Effect of adenoviral vector infection on cell proliferation in cultured primary human airway epithelial cells.

Although recombinant adenoviruses are used as vectors for delivering therapeutic genes to the airways of cystic fibrosis (CF) patients, the effects of these vectors on the kinetics of airway epithelial cell growth have not been investigated. We tested whether E1, E3-deleted Ad vectors (Ad5-CMV-lacZ) affect the kinetics of cell proliferation of human airway epithelial cells in primary culture. There was a dose-dependent relationship between the vector multiplicity of infection (moi) and the efficiency of Ad-mediated lacZ gene transfer. Growth curves of cells exposed to vector were shifted to the right as compared to vehicle in a dose-dependent manner. The vector-induced slowing of cell proliferation resulted from both (i) increased apoptotic cell death and (ii) lower recruitment into S phase. UV inactivation of the vector genes abolished the effects on cell proliferation. These data demonstrate that as the moi of vectors is increased to achieve effective gene transfer, apoptosis and slowing of the cell cycle of infected cells increases concomitantly. The identification and inactivation of these vector effects on human airway cells may be important for reducing the toxicity of adenovirus vectors for gene therapy of CF airways.

Mycoplasma pneumoniae as the causative agent for pneumonia in the immunocompromised host.

A young man undergoing chemotherapy for Ewing's sarcoma presented with fever, neutropenia, anemia, thrombocytopenia, and a new infiltrate on the chest roentgenogram. Routine cultures and cytopathologic examination of bronchoalveolar lavage fluid provided no evidence for an etiology; however, special cultures of the BAL fluid demonstrated heavy growth of Mycoplasma pneumoniae. We recommend that evaluation of pneumonia in the immuno-compromised host include appropriate cultures of BAL fluid for M pneumoniae, particularly when the patient is 5 to 25 years old, the age of high incidence of mycoplasmal pneumonia.

Preventive services received by adolescents with cystic fibrosis and sickle cell disease.

OBJECTIVE: To determine the proportion of adolescents with cystic fibrosis (CF) or sickle cell disease (SCD) who reported speaking with their physicians about health-promoting and risky behaviors and whether the rate of discussions varied by whether the main physician was a primary care provider or specialist. HYPOTHESIS: Adolescents reporting a primary care provider as their main physician would be more likely to have received risk behavior counseling and other preventive services. DESIGN: Survey. SETTING: Comprehensive CF and SCD centers in 5 North Carolina referral hospitals. PARTICIPANTS: Three hundred twenty-one (74%) of 437 adolescents aged 12 through 19 years (mean age, 15.6 years; 51% male) with CF or SCD identified through center registries. MAIN OUTCOME MEASURES: Sources of health care, main physician, and recall of discussions with physicians regarding sexual issues, substance use, weight or dieting, safety issues, depression, and violence. RESULTS: Adolescents with CF (53%) or SCD (46%) most commonly reported a specialist as their main physician. For those (83%) who saw their main physician in the past year, adolescents with SCD reported counseling rates ranging from 43% for sexuality to 15% for weapon carrying or fighting. For adolescents with CF, rates ranged from 65% for weight and dieting to 30% for sexuality and 6% for weapon carrying or fighting. Adolescents whose main physician was a primary care provider were no more or less likely to report counseling for any topic (all P>.05). CONCLUSIONS: Physicians, regardless of specialty, infrequently discussed common behavioral issues with these adolescents with CF or SCD. A coordinated effort between primary care physicians and specialists may be helpful in delivering optimal preventive services to this population.

Development of mucociliary transport in the postnatal ferret trachea.

Little is known of the developmental aspects of mucociliary transport. Previous studies have documented that newborn ferret trachea has very few ciliated cells but numerous immature secretory cells in the epithelium and only rudimentary submucosal glands. Rapid and complete maturation occurs in the first postnatal month. This study examines mucociliary transport during this period of rapid maturation. We made direct observations of particle movement across the epithelium of ferret tracheas. No mucus transport could be demonstrated on the first day of life. Transport was discernible, although sporadic and slow, by 7 days and reached adult levels (10.7 +/- 3.7 mm/min) by 28 postnatal days. The emergence of transport capability correlated well with previously described developmental changes in ciliation, mucus secretion, and ion permeability and transport. Threshold mucus transport occurred at 1 wk of age when 20-25% of the surface cells are ciliated. The neonatal ferret appears to be a useful model for assessing integrated epithelial structure-function relationships that are important not only during early development but also during repair after airway injury involving deciliation.

Comparison of physical activity in adolescents with cystic fibrosis versus age-matched controls.

The aims of this study were to compare self-reported vigorous physical activity and participation in sports among adolescents with cystic fibrosis (CF) to those of age matched peers from the general population, and to determine which CF patient characteristics are associated with regular physical activity. One hundred and sixteen of 141 (82%) adolescents aged 12-19 years identified through North Carolina CF Care Center registries confidentially completed a self-administered questionnaire (the CDC's Youth Risk Behavior Survey) addressing health-compromising and health-enhancing behaviors, including physical activity. They were age- and gender-matched to adolescents from North Carolina schools who completed the same survey. Adolescents with CF did not differ significantly from their matched peers with regard to participating three or more times a week in activities that "make you sweat or breathe hard" (63% vs. 67%, P = 0.37), physical education class (59% vs. 61%, P = 0.81), or team sports (52% vs. 61%, P = 0.10). For all categories of activity, participation declined among adolescents aged 17 years or older. After adjusting for gender and health status, early (14 years or younger) and middle (15-16 years) adolescents were more likely to participate in all three types of activity than older adolescents with CF. Like their peers in the general population, adolescents aged 17 and older are much less likely to report regular physical activity, or participation in physical education class or in team sports. Healthcare providers should be aware of this decline in activity in late adolescence and should consider efforts to maintain physical activity among older teenagers and young adults.

Mediastinal lymphadenopathy caused by Mycobacterium avium-intracellulare complex in a child with normal immunity: successful treatment with anti-mycobacterial drugs and laser bronchoscopy.

We report on the case of a 9-month-old Caucasian girl referred to our institution with a history of fever of unknown origin and wheezing, unresponsive to bronchodilator and anti-inflammatory therapy. Subsequent investigation led to a diagnosis of mediastinal lymphadenopathy caused by Mycobacterium avium-intracellulare (MAI). The infected lymph tissue infiltrated and obstructed the right bronchus and significantly compressed the left bronchus to the point of near closure. Given the high degree of morbidity and potential mortality from thoracic surgery in this patient, we treated her with a combination of anti-mycobacterial drugs (rifabutin, clarithromycin, ciprofloxacin, clofazimine, amikacin, ethambutol) and glucocorticoids to relieve airway compression. The endobronchial granulation tissue was resected by laser bronchoscopy. This combined approach led to eventual normalization of radiologic and endoscopic findings, and the anti-mycobacterial chemotherapy was discontinued 12 months after the first bronchoalveolar lavage culture was negative for MAI. The patient remains asymptomatic 1 year after completion of this course of therapy. We suggest that mediastinal lymphadenopathy with bronchial infiltration and extrinsic airway compression caused by MAI in otherwise healthy children can be successfully treated with aggressive chemotherapy, glucocorticoids, and laser bronchoscopy.

Chlamydial and mycoplasmal pneumonias.

Chlamydia species and Mycoplasma pneumoniae are among the most common agents of community-acquired pneumonia, as well as causes of various nonpneumonic syndromes. Both can be considered "exotic" bacteria: Chlamydiae because they depend on host cell energy, hence their obligate intracellular replication; and M pneumoniae because it is an extracellular parasite that lacks the standard protective bacterial cell wall. The unusual biology of these organisms complicates laboratory diagnosis, but because both are susceptible to selective antimicrobials, therapy often proceeds empirically on clinical suspicion. Generally the respiratory diseases produced are self-limited without significant complications or known sequelae.

Developmental changes of ferret tracheal mucin composition and biosynthesis.

We characterized the chemical composition of mucins secreted by ferret tracheal explants and the activities of key mucin glycosyltransferases in ferret tracheal epithelium during a period of rapid postnatal maturation of the mucin-secreting structures. Ferret tracheal explants secrete three major groups of high molecular weight glycoconjugates: (1) those susceptible to bovine testicular hyaluronidase; (2) those resistant to hyaluronidase and exhibiting high density (p greater than or equal to 1.60 g/mL); and (3) those resistant to hyaluronidase and exhibiting low density (1.45 less than or equal to p less than 1.60 g/mL). The hyaluronidase-resistant, low-density glycoconjugates have typical mucin properties and constitute 36% of total glycoconjugates released in newborns but only 8% in adult ferrets. Mucin secretory rate per unit surface area of trachea progressively decreases with age. Mucin amino acid and total carbohydrate contents do not vary; however, the sialic acid content increases, and fucose content as well as blood group A activity of the mucins decreases with age. Four glycosyltransferases involved in mucin biosynthesis [Gal beta 3GalNAc:(GlcNAc-GalNAc)beta 6 N-acetylglucosaminyl-, GalNAc:beta 3 galactosyl-, Gal:alpha 2 fucosyl-, and GalNAc alpha 2----6 neuraminyltransferase] are present in tracheal epithelium of ferrets at all ages. Activities of all but the neuraminyltransferase decrease with age. The relatively greater neuraminyltransferase activity is consistent with increased incorporation of sialic acid into secreted mucins over the same age span. Conversely, diminution of fucosyltransferase relative to galactosyltransferase activity may contribute to the lower fucose content and lower blood group A activity of mucins secreted by mature ferret tracheas.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathogenesis of respiratory infections due to influenza virus: implications for developing countries.

The influenza viruses have an important and distinctive place among respiratory viruses: they change antigenic character at irregular intervals, infect individuals of all ages, cause illnesses characterized by constitutional symptoms and tracheobronchitis, produce yearly epidemics associated frequently with excess morbidity and mortality, and predispose the host to bacterial superinfections. Much is known about influenza viruses, but their role in respiratory infections among children in developing countries is poorly understood, and the risk factors that lead to the excess morbidity and mortality have not been identified clearly. Among the many risk factors that may be important are alterations in host immunity, malnutrition, prior or coincident infections with other microorganisms, inhaled pollutants, and lack of access to medical care. There is a great need for research that can establish more precisely the role these and other unidentified factors play in the pathogenesis of influenza infections in children in the developing world.

Developmental changes in glycoconjugate secretion by ferret tracheas.

Surface epithelium and submucosal glands of the ferret trachea undergo extensive postnatal development. This study examined developmental changes in rates of release and types of high molecular weight glycoconjugates secreted by explanted ferret tracheas. Digestion with bovine testicular hyaluronidase separated the high molecular weight glycoconjugates into two types, hyaluronidase-resistant mucins and hyaluronidase-susceptible glycosaminoglycans. Release rates were measured under unstimulated conditions and in the presence of known secretagogues. The unstimulated rate of release of total 3H-glycoconjugates was 4-fold higher at birth than after complete maturation. The mucin content varied from 39 to 74% of total 3H-glycoconjugates; however, no age-related pattern was observed for mucin/glycosaminoglycan ratios. The rate of release of 3H-mucins was 6-fold higher at birth than in the adult but rapidly dropped to adult levels by 28 days of age. The secretory cells in the tracheal epithelium of newborn ferrets had more abundant rough endoplasmic reticulum than did mature goblet cells, suggesting increased synthesis of secretory product. Response to methacholine and trypsin, both known stimulators of mucin release, was not observed until 28 and 54 days of age, respectively. Incorporation of 35S-sulfate into mucins relative to that for 3H-glucosamine increased with age, consistent with increasingly acidic histochemical staining properties of secretory cells. These developmental differences in rates of release, modulation of release, and relative sulfation of mucins may represent changes in secretory and synthetic mechanisms of the secretory cells.

Attachment of influenza A virus to ferret tracheal epithelium at different maturational stages.

Influenza virus attaches primarily to ciliated cells in mature airways epithelium. This process is mediated by a viral envelope glycoprotein (hemagglutinin) that binds to sialic acid-containing receptors in the apical membrane of host cells. The purpose of this study was to determine the cellular distribution of these receptors as a function of tracheal epithelial maturation in the ferret, which is susceptible to influenza virus infection at all ages and undergoes postnatal ciliation. To assay for virus attachment, tracheal strips from ferrets at ages 0, 7, 14, and 28 d were incubated at 4 degrees C for 1 h with a concentrated suspension of influenza A virus. Transmission electron microscopy demonstrated virus attachment to the apical surface of 77 to 87% of ciliated cells, but only to 1 to 9% of nonciliated surface epithelial cells at all ages, including the newborn, which has few ciliated cells (less than 10% of total cells). Virions also attached to most of the preciliated cells identified. Pretreatment of tracheal strips with neuraminidase virtually eliminated viral attachment. These findings demonstrate preferential influenza virus binding to sialylated receptors on ciliated cells and their immediate precursors. The sparsity of ciliated cells with no evidence for increased influenza virus binding per cell in newborn ferret tracheas suggests that the previously demonstrated high risk of death from influenza infection in newborn ferrets is due to factors other than increased susceptibility to virus attachment. Influenza virus receptors appear to be selective membrane markers for ciliated cells and may be particularly useful for the identification of preciliated cells.

Receptor specificity of influenza virus influences severity of illness in ferrets.

Weanling ferrets were inoculated intranasally with either wild-type or receptor-variant clones of influenza A/Memphis/102/72 to determine if changes in receptor specificity influence virulence of influenza virus infection. Over the 5 days after inoculation, receptor-variant inoculated ferrets had a lower mean elevation in body temperature, greater weight gain and less sneezing than the wild-type group. Influenza virus was recovered from the lungs of fewer receptor-variant infected ferrets (5/12 vs 11/12) and in lower titers than in wild-type infected ferrets at 5 days after inoculation. The viruses recovered from lung homogenates retained the same receptor specificity as the inoculum. Serum hemagglutination inhibition titers for the two groups were similar. These findings suggest that the receptor-variant clone is less virulent but elicits a similar immunogenic response compared with the wild-type clone.

Cell proliferation in bronchial epithelium and submucosal glands of cystic fibrosis patients.

Integrative gene therapy typically requires dividing cells. This requirement has been perceived as an impediment for gene transfer to mature, uninjured airways where proliferation rates are very low. In diseases such as cystic fibrosis (CF) that may be candidates for integrative gene therapy, airway cell turnover is not known but may be increased as a result of chronic inflammation. To determine if cells in airway surface epithelium and submucosal glands of CF patients proliferate at an increased rate, paraffin sections of bronchial segments removed from CF patients (n = 6) at the time of lung transplantation or rapid autopsy and from non-CF patients (n = 4) undergoing lung resection or transplantation were immunostained with PC10, a monoclonal antibody to proliferating cell nuclear antigen (PCNA), a marker of proliferating cells. The PCNA index (percentage of nuclei immunostaining for PCNA) in CF bronchial surface epithelium was 17.0 +/- 4.6% (mean +/- SEM), substantially greater than in non-CF airways (less than 0.2%). Within submucosal glands, PCNA-positive cells were more prevalent in the collecting ducts of CF patients than in those of normal subjects, but only rare mucous or serous cells were PCNA positive. These studies show that airway epithelial cell proliferation rates can be very high in inflamed CF airways. This prevalence of proliferating cells suggests that CF airway epithelium and submucosal gland ducts may be amenable to gene transfer using vectors, such as retroviruses, that require cell replication for stable integrative expression. Further studies are needed to evaluate cell proliferation in CF airways with less extensive airway injury.

Quantitation of inflammatory responses to bacteria in young cystic fibrosis and control patients.

Recent studies suggest that inflammation plays a role in the pathogenesis of lung disease in cystic fibrosis (CF). The goal of the present study was to quantitatively compare bronchoalveolar lavage fluid (BALF) inflammation and its relation to bacterial infection, between children with CF and children with other chronic respiratory problems. Differential cell counts, immunoreactive interleukin 8 (IL-8), and quantitative bacterial cultures were done in BALF from 54 CF (median age 1.8 yr) and 55 control patients (median age 1.0 yr) who underwent bronchoscopy for clinical indications. Among infected CF patients, those with Pseudomonas aeruginosa did not have more inflammation than those without P. aeruginosa. The ratio of neutrophils or of IL-8 to bacteria in BALF was significantly greater for CF patients compared with control subjects, regardless of pathogen. Calculation of linear regression for either neutrophils or IL-8, as a function of bacterial quantity, yielded positive slopes for both CF and control patients, but with significant elevations for CF. We conclude that the inflammatory response to bacterial infection is increased or prolonged in CF compared with control patients, and that this increase is not necessarily due to pathogens specific for CF (e.g., P. aeruginosa). These data may provide further rationale for anti-inflammatory therapy early in CF.

Respiratory responses of vigorously exercising children to 0.12 ppm ozone exposure.

Changes in respiratory function have been suggested for children exposed to less than 0.12 ppm ozone (O3) while engaged in normal activities. Because the results of these studies have been confounded by other variables, such as temperature or the presence of other pollutants or have been questioned as to the adequacy of exposure measurements, we determined the acute response of children exposed to 0.12 ppm O3 in a controlled chamber environment. Twenty-three white males 8 to 11 yr of age were exposed once to clean air and once to 0.12 ppm O3 in random order. Exposures were for 2.5 h and included 2 h of intermittent heavy exercise. Measures of forced expiratory volume in one second (FEV1) and the symptom cough were determined prior to and after each exposure. A significant decline in FEV1 was found after the O3 exposure compared to the air exposure, and it appeared to persist for 16 to 20 h. No significant increase in cough was found due to O3 exposure. Forced vital capacity, specific airways resistance, respiratory frequency, tidal volume, and other symptoms were measured in a secondary exploratory analysis of this study.

Postnatal development of tracheal surface epithelium and submucosal glands in the ferret.

We explored the usefulness of the postnatal ferret as a model for early developmental events in the large airways, using light and scanning electron microscopy. In the first 28 postnatal days, ferret tracheal surface epithelium and glands undergo dramatic growth and development. Tracheal surface area increases 8-fold. At birth, ciliated cells are sparse (9.4 +/- 1.2% of total epithelial cells). A significant increase in ciliated cells is observed at weekly intervals and by day 28 the ciliated cell is the predominant cell type (54.2 +/- 2.8% of total epithelial cells). Secretory cells decrease from 66.4 +/- 1.0% at birth to 22.2 +/- 2.8% of total epithelial cells. Histochemical staining of the granules of the epithelial secretory cells changes from predominantly non-acidic (staining with PAS but not Alcian blue) to predominantly acidic (staining also with Alcian blue). During the same time interval, tracheal glands develop from intraepithelial cellular aggregates devoid of secretory granules at birth into complex, submucosal tubuloacinar structures composed predominantly of cells containing non-acidic secretory granules at 28 days. Therefore, infant ferrets offer an opportunity to examine the structural and functional components of the mucociliary clearance mechanism at developmental stages which occur prenatally in many laboratory animals and in humans.

Nasal and bronchoalveolar lavage fluid cytokines in early cystic fibrosis.

Cytokine levels in nasal and lower airways in young cystic fibrosis (CF) patients were compared with those in controls. Nasal (NLF) and bronchoalveolar (BALF) lavage fluids were obtained from children with or without CF who were undergoing bronchoscopy for clinical indications. In NLF, neither inflammatory cells nor cytokine concentrations differed between patients and controls. However, interleukin (IL)-8 levels in infected BALF from children with CF were markedly elevated compared with levels in infected and uninfected controls, even after standardization of IL-8 concentrations to bacterial counts. BALF IL-6 was modestly elevated in infected CF patients compared with uninfected but not infected controls; IL-10 did not differ among the groups. NLF and BALF IL-8 levels were not significantly correlated. Excessive airway inflammation in early CF thus appears to be confined to the lower respiratory tract, and IL-8 levels are markedly increased in children with CF compared with control children with a bacterial infection of the lower airways.

Cytokine production by cultured human bronchial epithelial cells infected with a replication-deficient adenoviral gene transfer vector or wild-type adenovirus type 5.

Exposure of animals to adenoviral gene transfer vectors has been associated with respiratory tract inflammation. The pathogenesis of this inflammation is unclear. One hypothesis is that viral vectors directly induce production of inflammatory cytokines by host cells in the airways. We exposed cultured human lung cells to an adenovirus-5--based vector containing the cytomegalovirus promoter and lacZ reporter gene (Ad.CMV.lacZ) and to wild-type adenovirus 5 (wtAd5) and measured subsequent release of cytokines into cell culture supernatants. Inoculation of human bronchial epithelial (HBE) cells with Ad.CMV. lacZ at 10(1) to 10(4) plaque-forming units (pfu)/cell resulted in dose-related expression of lacZ by both X-gal staining and immunohistochemistry but did not increase release of interleukin (IL)-8 or IL-6 at 24, 48, or 96 h after inoculation. In the same cultures, tumor necrosis factor-alpha induced marked increases in release of both IL-8 and IL-6 at 24 and 48 h after stimulation. Similar data were observed in the BEAS-2B HBE cell line. HBE cells incubated with wtAd5 at doses of 10(1) to 10(3) pfu/cell did not release increased amounts of IL-6 or IL-8 up to 48 h after inoculation, though wild-type respiratory syncytial virus (3 pfu/HBE cell) infection resulted in increases in both cytokines. Human alveolar macrophages obtained by bronchoalveolar lavage also showed no increases in cytokine release after incubation with Ad.CMV.lacZ, though relatively little gene transfer occurred in macrophages. These data do not support a role for direct induction of airway epithelial or alveolar macrophage inflammatory cytokines in the pathogenesis of inflammation associated with exposure of airways to adenovirus or to adenoviral gene transfer vectors.