A minimally invasive bronchoscopic approach for direct delivery to murine airways and application to models of pulmonary infection.

The laboratory mouse is used extensively for human disease modeling and preclinical therapeutic testing for efficacy, biodistribution, and toxicity. The variety of murine models available, and the ability to create new ones, eclipses all other species, but the size of mice and their organs create challenges for many in vivo studies. For pulmonary research, improved methods to access murine airways and lungs, and track substances administered to them, would be desirable. A nonsurgical endoscopic system with a camera, effectively a bronchoscope, coupled with a cryoimaging fluorescence microscopy technique to view the lungs in 3D, is described here that allows visualization of the procedure, including the anatomical location at which substances are instilled and fluorescence detection of those substances. We have applied it to bacterial infection studies to characterize better and optimize a chronic lung infection murine model in which we instill bacteria-laden agarose beads into the airways and lungs to extend the duration of the infection and inflammation. The use of the endoscope as guidance for placing a catheter into the airways is simple and quick, requiring only momentary sedation, and reduces post-procedural mortality compared with our previous instillation method that includes a trans-tracheal surgery. The endoscopic method improves speed and precision of delivery while reducing the stress on animals and the number of animals generated and used for experiments.

Serum inflammatory profiles in cystic fibrosis mice with and without Bordetella pseudohinzii infection.

Cystic fibrosis (CF) is an autosomal recessive disease caused by dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) protein, and is marked by an accumulation of mucus in affected airways resulting in persistent infection and chronic inflammation. Quantitative differences in inflammatory markers have been observed in CF patient serum, tracheal cells, and bronchoalveolar lavage fluid, in the absence of detectable infection, implying that absent CFTR function alone may result in dysregulated immune responses. To examine the relationship between absent CFTR and systemic inflammation, 22 analytes were measured in CF mice (F508del/F508del) sera using the MSD multiplex platform. Pro-inflammatory cytokines IL-2, TNF-α, IL-17α, IFN-γ, IL-1ß, and MIP-3α are significantly elevated in infection-naïve CF mice (p < 0.050). Anti-inflammatory cytokines IL-10 and IL-4 are also significantly increased (p = 0.00003, p = 0.004). Additionally, six general markers of inflammation are significantly different from non-CF controls (p < 0.050). To elucidate the effects of chronic infection on the CF inflammatory profile, we examined CF mice exposed to spontaneous Bordetella pseudohinzii infections. There are no statistical differences in nearly all inflammatory markers when compared to their infection-naïve CF counterparts, except in the Th2-derived IL-4 and IL-5 which demonstrate significant decreases following exposure (p = 0.046, p = 0.045). Lastly, following acute infection, CF mice demonstrate elevations in nearly all inflammatory markers, but exhibit a shortened return to uninfected levels over time, and suppression of Th1-derived IL-2 and IL-5 (p = 0.043, p = 0.011). These results imply that CF mice have a persistent inflammatory profile often indistinguishable from chronic infection, and a dysregulated humoral response during and following active infection.

Dynamic, Simultaneous Concentration Mapping of Multiple MRI Contrast Agents with Dual Contrast - Magnetic Resonance Fingerprinting.

Synchronous assessment of multiple MRI contrast agents in a single scanning session would provide a new "multi-color" imaging capability similar to fluorescence imaging but with high spatiotemporal resolution and unlimited imaging depth. This multi-agent MRI technology would enable a whole new class of basic science and clinical MRI experiments that simultaneously explore multiple physiologic/molecular events in vivo. Unfortunately, conventional MRI acquisition techniques are only capable of detecting and quantifying one paramagnetic MRI contrast agent at a time. Herein, the Dual Contrast - Magnetic Resonance Fingerprinting (DC-MRF) methodology was extended for in vivo application and evaluated by simultaneously and dynamically mapping the intra-tumoral concentration of two MRI contrast agents (Gd-BOPTA and Dy-DOTA-azide) in a mouse glioma model. Co-registered gadolinium and dysprosium concentration maps were generated with sub-millimeter spatial resolution and acquired dynamically with just over 2-minute temporal resolution. Mean tumor Gd and Dy concentration measurements from both single agent and dual agent DC-MRF studies demonstrated significant correlations with ex vivo mass spectrometry elemental analyses. This initial in vivo study demonstrates the potential for DC-MRF to provide a useful dual-agent MRI platform.

HDAC6 depletion improves cystic fibrosis mouse airway responses to bacterial challenge.

The hypothesis of this study was that Hdac6 depletion would restore cystic fibrosis (CF) responses to bacterial challenge to more wild type profiles using a CF mouse model. CF mice harboring the F508del Cftr mutation respond to bacterial challenge with 25,000 CFU Pseudomonas aeruginosa embedded into agarose beads to slow clearance. CF mice respond significantly more aggressively to this challenge compared to WT mice with respect to bacterial clearance, weight loss, neutrophil recruitment, and MIP-2 production. Depletion of Hdac6 expression in the CF mice (CF/Hdac6) significantly improves these responses to more WT levels. Weight loss in response to infection is most severe in CF mice and significantly attenuated in CF/Hdac6 mice. Bacterial levels are reduced at a faster rate in CF/Hdac6 mice compared to CF mice where infection persists. Percent neutrophils in lung lavage fluid post-infection are significantly higher in CF mice, but returned to WT levels with CF/Hdac6 mice. Similarly, CF Mip-2 levels are restored to WT levels in the absence of Hdac6 expression. These data demonstrate that Hdac6 depletion restores CF responses to bacterial challenge to WT-like profiles and offer a potential therapeutic avenue for addressing inflammation and infection in CF airways independently of Cftr correction.

Educating Nursing Scientists: Integrating Genetics and Genomics into PhD Curricula.

Nursing science is a diverse field of study, the scope of which has broadened to more fully incorporate genetics and genomics. In recent years, these topics have become focus areas for many nursing researchers. However, recent evidence suggests that doctoral level nursing students and nursing faculty may be underprepared to conduct independent research using genomic approaches. Furthermore, genetics and genomics are severely underrepresented in doctoral level nursing curricula across the United States. This article suggests a thorough, yet manageable three-part curriculum designed to educate doctoral level nursing students on genetics, genomics, and their use in nursing science. Recommendations are then given for the integration of the curriculum into existing nursing PhD programs.

AGTR2 absence or antagonism prevents cystic fibrosis pulmonary manifestations.

BACKGROUND: Pulmonary disease remains the primary cause of morbidity and mortality for individuals with cystic fibrosis (CF). Variants at a locus on the X-chromosome containing the type 2 angiotensin II receptor gene (AGTR2) were identified by a large GWAS as significantly associating with lung function in CF patients. We hypothesized that manipulating the angiotensin-signaling pathway may yield clinical benefit in CF. METHODS: Genetic subset analysis was conducted on a local CF cohort to extend the GWAS findings. Next, we evaluated pulmonary function in CF mice with a deleted AGTR2 gene, and in those who were given subcutaneous injections of PD123,319, a selective AGTR2 antagonist for 12 weeks beginning at weaning. RESULTS: The genetic subset analysis replicated the initial GWAS identified association, and confirmed the association of this locus with additional lung function parameters. Studies in genetically modified mice established that absence of the AGTR2 gene normalized pulmonary function indices in two independent CF mouse models. Further, we determined that pharmacologic antagonism of AGTR2 improved overall pulmonary function in CF mice to near wild-type levels. CONCLUSIONS: These results identify that reduced AGTR2 signaling is beneficial to CF lung function, and suggest the potential of manipulating the angiotensin-signaling pathway for treatment and/or prevention of CF pulmonary disease. Importantly, the beneficial effects were not CF gene mutation dependent, and were able to be reproduced with pharmacologic antagonism. As there are clinically approved drugs available to target the renin-angiotensin signaling system, these findings may be quickly translated to human clinical trials.

Early pulmonary disease manifestations in cystic fibrosis mice.

BACKGROUND: Altered pulmonary function is present early in the course of cystic fibrosis (CF), independent of documented infections or onset of pulmonary symptoms. New initiatives in clinical care are focusing on detection and characterization of preclinical disease. Thus, animal models are needed which recapitulate the pulmonary phenotype characteristic of early stage CF. METHODS: We investigated young CF mice to determine if they exhibit pulmonary pathophysiology consistent with the early CF lung phenotype. Lung histology and pulmonary mechanics were examined in 12- to 16-week-old congenic C57bl/6 F508del and R117H CF mice using a forced oscillation technique (flexiVent). RESULTS: There were no significant differences in the resistance of the large airways. However, in both CF mouse models, prominent differences in the mechanical properties of the peripheral lung compartment were identified including decreased static lung compliance, increased elastance and increased tissue damping. CF mice also had distal airspace enlargement with significantly increased mean linear intercept distances. CONCLUSIONS: An impaired ability to stretch and expand the peripheral lung compartment, as well as increased distances between gas exchange surfaces, were present in young CF mice carrying two independent Cftr mutations. This altered pulmonary histopathophysiology in the peripheral lung compartment, which develops in the absence of infection, is similar to the early lung phenotype of CF patients.

Increased Expression of RhoA in Epithelium and Smooth Muscle of Obese Mouse Models: Implications for Isoprenoid Control of Airway Smooth Muscle and Fibroblasts.

The simultaneous rise in the prevalence of asthma and obesity has prompted epidemiologic studies that establish obesity as a risk factor for asthma. The alterations in cell signaling that explain this link are not well understood and warrant investigation so that therapies that target this asthma phenotype can be developed. We identified a significant increase in expression of the small GTPase RhoA in nasal epithelial cells and tracheal smooth muscle cells from leptin-deficient (ob/ob) mice compared to their wild-type counterparts. Since RhoA function is dependent on isoprenoid modification, we sought to determine the role of isoprenoid-mediated signaling in regulating the viability and proliferation of human airway smooth muscle cells (ASM) and normal human lung fibroblasts (NHLF). Inhibiting isoprenoid signaling with mevastatin significantly decreased the viability of ASM and NHLF. This inhibition was reversed by geranylgeranyl pyrophosphate (GGPP), but not farnesyl pyrophosphate (FPP), suggesting specificity to the Rho GTPases. Conversely, increasing isoprenoid synthesis significantly increased ASM proliferation and RhoA protein expression. RhoA expression is inherently increased in airway tissue from ob/ob mice, and obesity-entrained alterations in this pathway may make it a novel therapeutic target for treating airway disease in the obese population.

Ventilatory pattern and energy expenditure are altered in cystic fibrosis mice.

BACKGROUND: Altered ventilatory pattern and increased energy expenditure are facets of the complex cystic fibrosis (CF) phenotype. It is not known whether these are inherent attributes of CF, secondary consequences of lung infection or other disease complications. METHODS: Studies were performed in congenic C57BL/6J, F508del (Cftr((tm1kth))) and CF gut-corrected (F508del) mice. Ventilatory patterns were measured using whole-body plethysmography. Indirect calorimetry was used to determine oxygen consumption, carbon dioxide production and resting energy expenditure. RESULTS: CF mice (F508del and F508del gut-corrected) have a significantly faster respiratory rate and increased ventilatory pattern variability as compared to non-CF mice. F508del but not CF gut-corrected mice had significantly increased energy expenditure per gram body weight. CONCLUSIONS: CF mice exhibit a faster, more variable ventilatory pattern. These changes were present in the absence of detectable infection or illness due to gastrointestinal obstruction. Increased resting energy expenditure does not completely account for these differences.

Genetic modifiers of lung disease in cystic fibrosis.

BACKGROUND: Polymorphisms in genes other than the cystic fibrosis transmembrane conductance regulator (CFTR) gene may modify the severity of pulmonary disease in patients with cystic fibrosis. METHODS: We performed two studies with different patient samples. We first tested 808 patients who were homozygous for the DeltaF508 mutation and were classified as having either severe or mild lung disease, as defined by the lowest or highest quartile of forced expiratory volume in one second (FEV1), respectively, for age. We genotyped 16 polymorphisms in 10 genes reported by others as modifiers of disease severity in cystic fibrosis and tested for an association in patients with severe disease (263 patients) or mild disease (545). In the replication (second) study, we tested 498 patients, with various CFTR genotypes and a range of FEV1 values, for an association of the TGFbeta1 codon 10 CC genotype with low FEV1. RESULTS: In the initial study, significant allelic and genotypic associations with phenotype were seen only for TGFbeta1 (the gene encoding transforming growth factor beta1), particularly the -509 and codon 10 polymorphisms (with P values obtained with the use of Fisher's exact test and logistic regression ranging from 0.006 to 0.0002). The odds ratio was about 2.2 for the highest-risk TGFbeta1 genotype (codon 10 CC) in association with the phenotype for severe lung disease. The replication study confirmed the association of the TGFbeta1 codon 10 CC genotype with more severe lung disease in comparisons with the use of dichotomized FEV1 for severity status (P=0.0002) and FEV1 values directly (P=0.02). CONCLUSIONS: Genetic variation in the 5' end of TGFbeta1 or a nearby upstream region modifies disease severity in cystic fibrosis.

Beta 2 adrenergic receptor polymorphisms in cystic fibrosis.

There has been a recent emphasis on identifying modifier genes that influence the severity of cystic fibrosis (CF) lung disease. The beta-2-adrenergic receptor is expressed on airway smooth muscle, is the target for inhaled beta agonists, and has several common polymorphisms in its gene, ADRB2. Polymorphisms changing glycine to arginine or glutamate to glutamine in codons 16 and 27, respectively, were associated with differences in clinical response to inhaled beta agonists in individuals with asthma. We compared acute airway responsiveness and 5-year decline in pulmonary function in CF patients with different ADRB2 genotypes. One hundred and six subjects performed spirometry before and after the administration of an inhaled bronchodilator, and had ADRB2 genotype determined for codons 16 and 27. Comparing the percent change in FEV(1) and FEF(25-75) continuously revealed differences in the degree of airway responsiveness to bronchodilator between ADRB2-genotyped groups. However, there was no significant relationship between the ADRB2 genotype at positions 16 and 27 and bronchodilator response when defined as 12% improvement in FEV(1). Five-year decline in percent predicted FEV(1) showed no association with ADRB2 genotype. These data are consistent with variants of the ADRB2 gene having different responses to bronchodilator, but the long-term effects, if any, are not apparent over a 5-year period.