Surfactant Protein A Stimulates Release of Neutrophil Chemotactic Factors by Alveolar Type II Pneumocytes

Mucosal immunity is an important mechanism in the response to injury. Our hypothesis is that surfactant protein A (SP-A) is an autocrine factor that stimulates alveolar type II epithelial cell release of neutrophil chemotactic factors by binding to the SP-A receptor expressed by these cells. We examined (1) the effect of SP-A (20 μg/ml) or IL-1β (10 ng/ml) on release of neutrophil chemotactic factors by primary cultures of type II cells or alveolar macrophages, and (2) the effect of intratracheal instillation of the blocking antibody to the SP-A receptor on the response to oleic acid-induced lung injury in vivo. All media and cell culture supernates were assayed for neutrophil chemotactic activity, and bronchoalveolar lavage fluid from the in vivo experiments was analyzed for inflammatory cell counts. While SP-A and media used for the cell cultures has no intrinsic neutrophil chemotactic activity, supernates from primary cultures of type II cells incubated in either SP-A or IL-1β had twofold higher neutrophil chemotactic factor activity compared to supernates from controls. SP-A had no effect on release of neutrophil chemotactic factor by alveolar macrophages. Oleic acid-induced lung injury resulted in a marked influx of neutrophils into BAL, and this influx was reduced by 70% by pretreatment with the antibody to SP-A receptor. We conclude that SP-A stimulates the release of neutrophil chemotactic factor by alveolar type II cells, and this effect is mediated by the receptor for SP-A specifically expressed by these cells.     

Effect of abdominal trauma on hemorrhagic shock-induced acute lung injury in rats

AIM: To evaluate the effects of abdominal trauma on hemorrhagic shock-induced acute lung injury in rats. METHODS: Five groups were allocated (n = 8) in the study. GroupⅠwas taken as the control group, groupⅡas the hemorrhagic shock group, groupⅢas hemorrhagic shock laparotomy, groupⅣas hemorrhagic shock splenectomy and groupⅤas splenec-tomy omentectomy hemorrhagic shock group. Hemorrhagic shock was induced by drawing blood and reducing mean arterial pressure (MAP) to 40 mmHg within 10 min. After a hypotensive period of 1 h, animals were resuscitated. Bronchoalveolar lavage (BAL) was performed to recover cells from the alveolar space with 40 ml of BAL fluid after resuscitation malondialdehyde (MDA) and L-γ-glutarnyl-L-cysteinyl-glycine (GSH) levels were measured in serum, eryth-rocytes and lung tissue. RESULTS: Serum, erythrocyte, lung tissue MDA and GSH levels were significantly increased in hemorrhagic shock groupsⅡ-Ⅴ(P < 0.05). Lymphocyte, neutrophil and alveolar macrophage counts in BAL fluid indicated a significant difference between control and shock groups (P < 0.05). CONCLUSION: The degree of trauma increases hemorrhagic shock-induced acute lung injury.     

The use of alveolar epithelial type I cell-selective markers to investigate lung injury and repair.

Alveolar epithelial type I cells cover most of the internal surface area of the lungs. Ultrastructural studies demonstrate that alveolar epithelial type I cell damage is frequently observed in acute and chronic lung diseases. This article discusses the use of cell-selective proteins as markers for the investigation of injury and repair of the alveolar epithelium. The utility of proteins specific to alveolar epithelial type I cells as diagnostic markers of alveolar epithelial injury in acute lung injury is considered, and expression of proteins selective for alveolar epithelial type I cells in lungs following injury and in fibrosis are discussed.     

Bronchoalveolar lavage lipids during development of bleomycin-induced fibrosis in rats. Relationship to altered epithelial cell morphology

Alterations in the structure and function of alveolar epithelial cells may contribute to the interstitial fibrosis that can develop following lung injury. The present studies were undertaken to determine if alterations observed in alveolar epithelial cell morphology and cytoskeletal composition are reflected in the profile of bronchoalveolar lavage (BAL) lipids recovered from injured lung. BAL protein and lipid analyses were performed on fluids recovered from control rats and from rats 7, 14, and 28 days after intratracheal instillation of bleomycin, an antineoplastic agent well-known to cause pulmonary interstitial fibrosis. There were increases in recovery of total protein, nonpolar lipid, polar lipid, and phospholipid following bleomycin treatment. The recovery of saturated phosphatidylcholine was increased, but recovery of a second surfactant phospholipid, phosphatidylglycerol, was unchanged, resulting in a significant change in their ratio. The recoveries of cholesterol, cholesterol ester, and triglyceride also were elevated. Changes in the proportional recoveries of neutral lipids, such as cholesterol and saturated phospholipids, could partly explain concurrent reductions in lung compliance that have been described. Changes in lavage lipids paralleled both the process of alveolar reepithelialization and altered expression of alveolar epithelial cell cytoskeletal proteins. Changes in lipid metabolism by alveolar epithelial cells following bleomycin-induced lung injury may be responsible for altered lavage lipid recovery and may directly be related to processes that take place during alveolar type II cell hyperplasia followed by transition to type I cells. BAL lipid analyses thus may provide a relatively noninvasive way of assessing these events.     

Intra-alveolar release of a competence-type growth factor after lung injury

Growth factors released by platelets, macrophages, and endothelial and smooth muscle cells have been recognized and characterized using in vitro tests of isolated cell populations. However, their production, secretion, and effects on target cells in situ after tissue injury remains largely presumptive. Alveolar macrophages cultured during acute and chronic lung injury release increased amounts of macrophage-derived growth factor (MDGF). In the present study, we sampled the alveolar lining fluid by lavage for the presence of macromolecular competence factor activity. We report that alveolar lavage fluid obtained following acute lung injury induced by bleomycin in the rat contains large amounts of soluble growth factor activity not found in lung lavage fluid from normal animals. We compared the properties of the growth factor found in fresh lavage fluid to MDGF and platelet-derived growth factor (PDGF). The amount of growth factor in lavage fluid paralleled the ability of cultured alveolar macrophages to release MDGF. Like PDGF and MDGF, lavage fluid growth factor served as a competence factor promoting the reentry of quiescent fibroblasts into the cell cycle rather than as a progression factor. Chromatography on DEAE-Sephacel yielded a single peak of growth factor activity eluting at 0.3 M NaCl. On the basis of these and other physical and biologic properties, we conclude that growth factor activity found in high levels in the alveolar space following acute lung injury resembles MDGF. Growth factor present in the alveolar space may provide the major local stimulus to lung structural cell replication after acute lung injury.     

Pathophysiologic changes induced by phospholipase A2 in the isolated, perfused guinea pig lung

Pathophysiologic changes induced by phospholipase A2 (PLA2), a lipolytic enzyme implicated in a variety of pulmonary diseases, were examined in the isolated, perfused guinea pig lung. Thirty microliters of vehicle (saline) or 30 units of PLA2 suspended in saline was given as a bolus injection into either the trachea or pulmonary artery, and the lungs were examined by combined light and transmission electron microscopy at 1, 10, and 30 min following administration. Pulmonary morphologic and physiologic changes were observed only in animals that received PLA2 via the trachea. Initially, morphologic evidence of airway constriction was accompanied by a significant increase in intratracheal pressure. No morphologic change was observed in alveolar parenchymal cell populations at this time. Later there was marked swelling of airway epithelial cells accompanied by pronounced blebbing of the apical cytoplasm with a subsequent decrease in airway lumen size. Interalveolar septa were multifocally thickened by edema and alveolar spaces contained sparse fibrogranular material 10 min after intratracheal PLA2. Significant increases in lung weight were observed at this time. Morphologic features of vasoconstriction, characterized by undulating and convoluted endothelium with prominent cytoplasmic processes resulting in decreased luminal size were observed in small caliber vessels and occurred in conjunction with significant increases in pulmonary arterial pressure. There was pronounced injury to type I alveolar epithelium, including marked cell swelling and fragmentation, accompanied by lesser injury in type II alveolar epithelium. This study demonstrates that the airway epithelium was the initial cell population having morphologic change following intratracheal PLA2, and that the airway epithelium and type I alveolar epithelium were the cell populations most severely affected following intratracheal PLA2. Results further suggest that normal circulating levels of blood-formed elements are not essential for the development of PLA2-induced morphologic change in the isolated, perfused guinea pig lung.     

Macrophages stimulate DNA synthesis in rat alveolar type II cells

Proliferation of alveolar type II cells after lung injury is crucial for repair of the epithelium. Because an influx of macrophages occurs as part of the inflammatory response associated with acute lung injury and macrophages produce mitogenic factors for a variety of cell types, experiments were conducted to determine if macrophages stimulated DNA synthesis in type II cells. Dialyzed medium conditioned by macrophages consistently stimulated type II cell DNA synthesis, whereas medium conditioned by a variety of other cell types did not. A SV40-transformed macrophage cell line, produced in our laboratory, also secreted substance(s) that enhanced 3H-thymidine incorporation into type II cells. In addition, coculturing rat alveolar macrophages with type II cells stimulated DNA synthesis in the epithelial cells. As determined by autoradiography, the addition of macrophages to type II cells cultured on plastic or on an endothelial cell extracellular matrix increased the labeling index of the epithelial cells from 1 to 15% and from 19 to 51%, respectively. The culture conditions that promoted the greatest increase in DNA synthesis, as well as an increase in cell number, occurred with type II cells plated on an extracellular matrix in medium containing macrophage-conditioned medium, cholera toxin, insulin, and epidermal growth factor. The results suggest that substances secreted by macrophages play a role in regulating alveolar type II cell proliferation in vivo.     

晚期糖基化终末产物受体在急性肺损伤中作用的研究进展

晚期糖基化终末产物受体是细胞表面模式识别受体,在Ⅰ型肺泡上皮细胞有丰富表达.近年研究表明急性肺损伤时,支气管肺泡灌洗液及血浆中晚期糖基化终末产物受体含量明显升高,且与肺损伤程度密切相关.另一方面,它与相应配体结合激活细胞内信号通路,参与急性肺损伤的炎症激活和放大过程.晚期糖基化终末产物受体将在判断急性肺损伤患者病情、预...     

Epithelial injury, inflammation, and repair in the hamster lung following intratracheal instillation of enzyme-generated oxidants

Oxidant-induced inflammatory and morphologic changes evoked by intratracheally instilled glucose, active glucose oxidase, and lactoperoxidase in hamsters are described. Similar solutions containing heat-denatured (DE) glucose oxidase served as a control treatment. During the 7-week post-treatment period, animals were sacrificed and lavaged, and their lungs were preserved for later study. Although a neutrophil-rich alveolitis was observed initially in both groups, the leukocytic influx and mild morphologic changes in DE-treated animals resolved within 7 days. In contrast, glucose oxidase mixtures caused necrotic alveolar epithelium, fibrin-like exudate, and significantly higher neutrophil and protein levels in bronchoalveolar lavage fluid (BALF) after 1 day. At 4 days, type II cell hyperplasia was extensive and BALF levels of all inflammatory cell types were significantly greater than BALF counts in DE animals. By 7 days, BALF macrophage and lymphocyte levels remained significantly elevated, and epithelial hyperplasia coincided with marked increases in lung hydroxyproline and interstitial cells. BALF parameters suggested that inflammation induced by glucose oxidase resolved within 15 days, yet total hydroxyproline levels remained elevated at 15 days and epithelial lesions were still detectable at 29 days. Oxidant-induced inflammation and epithelial injury were transient and preceded moderate collagen deposition.     

Sivelestat, a specific neutrophil elastase inhibitor, prevented phorbol myristate acetate-induced acute lung injury in conscious rabbits

The in vivo contribution of neutrophil elastase (NE) in phorbol myristate acetate (PMA)-induced acute lung injury has so far been unclear. This study examined the role of NE in PMA-induced acute lung injury in conscious rabbits, using a specific NE inhibitor, sivelestat sodium hydrate (Sivelestat). A single bolus injection of PMA (40 microg/kg) caused acute lung injury as indicated by an increase in protein concentration and hemorrhage in bronchoalveolar lavage fluid (BALF) 4h after PMA injection. These changes were associated with mild decrease in arterial oxygen pressure and peripheral white blood cell and platelet. When continuously infused starting 1h before and ending 4h post-PMA injection, Sivelestat at 3-30 mg/kg/h that are able to inhibit rabbit NE activity by 60-90%, dose-dependently attenuated both PMA-induced hemorrhagic pneumonitis and the increase in protein concentration in BALF without affecting myeloperoxidase activity in the lung. Histopathological study indicated that sivelestat (30 mg/kg/h) markedly attenuated lung histopathological changes, alveolar hemorrhage and white blood cells migration with evidence of inhibition of NE activity in BALF. These results suggest that NE plays a significant role in PMA-induced acute lung injury and further supports the importance of this enzyme in acute lung injury.     

Low tidal volume reduces epithelial and endothelial injury in acid-injured rat lungs.

Using a rat model of acid-induced lung injury, we tested the hypothesis that tidal volume reduction at the same level of PEEP (10 cm H(2)O) would diminish the degree of pulmonary edema by attenuating injury to the alveolar epithelial and endothelial barriers. Tidal volume reduction from 12 to 6 to 3 ml/kg significantly reduced the rate of lung water accumulation from 690 microl/h to 310 microl/h to 210 microl/h. Ventilation with either 6 or 3 ml/kg reduced endothelial injury equally as measured by plasma vWf:Ag and permeability to albumin. Plasma RTI40, a marker of type I epithelial cell injury, decreased 46% when tidal volume was reduced from 12 to 6 ml/kg and decreased an additional 33% with 3 ml/kg (p < 0.05). The rate of alveolar epithelial fluid clearance was significantly faster in the 3-ml/kg group (24 +/- 7%/h) compared with 6 ml/kg (15 +/- 11%/h) and 12 ml/kg (3 +/- 6%/h). We conclude that low tidal volume ventilation protects both the alveolar epithelium and the endothelium in this model of acute lung injury. The additional decrease in pulmonary edema with a tidal volume of 3 ml/kg is partly accounted for by greater protection of the alveolar epithelium.     

Alveolar epithelium: role in lung fluid balance and acute lung injury

The resolution of alveolar edema is regulated by active sodium and chloride transport across the pulmonary epithelium, including alveolar epithelial type I and II cells as well as distal airway epithelia. Catecholamine-dependent mechanisms can markedly upregulate alveolar fluid clearance even under pathological conditions, an effect that is mediated by both epithelial sodium channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR). Under pathological conditions, impaired alveolar fluid clearance is associated with worse survival in patients with acute lung injury. However, there is some experimental and clinical evidence that cAMP stimulation could accelerate the resolution of pulmonary edema in the presence of acute lung injury. Clinical trials are needed to test this potential therapeutic strategy in patients with acute lung injury.     

Doxycycline attenuated lung injury by its biological effect apart from its antimicrobial function

Antibiotics can have a biological effect apart from their anti-bacterial effect. We hypothesized that doxycycline could attenuate acute lung injury through its biological effect. Lipopolysaccharide or doxycycline-resistant Streptococcus pneumoniae was administered intratracheally into mice with the co-administration of doxycycline. Thereafter, the lung pathology, intraalveolar inflammatory cells, bacterial number, and matrix metalloproteinases were investigated. Matrix metalloproteinases, neutrophil migration, and alveolar destruction were induced by lipopolysaccharide. Doxycycline was thus found to improve all of these symptoms. In addition, an inhibitor of matrix metalloproteinases, CGS27023A, attenuated lipopolysaccharide-induced lung injury. Doxycycline also attenuated the lung injury induced by doxycycline-resistant S. pneumoniae and improved the mortality rate although the bacterial number in the lung did not change. Our data indicated that doxycycline could attenuate acute lung injury through a biological effect that was different from its antibiotic effect.     

Elevated histamine and tryptase levels in smokers' bronchoalveolar lavage fluid. Do lung mast cells contribute to smokers' emphysema?

Human lung mast cells have been reported recently to contain small amounts of the elastolytic protease present in the neutrophil and implicated in the pathogenesis of alveolar wall destruction in emphysema. Since mast cells are numerous within alveolar walls, release of inflammatory mediators (and possibly elastase) by cigarette smoking could contribute to alveolar injury in this disease. We therefore examined bronchoalveolar lavage (BAL) fluid for the mast cell granule constituents histamine and tryptase. The results, while not conclusive, supported the possibility that cigarette smoking increases secretion of histamine releasing activity by alveolar macrophages with subsequent degranulation of local mast cells. Mast cell discharge of inflammatory mediators (including neutrophil chemotactic factors and perhaps the elastolytic protease) could then participate in the destruction of alveolar walls.     

Angiotensin II receptor inhibition prevents pneumocyte apoptosis in surfactant-depleted rat lungs

Pneumocyte apoptosis is implicated in the pathophysiology of acute inflammatory lung injuries in newborns and adults. Pulmonary angiotensin (ANG) II contributes to lung epithelial apoptosis in vitro, but its role in acute lung injury in vivo is unclear. We therefore studied the effects of ANG II receptor action on the pulmonary inflammatory and apoptotic changes in surfactant-depleted lungs in rats. Lung injury was induced by repeated lung lavage with saline, and the rats were then ventilated with 60% oxygen for 1, 3, or 5 hr. Separate groups of rats were pretreated with a nonspecific ANG II receptor inhibitor saralasin, the specific ANG II type 1 receptor antagonist losartan, or ANG II type 2 receptor inhibitor PD123319, and were similarly studied. Lungs were studied histologically for tissue injury, and with terminal deoxynucleodityl transferase-mediated dUTP nick end-labeling (TUNEL) and cleaved caspase 3 antibody staining, and by electron microscopy for apoptotic cell death. Surfactant-depleted lungs showed an increased number of TUNEL-positive epithelial cells throughout the study, and intrapulmonary leukocyte migration and histological tissue injury scores were similarly elevated, compared to controls, from 1-5 hr of ventilation. Pretreatment with saralasin or losartan significantly prevented the increase of TUNEL positivity in pneumocytes, but had no effect on the amount of neutrophil influx or total injury score in lavaged lungs. In contrast, administration of PD123319 did not affect the number of TUNEL-positive epithelial cells or histological injury . The results suggest that increased epithelial apoptosis in surfactant-deficient lungs is mediated by ANG II receptor (specifically, subtype 1) action.     

The role of cytokine-induced neutrophil chemoattractant-1 in neutrophil-mediated remote lung injury after intestinal ischaemia/reperfusion in rats

OBJECTIVE: Remote lung injury is induced by ischaemia/reperfusion (I/R) of the gastrointestinal tract and the liver following hypovolaemic shock. In the present study, the role of cytokine-induced neutrophil chemoattractant (CINC), a member of the interleukin (IL)-8 family, in neutrophil-mediated remote lung injury following intestinal I/R was investigated in anaesthetized rats. METHODOLOGY: The I/R group was subjected to 60 min of occlusion of the superior mesenteric artery with laparotomy, followed by 240 min of intestinal reperfusion. The sham-operated (sham) group was subjected to the same procedures with the exception of intestinal I/R. RESULTS: In the I/R group, the permeability index of the lung, the neutrophil count in pulmonary vascular lavage fluid and bronchoalveolar lavage fluid (BALF), lung myeloperoxidase activity and neutrophil oxidative production were all significantly greater than those in the sham group. Cytokine-induced neutrophil chemoattractant-1 levels in blood and BALF were significantly increased at 240 min after intestinal reperfusion. There was a significant relationship between neutrophils in BALF and CINC-1 level in BALE CONCLUSION: These findings suggest that intestinal reperfusion was associated with activation and accumulation of neutrophils in the lung and resulted in remote lung injury with increased microvascular permeability. Thus, CINC-1 in BALF may induce neutrophil migration from the pulmonary vessels to the interstitium and alveolar spaces in remote lung injury after intestinal I/R.     

Bronchoalveolar lavage fluid from normal rats stimulates DNA synthesis in rat alveolar type II cells

Proliferation of alveolar type II cells after lung injury is important for the restoration of the alveolar epithelium. Bronchoalveolar lavage fluid (BALF) may represent an important source of growth factors for alveolar type II cells. To test this possibility, BALF fluid was collected from normal rats, concentrated 10-fold by Amicon filtration, and tested for its ability to stimulate DNA synthesis in rat alveolar type II cells in primary culture. BALF induced a dose-dependent increase in type II cell DNA synthesis resulting in a 6-fold increase in [3H]thymidine incorporation. Similar doses also stimulated [3H]thymidine incorporation into rat lung fibroblasts by 6- to 8-fold. Removal of pulmonary surface active material by centrifugation did not significantly reduce the stimulatory activity of BALF for type II cells. The stimulation of type II cell DNA synthesis by BALF was reduced by 100% after heating at 100 degrees C for 10 min, and by approximately 80% after reduction with dithiothreitol, and after trypsin treatment. Dialysis of BALF against 1 N acetic acid resulted in a 27% reduction in stimulatory activity. The effect of BALF in promoting type II cell DNA synthesis was more pronounced when tested in the presence of serum, although serum itself has very little effect on type II cell DNA synthesis. When BALF was tested in combination with other substances that stimulate type II cell DNA synthesis (cholera toxin, insulin, epidermal growth factor, and acidic fibroblast growth factor), additive effects or greater were observed. When BALF was chromatographed over Sephadex G150, the activity eluted with an apparent molecular weight of 100 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increase in the beta chain of hepatocyte growth factor (HGF beta) precedes c-met expression after bleomycin-induced lung injury in the rat

After lung injury the regeneration of the alveolar epithelium is highly dependent on the proliferation of type II alveolar epithelial cells (AECs). Hepatocyte growth factor (HGF), a potent epithelial cell mitogen, is present as a single chain in normal tissue, but after injury HGF is converted to an active form composed of an alpha and a beta chain. In this study it was demonstrated that there was an increase in the beta chain of HGF 4 days after bleomycin administration, coinciding with the time of maximal type II AEC proliferation. Bronchoalveolar lavage fluid (BALF) obtained 4 days after bleomycin administration was maximally mitogenic to L2 cells, a nontransformed rat alveolar epithelial cell line. Type II cells isolated from normal rats do not express the HGF receptor, c-met. However, 4 days after bleomycin injury, using Western blot analysis, an increase in c-met was detected in AEC protein extracts. HGF induced c-met expression by L2 cells and neutralizing antibodies to HGF inhibited the mitogenic activity in the BALF. These findings suggest that HGF may regulate its own receptor on AECs and is an important mitogen for AECs 4 days after bleomycin administration.     

Modulating the alveolar milieu to enhance resolution of fibrotic lung injury

Fibrotic lung injury is often attributed to a myriad of factors, including environmental exposure, age, genetic predisposition, epigenetics, coexisting conditions, acute lung injury, and viral infection. No effective therapies, other than lung transplantation, have proven effective against lung fibrosis. Loss of cellular homeostasis mechanisms in alveolar epithelial type I cells and any inability of type II progenitor cells to resist and repair epithelial injury are indicators that impaired response to injury and regeneration is a critical component of this disorder. The alveolar epithelium has a limited repertoire of responses to injury, which are dictated by the alveolar milieu, a repository of cytokines and growth factors that affect recruitment of other cells to the site of injury, or the proliferation of resident cells at the site of injury. The identification and characterization of the cytokines, growth factors, and other biomarkers that dictate the response to disease is key to understanding, diagnosing, treating, and determining the trajectory of various lung disorders. Corrective therapy of the alveolar milieu may therefore prove to be beneficial in many presently serious and incurable lung diseases that likely begin and progress with injury to the alveolar epithelium.     

Idiopathic pulmonary fibrosis. Abnormalities in bronchoalveolar lavage fluid phospholipids

Bronchoalveolar lavage has been used to sample cells and proteins in the distal lung. One of the major secretory products of the alveolar type II epithelial cells, pulmonary surfactant, can be recovered by lavage. Abnormalities in alveolar type II cells are found in biopsies of patients with idiopathic pulmonary fibrosis (IPF), and abnormalities of pulmonary surfactant phospholipids have been reported after diffuse lung injury in animals and in humans. Therefore, we questioned if abnormalities in lavage phospholipids might also occur in IPF, a chronic inflammatory disease of the alveolar epithelium and interstitium, and, if present, would these abnormalities reflect histopathologic changes or predict responsiveness to therapy. Fifteen untreated patients with IPF, diagnosed by open lung biopsy, were studied and were found to have less than half the amount of bronchoalveolar lavage phospholipid as that recovered from healthy volunteers (p less than 0.05). In addition, patients with IPF had a lower proportion of phosphatidylglycerol and a higher proportion of phosphatidylinositol in the recovered phospholipids than did healthy volunteers (p less than 0.05). The severity of these alterations in phospholipid composition correlated with more advanced fibrotic histopathologic changes. Patients with less depression of total phospholipids in lavage improved with corticosteroid therapy, whereas the patients with more severely decreased total phospholipid recovered in lavage did not.