U-BIOPRED clinical adult asthma clusters linked to a subset of sputum omics.

BACKGROUND: Asthma is a heterogeneous disease in which there is a differential response to asthma treatments. This heterogeneity needs to be evaluated so that a personalized management approach can be provided. OBJECTIVES: We stratified patients with moderate-to-severe asthma based on clinicophysiologic parameters and performed an omics analysis of sputum. METHODS: Partition-around-medoids clustering was applied to a training set of 266 asthmatic participants from the European Unbiased Biomarkers for the Prediction of Respiratory Diseases Outcomes (U-BIOPRED) adult cohort using 8 prespecified clinic-physiologic variables. This was repeated in a separate validation set of 152 asthmatic patients. The clusters were compared based on sputum proteomics and transcriptomics data. RESULTS: Four reproducible and stable clusters of asthmatic patients were identified. The training set cluster T1 consists of patients with well-controlled moderate-to-severe asthma, whereas cluster T2 is a group of patients with late-onset severe asthma with a history of smoking and chronic airflow obstruction. Cluster T3 is similar to cluster T2 in terms of chronic airflow obstruction but is composed of nonsmokers. Cluster T4 is predominantly composed of obese female patients with uncontrolled severe asthma with increased exacerbations but with normal lung function. The validation set exhibited similar clusters, demonstrating reproducibility of the classification. There were significant differences in sputum proteomics and transcriptomics between the clusters. The severe asthma clusters (T2, T3, and T4) had higher sputum eosinophilia than cluster T1, with no differences in sputum neutrophil counts and exhaled nitric oxide and serum IgE levels. CONCLUSION: Clustering based on clinicophysiologic parameters yielded 4 stable and reproducible clusters that associate with different pathobiological pathways.

Divergent gene activation in peripheral blood and tissues of patients with rheumatoid arthritis, psoriatic arthritis and psoriasis following infliximab therapy.

OBJECTIVE: The immune inflammatory disorders rheumatoid arthritis (RA), psoriatic arthritis (PsA) and psoriasis (Ps) share common pathologic features and show responsiveness to anti-tumor necrosis factor (TNF) agents yet they are phenotypically distinct. The aim of this study was to examine if anti-TNF therapy is associated with divergent gene expression profiles in circulating cells and target tissues of patients with these diseases. METHODS: Peripheral blood CD14+ and CD14- cells were isolated from 9 RA, 12 PsA and 10 Ps patients before and after infliximab (IFX) treatment. Paired synovial (n=3, RA, PsA) and skin biopsies (n=5, Ps) were also collected. Gene expression was analyzed by microarrays. RESULTS: 26 out of 31 subjects responded to IFX. The transcriptional response of CD14+ cells to IFX was unique for the three diseases, with little overlap (<25%) in significantly changed gene lists (with PsA having the largest number of changed genes). In Ps, altered gene expression was more pronounced in lesional skin (relative to paired, healthy skin) compared to blood (relative to healthy controls). Marked suppression of up-regulated genes in affected skin was noted 2 weeks after therapy but the expression patterns differed from uninvolved skin. Divergent patterns of expression were noted between the blood cells and skin or synovial tissues in individual patients. Functions that promote cell differentiation, proliferation and apoptosis in all three diseases were enriched. RA was enriched in functions in CD14- cells, PsA in CD14+ cells and Ps in both CD14+ and CD14- cells, however, the specific functions showed little overlap in the 3 disorders. CONCLUSION: Divergent patterns of altered gene expression are observed in RA, PsA and Ps patients in blood cells and target organs in IFX responders. Differential gene expression profiles in the blood do not correlate with those in target organs.

GLP-2 receptor agonism ameliorates inflammation and gastrointestinal stasis in murine postoperative ileus.

Glucagon-like peptide 2 (GLP-2) is a pleiotropic intestinotrophic hormone that we hypothesized could lessen gastrointestinal inflammation associated with postoperative ileus (POI). To test this idea, the prophylactic timing and dose of a long-acting variant of human GLP-2 linked to the Fc portion of murine immunoglobulin G (IgG) (GLP-2/IgG) was optimized in a murine model of POI. Surgically treated mice received a single dose of GLP-2/IgG, IgG isotype control, or phosphate-buffered saline 1 to 48 h before small bowel surgical manipulation. The distribution of orally fed fluorescein isothiocyanate-dextran and histological analyses of myeloperoxidase-positive immune cells were determined 24 and 48 h postoperatively. TaqMan quantitative polymerase chain reaction was used to determine early changes in mRNA expression in the muscularis or mucosa. In normal mice, prolonged exposure to GLP-2 increased upper gastrointestinal (GI) transit and mucosal weight. When administered 1 or 3 h before surgery, GLP-2/IgG reduced the leukocyte infiltrate 24 and 48 h postoperatively and improved GI transit 48 h postoperatively. Surgical manipulation rapidly increased gene expression of proinflammatory cytokines and enzymes for kinetically active mediators in the mucosa and muscularis. GLP-2/IgG2a affected the expression of genes associated with mucosal inflammation and barrier function. We conclude that prophylactic treatment with a long-acting GLP-2 agonist ameliorates inflammation and improves intestinal dysmotility associated with surgical manipulation of the bowel. The action of GLP-2 is consistent with a lessening of inflammation, leading to a more rapid recovery.

Codon engineering for improved antibody expression in mammalian cells.

While well established in bacterial hosts, the effect of coding sequence variation on protein expression in mammalian systems is poorly characterized outside of viral proteins or proteins from distant phylogenetic families. The potential impact is substantial given the extensive use of mammalian expression systems in research and manufacturing of protein biotherapeutics. We are studying the effect of codon engineering on expression of recombinant antibodies with an emphasis on developing manufacturing cell lines. CNTO 888, a human mAb specific for human MCP-1, was obtained by antibody phage display in collaboration with MorphoSys AG. The isolated DNA sequence of the antibody was biased towards bacterial codons, reflecting the engineering of the Fab library for phage display expression in Escherichia coli. We compared the expression of CNTO 888 containing the parental V-region sequences with two engineered coding variants. In the native codon exchanged (NCE) variant, the V-region codons were replaced with those used in naturally derived human antibody genes. In the human codon optimized (HCO) variant the V-region codons were those used at the highest frequency based on a human codon usage table. The antibody expression levels from stable transfections in mammalian host cells were measured. The HCO codon variant of CNTO 888 yielded the highest expressing cell lines and the highest average expression for the screened populations. This had a significant positive effect on the process to generate a CNTO 888 production cell line and indicates the potential to improve antibody expression in mammalian expression systems by codon engineering.

Phosphorylation by protein kinase CKII modulates the DNA-binding activity of a chloroplast nucleoid-associated protein.

Plastid DNA, like bacterial and mitochondrial DNA, is organized into protein-DNA complexes called nucleoids. Plastid nucleoids are believed to be associated with the inner envelope in developing plastids and the thylakoid membranes in mature chloroplasts, but the mechanism for this localization is unknown. MFP1 is a DNA-binding, coiled-coil protein associated with the thylakoid membranes of mature chloroplasts. It is also a component of nucleoids, suggesting a function at the interface of the chloroplast genome and the photosynthetic membranes. Several thylakoid proteins are phosphorylated by a protein kinase CKII-like activity and the alpha subunit of a chloroplast-located CKII has recently been identified as a component of the chloroplast transcription complex. Here, we show evidence for the phosphorylation of MFP1 in purified chloroplasts from tobacco (Nicotiana tabacum L.). We demonstrate that the DNA-binding domain of MFP1 is a substrate for CKII and that phosphorylation by CKII inhibits DNA binding. Using site-directed mutagenesis, we identify a conserved twin CKII site in the DNA-binding domain that is required for the inhibition of DNA binding. Phosphorylation of MFP1 by chloroplast CKII as a possible means to modulate its DNA-binding activity is discussed.