Senescence-Driven Inflammatory and Trophic Microenvironment Imprints Mesenchymal Stromal/Stem Cells in Osteoarthritic Patients.

Senescent cells promote progressive tissue degeneration through the establishment of a combined inflammatory and trophic microenvironment. The cellular senescence state has therefore emerged as a central driving mechanism of numerous age-related diseases, including osteoarthritis (OA), the most common rheumatic disease. Senescence hallmarks are detectable in chondrocytes, synoviocytes and sub-chondral bone cells. This study investigates how the senescence-driven microenvironment could impact the cell fate of resident osteoarticular mesenchymal stromal/stem cells (MSCs) that are hence contributing to OA disease progression. For that purpose, we performed a comparative gene expression analysis of MSCs isolated from healthy donors that were in vitro chronically exposed either to interferon-gamma (IFN-γ) or Transforming Growth Factor beta 1 (TGFß1), two archetypical factors produced by senescent cells. Both treatments reduced MSC self-renewal capacities by upregulating different senescence-driven cycle-dependent kinase inhibitors. Furthermore, a common set of differentially expressed genes was identified in both treated MSCs that was also found enriched in MSCs isolated from OA patients. These findings highlight an imprinting of OA MSCs by the senescent joint microenvironment that changes their matrisome gene expression. Altogether, this research gives new insights into OA etiology and points to new innovative therapeutic opportunities to treat OA patients.

Sputum Type 2 Markers Could Predict Remission in Severe Asthma Treated With Anti-IL-5.

BACKGROUND: Biotherapies targeting IL-5 allow a tangible improvement of asthma. However, all patients do not respond the same way to these treatments. Even if high blood eosinophil counts seem to be associated with a reduction in exacerbations with treatment targeting IL-5, we lack biomarkers for the prediction of remission after these very expensive treatments. RESEARCH QUESTION: Are there biomarkers of remission after therapy targeting IL-5 in the sputum of patients with severe eosinophilic asthma? STUDY DESIGN AND METHODS: This observational study included 52 patients with severe asthma initiated with anti-IL-5 therapy and recruited from the asthma clinic of the Centre Hospitalier Universitaire of Liege, Belgium. Remission was defined as patients who combined the following at 1 year after therapy: no chronic treatment with oral corticosteroids; no exacerbation; asthma control questionnaire score < 1.5, asthma control test score > 19, or both; FEV1 of ≥ 80% predicted, improvement of FEV1 of ≥ 10%, or both; and a blood eosinophil count < 300 cells/µL. Eosinophil peroxidase (EPX), IgE, IL-3, IL-4, IL-5, IL-13, IL-25, IL-33, granulocyte-macrophage colony-stimulating factor, thymic stromal lymphopoietin (TSLP), and eotaxin-1 levels were measured in the sputum of these patients before anti-IL-5 treatment. RESULTS: Among the 52 patients, 11 were classified as being in remission. These patients were characterized by higher sputum eosinophil, macrophage, and lymphocyte counts, whereas the sputum neutrophil percentage was lower than in the nonremission group. In addition, the sputum eotaxin-1, TSLP, IL-5, EPX, and IgE protein levels were higher at baseline in the remission group compared with the nonremission group. Univariate regression analysis revealed that male vs female sex, sputum neutrophil percentage, eotaxin-1, IL-5, and EPX were potential predictors of remission. INTERPRETATION: Sputum type 2 markers seemed to be potentially predictive of remission after anti-IL-5 therapy in a cohort of patients with severe eosinophilic asthma. These results need validation on a larger cohort.

Deeper insights into long-term survival heterogeneity of pancreatic ductal adenocarcinoma (PDAC) patients using integrative individual- and group-level transcriptome network analyses.

Pancreatic ductal adenocarcinoma (PDAC) is categorized as the leading cause of cancer mortality worldwide. However, its predictive markers for long-term survival are not well known. It is interesting to delineate individual-specific perturbed genes when comparing long-term (LT) and short-term (ST) PDAC survivors and integrate individual- and group-based transcriptome profiling. Using a discovery cohort of 19 PDAC patients from CHU-Liège (Belgium), we first performed differential gene expression analysis comparing LT to ST survivor. Second, we adopted systems biology approaches to obtain clinically relevant gene modules. Third, we created individual-specific perturbation profiles. Furthermore, we used Degree-Aware disease gene prioritizing (DADA) method to develop PDAC disease modules; Network-based Integration of Multi-omics Data (NetICS) to integrate group-based and individual-specific perturbed genes in relation to PDAC LT survival. We identified 173 differentially expressed genes (DEGs) in ST and LT survivors and five modules (including 38 DEGs) showing associations to clinical traits. Validation of DEGs in the molecular lab suggested a role of REG4 and TSPAN8 in PDAC survival. Via NetICS and DADA, we identified various known oncogenes such as CUL1 and TGFB1. Our proposed analytic workflow shows the advantages of combining clinical and omics data as well as individual- and group-level transcriptome profiling.

CEMIP (KIAA1199) regulates inflammation, hyperplasia and fibrosis in osteoarthritis synovial membrane.

Osteoarthritis (OA) synovial membrane is mainly characterized by low-grade inflammation, hyperplasia with increased cell proliferation and fibrosis. We previously underscored a critical role for CEMIP in fibrosis of OA cartilage. However, its role in OA synovial membrane remains unknown. An in vitro model with fibroblast-like synoviocytes from OA patients and an in vivo model with collagenase-induced OA mice were used to evaluate CEMIP-silencing effects on inflammation, hyperplasia and fibrosis. Our results showed that i. CEMIP expression was increased in human and mouse inflamed synovial membrane; ii. CEMIP regulated the inflammatory response pathway and inflammatory cytokines production in vitro and in vivo; iii. CEMIP induced epithelial to mesenchymal transition pathway and fibrotic markers in vitro and in vivo; iv. CEMIP increased cell proliferation and synovial hyperplasia; v. CEMIP expression was increased by inflammatory cytokines and by TGF-ß signaling; vi. anti-fibrotic drugs decreased CEMIP expression. All these findings highlighted the central role of CEMIP in OA synovial membrane development and underscored that targeting CEMIP could be a new therapeutic approach.

The Emerging Role of Non-Coding RNAs in Osteoarthritis.

Osteoarthritis (OS) is the most frequent degenerative condition in the joints, disabling many adults. Several abnormalities in the articular cartilage, subchondral bone, synovial tissue, and meniscus have been detected in the course of OA. Destruction of articular cartilage, the formation of osteophytes, subchondral sclerosis, and hyperplasia of synovial tissue are hallmarks of OA. More recently, several investigations have underscored the regulatory roles of non-coding RNAs (ncRNAs) in OA development. Different classes of non-coding RNAs, including long ncRNAs (lncRNAs), microRNAs (miRNAs), and circular RNAs (circRNAs), have been reported to affect the development of OA. The expression level of these transcripts has also been used as diagnostic tools in OA. In the present article, we aimed at reporting the role of these transcripts in this process. We need to give a specific angle on the pathology to provide meaningful thoughts on it.

Influence of Glucocorticoids on Cellular Senescence Hallmarks in Osteoarthritic Fibroblast-like Synoviocytes.

Osteoarthritis (OA) is recognized as being a cellular senescence-linked disease. Intra-articular injections of glucocorticoids (GC) are frequently used in knee OA to treat synovial effusion but face controversies about toxicity. We investigated the influence of GC on cellular senescence hallmarks and senescence induction in fibroblast-like synoviocytes (FLS) from OA patients and mesenchymal stem cells (MSC). METHODS: Cellular senescence was assessed via the proliferation rate, ß-galactosidase staining, DNA damage and CKI expression (p21, p16INK4A). Experimental senescence was induced by irradiation. RESULTS: The GC prednisolone did not induce an apparent senescence phenotype in FLS, with even higher proliferation, no accumulation of ß-galactosidase-positive cells nor DNA damage and reduction in p21mRNA, only showing the enhancement of p16INK4A. Prednisolone did not modify experimental senescence induction in FLS, with no modulation of any senescence parameters. Moreover, prednisolone did not induce a senescence phenotype in MSC: despite high ß-galactosidase-positive cells, no reduction in proliferation, no DNA damage and no CKI enhancement was observed. CONCLUSIONS: We provide reassuring in vitro data about the use of GC regarding cellular senescence involvement in OA: the GC prednisolone did not induce a senescent phenotype in OA FLS (the proliferation ratio was even higher) and in MSC and did not worsen cellular senescence establishment.

Correction: In Vivo Tumorigenesis Was Observed after Injection of In Vitro Expanded Neural Crest Stem Cells Isolated from Adult Bone Marrow.

[This corrects the article DOI: 10.1371/journal.pone.0046425.].

Differences in plasma microRNA content impair microRNA-based signature for breast cancer diagnosis in cohorts recruited from heterogeneous environmental sites.

Circulating microRNAs are non-invasive biomarkers that can be used for breast cancer diagnosis. However, differences in cancer tissue microRNA expression are observed in populations with different genetic/environmental backgrounds. This work aims at checking if a previously identified diagnostic circulating microRNA signature is efficient in other genetic and environmental contexts, and if a universal circulating signature might be possible. Two populations are used: women recruited in Belgium and Rwanda. Breast cancer patients and healthy controls were recruited in both populations (Belgium: 143 primary breast cancers and 136 healthy controls; Rwanda: 82 primary breast cancers and 73 healthy controls; Ntot = 434), and cohorts with matched age and cancer subtypes were compared. Plasmatic microRNA profiling was performed by RT-qPCR. Random Forest was used to (1) evaluate the performances of the previously described breast cancer diagnostic tool identified in Belgian-recruited cohorts on Rwandan-recruited cohorts and vice versa; (2) define new diagnostic signatures common to both recruitment sites; (3) define new diagnostic signatures efficient in the Rwandan population. None of the circulating microRNA signatures identified is accurate enough to be used as a diagnostic test in both populations. However, accurate circulating microRNA signatures can be found for each specific population, when taken separately.

New Proteins Contributing to Immune Cell Infiltration and Pannus Formation of Synovial Membrane from Arthritis Diseases.

An inflamed synovial membrane plays a major role in joint destruction and is characterized by immune cells infiltration and fibroblast proliferation. This proteomic study considers the inflammatory process at the molecular level by analyzing synovial biopsies presenting a histological inflammatory continuum throughout different arthritis joint diseases. Knee synovial biopsies were obtained from osteoarthritis (OA; n = 9), chronic pyrophosphate arthropathy (CPPA; n = 7) or rheumatoid arthritis (RA; n = 8) patients. The histological inflammatory score was determined using a semi-quantitative scale based on synovial hyperplasia, lymphocytes, plasmocytes, neutrophils and macrophages infiltration. Proteomic analysis was performed by liquid chromatography-mass spectrometry (LC-MS/MS). Differentially expressed proteins were confirmed by immunohistochemistry. Out of the 1871 proteins identified and quantified by LC-MS/MS, 10 proteins (LAP3, MANF, LCP1, CTSZ, PTPRC, DNAJB11, EML4, SCARA5, EIF3K, C1orf123) were differentially expressed in the synovial membrane of at least one of the three disease groups (RA, OA and CPPA). Significant increased expression of the seven first proteins was detected in RA and correlated to the histological inflammatory score. Proteomics is therefore a powerful tool that provides a molecular pattern to the classical histology usually applied for synovitis characterization. Except for LCP1, CTSZ and PTPRC, all proteins have never been described in human synovitis.

Exosomal Long Non-Coding RNAs in Lung Diseases.

Within the non-coding genome landscape, long non-coding RNAs (lncRNAs) and their secretion within exosomes are a window that could further explain the regulation, the sustaining, and the spread of lung diseases. We present here a compilation of the current knowledge on lncRNAs commonly found in Chronic Obstructive Pulmonary Disease (COPD), asthma, Idiopathic Pulmonary Fibrosis (IPF), or lung cancers. We built interaction networks describing the mechanisms of action for COPD, asthma, and IPF, as well as private networks for H19, MALAT1, MEG3, FENDRR, CDKN2B-AS1, TUG1, HOTAIR, and GAS5 lncRNAs in lung cancers. We identified five signaling pathways targeted by these eight lncRNAs over the lung diseases mentioned above. These lncRNAs were involved in ten treatment resistances in lung cancers, with HOTAIR being itself described in seven resistances. Besides, five of them were previously described as promising biomarkers for the diagnosis and prognosis of asthma, COPD, and lung cancers. Additionally, we describe the exosomal-based studies on H19, MALAT1, HOTAIR, GAS5, UCA1, lnc-MMP2-2, GAPLINC, TBILA, AGAP2-AS1, and SOX2-OT. This review concludes on the need for additional studies describing the lncRNA mechanisms of action and confirming their potential as biomarkers, as well as their involvement in resistance to treatment, especially in non-cancerous lung diseases.

Suitable reference genes determination for real-time PCR using induced sputum samples.

Induced sputum is a non-invasive method of collecting cells from airways. Gene expression analysis from sputum cells has been used to understand the underlying mechanisms of airway diseases such as asthma or chronic obstructive pulmonary disease (COPD). Suitable reference genes for normalisation of target mRNA levels between sputum samples have not been defined so far.The current study assessed the expression stability of nine common reference genes in sputum samples from 14 healthy volunteers, 12 asthmatics and 12 COPD patients.Using three different algorithms (geNorm, NormFinder and BestKeeper), we identified HPRT1 and GNB2L1 as the most optimal reference genes to use for normalisation of quantitative reverse transcriptase (RT) PCR data from sputum cells. The higher expression stability of HPRT1 and GNB2L1 were confirmed in a validation set of patients including nine healthy controls, five COPD patients and five asthmatic patients. In this group, the RNA extraction and RT-PCR methods differed, which attested that these genes remained the most reliable whatever the method used to extract the RNA, generate complementary DNA or amplify it.Finally, an example of relative quantification of gene expression linked to eosinophils or neutrophils provided more accurate results after normalisation with the reference genes identified as the most stable compared to the least stable and confirmed our findings.

Predictive and prognostic role of peripheral blood eosinophil count in triple-negative and hormone receptor-negative/HER2-positive breast cancer patients undergoing neoadjuvant treatment.

In current clinical practices, up to 27% of all breast cancer patients receive neoadjuvant chemotherapy. High pathological complete response rate is frequently associated with tumor-infiltrating lymphocytes. Additionally, circulating immune cells are also often linked to chemotherapy response. We performed a retrospective analysis on a cohort of 112 breast cancer patients (79 triple-negative, 33 hormone receptor-negative/HER2-positive) treated with standard neoadjuvant chemotherapy. Eosinophil and lymphocyte counts were collected from whole blood at baseline and during follow-ups and their associations with pathological complete response, relapse, disease-free and breast cancer-specific survival were analyzed. We observed a higher pathological complete response rate in patients who presented at baseline a relative eosinophil count ≥ 1.5% (55.6%) than in those with a relative eosinophil count < 1.5% (36.2%)(p = 0.04). An improvement in breast cancer-specific survival in patients with high relative eosinophil count (p = 0.05; HR = 0.336; 95% CI = 0.107-1.058) or with high relative lymphocyte count (threshold = 17.5%, p = 0.01; HR = 0.217; 95% CI = 0.060-0.783) were also observed. Upon combining the two parameters into the eosinophil x lymphocyte product with a threshold at 35.8, associations with pathological complete response (p = 0.002), relapse (p = 0.028), disease-free survival (p = 0.012) and breast cancer-specific survival (p = 0.001) were also recorded. In conclusion, the relative eosinophil count and eosinophil x lymphocyte product could be promising, affordable and accessible new biomarkers that are predictive for neoadjuvant chemotherapy response and prognostic for longer survival in triple-negative and hormone receptors-negative/HER2-positive breast cancers. Confirmation of these results in a larger patient population is needed.

Natural Antisense Transcripts: Molecular Mechanisms and Implications in Breast Cancers.

Natural antisense transcripts are RNA sequences that can be transcribed from both DNA strands at the same locus but in the opposite direction from the gene transcript. Because strand-specific high-throughput sequencing of the antisense transcriptome has only been available for less than a decade, many natural antisense transcripts were first described as long non-coding RNAs. Although the precise biological roles of natural antisense transcripts are not known yet, an increasing number of studies report their implication in gene expression regulation. Their expression levels are altered in many physiological and pathological conditions, including breast cancers. Among the potential clinical utilities of the natural antisense transcripts, the non-coding|coding transcript pairs are of high interest for treatment. Indeed, these pairs can be targeted by antisense oligonucleotides to specifically tune the expression of the coding-gene. Here, we describe the current knowledge about natural antisense transcripts, their varying molecular mechanisms as gene expression regulators, and their potential as prognostic or predictive biomarkers in breast cancers.

Genomic studies of multiple myeloma reveal an association between X chromosome alterations and genomic profile complexity.

The genomic profile of multiple myeloma (MM) has prognostic value by dividing patients into a good prognosis hyperdiploid group and a bad prognosis nonhyperdiploid group with a higher incidence of IGH translocations. This classification, however, is inadequate and many other parameters like mutations, epigenetic modifications, and genomic heterogeneity may influence the prognosis. We performed a genomic study by array-based comparative genomic hybridization on a cohort of 162 patients to evaluate the frequency of genomic gains and losses. We identified a high frequency of X chromosome alterations leading to partial Xq duplication, often associated with inactive X (Xi) deletion in female patients. This partial X duplication could be a cytogenetic marker of aneuploidy as it is correlated with a high number of chromosomal breakages. Patient with high level of chromosomal breakage had reduced survival regardless the region implicated. A higher transcriptional level was shown for genes with potential implication in cancer and located in this altered region. Among these genes, IKBKG and IRAK1 are members of the NFKB pathway which plays an important role in MM and is a target for specific treatments. © 2016 Wiley Periodicals, Inc.

Prognostic relevance of epilepsy at presentation in glioblastoma patients.

BACKGROUND: Epileptogenic glioblastomas are thought to convey a favorable prognosis, either due to early diagnosis or potential antitumor effects of antiepileptic drugs. We investigated the relationship between survival and epilepsy at presentation, early diagnosis, and antiepileptic drug therapy in glioblastoma patients. METHODS: Multivariable Cox regression was applied to survival data of 647 consecutive patients diagnosed with de novo glioblastoma between 2005 and 2013 in order to investigate the association between epilepsy and survival in glioblastoma patients. In addition, we quantified the association between survival and valproic acid (VPA) treatment. RESULTS: Epilepsy correlated positively with survival (HR: 0.75 (95% CI: 0.61-0.92), P < .01). This effect is independent of age, sex, performance status, type of surgery, adjuvant therapy, tumor location, and tumor volume, suggesting that this positive correlation cannot be attributed solely to early diagnosis. For patients who presented with epilepsy, the use of the antiepileptic drug VPA did not associate with survival when compared with patients who did not receive VPA treatment. CONCLUSION: Epilepsy is an independent prognostic factor for longer survival in glioblastoma patients. This prognostic effect is not solely explained by early diagnosis, and survival is not associated with VPA treatment.

Connexin 30 expression inhibits growth of human malignant gliomas but protects them against radiation therapy.

BACKGROUND: Glioblastomas remain ominous tumors that almost invariably escape treatment. Connexins are a family of transmembrane, gap junction-forming proteins, some members of which were reported to act as tumor suppressors and to modulate cellular metabolism in response to cytotoxic stress. METHODS: We analyzed the copy number and expression of the connexin (Cx)30 gene gap junction beta-6 (GJB6), as well as of its protein immunoreactivity in several public and proprietary repositories of glioblastomas, and their influence on patient survival. We evaluated the effect of the expression of this gap junction protein on the growth, DNA repair and energy metabolism, and treatment resistance of these tumors. RESULTS: The GJB6 gene was deleted in 25.8% of 751 analyzed tumors and mutated in 15.8% of 158 tumors. Cx30 immunoreactivity was absent in 28.9% of 145 tumors. Restoration of Cx30 expression in human glioblastoma cells reduced their growth in vitro and as xenografts in the striatum of immunodeficient mice. Cx30 immunoreactivity was, however, found to adversely affect survival in 2 independent retrospective cohorts of glioblastoma patients. Cx30 was found in clonogenic assays to protect glioblastoma cells against radiation-induced mortality and to decrease radiation-induced DNA damage. This radioprotection correlated with a heat shock protein 90-dependent mitochondrial translocation of Cx30 following radiation and an improved ATP production following this genotoxic stress. CONCLUSION: These results underline the complex relationship between potential tumor suppressors and treatment resistance in glioblastomas and single out GJB6/Cx30 as a potential biomarker and target for therapeutic intervention in these tumors.

In vivo tumorigenesis was observed after injection of in vitro expanded neural crest stem cells isolated from adult bone marrow.

Bone marrow stromal cells are adult multipotent cells that represent an attractive tool in cellular therapy strategies. Several studies have reported that in vitro passaging of mesenchymal stem cells alters the functional and biological properties of those cells, leading to the accumulation of genetic aberrations. Recent studies described bone marrow stromal cells (BMSC) as mixed populations of cells including mesenchymal (MSC) and neural crest stem cells (NCSC). Here, we report the transformation of NCSC into tumorigenic cells, after in vitro long-term passaging. Indeed, the characterization of 6 neural crest-derived clones revealed the presence of one tumorigenic clone. Transcriptomic analyses of this clone highlighted, among others, numerous cell cycle checkpoint modifications and chromosome 11q down-regulation (suggesting a deletion of chromosome 11q) compared with the other clones. Moreover, unsupervised analysis such as a dendrogram generated after agglomerative hierarchical clustering comparing several transcriptomic data showed important similarities between the tumorigenic neural crest-derived clone and mammary tumor cell lines. Altogether, it appeared that NCSC isolated from adult bone marrow represents a potential danger for cellular therapy, and consequently, we recommend that phenotypic, functional and genetic assays should be performed on bone marrow mesenchymal and neural crest stem cells before in vivo use, to demonstrate whether their biological properties, after ex vivo expansion, remain suitable for clinical application.

Mesenchymal stem cells and neural crest stem cells from adult bone marrow: characterization of their surprising similarities and differences.

The generation of neuronal cells from stem cells obtained from adult bone marrow is of significant clinical interest in order to design new cell therapy protocols for several neurological disorders. The recent identification in adult bone marrow of stem cells derived from the neural crest stem cells (NCSC) might explain the neuronal phenotypic plasticity shown by bone marrow cells. However, little information is available about the nature of these cells compared to mesenchymal stem cells (MSC), including their similarities and differences. In this paper, using transcriptomic as well as proteomic technologies, we compared NCSC to MSC and stromal nestin-positive cells, all of them isolated from adult bone marrow. We demonstrated that the nestin-positive cell population, which was the first to be described as able to differentiate into functional neurons, was a mixed population of NCSC and MSC. More interestingly, we demonstrated that MSC shared with NCSC the same ability to truly differentiate into Tuj1-positive cells when co-cultivated with paraformaldehyde-fixed cerebellar granule neurons. Altogether, those results suggest that both NCSC and MSC can be considered as important tools for cellular therapies in order to replace neurons in various neurological diseases.