KAR2, cannot account for the transcriptional regulation of most genes in this set. To address this puzzle, we analyzed the promoters of UPR target genes computationally, identifying as candidate UASs short sequences that are statistically overrepresented. We tested the most promising of these candidate UASs for biological activity, and identified two novel UPREs, which are necessary and sufficient for UPR activation of promoters. A genetic screen for activators of the novel motifs revealed that the transcription factor Gcn4p plays an essential and previously unrecognized role in the UPR: Gcn4p and its activator Gcn2p are required for induction of a majority of UPR target genes during ER stress. Both Hac1p and Gcn4p bind target gene promoters to stimulate transcriptional induction. Regulation of Gcn4p levels in response to changing physiological conditions may function as an additional means to modulate the UPR. The discovery of a role for Gcn4p in the yeast UPR reveals an additional level of complexity and demonstrates a surprising conservation of the signaling circuit between yeast and metazoan cells.Eukaryotic cells respond to accumulation of unfolded proteins in the endoplasmic reticulum (ER) by activating the unfolded protein response (UPR), a signal transduction pathway that communicates between the ER and the nucleus. In yeast, a large set of UPR target genes has been experimentally determined, but the previously characterized unfolded protein response element (UPRE), an upstream activating sequence (UAS) found in the promoter of the UPR target gene  The yeast unfolded protein response activates a large set of target genes, but a characterized element found in the promoter of one target, KAR2, cannot account for most targets. Using computational and experimental methods, the authors identify additional elements, as well a role for GCN4p in the response The vast majority of all cellular secretory and membrane proteins are folded and modified in the endoplasmic reticulum (ER), from which they are transported to their final destination in the secretory pathway. When the protein folding capacity of the ER is exceeded or experimentally impaired, unfolded proteins accumulate in the ER and activate the unfolded protein response (UPR). The UPR allows the ER to communicate with the nucleus , where aS. cerevisiae, unfolded ER proteins stimulate the ER-resident bifunctional transmembrane kinase/endoribonuclease Ire1p  . The UPRire1 or Δhac1 cells.The set of 381 genes was defined by microarray hybridization expression profiling, using a stringent quantitative “filter” that required the expression profile of each target gene to closely match that of previously known and well-characterized UPR target genes. In particular, the filter demanded that the expression profile of a target gene closely correlate to that of canonical UPR targets over a time course of UPR induction, and that induction be significantly greater in wild-type (WT) than in either ΔKAR2/BiP promoter , which is modeled as having been composed by concatenating “words” (short oligonucleotides) drawn from a probabilistic “dictionary” according to their frequencies. To infer the dictionary from the observed text, we employed the previously developed computational algorithm, MobyDick, which was developed based on a probabilistic segmentation model , 2000b. We first constructed a dictionary from the UPR target gene promoters. To this end, we compiled a text from the promoters of all 381 UPR target genes as previously defined . We defiBecause words with similar sequences are likely to possess similar biological activity, we considered groups of related words as units in our subsequent analysis. We grouped the dictionary into motifs by performing every possible pairwise alignment between all words, and then clustering words with high mutual alignment scores. A motif may contain two or more words, or just a single word. For a multiword motif, the words defining the motif are similar to one another and share common core sequences . The cluP of this many or more appearances occurring by chance. A small P value (high −log10P) indicates that the motif is overrepresented relative to the expectation. 10P for each motif plotted against this rank. We chose the eight highest-ranking motifs (open circles) as candidates for experimental testing  appeared in the approximately 6,000 promoters in the genome, and computed from this figure the frequency with which each motif would be expected to appear in a promoter if it were distributed randomly throughout the genome. We then counted the number of times the motif was actually found in the 381 target promoters and calculated the probability  testing B, analyzlacZ reporter construct that contains a crippled version of the CYC1 promoter that is transcriptionally silent in the absence of a UAS  A, or ditlacZ reporter constructs derived from two native promoters in which the motifs appear. We chose for UPRE-2 the promoter of ERO1, encoding an ER resident redox protein, and for UPRE-3 the promoter of DHH1, encoding an RNA helicase. Both genes are robust targets of the UPR . Similarly, WT but not Δire1 or Δhac1 cells bearing the UPRE-3-containing DHH1-promoter-driven reporter expressed higher levels of β-galactosidase after treatment with DTT (DHH1 promoter entirely eliminated induction by ER stress ( “− UPRE-3” columns).To test whether these motifs are also necessary for transcriptional activation, we designed  the UPR  and lackTaken together, the data presented so far indicate that, as with the classical UPRE-1, UPRE-2 and UPRE-3 are both sufficient A and neccis-regulatory elements that differ in sequence from the canonical UPRE-1 suggests that trans-activating factors other than Hac1p may bind these elements. Alternatively, Hac1p, alone or accompanied by another factor or factors, may be able to recognize multiple sequences. To distinguish between these possibilities and potentially reveal novel regulatory factors, we attempted to identify genes which, when overexpressed, activate transcription of the UPRE-2 reporter plasmid in the absence of an ER stress signal. The design of this screen recapitulates the approach which identified HAC1 as a high-copy activator of the UPRE-1  genomic DNA library  colonies were recovered and rescreened by the same assay. Plasmids from positively rescreened clones were retransformed into the Δire1 UPRE-2-lacZ strain to verify plasmid linkage of the activator phenotype.We transformed a strain bearing the UPRE-2- library . A Δire1IRE1 locus and surrounding sequences; IRE1 has been previously shown to be activated by overexpression and is a high-copy activator of UPRE-1  shared the f UPRE-1 . RecoverGCN4 locus. GCN4 encodes a bZIP transcription factor, which has been well-characterized as a component of the cellular response to amino acid starvation and other stresses , indicating that overexpression of GCN4 is sufficient to stimulate transcription from the UPRE-2-driven reporter gene in the absence of ER stress, Ire1p activity, or Hac1p production. We also starved cells for histidine by administering 3-aminotriazole (3-AT), which induces translation of Gcn4p . GCN4 overexpression alone did not activate transcription from either UPRE-1 or UPRE-3 reporter genes, emphasizing that these motifs are not synonymous with UPRE-2.The second class of positive inserts (eight plasmids) shared the stresses  but has activity A. GCN4 oof Gcn4p . As whenGCN4 overexpression is sufficient to activate transcription from a UPRE-2 reporter, we next asked whether GCN4 is also necessary to activate transcription in response to ER stress. We deleted GCN4 from strains bearing UPRE-1, UPRE-2, and UPRE-3 reporter constructs and assayed β-galactosidase activity in response to UPR activation. Upon UPR induction, HAC1 mRNA was spliced normally, and Hac1p was produced at WT levels in Δgcn4 mutants (unpublished data). However, Δgcn4 cells failed to induce transcription, not only of the UPRE-2-driven reporter but also of the UPRE-1- and UPRE-3-driven reporters , suggesting that GCN4 activity is limiting for UPR-dependent transcription from all three UPREs. Similarly, stimulation of Gcn4p production by amino acid starvation also increases the magnitude of the transcriptional response ; when eIF-2α is phosphorylated, scanning ribosomes fail to initiate at upstream ORFs encoded by the GCN4 5′ UTR and are able to initiate translation at the GCN4 ORF itself , and the set of genes previously identified as UPR-independent (“nontargets”) . The disire1 cells . This is the case both for target genes bearing any UPRE in the promoter (a–l) as well as the remainder of the target set for which a UPRE has not been identified (m–p). For those genes with an identified UPRE in their promoters, expression patterns in both Δgcn4  and Δgcn2 mutants  show trends similar to those in Δire1. In both mutants, the sets of genes whose promoters contain a UPRE are significantly less upregulated relative to their induction in the WT. Some UPR target genes exhibit residual upregulation in Δgcn4 and Δgcn2, suggesting that these promoters have only a partial requirement for GCN4/GCN2. This effect is most prominent for genes containing UPRE-1 in the Δgcn2 mutant (j), where the residual induction crosses the threshold into marginal statistical significance ; it is possible that the residual levels of Gcn4p present in a Δgcn2 mutant are sufficient to allow UPR transcription from these promoters, or alternatively that UPRE-1 promoters are relatively less sensitive to Gcn4p levels  for induction (see Discussion). In contrast, induction of the “no UPRE” genes is quite high in Δgcn4 and Δgcn2 cells (o and p versus m). As a population, these genes are not significantly less upregulated in the mutants than in the WT. It would appear that the UPREs identified to date define a special subset of UPR target genes that are responsive not only to IRE1 and HAC1 but that are particularly sensitive to the GCN4/GCN2 branch of the pathway.The majority of the genes in the nontarget set , as previously reported , Gcn4p levels are regulated via phosphorylation of eIF-2α by Gcn2p . Becausereported . We obsereported B.GCN4 by ER stress mediated by increased phospho-eIF-2α. Here, we observe increased Gcn4p levels under ER stress conditions even when phospho-eIF-2α levels are not detectably altered.These data are consistent with two possibilities: first, that Gcn2p is responsible for both basal levels of Gcn4p and its induction upon ER stress; or second, that Gcn2p is responsible only for maintaining basal levels of Gcn4p, while another pathway mediated by Ire1p/Hac1p further elevates Gcn4p levels during the UPR. If Gcn2p is responsible for upregulation of Gcn4p during the UPR, we should observe a concomitant increase in the level of eIF-2α phosphorylation. We did not observe such an increase . It is pGCN4 plays an essential role in the UPR, with a knockout phenotype closely resembling that of Δire1 and Δhac1: the absence of any of these genes prevents transcriptional activation by ER stress. This observation could be a consequence of one of several different mechanisms: Gcn4p might act upstream or downstream of Hac1p in the same linear pathway, or act in a parallel pathway that converges at target promoters. Two lines of evidence from data already introduced argue that Gcn4p does not act upstream of Hac1p. First, GCN4 overexpression is sufficient to activate transcription from UPRE-2 in a Δhac1 mutant . In the absence of GCN4, however, the constitutive expression of Hac1p does not activate transcription from any of the three reporter constructs , suggesting that Hac1p's function at promoters containing any one of the three UPREs requires the presence of Gcn4p. Thus, Gcn4p must act at the same point as or downstream of Hac1p. Following the same line of reasoning, for UPRE-1 and UPRE-3, GCN4 overexpression alone is insufficient to activate transcription in the absence of HAC1 , indicating that at UPRE-containing promoters Hac1p must act at the same point as or downstream of Gcn4p. Thus, the observations enumerated here are consistent with the interpretation that Gcn4p and Hac1p act together at target gene promoters.Further evidence that Gcn4p does not act upstream of Hac1p is provided by the observation that expression of Hac1p cannot activate transcription in a Δ4 mutant . In a WTKAR2 promoter (oligo 1), used in previous experiments demonstrating direct binding of Hac1p to UPRE-1 (ERO1 promoter (oligo 2). 32P-labeled oligonucleotides were incubated with cell extracts and subjected to native (nondenaturing) polyacrylamide gel electrophoresis, and visualized by autoradiography , in which Hac1p cannot be synthesized. Likewise, in both Δgcn4 and Δgcn2 mutants, the binding activity observed in WT cells was absent. In both Δgcn4 and Δgcn2 mutants, however, a faster migrating complex appeared, which likely represents Hac1p alone binding the oligos .As previously observed, oligo 1's mobility was retarded when incubated with crude extracts from UPR-induced cells, but not extracts from untreated cells . Although the two new UPRE sequences look at first glance entirely different from the well-characterized UPRE-1, one of them may share “half-site” similarity: UPRE-2 has a three base identity with UPRE-1 at the 3′ end (TACGTG versus CAGNGTG); whether these bases make equivalent contacts with the bound transcription factors remains to be determined. Taken together, the sequence diversity of the motifs conferring similar transcriptional control upon binding of the same transcriptional activators illustrates the difficulty of predicting biological regulation from promoter sequences alone, even if binding sites in one context are well defined experimentally.Beginning only with the set of genes induced by the UPR and the promoter sequences of all genes in the genome, we computationally identified candidate motifs that obeyed the statistical properties we would expect of regulatory sequences, i.e., high frequency in UPR target promoters, and enrichment in the target promoters relative to the rest of the promoters in the genome. Two of these motifs, UPRE-2 and UPRE-3, are both necessary and sufficient to confer ER stress responsiveness in an cis-acting elements known to act in the UPR.The identification of these novel sequences allows a greater proportion of UPR target gene regulation to be explained within the paradigm of modular transcriptional control, i.e., in which a “portable” sequence module (a UAS) located within a promoter confers pathway responsiveness on the gene in question. The two novel motifs described triple the number of target genes whose regulation can be described in terms of a modular control mechanism, thus adding significantly to the repertoire of And yet, the resulting description of UPR transcription remains incomplete, as approximately 50% of the target genes still lack a recognizable UPREs. It may be that more biologically active motifs exist among the 109 motifs that emerged from the overrepresentation analysis, as many of the untested motifs are overrepresented relative to chance in the UPR target set by many orders of magnitude. For the eight motifs tested, we tested whether a motif was necessary for promoter induction only if it had already been shown to be sufficient in the artificial promoter system. Because of this experimental approach, it remains possible that some motifs not found to be sufficient are dependent for their activity on some contextual parameter . Thus it may be that some UPREs are not generally portable to other contexts, but are nonetheless necessary for UPR responsiveness of the native promoters in which they reside. Also, particularly rare motifs would have been omitted from the dictionary; thus, it is possible that complementary computational approaches might allow detection of uncommon motifs that this analysis missed. Finally, some UASs may remain ultimately undiscoverable within the paradigm of modular regulation. Motifs that are particularly sensitive to chromatin structure or position relative to the transcription initiation site would not be detected by an approach that neglected these parameters.ERO1 after removal of UPRE-2 .A plausible resolution to this paradox may be that the UPRE-1 is heavily dependent on context. The experiments that defined the key core nucleotides proceeded by single point mutation at each position while holding constant the identity of all other nucleotides from the source 22-bp stretch of the  species A. This cDespite these qualifications, the approach has successfully uncovered novel information about how the UPR is regulated. The appealing aspect of the strategy described here is that such studies are not limited to the UPR but can be generally employed in the study of any transcriptional response in any organism for which promoter sequences for all genes are known and in which the comprehensive genomic output of the response can be measured by expression profiling. The sole requirement of the probabilistic segmentation/overrepresentation computations is that a partition of the genome (into “target genes” and “nontarget genes”) be made on the basis of some meaningful difference in expression levels under the conditions of interest; the analysis thereafter proceeds by comparing the distribution of candidate motifs in the target gene set and the remainder of the genome. Further refinement of the mathematical tools therefore promises to be of invaluable help in our quest for a comprehensive understanding of the logic and complex interactions of transcriptional programs in eukaryotic cells.The overexpression screen for activators of UPRE-2 revealed a role for the transcription factor Gcn4p, which we show to be required not only for activity of UPRE-2 but for all three known UPREs. Gcn4p and its upstream activator Gcn2p thus join Ire1p, Hac1p, and Rlg1p in the list of essential players in the yeast UPR.GCN4 encodes a well-characterized transcription factor acting in several distinct stress responses including amino acid starvation, glucose limitation, and ultraviolet irradiation , which retains some expression of Gcn4p. In the absence of Hac1p but in the presence of high concentrations of Gcn4p , Gcn4p can bind the UPRE-2 on its own, either as a homodimer or a monomer.The basis for this differential affinity for Gcn4p is strongly suggested by a refined consensus sequence for UPRE-2, and is illustrated by multiple species alignment of the promoter B. We seapromoter . The res compare A and 7B The transient upregulation of Gcn4p levels, which we observe upon UPR induction, may therefore serve to increase the transcriptional output of the response, especially early in the response. Most UPR target genes are robustly induced after 15 min of ER stress ; hence, GCN4 for normal UPR induction are those that are most urgently required by the cell under specific conditions, under which UPR is induced and Gcn4p levels are high for reasons unrelated to ER stress. The relationship between the cellular stress responses that regulate Gcn4p and the potentiation of UPR transcription will therefore be an important subject for future study.Gcn4p itself mediates a broad transcriptional program in response to a diverse set of cellular conditions and stresses . The recIRE1 and HAC1 mediate the transient increase in Gcn4p remains to be elucidated. Given that Hac1p and Gcn4p are observed in the same complex with DNA, one intriguing possibility is that association with Hac1p serves to stabilize Gcn4p.The mechanism by which HAC1 mRNA splicing retains its role as the “switch” that turns the UPR on or off. Gcn4p, whose levels appear to be limiting for the extent of gene regulation, would therefore play a role in setting the “gain” or “volume” of the response, perhaps allowing communication from other stress response pathways in the cell. Such a gain control could serve as an adjunct to the “Super-UPR” (S-UPR) gain control described in the accompanying paper  and of a mechanism for transcriptional regulation of Hac1p . MobyDick generated a dictionary of 328 words. We filtered out words that were too short, appeared in too many copies (such as AT-rich short repeats), or were of low quality . With the filters number_of_copies < 200, length > 4, and quality_factor > 0.2, we obtained 201 words.To build the dictionary of putative regulatory elements for UPR target genes, we first extracted the 600-bp upstream regions of all UPR target genes. To get rid of simple repeats unlikely to be regulatory elements (such as AT-rich repeats and transposable elements), we removed exact repeats of lengths 15 bp or longer, and kept the remaining fragments of lengths longer than 50 bp. What remained was the input sequence for the dictionary construction. We used the MobyDick algorithm based on probabilistic segmentation  to buildx mutates to any other given base y with probability p/3, and remains the same base with a probability (1 − p). The score for a pair x–y is given by the log-odds-ratio of observing the pair under the mutation model versus observing the pair at random. With the choice of p = 0.5 , a matching pair scores ln(2), and a mismatch scores ln(2/3). We normalized the scores to fall between 0 and 1 by the largest score. We then used the CAST algorithm to group words into clusters, with the threshold parameter set at 0.7 . This procedure generated 109 motifs.Using the filtered dictionary, we grouped similar words into motifs using the clustering algorithm CAST , as follexpN in the UPR target gene promoters based on the genome-wide frequencies. We then counted the observed number of occurrences obsN of the motif in the promoters of UPR target genes. We used Poisson statistics to calculate the probability P of observing a number of occurrences equal to or greater than obsN by chance, based on expN. The test based on Poisson statistics is a very good approximation of the more rigorous test based on the binomial distribution, where the probability P is the probability of seeing a specific instance of the motif in the UPR gene set and the total number of trials tN is the total number of copies of the motif in the genome. Since P is small (0.059) and tN is large (ranging from approximately ten to approximately 1000) but the product expN is finite, the resulting distribution is well approximated by a Poisson distribution with mean = expN.To test which motifs are significantly overrepresented in the promoters of UPR target genes, we counted for each motif the total number of occurrences in all promoters, and calculated the expected number of occurrences S. cerevisiae, S. bayanus, S. mikatae, S. paradoxus, and S. kudriavevii  UPRE. Intact promoter reporter constructs (pPW668–pPW671) used in ERO1 or DHH1 initiation codon, or by two fragments spanning the same sequence but with the UPRE motif replaced by a restriction site. The high-copy GCN4 plasmid (pPW672) used in GCN4 ORF. Source sequence contexts, olignucleotide sequences, and select PCR primers are compiled in HAC1 used in HAC1 (pPW353/pJC316) used in UPRE reporter constructs used in B, and 5  the UPR  and, if GCN4 and GCN2 and the integrated GCN4-myc were constructed by PCR cassette/generic primer mutagenesis  prior to addition of drug.HIS3 gene; for histidine-deprived cultures, overnight cultures were washed three times in SD-histidine, then diluted to low density in SD-histidine and grown to midlog phase before the addition of the drug.DTT  was added to cultures to a final concentration of 2 mM. Tm  was added to cultures to a final concentration of 1 μg/ml. 3-AT (Sigma) was added to cultures to a final concentration of 10 mM. All 3-AT treatments were performed on strains WT for the To assay β-galactosidase activity on solid growth media, we overlaid plates with buffered soft agar containing X-gal (Sigma) as described previously . For liqStrains were grown in YPD (pH 5.4) as in http://derisilab.ucsf.edu/more) and was performed as described previously  using a z-score scheme. For a given gene set and a given strain, we calculated the average fold induction for genes in the set and compared it to the value for the genome overall. The null hypothesis was that the selected gene set was no different from a randomly selected set  from the genome overall. Under this hypothesis, the average μ has a distribution well approximated by a normal distribution  with mean genomeμ and standard deviation σ/√(Nset), where setN is the total number of genes in the test set. We computed a z-score, z=√(Nset)(μ−μgenome)/σ, which should have a standard normal distribution (zero mean and unit variance) under the null hypothesis. The P value was calculated by integrating the standard normal curve from z to infinity.We tested the statistical significance of the induction for the three gene sets  in four different strains . For sequences, see Gel shifts were performed as previously described  except tTable S1N, the average number of times the string is delimited as a word among all segmentations of the data; Xi, the number of matches of the word anywhere in the text; p, the frequency of drawing the word from the dictionary, optimized over all words to give the maximum likelihood of observing the text; Z = p + sp, where sp is the probability with which the word can be made by combining shorter words from the dictionary; sig = significance = Np/sqrt(N[Z − p]).This table contains an alphabetical list of the dictionary “words” compiled by the MobyDick algorithm from the “text” comprising the promoters of UPRE target genes. Associated statistics for each word are as follows: (10 KB TXT).Click here for additional data file.Table S2tot is the number of times a given motif appeared in the promoters of the genome overall; Nexp is the number of times one would expect a given motif to appear in the 381 promoters of UPR target genes if the motif were distributed randomly throughout all promoters; Nobs is the number of times a given motif actually appears in the target gene promoters; and −log10P is a measure of overrepresentation based on Poisson statistics (P is the likelihood that a given observed distribution would occur by chance).N(3 KB TXT).Click here for additional data file.Table S3KAR2 and ERO1 promoters, derived from S. cerevisiae and related budding yeasts. Asterisk indicates 100% conserved residues. Scer, S. cerevisiae; Skud, S. kudriavevii; Spar, S. paradoxus; Smik, S. mikatae; Sbay, S. bayanus.This table contains CLUSTALW alignments for the (8 KB TXT).Click here for additional data file.Table S4This table contains the sequences of primers and olignonucleotide sequences used in construction of plasmids for this study, as well as oligonucleotide sequences used for probes in the gel-shift analysis.(38 KB DOC).Click here for additional data file.The GenBank accession numbers of the gene products discussed in this paper are Dhh1p (NP_010121), Ero1p (NP_013576), Gcn4p (NP_010907), Gcn2p (NP_010569), Hac1p (NP_011946), and Ire1p (NP_116622).Microarray data can be accessed at the Gene Expression Omnibus (GEO) at the National Center for Biotechnology Information (NCBI) database as platform number GPL1001 and sample numbers GSM16985–GSM1988.


Recently reported technologies for DNA-directed organic synthesis and for DNA computing rely on routing DNA populations through complex networks. The reduction of these ideas to practice has been limited by a lack of practical experimental tools. Here we describe a modular design for DNA routing genes, and routing machinery made from oligonucleotides and commercially available chromatography resins. The routing machinery partitions nanomole quantities of DNA into physically distinct subpools based on sequence. Partitioning steps can be iterated indefinitely, with worst-case yields of 85% per step. These techniques facilitate DNA-programmed chemical synthesis, and thus enable a materials biology that could revolutionize drug discovery. Resin-linked oligonucleotides are described that efficiently partition subpools of DNA based on sequence, enabling DNA- programmed chemical synthesis The information is amplifiable, so that a single molecule can be copied to produce a measurable quantity of nucleic acid. A large collection of enzymatic tools  and man-made tools  exist to manipulate DNA. Several technologies take advantage of these facts. For example, patterned DNA fragments have been used to direct self-assembly of nucleic acid objects , second position , and subsequent positions. Codons present in one coding position are mutually exclusive of the codons at any other position. The identity of the first codon determines the fate of the gene at the first decision point of the network, the second codon at the second decision point, and so forth.Our experimental scheme is illustrated in Genes are “read” by hybridization to a set of anticodon columns. Each anticodon column displays an oligonucleotide complementary to one codon sequence, and a complete set of columns comprises the complements to all codons at a single coding position. Genes bind to the columns by codon–anticodon base pairing, and are thus partitioned. To read a subsequent coding position, the genes are first transferred to a nonspecific DNA binding resin, regenerating an unhy-bridized state. This DNA is then hybridized to a new set of anticodon columns. By a series of such reading cycles, the sequence of a gene guides it through the network.8 unique genes contained routing instructions for eight decision points.For DNA routing genes, we adopted a modular design adaptable to networks of varying depth and width. We chose codons consisting of 20 bases, catenated to neighboring codons through 20-base noncoding regions . To prev1, a2, . . . , a8) into ten 340-base genes . The ten different genes were subcloned. Eight 60-base segments were then PCR amplified from an equimolar mixture of the parental plasmids. Each segment consisted of a coding position and two adjacent noncoding regions. The eight degenerate products were spliced together into 340-base fragments by primerless PCR, thus producing a library of 108 complexity. In principle, collective assembly of the 160 40mer oligonucleotides would have created the library in one step. In practice, the two-stage approach provided better control over codon distributions in the final gene population.Construction of the gene library proceeded in two stages A. InitiaThe noncoding regions play an instrumental role in the construction and handling of genes. By providing conserved crossover points, they facilitate the modular generation of highly complex populations from a small number of starting oligonucleotides. For the same reason, the noncoding regions make it possible to diversify existing gene sets by recombination. The noncoding regions also place codons in the correct coding position, ensuring that all genes incorporate one codon per branch point of a network. The existence of a well-defined “reading frame” results from the fact that anticodon columns only hybridize to DNA subsequences at a specified coding position, and not to codons elsewhere in the gene.To obtain hybridization-capable nucleic acid, the duplex DNA genes must be converted to a single-stranded form. . A numbSynthesis of anticodon columns involves covalent attachment of oligonucleotides to a solid phase. Thiol-containing and amine-containing oligonucleotide modification reagents are commercially available, and either should facilitate coupling to an appropriately activated resin. However, pilot experiments indicated that amide linkages were more easily prepared than thioether linkages. The deprotection protocols for oligonucleotide-linked sulfhydryl moieties were more complex than for amine moieties, and sulfhydryl-modified oligonucleotides were prone to oxidation and general loss during manipulation steps. Amine-modified oligonucleotides were easier to work with and were thus used for production of anticodon columns. It proved necessary to desalt crude oligonucleotides over reverse-phase cartridges before coupling.N-hydroxysuccinimide[NHS]-activated Sepharose, carbonyl diimidazole-activated Sephacryl S-1000). 20-base modified anticodon oligonucleotides were coupled to the resins. Quantification by reverse-phase chromatography of the uncoupled oligonucleotide remaining in solution provided a measure of reaction progress , methacrylate , and agarose  analysis of the load, flow-through, and elute fractions . By thisFollowing an observation that long polyethylene glycol (PEG) linkers dramatically improve hybridization to DNA on polypropylene surfaces , we syntWe next investigated how buffer conditions and temperature influenced the accuracy and yield of 340-base ssDNA hybridization to anticodon columns. For these experiments, a single radiolabeled DNA gene was diluted 10-fold into an excess (50 pmol) of an unlabeled routing gene library, and loaded onto a 250-μl diethylaminoethyl (DEAE) Sepharose column. The DEAE Sepharose column was placed in a closed 3-ml fluid circuit containing ten anticodon columns, of which only one complemented a codon within the radiolabeled DNA. Hybridization buffer was pumped over the system in a direction that placed the complementary anticodon column distal to the DEAE Sepharose column , left. ABy varying the temperature (25 °C to 70 °C), salt identity  and salt concentration (10 mM to 2 M) of the hybridization buffer, we determined that 1.5 M sodium chloride in a phosphate buffered solution with pH 6.5 at 45 °C provided the most robust hybridization behavior over multiple codon sequences. In addition, an initial high temperature step (70 °C) and the presence of a DEAE column inline proved critical to achieving uniformly high yields . The higIn order to route a DNA fragment through successive levels of a hierarchical tree, multiple hybridization steps are required. DNA from parental anticodon columns must be isolated and hybridized to anticodon columns corresponding to the daughter-node branches. The manipulations must be highly efficient to ensure good routing yields through trees with many levels.n for n hybridizations.We investigated several schemes for accomplishing iterative hybridizations. Our initial strategies utilized a multi-step procedure with three columns , linear transfer formats, and centrifugal evaporation. These three-column strategies did not prove to be high-yielding. We eventually observed that efficient iterative hybridizations could be accomplished with only two columns, using cyclic column-to-column transfers. Thus, a parental anticodon column with bound DNA was placed in a liquid circuit with a 250-μl DEAE-Sepharose column , right. The final requirement was to isolate DNA as a concentrated, salt-free aqueous solution upon completion of routing. For this purpose, DNA bound to anticodon columns was eluted with a small volume of an (ethylenedinitrilo)tetraacetic acid (EDTA) solution and precipitated. Alternatively, DNA bound to DEAE-Sepharose columns was transferred to a reverse-phase cartridge by cyclically pumping a high-salt buffer over the two columns in series. DNA on the reverse-phase cartridge was then washed with deionized water, eluted in acetonitrile/water, concentrated by evaporation, and desalted over a microcentrifuge gel-filtration column.Escherichia coli SSB or RecA  polyethylene glycol of average molecular weight 1500 Da  and all other chemicals and solvents were purchased from Sigma-Aldrich . DEAE columns were prepared by pipetting approximately 250 μl of DEAE Sepharose Fast Flow resin  into empty TWIST column housings .1–a8), b-type codons (b1–b8), and so forth. Each set of 16 oligonucleotides was assembled into a 340-base fragment by primerless PCR , 500 μM EDTA, and 500 μM EGTA. Acrylamide was removed by spin column filtration, and the solution volume was reduced to 800 μl by centrifugal evaporation. Samples were phenol/chlorofom extracted, ethanol precipitated, and resuspended in water.ssDNA was generated using a modified NASBA reaction . Duplex The crude Jeffamine material was purified by fast protein liquid chromatography cation-exchange chromatography over a 5-ml Hi-Trap SP column (Amersham Biosciences #17-1152-01). In early work, 1-ml batches of a 250-mg/ml aqueous solution were loaded onto the column in 50 mM acetic acid, washed with load buffer, and bumped off with 1 M lithium chloride. Subsequently, we developed a gradient protocol. The material was loaded in water, and the product was eluted with a linear water–hydrogen-chloride gradient (0–30 mM hydrogen chlo-ride over 15 column volumes) at 6 °C monitored by refractive index detection . After every fifth injection, the column was washed with 1.5 M sodium chloride to remove a yellow residue and was then reequilibrated in deionized water. Pooled fractions of the bisamine peak were brought to pH 10 by addition of solid sodium carbonate, and the purified Jeffamine product (compound 1) was extracted into methylene chloride. The combined organic layers were dried over sodium sulfate, and solvent was removed by rotary evaporation. Yields of the pale yellow solid were 40% based on the weight of crude starting material.One mole equivalent of 1.5 M glutaric anhydride in dioxane was added to a briskly stirred 250-mg/ml aqueous solution of purified Jeffamine (compound 1). After 30 min, the crude reaction product was injected in 1-ml batches over a 5-ml Hi-Trap SP column and eluted as described above. Pooled fractions of the monoamine peak were brought to pH 7 by addition of solid sodium bicarbonate, and the purified linker product (compound 2) was extracted into methylene chloride, dried over sodium sulfate, and obtained as a pale yellow solid by rotary evaporation. Yields were 35% to 50% based on the weight of purified Jeffamine starting material. A compound similar to the purified linker compound 2 is commercially available .To prepare resin compound 3, compound 1 or compound 2 was dissolved at 300 mg/ml in DMF/200 mM DIEA. The linker solution was incubated with one volume equivalent of drained NHS-activated Sepharose (Amersham Biosciences #17-0906-01). The suspension was rotated at 37 °C for 72 h, washed over a plastic frit with DMF to remove excess linker, and incubated with 1 M ethanolamine in DMF for an additional 12 h at 37 °C. The product resin was washed and stored at 6 °C. Resins coupled to compound 1 were further treated by incubation with an equal volume of DMF containing 100 mM glutaric anhydride and 15 mM pyridine at 37 °C under rotation for 48 h.Typically, TBTU (320 mg) and NHS (115 mg) were dissolved in 4 ml of DMF/500 mM DIEA, and drained compound 3 (1 ml) was added. The suspension was rotated at 37 °C for 1 h. Product resin was washed with the following sequence (20 ml of each): ethyl acetate, tetrahydrofuran, ethanol, water, 5 M sodium chloride, water, and DMF. Resin activation was performed just prior to oligonucleotide coupling.Twenty-base capture oligonucleotides were synthesized using standard phosphoramidite chemistry, with the addition of a C12-methoxytritylamine modifier at the 5′-end (Glen Research #10-1912). Following ammonia cleavage and drying, the oligonucleotides were desalted over C18 Sep-Pak cartridges . Purification proceeded according to the manufacturer's instructions, but a deionized water wash was inserted before the final elution step to remove residual TEAA. Coupling reactions of oligonucleotides to resin were carried out in low-binding 0.65-ml microcentrifuge tubes . Ten nanomoles of a capture oligonucleotide and 10 nmol of a nonaminated 10mer control oligonucleotide in a 40-μl aqueous solution were mixed with 160 μl of DMF/200 mM DIEA. Fifty microliters of drained resin compound 4 was added, and the suspension was rotated at 37 °C for 12 h. Reaction progress was monitored by HPLC. Supernatant aliquots (20 μl) were injected onto a 4.6-mm × 25-cm Varian Microsorb-MV 300-5 C18 column  and eluted with a linear water–acetonitrile gradient (0%–45% acetonitrile in five column volumes) in the presence of 0.1 M TEAA (pH 5.2) at 50 °C. After 12 h, the resin was pelleted by centrifugation at 100 g for 1 min, supernatant was removed, and the resin was incubated with 1 M ethanolamine in DMF for an additional 12 h at 37 °C. The derivatized resins were loaded into empty DNA synthesis column housings .Hybridization was performed in a closed system consisting of an anticodon column, male tapered luer couplers (Biosearch Technologies #CL-1504-1), capillary tubing (Amersham Biosciences #19-7477-01), silicone tubing , tubing connectors , and a peristaltic pump (Amersham Biosciences #18-1110-91). Approximately 1 ml of hybridization buffer (60 mM sodium phosphate (pH 6.5), 1.5 M sodium chloride, 10 mM EDTA, and 0.005% Triton X-100) containing 400 pmol of a complementary 20-base oligonucleotide and 400 pmol of a noncomplementary ten-base oligonucleotide was cyclically pumped through the system at 0.5 ml/min for 1 h in a 46-°C water bath. DNA was eluted off the anticodon column with 4 ml of 1 mM EDTA (pH 8.0) and 0.005% Triton X-100 heated to 80 °C. Flow-through and elute fractions were analyzed by HPLC as described above (0%–18% acetonitrile in five column volumes).35S preset channel of a scintillation counter .ssDNA was loaded onto a DEAE-Sepharose column as described . AnticodDEAE and anticodon columns were connected in series using male tapered luer couplers, 3.16-mm manifold tubing , and tygon tubing 3603. Using a peristaltic pump , approximately 7 ml of a 50% DMF solution was flowed cyclically over the columns at 3 ml/min for either 1 h at 45 °C or 12 h at 25 °C.DEAE columns were connected in series to C8 SepPak columns (Waters #WAT036775) using male tapered luer couplers, tygon tubing 3603, and tubing connectors. Approximately 6 ml of 50 mM ethanolamine (pH 10.0), 1.5 M sodium chloride, 1 mM EDTA, and 0.005% Triton X-100 was cyclically pumped over the columns at 1 ml/min for 1 h at 50 °C. The Sep-Pak columns were then washed with 12 ml of 100 mM TEAA (pH 6.5) followed by 12 ml of water. ssDNA was eluted from the Sep-Pak column with 4 ml of 50% acetonitrile heated to 80 °C. Samples were concentrated by centrifugal evaporation to a volume of approximately 30 μl and desalted over G25 Sephadex spin columns (Sigma-Aldrich #G-25-150).


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  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   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 . 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 . 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  and evening PEF  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  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  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  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 –50% of predicted and a clearly positive symptom score was required in most studies  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  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  . 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  . 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  and the median percentage of asthma free days was 56% higher  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  nocturnal awakenings, and significantly greater reductions in β2-agonist use , and morning PEF . 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  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