Paula C. Murphy

Eosinophil influx to the nasal airway after local, low-level LPS challenge in humans

BACKGROUND Recent observations show that atopic asthmatic subjects have increased sensitivity to respirable endotoxin (or LPS) compared with normal persons. In vitro studies demonstrate that LPS enhances eosinophil survival. These observations suggest that the effects of inhaled LPS in asthmatic subjects may include increases in the number of airway eosinophils. OBJECTIVE We sought to determine whether low-level nasal LPS challenge causes an increase in eosinophil numbers in the nasal airways of atopic or normal subjects. METHODS Sixteen volunteers (10 atopic asthmatic subjects and 6 normal subjects) underwent 2 nasal challenge sessions. In one session, one nostril was challenged with saline and the other with 0. 1 microg of LPS. During the second session, 0.3 microg and 1.0 microg of LPS was delivered to each nostril, respectively. Nasal lavage fluid was obtained from each nostril before challenge, as well as 4 and 24 hours after challenge, and examined for the percent of total cells that were eosinophils and neutrophils, as well as cytokine levels. RESULTS LPS (1.0 microg) increased the percent of eosinophils in nasal lavage fluid 4 hours after challenge in atopic subjects only. There was also a correlation between constitutive nasal GM-CSF and eosinophil response to LPS in atopic subjects. CONCLUSION LPS challenge increases eosinophils in the airways of atopic subjects.

The effect of respiratory synctial virus on chemokine release by differentiated airway epithelium

Respiratory synctial virus (RSV) infection of undifferentiated airway epithelial cells has been shown to induce the production of chemokines. The purpose of this study was to investigate the vectorial release of interleukin (IL-8) and Released on Activation, Normal T-cell Expressed and Secreted (RANTES) by polarized, well-differentiated respiratory epithelial cells after RSV infection. Human bronchial epithelial cultures were differentiated under air-liquid interface conditions and infected with RSV by the apical or basolateral route. RSV infection was specific to the apical surface. Supernatants were collected at 6 and 48 hours after RSV inoculation, and IL-8 and RANTES were measured by enzyme-linked immunosorbent assay (ELISA). Both IL-8 and RANTES were significantly released by 48 hours following inoculation with RSV. The secretion of each chemokine was greatest after apical inoculation, and secretion was polarized to the basolateral supernatant. Immunohistochemical staining confirmed that RSV infection was specific to ciliated cells, and immunohistochemical staining for chemokines was localized to RSV-infected ciliated cells. The authors conclude that, in differentiated human airway epithelium in vitro, RSV-induced increases in IL-8 and RANTES release are predominantly in the basolateral direction. In epithelial layers, virus-containing cells are the predominant source of the increased chemokine release. The authors speculate that similar processes in vivo influence recruitment of leukocytes to sites of RSV infection.

Immunoprecipitation and Virus Neutralization Assays Demonstrate Qualitative Differences between Protective Antibody Responses to Inactivated Hepatitis A Vaccine and Passive Immunization with Immune Globulin

Antibodies to hepatitis A virus (anti-HAV) were measured in children from two separate vaccine trials (n = 70) 4 weeks after a dose of inactivated hepatitis A vaccine (VAQTA). The geometric mean titers (GMTs) of anti-HAV were 49.3 and 45.2 mIU/mL by immunoassay, while reciprocal GMTs of neutralizing anti-HAV were 6.5 and 15.0 by an 80% radioimmunofocus inhibition test (RIFIT) and 55.6 and 92.0 by antigen reduction assay (HAVARNA). The GMT of antibody detected by radioimmunoprecipitation (RIPA) was > or =401. These data establish serologic correlates of protection against disease and show that RIPA is most sensitive for detection of early vaccine-induced antibody. Sera collected from adults (n = 20) 7 days after administration of immune globulin contained similar antibody levels by immunoassay (45.1 mIU/mL) and slightly higher GMTs of neutralizing antibody (27.5 by RIFIT and 146 by HAVARNA) but negligible precipitating antibody (GMT, 5.6). These results are best explained by differences in the affinity of antibodies for virus following active versus passive immunization.

Immunogenicity and antigenicity of chimeric picornaviruses which express hepatitis A virus (HAV) peptide sequences: evidence for a neutralization domain near the amino terminus of VP1 of HAV.

We evaluated the antigenic characteristics of chimeric picornaviruses created by inserting peptide sequences from hepatitis A virus (HAV) capsid proteins into the B-C loop of VP1 of Sabin strain type 1 poliovirus (PV-1). Fifteen viable chimeras were generated. Each retained the ability to be neutralized by polyclonal PV-1 antisera. Two chimeras (H15 and H2) stimulated production of low levels of HAV neutralizing antibodies in immunized rabbits or mice, although in both cases only a small fraction of immunized animals produced this response. The H15 chimera, which contains residues 13-24 of HAV VP1, elicited HAV neutralizing antibodies in three of nine rabbits and at least one of seven immunized mice. These results indicate that a neutralization domain exists in this region of VP1. However, human sera with high titers of antibodies to HAV failed to neutralize or immunoprecipitate this chimera, suggesting the absence of a significant antibody response to this neutralization domain following natural infection. Sera from rabbits immunized with H15 that did not develop HAV neutralizing antibodies contained antibodies reactive with the HAV peptide segment expressed by the H15 virus, indicating substantial differences in the specificities of antibodies elicited by this peptide segment among individual immunized rabbits. The H15 peptide insert was an effective antigen, as indicated by a high level of sensitivity of the H15 chimera to neutralization by a related anti-peptide antibody which was itself devoid of HAV neutralizing activity. One of 16 rabbits immunized with the H2 chimera (residues 101-108 of HAV VP1) developed HAV neutralizing antibodies, confirming both the presence and the highly conformational nature of a neutralization antigenic site involving these residues of HAV.

Release of Cytokines by Human Nasal Epithelial Cells and Peripheral Blood Mononuclear Cells Infected with Mycoplasma pneumoniae

Mycoplasma pneumoniae (Mp) infection is associated with asthma exacerbation in children. We hypothesized that Mp infection may cause airway inflammation by inducing the release of cytokines by respiratory epithelial cells. The levels of chemokines interleukin-8 (IL-8) and released upon activation, normal t cell expressed and secreted (RANTES) released by nasal epithelial cell (NEC) cultures established from asthmatic and nonasthmatic children were measured by ELISA at 4, 24, 48, and 72 hr after cells were inoculated with Mp, and were compared with baseline release of these factors. The presence of MP on apical membranes of NEC after infection was confirmed by transmission electron microscopy, and adherence was shown to be inhibited by erythromycin. Mp infection did not alter NEC release of IL-8 or RANTES at any time point. In contrast, tumor necrosis factor α (TNF-α) stimulated increased IL-8 at all time points, and respiratory syncytial virus (RSV) infection stimulated RANTES release at 48 and 72 hr by NEC. These results were not significantly different between NEC from asthmatic and nonasthmatic children. As a comparison, peripheral blood mononuclear cells from normal human volunteers were also incubated with Mp and had significantly increased release of IL-2, IL-6, and TNF-α. We conclude that Mp, unlike viral pathogens such as RSV, is unlikely to directly stimulate early airway surface cytokine responses via mechanisms involving epithelial cells. We speculate that the chronic presence of mononuclear cells at the airway surface of asthmatics provides a target for Mp-triggered cytokine production.

Effect of Broccoli Sprouts on Nasal Response to Live Attenuated Influenza Virus in Smokers: A Randomized, Double-Blind Study

Background Smokers have increased susceptibility and altered innate host defense responses to influenza virus infection. Broccoli sprouts are a source of the Nrf2 activating agentsulforaphane, and short term ingestion of broccoli sprout homogenates (BSH) has been shown to reduce nasal inflammatory responses to oxidant pollutants. Objectives Assess the effects of BSH on nasal cytokines, virus replication, and Nrf2-dependent enzyme expression in smokers and nonsmokers. Methods We conducted a randomized, double-blind, placebo-controlled trial comparing the effects of BSH on serially sampled nasal lavage fluid (NLF) cytokines, viral sequence quantity, and Nrf2-dependent enzyme expression in NLF cells and biopsied epithelium. Healthy young adult smokers and nonsmokers ingested BSH or placebo (alfalfa sprout homogenate) for 4 days, designated Days -1, 0, 1, 2. On Day 0 they received standard vaccine dose of live attenuated influenza virus (LAIV) intranasally. Nasal lavage fluids and nasal biopsies were collected serially to assess response to LAIV. Results In area under curve analyses, post-LAIV IL-6 responses (P = 0.03) and influenza sequences (P = 0.01) were significantly reduced in NLF from BSH-treated smokers, whileNAD(P)H: quinoneoxidoreductasein NLF cells was significantly increased. In nonsmokers, a similar trend for reduction in virus quantity with BSH did not reach statistical significance. Conclusions In smokers, short term ingestion of broccoli sprout homogenates appears to significantly reduce some virus-induced markers of inflammation, as well as reducing virus quantity. Nutritional antioxidant interventions have promise as a safe, low-cost strategy for reducing influenza risk among smokers and other at risk populations. Trial Registration ClinicalTrials.gov NCT01269723

Repeated measurement of nasal lavage fluid chemokines in school-age children with asthma

BACKGROUND Inflammatory processes at the mucosal surface may play a role in maintenance of asthma pathophysiology. Cross-sectional studies in asthmatic patients suggest that chemokines such as interleukin 8 (IL-8) are overproduced by respiratory epithelium. OBJECTIVE To test the hypothesis that chemokine levels are persistently elevated in the respiratory secretions of asthmatic children at a stable baseline. METHODS We measured nasal lavage fluid (NLF) levels of chemokines and other mediators at 3- to 4-month intervals in a longitudinal study of asthmatic children, with nonasthmatic siblings as controls. RESULTS In a linear mixed-model analysis, both family and day of visit had significant effects on nasal mediators. Thus, data for 12 asthmatic-nonasthmatic sibling pairs who had 3 or more same-day visits were analyzed separately. For sibling pairs, median eosinophil cationic protein levels derived from serial measurements in NLF were elevated in asthmatic patients compared with nonasthmatic patients, with a near-significant tendency for elevation of total protein and eotaxin levels as well. However, no significant differences were found for IL-8 or several other chemokines. Ratios of IL-13 or IL-5 to interferon-gamma released by house dust mite antigen-stimulated peripheral blood mononuclear cells, tested on a single occasion, were significantly increased for asthmatic patients. CONCLUSIONS Substantial temporal and family-related variability exists in nasal inflammation in asthmatic children. Although higher levels of eosinophil cationic protein are usually present in NLF of patients with stable asthma compared with patients without asthma, chemokines other than eotaxin are not consistently increased. Eosinophil activation at the mucosal surface is a more consistent predictor of asthmatic symptoms than nonspecific elevation of epithelium-derived inflammatory chemokine levels.