Genetic profiles of transcriptomic clusters of childhood asthma determine specific severe subtype.

BACKGROUND: Previous studies have defined transcriptomic subtypes of adult asthma using samples of induced sputum and bronchial epithelium; however, those procedures are not readily applicable in the clinic, especially for childhood asthma. OBJECTIVE: We aim to dissect the transcriptomic clusters of childhood asthma using highly variably expressed genes of peripheral blood mononuclear cells (PBMC) among patients. METHODS: Gene expression of PBMC from 133 asthmatic children and 11 healthy controls was measured with Illumina microarrays. We applied the k-means clustering algorithm of 2048 genes to assign asthmatic children into clusters. Genes with differential expression between asthma clusters and healthy controls were used to investigate whether they could identify severe asthma of children and adults. RESULTS: We identified 3 asthma clusters with distinct inflammatory profiles in peripheral blood. Cluster 1 had the highest eosinophil count. Cluster 2 showed lower counts of both eosinophils and neutrophils. Cluster 3 had the highest neutrophil count and the poorest treatment control. Compared with other patients, Cluster 3 exhibited a unique gene expression pattern which was associated with changes in the glucocorticoid signalling and activation of the T helper 1/T helper 17 (TH 1/TH 17) immune pathways. In the validation studies, an 84-gene signature could identify severe asthma in children on leucocytes, as well as severe asthma in adults on CD8+ T cells. CONCLUSIONS AND CLINICAL RELEVANCE: Gene expression profiling of PBMC is useful for the identification of TH 1/TH 17-mediated asthma with poor treatment control. PBMC and CD8+ T cells could be important targets for the investigation and identification of severe asthma.

Childhood asthma clusters reveal neutrophil-predominant phenotype with distinct gene expression.

BACKGROUND: Childhood asthma comprises different phenotypes with complex pathophysiology. Different asthma phenotypes evoke various clinical symptoms and vary in their responses to treatments. METHODS: We applied k-means clustering algorithm of twelve objective laboratory tests among 351 asthmatic children enrolled in the Taiwanese Consortium of Childhood Asthma Study (TCCAS). We constructed gene expression profiles of peripheral blood mononuclear cells (PBMC) from children with different asthma phenotypes. RESULTS: Five distinct phenotypes of childhood asthma were identified and can be characterized by either eosinophil-predominant or neutrophil-predominant inflammatory characteristics. In the gene expression profile analysis, significant differences were noted for neutrophil-predominant asthma, compared with samples from all the other asthma phenotypes. The vast majority of the differentially expressed genes in neutrophil-predominant asthma was associated with corticosteroid response. From an independent inhaled corticosteroid (ICS) response cohort, we also found neutrophils could be activated in this severe asthma phenotype and neutrophil-predominant asthma may be associated with corticosteroid nonresponsiveness. CONCLUSION: Phenotype clustering of childhood asthma can be helpful to identify clinically relevant patients and reveal different inflammatory characteristics in asthmatic children. Neutrophil-predominant asthma is the most severe asthma phenotype with poor corticosteroid response. Gene expression profile of different asthma phenotypes not only improve our knowledge of childhood asthma, but also can guide asthma precision medicine.

GSTP1 is a hub gene for gene-air pollution interactions on childhood asthma.

There is growing evidence that multiple genes and air pollutants are associated with asthma. By identifying the effect of air pollution on the general population, the effects of air pollution on childhood asthma can be better understood. We conducted the Taiwan Children Health Study (TCHS) to investigate the influence of gene-air pollution interactions on childhood asthma. Complete monitoring data for the ambient air pollutants were collected from Taiwan Environmental Protection Agency air monitoring stations. Our results show a significant two-way gene-air pollution interaction between glutathione S-transferase P (GSTP1) and PM10 on the risk of childhood asthma. Interactions between GSTP1 and different types of air pollutants have a higher information gain than other gene-air pollutant combinations. Our study suggests that interaction between GSTP1 and PM10 is the most influential gene-air pollution interaction model on childhood asthma. The different types of air pollution combined with the GSTP1 gene may alter the susceptibility to childhood asthma. It implies that GSTP1 is an important hub gene in the anti-oxidative pathway that buffers the harmful effects of air pollution.

Inhibitory effects of schisandrin A and schisandrin B on CYP3A activity.

Our aim was to investigate the inhibitory effects of schisandrin A and schisandrin B on cytochrome P450 (CYP3A) activity in rat liver microsomes and the mechanism of this interaction. 1'-Hydroxy midazolam and midazolam 1-hydroxylation catalyzed by CYP3A were analyzed by high performance liquid chromatography (HPLC). Results showed that schisandrin A and schisandrin B inhibited CYP3A activity with IC(50) values of 6.60 and 5.51 microM and K(i) values of 5.83 and 4.24 microM, respectively. A dilution assay plot of each inhibitor gave a slope value of up to 91% that of the control samples. The inactivation of CYP3A activity by schisandrin A and schisandrin B was found to be both time-and concentration-dependent (schisandrin A: K(I) = 4.51 microM, K(inact) = 0.134/min; schisandrin B: K(I) = 3.01 microM, K(inact) = 0.112/min). We conclude that the inhibition of CYP3A activity in rat liver microsomes by schisandrin A and schisandrin B is mostly attributed to a mixed noncompetitive and complete inhibition.

Expression of two nonallelic type II procollagen genes during Xenopus laevis embryogenesis is characterized by stage-specific production of alternatively spliced transcripts.

The pattern of type II collagen expression during Xenopus laevis embryogenesis has been established after isolating specific cDNA and genomic clones. Evidence is presented suggesting that in X. laevis there are two transcriptionally active copies of the type II procollagen gene. Both genes are activated at the beginning of neurula stage and steady-state mRNA levels progressively increase thereafter. Initially, the transcripts are localized to notochord, somites, and the dorsal region of the lateral plate mesoderm. At later stages of development and parallel to increased mRNA accumulation, collagen expression becomes progressively more confined to chondrogenic regions of the tadpole. During the early period of mRNA accumulation, there is also a transient pattern of expression in localized sites that will later not undergo chondrogenesis, such as the floor plate in the ventral neural tube. At later times and coincident with the appearance of chondrogenic tissues in the developing embryo, expression of the procollagen genes is characterized by the production of an additional, alternatively spliced transcript. The alternatively spliced sequences encode the cysteine-rich globular domain in the NH2-propeptide of the type II procollagen chain. Immunohistochemical analyses with a type II collagen monoclonal antibody documented the deposition of the protein in the extracellular matrix of the developing embryo. Type II collagen expression is therefore temporally regulated by tissue-specific transcription and splicing factors directing the synthesis of distinct molecular forms of the precursor protein in the developing Xenopus embryo.

Tumor necrosis factor-alpha and interferon-gamma suppress the activation of human type I collagen gene expression by transforming growth factor-beta 1. Evidence for two distinct mechanisms of inhibition at the transcriptional and posttranscriptional levels.

Regulation of human type I procollagen gene expression was studied in cultured fibroblasts both at the transcriptional and posttranscriptional level. Transcriptional regulation was examined in cultures transfected with a human pro alpha 2(I) collagen promoter/reporter gene (chloramphenicol acetyltransferase) construct, while posttranscriptional regulation was assessed by parallel determinations of type I procollagen mRNA steady-state levels. Transforming growth factor-beta 1 (TGF-beta 1) elicited a marked, approximately 5-23-fold, enhancement of pro alpha 2(I) collagen promoter activity, which was accompanied by an elevation of type I procollagen mRNA levels. This enhancement of gene expression was suppressed by tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), as determined at mRNA steady-state level, but two distinct mechanisms were involved. TNF-alpha suppressed the pro alpha 2(I) collagen promoter activity, whereas IFN-gamma had only a minimal effect at transcriptional level. The effects of TNF-alpha and IFN-gamma were synergistic, suggesting that combination of these two factors may potentially provide pharmacologic means to counteract tissue deposition of collagen in diseases involving TGF-beta.

Evidence for an oncogenic role of AHI-1 in Sezary syndrome, a leukemic variant of human cutaneous T-cell lymphomas.

Ahi-1 (Abelson helper integration site 1) is a novel gene frequently activated by provirus insertional mutagenesis in murine leukemias and lymphomas. Its involvement in human leukemogenesis is demonstrated by gross perturbations in its expression in human leukemia cells, particularly in cutaneous T-cell lymphoma cell lines where increases in AHI-1 transcripts of 40-fold are seen. To test directly whether deregulated expression of AHI-1 contributes to their transformed properties, knockdown of AHI-1 expression in Hut78 cells, a cell line derived from a patient with Sezary syndrome (SS), was performed using retroviral-mediated RNA interference. Retroviral-mediated suppression specifically inhibited expression of AHI-1 and its isoforms in transduced cells by 80% and also reduced autocrine production of interleukin (IL)-2, IL-4 and tumor necrosis factor-alpha (TNFalpha) by up to 85%. It further significantly reduced their growth factor independence in vitro and the ability to produce tumors in immunodeficient mice. Interestingly, aberrant expression of AHI-1, particularly truncated isoforms, was present in CD4+CD7- Sezary cells from some patients with SS. Elevated expression of IL-2 and TNFalpha was also found in these cells. These findings provide strong evidence of the oncogenic activity of AHI-1 in human leukemogenesis and demonstrate that its deregulation may contribute to the development of SS.

BIN1 tumor suppressor regulates Fas/Fas ligand-mediated apoptosis through c-FLIP in cutaneous T-cell lymphoma.

The bridging integrator 1 (BIN1) tumor suppressor encodes multiple alternatively spliced isoforms implicated in DNA repair, cell-cycle control, apoptosis and membrane dynamics. BIN1 attenuation has been reported in several solid tumors; however, the role of BIN1 in lymphomagenesis remains unexplored. We recently demonstrated that BIN1 transcript levels are significantly downregulated in CD4(+)CD7(-) Sezary cells from patients with Sezary syndrome (SS), a subtype of cutaneous T-cell lymphoma (CTCL). We have now demonstrated that restored BIN1 expression in CTCL cells leads to a significant reduction in cell proliferation, an increase in spontaneous and Fas/Fas ligand (Fas/FasL)-induced apoptosis in vitro and inhibition of tumorigenic activity of CTCL cells in vivo. Interestingly, restoration of BIN1 expression in CTCL cells downregulates the expression of c-FLIP, an important inhibitor of Fas/FasL-mediated apoptosis, and activates the caspase cascade; these phenotypes can be rescued by knockdown of BIN1. Importantly, significantly reduced BIN1 expression and increased c-FLIP expression are observed in primary CTCL patient samples, and high BIN1 and low c-FLIP mRNA levels correlate with better survival rate in SS patients. These results indicate that BIN1 regulates Fas/FasL-mediated apoptosis through c-FLIP and that BIN1 deficiency may have an important role in CTCL pathogenesis by causing apoptosis resistance. Thus BIN1 and c-FLIP represent potential therapeutic targets in CTCL.

Cloning and analysis of the 5' portion of the human type-III procollagen gene (COL3A1).

We have isolated overlapping clones containing the 5'-terminal portion of the human pro-alpha 1(III) collagen gene (COL3A1). This has enabled us to extend our previous studies and thus generate a restriction map of nearly 64 kb of DNA encompassing all of COL3A1 and more than 20 kb of flanking sequences. Aside from the complete nucleotide and amino acid sequences of type-III N-pre-propeptide, this study has established the number of the corresponding exons, whose relative organization deviates from the pattern observed in the analogous regions of type-I procollagen genes, COL1A1 and COL1A2. Moreover, we have sequenced 1628 bp of the 5'-flanking region of COL3A1, from the transcription start point (tsp) to an AluI repetitive element. Pairwise comparison with the analogous segment of the mouse gene has showed 78% sequence similarity in nearly 270 bp immediately preceding the tsp and including the TATA element and a presumptive NF-1 binding site. Relatively close to the tsp, but upstream from the region of homology with the murine gene, a potential AP-1 binding site has also been identified.

Expression of the UNC-5 guidance receptor in the touch neurons of C. elegans steers their axons dorsally.

Growth cones in developing nervous systems encounter a sequence of extracellular cues during migration. In theory, a growth cone can navigate by selectively expressing or activating surface receptor(s) that recognize extracellular cues appropriate to each migratory phase. Using the simple Caenorhabditis elegans nervous system, we attempted to demonstrate that path selection by migrating growth cones can be predictably altered by ectopic expression of a single receptor. The unc-5 gene of C. elegans encodes a unique receptor of the immunoglobulin superfamily (UNC-5), required cell-autonomously to guide growth cone and mesodermal cell migrations in a dorsal direction on the epidermis. We report here that the UNC-5 receptor induces dorsally oriented axon trajectories when ectopically expressed in the touch receptor neurons which normally extend pioneer axons longitudinally or ventrally on the epidermis. These errant trajectories depend on unc-6, which encodes a putative epidermal path cue, just as normal dorsally oriented axon trajectories do (such as those of certain motor neurons), suggesting that UNC-5 acts to reorient the touch cell growth cones by using its normal guidance mechanisms. These results support previous evidence that UNC-5 and UNC-6 play instructive rules in guiding growth cone migrations on the epidermis in C. elegans, and indicate that pioneering growth cones, which normally migrate in different directions, may use equivalent intracellular signalling mechanisms for guidance.

Cognate peptide-induced destruction of CD8+ cytotoxic T lymphocytes is due to fratricide.

In the absence of other cells, cloned CTL in culture can undergo massive destruction upon the addition of a peptide that is recognized, in association with the CTL's class I MHC proteins, by the CTL's Ag-specific TCR. To determine whether the destruction is a result of the individual CTL's recognition via its own TCR of peptide-MHC-I complexes on its own surface ("suicide"), or to cytolytic attack by some CTL on others in the same culture ("fratricide"), we compared the rate of peptide-induced cell death in conventional cultures, where CTL are free to establish cell-cell contacts, with other cultures in which individual CTL were prevented from forming cell-cell contacts by encasing them individually in agarose gel microdrops. The differences were dramatic: in the presence of high concentrations of peptide (10 millionfold greater than is necessary to support 50% lysis of conventional target cells by these CTL) cell death was linear over 0 to 8 h in conventional cultures, at a rate of about 10% per hour, whereas in the presence of the same high concentration of peptide over the same time course, no death was detected among the cells encased in agarose gel microdroplets. The results demonstrate an absolute requirement for cell-cell contact in the destruction of cloned CTL in culture with their cognate peptides at high concentration. Using an increase of intracellular calcium ion concentration ([Ca2+]i) as a measure of T-cell activation, we also found that peptide-dependent activation of CTL likewise depends upon cell-cell contact.

Functional analysis of cis-acting DNA sequences controlling transcription of the human type I collagen genes.

The 3500-base pair region located immediately upstream of the transcriptional start site of the human pro-alpha 2(I) collagen gene contains all the sequences necessary for cell-specific transcription. In transient expression assays, the pro-alpha 2(I) collagen promoter directed the production of high levels of bacterial chloramphenicol acetyltransferase in collagen-producing human fetal fibroblasts. Enzyme activity, on the other hand, was nearly undetectable in extracts from collagen-nonproducing immortalized lymphoblasts. Deletion experiments narrowed the active segment of the human promoter to a phylogenetically conserved sequence comprised between nucleotides-376 and -108, relative to the initiation site of transcription. In similar analyses, the pro-alpha 1(I) collagen gene failed to direct cell-specific transcription. As part of this study, the controversial issue surrounding the putative enhancer element in the first intron of the human pro-alpha 1(I) collagen gene also has been reconsidered. Accordingly, we now propose a more restricted definition of this cis-acting DNA element since its action is exerted in an orientation-preferred manner and with a strong specificity for its own promoter. Moreover, stimulation does not appear to be tissue-specific. Finally, evidence is presented supporting the notion that although structurally different and distinctly arranged, the regulatory sequences of the type I collagen genes may bind similar trans-acting factors.

Organization of the exons coding for pro alpha 1(II) collagen N-propeptide confirms a distinct evolutionary history of this domain of the fibrillar collagen genes.

The organization of the exons coding for the N-terminal portion of human type II procollagen has been determined. Aside from inferring the previously unknown primary structure of type II N-propeptide, this study has revealed that this coding domain of the gene exhibits an organization uniquely distinct from those of type I and type III collagens. This finding substantiates the notion that the N-propeptide coding domains of the fibrillar collagen genes evolved under less stringent selection than those encoding the C-propeptide and triple helical regions.

Molecular pathobiology of human collagens.

The fibril-forming collagens (types I-III, V and XI) represent a homogeneous and evolutionary related group of proteins and genes. In addition to serving as supportive elements, these macromolecules influence the spatial and ontogenic diversity of extracellular matrices, for they regulate a number of developmental programs and cellular activities, such as adhesion, proliferation and migration. Deranged expression of fibrillar collagen genes results in a number of inherited and acquired disorders which greatly affect the structural integrity of the organism. An understanding of collagen biosynthesis and regulation in normal and diseased states provides an opportunity to dissect biological problems which relate to a wide variety of subjects, including morphogenesis, relationships between structure and function of proteins, gene expression and human mutations.

Progressively restricted expression of a new homeobox-containing gene during Xenopus laevis embryogenesis.

We have isolated cDNAs encoding a novel Xenopus homeodomain-containing protein homologous to the mouse Hox-7.1 and the Drosophila muscle segment homebox (msh). Northern blot and RNAase protection experiments established that transcripts of the frog gene, termed Xhox-7.1, first appear at about the beginning of gastrulation. After a rapid increase, mRNA levels plateau between the neurula and middle-tailbud stages, and decrease steadily thereafter. In situ hybridization localized the Xhox-7.1 message to the dorsal mesodermal mantle of gastrula stage embryos. Comparison of the hybridization patterns of progressively more anterior cross-section of tailbud stage embryos localized the signal to the dorsal neural tube and neural crest, to specific regions of the lateral plate mesoderm, and to the cardiogenic region. By the tadpole stage, the Xhox-7.1 message appears only at specific sites in the central nervous system, such as in the dorsal hindbrain. Thus, during embryonic development levels of Xhox-7.1 expression decrease as the transcript becomes more progressively localized. Finally, evidence is presented of a distinct msh-like transcript (provisionally termed Xhox-7.1') which begins to accumulate at early-gastrula stage, as well.

Targeting Src homology 2 domain-containing tyrosine phosphatase (SHP-1) into lipid rafts inhibits CD3-induced T cell activation.

To study the mechanism by which protein tyrosine phosphatases (PTPs) regulate CD3-induced tyrosine phosphorylation, we investigated the distribution of PTPs in subdomains of plasma membrane. We report here that the bulk PTP activity associated with T cell membrane is present outside the lipid rafts, as determined by sucrose density gradient sedimentation. In Jurkat T cells, approximately 5--10% of Src homology 2 domain-containing tyrosine phosphatase (SHP-1) is constitutively associated with plasma membrane, and nearly 50% of SHP-2 is translocated to plasma membrane after vanadate treatment. Similar to transmembrane PTP, CD45, the membrane-associated populations of SHP-1 and SHP-2 are essentially excluded from lipid rafts, where other signaling molecules such as Lck, linker for activation of T cells, and CD3 zeta are enriched. We further demonstrated that CD3-induced tyrosine phosphorylation of these substrates is largely restricted to lipid rafts, unless PTPs are inhibited. It suggests that a restricted partition of PTPs among membrane subdomains may regulate protein tyrosine phosphorylation in T cell membrane. To test this hypothesis, we targeted SHP-1 into lipid rafts by using the N-terminal region of Lck (residues 1--14). The results indicate that the expression of Lck/SHP-1 chimera inside lipid rafts profoundly inhibits CD3-induced tyrosine phosphorylation of CD3 zeta/epsilon, IL-2 generation, and nuclear mobilization of NF-AT. Collectively, these results suggest that the exclusion of PTPs from lipid rafts may be a mechanism that potentiates TCR/CD3 activation.

Analysis of six distinct integrated hepatitis B virus sequences cloned from the cellular DNA of a human hepatocellular carcinoma.

Six distinct hepatitis B virus (HBV) integrations and the flanking cellular sequences were cloned from a hepatoma DNA preparation. None of the cloned fragments retains the entire HBV sequences but the surface antigen (HBsAg) gene and the HBV enhancer are retained in three of the six clones. The other three clones carry only short and possibly highly rearranged HBV genomic sequences and seem to contain some GC-rich clusters. Members of the repetitive Alu family are also found in the vicinity of five of the six integration regions which may have contributed to genome instability. In these six clones, the preferred integration sites are shown to lie within the single-strand region of the HBV genome. None of the clones carries in the flanking cellular sequences any of the 17 oncogenes tested, although the possibility still exists that an oncogene may be found on the side of the genome which has not been cloned. This work thus paves the way for detailed sequence analysis of virus-host junctions, for transfection studies of the HBV integration events, and for a search of genes in the flanking cellular sequences which may have been activated by the retained HBV enhancer using the clones described.

Restriction fragment length polymorphism of the human beta-globin gene cluster: analysis of a beta-thalassemic family in Taiwan.

We have analysed seven polymorphic restriction sites of the human beta-globin gene cluster of six members of a Chinese family with a beta +-thalassemic sibling. The seven polymorphic sites analysed are the HincII site at the 5'-end of the epsilon-globin gene, the HindIII sites in the two gamma-globin genes, two HincII sites within and at the 3'-end of the psi beta 1 pseudogene, the AvaII site in the beta-globin gene and the BamHI site located at the 3' side of the beta-globin gene. The beta thal chromosome has been identified to have a haplotype of +----++ with respect to these seven polymorphic sites. This is also the most predominant haplotype associated with beta +-thalassemia in Mediterranean and Chinese populations (Chen et al., 1984; Orkin et al., 1982). Of the seven sites analysed in this family, four will be useful in prenatal diagnosis of beta-thalassemia in subsequent pregnancies in the family.

UNC-5, a transmembrane protein with immunoglobulin and thrombospondin type 1 domains, guides cell and pioneer axon migrations in C. elegans.

The unc-5 gene is required for guiding pioneering axons and migrating cells along the body wall in C. elegans. In mutants, dorsal migrations are disrupted, but ventral and longitudinal movements are largely unaffected. The gene was tagged for molecular cloning by transposon insertions. Based on genomic and cDNA sequencing, the gene encodes UNC-5, a transmembrane protein of 919 aa. The predicted extracellular N-terminus comprises two immunoglobulin and two thrombospondin type 1 domains. Except for an SH3-like motif, the large intracellular C-terminus is novel. Mosaic analysis shows that unc-5 acts in migrating cells and pioneering neurons. We propose that UNC-5 is a transmembrane receptor expressed on the surface of motile cells and growth cones to guide dorsal movements.