Vimentin fragments are potential markers of rheumatoid synovial fibroblasts.

OBJECTIVES: To study the protein expression differences between primary fibroblasts explanted from synovial membranes of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: Fibroblast cultures were obtained from 10 patients with RA and 5 patients with OA. After two-dimensional gel electrophoresis, proteins were excised and identified using peptide mass fingerprint. Expression of selected proteins was subsequently examined by immunoblot. Furthermore, we examined the cellular lysates for the presence of citrullinated proteins. RESULTS: The study was designed to compare expression changes of the common proteins detected in all studied fibroblast cultures (i.e. detected in all patients samples). We totally identified 191 shared proteins between RA and OA fibroblasts. A significant difference was defined as at least 2-fold upregulation or 0.6-fold downregulation of protein expression. The most obvious alteration observed in RA was the appearance of several vimentin fragments not present in OA. We did not detect citrullinated proteins in lysates from RA fibroblasts. This corroborates the current assumption that fibroblasts are not able to citrullinate proteins by themselves and that invading macrophages play a central role in this process. CONCLUSIONS: We demonstrated that fibroblasts from patients with RA, despite being grown under identical conditions, preserve a particular feature and generate vimentin fragments not present in fibroblasts from OA. Elevated levels of different vimentin fragments have been recently reported in several rheumatic conditions. Further studies are needed to elucidate the pathogenic mechanisms induced by vimentin fragments in RA.

Stimulated emission depletion live-cell super-resolution imaging shows proliferative remodeling of T-tubule membrane structures after myocardial infarction.

RATIONALE: Transverse tubules (TTs) couple electric surface signals to remote intracellular Ca(2+) release units (CRUs). Diffraction-limited imaging studies have proposed loss of TT components as disease mechanism in heart failure (HF). OBJECTIVES: Objectives were to develop quantitative super-resolution strategies for live-cell imaging of TT membranes in intact cardiomyocytes and to show that TT structures are progressively remodeled during HF development, causing early CRU dysfunction. METHODS AND RESULTS: Using stimulated emission depletion (STED) microscopy, we characterized individual TTs with nanometric resolution as direct readout of local membrane morphology 4 and 8 weeks after myocardial infarction (4pMI and 8pMI). Both individual and network TT properties were investigated by quantitative image analysis. The mean area of TT cross sections increased progressively from 4pMI to 8pMI. Unexpectedly, intact TT networks showed differential changes. Longitudinal and oblique TTs were significantly increased at 4pMI, whereas transversal components appeared decreased. Expression of TT-associated proteins junctophilin-2 and caveolin-3 was significantly changed, correlating with network component remodeling. Computational modeling of spatial changes in HF through heterogeneous TT reorganization and RyR2 orphaning (5000 of 20 000 CRUs) uncovered a local mechanism of delayed subcellular Ca(2+) release and action potential prolongation. CONCLUSIONS: This study introduces STED nanoscopy for live mapping of TT membrane structures. During early HF development, the local TT morphology and associated proteins were significantly altered, leading to differential network remodeling and Ca(2+) release dyssynchrony. Our data suggest that TT remodeling during HF development involves proliferative membrane changes, early excitation-contraction uncoupling, and network fracturing.

Codon 129 polymorphism specific cerebrospinal fluid proteome pattern in sporadic Creutzfeldt-Jakob disease and the implication of glycolytic enzymes in prion-induced pathology.

Cerebrospinal fluid (CSF) contains a dynamic and complex mixture of proteins, which can reflect a physiological and pathological state of the central nervous system. In our present study, we show CSF protein patterns from patients with the two most frequent subtypes of sporadic Creutzfeldt-Jakob disease (sCJD) defined by the codon 129 genotype (MM, MV, and VV) and the protease-resistant form of prion protein (type 1 and type 2). The densitometric analysis of 2D gels showed up-regulation of 27 and down-regulation of 3 proteins in the MM-sCJD as well as the up-regulation of 24 proteins in the VV-sCJD as compared to nondemented control. Almost 40% of sCJD specific regulated proteins in CSF are involved in glucose metabolism, regardless of the codon 129 polymorphism. The increase in CSF levels of lactate dehydrogenase (LDH), glucose-6-phosphate isomerase (G6PI), and fructose-bisphosphate aldolase A (ALDOA) were validated on a larger group of sCJD patients including three possible codon 129 polymorphism carriers and three control groups consisting of nondemented, neurological cases as well as patients suffering from Alzheimer's disease or vascular dementia. Subsequently, the abundance of these glycolytic enzymes in the brain as well as their cellular localization were determined. This study demonstrates for the first time the implication of G6PI in prion-induced pathology as well as its cellular translocalization in sCJD. The identification of sCJD-regulated proteins in CSF of living symptomatic patients in our study can broaden our knowledge about pathological processes occurring in sCJD, as they are still not fully understood.

Low-abundant cerebrospinal fluid proteome alterations in dementia with Lewy bodies.

Dementia with Lewy bodies (DLB) is one of the most common neurodegenerative diseases and shares multiple clinical and neuropathological parallels with Alzheimer's (AD) and Parkinson's disease (PD). A variety of clinical signs are suggestive for the diagnosis, and imaging (ßCIT SPECT) contributes substantially to the diagnosis. The study reported here was performed in search for a biomarker in the cerebrospinal fluid (CSF) of these patients. We applied 2D fluorescence difference gel electrophoresis and mass spectrometry to analyze the CSF proteome pattern of DLB patients after depleting twelve high-abundant proteins. The densitometric analysis of 2D gels showed the up- or down-regulation of 44 protein spots. Subsequently, 23 different proteins were identified. The majority is involved in acute phase and immune response. Many of these proteins were previously reported before as being associated with AD or PD, which strongly suggests a molecular cross-talk and may explain clinical and pathological overlap of these disease entities. Among the identified proteins are two highly upregulated proteins-inter alpha trypsin inhibitor heavy chain (ITIH4) and calsyntenin 1-that may have the potential to serve as molecular biomarkers specific for DLB. The identification of DLB-associated proteome changes will help to further understand pathological processes occurring in DLB and may provide future prospects to diagnostic and therapeutic options.