👤 Katja M Arndt

Thoracic aortic aneurysms frequently go undetected until serious complications like acute dissections or ruptures arise. Therefore, this study aims to identify potential blood circulating biomarkers enabling an easy and early diagnosis of thoracic aortic disease. Potential biomarker candidates were identified through two different techniques, untargeted and targeted proteomic as well as extracellular vesicle (EV) analyses. The biomarker levels were compared between two patient groups with thoracic aortic aneurysms and two control groups without thoracic aortic disease. In total, 80 patients (TAA group (n = 40) vs. control group (n = 40)) were matched for untargeted and targeted proteome analysis, and 85 for EV analysis (TAA group (n = 42) vs. control group (n = 43)), based on the availability of blood samples and excised aortic tissue. Levels of biomarker candidates were correlated with aortic diameter, patient age, and histological alterations in aortic tissue using linear and logistic regression models. The untargeted proteomic and EV analysis identified 1,037 and 1,077 proteins, respectively, of which 11 and 28 proteins showed significant differences in concentration between the study groups. Of these, 9 proteins correlated with the aortic diameter: ACTN1 (Regression coefficient B = 1.633, p < 0.001), CRP (B = 0.001, p = 0.004), TGM3 (B=-0.293, p = 0.010), KRT84 (B=-0.477, p = 0.010), IGHG3 (-0.267, p = 0.018), DPYSL2 (B = 0.644, p = 0.020), TSPAN8 (B-0.838, p = 0.042), IGKV3D-11 (B=-0.242, p = 0.046), and VDAC1 (B=-0.491, p = 0.047). Moreover, IGKV3D-11 (B=-3.257, p = 0.029), IGHG3 (B=-0.003, p = 0.034), and APOC3 (B=-2.104, p = 0.037) showed significant correlations with the grade of aortic medial layer degeneration. None of the proteins correlated with patient age. The study identified 9 biomarker candidates correlating with the aortic diameter. To enable the clinical use for diagnosis and prognostic assessment, these biomarkers need to be validated in larger external cohorts. Show less

Congenital myasthenic syndromes (CMS) have some phenotypic overlap with seronegative myasthenia gravis (SNMG). The aim of this single center study was to assess the minimum occurrence of CMS misdiagnosed as double SNMG in a Brazilian cohort. The genetic analysis of the most common mutations in CHRNE, RAPSN, and DOK7 genes was used as the main screening tool. We performed genetic analysis in 22 patients with a previous diagnosis of 'double' SNMG. In this study, one CMS patient was confirmed due to the presence of compound heterozygous variants in the CHRNE gene (c.130insG/p.Cys210Phe). This study confirmed that CMS due to CHNRE mutations can be mistaken for SNMG. In addition, our study estimated the prevalence of misdiagnosed CMS to be 4.5% in 'double' SNMG patients of our center. Based on our findings, genetic screening could be helpful in the diagnostic workup of patients with 'double' SNMG in whom differential diagnosis is recommended. Show less

LINGO-1 is a membrane protein of the central nervous system (CNS) that suppresses myelination of axons. Preclinical studies have revealed that blockade of LINGO-1 function leads to CNS repair in demyelinating animal models. The anti-LINGO-1 antibody Li81 (opicinumab), which blocks LINGO-1 function and shows robust remyelinating activity in animal models, is currently being investigated in a Phase 2 clinical trial as a potential treatment for individuals with relapsing forms of multiple sclerosis (AFFINITY: clinical trial.gov number NCT03222973). Li81 has the unusual feature that it contains two LINGO-1 binding sites: a classical site utilizing its complementarity-determining regions and a cryptic secondary site involving Li81 light chain framework residues that recruits a second LINGO-1 molecule only after engagement of the primary binding site. Concurrent binding at both sites leads to formation of a 2:2 complex of LINGO-1 with the Li81 antigen-binding fragment, and higher order complexes with intact Li81 antibody. To elucidate the role of the secondary binding site, we designed a series of Li81 variant constructs that eliminate it while retaining the classic site contacts. These Li81 mutants retained the high affinity binding to LINGO-1, but lost the antibody-induced oligodendrocyte progenitor cell (OPC) differentiation activity and myelination activity in OPC- dorsal root ganglion neuron cocultures seen with Li81. The mutations also attenuate antibody-induced internalization of LINGO-1 on cultured cortical neurons, OPCs, and cells over-expressing LINGO-1. Together these studies reveal that engagement at both LINGO-1 binding sites of Li81 is critical for robust functional activity of the antibody. Show less

Multiple sclerosis (MS) is an autoimmune-inflammatory disease of the central nervous system (CNS) with prominent demyelination and axonal injury. While most MS therapies target the immunologic response, there is a large unmet need for treatments that can promote CNS repair. LINGO-1 (leucine-rich repeat and Ig-containing Nogo receptor interacting protein-1) is a membrane protein selectively expressed in the CNS that suppresses myelination, preventing the repair of damaged axons. We are investigating LINGO-1 antagonist antibodies that lead to remyelination as a new paradigm for treatment of individuals with MS. The anti-LINGO-1 Li81 antibody,BIIB033, is currently in clinical trials and is the first MS treatment targeting CNS repair. Here, to elucidate the mechanism of action of the antibody, we solved the crystal structure of the LINGO-1-Li81 Fab complex and used biochemical and functional studies to investigate structure-function relationships. Li81 binds to the convex surface of the leucine-rich repeat domain of LINGO-1 within repeats 4-8. Fab binding blocks contact points used in the oligomerization of LINGO-1 and produces a stable complex containing two copies each of LINGO-1 and Fab that results from a rearrangement of contacts stabilizing the quaternary structure of LINGO-1. The formation of the LINGO-1-Li81 Fab complex masks functional epitopes within the Ig domain of LINGO-1 that are important for its biologic activity in oligodendrocyte differentiation. These studies provide new insights into the structure and biology of LINGO-1 and how Li81 monoclonal antibody can block its function. Show less

The non-random chromosomal translocations t(10;11)(p13;q23) and t(10;11)(p13;q14-21) result in leukemogenic fusion proteins comprising the coiled coil domain of the transcription factor AF10 and the proteins MLL or CALM, respectively, and subsequently cause certain types of acute leukemia. The AF10 coiled-coil domain, which is crucial for the leukemogenic effect, has been shown to interact with GAS41, a protein previously identified as the product of an amplified gene in glioblastoma. Using sequential synthetic peptides, we mapped the potential AF10/GAS41 interaction site, which was subsequently be used as scaffold for a library targeting the AF10 coiled-coil domain. Using phage display, we selected a peptide that binds the AF10 coiled-coil domain with higher affinity than the respective coiled-coil region of wild-type GAS41, as demonstrated by phage ELISA, CD, and PCAs. Furthermore, we were able to successfully deploy the inhibitory peptide in a mammalian cell line to lower the expression of Hoxa genes that have been described to be overexpressed in these leukemias. This work dissects molecular determinants mediating AF10-directed interactions in leukemic fusions comprising the N-terminal parts of the proteins MLL or CALM and the C-terminal coiled-coil domain of AF10. Furthermore, it outlines the first steps in recognizing and blocking the leukemia-associated AF10 interaction in histiocytic lymphoma cells and therefore, may have significant implications in future diagnostics and therapeutics. Show less

Initiation and progression of laryngeal cancer is associated with tobacco smoking and abusing of strong alcoholic beverages. A significance of genetic factor, although not defined sufficiently yet has been raised as well. The studies were focused on an influence of AHR gene polymorphism on survival of squamous cell carcinoma laryngeal subjects. The study material was 65 archival DNA samples analyzed by RLP-PCR. The samples varying with electrophoretic mobility were DNA sequenced. In the study group 9 heterozygotic variants Arg554Lys (codon 554) were detected. One case was a carrier of two other mutations in codon: 490 (1468 A > G) and 570 (1708 G > A). Survival time, metastasis and occurrence of second primary tumors were compared in carriers of wild type and Arg554Lys variant AHR. Preliminary results indicate for a necessity of further studies as until now the study group is too small to find a conclusive association. Show less

Thyroid-associated ophthalmopathy (TAO) is a potentially severe autoimmune disease, in and around the orbit, usually accompanied by Graves' disease. It was the goal of this study to develop a serological indicator for TAO and to characterize its expression in human thyroid and eye muscle tissue. Thus, we have recloned the full-length 1D-complementary DNA and assessed its expression levels in 90 healthy and diseased human thyroids. Only Graves' patients suffering from TAO (n = 29) displayed a significant, 2.1-fold increase of 1D expression levels (P = 0.029), compared with normal controls (n = 9), as assessed using the Mann-Whitney U-test for paired, nonnormally distributed samples. In contrast, a decrease of 1D expression (to 40% of control normal values) was confined to thyroid autonomy (n = 19, P = 0.032). In all other diseased human thyroids, including Graves' thyroids from patients not suffering from clinically overt TAO (n = 9), 1D expression levels were not different from the healthy controls. 1D gene expression was demonstrated in both healthy (n = 10) and diseased (n = 10) eye muscle tissues. Furthermore, a recombinant protein derived from baculovirus-infected Sf9 insect cells was purified under both nondenaturing and denaturing conditions. While under nondenaturing conditions, the molecular mass of recombinant 1D was determined to be 85 kDa; denaturing isolation yielded the expected 64-kDa protein. Autoantibodies against denatured 1D protein were not detectable in sera of diseased or healthy subjects. Immunoreactivity against the 85-kDa, nondenatured protein, evaluated in a panel of 222 different human sera, showed that 82% of Graves' patients suffering from TAO had autoantibodies against recombinant 1D, whereas only 5% of the healthy controls were positive for antibodies against 1D. Taken together, our results demonstrate a high disease sensitivity and specificity of recombinant, nondenatured 1D, to distinguish Graves' disease with or without TAO from other forms of thyroid and/or eye disease. Prospective studies will have to show whether autoantibodies against 1D can also be used as a prognosticator of TAO. Show less

The Saccharomyces cerevisiae CLN3 protein, a G1 cyclin, positively regulates the expression of CLN1 and CLN2, two additional G1 cyclins whose expression during late G1 is activated, in part, by the transcription factors SWI4 and SWI6. We isolated 12 complementation groups of mutants that require CLN3. The members of one of these complementation groups have mutations in the BCK2 gene. In a wild-type CLN3 genetic background, bck2 mutants have a normal growth rate but have a larger cell size, are more sensitive to alpha-factor, and have a modest defect in the accumulation of CLN1 and CLN2 RNA. In the absence of CLN3, bck2 mutations cause an extremely slow growth rate: the cells accumulate in late G1 with very low levels of CLN1 and CLN2 RNA. The slow growth rate and long G1 delay of bck2 cln3 mutants are cured by heterologous expression of CLN2. Moreover, overexpression of BCK2 induces very high levels of CLN1, CLN2, and HCS26 RNAs. The results suggest that BCK2 and CLN3 provide parallel activation pathways for the expression of CLN1 and CLN2 during late G1. Show less

The Saccharomyces cerevisiae SIS2 gene was identified by its ability, when present on a high copy number plasmid, to increase dramatically the growth rate of sit4 mutants. SIT4 encodes a type 2A-related protein phosphatase that is required in late G1 for normal G1 cyclin expression and for bud initiation. Overexpression of SIS2, which contains an extremely acidic carboxyl terminal region, stimulated the rate of CLN1, CLN2, SWI4 and CLB5 expression in sit4 mutants. Also, overexpression of SIS2 in a CLN1 cln2 cln3 strain stimulated the growth rate and the rate of CLN1 and CLB5 RNA accumulation during late G1. The SIS2 protein fractionated with nuclei and was released from the nuclear fraction by treatment with either DNase I or micrococcal nuclease, but not by RNase A. This result, combined with the finding that overexpression of SIS2 is extremely to a strain containing lower than normal levels of histones H2A and H2B, suggests that SIS2 might function to stimulate transcription via an interaction with chromatin. Show less

In Saccharomyces cerevisiae, the RNA levels of the G1 cyclins CLN1, CLN2, and HCS26 increase dramatically during the late G1 phase of the cell cycle. The SIT4 gene, which encodes a serine/threonine protein phosphatase, is required for the normal accumulation of CLN1, CLN2, and HCS26 RNAs during late G1. This requirement for SIT4 in normal G1 cyclin RNA accumulation is at least partly via SWI4. Strains containing mutations in SIT4 are sensitive to the loss of either CLN2 or CLN3 function. At the nonpermissive temperature, temperature-sensitive sit4 strains are blocked for both bud emergence and DNA synthesis. Heterologous expression of CLN2 in the absence of SIT4 function results in DNA synthesis, but most of the cells are still blocked for bud emergence. Therefore, SIT4 is required for at least two late G1 or G1/S functions: the normal accumulation of G1 cyclin RNAs (which is required for DNA synthesis) and some additional function that is required for bud emergence or cell cycle progression through late G1 or G1/S. Show less