Bronchoalveolar Lavage Markers Research Articles

Feasibility study of aerosolized prostaglandin E1 microspheres as a noninvasive therapy for pulmonary arterial hypertension

This study was designed to test the feasibility of polymeric microspheres as an inhalable carrier for prostaglandin E1 (PGE1) for treatment of pulmonary arterial hypertension. Poly(lactic‐co‐glycolic acid) (PLGA) microspheres were prepared by a double emulsion–solvent evaporation method. Six different microspheric formulations were prepared using two different blends of PLGA (50:50 and 85:15) and varying concentrations of polyvinyl alcohol (PVA) in the external aqueous phase (EAP). The particles were characterized for morphology, size, aerodynamic diameter, entrapment efficiency, release patterns, and metabolic stability. Pulmonary absorption was studied in a rat model, and safety of the formulations was evaluated by measuring cytotoxicity in Calu‐3 cells and assessing injury markers in bronchoalveolar lavage (BAL) fluid. Both actual particle size and aerodynamic diameter of the formulations decreased with increasing PVA concentration. The mass median aerodynamic diameter of the particles was within the respirable range. Entrapment efficiency increased with increasing PVA concentration; PLGA 85:15 showed better entrapment due to hydrophobic interactions with the drug. Compared to intravenously administered PGE1, microspheres prepared with PLGA 85:15 produced a 160‐fold increase in the half‐life of PGE1 following pulmonary administration. Although plain PGE1 showed rapid degradation in rat lung homogenate, PGE1 entrapped in the particles remained intact for about 8 h. Optimized formulations were demonstrated to be safe, based on analysis of cytotoxicity and lung‐injury markers in BAL fluid. Overall, the data suggest that microspheric PGE1 formulations have the potential to be used as a noninvasive and controlled‐release alternative to the current medications used for treatment of pulmonary arterial hypertension that are administered by continuous infusion or require multiple inhalations. © 2009 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 1774–1789, 2010

Impact of Topical Cooling Solution and Prediction of Pulmonary Graft Viability From Non–heart-beating Donors

Functional assessment of the potentially damaged graft from a non-heart-beating donor (NHBD) is mandatory for successful outcome after transplantation. We investigated the impact of the topical cooling solution on graft preservation and whether inflammatory markers in bronchoalveolar lavage (BAL) can predict pulmonary graft viability in a pig ex vivo lung perfusion model. Pigs were euthanized and left untouched for 1 (SAL-1, PER-1) or 3 (SAL-3, PER-3) hours. Topical cooling was done with saline or low-potassium dextran solution (Perfadex) for 1 or 3 hours. In the heart-beating donor control group, the lungs were flushed, explanted and stored for 4 hours. BAL samples were taken from right lungs after explantation and assessed for nitrite, interleukin-8 (IL-8) and protein levels. Left lungs were prepared for ex vivo evaluation. Hemodynamic and oxygenation parameters were measured. Pulmonary vascular resistance (PVR), oxygenation index and Pao(2)/Fio(2) ratio differed significantly between the SAL-3 (42.2 +/- 6.0, 15.9 +/- 3.2 and 148 +/- 14.6 Wood units, respectively) and PER-3 (23.9 +/- 2.7, 6.4 +/- 0.8 and 221.7 +/- 15.06 Wood units, respectively) groups (p < 0.05). BAL IL-8 levels were higher in the SAL-3 group compared with the PER-3 group. BAL nitrite and protein levels were statistically higher in the SAL-3 group (0.98 +/- 0.17 micromol/liter, 728.3 +/- 75.7 microg/ml) than in the PER-3 (0.22 +/- 0.09 micromol/liter, 393.3 +/- 51.1 microg/ml) group (p < 0.05) and correlated with an increase in PVR (r = 0.623, p = 0.001; r = 0.530, p = 0.006, respectively). After 3 hours of warm ischemia topical cooling with Perfadex resulted in better graft function. Nitrite and protein levels in BAL correlated well with PVR and may therefore be used as a non-invasive marker to predict graft function for NHBDs.

Nanoparticle inhalation augments particle-dependent systemic microvascular dysfunction

BackgroundWe have shown that pulmonary exposure to fine particulate matter (PM) impairs endothelium dependent dilation in systemic arterioles. Ultrafine PM has been suggested to be inherently more toxic by virtue of its increased surface area. The purpose of this study was to determine if ultrafine PM (or nanoparticle) inhalation produces greater microvascular dysfunction than fine PM. Rats were exposed to fine or ultrafine TiO2 aerosols (primary particle diameters of ~1 μm and ~21 nm, respectively) at concentrations which do not alter bronchoalveolar lavage markers of pulmonary inflammation or lung damage.ResultsBy histopathologic evaluation, no significant inflammatory changes were seen in the lung. However, particle-containing macrophages were frequently seen in intimate contact with the alveolar wall. The spinotrapezius muscle was prepared for in vivo microscopy 24 hours after inhalation exposures. Intraluminal infusion of the Ca2+ ionophore A23187 was used to evaluate endothelium-dependent arteriolar dilation. In control rats, A23187 infusion produced dose-dependent arteriolar dilations. In rats exposed to fine TiO2, A23187 infusion elicited vasodilations that were blunted in proportion to pulmonary particle deposition. In rats exposed to ultrafine TiO2, A23187 infusion produced arteriolar constrictions or significantly impaired vasodilator responses as compared to the responses observed in control rats or those exposed to a similar pulmonary load of fine particles.ConclusionThese observations suggest that at equivalent pulmonary loads, as compared to fine TiO2, ultrafine TiO2 inhalation produces greater remote microvascular dysfunction.

Endpoints for clinical trials in young children with cystic fibrosis.

The availability of sensitive, reproducible, and feasible outcome measures for quantifying lung disease in children with cystic fibrosis (CF) younger than 6 years is critical to the conduct of clinical trials in this important population. Historically, identifying and quantifying the presence of lung disease in very young children with CF was hampered by a lack of reproducible measures of lung function or lung pathology. Over the past 10 years, significant progress has led to physiologic, anatomic, and bronchoscopic measures that may serve as endpoints for future intervention trials. These endpoints include infant and preschool lung function testing, computed tomography of the chest, and bronchoalveolar lavage markers of inflammation and infection. Much progress has occurred in standardizing lung function testing, which is essential for multicenter collaboration. Pulmonary exacerbation has the potential to serve as a clinical endpoint; however, there is currently no standardized definition in children with CF younger than 6 years. Further development of these outcomes measures will enable clinical trials in the youngest CF population with the objective of improving long-term prognosis.

Comparison of Biomarkers in Exhaled Breath Condensate and Bronchoalveolar Lavage

Exhaled breath condensate (EBC) is increasingly studied as a noninvasive research method of sampling the lungs, measuring several biomarkers. The exact site of origin of substances measured in EBC is unknown, as is the clinical applicability of the technique. Special techniques might be needed to measure EBC biomarkers. To assess biomarker concentrations in clinical disease and investigate the site of origin of EBC, we compared EBC and bronchoalveolar lavage (BAL) biomarkers in 49 patients undergoing bronchoscopy for clinical indications. We measured exhaled nitric oxide, 8-isoprostane, hydrogen peroxide, total nitrogen oxides, pH, total protein, and phospholipid (n = 33) and keratin (n = 15) to assess alveolar and mucinous compartments, respectively. EBC was collected over 10 min using a refrigerated condenser according to European Respiratory Society/American Thoracic Society recommendations, and BAL performed immediately thereafter. 8-Isoprostane, nitrogen oxides, and pH were significantly higher in EBC than in BAL (3.845 vs. 0.027 ng/ml, 28.4 vs. 3.8 microM, and 7.35 vs. 6.4, respectively; p < 0.001). Hydrogen peroxide showed no difference between EBC and BAL (17.5 vs. 20.6 microM, p = not significant), whereas protein was significantly higher in BAL (33.8 vs. 183.2 microg/ml, p < 0.001). Total phospholipid was also higher in EBC, but keratin showed no difference. No significant correlation was found between EBC and BAL for any of the biomarkers evaluated either before or after correction for dilution. In clinical disease, markers of inflammation and oxidative stress are easily measurable in EBC using standard laboratory techniques and EBC is readily obtained. However, EBC and BAL markers do not correlate.

Acute Nose-Only Exposure of Rats to Phosgene. Part II. Concentration × Time Dependence of Changes in Bronchoalveolar Lavage During a Follow-Up Period of 3 Months

Groups of young adult male Wistar rats were acutely exposed to phosgene gas for either 30 or 240 min using a directed-flow nose-only mode of exposure. In 30-min exposed rats the concentrations were 0.94, 2.02, 3.89, 7.35, and 15.36 mg/m3, which relate to C × t products of 28.2, 60.6, 116.7, 220.5, and 460.8 mg/m3 × min. In 240-min exposed rats the concentrations were 0.96, 0.387, 0.786, 1.567, and 4.2 mg/m3, which relate C × t products of 47.0, 92.9, 188.6, 376, and 1008 mg/m3 × min. Six rats/group were sacrificed on postexposure days 1, 3, 7, 14, and 84, while the rats of the 1008 mg/m3 × min group where sacrificed on postexposure days 1, 7, 14, and 28. The focus of measurements was directed toward indicators of inflammatory response and increased transmucosal permeability in bronchoalveolar lavage (BAL), including lung weights. Lungs from rats sacrificed at the end of the postexposure period were additionally examined by histopathology. Mortality did not occur at any C × t product. The most pronounced changes were related to C × t-dependent increases in the following markers in BAL: protein, soluble collagen, polymorphonuclear leukocytes (PMN) counts, and alveolar macrophages with foamy appearance. These indicators were maximal on the first postexposure day, while total cell counts and alveolar macrophages containing increased phospholipids reached their climax around post-exposure day 3. At 1008 mg/m3 × min the most sensitive indicators in BAL, that is, protein, PMN, and collagen, resolved within 2 wk, whereas at lower C × t products they reached the level of the control by postexposure day 7. At 1008 mg/m3 × min (day 28), histopathology revealed a minimal to slight hypercellularity in terminal bronchioles with focal peribronchiolar inflammatory infiltrates and focal septal thickening. At lower C × t products (day 84) the rats from all groups were indistinguishable and Sirius red-stained lungs did not provide evidence of late-onset sequelae, such as fibrotic changes or collagen deposition. At similar C × t products the changes in BAL were slightly less pronounced using 30-min exposure periods when compared to 240-min exposure periods. In summary, the phosgene-induced transmucosal permeability caused a C × t-dependent increase of several BAL indicators, of which those of protein, PMN, and soluble collagen were most pronounced. Exposure intensities up to 116.7 mg/m3 × min did not cause changes different from those observed in controls, while at 188.6 mg/m3 × min distinct differences to the control existed. Despite the extensively increased airway permeability, histopathology did not provide evidence of lung tissue remodeling or irreversible sequelae.

Systemic Microvascular Dysfunction and Inflammation after Pulmonary Particulate Matter Exposure

The epidemiologic association between pulmonary exposure to ambient particulate matter (PM) and cardiovascular dysfunction is well known, but the systemic mechanisms that drive this effect remain unclear. We have previously shown that acute pulmonary exposure to PM impairs or abolishes endothelium-dependent arteriolar dilation in the rat spinotrapezius muscle. The purpose of this study was to further characterize the effect of pulmonary PM exposure on systemic microvascular function and to identify local inflammatory events that may contribute to these effects. Rats were intratracheally instilled with residual oil fly ash (ROFA) or titanium dioxide at 0.1 or 0.25 mg/rat 24 hr before measurement of pulmonary and systemic microvascular responses. In vivo microscopy of the spinotrapezius muscle was used to study systemic arteriolar responses to intraluminal infusion of the Ca2+ ionophore A23187 or iontophoretic abluminal application of the adrenergic agonist phenylephrine (PHE). Leukocyte rolling and adhesion were quantified in venules paired with the studied arterioles. Histologic techniques were used to assess pulmonary inflammation, characterize the adherence of leukocytes to systemic venules, verify the presence of myeloperoxidase (MPO) in the systemic microvascular wall, and quantify systemic microvascular oxidative stress. In the lungs of rats exposed to ROFA or TiO2, changes in some bronchoalveolar lavage markers of inflammation were noted, but an indication of cellular damage was not found. In rats exposed to 0.1 mg ROFA, focal alveolitis was evident, particularly at sites of particle deposition. Exposure to either ROFA or TiO2 caused a dose-dependent impairment of endothelium-dependent arteriolar dilation. However, exposure to these particles did not affect microvascular constriction in response to PHE. ROFA and TiO2 exposure significantly increased leukocyte rolling and adhesion in paired venules, and these cells were positively identified as polymorphonuclear leukocytes (PMNLs). In ROFA- and TiO2-exposed rats, MPO was found in PMNLs adhering to the systemic microvascular wall. Evidence suggests that some of this MPO had been deposited in the microvascular wall. There was also evidence for oxidative stress in the microvascular wall. These results indicate that after PM exposure, the impairment of endothelium-dependent dilation in the systemic microcirculation coincides with PMNL adhesion, MPO deposition, and local oxidative stress. Collectively, these microvascular observations are consistent with events that contribute to the disruption of the control of peripheral resistance and/or cardiac dysfunction associated with PM exposure.

How to express tumor markers in bronchoalveolar lavage

Bronchoalveolar lavage (BAL) is a fundamental technique in the diagnosis of different respiratory diseases including lung cancer. Tumor marker values can be determined in the BAL fluid, but controversy still exists about how to express the results. The aim of this study was to determine the best method of expressing tumor markers in BAL, either referring to total proteins or volume of fluid recovered. A prospective, randomized, non-blind study was carried out. Seventy-six patients (72 men and 4 women) diagnosed with lung cancer and 17 subjects without respiratory disease were included. BAL was performed in all patients and the fluid retrieved was divided into two fractions: a bronchiolar fraction (F0) and an alveolar fraction (F1). Five tumor markers: cytokeratin fragment 19 (CYFRA 21-1), squamous cell carcinoma antigen (SCC), tissue polypeptide antigen (TPA), tissue polypeptide-specific antigen (TPS) and neuron-specific enolase (NSE) as well as total protein were measured in both fractions. The concentrations were expressed in relation to the volume of BAL fluid recovered (ng or mU/mL) and in milligrams of total protein of lavage fluid (ng or mU/mg TP). The SPSS 11.01 software was used for statistical analysis. Mann-Whitney U test and ROC curves were developed when significant differences were found. We found significant differences in the CYFRA 21-1 values in the two BAL fractions and in both ways of expressing its concentration; in SCC in F1 expressed in ng/mg TP; in TPA in F0 expressed in mU/mg TP; in TPS in both fractions expressed in mU/mg TP, and in NSE in both fractions in ng/mg TP. The markers that best differentiated tumors from controls (ROC curves) were CYFRA 21-1 in F0 and NSE in both fractions in ng/mg TP. Our study demonstrates that the concentrations of tumor markers in BAL expressed in relation to total protein were more effective than if expressed in mL of BAL fluid collected.

How to Express Tumor Markers in Bronchoalveolar Lavage

Introduction Bronchoalveolar lavage (BAL) is a fundamental technique in the diagnosis of different respiratory diseases including lung cancer. Tumor marker values can be determined in the BAL fluid, but controversy still exists about how to express the results. Objective The aim of this study was to determine the best method of expressing tumor markers in BAL, either referring to total proteins or volume of fluid recovered. Patients and Methods A prospective, randomized, non-blind study was carried out. Seventy-six patients (72 men and 4 women) diagnosed with lung cancer and 17 subjects without respiratory disease were included. BAL was performed in all patients and the fluid retrieved was divided into two fractions: a bronchiolar fraction (F0) and an alveolar fraction (F1). Five tumor markers: cytokeratin fragment 19 (CYFRA 21–1), squamous cell carcinoma antigen (SCC), tissue polypeptide antigen (TPA), tissue polypeptide-specific antigen (TPS) and neuron-specific enolase (NSE) as well as total protein were measured in both fractions. The concentrations were expressed in relation to the volume of BAL fluid recovered (ng or mU/mL) and in milligrams of total protein of lavage fluid (ng or mU/mg TP). The SPSS 11.01 software was used for statistical analysis. Mann-Whitney U test and ROC curves were developed when significant differences were found. Results We found significant differences in the CYFRA 21-1 values in the two BAL fractions and in both ways of expressing its concentration; in SCC in F1 expressed in ng/mg TP; in TPA in F0 expressed in mU/mg TP; in TPS in both fractions expressed in mU/mg TP, and in NSE in both fractions in ng/mg TP. The markers that best differentiated tumors from controls (ROC curves) were CYFRA 21-1 in F0 and NSE in both fractions in ng/mg TP. Conclusions Our study demonstrates that the concentrations of tumor markers in BAL expressed in relation to total protein were more effective than if expressed in mL of BAL fluid collected.

Proteomics as the tool to search for lung disease markers in bronchoalveolar lavage.

Most lung disorders are known to be associated to considerable modifications of surfactant composition. Numerous of these abnormalities have been exploited in the past to diagnose lung diseases, allowing proper treatment and follow-up. Diagnosis was then based on phospholipid content, surface tension and cytological features of the epithelial lining fluid (ELF), sampled by bronchoalveolar lavage (BAL) during fiberoscopic bronchoscopy. Today, it appears that the protein content of ELF displays a remarkably high complexity, not only due to the wide variety of the proteins it contains but also because of the great diversity of their cellular origins. The significance of the use of proteome analysis of BAL fluid for the search for new lung disease marker proteins and for their simultaneous display and analysis in patients suffering from lung disorders has been examined.

Neutrophil Inflammation and Activation in Bronchiectasis: Comparison with Pneumonia and Idiopathic Pulmonary Fibrosis

Background: Pulmonary inflammation in bronchiectasis, pneumonia and idiopathic pulmonary fibrosis (IPF) is dominated by neutrophils. Pathophysiologic differences are seen in the degree of airway and tissue destruction. Neutrophil activation and neutrophil proteolytic activity might differ between bronchiectasis, pneumonia and IPF. Objective: The aim of this study was to determine whether levels of inflammatory and protective markers in bronchoalveolar lavage (BAL) differed among cases of bronchiectasis, pneumonia and IPF. Methods: We studied 11 bronchiectasis patients (group 1), 30 pneumonia patients (group 2), 15 IPF patients (group 3) and 12 healthy volunteers (group 4). In the bronchoalveolar lavage fluid, concentrations of α₁-proteinase inhibitor, myeloperoxidase (MPO) and elastase-α₁PI complex were determined using immunoluminometric assays. Elastase inhibition capacity (EIC) and elastase activity were determined using a colorimetric assay. Results: No EIC, but free elastase activity, was found in 82% of group 1, 20% of group 2, 20% of group 3 and 0% of group 4. Median MPO concentration was highest in group 1: 7,951 ng/ml (16th–84th percentile [16–84%]: 256–36,342) vs. 692 ng/ml (106–2,279; group 2), 332 ng/ml (98–1,657; group 3), and 0.12 ng/ml (0.08–0.26; group 4). Bronchiectasis patients with bronchial Pseudomonas infection showed higher amounts of neutrophils (p < 0.01) and higher elastase activity (p < 0.05) than patients with sterile lavage. Conclusion: Bronchiectasis patients show a severe imbalance between neutrophil activity and protective molecules leading to possible lung destruction. Chronic Pseudomonas infection might trigger neutrophil activation. Future research and treatment strategies should focus on increased bacterial clearance and inhibition of neutrophil toxicity.

Persistent high BAL fluid granulocyte activation marker levels as early indicators of bronchiolitis obliterans after lung transplant.

The major cause of mortality in the long-term in lung transplant recipients is chronic rejection. This is a fibroproliferative process in the small airways leading to obliterative bronchiolitis and progressive loss of lung function, both constituting the clinical entity bronchiolitis obliterans syndrome (BOS). Granulocyte activation has been implicated as one factor behind BOS. Granulocyte markers in bronchoalveolar lavage (BAL) fluid were prospectively and longitudinally studied in order to identify possible association with BOS. BAL fluid from 266 bronchoscopy procedures performed in twelve single lung, eight bilateral lung and five heart/lung transplant recipients were analysed. The majority (19 of 25) were studied for a period of 2 yrs after surgery. Myeloperoxidase (MPO), eosinophil cationic protein (ECP) and interleukin-8 (IL-8) levels were used as indirect markers of activation and attraction of granulocytes. Five patients developed BOS. Ninety-eight episodes of acute rejection, nine of bacterial infection, 19 of cytomegalovirus pneumonitis, nine of Pneumocystis carinii infection, two of aspergillus infection and two of respiratory syncytial virus infection were diagnosed. BOS patients had significantly higher mean levels of MPO, ECP and IL-8 compared to patients without BOS, irrespective of acute rejection status. Over time, the five patients with BOS had significantly elevated BAL fluid levels of MPO and ECP as well as neutrophil percentages, and in four patients this increase preceded the clinical diagnosis of BOS by several months. Elevated bronchoalveolar lavage fluid neutrophil percentage as well as levels of the granulocyte activation markers myeloperoxidase and eosinophil cationic protein appear to be early signs of development of BOS in lung transplant recipients.

Inflammatory cells and activation markers in BAL during acute rejection and infection in lung transplant recipients: a prospective, longitudinal study

Acute rejection of the transplanted lung is a clinical problem, since it decreases graft survival and predisposes the patient to chronic rejection and obliterative bronchiolitis (OB). In an earlier study, we had indications that eosinophil cationic protein (ECP) from activated eosinophils and hyaluronan (HYA) from fibroblasts were associated with acute pulmonary rejection. This prospective longitudinal study was designed to investigate whether molecules from activated inflammatory cells in bronchoalveolar lavage (BAL) fluid could serve as clinically useful diagnostic markers for acute rejection. BAL fluid from 138 bronchoscopies performed in 10 single lung, four bilateral lung and five heart-lung transplant recipients were analysed. Nine patients were studied for a period of more than 1 yr (mean 13.4 months) after surgery. Differential cell counts were made from the BAL fluid. ECP, myeloperoxidase (MPO), HYA and interleukin-8 (IL-8) were used as indirect markers for activation and attraction of eosinophils, neutrophils and fibroblasts, respectively. Fifty four episodes of acute rejection were diagnosed. Two patients developed OB. Nine episodes of bacterial infection, 13 episodes of cytomegalovirus (CMV) pneumonitis, three of Pneumocystis carinii infection and one of respiratory syncytial virus (RSV) infection were diagnosed. The mean levels of ECP, MPO, HYA and IL-8 were all higher during rejection episodes, but differences were not statistically significant compared to no rejection, when the confounding factors of time, concomitant infection, and repeated measures in the same individual had been accounted for. We could not confirm that measurements of eosinophil cationic protein, myeloperoxidase, hyaluronan and interleukin-8 in bronchoalveolar lavage fluid can be used as diagnostic markers for acute rejection in the postoperative follow-up of lung transplant recipients.

EFFECTS OF REPEATED EXPOSURES OF GERIATRIC RATS TO OZONE AND PARTICLE-CONTAINING ATMOSPHERES: AN ANALYSIS OF BRONCHOALVEOLAR LAVAG E AND PLASMA PROTEINS

Inhalation of ozone (O3) and airborne particles less than 10 m (PM-10) in mass median aerodynamic diameter (MMAD) is associated with adverse health effects in sensitive human populations, and pulmonary injury in laboratory animals. In order to simulate environmental exposures of sensitive individuals, such as the elderly, geriatric rats were exposed to O and particulate mixtures. This pilot study determined whether the pul3 monary effects of higher O3 concentrations reported in earlier studies are also seen after repeated exposures to lower O3 concentration used in this study, whether the O3 effects are modified by PM-10 components, and whether plasma biomarkers can be developed as noninvasive tests of pulmonary injury. Male Fischer 344 rats, 22-24 mo old, were exposed 4 h/ day, 3 days/wk for 4 wk to (a) purified air, (b) O3 (0.2 ppm), (c) low-level mixture of carbon (C, 50 g/ m3) plus ammonium bisulfate (ABS, 70 g/m3) plus O3, and (d) high-level mixture of C (100 g/m3) plus ABS (140 g/m3) plus O3. Twenty-four hours after the last exposure, groups of rats were prepared for measurement of protein and albumin concentrations in bronchoalveolar lavage (BAL) as markers of airways permeability, and blood was drawn for analysis of plasma immunoreactive prolyl 4-hydroxylase and fibronectin. Exposures to O3 and O3 /particle mixtures did not produce a consistent, significant change in the BAL markers of permeability. Nonsignificant changes represented individual animal variations. On the other hand, a significant increase in plasma fibronectin was observed in the group exposed to O3, but not in the rats exposed to O3/particle combinations. Such an effect was not observed for plasma immunoreactive prolyl 4- hydroxylase. The inability of low-level O3 and O3/particle combinations to produce intra pulmonary effects suggests potential utility of plasma biomarkers for the evaluation of pulmonary toxicity. These results also suggest modification of O3 effects upon its combination with PM-10.

Early changes in T lymphocytes recovered by bronchoalveolar lavage after local allergen challenge of asthmatic airways.

To assess the role of T lymphocytes in the initiation of the allergic asthmatic response we have investigated T-cells subsets and their activation markers in bronchoalveolar lavage (BAL) fluid recovered 10 min after local challenge of the bronchial mucosa with allergen or saline. Endobronchial challenge was performed in 13 mildly atopic asthmatic patients (FEV1% predicted range, 78.2 to 116.5) and 10 normal volunteers. In all of the asthmatics but in none of the normal subjects allergen but not saline exposure resulted in visible bronchoconstriction. Analysis of BAL by flow cytometry showed no differences in the overall number of T cells (CD3+) and their CD4+ and CD8+ subsets per milliliter of BAL between the groups of normal subjects and asthmatics. However, within 10 min of allergen challenge, in the asthmatics but not in the normal subjects, there occurred a significant loss of CD3+ cells (p less than 0.01) comprising mostly CD4+ (p less than 0.05) but also CD8+ cells, with a consequent decrease in the CD4:CD8 ratio. At this early time point no differences in the extent of expression of the T-cell activation markers, IL-2 receptor, and HLA-DR were found. These results provide evidence to support a role of T lymphocytes early in the allergen-induced inflammatory response in asthma.

Inflammatory Markers in Bronchoalveolar Lavage and in Bronchial Biopsy in Asthma during Remission

Bronchoalveolar lavage and BB were performed in 13 asthmatic and six healthy subjects to characterize cellular markers of inflammation in BAL and BB; to compare cellular profile of BAL with cell infiltration in BB; to examine the relationship between bronchial responsiveness and markers of inflammation in BAL and BB. Eosinophils and mast cells were increased in BAL in asthmatic subjects; eosinophils were positively correlated with neutrophils and mast cells. Epithelial shedding was present in nine asthmatic and five control subjects. Intraepithelial cells and cells in submucosa were increased in asthmatic subjects. Eosinophils and intraepithelial mast cells were higher. Thickened basement membrane was associated with more marked cell infiltration in submucosa. Ciliated cells in BAL relate to intraepithelial cells; cells in BAL broadly reflect cell infiltration of submucosa. In the asthmatic group, the degree of bronchial responsiveness correlated with ciliated cells in BAL and with intra-epithelial cells in BB. Marked airway inflammation is associated with stable asthma; inflammatory changes within bronchial epithelium may be linked to the development of bronchial hyperresponsiveness.