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Vassiliadis E.,Assay Development | Vassiliadis E.,University of Southern Denmark | Oliveira C.P.,University of Sao Paulo | Alvares-da-Silva M.R.,Federal University of Rio Grande do Sul | And 15 more authors.
American Journal of Translational Research

Aim: To investigate whether increased levels of vimentin citrullinated peptides identified by MS in articular cartilage can be measured in pathologies other than rheumatoid arthritis and be utilised for diagnostic purposes. Methods: A monoclonal antibody against the sequence RLRSSVPGV-citrulline (VICM) was developed and evaluated in a carbon tetrachloride (CCI4) (n=52 + 28 controls) rat model of liver fibrosis and two clinical cohorts of adult patients with hepatitis C (HCV) (n=92) and non-alcoholic fatty liver disease (NAFLD) (n=62), and compared to healthy controls. Results: In CCI4-treated rats, mean systemic VICM levels increased 31% at week 12 (176 ng/mL, P<0.001), 41.7% at weeks 16 (190 ng/mL, P<0.001), 49.2% at weeks 20 (200 ng/ml, P<0.001), compared to controls (134 ng/mL). VICM levels correlated with total hepatic collagen determined by Sirius red staining of rat livers (r=0.75, P<0.05). In the HCV cohort, when stratified according to the METAVIR F score, VICM levels were 63% higher in FO (632 ng/mL ±75, p<0.05), 54% in F1 (597 ng/mL ±41.3, p<0.05) and 62% in F2 (628 ng/mL ±59, p<0.05) all compared to controls. In the NAFLD cohort, VICM levels were 20.6% higher in FO (339 ±12 ng/mL, P<0.05), 23.8% in F1 (348 ±12 ng/mL, P<0.05) and 28.8% in F2 (362 ±25 P<0.05). Conclusion: We demonstrated increased serological levels of citrullinated and MMP degraded vimentin in an animal model of liver fibrosis and in early fibrosis associated with HCV and NAFLD patients. These data suggest that citrullinated and MMP degraded proteins are also present in liver fibrosis. Source

Vassiliadis E.,Assay Development | Vassiliadis E.,University of Southern Denmark | Veidal S.S.,Assay Development | Veidal S.S.,University of Southern Denmark | And 12 more authors.
BMC Dermatology

Background: The current study utilized a Bleomycin-induced model of skin fibrosis to investigate the neo-epitope CO3-610 (KNGETGPQGP), a fragment of collagen III released during matrix metalloproteinase-9 (MMP9) degradation of the protein, we have previously described as a novel biomarker for liver fibrosis. The aim was to investigate CO3-610 levels in another well characterised model of fibrosis, to better describe the biomarker in relation to additional fibrotic pathologies.Methods: Skin fibrosis was induced by daily injections of Bleomycin to a total of 52 female C3 H mice, while control mice (n = 28) were treated with phosphate buffered saline (PBS), for 2, 4, 6 or 8 weeks. Skin fibrosis was evaluated using Visiopharm software on Sirius-red stained skin sections. Urine ELISA assays and creatinine corrections were performed to measure CO3-610 levels.Results: CO3-610 levels were significantly higher in Bleomycin-treated vs. PBS-treated mice at each time point of termination. The mean increases were: 59.2%, P < 0.0008, at 2 weeks; 113.5%, P < 0.001, at 4 weeks; 136.8%, P < 0.0001 at 6 weeks; 157.2%, P < 0.0001 at 8 weeks). PBS-treated mice showed a non-significant increase in CO3-610 levels (mean increase for weeks 2-8 = 1.7%, P = 0.789) CO3-610 levels assayed in urine were statistically significantly correlated with Western blot analysis showing increased skin fibrosis (P < 0.0001, r = 0.65).Conclusion: Increased levels in mouse urine of the MMP-9 mediated collagen III degradation fragment CO3-610 were correlated with skin fibrosis progression, suggesting that CO3-610 may be a potential positive biomarker to study the pathogenesis of skin fibrosis in mice. © 2011 Vassiliadis et al; licensee BioMed Central Ltd. Source

Bay-Jensen A.-C.,Cartilage Biology and Biomarkers R and D | Liu Q.,Assay Development | Byrjalsen I.,Cartilage Biology and Biomarkers R and D | Li Y.,Assay Development | And 7 more authors.
Clinical Biochemistry

Objectives: In joint degenerative diseases, the collagens are degraded by matrix metalloproteinases and protein fragments are released to serum as potential biomarkers. Methods: A collagen type II specific neoepitope, CIIM, was identified (RDGAAG1053) by mass spectrometry. Two ELISAs against the neoepitope were developed. CIIM was measured in cartilage explants in the presence or absence of protease inhibitors. CIIM was measured in OA synovial fluid (n=51) and serum (n=156). Knee OA was graded by standard Kellgren-Lawrence (KL) score. Results: The ELISAs showed good technical performance; CV%, < 13%. CIIM release from cartilage explants was blocked by the MMP inhibitor. CIIM was detected in synovial fluid. Furthermore, serum CIIM levels were significantly higher (P< 0.05) in those individuals with mild or severe OA than in those with no OA. Conclusion: We developed a new biomarker for joint degenerative diseases, which we demonstrated was derived from MMP-degraded type II collagen. © 2011 The Canadian Society of Clinical Chemists. Source

Vassiliadis E.,Assay Development | Vassiliadis E.,University of Southern Denmark | Veidal S.S.,Assay Development | Veidal S.S.,University of Southern Denmark | And 5 more authors.
American Journal of Translational Research

Having previously shown that levels of the citrullinated vimentin peptide VICM are raised in liver fibrosis in rats, we aimed to investigate whether inhibition of citrullination as measured by VICM levels could affect fibrogenesis. Methods: Fibrogenesis was evaluated by quantitative histology and circulating levels of collagen type III in a carbon tetrachloride (CCl4) rat model of liver fibrosis for 6 weeks (n=40+10 untreated controls). The first treatment group (n=20) was treated exclusively with CCl4 for the duration of the study.The second treatment group (n=20) was additionally treated, for the same period, with N-a-benzoyl-N5-(2 Chloro-1-iminoethyl)-L-Ornithine amide, a known PAD inhibitor. Results: All 40 CCl4 treated animals showed a statistically significant increase in total collagen (p<0.0001) and C3M levels (p<0.001) compared with controls assessed by quantitative histology. Animals additionally treated with the PAD inhibitor showed a statistically significant increase when compared with controls for both total collagen (p<0.001) and C3M levels (p<0.0001) but no statistically difference when compared with animals treated only with CCl4. The mean systemic level of VICM in control animals was 115 ng/ml at 6 weeks. In CCl4-treated animals, mean systemic VICM levels increased 324% at week 6 (p<0.001). The mean level of the marker in CCl4-treated rats was not statistically significant from that in controls (P>0.05). In PAD-treated animals VICM levels were 51% (P<0.05) lower than in non-PAD CCl4-treated animals. Conclusion: The PAD inhibitor did not reduce fibrogenesis in this preclinical model. However circulating VICM marker levels were decreased in the presence of the PAD inhibitor. Source

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