Gene therapy for lung diseases: development in the vector biology and novel concepts for gene therapy applications.

The lung represents an attractive target organ for somatic gene therapy strategy in that, (1) it is easily accessible by vectors, (2) most frequent hereditary disorders, cystic fibrosis (CF) and alpha1-antitrypsin deficiency (alpha1AT), occur in the lung, and (3) carcinoma of the lung is apparently a most common cause of death in humans. To date, approximately 400 clinical protocols for human gene therapy have been approved, and approximately 10% of the protocols target lung diseases such as cystic fibrosis (CF) and lung cancer. Currently available data from some of these human trials have successfully demonstrated that gene transfer to the human lung is possible, and that the strategy of overexpressing exogenous genes for curing or controlling lung diseases is potentially promising. In this manuscript, focusing on gene therapy of lung disorders, we aim to give an overview of the hurdles of current gene transfer strategies to overcome, then and also we aim to review recent, remarkable progresses in the vector biology that are potentially promising to maximize safety and efficiency of gene therapy. In addition, based on the most recent advances in the understanding of the molecular biological aspects of the pathogenesis of lung cancer, asthma, pulmonary fibrosis, and acute lung injury, novel therapeutic strategies of gene therapy for inflammatory and malignant diseases of the lung are discussed.

Transfer of heme oxygenase 1 cDNA by a replication-deficient adenovirus enhances interleukin 10 production from alveolar macrophages that attenuates lipopolysaccharide-induced acute lung injury in mice.

By using a direct, intratracheal inoculation of an adenovirus encoding heme oxygenase 1 (Ad.HO-1), model gene therapy for acute lung injury induced by inhaled pathogen was performed. Data demonstrated that Ad.HO-1 administration is as effective as the pharmacologic upregulation of the endogenous HO-1 gene expression by hemin to attenuate neutrophilic inflammations of the lung after aerosolized lipopolysaccharide (LPS) exposure. Interestingly, immunohistochemical analysis revealed that the HO-1 gene was transferred not only to the airway epithelium, but to the alveolar macrophages (AMs). Moreover, overexpression of exogenous HO-1 in the macrophages provided a high level of endogenous interleukin 10 (IL-10) production from the macrophages, and additional experiments using IL-10 knockout mice demonstrated that the increase in IL-10 in the macrophages was critical for the resolution of neutrophilic migration in the lung after LPS exposure. These results suggest that AMs not only are barriers for efficient gene transfer to the respiratory epithelium, but also represent logical targets for Ad-mediated, direct, in vivo gene therapy strategies for inflammatory disorders in humans.

The effects of aging on the function of alveolar macrophages in mice.

In order to determine whether the function of alveolar macrophages (AM) is modulated by aging, we measured the TNF-alpha production, phagocytic function, and surface antigen expression of AM from young and old mice. When AM were primed by IFN-gamma (500 units/ml) and triggered by LPS (100 micrograms/ml), TNF-alpha production by AM was significantly smaller in old mice as compared with young mice (young mice: 161.7 +/- 28.2 units/ml; old mice: 89.3 +/- 13.6 units/ml, P < 0.05). The percentage of AM which phagocytosed latex particles (more than one particle) in old mice was significantly lower than in young mice (young: 78.1 +/- 2.5%; old: 62.8 +/- 3.4%, P < 0.05). Ia antigen expression of the AM was significantly higher and asialo-GM1 antigen expression was significantly lower in old mice than in young mice (Ia: young, 0.030 +/- 0.005; old, 0.092 +/- 0.024, P < 0.05; asialo-GM1: young, 0.-9 +/- 0.01; old, 0.75 +/- 0.07, P < 0.01). These results suggest that alveolar macrophage function is at least decreased in part with aging in mice.

Importance of diffuse aspiration bronchiolitis caused by chronic occult aspiration in the elderly.

Diffuse aspiration bronchiolitis (DAB) is a new term that we proposed to define a clinical entity that is characterized by a chronic inflammation of bronchioles caused by recurrent aspiration of foreign particles. In the present study, a total of 4,880 consecutive autopsies were reviewed and we found 31 patients with DAB (0.64%). To investigate the clinicopathologic features of DAB, the 23 patients with DAB (age, 81.2 +/- 6.2 years [mean +/- SD]), from whom clinical information was available, had their features compared to those of 40 randomly selected patients with aspiration pneumonia (age, 81.9 +/- 8.3 years [mean +/- SD]). Oropharyngeal dysphagia was observed in half of the patients with DAB, and two thirds of patients with DAB were bedridden. The onset of DAB was more insidious than aspiration pneumonia, and in half of the patients with DAB episodes of aspiration were unrecognized. Neurologic disorders (52.2%) and dementia (47.8%) were common associated diseases. Most patients with DAB showed signs of bronchorrhea, bronchospasm, and dyspnea. The macroscopic appearance of the cut surface of DAB lung showed diffusely scattered miliary yellowish nodules that resembled those of diffuse panbronchiolitis (DPB). Histologic findings of DAB were characterized by localization of chronic mural inflammation with foreign body reaction in bronchioles. Recurrence of small amounts of aspiration might play a role in the pathogenesis of DAB. In view of possible therapeutic intervention, we emphasized the importance of recognizing this entity and differentiating DAB from pulmonary diseases associated with bronchospasm in the elderly, in particular, late-onset asthma and DPB.

Effect of cigarette smoking on pulmonary function in each phenotype M of alpha-1-protease inhibitor.

Human alpha-1-protease inhibitor (alpha-1-Pi) has been known to be a highly polymorphic protein. We hypothesized that antiprotease activity of each phenotype M of alpha 1-protease inhibitor (PiM) might be different among smokers and that a variation of decrease in pulmonary function for a given amount of cigarette smoking might be associated with PiM phenotypes. To test this, we investigated the effect of cigarette smoking on pulmonary function in each PiM phenotype. The serum level of alpha 1-Pi was measured by the turbidimetric immunoassay and the distribution of PiM phenotypes was determined using isoelectric focusing technique in 247 healthy subjects and 20 COPD patients. Serum levels of alpha-1-antitrypsin of healthy and COPD subjects were 205.1 +/- 31.1 and 179.2 +/- 44.4 (+/- SD) mg/dL, respectively (p > 0.01). The frequency of each PiM phenotype in healthy subjects was shown as follows: M1, 0.555; M1M2, 0.328; M2, 0.041; M1M3, 0.057; M2M3, 0.016; M3, 0.004. The difference in the distribution of PiM phenotypes between healthy and COPD subjects was not significant. Single- and multiple-regression analyses showed that the ratio of FEV1 to forced vital capacity (FVC), in which FEV1 is expressed as percentage of FVC, the maximum flow rate at 50% of FVC divided by measured body height (V50/Ht), and the maximum flow rate at 25% of FVC divided by body height (V25/Ht) were closely related to age and that V25/Ht also was related to smoking index. However, PiM phenotype was unrelated to those pulmonary function variables. We conclude that PiM phenotype is not a major determinant of difference in magnitude of pulmonary impairments caused by cigarette smoking in each individual.

Quantitative assessment of dyspnea during exercise before and after bullectomy for giant bulla.

Incremental exercise testing using a cycle ergometer was performed in eight patients with giant bulla before and after bullectomy to assess dyspnea. There was a significant positive linear relationship between dyspnea expressed in the Borg scale (BS) and oxygen consumption (VO2) during exercise in all subjects. From these correlations, we introduced the following three new parameters for quantitative assessment of dyspnea: the Borg scale slope (BSS); the threshold load of dyspnea (TLD); and the breakpoint load of dyspnea (BLD), representing the slope of the regression line, onset of dyspnea on the regression line, and the maximum oxygen consumption before the subjects interrupted exercise, respectively. After surgery, the BSS showed marked decrease, and the TLD and BLD showed significant increase. Therefore, the reduction in the dyspnea with peak exercise after surgery was thought to be, at least in part, based on the delay of dyspnea onset, the decrease in dyspnea sensitivity, and the improvement in exercise capacity. The improvement in dyspnea during exercise in patients with giant bulla after surgery was extensively evaluated by newly introduced parameters based on BS-VO2 regression line.

Impaired swallowing reflex in patients with obstructive sleep apnea syndrome.

BACKGROUND: The swallowing reflex is well coordinated with breathing patterns in normal humans. However, patients with obstructive sleep apnea syndrome (OSAS) may have a swallowing disorder that reflects the abnormal function of nerves and muscles in the suprapharynx. OBJECTIVE: To examine the relationship between the swallowing function and sleep-disordered breathing in patients with OSAS. PARTICIPANTS: Twenty patients with OSAS with a mean (+/-SD) age of 53.4+/-8.9 years old, and 20 age-matched control subjects with a mean age of 51.4+/-9.1 years old. METHODS: OSAS was diagnosed using the recordings of overnight polysomnography. The swallowing function in the subject was tested using a swallowing provocation test. The swallowing reflex was determined according to the following criteria: latent time (LT), the time following a bolus injection of distilled water at the suprapharynx to the onset of swallowing; inspiratory suppression time (IST), the time from the termination of swallowing to the next onset of inspiration; and threshold volume, the minimum volume of water (range, 0.4 to 2 mL) that could evoke the swallowing response. RESULTS: Whereas the LT values in patients with OSAS were larger than the LT values in the control subjects, the IST values (which may reflect the switching mechanism from deglutition apnea to breathing) were actually shorter. In addition, a greater bolus volume was necessary to elicit swallowing in patients with OSAS than was necessary in the control subjects. CONCLUSION: Patients with OSAS are likely to exhibit an impaired swallowing reflex, probably due to the perturbed neural and muscular function of the upper airways.

Immunohistochemical and in situ hybridisation detection of adenovirus early region 1A (E1A) gene in the microglia of human brain tissue.

To investigate whether brain tissue is infected latently by adenovirus via a monocyte/microglia-mediated entry mechanism, brain tissue resected at necropsy from seven senile subjects (five with senile dementia of Alzheimer type (SDAT) and two subjects without pathological changes) was examined for adenovirus early region 1A (E1A) gene and its expression using in situ hybridisation and immunohistochemical staining. HLA-DR positive, reactive microglial cells in both SDAT and normal brain tissue showed positive hybridisation and immunoreactive expression of adenovirus E1A. Thus there may be monocyte/microglia-mediated entry of adenovirus in the central nervous system which would be a novel and presumably common interaction between brain tissue and adenovirus.

Immunohistochemical localisation of advanced glycation end products in pulmonary fibrosis.

AIM: To investigate the presence and distribution of advanced glycation end products (AGE) in pulmonary fibrosis. METHODS: Lung tissue samples obtained from seven necropsy cases with idiopathic pulmonary fibrosis and seven with normal pulmonary parenchyma were examined immunohistochemically with a monoclonal antibody specific for AGE: 6D12. We also tested three cases with diffuse alveolar damage. RESULTS: All the specimens from cases with pulmonary fibrosis and diffuse alveolar damage showed strong AGE expression on macrophages. Lung specimens from normal parenchyma showed positive AGE immunoreactivity on macrophages from only two of seven cases. CONCLUSIONS: These findings suggest that AGE modified proteins accumulate in alveolar macrophages in patients with diffuse alveolar damage and idiopathic pulmonary fibrosis.

Adenovirus pulmonary infections identified by PCR and in situ hybridisation in bone marrow transplant recipients.

AIMS: To investigate adenovirus pulmonary infections in bone marrow transplant (BMT) recipients. METHODS: Formalin fixed, paraffin wax embedded lung tissue was examined from 13 necropsy cases after BMT using PCR and in situ hybridisation to detect adenovirus DNA. The E1A region of the adenoviral genome was targeted for PCR. In situ hybridisation was performed only in the PCR positive cases. RESULTS: Of the 13 lung specimens analysed, nine cases were negative for adenoviral nucleic acid. Four (30%) PCR and two (15%) in situ hybridisation positive cases were found. In some of the patients there were clinical and pathological indications that some diseases might be associated with adenovirus infection--haemorrhagic cystitis (three cases); necrotising pneumonia (one case). In necrotising pneumonia in which no pathogenic agents had been shown by conventional histological study, the in situ hybridisation technique showed positive staining for adenovirus. In a patient who died of renal failure caused by adenovirus nephritis, both PCR and in situ hybridisation were positive in the lung as well as in the kidney, although no histological change was found. Two PCR positive cases lacked positive sites for adenovirus by in situ hybridisation. CONCLUSIONS: The combination of PCR and in situ hybridisation could be useful for diagnosing adenovirus infection of the lung in BMT recipients. These results provide a basis for exploring further the clinical use of PCR and in situ hybridisation to diagnose adenovirus infection.

A comparison of ventilation components in young and elderly men during exercise.

To elucidate the influence of age on ventilation components during exercise, we investigated the change in fractional contribution of abdomen or thorax during exercise in 12 elderly (71.9 +/- 5.3, mean +/- SD years) and 12 young (25.0 +/- 4.9 years) normal male subjects using respiratory-inductive plethysmography. At rest, abdominal/thoracic contribution was not different between elderly and young. During exercise, abdominal contribution to total ventilation was decreased in the young compared to that at rest (rest: 53.6 +/- 2.9% vs exercise: 50.4 +/- 1.9-48.9 +/- 1.8%; p < .01), but significantly increased in the elderly (rest: 53.9 +/- 1.8% vs exercise: 57.3 +/- 1.7-59.8 +/- 2.0%; p < .01). Only in the elderly, respiratory frequency was increased during exercise compared to that at rest (rest: 20.1 +/- 0.8 [/min] vs exercise; 25.6 +/- 1.5-27.8 +/- 1.6 [/min]; p < .05). The breathing pattern in the elderly during exercise was partly simulated in the young by reducing thoracic compliance using chest strapping. This study demonstrates the greater participation of diaphragmatic motion together with rapid shallow breathing during lower graded exercise in the elderly as compared with the young. This ventilatory pattern during exercise may result from a stiffening of the thorax with advancing age.

Medullary swallowing-related neurons in the anesthetized cat.

Swallowing-related neurons (SRNs) were recorded systematically in the medulla oblongata of urethane-anesthetized cats. The SRNs received orthodromic inputs from the superior laryngeal nerve (SLN) and showed transient changes in their activity synchronous with swallowing. These neurons could be divided into three types. Type I SRNs are sensory-relay neurons from the SLN in the nucleus of the tractus solitarius (NTS), type II are interneurons located diffusely in the parvocellular reticular formation ventral to the NTS, which received oligosynaptic inputs from the SLN, and type III are motoneurons in the nucleus ambiguus. Some type II neurons still showed the swallowing activity after the animals were paralysed, which suggests that they could be involved in the generation of swallowing outputs.

Mechanical interdependence in relation to age: effects of lung volume on airway resistance in rats.

Elastic recoil has been reported to decrease with increasing age in human subjects. In the current study, we hypothesized that aging influences mechanical interdependence, which affects the magnitude of agonist-induced airway constriction. To examine this hypothesis, we compared the effects of changing lung volume on airway resistance (Raw) under baseline conditions and during methacholine-induced constriction in adult (14 mo old, 624 +/- 14 g; n = 11) and aged (29 mo old, 629 +/- 9 g; n = 11) Sprague-Dawley rats. With use of alveolar capsules, Raw was directly measured under baseline conditions at different levels of end-expiratory transpulmonary pressure (PL; 3-11 cmH2O). Then aerosolized methacholine was delivered (125 mg/ml), and measurements were performed at different levels of PL (3 and 11 cmH2O). From measured tracheal flow and tracheal and alveolar pressures in open-chest animals during mechanical ventilation (6 ml/kg tidal volume, 1 Hz frequency), we calculated dynamic lung elastance and resistance of lung, tissue, and airway. In the baseline conditions, we found that increasing lung volume decreased Raw similarly in both groups but that lung elastance was reduced in the aged group at PL > or = 7 cmH2O. The shape constant obtained from the pressure-volume curve in the aged rats was significantly greater than that in the adult rats. During induced constriction, higher lung volume significantly lowered Raw in the adult group, whereas Raw was not significantly reduced by increasing PL in the aged group. These observations suggest that increasing age may affect the mechanical properties of the airway and/or airway-parenchymal interdependence.

Effects of lung volume on airway resistance during induced constriction in papain-treated rabbits.

It has been reported that both the elasticity of the cartilage and airway-parenchymal interdependence can modify shortening of the airway smooth muscle and airway narrowing during induced constriction. We hypothesized that induced softening of the cartilage could alter airway compliance and/or the forces of mechanical interdependence, resulting in an increased degree of airway narrowing in response to a contractile stimulus. To test this hypothesis, we compared the effects of changing lung volume on airway resistance (Raw) under baseline conditions and during methacholine (MCh)-induced constriction in papain-treated (n = 6) and control rabbits (n = 6). With use of the alveolar capsule technique, Raw was directly measured under baseline conditions at different levels of end-expiratory transpulmonary pressure (Ptp = 4-12 cmH2O). Then aerosolized MCh was delivered (0.2-25 mg/ml) and measurements were performed at different levels of Ptp (4 and 12 cmH2O). From measured tracheal flow and tracheal and alveolar pressure in open-chest animals during mechanical ventilation (tidal volume = 6 ml/kg, breathing frequency = 1 Hz), we calculated Raw by subtracting tissue resistance from lung resistance. Papain treatment significantly increased Raw both under baseline conditions and after induced constriction. We found that increasing Ptp decreased Raw before and after MCh in both groups; however, the effects of changing Ptp on Raw were less in papain-treated animals. These observations suggest that both cartilage elasticity and mechanical interdependence are important determinants of airway smooth muscle shortening. The observation that volume dependence of Raw was less in papain-treated animals is consistent with the hypothesis that papain effects significant changes in the parenchymal attachments.

Increased levels of circulating ICAM-1, VCAM-1, and L-selectin in obstructive sleep apnea syndrome.

Obstructive sleep apnea syndrome (OSAS) may be one of the most important risk factors of cardiovascular disorders, although the exact mechanism remains to be elucidated. In the present study, we hypothesized that OSAS-induced hypoxic stress might be involved in the etiology of cardiovascular disorders by activating adhesion molecules, including intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and L-selectin. To examine this hypothesis, we measured circulating ICAM-1, VCAM-1, and L-selectin levels before and after sleep in OSAS patients and age-matched controls. The circulating ICAM-1, VCAM-1, and L-selectin levels increased in the OSAS patients before sleep compared with the normal subjects (ICAM-1: 392.9 +/- 48.5 vs. 201.2 +/- 55.0 ng/ml, P < 0.05; VCAM-1: 811.0 +/- 87.8 vs. 574.2 +/- 42.7 ng/ml, P < 0.05; L-selectin: 1,386.6 +/- 77.9 vs. 1,038.8 +/- 78.6 ng/ml, P < 0.01, respectively). After sleep, significantly greater levels of ICAM-1 and L-selectin, but not VCAM-1, were observed in the OSAS group. These observations suggest that OSAS-induced hypoxia activates adhesion molecules, resulting in the important risk factor of cardiovascular disorders. Treatment of OSAS can be, therefore, a potential approach to prevention of cardiovascular events.

Effect of endothelin-1 on pulmonary resistance in rats.

We examined the effect of endothelin-1 (ET-1), a novel 21-residue vasoconstrictor peptide, on pulmonary resistance (RL) in Wistar rats. The lung volume, tracheal flow, and transpulmonary pressure of tracheotomized and paralyzed rats were measured with a fluid-filled esophageal catheter and a pressure-sensitive body plethysmograph. RL was calculated by the method of von Neergaard. The femoral artery was cannulated to measure the mean arterial blood pressure. Intravenous bolus administration of synthetic ET-1 provoked a dose-dependent increase in RL in rats. The bronchoconstricting effect reached maximum at 500 pmol/kg. This bronchoconstriction was observed in less than 5 min, increased up to 15 min, and was sustained for 60 min. ET-1 increased the mean arterial blood pressure in a dose-dependent manner. We conclude that ET-1 is a hitherto unknown potent bronchoconstrictor that has a sustained effect in vivo. The potential physiological and pathophysiological role of this new peptide in the development of respiratory disease warrants further investigation.

Variables pertinent to the efficiency of adeno-associated virus (AAV) vectors mediated gene transfer to human vascular endothelial cells.

Factors influencing adeno-associated virus (AAV) - mediated gene transfer to endothelial cells are not fully determined. We tested the variables pertinent to the efficiency of AAV-mediated gene transfer to human vascular endothelial cells (HUVEC) including: (i) kinetics of transduction efficiency of LacZ gene to HUVEC, (ii) the concentration and volume of vector-containing medium, (iii) the period of incubation time of AAV vectors with HUVEC, (iv) the target cell density/proliferation, (v) the duration of transgene expression. There is a dose-response relationship between moi of vectors and transduction efficiency in HUVEC. The higher moi of AAV vectors achieved more than 80% of transduction efficiency in cultured HUVEC. AAV vectors showed incubation time dependent increase in transduction efficiency of LacZ gene to the HUVEC up to 24 h of vector exposure. The foreign gene of AAV vectors preferably transduces the lower density of cells being proliferated. These results indicate that AAV-vector is efficient for gene transfer to HUVEC, and higher moi of vectors or a longer period exposure of vectors to proliferating HUVEC can facilitate efficient tranduction of foreign gene into human vascular endothelial cells in vitro.

Reduction of 15-hydroxyeicosatetraenoic acid (15-HETE) in tracheal fluid by high frequency oscillatory ventilation.

The effects of high frequency oscillatory ventilation (HFOV) and conventional mechanical ventilation (CMV) on tracheal secretion were compared in 6 anesthetized dogs. Using a double-balloon endotracheal catheter, 5 ml of saline was instilled into an isolated tracheal segment during HFOV and CMV for 10 min respectively. Two eicosanoids, 15-hydroxyeicosatetraenoic acid (15-HETE) and 11-dehydrothromboxane B2 (11-dehydro-TXB2) were measured by radioimmunoassay in each sample. HFOV (stroke volume: 6 ml/kg, f: 10 Hz, bias flow: 5 l/min) and CMV (stroke volume: 12 ml/kg, f: 15/min) were performed in random sequence and achieved comparable gas exchange. The concentration of 15-HETE in tracheal fluid during HFOV (87 +/- 67 pg/ml) was decreased to less than half of that during CMV (286 +/- 184 pg/ml, P less than 0.05), while there was no significant change of 11-dehydro-TXB2 either in tracheal fluid or in plasma. This reduction of 15-HETE was tended to be enhanced by vagotomy (HFOV: 42 +/- 14, CMV: 120 +/- 103 pg/ml) with the concentration ratio of CMV/HFOV remaining unchanged. HFOV may provide hitherto unrecognized advantage over CMV by reducing airway secretion of 15-HETE, a potent inflammatory mediator.

Activin receptors are expressed on human lung fibroblast and activin A facilitates fibroblast-mediated collagen gel contraction.

Activin A is a member of the transforming growth factor-beta superfamily that exerts its diverse biological effects through bindings to activin specific transmembrane serine/threonine kinase receptors. The fibroblast-mediated contraction of a collagen gel is thought to be a model of part of the wound-repair response and tissue contraction. In this study, we found the expression of activin type I receptors (ActR-I and ActR-IB) and type II receptor (ActR-II) on human fetal lung fibroblasts (HFL-1) by RT-PCR and immunocytochemistry. We also examined the effects of activin A on the HFL-1-mediated collagen gel contraction. Activin A stimulated collagen gel contraction in a dose dependent manner and its effect was abolished by an activin-binding protein, follistatin, that specifically suppresses activin A activities. This study demonstrated that ActR-I, ActR-1B and ActR-II are expressed on human fetal lung fibroblast and that activin A regulates fibroblast-mediated collagen gel contraction, suggesting that activin A might contribute to human lung fibroblast activities and structural remodeling observed in pulmonary fibrosis.

Kinetics of adenovirus-mediated gene transfer to human lung fibroblasts.

Many features of Ad-mediated gene transfer to human lung fibroblasts are not well understood. We tested kinetics of transduction efficiency of LacZ gene to human lung fibroblasts by E1-deleted adenovriuses containing two different promoters, i.e. CMV and RSV LTR. A dose-dependent relationship between the vector multiplicity of infection (moi) and the efficiency of LacZ gene transfer to fibroblasts was observed with each vector, and higher moi of vectors achieved 100% of transduction efficiency. Further, Ad-mediated gene transfer was enhanced by a long period of vector exposure to fibroblasts up to 6 hours. There were no differences in transduction efficiency between the two Ad vectors. LacZ gene expression by Ad vectors consistently decreased one day after infection. These results indicate that both Ad vectors are equally effective for gene transfer to human lung fibroblasts, and higher moi of vectors and/or a longer period exposure of fibroblasts to vectors can facilitate more efficient transduction of LacZ reporter gene into human lung fibroblasts.