PTTG1IP and MAML3, novel genomewide association study genes for severity of hyperresponsiveness in adult asthma.

BACKGROUND: The severity of bronchial hyperresponsiveness (BHR) is a fundamental feature of asthma. The severity of BHR varies between asthmatics and is associated with lack of asthma control. The mechanisms underlying this trait are still unclear. This study aimed to identify genes associated with BHR severity, using a genomewide association study (GWAS) on the slope of BHR in adult asthmatics. METHODS: We performed a GWAS on BHR severity in adult asthmatics from the Dutch Asthma GWAS cohort (n = 650), adjusting for smoking and inhaled corticosteroid use, and verified results in three other cohorts. Furthermore, we performed eQTL and co-expression analyses in lung tissue. RESULTS: In the discovery cohort, one genomewide significant hit located in phosphodiesterase 4D, cAMP-specif (PDE4D) and 26 SNPs with P-values < 1*10-5 were found. None of our findings replicated in adult and childhood replication cohorts jointly. In adult cohorts separately, rs1344110 in pituitary tumour-transforming 1 interacting protein (PTTG1IP) and rs345983 in Mastermind-like 3 (MAML3) replicated nominally; minor alleles of rs345983 and rs1344110 were associated with less severe BHR and higher lung tissue gene expression. PTTG1IP showed significant co-expression with pituitary tumour-transforming 1, the binding factor of PTTG1lP, and with vimentin and E-cadherin1. MAML3 co-expressed significantly with Mastermind-like 2 (MAML2), both involved in Notch signalling. CONCLUSIONS: PTTG1IP and MAML3 are associated with BHR severity in adult asthma. The relevance of these genes is supported by the eQTL analyses and co-expression of PTTG1lP with vimentin and E-cadherin1, and MAML3 with MAML2.

Longitudinal stability of asthma characteristics and biomarkers from the Airways Disease Endotyping for Personalized Therapeutics (ADEPT) study.

BACKGROUND: Asthma is a biologically heterogeneous disease and development of novel therapeutics requires understanding of pathophysiologic phenotypes. There is uncertainty regarding the stability of clinical characteristics and biomarkers in asthma over time. This report presents the longitudinal stability over 12 months of clinical characteristics and clinically accessible biomarkers from ADEPT. METHODS: Mild, moderate, and severe asthma subjects were assessed at 5 visits over 12 months. Assessments included patient questionnaires, spirometry, bronchodilator reversibility, fractional exhaled nitric oxide (FENO), and biomarkers measured in induced sputum. RESULTS: Mild (n = 52), moderate (n = 55), and severe (n = 51) asthma cohorts were enrolled from North America and Western Europe. For all clinical characteristics and biomarkers, group mean data showed no significant change from visit to visit. However, individual data showed considerable variability. FEV1/FVC ratio showed excellent reproducibility while pre-bronchodilator FEV1 and FVC were only moderately reproducible. Of note bronchodilator FEV1 reversibility showed low reproducibility, with the nonreversible phenotype much more reproducible than the reversible phenotype. The 7-item asthma control questionnaire (ACQ7) demonstrated moderate reproducibility for the combined asthma cohorts, but the uncontrolled asthma phenotype (ACQ7 > 1.5) was inconstant in mild and moderate asthma but stable in severe asthma. FENO demonstrated good reproducibility, with the FENO-low phenotype (FENO < 35 ppb) more stable than the FENO-high phenotype (FENO ≥ 35 ppb). Induced sputum inflammatory phenotypes showed marked variability across the 3 sputum samples taken over 6 months. CONCLUSIONS: The ADEPT cohort showed group stability, individual stability in some parameters e.g. low FEV1/FVC ratio, and low FENO, but marked individual variability in other clinical characteristics and biomarkers e.g. type-2 biomarkers over 12 months. This variability is possibly related to seasonal variations in climate and allergen exposure, medication changes and acute exacerbations. The implications for patient selection strategies based on clinical biomarkers may be considerable.

Asthma characteristics and biomarkers from the Airways Disease Endotyping for Personalized Therapeutics (ADEPT) longitudinal profiling study.

BACKGROUND: Asthma is a heterogeneous disease and development of novel therapeutics requires an understanding of pathophysiologic phenotypes. The purpose of the ADEPT study was to correlate clinical features and biomarkers with molecular characteristics, by profiling asthma (NCT01274507). This report presents for the first time the study design, and characteristics of the recruited subjects. METHODS: Patients with a range of asthma severity and healthy non-atopic controls were enrolled. The asthmatic subjects were followed for 12 months. Assessments included history, patient questionnaires, spirometry, airway hyper-responsiveness to methacholine, fractional exhaled nitric oxide (FENO), and biomarkers measured in induced sputum, blood, and bronchoscopy samples. All subjects underwent sputum induction and 30 subjects/cohort had bronchoscopy. RESULTS: Mild (n = 52), moderate (n = 55), severe (n = 51) asthma cohorts and 30 healthy controls were enrolled from North America and Western Europe. Airflow obstruction, bronchodilator response and airways hyperresponsiveness increased with asthma severity, and severe asthma subjects had reduced forced vital capacity. Asthma control questionnaire-7 (ACQ7) scores worsened with asthma severity. In the asthmatics, mean values for all clinical and biomarker characteristics were stable over 12 months although individual variability was evident. FENO and blood eosinophils did not differ by asthma severity. Induced sputum eosinophils but not neutrophils were lower in mild compared to the moderate and severe asthma cohorts. CONCLUSIONS: The ADEPT study successfully enrolled asthmatics across a spectrum of severity and non-atopic controls. Clinical characteristics were related to asthma severity and in general asthma characteristics e.g. lung function, were stable over 12 months. Use of the ADEPT data should prove useful in defining biological phenotypes to facilitate personalized therapeutic approaches.

OX40L blockade and allergen-induced airway responses in subjects with mild asthma.

BACKGROUND: The OX40/OX40L interaction contributes to an optimal T cell response following allergic stimuli and plays an important role in the maintenance and reactivation of memory T effector cells. OBJECTIVE: We tested whether treatment with an anti-OX40L monoclonal antibody (MAb) would inhibit allergen-induced responses in subjects with asthma. METHODS: Twenty-eight mild, atopic asthmatic subjects were recruited for a double-blind, randomized, placebo-controlled, parallel-group trial (ClinicalTrials.gov identifier NCT00983658) to compare blockade of OX40L using a humanized anti-OX40L MAb to placebo-administered intravenously in 4 doses over 3 months. Allergen inhalation challenges were carried out 56 and 113 days after the first dose of study drug. The primary outcome variable was the late-phase asthmatic response. Other outcomes included the early-phase asthmatic response, airway hyperresponsiveness, serum IgE levels, blood and sputum eosinophils, safety and tolerability. RESULTS: Treatment with anti-OX40L MAb did not attenuate the early- or late-phase asthmatic responses at days 56 or 113 compared with placebo. In the anti-OX40L MAb treatment group, total IgE was reduced 17% from pre-dosing levels, and sputum eosinophils decreased 75% by day 113 (both P = 0.04). There was no effect of anti-OX40L MAb on airway hyperresponsiveness or blood eosinophils. The frequency of AEs was similar in both groups. CONCLUSION AND CLINICAL RELEVANCE: Pharmacological activity of anti-OX40L MAb was observed by decreases in serum total IgE and airway eosinophils at 16 weeks post-dosing, but there was no effect on allergen-induced airway responses. It is possible that the treatment duration or dose of antibody was insufficient to impact the airway responses.

The relationship between eosinophilia and airway remodelling in mild asthma.

BACKGROUND: Eosinophilia is a marker of corticosteroid responsiveness and risk of exacerbation in asthma; although it has been linked to submucosal matrix deposition, its relationship with other features of airway remodelling is less clear. OBJECTIVE: The aim of this study was to investigate the relationship between airway eosinophilia and airway remodelling. METHODS: Bronchial biopsies from subjects (n = 20 in each group) with mild steroid-naïve asthma, with either low (0-0.45 mm(-2)) ) or high submucosal eosinophil (23.43-46.28 mm(-2) ) counts and healthy controls were assessed for in vivo epithelial damage (using epidermal growth factor receptor staining), mucin expression, airway smooth muscle (ASM) hypertrophy and inflammatory cells within ASM. RESULTS: The proportion of in vivo damaged epithelium was significantly greater (P = 0.02) in the high-eosinophil (27.37%) than the low-eosinophil (4.14%) group. Mucin expression and goblet cell numbers were similar in the two eosinophil groups; however, MUC-2 expression was increased (P = 0.002) in the high-eosinophil group compared with controls. The proportion of submucosa occupied by ASM was higher in both asthma groups (P = 0.021 and P = 0.046) compared with controls. In the ASM, eosinophil and T-lymphocyte numbers were higher (P < 0.05) in the high-eosinophil group than both the low-eosinophil group and the controls, whereas the numbers of mast cells were increased in the high-eosinophil group (P = 0.01) compared with controls. CONCLUSION: Submucosal eosinophilia is a marker (and possibly a cause) of epithelial damage and is related to infiltration of ASM with eosinophils and T lymphocytes, but is unrelated to mucus metaplasia or smooth muscle hypertrophy.

Monitoring sputum eosinophils in mucosal inflammation and remodelling: a pilot study.

Normalisation of eosinophil counts in sputum of asthmatic patients reduces eosinophilic exacerbations. However, the effect of this strategy on airway remodelling remains to be determined. We compared bronchial inflammation and collagen deposition after 2 yrs of treatment guided by either sputum eosinophils (sputum strategy, SS) or by clinical criteria (clinical strategy, CS). As a pilot study, 20 mild asthmatic patients were randomly assigned to CS or SS strategies. Bronchial biopsies were obtained when minimum treatment needed to maintain control was identified and this was continued for 2 yrs. Biopsies were immunostained for inflammatory cells, mucin 5A (MUC5A) and collagen. The mean dose of inhaled corticosteroids at the start and end of the study was similar in both SS and CS groups. Forced expiratory volume in 1 s increased in both groups at the study end. In SS, mucosal lymphocyte and eosinophil counts, but not neutrophils, were reduced at the end of the study. In CS, only activated eosinophil and neutrophil counts decreased. MUC5A staining decreased in SS but not CS. No change in collagen deposition underneath the basement membrane was observed in either strategy. Treatment strategies that normalise sputum eosinophils also reduce mucosal inflammatory cells and MUC5A expression, but do not change subepithelial collagen deposition in mild to moderate asthma.

Regulation of epithelial cell proliferation by bronchial fibroblasts obtained from mild asthmatic subjects.

BACKGROUND: Bronchial epithelium is considered a key player in coordinating airway wall remodelling. The function of epithelial cells can be modulated by the underlying fibroblasts through autocrine and paracrine mechanisms. OBJECTIVE: To investigate the effect of phenotypic changes in bronchial fibroblasts from asthmatic subjects on epithelial cell proliferation. METHODS: Epithelial cells and fibroblasts derived from bronchial biopsies of asthmatic and healthy controls were cultured in an engineered model. Proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromid (MTT). Epidermal growth factor receptor (EGFR), cyclin-dependent kinase inhibitors p21 and p27 were measured by western blots. Total and active forms of transforming growth factor (TGF)-ß1 were measured using ELISA and bioassay. TGF-ß was inhibited using a recombinant TGF-ß soluble receptor II protein. RESULTS: Proliferation of epithelial cells from asthmatics (AE) is increased when cells were cultured with fibroblasts from normal controls (NF). Fibroblasts from asthmatics (AF) significantly decreased the proliferation of epithelial cells from healthy subjects (NE). Activation of p21, p27, EGFR and TGF-ß1 reflects the proliferation data by decreasing in AE cultured with NF and increasing in NE cultured with AF. Neutralization of TGF-ß increased proliferation of epithelial cells cultured in the asthmatic model. CONCLUSION: Fibroblasts from asthmatic subjects regulate epithelial cell prolifearation, and TGF-ß signalling may represent one of the pathway involved in these interactions.

Normal weight men and women overestimate exercise energy expenditure.

AIM: The limited potential of exercise to induce weight loss could be partly due to the overestimation of the energy cost of exercise. The objectives of this study were twofold: 1) to investigate whether men and women are able to accurately estimate exercise energy expenditure (EE); and 2) to determine whether they have the ability to accurately compensate for the EE of exercise during a buffet-type meal. METHODS: Sixteen (8 men, 8 women) moderately active (VO2 peak=45.4±7.7 mL.kg-1.min-1), normal weight (BMI=22.8±3.3 kg/m2) individuals, aged 20-35 years, were studied. They were blinded to two randomly assigned experimental conditions: a 200 and a 300 kcal (measured by indirect calorimetry) exercise sessions that were performed on a treadmill at the same intensity (50% of VO2 peak). At the end of each exercise session individuals were asked to estimate EE of the exercise sessions and to then eat the caloric equivalent of their estimated exercise EE from a buffet-type meal. RESULTS: Estimated EE was higher than measured EE for both the 200 kcal (825.0±1061.8 vs. 200.1±0.7 kcal, P<0.05) and 300 kcal (896.9±952.4 vs. 300.2±0.7 kcal, P<0.05) sessions. Further, post-exercise energy intake was higher than measured EE for the 200 kcal (556.8±204.4 vs. 200.1±0.7 kcal, P<0.001) and the 300 kcal (607.2±166.5 vs. 300.2±0.7 kcal, P<0.001) sessions. Although post-exercise energy intake was lower than estimated EE, no significant differences were noted. CONCLUSION: These results suggest that normal weight individuals overestimate EE during exercise by 3-4 folds. Further, when asked to precisely compensate for exercise EE with food intake, the resulting energy intake is still 2 to 3 folds greater than the measured EE of exercise.

Budesonide/formoterol maintenance and reliever therapy: impact on airway inflammation in asthma.

The aim of the present study was to compare the effectiveness, safety and health economics of budesonide/formoterol maintenance and a novel reliever therapy with conventional best practice in patients with persistent asthma in Canada. After 2 weeks of usual therapy, 1,538 patients were randomised for 6 months to open-label budesonide/formoterol maintenance and reliever therapy 160/4.5 microg twice daily and as needed, or to guideline-based conventional best practice. Severe asthma exacerbations, reliever medication use and total inhaled corticosteroid dose were analysed in all patients and airway inflammation was assessed in a sub-study of 115 patients. No differences were seen in time to first severe exacerbation and severe asthma exacerbation rate. There were numerically fewer emergency room visits or hospitalisations with budesonide/formoterol maintenance and reliever therapy (4.4 versus 7.5 events per 100 patients x yr(-1), 41% reduction); however, this did not reach statistical significance. Mean total inhaled corticosteroid dose, reliever use, asthma medication costs and total annual costs per patient were all significantly lower with budesonide/formoterol maintenance and reliever therapy. Mean sputum eosinophil cell counts remained in the range for controlled inflammation in both groups. In conclusion, budesonide/formoterol maintenance and reliever therapy achieved similar or improved clinical control compared with conventional best practice, with significantly lower total inhaled corticosteroid dose and lower cost, while maintaining similar control of eosinophilic inflammation.

Influence of smoking on airway inflammation and remodelling in asthma.

BACKGROUND: Although exposure to tobacco smoke has been associated with increased morbidity and mortality, cigarette smoking is still common in the asthmatic population. Induced sputum neutrophilia has been observed in asthmatic smokers, but the effects of regular smoking on their bronchial mucosa morphology remain to be defined. This study documents the inflammatory and remodelling features in bronchial biopsies of smoking compared with non-smoking asthmatics. METHODS: We analysed bronchial biopsies from 24 steroid-naïve young subjects with mild asthma: 12 non-smoking and 12 currently smoking subjects. In addition to airway morphology assessment, inflammation and remodelling were analysed by immunohistochemistry using antibodies against CD3, CD68, major basic protein, neutrophil elastase, and tryptase. Expression of the cytokines IL-4, IL-5, IL-8, IFN-gamma, transforming growth factor-beta, and TNF was determined by in situ hybridization. RESULTS: Compared with non-smoking asthmatic subjects, smoking asthmatics' bronchial mucosa showed squamous cell metaplasia, in addition to increased expression of subepithelial neutrophil elastase, IFN-gamma, and intraepithelial IL-8. CONCLUSIONS: Smoking status modifies morphological and inflammatory processes in young subjects with mild asthma. The changes may possibly affect asthma treatment responses and clinical outcomes.

Decreased leukotriene B4 synthesis in smokers' alveolar macrophages in vitro.

Recent studies have shown that alveolar macrophages (AM) are able to release leukotrienes (LTs). Since cigarette smoking inhibits the cyclooxygenase pathway of arachidonic acid metabolism in the AM, we evaluated the LT production by AM from smokers and nonsmokers. AM were obtained from 35 volunteers, 16 nonsmokers, and 19 smokers. The cells were incubated under various conditions including stimulation with 30 microM arachidonic acid, 2 microM ionophore A23187, or both. Each experiment was performed in parallel using cells from a smoker and a nonsmoker. Lipoxygenase products were analyzed by reverse-phase high performance liquid chromatography. After stimulation, nonsmokers' AM produced LTB4 and 5-hydroxy-eicosatetraenoic acid (5-HETE). In incubations of AM with arachidonic acid and ionophore, the amounts of products formed were: LTB4, 317 +/- 56 pmol/10(6) cells and 5-HETE, 1,079 +/- 254, mean +/- SEM. No metabolites were generated under control conditions (no stimulation). In all incubations performed, the peptido-LTs (LTC4, LTD4, and LTE4) were undetectable. In comparison with AM from nonsmokers, those from smokers showed a 80-90% reduction of 5-HETE and LTB4 synthesis (P less than 0.05 to P less than 0.001 according to stimulatory conditions). This defective lipoxygenase metabolite production in AM from smokers was observed over a wide range of stimuli concentrations and incubation times; AM from smokers also had lower levels of intracellular (esterified) 5-HETE than nonsmokers' AM. We also studied blood polymorphonuclear leukocytes (PMNL) and no difference in the synthesis of 5-lipoxygenase products in these cells was noticed between smokers and nonsmokers. These data show that cigarette smoking causes a profound inhibition of the 5-lipoxygenase pathway in AM but not in blood PMNL.

Characteristics of leukotriene biosynthesis by human granulocytes in presence of plasma.

The formation of leukotriene B4 (LTB4) by neutrophils stimulated with the ionophore A23187 or physiological stimuli in heparinized plasma was investigated. In comparison with neutrophils stimulated (A23187) in a protein-free buffered salt solution, neutrophils stimulated in plasma produced only trace amounts of LTB4. The addition of human recombinant LTA4-hydrolase or erythrocytes to plasma prior to A23187 stimulation strongly and selectively stimulated (> 4-fold) the formation of LTB4 supporting that neutrophils activated in plasma with A23187 release in the extracellular milieu most of LTA4 formed by the cells, and indicating that plasma proteins drastically slow down the further metabolism of LTA4 released by neutrophils. The formation of LTB4 was then investigated in GM-CSF-primed neutrophils stimulated with fMLP in plasma; levels of synthesis were very low and the addition of erythrocytes prior to stimulation strongly enhanced LTB4 synthesis, demonstrating that agonist-stimulated neutrophils also release most of LTA4 generated in the extracellular milieu. Investigations on the fate of LTA4 in plasma revealed that LTA4 was slowly degraded through an unknown process, i.e. not through the previously described non-enzymic hydrolysis resulting in the formation of dihydroxy derivatives of LTA4. Using neutrophils labeled with tritiated arachidonate, we also demonstrated that neutrophils stimulated in plasma with fMLP or A23187, almost exclusively use endogenous arachidonate, as opposed to plasma arachidonate, to generate 5-lipoxygenase products. Finally, experiments performed with purified eosinophils indicated that contrary to neutrophils, the eosinophils do not release LTA4, but directly release LTC4.

Eotaxin promotes eosinophil transmigration via the activation of the plasminogen-plasmin system.

The effect of eotaxin, a potent eosinophil chemotactic factor, on eosinophil transmigration through a reconstituted basal membrane (Matrigel) was evaluated. Eotaxin induced significant eosinophil transmigration in the presence of 10% fetal bovine serum (FBS) and interleukin-5. Its effect was optimal at 0.01 microM, and it plateaued at 18 h. Eotaxin's effect was greater with eosinophils from asthmatic subjects (61.1 +/- 3.4%) than with eosinophils from normal subjects (38.7 +/- 4.2%) (P < 0.001). Inhibition of metalloproteinases decreased eotaxin-induced transmigration by < or = 10.4%, whereas inhibition of the plasminogen-plasmin system decreased eotaxin's effect by < or = 44.4% (P = 0.0002). Moreover, eotaxin-induced transmigration was largely diminished in medium with low concentrations of serum [0.5% FBS: 6.1 +/- 2.4%; 10% FBS: 40.2 +/- 5.8% (P = 0.0001)] but returned to its initial level with the addition of plasminogen (2 U/mL) to 0.5% FBS (43.1 +/- 6.5%). These data show that eotaxin is an efficient promoter of eosinophil transmigration in vitro, that it is more potent with cells from asthmatics than with normal cells, and that its effect depends predominantly on the activation of the plasminogen-plasmin system.

Relationship among antigen contact, alveolitis, and clinical status in farmer's lung disease.

We studied 41 subjects with a history of farmer's lung disease who had been free of acute episodes for at least one year. Twenty-six were still in daily contact with hay (group 1), and 15 had ceased all antigenic exposure (group 2). While the incidence of dyspnea was similar in both groups, coughing and sputum were more frequent in group 1. Inspiratory crackles were frequent in group 1 subjects (15 of 26) and absent in all group 2 subjects. In both groups, a high percentage of lymphocytes was demonstrated by bronchoalveolar lavage: 52.5% +/- 21.1% (mean +/- SD) and 26.3% +/- 18.7%, respectively. Lymphocytic alveolitis (greater than 22% lymphocytes) was more common in group 1 (23 of 26) than in group 2 (6 of 15). There was no relationship between functional abnormalities and the intensity of the alveolitis. We conclude that lymphocytic alveolitis may persist after an acute episode of farmer's lung disease, but the intensity of the alveolitis is not associated with functional alterations.

[Bronchial thermoplasty in the treatment of severe adult asthma]. / La thermoplastie bronchique dans le traitement de l'asthme sévère de l'adulte.

Bronchial thermoplasty is a recent endoscopic technique for the treatment of severe asthma. It is an innovative treatment whose clinical efficacy and safety are beginning to be better understood. Since this is a device-based treatment, the evaluation procedure of risks and benefits is different that for pharmaceutical products; safety aspects, regulatory requirements, study design and the assessment of the magnitude of effects may all be different. The mechanism of action and optimal patient selection need to be assessed further in rigorous clinical and scientific studies. This technique is in harmony with the development of personalised medicine in the 21st century. It should be developed further in response to the numerous challenges and needs not yet met in the management of severe asthma.

Comparison of two modified techniques for purifying blood eosinophils.

As there is much heterogeneity in the morphology and function of blood eosinophils, comparison of their properties between groups of subjects requires recovering the majority of these cells. In two currently used techniques to isolate eosinophils, blood granulocytes are processed either on Percoll gradients after an incubation of granulocytes with 10(-8) M N-formyl-methionyl-leucyl-phenylalanine (fMLP) or on a magnetic cell sorter (MACS). In this study, these techniques were modified to increase the efficiency of eosinophil recovery. With the Percoll gradients, using 1.078 g/ml as the top gradient instead of 1.082 g/ml doubled the eosinophil recovery from 43 +/- 5.3% (mean +/- SEM) to 86.9 +/- 2.9%, without decreasing the purity (96.1 +/- 1.4% versus 96.2 +/- 0.9%). With a MACS, the neutrophils in granulocytes obtained on Ficoll-Paque (1.077 g/ml) instead of on Percoll gradient 1.082-1.094 g/ml, were tagged with anti-CD16 antibodies and eliminated by passing them through a magnetic field. When blood eosinophils of the same subjects were isolated using the two techniques, similar recovery and purity levels were obtained: Percoll gradients, 72.7 +/- 6.8% and 92.5 +/- 2.2%; MACS, 80.2 +/- 5.1% and 90.4 +/- 3.8%. Eosinophils isolated through the two techniques were also compared for their production of superoxide anion and leukotriene (LT) C4, with and without pre-incubation with cytokines interleukin-3, interleukin-5 and granulocyte-macrophage colony stimulating factor. The release of these products was similar between the two eosinophil preparations under all conditions tested except for interleukin-3 where eosinophils isolated with a MACS produced more LTC4. These results show that both techniques efficiently recover pure eosinophils. Furthermore, cell incubation with 10(-8) M fMLP did not enhance superoxide anion and LTC4 production nor modify the response to cytokines. The two modified techniques are therefore suitable for comparative studies of eosinophils from different groups of subjects.

Inhibitory effects of MK-886 on arachidonic acid metabolism in human phagocytes.

1. We have investigated the inhibitory activity of compound MK-886 (formerly L-663,536), an indole derivative, on 5-lipoxygenase product synthesis in various human phagocytes stimulated with either the ionophore A23187, in the presence and absence of exogenous arachidonic acid, or platelet-activating factor (PAF). The lipoxygenase products were analysed by reversed-phase h.p.l.c. 2. MK-886 inhibited the formation of 5-hydroxy-eicosatetraenoic acid (5-HETE), leukotriene B4 (LTB4), its omega-oxidation products and 6-trans-isomers with an IC50 value of 10-14 nM in A23187-stimulated neutrophils. In the same system, nordihydroguaiaretic acid (NDGA), AA-861 and L-655,240 showed IC50 values of 250-510, 110-420 nM and 1.7-3.9 microM, respectively. 3. MK-886 inhibited 5-lipoxygenase product synthesis in A23187-stimulated blood eosinophils and monocytes, and in neutrophils primed with granulocyte-macrophage colony-stimulating factor and stimulated with PAF with IC50 values of 1-13 nM. 4. The inhibitory activity of MK-886 was not reversed by addition of 10 microM arachidonic acid to A23187-stimulated neutrophils. 5. Compound MK-886 had no effect on 15-lipoxygenase product synthesis in blood eosinophils and neutrophils up to a concentration of 1 microM. 6. At 100 nM compound MK-886 had no significant effects on calcium ion mobilization, superoxide anion production and actin polymerization in neutrophils. 7. In conclusion, MK-886 is a very potent and specific inhibitor of both LTB4 and LTC4 synthesis in various types of human phagocytes.

A randomized, double-blind, placebo-controlled study of lorazepam as premedication for bronchoscopy.

To our knowledge, no study has clearly demonstrated the advantage of sedative premedication for bronchoscopy. In a double-blind study, we evaluated the efficacy of oral lorazepam as premedication for bronchoscopy. One hundred patients were randomly assigned to receive placebo (group A) or lorazepam (2 mg) (group B) approximately 1.5 h before bronchoscopy. Immediately after the procedure and the following day, a questionnaire addressing the patient's perception of the procedure was administered. Specifically, subjects were asked to grade the bronchoscopy as very easy, easy, difficult, or very difficult to tolerate and if they would agree to a second bronchoscopy if believed necessary. In addition, their recollection of the procedure was graded as clear, indistinct, or not at all. No difference was found between the two groups for age, duration of the bronchoscopy, and the answers to the questionnaire administered immediately after the procedure. Most patients from both groups found their level of sedation adequate. On the following day, however, group B reported with lower frequency that the technique was difficult or very difficult (38.0% vs 65.3% for group A; p < 0.005) and that they would be less reluctant to a repeated bronchoscopy (30.0% vs 57.1% for group A; p < 0.015). Moreover, their recollection of the procedure was now less precise than for those who had received the placebo (p < 0.005). This suggests that the difference observed between the two groups at 24 h was related to the amnesic effect of lorazepam. We conclude that lorazepam, by improving patient's perception of the bronchoscopy, is a useful premedication and may facilitate patient's investigation when a second bronchoscopy becomes necessary.

Farmer's fever. Systemic manifestation of farmer's lung without lung involvement.

A 35-year-old dairy farmer presented with recurrent febrile episodes over more than three years. Fever up to 39 degrees C occurred only if he was in contact with the dairy barn and typically appeared at 4 to 8 h after an otherwise usual barn exposure. He had no dyspnea and throughout this period repeated physical examinations, chest roentgenograms, and lung functions remained normal. He was bronchoscoped and lavaged on three occasions, a lymphocytic alveolitis was always present and once, immediately after an acute episode, an important increase in lavage neutrophils was also found (47 percent). Transbronchial lung biopsy specimens showed a moderate cellular infiltrate but no granuloma or fibrosis. Three other persons who worked at the same farm had no similar manifestations or respiratory illnesses. The patient therefore had typical systemic manifestations of acute farmer's lung, but without the lung involvement required to confirm that diagnosis. Furthermore, his disease does not fit the entities known as grain fever and organic dust toxic syndrome. We believe that this patient presented a different entity that we coined "farmer's fever."

Prognostic value of bronchoalveolar lavage lymphocyte count in recently diagnosed pulmonary sarcoidosis.

We prospectively looked at the prognostic value of bronchoalveolar lavage (BAL) lymphocyte count in 98 patients with recently diagnosed (less than 4 months) untreated sarcoidosis. These 50 men and 48 women (mean age, 37.4) were followed up for a period of 6 to 60 months (mean, 25.6), and were clinically evaluated every three to six months with repeated chest roentgenograms and pulmonary function tests. Twenty-four patients required steroid treatment during the study period. The proportion of treated patients was not significantly higher in the group presenting a BAL lymphocyte count less than or equal to 30 percent at diagnosis than in the group with fewer lymphocytes (31.9 and 17.7 percent of total group respectively, p = 0.10). No significant change in TLC, FRC, FVC, FEV1 or DLCO was found at follow-up between the groups with or without an initial high lymphocyte count. In the treated group, BAL lymphocyte percent weakly correlated with the improvement of FEV1 and FVC while on steroid treatment (mean duration: 3.5 months): r = 0.41, p = 0.031 and r = 0.36, p = 0.05 respectively; no correlation was found with lung volumes and DCO. We conclude that BAL lymphocyte count at the time of diagnosis is not a helpful predictor of lung function deterioration in recently diagnosed sarcoidosis and is not very useful in predicting response to treatment.