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Neonatal chronic lung disease is characterized by failed formation of alveoli and capillaries, and excessive deposition of matrix elastin, which are linked to lengthy mechanical ventilation (MV) with [O.sub.2]-rich gas. Vitamin A supplementation has improved respiratory outcome of premature infants, but there is little information about the structural and molecular manifestations in the lung that occur with vitamin A treatment. We hypothesized that vitamin A supplementation during prolonged MV, without confounding by antenatal steroid treatment, would improve alveolar secondary septation, decrease thickness of the mesenchymal tissue cores between distal air space walls, and increase alveolar capillary growth. We further hypothesized that these structural advancements would be associated with modulated expression of tropoelastin and deposition of matrix elastin, phosphorylated Smad2 (pSmad2), cleaved caspase 3, proliferating cell nuclear antigen (PCNA), VEGF, VEGF-R2, and midkine in the parenchyma of the immature lung. Eight preterm lambs (125 days' gestation, term ~150 days) were managed by MV for 3 wk: four were treated with daily intramuscular Aquasol A (vitamin A), 5,000 IU/kg, starting at birth; four received vehicle alone. Postmortem lung assays included quantitative RT-PCR and in situ hybridization, immunoblot and immunohistochemistry, and morphometry and stereology. Daily vitamin A supplementation increased alveolar secondary septation, decreased thickness of the mesenchymal tissue cores between the distal air space walls, and increased alveolar capillary growth. Associated molecular changes were less tropoelastin mRNA expression, matrix elastin deposition, pSmad2, and PCNA protein localization in the mesenchymal tissue core of the distal air space walls. On the other hand, mRNA expression and protein abundance of VEGF, VEGF-R2, midkine, and cleaved caspase 3 were increased. We conclude that vitamin A treatment partially improves lung development in chronically ventilated preterm neonates by modulating expression of tropoelastin, deposition of elastin, and expression of vascular growth factors. bronchopulmonary dysplasia; VEGF; VEGF-R2; midkine; tropoelastin; elastin; TGF-[beta]; Smad; lung growth and development: alveolar formation; alveolar capillary growth doi: 10.1152/ajplung.00380.2009.

Collagen VII anchoring fibrils in the basement membrane zone (BMZ) are part of a supracellular anchoring network that attaches the epithelium to the BMZ. Sloughing of airway epithelium in asthmatics (creola bodies) is a pathology associated with the supracellular anchoring network. In a rhesus monkey model of house dust mite (HDM)-induced allergic asthma, we found increased deposition of collagen I in the BMZ. In this study, we determine whether HDM also affected deposition of collagen VII in the BMZ. In the developing airway of rhesus monkeys, the width of collagen VII anchoring fibrils in the BMZ was 0.02 [+ or -] 0.04 [micro]m at 1 mo of age. At 6 mo the width had increased to 1.28 [+ or -] 0.34 [micro]m and at 12 mo 2.15 [+ or -] 0.13 [micro]m. In animals treated with HDM, we found a 42.2% reduction in the width of collagen VII layer in the BMZ at 6 mo (0.74 [+ or -] 0.15 [micro]m; P < 0.05). During recovery, the rate of collagen VII deposition returned to normal. However, the amount of collagen VII lost was not recovered after 6 mo. We concluded that normal development of the collagen VII attachment between the epithelium and BMZ occurs in coordination with development of the BMZ. However, in HDM-treated animals, the collagen VII attachment with the epithelium was significantly reduced. Such a reduction in collagen VII may weaken the supracellular anchoring network and be associated with sloughing of the epithelium and formation of creola bodies in asthmatics. doi: 10.1152/aiplung.00337.2009 supracellular anchoring network; intermediate filaments; hemidesmosomes; desmosomes

Thymic stromal lymphopoietin (TSLP) potently induces deregulation of Th2 responses, a hallmark feature of allergic inflammatory diseases such as asthma, atopic dermatitis, and allergic rhinitis. However, direct downstream in vivo mediators in the TSLP-induced atopic immune cascade have not been identified. In our current study, we have shown that OX40 ligand (OX40L) is a critical in vivo mediator of TSLP-mediated Th2 responses. Treating mice with OX40L-blocking antibodies substantially inhibited immune responses induced by TSLP in the lung and skin, including Th2 inflammatory cell infiltration, cytokine secretion, and IgE production. OX40L-blocking antibodies also inhibited antigen-driven Th2 inflammation in mouse and nonhuman primate models of asthma. This treatment resulted in both blockade of the OX40-OX40L receptor-ligand interaction and depletion of OX40L-positive cells. The use of a blocking, OX40L-specific mAb thus presents a promising strategy for the treatment of allergic diseases associated with pathologic Th2 immune responses., Introduction Studies of allergic inflammatory disease pathogenesis such as asthma have shown chronic inflammation resulting from a hyperresponse to innocuous environmental antigens. The pathophysiology of asthma includes mucus hypersecretion, bronchial [...]

Postnatal developmental stages of lung parenchyma in rhesus monkeys is about one-third that of humans. Alveoli in humans are reported to be formed up to 8 yr of age. We used design-based stereological methods to estimate the number of alveoli ([N.sub.alv]) in male and female rhesus monkeys over the first 7 yr of life. Twenty-six rhesus monkeys (13 males ranging in age from 4 to 1,920 days and lung volumes from 41.7 to 602 [cm.sup.3], 13 females ranging in age from 22 to 2,675 days and lung volumes from 43.5 to 380 [cm.sup.3]) were necropsied and lungs fixed, isotropically oriented, fractionated, sampled, embedded, and sectioned for alveolar counting. Parenchymal, alveolar, alveolar duct core air, and interalveolar septal tissue volumes increased rapidly during the first 2 yr with slowed growth from 2 to 7 yr. The rate of change was greater in males than females. [N.sub.lv] also showed consistent growth throughout the study, with increases in [N.sub.lv] best predicted by increases in lung volume. However, mean alveolar volume showed little relationship with age, lung volume, or body weight but was larger in females and showed a greater size distribution than in males. Alveoli increase in number but not volume throughout postnatal development in rhesus monkeys. stereology; parenchyma; alveolar ducts; postnatal development

Inner city children exposed to high levels of ozone suffer from an increased prevalence of respiratory diseases. Lung development in children is a long-term process, and there is a significant period of time during development when children growing up in urban areas are exposed to oxidant air pollution. This study was designed to test whether repeating cycles of injury and repair caused by episodes of ozone exposure lead to chronic airway disease and decreased lung function by altering normal lung maturation. We evaluated postnatal lung morphogenesis and function of infant monkeys after 5 mo of episodic exposure of 0.5 parts per million ozone beginning at 1 mo of age. Nonhuman primates were chosen because their airway structure and postnatal lung development is similar to those of humans. Airway morphology and structure were evaluated at the end of the 5-too exposure period. Compared with control infants, ozone-exposed animals had four fewer nonalveolarized airway generations, hyperplastic bronchiolar epithelium, and altered smooth muscle bundle orientation in terminal and respiratory bronchioles. These results suggest that episodic exposure to environmental ozone compromises postnatal morphogenesis of tracheobronchial airways. oxidant air pollution; adverse effects; growth and development; lung

Mucous cell hypersecretion and increased neuropeptide production play a role in the exacerbation of symptoms associated with asthma. The source J of these neuropeptides have been confined to the contributions of small afferent nerves or possibly neuroendocrine cells. We tested the hypothesis that repeated exposure to allergen would alter the sources and abundance of neuropeptides in airways. Right middle lobes from rats (8 wk old) exposed to 2.5% ovalbumin (OVA) for five episodes (30 min each) or filtered air were inflation fixed with paraformaldehyde. The lobes were dissected to expose the airway tree, permeabilized with DMSO, and incubated in antibody to rat calcitonin generelated peptide (CGRP), followed with a fluorochrome-labeled second antibody. CGRP-positive structures were imaged via confocal microscopy. Airways were later embedded in plastic and sectioned for cell identification. In animals challenged with OVA, CGRP-positive cells, not neuroendocrine or neuronal in origin (confirmed by a lack of protein gene product 9.5 signal), were recorded along the axial path. In section, this fluorescent signal was localized to granules within epithelial cells. Alcian blue/periodic acid-Schiff staining of these same sections positively identify these cells as mucous cells. Mucous cells of animals not challenged with OVA were not positive for CGRP. We conclude that episodic allergen exposure results in the accumulation of CGRP within mucous cells, creating a new source for the release of this neuropeptide within the airway. asthma; protein gene product

Davie, Neil J., Joseph T. Crossno, Jr., Maria G. Frid, Stephen E. Hofmeister, John T. Reeves, Dallas M. Hyde, Todd C. Carpenter Jacqueline A. Brunetti, Ian K. McNiece, and Kurt R. Stenmark Hypoxia-induced pulmonary artery adventitial remodeling and neovascularization: contribution of progenitor cells. Am J Physiol Lung Cell Mol Physiol 286: L668-L678, 2004. First published May 16, 2003; 10.1152/ajplung.00108.2003.--Information is rapidly emerging regarding the important role of the arterial vasa vasorum in a variety of systemic vascular diseases. In addition, increasing evidence suggests that progenitor cells of bone marrow (BM) origin may contribute to postnatal neovascularization and/or vascular wall thickening that is characteristic in some forms of systemic vascular disease. Little is known regarding postnatal vasa formation and the role of BM-derived progenitor cells in the setting of pulmonary hypertension (PH). We sought to determine the effects of chronic hypoxia on the density of vasa vasorum in the pulmonary artery and to evaluate if BM-derived progenitor cells contribute to the increased vessel wall mass in a bovine model of hypoxia-induced PH. Quantitative morphometric analyses of lung tissue from normoxic and hypoxic calves revealed that hypoxia results in a dramatic expansion of the pulmonary artery adventitial vasa vasorum. Flow cytometric analysis demonstrated that cells expressing the transmembrane tyrosine kinase receptor for stem cell factor, c-kit, are mobilized from the BM in the circulation in response to hypoxia. Immunohistochemistry revealed an increase in the expression of c-ki[t.sup.+] cells together with vascular endothelial growth factor, fibronectin, and thrombin in the hypoxia-induced remodeled pulmonary artery vessel wall. Circulating mononuclear cells isolated from neonatal calves exposed to hypoxia were found to differentiate into endothelial and smooth muscle cell phenotypes depending on culture conditions. From these observations, we suggest that the vasa vasorum and circulating progenitor cells could be involved in vessel wall thickening in the setting of hypoxia-induced PH. vascular remodeling; bone marrow

Development of the basement membrane zone (BMZ) occurs postnatally in the rhesus monkey. The purpose of this study was to determine whether house dust mite allergen (HDMA) plus ozone altered this process. Rhesus monkeys were exposed to a regimen of HDMA and/or ozone or filtered air for 6 mo. To detect structural changes in the BMZ, we measured immunoreactivity of collagen I. To detect functional changes in the BMZ, we measured perlecan and fibroblast growth factor-2 (FGF-2). We also measured components of the FGF-2 ternary signaling complex [fibroblast growth factor receptor-1 (FGFR-1) and syndecan-4]. The width of the BMZ was irregular in the ozone groups, suggesting atypical development of the BMZ. Perlecan was also absent from the BMZ. In the absence of perlecan, FGF-2 was not bound to the BMZ. However, FGF-2 immunoreactivity was present in basal cells, the lateral intercellular space (LIS), and attenuated fibroblasts. FGFR-1 immunoreactivity was downregulated, and syndecan-4 immunoreactivity was upregulated in the basal cells. This suggests that FGF-2 in basal cells and LIS may be bound to the syndecan-4. We conclude that ozone and HDMA plus ozone effected incorporation of perlecan into the BMZ, resulting in atypical development of the BMZ. These changes are associated with specific alterations in the regulation of FGF-2, FGFR-1, and syndecan-4 in the airway epithelial-mesenchyreal trophic unit, which may be associated with the developmental problems of lungs associated with exposure to ozone. perlecan; fibroblast growth factor-2; fibroblast growth factor receptor-1; syndecan-4; epithelial-mesenchymal trophic unit

ABSTRACT This study tested the hypothesis that exposure to mixtures containing fine particles and ozone ([O.sub.3]) would cause pulmonary injury and decrements in functions of immunological cells in exposed rats [...]

Byline: Swarnalatha N. Iyer (1), Dallas M. Hyde (2), Shri N. Giri (1) Abstract: We have previously reported the antifibrotic effects of pirfenidone (PD) in the bleomycin (BL)-hamster model of lung fibrosis. Since the development of fibrosis is generally preceded by acute lung inflammation, the present study was conducted to find out if dietary intake of PD (0.5%) has any effects on BL-induced lung inflammation. In this regard, we evaluated the effects of PD on BL-induced increased pulmonary vascular permeability, increased influx of inflammatory cells and increased levels of TGF-[beta] in the bronchoalveolar lavage fluid (BALF). Hamsters were intratracheally (IT) instilled with saline (SA) or BL (5.5 units/kg/5 ml). The animals were fed the control diet (CD) or the same diet containing 0.5% PD 2 days prior to IT instillation and throughout the study. The bronchoalveolar lavage was carried out at different times after IT instillation. Lavage fluid was used for total and differential cell counts and BALF-supernatant for measurement of total protein and TGF-[beta]. IT instillation of BL caused significant increases in total cells, neutrophils, macrophages and lymphocytes and in the levels of total protein and TGF-[beta] in BALF from hamsters in the BL + CD groups as compared to the corresponding SA + CD control groups. In contrast, treatment with pirfenidone in general, suppressed the BL-induced increases in the levels of proteins and TGF-[beta] and in the influx of neutrophils, macrophages and lymphocytes in BALF at the early time points in BL + PD groups. Based on the data reported in this study, we conclude that the anti-inflammatory effects of pirfenidone as evident by suppressions of BL-induced increased pulmonary vascular permeability and increased influx of inflammatory cells in the lung contribute additionally to its inherent anti-fibrotic effect. Author Affiliation: (1) Department of Molecular Biosciences, Physiology and Cell Biology School of Veterinary Medicine University of California, Davis (2) Department of Anatomy, Physiology and Cell Biology School of Veterinary Medicine University of California, Davis Article History: Registration Date: 05/10/2004

Byline: Lisa A. Miller (1), Congfen Li (1), Dallas M. Hyde (1) Abstract: Interferon gamma (IFNI3)-mediated activation of the myelomonocytic cell line HL-60 and peripheral blood monocytes will induce expression of an epitope for the monoclonal antibody which recognizes human [alpha]E[beta]7 integrin, HML-1. Here, we found that the anti-human [alpha]E[beta]7 monoclonal antibody Ber-ACT8 did not recognize IFNI3-stimulated, HML-1 positive HL-60 cells. Culture of blood monocytes with IFNI3 also induced expression of HML-1 but not Ber-ACT8 epitopes. Moreover, migration of monocytes across a monolayer of human airway epithelial cells rapidly induced expression of HML-1, with no detectable Ber-ACT8 staining. Using two different sets of primers specific for the human [alpha]E integrin gene, we were unable to detect [alpha]E mRNA within HL-60 cells or isolated monocytes by reverse transcriptase polymerase chain reaction methods. We conclude that reactivity of the HML-1 antibody for HL-60 cells and monocytes does not correspond with the expression of the [alpha]E integrin subunit, and instead detects a marker for cellular activation. Author Affiliation: (1) The Center for Comparative Respiratory Biology and Medicine, Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, UC Davis, Davis, California, 95616 Article History: Registration Date: 09/10/2004

A rhesus monkey interleukin-8 (IL-8) cDNA clone almost homologous to the human IL-8 was isolated by differential hybridization from a cDNA library of distal airways after ozone inhalation. The appearance of IL-8 in airway epithelium correlated well with neutrophil influx into airway epithelia and lumens. A transient increase in IL-8 secretion was observed after ozone exposure and a dose-dependent increase in IL-8 secretion and mRNA production. Results suggested that IL-8 is an important chemokine in acute ozone-induced airway inflammation in primates.

The interaction between acute pulmonary inflammation and deficient neonatal netrophil function and blood flow responsiveness was analyzed in adult and four-week-old rabbits. Analysis of local inflammatory response after the administration of C5a in adult and four-week-old rabbits indicated the presence of different responses to inflammatory stimulus in adult and neonatal rabbit lungs. Adult rabbit lungs responded to inflammatory stimulus by diverting blood away from the inflammatory site while neonatal rabbit lungs responded passively to inflammatory stimulus. Furthermore, neonatal rabbit lungs exhibited impaired neutrophil chemotaxis.

Neutrophils (PMN) in the lungs preferentially target epithelial cells damaged by ozone (O3) exposure, and help in the removal of the damaged cells and recovery of the centriacinar epithelium. The effect of PMN depends on O3 concentration. The PMN-aided epithelium cells are able to recover permeability after exposure to low levels of O3 better than after exposure to high levels. However, PMN increases the damage caused by high concentrations of O3, and decreases epithelial cell survival.

Mechanism of dexamethasone-mediated interleukin-8 gene suppression in cultured airway epithelial cells. Am J Physiol Lung Cell Mol Physiol 280: L107-L115, 2001.--The effects of dexamethasone, a glucocorticoid analog, on interleukin 8 (IL-8) gene expression were studied in cultures of primary human tracheobronchial epithelial cells and an immortalized human bronchial epithelial cell line, HBE1 cells. Dexamethasone inhibited IL-8 mRNA and protein expression in a concentration- and time-dependent manner. The inhibition did not occur at the transcriptional level since both nuclear run-on activity and IL-8 promoter-reporter gene expression assay revealed no significant effect. Instead, there was a change in IL-8 mRNA stability in dexamethasone-treated cultures. Under actinomycin D treatment, IL-8 mRNA was quite stable in dexamethasone-depleted cultures, while in dexamethasone-pretreated cultures, IL-8 message was rapidly degraded within the first hour, then leveled off. When dexamethasone and actinomycin D were added simultaneously to dexamethasone-depleted cultures, IL-8 mRNA remained rather stable. When cycloheximide was used to inhibit new protein synthesis, dexamethasone-dependent inhibition was not observed. These results suggest that a posttranscriptional mechanism, which requires dexamethasone-dependent new protein synthesis, is involved in the regulation of IL-8 mRNA by dexamethasone in airway epithelial cells. mRNA stability; transcription; posttranscriptional regulation

Hyde, Dallas M., Lisa A. Miller, Ruth J. McDonald, Mary Y. Stovall, Viviana Wong, Kent E. Pinkerton, Craig D. Wegner, Robert Rothlein, and Charles G. Plopper. Neutrophils enhance clearance of necrotic epithelial cells in ozone-induced lung injury in rhesus monkeys. Am. J. Physiol. 277 (Lung Cell. Mol. Physiol. 21): L1190-L1198, 1999.--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. epithelial repair; morphometry; CD18 monoclonal antibody; complement 5a; bronchoalveolar lavage

Nutritional Importance of Pyrroloquinoline Quinone THE QUINOPROTEINS WERE RECOGNIZED in the late 1970s as a novel class of bacterial oxidoreductases that utilize PQQ (or Methoxatin) as a cofactor [1]. PQQ [...]