Matrix gla protein, a potential marker of tissue remodelling and physiological ageing of the gut in crohn's disease.

BACKGROUND: The inactive dephosphorylated and uncarboxylated form of the matrix Gla protein (dp-ucMGP) has been shown to be increased in plasma of inflammatory bowel disease (IBD) patients. Our aim was to assess if the plasmatic level of dp-ucMGP could reflect disease endoscopic activity, presence of strictures and cumulative structural bowel damage in Crohn's disease (CD) patients. METHODS: The plasmatic level of dp-ucMGP was measured in a monocentric cohort of prospectively recruited patients. The analysis was done by chemiluminescent immunoassay on blood samples collected the day of a planned ileocolonoscopy. In addition to classical clinical data (gender, age, body mass index (BMI), disease duration, current treatment), endoscopic data (disease location, Crohn's Disease Endoscopic Index of Severity (CDEIS), mucosal healing (MH), presence of 9 CD lesion types) and biological markers (faecal calprotectin and C-reactive protein (CRP)) were collected. The association between dp-ucMGP level and Lémann index was also investigated. Univariate linear regression was used to investigate the relationship between dp-ucMGP level and different parameters collected. RESULTS: A total of 82 ileocolonoscopies and dp-ucMGP assays were performed in 75 CD patients (45 females; 37 ileocolonic, 19 ileal and 19 colonic diseases) between October 2012 and November 2019. A total of 24 patients (29.3%) showed MH. The dp-ucMGP levels were not associated with MH, CDEIS, faecal calprotectin or CRP levels. Plasmatic dp-ucMGP levels increased significantly with age (p = 0.0032), disease duration (p = 0.0033), corticosteroids use (p = 0.019) and tended to increase in patients with intestinal strictures (p = 0.086) but not with the Lémann index. CONCLUSION: The significant increase of plasmatic dp-ucMGP levels with age, disease duration and the trend observed in patients with non-ulcerated strictures may suggest that this extracellular matrix protein could be a marker of tissue remodelling and physiological ageing of the gut.

Distinct blood protein profiles associated with the risk of short-term and mid/long-term clinical relapse in patients with Crohn's disease stopping infliximab: when the remission state hides different types of residual disease activity.

OBJECTIVE: Despite being in sustained and stable remission, patients with Crohn's disease (CD) stopping anti-tumour necrosis factor α (TNFα) show a high rate of relapse (~50% within 2 years). Characterising non-invasively the biological profiles of those patients is needed to better guide the decision of anti-TNFα withdrawal. DESIGN: Ninety-two immune-related proteins were measured by proximity extension assay in serum of patients with CD (n=102) in sustained steroid-free remission and stopping anti-TNFα (infliximab). As previously shown, a stratification based on time to clinical relapse was used to characterise the distinct biological profiles of relapsers (short-term relapsers: <6 months vs mid/long-term relapsers: >6 months). Associations between protein levels and time to clinical relapse were determined by univariable Cox model. RESULTS: The risk (HR) of mid/long-term clinical relapse was specifically associated with a high serum level of proteins mainly expressed in lymphocytes (LAG3, SH2B3, SIT1; HR: 2.2-4.5; p<0.05), a low serum level of anti-inflammatory effectors (IL-10, HSD11B1; HR: 0.2-0.3; p<0.05) and cellular junction proteins (CDSN, CNTNAP2, CXADR, ITGA11; HR: 0.4; p<0.05). The risk of short-term clinical relapse was specifically associated with a high serum level of pro-inflammatory effectors (IL-6, IL12RB1; HR: 3.5-3.6; p<0.05) and a low or high serum level of proteins mainly expressed in antigen presenting cells (CLEC4A, CLEC4C, CLEC7A, LAMP3; HR: 0.4-4.1; p<0.05). CONCLUSION: We identified distinct blood protein profiles associated with the risk of short-term and mid/long-term clinical relapse in patients with CD stopping infliximab. These findings constitute an advance for the development of non-invasive biomarkers guiding the decision of anti-TNFα withdrawal.

Correlations for untargeted GC × GC-HRTOF-MS metabolomics of colorectal cancer.

INTRODUCTION: Modern comprehensive instrumentations provide an unprecedented coverage of complex matrices in the form of high-dimensional, information rich data sets. OBJECTIVES: In addition to the usual biomarker research that focuses on the detection of the studied condition, we aimed to define a proper strategy to conduct a correlation analysis on an untargeted colorectal cancer case study with a data set of 102 variables corresponding to metabolites obtained from serum samples analyzed with comprehensive two-dimensional gas chromatography coupled to high-resolution time-of-flight mass spectrometry (GC × GC-HRTOF-MS). Indeed, the strength of association existing between the metabolites contains potentially valuable information about the molecular mechanisms involved and the underlying metabolic network associated to a global perturbation, at no additional analytical effort. METHODS: Following Anscombe's quartet, we took particular attention to four main aspects. First, the presence of non-linear relationships through the comparison of parametric and non-parametric correlation coefficients: Pearson's r, Spearman's rho, Kendall's tau and Goodman-Kruskal's gamma. Second, the visual control of the detected associations through scatterplots and their associated regressions and angles. Third, the effect and handling of atypical samples and values. Fourth, the role of the precision of the data on the attribution of the ranks through the presence of ties. RESULTS: Kendall's tau was found the method of choice for the data set at hand. Its application highlighted 17 correlations significantly altered in the active state of colorectal cancer (CRC) in comparison to matched healthy controls (HC), from which 10 were specific to this state in comparison to the remission one (R-CRC) investigated on distinct patients. 15 metabolites involved in the correlations of interest, on the 25 unique ones obtained, were annotated (Metabolomics Standards Initiative level 2). CONCLUSIONS: The metabolites highlighted could be used to better understand the pathology. The systematic investigation of the methodological aspects that we expose allows to implement correlation analysis to various fields and many specific cases.

Comprehensive Insight into Colorectal Cancer Metabolites and Lipids for Human Serum: A Proof-of-Concept Study.

Colorectal cancer (CRC) ranks as the third most frequently diagnosed cancer and the second leading cause of cancer-related deaths. The current endoscopic-based or stool-based diagnostic techniques are either highly invasive or lack sufficient sensitivity. Thus, there is a need for less invasive and more sensitive screening approaches. We, therefore, conducted a study on 64 human serum samples representing three different groups (adenocarcinoma, adenoma, and control) using cutting-edge GC×GC-LR/HR-TOFMS (comprehensive two-dimensional gas chromatography coupled with low/high-resolution time-of-flight mass spectrometry). We analyzed samples with two different specifically tailored sample preparation approaches for lipidomics (fatty acids) (25 µL serum) and metabolomics (50 µL serum). In-depth chemometric screening with supervised and unsupervised approaches and metabolic pathway analysis were applied to both datasets. A lipidomics study revealed that specific PUFA (ω-3) molecules are inversely associated with increased odds of CRC, while some PUFA (ω-6) analytes show a positive correlation. The metabolomics approach revealed downregulation of amino acids (alanine, glutamate, methionine, threonine, tyrosine, and valine) and myo-inositol in CRC, while 3-hydroxybutyrate levels were increased. This unique study provides comprehensive insight into molecular-level changes associated with CRC and allows for a comparison of the efficiency of two different analytical approaches for CRC screening using same serum samples and single instrumentation.

Discovery of biomarker candidates associated with the risk of short-term and mid/long-term relapse after infliximab withdrawal in Crohn's patients: a proteomics-based study.

OBJECTIVE: A subset of Crohn's disease (CD) patients experiences mid/long-term remission after infliximab withdrawal. Biomarkers are needed to identify those patients. DESIGN: New biomarkers of relapse were searched in the baseline serum of CD patients stopping infliximab when they were under combined therapy (antimetabolite and infliximab) and stable clinical remission (diSconTinuation in CrOhn's disease patients in stable Remission on combined therapy with Immunosuppressors cohort, n=102). From shotgun proteomics experiment (discovery step), biomarker candidates were identified and further targeted by selected reaction monitoring (verification step). The dataset was stratified to search for markers of short-term (<6 months) or mid/long-term relapse (>6 months). The risk of relapse and the predicting capacity associated with biomarker candidates were evaluated using univariate Cox model and log-rank statistic, respectively. To test their complementary predicting capacity, biomarker candidates were systematically combined in pairs. RESULTS: Distinct biomarker candidates were associated with the risk (HR) of short-term (15 proteins, 2.9

Untargeted Serum Metabolic Profiling by Comprehensive Two-Dimensional Gas Chromatography-High-Resolution Time-of-Flight Mass Spectrometry.

While many laboratories take appropriate care, there are still cases where the performances of untargeted profiling methods suffer from a lack of design, control, and articulation of the various steps involved. This is particularly harmful to modern comprehensive analytical instrumentations that otherwise provide an unprecedented coverage of complex matrices. In this work, we present a global analytical workflow based on comprehensive two-dimensional gas chromatography coupled to high-resolution time-of-flight mass spectrometry. It was optimized for sample preparation and chromatographic separation and validated on in-house quality control (QC) and NIST SRM 1950 samples. It also includes a QC procedure, a multiapproach data (pre)processing workflow, and an original bias control procedure. Compounds of interest were identified using mass, retention, and biological information. As a proof of concept, 35 serum samples representing three subgroups of Crohn's disease (with high, low, and quiescent endoscopic activity) were analyzed along with 33 healthy controls. This led to the selection of 33 unique candidate biomarkers able to classify the Crohn's disease and healthy samples with an orthogonal partial least-squares discriminant analysis Q2 of 0.48 and a receiver-operating-characteristic area under the curve of 0.85 (100% sensitivity and 82% specificity in cross validation). Fifteen of these 33 candidates were reliably annotated (Metabolomics Standards Initiative level 2).

Specificity of metabolic colorectal cancer biomarkers in serum through effect size.

INTRODUCTION: Colorectal cancer is one of the most diagnosed cancers, leading to numerous deaths. In addition to existing screening methods, metabolic profiling could help both to diagnose and to understand the various states of the disease. OBJECTIVES: Find specific candidate biomarkers (CB) in serum of patients with colorectal cancer (CRC), in comparison to the situation after remission (R-CRC), evaluated on distinct patients. METHODS: All serum samples were analyzed using comprehensive two-dimensional gas chromatography (GC × GC) coupled to high resolution time of flight mass spectrometry (TOF-MS) through an optimized and validated untargeted analytical method regulated by a quality control (QC) system. First, we used a specific multi-approaches data (pre)processing workflow to highlight, annotate and assess the performances of the most altered metabolites between CRC patients (n = 18) and healthy control samples (HC, n = 19) specifically matched for age and gender, two of the most influential confounding factors. On the contrary, due to the difficulty to control for all clinical and demographic traits when sampling small cohorts, the samples from patients in remission (n = 17) were not matched. Because of the consequent risk of bias, the usual null hypothesis significance tests (NHST) could not be applied reliably. Therefore, we compared the R-CRC samples to another specifically matched group of healthy controls (R-HC, n = 17), and used this comparison to indirectly address the difference between patients with colorectal cancer and patients in remission through a measure called effect size (ES) whose methodological aspects were investigated. RESULTS: 24 candidate biomarkers were found significantly altered and able to discriminate the CRC and HC samples efficiently (Receiver Operating Characteristic (ROC) area under the curve (AUC) of 0.86, sensitivity and specificity of 0.72 and 0.78). 10 of those were found to have signals close to healthy levels in the R-CRC samples and were therefore specific to colorectal cancer. In the point-biserial case studied here, r-like (strength of association) and d-like (standardized mean difference) ES were directly convertible and only linear and rank-based ES were different. We therefore used and recommend Hedges' g, Spearman's rho and Kendall's tau, along with an unstandardized ES. The confidence intervals, that quantify the uncertainty of the measure, were well represented through scatterplots and distribution curves. CONCLUSION: The candidate biomarkers found, along with their specificity, could help for the detection of colorectal cancer, the diagnosis of remission, and for the understanding of its pathophysiology, after proper validation on independent cohorts. The effect size, here applied on a MS global profiling data set, is an ideal complement to NHST and a useful tool to compare and combine distinct cohorts, within a study as well as between studies (meta-analysis).

Multi-Enzymatic Limited Digestion: The Next-Generation Sequencing for Proteomics?

Over the past 40 years, proteomics, generically defined as the field dedicated to the identification and analysis of proteins, has tremendously gained in popularity and potency through advancements in genome sequencing, separative techniques, mass spectrometry, and bioinformatics algorithms. As a consequence, its scope of application has gradually enlarged and diversified to meet specialized topical biomedical subjects. Although the tryptic bottom-up approach is widely regarded as the gold standard for rapid screening of complex samples, its application for precise and confident mapping of protein modifications is often hindered due to partial sequence coverage, poor redundancy in indicative peptides, and lack of method flexibility. We here show how the synergic and time-limited action of a properly diluted mix of multiple enzymes can be exploited in a versatile yet straightforward protocol to alleviate present-day drawbacks. Merging bottom-up and middle-down ideologies, our results highlight broad assemblies of overlapping peptides that enable refined and reliable characterizations of proteins, including variant identification, and their carried modifications, including post-translational modifications, truncations, and cleavages. Beyond this boost in performance, our methodology also offers efficient de novo sequencing capabilities, in view of which we here present a dedicated custom assembly algorithm.

Treatments for Crohn's Disease-Associated Bowel Damage: A Systematic Review.

BACKGROUND & AIMS: Despite significant advances in the treatment of Crohn's disease (CD), most patients still develop stricturing or penetrating complications that require surgical resections. We performed a systematic review of mechanisms and potential treatments for tissue damage lesions in CD patients. METHODS: We searched the PubMed, MBASE, and Cochrane databases from January 1960 to July 2017 for full-length articles on CD, fibrosis, damage lesions, mesenchymal stem cells, and/or treatment. We also searched published conference abstracts and performed manual searches of all reference lists of relevant articles. RESULTS: Mechanisms of intestinal damage in patients with CD include fibroblast proliferation and migration, activation of stellate cells, recruitment of intestinal or extra-intestinal fibroblast, and cell trans-differentiation. An altered balance of metalloproteinases and tissue inhibitors of metalloproteinases might contribute to fistula formation. Treatment approaches that reduce excessive transforming growth factor beta (TGFB) activation might be effective in treating established intestinal damage. Stem cell therapies have been effective in tissue damage lesions in CD. Particularly, randomized controlled trials have shown local injections of mesenchymal stem cells to heal perianal fistulas. CONCLUSION: In a systematic review of mechanisms and treatments of bowel wall damage in patients with CD, we found a need to test drugs that reduce TGFB and increase healing of transmural damage lesions and to pursue research on local injection of mesenchymal stem cells.

OLFM4, KNG1 and Sec24C identified by proteomics and immunohistochemistry as potential markers of early colorectal cancer stages.

BACKGROUND: Despite recent advances in colorectal cancer (CRC) diagnosis and population screening programs, the identification of patients with preneoplastic lesions or with early CRC stages remains challenging and is important for reducing CRC incidence and increasing patient's survival. METHODS: We analysed 76 colorectal tissue samples originated from early CRC stages, normal or inflamed mucosa by label-free proteomics. The characterisation of three selected biomarker candidates was performed by immunohistochemistry on an independent set of precancerous and cancerous lesions harbouring increasing CRC stages. RESULTS: Out of 5258 proteins identified, we obtained 561 proteins with a significant differential distribution among groups of patients and controls. KNG1, OLFM4 and Sec24C distributions were validated in tissues and showed different expression levels especially in the two early CRC stages compared to normal and preneoplastic tissues. CONCLUSION: We highlighted three proteins that require further investigations to better characterise their role in early CRC carcinogenesis and their potential as early CRC markers.

Role of endoscopy, cross-sectional imaging and biomarkers in Crohn's disease monitoring.

Crohn's disease is characterised by recurrent and/or chronic inflammation of the gastrointestinal tract leading to cumulative intestinal tissue damage. Treatment tailoring to try to prevent this tissue damage as well as achieve optimal benefit/risk ratio over the whole disease course is becoming an important aspect of Crohn's disease management. For decades, clinical symptoms have been the main trigger for diagnostic procedures and treatment strategy adaptations. However, the correlation between symptoms and intestinal lesions is only weak. Furthermore, preliminary evidence suggests that a state of remission beyond the simple control of clinical symptoms, and including mucosal healing, may be associated with better disease outcome. Therefore monitoring the disease through the use of endoscopy and cross-sectional imaging is proposed. However, the degree of mucosal or bowel wall healing that needs to be reached to improve disease outcome has not been appropriately studied. Furthermore, owing to their invasive nature and cost, endoscopy and cross-sectional imaging are not optimal tools for the patients or the payers. The use of biomarkers as surrogate markers of intestinal and systemic inflammation might help. Two biomarkers have been most broadly assessed in Crohn's disease: C-reactive protein and faecal calprotectin. These markers correlate significantly with endoscopic lesions, with the risk of relapse and with response to therapy. They could be used to help make decisions about diagnostic procedures and treatment. In particular, with the use of appropriate threshold values, they could determine the need for endoscopic or medical imaging procedures to confirm the disease activity state.

Serum proteome signatures associated with ileal and colonic ulcers in Crohn's disease.

At a clinical level, ileal and colonic Crohn's disease (CD) are considered as separate entities. These subphenotypes need to be better supported by biological data to develop personalised medicine in CD. To this end, we combined different technologies (proximity extension assay, selected reaction monitoring, and high-sensitivity turbidimetric immunoassay (hsCRP)) to measure 207 immune-related serum proteins in CD patients presenting no endoscopic lesions (endoscopic remission) (n = 23), isolated ileal ulcers (n = 17), or isolated colonic ulcers (n = 16). We showed that isolated ileal ulcers and isolated colonic ulcers were specifically associated with 6 and 18 serum proteins, respectively: (high level: JUN, CNTNAP2; low level: FCRL6, LTA, CLEC4A, NTF4); (high level: hsCRP, IL6, APCS, CFB, MBL2, IL7, IL17A, CCL19, CXCL10, CSF3, IL10, CLEC4G, MMP12, VEGFA; low level: CLEC3B, GSN, TNFSF12, TPSAB1). Isolated ileal ulcers and isolated colonic ulcers were detected by hsCRP with an area under the receiver operating characteristics curve of 0.64 (p-value = 0.07) and 0.77 (p-value = 0.001), respectively. We highlighted distinct serum proteome profiles associated with ileal and colonic ulcers in CD, this finding might support the development of therapeutics and biomarkers tailored to disease location. SIGNIFICANCE: Although ileal and colonic Crohn's disease present important clinical differences (eg, progression, response to treatment and reliability of biomarkers), these two entities are managed with the same therapeutic strategy. The biological specificities of ileal and colonic Crohn's disease need to be better characterised to develop more personalised approaches. The present study used robust technologies (selected reaction monitoring, proximity extension assays and turbidimetric immunoassay) to quantify precisely 207 serum immune-related proteins in three groups of Crohn's disease patients presenting: 1) no endoscopic lesions (endoscopic remission) (n = 23); 2) isolated ileal ulcers (n = 17); 3) isolated colonic ulcers (n = 16). We found distinct serum proteome signatures associated with ileal and colonic ulcers. Our findings could foster the development of biomarkers and treatments tailored to Crohn's disease location.

Defining Biological Remission in Crohn's Disease: Interest, Challenges and Future Directions.

In Crohn's disease, the treat-to-target strategy has been greatly encouraged and has become a standard of care. In this context, defining the target [remission] constitutes a major stake and is fuelling the literature. Currently, clinical remission [symptom control] is no longer the only objective of treatments since it does not allow to closely control inflammation-induced tissue damage. The introduction of endoscopic remission as a therapeutic target clearly represented progress but this examination remains invasive, costly, not well accepted by patients and does not allow tight control of disease activity. More fundamentally, morphological techniques [e.g. endoscopy, histology, ultrasonography] are limited since they do not evaluate the biological activity of the disease but only its consequences. Besides, emerging evidence suggests that biological signs of disease activity could better guide treatment decisions than clinical parameters. In this context, we stress the necessity to define a novel treatment target: biological remission. Based on our previous work, we propose a conceptual definition of biological remission which goes beyond the classical normalization of inflammatory markers [C-reactive protein and faecal calprotectin]: absence of biological signs associated with the risk of short-term relapse and mid-/long-term relapse. The risk of short-term relapse seems essentially to be characterized by a persistent inflammatory state while the risk of mid-/long-term relapse implies a more heterogeneous biology. We discuss the value of our proposal [guiding treatment maintenance, escalation or de-escalation] but also the fact that its clinical implementation would require overcoming major challenges. Finally, future directions are proposed to better define biological remission.

Discovery and biochemical characterisation of four novel biomarkers for osteoarthritis.

OBJECTIVE: Knee osteoarthritis (OA) is a heterogeneous, complex joint pathology of unknown aetiology. Biomarkers have been widely used to investigate OA but currently available biomarkers lack specificity and sensitivity. Therefore, novel biomarkers are needed to better understand the pathophysiological processes of OA initiation and progression. METHODS: Surface enhanced laser desorption/ionisation-time of flight-mass spectrometry proteomic technique was used to analyse protein expression levels in 284 serum samples from patients with knee OA classified according to Kellgren and Lawrence (K&L) score (0-4). OA serum samples were also compared to serum samples provided by healthy individuals (negative control subjects; NC; n=36) and rheumatoid arthritis (RA) patients (n=25). Proteins that gave similar signal in all K&L groups of OA patients were ignored, whereas proteins with increased or decreased levels of expression were selected for further studies. RESULTS: Two proteins were found to be expressed at higher levels in sera of OA patients at all four K&L scores compared to NC and RA, and were identified as V65 vitronectin fragment and C3fpeptide. Of the two remaining proteins, one showed increased expression (unknown protein at m/z of 3762) and the other (identified as connective tissue-activating peptide III protein) was decreased in K&L scores >2 subsets compared to NC, RA and K&L scores 0 or 1 subsets. CONCLUSION: The authors detected four unexpected biomarkers (V65 vitronectin fragment, C3f peptide, CTAP-III and m/z 3762 protein) that could be relevant in the pathophysiological process of OA as having significant correlation with parameters reflecting local inflammation and bone remodelling, as well as decrease in cartilage turnover.

Review article: distinctions between ileal and colonic Crohn's disease: from physiology to pathology.

BACKGROUND: Ileal and colonic Crohn's disease seem to be two separate entities. AIMS: To describe the main physiological distinctions between the small and the large intestine and to analyse the differences between ileal and colonic Crohn's disease. METHODS: The relevant literature was critically examined and synthesised. RESULTS: The small and large intestine have fundamental distinctions (anatomy, cellular populations, immune defence, microbiota). The differences between ileal and colonic Crohn's disease are highlighted by a heterogeneous body of evidence including clinical features (natural history of the disease, efficacy of treatments, and monitoring), epidemiological data (smoking status, age, gender) and biological data (genetics, microbiota, immunity, mesenteric fat). However, the contribution of these factors to disease location remains poorly understood. CONCLUSION: The classification of ileal and colonic Crohn's disease as distinct subphenotypes is well supported by the literature. Understanding of these differences could be exploited to develop more individualised patient care.

Potential Role of Epithelial Endoplasmic Reticulum Stress and Anterior Gradient Protein 2 Homologue in Crohn's Disease Fibrosis.

BACKGROUND AND AIMS: Intestinal fibrosis is a common complication of Crohn's disease [CD]. It is characterised by an accumulation of fibroblasts differentiating into myofibroblasts secreting excessive extracellular matrix. The potential role of the intestinal epithelium in this fibrotic process remains poorly defined. METHODS: We performed a pilot proteomic study comparing the proteome of surface epithelium, isolated by laser-capture microdissection, in normal and fibrotic zones of resected ileal CD strictures [13 zones collected in five patients]. Proteins of interests were validated by immunohistochemistry [IHC] in ileal and colonic samples of stricturing CD [n = 44], pure inflammatory CD [n = 29], and control [n = 40] subjects. The pro-fibrotic role of one selected epithelial protein was investigated through in-vitro experiments using HT-29 epithelial cells and a CCD-18Co fibroblast to myofibroblast differentiation model. RESULTS: Proteomic study revealed an endoplasmic reticulum [ER] stress proteins increase in the epithelium of CD ileal fibrotic strictures, including anterior gradient protein 2 homologue [AGR2] and binding-immunoglobulin protein [BiP]. This was confirmed by IHC. In HT-29 cells, tunicamycin-induced ER stress triggered AGR2 intracellular expression and its secretion. Supernatant of these HT-29 cells, pre-conditioned by tunicamycin, led to a myofibroblastic differentiation when applied on CCD-18Co fibroblasts. By using recombinant protein and blocking agent for AGR2, we demonstrated that the secretion of this protein by epithelial cells can play a role in the myofibroblastic differentiation. CONCLUSIONS: The development of CD fibrotic strictures could involve epithelial ER stress and particularly the secretion of AGR2.

Challenges for biomarker discovery in body fluids using SELDI-TOF-MS.

Protein profiling using SELDI-TOF-MS has gained over the past few years an increasing interest in the field of biomarker discovery. The technology presents great potential if some parameters, such as sample handling, SELDI settings, and data analysis, are strictly controlled. Practical considerations to set up a robust and sensitive strategy for biomarker discovery are presented. This paper also reviews biological fluids generally available including a description of their peculiar properties and the preanalytical challenges inherent to sample collection and storage. Finally, some new insights for biomarker identification and validation challenges are provided.

Marked Increased Production of Acute Phase Reactants by Skeletal Muscle during Cancer Cachexia.

Loss of skeletal muscle mass in cancer cachexia is recognized as a predictor of mortality. This study aimed to characterize the changes in the muscle secretome associated with cancer cachexia to gain a better understanding of the mechanisms involved and to identify secreted proteins which may reflect this wasting process. The changes in the muscle proteome of the C26 model were investigated by label-free proteomic analysis followed by a bioinformatic analysis in order to identify potentially secreted proteins. Multiple reaction monitoring and Western blotting were used to verify the presence of candidate proteins in the circulation. Our results revealed a marked increased muscular production of several acute phase reactants (APR: Haptoglobin, Serine protease inhibitor A3N, Complement C3, Serum amyloid A-1 protein) which are released in the circulation during C26 cancer cachexia. This was confirmed in other models of cancer cachexia as well as in cancer patients. Glucocorticoids and proinflammatory cytokines are responsible for an increased production of APR by muscle cells. Finally, their muscular expressions are strongly positively correlated with body weight loss as well as the muscular induction of atrogens. Our study demonstrates therefore a marked increased production of APR by the muscle in cancer cachexia.

Proteomics Highlights Common and Distinct Pathophysiological Processes Associated with Ileal and Colonic Ulcers in Crohn's Disease.

BACKGROUND AND AIMS: Based on genetics and natural history, Crohn's disease can be separated into two entities, an ileal and a colonic disease. Protein-based approaches are needed to elucidate whether such subphenotypes are related to distinct pathophysiological processes. METHODS: The proteome of ulcer edges was compared with that of paired control tissue samples [n = 32 biopsies] by differential proteomics in the ileum and the colon of Crohn's disease patients [n = 16]. The results were analysed using a hypothesis-driven approach [based on the literature] and a hypothesis-free approach [pathway enrichment analyses] to determine common and segment-specific pathophysiological processes associated with ileal and colonic CD ulcer edges. To confirm the involvement of a key pathway highlighted by proteomics, two proteins were also studied by immunochemistry. RESULTS: In the ileum and the colon, 4428 and 5204 proteins, respectively, were identified and quantified. Ileal and colonic ulcer edges differed in having a distinct distribution of proteins associated with epithelial-mesenchymal transition, neutrophil degranulation, and ribosomes. Ileal and colonic ulcer edges were similarly characterized by an increase in the proteins implicated in the endoplasmic reticulum protein-processing pathway and a decrease in mitochondrial proteins. Immunochemistry confirmed the presence of endoplasmic reticulum stress in the mucosa of ileal and colonic ulcer edges. CONCLUSION: This study provides protein-based evidence for partially distinct pathophysiological processes being associated with ileal and colonic ulcer edges in Crohn's disease patients. This could constitute a first step toward the development of gut segment-specific diagnostic markers and therapeutics.