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.

Sweat Proteomics in Cystic Fibrosis: Discovering Companion Biomarkers for Precision Medicine and Therapeutic Development.

In clinical routine, the diagnosis of cystic fibrosis (CF) is still challenging regardless of international consensus on diagnosis guidelines and tests. For decades, the classical Gibson and Cooke test measuring sweat chloride concentration has been a keystone, yet, it may provide normal or equivocal results. As of now, despite the combination of sweat testing, CFTR genotyping, and CFTR functional testing, a small fraction (1-2%) of inconclusive diagnoses are reported and justifies the search for new CF biomarkers. More importantly, in the context of precision medicine, with a view to early diagnosis, better prognosis, appropriate clinical follow-up, and new therapeutic development, discovering companion biomarkers of CF severity and phenotypic rescue are of utmost interest. To date, previous sweat proteomic studies have already documented disease-specific variations of sweat proteins (e.g., in schizophrenia and tuberculosis). In the current study, sweat samples from 28 healthy control subjects and 14 patients with CF were analyzed by nanoUHPLC-Q-Orbitrap-based shotgun proteomics, to look for CF-associated changes in sweat protein composition and abundance. A total of 1057 proteins were identified and quantified at an individual level, by a shotgun label-free approach. Notwithstanding similar proteome composition, enrichment, and functional annotations, control and CF samples featured distinct quantitative proteome profiles significantly correlated with CF, accounting for the respective inter-individual variabilities of control and CF sweat. All in all: (i) 402 sweat proteins were differentially abundant between controls and patients with CF, (ii) 68 proteins varied in abundance between F508del homozygous patients and patients with another genotype, (iii) 71 proteins were differentially abundant according to the pancreatic function, and iv) 54 proteins changed in abundance depending on the lung function. The functional annotation of pathophysiological biomarkers highlighted eccrine gland cell perturbations in: (i) protein biosynthesis and trafficking, (ii) CFTR proteostasis and membrane stability, and (iii) cell-cell adherence, membrane integrity, and cytoskeleton crosstalk. Cytoskeleton-related biomarkers were of utmost interest because of the consistency between variations observed here in CF sweat and variations previously documented in other CF tissues. From a clinical stance, nine candidate biomarkers of CF diagnosis (CUTA, ARG1, EZR, AGA, FLNA, MAN1A1, MIA3, LFNG, SIAE) and seven candidate biomarkers of CF severity (ARG1, GPT, MDH2, EML4 (F508del homozygous), MGAT1 (pancreatic insufficiency), IGJ, TOLLIP (lung function impairment)) were deemed suitable for further verification.

HPV infection alters vaginal microbiome through down-regulating host mucosal innate peptides used by Lactobacilli as amino acid sources.

Despite the high prevalence of both cervico-vaginal human papillomavirus (HPV) infection and bacterial vaginosis (BV) worldwide, their causal relationship remains unclear. While BV has been presumed to be a risk factor for HPV acquisition and related carcinogenesis for a long time, here, supported by both a large retrospective follow-up study (n = 6,085) and extensive in vivo data using the K14-HPV16 transgenic mouse model, we report a novel blueprint in which the opposite association also exists. Mechanistically, by interacting with several core members (NEMO, CK1 and ß-TrCP) of both NF-κB and Wnt/ß-catenin signaling pathways, we show that HPV E7 oncoprotein greatly inhibits host defense peptide expression. Physiologically secreted by the squamous mucosa lining the lower female genital tract, we demonstrate that some of these latter are fundamental factors governing host-microbial interactions. More specifically, several innate molecules down-regulated in case of HPV infection are hydrolyzed, internalized and used by the predominant Lactobacillus species as amino acid source sustaining their growth/survival. Collectively, this study reveals a new viral immune evasion strategy which, by its persistent/negative impact on lactic acid bacteria, ultimately causes the dysbiosis of vaginal microbiota.

Characterization of the Human Eccrine Sweat Proteome-A Focus on the Biological Variability of Individual Sweat Protein Profiles.

The potential of eccrine sweat as a bio-fluid of interest for diagnosis and personalized therapy has not yet been fully evaluated, due to the lack of in-depth sweat characterization studies. Thanks to recent developments in omics, together with the availability of accredited sweat collection methods, the analysis of human sweat may now be envisioned as a standardized, non-invasive test for individualized monitoring and personalized medicine. Here, we characterized individual sweat samples, collected from 28 healthy adult volunteers under the most standardized sampling methodology, by applying optimized shotgun proteomics. The thorough characterization of the sweat proteome allowed the identification of 983 unique proteins from which 344 were identified across all samples. Annotation-wise, the study of the sweat proteome unveiled the over-representation of newly addressed actin dynamics, oxidative stress and proteasome-related functions, in addition to well-described proteolysis and anti-microbial immunity. The sweat proteome composition correlated with the inter-individual variability of sweat secretion parameters. In addition, both gender-exclusive proteins and gender-specific protein abundances were highlighted, despite the high similarity between human female and male sweat proteomes. In conclusion, standardized sample collection coupled with optimized shotgun proteomics significantly improved the depth of sweat proteome coverage, far beyond previous similar studies. The identified proteins were involved in many diverse biological processes and molecular functions, indicating the potential of this bio-fluid as a valuable biological matrix for further studies. Addressing sweat variability, our results prove the proteomic profiling of sweat to be a promising bio-fluid analysis for individualized, non-invasive monitoring and personalized medicine.

Human Liver-Derived Extracellular Matrix for the Culture of Distinct Human Primary Liver Cells.

The lack of robust methods to preserve, purify and in vitro maintain the phenotype of the human liver's highly specialized parenchymal and non-parenchymal cell types importantly hampers their exploitation for the development of research and clinical applications. There is in this regard a growing interest in the use of tissue-specific extracellular matrix (ECM) to provide cells with an in vitro environment that more closely resembles that of the native tissue. In the present study, we have developed a method that allows for the isolation and downstream application of the human liver's main cell types from cryopreserved material. We also isolated and solubilized human liver ECM (HL-ECM), analyzed its peptidomic and proteomic composition by mass spectrometry and evaluated its interest for the culture of distinct primary human liver cells. Our analysis of the HL-ECM revealed proteomic diversity, type 1 collagen abundance and partial loss of integrity following solubilization. Solubilized HL-ECM was evaluated either as a coating or as a medium supplement for the culture of human primary hepatocytes, hepatic stellate cells and liver sinusoidal endothelial cells. Whereas the solubilized HL-ECM was suitable for cell culture, its impact on the phenotype and/or functionality of the human liver cells was limited. Our study provides a first detailed characterization of solubilized HL-ECM and a first report of its influence on the culture of distinct human primary liver cells.

Tissue proteomics for the next decade? Towards a molecular dimension in histology.

The concept of tissues appeared more than 200 years ago, since textures and attendant differences were described within the whole organism components. Instrumental developments in optics and biochemistry subsequently paved the way to transition from classical to molecular histology in order to decipher the molecular contexts associated with physiological or pathological development or function of a tissue. In 1941, Coons and colleagues performed the first systematic integrated examination of classical histology and biochemistry when his team localized pneumonia antigens in infected tissue sections. Most recently, in the early 21(st) century, mass spectrometry (MS) has progressively become one of the most valuable tools to analyze biomolecular compounds. Currently, sampling methods, biochemical procedures, and MS instrumentations allow scientists to perform "in depth" analysis of the protein content of any type of tissue of interest. This article reviews the salient issues in proteomics analysis of tissues. We first outline technical and analytical considerations for sampling and biochemical processing of tissues and subsequently the instrumental possibilities for proteomics analysis such as shotgun proteomics in an anatomical context. Specific attention concerns formalin fixed and paraffin embedded (FFPE) tissues that are potential "gold mines" for histopathological investigations. In all, the matrix assisted laser desorption/ionization (MALDI) MS imaging, which allows for differential mapping of hundreds of compounds on a tissue section, is currently the most striking evidence of linkage and transition between "classical" and "molecular" histology. Tissue proteomics represents a veritable field of research and investment activity for modern biomarker discovery and development for the next decade.

Proteome alteration induced by hTERT transfection of human fibroblast cells.

BACKGROUND: Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human telomerase reverse transcriptase (hTERT) gene. hTERT transfected cell lines may be of outstanding interest to monitor the effect of drugs targeting the telomerase activity. The incidence of hTERT gene transfection at the proteome level is a prerequisite to that purpose. The effect of the transfection has been studied on the proteome of human fibroblast (WI38). Cytosolic and nuclear fractions of WI38 cells, empty vector transfected WI38 (WI38-HPV) and hTERT WI38 cells were submitted to a 2D-DIGE (Two-Dimensional Differential In-Gel Electrophoresis) analysis. Only spots that had a similar abundance in WI38 and WI38-HPV, but were differentially expressed in WI38 hTERT were selected for MS identification. This method directly points to the proteins linked with the hTERT expression. Number of false positive differentially expressed proteins has been excluded by using control WI38-HPV cells. The proteome alteration induced by hTERT WI38 transfection should be taken into account in subsequent use of the cell line for anti-telomerase drugs evaluation. RESULTS: 2D-DIGE experiment shows that 57 spots out of 2246 are significantly differentially expressed in the cytosolic fraction due to hTERT transfection, and 38 were confidently identified. In the nuclear fraction, 44 spots out of 2172 were selected in the differential proteome analysis, and 14 were identified. The results show that, in addition to elongating telomeres, hTERT gene transfection has other physiological roles, among which an enhanced ER capacity and a potent cell protection against apoptosis. CONCLUSION: We show that the methodology reduces the complexity of the proteome analysis and highlights proteins implicated in other processes than telomere elongation. hTERT induced proteome changes suggest that telomerase expression enhances natural cell repair mechanisms and stress resistance probably required for long term resistance of immortalized cells. Thus, hTERT transfected cells can not be only consider as an immortal equivalent to parental cells but also as cells which are over-resistant to stresses. These findings are the prerequisite for any larger proteomics aiming to evaluate anti-telomerase drugs proteome alteration and thus therapeutics induced cell reactions.