An integrated nano-scale approach to profile miRNAs in limited clinical samples

Profiling miRNA expression in cells that directly contribute to human disease pathogenesis is likely to aid the discovery of novel drug targets and biomarkers. However, tissue heterogeneity and the limited amount of human diseased tissue available for research purposes present fundamental difficulties that often constrain the scope and potential of such studies. We established a flow cytometry-based method for isolating pure populations of pathogenic T cells from bronchial biopsy samples of asthma patients, and optimized a high-throughput nano-scale qRT-PCR method capable of accurately measuring 96 miRNAs in as little as 100 cells. Comparison of circulating and airway T cells from healthy and asthmatic subjects revealed asthma-associated and tissue-specific miRNA expression patterns. These results establish the feasibility and utility of investigating miRNA expression in small populations of cells involved in asthma pathogenesis, and set a precedent for application of our nano-scale approach in other human diseases. The microarray data from this study (Figure 7) has been submitted to the NCBI Gene Expression Omnibus (GEO; http://ncbi.nlm.nih.gov/geo) under accession no. GSE31030.

Pediatric severe asthma is characterized by eosinophilia and remodeling without T(H)2 cytokines

BACKGROUND: The pathology of pediatric severe therapy-resistant asthma (STRA) is little understood. OBJECTIVES: We hypothesized that STRA in children is characterized by airway eosinophilia and mast cell inflammation and is driven by the T(H)2 cytokines IL-4, IL-5, and IL-13. METHODS: Sixty-nine children (mean age, 11.8 years; interquartile range, 5.6-17.3 years; patients with STRA, n = 53; control subjects, n = 16) underwent fiberoptic bronchoscopy, bronchoalveolar lavage (BAL), and endobronchial biopsy. Airway inflammation, remodeling, and BAL fluid and biopsy specimen T(H)2 cytokines were quantified. Children with STRA also underwent symptom assessment (Asthma Control Test), spirometry, exhaled nitric oxide and induced sputum evaluation. RESULTS: Children with STRA had significantly increased BAL fluid and biopsy specimen eosinophil counts compared with those found in control subjects (BAL fluid, P < .001; biopsy specimen, P < .01); within the STRA group, there was marked between-patient variability in eosinophilia. Submucosal mast cell, neutrophil, and lymphocyte counts were similar in both groups. Reticular basement membrane thickness and airway smooth muscle were increased in patients with STRA compared with those found in control subjects (P < .0001 and P < .001, respectively). There was no increase in BAL fluid IL-4, IL-5, or IL-13 levels in patients with STRA compared with control subjects, and these cytokines were rarely detected in induced sputum. Biopsy IL-5(+) and IL-13(+) cell counts were also not higher in patients with STRA compared with those seen in control subjects. The subgroup (n = 15) of children with STRA with detectable BAL fluid T(H)2 cytokines had significantly lower lung function than those with undetectable BAL fluid T(H)2 cytokines. CONCLUSIONS: STRA in children was characterized by remodeling and variable airway eosinophil counts. However, unlike in adults, there was no neutrophilia, and despite the wide range in eosinophil counts, the T(H)2 mediators that are thought to drive allergic asthma were mostly absent.

High rhinovirus burden in lower airways of children with cystic fibrosis

Rhinovirus (RV)-induced pulmonary exacerbations are common in cystic fibrosis (CF) and have been associated with impaired virus clearance by the CF airway epithelium in vitro. Here, we assess in vivo the association of RV prevalence and load with antiviral defense mechanisms, airway inflammation, and lung function parameters in children with CF compared with a control group and children with other chronic respiratory diseases.

Identification of SNPs in the cystic fibrosis interactome influencing pulmonary progression in cystic fibrosis

There is growing evidence that the great phenotypic variability in patients with cystic fibrosis (CF) not only depends on the genotype, but apart from a combination of environmental and stochastic factors predominantly also on modifier gene effects. It has been proposed that genes interacting with CF transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) are potential modifiers. Therefore, we assessed the impact of single-nucleotide polymorphisms (SNPs) of several of these interacters on CF disease outcome. SNPs that potentially alter gene function were genotyped in 95 well-characterized p.Phe508del homozygous CF patients. Linear mixed-effect model analysis was used to assess the relationship between sequence variants and the repeated measurements of lung function parameters. In total, we genotyped 72 SNPs in 10 genes. Twenty-five SNPs were used for statistical analysis, where we found strong associations for one SNP in PPP2R4 with the lung clearance index (P ≤ 0.01), the specific effective airway resistance (P ≤ 0.005) and the forced expiratory volume in 1 s (P ≤ 0.005). In addition, we identified one SNP in SNAP23 to be significantly associated with three lung function parameters as well as one SNP in PPP2R1A and three in KRT19 to show a significant influence on one lung function parameter each. Our findings indicate that direct interacters with CFTR, such as SNAP23, PPP2R4 and PPP2R1A, may modify the residual function of p.Phe508del-CFTR while variants in KRT19 may modulate the amount of p.Phe508del-CFTR at the apical membrane and consequently modify CF disease.

Exhaled nitric oxide in symptomatic children at preschool age predicts later asthma

Prediction of asthma in young children with respiratory symptoms is hampered by the lack of objective measures applicable in clinical routine. In this prospective study in a preschool children cohort, we assessed whether the fraction of exhaled nitric oxide (FeNO), a biomarker of airway inflammation, is associated with asthma at school age.

Respiratory function during anesthesia: effects on gas exchange

Anaesthesia causes a respiratory impairment, whether the patient is breathing spontaneously or is ventilated mechanically. This impairment impedes the matching of alveolar ventilation and perfusion and thus the oxygenation of arterial blood. A triggering factor is loss of muscle tone that causes a fall in the resting lung volume, functional residual capacity. This fall promotes airway closure and gas adsorption, leading eventually to alveolar collapse, that is, atelectasis. The higher the oxygen concentration, the faster will the gas be adsorbed and the aleveoli collapse. Preoxygenation is a major cause of atelectasis and continuing use of high oxygen concentration maintains or increases the lung collapse, that typically is 10% or more of the lung tissue. It can exceed 25% to 40%. Perfusion of the atelectasis causes shunt and cyclic airway closure causes regions with low ventilation/perfusion ratios, that add to impaired oxygenation. Ventilation with positive end-expiratory pressure reduces the atelectasis but oxygenation need not improve, because of shift of blood flow down the lung to any remaining atelectatic tissue. Inflation of the lung to an airway pressure of 40 cmH2O recruits almost all collapsed lung and the lung remains open if ventilation is with moderate oxygen concentration (< 40%) but recollapses within a few minutes if ventilation is with 100% oxygen. Severe obesity increases the lung collapse and obstructive lung disease and one-lung anesthesia increase the mismatch of ventilation and perfusion. CO2 pneumoperitoneum increases atelectasis formation but not shunt, likely explained by enhanced hypoxic pulmonary vasoconstriction by CO2. Atelectasis may persist in the postoperative period and contribute to pneumonia.

Fate of inhaled particles after interaction with the lung surface

Inhaled particles may cause increased pulmonary and cardiovascular morbidity and mortality. The wall structures of airways and alveoli act as a series of structural and functional barriers against inhaled particles. Deposited particles are displaced and come into close association with epithelial cells, macrophages and dendritic cells. The cellular interplay after particle deposition in a triple cell co-culture model of the human airway wall was investigated by laser scanning microscopy. Furthermore, the cellular response was determined by measurement of TNF-alpha. Dendritic cells gained access to the apical side of the epithelium where they sampled particles and interacted with macrophages.

Epilepsy and obstructive sleep apnea

A few publications documented the coexistence of epilepsy and obstructive sleep apnea (OSA). The extent, nature, and clinical relevance of this association remain poorly understood. We retrospectively reviewed the database of our sleep center to identify patients with both sleep apnea and epilepsy. Characteristics of epilepsy, sleep history, presence of excessive daytime sleepiness [Epworth Sleepiness Scale (ESS)] and polysomnographic data were assessed. The effect of continuous positive airway pressure (CPAP) on seizure reduction was prospectively analyzed after a median interval of 26 months (range: 2-116 months) from the diagnosis of OSA. OSA was found in 29 epilepsy patients (25 men and 4 women) with a median age of 56 years (range: 37-79). The median apnea hypopnea index was 33 (range: 10-85), the oxygen desaturation index was 12 (range 0-92), and 52% of the patients had an ESS score >10. In 27 patients, epilepsy appeared 1 month to 44 years prior to the diagnosis of OSA. In 21 patients, the appearance of OSA symptoms coincided with a clear increase in seizure frequency or the first appearance of a status epilepticus. Treatment with CPAP was continued with good compliance in 12 patients and led to a significant reduction of both ESS scores and seizure frequency in 4 patients. Our data suggest the importance of considering diagnosis and treatment of OSA in epilepsy patients with poor seizure control and/or reappearance of seizures after a seizure-free interval.

Sleep-disordered breathing and acute ischemic stroke: diagnosis, risk factors, treatment, evolution, and long-term clinical outcome

BACKGROUND AND PURPOSE: Sleep-disordered breathing (SDB) is frequent in stroke patients. Risk factors, treatment response, short-term and long-term outcome of SDB in stroke patients are poorly known. METHODS: We prospectively studied 152 patients (mean age 56+/-13 years) with acute ischemic stroke. Cardiovascular risk factors, Epworth sleepiness score (ESS), stroke severity/etiology, and time of stroke onset were assessed. The apnea-hypopnea index (AHI) was determined 3+/-2 days after stroke onset and 6 months later (subacute phase). Continuous positive airway pressure (CPAP) treatment was started acutely in patients with SDB (AHI > or =15 or AHI > or =10+ESS >10). CPAP compliance, incidence of vascular events, and stroke outcome were assessed 60+/-16 months later (chronic phase). RESULTS: Initial AHI was 18+/-16 (> or =10 in 58%, > or =30 in 17% of patients) and decreased in the subacute phase (P<0.001). Age, diabetes, and nighttime stroke onset were independent predictors of AHI (r2=0.34). In patients with AHI > or =30, age, male gender, body mass index, diabetes, hypertension, coronary heart disease, ESS, and macroangiopathic etiology of stroke were significantly higher/more common than in patients with AHI <10. Long-term incidence of vascular events and stroke outcome were similar in both groups. CPAP was started in 51% and continued chronically in 15% of SDB pts. Long-term stroke mortality was associated with initial AHI, age, hypertension, diabetes, and coronary heart disease. CONCLUSIONS: SDB is common particularly in elderly stroke male patients with diabetes, nighttime stroke onset, and macroangiopathy as cause of stroke; it improves after the acute phase, is associated with an increased poststroke mortality, and can be treated with CPAP in a small percentage of patients.

Fluorescent microangiography (FMA): an improved tool to visualize the pulmonary microvasculature

Visualization of the complex lung microvasculature and resolution of its three-dimensional architecture remains a difficult experimental challenge. We present a novel fluorescent microscopy technique to visualize both the normal and diseased pulmonary microvasculature. Physiologically relevant pulmonary perfusion conditions were applied using a low-viscosity perfusate infused under continuous airway ventilation. Intensely fluorescent polystyrene microspheres, confined to the vascular space, were imaged through confocal optical sectioning of 200 microm-thick lung sections. We applied this technique to rat lungs and the markedly enhanced depth of field in projected images allowed us to follow vascular branching patterns in both normal lungs and lungs from animals with experimentally induced pulmonary arterial hypertension. In addition, this method allowed complementary immunostaining and identification of cellular components surrounding the blood vessels. Fluorescent microangiography is a widely applicable and quantitative tool for the study of vascular changes in animal models of pulmonary disease.

Progression of pulmonary hyperinflation and trapped gas associated with genetic and environmental factors in children with cystic fibrosis

BACKGROUND: Functional deterioration in cystic fibrosis (CF) may be reflected by increasing bronchial obstruction and, as recently shown, by ventilation inhomogeneities. This study investigated which physiological factors (airway obstruction, ventilation inhomogeneities, pulmonary hyperinflation, development of trapped gas) best express the decline in lung function, and what role specific CFTR genotypes and different types of bronchial infection may have upon this process. METHODS: Serial annual lung function tests, performed in 152 children (77 males; 75 females) with CF (age range: 6-18 y) provided data pertaining to functional residual capacity (FRCpleth, FRCMBNW), volume of trapped gas (VTG), effective specific airway resistance (sReff), lung clearance index (LCI), and forced expiratory indices (FVC, FEV1, FEF50). RESULTS: All lung function parameters showed progression with age. Pulmonary hyperinflation (FRCpleth > 2SDS) was already present in 39% of patients at age 6-8 yrs, increasing to 67% at age 18 yrs. The proportion of patients with VTG > 2SDS increased from 15% to 54% during this period. Children with severe pulmonary hyperinflation and trapped gas at age 6-8 yrs showed the most pronounced disease progression over time. Age related tracking of lung function parameters commences early in life, and is significantly influenced by specific CFTR genotypes. The group with chronic P. aeruginosa infection demonstrated most rapid progression in all lung function parameters, whilst those with chronic S. aureus infection had the slowest rate of progression. LCI, measured as an index of ventilation inhomogeneities was the most sensitive discriminator between the 3 types of infection examined (p < 0.0001). CONCLUSION: The relationships between lung function indices, CFTR genotypes and infective organisms observed in this study suggest that measurement of other lung function parameters, in addition to spirometry alone, may provide important information about disease progression in CF.

Elevated exhaled nitric oxide in newborns of atopic mothers precedes respiratory symptoms

RATIONALE: Exhaled nitric oxide (NO) is a well-known marker of established airway inflammation in asthma. Its role in the disease process before the onset of respiratory symptoms remains unclear. Objectives: To examine whether elevated NO in newborns with clinically naive airways is associated with subsequent respiratory symptoms in infancy. METHODS: We measured exhaled NO concentration and output after birth and prospectively assessed respiratory symptoms during infancy in a birth cohort of 164 unselected healthy neonates. We examined a possible association between NO and respiratory symptoms using Poisson regression analysis. RESULTS: In infants of atopic mothers, elevated NO levels after birth were associated with increased risk of subsequent respiratory symptoms (risk ratio [RR], 7.5; 95% confidence interval [CI], 1.7-32.4 for each nl/s increase in NO output; p = 0.007). Similarly, a positive association between NO and symptoms was seen in infants of smoking mothers (RR, 6.6; 95% CI, 2.3-19.3; p = 0.001), with the strongest association in infants whose mothers had both risk factors (RR, 21.8; 95% CI, 5.8-81.3; p < 0.001). CONCLUSIONS: The interaction of NO with maternal atopy and smoking on subsequent respiratory symptoms is present early in life. Clinically, noninvasive NO measurements in newborns may prove useful as a new means to identify high-risk infants. Future confirmation of a role for NO metabolism in the evolution of respiratory disease may provide an avenue for preventative strategies.

Effects of an alveolar recruitment maneuver on cardiovascular and respiratory parameters during total intravenous anesthesia in ponies

OBJECTIVE: To evaluate pulmonary and cardiovascular effects of a recruitment maneuver (RM) combined with positive end-expiratory pressure (PEEP) during total intravenous anesthesia in ponies. ANIMALS: 6 healthy adult Shetland ponies. PROCEDURE: After premedication with detomidine (10 microg/kg, IV), anesthesia was induced with climazolam (0.06 mg/kg, IV) and ketamine (2.2 mg/kg, IV) and maintained with a constant rate infusion of detomidine (0.024 mg/kg/h), climazolam (0.036 mg/kg/h), and ketamine (2.4 mg/kg/h). The RM was preceded by an incremental PEEP titration and followed by a decremental PEEP titration, both at a constant airway pressure difference (deltaP) of 20 cm H2O. The RM consisted of a stepwise increase in deltaP by 25, 30, and 35 cm H2O obtained by increasing peak inspiratory pressure (PIP) to 45, 50, and 55 cm H2O, while maintaining PEEP at 20 cm H2O. Hemodynamic and pulmonary variables were analyzed at every step of the PEEP titration-RM. RESULTS: During the PEEP titration-RM, there was a significant increase in PaO 2 (+12%), dynamic compliance (+ 62%), and heart rate (+17%) and a decrease in shunt (-19%) and mean arterial blood pressure (-21%) was recorded. Cardiac output remained stable. CONCLUSIONS AND CLINICAL RELEVANCE: Although baseline oxygenation was high, Pa(O2) and dynamic compliance further increased during the RM. Despite the use of high PIP and PEEP and a high tidal volume, limited cardiovascular compromise was detected. A PEEP titration-RM may be used to improve oxygenation in anesthetized ponies. During stable hemodynamic conditions, PEEP titration-RM can be performed with acceptable adverse cardiovascular effects.

Quantification of atelectatic lung volumes in two different porcine models of ARDS

BACKGROUND: Cyclic recruitment during mechanical ventilation contributes to ventilator associated lung injury. Two different pathomechanisms in acute respiratory distress syndrome (ARDS) are currently discussed: alveolar collapse vs persistent flooding of small airways and alveoli. We compare two different ARDS animal models by computed tomography (CT) to describe different recruitment and derecruitment mechanisms at different airway pressures: (i) lavage-ARDS, favouring alveolar collapse by surfactant depletion; and (ii) oleic acid ARDS, favouring alveolar flooding by capillary leakage. METHODS: In 12 pigs [25 (1) kg], ARDS was randomly induced, either by saline lung lavage or oleic acid (OA) injection, and 3 animals served as controls. A respiratory breathhold manoeuvre without spontaneous breathing at different continuous positive airway pressure (CPAP) was applied in random order (CPAP levels of 5, 10, 15, 30, 35 and 50 cm H(2)O) and spiral-CT scans of the total lung were acquired at each CPAP level (slice thickness=1 mm). In each spiral-CT the volume of total lung parenchyma, tissue, gas, non-aerated, well-aerated, poorly aerated, and over-aerated lung was calculated. RESULTS: In both ARDS models non-aerated lung volume decreased significantly from CPAP 5 to CPAP 50 [oleic acid lung injury (OAI): 346.9 (80.1) to 96.4 (48.8) ml, P<0.001; lavage-ARDS: 245 17.6) to 42.7 (4.8) ml, P<0.001]. In lavage-ARDS poorly aerated lung volume decreased at higher CPAP levels [232 (45.2) at CPAP 10 to 84 (19.4) ml at CPAP 50, P<0.001] whereas in OAI poorly aerated lung volume did not vary at different airway pressures. CONCLUSIONS: In both ARDS models well-aerated and non-aerated lung volume respond to different CPAP levels in a comparable fashion: Thus, a cyclical alveolar collapse seems to be part of the derecruitment process also in the OA-ARDS. In OA-ARDS, the increase in poorly aerated lung volume reflects the specific initial lesion, that is capillary leakage with interstitial and alveolar oedema.

Initiation of high-frequency oscillatory ventilation and its effects upon cerebral circulation in pigs: an experimental study

BACKGROUND: Current practice at high-frequency oscillatory ventilation (HFOV) initiation is a stepwise increase of the constant applied airway pressure to achieve lung recruitment. We hypothesized that HFOV would lead to more adverse cerebral haemodynamics than does pressure controlled ventilation (PCV) in the presence of experimental intracranial hypertension (IH) and acute lung injury (ALI) in pigs with similar mean airway pressure settings. METHODS: In 12 anesthetized pigs (24-27 kg) with IH and ALI, mean airway pressure (P(mean)) was increased (to 20, 25, 30 cm H(2)O every 30 min), either with HFOV or with PCV. The order of the two ventilatory modes (cross-over) was randomized. Mean arterial pressure (MAP), intracranial pressure (ICP), cerebral perfusion pressure (CPP), cerebral blood flow (CBF) (fluorescent microspheres), cerebral metabolism, transpulmonary pressures (P(T)), and blood gases were determined at each P(mean) setting. Our end-points of interest related to the cerebral circulation were ICP, CPP and CBF. RESULTS: CBF and cerebral metabolism were unaffected but there were no differences between the values for HFOV and PCV. ICP increased slightly (HFOV median +1 mm Hg, P<0.05; PCV median +2 mm Hg, P<0.05). At P(mean) setting of 30 cm H(2)O, CPP decreased during HFOV (median -13 mm Hg, P<0.05) and PCV (median -17 mm Hg, P<0.05) paralleled by a decrease of MAP (HFOV median -11 mm Hg, P<0.05; PCV median -13 mm Hg, P<0.05). P(T) increased (HFOV median +8 cm H(2)O, P<0.05; PCV median +8 cm H(2)O, P<0.05). Oxygenation improved and normocapnia maintained by HFOV and PCV. There were no differences between both ventilatory modes. CONCLUSIONS: In animals with elevated ICP and ALI, both ventilatory modes had effects upon cerebral haemodynamics. The effects upon cerebral haemodynamics were dependent of the P(T) level without differences between both ventilatory modes at similar P(mean) settings. HFOV seems to be a possible alternative ventilatory strategy when MAP deterioration can be avoided.