, we compared four immunoglobulin G1-based (IgG1) TcE Formats (A–D) targeting CD3ε and Her2, designed to create varying intermembrane distances (A < B < C < D). Small-angle X-ray scattering (SAXS) and modeling of the conformational states of isolated TcEs and TcE–antigen complexes predicted close contacts (≤13 nm) for Formats A and B and far contacts (≥18 nm) for Formats C and D. In supported lipid bilayer (SLB) model interfaces, Formats A and B recruited, whereas Formats C and D repelled, CD2–CD58 interactions. Formats A and B also excluded bulky Quantum dots more effectively. SAXS also revealed that TcE–antigen complexes formed by Formats A and C were less flexible than complexes formed by Formats B and D. Functional data with Her2-expressing tumor cells showed cytotoxicity, surface marker expression, and cytokine release following the order A > B = C > D. In a minimal system for IS formation on SLBs, TcE performance followed the trend A = B = C > D. Addition of close contact requiring CD58 costimulation revealed phospholipase C-γ activation matching cytotoxicity with A > B = C > D. Our findings suggest that when adhesion is equivalent, TcE potency is determined by two parameters: contact distance and flexibility. Both the close/far-contact formation axis and the low/high flexibility axis significantly impact TcE potency, explaining the similar potency of Format B (close contact/high flexibility) and C (far contact/low flexibility).
The perceived time can shrink or expand for emotional stimuli. Converging evidence suggests that emotional time distortions are rooted in the emotional states of the timing agents because emotional stimuli can influence the timing of simultaneous neutral events. As emotional states are transitory, we investigated if time modulating emotional states also influence timing of subsequent neutral events. In each trial, we induced different valence and arousal levels by using affective vibrotactile patterns before participants judged the duration of neutral auditory tones. Compared to neutral patterns, affective patterns modulated participants’ time perception of the subsequent tones. We observed an interaction between arousal and valence: Pleasant-Low arousal patterns expanded the timing of subsequent neutral events more than Unpleasant-Low arousal patterns while Pleasant and Unpleasant-High arousal led to a similar temporal expansion. Our results indicate time modulating effects of emotional stimuli are due to changed emotional states and influence time perception likely until the underlying state decays.
Menadione as Antibiotic Adjuvant Against P. aeruginosa: Mechanism of Action, Efficacy and Safety
(2025)
Antibiotic resistance in chronic lung infections caused by Pseudomonas aeruginosa requires alternative approaches to improve antibiotic efficacy. One promising approach is the use of adjuvant compounds that complement antibiotic therapy. This study explores the potential of menadione as an adjuvant to azithromycin against planktonic cells and biofilms of P. aeruginosa, focusing on its mechanisms of action and cytotoxicity in pulmonary cell models. Methods: The effect of menadione in improving the antibacterial and antibiofilm potency of azithromycin was tested against P. aeruginosa. Mechanistic studies in P. aeruginosa and AZMr-E. coli DH5α were performed to probe reactive oxygen species (ROS) production and bacterial membrane disruption. Cytotoxicity of antibacterial concentrations of menadione was assessed by measuring ROS levels and membrane integrity in Calu-3 and A549 lung epithelial cells. Results: Adding 0.5 µg/mL menadione to azithromycin reduced the minimum inhibitory concentration (MIC) by four-fold and the minimum biofilm eradication concentration (MBEC) by two-fold against P. aeruginosa. Adjuvant mechanisms of menadione involved ROS production and disruption of bacterial membranes. Cytotoxicity tests revealed that antibacterial concentrations of menadione (≤64 µg/mL) did not affect ROS levels or membrane integrity in lung cell lines. Conclusions: Menadione enhanced the efficacy of azithromycin against P. aeruginosa while exhibiting a favorable safety profile in lung epithelial cells at antibacterial concentrations. These findings suggest that menadione is a promising antibiotic adjuvant. However, as relevant data on the toxicity of menadione is sparse, further toxicity studies are required to ensure its safe use in complementing antibiotic therapy.
NK cells are one of the major immune killer cell types exhibiting anti-tumor activity. During immune surveillance NK cells infiltrate into tissues and come in contact with cells and organs with varied stiffness. It has been shown that tumor cells with a lower elasticity modulus than their counterparts to have a higher metastatic potential. Whether the change in tumor cell stiffness affects the functionality of NK cells is not well understood. In this work, to test the effect of substrate stiffness on NK responses, PAAm-co-AA hydrogels of varied stiffness (2 kPa,12 kPa, 50 kPa) functionalized with biotinylated NKp46 activating antibody, prepared by Dr. Jingnan Zhang (research group of Prof. del Campo, INM-Leibniz Institute for New Materials) were used. Stiffness of substrate indeed played a huge role in modulating NK responses with stiffer substrates (12 kPa, 50 kPa) eliciting a stronger response in most donors whereas the soft substrates (2 kPa) failed to do so. To further test the impact of target cell stiffness on NK cytolytic activity, stiffness of target cells was altered using blebbistatin (made cells stiffer) and DMSO (made cells softer). Cytotoxicity of NK cells was boosted against stiffened tumor cells and impaired against softened tumor cells. In addition, the time required for NK cell to detach from the stiffened target cell after apoptosis or necrosis of the latter was significantly shorter, also contributing to a more effective cytotoxicity. To further decipher the role of mechanosensing in killing processes, functions of mechanosensitive ion channels was blocked using unspecific antagonizers (gadolinium and nifedipine), and it was found that blockage of mechanosensing substantially impaired NK mediated cytotoxicity as determined by 2D and 3D killing assays. Regarding the responsible mechanosensor, we have identified from the microarray data of our lab (by Dr. Eva Schwarz) that PIEZO1 are the predominately expressed mechanosensitive ion channels in NK cells. Blockade of PIEZO1 in NK cells by GsMTx4 impaired NK mediated cytotoxicity and activation of PIEZO1, using its specific agonist Yoda-1, potentiated NK mediated killing of the target cells. Blockade of PIEZO1 was shown to decrease the infiltration of NK cells into 3D collagen matrices, and activation of PIEZO1 boosted the infiltration of NK cells into 3D collagen matrix. As the role of PIEZO1 to be a major mechanosensor in NK cells was established, its role in sensing the stiffness of substrates was explored. Perturbation of PIEZO1 channels abrogated NK responses to substrate stiffness. Together, these data emphasize the role of mechanosensing in regulating NK cytotoxicity and the central role of tumor cell stiffness in evading immune surveillance. To fight cancer and other infective diseases, living therapeutic materials (LTMs) offer possibilities to release therapeutics in a sustainable and tunable manner. LTMs contain genetically engineered biological component encapsulated in a polymeric material such as hydrogels to contain their exposure in the host. LTMs are being extensively researched for their use in treatment of cancer with many studies reinforcing the beneficial effects of using smart materials. To contain the exposure of living component such as bacteria and to protect it from adverse environmental conditions of the host and to avoid a direct contact with immune cells, they are often encapsulated. However, one major concern for LTMs is that they may trigger an immune response and create a pro-inflammatory milieu in the host which could lead to critical situations if unregulated. So, the second part of my thesis is to characterize the immune response of PBMCs to PluDA hydrogel encapsulated E.coli and ClearColi bacteria. This work was carried out in collaboration with the group of Dr. Shrikrishnan Sankaran, Bioprogramable materials, INM-Leibniz Institute for New Materials, Saarbrücken. The ClearColi strain was produced from E.coli after genetically deleting LPS. ClearColi was encapsulated in Pluronic F127-based hydrogels (PluDA). It has to be noted that the bacteria were not in direct contact with the host so any immune reaction elicited would be due to the release of soluble factors and metabolites. The release of pro-inflammatory cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, TNFα and IFNγ) and cytotoxic proteins (granzyme A, granzyme B, perforin, granulysin, sFas, and sFasL) by PBMCs in response to bacteria and bacteria encapsulated gels was checked along with its influence on immune killer cells’ subtypes. Interestingly, PBMCs from the blood donors could be grouped in to two groups, donors with low spontaneous IL-2 and high spontaneous IL-2 release, based on the IL-2 release when PBMCs were cultured alone. Our results show that co-incubation of PluDA blank gels with PBMCs did not alter their profiles of cytokines and cytotoxic proteins, and had no influence on differentiation of NK cells, CD4+ and CD8+ T cells in donors with low spontaneous release of IL-2. ClearColi elicited release of IL-6 and IFNγ from PBMCs. Interestingly, the predominantly released cytokine was IL-6 for low spontaneous IL-2 release donors, but IFNγ for high spontaneous IL-2 release donors. Both the transwell condition and the encapsulated gel condition showed the same tendency. When the bacteria were in direct contact with the PBMCs they triggered the apoptosis of PBMCs on day 3 but encapsulation of the bacteria in PluDA gels completely abolished this effect.
Signal-amplifying Biohybrid Material Circuits for CRISPR/Cas-based single-stranded RNA Detection
(2024)
The functional integration of biological switches with synthetic building blocks enables the design of modular, stimulus-responsive biohybrid materials. By connecting the individual modules via diffusible signals, information-processing circuits can be designed. Such systems are, however, mostly limited to respond to either small molecules, proteins, or optical input thus limiting the sensing and application scope of the material circuits. Here, we design a highly modular biohybrid material based on CRISPR-Cas13a to translate arbitrary single-stranded RNAs into a biomolecular material response. We exemplify this system by the development of a cascade of communicating materials that can detect the tumor biomarker microRNA miR19b in patient samples or sequences specific for COVID-19. Specificity of the system is further demonstrated by discriminating between input miRNA sequences with single-nucleotide differences. To quantitatively understand information processing in the materials cascade, we developed a mathematical model. The model was used to guide systems design for enhancing signal amplification functionality of the overall materials system. The newly designed modular materials can be used to interface desired RNA input with stimulus-responsive and information-processing materials for building point-of-care suitable sensors as well as multi-input diagnostic systems with integrated data processing and interpretation.
Neutrophils play a crucial role in the tumor microenvironment (TME) of head and neck squamous cell carcinomas (HNSCC) and significantly influence treatment outcomes. Phenotypic and functional properties of neutrophils adapt to the TME with distinct subsets modulating disease progression and therapeutic interventions. Here, we evaluated phenotypic and functional differences of neutrophils derived from HNSCC patients and healthy donors. We observed significant phenotypic differences between neutrophils from healthy donors and HNSCC patient-derived neutrophils. Gender and tumor stage influenced neutrophil phenotypes and their ability to lyse tumor cells through antibody-dependent cell-mediated cytotoxicity (ADCC). Patients with advanced HNSCC and males may benefit less from neutrophil-centered immunotherapy. An engineered IgA2 antibody specific for the epidermal growth factor receptor (EGFR) demonstrated superior efficacy in activating neutrophils for ADCC compared to Panitumumab using healthy and patient-derived neutrophils, underscoring the potential of the IgA isotype as a therapeutic alternative. The distinct behavior and antibody-isotype dependent ADCC competence of CD177+/- neutrophils of healthy but not HNSCC donors warrants further exploration. Our study emphasizes the importance of personalized immunotherapy treatments that consider the characteristics of neutrophils, patient demographics, and the type of antibody to improve ADCC and ultimately enhance treatment outcomes for HNSCC.
Microfluidics plays a pivotal role in organ-on-chip technologies and in the study of synthetic cells, especially in the development and analysis of artificial cell models. However, approaches that use synthetic cells as integral functional components for microfluidic systems to shape the microenvironment of natural living cells cultured on-chip are not explored. Here, colloidosome-based synthetic cells are integrated into 3D microfluidic devices, pioneering the concept of synthetic cell-based microenvironments for organs-on-chip. Methods are devised to create dense and stable networks of silica colloidosomes, enveloped by supported lipid bilayers, within microfluidic channels. These networks promote receptor-ligand interactions with on-chip cultured cells. Furthermore, a technique is introduced for the controlled release of growth factors from the synthetic cells into the channels, using a calcium alginate-based hydrogel formation within the colloidosomes. To demonstrate the potential of the technology, a modular plug-and-play lymph-node-on-a-chip prototype that guides the expansion of primary human T cells by stimulating receptor ligands on the T cells and modulating their cytokine environment is presented. This integration of synthetic cells into microfluidic systems offers a new direction for organ-on-chip technologies and suggests further avenues for exploration in potential therapeutic applications.
Glioblastoma (GB) is the most common and aggressive brain tumor. The treatment for newly diagnosed glioblastoma is surgical resection of the primary tumor mass, followed by radiotherapy and chemotherapy. However, recurrences frequently occur in proximity to the surgical resection area. In these cases, none of the current therapies is effective. Recently, implantable biomaterials seem to be a promising strategy against GB recurrence. Here, for the first time we combined the tailorable properties of soy-protein hydrogels with the versatility of drug-loaded liposomes to realize a hybrid biomaterial for controlled and sustained nanoparticles release. Hydrogel consisting of 18–20 % w/v soy-protein isolated were fabricated in absence of chemical cross-linkers. They were biodegradable (−10 % and −30 % of weight by hydrolytic and enzymatic degradation, respectively in 3 days), biocompatible (>95 % of cell viability after treatment), and capable of sustained release of intact doxorubicin-loaded liposomes (diffusion coefficient between 10−18 and 10 −19 m2 s−1). A proof-of-concept in a “donut-like” 3D-bioprinted model shows that liposomes released by hydrogels were able to diffuse in a model with a complex extracellular matrix-like network and a 3D structural organization, targeting glioblastoma cells.The combination of nanoparticles' encapsulation capabilities with the hydrogels' structural support and controlled release properties will provide a powerful tool with high clinical relevance that could be applicable for the treatment of other cancers, realizing patient-specific interventions.
An unresolved issue in contemporary biomedicine is the overwhelming
number and diversity of complex images that require annotation, analysis and interpretation. Recent advances in Deep Learning have revolutionized the field of computer vision, creating algorithms that compete with human experts in image segmentation tasks. However, these frameworks require large human-annotated datasets for training and the resulting “black box” models are difficult to interpret. In this study, we introduce Kartezio, a modular Cartesian Genetic Programming-based computational strategy that generates fully transparent and easily interpretable image processing pipelines by iteratively assembling and parameterizing computer vision functions. The pipelines thus generated exhibit comparable precision to state-of-the-art Deep Learning approaches on instance segmentation tasks, while requiring drastically smaller training datasets. This Few-Shot Learning method confers tremendous flexibility, speed, and functionality to this approach. We then deploy Kartezio to solve a series of semantic and instance segmentation problems, and demonstrate its utility across diverse images ranging from multiplexed tissue histo-pathology images to high resolution microscopy images. While the flexibility, robustness and practical utility of Kartezio make this fully explicable evolutionary designer a potential game-changer in the field of biomedical image processing, Kartezio remains complementary and potentially auxiliary to mainstream Deep Learning approaches.
