Metabolic profiling of murine radiation-induced lung injury with Raman spectroscopy and comparative machine learning.

Radiation-induced lung injury (RILI) is a dose-limiting toxicity for cancer patients receiving thoracic radiotherapy. As such, it is important to characterize metabolic associations with the early and late stages of RILI, namely pneumonitis and pulmonary fibrosis. Recently, Raman spectroscopy has shown utility for the differentiation of pneumonitic and fibrotic tissue states in a mouse model; however, the specific metabolite-disease associations remain relatively unexplored from a Raman perspective. This work harnesses Raman spectroscopy and supervised machine learning to investigate metabolic associations with radiation pneumonitis and pulmonary fibrosis in a mouse model. To this end, Raman spectra were collected from lung tissues of irradiated/non-irradiated C3H/HeJ and C57BL/6J mice and labelled as normal, pneumonitis, or fibrosis, based on histological assessment. Spectra were decomposed into metabolic scores via group and basis restricted non-negative matrix factorization, classified with random forest (GBR-NMF-RF), and metabolites predictive of RILI were identified. To provide comparative context, spectra were decomposed and classified via principal component analysis with random forest (PCA-RF), and full spectra were classified with a convolutional neural network (CNN), as well as logistic regression (LR). Through leave-one-mouse-out cross-validation, we observed that GBR-NMF-RF was comparable to other methods by measure of accuracy and log-loss (p > 0.10 by Mann-Whitney U test), and no methodology was dominant across all classification tasks by measure of area under the receiver operating characteristic curve. Moreover, GBR-NMF-RF results were directly interpretable and identified collagen and specific collagen precursors as top fibrosis predictors, while metabolites with immune and inflammatory functions, such as serine and histidine, were top pneumonitis predictors. Further support for GBR-NMF-RF and the identified metabolite associations with RILI was found as CNN interpretation heatmaps revealed spectral regions consistent with these metabolites.

Human antigen R promotes lung fibroblast differentiation to myofibroblasts and increases extracellular matrix production.

Idiopathic pulmonary fibrosis (IPF) is a disease of progressive scarring caused by excessive extracellular matrix (ECM) deposition and activation of α-SMA-expressing myofibroblasts. Human antigen R (HuR) is an RNA binding protein that promotes protein translation. Upon translocation from the nucleus to the cytoplasm, HuR functions to stabilize messenger RNA (mRNA) to increase protein levels. However, the role of HuR in promoting ECM production, myofibroblast differentiation, and lung fibrosis is unknown. Human lung fibroblasts (HLFs) treated with transforming growth factor ß1 (TGF-ß1) showed a significant increase in translocation of HuR from the nucleus to the cytoplasm. TGF-ß-treated HLFs that were transfected with HuR small interfering RNA had a significant reduction in α-SMA protein as well as the ECM proteins COL1A1, COL3A, and FN1. HuR was also bound to mRNA for ACTA2, COL1A1, COL3A1, and FN. HuR knockdown affected the mRNA stability of ACTA2 but not that of the ECM genes COL1A1, COL3A1, or FN. In mouse models of pulmonary fibrosis, there was higher cytoplasmic HuR in lung structural cells compared to control mice. In human IPF lungs, there was also more cytoplasmic HuR. This study is the first to show that HuR in lung fibroblasts controls their differentiation to myofibroblasts and consequent ECM production. Further research on HuR could assist in establishing the basis for the development of new target therapy for fibrotic diseases, such as IPF.

Fine mapping of the major bleomycin-induced pulmonary fibrosis susceptibility locus in mice.

Susceptibility to fibrotic lung disease differs among people and among inbred strains of mice exposed to bleomycin where C57BL/6J mice are susceptible and C3H/HeJ mice are spared fibrotic disease. Genetic mapping studies completed in offspring derived from these inbred strains revealed the inheritance of C57BL/6J alleles at loci, including the major locus on chromosome 17, called Blmpf1 bleomycin-induced pulmonary fibrosis 1, to be linked to pulmonary fibrosis in treated mice. In the present study, to reduce the interval of Blmpf1, we bred and phenotyped a panel of subcongenic mice with C3H/HeJ alleles in a C57BL/6J background. Subcongenic mice received bleomycin via osmotic minipump and the fibrosis phenotype was measured histologically. Inheritance of C3H/HeJ alleles from 34.31 to 35.02 Mb was revealed to spare bleomycin-induced pulmonary fibrosis of C57BL/6J mice. From database analysis, 40 protein coding genes have been mapped to this reduced Blmpf1 interval, 18 of which contain C57BL/6J:C3H/HeJ sequence polymorphisms predicted to affect protein structure or to confer allele-dependent expression, and by RT-PCR analysis of lung tissue, we show 6 of these genes to differ in expression between C57BL/6J and C3H/HeJ mice. Genes known to regulate T cell numbers and activation (Btnl family, Notch4) are among the limited list of potential causal variants leading to lung disease in this model and the bronchoalveolar lavage of protected subcongenic mice had fewer lymphocytes, post bleomycin, than did C57BL/6J mice. We conclude that Blmpf1genes contributing to the susceptibility to bleomycin-induced pulmonary fibrosis could alter the adaptive immune response of C57BL/6J mice.

Positional cloning reveals strain-dependent expression of Trim16 to alter susceptibility to bleomycin-induced pulmonary fibrosis in mice.

Pulmonary fibrosis is a disease of significant morbidity, with no effective therapeutics and an as yet incompletely defined genetic basis. The chemotherapeutic agent bleomycin induces pulmonary fibrosis in susceptible C57BL/6J mice but not in mice of the C3H/HeJ strain, and this differential strain response has been used in prior studies to map bleomycin-induced pulmonary fibrosis susceptibility loci named Blmpf1 and Blmpf2. In this study we isolated the quantitative trait gene underlying Blmpf2 initially by histologically phenotyping the bleomycin-induced lung disease of sublines of congenic mice to reduce the linkage region to 13 genes. Of these genes, Trim16 was identified to have strain-dependent expression in the lung, which we determined was due to sequence variation in the promoter. Over-expression of Trim16 by plasmid injection increased pulmonary fibrosis, and bronchoalveolar lavage levels of both interleukin 12/23-p40 and neutrophils, in bleomycin treated B6.C3H-Blmpf2 subcongenic mice compared to subcongenic mice treated with bleomycin only, which follows the C57BL/6J versus C3H/HeJ strain difference in these traits. In summary we demonstrate that genetic variation in Trim16 leads to its strain-dependent expression, which alters susceptibility to bleomycin-induced pulmonary fibrosis in mice.

Cystic fibrosis mouse model-dependent intestinal structure and gut microbiome.

Mice with a null mutation in the cystic fibrosis transmembrane conductance regulator (Cftr) gene show intestinal structure alterations and bacterial overgrowth. To determine whether these changes are model-dependent and whether the intestinal microbiome is altered in cystic fibrosis (CF) mouse models, we characterized the ileal tissue and intestinal microbiome of mice with the clinically common ΔF508 Cftr mutation (FVB/N Cftr(tm1Eur)) and with Cftr null mutations (BALB/c Cftr(tm1UNC) and C57BL/6 Cftr(tm1UNC)). Intestinal disease in 12-week-old CF mice, relative to wild-type strain controls, was measured histologically. The microbiome was characterized by pyrosequencing of the V4-V6 region of the 16S rRNA gene and intestinal load was measured by RT-PCR of the 16S rRNA gene. The CF-associated increases in ileal crypt to villus axis distention, goblet cell hyperplasia, and muscularis externa thickness were more severe in the BALB/c and C57BL/6 Cftr(tm1UNC) mice than in the FVB/N Cftr(tm1Eur) mice. Intestinal bacterial load was significantly increased in all CF models, compared to levels in controls, and positively correlated with circular muscle thickness in CF, but not wild-type, mice. Microbiome profiling identified Bifidobacterium and groups of Lactobacillus to be of altered abundance in the CF mice but overall bacterial frequencies were not common to the three CF strains and were not correlative of major histological changes. In conclusion, intestinal structure alterations, bacterial overgrowth, and dysbiosis were each more severe in BALB/c and C57BL/6 Cftr(tm1UNC) mice than in the FVB/N Cftr(tm1Eur) mice. The intestinal microbiome differed among the three CF mouse models.

Basal levels of glutathione peroxidase correlate with onset of radiation induced lung disease in inbred mouse strains.

Biomarkers predicting for the radiation-induced lung responses of pneumonitis or fibrosis are largely unknown. Herein we investigated whether markers of oxidative stress and intracellular antioxidants, measured within days of radiation exposure, are correlated with the lung tissue injury response occurring weeks later. Mice of the eight inbred strains differing in their susceptibility to radiation-induced pulmonary fibrosis, and in the duration of asymptomatic survival, received 18 Gy whole thorax irradiation and were killed 6 h, 24 h, or 7 days later. Control mice were not irradiated. Lung levels of antioxidants superoxide dismutase, catalase, glutathione peroxidase (GPx), and glutathione, and of oxidative damage [reactive oxygen species (ROS) and 8-hydroxydeoxyguanosine (8-OHdG)], were biochemically determined. GPx was additionally measured through gene expression and immunohistochemical assessment of lung tissue, and activity in serum. ROS and 8-OHdG were increased postirradiation and exhibited significant strain and time-dependent variability, but were not strongly predictive of radiation-induced lung diseases. Antioxidant measures were not dramatically changed postirradiation and varied significantly among the strains. Basal GPx activity (r = 0.73, P = 0.04) in the lung and the pulmonary expression of GPx2 (r = 0.94, P = 0.0003) correlated with postirradiation asymptomatic survival, whereas serum GPx activity was inversely correlated (r = -0.80, P = 0.01) with fibrosis development. In conclusion, pulmonary oxidative stress and antioxidant markers were more affected by inbred strain than radiation over 7 days posttreatment. Lung GPx activity, and GPx2 expression, predicted for survival from lethal pneumonitis, and serum GPx for fibrosis, in this panel of mice.

Strain-dependent airway hyperresponsiveness and a chromosome 7 locus of elevated lymphocyte numbers in cystic fibrosis transmembrane conductance regulator-deficient mice.

We previously observed the lungs of naive BALB/cJ Cftr(tm1UNC) mice to have greater numbers of lymphocytes, by immunohistochemical staining, than did BALB wild type littermates or C57BL/6J Cftr(tm1UNC) mice. In the present study, we initially investigated whether this mutation in Cftr alters the adaptive immunity phenotype by measuring the lymphocyte populations in the lungs and spleens by FACS and by evaluating CD3-stimulated cytokine secretion, proliferation, and apoptosis responses. Next, we assessed a potential influence of this lymphocyte phenotype on lung function through airway resistance measures. Finally, we mapped the phenotype of pulmonary lymphocyte counts in BALB × C57BL/6J F2 Cftr(tm1UNC) mice and reviewed positional candidate genes. By FACS analysis, both the lungs and spleens of BALB Cftr(tm1UNC) mice had more CD3(+) (both CD4(+) and CD8(+)) cells than did littermates or C57BL/6J Cftr(tm1UNC) mice. Cftr(tm1UNC) and littermate mice of either strain did not differ in anti-CD3-stimulated apoptosis or proliferation levels. Lymphocytes from BALB Cftr(tm1UNC) mice produced more IL-4 and IL-5 and reduced levels of IFN-γ than did littermates, whereas lymphocytes from C57BL/6J Cftr(tm1UNC) mice demonstrated increased Il-17 secretion. BALB Cftr(tm1UNC) mice presented an enhanced airway hyperresponsiveness to methacholine challenge compared with littermates and C57BL/6J Cftr(tm1UNC) mice. A chromosome 7 locus was identified to be linked to lymphocyte numbers, and genetic evaluation of the interval suggests Itgal and Il4ra as candidate genes for this trait. We conclude that the pulmonary phenotype of BALB Cftr(tm1UNC) mice includes airway hyperresponsiveness and increased lymphocyte numbers, with the latter trait being influenced by a chromosome 7 locus.

Association analysis reveals genetic variation altering bleomycin-induced pulmonary fibrosis in mice.

Pulmonary fibrosis is a disease of significant morbidity, with an incompletely defined genetic basis. Here, we combine linkage and association studies to identify genetic variations associated with pulmonary fibrosis in mice. Mice were treated with bleomycin by osmotic minipump, and pulmonary fibrosis was histologically assessed 6 weeks later. Fibrosis was mapped in C57BL6/J (fibrosis-susceptible) × A/J (fibrosis-resistant) F2 mice, and the major identified linkage intervals were evaluated in consomic mice. Genome-wide and linkage-interval genes were assessed for their association with fibrosis, using phenotypic data from 23 inbred strains and the murine single-nucleotide polymorphism map. Susceptibility to pulmonary fibrosis mapped to a locus on chromosome 17, which was verified with consomic mice, and to three additional suggestive loci that may interact with alleles on chromosome 17 to affect the trait in F2 mice. Two of the loci, including the region on chromosome 17, are homologous to previously mapped loci of human idiopathic fibrosis. Of the 23 phenotyped murine strains, four developed significant fibrosis, and the majority presented minimal disease. Genome-wide and linkage region-specific association studies revealed 11 pulmonary expressed genes (including the autophagy gene Cep55, and Masp2, which is a complement component) to contain polymorphisms significantly associated with bleomycin-induced fibrotic lung disease. In conclusion, genomic approaches were used to identify linkage intervals and specific genetic variations associated with pulmonary fibrosis in mice. The common loci and similarities in phenotype suggest these findings to be of relevance to clinical pulmonary fibrosis.

A selenocysteine derivative therapy affects radiation-induced pneumonitis in the mouse.

The mechanism leading to the radiation-induced lung response of pneumonitis is largely unknown. Here we investigated whether treatment with 3,3'-diselenodipropionic acid (DSePA), which reduces radiation-induced oxidative stress in acute response models, decreases the lung response to irradiation. Mice of the C3H/HeJ (alveolitis/pneumonitis-responding) strain received 18 Gy whole-thorax irradiation, and a subset of these mice was treated with DSePA (2 mg/kg) three times per week, beginning at 2 hours after radiation treatment, and continuing in the postirradiation period until death because of respiratory distress symptoms. DSePA treatment increased the postirradiation survival time of mice by an average of 32 days (P = 0.0002). Radiation-treated and DSePA-treated mice presented lower levels of lipid peroxidation and augmented glutathione peroxidase in the lungs, compared with those levels measured in mice receiving radiation only, when mice receiving radiation only were killed because of distress symptoms, whereas catalase and superoxide dismutase levels did not show consistent differences among treatment groups. DSePA treatment decreased pneumonitis and the numbers of mast cells, neutrophils, and lymphocytes in the lungs and bronchoalveolar lavage, respectively, of irradiated mice relative to mice exposed to radiation alone. DSePA treatment also decreased the radiation-induced increase in granulocyte colony-stimulating factor levels in the bronchoalveolar lavage and lung-tissue expression of intercellular adhesion molecule-1 and E-selectin, while increasing the expression of glutathione peroxidase-4. We conclude that DSePA treatment reduces radiation-induced pneumonitis in mice by delaying oxidative damage and the inflammatory cell influx.

The effect of spatial resolution on deep learning classification of lung cancer histopathology.

Objective: The microscopic analysis of biopsied lung nodules represents the gold-standard for definitive diagnosis of lung cancer. Deep learning has achieved pathologist-level classification of non-small cell lung cancer histopathology images at high resolutions (0.5-2 µm/px), and recent studies have revealed tomography-histology relationships at lower spatial resolutions. Thus, we tested whether patterns for histological classification of lung cancer could be detected at spatial resolutions such as those offered by ultra-high-resolution CT. Methods: We investigated the performance of a deep convolutional neural network (inception-v3) to classify lung histopathology images at lower spatial resolutions than that of typical pathology. Models were trained on 2167 histopathology slides from The Cancer Genome Atlas to differentiate between lung cancer tissues (adenocarcinoma (LUAD) and squamous-cell carcinoma (LUSC)), and normal dense tissue. Slides were accessed at 2.5 × magnification (4 µm/px) and reduced resolutions of 8, 16, 32, 64, and 128 µm/px were simulated by applying digital low-pass filters. Results: The classifier achieved area under the curve ≥0.95 for all classes at spatial resolutions of 4-16 µm/px, and area under the curve ≥0.95 for differentiating normal tissue from the two cancer types at 128 µm/px. Conclusions: Features for tissue classification by deep learning exist at spatial resolutions below what is typically viewed by pathologists. Advances in knowledge: We demonstrated that a deep convolutional network could differentiate normal and cancerous lung tissue at spatial resolutions as low as 128 µm/px and LUAD, LUSC, and normal tissue as low as 16 µm/px. Our data, and results of tomography-histology studies, indicate that these patterns should also be detectable within tomographic data at these resolutions.

Radiomic analysis for early differentiation of lung cancer recurrence from fibrosis in patients treated with lung stereotactic ablative radiotherapy.

Objective. The development of radiation-induced fibrosis after stereotactic ablative radiotherapy (SABR) can obscure follow-up images and delay detection of a local recurrence in early-stage lung cancer patients. The objective of this study was to develop a radiomics model for computer-assisted detection of local recurrence and fibrosis for an earlier timepoint (<1 year) after the SABR treatment.Approach. This retrospective clinical study included CT images (n= 107) of 66 patients treated with SABR. A z-score normalization technique was used for radiomic feature standardization across scanner protocols. The training set for the radiomics model consisted of CT images (66 patients; 22 recurrences and 44 fibrosis) obtained at 24 months (median) follow-up. The test set included CT-images of 41 patients acquired at 5-12 months follow-up. Combinations of four widely used machine learning techniques (support vector machines, gradient boosting, random forests (RF), and logistic regression) and feature selection methods (Relief feature scoring, maximum relevance minimum redundancy, mutual information maximization, forward feature selection, and LASSO) were investigated. Pyradiomics was used to extract 106 radiomic features from the CT-images for feature selection and classification.Main results. An RF + LASSO model scored the highest in terms of AUC (0.87) and obtained a sensitivity of 75% and a specificity of 88% in identifying a local recurrence in the test set. In the training set, 86% accuracy was achieved using five-fold cross-validation. Delong's test indicated that AUC achieved by the RF+LASSO is significantly better than 11 other machine learning models presented here. The top three radiomic features: interquartile range (first order), Cluster Prominence (GLCM), and Autocorrelation (GLCM), were revealed as differentiating a recurrence from fibrosis with this model.Significance. The radiomics model selected, out of multiple machine learning and feature selection algorithms, was able to differentiate a recurrence from fibrosis in earlier follow-up CT-images with a high specificity rate and satisfactory sensitivity performance.

Lack of CFTR in skeletal muscle predisposes to muscle wasting and diaphragm muscle pump failure in cystic fibrosis mice.

Cystic fibrosis (CF) patients often have reduced mass and strength of skeletal muscles, including the diaphragm, the primary muscle of respiration. Here we show that lack of the CF transmembrane conductance regulator (CFTR) plays an intrinsic role in skeletal muscle atrophy and dysfunction. In normal murine and human skeletal muscle, CFTR is expressed and co-localized with sarcoplasmic reticulum-associated proteins. CFTR-deficient myotubes exhibit augmented levels of intracellular calcium after KCl-induced depolarization, and exposure to an inflammatory milieu induces excessive NF-kB translocation and cytokine/chemokine gene upregulation. To determine the effects of an inflammatory environment in vivo, sustained pulmonary infection with Pseudomonas aeruginosa was produced, and under these conditions diaphragmatic force-generating capacity is selectively reduced in Cftr(-/-) mice. This is associated with exaggerated pro-inflammatory cytokine expression as well as upregulation of the E3 ubiquitin ligases (MuRF1 and atrogin-1) involved in muscle atrophy. We conclude that an intrinsic alteration of function is linked to the absence of CFTR from skeletal muscle, leading to dysregulated calcium homeostasis, augmented inflammatory/atrophic gene expression signatures, and increased diaphragmatic weakness during pulmonary infection. These findings reveal a previously unrecognized role for CFTR in skeletal muscle function that may have major implications for the pathogenesis of cachexia and respiratory muscle pump failure in CF patients.

Raman microspectroscopy and machine learning for use in identifying radiation-induced lung toxicity.

OBJECTIVE: In this work, we explore and develop a method that uses Raman spectroscopy to measure and differentiate radiation induced toxicity in murine lungs with the goal of setting the foundation for a predictive disease model. METHODS: Analysis of Raman tissue data is achieved through a combination of techniques. We first distinguish between tissue measurements and air pockets in the lung by using group and basis restricted non-negative matrix factorization. We then analyze the tissue spectra using sparse multinomial logistic regression to discriminate between fibrotic gradings. Model validation is achieved by splitting the data into a training set containing 70% of the data and a test set with the remaining 30%; classification accuracy is used as the performance metric. We also explore several other potential classification tasks wherein the response considered is the grade of pneumonitis and fibrosis sickness. RESULTS: A classification accuracy of 91.6% is achieved on the test set of fibrotic gradings, illustrating the ability of Raman measurements to detect differing levels of fibrotic disease among the murine lungs. It is also shown via further modeling that coarser consideration of fibrotic grading via binning (ie. 'Low', 'Medium', 'High') does not degrade performance. Finally, we consider preliminary models for pneumonitis discrimination using the same methodologies.

MicroRNA profiling of cystic fibrosis intestinal disease in mice.

Cystic fibrosis (CF) intestinal disease is characterized by alterations in processes such as proliferation and apoptosis which are known to be regulated in part by microRNAs. Herein, we completed microRNA expression profiling of the intestinal tissue from the cystic fibrosis mouse model of cystic fibrosis transmembrane conductance regulator (Cftr) deficient mice (BALBc/J Cftr(tm1UNC)), relative to that of wildtype littermates, to determine whether changes in microRNA expression level are part of this phenotype. We identified 24 microRNAs to be significantly differentially expressed in tissue from CF mice compared to wildtype, with the higher expression in tissue from CF mice. These data were confirmed with real time PCR measurements. A comparison of the list of genes previously reported to have decreased expression in the BALB×C57BL/6J F2 CF intestine to that of genes putatively targeted by the 24 microRNAs, determined from target prediction software, revealed 155 of the 759 genes of the expression profile (20.4%) to overlap with predicted targets, which is significantly more than the 100 genes expected by chance (p=1×10(-8)). Pathway analysis identified these common genes to function in phosphatase and tensin homolog-, protein kinase A-, phosphoinositide-3 kinase/Akt- and peroxisome proliferator-activated receptor alpha/retinoid X receptor alpha signaling pathways, among others, and through real time PCR experiments genes of these pathways were demonstrated to have lower expression in the BALB CF intestine. We conclude that altered microRNA expression is a feature which putatively influences both metabolic abnormalities and the altered tissue homeostasis component of CF intestinal disease.

IGF binding protein-3 treatment alters intestinal cell proliferation but not body weight of adult cystic fibrosis transmembrane conductance regulator deficient mice.

The intestinal phenotype of cystic fibrosis (CF) transmembrane conductance regulator deficient mice includes altered cell homeostasis and a distended crypt-villus axis, which, in previous work, was inversely proportional to body weight. To investigate this correlation, herein, we treated CF mice with IGF binding protein-3 (IGFBP-3), a protein which, as it has potent effects on cell proliferation and apoptosis, we hypothesized would alter the intestinal cell homeostasis, and assessed body weight. Six-week-old C57BL/6JxBALB F2 CF and WT mice received recombinant human IGFBP-3 (rhIGFBP-3, 20 mg/kg) or vehicle treatment, and weight gain, serum protein levels, and intestinal histology were assessed. Administration of rhIGFBP-3 to CF mice significantly increased the number of Igfbp-3 positive cells in the intestine and partially reversed the hyperproliferative phenotype of intestinal crypts and muscularis externa, while not affecting apoptosis. Serum Igfbp-3 levels were increased, and Igf-I, albumin, and triglycerides measures were decreased in CF compared with WT mice. rhIGFBP-3 treatment significantly increased serum albumin and triglycerides but did not affect weight gain in CF mice. We have identified rhIGFBP-3 treatment to reduce intestinal cell proliferation, resulting in decreases in crypt depth and muscularis externa thickness in CF mice.

Acute Radiation-Induced Hematopoietic Depletion Does Not Alter the Onset or Severity of Pneumonitis in Mice.

Survival from partial-body irradiation (PBI) may be limited by the development of the late lung injury response of pneumonitis. Herein we investigated the hypothesis that acute hematopoietic depletion alters the onset and severity of lung disease in a mouse model. To establish depletion, C3H/HeJ mice received 8 Gy PBI with shielding of only the tibiae, ankles and feet. One week after irradiation, blood lymphocyte and neutrophil counts were each significantly reduced (P < 0.04) in these mice compared to levels in untreated controls or in mice receiving 16 Gy to the whole thorax only. All 8 Gy PBI mice survived to the experimental end point of 16 weeks postirradiation. To determine whether the hematopoietic depletion affects lung disease, groups of mice received 8 Gy PBI plus 8 Gy whole-thorax irradiation (total lung dose of 16 Gy) or 16 Gy whole-thorax irradiation only. The weight loss, survival to onset of respiratory distress (P = 0.17) and pneumonitis score (P = 0.96) of mice that received 8 Gy PBI plus 8 Gy whole-thorax irradiation were not significantly different from those of mice receiving 16 Gy whole-thorax irradiation only. Mice in respiratory distress from PBI plus whole-thorax irradiation had significantly reduced (P = 0.02) blood monocyte counts compared to levels in distressed, whole-thorax irradiated mice, and symptomatic pneumonitis was associated with increased blood neutrophil counts (P = 0.04) relative to measures from irradiated, non-distressed mice. In conclusion, survivable acute hematopoietic depletion by partial-body irradiation did not alter the onset or severity of lethal pneumonitis in the C3H/HeJ mouse model.

Toll-like receptor-4 genotype influences the survival of cystic fibrosis mice.

Toll-like receptor (Tlr) 4 is a lipopolysaccharide (LPS) receptor that contributes to the regulation of intestinal cell homeostasis, a condition that is altered in the intestines of cystic fibrosis mice. Herein, we assessed whether Tlr4 genotype influences cystic fibrosis intestinal disease by producing and phenotyping 12-wk (adult)- and 4-day (neonate)-old mice derived from BALB cystic fibrosis transmembrane conductance regulator, Cftr(+/tm1Unc) and C.C3-Tlr4(Lps-d)/J (Tlr4(-/-)), progenitors. Intestinal disease was assayed through mouse survival, crypt-villus axis (CVA) length, cell proliferation, bacterial load, bacterial classification, inflammatory cell infiltrate, and mucus content measures. Of the 77 Cftr(-/-) (CF) mice produced, only one Cftr/Tlr4 double-mutant mouse lived to the age of 12 wk while the majority of the remainder succumbed at approximately 4 days of age. The survival of CF Tlr4(+/-) mice exceeded that of both CF Tlr4(+/+) and Cftr/Tlr4 double-mutant mice. Adult CF mice presented increased Tlr4 expression, CVA length, crypt cell proliferation, and bacterial load relative to non-CF mice, but no differences were detected in Tlr4(+/-) compared with Tlr4(+/+) CF mice. The double-mutant neonates did not differ from Tlr4(+/+) or Tlr4(+/-) CF mice by intestinal CVA length or bacterial load, but fewer Tlr4(+/-) CF neonates presented with luminal mucus obstruction in the distal ileum, and the intestinal mast cell increase of CF mice was not evident in double-mutant neonates. We conclude that Tlr4 deficiency reduces the survival, but does not alter the intestinal phenotypes, of extended CVA or increased bacterial load in BALB CF mice.

Role of the cystic fibrosis transmembrane conductance channel in human airway smooth muscle.

Patients with cystic fibrosis (CF) suffer from asthma-like symptoms and gastrointestinal cramps, attributed to a mutation in the CF transmembrane conductance regulator (CFTR) gene present in a variety of cells. Pulmonary manifestations of the disease include the production of thickened mucus and symptoms of asthma, such as cough and wheezing. A possible alteration in airway smooth muscle (ASM) cell function of patients with CF has not been investigated. The aim of this study was to determine whether the (CFTR) channel is present and affects function of human ASM cells. Cell cultures were obtained from the main or lobar bronchi of patients with and without CF, and the presence of the CFTR channel detected by immunofluorescence. Cytosolic Ca(2+) was measured using Fura-2 and dual-wavelength microfluorimetry. The results show that CFTR is expressed in airway bronchial tissue and in cultured ASM cells. Peak Ca(2+) release in response to histamine was significantly decreased in CF cells compared with non-CF ASM cells (357 +/- 53 nM versus 558 +/- 20 nM; P < 0.001). The CFTR pharmacological blockers, glibenclamide and N-phenyl anthranilic acid, significantly reduced histamine-induced Ca(2+) release in non-CF cells, and similar results were obtained when CFTR expression was varied using antisense oligonucleotides. In conclusion, these data show that the CFTR channel is present in ASM cells, and that it modulates the release of Ca(2+) in response to contractile agents. In patients with CF, a dysfunctional CFTR channel could contribute to the asthma diathesis and gastrointestinal problems experienced by these patients.

Persistent osteopenia in adult cystic fibrosis transmembrane conductance regulator-deficient mice.

RATIONALE: A loss of function mutation in the cystic fibrosis transmembrane conductance regulator gene is believed to be an independent risk factor for bone disease in patients with cystic fibrosis. OBJECTIVES: The objective of this work was to use congenic mice as a preclinical model to examine the bone phenotype of Cftr(-/-) mice and control littermates at 8, 12, and 28 weeks of age. METHODS: The bone phenotype of control and Cftr(-/-) mice was evaluated by quantitative imaging, histologic and histomorphometric analyses, and serum levels of bone biomarkers. MEASUREMENTS AND MAIN RESULTS: At 12 weeks of age, Cftr(-/-) mice were smaller, had lower bone mineral density, cortical bone thinning, and altered trabecular architecture compared with Cftr(+/+) or Cftr(+/-) control mice. In skeletally mature 28-week-old mice, there were persistent deficits in cortical and trabecular bone structure in Cftr(-/-) mice despite significant, quantifiable improvements. Cftr(-/-) mice also had lower serum insulin-like growth factor-I levels at 12 weeks of age than did control mice, whereas parathyroid hormone and 25-hydroxyvitamin D levels were not significantly different. CONCLUSIONS: Persistent osteopenia and structural abnormalities in adult Cftr(-/-) mice, in the absence of overt respiratory and gastrointestinal disease, suggest that loss of Cftr function has a direct impact on bone metabolism in Cftr(-/-) mice that is not sex specific or subject to haplotype insufficiency.

Radiation-induced lung response of AcB/BcA recombinant congenic mice.

The genetic factors that influence the development of radiotherapy-induced lung disease are largely unknown. Herein we identified a strain difference in lung response to radiation wherein A/J mice developed alveolitis with increased levels of pulmonary mast cells and cells in bronchoalveolar lavage while the phenotype in C57BL/6J mice was fibrosis with fewer inflammatory cells. To identify genomic loci that may influence these phenotypes, we assessed recombinant congenic (RC) mice derived from the A/J and C57BL/6J strains for their propensity to develop alveolitis or fibrosis after 18 Gy whole-thorax irradiation. Mouse survival, lung histopathology and bronchoalveolar lavage cell types were recorded. Informative strains for each of mast cell influx, bronchoalveolar cell numbers, alveolitis and fibrosis were identified. In mice with the A/J strain background, the severity of alveolitis correlated with increased mast cell numbers while in C57BL/6J background strain mice fibrosis was correlated with the percentage of neutrophils in lavage. The data for RC mice support the association of specific inflammatory cells with the development of radiation-induced lung disease and provide informative strains with which to dissect the genetic basis of these complex traits.