Ruth J. McDonald

Five-fold increase in pediatric parapneumonic empyema since introduction of pneumococcal conjugate vaccine.

A retrospective review of medical records for all pediatric parapneumonic empyema (PPE) patients admitted to our hospital from 1996 to 2006 revealed that PPE increased 5-fold in the post-heptavalent pneumococcal conjugate vaccine (PCV7) period (2001–2005) relative to the pre-PCV7 period (1996–2000), from 13 cases to 65. Most of this increase was associated with culture-negative empyema, which accounted for 61% of all post-2000 cases; 19% was culture-positive pneumococcal empyema. Our analysis indicates that non-PCV7 serotypes became more prevalent at our institution after introduction of the vaccine.

Aerosol Delivery of Lipid:DNA Complexes to Lungs of Rhesus Monkeys

AbstractPurpose. The potential use of aerosol delivery for non-viral gene therapy was tested by nebulization of lipid:DNA complexes to the lungs of rhesus monkeys. Methods. Four female rhesus monkeys were dosed with lipid:DNA formulations via aerosol inhalation, where the DNA coded for the human Cystic Fibrosis Transmembrane Conductance Regulator (hCFTR) protein. Delivery of DNA was determined in lung samples by polymerase chain reaction (PCR) by qualitative and quantitative methods. Transgene specific messenger RNA was measured by reverse transcriptase PCR (RT-PCR) and protein expression and localization were evaluated by immunohistochemistry (IHC). Results. Approximately four mg of DNA, complexed with cationic lipid 1,2-dimyristoyl-sn-glycero-3-ethylphosphatidylcholine (EDMPC) and cholesterol were delivered to the lungs of animals by air jet nebulizer. Three days after dosing, tissue samples from the lung were collected and shown to have vector specific DNA, RNA and the presence of CFTR protein. Specifically, the hCFTR protein was distributed widely, although non-uniformly, throughout airway epithelium being located on the apical surface of epithelial cells. Importantly, no adverse clinical effects were observed and the lungs showed no histological abnormalities or signs of acute inflammation. Conclusions. This study shows that lipid:DNA formulations based on EDMPC and cholesterol can be administered to primates by nebulization resulting in measurable expression of the hCFTR protein. The absence of inflammation is also encouraging and such systems may have utility for delivery of genes to the lungs for the treatment of a variety of pulmonary diseases including cystic fibrosis.

Substance P primes the formation of hydrogen peroxide and nitric oxide in human neutrophils.

Substance P (SP), a neurotransmitter of the central and peripheral nervous system, has been implicated as a mediator of the pulmonary inflammatory response through its stimulatory effects on neutrophils. We investigated the role of SP in priming the production of reactive oxygen species by human neutrophils with the cytochrome c reduction assay and by flow cytometry using the intracellular oxidizable probe dichlorofluorescein. We also investigated SP‐induced formation of nitrite and nitrate as an index of nitric oxide (NO) production. Our results indicate that SP primes two distinct pathways with respect to the induction of reactive oxygen species in the human neutrophil: the production of superoxide anion and hydrogen peroxide by the calmodulin‐dependent NADPH oxidase, and the generation of NO by a constitutive NO synthase. Preincubation of neutrophils with inhibitors of calmodulin and NO synthase diminished the oxidative response in an additive fashion. These results give insight into distinct signal transduction pathways in the SP‐primed neutrophil with respect to the formation of superoxide anion, hydrogen peroxide, and NO. J. Leukoc. Biol. 65: 834–840; 1999.

DNA transfection of macaque and murine respiratory tissue is greatly enhanced by use of a nuclease inhibitor

Nuclease activity present within respiratory tissues contributes to the rapid clearance of injected DNA and therefore may reduce the transfection activity of directly injected transgenes. Most gene transfer technologies transduce or transfect murine tissues more efficiently than corresponding primate tissues. Therefore, it is prudent to assess the utility of novel gene transfer strategies in both rodent and primate models before proceeding with further development.

Neutrophils enhance clearance of necrotic epithelial cells in ozone-induced lung injury in rhesus monkeys

To test the hypothesis that neutrophil influx is important for the removal of necrotic airway epithelial cells, rhesus monkeys were treated with a function-blocking monoclonal antibody (MAb) against CD18 followed by exposure to ozone or filtered air. CD18 MAb-treated, ozone-exposed monkeys showed a significant inhibition of neutrophil emigration and an accumulation of necrotic airway epithelial cells. In a subsequent experiment, monkeys were given CD18 MAb or an isotype control immunoglobulin before ozone or filtered-air exposure. Complement 5a was instilled into lobes of the right lung at the end of the exposure. Lavage neutrophils were significantly elevated in the right lobes compared with those in the contralateral left lobes; consequently, there were significantly fewer necrotic cells in the airways of the right lung, whereas large aggregations of necrotic cells were observed in the contralateral airways of the left lung. These data indicate that neutrophil influx in ozone-induced injury in primates is CD18 dependent and that neutrophils contribute to the repair of airway epithelium by removal of injured epithelial cells.

Hydrogen peroxide induces DNA single strand breaks in respiratory epithelial cells.

The respiratory epithelium is often exposed to oxidant gases, including ozone from photochemical smog and toxic oxygen metabolites released from neutrophils recruited in conditions of airway inflammation. We evaluated DNA single strand break formation by alkaline elution as a measure of oxidant-induced DNA damage to bronchial epithelial cells. Human AdenoSV-40-transformed bronchial epithelial cells (BEAS), subclone R1.4 or nonhuman primate bronchial epithelial cells were cultured in growth factor supplemented Hams F12 medium on polycarbonate filters. DNA was labeled by incubation with [3H]thymidine. Cells were incubated for 1 h in HBSS or HBSS and increasing concentrations of hydrogen peroxide (H2O2). Cells incubated in H2O2 demonstrated dose-dependent increases in strand break formation, and BEAS cells were more sensitive to H2O2-induced injury than primary bronchial epithelial cells. The addition of catalase or preincubation of cells with the iron chelator desferoxamine prevented H2O2-induced strand breakage. DNA strand break formation may be an important mechanism of oxidant injury in respiratory epithelial cells.

Neutrophil adherence to airway epithelium is reduced by antibodies to the leukocyte CD11/CD18 complex

Airway inflammation, including neutrophil influx is commonly seen in human pulmonary diseases. We developed an in vitro system where the adherence of neutrophils to bronchial epithelial cells could be examined. Primary cultures of nonhuman primate brenchial epithelial cells or transformed BEAS human bronchial epithelial cells were grown to confluence on collagen-coated culture plates. Cells were cocultured for 30 min following the addition of human neutrophils and PMA. Cultures were then inverted, fixed with methanol, and adherent neutrophils labeled with 1B4 mouse monoclonal anti-human neutrophil antibody followed by fluoresceinlabeled sheep anti-mouse IgG. Slides were examined using fluorescence microscopy. The 1B4 antibody allowed rapid identification of neutrophils adherent to the epithelial cell monolayers, which were not labeled by this technique. PMA increased the adherence of neutrophils to bronchial epithelial cells. Pretreatment of the neutrophils with anti-CD11/CD18 antibodies prevented the increase in PMA-stimulated adherence. We conclude that PMA-stimulated adherence to airway epithelial cells is in part dependent on the neurrophil CD11/CD18 adherence complex.

Effect of secondhand cigarette smoke, RSV bronchiolitis and parental asthma on urinary cysteinyl LTE4

Cysteinyl leukotrienes promote airway inflammation, bronchoconstriction and mucus hypersecretion. Cigarette smoking and respiratory syncytial virus (RSV) bronchiolitis are known to increase urinary cysteinyl leukotriene E4 (uLTE4), the end product of the cysteinyl leukotriene biosynthetic pathway. We tested the following hypotheses: (1) Secondhand smoke (SHS) exposure increases uLTE4 in well infants and in those hospitalized for RSV bronchiolitis; (2) Length of hospital stay for those with RSV bronchiolitis correlates with uLTE4; and (3) Infants with parent(s) with asthma will have higher uLTE4. Parental asthma for infants hospitalized with RSV bronchiolitis (n = 79) and Well babies (n = 31) was determined by questionnaire. Urine was analyzed for LTE4, cotinine, and creatinine. SHS exposure was determined by cotinine to creatinine ratio. Chi square, or t‐tests were used to determine significant differences between two groups. A three‐way analysis of variance compared the effects of SHS exposure and parental asthma on uLTE4 in Well versus RSV babies. Independent variables predicting length of hospital stay were determined by stepwise multiple regression. High SHS exposure and RSV significantly increased uLTE4. The SHS induced increase in uLTE4 was seen in infants with no parental asthma but not in those with parental asthma. Length of hospital stay positively correlated with uLTE4. We concluded that SHS exposure may increase the severity of bronchiolitis in RSV‐infected infants by enhancing production of cysLTs in infants with no parental asthma. Pediatr Pulmonol. 2008; 43:760–766.

Postnatal episodic ozone results in persistent attenuation of pulmonary and peripheral blood responses to LPS challenge.

Early life is a dynamic period of growth for the lung and immune system. We hypothesized that ambient ozone exposure during postnatal development can affect the innate immune response to other environmental challenges in a persistent fashion. To test this hypothesis, we exposed infant rhesus macaque monkeys to a regimen of 11 ozone cycles between 30 days and 6 mo of age; each cycle consisted of ozone for 5 days (0.5 parts per million at 8 h/day) followed by 9 days of filtered air. Animals were subsequently housed in filtered air conditions and challenged with a single dose of inhaled LPS at 1 yr of age. After completion of the ozone exposure regimen at 6 mo of age, total peripheral blood leukocyte and polymorphonuclear leukocyte (PMN) numbers were reduced, whereas eosinophil counts increased. In lavage, total cell numbers at 6 mo were not affected by ozone, however, there was a significant reduction in lymphocytes and increased eosinophils. Following an additional 6 mo of filtered air housing, only monocytes were increased in blood and lavage in previously exposed animals. In response to LPS challenge, animals with a prior history of ozone showed an attenuated peripheral blood and lavage PMN response compared with controls. In vitro stimulation of peripheral blood mononuclear cells with LPS resulted in reduced secretion of IL-6 and IL-8 protein in association with prior ozone exposure. Collectively, our findings suggest that ozone exposure during infancy can result in a persistent effect on both pulmonary and systemic innate immune responses later in life.

Neutrophils Injure Bronchial Epithelium after Ozone Exposure

Neutrophil (PMN) influx is an early, prominent finding in the airways of humans after experimental inhalation of ozone (O3), however the potential for PMN to contribute to epithelial injury in this setting is unknown. Bronchial epithelial cells of the human BEAS 2B R1.4 cell line or primary human bronchial epithelial cells underwent DNA labeling by incubation with BrdU. Monolayers were exposed to O3 (0.05 to 1 ppm) or filtered air for 60 min., and subsequently incubated with PMN for 2 h. Epithelial cell cytolysis was significant only in BEAS exposed to O3 and co-cultured with PMN. Apoptosis was maximal in BEAS exposed to O3 + PMN. Primary bronchial epithelial cells were resistant to injury; no cytolysis was detected, and apoptosis was detected only after treatment with 10 mM H2O2. Neutrophils may increase damage to the respiratory epithelium after O3 exposure, but primary bronchial epithelial cells are resistant to PMN and ozone induced injury.