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The International Congress on Autoimmune Pre-Disease was organized by the German Research Foundation-founded Research Training Group "Autoimmune Pre-Disease" and took place at the University of Lübeck, Germany, on September 16-17, 2024. The event featured various talks and posters from young researchers and international experts and emphasized early interventions and prevention in autoimmune diseases with a focus on systemic rheumatic diseases, pemphigus, and pemphigoid diseases., (© 2024 The Authors.)

Introduction: Progressive loss of regulatory T cell (Treg)-mediated control over autoreactive effector T cells contributes to the development of systemic lupus erythematosus (SLE). Accordingly, we hypothesized that Treg may also have the capacity to suppress the activation of autoreactive CD4 + T cells that are considered to drive autoimmunity., Methods: To investigate whether Treg are involved in the control of autoreactive CD4 + T cells, we depleted CD25 + Treg cells either in vivo or in vitro , or combined both approaches before antigen-specific stimulation with the SLE-associated autoantigen SmD1(83-119) in the NZB/W F1 mouse model either after immunization against SmD1(83-119) or during spontaneous disease development. Frequencies of autoantigen-specific CD4 + T cells were determined by flow cytometry using the activation marker CD154., Results: Both in vitro and in vivo depletion of CD25 + Treg, respectively, increased the frequencies of detectable autoantigen-specific CD4 + T cells by approximately 50%. Notably, the combined in vivo and in vitro depletion of CD25 + Treg led almost to a doubling in their frequencies. Frequencies of autoantigen-specific CD4 + T cells were found to be lower in immunized haploidentical non-autoimmune strains and increased frequencies were detectable in unmanipulated NZB/W F1 mice with active disease. In vitro re-addition of CD25 + Treg after Treg depletion restored suppression of autoantigen-specific CD4 + T cell activation., Discussion: These results suggest that the activation and expansion of autoantigen-specific CD4 + T cells are partly controlled by Treg in murine lupus. Depletion of Treg therefore can be a useful approach to increase the detectability of autoantigen-specific CD4 + T cells allowing their detailed characterization including lineage determination and epitope mapping and their sufficient ex vivo isolation for cell culture., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Rosenberger, Undeutsch, Akbarzadeh, Ohmes, Enghard, Riemekasten and Humrich.)

The endothelial cell lining creates an interface between circulating blood and adjoining tissue and forms one of the most critical barriers and targets for therapeutical intervention. Recent studies suggest that fucoidans, sulfated and fucose-rich polysaccharides from brown seaweed, show multiple promising biological effects, including anti-inflammatory properties. However, their biological activity is determined by chemical characteristics such as molecular weight, sulfation degree, and molecular structure, which vary depending on the source, species, and harvesting and isolation method. In this study, we investigated the impact of high molecular weight (HMW) fucoidan extract on endothelial cell activation and interaction with primary monocytes (MNCs) in lipopolysaccharide (LPS)-induced inflammation. Gentle enzyme-assisted extraction combined with fractionation by ion exchange chromatography resulted in well-defined and pure fucoidan fractions. FE_F3, with a molecular weight ranging from 110 to 800 kDa and a sulfate content of 39%, was chosen for further investigation of its anti-inflammatory potential. We observed that along with higher purity of fucoidan fractions, the inflammatory response in endothelial mono- and co-cultures with MNCs was reduced in a dose-dependent manner when testing two different concentrations. This was demonstrated by a decrease in IL-6 and ICAM-1 on gene and protein levels and a reduced gene expression of TLR-4, GSK3β and NF-kB. Expression of selectins and, consequently, the adhesion of monocytes to the endothelial monolayer was reduced after fucoidan treatment. These data indicate that the anti-inflammatory effect of fucoidans increases with their purity and suggest that fucoidans might be useful in limiting the inflammatory response of endothelial cells in cases of LPS-induced bacterial infection.

The human xylosyltransferase isoform XT-I catalyzes the initial step in proteoglycan biosynthesis and represents a biomarker of myofibroblast differentiation. Furthermore, XT-I overexpression is associated with fibrosis, whereby a fibrotic process initially develops from a dysregulated wound healing. In a physiologically wound healing process, extracellular matrix-producing myofibroblasts enter acute senescence to protect against fibrosis. The aim of this study was to determine the role of XT-I in acute senescent proto-myofibroblasts. Normal human dermal fibroblasts were seeded in a low cell density to promote myofibroblast differentiation and treated with H 2 O 2 to induce acute senescence. Initiation of the acute senescence program in human proto-myofibroblasts resulted in a suppression of XYLT mRNA expression compared to the control, whereby the isoform XYLT1 was more affected than XYLT2 . Moreover, the XT-I protein expression and enzyme activity were also reduced in H 2 O 2 -treated cells compared to the control. The examination of extracellular matrix remodeling revealed reduced expression of collagen I, fibronectin and decorin. In summary, acute senescent proto-myofibroblasts formed an anti-fibrotic phenotype, and suppression of XT-I during the induction process of acute senescence significantly contributed to subsequent ECM remodeling. XT-I therefore plays an important role in the switch between physiological and pathological wound healing.

In the normal immune system, T cell activation is tightly regulated and controlled at several levels to ensure that activation occurs in the right context to prevent the development of pathologic conditions such as autoimmunity or other harmful immune responses. CD4 + FoxP3 + regulatory T cells (Treg) are crucial for the regulation of T cell responses in the peripheral lymphatic organs and thus for the prevention and control of autoimmunity. In systemic lupus erythematosus (SLE), a prototypic systemic autoimmune disease with complex etiology, a disbalance between Treg and pathogenic effector/memory CD4 + T cells develops during disease progression indicating that gradual loss of control over T cell activation is an important event in the immune pathogenesis. This progressive failure to adequately regulate the activation of autoreactive T cells facilitates chronic activation and effector/memory differentiation of pathogenic T cells, which are considered to contribute significantly to the induction and perpetuation of autoimmune processes and tissue inflammation in SLE. However, in particular in humans, little is known about the factors which drive the escape from immune regulation and the chronicity of pathogenic T cell responses in an early stage of autoimmune disease when clinical symptoms are still unapparent. Here we briefly summarize important findings and discuss current views and models on the mechanisms related to the dysregulation of T cell responses which promotes chronicity and pathogenic memory differentiation with a focus on the early stage of disease in lupus-prone individuals., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ohmes, Comdühr, Akbarzadeh, Riemekasten and Humrich.)

Fucoidans, sulfated polysaccharides from brown algae, possess multiple bioactivities in regard to osteogenesis, angiogenesis, and inflammation, all representing key molecular processes for successful bone regeneration. To utilize fucoidans in regenerative medicine, a delivery system is needed which temporarily immobilizes the polysaccharide at the injured site. Hydrogels have become increasingly interesting biomaterials for the support of bone regeneration. Their structural resemblance with the extracellular matrix, their flexible shape, and capacity to deliver bioactive compounds or stem cells into the affected tissue make them promising materials for the support of healing processes. Especially injectable hydrogels stand out due to their minimal invasive application. In the current study, we developed an injectable thermosensitive hydrogel for the delivery of fucoidan based on chitosan, collagen, and β-glycerophosphate (β-GP). Physicochemical parameters such as gelation time, gelation temperature, swelling capacity, pH, and internal microstructure were studied. Further, human bone-derived mesenchymal stem cells (MSC) and human outgrowth endothelial cells (OEC) were cultured on top (2D) or inside the hydrogels (3D) to assess the biocompatibility. We found that the sol-gel transition occurred after approximately 1 min at 37 °C. Fucoidan integration into the hydrogel had no or only a minor impact on the mentioned physicochemical parameters compared to hydrogels which did not contain fucoidan. Release assays showed that 60% and 80% of the fucoidan was released from the hydrogel after two and six days, respectively. The hydrogel was biocompatible with MSC and OEC with a limitation for OEC encapsulation. This study demonstrates the potential of thermosensitive chitosan-collagen hydrogels as a delivery system for fucoidan and MSC for the use in regenerative medicine.

Fucoidans are polysaccharides from brown macroalgae, showing multiple bioactivities important for bone regeneration and bone health. However, the use of fucoidans in medical applications remains sparse due to the heterogeneity in their chemical properties and unclear structure-function relationships. Innovations in extraction techniques and post processing steps are needed to produce homogeneous fucoidan molecules with tailorable bioactivities. Here, we applied enzyme-assisted extraction coupled with enzymatic hydrolysis by Fhf1 fucoidanase to generate low (LMW) and medium molecular weight (MMW) fucoidans from Fucus evanescens. In contrast to the anti-angiogenic properties of the high molecular weight fucoidan, LMW and MMW no longer suppressed the production of pro-angiogenic molecules by bone stem cells, nor impaired the formation of prevascular structures in vitro. In contrast to LMW, a pro-inflammatory response of OEC was observed after treatment with high concentrations of MMW. Thus, fucoidanase hydrolysis could be a useful tool to tailor the bioactivity of fucoidans., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The need for a substitute for allograft and autograft is rising as bone graft surgeries exceed available supplies. We investigated the efficacy of the low-molecular weight marine bioactive compound fucoidan (FUC) on bone regeneration and implant fixation in seven female sheep, as FUC has shown great promise as a bone substitute. Titanium implants were inserted bilaterally in the distal femurs to test three hydroxyapatite/fucoidan (HA/FUC) groups and compared to allograft. The HA was coated with either 500 or 1500 μg of FUC, obtained by microwave-assisted chemical extraction, or 500 μg of FUC obtained by an enzyme-assisted extraction method. The concentric 2-mm gap around the implant was filled with either one of the HA/FUCs or allograft from the donor sheep. After 12 weeks, implant-bone blocks were harvested and divided into three parts for mechanical push-out testing, immunohistochemistry, and micro-CT and histomorphometry. Pronounced bone formations were observed by micro-CT and histomorphometry in all groups, but higher bone volume fractions were seen in the allograft group compared to the three HA/FUC groups. The trabecular thickness, trabecular separation, and architectural anisotropy were all significantly higher in the allograft group compared to the three HA/FUC groups. In conclusion, adequate bone formation was observed in all groups, although the bone formation was significantly greater in the allograft group. Also, no significant differences existed in the shear mechanical properties between groups, suggesting that the combination of HA and FUC can achieve a similar fixation strength to allograft in this model., (© 2021 Wiley Periodicals LLC.)

Fucoidans, sulfated polysaccharides extracted from brown algae, are marine products with the potential to modulate bone formation and vascularization processes. The bioactivity and safety of fucoidans are highly associated with their chemical structure, which may vary with algae species and extraction method. Thus, in depth evaluation of fucoidan extracts in terms of endotoxin content, cytotoxicity, and their detailed molecular biological impact on the individual cell types in bone is needed. In this study, we characterized fucoidan extracts from three different Fucus species including Fucus vesiculosus (Fv), Fucus serratus (Fs), and Fucus distichus subsp. evanescens (Fe) for their chemical features, endotoxin content, cytotoxicity, and bioactive effects on human outgrowth endothelial cells (OEC) and human mesenchymal stem cells (MSC) as in vitro models for bone function and vascularization. Extracts contained mainly high molecular weight (HMW) fucoidans and were free of endotoxins that may cause inflammation or influence vascularization. OEC tolerated fucoidan concentrations up to 200 µg/mL, and no indication of cytotoxicity was observed. The inflammatory response, however, investigated by real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) and endothelial barrier assessed by impedance measurement differed for the individual extracts. MSC in comparison with endothelial cells were more sensitive to fucoidans and showed partly reduced metabolic activity and proliferation at higher doses of fucoidans. Further results for MSC indicated impaired osteogenic functions in alkaline phosphatase and calcification assays. All tested extracts consistently lowered important molecular mediators involved in angiogenesis, such a VEGF (vascular endothelial growth factor), ANG-1 (angiopoietin 1), and ANG-2 (angiopoietin 2), as indicated by RT-PCR and ELISA. This was associated with antiangiogenic effects at the functional level using selected extracts in co-culture models to mimic bone vascularization processes during bone regeneration or osteosarcoma.

Angiogenesis, the formation of new blood vessels from existing ones, is an essential process for successful bone regeneration. Further, angiogenesis is a key factor for the development of bone-related disorders like osteosarcoma or arthritis. Fucoidans, sulfated polysaccharides from brown algae, have been shown to affect angiogenesis as well as a series of other physiological processes including inflammation or infection. However, the chemical properties of fucoidan which define the biological activity vary tremendously, making a prediction of the bioactivity or the corresponding therapeutic effect difficult. In this study, we compare the effect of four chemically characterized high molecular weight fucoidan extracts from Fucus distichus subsp. evanescens (FE_crude and fractions F1, F2, F3) on angiogenic and osteogenic processes in bone-related primary mono- and co-culture cell systems. By determining the gene expression and protein levels of the regulatory molecules vascular endothelial growth factor (VEGF), angiopoietin-1 (ANG-1), ANG-2 and stromal-derived factor 1 (SDF-1), we show that the extracted fucoidans negatively influence angiogenic and osteogenic processes in both the mono- and co-culture systems. We demonstrate that purer fucoidan extracts with a high fucose and sulfate content show stronger effects on these processes. Immunocytochemistry of the co-culture system revealed that treatment with FE_F3, containing the highest fucose and sulfate content, impaired the formation of angiogenic tube-like structures, indicating the anti-angiogenic properties of the tested fucoidans. This study highlights how chemical properties of fucoidan influence its bioactivity in a bone-related context and discusses how the observed phenotypes can be explained on a molecular level-knowledge that is indispensable for future therapies based on fucoidans.

Many intracellular pathogens, such as mammalian reovirus, mimic extracellular matrix motifs to specifically interact with the host membrane. Whether and how cell-matrix interactions influence virus particle uptake is unknown, as it is usually studied from the dorsal side. Here we show that the forces exerted at the ventral side of adherent cells during reovirus uptake exceed the binding strength of biotin-neutravidin anchoring viruses to a biofunctionalized substrate. Analysis of virus dissociation kinetics using the Bell model revealed mean forces higher than 30 pN per virus, preferentially applied in the cell periphery where close matrix contacts form. Utilizing 100 nm-sized nanoparticles decorated with integrin adhesion motifs, we demonstrate that the uptake forces scale with the adhesion energy, while actin/myosin inhibitions strongly reduce the uptake frequency, but not uptake kinetics. We hypothesize that particle adhesion and the push by the substrate provide the main driving forces for uptake.