Thirdly, studies comparing the different "add-on" options were identified. The addition of a LABA is more effective than increasing the dose of ICS in improving asthma control. By increasing the dose of ICS, clinical improvement is likely to be of small magnitude. Addition of a LTRA or theophylline to the treatment regimen appears to be equivalent to doubling the dose of ICS. Addition of a LABA seems to be superior to an LTRA in improving lung function. However, addition of LABA and LTRA may be equal with respect to asthma exacerbations. However, more and longer studies are needed to better clarify the role of LTRAs and theophylline as add-on therapies.Many patients with persistent asthma can be controlled with inhaled corticosteroids (ICS). However, a considerable proportion of patients remain symptomatic, despite the use of ICS. We present systematically evidence that supports the different treatment options. A literature search was made of Medline/PubMed to identify randomised and blinded trials. To demonstrate the benefit that can be obtained by increasing the dose of ICS, dose-response studies with at least three different ICS doses were identified. To demonstrate whether more benefit can be obtained by adding long-acting β Daily rescue therapy is usually needed. Typical findings include low values of PEF or FEV1, a marked variation in daily PEF recordings and/or a significant response to bronchodilators. Thus, asthma is not adequately controlled, and the treatment needs to be optimized.Inhaled corticosteroids (ICS) are the mainstay of current asthma management and should be used in all patients with persistent asthma. Many patients with persistent asthma can be controlled with regular ICS. However, a considerable proportion of patients treated with ICS remain symptomatic, despite the use of low to moderate doses , 3. Addition of a leukotriene receptor antagonist  and 4. Addition of theophylline. Currently, the National Heart, Lung and Blood Institute guideline [According to current guidelines the therapeutic options in the treatment of asthma not adequately controlled by low to moderate doses of ICS are as follows: 1. Increase in the dose of the ICS, 2. Addition of long-acting βuideline ,8. Severuideline ,9-16. HoThe paper reviews studies where participants were adults or adolescents (≥12 years) with clinical evidence of asthma not adequately controlled with ICS. The general inclusion criteria in this review were: randomized, blinded and controlled trials with either parallel group or cross-over design published as a full-length paper. Steroid-tapering studies were not included as they are difficult to interpret. Studies published in abstract form only were not included. Similarly, studies lasting less than 4 weeks, containing less than 10 patients per group or studies containing a significant proportion (>10%) of patients using systemic steroids were excluded. Similarly "add-on" studies where a significant proportion (>10%) of patients were not using inhaled steroids were excluded.in vitro results or results from open, non-randomized or uncontrolled trials or meta-analysis of particular relevance to the present topic may be cited.We made a search of Medline from January 1 1966 to October 2001. All searches were limited to studies published in the English language. To identify the latest studies published, another search was made by using the drug names  from Medline on October 2003. The searches were manually (HK) evaluated to identify studies fulfilling the inclusion criteria and full papers were retrieved. In the case of uncertainty based on the abstract full papers were retrieved. All studies fulfilling the inclusion criteria for the ICS dose-response studies or "add-on" studies (see below) were scored for quality using the method described by Jadad et al. . FurtherTo find the dose-response studies with ICS the term "anti-inflammatory agents, steroidal" was combined with the term: "dose-response relationship, drug" (MeSH), which combination produced 249 papers. To demonstrate the dose-response effect of ICS only controlled studies with at least three different ICS doses and a parallel-group design were included. Studies using consecutive doses of steroids were not included because it makes it impossible to differentiate the dose-response relation from the time course relation of efficacy.When the basic search done with the term "anti-inflammatory agents, steroidal" was combined with another made with terms: "salmeterol OR formoterol" it produced 97 papers, when combined with a search made with a term "leukotriene antagonists" (MeSH), it produced 26 papers and when combined with a search with a term "theophylline" (MeSH) it produced 342 papers. Only studies where the addition of LABA, LTRA or theophylline to the treatment with inhaled steroid was compared with the addition of a corresponding placebo to an increased dose  of inhaled steroid were included. In addition, studies comparing the different "add-on" options were identified.Table 1 . In this review, both ways of analysis are accepted as evidence for the presence of dose-response. In the following discussion the important difference between the formal dose-response studies presented in this review and the results reported in some meta-analysis is that the data of the meta-analyses may result from studies assessing one or more doses of ICS and comparing their effects with placebo or baseline. Thus, the data derived from some the published meta-analyses [We identified 14 studies -31 assesle 1 see . The incanalyses ,11,14,321 in the treatment of chronic asthma in 9 (69%) and 5 (31%) studies of the 14 studies included, respectively (Table 2a, see Table 2a, see 2-agonist use was found in 4 (33%) out of 12 studies where it was analyzed , night-time symptom score and rescue β2-agonist use  and FVC . Similarly, the difference between the two consecutive doses of ICS was very seldom statistically significant  was evaluated in asthmatic subjects who had deterioration in asthma control after discontinuation of ICS . There w1 and morning PEF and reduction in night-time symptom score compared to baseline. Studies that assessed BDP 1000 v 500 μg/d and BDP 1600 v 400 μg/d demonstrated a significant advantage of the higher dose compared to the lower dose for percentage improvement in airway responsiveness to histamine and FEV1 compared to baseline. No differences between higher and lower daily doses of BDP were apparent for daytime symptoms, withdrawals due to asthma exacerbations or oropharyngeal side effects.A recent meta-analysis  analysed1. In this study, there was no statistically significant difference between the doubling doses of budesonide (Table 2ab, see 1.A 6 weeks dose-response study in Japanese asthmatics previously not on ICS showed that increasing the dose of budesonide (200–800 μg/d)  results 1, but not in evening PEF, FVC, symptom scores or rescue β2-agonist use. Instead, even the lowest dose of budesonide (200 μg/d) was superior to placebo for all parameters studied. The improvement induced by these low doses is much greater than the difference between the lowest and highest doses of budesonide studied, despite the 8-fold difference in the dose  or the highest dose (1600 μg/d) did not differ from the two medium doses (400–800 μg/d). When evening PEF, FVC, daytime or nighttime asthma symptom scores or the use of rescue medication were analysed, there was no significant differences between any of the studied budesonide doses [In a randomised, double-blind, placebo-controlled study of parallel-group design lasting 12 weeks four different doses of budesonide . Increasing the dose of budesonide  results de doses .1, morning PEF, symptom scores or rescue β2-agonist use were apparent across a dose range of 200–1600 μg/d. However, in moderate to severe asthma there was a significant reduction in the likelihood of trial withdrawal due to asthma exacerbation with budesonide 800 μg/d compared with budesonide 200 μg/d. The reviewers also conclude that budesonide exhibits a significant improvements favouring high dose (1600 μg/d) over low dose (200 μg/d) for improvement in FEV1 in severe asthma [1 but not for evening PEF [The dose-relationship of budesonide in the treatment of chronic asthma is a subject of a recent Cochrane review . In thise asthma . Anotherning PEF .Table 1, see 1, FVC and daytime symptom score (Table 2ab, see 1, FVC, evening PEF, symptom scores, use of rescue medication or the number of night awakenings between the lowest and highest FP dose, despite a 20-fold difference in the dose [The dose-dependency of FP has been studied in seven studies in patients with mild to moderate asthma. In two of the studies, patients were previously not on ICS (the dose . Only fo1 increased 0.03 L (95% CI 0–0.05 in two weeks). How does this translate into clinical practice? When assessing a response to a bronchodilator or when assessing a response to inhaled or oral steroid an improvement of 10–20% above the previous values is often considered significant. Thus, in the above study, this would mean >36 L/min increase in morning PEF values. Recently, the average minimal patient perceivable improvements have been estimated as 18.8 L/min for PEF and 0.23 L for FEV1 [In a dose-response study  with patfor FEV1 . Based o1, morning PEF, evening PEF, nighttime awakening score and physician-rated efficacy. It is important to appreciate that this was only evident when improvements over placebo were compared for the highest dose of FP (1000 μg/d) and lowest dose of FP (100 μg/d). There were no significant differences when any other doses were compared (e.g. FP 200 v 100 μg/d, FP 500 v 200 μg/d, FP 1000 v 500 μg/d). Sixty percent  of the effect on FEV1 with FP 1000 μg/d  was achieved with tenth of the dose. No dose-response effect was apparent for change in symptom score or for rescue β2-agonist use [1, daily symptom score, night-time awakenings and daily use of rescue β2-agonist use. No significant advantage was obtained with the use of FP at doses of 400–500 μg/d over 200 μg/d for morning or evening PEF, FEV1, daily symptom score or rescue β2-agonist use. Patients treated with higher dose (800 – 1000 μg/d) of FP achieved significantly greater improvements in morning PEF (22 L/min, 95% CI 15–29 L/min) and evening PEF (13 L/min, 95% CI 6–19 L/min) compared to the lower dose (50–100 μg/d). Another recent meta-analysis [1 or β2-agonist use.In a recent meta-analysis  the dosenist use . Anothernist use  found a analysis  includinTable 2a, see Table 2a, see 1 or daytime or total symptom score. In contrast, no significant dose-dependency was found for FVC, nighttime symptom score or rescue β2-agonist use  in the treatment of moderate to severe asthma across the dose-range of 150 to 600 μg/d or 200 to 1600 μg/d for most variables in the two studies included in this review  shows that even the lowest dose significantly differs from placebo, and there appears to be no clear dose-response. However, no formal statistical analysis was reported for the presence of a dose-response and thus this study is not included in Tables 1–2. A recent meta-analysis [1.A placebo-controlled, double-blind parallel-group study assessed the effects of three different doses of TAA  in patients with chronic symptomatic asthma and using ICS . The datanalysis  includin2-agonists, PEF or lung function. However, the steepness of the dose-response curve for different outcomes may vary. For example, an open dose-response evaluation of different sequential doses of budesonide in patients with mild-to-moderate asthma (38) shows that the dose-response curves for FEV1/PEF and FEF25–75 are not identical. Similarly, the dose-response curves of budesonide on adenosine monophosphate (AMP) and methacholine bronchial challenges were significantly different [Taken together these results indicate that the change in the ICS dose from low dose to moderate dose is at the flat part of the ICS dose-response curve for most lung function and symptom parameters studied Figure . Furthere asthma  shows the asthma 8 shows tThe studies discussed above present mean data for groups of patients, but do not address the issue of differences in responsiveness to the anti-inflammatory effects of corticosteroids between individual patients. It may be possible that increasing the dose of ICS may be beneficial for some patients.We were not able to identify any studies that would have studied the dose-dependency of the anti-inflammatory effects of ICS in asthma and would have satisfied the inclusion criteria for the present review.20 from 259 to 467 μg and FP from 271 to 645 μg, both showing a dose-dependency. However, no statistical comparison was made between individual doses. The PD20 was increased 1.67-fold and 1.96-fold when the patients were switched from the lowest dose to the highest dose of budesonide and FP, respectively. An apparently dose-dependent decrease in the blood eosinophil count was obtained with budesonide but not with FP treatment [2-agonist were analysed. Allergen PC15 and methacholine PC20 values were determined before and after treatment with budesonide at 200, 400 and 800 μg/d for 7 days in a double-blind, randomized and cross-over study (6 day washout period) in eleven atopic subjects with inhalation allergy [15 and methacholine PC20 were significantly larger for all doses of budesonide as compared with placebo, but there was no significant difference between the 3 doses of budesonide. In an open trial with patients with moderate to severe asthma the effects of progressively increasing doses of budesonide (400, 800, 1600 and 2400 μg/d) were studied [20 to inhaled budesonide but a plateau response of exhaled NO was found at a dose of 400 μg/d [1, exhaled nitric oxide (FENO) and methacholine PC20 [1 and methacholine PC20 from baseline or placebo treatment, but with no significant separation of active BDP doses. All doses of BDP resulted in a significant change in FENO from placebo treatment, but with significant separation of only the 100 μg and 800 μg doses by FENO. Another study assessed the dose-response relationship of the anti-inflammatory effects of BDP (50, 100, 200 and 500 μg/d) in the treatment of mild to moderate asthma for 8 weeks in a randomised, placebo-controlled, double-blind trial of parallel-group design [15 to saline were followed. There was a significant linear relationship between BDP dose and exhaled NO concentration, FEV1 and changes in sputum eosinophils at the end of treatment. In contrast no relationship was found between BDP dose and PD15 to saline. However, the results of this study may be confounded because the patients were treated with oral prednisolone for two days in the beginning of the study.In a study  with patreatment . In cont studied . Budeson400 μg/d . In a st400 μg/d . Compareine PC20 . All dos1 after placebo treatment was 24% and was significantly reduced in a dose-dependent manner to 12%, 11% and 6% for the 100, 200 and 800 μg daily treatments. The allergen-induced sputum eosinophilia (×104 cells/ml) 24 h after challenge during placebo treatment was 60.2 and was significantly reduced to 24.0, 15.3 and 6.2 for the 100, 200 and 800 μg daily treatments, respectively. Although a statistically significant dose-response relationship was present, the difference between the lowest and highest dose (8-fold difference) for late maximal fall in FEV1 or allergen-induced sputum eosinophilia was less than the difference between placebo and the lowest dose of MF.In a recent randomized and double-blinded study, 12 atopic mild stable asthmatic subjects were treated with placebo or mometasone furoate (100, 200 and 800 μg/d) for six days  in a croTaken together, the results suggest that there is tendency towards slightly higher anti-inflammatory efficacy with higher doses of ICS. At the moment there are only a few studies that assess the dose-dependency of the anti-inflammatory effects of ICS. Most of these studies included only small numbers of patients. However, despite the 4–8–16-fold differences in the doses of ICS studied, it has not been easy to demonstrate the dose-dependency of the anti-inflammatory effects of inhaled glucocorticoids. Thus, based on the scarce published evidence we would predict that doubling of the commonly used low to moderate doses of ICS is likely to produce only a small increase in the anti-inflammatory effect, suggesting that inflammation may be suppressed in most patients by relatively low doses of ICS.Glucocorticoids suppress corticotrophin levels, which may eventually lead to atrophy of the adrenal cortex and diminished levels of endogenous cortisol. The diminished levels of endogenous cortisol or reduced cortisol excretion have been used as markers of systemic activity of ICS. These systemic effects may include osteoporosis, behavioural effects, growth suppression, posterior subcapsular cataracts, risk for ocular hypertension and glaucoma as well as skin thinning and bruising . In the Table 2b, see Table 2b, see Of the 14 studies included in this review, in 8 the effects on HPA-axis suppression were analysed. No data on the effects of BDP, budesonide or TAA on HPA-axis were reported. Six of the 7 randomised, double-blind dose-response studies with FP also analysed its effect on HPA axis, measuring either basal morning cortisol levels, post-cosyntropin stimulation test levels or urinary excretion of cortisol metabolites (The dose-related effects of HFA-BDP (200–800 μg/d) were studied in 43 steroid-naïve asthmatic patients in a randomized double-blind fashion for 14 days . When thA randomized double-blind study with consecutive dose design  comparinFP has also been shown to suppress 8 am serum cortisol and urinary cortisol/creatinine ratio in a dose-dependent manner in a single-blind placebo-controlled cross-over study for 9 days in patients (n = 12) with mild to moderate asthma . SimilarA Cochrane review  collecteBased on the recent Cochrane review and meta-analysis  it seemsIn two randomized studies, TAA in the dose range of 400–1600 μg/d ,51 did nTaken together, the data on the systemic adverse effects of ICS is conflicting and seems also to reflect the study design. Several studies have measured only the basal morning cortisol levels or levels after stimulation with high cosyntrophin doses. However, these may be insensitive markers for HPA-axis suppression . Differe2-agonists may have several other actions that may contribute to their efficacy in relieving asthma symptoms. β2-Agonists inhibit plasma exudation in the airways by acting on β2-receptors on postcapillary venule cells. They inhibit the secretion of bronchoconstrictor mediators from airway mast cells and may inhibit release of mediators from eosinophils, macrophages, T-lymphocytes and neutrophils. In addition, β2-agonists may have an inhibitory effect on the release of neuropeptides from sensory nerves [2-receptors in inflammatory cells to overcome the desensitisation in response to chronic β2-agonist exposure [LABA provide long-lasting relaxation of airway smooth muscle, while the ICS provide potent topical anti-inflammatory action. In addition to these complementary actions, βy nerves . Corticoexposure . In addiexposure ,66.1 reversibility of at least 10–15% was required (Table 3, see 1 of >(40)–50% of predicted and a clearly positive symptom score was required in most studies (Table 3, see 1 (% predicted) varied between 61 and 87% in different studies, being 61–70% in 4 studies, 70–80% in 3 studies, 81–87% in two studies and was not reported in three studies. The mean absolute PEF values varied from 299 to 404 L/min and FEV1 from 2.12 to 2.54 L (Table 5, see Table 4, see Table 3, see The literature search identified 3 studies with formoterol -69 and 91 of at least 50% of predicted (mean 75–76%) with an increase in FEV1 ≥15% after inhalation of terbutaline. Addition of formoterol was superior to the increase in steroid dose in increasing FEV1 and morning PEF  in the dose of inhaled budesonide (from 200 μg/d to 800 μg/d) in patients with moderate to severe symptomatic chronic asthma . The pat®) with an increased dose of budesonide (400 μg/d) in adults with mild to moderate asthma (mean FEV1 81–82%) not fully controlled on low doses of ICS alone. The increase in mean morning and evening PEF was significantly higher for budesonide/formoterol compared with budesonide alone. In addition, the percentage of symptom-free days and asthma control days were significantly improved in the budesonide/formoterol group. Budesonide and formoterol decreased the relative risk of an asthma exacerbation by 26% as compared with higher dose budesonide alone.Another study  compared1 86–87% of predicted and using approximately 1 rescue inhalation per day) [1 and morning PEF in the patients already treated with a low dose of ICS, but not in steroid-naïve patients (Table 5), or in reducing the percentage of days with symptoms, number of rescue inhalations or nights with awakenings in the patients with mild persistent asthma already treated with low doses of ICS (Table 6, see The results of the formoterol study  on the bper day) . In thisA subgroup of the patients participating in the formoterol study  was analAs compared with the abovementioned three studies, similar superiority of addition of formoterol on morning PEF, rescue medication use and asthma symptoms were reported in an open randomised parallel-group study comparing the addition of formoterol to the low-dose BDP with 2-fold higher dose of BDP in patients suffering from symptomatic asthma, despite the use of inhaled BDP .Tables 3 and 4, see 1 better than increasing the dose of ICS 2–4-fold in 5 studies  and mean morning PEF in 7 studies , respectively , have r2-agonists may mask an increase in the underlying airway inflammation in asthma. Also, some proinflammatory effects have been described for β2-agonists such as delay of constitutive eosinophil apoptosis [The results of the above mentioned studies favour the addition of a LABA instead of increasing the dose of ICS in patients not adequately controlled with low to moderate doses of ICS. However, there have been concerns that regular use of inhaled βpoptosis  or reverpoptosis . Furtherpoptosis -86 do nopoptosis .In a randomised, double-blind and parallel-group study (n = 61) with similar inclusion and exclusion criteria than in the formoterol add-on study , the eff+ and CD8+ lymphocytes and lymphocyte activation as assessed as proportions of lymphocytes expressing HLA-DR were measured in BAL samples before and after treatment. There were no significant changes after salmeterol treatment. In another double-blind, parallel-group, placebo-controlled study [1 and PEF. In the week before clinical exacerbation, sputum eosinophil counts were higher in the salmeterol-treatment arm as compared with placebo, whereas there were no differences in PC20 or serum ECP. Five subjects showed >10% sputum eosinophilia before exacerbation during salmeterol treatment, compared to two receiving placebo. This suggests that the use of salmeterol allowed subjects to tolerate a greater degree of inflammation without increased symptoms or reduced lung function. Thus, during progressive reduction of ICS the bronchodilator and symptom-relieving effects of salmeterol may mask increasing inflammation and delay awareness of worsening asthma. These findings strengthen guideline recommendations that LABA should not be described as sole anti-asthma medication and that they should be used as "add-on" therapy rather than for steroid tapering purposes.In a small study (n = 9) with asthma patients using regular inhaled glucocorticoids and inhaled salbutamol for symptom relief, the addition of salmeterol for 8 weeks was studied in a double-blind crossover placebo-controlled protocol . Bronchoed study  the effeed study . SubjectThe effect of addition of salmeterol (50 μg bd), FP (200 μg/d) or placebo for 3 months on airway wall vascular remodelling has been studied in 45 symptomatic patients with asthma who were receiving treatment with ICS (range 400–1000 μg/d) . BronchiIf there were a marked masking of pulmonary inflammation by LABA, one would expect to see an increase in the number and severity of asthma exacerbations during their long-term use. There is some difficulty in comparing the different studies done with formoterol and salmeterol as the definition of exacerbation varies. In formoterol studies ,68 a sevTable 7, see In the formoterol study  the mainTable 7, see In contrast to that described in moderate to severe asthma, in the other formoterol study  addition®) with an increased dose of budesonide (400 μg/d) in adults with mild to moderate asthma (mean FEV1 81–82%) not fully controlled on low doses of ICS alone. Budesonide/formoterol combination significantly decreased the relative risk of an asthma exacerbation by 26% as compared with higher dose budesonide alone. In contrast, the estimated risk of having a severe exacerbation was 6% lower in patients treated with budesonide/formoterol compared with those receiving budesonide alone, but this was not statistically significant.Another study  comparedOnly two studies ,78 of thTable 7, see In the salmeterol studies lasting 3–6 months the numbers of patients with exacerbations were analysed. The meta-analysis  revealedRecently, the exacerbation rates and clinical measures of asthma worsening were assessed in an analysis combining results from two double-blind studies (n = 925) comparing addition of salmeterol to low-dose-FP with increasing the dose of FP 2.5-fold . The addThe addition of LABA to the treatment regimen usually results in a slight increase in those pharmacologically predictable adverse events such as tremor and tachycardia. However, generally these do not lead to the discontinuation of the treatment. In the formoterol studies -69, no sCysteinyl leukotriene receptor-antagonists (LTRA), such as montelukast, pranlukast and zafirlukast, are a new class of asthma medication, whose role in the stepwise management of asthma has not yet been fully established. Leukotriene antagonists blunt the obstructive response and have weak anti-inflammatory activity. In some studies corticosteroids are not very effective inhibitors of cysteinyl leukotriene pathways, at least when assessed by their inability to reduce cysteinyl leukotriene concentrations ,92 and t1 ≥50% predicted at visits 1 and 3, with a ≥12% bronchodilator response and symptoms requiring β-agonist treatment of at least 1 puff/day during the last 2 weeks of the run in period . Both groups showed progressive improvement in several measures of asthma control compared with baseline. Mean morning PEF improved similarly in the last 10 weeks of treatment compared with baseline in both the montelukast + budesonide group and in the double dose budesonide group (33.5 vs 30.1 L/min). The improvement in montelukast + budesonide group was faster as the mean morning PEF was significantly higher during days 1–3 after start of treatment in this group as compared with the double dose budesonide group (20.1 vs 9.6 L/min) .We identified one randomised, double-blind, parallel-group 16 week study (Jadad score 3) comparing the addition of montelukast (10 mg/d) to budesonide (800 μg/d) with doubling the dose of budesonide (1600 μg/d) in patients inadequately controlled on inhaled budesonide (800 μg/d, n = 448) . The inc1 ≥55%, a bronchodilator response greater than 12%, symptoms and rescue β2 agonist use of at least 1 puff/day. The mean FEV1 at baseline was 81% predicted. The patients were randomised to obtain either montelukast (10 mg/d) or placebo in a double-blind manner. The ICS dose remained constant throughout the study. The primary efficacy end point was the percentage of asthma exacerbation days. The major advantage of this study is that this study adopted several different definitions for asthma exacerbation days from previously published other studies, making comparison to other studies more easy. The median percentage of asthma exacerbation days was 35% lower (3.1% vs 4.8%, p = 0.03) and the median percentage of asthma free days was 56% higher (66.1% vs 42.3%, p = 0.001) in the montelukast group than in the placebo group. Thus, the NNT with montelukast to avoid one exacerbation day was 13, and the NNT to avoid one day not free of asthma – that is, to gain an asthma free day – was 10. Patients receiving concomitant treatment with montelukast had significantly less (25.6% vs 32.2%, p = 0.01) nocturnal awakenings, and significantly greater reductions in β2-agonist use (17.26% v 4.92%, p = 0.05, baseline use was 3.2–3.3 puffs/day), and morning PEF (16.86 L/min vs 11.30 L/min, p = 0.05, baseline 365–373 L/min). No significant difference was found in asthma specific quality of life or in morning FEV1. The results of this study suggest that although the effect of montelukast on endpoints such as morning PEF, FEV1 and rescue β2-agonist use are only small or modest, addition of montelukast may produce a significant improvement of asthma control by reducing the number of asthma exacerbation days.A large (n = 639) study  recruite1 and PEF, asthma symptom score and the percentage of asthma exacerbation free days better than placebo during 16 week treatment period [1 was approximately 140 mL and in morning PEF 10 L/min. There was a tendency towards reduced rescue medication use with the combination therapy, but the reduction was only 0.2 puffs/day. Addition of montelukast to ICS seemed to prevent the increase in the number of peripheral blood eosinophils seen in other treatment groups.In another study with patients (n = 642) with symptomatic persistent asthma despite the treatment with BDP (400 μg/d), addition of montelukast (10 mg/d), improved morning FEVt period . The inc2-agonist use, or morning or evening PEF. When treatment response was defined as a 15% or greater increase in mean PEF recordings, there were four responders to montelukast and seven responders to placebo. Although several points in this study may be criticised , the results suggest that the effects of montelukast are not as evident in unselected population than in the more clearly defined patients included in other trials [In an atypical "add-on" study , addition of montelukast (10 mg/d) was compared with placebo in patients with asthma (n = 72) and symptoms despite treatment with ICS and additional therapy . Most ofr trials -95.1 or histamine PC20 between the two treatment regimens. There was no difference in the efficacy of either treatment in decreasing T cell, CD45RO+, mast cell or activated eosinophil numbers in bronchial biopsies. In a third study [The additional anti-inflammatory activity obtained by adding montelukast to the treatment regimen has been assessed in three randomised, double-blind, cross-over studies lasting 10 days–8 weeks. In one study , additiord study , the addAddition of high-dose zafirlukast (80 mg b.i.d.: 4-fold greater than the approved dose) improved asthma control better than placebo in patients (n = 368) on high-dose ICS (1000 – 4000 μg/d) ,102, in According to recent meta-analyses (12 adult studies and 1 in children) ,102, leuAlthough theophylline has traditionally been classified as a bronchodilator, its ability to control chronic asthma is greater than can be explained by its relatively small degree of bronchodilator activity. In fact, theophylline has immunomodulatory, anti-inflammatory and bronchoprotective effects that may contribute to its efficacy as an anti-asthma drug . There iThe addition of theophylline has been compared with doubling the dose of ICS  in two separate studies with 195 patients with symptomatic asthma for 6 to 12 weeks ,107. The1 predicted normal ≥50%, bronchodilator response of at least 15% and to have symptoms despite the use of ICS . The overall treatment effect of addition of theophylline was superior to double-dose budesonide in improving FVC and FEV1  or non-inferiority between the treatments.In the study (Jadad score 4) of Evans and coworkers  addition1 Figure , althoug1 50–85% predicted normal and a documented reversibility of at least 15% of FEV1 over baseline and to be not controlled by BDP (400 μg/d) or equivalent. The sample size of this study was powered to detect equivalence. No significant differences were found between the high-dose BDP and low-dose BDP plus theophylline groups in outcomes such as morning or evening PEF, PEF variability, FEV1, daytime or nighttime symptom scores or rescue medication use. Both treatments were well tolerated.A randomised, double-blind parallel-group study (Jadad score 3) by Ukena and coworkers  comparedet al. [2-agonist use, symptom scores or in the number of exacerbations. Of note is that there were no difference between the low dose BDP alone and high dose BDP groups in any of the parameters. This study was powered to detect superiority of theophylline plus BDP as compared with high-dose BDP. There were no significant differences between the treatment groups for any of the commonly reported adverse effects. The results of this study suggest that when the benefit of an "add-on" therapy is evaluated as compared with double-dose inhaled steroid, additional group using low-dose steroid alone should be included to see whether even the doubling of the dose of steroid produces any benefit to the patient.Lim et al.  recruiteTaken together, the results from two relatively small studies suggest that addition of low-dose theophylline may be equal to doubling the dose of ICS in the treatment of asthma not adequately controlled by low dose of ICS. However, one needs to remember that the effect of doubling the dose of ICS on asthma control is generally small or negligible (see above). Furthermore, a placebo group should be included in these studies to see whether an improvement in asthma control is obtained by doubling the dose of ICS. Thus, more data is needed to confirm the present results. Use of theophylline at concentrations at the lower limit or slightly below the recommended therapeutic range may help to limit the adverse effects.1 between 50% and 80% predicted normal, and at least 1 additional sign of inadequate asthma control during the 7 preceding days. Salmeterol/FP combination was superior to montelukast/FP in improving morning PEF (24.9 vs 13.0 L/min), evening PEF (18.9 vs 9.6 L/min), FEV1 (0.34 vs 0.20 L) and shortness of breath symptom score (-0.56 vs -0.40) as well as increasing the percentage of days without rescue medication (26.3 vs 19.1%). In contrast, there was no significant difference in outcomes such as chest tightness, wheeze and overall symptom scores. Asthma exacerbation rates were significantly (P = 0.031) lower in the FP + salmeterol group (2%) than in the FP+ montelukast group (6%). Adverse event profiles were reported to be similar.Combination of fluticasone (100 μg bid) and salmeterol (50 μg bid) in a single inhaler has recently been shown to provide more effective asthma control than montelukast (10 mg daily) combined with FP (100 μg bid) in a 12 weeks study (randomised, double-blind, double-dummy, Jadad score 3) in patients (n = 447) whose symptoms were suboptimally controlled by ICS only . The inc1 above 50% and at least 15% bronchodilator response, and asthma symptoms at least at 4/7 days during run-in. Salmeterol/FP combination was superior to montelukast/FP in improving morning PEF (36 vs 19 L/min), evening PEF (29 vs 14 L/min), FEV1 (0.26 vs 0.17 L), percentage of symptom-free days (42.9 vs 31.5%), percentage of symptom-free nights (46.5 vs 41.1%) as well as increasing the percentage of days without rescue medication (47.9 vs 46%). In contrast, there was no significant difference in percentage of rescue free nights. The number of patients experiencing at least one asthma exacerbation (any severity) was significantly (P < 0.05) lower in the FP + salmeterol group (9.6%) than in the FP+ montelukast group (14.6%). The percentage of patients who had at least one asthma exacerbation of either moderate or severe intensity was 4.8% in the salmeterol + FP group and 8.4% in the montelukast + FP group, but this difference did not reach statistical significance. The time to the first exacerbation was significantly (P < 0.05) longer in the salmeterol + FP group than in the montelukast + FP group. Adverse event profiles were reported to be similar.A similar study  comparin1 50–90% predicted and at least 12% bronchodilator response, short-acting β2-agonist use of one puff/day or more and asthma symptoms. Salmeterol/FP combination was superior to montelukast/FP in improving morning PEF (34.6 vs 17.7 L/min), FEV1 (0.19 vs 0.11 L). In contrast, there was no significant difference in nocturnal awakenings and asthma specific quality of life score. The percentage of patients experiencing at least one asthma exacerbation (any severity) was shown to be similar in the FP + salmeterol group (19.1%) than in the FP+ montelukast group (20.1%). Also there was no difference in the time to the first exacerbation between the salmeterol + FP and the montelukast + FP groups. Peripheral blood eosinophils were reported to be reduced significantly more in the montelukast + FP group (-0.04 × 103/μl) than in the salmeterol + FP group (-0.01 × 103/μl). Interestingly more serious adverse events were reported in the salmeterol + FP group.Another very similar study  was desi1 between 50% and 80% predicted and at least 12% bronchodilator response. Treatment with salmeterol resulted in significantly greater improvements from baseline compared with montelukast for most efficacy measurements, including morning PEF (35.0 vs 21.7 L/min), percentage of symptom-free days (24 v 16%) and percentage of rescue-free days (27 vs 20%). Also total supplemental salbutamol use (-1.90 vs -1.66 puffs per day) and nighttime awakenings per week (-1.42 vs -1.32) decreased significantly more with salmeterol than with montelukast. Six percent of patients in the salmeterol group experienced a total of 27 asthma exacerbations compared with 5% of patients in the montelukast group who experienced 24 asthma exacerbations during the 12 weeks treatment period. However, the patients experiencing an asthma exacerbation were withdrawn from the study. Thus, annualised incidences of exacerbations cannot be compared [In another randomised, double-blind, double-dummy, parallel-group study (Jadad score 3) in patients (n = 948) with symptomatic asthma despite treatment with ICS, addition of montelukast (10 mg daily) was compared with addition of salmeterol (50 μg bid) for 12 weeks . Patientcompared . The saf1 in response to β2-agonist were 23–24% [2-agonist. However, one needs to remember that many of those studies done with leukotriene receptor antagonist to prove their efficacy in the treatment of asthma have been performed with patients displaying a significant response to β2-agonist. Another factor that might be considered to produce bias is that all the above three studies that report salmeterol to be better have been sponsored by the producer of salmeterol and that study reporting the non-inferiority of montelukast as compared with salmeterol has been sponsored by producer of montelukast.Taken together, addition of salmeterol seems to produce better improvement of asthma control when lung function is assessed than addition of montelukast in patients with asthma suboptimally controlled by small to moderate doses of ICS. However, in one long-term study  additione 23–24% , 27.0–27e 23–24%  and 18.4e 23–24%  in the s1, compared to placebo, there were significant differences with the first and last doses of montelukast as well as the first but not the last dose of salmeterol, thus indicating the development of some tolerance with salmeterol. Only montelukast produced a significant, albeit trivial, suppression of blood eosinophil count. There were significant improvements with the first doses of salmeterol for all parameters of lung function. After 2 weeks of treatment, there were significant improvements with both drugs on rescue bronchodilator requirement and morning PEF. There were no significant differences between drugs for any endpoints except blood eosinophils. Thus, the results suggest some anti-inflammatory activity for montelukast when used as an "add-on" therapy.In addition to the normal clinical endpoints, the effects of addition of salmeterol (50 μg bid) or montelukast (10 mg/d) to the treatment regimen were analysed on AMP bronchial challenge, blood eosinophil counts and exhaled NO in a placebo-controlled, double-dummy, crossover study in patients (n = 20) with persistent asthma not controlled with ICS . For the1 percentage predicted normal between 50 and 70% with or without asthma symptoms, or FEV1 of 70.1% to 80% of predicted normal values and symptoms or requirement for rescue β2-agonist use ≥4 puffs/day or diurnal PEF-variation of more than 20% at two days during 6 days run-in. Both inhaled salmeterol and oral zafirlukast resulted in within-group improvements from baseline in measures of pulmonary function (morning and evening PEF and FEV1), asthma symptoms, and supplemental salbutamol use. Salmeterol treatment resulted in significantly greater improvements from baseline compared with zafirlukast for most efficacy measurements, including morning PEF (28.8 vs 13.0 L/min), evening PEF (21.8 vs 11.2 L/min), combined patient-rated symptom scores for all symptoms (-35 vs 21%), daytime albuterol use (41 vs 25%) and night-time salbutamol use (42% vs 16%). Also, statistically significant differences favouring the addition of salmeterol were noted on patient-rated symptom scores for shortness of breath and chest tightness, percentage of symptom-free days, sleep symptoms, nighttime awakenings and percentage of days and nights with no albuterol use. There was no difference between the groups in symptom score for wheezing. Interestingly, the difference between salmeterol and zafirlukast was clear at week 1, but not at 4 weeks when the effect on FEV1 was analysed. One factor that may affect the results of this study is that there may be a randomisation bias as the proportions of patients using FP or TAA were not similar in the salmeterol and zafirlukast groups. This study was funded by the producer of salmeterol.In a randomised, double-blind, double-dummy parallel-group trial (Jadad score 3) addition of zafirlukast (20 mg bid) was compared with the addition of salmeterol (50 μg bid via MDI) for 4 weeks in adult and adolescent patients (n = 429) with persistent asthma . Patient1), asthma symptoms, and supplemental salbutamol use. Salmeterol treatment resulted in significantly greater improvements from baseline compared with zafirlukast for most efficacy measurements, including morning PEF (29.6 vs 13.0 L/min), percentage of symptom-free days (22.2% vs 8.8%) and percentage of days and nights with no supplemental albuterol use (30.5% vs. 11.3%).As a part of the above study , a rando1 reversibility of at least 15%. Patients (n = 64) were randomised to three different treatments budesonide (800 μg/d) plus formoterol (9 μg bid), budesonide (800 μg/d) plus zafirlukast (20 mg bid) or budesonide (800 μg/d) plus sustained-release theophylline (400 mg/d) for three months. After three months there were no between group differences in endpoints such as morning and evening PEF, PEF variability, FEV1, daytime or nighttime symptom scores and rescue terbutaline use. However, the addition of formoterol produced earlier improvements compared with the two other groups in criteria such as PEF variability, day- and night-time asthma symptom scores and supplemental terbutaline use. Patients in budesonide plus zafirlukast group experienced most adverse effects, but no statistical analysis was presented. The authors conclude that in patients who still have symptoms despite the treatment with ICS, the addition of any of these medications to the treatment is a logical approach and may be chosen.An open, randomised Turkish study  recruiteLABA  seem to have superior efficacy as add-on therapy in persistent asthma not controlled by low to moderate doses of ICS as compared with LTRA . More studies comparing the different add-on options are needed as well as studies with longer duration as the current evidence is mostly limited to follow-up period of 3 months.When assessing a patient with persistent asthma who is not adequately controlled by low to moderate doses of ICS:• It is important to find out whether the patient is using the prescribed medication correctly. Poor compliance in asthma patients treated with ICS is a very common reason for treatment failure. Compliance with ICS is often less than 50% ,118. Ora• Secondly, it is important to check whether the inhalation technique is adequate. Problems with the inhalation techniques are very common, especially among children and the elderly . Good pa• Thirdly, it is important to search for possible environmental factors, such as changes in home and working environment, hobbies and pets.If asthma exacerbations are the dominant problem, guided self-management of asthma has been proven to be an efficient treatment strategy. In a Cochrane review  self-manAddition of formoterol or salmeterol seems to be superior as compared with the increase in the dose of the ICS in improving lung function, controlling asthma symptoms and reducing the use of rescue bronchodilator treatment. By increasing (doubling) the dose of the ICS the clinical improvement is likely to be of small magnitude. However, if frequent exacerbations are the major problem, increasing the dose of ICS may significantly help to reduce the number of exacerbations. By avoiding doses above 1000 – 1500 μg/d (budesonide and BDP) or 500 – 750 μg/d (FP) the risk of systemic adverse effects remains low. However, it should be noted that the evidence on the superiority of LABA is limited to symptomatic patients with mild to severe persistent asthma currently treated with low to moderate doses of ICS and presenting with a significant bronchodilator response. Also, addition of the LTRA montelukast or zafirlukast may improve asthma control in patients remaining symptomatic with ICS and addition of montelukast may be equal to double-dose ICS. Addition of LABA  seems to produce better asthma control as compared with a LTRA (montelukast or zafirlukast) whereas the long-term efficacy of LTRA (montelukast) on asthma exacerbations may be equal to LABA . There is evidence that addition of low-dose theophylline to the treatment regimen may be equal to doubling of the dose of ICS. However, more studies are needed to better clarify the role of leukotriene antagonists and theophylline as "add on"-therapies. For patients with inappropriate inhalation technique the value of LTRA or theophylline are especially worth considering. More studies are now needed to compare between different add-on therapies and to explore the effect of more than one add-on therapy in patients with more severe asthma as well as in those having symptoms but not significant bronchodilator response.Another issue not addressed by these studies of large patient groups are the different responses of patients to the different add-on therapies. This needs to be studied by comparing add-on treatments in the same patients, but these studies are difficult and prolonged. In the future it may be possible to predict factors that predict the value of a particular add-on therapy in a particular patient, but the currently published studies unfortunately provide no guidance.50: forced expiratory flow when 50% of vital capacity has been exhaled, FENO: exhaled nitric oxide, FEV1: forced expiratory volume in one second, FP: fluticasone propionate, FVC: forced vital capacity, HFA: hydrofluoroalkane-134a formulation, HPA: hypothalamic-pituitary-adrenal, ICS: inhaled corticosteroid, LABA: long-acting β2-agonist, LTRA: leukotriene receptor antagonist, MDI: metered dose inhaler, NNH: number needed to harm, NNT: number needed to treat, PC20: provocative concentration causing a 20% fall in FEV1, PD20: provocative dose causing a 20% fall in FEV1, PEF: peak expiratory flow, TAA: triamcinolone acetonideACTH: corticotrophin, AMP: adenosine monophosphate, AQLQ: asthma quality of life questionnaire, BAL: bronchoalveolar lavage, BDP: beclomethasone dipropionate, ECP: eosinophil cationic protein, FEFHK carried out the literature searches, evaluated the studies, conceived the review and drafted the manuscript. AL, EM and PJB participated in the design and writing of the review. All authors read and approved the final manuscript.Tables 1–7-Kankaanranta.doc contains tables 1–7 of this review.Click here for file


A substantial proportion of protein interactions relies on small domains binding to short peptides in the partner proteins. Many of these interactions are relatively low affinity and transient, and they impact on signal transduction. However, neither the number of potential interactions mediated by each domain nor the degree of promiscuity at a whole proteome level has been investigated. We have used a combination of phage display and SPOT synthesis to discover all the peptides in the yeast proteome that have the potential to bind to eight SH3 domains. We first identified the peptides that match a relaxed consensus, as deduced from peptides selected by phage display experiments. Next, we synthesized all the matching peptides at high density on a cellulose membrane, and we probed them directly with the SH3 domains. The domains that we have studied were grouped by this approach into five classes with partially overlapping specificity. Within the classes, however, the domains display a high promiscuity and bind to a large number of common targets with comparable affinity. We estimate that the yeast proteome contains as few as six peptides that bind to the Abp1 SH3 domain with a dissociation constant lower than 100 μM, while it contains as many as 50–80 peptides with corresponding affinity for the SH3 domain of Yfr024c. All the targets of the Abp1 SH3 domain, identified by this approach, bind to the native protein in vivo, as shown by coimmunoprecipitation experiments. Finally, we demonstrate that this strategy can be extended to the analysis of the entire human proteome. We have developed an approach, named WISE (whole interactome scanning experiment), that permits rapid and reliable identification of the partners of any peptide recognition module by peptide scanning of a proteome. Since the SPOT synthesis approach is semiquantitative and provides an approximation of the dissociation constants of the several thousands of interactions that are simultaneously analyzed in an array format, the likelihood of each interaction occurring in any given physiological settings can be evaluated. WISE can be easily extended to a variety of protein interaction domains, including those binding to modified peptides, thereby offering a powerful proteomic tool to help completing a full description of the cell interactome. By combining phage display and SPOT selection, the binding partners of any peptide recognition motif can be identified, thus facilitating the identification of all protein-protein interactions within a proteome Saccharomyces cerevisiae emphasized the importance of confirming any interaction by different methods  that act as receptors to accommodate, in their binding pockets, short peptides in an extended conformation .We have recently shown that the peptide sequences, obtained by panning phage-displayed random peptide libraries with SH3 domains, can be used to derive position-specific scoring matrices to computationally scan the entire proteome in search of putative partners . This ap7. This figure is far beyond the limits of the current technology for peptide synthesis. On the other hand, one could use the information obtained from screening random peptide repertoires to filter out the amino acid sequences that are highly unlikely to bind, thereby decreasing the peptide sequence space to be tested experimentally. We will refer to this approach with the acronym WISE (whole interactome scanning experiment)  .It should be pointed out that WISE only addresses the problem of identifying natural peptides with the potential for binding to any given recognition domain. Although we use this information to infer the formation of protein complexes in vivo, there are a number of reasons why this inference could turn out to be incorrect. For instance, a peptide could be unavailable for interaction in the native protein structural context. Alternatively, the two inferred partners could be located in different cell compartments or expressed in different tissues or at different times during development. Finally, all the interactions that are mediated by an extended region of a protein surface and that cannot be supported by a relatively short peptide will be missed by this approach.S. cerevisaie proteins that contain SH3 domains belonging to five different specificity classes, as determined by phage display experiments . A detailed description of the relaxed patterns can be found in For each SH3 domain, we have defined a “relaxed pattern,” less selective than the pattern identified by comparing the most frequent ligands discovered by phage display. We have then used this to scan the whole  TM instrument)This approach was repeated for the eight SH3 domains. For each domain, approximately 1,500 peptides, matching the relaxed patterns, were selected for synthesis see . The pep−4 M or above give a positive signal in the assay  that the intersection of a protein list with any given functional category occurs by chance. The inferred ligands of the Ysc84, Yfr024c, and Rvs167 SH3 domains were significantly enriched for the GO biological process term “actin cytoskeleton organization and biogenesis” (p < 5.46 × 10−7, p < 7.06 × 10−6, and p < 5.50 × 10−5, respectively). By contrast, the partners of Abp1 and Myo5 SH3 domains were found to be enriched for the GO terms “actin cortical patch assembly” (p < 3.49 × 10−7) and “actin cytoskeleton” (p < 7.14 × 10−6). These results are in accord with the available information about the participation of these bait proteins in the organization of the yeast cytoskeleton, whereas arbitrarily selected gene groups of similar size showed no comparable enrichments for any of the GO terms (best result, p < 10−3).Interacting proteins often share similar functions and participate in common processes. Hence, we examined whether the proteins, found in our approach to bind to a specific SH3 domain, could be preferentially associated to a biological process. For this analysis we considered, as putative ligands, all the proteins containing at least one peptide with an intensity higher than an arbitrarily chosen threshold of 20,000 (in BLUs, corresponding to a dissociation constant of approximately 100 μM). We then used the FunSpec software  to identWe have previously shown that the information obtained by panning random peptide libraries can be used to draw an interaction network that recapitulates a fraction of the SH3-mediated interaction network determined by the two-hybrid approach . By usinThe Abp1 SH3 domain, compared to most of the remaining SH3 domains that we have studied, has a narrower peptide recognition specificity and, as a consequence, fewer inferred partners. Our analysis confirmed that Srv2 and Ark1, previously identified Abp1 SH3 functional partners , containIn order to assess how accurately peptide binding affinity permits us to infer physiological partners, we investigated whether the putative partners can be copurified with Abp1 in vivo. We used the tandem affinity purification (TAP) technology  to tag tTo establish whether Abp1 forms a complex with these proteins in vivo, we next affinity-purified the four tagged proteins on an IgG resin. We next probed the purified complexes with an anti-Abp1 antibody. As shown in We have also investigated whether the fraction of coimmunoprecpitated Abp1 protein correlates with either the BLU intensity or the dissociation constant of the corresponding SH3–peptide interaction. The observed lack of correlation indicates that other factors, as, for instance, local protein concentration (mediated by different interactions), are important in determining the efficiency of complex formation.Finally, we asked whether this approach could be extended to the analysis of a mammalian proteome that is approximately five to six times more complex than the yeast one. To this end, we selected two proteins involved in membrane recycling, amphiphysin-1 (P49418) and endophilin-1 (Q99962), and whose SH3 domains we had previously characterized by phage display . These tDynamin and synaptojanin, two proteins involved in endocytosis, form an SH3-mediated complex with amphiphysin and endophilin in vivo . Our appThe WISE strategy described here has the merit of combining the strengths of a selective approach  with a quantitative analysis that can be achieved by screening a large number of peptides arrayed at high density on a solid support. This makes it possible to identify rapidly and directly the tighest ligands of a peptide-binding receptor among all the peptides in an entire proteome. We have demonstrated the approach by applying it to the family of SH3 domains. However, WISE can also be extended to all those domain families (WW, PDZ, EH, GYF, VHS, SH2 PTB, 14-3-3, FHA, WD40, etc.) that mostly recognize short peptides in their partner proteins.Our approach, as any in vitro approach, suffers from some simplifications when it comes to inferring the physiological partners from the domain–peptide interaction data. According to a naive strategy, we would assimilate the cell to a cellulose membrane, where all the peptides are equally represented and accessible to the bait domain, and we would be tempted to conclude that all the proteins containing the identified peptide ligands were likely to be physiological partners. In the real cell, however, the target peptides may be hidden inside the core of the folded proteins, and the protein partners may not be equally represented. Furthermore, the partner proteins may be expressed in different cell types or segregated in different macromolecular complexes or cell compartments. In order to obtain more reliable inferences, the peptide interaction information obtained by a WISE approach should be complemented by information about peptide accessibility obtained by structural predictors  and dataNevertheless, the average number of peptides in the yeast proteome that have the potential to bind SH3 domains with an affinity that may have physiological relevance was found to be surprisingly high, ranging from a few peptides, in the case of the Abp1 and Boi2 SH3 domains, to several tenths, in the case, for instance, of the Yfr024w SH3 domain. Given the hypothesis that all (or most of) these peptides are equally expressed inside the cell and exposed to the solvent in the folded protein structure as most Pro-rich peptides are, these findings raise the question of whether the observed binding promiscuity has any physiological implication. Recent proteome-wide analyses of yeast protein complexes have revealed that many proteins are organized in discrete complexes . Yet thiAll the same, we have identified a larger number of target peptides that bind with affinities that are comparable to the ones of the validated physiological targets. Some of these peptides may never encounter the cognate SH3 domain, while some will only meet partners in specific physiological conditions. Others may contribute to add specificity to the formation of a complex by cooperating with other associated low-specificity binding domains. Finally, we have to consider a new scenario in which proteins, even when not forming stable complexes, are seldom isolated in solution, but navigate in the cell by moving from one weak partner to another. These weak interactions may be important in modulating cell architecture even when they are not instrumental in the nucleation of a stable complex. Although this is difficult to prove, the semiquantitative data provided by our approach, complemented with the results of large-scale expression and localization studies, may eventually allow one to model these different settings.The in vitro approach that we have described, albeit limited to interactions in which one of the partners can be reduced to a relatively short peptide, presents a number of interesting features that complement other strategies aimed at revealing the details of the protein interaction network within cells.First, it takes full advantage both of the genomic information that is being accumulated and of the array format in which all the possible targets are equally represented. Second, it is comprehensive and provides a high level of detail on the interaction topology. Third, it is not affected by protein concentrations inside the cell and is very sensitive (interactions up to 100 μM can be detected). Fourth, interactions that depend on peptide modifications, for instance, phosphorylation, can also be studied. Fifth, the output is semiquantitative. Finally, the identified target peptide can be used as a lead to develop tighter binding molecules in order to interfere with complex formation in vivo.We have shown that the current implementation of the SPOT synthesis technology is sufficient to carry out WISE screening of a proteome as complex as the one of a mammalian organism. Foreseeable technological improvements of the SPOT synthesis technology will permit the assembly of relatively cheap microarrays containing up to 15,000 peptides. This will extend the approach's power by relieving, in some cases, the requirement for an experimental filtering step, as performed here by the phage display approach, thereby allowing more freedom in the design of the relaxed pattern.Yeast PJ694a strains expressing TAP-tagged ORFs were constructed as described . PrimersThe polymerase chain reaction (PCR) fragments were used to transform the yeast strain. Positive clones were selected on selective plates and checked by PCR analysis and Western blot analysis. For the PCR analysis, we used a new forward primer together with the reverse ones used for the construction: forward for Yir003, AGCAGATGGAGGACCAAATGGAGGTTG; forward for Scp1, CGGTTATATGAAAGGTGCATCTCAGGC; forward for Prk1, CGTTTACAATCAAAGAAACTGCCGATTG; and forward for Ynl094w, GGACTCAATTCAAAAATTGAGCAATCAAG.Yeast strains expressing TAP-tagged Yir003w, Scp1, Prk1, Ynl094c, or Bmh1 as a control, were cultured at 30°C in 5-l flasks containing 2 l of YPD medium, collected in the exponential growth phase, and lysed mechanically with glass beads in 5 ml of IPP-150 buffer (10 mM Tris–HCl [pH 8.0], 150 mM NaCl, 0.1% NP-40) in the presence of protease inhibitors (2 mM benzamidine, 0.5 mM PMSF, 1 mM leupeptina, 2.6 mM aprotinin). Half of the extract was incubated for 2 h at 4°C with 100 μg of GST–Abp1SH3 (bound to glutathione–sepharose), while the remaining half was incubated with 100 μg of GST as a control. The resins were washed four times with 5 ml of IPP-150 buffer and the bound proteins recovered (by boiling in SDS–BLU–dye) and analyzed on a 10% SDS–polyacrylamide gel. They were transferred onto nitrocellulose membranes. Filters were blocked overnight at 4°C in PBS containing 5% milk powder (blocking solution), and then incubated with peroxidase (POD)-conjugated anti-POD antibody (PAP) antibody  diluted 1:1,000 for 2 h at room temperature (RT), washed five times for 15 min with PBS–0.05% Tween, and revealed by chemoluminescence. GST fusion proteins were expressed and purified by standard procedures.Yeast cultures expressing TAP-tagged Yir003, Scp1, Prk1, Ynl094c, or Bmh1 were cultured, collected, and lysed as described for the pulldown experiments. Each extract was incubated with 500 μl of IgG–sepharose  for 2 h at 4°C. The resins were washed four times in 5 ml of IPP-150 buffer, resuspended in 300 ml of 50 mM Tris–HCl (pH 8), 0.5 mM EDTA, 5 mM DTT, transferred to Eppendorf tubes, and incubated with 30 U of recombinant TEV protease  for 1 h at 20°C. After centrifugation for 2 min at 2,300 rpm, the supernatants were loaded on 10% SDS–polyacrylamide gels and then transferred onto nitrocellulose membranes. Filters were blocked overnight at 4°C in blocking solution, incubated for 2 h at RT with anti-Abp1 antibody (diluited 1:1,000), and washed five times with PBS–0.05% Tween. They were then incubated for 1 h at RT with anti-rabbit POD coniugated, washed ten times with PBS–0.05% Tween, and detected by chemoluminescence.Cellulose membrane-bound peptides were automatically prepared according to standard SPOT synthesis protocols  using a p-toluenesulfonic acid in methanol (50 ml) and shaken for 3 min. The membrane was removed from the tray and air-dried. Meanwhile a solution of 7.8 g of N-(2,3-epoxypropyl)-phathalimid in dioxane (60 ml) was heated up to 80°C in a covered stainless steel dish placed on a shaking platform using a heater mat. Then, a solution of 400 mg of p-toluenesulfonic acid in 5 ml of dioxane was added. Immediately, the membrane was placed in this solution and shaken at 80°C for 3–5 h. Afterwards, the membrane was washed three times with 50 ml of dioxane and ethanol (twice, 50 ml each) and subsequently incubated with a 10% (v/v) solution (50 ml) of hydrazine hydrate (80%) in ethanol for approximately 6 h. Finally, the membrane was washed twice with ethanol, three times with dimethylacetamide, and once again with ethanol (twice, 50 ml each), and dried. The loading of this type of amino-functionalized cellulose membrane is about 120–200 nmol/cm2.A 18 × 28 cm Whatman 50 paper (Whatman, Maidstone, United Kingdom) was immersed in a stainless steel dish containing a solution of 400 mg of TM instrument (Roche Diagnostics, Basel, Switzerland). For quantification, the SPOT signal intensities were measured in BLUs. To exclude false-positive results, in the SH3-incubation experiment, each membrane was preexamined with GST/anti-GST mAb/anti-mouse mAb. The data obtained with the different membranes were normalized by using as a reference the intensity of three control peptides that bind to the anti-GST antibody. The sequence of these peptides were QRALAKDLIVPRRP, LAKDLIVPRRPEWN, and DLVIRPPRPPKVLGL.Generally, all incubations and washing steps were carried out under gentle shaking. After washing the membrane once with ethanol (10 min) and three times for 10 min with Tris-buffered saline (TBS: 50 mM Tris-(hydroxymethyl)-aminomethane, 137 mM NaCl, 2.7 mM KCl, adjusted to pH 8 with HCl), the membrane-bound peptide arrays were blocked (3 h) with blocking buffer. Blocking reagent (CRB, Northwich, United Kingdom) was diluted 1:10 in TBS containing 5% (w/v) sucrose. After washing with TBS (10 min), 10 μg/ml of the corresponding GST-fused SH3 domain (in blocking buffer) was added and incubated overnight at 4°C. After washing three times for 10 min with TBS, the anti-GST monoclonal antibody (mAb)  was added at a concentration of 1 μg/ml in blocking buffer for 2 h at RT. Subsequently, the membrane was washed three times with TBS (10 min each) and the POD-labeled anti-mouse mAb (1 μg/ml in blocking buffer) was applied for 1.5 h at RT, followed by washing three times with TBS. Analysis and quantification of peptide-bound SH3 domains were carried out using a chemoluminescence substrate and the Lumi-ImagerSurface plasmon resonance measurements were carried out with a BIAcoreX instrument . Experiments were carried out on sensor chips with GST-fused SH3 domains and GST as a control. GST-fused SH3 domains and the GST were coupled to CM5 sensor chips using the EDC/NHS (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide and N-hydroxysuccinimide)amine-coupling kit, yielding approximately 4,350 resonance units in the case of the GST-fused SH3 domain and 4,330 resonance units for GST. Interaction analysis was performed at 25°C with the peptides dissolved in 10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, and 0.005% surfactant P20 (pH 7.4), at 15 μl/min flow rate, using six to seven dilutions, ranging from 500 μM to 65 nM. Dissociation constant values were evaluated by applying the steady-state model using BIAcore evalution 3.1 software.Dataset S1(102 KB TXT).Click here for additional data file.Dataset S2(104 KB TXT).Click here for additional data file.Dataset S3(103 KB TXT).Click here for additional data file.Dataset S4(86 KB TXT).Click here for additional data file.Dataset S5(104 KB TXT).Click here for additional data file.Dataset S6(104 KB TXT).Click here for additional data file.Dataset S7(104 KB TXT).Click here for additional data file.Dataset S8(76 KB TXT).Click here for additional data file.Dataset S9(183 KB TXT).Click here for additional data file.Dataset S10(184 KB TXT).Click here for additional data file.Table S1(66 KB PDF).Click here for additional data file.


Melanoma inhibitory activity (MIA) is a small secreted protein that interacts with extracellular matrix proteins. Its over-expression promotes the metastatic behavior of malignant melanoma, thus making it a potential prognostic marker in this disease. In the present study, the expression and functional role of MIA was analyzed in pancreatic cancer by quantitative real-time PCR (QRT-PCR), immunohistochemistry, immunoblot analysis and ELISA. To determine the effects of MIA on tumor cell growth and invasion, MTT cell growth assays and modified Boyden chamber invasion assays were used.The mRNA expression of MIA was 42-fold increased in pancreatic cancers in comparison to normal pancreatic tissues (p < 0.01). In contrast, MIA serum levels were not significantly different between healthy donors and pancreatic cancer patients. In pancreatic tissues, MIA was predominantly localized in malignant cells and in tubular complexes of cancer specimens, whereas normal ductal cells, acinar cells and islets were devoid of MIA immunoreactivity. MIA significantly promoted the invasiveness of cultured pancreatic cancer cells without influencing cell proliferation.MIA is over-expressed in pancreatic cancer and has the potential of promoting the invasiveness of pancreatic cancer cells. Despite improvements in diagnosis and treatment, pancreatic cancer remains one of the most common causes of cancer-related deaths in the world . One of To quantify the mRNA expression of MIA in pancreatic tissues, QRT-PCR was performed using RNA from pancreatic cancer tissues (n = 23) and normal pancreatic tissue samples (n = 17). The analysis revealed a 42 ± 28-fold increase (p = 0.0013) of MIA mRNA levels in pancreatic cancer tissues compared to the normal pancreas Fig. . To deteTo determine the functional role of MIA in pancreatic cancer, we first investigated MIA expression in pancreatic cell lines. QRT-PCR analysis revealed relatively high MIA mRNA levels in Mia PaCa-2, Panc-1, and SU8686 pancreatic cancer cells compared to the other cell lines Fig. . ImmunobMethods section, MIA significantly increased the invasion of Aspc-1 by 2.4-fold (p < 0.001) and the invasion of T3M4 by 3.1-fold (p < 0.0001) . To analyze whether MIA may promote invasiveness of pancreatic cancer cells, Matrigel-based invasion assays using a modified Boyden chamber were carried out in T3M4 and Aspc-1 pancreatic cancer cell lines that exhibited relatively low MIA expression levels according to QRT-PCR and Western blot data. Added to the top chamber as described in 01) Fig. . To inveOne of the most devastating aspects of malignant growth is the emergence of cancer foci in organs distant from the primary tumor, with most cancer mortality being related to metastases. Thus, understanding the molecular mechanisms underlying the metastatic process is one of the most important issues in cancer research.Pancreatic cancer is characterized by aggressive local tumor growth and early systemic tumor spread -10. ManyAnother important aspect of the metastasis process is neo-angiogenesis. Angiogenesis itself encompasses a cascade of sequential processes emanating from microvascular endothelial cells, which are stimulated to proliferate, degrade the endothelial basement membranes of parental vessels, migrate, penetrate host stroma, and initiate a capillary sprout . NumerouMelanoma inhibitory activity (MIA) increases cell motility by decreasing the attachment of the cells to the extracellular matrix (ECM). Overexpression of MIA leads to increased metastasis of malignant melanoma cells by enhancing invasion and extravasation ,23. In tIn contrast to observations in malignant melanoma, where MIA has been established as a reliable marker for prognosis ,24, we cThe reason why high MIA mRNA levels lead to high serum levels in malignant melanoma, but not in pancreatic cancer, is currently not known. As to the possible role of MIA in pancreatic cancer pathogenesis, MIA had no effect on the proliferation of pancreatic cancer cells, similar to previous experiments employing fibroblasts, keratinocytes, endothelial cells and lymphocytes. The only cellular system in which MIA has been found to influence cell growth is melanoma cells; in these cells, MIA exerts anti-proliferative effects .Although MIA did not affect the growth of pancreatic cancer cells in vitro, its impact on the invasion was striking. Interestingly, promotion of invasiveness of pancreatic cancer contrasts the previously demonstrated decrease in the invasion of MIA-treated malignant melanoma cells . A possiIn conclusion, our study shows a striking overexpression of MIA in cancerous pancreatic tissues without consequent elevation of MIA in the circulation. Involvement of MIA in regulation of invasiveness of pancreatic cancer cells indicates that this protein may serve as a novel therapeutic target in the search for anti-metastatic drugs.Pancreatic cancer tissue samples were collected from 55 patients who underwent pancreatic cancer resection in the Department of General Surgery at the University of Heidelberg, Germany, and in the Department of Visceral and Transplantation Surgery at the University of Bern, Switzerland. Twelve cases were stage I, 13 cases stage II, 23 cases stage III, and 7 cases stage IV pancreatic adenocarcinomas, according to the Union International Contre Le Cancer (UICC) system. According to routine pathological grading, 16 cases were well-differentiated, 22 moderately differentiated, and 17 poorly differentiated. Normal pancreatic tissues were collected from 34 healthy organ donors. Pancreatic tissues were either frozen in liquid nitrogen and stored at -80°C (for RNA and protein extraction) or immediately fixed in 4% paraformaldehyde solution and subsequently embedded in paraffin. In order to determine MIA serum concentrations, sera from 50 pancreatic cancer patients  and healthy volunteers  were collected at the Department of General Surgery, University of Heidelberg, Germany. Written informed consent was obtained from all patients. The study was approved by the Ethics Committees of the Universities of Bern, Switzerland, and Heidelberg, Germany.2. For TGF-β1 induction experiments, pancreatic cancer cells were seeded in 10 cm dishes in 10% FBS growth medium and allowed to attach for 12 hrs. Growth medium was replaced by serum-reduced medium (0.5% FBS), supplemented with 200 pM TGF-β1 for the indicated time periods. For experimental hypoxia, cells were subjected to a hypoxic microenvironment by one hour-long flushing in a special incubator chamber with an anoxic gas mixture (89.25% N2, 10% CO2, 0.75%O2) and sealing of the unit.Mia PaCa-2, T3M4, Aspc-1, Bxpc-3, Capan-1, Colo-357, SU8686 and Panc-1 pancreatic cancer cells and B16 (cloneB78/H1) mouse melanoma cells were grown in RPMI 1640 medium containing 10% FBS , 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen, Karlsruhe, Germany). Cells were maintained in a 37°C humidified atmosphere saturated with 5% COAll reagents and equipment for mRNA/cDNA preparation were purchased from Roche Applied Science (Mannheim, Germany). mRNA was prepared by automated isolation using MagNA Pure LC instrument and isolation kits I (for cells) and II (for tissue). cDNA was prepared using a 1st strand cDNA synthesis kit for RT-PCR according to the manufacturer's instructions. Real-time PCR was performed with the Light Cycler Fast Start DNA SYBR Green kit . The numBriefly, consecutive paraffin-embedded tissue sections (5 μm thick) were deparaffinized and rehydrated. Antigen retrieval was performed by pretreatment of the slides in citrate buffer (pH 6.0) in a microwave oven for 10 min. Thereafter, slides were cooled to room temperature in deionized water for 5 min. After blocking of endogenous peroxidase activity with 0.3% hydrogen peroxide and washing in deionized water 3 times for 10 min, the sections were blocked for 1 h at room temperature with normal rabbit serum (DAKO, Hamburg, Germany), then incubated with primary goat polyclonal anti-MIA antibody  overnight at 4°C. The slides were rinsed with washing buffer  and incubated with secondary rabbit anti-goat HRPO-labeled IgG (Sigma-Aldrich, Taufkirchen, Germany), diluted 1:200 for 45 min at room temperature. After color reaction, tissues were counterstained with Mayer's hematoxylin. For negative control, appropriately diluted goat IgG was used instead of the primary antibody.The amount of secreted MIA protein in cell culture supernatants and serum samples was determined using a one-step MIA ELISA (Roche Diagnostic GmbH, Mannheim, Germany) according to the manufacturer's instructions.Cells were washed with ice-cold PBS and collected in lysis buffer (50 mM Tris-HCl, 100 mM NaCl, 2 mM EDTA, 1% SDS) containing the Complete mini-EDTA-free protease inhibitor cocktail tablets from Roche (Roche Applied Science, Mannheim, Germany). Lysates were centrifuged at 13,000 rpm at 4°C for 30 min, the supernatants were collected, and protein concentrations were measured with the BCA protein assay  using BSA as protein standard. 20 μg of protein were mixed with loading buffer, heated at 95°C for 5 min, separated on 12% SDS polyacrylamide gels, and transferred onto nitrocellulose membrane at 100 V for 90 min. Membranes were blocked in 5% non-fat milk in TBS-T (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20) for 1 h, incubated overnight at 4°C with anti-MIA antibody (A-20, Santa Cruz) and exposed to secondary HRPO-labeled donkey anti-goat antibody (Santa Cruz) for 1 h at room temperature. The signal detection was performed using the ECL system (Amersham Life Science, Amersham, UK).For immunoprecipitation, pancreatic cell lines (Mia PaCa-2 and SU8686) were suspended in lysis buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 25 mM NaF, 10% glycerol, 1 mM PMSF) supplemented with the Complete-TM mixture of proteinase inhibitors (Roche Diagnostic, Mannheim, Germany) and incubated for 30 min on ice. After centrifugation, the supernatant was transferred into a fresh vial, pre-cleared with protein A-Sepharose beads (Santa Cruz) and incubated with 50 μl anti-MIA antibody (A-20, Santa Cruz) overnight at 4°C. Following addition of 30 μl of protein A-Sepharose for 1 h at 4°C, the mixture was pelleted, washed three times with lysis buffer, and resuspended in Laemmli sample buffer.Cell growth experiments were performed using the 3-(4, 5-methylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Pancreatic cancer cells were seeded at a density of 5000 cells/well in 96-well plates, grown overnight, and then exposed to different concentrations of recombinant MIA protein as indicated. After 24 h, MTT was added (50 μg/well) for 4 hours. Formazan products were solubilized with acidic isopropanol, and the optical density was measured at 570 nm.4 cells were incubated for 24 h and subsequently treated with MIA (100 ng/ml) [Invasion assays were performed in a BD Biocoat Matrigel Invasion Chamber with 8-μm pore size (BD Biosciences, Heidelberg, Germany) according to the manufacturer's instructions. The Matrigel was rehydrated with 500 μl DMEM (serum-free) and incubated in a 37°C, 5% CO2 atmosphere for 2 h. 5 × 100 ng/ml) , which wThe author(s) declare that they have no competing interests.JEF, NG, and AG carried out all the experiments, and participated in data analysis and interpretation. JK, MWB, and HF conceived of the study, and participated in its design and coordination. JK, NG, and AKB analyzed and interpreted the data and drafted the manuscript. All authors read and approved the final version.