@article{LauMaierBrauneetal.2021, author = {Lau, Skadi and Maier, Anna and Braune, Steffen and Gossen, Manfred and Lendlein, Andreas}, title = {Effect of endothelial culture medium composition on platelet responses to polymeric biomaterials}, series = {International journal of molecular sciences}, volume = {22}, journal = {International journal of molecular sciences}, number = {13}, publisher = {Molecular Diversity Preservation International}, address = {Basel}, issn = {1422-0067}, doi = {10.3390/ijms22137006}, pages = {13}, year = {2021}, abstract = {Near-physiological in vitro thrombogenicity test systems for the evaluation of blood-contacting endothelialized biomaterials requires co-cultivation with platelets (PLT). However, the addition of PLT has led to unphysiological endothelial cell (EC) detachment in such in vitro systems. A possible cause for this phenomenon may be PLT activation triggered by the applied endothelial cell medium, which typically consists of basal medium (BM) and nine different supplements. To verify this hypothesis, the influence of BM and its supplements was systematically analyzed regarding PLT responses. For this, human platelet rich plasma (PRP) was mixed with BM, BM containing one of nine supplements, or with BM containing all supplements together. PLT adherence analysis was carried out in six-channel slides with plasma-treated cyclic olefin copolymer (COC) and poly(tetrafluoro ethylene) (PTFE, as a positive control) substrates as part of the six-channel slides in the absence of EC and under static conditions. PLT activation and aggregation were analyzed using light transmission aggregometry and flow cytometry (CD62P). Medium supplements had no effect on PLT activation and aggregation. In contrast, supplements differentially affected PLT adherence, however, in a polymer- and donor-dependent manner. Thus, the use of standard endothelial growth medium (BM + all supplements) maintains functionality of PLT under EC compatible conditions without masking the differences of PLT adherence on different polymeric substrates. These findings are important prerequisites for the establishment of a near-physiological in vitro thrombogenicity test system assessing polymer-based cardiovascular implant materials in contact with EC and PLT.}, language = {en} } @article{MoradianRochAnthoferetal.2022, author = {Moradian, Hanieh and Roch, Toralf and Anthofer, Larissa and Lendlein, Andreas and Gossen, Manfred}, title = {Chemical modification of uridine modulates mRNA-mediated proinflammatory and antiviral response in primary human macrophages}, series = {Molecular therapy}, volume = {27}, journal = {Molecular therapy}, publisher = {Cell Press}, address = {Cambridge}, issn = {2162-2531}, doi = {10.1016/j.omtn.2022.01.004}, pages = {854 -- 869}, year = {2022}, abstract = {In vitro transcribed (IVT)-mRNA has been accepted as a promising therapeutic modality. Advances in facile and rapid production technologies make IVT-mRNA an appealing alternative to protein- or virus-based medicines. Robust expression levels, lack of genotoxicity, and their manageable immunogenicity benefit its clinical applicability. We postulated that innate immune responses of therapeutically relevant human cells can be tailored or abrogated by combinations of 5'-end and internal IVT-mRNA modifications. Using primary human macrophages as targets, our data show the particular importance of uridine modifications for IVT-mRNA performance. Among five nucleotide modification schemes tested, 5-methoxy-uridine outperformed other modifications up to 4-fold increased transgene expression, triggering moderate proinflammatory and non-detectable antiviral responses. Macrophage responses against IVT-mRNAs exhibiting high immunogenicity (e.g., pseudouridine) could be minimized upon HPLC purification. Conversely, 5'-end modifications had only modest effects on mRNA expression and immune responses. Our results revealed how the uptake of chemically modified IVT-mRNA impacts human macrophages, responding with distinct patterns of innate immune responses concomitant with increased transient transgene expression. We anticipate our findings are instrumental to predictively address specific cell responses required for a wide range of therapeutic applications from eliciting controlled immunogenicity in mRNA vaccines to, e.g., completely abrogating cell activation in protein replacement therapies.}, language = {en} } @article{LauLiuMaieretal.2021, author = {Lau, Skadi and Liu, Yue and Maier, Anna and Braune, Steffen and Gossen, Manfred and Neffe, Axel T. and Lendlein, Andreas}, title = {Establishment of an in vitro thrombogenicity test system with cyclic olefin copolymer substrate for endothelial layer formation}, series = {MRS communications / a publication of the Materials Research Society}, volume = {11}, journal = {MRS communications / a publication of the Materials Research Society}, number = {5}, publisher = {Springer}, address = {Berlin}, issn = {2159-6867}, doi = {10.1557/s43579-021-00072-6}, pages = {559 -- 567}, year = {2021}, abstract = {In vitro thrombogenicity test systems require co-cultivation of endothelial cells and platelets under blood flow-like conditions. Here, a commercially available perfusion system is explored using plasma-treated cyclic olefin copolymer (COC) as a substrate for the endothelial cell layer. COC was characterized prior to endothelialization and co-cultivation with platelets under static or flow conditions. COC exhibits a low roughness and a moderate hydrophilicity. Flow promoted endothelial cell growth and prevented platelet adherence. These findings show the suitability of COC as substrate and the importance of blood flow-like conditions for the assessment of the thrombogenic risk of drugs or cardiovascular implant materials.}, language = {en} } @article{PeterWenderingSchlickeiseretal.2022, author = {Peter, Lena and Wendering, D{\´e}sir{\´e}e Jacqueline and Schlickeiser, Stephan and Hoffmann, Henrike and Noster, Rebecca and Wagner, Dimitrios Laurin and Zarrinrad, Ghazaleh and M{\"u}nch, Sandra and Picht, Samira and Schulenberg, Sarah and Moradian, Hanieh and Mashreghi, Mir-Farzin and Klein, Oliver and Gossen, Manfred and Roch, Toralf and Babel, Nina and Reinke, Petra and Volk, Hans-Dieter and Amini, Leila and Schmueck-Henneresse, Michael}, title = {Tacrolimus-resistant SARS-CoV-2-specific T cell products to prevent and treat severe COVID-19 in immunosuppressed patients}, series = {Molecular therapy methods and clinical development}, volume = {25}, journal = {Molecular therapy methods and clinical development}, publisher = {Cell Press}, address = {Cambridge}, issn = {2329-0501}, doi = {10.1016/j.omtm.2022.02.012}, pages = {52 -- 73}, year = {2022}, abstract = {Solid organ transplant (SOT) recipients receive therapeutic immunosuppression that compromises their immune response to infections and vaccines. For this reason, SOT patients have a high risk of developing severe coronavirus disease 2019 (COVID-19) and an increased risk of death from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Moreover, the efficiency of immunotherapies and vaccines is reduced due to the constant immunosuppression in this patient group. Here, we propose adoptive transfer of SARS-CoV-2-specific T cells made resistant to a common immunosuppressant, tacrolimus, for optimized performance in the immunosuppressed patient. Using a ribonucleoprotein approach of CRISPR-Cas9 technology, we have generated tacrolimus-resistant SARS-CoV-2-specific T cell products from convalescent donors and demonstrate their specificity and function through characterizations at the single-cell level, including flow cytometry, single-cell RNA (scRNA) Cellular Indexing of Transcriptomes and Epitopes (CITE), and T cell receptor (TCR) sequencing analyses. Based on the promising results, we aim for clinical validation of this approach in transplant recipients. Additionally, we propose a combinatory approach with tacrolimus, to prevent an overshooting immune response manifested as bystander T cell activation in the setting of severe COVID-19 immunopathology, and tacrolimus-resistant SARS-CoV-2-specific T cell products, allowing for efficient clearance of viral infection. Our strategy has the potential to prevent severe COVID-19 courses in SOT or autoimmunity settings and to prevent immunopathology while providing viral clearance in severe non-transplant COVID-19 cases.}, language = {en} }