Crizotinib inhibition of ROS1-positive tumours in advanced non-small-cell lung cancer: a Canadian perspective.

The ros1 kinase is an oncogenic driver in non-small-cell lung cancer (nsclc). Fusion events involving the ROS1 gene are found in 1%-2% of nsclc patients and lead to deregulation of a tyrosine kinase-mediated multi-use intracellular signalling pathway, which then promotes the growth, proliferation, and progression of tumour cells. ROS1 fusion is a distinct molecular subtype of nsclc, found independently of other recognized driver mutations, and it is predominantly identified in younger patients (<50 years of age), women, never-smokers, and patients with adenocarcinoma histology. Targeted inhibition of the aberrant ros1 kinase with crizotinib is associated with increased progression-free survival (pfs) and improved quality-of-life measures. As the sole approved treatment for ROS1-rearranged nsclc, crizotinib has been demonstrated, through a variety of clinical trials and retrospective analyses, to be a safe, effective, well-tolerated, and appropriate treatment for patients having the ROS1 rearrangement. Canadian physicians endorse current guidelines which recommend that all patients with nonsquamous advanced nsclc, regardless of clinical characteristics, be tested for ROS1 rearrangement. Future integration of multigene testing panels into the standard of care could allow for efficient and cost-effective comprehensive testing of all patients with advanced nsclc. If a ROS1 rearrangement is found, treatment with crizotinib, preferably in the first-line setting, constitutes the standard of care, with other treatment options being investigated, as appropriate, should resistance to crizotinib develop.

Improving molecular testing and personalized medicine in non-small-cell lung cancer in Ontario.

BACKGROUND: Although molecular testing has become standard in managing advanced nonsquamous non-small-cell lung cancer (nsclc), most patients undergo minimally invasive procedures, and the diagnostic tumour specimens available for testing are usually limited. A knowledge translation initiative to educate diagnostic specialists about sampling techniques and laboratory processes was undertaken to improve the uptake and application of molecular testing in advanced lung cancer. METHODS: A multidisciplinary panel of physician experts including pathologists, respirologists, interventional thoracic radiologists, thoracic surgeons, medical oncologists, and radiation oncologists developed a specialty-specific education program, adapting international clinical guidelines to the local Ontario context. Expert recommendations from the program are reported here. RESULTS: Panel experts agreed that specialists procuring samples for lung cancer diagnosis should choose biopsy techniques that maximize tumour cellularity, and that conservation strategies to maximize tissue for molecular testing should be used in tissue processing. The timeliness of molecular reporting can be improved by pathologist-initiated reflex testing upon confirmation of nonsquamous nsclc and by prompt transportation of specimens to designated molecular diagnostic centres. To coordinate timely molecular testing and optimal treatment, collaboration and communication between all clinicians involved in diagnosing patients with advanced lung cancer are mandatory. CONCLUSIONS: Knowledge transfer to diagnostic lung cancer specialists could potentially improve molecular testing and treatment for advanced lung cancer patients.

Solitary fibrous tumour of pleura: CT differentiation of benign and malignant types.

AIM: To analyse and compare the computed tomography (CT) features of benign and malignant types of histopathologically proven cases of solitary fibrous tumours of pleura (SFTP). MATERIALS AND METHODS: Retrospective analysis of preoperative CT images of 28 cases of histopathologically proven and classified SFTP from three participating institutions was performed. Patient demographics and lesion characteristics including size, borders, presence of a pedicle, extension into the fissure, attenuation, enhancement, pleural effusion, and calcifications were recorded and correlated with the final histopathological diagnosis. Type and results of preoperative biopsy were also recorded. Follow-up imaging and the clinical charts were reviewed to identify recurrence. RESULTS: Out of 28 cases (15 women and 13 men), 18 were proven to be benign and 10 were malignant. The mean age of patients was 58.1±15.9 and 66.5±11.8 years (p=0.1564) for benign and malignant tumours, respectively. The median (interquartile range) diameter was 6.05 (3.2-10.9) cm for benign and 15.7 (7.1-17.5) cm for malignant type tumours (p=0.0291). Tumours had lobulate borders in 28% (5/18) of benign cases and in 80% (8/10) of malignant cases (p=0.0163). Extension into adjacent fissure was seen in 22% (4/18) of benign lesions and 40% (4/10) of malignant lesions (p=0.40). A pedicle was present in 17% (3/18) of benign and 10% (1/10) of malignant lesions (p=1). Heterogeneous attenuation was present in 61% (11/18) of benign and 90% (9/10) of malignant lesions (p=0.19). Calcification was present in 17% (3/18) of benign tumours and in 70% (7/10) of malignant tumours (p=0.0113). Pleural effusion was present in 6% (1/18) of benign and 40% (4/10) of malignant lesions (p=0.04). Only 1/13 preoperative fine-needle aspirates yielded diagnosis of SFTP. Preoperative diagnosis of SFTP was made in all cases (11/11) with core biopsies. At follow-up (1-10 years, mean 3 years), local recurrence occurred in 3/6 (50%) patients with malignant SFTP and in none of the 10 patients with benign SFTP. CONCLUSION: No definite imaging feature to differentiate benign from malignant SFTP was found. Large size, lobulate borders, presence of calcification, and ipsilateral pleural effusion were the only CT features predictive of malignancy. In suspected cases, core biopsies should be performed rather than fine-needle aspiration.

Prenatal nicotine exposure increases connective tissue expression in foetal monkey pulmonary vessels.

Among the many deleterious effects of maternal smoking during pregnancy on foetal development, is a higher incidence of persistent pulmonary hypertension. The recent identification of nicotinic acetylcholine receptors (nAChR) on cells of the pulmonary vessel walls suggests that maternal smoking during pregnancy may produce morphological alterations in foetal pulmonary vasculature. Timed-pregnant rhesus monkeys were treated with nicotine (1 mg x kg(-1) x day(-1)) delivered by subcutaneous osmotic mini-pumps from days 26-134 of gestation (term: 165 days). Lung sections from 134-day foetal monkeys were used for morphometric analysis, in situ hybridisation and immunohistochemical staining. Following nicotine treatment, total wall and tunica adventitia thickness of airway associated vessels (AAV) increased significantly. Nicotine exposure significantly increased collagen I and III mRNA and protein in tunica adventitia in all AAV but not in tunica media. By contrast, levels of elastin protein were significantly decreased. alpha7 nAChR were detected in AAV fibroblasts that expressed collagen mRNA. Choline acetyltransferase, the enzyme which synthesises acetylcholine, the ligand for alpha7 nAChR was also detected in endothelium and fibroblasts. These findings suggest that with smoking during pregnancy, nicotine is transported across the placenta and directly interacts with nicotinic acetylcholine receptors in pulmonary vessels to alter connective tissue expression and therefore produce vascular structural alterations.

Late relapse of myelodysplasia after allogeneic transplantation concomitant with new presentation of invasive liposarcoma as a secondary neoplasm.

Second malignancies are uncommon events in the survivors of allogeneic transplant procedures, although they are increased compared to normal control populations. Among these malignancies, sarcomas are exceedingly rare. In addition, relapse of primary myelodysplasia rarely occurs after 5 years from the time of allogeneic transplantation. This report describes an unusual presentation of liposarcoma with concomitant relapse of underlying myelodysplasia developing in a patient 9 years after the first of two allogeneic transplantations.

Synthesis of acetylcholine by lung cancer.

The role of autocrine growth factors in the stimulation of lung cancer growth is well established. Nicotine is an agonist for acetylcholine receptors and stimulates lung cancer growth. This suggests that if lung cancers synthesize acetylcholine (ACh), then ACh may be an autocrine growth factor for lung cancer. Analysis of normal lung demonstrated that the cells of origin of lung cancers express the proteins necessary for non-neuronal ACh storage and synthesis. Analysis of mRNA from squamous cell lung carcinoma, small cell lung carcinoma (SCLC) and adenocarcinoma showed synthesis of choline acetyltransferase (ChAT) and nicotinic receptors. Immunohistochemical analysis of a retrospective series of SCLC and adenocarcinomas showed that more than 50% of the lung cancers screened expressed ChAT and nicotinic receptors. To study the effect of endogenous ACh synthesis on growth, SCLC cell lines were studied. SCLC cell lines were found to express ChAT mRNA and to secrete ACh into the medium as measured by HPLC separation and enzymatically-coupled electrochemical detection. The SCLC cell line NCI-H82 synthesized highest levels of ACh. Showing that the endogenously synthesized ACh interacted with its receptors to stimulate cell growth, addition of muscarinic and nicotinic antagonists slowed H82 cell proliferation. These findings demonstrate that lung cancer cell lines synthesize and secrete ACh to act as an autocrine growth factor. The existence of a cholinergic autocrine loop in lung cancer provides a basis for understanding the effects of nicotine in cigarette smoke on lung cancer growth and provides a new pathway to investigate for potential therapeutic approaches to lung cancer.

Chronic maternal nicotine exposure alters neuronal systems in the arcuate nucleus that regulate feeding behavior in the newborn rhesus macaque.

It is well known that maternal smoking during pregnancy can lead to low birth weight and low body fat in human newborns. The purpose of this study was to determine whether chronic maternal nicotine treatment alters levels of known regulators of energy balance in the newborn offspring. Pregnant rhesus monkeys were treated with nicotine tartrate (1.5 mg/kg x d) starting on d 26 of pregnancy and maintained through d 160 of gestation. Nicotine exposure had no significant effect on absolute birth weights of the neonatal monkeys, although there was a 10% reduction in birth weights with nicotine exposure when they were normalized to maternal weight. Postnatal d 1 plasma leptin levels were significantly reduced by about 50% in the nicotine treatment group compared with saline controls, suggesting that the infant monkeys exposed to nicotine may also have lower body fat levels. In situ hybridization studies demonstrated that chronic nicotine exposure resulted in a significant decrease in arcuate NPY mRNA expression in the neonatal monkeys. In addition, there was a 2-fold increase in POMC mRNA in the arcuate nucleus in the nicotine-exposed group. These data suggest that nicotine exposure during pregnancy may increase energy expenditure in the developing fetus through actions on hypothalamic systems, resulting in lower birth weights and body fat levels.

Prenatal nicotine exposure alters pulmonary function in newborn rhesus monkeys.

Epidemiological studies have shown that offspring of women who smoke during pregnancy have abnormal lung function and associated higher incidences of lower respiratory disorders. The recent identification of nicotinic acetylcholine receptors (nAChR) in fetal lung suggests that the direct interaction between nicotine and nAChR in fetal lung may underlie the postnatal pulmonary abnormalities seen in such infants. This hypothesis was tested in monkeys to determine if maternal nicotine exposure would produce changes in lung mechanics in newborn monkeys similar to those observed in human infants whose mothers smoked during pregnancy. Timed pregnant rhesus monkeys were infused with either nicotine (1.5 mg/kg/d, n = 7) or saline (n = 7) using subcutaneous osmotic pumps from Day 26 to 160 of gestation. On Day 160 of pregnancy (term = 165 d), fetuses were delivered by C-section, and the following day were subjected to pulmonary function testing. After testing, animals were sacrificed, and lungs weighed and fixed. Lung weight and fixed lung volume decreased (16% and 14%, respectively) significantly following in utero nicotine exposure. Peak tidal expiratory flow, FEV(0.2), mean mid-expiratory flow, forced expiratory volume at peak expiratory flow (FEV(PEF)), and FEV(PEF)/FVC% were significantly lower in newborns exposed to nicotine during gestation. Absolute and specific pulmonary resistance increased significantly whereas absolute and specific dynamic compliance remained unchanged in prenatally nicotine-treated pups. These changes in pulmonary function are strikingly similar to the changes observed in offspring of human smokers. This suggests that the interaction of nicotine with nAChR in developing lung is responsible for the altered pulmonary mechanics observed in human infants whose mothers smoked during pregnancy.

Prenatal nicotine increases pulmonary alpha7 nicotinic receptor expression and alters fetal lung development in monkeys.

It is well established that maternal smoking during pregnancy is a leading preventable cause of low birth weight and prematurity. Less appreciated is that maternal smoking during pregnancy is also associated with alterations in pulmonary function at birth and greater incidence of respiratory illnesses after birth. To determine if this is the direct result of nicotine interacting with nicotinic cholinergic receptors (nAChRs) during lung development, rhesus monkeys were treated with 1 mg/kg/day of nicotine from days 26 to 134 of pregnancy. Nicotine administration caused lung hypoplasia and reduced surface complexity of developing alveoli. Immunohistochemistry and in situ alpha-bungarotoxin (alphaBGT) binding showed that alpha7 nAChRs are present in the developing lung in airway epithelial cells, cells surrounding large airways and blood vessels, alveolar type II cells, free alveolar macrophages, and pulmonary neuroendocrine cells (PNEC). As detected both by immunohistochemistry and by alphaBGT binding, nicotine administration markedly increased alpha7 receptor subunit expression and binding in the fetal lung. Correlating with areas of increased alpha7 expression, collagen expression surrounding large airways and vessels was significantly increased. Nicotine also significantly increased numbers of type II cells and neuroendocrine cells in neuroepithelial bodies. These findings demonstrate that nicotine can alter fetal monkey lung development by crossing the placenta to interact directly with nicotinic receptors on non-neuronal cells in the developing lung, and that similar effects likely occur in human infants whose mothers smoke during pregnancy.

Modification of development by the CFTR gene in utero.

The in utero infection of rats at 16-17 days gestation with a recombinant adenovirus carrying the human cystic fibrosis transmembrane conductance regulator (cftr) gene resulted in altered lung development and morphology. These structural alterations prompted an evaluation of concurrent functional changes in the cftr-treated lung. CFTR protein could be detected in treated lungs for up to 30 days postinfection, although it was not detected in the intestines at this time. Increased levels of secreted glycoconjugates and lipids were found in lungs treated in utero with human cftr and large vacuoles containing glycoconjugates were detected within cells of the intestines. The scope and durability of these changes suggested that in utero cftr treatment influenced the activity of secretory cells in the developing lung. Altered secretory products in the lungs of cystic fibrosis patients are thought to be associated with increased susceptibility to Pseudomonas aeruginosa infection. We challenged 3-month-old rats (treated in utero with the human cftr gene) with a lethal, intratrachial dose of this bacteria. Rats treated with cftr exhibited enhanced resistance to Pseudomonas infection when compared to controls. These animals displayed little or no associated inflammatory response. No evidence of the adenovirus transgene was detectable at the time of P. aeruginosa inoculation, indicating that continuous ectopic expression of hcftr was not required for enhanced protection. These data demonstrate that in utero, cftr expression influenced the development and function of cells involved in the primary host defense against bacterial infection in the lung.

Expression of the insulin-like growth factor system in postpneumonectomy lung growth.

The insulin-like growth factors (IGF-I and IGF-II) may play an important role in postpneumonectomy compensatory lung growth by translating hormonal inputs and mechanical forces into cellular proliferation signals. We examined the mRNA abundance of IGF-I, IGF-II, and IGF binding proteins (IGFBPs) in lungs of rats on postoperative days 1, 2, 3, 5, and 7 following left pneumonectomy (PNX) or shamoperation (SC) and in normal animals (CON). There was no difference in the abundance of lung IGF-I mRNA (measured by Northern analysis) or serum IGF-I (measured by radioimmunoassay (RIA)) between SC and PNX animals. IGF-II mRNA abundance was initially decreased following PNX (73% decrease compared to SC animals on day 1, p < .05) and then rose to approach SC group values on subsequent days. Transcripts for IGFBP-2, -3, -4, -5, and -6 were decreased in both the SC and PNX groups compared to CON animals on the day following pneumonectomy, then rose back to baseline by postoperative day 2-3. Tissue IGFBPs, measured by ligand blot analyses, were not different in either the SC or PNX groups. In contrast, all serum IGFBP bands were increased on postoperative day 1 following either sham or PNX surgery. In addition, serum IGFBP-4 was increased in PNX animals compared to the SC group on days 1 and 2 (increase of 38% and 78%, respectively, p < .05). We conclude that the changes observed in lung IGF and IGFBP expression following pneumonectomy do not represent major.

Comparative biochemistry of gestational and postnatal lung growth and development in the rat and human.

We compared the ontogeny of collagen (hydroxyproline), elastin (desmosine), soluble protein, and DNA in the lungs of rate and humans during gestation and postnatal life. In humans, lung weight/body weight ratios declined faster during gestation than postnatally, whereas in rats lung weight/body weight ratio declined little during gestation and then suddenly on the first day of life. Lung weight/body weight ratios may be lower than expected around term in humans, and prediction data are given to assess human pulmonary hypoplasia. Rats and humans differed in water content of their lungs, with rats showing a sharper decline during gestation. In the human lung, collagen and elastin made their appearance at an early stage of gestation; elastin. In particular, increased rapidly during gestation, suggesting a role in intrauterine alveolar formation. In the rat, elastin accumulation is primarily a postnatal event, as is alveolar formation. Hydroxyproline concentrations increased with conceptual age and continued to increase rapidly postnatally between 4 and 7 weeks in the rat, but slowed in the human after 60 weeks of conceptual age. Desmosine concentrations level off at the end of the study period in rats, while these are still increasing, although slowly, in humans. Overall lung growth, as assessed by weight, was linear in humans, but phases of lung growth were apparent in the rat, including one of minimal growth in the immediate postnatal period.

Effect of triamcinolone acetonide on the development of the pulmonary airways in the fetal rat.

Triamcinolone acetonide (TAC) has a potent teratogenic effect on various mammalian fetal tissues as well as a steroid effect on the lung. Less well documented is the fact that it produces profound oligohydramnios. We wished to determine what effect TAC would have on branching morphogenesis and other aspects of lung development, using an in vivo model described previously. Thirty rats were randomized to receive 0.6 mg/kg of TAC or saline on days 12, 13, and 14 of gestation. At gestational days 15, 17, 18, and 21, the left lungs of 365 fetuses were studied by dissecting microscopy, histology, and morphometry. TAC produced profound pulmonary hypoplasia (dry Jung weight/body weight 0.025, compared with 0.06 in controls) on day 21. TAC decreased maternal weight gain, fetal weight, placental weight, aminiotic fluid, and pole to pole length (PTP), while it increased the peripheral airway count (PAC). The number of central and intermediate airway branches was reduced, and they were dilated. Growth of peripheral airways was enhanced. In treated fetuses epithelial cells lining these airspaces were histologically more mature and the mesenchyme thinner than in controls. These findings were confirmed by the morphometric measurements. We conclude that when TAC is administered in the early phase of fetal rat lung development, the lungs become hypoplastic, with hypoplasia of the intermediate airways, an increase in the number of peripheral airways, and increased differentiation. We speculate that these effects are primarily due to the steroid action of TAC and that the mechanisms of monopodial branching are different from those of dichotomous branching.

Two different patterns of airway branching regulated by different components of the extracellular matrix in vitro.

This study examined the effects of beta-D-xyloside (an inhibitor of proteoglycan synthesis) and cis-4-hydroxyl-L-proline (an inhibitor of collagen synthesis) on branching morphogenesis in cultures of fetal rat lung. Lungs from day 15 gestation were incubated for 4 days in (1) the control medium (Dulbecco's Modified Eagle Medium + 10% Fetal Bovine Serum) alone (control), (2) control medium plus 2 mM beta-D-xyloside (beta-XYL), (3) control medium plus 2 mM alpha-D-xyloside (alpha-XYL), (4) control medium plus 50 micrograms/mL cis-4-hydroxy-L-proline (cis-HYP). The number of peripheral buds of left lungs was counted daily. Histological examination was performed on lungs on days 2 and 4. beta-XYL inhibited proximal monopodial branching on day 2 without affecting lung size, but produced numerous peripheral buds on day 4 which were of abnormal appearance, suggesting that lung airway branching and growth may be regulated by different mechanisms. Histology and morphometry showed significantly enlarged airspaces and diminished mesenchyme. cis-HYP did not affect proximal branching on 2 days in culture, but inhibited further dichotomous branching and lung growth after 2 days. On day 4, diminished branching and lung growth was accompanied by a proportional decrease in mesenchyme. The difference between effects of beta-XYL and cis-HYP on the branching pattern suggests that proteoglycans and collagen are involved in different patterns of branching morphogenesis.

Lung morphometric changes after exposure to hypobaria and/or hypoxia and undernutrition.

Lung morphometry was studied in rats between 4 and 7 weeks of age. The animals were divided into 5 groups: general controls (fed ad libitum), hypobaric normoxic, normobaric hypoxic, hypobaric hypoxic, and weight-matched controls (weight matched to the hypobaric hypoxic group). In both hypobaric and normobaric hypoxia, lung volume, alveolar surface area and total alveolar number increased compared to weight-matched controls. In normobaric hypoxia, mean linear intercept, mean chord length of alveoli increased and number of alveoli/unit volume decreased compared to weight-matched animals. In hypobaric hypoxia, only mean chord length increased. Dysanaptic index decreased in both. In hypobaric normoxia, alveolar size and lung volume diminished compared to general controls. Lung growth was impaired in weight-matched controls without affecting airspace dimensions. Hypobaric and normobaric hypoxia increase lung growth overcoming nutritional effects but is dysanaptic. Lung growth in hypobaric hypoxia is mainly determined by low oxygen but low pressure may also produce subtle structural alterations.

Time course of lung growth following exposure to hypobaria and/or hypoxia in rats.

Four-week-old rats were divided into five groups: general controls, weight-matched controls (weight matched to hypobaric hypoxia), hypobaric hypoxia, normobaric hypoxia, and hypobaric normoxia. Lung growth impairment in weight-matched animals occurred by reduction in cell number and size. In both hypoxic groups, lung weight, RNA and protein were significantly higher on day 3, and DNA on day 5 and remained higher thereafter. Maximum 3H-TdR incorporation occurred on day 3 in both hypoxic groups. Hypoxia increased RNA/DNA ratio on day 1 and protein/DNA on day 3. Following 3 days of recovery, DNA synthesis and RNA/DNA ratio of hypoxic groups and controls were identical. DNA synthesis also doubled on day 5 in hypobaric normoxia compared to general controls. Hypoxia up regulates lung growth despite down regulation by undernutrition. Maximum lung growth stimulation occurs during early exposure by cellular hypertrophy followed by hyperplasia. Low pressure by itself also stimulates lung growth. Cellular activity returns immediately to normal levels after removal of hypoxic stimulus.

Development of the pulmonary airways in the fetal rat and its relation to the prenatal environment.

We studied the left lung using multi-focus microphotography in 378 rat fetuses, assessing airway branching from day 13 to day 19 of gestation, and lung growth variables from day 13 to day 21. Longitudinal growth, and monopodial and dichotomous branching brought about a consistent airway pattern with variations within each day of gestation and a small overlap between adjacent days. Amniotic fluid weight and pole to pole (PTP) distance of the lung increased quadratically with age, while fetal weight and the peripheral airway count (PAC) increased exponentially. The location of the fetus within the uterus had no effect on fetal variables, but correlations were found between maternal weight gain and both fetal weight and PTP. Fetal weight was the best predictor of PAC from gestational ages 15 to 19 days (P < 0.008). The method described allows for observations that are reproducible within the environmental variations present in normal gestation and can be used to study the effect of external factors on lung development.

Cytodynamics of in vitro developing airways and interaction with extracellular matrix proteins.

We studied epithelial and mesenchymal cell kinetics during lung branching and the possible importance of proteoglycan and collagen in rat fetal organ culture for 5 days, starting from 15 days gestation. The lungs were randomly divided into three groups: (1) basal medium (controls); (2) basal medium + beta-D-xyloside (beta-XYL); and (3) basal medium+cis-4-hydroxy-L-proline (cis-HYP). The labeling index of incorporation of 5-bromo-2-deoxyuridine (BrdU) was used as an indicator of DNA synthesis and assessed in the parent airways and in the tips and sides of peripheral buds. In controls, BrdU incorporation was highest in bud tips, followed by bud sides and then parent airway for both epithelium and mesenchyme. These data suggest that preferential cell proliferation occurs in normal airway branching. In parent airways, labeling of epithelium and mesenchyme was consistently diminished throughout the culture period in beta-XYL, whereas in cis-HYP this was not the case. In the peripheral buds, the DNA synthetic activity in both epithelium and mesenchyme was impaired in beta-XYL and cis-HYP, but the effect was more marked in beta-XYL. We conclude that although collagen may be required in peripheral bud morphogenesis, the proteoglycans appear to be essential in growth and development of both parent airways and peripheral buds in the lung.

In utero gene transfer into the pulmonary epithelium.

In early gestation the internal surface of the lung is structurally simple and an ideal target for somatic gene transfer. The transfer of genes into the growing lung would be particularly useful in the prenatal correction of cystic fibrosis, which has devastating pulmonary complications. In addition, in utero gene therapy has the potential to immunotolerize the individual, and thereby to avoid the immune reactions now seen with the current generation of adenoviral vectors. We injected a replication-defective adenoviral vector containing the lacZ reporter gene (Ad5.CMVlacZ) into the amniotic fluid of rat pups on the 16th day of gestation. At 16 days of gestation, rat lungs are equivalent in maturity to those of a 22-week human fetus as their airways are lined with undifferentiated multipotential stem cells. The pups showed high-level reporter gene expression in their airways a week following birth (13 days following infection). The expression was maintained during a time when the lung volume increased approximately 20-fold, alveolarization occurred, and the epithelial cells differentiated. These data establish gene targeting of undifferentiated fetal cells as an effective means of gene therapy.

Lung cytokinetics after exposure to hypobaria and/or hypoxia and undernutrition in growing rats.

Lung cellular dynamics were examined in growing rats from 4 to 7 wk of age after exposure to 1) room air (general controls), 2) hypobaric normoxia, 3) normobaric hypoxia, 4) hypobaric hypoxia, and 5) room air and restricted food intake (weight-matched controls). Tritiated thymidine ([3H]TdR) incorporation diminished in weight-matched controls. In both hypoxic groups, maximum [3H]TdR incorporation occurred on day 3 in all cells of peripheral alveoli, capillary endothelium of central alveoli, airway walls and epithelium, and arterial wall and endothelium, but maximum [3H]TdR labeling of interstitial and unidentifiable cells of central alveoli occurred on day 5. The percent labeling with [3H]TdR was higher in cells of peripheral alveolar walls than in cells of central alveolar walls. Labeling of the interstitium was higher in hypobaric hypoxic than normobaric hypoxic rats. In hypobaric normoxia, DNA synthetic activity increased in alveolar wall cells, except for capillary endothelium. In hypobaric hypoxia, DNA synthesis occurred primarily because of low O2, but low pressure may also affect cytokinetics. [3H]TdR incorporation is greater and earlier in the alveoli of the peripheral part of the lung than central alveoli, and the cellular response of the various cells types is not synchronous.