Novel fibro-inflammatory biomarkers associated with disease activity in patients with Crohn's disease.

INTRODUCTION: Crohn's disease (CD) is a complex disease, and assessing activity is challenging due to pathobiologic process e.g. ECM remodeling, mucosal damage, and intestinal fibrosis, which greatly limits current disease activity assessments through e.g. endoscopy and imaging techniques. AREAS COVERED: The review highlights the importance of novel biomarkers reflecting ECM remodeling and immune cell activity that accurately reflect CD activity and progression. Such biomarkers could include collagen formation and degradation fragments and a serum fragment of calprotectin, reflecting neutrophil activity. A new concept, fibro-inflammation, is also introduced in the review, in which all aspects of mucosal damage, such as inflammation, mucosal damage, tissue remodeling, intestinal fibrosis, and fibrosis resolution, should be assessed. PubMed searches performed from July 2022 - November 2022 provided the scientific information included in the review. EXPERT OPINION: Current data suggest intestinal fibrosis may sustain and exacerbate chronic inflammation, leading to non-response to anti-inflammatory treatments. Therefore, evaluating novel biomarkers reflecting different stages of fibro-inflammatory disease activity should be done in a clinical setting and considered for clinical trials. This approach will help accurately assess disease activity, leading to better management and treatment of CD.

Does receptor balance matter? - Comparing the efficacies of the dual amylin and calcitonin receptor agonists cagrilintide and KBP-336 on metabolic parameters in preclinical models.

Cagrilintide is a novel long-acting amylin receptor agonist, which has shown a potent induction of weight loss. Interestingly, cagrilintide is a Dual Amylin and Calcitonin Receptor Agonist (DACRA) derived from an amylin backbone. Another class of long-acting DACRAs exists, namely the KBPs. These are salmon calcitonin-based and have shown preclinical potential; however, how and if they differentiate from amylin-derived molecules remain to be studied. Here, we compare cagrilintide to the DACRA KBP-336 with respect to receptor activation balance in vitro and using metabolic in vivo models. Peptide potencies were assessed using receptor-specific assays in vitro and in vivo. In vivo efficacies on body weight and glucose homeostasis were investigated head-to-head in high-fat diet (HFD) fed obese and T2D (ZDF) rat models. Both peptides activate the amylin and the calcitonin receptor in vitro and in vivo, with KBP-336 being more potent, and showing a CTR bias. KBP-336 and cagrilintide induced a potent and dose-dependent weight loss in HFD rats, with the highest dose of KBP-336 being superior to cagrilintide. In diabetic ZDF rats, DACRA treatment improved fasting blood glucose, HbA1c levels, and insulin action, with KBP-336 being superior to cagrilintide in improving glucose control. In summary, both KBP-336 and cagrilintide are DACRAs, however with KBP-336 being biased towards the CTR resulting in a different receptor activation balance. Interestingly, KBP-336 showed superior long-term efficacy on both weight loss and glucose control, supporting relevance of the receptor balance, and highlighting KBP-336 as a promising agent for the treatment of obesity and T2D.

Considerations for understanding protein measurements: Identification of formation, degradation and more pathological relevant epitopes.

OBJECTIVES: There is a need for precision medicine and an unspoken promise of an optimal approach for identification of the right patients for value-based medicine based on big data. However, there may be a misconception that measurement of proteins is more valuable than measurement of fewer selected biomarkers. In population-based research, variation may be somewhat eliminated by quantity. However, this fascination of numbers may limit the attention to and understanding of the single. This review highlights that protein measurements (with collagens as examples) may mean different things depending on the targeted epitope - formation or degradation of tissues, and even signaling potential of proteins. DESIGN AND METHODS: PubMed was searched for collagen, neo-epitope, biomarkers. RESULTS: Ample examples of assays with specific epitopes, either pathological such as HbA1c, or domain specific such as pro-peptides, which total protein arrays would not have identified were evident. CONCLUSIONS: We suggest that big data may be considered as the funnel of data points, in which most important parameters will be selected. If the technical precision is low or the biological accuracy is limited, and we include suboptimal quality of biomarkers, disguised as big data, we may not be able to fulfill the promise of helping patients searching for the optimal treatment. Alternatively, if the technical precision of the total protein quantification is high, but we miss the functional domains with the most considerable biological meaning, we miss the most important and valuable information of a given protein. This review highlights that measurements of the same protein in different ways may provide completely different meanings. We need to understand the pathological importance of each epitope quantified to maximize protein measurements.

KBP-066A, a long-acting dual amylin and calcitonin receptor agonist, induces weight loss and improves glycemic control in obese and diabetic rats.

OBJECTIVE: Dual amylin and calcitonin receptor agonists (DACRAs) are novel therapeutic agents that not only improve insulin sensitivity but also work as an adjunct to established T2DM therapies. DACRAs are currently administered once daily, though it is unknown whether DACRAs with increased plasma half-life can be developed as a once-weekly therapy. METHODS: The in vitro potencies of the KBP-066A and KBP-066 (non-acylated) were assessed using reporter assays. Acylation functionality was investigated by a combination of pharmacokinetics and acute food intake in rats. in vivo efficacies were investigated head-to-head in obese (HFD) and T2D (ZDF) models. RESULTS: In in vitro, KBP-066A activated the CTR and AMY-R potently, with no off-target activity. Acylation functionality was confirmed by acute tests, as KBP-066A demonstrated a prolonged PK and PD response compared to KBP-066. Both compounds induced potent and dose-dependent weight loss in the HFD rat model. In ZDF rats, fasting blood glucose/fasting insulin levels (tAUC) were reduced by 39%/50% and 36%/47% for KBP-066 and KBP-066A, respectively. This effect resulted in a 31% and 46% vehicle-corrected reduction in HbA1c at the end of the study for KBP-066 and KBP-066A, respectively. CONCLUSIONS: Here, we present pre-clinical data on an acylated DACRA, KBP-066A. The in vivo efficacy of KBP-066A is significantly improved compared to its non-acylated variant regarding weight loss and glycemic control in obese (HFD) and obese diabetic rats (ZDF). This compendium of pre-clinical studies highlights KBP-066A as a promising, once-weekly therapeutic agent for treating T2DM and obesity.

Turnover of type I and III collagen predicts progression of idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is characterized by the accumulation of fibrillar collagens in the alveolar space resulting in reduced pulmonary function and a high mortality rate. Biomarkers measuring the turnover of type I and III collagen could provide valuable information for prognosis and treatment decisions in IPF. METHODS: Serological biomarkers reflecting the formation of type III collagen (PRO-C3) and degradation of type I (C1M) and III collagen (C3M) were evaluated in a real-world cohort of 178 newly diagnosed IPF patients. Blood samples and clinical data were collected at baseline, six, and 12 months. Baseline and longitudinal biomarker levels were related to disease progression of IPF (defined as ≥ 5% decline in forced vital capacity (FVC) and/or ≥ 10% decline in diffusing capacity for carbon monoxide (DLco) and/or all-cause mortality at 12 months). Furthermore, we analysed differences in percentage change of biomarker levels from baseline between patients receiving antifibrotic treatment or not. RESULTS: Increased baseline levels of type I and III collagen turnover biomarkers were associated with a greater risk of disease progression within 12 months compared to patients with a low baseline type I and III collagen turnover. Patients with progressive disease had higher serum levels of C1M (P = 0.038) and PRO-C3 (P = 0.0022) compared to those with stable disease over one year. There were no differences in biomarker levels between patients receiving pirfenidone, nintedanib, or no antifibrotics. CONCLUSION: Baseline levels of type I and III collagen turnover were associated with disease progression within 12 months in a real-world cohort of IPF patients. Longitudinal biomarker levels of type I and III collagen turnover were related to progressive disease. Moreover, antifibrotic therapy did not affect type I and III collagen turnover biomarkers in these patients. PRO-C3 and C1M may be potential biomarkers for a progressive disease behavior in IPF.

Basement membrane collagen IV deficiency promotes abdominal aortic aneurysm formation.

Abdominal aortic aneurysm (AAA) is a complex disease which is incompletely accounted for. Basement membrane (BM) Collagen IV (COL4A1/A2) is abundant in the artery wall, and several lines of evidence indicate a protective role of baseline COL4A1/A2 in AAA development. Using Col4a1/a2 hemizygous knockout mice (Col4a1/a2+/-, 129Svj background) we show that partial Col4a1/a2 deficiency augmented AAA formation. Although unchallenged aortas were morphometrically and biomechanically unaffected by genotype, explorative proteomic analyses of aortas revealed a clear reduction in BM components and contractile vascular smooth muscle cell (VSMC) proteins, suggesting a central effect of the BM in maintaining VSMCs in the contractile phenotype. These findings were translated to human arteries by showing that COL4A1/A2 correlated to BM proteins and VSMC markers in non-lesioned internal mammary arteries obtained from coronary artery bypass procedures. Moreover, in human AAA tissue, MYH11 (VSMC marker) was depleted in areas of reduced COL4 as assessed by immunohistochemistry. Finally, circulating COL4A1 degradation fragments correlated with AAA progression in the largest Danish AAA cohort, suggesting COL4A1/A2 proteolysis to be an important feature of AAA formation. In sum, we identify COL4A1/A2 as a critical regulator of VSMC phenotype and a protective factor in AAA formation.

Elevated ectodomain of type 23 collagen is a novel biomarker of the intestinal epithelium to monitor disease activity in ulcerative colitis and Crohn's disease.

BACKGROUND: Impaired intestinal epithelial barrier is highly affected in inflammatory bowel disease. Transmembrane collagens connecting the epithelial cells to the extracellular matrix have an important role in epithelial cell homeostasis. Thus, we sought to determine whether the transmembrane type 23 collagen could serve as a surrogate marker for disease activity in patients with Crohn's disease and ulcerative colitis. METHODS: We developed an enzyme-linked immunosorbent assay to detect the ectodomain of type 23 collagen (PRO-C23) in serum, followed by evaluation of its levels in both acute and chronic dextran sulphate sodium colitis models in rats and human inflammatory bowel disease cohorts. Serum from 44 Crohn's disease and 29 ulcerative colitis patients with active and inactive disease was included. RESULTS: In the acute and chronic dextran sulphate sodium-induced rat colitis model, the PRO-C23 serum levels were significantly increased after colitis and returned to normal levels after disease remission. Serum levels of PRO-C23 were elevated in Crohn's disease (p < 0.05) and ulcerative colitis (p < 0.001) patients with active disease compared to healthy donors. PRO-C23 differentiated healthy donors from ulcerative colitis (area under the curve [AUC]: 0.81, p = 0.0009) and Crohn's disease (AUC: 0.70, p = 0.0124). PRO-C23 differentiated ulcerative colitis patients with active disease from those in remission (AUC: 0.75, p = 0.0219) and Crohn's disease patients with active disease from those in remission (AUC: 0.68, p = 0.05). CONCLUSION: PRO-C23 was elevated in rats with active colitis, and inflammatory bowel disease patients with active disease. Therefore, PRO-C23 may be used as a surrogate marker for monitoring disease activity in ulcerative colitis and Crohn's disease.

Inflammation and joint destruction may be linked to the generation of cartilage metabolites of ADAMTS-5 through activation of toll-like receptors.

OBJECTIVE: Links between pain and joint degradation are poorly understood. We investigated the role of activation of Toll-like receptors (TLR) by cartilage metabolites in initiating and maintaining the inflammatory loop in OA causing joint destruction. METHODS: Synovial membrane explants (SMEs) were prepared from OA patients' synovial biopsies. SMEs were cultured for 10 days under following conditions: culture medium alone, OSM + TNFα, TLR2 agonist - Pam2CSK4, Pam3CSK4 or synthetic aggrecan 32-mer, TLR4 agonist - Lipid A. Release of pro-inflammatory and degradation biomarkers (acMMP3 and C3M) were measured by ELISA in conditioned media along with IL-6. Additionally, human cartilage was digested with ADAMTS-5, with or without the ADAMTS-5 inhibiting nanobody - M6495. Digested cartilage solution (DCS) and synthetic 32-mer were tested for TLR activation in SEAP based TLR reporter assay. RESULTS: Western blotting confirmed TLR2 and TLR4 in untreated OA synovial biopsies. TLR agonists showed an increase in release of biomarkers - acMMP3 and C3M in SME. Synthetic 32-mer showed no activation in the TLR reporter assay. ADAMTS-5 degraded cartilage fragments activated TLR2 in vitro. Adding M6495 - an anti-ADAMTS-5 inhibiting nanobody®, blocked ADAMTS-5-mediated DCS TLR2 activation. CONCLUSION: TLR2 is expressed in synovium of OA patients and their activation by synthetic ligands causes increased tissue turnover. ADAMTS-5-mediated cartilage degradation leads to release of aggrecan fragments which activates the TLR2 receptor in vitro. M6495 suppressed cartilage degradation by ADAMTS-5, limiting the activation of TLR2. In conclusion, pain and joint destruction may be linked to generation of ADAMTS-5 cartilage metabolites.

The intestinal tissue homeostasis - the role of extracellular matrix remodeling in inflammatory bowel disease.

Introduction: Extracellular matrix (ECM) remodeling of the intestinal tissue is important in inflammatory bowel disease (IBD) due to the extensive mucosal remodeling. There are still gaps in our knowledge as to how ECM remodeling is related to intestinal epithelium homeostasis and healing of the intestinal mucosa.Areas covered: The aim of this review is to highlight the importance of the ECM in relation to the pathogenesis of IBD, while addressing basement membrane and interstitial matrix remodeling, and the processes of wound healing of the intestinal tissue in IBD.Expert opinion: In IBD, basement membrane remodeling may reflect the integrity of the intestinal epithelial-cell homeostasis. The interstitial matrix remodeling is associated with deep inflammation such as the transmural inflammation as seen in fistulas and intestinal fibrosis leading to fibrostenotic strictures, in patients with CD. The interplay between wound healing processes and ECM remodeling also affects the tissue homeostasis in IBD. The interstitial matrix, produced by fibroblasts, holds a very different biology as compared to the epithelial basement membrane in IBD. In combination with integration of wound healing, quantifying the interplay between damage and repair to these sub compartments may provide essential information in IBD patient profiling, mucosal healing and disease management.

A dual amylin and calcitonin receptor agonist inhibits pain behavior and reduces cartilage pathology in an osteoarthritis rat model.

OBJECTIVES: Pain and disability are the main clinical manifestations of osteoarthritis, for which only symptomatic therapies are available. Hence, there is a need for therapies that can simultaneously alter disease progression and provide pain relief. KBP is a dual amylin- and calcitonin-receptor agonist with antiresorptive and chondroprotective properties. In this study we investigated the effect of KBP in a rat model of osteoarthritis. METHODS: Medial meniscectomy (MNX) was performed in 39 rats, while 10 underwent sham surgery. Rats were treated with KBP and/or naproxen. Nociception was assessed by mechanical and cold allodynia, weight bearing asymmetry, and burrowing behavior. Blood samples were collected for biomarker measurements, and knees for histology. Cartilage histopathology was evaluated according to the advanced Osteoarthritis Research International (OARSI) score and KBPs in vitro antiresorptive effects were assessed using human osteoclasts cultured on bone. RESULTS: The MNX animals displayed an increased nociceptive behavior. Treatment with KBP attenuated the MNX-induced osteoarthritis-associated joint pain. The cartilage histopathology was significantly lower in rats treated with KBP than in MNX animals. Bone and cartilage degradation, assessed by CTX-I and CTX-II plasma levels, were decreased in all KBP-treated groups and KBP potently inhibited bone resorption in vitro. CONCLUSIONS: Our study demonstrates the effectiveness of KBP in ameliorating osteoarthritis-associated joint pain and in protecting the articular cartilage, suggesting KBP as a potential drug candidate for osteoarthritis.

A serological marker of the N-terminal neoepitope generated during LOXL2 maturation is elevated in patients with cancer or idiopathic pulmonary fibrosis.

OBJECTIVES: Lysyl oxidase like 2 (LOXL2) is associated with poor prognosis in idiopathic pulmonary disease (IPF) and cancer. We developed an Enzyme-linked immunosorbent assay (ELISA) targeting the LOXL2 neo-epitope generated through the release of the signal peptide during LOXL2 maturation. DESIGN AND METHODS: An ELISA targeting the N-terminal site of the human LOXL2 was developed including technical optimization and validation steps. Serum LOXL2 was measured in patients with breast, colorectal, lung, ovarian, pancreatic and prostate cancer, melanoma, IPF and in healthy controls (n = 16). RESULTS: A technically robust and specific assay was developed. LOXL2 was detectable in serum from healthy controls and showed reactivity towards recombinant LOXL2. Compared to controls, LOXL2 levels were significantly (p < 0.001-0.05) elevated in serum from patients with breast, colerectal, lung, ovarian and pancreatic cancer (mean range: 49-84 ng/mL), but not in prostate cancer (mean: 36 ng/mL) and malignant melanoma patients (41 ng/mL). Serum LOXL2 was elevated in IPF patients compared to healthy controls (mean: 76.5 vs 46.8 ng/mL; p > 0.001). CONCLUSIONS: A specific ELISA towards the N-terminal neo-epitope site in LOXL2 was developed which detected significantly elevated serum levels from patients with above-mentioned cancer types or IPF compared to healthy controls.

A fragment of SPARC reflecting increased collagen affinity shows pathological relevance in lung cancer - implications of a new collagen chaperone function of SPARC.

The matricellular protein SPARC (secreted proteome acidic and rich in cysteine) is known to bind collagens and regulate fibrillogenesis. Cleavage of SPARC at a single peptide bond, increases the affinity for collagens up to 20-fold. To investigate if this specific cleavage has pathological relevance in fibrotic disorders, we developed a competitive ELISA targeting the generated neo-epitope on the released fragment and quantified it in serum from patients with lung cancer, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD) and healthy subjects. Furthermore, the ability of SPARC to protect fibrillar collagens from proteolytic degradation was investigated in vitro, potentially adding a new collagen chaperone function to SPARC. The fragment was significantly elevated in lung cancer patients when compared to healthy subjects measured in a discovery cohort (p = 0.0005) and a validation cohort (p < 0.0001). No significant difference was observed for IPF and COPD patients compared to healthy subjects. When recombinant SPARC was incubated with type I or type III collagen and matrix metalloproteinase-9, collagen degradation was completely inhibited. Together, these data suggest that cleavage of SPARC at a specific site, which modulates collagen binding, is a physiological mechanism increased during pathogenesis of lung cancer. Furthermore, inhibition of fibrillar collagen degradation by SPARC adds a new chaperone function to SPARC which may play additional roles in the contribution to increased collagen deposition leading to a pro-fibrotic and tumorigenic environment.

Type V Collagen is Persistently Altered After Inguinal Hernia Repair.

BACKGROUND AND AIMS: Hernia formation is associated with alterations of collagen metabolism. Collagen synthesis and degradation cause a systemic release of products, which are measurable in serum. Recently, we reported changes in type V and IV collagen metabolisms in patients with inguinal and incisional hernia. The aim of this study was to determine if the altered collagen metabolism was persistent after hernia repair. MATERIAL AND METHODS: Patients who had undergone repairs for inguinal hernia (n = 11) or for incisional hernia (n = 17) were included in this study. Patients who had undergone elective cholecystectomy served as controls (n = 10). Whole venous blood was collected 35-55 months after operation. Biomarkers for type V collagen synthesis (Pro-C5) and degradation (C5M) and those for type IV collagen synthesis (P4NP) and degradation (C4M2) were measured by a solid-phase competitive assay. RESULTS: The turnover of type V collagen (Pro-C5/C5M) was slightly higher postoperatively when compared to preoperatively in the inguinal hernia group (P = 0.034). In addition, the results revealed a postoperatively lower type V collagen turnover level in the inguinal hernia group compared to controls (P = 0.012). In the incisional hernia group, the type V collagen turnover was higher after hernia repair (P = 0.004) and the postoperative turnover level was not different from the control group (P = 0.973). CONCLUSION: Patients with an inguinal hernia demonstrated a systemic and persistent type V collagen turnover alteration. This imbalance of the collagen metabolism may be involved in the development of inguinal hernias.

Safety, tolerability, and pharmacodynamics of an anti-interleukin-1α/ß dual variable domain immunoglobulin in patients with osteoarthritis of the knee: a randomized phase 1 study.

OBJECTIVE: To investigate the safety, tolerability, pharmacokinetics, and pharmacodynamics of ABT-981, a human dual variable domain immunoglobulin simultaneously targeting interleukin (IL)-1α and IL-1ß, in patients with knee osteoarthritis (OA). METHOD: This was a randomized, double-blind, placebo-controlled, single-center study of multiple subcutaneous (SC) injections of ABT-981 in patients with mild-to-moderate OA of the knee (NCT01668511). Three cohorts received ABT-981 (0.3, 1, or 3 mg/kg) or placebo every other week for a total of four SC injections, and one cohort received ABT-981 (3 mg/kg) or placebo every 4 weeks for a total of three SC injections. Assessment of safety and tolerability were the primary objectives. A panel of serum and urine biomarkers of inflammation and joint degradation were evaluated. RESULTS: A total of 36 patients were randomized (ABT-981, n = 28; placebo, n = 8); 31 (86%) completed the study. Adverse event (AE) rates were comparable between ABT-981 and placebo (54% vs 63%). The most common AE reported with ABT-981 vs placebo was injection site erythema (14% vs 0%). ABT-981 significantly reduced absolute neutrophil count and serum concentrations of IL-1α/IL-1ß, high-sensitivity C-reactive protein, and matrix metalloproteinase (MMP)-derived type 1 collagen. Serum concentrations of MMP-derived type 3 collagen and MMP-degraded C-reactive protein demonstrated decreasing trends with ABT-981. Antidrug antibodies were found in 37% of patients but were not associated with the incidence or severity of AEs. CONCLUSION: ABT-981 was generally well tolerated in patients with knee OA and engaged relevant tissue targets, eliciting an anti-inflammatory response. Consequently, ABT-981 may provide clinical benefit to patients with inflammation-driven OA.

Cathepsin-S degraded decorin are elevated in fibrotic lung disorders - development and biological validation of a new serum biomarker.

BACKGROUND: Decorin is one of the most abundant proteoglycans of the extracellular matrix and is mainly secreted and deposited in the interstitial matrix by fibroblasts where it plays an important role in collagen turnover and tissue homeostasis. Degradation of decorin might disturb normal tissue homeostasis contributing to extracellular matrix remodeling diseases. Here, we present the development and validation of a competitive enzyme-linked immunosorbent assay (ELISA) quantifying a specific fragment of degraded decorin, which has potential as a novel non-invasive serum biomarker for fibrotic lung disorders. METHODS: A fragment of decorin cleaved in vitro using human articular cartilage was identified by mass-spectrometry (MS/MS). Monoclonal antibodies were raised against the neo-epitope of the cleaved decorin fragment and a competitive ELISA assay (DCN-CS) was developed. The assay was evaluated by determining the inter- and intra-assay precision, dilution recovery, accuracy, analyte stability and interference. Serum levels were assessed in lung cancer patients, patients with idiopathic pulmonary fibrosis (IPF), patients with chronic obstructive pulmonary disease (COPD) and healthy controls. RESULTS: The DCN-CS ELISA was technically robust and was specific for decorin cleaved by cathepsin-S. DCN-CS was elevated in lung cancer patients (p < 0.0001) and IPF patients (p < 0.001) when compared to healthy controls. The diagnostic power for differentiating lung cancer patients and IPF patients from healthy controls was 0.96 and 0.77, respectively. CONCLUSION: Cathepsin-S degraded decorin could be quantified in serum using the DCN-CS competitive ELISA. The clinical data indicated that degradation of decorin by cathepsin-S is an important part of the pathology of lung cancer and IPF.

Misbalance in type III collagen formation/degradation as a novel serological biomarker for penetrating (Montreal B3) Crohn's disease.

BACKGROUND: Misbalances in extracellular matrix turnover are key factors in the development of stricturing (Montreal B2) and penetrating (Montreal B3) Crohn's disease. AIM: To determine whether serological markers for collagen formation and degradation could serve as biomarkers for complications of Crohn's disease. METHODS: Serum biomarkers for type I, III, V and VI collagen formation (P1NP, Pro-C3, Pro-C5, Pro-C6) and matrix metalloproteinase mediated degradation (C1M, C3M, C5M and C6M) were measured in a retrospective, single centre cohort of 112 patients with Crohn's disease in the terminal ileum (nonstricturing/nonpenetrating: n=40, stricturing: n=55, penetrating: n=17) and 24 healthy controls. Active inflammation was defined as CRP >5 mg/L. RESULTS: C3M and Pro-C5 levels were higher in penetrating vs nonpenetrating/nonstricturing and stricturing disease (33.6±5 vs 25.8±2.2 [P=.004] and 27.2±2.3 [P=.018] nmol/L C3M, 1262.7±259.4 vs 902.9±109.9 [P=.005] and 953.0±106.4 [P=.015] nmol/L Pro-C5). C1M (71.2±26.1 vs 46.2±6.2 nmol/L [P<.001]), C3M (31.6±3.9 vs 26.1±1.6 nmol/L [P=.002] and Pro-C5 levels (1171.7±171.5 vs 909.6±80.4 nmol/L [P=.002]) were higher in patients with active inflammation vs without active inflammation. Pro-C3/C3M-ratios were best to differentiate between penetrating vs nonstricturing/nonpenetrating and stricturing disease with area under the curves of 0.815±0.109 (P<.001) and 0.746±0.114 (P=.002) respectively. CONCLUSIONS: Serological biomarkers show that penetrating Crohn's disease is characterised by increased matrix metalloproteinase-9 degraded type III collagen and formation of type V collagen. Active inflammation in Crohn's disease is characterised by increased formation of type V collagen and increased matrix metalloproteinase mediated breakdown of type I, III collagen. Pro-C3/C3M ratios are superior in differentiating between penetrating Crohn's disease vs inflammatory and stricturing Crohn's disease.

Identification of serological biomarker profiles associated with total joint replacement in osteoarthritis patients.

OBJECTIVE: Establish a biomarker panel associated with all-cause total joint replacement (TJR) through identification of patients with osteoarthritis (OA) who do or do not progress to TJR and investigate effects of nonsteroidal anti-inflammatory drugs (NSAIDs). DESIGN: Serum samples from patients enrolled in phase III trials of tanezumab who experienced TJR (n = 174) or matched patients who did not (n = 321) were analyzed for bone, cartilage, soft tissue, and inflammation markers. Classification and Regression Tree (CART) analysis was used to identify biomarker phenotypes associated with TJR. RESULTS: At baseline, biomarker combinations for patients who did not use NSAIDs before starting tanezumab and used NSAIDs during tanezumab treatment <90 days ("nonNSAID"), identified 77% (95% confidence interval [CI]: 71-84%) of patients who experienced TJR and 77% (95% CI: 65-86%) who did not over a 6-month study period (on average). These biomarker combinations increased odds of identifying patients to remain free of a TJR by 3.3-fold. In patients who used NSAIDs continuously (during screening and ≥90 days during tanezumab treatment), 64% (95% CI: 54-73%) who had TJR and 75% (95% CI: 68-83%) who did not were identified by biomarker combinations different from nonNSAID patients, with an increase in odds of identifying patients to remain free of a TJR by two-fold. CONCLUSIONS: Although validation on other cohorts is necessary, biomarkers may assist in identifying patients who will need TJR. The profiles suggest NSAID use increases importance of bone metabolism in TJR pathology.

Neo-epitope Peptides as Biomarkers of Disease Progression for Muscular Dystrophies and Other Myopathies.

For several decades, serological biomarkers of neuromuscular diseases as dystrophies, myopathies and myositis have been limited to routine clinical biochemistry panels. Gauging the pathological progression is a prerequisite for proper treatment and therefore identifying accessible, easy to monitor biomarkers that can predict the disease progression would be an important advancement. Most muscle diseases involve accelerated muscle fiber degradation, inflammation, fatty tissue substitution and/or fibrosis. All these pathological traits have been shown to give rise to serological peptide biomarkers in other tissues, underlining the potential application of existing biomarkers of such traits in muscle disorders. A significant quantity of tissue is involved in these pathological mechanisms alongside with qualitative changes in protein turnover in myofibrillar, extra-cellular matrix and immunological cell protein fractions accompanied by alterations in body fluids. We propose that protein and peptides can leak out of the afflicted muscles and can be of use in diagnosis, prediction of pathology trajectory and treatment efficacy. Proteolytic cleavage systems are especially modulated during a range of muscle pathologies, thereby giving rise to peptides that are differentially released during disease manifestation. Therefore, we believe that pathology-specific post-translational modifications like cleavages can give rise to neoepitope peptides that may represent a promising class of peptides for discovery of biomarkers pertaining to neuromuscular diseases.

Quantification of fibronectin as a method to assess ex vivo extracellular matrix remodeling.

Altered architecture, composition and quality of the extracellular matrix (ECM) are pathological hallmarks of several inflammatory and fibro-proliferative pathological processes such as osteoarthritis (OA), rheumatoid arthritis (RA), fibrosis and cancer. One of the most important components of the ECM is fibronectin. Fibronectin serves as an adhesion molecule anchoring cells to the underlying basement membrane through direct interaction with integrin receptors. Fibronectin hereby modulates the properties of the ECM and affects cellular processes. Quantification of fibronectin remodeling could therefore be used to assess the changes in the ECM that occur during progression of fibro-proliferative pathologies. Ex vivo models are becoming state-of-the-art tools to study ECM remodeling as the cellular composition and the organization of the ECM are preserved. Ex vivo models may therefore be a valuable tool to study the ECM remodeling that occurs during progression of fibro-proliferative pathologies. The aim of this study was to quantify fibronectin remodeling in ex vivo models of cartilage and cancer. A competitive The enzyme-linked immunosorbent assay (ELISA) against the C-terminus of fibronectin was developed (FBN-C). The assay was evaluated in relation to specificity, technical performance and as a marker for quantification of fibronectin in cartilage and cancer ex vivo models. The ELISA was specific and technically stable. Cleavage of tumor tissue with MMP-2 released significantly higher levels of FBN-C compared to tissue with buffer only and western blot analysis revealed that FBN-C recognizes both full length and degraded fibronectin. When ex vivo cartilage cultures were stimulated with the anabolic factor TGFß and catabolic factors TNF-α and OSM, significantly higher levels of FBN-C were found in the conditioned media. Lastly, FBN-C was released from a cancer ex vivo model. In conclusion, we were able to quantify fibronectin remodeling in ex vivo models of cartilage and cancer. Quantification of fibronectin remodeling could be a valuable tool to understand ECM remodeling in ex vivo models of fibro-proliferative pathologies.

Excessive collagen turnover products are released during colorectal cancer progression and elevated in serum from metastatic colorectal cancer patients.

During cancer progression, the homeostasis of the extracellular matrix becomes imbalanced with an excessive collagen remodeling by matrix metalloproteinases. As a consequence, small protein fragments of degraded collagens are released into the circulation. We have investigated the potential of protein fragments of collagen type I, III and IV as novel biomarkers for colorectal cancer. Specific fragments of degraded type I, III and IV collagen (C1M, C3M, C4M) and type III collagen formation (Pro-C3) were assessed in serum from colorectal cancer patients, subjects with adenomas and matched healthy controls using well-characterized and validated ELISAs. Serum levels of the biomarkers were significantly elevated in colorectal cancer patients compared to subjects with adenomas (C1M, Pro-C3, C3M) and controls (C1M, Pro-C3). When patients were stratified according to their tumour stage, all four biomarkers were able to differentiate stage IV metastatic patients from all other stages. Combination of all markers with age and gender in a logistic regression model discriminated between metastatic and non-metastatic patients with an AUROC of 0.80. The data suggest that the levels of these collagen remodeling biomarkers may be a measure of tumour activity and invasiveness and may provide new clinical tools for monitoring of patients with advanced stage colorectal cancer.