Eosinophils-from cradle to grave: An EAACI task force paper on new molecular insights and clinical functions of eosinophils and the clinical effects of targeted eosinophil depletion.

Over the past years, eosinophils have become a focus of scientific interest, especially in the context of their recently uncovered functions (e.g. antiviral, anti-inflammatory, regulatory). These versatile cells display both beneficial and detrimental activities under various physiological and pathological conditions. Eosinophils are involved in the pathogenesis of many diseases which can be classified into primary (clonal) and secondary (reactive) disorders and idiopathic (hyper)eosinophilic syndromes. Depending on the biological specimen, the eosinophil count in different body compartments may serve as a biomarker reflecting the underlying pathophysiology and/or activity of distinct diseases and as a therapy-driving (predictive) and monitoring tool. Personalized selection of an appropriate therapeutic strategy directly or indirectly targeting the increased number and/or activity of eosinophils should be based on the understanding of eosinophil homeostasis including their interactions with other immune and non-immune cells within different body compartments. Hence, restoring as well as maintaining homeostasis within an individual's eosinophil pool is a goal of both specific and non-specific eosinophil-targeting therapies. Despite the overall favourable safety profile of the currently available anti-eosinophil biologics, the effect of eosinophil depletion should be monitored from the perspective of possible unwanted consequences.

Changes of Motile Ciliary Phenotype in Patients with Primary Ciliopathies.

Primary ciliopathies are a group of disorders associated with abnormal formation and function of primary cilia. Many cilia-associated proteins found in primary cilia are also present in motile cilia. Such proteins are important for the ciliary base, such as the transition zone or basal bodies, and the intraflagellar transport. Their exact role in the respiratory motile cilia is unsettled. In this prospective clinical single-center study, we investigated the hypothesis that these proteins regulate the function of motile cilia. We addressed the issue by defining the motile cilia beat frequency in the respiratory tract of patients with primary ciliopathies accompanied by chronic kidney disease and comparing it in those without kidney involvement. Ciliary beat frequency in the nasal mucosa samples was evaluated by the ciliary analysis software LabVIEW. Both children and their parents with primary ciliopathies and kidney involvement had significantly lower median airway ciliary beat frequencies than those without kidney involvement who have normal ciliary motility. Further, the ciliary beat frequency is inversely associated with the serum creatinine level. These findings strongly suggest that kidney involvement in patients with primary ciliopathy may underlie the development of motile cilia dysfunction in the respiratory tract, potentially increasing respiratory morbidity.

Influence of combined voltage-gated sodium channel NaV1.7 and NaV1.8 inhibitors on cough in a guinea pig model.

Pathological excessive cough is a serious clinical problem in many patients. It is no doubt that an increased activation and sensitization of airway vagal C-fibres in disease stems from dysregulation of the neural pathways that control cough. Due to the limited efficacy and unwanted side effects of current antitussives, there is a continual demand for the development of a novel more effective antitussive. Since voltage-gated sodium channels (NaVs) are absolutely required for action potentials initiation and conduction irrespective of the stimulus, NaVs became a promising and attractive neural target. Current studies establish that NaV1.7 and NaV1.8 inhibitors have the potential to suppress cough. In this study, we demonstrated that inhaled aerosol of NaV1.7 inhibitor PF-05089771 (10 µM) and NaV1.8 inhibitor A-803467 (1 mM) mixture inhibited the capsaicin-induced cough by ≈ 60 % and citric acid-induced cough by ≈ 65 % at doses that did not modify respiratory rate. Our previous and present studies indicate that NaV1.7 and NaV1.8 may present promising therapeutic targets for antitussive therapy.

Modification of oestrogen signalling pathways influences cough induced by citric acid but not capsaicin in the animal model of both sexes.

To clarify the role of oestrogen signalling and the role of oestrogen receptor alpha (ERα) in the cough pathways we performed a study in which coughing was observed in both sexes animal models after the treatment by selective ERα degrader fulvestrant (ICI 182-780) and inhibitor of oestrogen synthesis danazol. Degradation of ERα with the normal plasma oestrogen levels induced by fulvestrant, significantly augments the cough response of female but not male guinea pigs. These changes were observed in citric acid-induced cough. Female guinea pigs responded with an increased count of cough expulsions per challenge time and we also detected shorter cough latency. The capsaicin-induced cough did not change. A similar response was observed after danazol treatment, which decreased the plasma oestrogen level. Our results indicate that the transient receptor potential vanilloid-1 (TRPV1) channel-mediated cough is resistant to the hypoestrous state, while the citric acid-mediated cough is oestrogen-dependent and hypersensitive during the hypoestrous state.

Cough response in specific pathogen-free guinea pig animal model.

Laboratory animal science associations recommend the usage of specific pathogen-free (SPF) animals in experiments to achieve appropriate results. However, their non-natural microbiome might represent an obstacle in the successful translation of obtained results into clinical practice, since the microbiome contributes to the regulation of the host's physiology. Inconsistent results, even in repeated experiments in separate animal groups, irrespective of the season or sex led us to assess the cough response of SPF animal variables and compare it to wild-type animals. The dose-response curves in citric acid and capsaicin cough challenge construction shows significantly increased cough threshold in SPF animals compared to wild-type animals of both sexes. The cause of cough response alteration in SPF animals is not presently known and thus their usage in basic cough research remains questionable. The relationship between airway reflexes and the airway microbiome has never been studied hence we propose that it might be microbiome-related.

The effect of the voltage-gated sodium channel NaV1.7 blocker PF-05089771 on cough in the guinea pig.

Cough in chronic respiratory diseases is a common symptom associated with significant comorbidities including visceral pain. Available antitussive therapy still has limited efficacy. Recent advances in the understanding of voltage-gated sodium channels (NaVs) lead to the rational hypothesis that subtype NaV1.7 is involved in initiating cough and thus may present a promising therapeutic target for antitussive therapy. We evaluated the antitussive effect of NaV1.7 blocker PF-05089771 administered systemically and topically in awake guinea pigs using capsaicin cough challenge. Compared to vehicle, peroral or inhaled PF-05089771 administration caused about 50-60 % inhibition of cough at the doses that did not alter respiratory rate. We conclude that the NaV1.7 blocker PF-05089771 inhibits cough in a manner consistent with its electrophysiological effect on airway C-fibre nerve terminals.

Animal models of cough.

Cough is a vital airway reflex that keeps the respiratory tract wisely protected. It is also a sign of many diseases of the respiratory system and it may become a disease in its own right. Even though the efficacy of antitussive compounds is extensively studied in animal models with promising results, the treatment of pathological cough in humans is insufficient at the moment. The limited translational potential of animal models used to study cough causes, mechanisms and possible therapeutic targets stems from multiple sources. First of all, cough induced in the laboratory by mechanical or chemical stimuli is far from natural cough present in human disease. The main objective of this review is to provide a comprehensive summary of animal models currently used in cough research and to address their advantages and disadvantages. We also want to encourage cough researchers to call for precision is research by addressing the sex bias which has existed in basic cough research for decades and discuss the role of specific pathogen-free (SPF) animals.

Volume feedback during cough in anesthetized cats, effects of occlusions and modulation summary.

The study investigates the effects of 6 occlusion conditions on the mechanically induced cough reflex in 15 anesthetized (pentobarbital) spontaneously breathing cats (14♂, 1♀). Esophageal pressure and integrated EMG activities of inspiratory (I) diaphragm and expiratory (E) abdominal muscles were recorded and analyzed. Occlusions: inspiratory (Io), continual I (cIo), during I and active E (I+Eo) cough phase, during I and then E phase with short releasing of airflow before each phase (I-Eo), and E occlusion (Eo) had little influence on cough number. Only continual E occlusion (cEo) reduced the number of coughs by 19 % (to 81 %, p < 0.05). Cough I esophageal pressure reached higher amplitudes under all conditions, but only Eo caused increased I diaphragm motor drive (p < 0.05). Cough E efforts (abdominal motor drive and E amplitudes of esophageal pressure) increased during Eo, decreased during I+Eo (p < 0.05), and did not change significantly under other conditions (p > 0.05). All I blocks resulted in prolonged I cough characteristics (p < 0.05) mainly cough I phase (incrementing part of the diaphragm activity). Shorter I phase occurred with cEo (p < 0.05). Cough cycle time and active E phase (from the I maximum to the end of cough E motor drive) prolonged (p < 0.05) during all occlusions (E phase duration statistically non-significantly for I+Eo). Airflow block during cough (occlusions) results in secondary changes in the cough response due to markedly altered function of cough central pattern generator and cough motor pattern produced. Cough compensatory effects during airflow resistances are more favorable compared to occlusions. Volume feedback represents significant factor of cough modulation under various pathological obstruction and/or restriction conditions of the respiratory system.

Retrospective Study of Factors Potentially Influencing Occurrence of Cough in Slovak Patients with Sarcoidosis.

Introduction: Sarcoidosis is a multisystem granulomatous disease of unknown aetiology, commonly involving the lungs.  Cough is a frequent and troublesome symptom of sarcoidosis that reduces patients' quality of life. Aim: Retrospective analysis of different factors-smoking history, Scadding stage, results of lung function testing, calcium metabolism, endobronchial finding, CD4+/CD8+ T-cell ratio in bronchoalveolar lavage fluid (BALF), and other sarcoidosis symptoms in relationship to presence/absence of cough in sarcoidosis patients. Methods: We retrospectively studied sarcoidosis patients diagnosed at the Clinic of Pneumology and Phthisiology of Martin University Hospital between 1998 and 2018. Patients with a history of cough-relevant comorbidities were excluded from the study. GraphPad Prism 7.0 software was used to perform statistical analysis. Results: 101 sarcoidosis patients were included to the study: 65 patients reporting from cough and 36 without cough. The cough was slightly more frequent in nonsmokers (p=0.166) and in women (p=0.688). Cough was associated with dyspnoea (p=0.0007), fever (p=0.0324), and chest pain (p=0.0206) and did not associate with arthralgia (p=0.317) and erythema nodosum (p=0.505). Patients with cough had significantly a lower average value of calciuria (p=0.0014) and lower MEF25 (p=0.0304), MEF50 (p=0.0061), FEV1 (p=0.0025), and FVC (p=0.0025) in % of predicted values, and more often positive endobronchial finding (p=0.0206), compared to patients without cough. Calcemia, FEV1/FVC, DLCO, and CD4+/CD8+ T-cell ratio in BALF and occurrence of cough did not differ between different stages of the disease. Conclusions: We found significant differences between sarcoidosis patients with/without cough regarding symptoms, results of lung function tests, endobronchial finding, and calcium metabolism. Further research is needed to understand the etiopathogenesis of cough in sarcoidosis patients.

Cough in sarcoidosis patients.

Sarcoidosis is a multi-system disease of unknown aetiology characterized by presence of non-caseating granulomatous inflammation. Cough is a common and significant symptom in sarcoidosis, reducing quality of life. Objective 24 h cough monitoring proved that sarcoidosis patients have significantly higher cough frequency compared to controls and their cough has diurnal variation, it is gender-specific and shows racial differences. It correlates with the presence of inflammation in the airways, but is not influenced by the X-ray staging of the disease, nor the degree of airway obstruction. Subjects with sarcoidosis have heightened cough reflex sensitivity, which is a result of interaction between the airway cough sensors and consequences of pathological process, detailed pathogenesis of cough in this demographic is unclear. The airway hyperresponsiveness, sarcoidosis of the upper airways and sensitivity to biomechanical forces play a role. More studies should be performed to understand pathogenesis of cough in sarcoidosis patients to improve the management of this troublesome symptom.

Sensitivity of airway cough-related afferents is influenced by female sex hormones.

Chronic hypersensitivity cough syndrome affects mainly postmenopausal women; however, the pathogenesis of cough hypersensitivity in this demographic is not entirely understood. The role of sex hormones in cough has never been studied in detail; however, sex hormones seem to play an important role in the lung health of women. Our study was aimed to analyse the effect of female sex hormones (oestrogen - E2 and progesterone - Pg) on cough sensitivity measured by inhalation of capsaicin in follicular and luteal phases of menstrual cycle, characterized by significantly different concentrations of sex hormones. These data were compared with a matched group of women taking oral contraceptives. Cough sensitivity to capsaicin increased in luteal phase in subjects with normal menstrual cycle, and this functional change was not present in group with contraceptive pills. The cough sensitivity correlates with the Pg/E2 ratio, and relative lack of oestrogen in luteal phase is associated with higher cough sensitivity to capsaicin.

TRPV4 antagonist GSK2193874 does not modulate cough response to osmotic stimuli.

Osmolarity changes of airway superficial fluid are associated with cough and are used in research. TRPV4 is calcium channel initially described as osmosensor. In the airways, it can play role in increasing cough reflex sensitivity. The aim of our study was to test whether cough to osmotic stimuli is mediated via TRPV4 channel. Cough response was measured in 12 male guinea pigs by inhalation of saline, distilled water, hypertonic solution and citric acid for 10min in whole-body plethysmograph. Data were obtained in naïve animals and after pre-treatment with selective TRPV4 antagonist GSK2193874 in doses 300µg/kg (GSK300) and 900µg/kg (GSK900). Cough response to all tested aerosols was significantly higher than to saline. Pre-treatment with GSK300 did not influence response to osmotic stimuli - only reduced cough to citric acid. GSK900 reduced cough response to hypotonic stimuli and citric acid. TRPV4 mediated activation of airway afferents does not seem to be the exclusive mechanism responsible for cough to osmotic stimuli.

Role of gender in basic cough research.

Cough hypersensitivity affects mainly postmenopausal women. Pathogenesis remains unknown in their case; therefore, an optimal treatment is unavailable. Only male guinea pigs are used in basic cough research and exclusion of females leads to gender bias. Nowadays, the efforts of grant agencies aim to support projects which take gender into account and involve both sexes to reduce gender bias. The aim of our pilot study is to assess the suitability of female guinea pig model in cough research. Cough response to citric acid (0.4M) was obtained in female and male guinea pigs (n=12 each). Reproducibility of cough response was tested four times a week apart. The cough was detected from the airflow traces and cough sound analysis. Our initial results show that cough response of female guinea pigs is higher than in males. Variability of females is also higher than in males. Further investigations are necessary to validate this model in different conditions to achieve gender equality in cough studies.

Impact of Air Pollution on Age and Gender Related Increase in Cough Reflex Sensitivity of Healthy Children in Slovakia.

BACKGROUND: Numerous studies show higher cough reflex sensitivity (CRS) and cough outcomes in children compared to adults and in females compared to males. Despite close link that exists between cough and environment the potential influence of environmental air pollution on age- and gender -related differences in cough has not been studied yet. PURPOSE: The purpose of our study was to analyse whether the effects of exposure to environmental tobacco smoke (ETS) from parental smoking and PM10 from living in urban area are implied in age- and gender-related differences in cough outcomes of healthy, non-asthmatic children. Assessment of CRS using capsaicin and incidence of dry and wet cough was performed in 290 children (mean age 13.3 ± 2.6 years (138 females/152 males). RESULTS: CRS was significantly higher in girls exposed to ETS [22.3 µmol/l (9.8-50.2 µmol/l)] compared to not exposed girls [79.9 µmol/l (56.4-112.2 µmol/l), p = 0.02] as well as compared to exposed boys [121.4 µmol/l (58.2-253.1 µmol/l), p = 0.01]. Incidence of dry cough lasting more than 3 weeks was significantly higher in exposed compared to not exposed girls. CRS was significantly higher in school-aged girls living in urban area [22.0 µmol/l (10.6-45.6 µmol/l)] compared to school-aged girls living in rural area [215.9 µmol/l (87.3-533.4 µmol/l); p = 0.003], as well as compared to teenage girls living in urban area [108.8 µmol/l (68.7-172.9 µmol/l); p = 0.007]. No CRS differences were found between urban and rural boys when controlled for age group. No CRS differences were found between school-aged and teenage boys when controlled for living area. CONCLUSIONS: Our results have shown that the effect of ETS on CRS was gender specific, linked to female gender and the effect of PM10 on CRS was both gender and age specific, related to female gender and school-age. We suggest that age and gender related differences in incidence of cough and CRS might be, at least partially, ascribed to the effect of environmental pollutants. The role of age and gender in the effect of air pollution on cough strongly suggest some interplay of development with biological and behavioral factors.

The course of lung inflation alters the central pattern of tracheobronchial cough in cat-The evidence for volume feedback during cough.

The effect of volume-related feedback and output airflow resistance on the cough motor pattern was studied in 17 pentobarbital anesthetized spontaneously-breathing cats. Lung inflation during tracheobronchial cough was ventilator controlled and triggered by the diaphragm electromyographic (EMG) signal. Altered lung inflations during cough resulted in modified cough motor drive and temporal features of coughing. When tidal volume was delivered (via the ventilator) there was a significant increase in the inspiratory and expiratory cough drive (esophageal pressures and EMG amplitudes), inspiratory phase duration (CTI), total cough cycle duration, and the duration of all cough related EMGs (Tactive). When the cough volume was delivered (via the ventilator) during the first half of inspiratory period (at CTI/2-early over inflation), there was a significant reduction in the inspiratory and expiratory EMG amplitude, peak inspiratory esophageal pressure, CTI, and the overlap between inspiratory and expiratory EMG activity. Additionally, there was significant increase in the interval between the maximum inspiratory and expiratory EMG activity and the active portion of the expiratory phase (CTE1). Control inflations coughs and control coughs with additional expiratory resistance had increased maximum expiratory esophageal pressure and prolonged CTE1, the duration of cough abdominal activity, and Tactive. There was no significant difference in control coughing and/or control coughing when sham ventilation was employed. In conclusion, modified lung inflations during coughing and/or additional expiratory airflow resistance altered the spatio-temporal features of cough motor pattern via the volume related feedback mechanism similar to that in breathing.

Defining Chronic Cough: A Systematic Review of the Epidemiological Literature.

PURPOSE: Recent evidence suggests a global burden of chronic cough in general populations. However, the definitions vary greatly among epidemiological studies, and none have been validated for clinical relevance. We aimed to examine previous epidemiological definitions in detail and explore the operational characteristics. METHODS: A systematic review was conducted for epidemiological surveys that reported the prevalence of chronic cough in general adult populations during the years 1980 to 2013. A literature search was performed on Pubmed and Embase without language restriction. Epidemiological definitions for chronic cough were classified according to their components, such as cutoff duration. Meta-analyses were performed for the male-to-female ratio of chronic cough prevalence to explore operational characteristics of epidemiological definitions. RESULTS: A total of 70 studies were included in the systematic review. The most common epidemiological definition was identified as 'cough ≥3 months' duration without specification of phlegm (n=50); however, it conflicted with the cutoff duration in current clinical guidelines (cough ≥8 weeks). Meta-analyses were performed for the male-to-female ratio of chronic cough among 28 studies that reported sex-specific prevalence using the most common definition. The pooled male-to-female odds ratio was 1.26 (95% confidence interval 0.92-1.73) with significant heterogeneity (I²=96%, P<0.001), which was in contrast to clinical observations of female predominance from specialist clinics. Subgroup analyses did not reverse the ratio or reduce the heterogeneity. CONCLUSIONS: This study identified major issues in defining chronic cough in future epidemiological studies. The conflict between epidemiological and clinical diagnostic criteria needs to be resolved. The unexpected difference in the gender predominance between the community and clinics warrants further studies. Clinical validation of the existing definition is required.

Interactions of mechanically induced coughing and sneezing in cat.

Mutual interactions of cough and sneeze were studied in 12 spontaneously breathing pentobarbitone anesthetized cats. Reflexes were induced by mechanical stimulation of the tracheobronchial and nasal airways, respectively. The amplitude of the styloglossus muscle EMG moving average during the sneeze expulsion was 16-fold higher than that during cough (p<0.01). Larger inspiratory efforts occurred during coughing (p<0.01) vs. those in sneeze. The number of reflexes during simultaneous mechanical stimulation of the nasal and tracheal airways was not altered significantly compared to controls (p>0.05) and there was no modulation in temporal characteristics of the behaviors. When both reflexes occurred during simultaneous stimuli the responses were classified as either sneeze or cough (no hybrid responses occurred). During simultaneous stimulation of both airway sites, peak diaphragm EMG and inspiratory esophageal pressures during sneezes were significantly increased. The expiratory maxima of esophageal pressure and amplitudes of abdominal EMGs were increased in coughs and sneezes during simultaneous mechanical stimulation trials compared to control reflexes.

Control of coughing by medullary raphé.

The medullary raphé nuclei participate in the regulation of breathing and airway defensive reflexes. Our focus was to analyze the effects of codeine and kainic acid within the medullary raphé on coughing, sneezing, solitary expulsions, and concomitant breathing changes using models of anesthetized cats (n=12) and rabbits (n=15) and microinjection techniques. Our findings are consistent with (1) involvement of neurons within the medullary raphé in the generation of the cough motor pattern by a codeine-sensitive and -insensitive mechanisms, (2) a contribution of raphé nuclei to the control of expiratory efforts, (3) limited contribution of the medullary raphé to the cough-gating mechanism, (4) minor contribution of respiration/coughing neuronal network, including the portion within raphé nuclei and the respiratory/cough central pattern generator, to the production of the sneeze reflex motor pattern.

What is chronic cough in children?

The cough reflex is modulated throughout growth and development. Cough-but not expiration reflex-appears to be absent at birth, but increases with maturation. Thus, acute cough is the most frequent respiratory symptom during the first few years of life. Later on, the pubertal development seems to play a significant role in changing of the cough threshold during childhood and adolescence resulting in sex-related differences in cough reflex sensitivity in adulthood. Asthma is the major cause of chronic cough in children. Prolonged acute cough is usually related to the long-lasting effects of a previous viral airway infection or to the particular entity called protracted bacterial bronchitis. Cough pointers and type may orient toward specific etiologies, such as barking cough in croup or tracheomalacia, paroxystic whooping cough in Pertussis. Cough is productive in protracted bacterial bronchitis, sinusitis or bronchiectasis. Cough is usually associated with wheeze or dyspnea on exertion in asthma; however, it may be the sole symptom in cough variant asthma. Thus, pediatric cough has particularities differentiating it from adult cough, so the approach and management should be developmentally specific.