Characterization of CD28null T cells in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fibrotic lung disease, with unknown etiopathogenesis and suboptimal therapeutic options. Previous reports have shown that increased T-cell numbers and CD28null phenotype is predictive of prognosis in IPF, suggesting that these cells might have a role in this disease. Flow cytometric analysis of explanted lung cellular suspensions showed a significant increase in CD8+ CD28null T cells in IPF relative to normal lung explants. Transcriptomic analysis of CD3+ T cells isolated from IPF lung explants revealed a loss of CD28-transcript expression and elevation of pro-inflammatory cytokine expression in IPF relative to normal T cells. IPF lung explant-derived T cells (enriched with CD28null T cells), but not normal donor lung CD28+ T cells induced dexamethasone-resistant lung remodeling in humanized NSG mice. Finally, CD28null T cells expressed similar CTLA4 and significantly higher levels of PD-1 proteins relative to CD28+ T cells and blockade of either proteins in humanized NSG mice, using anti-CTLA4, or anti-PD1, mAb treatment-accelerated lung fibrosis. Together, these results demonstrate that IPF CD28null T cells may promote lung fibrosis but the immune checkpoint proteins, CTLA-4 and PD-1, appears to limit this effect.

Development of a pharmacodynamic assay based on PLCγ2 phosphorylation for quantifying spleen tyrosine kinase (SYK)-Bruton's tyrosine kinase (BTK) signaling.

Spleen tyrosine kinase (SYK) and Bruton's tyrosine kinase (BTK) are key mediators in coupling cell surface receptors, such as the B-cell receptor (BCR), to downstream signaling events affecting diverse biological functions. There is therefore tremendous interest in the development of pharmacological inhibitors targeting the SYK-BTK axis for the treatment of inflammatory disorders and hematological malignancies. A good pharmacodynamic (PD) assay, ideally a blood-based assay that measures proximal events, is warranted for evaluation of such inhibitors. In platelets, collagen-induced activation of membrane glycoprotein GPVI is dependent on the SYK-BTK axis. Here, we report the development of a novel immunoassay that uses the dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) to measure GPVI-mediated phosphorylation of phospholipase C γ2 (PLCγ2), a direct substrate of SYK and BTK, in platelets. The assay was validated using SYK or BTK inhibitors and generated IC50 correlated with those from the BCR-induced B-cell activation assay. Furthermore, this assay showed good stability and uniformity over a period of 24 h in different donors. Interestingly, compound IC50 values using blood from patients with rheumatoid arthritis were slightly higher compared with those produced using samples from healthy donors. This novel platelet PLCγ2 phosphorylation-based immunoassay should serve as a promising PD assay for preclinical and clinical development of inhibitors targeting the SYK-BTK axis.

Bleomycin induces molecular changes directly relevant to idiopathic pulmonary fibrosis: a model for "active" disease.

The preclinical model of bleomycin-induced lung fibrosis, used to investigate mechanisms related to idiopathic pulmonary fibrosis (IPF), has incorrectly predicted efficacy for several candidate compounds suggesting that it may be of limited value. As an attempt to improve the predictive nature of this model, integrative bioinformatic approaches were used to compare molecular alterations in the lungs of bleomycin-treated mice and patients with IPF. Using gene set enrichment analysis we show for the first time that genes differentially expressed during the fibrotic phase of the single challenge bleomycin model were significantly enriched in the expression profiles of IPF patients. The genes that contributed most to the enrichment were largely involved in mitosis, growth factor, and matrix signaling. Interestingly, these same mitotic processes were increased in the expression profiles of fibroblasts isolated from rapidly progressing, but not slowly progressing, IPF patients relative to control subjects. The data also indicated that TGFß was not the sole mediator responsible for the changes observed in this model since the ALK-5 inhibitor SB525334 effectively attenuated some but not all of the fibrosis associated with this model. Although some would suggest that repetitive bleomycin injuries may more effectively model IPF-like changes, our data do not support this conclusion. Together, these data highlight that a single bleomycin instillation effectively replicates several of the specific pathogenic molecular changes associated with IPF, and may be best used as a model for patients with active disease.

A model to identify novel targets involved in oxidative stress-induced apoptosis in human lung epithelial cells by RNA interference.

Chronic obstructive pulmonary disease (COPD) is an increasing health problem primarily associated with cigarette smoking, and one of the leading causes of morbidity and mortality worldwide. Despite recent advances in understanding the pathogenesis of the disease, overall patient outcome remains poor with limited therapeutic intervention. Chronic inflammation, an imbalance between proteolytic and anti-proteolytic activities (leading to lung parenchyma destruction) and excessive oxidative stress contribute to COPD pathophysiology. Oxidative stress-triggered apoptosis of alveolar structural cells, including epithelial cells, may be an important event in the development of COPD. In this study, we developed a cell-based oxidative stress-induced apoptosis assay and performed a high-throughput screen (HTS) using a human druggable genome siRNA library. Our results have identified potential novel pathways (e.g. unfolded protein response, proteosomal activity) and targets (e.g. MAP3K14, HMGB2) that regulate the response of lung epithelial cells to oxidative stress. This assay has proven to be a useful tool for the identification of potential new therapeutic targets for lung disease.

Relating chemical and biological diversity space: a tunable system for efficient gene transfection.

Polyethyleneimine (PEI), a well-established nonviral transfection reagent, was combinatorially modified with varying proportions of methyl, benzyl, and n-dodecyl groups to create a library of 435 derivatized polymers. Screening of this library for transfection, DNA binding, and toxicity allows systematic correlation of the biological properties of our polymers to their derivatizations. Combinations of derivatizations bring about a 100-fold variation in transfection efficiency between library members. The best PEI derivatives exhibit increases in transfection efficiency of more than 80-fold over unmodified PEI (up to 28+/-7 % of cells transfected) and rival commercial reagents such as Lipofectamine 2000 (21+/-10 %) and JetPEI (32+/-5.0 %). In addition, we can identify compounds that are specifically tuned for efficient transfection in CHO-K1 over Ishikawa cells and vice versa, demonstrating that the approach can lead to cell-type selectivity of at least one order of magnitude. This work demonstrates that multivalent derivatization of a polymeric framework can create functional diversity substantially greater than the structural diversity of the derivatization building blocks and suggests an approach to a better understanding of the molecular underpinnings of transfection as well as their exploitation.

Inefficient cationic lipid-mediated siRNA and antisense oligonucleotide transfer to airway epithelial cells in vivo.

BACKGROUND: The cationic lipid Genzyme lipid (GL) 67 is the current "gold-standard" for in vivo lung gene transfer. Here, we assessed, if GL67 mediated uptake of siRNAs and asODNs into airway epithelium in vivo. METHODS: Anti-lacZ and ENaC (epithelial sodium channel) siRNA and asODN were complexed to GL67 and administered to the mouse airway epithelium in vivo Transfection efficiency and efficacy were assessed using real-time RT-PCR as well as through protein expression and functional studies. In parallel in vitro experiments were carried out to select the most efficient oligonucleotides. RESULTS: In vitro, GL67 efficiently complexed asODNs and siRNAs, and both were stable in exhaled breath condensate. Importantly, during in vitro selection of functional siRNA and asODN we noted that asODNs accumulated rapidly in the nuclei of transfected cells, whereas siRNAs remained in the cytoplasm, a pattern consistent with their presumed site of action. Following in vivo lung transfection siRNAs were only visible in alveolar macrophages, whereas asODN also transfected alveolar epithelial cells, but no significant uptake into conducting airway epithelial cells was seen. SiRNAs and asODNs targeted to beta-galactosidase reduced betagal mRNA levels in the airway epithelium of K18-lacZ mice by 30% and 60%, respectively. However, this was insufficient to reduce protein expression. In an attempt to increase transfection efficiency of the airway epithelium, we increased contact time of siRNA and asODN using the in vivo mouse nose model. Although highly variable and inefficient, transfection of airway epithelium with asODN, but not siRNA, was now seen. As asODNs more effectively transfected nasal airway epithelial cells, we assessed the effect of asODN against ENaC, a potential therapeutic target in cystic fibrosis; no decrease in ENaC mRNA levels or function was detected. CONCLUSION: This study suggests that although siRNAs and asODNs can be developed to inhibit gene expression in culture systems and certain organs in vivo, barriers to nucleic acid transfer in airway epithelial cells seen with large DNA molecules may also affect the efficiency of in vivo uptake of small nucleic acid molecules.

Effect of chain length on transfection properties of spermine-based gemini surfactants.

The synthesis and associated structure-activity relationships for gene transfection of a series of spermine-derived cationic gemini surfactants incorporating diamino acid headgroups and either identical (symmetrical) or different (unsymmetrical) lipophilic tailgroups is described. Transfection activity is found to depend critically upon the structural elements present.