Biomarkers and severe asthma: a critical appraisal.

Severe asthma (SA) is a clinically and etiologically heterogeneous respiratory disease which affects among 5-10 % of asthmatic patients. Despite high-dose therapy, a large patients percentage is not fully controlled and has a poor quality of life. In this review, we describe the biomarkers actually known in scientific literature and used in clinical practice for SA assessment and management: neutrophils, eosinophils, periostin, fractional exhaled nitric oxide, exhaled breath condensate and galectins. Moreover, we give an overview on clinical and biological features characterizing severe asthma, paying special attention to the potential use of these ones as reliable markers. We finally underline the need to define different biomarkers panels to select patients affected by severe asthma for specific and personalized therapeutic approach.

Severe asthma: One disease and multiple definitions.

INTRODUCTION: There is, so far, no universal definition of severe asthma. This definition usually relies on: number of exacerbations, inhaled therapy, need for oral corticosteroids, and respiratory function. The use of such parameters varies in the different definitions used. Thus, according to the parameters chosen, each patient may result in having severe asthma or not. The aim of this study was to evaluate how the choice of a specific definition of severe asthma can change the allocation of patients. METHODS: Data collected from the Severe Asthma Network Italy (SANI) registry were analyzed. All the patients included were then reclassified according to the definitions of U-BIOPRED, NICE, WHO, ATS/ERS, GINA, ENFUMOSA, and TENOR. RESULTS: 540 patients, were extracted from the SANI database. We observed that 462 (86%) met the ATS/ERS criteria as well as the GINA criteria, 259 (48%) the U-Biopred, 222 (41%) the NICE, 125 (23%) the WHO, 313 (58%) the Enfumosa, and 251 (46%) the TENOR criteria. The mean eosinophil value were similar in the ATS/ERS, U-Biopred, and Enfumosa (528, 532 and 516 cells/mcl), higher in WHO and Tenor (567 and 570 cells/mcl) and much higher in the NICE classification (624 cells/mcl). Lung function tests resulted similarly in all groups, with WHO (67%) and ATS/ERS-GINA (73%), respectively, showing the lower and upper mean FEV1 values. CONCLUSIONS: The present observations clearly evidence the heterogeneity in the distribution of patients when different definitions of severe asthma are used. However, the recent definition of severe asthma, provided by the GINA document, is similar to that indicated in 2014 by ATS/ERS, allowing mirror reclassification of the patients examined. This lack of homogeneity could complicate the access to biological therapies. The definition provided by the GINA document, which reflects what suggested by ATS/ERS, could partially overcome the problem.

Evolving phenotypes to endotypes: is precision medicine achievable in asthma?

Introduction: The development of biologic molecules led to a drastic change in the therapeutic approach to asthma. With the prospect of acting on different pathophysiological mechanisms of the disease, the idea of precision medicine was developed, in which a single molecule is able to modify a specific triggering mechanism. Thus, it seemed limiting to stop at the distinction of patients phenotypes and the concept of endotypes became more relevant in the therapeutic approach.Areas covered: This review deepened the topic of precision medicine through the transition from phenotyping to endotyping. We performed a review of the literature, preferring articles quoted in Medline and published in journals with an impact factor. Results showed that it is fundamental to take into consideration the role of biomarkers and the related therapies currently available for precision medicine.Expert opinion: The possible overlap of patients in different phenotypes requires a more precise classification, which considers endotypization. With the development of biological drugs able to modify and modulate some pathophysiological mechanisms of the disease, the theoretical concept of endotyping becomes practical, allowing the clinician to choose the specific mechanism to 'attack' in order to control the disease.

Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin-induced intestinal fluid secretion.

Secretory diarrhea is the leading cause of infant death in developing countries and a major cause of morbidity in adults. The cystic fibrosis transmembrane conductance regulator (CFTR) protein is required for fluid secretion in the intestine and airways and, when defective, causes the lethal genetic disease cystic fibrosis. We screened 50,000 chemically diverse compounds for inhibition of cAMP/flavone-stimulated Cl(-) transport in epithelial cells expressing CFTR. Six CFTR inhibitors of the 2-thioxo-4-thiazolidinone chemical class were identified. The most potent compound discovered by screening of structural analogs, CFTR(inh)-172, reversibly inhibited CFTR short-circuit current in less than 2 minutes in a voltage-independent manner with K(I) approximately 300 nM. CFTR(inh)-172 was nontoxic at high concentrations in cell culture and mouse models. At concentrations fully inhibiting CFTR, CFTR(inh)-172 did not prevent elevation of cellular cAMP or inhibit non-CFTR Cl(-) channels, multidrug resistance protein-1 (MDR-1), ATP-sensitive K(+) channels, or a series of other transporters. A single intraperitoneal injection of CFTR(inh)-172 (250 micro g/kg) in mice reduced by more than 90% cholera toxin-induced fluid secretion in the small intestine over 6 hours. Thiazolidinone CFTR inhibitors may be useful in developing large-animal models of cystic fibrosis and in reducing intestinal fluid loss in cholera and other secretory diarrheas.

Clara cell 16 protein in COPD sputum: a marker of small airways damage?

RATIONALE: The development of chronic obstructive pulmonary disease (COPD) in smokers and their susceptibility to infections is not fully understood. Recent evidences suggest that Clara cells play a part in host defense, immunomodulatory response and airways remodelling through the production of specific factors such as Clara cell 16 (CC-16). This protein has never been related to patients' lung function tests, blood gases parameters and diseases severity. OBJECTIVES: To evaluate a possible correlation between CC-16 expression in sputum, measured by a new methodological approach, and the degree of severity in patients with moderate and severe COPD. We also analyzed possible correlations between CC-16 and cytological sputum population, arterial blood gases and lung function. MAIN FINDINGS: We analyzed 20 patients, mean age 72.95, classified on the basis of the global initiative for chronic obstructive lung disease guidelines (GOLD 2006). The samples were processed for cytological analysis and CC-16 levels were assessed by Western blot. We found lower levels of CC-16 in severe COPD compared to moderate ones (p<0.027). No statistically significant differences were found between CC-16 expression and sputum cellularity (except for macrophages), arterial blood gases, and spirometric parameters. Multiple linear regression analysis of CC-16 versus functional and cytological parameters showed no significance. CONCLUSIONS: We found a significantly different expression of CC-16 in COPD patients, according to their stage of severity, as defined by the GOLD 2006 guidelines. Considering CC-16 properties in innate immunity, a possible link between protein expression, innate immune system, and COPD infectious exacerbations may be hypothesized but further investigation are needed.

CD4(+)CD25(high)CD127(-) regulatory T-cells in COPD: smoke and drugs effect.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a progressive lung disorder characterized by poorly reversible airway obstruction and its pathogenesis remains largely misunderstood. Local changes of regulatory T-cell populations in the lungs of COPD patients have been demonstrated although data concerning their pathologic role are contrasting. The aim of our study was to evaluate the relative percentage of regulatory T-cells in the peripheral blood of current and former smoker subjects, affected or not by COPD. Furthermore, the effect of different concentrations of budesonide and formoterol, on regulatory T-cells has been investigated. METHODS: T regulatory lymphocytes were isolated and assessed as CD4(+)CD25(high)CD127(-) cells by flow cytometry and cultured for 48 hours in the absence or in the presence of budesonide and/or formoterol at different doses. RESULTS: CD4(+)CD25(high)CD127(-) regulatory T-cells percentage was significantly reduced in COPD patients, both current and former smokers, with respect to volunteers. Furthermore, CD4(+)CD25(high)CD127(-) cells of COPD patients showed a not statistically significant response to drugs compared to healthy subjects. DISCUSSION: Our results evidenced a different behaviour of CD4(+)CD25(high)CD127(-) Treg cells in COPD patients after in vitro treatments. CONCLUSIONS: Based on our data, we suggested a possible role of CD4 CD25(high)CD127 T-cells in COPD pathogenesis.

Altered channel gating mechanism for CFTR inhibition by a high-affinity thiazolidinone blocker.

The thiazolidinone CFTR(inh)-172 was identified recently as a potent and selective blocker of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. Here, we characterized the CFTR(inh)-172 inhibition mechanism by patch-clamp and short-circuit analysis using cells stably expressing wild-type and mutant CFTRs. CFTR(inh)-172 did not alter CFTR unitary conductance (8 pS), but reduced open probability by >90% with K(i) approximately 0.6 microM. This effect was due to increased mean channel closed time without changing mean channel open time. Short-circuit current experiments indicated similar CFTR(inh)-172 inhibitory potency (K(i) approximately 0.5 microM) for inhibition of Cl(-) current in wild-type, G551D, and G1349D CFTR; however, K(i) was significantly reduced to 0.2 microM for DeltaF508 CFTR. Our studies provide evidence for CFTR inhibition by CFTR(inh)-172 by a mechanism involving altered CFTR gating.

Correction of G551D-CFTR transport defect in epithelial monolayers by genistein but not by CPX or MPB-07.

1. This study compares the effect of three chemically unrelated cystic fibrosis transmembrane conductance regulator (CFTR) activators on epithelial cell monolayers expressing the G551D-CFTR mutant. 2. We measured Cl(-) transport as the amplitude of short-circuit current in response to the membrane permeable cAMP analogue 8-(4-chlorophenylthio)adenosine-3'-5'-cyclic monophosphate (CPT-cAMP) alone or in combination with a CFTR opener. The correction of G551D-CFTR defect was quantified by comparison with maximal activity elicited in cells expressing wild type CFTR. To this end we used Fisher rat thyroid (FRT) cells transfected with wild type or G551D CFTR, and primary cultures of human nasal epithelial cells. 3. In both types of epithelia, cAMP caused activation of Cl(-) transport that was inhibited by glibenclamide and not by 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid. After normalising for CFTR expression, the response of FRT-G551D epithelia was 1% that of wild type monolayers. 4. Addition of genistein (10-200 micro M), but not of 8-cyclopentyl-1,3-dipropylxanthine (CPX, 1-100 micro M) or of the benzo[c]quinolizinium MPB-07 (10-200 micro M) to FRT-G551D epithelia pre-treated with cAMP, stimulated a sustained current that at maximal genistein concentration corresponded to 30% of the response of wild type epithelia. 5. The genistein dose-response curve was bell-shaped due to inhibitory activity at the highest concentrations. The dose-dependence in G551D cells was shifted with respect to wild type CFTR so that higher genistein concentrations were required to observe activation and inhibition, respectively. 6. On human nasal epithelia the correction of G551D-CFTR defective conductance obtained with genistein was 20% that of wild type. The impressive effect of genistein suggests that it might correct the Cl(-) transport defect on G551D patients.

2-(dialkylamino)-4H-1-benzopyran-4-one derivatives modify chloride conductance in CFTR expressing cells.

Some 2-(diethylamino)-7-hydroxy-4H-1-benzopyran-4-one derivatives, potentially useful as activators of the cystic fibrosis transmembrane conductance regulator (CFTR), were prepared. The synthesized compounds were tested, together with others 2-(dialkylamino)-7-hydroxybenzopyran-4-one derivatives, by measuring their capacity to modify the kinetics of iodide influx in Fisher rat thyroid cells expressing wild type CFTR and the halide-sensitive yellow fluorescent protein. Among the tested compounds the dinitrile derivatives 8 and 9 are endowed with an activity comparable to the reference compound apigenin.

Administration of a polyvalent mechanical bacterial lysate to elderly patients with COPD: Effects on circulating T, B and NK cells.

The modifications of the subsets of circulating lymphocytes were evaluated in a group of patients with COPD undergoing treatment with a polyvalent mechanical bacterial lysate (PMBL), a drug that is able to significantly modify the natural history of these patients. Using multicolor immune-florescence and flow cytometry, T, B subsets and NK cells were extensively studied both in the group of treated patients and in a disease and age matched controls. Despite the age, in treated patients, T and NK cells were significantly increased in numbers of circulating cells, but not in percentages, while B cells remained unmodified. CD3+4+T cells were increased in treated patients, while CD3+CD8T cells were unmodified by the treatment. Activated T cells were increased but Treg, resulted reduced both in percentage than in absolute numbers. Transitional B cells resulted increased (in percentage and in absolute numbers) in their late maturation step (T3), while only early Naïve B cells were increased by the treatment, while other naïve subpopulations were unmodified. Memory B cells were reduced in percentage (but remained unmodified as absolute numbers), while the most immature form of memory B cells was significantly increased. Finally, both switch memory B cells and plasma cells resulted unmodified by the PMBL treatment. These results clearly indicated that the administration of the PMBL, even in elderly patients with COPD, was able to induce a significant immune-stimulation and these results, at cellular level, clearly support the evidence that the mechanism of action of PMBL is strictly related to a direct effect on immune-competent cells.

New insights into airway remodelling in asthma and its possible modulation.

PURPOSE OF REVIEW: Airway remodelling, a central feature of asthma, is characterized by an alteration in the size, mass or number of tissue components which occur in and around the trachea, bronchi and bronchioles in the airways in response to injury and/or inflammation. The present review focuses on the most recent literature on airway remodelling and on the different drugs commonly used or potentially useful in the treatment of asthma with a particular attention to the studies conducted by our group in the last few years. RECENT FINDINGS: The interaction between the epithelium and mesenchymal elements such as fibroblasts is essential for normal airway repair. An abnormal response of this epithelial-mesenchymal trophic unit has been proposed to be central to the airway pathology and physiology characteristic of asthma. Current treatments may indirectly control airway remodelling through a reduction of inflammation but such a kind of approach is only in part successful. SUMMARY: The clear understanding of the events that take place during remodeling and the targeting of its specific components will be helpful in the development of novel therapies that might restore lung function.

Importance of fibroblasts-myofibroblasts in asthma-induced airway remodeling.

Asthma is a chronic airway disorder principally characterized by bronchial hyperreactivity and airflow obstruction. Increased epithelial and smooth muscle thickness, goblet cell hyperplasia, increased mucus secretion, abnormal deposition of extracellular matrix (ECM) components in the basement membrane (BM) layer and angiogenesis are all events which occur in asthma and are defined with the general term of remodeling. This is an important feature whose repetition and regeneration may bring to an abnormal or exaggerated response to airway insults. One of the characteristic aspects of asthma is an alteration in structural cell function. Airway smooth muscle cells (ASM), myofibroblasts and fibroblasts have the ability to secrete immunomodulatory cytokines and chemokines and to express cell surface receptors. These elements are all important for cell adhesion and leukocyte activation and may be integral components of the inflammatory response as well. In particular cells such as fibroblasts and myofibroblasts, important regulators in the development and maintenance of allergic airway inflammation, have been studied in depth by our group and several studies regarding their role in asthma therapy have been analyzed.

Effect of inflammatory stimuli on airway ion transport.

The airway epithelium controls the chemical and physical properties of airway surface fluid and consequently mucociliary clearance. The treatment for 24-48 hours of human bronchial epithelial cells with interferon-gamma or interleukin-4 leads to marked changes in transepithelial ion transport properties. Both cytokines downregulate the activity of the epithelial Na+ channel and, at the same time, upregulate Ca2+-dependent Cl- secretion. Interleukin-4 also increases the expression and function of the cystic fibrosis transmembrane conductance regulator Cl- channel. These results suggest that some inflammatory stimuli may change the balance between fluid absorption and secretion to favor hydration of the airway surface and consequently mucus clearance.

Double mechanism for apical tryptophan depletion in polarized human bronchial epithelium.

Indoleamine 2,3-dioxygenase is an enzyme that catabolizes tryptophan to kynurenine. We investigated the consequences of IDO induction by IFN-gamma in polarized human bronchial epithelium. IDO mRNA expression was undetectable in resting conditions, but strongly induced by IFN-gamma. We determined the concentration of tryptophan and kynurenine in the extracellular medium, and we found that apical tryptophan concentration was lower than the basolateral in resting cells. IFN-gamma caused a decrease in tryptophan concentration on both sides of the epithelium. Kynurenine was absent in control conditions, but increased in the basolateral medium after IFN-gamma treatment. The asymmetric distribution of tryptophan and kynurenine suggested the presence of a transepithelial amino acid transport. Uptake experiments with radiolabeled amino acids demonstrated the presence of a Na(+)-dependent amino acid transporter with broad specificity that was responsible for the tryptophan/kynurenine transport. We confirmed these data by measuring the short-circuit currents elicited by direct application of tryptophan or kynurenine to the apical surface. The rate of amino acid transport was dependent on the transepithelial potential, and we established that in cystic fibrosis epithelia, in which the transepithelial potential is significantly more negative than in noncystic fibrosis epithelia, amino acid uptake was reduced. This work suggests that human airway epithelial cells maintain low apical tryptophan concentrations by two mechanisms, a removal through a Na(+)-dependent amino acid transporter and an IFN-gamma-inducible degradation by IDO.

IL-4 is a potent modulator of ion transport in the human bronchial epithelium in vitro.

Recent data show that proinflammatory stimuli may modify significantly ion transport in the airway epithelium and therefore the properties of the airway surface fluid. We have studied the effect of IL-4, a cytokine involved in the pathogenesis of asthma, on transepithelial ion transport in the human bronchial epithelium in vitro. Incubation of polarized bronchial epithelial cells with IL-4 for 6-48 h causes a marked inhibition of the amiloride-sensitive Na(+) channel as measured in short circuit current experiments. On the other hand, IL-4 evokes a 2-fold increase in the current activated by a cAMP analog, which reflects the activity of the cystic fibrosis transmembrane conductance regulator (CFTR). Similarly, IL-4 enhances the response to apical UTP, an agonist that activates Ca(2+)-dependent Cl(-) channels. These effects are mimicked by IL-13 and blocked by an antagonist of IL-4Ralpha. RT-PCR experiments show that IL-4 elicits a 7-fold decrease in the level of the gamma amiloride-sensitive Na(+) channel mRNA, one of the subunits of the amiloride-sensitive Na(+) channel, and an increase in CFTR mRNA. Our data suggest that IL-4 may favor the hydration of the airway surface by decreasing Na(+) absorption and increasing Cl(-) secretion. This could be required to fluidify the mucus, which is hypersecreted during inflammatory conditions. On the other hand, the modifications of ion transport could also affect the ion composition of airway surface fluid.

CFTR activation in human bronchial epithelial cells by novel benzoflavone and benzimidazolone compounds.

Activators of the CFTR Cl- channel may be useful for therapy of cystic fibrosis. Short-circuit current (Isc) measurements were done on human bronchial epithelial cells to characterize the best flavone and benzimidazolone CFTR activators identified by lead-based combinatorial synthesis and high-throughput screening. The 7,8-benzoflavone UCcf-029 was a potent activator of Cl- transport, with activating potency (<1 microM) being much better than other flavones, such as apigenin. The benzimidazolone UCcf-853 gave similar Isc but with lower potency (5-20 microM). In combination, the effect induced by maximal UCcf-029 and UCcf-029, UCcf-853, and apigenin increased strongly with increasing basal CFTR activity: for example, Kd for activation by UCcf-029 decreased from >5 to <0.4 microM with increasing basal Isc from approximately 4 microA/cm2 to approximately 12 microA/cm2. This dependence was confirmed in permeabilized Fischer rat thyroid cells stably expressing CFTR. Our results demonstrate efficacy of novel CFTR activators in bronchial epithelia and provide evidence that activating potency depends on basal CFTR activity.

Nanomolar affinity small molecule correctors of defective Delta F508-CFTR chloride channel gating.

Deletion of Phe-508 (Delta F508) is the most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) causing cystic fibrosis. Delta F508-CFTR has defects in both channel gating and endoplasmic reticulum-to-plasma membrane processing. We identified six novel classes of high affinity potentiators of defective Delta F508-CFTR Cl- channel gating by screening 100,000 diverse small molecules. Compounds were added 15 min prior to assay of iodide uptake in epithelial cells co-expressing Delta F508-CFTR and a high sensitivity halide indicator (YFP-H148Q/I152L) in which Delta F508-CFTR was targeted to the plasma membrane by culture at 27 degrees C for 24 h. Thirty-two compounds with submicromolar activating potency were identified; most had tetrahydrobenzothiophene, benzofuran, pyramidinetrione, dihydropyridine, and anthraquinone core structures (360-480 daltons). Further screening of >1000 structural analogs revealed tetrahydrobenzothiophenes that activated DeltaF508-CFTR Cl- conductance reversibly with Kd < 100 nm. Single-cell voltage clamp analysis showed characteristic CFTR currents after Delta F508-CFTR activation. Activation required low concentrations of a cAMP agonist, thus mimicking the normal physiological response. A Bayesian computational model was developed using tetrahydrobenzothiophene structure-activity data, yielding insight into the physical character and structural features of active and inactive potentiators and successfully predicting the activity of structural analogs. Efficient potentiation of defective Delta F508-CFTR gating was also demonstrated in human bronchial epithelial cells from a Delta F508 cystic fibrosis subject after 27 degrees C temperature rescue. In conjunction with correctors of defective Delta F508-CFTR processing, small molecule potentiators of defective Delta F508-CFTR gating may be useful for therapy of cystic fibrosis caused by the Delta F508 mutation.