Biology subjects

Thiel, S.

Publications and source records attributed to Thiel, S..

4 recordsLinked to original sources

bioRxiv2025

Targeting Langerhans cells using a modular mannosylated nucleic acid-based vaccine platform

The skin is a diverse reservoir of immune cells with strong potential for immunotherapeutic delivery. Langerhans cells (LCs) 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 LCs. 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 LCs. Finally, in an antigen-presentation assay, in vitro differentiated LCs loaded with mannosylated and peptide-conjugated scaffolds significantly enhanced T cell activation. Overall, our study presents a promising nucleic acid-based platform for precise LC targeting and drug delivery, with broad potential for skin-directed immunotherapies.

molecular biology↗

bioRxiv2025

Galactan mobilization during carbon starvation compromises cell wall-mediated fungal resistance in Arabidopsis

Polysaccharides are the main components present in plant cell walls. They form a network that is dynamically modified during growth and upon both abiotic and biotic stress. We investigated how the cell wall of Arabidopsis rosettes is remodeled during periods of dark-induced starvation in the wild type and in plastidic phosphoglucomutase (pgm) mutants, which suffer from periodic starvation due to starch deficiency. Time-course analysis demonstrated that up to one fifth of the galactose present in leaf cell walls is reversibly released upon starvation, while other cell wall monosaccharides were less affected. An investigation of BETA-GALACTOSIDASE (BGAL) expression and the analysis of bgal mutants indicated that BGAL1 and BGAL4 contribute to the release of cell wall galactose upon starvation. Increased transcript abundance of UDP-GLUCOSE 4-EPIMERASE (UGE) 1 and 3 under starvation proposed an increased flux through the galactose salvage pathway, however an analysis of the UDP- galactose pool in mutant plants indicated redundancy with other UGEs. Simultaneously to galactan degradation, GALACTAN SYNTHASE1 (GALS1) expression was reduced, attenuating the synthesis of new galactan chains. We show that overexpression of GALS1 prevents depletion of the recyclable cell wall galactose pool and is sufficient to rescue impaired penetration resistance to the hemibiotrophic fungal pathogen Colletotrichum higginsianum upon dark-induced and periodic starvation. Our data suggest that pectic galactan in the plant cell wall serves as a sugar resource during starvation conditions. However, galactose release from the wall leads to impaired penetration resistance against a fungal pathogen, causing a tradeoff between sugar supply for plant metabolism and preformed defense. Significance statementPolysaccharides present in plant cell walls must be dynamically modified in response to external stimuli, but it is not well understood how cell wall remodeling is adjusted in response to metabolic cues and how this affects defense against pathogens. Here, we report that galactose is reversibly released from cell walls upon carbon starvation, which critically affects penetration resistance to a fungal pathogen.

plant biology↗

bioRxiv2025

Lock, Protect, and Bind: In Vitro Selection of LNA-modified Aptamers Using a Mutant T7 RNA Polymerase

RNA therapeutics are powerful tools for gene modulation and targeted therapies, but their clinical application is hindered by nuclease degradation and immunogenicity. Incorporating chemical modifications, like locked nucleic acids (LNAs), can enhance nuclease resistance, targeting properties, and thermal stability. Traditionally, LNA incorporation has relied on solid-phase synthesis of short RNAs. Engineered polymerases capable of incorporating xenonucleic acids (XNAs), including LNA, into longer RNAs have been described. However, their XNA yield is limited by primer and template copy numbers, and the generated DNA-XNA duplexes can be difficult to purify. We present a novel approach for incorporating LNA-ATP and LNA-TTP alongside 2Fluoro (2F)-modified pyrimidines via in vitro transcription using a mutant T7 RNA polymerase. This method enables efficient, primer-independent synthesis and amplification of LNA-modified RNA with low error rates. To demonstrate its utility, we performed in vitro selection (SELEX) of LNA- and 2F-modified aptamers targeting Influenza hemagglutinin (HA) and human CD40 ligand (hCD40L), two therapeutically relevant proteins. Iterative SELEX cycles yielded aptamers with low-nanomolar affinities, high specificity, and high nuclease resistance. Overall, this approach provides a scalable and versatile platform for generating chemically stabilized RNAs, fully compatible with SELEX, and holds potential for developing next-generation RNA-based therapeutics with improved pharmacokinetics.

molecular biology↗

bioRxiv2019

Adding MASP1 to the lectin pathway - leprosy association puzzle: hints from gene polymorphisms and protein levels.

BackgroundDeposition of complement factors on Mycobacterium leprae may enhance phagocytosis. Such deposition may occur through the lectin pathway of complement. Three proteins of the lectin pathway are produced from the gene MASP1: Mannan-binding lectin-associated serine protease 1 (MASP-1) and MASP-3 and mannan-binding lectin-associated protein of 44 kDa (MAp44). Despite their obvious importance, the roles played by these proteins have never been investigated in leprosy disease.\n\nMethodologyWe haplotyped five MASP1 polymorphisms by multiplex sequence-specific PCR (intronic rs7609662*G>A and rs13064994*C>T, exon 12 3-untranslated rs72549262*C>G, rs1109452*C>T and rs850314*G>A) and measured MASP-1, MASP-3 and MAp44 serum levels in 196 leprosy patients (60%, lepromatous) and 193 controls.\n\nPrincipal findingsLower MASP-3 and MAp44 levels were observed in patients, compared with controls (P=0.0002 and P<0.0001, respectively) and in lepromatous, compared with non-lepromatous patients (P=0.008 and P=0.002, respectively). Higher MASP-3 levels occurred in controls carrying variants/haplotypes associated with leprosy resistance (rs13064994*T, rs1109452_rs850314*CG within GT_CCG and rs850314*A: OR=0.5-0.6, Pcorr=0.01-0.04). Controls with rs1109452*T, included in susceptibility haplotypes (GT_GTG/GT_CTG: OR=2.0, Pcorr=0.03), had higher MASP-1 and lower MASP-3 levels (P[≤]0.009). Those with GC_CCG, presented increasing susceptibility (OR=1.7, Pcorr=0.006) and had higher MAp44 levels (P=0.015). MASP-3 expression decreased in patients, compared with controls carrying rs1109452_rs850314*CA or CG (P[≤]0.02), which may rely on exon 12 CpG methylation and/or miR-2861/miR-3181 mRNA binding.\n\nConclusionPolymorphisms regulating MASP-3/MAp44 availability in serum modulate leprosy susceptibility, underlining the importance of lectin pathway regulation against pathogens that exploit phagocytosis to parasitize host macrophages.\n\nAuthor summarySince immemorial times, Mycobacterium leprae inflicts permanent injuries in human kind, within a wide symptomatic spectrum ranging from insensitive skin patches to disabling physical lesions. Innate resistance to this parasite is well recognized, but poorly understood. The complement system is one of the most important arms of the innate response, and several lines of evidence indicate that it may be usurped by the parasite to enhance its entrance into host cells. These include our recent work on genetic association of the disease with lectin pathway components and the complement receptor CR1, whose polymorphisms modulate susceptibility to infection and clinical presentation. Here, we add another pivotal piece in the leprosy parasite-host interaction puzzle: polymorphisms and serum levels of three different lectin pathway proteins, all encoded by the same gene, namely mannan-binding lectin-associated serine protease 1 (MASP1). We found lower levels of two of these proteins, MASP-3 and MAp44, in leprosy patients. Higher MASP-3/lower MASP-1 levels were associated with protective haplotypes, containing two side-by-side polymorphisms located in the exclusive untranslated region of MASP-3 exon 12, which may regulate exon splicing and/or translation efficiency. The associations revealed in this study reflect the pleiotropic nature of this gene. They further illustrate the complexity of the response mounted against the parasite, which places MASP1 products in the regulatory crossroad between the innate and adaptive arms of the immunological system, modulating leprosy susceptibility.

immunology↗