Hyaluronate and type III procollagen peptide concentrations in bronchoalveolar lavage fluid as markers of disease activity in farmer's lung.

Ten patients were studied during an acute episode of farmer''s lung. Prominent findings were an impaired diffusion capacity (on average only 51% of predicted) and substantially increased amounts of hyaluronate and type III procollagen peptide recovered during bronchoalveolar lavage; mean concentrations of these constituents in lavage fluid were 547 (range 137-1125) and 9.7 (2.8-19.4) micrograms/l, respectively. In bronchoalveolar lavage fluid from healthy controls (n = 21) hyaluronate concentrations were less than 15 micrograms/l and procollagen peptide concentrations less than 0.2 micrograms/l. Lavage fluid concentrations of these potential markers of fibroblast activation declined during the recovery phase of farmer''s lung; four to 10 weeks after admission (n = 7) mean concentrations of hyaluronate and procollagen peptide were 154 (range 38-650) and 4.4 (0.6-15.8) micrograms/l, respectively. At clinical remission six to 14 months after admission concentrations of these markers had returned almost to normal, though slightly increased concentrations were still evident in about half the patients (n = 7). At that time lung volumes were normal but diffusion capacity remained slightly subnormal. It was concluded that in farmer''s lung release of hyaluronate and type III procollagen peptide reflects activity of the disease. Increased synthesis of these connective tissue components continuing in a patient avoiding mouldy plant material may signal an increased risk of developing fibrotic lung disease. The abnormal accumulation of hyaluronate in the smaller airways in acute farmer''s lung may be expected to immobilize water and thereby provide a possible mechanism of the interstitial inflammatory lung oedema with associated impaired gas diffusion. This hypothesis is supported by the relation found between hyaluronate in lavage fluid and reduced diffusion capacity.     

Asbestos bodies in bronchoalveolar lavage fluid. A study of 20 asbestos-exposed individuals and comparison to patients with other chronic interstitial lung diseases

We studied the asbestos body (AB) content of bronchoalveolar lavage fluid from 20 patients with a history of occupational asbestos exposure, 31 patients with sarcoidosis and 5 patients with idiopathic pulmonary fibrosis. The cellular lavage pellet was digested in sodium hypochlorite and filtered onto Nuclepore filters for AB quantification by light microscopy. ABs were found in 15 of 20 asbestos-exposed individuals, 9 of 31 sarcoidosis cases and 2 of 5 patients with idiopathic pulmonary fibrosis. There was a statistically significant difference in the number of ABs per million cells recovered or per milliliter of recovered lavage fluid in the asbestos-exposed group as compared to the other categories of chronic interstitial lung disease. The highest levels occurred in patients with asbestosis. Large numbers of asbestos bodies in the lavage fluid (greater than 1 AB/10(6) cells) were indicative of considerable occupational asbestos exposure, whereas occasional bodies were a nonspecific finding.     

Soluble Vascular Cell Adhesion Molecule-1 (sVCAM-1) Is Elevated in Bronchoalveolar Lavage Fluid of Patients with Acute Respiratory Distress Syndrome

Introduction

Pulmonary vascular endothelial activation has been implicated in acute respiratory distress syndrome (ARDS), yet little is known about the presence and role of endothelial activation markers in the alveolar space in ARDS. We hypothesized that endothelial activation biomarkers would be differentially expressed in bronchoalveolar lavage fluid from patients with ARDS compared with healthy volunteers, and that biomarker concentrations would be associated with ARDS severity.

Methods

We performed a cross-sectional analysis of data from 26 intubated patients with ARDS undergoing evaluation for clinically suspected ventilator-associated pneumonia and five healthy volunteers. Patients underwent bronchoalveolar lavage a median of five days after intubation. Healthy volunteers also underwent bronchoalveolar lavage. Endothelial activation biomarkers (soluble vascular cell adhesion molecule-1 [sVCAM-1], soluble endothelial selectin [sESEL], angiopoietin-1 [Ang-1] and angiopoietin-2 [Ang-2]) were measured in bronchoalveolar lavage fluid. Clinically suspected ventilator-associated pneumonia was confirmed with microbiologic culture data.

Results

Patients with ARDS had significantly higher median sVCAM-1 concentrations in the bronchoalveolar lavage fluid compared with healthy volunteers (985 vs 119 pg/mL, p = 0.03). Additionally, there was a trend toward greater bronchoalveolar lavage fluid sVCAM-1 concentrations among patients with moderate/severe compared to mild ARDS (1395 vs 209 pg/mL, p = 0.06). We did not detect significant differences in bronchoalveolar lavage fluid levels of sESEL, Ang-1 or Ang-2 between patients with ARDS and healthy volunteers. Median bronchoalveolar lavage fluid biomarker levels did not differ between patients with and without microbiologically-confirmed ventilator-associated pneumonia.

Conclusions

sVCAM-1 concentrations were significantly higher in the bronchoalveolar lavage fluid of patients with ARDS compared to healthy controls, and tended to be higher in moderate/severe ARDS compared to mild ARDS. Our findings add to the growing evidence supporting the concept that endothelial activation plays an important mechanistic role in the pathogenesis of ARDS. Further studies are necessary to characterize the role and/or clinical significance of sVCAM-1 and other endothelial activation markers present in the alveolar space in ARDS.     

Spontaneous resolution of pulmonary edema caused by short periods of cyclic overinflation.

Mechanical ventilation with high or even moderate peak inspiratory pressure produces pulmonary permeability edema. Besides the level of overinflation, duration may affect both severity and type of edema. We studied the effect of 2 min of 35-mmHg peak pressure mechanical ventilation (HV) on microvascular permeability and deep lung fluid balance in rats. It resulted in increased extravascular lung water (+50%), bloodless dry lung weight (+25%), and albumin uptake in lungs (+450%). The increase in dry lung weight and albumin uptake compared with that of lung water suggested major permeability alterations. Ultrastructural examination showed the presence of numerous endothelial blebs. Epithelial lining fluid (ELF) volume, its potassium and protein concentrations, and cellular composition were assessed by bronchoalveolar lavage. There was an increase in ELF volume (+180%), a decrease in ELF potassium concentration (-50%), and an increase in ELF protein content (+76%). A few blood cells were recovered, suggesting the presence of a few large epithelial breaks. Some animals were allowed to recover for periods less than or equal to 180 min after HV. Extravascular lung water, dry lung weight, and albumin distribution space returned to control levels within 45 min. ELF volume diminished but remained larger than in controls, and ELF protein concentration increased probably because of alveolar fluid resorption. No further hemorrhage was observed. These results indicate that periods of HV as short as 2 min transiently alter microvascular permeability in rats.     

Vascular endothelial growth factor in patients with high-altitude pulmonary edema.

To examine the role of VEGF in the pathogenesis of high-altitude pulmonary edema (HAPE), we measured the concentrations of VEGF in venous serum and bronchoalveolar lavage fluid in patients with HAPE and in healthy volunteers. The VEGF in venous serum of the patients was normal at admission and significantly increased at recovery. Similarly, the VEGF in bronchoalveolar lavage fluid of the patients was increased at recovery compared with admission, but values at both admission and recovery were significantly lower than those of the controls. The present finding suggests that VEGF probably is destroyed in the lung of HAPE, and it appears less likely to have a critical part in the pathogenesis of HAPE but has rather an important role in the repair process for the impaired cell layer.     

Leukocyte immunophenotypes in bronchoalveolar lavage fluid and peripheral blood of paracoccidioidomycosis, sarcoidosis and silicosis.

Leukocyte subsets in bronchoalveolar lavage (BAL) fluid and peripheral blood of patients with paraccoccidioidomycosis, sarcoidosis and silicosis were characterized using monoclonal antibodies and an immunoperoxidase technique. In paraccocidioidomycosis, the number of T-helper/inducer CD4-positive lymphocytes was lower in peripheral blood than in BAL fluid. Additional analysis showed that the expression of HLA-DR was very similar in alveolar macrophages, lung and blood T-cells. In sarcoidosis and silicosis there were higher proportions of T-helper/inducer cells in peripheral blood than in BAL fluid. The alterations in the T-helper/inducer/T-suppressor/cytoxic CD4/CD8 ratio in sarcoidosis and silicosis were more appreciable in peripheral blood than in BAL fluid, contrasting with the results in paracoccidioidomycosis. The expression of HLA-DR by alveolar macrophages in sarcoidosis was the highest of all the disease studied. No statistically significant differences were observed between chronic multifocal and chronic unifocal paracoccidioidomycosis disease, stage II and stage III sarcoidosis, and chronic and accelerated silicosis. The three granulomatous diseases analyzed had a few alveolar macrophages expressing the CD4 molecule on their surface. These findings and the technique of analyzing both peripheral blood and BAL leukocyte subsets may help to understand the pathogenesis of interstitial lung diseases.     

Lung injury in the neonatal piglet caused by hyperoxia and mechanical ventilation

Neonatal lung injury from hyperoxia and mechanical hyperventilation was studied in newborn piglets hyperventilated (arterial PCO2 15-20 Torr) for 24-48 h with 100% O2 and compared with unventilated controls. Pulmonary function testing was performed, and biochemical indicators of lung injury were analyzed from tracheobronchial aspirates at 0, 24, and 48 h. Lung sections were obtained for light and electron microscopy, and bronchoalveolar lavage fluid was analyzed for surfactant composition and activity. At 24 h significant changes in tracheobronchial aspirate albumin concentrations (up 78%) and percent of polymorphonuclear cells (up 16%) were demonstrated. At 48 h a 35% decrease in dynamic lung compliance (P less than 0.05) and a 36% increase in pulmonary resistance (P less than 0.05) were noted. Further biochemical abnormalities occurred with total cell counts increased by 271% (P less than 0.02), albumin 163% (P less than 0.05), total protein 217% (P less than 0.01), and elastase 108% (P less than 0.02). Pathological analyses revealed mild lung injury at 24 h and marked inflammation, abnormal inflation patterns, flattening of Clara cells, fibrinous exudate and edema, early collagen formation, and cell necrosis observed at 48 h. Bronchoalveolar lavage surfactant had normal biophysical activity. Results demonstrate that exposure of neonatal piglets to O2 and mechanical hyperventilation for 48 h cause severe progressive lung injury.     

Analysis of Trace Elements in Bronchoalveolar Lavage of Patients with Diffuse Lung Diseases

Airborne trace elements are implicated in the etio-pathogenesis of a large number of pulmonary diseases. The aim of this study was to evaluate the reliability and effectiveness of direct determination of Cd, Cr, Cu, Fe, Mn, Ni, Pb, V, and Zn concentrations in bronchoalveolar lavage (BAL) samples from patients with sarcoidosis, idiopathic pulmonary fibrosis, and Langerhans cell histiocytosis and healthy (smoking and non-smoking) controls. A total of 44 individuals were recruited among sarcoidosis, idiopathic pulmonary fibrosis, and Langerhans cell histiocytosis patients and healthy (smoking and non-smoking) controls. Average Mn concentrations in BAL from patients were 45% lower than in controls (p < 0.01) and remarkable decreases in average concentrations of Cr, Ni and Zn were also found in BAL from patients with idiopathic pulmonary fibrosis and Langerhans cell histiocytosis. As these diseases are characterized by the enhanced activation of certain immunomodulatory cells and by generation of free radicals, the depressed Mn, Zn, Cr and Ni concentrations in BAL from patients may be due to oxidative stress. These preliminary results indicate that assessment of the elemental composition of BAL is a promising approach to study the pathogenesis of diffuse lung diseases and Langerhans cell histiocytosis.     

Bronchoalveolar lavage in interstitial lung disease: effect of volume of fluid infused

To evaluate the effect of varying infusate volume on the results of bronchoalveolar lavage (BAL) in patients with interstitial lung disease, 55 patients underwent 58 BAL during which both a 100- and 250-ml lavage was performed in the same lobe of the lung. Although the percent of the fluid that was returned and the total numbers of cells were greater in the 250- vs. the 100-ml lavage, there were no significant differences in cell differentials or numbers of cells per milliliter between the 100- and 250-ml BAL. We conclude that infusate volume does not affect cell differentials or numbers of cells per milliliter of bronchoalveolar lavage fluid in patients with interstitial lung disease.     

Bronchoalveolar mast cells in extrinsic asthma: a mechanism for the initiation of antigen specific bronchoconstriction.

Bronchoalveolar lavage performed in 10 patients with extrinsic asthma and 14 controls yielded similar recoveries of fluid and cells. Mast cells and eosinophils, however, formed a greater proportion of the cells recovered from the asthmatic subjects (p less than 0.001 for mast cells; p less than 0.01 for eosinophils), the histamine content of the lavage cells being correspondingly increased (p less than 0.01). Both the percentage of mast cells and the histamine content of lavage cells were significantly inversely correlated with the forced expiratory volume in one second (FEV1; expressed as percentage of predicted) and with the ratio of FEV1 to forced vital capacity before lavage. There was also a significant inverse correlation between the concentration of histamine required to produce a 20% fall in FEV1 and the percentage of mast cells recovered (p less than 0.05). When incubated with antihuman IgE bronchoalveolar mast cells from asthmatic subjects released a significantly increased proportion of total cellular histamine than cells from control subjects at all effective doses of anti-IgE. By contrast, dose response curves for IgE dependent histamine release from peripheral blood leucocytes were similar in asthmatics and controls. Specific antigen led to release of histamine from bronchoalveolar cells and peripheral blood leucocytes of asthmatic subjects but not controls. Lying superficially within the airways, bronchoalveolar mast cells would be readily exposed to inhaled antigen and would release mediators directly on to the airway surface. Their immunological response suggests that they are likely to be important in the pathogenesis of airflow obstruction in asthma.     

Effect of adenosine A2A receptor activation in murine models of respiratory disorders

Activation of the adenosine A(2A) receptor has been postulated as a possible treatment for lung inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD). In this report, we have studied the anti-inflammatory properties of the reference A(2A) agonist CGS-21680, given intranasally at doses of 10 and 100 microg/kg, in a variety of murine models of asthma and COPD. After an acute ovalbumin challenge of sensitized mice, prophylactic administration of CGS-21680 inhibited the bronchoalveolar lavage fluid inflammatory cell influx but not the airway hyperreactivity to aerosolized methacholine. After repeated ovalbumin challenges, CGS-21680 given therapeutically inhibited the bronchoalveolar lavage fluid inflammatory cell influx but had no effect on the allergen-induced bronchoconstriction, the airway hyperreactivity, or the bronchoalveolar lavage fluid mucin levels. As a comparator, budesonide given intranasally at doses of 0.1-1 mg/kg fully inhibited all the parameters measured in the latter model. In a lipopolysaccharide-driven model, CGS-21680 had no effect on the bronchoalveolar lavage fluid inflammatory cell influx or TNF-alpha, keratinocyte chemoattractant, and macrophage inflammatory protein-2 levels, but potently inhibited neutrophil activation, as measured by bronchoalveolar lavage fluid elastase levels. With the use of a cigarette smoke model of lung inflammation, CGS-21680 did not significantly inhibit bronchoalveolar lavage fluid neutrophil infiltration but reversed the cigarette smoke-induced decrease in macrophage number. Together, these results suggest that activation of the A(2A) receptor would have a beneficial effect by inhibiting inflammatory cell influx and downregulating inflammatory cell activation in asthma and COPD, respectively.     

Tracheal insufflation of tumor necrosis factor protects rats against oxygen toxicity

Tracheal insufflation of tumor necrosis factor (TNF; 5 micrograms or 1.2 x 10(5) U) markedly enhanced the survival of adult rats exposed to 100% O2: 12 of 17 rats (71%) survived for greater than 11 days, whereas 30 of 30 control (Hanks' balanced salt solution) insufflated rats (100%) died within 3 days of O2 exposure. Insufflation of gamma-interferon (5 micrograms) or intraperitoneal injection of up to 40 micrograms TNF did not afford any protection. At 55 h after O2 exposure, TNF-insufflated rats showed less pulmonary edema, as determined by the extravascular lung water content-to-bloodless lung dry weigh ratio and less alveolar capillary leak as determined by the protein content in the bronchoalveolar lavage fluid, than control insufflated rats similarly exposed. This protection against O2 toxicity by TNF insufflation was associated with increased lung superoxide dismutase, catalase, and glutathione peroxidase activities. The enhancement of lung antioxidant enzyme activities was noted at 55 h of O2 exposure, when control animals began to die of O2 toxicity. This temporal relationship suggests that TNF-induced increase in antioxidant enzyme activities contributes, at least in part, to the observed protection.     

Determinants of restrictive lung function in asbestos-induced pleural fibrosis

We evaluated whether restrictive lung function among asbestos-exposed individuals with pleural fibrosis was caused by radiographically inapparent parenchymal inflammation and/or parenchymal fibrosis. All 24 study participants were sheet metal workers who were nonsmokers with normal parenchyma on posteroanterior chest radiograph. These subjects had either normal pleura (n = 7), circumscribed plaques (n = 9), or diffuse pleural thickening (n = 8). After controlling for age, years in the trade, and pack-years of smoking, we found that sheet metal workers with diffuse pleural thickening had a lower forced vital capacity (P less than 0.001), total lung capacity (P less than 0.01), and CO-diffusing capacity of the lung (P less than 0.05) than those with normal pleura. Similarly, sheet metal workers with circumscribed plaques were found to have a reduced forced vital capacity; however, because of the small number of study subjects, this difference (regression coefficient = -11.0) was only marginally significant (P = 0.06). Although circumscribed plaque and diffuse pleural thickening were both associated with a lymphocytic alveolitis and a higher prevalence of parenchymal fibrosis on high-resolution computerized tomography (HRCT) scan, neither a lymphocytic alveolitis nor the finding of parenchymal fibrosis on HRCT scan influenced the relationship between pleural fibrosis and restrictive lung function. We conclude that pleural fibrosis is associated with restrictive lung function and abnormally low diffusion that appears to be independent of our measures of parenchymal injury (chest X-ray, bronchoalveolar lavage, and HRCT scan).(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid metabolism in rabbits exposed to paraquat aerosol

In order to examine the effects of paraquat on pulmonary lipid metabolism rabbits were exposed to double distilled water by aerosol, or 250 mg paraquat in double distilled water. One hour prior to sacrifice, a group of rabbits were injected with [2?¹⁴C]-acetate. The levels of phospholipids, fatty acids, cholesterol, and triglycerides were determined as were their ¹⁴C-contents in the lung, broncoalveolar lavage fluid, serum, and liver. The serum of paraquat-exposed animals contained significantly increased levels of phospholipids, cholesterol, and triglycerides. Liver, lung, and bronchoalveolar lavage contained less than or equal to control levels of phospholipids, cholesterol, and triglycerides. Percent of palmitate in the hepatic fatty acid profile was slightly increased in liver but not in lung. The source of the increased lipids in the serum is unknown.     

Procoagulant,Tissue Factor-Bearing Microparticles in Bronchoalveolar Lavage of Interstitial Lung Disease Patients: An Observational Study

Coagulation factor Xa appears involved in the pathogenesis of pulmonary fibrosis. Through its interaction with protease activated receptor-1, this protease signals myofibroblast differentiation in lung fibroblasts. Although fibrogenic stimuli induce factor X synthesis by alveolar cells, the mechanisms of local posttranslational factor X activation are not fully understood. Cell-derived microparticles are submicron vesicles involved in different physiological processes, including blood coagulation; they potentially activate factor X due to the exposure on their outer membrane of both phosphatidylserine and tissue factor. We postulated a role for procoagulant microparticles in the pathogenesis of interstitial lung diseases. Nineteen patients with interstitial lung diseases and 11 controls were studied. All subjects underwent bronchoalveolar lavage; interstitial lung disease patients also underwent pulmonary function tests and high resolution CT scan. Microparticles were enumerated in the bronchoalveolar lavage fluid with a solid-phase assay based on thrombin generation. Microparticles were also tested for tissue factor activity. In vitro shedding of microparticles upon incubation with H₂O₂ was assessed in the human alveolar cell line, A549 and in normal bronchial epithelial cells. Tissue factor synthesis was quantitated by real-time PCR. Total microparticle number and microparticle-associated tissue factor activity were increased in interstitial lung disease patients compared to controls (84±8 vs. 39±3 nM phosphatidylserine; 293±37 vs. 105±21 arbitrary units of tissue factor activity; mean±SEM; p<.05 for both comparisons). Microparticle-bound tissue factor activity was inversely correlated with lung function as assessed by both diffusion capacity and forced vital capacity (r² = .27 and .31, respectively; p<.05 for both correlations). Exposure of lung epithelial cells to H₂O₂ caused an increase in microparticle-bound tissue factor without affecting tissue factor mRNA.Procoagulant microparticles are increased in interstitial lung diseases and correlate with functional impairment. These structures might contribute to the activation of factor X and to the factor Xa-mediated fibrotic response in lung injury.     

Reactive oxygen species and elastase mediate lung permeability after acid aspiration.

Acid aspiration leads to increased neutrophil (PMN) oxidative metabolism, an event associated with lung leukosequestration and permeability increase. Neutropenia protected the vascular barrier function against acid injury. This study tests whether active oxygen species and elastase (which are presumably released by adherent PMNs) affect the microvascular barrier. Anesthetized rats underwent tracheostomy and insertion of a cannula into a lung segment. This was followed by localized instillation of 0.1 N HCl (n = 18) or saline (n = 18). Sequestration of PMNs in acid-aspirated and nonaspirated segments was 77 and 46 PMNs/high-power field (HPF), respectively, which was higher than control values of 11 and 8 PMNs/10 HPF in saline-aspirated and nonaspirated regions (P less than 0.05). Acid aspiration was associated with increased protein concentration in bronchoalveolar lavage (BAL) fluid to 3,550 and 2,900 micrograms/ml in the aspirated and nonaspirated lungs, respectively, which were higher than control values of 420 and 400 micrograms/ml (P less than 0.05). Acid aspiration also led to increased lung wet-to-dry weight ratios (W/D) of 6.6 and 5.4, which were higher than control values of 3.4 and 3.3 (P less than 0.05). Intravenous treatment of rats (n = 18) 90 min after aspiration with scavengers of reactive oxygen species, superoxide dismutase (1,500 U/kg), and catalase (5,000 U/kg), both conjugated to polyethylene glycol, did not reduce PMN sequestration but attenuated acid aspiration-induced increase in protein accumulation in BAL fluid in the aspirated and nonaspirated segments (990 and 610 micrograms/ml) as well as the increased lung W/D (4.6 and 4.0; all P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

MMP1 and MMP7 as potential peripheral blood biomarkers in idiopathic pulmonary fibrosis

Background

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic lung disease associated with substantial morbidity and mortality. The objective of this study was to determine whether there is a peripheral blood protein signature in IPF and whether components of this signature may serve as biomarkers for disease presence and progression.

Methods and Findings

We analyzed the concentrations of 49 proteins in the plasma of 74 patients with IPF and in the plasma of 53 control individuals. We identified a combinatorial signature of five proteins—MMP7, MMP1, MMP8, IGFBP1, and TNFRSF1A—that was sufficient to distinguish patients from controls with a sensitivity of 98.6% (95% confidence interval [CI] 92.7%–100%) and specificity of 98.1% (95% CI 89.9%–100%). Increases in MMP1 and MMP7 were also observed in lung tissue and bronchoalveolar lavage fluid obtained from IPF patients. MMP7 and MMP1 plasma concentrations were not increased in patients with chronic obstructive pulmonary disease or sarcoidosis and distinguished IPF compared to subacute/chronic hypersensitivity pneumonitis, a disease that may mimic IPF, with a sensitivity of 96.3% (95% CI 81.0%–100%) and specificity of 87.2% (95% CI 72.6%–95.7%). We verified our results in an independent validation cohort composed of patients with IPF, familial pulmonary fibrosis, subclinical interstitial lung disease (ILD), as well as with control individuals. MMP7 and MMP1 concentrations were significantly higher in IPF patients compared to controls in this cohort. Furthermore, MMP7 concentrations were elevated in patients with subclinical ILD and negatively correlated with percent predicted forced vital capacity (FVC%) and percent predicted carbon monoxide diffusing capacity (DL_CO%).

Conclusions

Our experiments provide the first evidence for a peripheral blood protein signature in IPF to our knowledge. The two main components of this signature, MMP7 and MMP1, are overexpressed in the lung microenvironment and distinguish IPF from other chronic lung diseases. Additionally, increased MMP7 concentration may be indicative of asymptomatic ILD and reflect disease progression.     

Pulmonary bioavailability of ascorbic acid in an ascorbate-synthesising species,the horse

Vitamin C (ascorbic acid) is a non-enzymatic antioxidant important in protecting the lung against oxidative damage and is decreased in lung lining fluid of horses with airway inflammation. To examine possible therapeutic regimens in a species with ascorbate-synthesising capacity, we studied the effects of oral supplementation of two forms of ascorbic acid, (each equivalent to 20 mg ascorbic acid per kg body weight) on the pulmonary and systemic antioxidant status of six healthy ponies in a 3 x 3 Latin square design. Two weeks supplementation with ascorbyl palmitate significantly increased mean plasma ascorbic acid concentrations compared to control (29 +/- 5 and 18 +/- 7 micromol/l, respectively; p < 0.05). Calcium ascorbyl-2-monophosphate, a more stable form of ascorbic acid, also increased mean plasma ascorbic acid concentrations, but not significantly (23 +/- 1 micromol/l; p = 0.07). The concentration of ascorbic acid in bronchoalveolar lavage fluid increased in five out of six ponies following supplementation with either ascorbyl palmitate or calcium ascorbyl-2-monophosphate compared with control (30 +/- 10, 25 +/- 4 and 18 +/- 8 micromol/l, respectively; p < 0.01). Neither supplement altered the concentration of glutathione, uric acid or alpha-tocopherol in plasma or bronchoalveolar lavage fluid. In conclusion, the concentration of lung lining fluid ascorbic acid is increased following ascorbic acid supplementation (20 mg/kg body weight) in an ascorbate-synthesising species.     

Diminished injury in hypotransferrinemic mice after exposure to a metal-rich particle

Using the hypotransferrinemic (Hp) mouse model, we studied the effect of altered iron homeostasis on the defense of the lung against a catalytically active metal. The homozygotic (hpx/hpx) Hp mice had greatly diminished concentrations of both serum and lavage fluid transferrin relative to wild-type mice and heterozygotes. Fifty micrograms of a particle containing abundant concentrations of metals (a residual oil fly ash) was instilled into wild-type mice and heterozygotic and homozygotic Hp animals. There was an oxidative stress associated with particle exposure as manifested by decreased lavage fluid concentrations of ascorbate. However, rather than an increase in lung injury, diminished transferrin concentrations in homozygotic Hp mice were associated with decreased indexes of damage, including concentrations of relevant cytokines, inflammatory cell influx, lavage fluid protein, and lavage fluid lactate dehydrogenase. Comparable to other organs in the homozygotic Hp mouse, siderosis of the lung was evident, with elevated concentrations of lavage fluid and tissue iron. Consequent to these increased concentrations of iron, proteins to store and transport iron, ferritin, and lactoferrin, respectively, were increased when assayed by immunoprecipitation and immunohistochemistry. We conclude that the lack of transferrin in Hp mice did not predispose the animals to lung injury after exposure to a particle abundant in metals. Rather, these mice demonstrated a diminished injury that was associated with an increase in the metal storage and transport proteins.