Biology subjects

Fichtner, M.

Publications and source records attributed to Fichtner, M..

5 recordsLinked to original sources

bioRxiv2026

A primary human muscle cell-based assay for detecting myasthenia gravis autoantibody binding and assessing AChR cluster impairment

Myasthenia gravis (MG) is an autoimmune disease caused by pathogenic autoantibodies against proteins at the neuromuscular junction (NMJ). The diagnosis and clinical management of MG patients largely relies on the detection of antigen-specific autoantibodies targeting acetylcholine receptor (AChR) or muscle-specific kinase (MuSK). Yet a subset of patients remains seronegative for known MG autoantibodies, highlighting a critical need for alternative approaches to identify pathogenic NMJ antibodies. We established a new human in vitro model of the NMJ based on primary human muscle cells that recapitulates key features of the NMJ: differentiation to myotubes, expression of key NMJ proteins and formation of postsynaptic AChR clusters in response to agrin stimulation. The model allows new insights into myogenesis and genetic muscle diseases, and the new muscle cell-based assay (CBA) detected autoantibodies in sera from patients with AChR- and MuSK-positive MG with 96.43% sensitivity and 100% specificity, while healthy control sera showed no reactivity. Incubation with patient sera significantly reduced AChR clustering compared to controls, demonstrating functional pathogenic effects. Thus, we established a physiologically relevant human NMJ model that enables detection and functional characterization of neuromuscular autoantibodies. This novel approach addresses a key limitation of current antigen-specific diagnostics and provides a method for improved detection and characterization of MG antibodies, independent of antigen specificity. One Sentence SummaryWe established a postsynaptic human in vitro neuromuscular junction model to assess binding and pathogenicity of MG autoantibodies. Key messagesO_ST_ABSWhat is already known on this topic?C_ST_ABSCurrent diagnosis of myasthenia gravis (MG) relies largely on the detection of antigen-specific autoantibodies against AChR and MuSK, leaving a clinically relevant subset of patients seronegative. What are the new findings?We established a physiologically relevant human in vitro neuromuscular junction model based on primary human muscle cells and developed a novel muscle cell-based assay (CBA) for the detection of neuromuscular autoantibodies. How might this impact on clinical practice or future developments?The CBA detected autoantibodies in patients with AChR- or MuSK-positive MG with high sensitivity and specificity and demonstrated their functional pathogenic effects on AChR clustering. This antigen-independent approach may improve the detection and functional characterization of MG autoantibodies, particularly in patients who are seronegative in current diagnostic assays. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/743478v1_ufig1.gif" ALT="Figure 1000"> View larger version (38K): org.highwire.dtl.DTLVardef@c2e1fforg.highwire.dtl.DTLVardef@8336fforg.highwire.dtl.DTLVardef@8fa2b0org.highwire.dtl.DTLVardef@1d94e_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

Permissive central tolerance plus defective peripheral checkpoints licence pathogenic memory B cells in CASPR2-antibody encephalitis

Autoimmunity affects 10% of the population. Within this umbrella, autoantibody-mediated diseases targeting one autoantigen provide a unique opportunity to comprehensively understand the developmental pathway of disease-causing B cells and autoantibodies. While such autoreactivities are believed to be generated during germinal centre reactions, the roles of earlier immune checkpoints in autoantigen-specific B cell tolerance are poorly understood. We address this concept in patients with CASPR2-autoantibody encephalitis and healthy controls. In both groups, comparable and high ([~]0.5%) frequencies of unmutated CASPR2-reactive naive B cells were identified. By contrast, CASPR2-reactive memory B cells were exclusive to patients, and their B cell receptors demonstrated affinity-enhancing somatic mutations with heterogenous binding kinetics. These effector molecules possessed epitope-dependent pathogenic effects in vitro neuronal cultures and in vivo. The unmutated common ancestors of these memory B cells showed a distinctive balance between strong CASPR2 reactivity and very limited binding across the remaining human proteome. Our results are the first to propose mechanisms underlying autoantigen-specific tolerance in humans. We identify permissive central tolerance, defective peripheral tolerance and heterogenous autoantibody binding properties as sequential pathogenic steps which licence CASPR2-directed pathology. By leveraging the basic immunobiology, we rationally direct tolerance-restoring approaches in CASPR2-antibody diseases. This paradigm is applicable across autoimmune conditions.

immunology↗

bioRxiv2021

An atlas of inter- and intra-tumor heterogeneityof apoptosis competency in colorectal cancertissue at single cell resolution

Cancer cells ability to inhibit apoptosis is key to malignant transformation and limits response to therapy. Here, we performed multiplexed immunofluorescence analysis on tissue microarrays with 373 cores from 168 patients, segmentation of 2.4 million individual cells and quantification of 20 cell lineage and apoptosis proteins. Ordinary differential equation-based modelling of apoptosis sensitivity at single cell resolution was conducted and an atlas of inter- and intra-tumor heterogeneity in apoptosis susceptibility generated. We identified an enrichment for BCL2 in immune, and BAK, SMAC and XIAP in cancer cells. ODE-based modelling at single cell resolution identified an enhanced sensitivity of cancer cells to mitochondrial permeabilization and executioner caspase activation compared to immune and stromal cells, with significant inter- and intra-tumor heterogeneity. However, we did not find increased spatial heterogeneity of apoptosis signaling in cancer cells, suggesting that such heterogeneity is an intrinsic, non-genomic property not increased by the process of malignant transformation.

cancer biology↗

bioRxiv2020

Influence of spatial structure on protein damage susceptibility - A bioinformatics approach

Aging research is a very popular field of research in which the gradual transformation of functional states into dysfunctional states are studied. Here we only consider the molecular level, which can also have effects on the macroscopic level. It is known that the proteinogenic amino acids differ in their modification susceptibilities and this can affect the function of proteins. For this it is important to know the distribution of amino acids between the protein surface/shell and the core. This was investigated in this study for all known structural data of peptides and proteins. As a result it is shown that the surface contains less susceptible amino acids than the core with the exception of thermophilic organisms. Furthermore, proteins could be classified according to their susceptibility. This can then be used in applications such as phylogeny, aging research, molecular medicine and synthetic biology.

bioinformatics↗

bioRxiv2018

Isolation and characterization of rare circulating autoantibody-producing cells from patients with muscle-specific kinase myasthenia gravis

Myasthenia gravis (MG) is a chronic autoimmune disorder characterized by muscle weakness and caused by autoantibodies that bind to functional membrane proteins at the neuromuscular junction. Most patients have autoantibodies to the acetylcholine receptor (AChR), but a subset of patients instead have autoantibodies to muscle specific tyrosine kinase (MuSK). MuSK is an essential component of the Agrin/Lipoprotein receptor-related protein 4(LRP4)/MuSK/downstream of tyrosine kinase 7 (DOK7) pathway that is responsible for synaptic differentiation, including clustering of AChRs at the neuromuscular junction, both during development and in adult muscle. Nerve-released Agrin binds to LRP4 which then binds to MuSK, stimulating autophosphorylation and recruitment of DOK7 to complete the membrane component of the pathway. Serum-derived IgG4 subclass MuSK autoantibodies prevent the binding of LRP4 to MuSK, subsequently impairing autophosphorylation, resulting in the loss of Agrin-induced AChR clustering in the mouse myotube-forming C2C12 line. Although this autoimmune mechanism appears well understood, MuSK autoantibodies from patients are polyclonal. Most are IgG4 but IgG1, 2 and 3 are also present and can achieve similar results on AChR clustering. In addition, most bind the first Ig-like domain in MuSK, however some patients harbor serum autoantibodies that recognize other domains in MuSK. We sought to establish individual MuSK IgG clones so that the disease mechanisms could be better understood. We isolated MuSK autoantibody-expressing B cells from MuSK MG patients with a fluorescent-tagged MuSK antigen multimer and generated a panel of human monoclonal autoantibodies (mAbs) from these cells. We produced 77 mAbs from single B cells collected from six MuSK MG patients. Here we focused on three highly specific mAbs that bound quantitatively to MuSK in solution, to MuSK-expressing HEK cells and at mouse neuromuscular junctions where they co-localized with AChRs. These three IgG isotype mAbs (two IgG4 and one IgG3 subclass) recognized the Ig-like domain 2 of MuSK. The three MuSK mAbs inhibited Agrin induced-AChR clustering in C2C12 myotubes, but intriguingly, they enhanced rather than inhibited MuSK phosphorylation. This approach for ex vivo isolation of MuSK MG autoantibody-producing cells and production of human recombinant mAbs has identified distinct autoantibody specificities and likely divergent effector mechanisms. Collectively, these findings will enable a better understanding of MuSK autoantibody-mediated pathology.

immunology↗