Background Sputum analysis in asthmatic patients is used to define airway inflammatory processes and might guide therapy. Objective We sought to determine differential gene and protein expression in ...sputum samples from patients with severe asthma (SA) compared with nonsmoking patients with mild/moderate asthma. Methods Induced sputum was obtained from nonsmoking patients with SA, smokers/ex-smokers with severe asthma, nonsmoking patients with mild/moderate asthma (MMAs), and healthy nonsmoking control subjects. Differential cell counts, microarray analysis of cell pellets, and SOMAscan analysis of sputum analytes were performed. CRID3 was used to inhibit the inflammasome in a mouse model of SA. Results Eosinophilic and mixed neutrophilic/eosinophilic inflammation were more prevalent in patients with SA compared with MMAs. Forty-two genes probes were upregulated (>2-fold) in nonsmoking patients with severe asthma compared with MMAs, including IL-1 receptor (IL-1R) family and nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3 (NRLP3) inflammasome members (false discovery rate < 0.05). The inflammasome proteins nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 1 (NLRP1), NLRP3, and nucleotide-binding oligomerization domain (NOD)-like receptor C4 (NLRC4) were associated with neutrophilic asthma and with sputum IL-1β protein levels, whereas eosinophilic asthma was associated with an IL-13–induced TH 2 signature and IL-1 receptor–like 1 (IL1RL1) mRNA expression. These differences were sputum specific because no activation of NLRP3 or enrichment of IL-1R family genes in bronchial brushings or biopsy specimens in patients with SA was observed. Expression of NLRP3 and of the IL-1R family genes was validated in the Airway Disease Endotyping for Personalized Therapeutics cohort. Inflammasome inhibition using CRID3 prevented airway hyperresponsiveness and airway inflammation (both neutrophilia and eosinophilia) in a mouse model of severe allergic asthma. Conclusion IL1RL1 gene expression is associated with eosinophilic SA, whereas NLRP3 inflammasome expression is highest in patients with neutrophilic SA. TH 2-driven eosinophilic inflammation and neutrophil-associated inflammasome activation might represent interacting pathways in patients with SA.
Background
Whether the clinical or pathophysiologic significance of the “treatable trait” high blood eosinophil count in COPD is the same as for asthma remains controversial. We sought to determine ...the relationship between the blood eosinophil count, clinical characteristics and gene expression from bronchial brushings in COPD and asthma.
Methods
Subjects were recruited into a COPD (emphysema versus airway disease EvA) or asthma cohort (Unbiased BIOmarkers in PREDiction of respiratory disease outcomes, U‐BIOPRED). We determined gene expression using RNAseq in EvA (n = 283) and Affymetrix microarrays in U‐BIOPRED (n = 85). We ran linear regression analysis of the bronchial brushings transcriptional signal versus blood eosinophil counts as well as differential expression using a blood eosinophil > 200 cells/μL as a cut‐off. The false discovery rate was controlled at 1% (with continuous values) and 5% (with dichotomized values).
Results
There were no differences in age, gender, lung function, exercise capacity and quantitative computed tomography between eosinophilic versus noneosinophilic COPD cases. Total serum IgE was increased in eosinophilic asthma and COPD. In EvA, there were 12 genes with a statistically significant positive association with the linear blood eosinophil count, whereas in U‐BIOPRED, 1197 genes showed significant associations (266 positive and 931 negative). The transcriptome showed little overlap between genes and pathways associated with blood eosinophil counts in asthma versus COPD. Only CST1 was common to eosinophilic asthma and COPD and was replicated in independent cohorts.
Conclusion
Despite shared “treatable traits” between asthma and COPD, the molecular mechanisms underlying these clinical entities are predominately different.
In a chronic obstructive pulmonary disease (COPD) cohort (EvA, n = 283), 12 genes, whereas in asthma cohort (UBIOPRED, n = 85), 1197 genes in bronchial epithelial brushes were correlated with a blood eosinophil count. The gene CST1 was common to eosinophilic asthma and COPD and was replicated in independent cohorts. Despite shared “treatable traits” between asthma and COPD, the molecular mechanisms underlying these clinical entities are predominately different.
Nerve growth factor (NGF) is the prototype member of the neurotrophin family. Identification of transcript structures and promoter regions is described here in view of clarifying the molecular basis ...of chicken
NGF gene regulation. Chicken
NGF complementary DNA (cDNA) was amplified from heart and brain mRNA using the single-strand ligation to cDNA (SLIC) procedure. Several cloning and sequencing rounds were necessary to elucidate the diversity of
NGF transcripts. The chicken
NGF gene was shown to possess, in addition to its unique 3′ coding exon, five 5′ exons grouped into two clusters that have been entirely sequenced. The first cluster encompasses three leader exons (1a, 1b and 1c) and is separated from the second cluster by a ∼15 kilobases (kb) intronic sequence. “Exon walking” based on reverse transcription–polymerase chain reaction (RT–PCR) allowed to ascertain the length of the three leader exons. The second cluster contains exons 2 and 3, separated from each other by a ∼2.4 kb intron, and lies ∼0.5 kb upstream from coding exon 4. Combination of several mechanisms, such as differential usage of leader and internal exons, alternative transcription start inside exon 1b, second donor and acceptor sites in exon 1c and 4, respectively, leads to the production of at least 21 different transcripts. This remarkable diversity may represent a common feature largely underestimated for other weakly expressed genes. Preliminary RT–PCR expression study in a panel of chicken tissues shows that transcripts containing exon 1b are present in most tissues tested. Transcripts containing exon 1a are represented mainly in heart and reproductive organs, whereas transcripts containing exon 1c are mostly represented in peripheral organs other than heart. Complementary data are published as a Web supplement available at
http://www.loria.fr/~devignes/NGF/index.html.
Expression profiles of 5058 human gene transcripts represented by an array of 7451 clones from the first IMAGE Consortium cDNA library from infant brain have been collected by semiquantitative ...hybridization of the array with complex probes derived by reverse transcription of mRNA from brain and five other human tissues. Twenty-one percent of the clones corresponded to transcripts that could be classified in general categories of low, moderate, or high abundance. These expression profiles were integrated with cDNA clone and sequence clustering and gene mapping information from an upgraded version of the Genexpress Index. For seven gene transcripts found to be transcribed preferentially or specifically in brain, the expression profiles were confirmed by Northern blot analyses of mRNA from eight adult and four fetal tissues, and 15 distinct regions of brain. In four instances, further documentation of the sites of expression was obtained by in situ hybridization of rat-brain tissue sections. A systematic effort was undertaken to further integrate available cytogenetic, genetic, physical, and genic map informations through radiation-hybrid mapping to provide a unique validated map location for each of these genes in relation to the disease map. The resulting Genexpress IMAGE Knowledge Base is illustrated by five examples presented in the printed article with additional data available on a dedicated Web site at the address http://idefix.upr420.vjf.cnrs.fr/EXPR++ +/ welcome.html.
Macro-array differential hybridization of a collection of 5058 human gene transcripts represented in an IMAGE infant brain cDNA library has led to the identification of transcripts displaying ...preferential or specific expression in brain (Genome Res. 9 (1999) 195; http://idefix.upr420.vjf.cnrs.fr/IMAGE). Most of these genes correspond to as yet undescribed functions. Detailed characterization of the expression, sequence, and genome assignment of one of these genes named C3orf4, is reported here. The full-length sequence of the transcript was obtained by 5' extension RT-PCR. The gene transcript (2.8 kb) encodes a 253 amino acid long protein, with four transmembrane domains. The position of the C3orf4 gene was determined at 3q12.1 thanks to the draft sequence of the human genome. It is composed of five exons spanning more than 7 kb. No TATAA box but a CpG island was found upstream of the beginning of the gene. Northern blot analysis and in situ hybridization revealed a predominant expression in myelinated structures such as corpus callosum and spinal cord. RT-PCR showed expression of the C3orf4 gene in rat optic nerve and cultured oligodendrocytes, the myelinating cells of the central nervous system, but not in astrocytes. This work supports further investigations aimed at determining the role of the C3orf4 gene in myelinating cells.
Sequence, gene mapping, and expression data corresponding to 910 genes transcribed in human skeletal muscle have been integrated to form the muscle module of the Genexpress IMAGE Knowledge Base. ...Based on cDNA array hybridization, a set of 14 transcripts preferentially or specifically expressed in muscle have been selected and characterized in more detail: Their pattern of expression was confirmed by Northern blot analysis; their structure was further characterized by full-insert cDNA sequencing and cDNA extension; the map location of the corresponding genes was refined by radiation hybrid mapping. Five of the 14 selected genes appear as interesting positional and functional candidate genes to study in relation with muscle physiology and/or specific orphan muscular pathologies. One example is discussed in more detail. The expression profiling data and the associated Genexpress Index2 entries for the 910 genes and the detailed characterization of the 14 selected transcripts are available from a dedicated Web server at. The database has been organized to provide the users with a working space where they can find curated, annotated, integrated data for their genes of interest. Different navigation routes to exploit the resource are discussed.
Natural killer (NK) cell activity appears to be conserved throughout vertebrate development but NK cells have only been well characterized in mammals. Candidate NK cells have been identified in the ...chicken as cytoplasmic CD3+ and surface T cell receptor (TCR)/CD3- (TCRO) lymphocytes that often express CD8. The fact that the TCRO cells are abundant in the embryonic spleen before T cells enter this organ allowed us to cultivate the embryonic TCRO cells using growth factors derived from activated adult lymphocytes. These TCRO cells were cytotoxic for an NK target cell line. They expressed cell surface CD8, a putative interleukin-2 receptor, CD45 and a receptor for IgG, but did not express CD4, major histocompatibility complex class II or immunoglobulin. Biochemical analysis of the cytoplasmic CD3 antigen revealed two of the three CD3 gamma, delta and epsilon homologues, and RNA transcripts for the third. The CD3 monoclonal antibody also precipitated a 32-kDa dimer that may represent a heterodimer of different CD3 constituents. TCR alpha and beta gene transcripts were not detected in the TCRO cells. These results indicate that the avian TCRO cell is the mammalian NK cell homologue. The shared evolutionary features of T cells and NK cells in birds and mammals support the idea that they derive from a common progenitor.
A limited number of genes, including the human brain-derived neutrotrophic factor (BDNF) gene, have been identified in the human chromosome 11p14 region. Since this area is involved in a genetic ...disorder (WAGR syndrome) and because of interest in studying the regulation of the human BDNF gene, we have established a detailed transcript map of a 810-kb region clone in a yeast artificial chromosome (YAC), corresponding to a portion of this genomic locus. A set of nested deletion mutants has been generated to map genes at a mean resolution of 75kb. Four genic markers from available mapping databases have been mapped on the YAC. Ten potential novel human exons have been isolated by a 3' terminal exon trapping procedure directly applied to purified YAC DNA. Most of these exons display polyadenylation signals and they all yield positive signals in RT-PCR experiments, confirming their status of transcribed sequences. The BDNF gene is now co-localised with three other genes on a 120 kb DNA fragment.
In this report, we describe a refinement of the human transcript map of chromosome 1p13.1, a subregion undergoing many aberrations in various types of human cancers. Publicly available genetic ...linkage, radiation hybrid and physical maps, as well as cytogenetic and sequence data were used to establish the relative order and orientation of ten known intragenic markers. The complete sequence of genomic clones of the region, available at the Sanger Centre, provided the tool for further studies performed by BLAST analysis against all cDNA sequences registered in the Genexpress Index2. This allowed us to assign to subband 1p13.1 nine of the ten known genes, an additional member of the gene family of one of these genes and eleven new transcripts. The remaining known gene and one additional new transcript map at the 1p13.1 and 1p13.2 boundary. The corresponding genes may be responsible for disorders related to this region. The resulting transcript map of 1p13.1 is presented in the printed article with additional data available on a dedicated Web site at the address http://idefix.upr420.vjf.cnrs.fr/CARTO.
WAGR (Wilms tumor, aniridia, genito-urinary abnormalities, mental retardation) syndrome in humans is associated with deletions of the 11p13 region. The brain-derived neurotrophic factor (BDNF) gene ...maps to this region, and its deletion seems to contribute to the severity of the patients' mental retardation. Yeast artificial chromosomes (YACs) carrying the BDNF gene have been isolated and characterized. Localization of two known exons of this gene leads to a minimal estimation of its size of about 40 kb. Chimerism of the BDNF YACs has been investigated by fluorescence in situ hybridization and chromosome assignment on somatic cell hybrids. Using the BDNF gene, YAC end sequence tagged sites (STS), and Généthon microsatellite markers, we constructed a 1.7-Mb contig and refined the cytogenetic map at 11p13. The resulting integrated physical, genetic, and cytogenetic map constitutes a resource for the characterization of genes that may be involved in the WAGR syndrome.
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