Microvascular maturation of the septal capillary layers takes place in parallel to alveolarization in human lungs.

Primary and secondary septa formed during lung development contain a double-layered capillary network. To improve gas exchange, the capillary network is remodeled into a single-layered one, a process that is called microvascular maturation (MVM). It takes place during classical and continued alveolarization. Classical alveolarization is defined as a formation of new septa from immature septa and continued alveolarization as a formation from mature septa. Until now, MVM was never quantitatively evaluated in human lungs. To correlate alveolarization and MVM, and to determine the transition point from classical to continued alveolarization, the degree of MVM was stereologically estimated. In 12 human lungs (0.1-15 yr), the alveolar surface area of immature and mature septa was estimated stereologically by intersection counting. An MVM-quotient (RMVM) was defined as the mature alveolar surface area over total alveolar surface area. The MVM-quotient increased logarithmically over age and showed a biphasic increase similar to alveolarization. It did not reach 100% maturity in these samples. A linear correlation between the MVM-quotient and the logarithm of the number of alveoli was observed. We conclude that MVM increased logarithmically and biphasically in parallel to alveolarization until alveolarization ceased. However, at 2-3 yr of age three-quarters of the alveolar microvasculature are mature. This result may explain a previous postulate that MVM is finished at this age. We hypothesize that as long as alveolarization takes place, MVM will take place in parallel. We propose that the transition from classical to continued alveolarization takes place between the ages of 1-3 yr in humans.NEW & NOTEWORTHY Newly formed alveolar septa contain a double-layered capillary network. To optimize gas exchange, the two layers fuse to a single-layered capillary network during microvascular maturation. Because its timing is unknow in humans, microvascular maturation was stereologically estimated throughout postnatal human lung development. It is shown that maturation of the microvascular and alveolar septa takes place in parallel to alveolarization. At an age of 2-3 yr three-quarters of the septa are mature.

Unbiased Quantitation of Alveolar Type II to Alveolar Type I Cell Transdifferentiation during Repair after Lung Injury in Mice.

The alveolar epithelium consists of squamous alveolar type (AT) I and cuboidal ATII cells. ATI cells cover 95-98% of the alveolar surface, thereby playing a critical role in barrier integrity, and are extremely thin, thus permitting efficient gas exchange. During lung injury, ATI cells die, resulting in increased epithelial permeability. ATII cells re-epithelialize the alveolar surface via proliferation and transdifferentiation into ATI cells. Transdifferentiation is characterized by down-regulation of ATII cell markers, up-regulation of ATI cell markers, and cell spreading, resulting in a change in morphology from cuboidal to squamous, thus restoring normal alveolar architecture and function. The mechanisms underlying ATII to ATI cell transdifferentiation have not been well studied in vivo. A prerequisite for mechanistic investigation is a rigorous, unbiased method to quantitate this process. Here, we used SPCCreERT2;mTmG mice, in which ATII cells and their progeny express green fluorescent protein (GFP), and applied stereologic techniques to measure transdifferentiation during repair after injury induced by LPS. Transdifferentiation was quantitated as the percent of alveolar surface area covered by ATII-derived (GFP+) cells expressing ATI, but not ATII, cell markers. Using this methodology, the time course and magnitude of transdifferentiation during repair was determined. We found that ATI cell loss and epithelial permeability occurred by Day 4, and ATII to ATI cell transdifferentiation began by Day 7 and continued until Day 16. Notably, transdifferentiation and barrier restoration are temporally correlated. This methodology can be applied to investigate the molecular mechanisms underlying transdifferentiation, ultimately revealing novel therapeutic targets to accelerate repair after lung injury.

Early life allergen and air pollutant exposures alter longitudinal blood immune profiles in infant rhesus monkeys.

Early life is a critical period for the progressive establishment of immunity in response to environmental stimuli; the impact of airborne challenges on this process is not well defined. In a longitudinal fashion, we determined the effect of episodic house dust mite (HDM) aerosol and ozone inhalation, both separately and combined, on peripheral blood immune cell phenotypes and cytokine expression from 4 to 25weeks of age in an infant rhesus monkey model of childhood development. Immune profiles in peripheral blood were compared with lung lavage at 25weeks of age. Independent of exposure, peripheral blood cell counts fluctuated with chronologic age of animals, while IFNγ and IL-4 mRNA levels increased over time in a linear fashion. At 12weeks of age, total WBC, lymphocyte numbers, FoxP3 mRNA and IL-12 mRNA were dramatically reduced relative to earlier time points, but increased to a steady state with age. Exposure effects were observed for monocyte numbers, as well as CCR3, FoxP3, and IL-12 mRNA levels in peripheral blood. Significant differences in cell surface marker and cytokine expression were detected following in vitro HDM or PMA/ionomycin stimulation of PBMC isolated from animals exposed to either HDM or ozone. Lavage revealed a mixed immune phenotype of FoxP3, IFNγ and eosinophilia in association with combined HDM plus ozone exposure, which was not observed in blood. Collectively, our findings show that airborne challenges during postnatal development elicit measureable cell and cytokine changes in peripheral blood over time, but exposure-induced immune profiles are not mirrored in the lung.

SMAD Signaling in the Airways of Healthy Rhesus Macaques versus Rhesus Macaques with Asthma Highlights a Relationship Between Inflammation and Bone Morphogenetic Proteins.

Bone morphogenetic protein (BMP) signaling is important for correct lung morphogenesis, and there is evidence of BMP signaling reactivation in lung diseases. However, little is known about BMP signaling patterns in healthy airway homeostasis and inflammatory airway disease and during epithelial repair. In this study, a rhesus macaque (Macaca mulatta) model of allergic airway disease was used to investigate BMP signaling throughout the airways in health, disease, and regeneration. Stereologic quantification of immunofluorescent images was used to determine the expression of BMP receptor (BMPR) Ia and phosphorylated SMAD (pSMAD) 1/5/8 in the airway epithelium. A pSMAD 1/5/8 expression gradient was found along the airways of healthy juvenile rhesus macaques (n = 3, P < 0.005). Membrane-localized BMPRIa expression was also present in the epithelium of the healthy animals. After exposure to house dust mite allergen and ozone, significant down-regulation of nuclear pSMAD 1/5/8 occurs in the epithelium. When the animals were provided with a recovery period in filtered air, proliferating cell nuclear antigen, pSMAD 1/5/8, and membrane-localized BMPRIa expression were significantly increased in the epithelium of conducting airways (P < 0.005). Furthermore, in the asthmatic airways, altered BMPRIa localization was evident. Because of the elevated eosinophil presence in these airways, we investigated the effect of eosinophil-derived proteins on BMPRIa trafficking in epithelial cells. Eosinophil-derived proteins (eosinophil-derived neurotoxin, eosinophil peroxidase, and major basic protein) induced transient nuclear translocation of membrane-bound BMPRIa. This work mapping SMAD signaling in the airways of nonhuman primates highlights a potential mechanistic relationship between inflammatory mediators and BMP signaling and provides evidence that basal expression of the BMP signaling pathway may be important for maintaining healthy airways.

Increased density of intraepithelial mast cells in patients with exercise-induced bronchoconstriction regulated through epithelially derived thymic stromal lymphopoietin and IL-33.

BACKGROUND: Exercise-induced bronchoconstriction (EIB) is a prototypical feature of indirect airway hyperresponsiveness. Mast cells are implicated in EIB, but the characteristics, regulation, and function of mast cells in patients with EIB are poorly understood. OBJECTIVES: We sought to examine mast cell infiltration of the airway epithelium in patients with EIB and the regulation of mast cell phenotype and function by epithelially derived cytokines. METHODS: Endobronchial biopsy specimens, epithelial brushings, and induced sputum were obtained from asthmatic patients with and without EIB and healthy control subjects. Mast cell proteases were quantified by using quantitative PCR, and mast cell density was quantified by using design-based stereology. Airway epithelial responses to wounding and osmotic stress were assessed in primary airway epithelial cells and ex vivo murine lung tissue. Mast cell granule development and function were examined in cord blood-derived mast cells. RESULTS: Tryptase and carboxypeptidase A3 expression in epithelial brushings and epithelial mast cell density were selectively increased in the asthma group with EIB. An in vitro scratch wound initiated the release of thymic stromal lymphopoietin, which was greater in epithelial cells derived from asthmatic patients. Osmotic stress induced the release of IL-33 from explanted murine lungs, which was increased in allergen-treated mice. Thymic stromal lymphopoietin combined with IL-33 increased tryptase and carboxypeptidase A3 immunostaining in mast cell precursors and selectively increased cysteinyl leukotriene formation by mast cells in a manner that was independent of in vitro sensitization. CONCLUSIONS: Mast cell infiltration of the epithelium is a critical determinant of indirect airway hyperresponsiveness, and the airway epithelium might serve as an important regulator of the development and function of this mast cell population.

Persistence of serotonergic enhancement of airway response in a model of childhood asthma.

The persistence of airway hyperresponsiveness (AHR) and serotonergic enhancement of airway smooth muscle (ASM) contraction induced by ozone (O3) plus allergen has not been evaluated. If this mechanism persists after a prolonged recovery, it would indicate that early-life exposure to O3 plus allergen induces functional changes predisposing allergic individuals to asthma-related symptoms throughout life, even in the absence of environmental insult. A persistent serotonergic mechanism in asthma exacerbations may offer a novel therapeutic target, widening treatment options for patients with asthma. The objective of this study was to determine if previously documented AHR and serotonin-enhanced ASM contraction in allergic monkeys exposed to O3 plus house dust mite allergen (HDMA) persist after prolonged recovery. Infant rhesus monkeys sensitized to HDMA were exposed to filtered air (FA) (n = 6) or HDMA plus O3 (n = 6) for 5 months. Monkeys were then housed in a FA environment for 30 months. At 3 years, airway responsiveness was assessed. Airway rings were then harvested, and ASM contraction was evaluated using electrical field stimulation with and without exogenous serotonin and serotonin-subtype receptor antagonists. Animals exposed to O3 plus HDMA exhibited persistent AHR. Serotonin exacerbated the ASM contraction in the exposure group but not in the FA group. Serotonin subtype receptors 2, 3, and 4 appear to drive the response. Our study shows that AHR and serotonin-dependent exacerbation of cholinergic-mediated ASM contraction induced by early-life exposure to O3 plus allergen persist for at least 2.5 years and may contribute to a persistent asthma phenotype.

Growth of alveoli during postnatal development in humans based on stereological estimation.

Alveolarization in humans and nonhuman primates begins during prenatal development. Advances in stereological counting techniques allow accurate assessment of alveolar number; however, these techniques have not been applied to the developing human lung. Based on the recent American Thoracic Society guidelines for stereology, lungs from human autopsies, ages 2 mo to 15 yr, were fractionated and isometric uniform randomly sampled to count the number of alveoli. The number of alveoli was compared with age, weight, and height as well as growth between right and left lungs. The number of alveoli in the human lung increased exponentially during the first 2 yr of life but continued to increase albeit at a reduced rate through adolescence. Alveolar numbers also correlated with the indirect radial alveolar count technique. Growth curves for human alveolarization were compared using historical data of nonhuman primates and rats. The alveolar growth rate in nonhuman primates was nearly identical to the human growth curve. Rats were significantly different, showing a more pronounced exponential growth during the first 20 days of life. This evidence indicates that the human lung may be more plastic than originally thought, with alveolarization occurring well into adolescence. The first 20 days of life in rats implies a growth curve that may relate more to prenatal growth in humans. The data suggest that nonhuman primates are a better laboratory model for studies of human postnatal lung growth than rats.

Ozone-induced airway epithelial cell death, the neurokinin-1 receptor pathway, and the postnatal developing lung.

Children are uniquely susceptible to ozone because airway and lung growth continue for an extensive period after birth. Early-life exposure of the rhesus monkey to repeated ozone cycles results in region-specific disrupted airway/lung growth, but the mediators and mechanisms are poorly understood. Substance P (SP), neurokinin-1 receptor (NK-1R); and nuclear receptor Nur77 (NR4A1) are signaling pathway components involved in ozone-induced cell death. We hypothesize that acute ozone (AO) exposure during postnatal airway development disrupts SP/NK-1R/Nur77 pathway expression and that these changes correlate with increased ozone-induced cell death. Our objectives were to 1) spatially define the normal development of the SP/NK-1R/Nur77 pathway in conducting airways; 2) compare how postnatal age modulates responses to AO exposure; and 3) determine how concomitant, episodic ozone exposure modifies age-specific acute responses. Male infant rhesus monkeys were assigned at age 1 mo to two age groups, 2 or 6 mo, and then to one of three exposure subgroups: filtered air (FA), FA+AO (AO: 8 h/day × 2 days), or episodic biweekly ozone exposure cycles (EAO: 8 h/day × 5 days/14-day cycle+AO). O3 = 0.5 ppm. We found that 1) ozone increases SP/NK-1R/Nur77 pathway expression in conducting airways, 2) an ozone exposure cycle (5 days/cycle) delivered early at age 2 mo resulted in an airway that was hypersensitive to AO exposure at the end of 2 mo, and 3) continued episodic exposure (11 cycles) resulted in an airway that was hyposensitive to AO exposure at 6 mo. These observations collectively associate with greater overall inflammation and epithelial cell death, particularly in early postnatal (2 mo), distal airways.

Kinetics of the angiogenic response in lung endothelium following acute inflammatory injury with bleomycin.

PURPOSE/AIM: Angiogenesis is a central component of normal wound healing but it has not been fully characterized in lung repair following acute inflammatory injury. The current literature lacks vital information pertaining to the extent, timing, and location of this process. This information is necessary for examining mechanisms that drive normal lung repair in resolving acute inflammatory injury. The goal of our study was to formally characterize lung angiogenesis over a time course of bleomycin-induced lung injury. MATERIALS AND METHODS: Female C57BL/6 mice age 8-12 weeks were treated with a single dose of intratracheal bleomycin. Total lung endothelial cells were quantified with flow cytometry 0, 7, 14, 21, and 28 days following bleomycin administration, and endothelial cell replication was assessed using bromodeoxyuridine (BrdU) incorporation. RESULTS: Endothelial cell replication was maximal 14 days after bleomycin administration, while total lung endothelial cells peaked at day 21. Tissue analysis with stereology was performed to measure total lung vascular surface area in bleomycin at day 21 relative to controls and demonstrated a trend toward increased vasculature in the bleomycin group. CONCLUSIONS: Angiogenesis begins shortly after injury in the bleomycin model and leads to an expansion in the lung endothelial cell population that peaks at day 21. This study offers the first longitudinal examination of angiogenesis following acute inflammatory lung injury induced by bleomycin. Information provided in this study will be vital for further investigating mechanisms of angiogenesis in both normal and abnormal lung repair.

Regulation and function of epithelial secreted phospholipase A2 group X in asthma.

RATIONALE: Indirect airway hyperresponsiveness (AHR) is a fundamental feature of asthma that is manifest as exercise-induced bronchoconstriction (EIB). Secreted phospholipase A2 group X (sPLA2-X) plays a key role in regulating eicosanoid formation and the development of inflammation and AHR in murine models. OBJECTIVES: We sought to examine sPLA2-X in the airway epithelium and airway wall of patients with asthma, the relationship to AHR in humans, and the regulation and function of sPLA2-X within the epithelium. METHODS: We precisely phenotyped 34 patients with asthma (19 with and 15 without EIB) and 10 normal control subjects to examine in vivo differences in epithelial gene expression, quantitative morphometry of endobronchial biopsies, and levels of secreted protein. The regulation of sPLA2-X gene (PLA2G10) expression was examined in primary airway epithelial cell cultures. The function of epithelial sPLA2-X in eicosanoid formation was examined using PLA2 inhibitors and murine tracheal epithelial cells with Pla2g10 deletion. MEASUREMENTS AND MAIN RESULTS: We found that sPLA2-X protein is increased in the airways of patients with asthma and that epithelial-derived sPLA2-X may be increased in association with indirect AHR. The expression of sPLA2-X increases during in vitro epithelial differentiation; is regulated by inflammatory signals including tumor necrosis factor, IL-13, and IL-17; and is both secreted from the epithelium and directly participates in the release of arachidonic acid by epithelial cells. CONCLUSIONS: These data reveal a relationship between epithelial-derived sPLA2-X and indirect AHR in asthma and that sPLA2-X serves as an epithelial regulator of inflammatory eicosanoid formation. Therapies targeting epithelial sPLA2-X may be useful in asthma.

Accelerated structural decrements in the aging female rhesus macaque lung compared with males.

Aging is associated with morphometric changes in the lung that lead to decreased lung function. The nonhuman primate lung has been shown to have similar architectural, morphological, and developmental patterns to that of humans. We hypothesized that the lungs of rhesus monkeys age in a pattern similar to human lungs. Thirty-four rhesus monkeys from the California National Primate Research Center were euthanized, necropsied, and the whole lungs sampled. Stereological analysis was performed to assess the morphological changes associated with age. The number of alveoli declined significantly from age 9 to 33 yr with a greater decline in females compared with males. Lungs of females contained roughly 20% more alveoli at age 9 yr than males, but by ∼30 yr of age, females had 30% fewer alveoli than males. The volume of alveolar air also showed a significant linear decrease in females relative to age, while males did not. The number-weighted mean volume of alveoli showed a significant positive correlation with age in females but not in males. The volume of alveolar duct showed a significant positive correlation with age in females, but not in males. Structural decrements due to aging in the lung were increased in the female compared with male rhesus monkey.

Prenatal nicotine exposure alters lung function and airway geometry through α7 nicotinic receptors.

Maternal smoking during pregnancy has been associated with adverse effects on respiratory health. Whereas the epidemiologic link is incontrovertible, the mechanisms responsible for this association are still poorly understood. Although cigarette smoke has many toxic constituents, nicotine, the major addictive component in cigarette smoke, may play a more significant role than previously realized. The objectives of this study were to determine whether exposure to nicotine prenatally leads to alterations in pulmonary function and airway geometry in offspring, and whether α7 nicotinic acetylcholine receptors (nAChRs) mediate these effects. In a murine model of in utero nicotine exposure, pulmonary function, airway size and number, methacholine response, and collagen deposition were examined. Exposure periods included Gestation Days 7-21, Gestation Day 14 to Postnatal Day 7, and Postnatal Days 3-15. Prenatal nicotine exposure decreases forced expiratory flows in offspring through α7 nAChR-mediated signals, and the critical period of nicotine exposure was between Prenatal Day 14 and Postnatal Day 7. These physiologic changes were associated with increased airway length and decreased diameter. In addition, adult mice exposed to prenatal nicotine exhibit an increased response to methacholine challenge, even in the absence of allergic sensitization. Collagen expression was increased between adjacent airways and vessels, which was absent in α7 nAChR knockout mice. These observations provide a unified mechanism of how maternal smoking during pregnancy may lead to lifelong alterations in offspring pulmonary function and increased risk of asthma, and suggest potential targets to counteract those effects.

Postnatal exposure history and airways: oxidant stress responses in airway explants.

Postnatally, the lung continues to grow and differentiate while interacting with the environment. Exposure to ozone (O(3)) and allergens during postnatal lung development alters structural elements of conducting airways, including innervation and neurokinin abundance. These changes have been linked with development of asthma in a rhesus monkey model. We hypothesized that O(3) exposure resets the ability of the airways to respond to oxidant stress and that this is mediated by changes in the neurokinin-1 receptor (NK-1R). Infant rhesus monkeys received episodic exposure to O(3) biweekly with or without house dust mite antigen (HDMA) from 6 to 12 months of age. Age-matched monkeys were exposed to filtered air (FA). Microdissected airway explants from midlevel airways (intrapulmonary generations 5-8) for four to six animals in each of four groups (FA, O(3), HDMA, and HDMA+O(3)) were tested for NK-1R gene responses to acute oxidant stress using exposure to hydrogen peroxide (1.2 mM), a lipid ozonide (10 µM), or sham treatment for 4 hours in vitro. Airway responses were measured using real-time quantitative RT-PCR of NK-1R and IL-8 gene expression. Basal NK-1R gene expression levels were not different between the exposure groups. Treatment with ozonide or hydrogen peroxide did not change NK-1R gene expression in animals exposed to FA, HDMA, or HDMA+O(3). However, treatment in vitro with lipid ozonide significantly increased NK-1R gene expression in explants from O(3)-exposed animals. We conclude that a history of prior O(3) exposure resets the steady state of the airways to increase the NK-1R response to subsequent acute oxidant stresses.

Lung gene expression in a rhesus allergic asthma model correlates with physiologic parameters of disease and exhibits common and distinct pathways with human asthma and a mouse asthma model.

Experimental nonhuman primate models of asthma exhibit multiple features that are characteristic of an eosinophilic/T helper 2 (Th2)-high asthma subtype, characterized by the increased expression of Th2 cytokines and responsive genes, in humans. Here, we determine the molecular pathways that are present in a house dust mite-induced rhesus asthma model by analyzing the genomewide lung gene expression profile of the rhesus model and comparing it with that of human Th2-high asthma. We find that a prespecified human Th2 inflammation gene set from human Th2-high asthma is also present in rhesus asthma and that the expression of the genes comprising this gene set is positively correlated in human and rhesus asthma. In addition, as in human Th2-high asthma, the Th2 gene set correlates with physiologic markers of allergic inflammation and disease in rhesus asthma. Comparison of lung gene expression profiles from human Th2-high asthma, the rhesus asthma model, and a common mouse asthma model indicates that genes associated with Th2 inflammation are shared by all three species. However, some pathophysiologic aspects of human asthma (ie, subepithelial fibrosis, angiogenesis, neural biology, and immune host defense biology) are better represented in the gene expression profile of the rhesus model than in the mouse model. Further study of the rhesus asthma model may yield novel insights into the pathogenesis of human Th2-high asthma.

Satratoxin-G from the black mold Stachybotrys chartarum induces rhinitis and apoptosis of olfactory sensory neurons in the nasal airways of rhesus monkeys.

Satratoxin-G (SG) is a trichothecene mycotoxin of Stachybotrys chartarum, the black mold suggested to contribute etiologically to illnesses associated with water-damaged buildings. We have reported that intranasal exposure to SG evokes apoptosis of olfactory sensory neurons (OSNs) and acute inflammation in the nose and brain of laboratory mice. To further assess the potential human risk of nasal airway injury and neurotoxicity, we developed a model of SG exposure in monkeys, whose nasal airways more closely resemble those of humans. Adult, male rhesus macaques received a single intranasal instillation of 20 µg SG (high dose, n = 3), or 5 µg SG daily for four days (repeated low dose, n = 3) in one nasal passage, and saline vehicle in the contralateral nasal passage. Nasal tissues were examined using light and electron microscopy and morphometric analysis. SG induced acute rhinitis, atrophy of the olfactory epithelium (OE), and apoptosis of OSNs in both groups. High-dose and repeated low-dose SG elicited a 13% and 66% reduction in OSN volume density, and a 14-fold and 24-fold increase in apoptotic cells of the OE, respectively. This model provides new insight into the potential risk of nasal airway injury and neurotoxicity caused by exposure to water-damaged buildings.

A flexible, open, decentralized system for digital pathology networks.

High-resolution digital imaging is enabling digital archiving and sharing of digitized microscopy slides and new methods for digital pathology. Collaborative research centers, outsourced medical services, and multi-site organizations stand to benefit from sharing pathology data in a digital pathology network. Yet significant technological challenges remain due to the large size and volume of digitized whole slide images. While information systems do exist for managing local pathology laboratories, they tend to be oriented toward narrow clinical use cases or offer closed ecosystems around proprietary formats. Few solutions exist for networking digital pathology operations. Here we present a system architecture and implementation of a digital pathology network and share results from a production system that federates major research centers.

Stereological analysis of bacterial load and lung lesions in nonhuman primates (rhesus macaques) experimentally infected with Mycobacterium tuberculosis.

Infection with Mycobacterium tuberculosis primarily produces a multifocal distribution of pulmonary granulomas in which the pathogen resides. Accordingly, quantitative assessment of the bacterial load and pathology is a substantial challenge in tuberculosis. Such assessments are critical for studies of the pathogenesis and for the development of vaccines and drugs in animal models of experimental M. tuberculosis infection. Stereology enables unbiased quantitation of three-dimensional objects from two-dimensional sections and thus is suited to quantify histological lesions. We have developed a protocol for stereological analysis of the lung in rhesus macaques inoculated with a pathogenic clinical strain of M. tuberculosis (Erdman strain). These animals exhibit a pattern of infection and tuberculosis similar to that of naturally infected humans. Conditions were optimized for collecting lung samples in a nonbiased, random manner. Bacterial load in these samples was assessed by a standard plating assay, and granulomas were graded and enumerated microscopically. Stereological analysis provided quantitative data that supported a significant correlation between bacterial load and lung granulomas. Thus this stereological approach enables a quantitative, statistically valid analysis of the impact of M. tuberculosis infection in the lung and will serve as an essential tool for objectively comparing the efficacy of drugs and vaccines.

Behavioral inhibition is associated with airway hyperresponsiveness but not atopy in a monkey model of asthma.

OBJECTIVE: To determine whether indicators of behavioral inhibition and cortisol responses to stressful situations, obtained in infancy, were associated with asthma-related measures (atopy and airway hyperresponsiveness [AHR]) approximately 2 years later. METHODS: Measures reflecting inhibited temperament and cortisol response after a 25-hour separation from mother and relocation to a novel room were obtained for 21 rhesus monkeys (mean age, 109 days; range, 91-122 days). Inhibited temperament was measured by reduced emotionality and increased vigilance. Atopy and AHR were assessed after 2 years (age range, 19-35 months) using skin tests to common aeroallergens and inhaled methacholine challenge, respectively. RESULTS: No associations were found between atopy and either behavioral inhibition or cortisol levels (p > .56). Low emotionality was associated with AHR (r = 0.47, p = .03), and a trend was found for blunted cortisol responsiveness and AHR (r = 0.42, p = .06). CONCLUSIONS: Inhibited temperament and blunted cortisol responsiveness may be related to the development of AHR that is common to both nonatopic and atopic asthma phenotypes and may indicate risk for nonatopic asthma specifically.

Increased CCL24/eotaxin-2 with postnatal ozone exposure in allergen-sensitized infant monkeys is not associated with recruitment of eosinophils to airway mucosa.

Epidemiology supports a causal link between air pollutant exposure and childhood asthma, but the mechanisms are unknown. We have previously reported that ozone exposure can alter the anatomic distribution of CD25+ lymphocytes in airways of allergen-sensitized infant rhesus monkeys. Here, we hypothesized that ozone may also affect eosinophil trafficking to allergen-sensitized infant airways. To test this hypothesis, we measured blood, lavage, and airway mucosa eosinophils in 3-month old monkeys following cyclical ozone and house dust mite (HDM) aerosol exposures. We also determined if eotaxin family members (CCL11, CCL24, CCL26) are associated with eosinophil location in response to exposures. In lavage, eosinophil numbers increased in animals exposed to ozone and/or HDM. Ozone+HDM animals showed significantly increased CCL24 and CCL26 protein in lavage, but the concentration of CCL11, CCL24, and CCL26 was independent of eosinophil number for all exposure groups. In airway mucosa, eosinophils increased with exposure to HDM alone; comparatively, ozone and ozone+HDM resulted in reduced eosinophils. CCL26 mRNA and immunofluorescence staining increased in airway mucosa of HDM alone animals and correlated with eosinophil volume. In ozone+HDM animal groups, CCL24 mRNA and immunofluorescence increased along with CCR3 mRNA, but did not correlate with airway mucosa eosinophils. Cumulatively, our data indicate that ozone exposure results in a profile of airway eosinophil migration that is distinct from HDM mediated pathways. CCL24 was found to be induced only by combined ozone and HDM exposure, however expression was not associated with the presence of eosinophils within the airway mucosa.