Blood mRNA biomarkers distinguish variable systemic and sputum inflammation at treatment initiation of inhaled antibiotics in cystic fibrosis: A prospective non-randomized trial.

Inhaled antibiotics control chronic airway infection and maintain respiratory health in cystic fibrosis (CF). Given variation in patient responses to inhaled antibiotics, the ability to identify distinct responder phenotypes would facilitate the delivery of personalized care. Previously, a 10-gene panel was identified, measured directly from blood leukocytes, which predicted host response to intravenous antibiotic treatment during pulmonary exacerbations. In the current study, we tested whether the same panel predicted clinical response in subjects receiving a month of inhaled antibiotic therapy with aztreonam lysine (AZLI; Cayston®). A small cohort of CF subjects infected with Pseudomonas aeruginosa were enrolled at baseline health, prior to initiating one month's treatment with AZLI using the Altera® nebulizer system. Eighteen CF subjects underwent blood leukocyte gene panel measurements, sputum quantitative microbiology, spirometry, and C-reactive protein (CRP) measurement prior to onset and at completion of 4 weeks of AZLI therapy. Mean absolute improvement in percent predicted Forced Expiratory Volume in one second (ppFEV1) was 3%. Significant reductions in sputum bacterial colony counts were detected with treatment. CRP increased following treatment. While single genes within the panel did not change significantly following treatment, the analysis of multigene panel data demonstrated that HCA112 gene predicted ppFEV1 improvement. Hierarchical clustering based on gene expression yielded two distinctive molecular clusters before and after AZLI therapy. In conclusion, peripheral blood leukocyte genes quantifying inflammation are associated with responses to inhaled antibiotic therapy. Molecular quantification of systemic inflammation may indicate subgroups of CF subjects with variations in underlying inflammation and with variable clinical responses to inhaled antibiotics. Given the size limitation of the study, larger studies are needed in order to evaluate whether molecular measures may add precision to the determination of infectious and inflammatory outcomes following courses of inhaled antimicrobial therapies. Clinical Trials.gov Identifier: NCT01736839.

Whole Blood Gene Expression Profiling Predicts Severe Morbidity and Mortality in Cystic Fibrosis: A 5-Year Follow-Up Study.

RATIONALE: Cystic fibrosis pulmonary exacerbations accelerate pulmonary decline and increase mortality. Previously, we identified a 10-gene leukocyte panel measured directly from whole blood, which indicates response to exacerbation treatment. We hypothesized that molecular characteristics of exacerbations could also predict future disease severity. OBJECTIVES: We tested whether a 10-gene panel measured from whole blood could identify patient cohorts at increased risk for severe morbidity and mortality, beyond standard clinical measures. METHODS: Transcript abundance for the 10-gene panel was measured from whole blood at the beginning of exacerbation treatment (n = 57). A hierarchical cluster analysis of subjects based on their gene expression was performed, yielding four molecular clusters. An analysis of cluster membership and outcomes incorporating an independent cohort (n = 21) was completed to evaluate robustness of cluster partitioning of genes to predict severe morbidity and mortality. RESULTS: The four molecular clusters were analyzed for differences in forced expiratory volume in 1 second, C-reactive protein, return to baseline forced expiratory volume in 1 second after treatment, time to next exacerbation, and time to morbidity or mortality events (defined as lung transplant referral, lung transplant, intensive care unit admission for respiratory insufficiency, or death). Clustering based on gene expression discriminated between patient groups with significant differences in forced expiratory volume in 1 second, admission frequency, and overall morbidity and mortality. At 5 years, all subjects in cluster 1 (very low risk) were alive and well, whereas 90% of subjects in cluster 4 (high risk) had suffered a major event (P = 0.0001). In multivariable analysis, the ability of gene expression to predict clinical outcomes remained significant, despite adjustment for forced expiratory volume in 1 second, sex, and admission frequency. The robustness of gene clustering to categorize patients appropriately in terms of clinical characteristics, and short- and long-term clinical outcomes, remained consistent, even when adding in a secondary population with significantly different clinical outcomes. CONCLUSIONS: Whole blood gene expression profiling allows molecular classification of acute pulmonary exacerbations, beyond standard clinical measures, providing a predictive tool for identifying subjects at increased risk for mortality and disease progression.

Rtp801 suppression of epithelial mTORC1 augments endotoxin-induced lung inflammation.

The mechanistic target of rapamycin (mTOR) is a central regulator of cellular responses to environmental stress. mTOR (and its primary complex mTORC1) is, therefore, ideally positioned to regulate lung inflammatory responses to an environmental insult, a function directly relevant to disease states such as the acute respiratory distress syndrome. Our previous work in cigarette smoke-induced emphysema identified a novel protective role of pulmonary mTORC1 signaling. However, studies of the impact of mTORC1 on the development of acute lung injury are conflicting. We hypothesized that Rtp801, an endogenous inhibitor of mTORC1, which is predominantly expressed in alveolar type II epithelial cells, is activated during endotoxin-induced lung injury and functions to suppress anti-inflammatory epithelial mTORC1 responses. We administered intratracheal lipopolysaccharide to wild-type mice and observed a significant increase in lung Rtp801 mRNA. In lipopolysaccharide-treated Rtp801(-/-) mice, epithelial mTORC1 activation significantly increased and was associated with an attenuation of lung inflammation. We reversed the anti-inflammatory phenotype of Rtp801(-/-) mice with the mTORC1 inhibitor, rapamycin, reassuring against mTORC1-independent effects of Rtp801. We confirmed the proinflammatory effects of Rtp801 by generating a transgenic Rtp801 overexpressing mouse, which displayed augmented inflammatory responses to intratracheal endotoxin. These data suggest that epithelial mTORC1 activity plays a protective role against lung injury, and its inhibition by Rtp801 exacerbates alveolar injury caused by endotoxin.

Circulating RNA transcripts identify therapeutic response in cystic fibrosis lung disease.

RATIONALE: Circulating leukocyte RNA transcripts are systemic markers of inflammation, which have not been studied in cystic fibrosis (CF) lung disease. Although the standard assessment of pulmonary treatment response is FEV(1), a measure of airflow limitation, the lack of systemic markers to reflect changes in lung inflammation critically limits the testing of proposed therapeutics. OBJECTIVES: We sought to prospectively identify and validate peripheral blood leukocyte genes that could mark resolution of pulmonary infection and inflammation using a model by which RNA transcripts could increase the predictive value of spirometry. METHODS: Peripheral blood mononuclear cells were isolated from 10 patients with CF and acute pulmonary exacerbations before and after therapy. RNA expression profiling revealed that 10 genes significantly changed with treatment when compared with matched non-CF and control subjects with stable CF to establish baseline transcript abundance. Peripheral blood mononuclear cell RNA transcripts were prospectively validated, using real-time polymerase chain reaction amplification, in an independent cohort of acutely ill patients with CF (n = 14). Patients who responded to therapy were analyzed using general estimating equations and multiple logistic regression, such that changes in FEV(1)% predicted were regressed with transcript changes. MEASUREMENTS AND MAIN RESULTS: Three genes, CD64, ADAM9, and CD36, were significant and independent predictors of a therapeutic response beyond that of FEV(1) alone (P < 0.05). In both cohorts, receiver operating characteristic analysis revealed greater accuracy when genes were combined with FEV(1). CONCLUSIONS: Circulating mononuclear cell transcripts characterize a response to the treatment of pulmonary exacerbations. Even in small patient cohorts, changes in gene expression in conjunction with FEV(1) may enhance current outcomes measures for treatment response.

Characterization of rat parotid and submandibular acinar cell apoptosis in primary culture.

Apoptosis is a highly organized cellular process that is critical for maintaining glandular homeostasis. We have used primary rat salivary acinar cells from the parotid and submandibular glands to investigate the critical regulatory events involved in apoptosis. Caspase-3 activity, cleavage of caspase substrates, and deoxyribonucleic acid (DNA) fragmentation were assayed in cells treated with etoposide, a DNA-damaging agent, or brefeldin A (BFA), a Golgi toxin. Dose-response studies showed that the sensitivity of both cell types to etoposide and BFA was similar, with 150 microM etoposide or 1.5 microM BFA inducing maximal caspase activation. However, BFA induced a more robust activation of caspase and DNA fragmentation in both cell types. Similar results were observed when the caspase cleavage of poly(adenosine 5'-diphosphate ribose) polymerase and protein kinase C delta were analyzed by Western blot. Analysis of the kinetics of apoptosis showed that caspase-3 activation was maximal at 8 h of etoposide or BFA treatment in the parotid cells and at 8-18 h in the submandibular cells. A similar time course was observed when DNA fragmentation was assayed, although maximal DNA fragmentation in BFA-treated cells was two- to threefold higher than that observed in etoposide-treated cells. Despite slight kinetic differences, it would appear that the apoptotic cascade is very similar in both primary parotid and submandibular acinar cells. Although limited in their long-term stability in culture, the use of primary, nonimmortalized salivary acinar cultures will also permit the use of specific transgenic animals to further characterize the molecular events involved in the regulation of salivary gland acinar cell apoptosis.

Estrogen restores cognition and cholinergic phenotype in an animal model of Down syndrome.

Estrogen maintains normal function of basal forebrain (BF) cholinergic neurons and estrogen replacement therapy (ERT) has therefore been proposed as a therapy for Alzheimer's disease (AD). We provide evidence to support this hypothesis in an animal model of Down syndrome (DS), a chromosome 16 segmental trisomy (Ts65Dn) mouse. These mice develop cholinergic degeneration similar to young adults with DS and AD patients. ERT has not been tested in women with DS, even though they are more likely than normosomic women to develop early menopause and AD. Female Ts65Dn and normosomic mice (11-15 months) received a subcutaneous estrogen pellet or a sham operation. After 60 days, estrogen treatment improved learning of a T-maze task and normalized behavior in the Ts65Dn mice in reversal learning of the task, a measure of cognitive flexibility. Stereological evaluation of choline acetyltransferase (ChAT) immunopositive BF neurons showed that estrogen increased cell size and total number of cholinergic neurons in the medial septum of Ts65Dn mice. In addition, estrogen increased NGF protein levels in the BF of trisomic mice. These findings support the emerging hypothesis that estrogen may play a protective role during neurodegeneration and cognitive decline, particularly in cholinergic BF neuronal systems underlying cognition. The findings also indicate that estrogen may act, at least partially, via endogenous growth factors. Collectively, the data suggest that ERT may be a viable therapeutic approach for women with DS coupled with dementia.