Genome Microarray Research Articles

Abstract P646: The Aging Immune Transcriptome is Linked to Ischemic Stroke Outcome

Background: Advancing age is associated with changes to the immune system, which affect stroke outcome. We previously demonstrated an age-associated alteration in leukocyte gene expression in patients with ischemic stroke. The aim of this study is to validate these findings in an independent stroke cohort and assess the relationship to stroke outcome. Methods: Genes associated with age were identified in a cohort of 57 patients with acute ischemic stroke. Peripheral blood RNA was measured using whole genome microarrays and genes associated with advancing age identified (FDR-corrected p < 0.05, partial correlation coefficient r > |0.3|); age-associated gene expression differences in patients with poor 90-day outcome (mRS < 2) were also compared. Genes were functionally characterized by pathway and enrichment analysis and verified against age-associated genes from two additional stroke cohorts containing a total of 173 patients. Results: There were 536 genes associated with age in the new stroke cohort, of which 286 decreased and 253 increased with age. Thirty-nine (39) of the age-associated genes were present in previous stroke cohorts analyzed, including a decrease in CCR6, CXCR5, BLNK and NT5E. A decrease in CXCR5 and CD79B was also identified in patients with poor outcome. Pathways and enriched terms relating to the humoral adaptive system immune system, including B-cell and lymphocyte activation, were among those represented in age-associated genes. Conclusions: Age-related changes in leukocyte gene expression were validated in an independent cohort of patients with ischemic stroke. Verified changes include alterations to the humoral immune system and a relationship to stroke outcome. Further investigation of the aging immune system in stroke is warranted.

P65.02 LINC01234 acts as an Oncogenic lncRNA that Interacts with HNRNPA2B1 and Regulates miR-106b Biogenesis

The discovery of long non-coding RNAs (lncRNAs) has dramatically deepened our understanding of the tumorigenesis and cancer progression, but their contributions to non-small-cell lung cancer (NSCLC) remain poorly understood. Here, we conducted a comprehensive analyses of lncRNA alterations in NSCLC by interrogating RNA sequencing data from The Cancer Genome Atlas and microarray data from the Gene Expression Omnibus.

Abstract PS6-30: Combined use of MammaPrint and BluePrint assays to evaluate patients for response to neo-adjuvant chemotherapy for locally advanced breast cancer

Abstract Background: The main goal of the Multi-Institutional Neo-Adjuvant Therapy MammaPrint (MINT) project was to determine the predictive power of the combination of the molecular assays MammaPrint and BluePrint (Agendia Inc., Irvine, CA) for chemosensitivity as measured by complete pathological response (pCR) in patients with locally advanced breast cancer (LABC). MammaPrint is a 70-gene microarray-based assay that classifies each breast cancer patient as low or high risk to develop metastases within 10 years after diagnosis. The BluePrint test is an 80-gene molecular subtyping profile that discriminates between luminal, basal and HER-2 subtypes. Methods: After appropriate IRB approval, 270 female patients with histologically-proven invasive breast cancer and no distant metastases were enrolled in this study. Patients had a clinical tumor classification of T2-T4 with 0-3 positive lymph nodes. DCIS or LCIS was allowed in addition to invasive cancer at the T2 or T3 levels. At least one lesion had to be accurately measured in two dimensions utilizing mammogram, ultrasound, or MRI images to define specific size and validate pCR. Patients were required to have adequate bone marrow reserves, renal function and hepatic function, as determined by standard blood and serum measurements. Patients under 18 years of age or those with confirmed metastatic disease, inflammatory breast cancer, any serious uncontrolled intercurrent infections, or other serious uncontrolled concomitant disease were excluded. Patients with any prior chemotherapy, radiotherapy, or endocrine therapy for the treatment of breast cancer were also excluded. Tumor samples were collected via incisional or core needle biopsy and shipped to Agendia for processing of the MammaPrint and BluePrint gene panels, as well as whole human genome expression microarrays. Comparison of response rates between MammaPrint and BluePrint molecular subtypes was conducted using Pearson Chi-square test with chemo-responsiveness measured as a binary response: pCR or residual disease. Results: Of 270 patients enrolled, 56 did not have TNM or RCB staging information reported in the case report form and/or were not submitted for central pathology review. Of 214 patients evaluated by central pathology review, 68 (32%) exhibited a pCR. Patients with a high risk MammaPrint result had a higher pCR rate (37%) compared to patients with a low risk MammaPrint (0%). And patients with a HER-2 or basal molecular subtype by BluePrint had significantly higher rates of pCR (62%, 37%, respectively). Conclusion: Upfront evaluation of LABC tumors using the combination of MammaPrint and BluePrint can help in the clinico-pathologic evaluation to determine which patients are more likely to benefit from neo-adjuvant chemotherapy, ranging from expected minimal response in Luminal A to substantial responses for HER2-type and Basal-type tumors. Additional studies to evaluate the prognostic and/or predictive values of additional gene panels from the whole human genome microarrays are underway. Rate of pCR in MammaPrint and MammaPrint/BluePrint groupsGroupsResidual DiseasepCRP valueMP Risk GroupLow Risk27 (100%)0 (0%)0.0004High Risk114 (63%)67 (37%)BP subtypeBasal42 (63%)25 (37%)2 x 10-9HER220 (38%)33 (62%)Luminal A26 (100%)0 (0%)Luminal B53 (85%)9 (15%) Citation Format: Brian Longbottom, Steven C Shivers, Geza Acs, Charles E Cox. Combined use of MammaPrint and BluePrint assays to evaluate patients for response to neo-adjuvant chemotherapy for locally advanced breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS6-30.

Securidaca inappendiculata-Derived Xanthones Protected Joints from Degradation in Male Rats with Collagen-Induced Arthritis by Regulating PPAR-γ Signaling.

BackgroundThe bark of Securidaca inappendiculata Hassk. is traditionally used for treating inflammatory diseases and bone fractures in China. We have previously validated the xanthone-enriched fraction (XRF) of S. inappendiculata with anti-rheumatic potentials, but mechanism underlying the joints protective effects is still largely unknown.Materials and MethodsThe male rats with collagen-induced arthritis (CIA) were treated with XRF. The therapeutic efficacy of XRF was evaluated by arthritis score changes, morphological observation of paws, histological examinations and serological analyses. Protein expression in tissues and cells was investigated by either immunohistochemical or immunoblotting methods, while levels of mRNA expression were investigated by RT-qPCR. Metabolites in serum were detected by LC-MS approach. The joints homogenates were used for analyzing possible targeted genes by genome microarray analyses.ResultsTreatment with XRF and methotrexate (MTX) led to significant decrease in arthritis scores, and alleviated deformation of paws in CIA rats. In addition, XRF and MTX reduced circulating TNF-α, IL-1β and IL-17α in the serum and down-regulated TLR4/NF-κB and JNK pathways in joints of CIA rats. Compared to MTX, XRF-loading microemulsion significantly protected joints, which was accompanied by dramatic decrease in MMP3. Differential genes-based KEGG enrichment and metabolomics analysis suggested that XRF reduced fatty acids biosynthesis by regulating PPAR-γ signaling. S inappendiculata-derived 1,7-dihydroxy-3,4-dimethoxyxanthone (XAN) up-regulated PPAR-γ expression in macrophages, but suppressed it in pre-adipocytes in vitro, which was synchronized with SIRT1 changes. Adiponectin production and SCD-1 expression in pre-adipocytes were also decreased. Aside the direct inhibition on MMP3 expression in synovioblast, the presence of XAN in macrophages-pre-adipocytes co-culture system further reinforced this effect.ConclusionThis study revealed the joint protective advantages of the bioactive fraction from S. inappendiculata in CIA rats over MTX, and demonstrated that S. inappendiculata-derived xanthones suppressed the erosive nature of synovioblast acquired under inflammatory circumstances by regulating PPAR-γ signaling-controlled metabolism-immunity feedback.

Evaluation of Fluorescent Light Energy for the Treatment of Acute Second-degree Burns.

The use of photobiomodulation has been proposed to improve wound healing for the last two decades. Recent development in photobiomodulation has led to the development of a novel biophotonic platform that utilizes fluorescent light energy (FLE) within the visible spectrum of light for healing of skin inflammation and wounds. In this article, FLE was used in preliminary analysis on 18 case studies of acute second-degree burns and in a pilot study using an ex vivo human skin model. Efficacy of FLE on wound healing and tissue remodeling was evaluated by monitoring improvements in the treated tissues, assessing pain for the patients, and by performing human genome microarray analysis of FLE-treated human skin samples. Healing was reported for all 18 patients treated with FLE for acute second-degree burns without reported adverse effects or development of infections. Furthermore, preliminary ex vivo skin model data suggest that FLE impacts different cellular pathways including essential immune-modulatory mechanisms. The results presented in this article are encouraging and suggest that FLE balances different stages of wound healing, which opens the door to initiating randomized controlled clinical trials for establishing the efficacy of FLE treatment in different phases of wound healing of second-degree burns.

Perchlorate detection via an invertebrate biosensor.

Improvised explosive devices (IEDs) are constructed from easily obtainable ingredients that are often unregulated and difficult to trace. Salts of the oxyhalide perchlorate are frequently used as oxidisers in IEDs and in commercially available munitions, thus a reliable detection is needed to aid forensic investigations and the tracing of environmental ground or surface water contamination. We introduce the nematode Caenorhabditis elegans as a biosensor for the presence of perchlorate, a promising alternative to the costly, technically challenging and time-consuming current perchlorate detection methods. Perchlorate uptake dynamics in C. elegans were first validated using ion exchange chromatography followed by assessing the effects of perchlorate on key life-point indices to verify the suitability of the nematodes as a forensic biosensor. Whole genome microarrays and qPCR analyses established that a set of immune and stress response genes were enriched during perchlorate exposure. A nematode strain (agIs219) containing an integrated copy of the significantly overexpressed t24b8.5 gene promoter followed by a GFP reporter gene was shown to fluoresce in a perchlorate dose dependent manner with a limit of detection (LOD) of 0.5 mg mL-1. Whilst chemicals commonly used in the construction of IEDs did not induce fluorescence, exposure to other oxyhalides did, highlighting the presence of possible shared stress response pathways. Burnt wire sparklers containing potassium perchlorate elicited fluorescence while other non-perchlorate containing post-blast explosion matrices did not. This demonstrates how C. elegans can be used to screen for perchlorate at environmental hotspots, an optimization, possibly with other target transgenes, is required to enable the detection of perchlorate at concentrations below 0.5 mg mL-1.

A Hybrid Feature Selection Optimization Model for High Dimension Data Classification

Feature selection is an NP-hard combinatorial problem, in which the number of possible feature subsets increases exponentially with the number of features. In the case of large dimensionality, the goal of feature selection is to determine the smallest possible features considering the most informative subset. In this paper, we proposed a hybrid feature selection optimization model for Cancer Classification called, ENSVM. Our model is based on using the Elastic Net (EN) method that regulates and selects variables for gene selection of genomic microarray data. We applied three different optimization techniques namely Social Ski-Driver (SSD), Randomized SearchCV (RS) and Elastic NetCV (ENCV) for determining Elastic Net with traditional Support Vector Machines for classification. To evaluate the model, we compared the results of applying ENSVM to seven genomic microarray data with the SSD-SVM model and SVM with (RBF) kernel without any feature selection method. The results of the comparison revealed the effect of ENSVM in selecting the optimal feature subset that maximized the classification performance. Accordingly, minimizing the number of features is significant when analyzing high dimensional data for performance nevertheless accuracy. Moreover, the ENSVM model is superior compared with the SSD-SVM model.

Gene Network Analysis Reveals Association between Somatic Loss of Y and Genes Involved in Hemostasis

Background and Hypothesis: 
 Somatic loss of the Y chromosome (LOY) is associated with diagnoses of various cancers, Alzheimer’s disease, and cardiovascular events. In the case of cardiovascular disorders, no mechanistic link to LOY has been elucidated. To explore this relationship, we utilized Weighted Gene Co-Expression Analysis (WGCNA), a powerful technique that can reveal associations between modules of co-expressed genes and clinical traits. We hypothesized WGCNA would reveal a module that illuminates the relationship between LOY and cardiovascular disease. 
 
 Methods: 
 Information about 403 male participants was obtained from Alzheimer’s Disease Neuroimaging Initiative (ADNI), including Genome Wide Association Study (GWAS), microarray, and patient demographic data. GWAS data was obtained from each participant within one year of collecting gene expression data. Using GWAS data for each participant, a median log R ratio was calculated for probes in the male specific region of the Y (MSY), which showed detectable LOY in 16 participants. Microarray data was used for WGCNA. Genes with correlated expression levels were organized into modules which were tested for associations with age, Alzheimer’s disease status, APOE-e4 allele status, and LOY. Ensembl IDs were used to investigate Gene Ontology (GO) enrichment in the GO database. 
 
 Results: 
 One module was associated with LOY (p=0.001). GO enrichment revealed significant representation of 47 GO terms within this module. Highly enriched GO terms included “platelet aggregation” (FDR = 8.05 x 10-6), “blood coagulation, fibrin clot formation” (FDR= 0.0328), “regulation of megakaryocyte differentiation” (FDR=3.01 x 10-5), and other terms related to the vascular response to injury. 
 
 Potential Impact: 
 WGNCA revealed a module associated with LOY that is related to vascular injury response. While LOY is associated with cardiovascular events, more work is needed to explore its association with biomarkers for cardiovascular disease. Our findings warrant more investigation into the relationship between LOY and cardiovascular dysfunction in males.

LGG-28. NOVEL BRAF INTRAGENIC DELETION IN A GLIOMA: A CASE REPORT OF A PEDIATRIC PATIENT

BACKGROUNDNumerous variant BRAF genetic alterations have been associated with malignancies. BRAF activating fusions/mutations are frequently present in low grade gliomas. BRAF intragenic deletions have been reported in melanoma, but have not previously been reported in gliomas.OBJECTIVETo report a BRAF intragenic deletion in a pediatric patient with recurrent low-grade glioma.RESULTSA 3-year-old female underwent a complete resection of a posterior fossa pilocytic astrocytoma. She had recurrences at age 4, and then at age 9; pathology was consistent with pilocytic astrocytoma. Microarray analysis on sample from the first recurrence showed one region of loss encompassing 86 Kbp within the BRAF gene. The deletion breakpoints are within intron 1 and 9, resulting in loss of exons 2 through 9, inclusive. This has been previously described melanoma, but appears to be a novel finding in glioma. It is hypothesized that, since the loss retains the kinase and ATP binding pocket domains but deletes the N-terminal conserved region 1 and 2 (CR1, CR2) of the BRAF gene, it is likely functionally similar to the loss and activation resulting from the more usually described KIAA1549 and BRAF gene fusion.CONCLUSIONThis is the first BRAF intragenic deletion involving exons 2–9 reported in a glioma. Although 86kbp is small using whole genome microarray technology, it is large using sequencing strategies, and a targeted sequencing approach to investigate the BRAF gene would not readily identify this deletion. It is speculated that the deletion may be under ascertained in the pediatric population.

Nanoemulsion adjuvantation strategy of tumor-associated antigen therapy rephrases mucosal and immunotherapeutic signatures following intranasal vaccination

BackgroundEmulsion adjuvants are a potent tool for effective vaccination; however, the size matters on mucosal signatures and the mechanism of action following intranasal vaccination remains unclear. Here, we launch a...

Analysis of the Molecular Diversity Among Cronobacter Species Isolated From Filth Flies Using Targeted PCR, Pan Genomic DNA Microarray, and Whole Genome Sequencing Analyses.

Cronobacter species are opportunistic pathogens capable of causing life-threatening infections in humans, with serious complications arising in neonates, infants, immuno-compromised individuals, and elderly adults. The genus is comprised of seven species: Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter muytjensii, Cronobacter dublinensis, Cronobacter universalis, and Cronobacter condimenti. Despite a multiplicity of genomic data for the genus, little is known about likely transmission vectors. Using DNA microarray analysis, in parallel with whole genome sequencing, and targeted PCR analyses, the total gene content of two C. malonaticus, three C. turicensis, and 14 C. sakazaki isolated from various filth flies was assessed. Phylogenetic relatedness among these and other strains obtained during surveillance and outbreak investigations were comparatively assessed. Specifically, microarray analysis (MA) demonstrated its utility to cluster strains according to species-specific and sequence type (ST) phylogenetic relatedness, and that the fly strains clustered among strains obtained from clinical, food and environmental sources from United States, Europe, and Southeast Asia. This combinatorial approach was useful in data mining for virulence factor genes, and phage genes and gene clusters. In addition, results of plasmidotyping were in agreement with the species identity for each strain as determined by species-specific PCR assays, MA, and whole genome sequencing. Microarray and BLAST analyses of Cronobacter fly sequence datasets were corroborative and showed that the presence and absence of virulence factors followed species and ST evolutionary lines even though such genes were orthologous. Additionally, zebrafish infectivity studies showed that these pathotypes were as virulent to zebrafish embryos as other clinical strains. In summary, these findings support a striking phylogeny amongst fly, clinical, and surveillance strains isolated during 2010–2015, suggesting that flies are capable vectors for transmission of virulent Cronobacter spp.; they continue to circulate among United States and European populations, environments, and that this “pattern of circulation” has continued over decades.

Dose and Dose-Rate Effects in a Mouse Model of Internal Exposure to 137Cs. Part 1: Global Transcriptomic Responses in Blood.

Internal contamination by radionuclides may constitute a major source of exposure and biological damage after radiation accidents and potentially in a dirty bomb or improvised nuclear device scenario. We injected male C57BL/6 mice with radiolabeled cesium chloride solution (137CsCl) to evaluate the biological effects of varying cumulative doses and dose rates in a two-week study. Injection activities of 137CsCl were 5.71, 6.78, 7.67 and 9.29 MBq, calculated to achieve a target dose of 4 Gy at days 14, 7, 5 and 3, respectively. We collected whole blood samples at days 2, 3, 5, 7 and 14 so that we can publish the issue in Decemberfrom all injection groups and measured gene expression using Agilent Mouse Whole Genome microarrays. We identified both dose-rate-independent and dose-rate-dependent gene expression responses in the time series. Gene Ontology analysis indicated a rapid and persistent immune response to the chronic low-dose-rate irradiation, consistent with depletion of radiosensitive B cells. Pathways impacting platelet aggregation and TP53 signaling appeared activated, but not consistently at all times in the study. Clustering of genes by pattern and identification of dose-rate-independent and -dependent genes provided insight into possible drivers of the dynamic transcriptome response in vivo, and also indicated that TP53 signaling may be upstream of very different transcript response patterns. This characterization of the biological response of blood cells to internal radiation at varying doses and dose rates is an important step in understanding the effects of internal contamination after a nuclear event.

The tumor immune microenvironment transcriptomic subtypes of colorectal cancer for prognosis and development of precise immunotherapy.

BackgroundBiomarkers based on immune context may guide prognosis prediction. T-cell inactivation, exclusion, or dysfunction could cause unfavorable tumor microenvironments, which affect immunotherapy and prognosis. However, none of the immuno-biomarkers reported to date can differentiate colorectal-cancer (CRC) patients. Thus, we aimed to classify CRC patients according to the levels of T-cell activation, exclusion, and dysfunction in the tumor microenvironment.MethodsRNAseq data of 618 CRC patients from The Cancer Genome Atlas and microarray data of 316 CRC patients from Gene Expression Omnibus were analysed using the Tumor Immune Dysfunction and Exclusion algorithm. Unsupervised clustering was used to classify patients.ResultsBased on the expression signatures of myeloid-derived suppressor cells, cancer-associated fibroblasts, M2-like tumor-associated macrophages, cytotoxic T-lymphocytes, and PD-L1, all patients were clustered into four subtypes: cluster 1 had a high level of immune dysfunction, cluster 2 had a low level of immune activation, cluster 3 had intense immune exclusion, and cluster 4 had a high level of immune activation and a moderate level of both dysfunction and exclusion signatures. Compared with cluster 1, the hazard ratios and 95% confidential intervals for overall survival were 0.63 (0.35–1.13) for cluster 2, 0.55 (0.29–1.03) for cluster 3, and 0.30 (0.14–0.64) for cluster 4 in multivariate Cox regression. Similar immune clustering and prognosis patterns were obtained upon validation in the GSE39582 cohort. In subgroup analysis, immune clustering was significantly associated with overall survival among stage I/II patients, microsatellite stable/instability-low patients, and patients not treated with adjuvant therapy.ConclusionsOur findings demonstrated that classifying CRC patients into different immune subtypes serves as a reliable prognosis predictor and may help to refine patient selection for personalized cancer immunotherapy.

Identification of TYROBP and C1QB as Two Novel Key Genes With Prognostic Value in Gastric Cancer by Network Analysis.

Background: Gastric cancer (GC) is the fifth most frequently diagnosed malignancy, and the third leading cause of tumor-related mortalities worldwide. Due to a high heterogeneity in GC, its treatment and prognosis are challenging, necessitating urgent identification of novel prognostic predictors for GC patients.Methods: We downloaded RNA sequence data, from the Cancer Genome Atlas and microarray data from Gene Expression Omnibus database, then identified common differentially-expressed genes (DEGs) between GC and normal gastric tissues across four datasets. We then used a combination of protein-protein interaction (PPI) network and weighted gene co-expression network analysis (WGCNA) to identify key genes with prognostic value in GC. Thereafter, we used quantitative real time polymerase chain reaction (qRT-PCR) to validate expression of the identified key genes in the Zhejiang University (ZJU) cohort. Finally, we evaluated the relationships between gene expression and immune factors, including immune cells and biomarkers of immunotherapy.Results: Among 426 common DEGs screened, 333 and 93 were upregulated and downregulated, respectively. PPI network and WGCNA successfully identified the top 30 hub genes, among which PTPRC, TYROBP, CCR1, CYBB, LCP2, and C1QB were common. Furthermore, TYROBP and C1QB were negatively associated with prognosis of GC patients, implying that they were key GC predictors. Interestingly, TYROBP and C1QB were positively correlated with predictive biomarkers for GC immunotherapy, including PD-L1 expression, CD8+ T cells infiltration, and EBV status.Conclusions: TYROBP and C1QB were identified as two novel key genes with prognostic value in GC by network analysis.

A 70-Gene Signature for Predicting Treatment Outcome in Advanced-Stage Cervical Cancer

Cervical cancer is the fourth most common cancer in women worldwide. The current approaches still have limitations in predicting the therapy outcome of each individual because of cancer heterogeneity. The goal of this study was to establish a gene expression signature that could help when choosing the right therapeutic method for the treatment of advanced-stage cervical cancer. The 666 patients were collected from four independent datasets. The 70-gene expression signature was established using univariate Cox proportional hazard regression analysis. The 70-gene signature was significantly different between low- and high-risk groups in the training dataset (p = 4.24e−6) and in the combined three validation datasets (p = 4.37e−3). Treatment of advanced-stage cancer patients in the high-risk group with molecular-targeted therapy combined with chemoradiotherapy yielded a better survival rate than with only chemoradiotherapy (p = 0.0746). However, treatment of the patients in the low-risk group with the combined therapy resulted in significantly lower survival (p = 0.00283). Functional classification of 70 genes revealed involvement of the angiogenesis pathway, specifically phosphatidylinositol 3-kinase signaling (p = 0.040), extracellular matrix organization (p = 0.0452), and cell adhesion (p = 0.011). The 70-gene signature could predict the prognosis and indicate an optimal therapeutic modality in molecular-targeted therapy or chemotherapy for advanced-stage cervical cancer.

Dataset on transcriptomic profiling of cholestatic liver injury in an in vitro and in vivo animal model.

The transcriptomic dataset (whole genome microarray Affymetrix Human U133 plus 2.0 and Affymetrix Mouse Genome 430 2.0) presented in this paper describes the differential gene expression profile of a human in vitro model of drug-induced cholestasis and a well-known mouse in vivo model of cholestasis. The in vitro model consists of human hepatoma HepaRG cells in monolayer configuration exposed to 3 different cholestatic drugs with or without bile acids. For in vivo modelling of cholestasis, mice were subjected to bile duct ligation surgery. Consecutive normalization, summarization and background adjustments have been made by means of Robust Multichip Average Express software.

Gene expression regulated by abatacept associated with methotrexate and correlation with disease activity in rheumatoid arthritis

ObjectivesAbatacept acts as a competitive inhibitor of the CD28/(CD80/86) costimulation signal required for T cell activation. Mechanisms of action of abatacept have not been fully investigated. The objective of this study was to provide detailed insight into the mode of action of Abatacept based on gene expression data.MethodsIn this ancillary study from the APPRAISE trial, we investigated the global molecular effects of Abatacept in whole blood samples collected prospectively in biologic naive rheumatoid arthritis patients (n = 19) at baseline and 6 months after the initiation of Abatacept therapy concomitant with methotrexate. Whole human genome microarrays (4x44K) were performed on both baseline and 6-month samples from responders and non-responders patients categorized according to EULAR criteria. T-test with Benjamini-Hochberg correction was performed to identify significant gene expression changes. Gene Ontology and Single Experiment Analysis tools allowed us to highlight specific biological mechanisms involved in methotrexate/Abatacept.ResultsIn methotrexate/Abatacept responders, 672 genes were significantly (q<0.05) dysregulated at 6 months compared to baseline. Correlation analysis highlighted 19 genes whose dysregulations were significantly associated with disease activity variation (p<0.05) and whose functions were associated with proliferation, apoptosis of cells and mitochondrial metabolism, suggesting a restoration of oxidative signaling. The other 653 gene expression changes were relative to direct or indirect effects of methotrexate/Abatacept treatment and were significantly (p<0.005) involved in pathways relative to mRNA processing, proteasome, angiogenesis, apoptosis and TCR signaling. This study highlights new mechanisms of action of methotrexate/Abatacept and may provide new therapeutic targets to prevent autoimmunity in rheumatoid arthritis.

Detection of mosaicism for segmental and whole chromosome imbalances by targeted sequencing.

Mosaic segmental and whole chromosome copy number alterations are postzygotic variations known to be associated with several disorders. We have previously presented an efficient targeted sequencing approach to simultaneously detect point mutations and copy number variations (CNVs). In this study, we evaluated the efficiency of this approach to detect mosaic CNVs, using seven postnatal and 19 tumor samples, previously characterized by chromosomal microarray analyses (CMA). These samples harbored a total of 28 genomic imbalances ranging in size from 0.68 to 171 Mb, and present in 10-80% of the cells. All CNV regions covered by the platform were correctly identified in postnatal samples, and only seven out of 19 CNVs from tumor samples were not identified either because of a lack of target probes in the affected genomic regions or an absence of minimum reads for an alteration call. These results demonstrate that, in a research setting, this is a robust approach for detecting mosaicism in cases of segmental and whole chromosome alterations. Although the current sequencing platform presented a resolution similar to genomic microarrays, it is still necessary to further validate this approach in a clinical setting in order to replace CMA and sequencing analyses by a single test.

H2 O2 -induced oxidative stress disrupts mitochondrial functions and impairs migratory potential of human epidermal melanocytes.

Reactive oxygen species (ROS) have already been demonstrated to impede the migratory ability in non-melanocytic cell lines by depleting mitochondrial ATP production. Therefore, understanding the mitochondrial metabolic response to migration in the presence of ROS should be a key to understanding repigmentation in vitiligo. This study aimed to investigate the energy mechanism associated with the ROS-mediated attenuation of melanocyte migration. After melanocytes were pretreated with H2 O2 , their ATP production, migratory ability, ultrastructural changes and Mitochondrial Permeability Potential were analysed. The results showed that, in parallel with the decreased ATP production, the migratory ability of melanocytes was significantly inhibited by oxidative stress. Supplementation with exogenous ATP reversed the suppressed ATP-dependent migration of melanocytes. Melanocytes were then stressed with H2 O2 and Agilent Whole Human Genome microarray analysis identified 763 up-regulated mRNAs and 1117 down-regulated mRNAs. Among them, 11 of the encoded proteins were involved in mitochondrial ATP production and their expression levels were verified. The decreased expression of NADH dehydrogenase 2(ND2) , cytochrome c oxidase 1(COX1) and cytochrome c oxidase 3(COX3) was shown to be involved in the depletion of mitochondrial ATP production, which was coupled with the impaired migratory potential. These results indicate that the migration of melanocytes relies heavily on an inexhaustible supply of ATP from mitochondria.

Assessment of candidate biomarkers to detect resistance to Mycobacterium bovis in Holstein-Friesian cattle

Bovine tuberculosis (bTB) caused by Mycobacterium bovis has a significant economic impact worldwide each year. Control of bTB is based on skin testing and removal of reactors. However, additional strategies are required to control this disorder. Natural disease resistance has been defined as the inherent capacity of an individual to resist disease when exposed to pathogens without previous exposure or immunization. However, little is known about natural disease resistance against Mycobacterium bovis in cattle. In this study, we aimed to identify candidate biomarkers to detect host resistance to M. bovis. We used a microbicidal assay to identify the resistance phenotype. A genomic microarray analysis was carried out on RNA from 2 resistant (R) and 2 susceptible (S) cows. Our results evidenced 69 differentially expressed genes. A subset of six genes that showed differential up (IL1RN), and down-regulation (VNN, GATM, ARHGEF11, NAAA and HSPA2) were selected for further analysis. To further validate the candidate biomarkers, we identified the R phenotype in 31 cattle (9 R and 22 S). Macrophage mRNA was isolated from this group of cattle. Expression of candidate biomarkers was evaluated by qPCR 2-ΔCt and ROC curves to determine their diagnostic potential. Candidates IL1RN and ARHGEF11 discriminates between R and S cattle. Furthermore, combination of candidates ARHGEF11: VNN: HSPA2 discriminate between R from S with AUC 0.7993 and agreement index of 0.853 (p ≤ 0.01). Our data suggest that candidate biomarkers may support the preliminary screening to identify natural resistance in herds against Mycobacterium bovis in Holstein-Friesian cattle.