Ivacaftor restores CFTR-dependent sweat gland fluid secretion in cystic fibrosis subjects with S945L alleles.

BACKGROUND: To determine in vivo effects of CFTR modulators on mutation S945L. METHODS: We measured effects of CFTR modulators on CFTR-dependent sweating ('C-sweat') in two pancreatic sufficient cystic fibrosis (CF) subjects. S1 (S945L/G542X) took ivacaftor and S2 (S945L/F508del) took ivacaftor+tezacaftor. Sweating was stimulated pharmacologically to produce sequentially both CFTR-independent (methacholine stimulated) M-sweat and C-sweat; and the ratio of these was compared. Sweat secretion was measured with two methods: real time secretory rate quantitative recording and by optically measuring the growth of sweat bubbles under oil from multiple identified glands. RESULTS: Using the quantitative recorder, we saw zero C-sweat secretion off-drug, but when on-drug the C-sweat responses for both subjects were comparable to those seen in carriers. The on-drug response was further quantified using the sweat bubble method. Each subject again showed robust C-sweat responses, with C-sweat/M-sweat ratios~half of the ratio determined for a cohort of 40 controls tested under identical conditions. CONCLUSION: These in vivo results, consistent with prior in vitro findings, indicate that the drug treatments restore near-normal function to S945L-CFTR, and support the use of ivacaftor as a treatment for CF patients who carry this allele.

Autonomous sweat extraction and analysis applied to cystic fibrosis and glucose monitoring using a fully integrated wearable platform.

Perspiration-based wearable biosensors facilitate continuous monitoring of individuals' health states with real-time and molecular-level insight. The inherent inaccessibility of sweat in sedentary individuals in large volume (≥10 µL) for on-demand and in situ analysis has limited our ability to capitalize on this noninvasive and rich source of information. A wearable and miniaturized iontophoresis interface is an excellent solution to overcome this barrier. The iontophoresis process involves delivery of stimulating agonists to the sweat glands with the aid of an electrical current. The challenge remains in devising an iontophoresis interface that can extract sufficient amount of sweat for robust sensing, without electrode corrosion and burning/causing discomfort in subjects. Here, we overcame this challenge through realizing an electrochemically enhanced iontophoresis interface, integrated in a wearable sweat analysis platform. This interface can be programmed to induce sweat with various secretion profiles for real-time analysis, a capability which can be exploited to advance our knowledge of the sweat gland physiology and the secretion process. To demonstrate the clinical value of our platform, human subject studies were performed in the context of the cystic fibrosis diagnosis and preliminary investigation of the blood/sweat glucose correlation. With our platform, we detected the elevated sweat electrolyte content of cystic fibrosis patients compared with that of healthy control subjects. Furthermore, our results indicate that oral glucose consumption in the fasting state is followed by increased glucose levels in both sweat and blood. Our solution opens the possibility for a broad range of noninvasive diagnostic and general population health monitoring applications.

The magnitude of ivacaftor effects on fluid secretion via R117H-CFTR channels: Human in vivo measurements.

We optically measured effects of orally available ivacaftor (Kalydeco®) on sweat rates of identified glands in 3 R117H subjects, each having a unique set of additional mutations, and compared them with 5 healthy control subjects tested contemporaneously. We injected ß-adrenergic agonists intradermally to stimulate CFTR-dependent 'C-sweat' and methacholine to stimulate 'M-sweat', which persists in CF subjects. We focused on an R117H-7T/F508del subject who produced quantifiable C-sweat off ivacaftor and was available for 1 blinded, 3 off ivacaftor, and 3 on ivacaftor tests, allowing us to estimate in vivo fold-increase in sweat rates produced by ivacaftor's effect on the open probability (PO) of R117H-CFTR. Measured sweat rates must be corrected for sweat losses. With estimated sweat losses of 0.023 to 0.08 nl·gland-1·min-1, ivacaftor increased the average C-sweat rates 3-7 fold, and estimated function as % of WT were 4.1-12% off ivacaftor and 21.9-32% on ivacaftor (larger values reflect increased loss estimates). Based on single tests, an R117H-7T/ R117H-7T subject showed 6-9% WT function off ivacaftor and 28-43% on ivacaftor. Repeat testing of an R117H-5T/F508del subject detected only trace responding to ivacaftor. We conclude that in vivo, R117H PO is strongly increased by ivacaftor, but channel number, mainly determined by variable deletion of exon 10, has a marked influence on outcomes.

Evaporimeter and Bubble-Imaging Measures of Sweat Gland Secretion Rates.

Beta-adrenergically-stimulated sweat rates determined by evaporimetry or by sweat bubble imaging are useful for measuring CFTR function because they provide a near-linear readout across almost the full range of CFTR function. They differentiate cystic fibrosis (CF) subjects from CF carriers and carriers from controls. However, evaporimetry, unlike bubble imaging, appears to be unable to detect improved levels of CFTR function in G551D subjects taking the CFTR modulator ivacaftor. Here, we quantify the sensitivity of evaporimetry and bubble imaging methods for assessing low levels of CFTR-dependent sweat rates. To establish sensitivity, we did dose-ranging studies using intradermally injected [cAMP]i-elevating cocktails. We reduced isoproterenol/aminophylline levels while maintaining a high level of atropine to block muscarinic elevation of [Ca2+]i. We stimulated the same sets of glands for both assays and recorded responses for 20 min. In response to a 3-log dilution of the stimulating cocktail (0.1%), bubble responses were detected in 12/12 tests (100%), with 49% ± 3% of glands secreting to produce an aggregate volume of 598 nl across the 12, 20-min tests. This was ~5% of the response to full cocktail. Evaporimetry detected responses in 3/12 (25%) tests with an aggregate secretion volume of 175 nl. After stimulation with a still more dilute cocktail (0.03%), bubble imaging detected 15 ± 13% of glands secreting at a rate ~0.9% of the response to full cocktail, while zero responding was seen with evaporimetry. The bubble imaging method detected secretion down to aggregate rates of <0.2 nl/(cm2·min), or ~1/30th of the average basal transepithelial water loss (TEWL) in the test subject of 4 g/m2·hr or 6.7 nl/(cm2·min). The increased sensitivity of bubble imaging may be required to detect small but physiologically important increases in secretion rates produced by CFTR modulators.

Blood basophil activation is a reliable biomarker of allergic bronchopulmonary aspergillosis in cystic fibrosis.

The diagnosis of cystic fibrosis (CF) patients with allergic bronchopulmonary aspergillosis (ABPA) is clinically challenging, due to the absence of an objective biological test. Since blood basophils play a major role in allergic responses, we hypothesised that changes in their surface activation pattern discriminate between CF patients with and without ABPA.We conducted a prospective longitudinal study (Stanford cohort) comparing basophil activation test CD203c levels by flow cytometry before and after activation with Aspergillus fumigatus allergen extract or recombinant Asp f1 in 20 CF patients with ABPA (CF-ABPA) and in two comparison groups: CF patients with A. fumigatus colonisation (AC) but without ABPA (CF-AC; n=13) and CF patients without either AC or ABPA (CF; n=12). Patients were tested every 6 months and when ill with pulmonary exacerbation. We also conducted cross-sectional validation in a separate patient set (Dublin cohort).Basophil CD203c surface expression reliably discriminated CF-ABPA from CF-AC and CF over time. Ex vivo stimulation with A. fumigatus extract or recombinant Asp f1 produced similar results within the Stanford (p<0.0001) and the Dublin cohorts. CF-ABPA patients were likelier to have elevated specific IgE to A. fumigatus and were less frequently co-infected with Staphylococcus aureus.Basophil CD203c upregulation is a suitable diagnostic and stable monitoring biomarker of ABPA in CF.

Experiences of recruiting to a pilot trial of Cardiac Rehabilitation In patients with Bowel cancer (CRIB) with an embedded process evaluation: lessons learned to improve recruitment.

BACKGROUND: Recruitment to randomised controlled trials (RCTs) is a perennial problem. Calls have been made for trialists to make recruitment performance publicly available. This article presents our experience of recruiting to a pilot RCT of cardiac rehabilitation for patients with bowel cancer with an embedded process evaluation. METHODS: Recruitment took place at three UK hospitals. Recruitment figures were based on the following: i) estimated number of patient admissions, ii) number of patients likely to meet inclusion criteria from clinician input and iii) recruitment rates in previous studies. The following recruitment procedure was used:Nurse assessed patients for eligibility.Patients signed a screening form indicating interest in and agreement to be approached by a researcher about the study.An appointment was made at which the patient signed a consent form and was randomised to the intervention or control group. Information about all patients considered for the study and subsequently included or excluded at each stage of the recruitment process and reasons given were recorded. RESULTS: There were variations in the time taken to award Research Management approval to run the study at the three sites (45-359 days). Sixty-two percent of the original recruitment estimate was reached. The main reason for under-recruitment was due to over-estimation of the number of patient admissions; other reasons were i) not assessing all patients for eligibility, ii) not completing a screening form for eligible patients and iii) patients who signed a screening form being lost to the study before consenting and randomisation. CONCLUSIONS: Pilot trials should not simply aim to improve recruitment estimates but should also identify factors likely to influence recruitment performance in a future trial and inform the development of that trial's recruitment strategies. Pilot trials are a crucial part of RCT design. Nevertheless, pilot trials are likely to be small scale, involving only a small number of sites, and contextual differences between sites are likely to impact recruitment performance in any future trial. This means that ongoing monitoring and evaluation in trials are likely to be required. TRIAL REGISTRATION: ISRCTN63510637; UKCRN id 14092.

A little CFTR goes a long way: CFTR-dependent sweat secretion from G551D and R117H-5T cystic fibrosis subjects taking ivacaftor.

To determine if oral dosing with the CFTR-potentiator ivacaftor (VX-770, Kalydeco) improves CFTR-dependent sweating in CF subjects carrying G551D or R117H-5T mutations, we optically measured sweat secretion from 32-143 individually identified glands in each of 8 CF subjects; 6 F508del/G551D, one G551D/R117H-5T, and one I507del/R117H-5T. Two subjects were tested only (-) ivacaftor, 3 only (+) ivacaftor and 3 (+/-) ivacaftor (1-5 tests per condition). The total number of gland measurements was 852 (-) ivacaftor and 906 (+) ivacaftor. A healthy control was tested 4 times (51 glands). For each gland we measured both CFTR-independent (M-sweat) and CFTR-dependent (C-sweat); C-sweat was stimulated with a ß-adrenergic cocktail that elevated [cAMP]i while blocking muscarinic receptors. Absent ivacaftor, almost all CF glands produced M-sweat on all tests, but only 1/593 glands produced C-sweat (10 tests, 5 subjects). By contrast, 6/6 subjects (113/342 glands) produced C-sweat in the (+) ivacaftor condition, but with large inter-subject differences; 3-74% of glands responded with C/M sweat ratios 0.04%-2.57% of the average WT ratio of 0.265. Sweat volume losses cause proportionally larger underestimates of CFTR function at lower sweat rates. The losses were reduced by measuring C/M ratios in 12 glands from each subject that had the highest M-sweat rates. Remaining losses were estimated from single channel data and used to correct the C/M ratios, giving estimates of CFTR function (+) ivacaftor  = 1.6%-7.7% of the WT average. These estimates are in accord with single channel data and transcript analysis, and suggest that significant clinical benefit can be produced by low levels of CFTR function.

Blood basophils from cystic fibrosis patients with allergic bronchopulmonary aspergillosis are primed and hyper-responsive to stimulation by aspergillus allergens.

INTRODUCTION: Fifteen to sixty percent of cystic fibrosis patients harbor Aspergillus fumigatus (Af) in their airways (CF-AC) and some will develop allergic bronchopulmonary aspergillosis (CF-ABPA). Since basophils play a key role in allergy, we hypothesized that they would display alterations in CF-ABPA patients compared to CF-AC or patients without Af colonization (CF). METHODS: Using flow cytometry, we measured CD203c, CD63 and CD123 levels on basophils from CF-ABPA (N=11), CF-AC (N=14), and CF (N=12) patients before and after ex vivo stimulation with Af allergens. RESULTS: Baseline CD203c was increased in basophils from CF-ABPA compared to CF-AC and CF patients. Af extract and recombinant Aspf1 stimulated basophils from CF-ABPA patients to markedly upregulate CD203c, along with modest upregulation of CD63 and a CD123 downward trend. Plasma TARC/CCL17 at baseline and post-stimulation cell supernatant histamine levels were similar in the three groups. CONCLUSIONS: In CF-ABPA, blood basophils are primed and hyperresponsive to Af allergen stimulation.

Activation of critical, host-induced, metabolic and stress pathways marks neutrophil entry into cystic fibrosis lungs.

Cystic fibrosis (CF) patients undergo progressive airway destruction caused in part by chronic neutrophilic inflammation. While opportunistic pathogens infecting CF airways can cause inflammation, we hypothesized that host-derived metabolic and stress signals would also play a role in this process. We show that neutrophils that have entered CF airways have increased phosphorylation of the eukaryotic initiation factor 4E and its partner the 4E-binding protein 1; 2 key effectors in the growth factor- and amino acid-regulated mammalian target of rapamycin (mTOR) pathway. Furthermore CF airway neutrophils display increased phosphorylation of the cAMP response element binding protein (CREB), a major transcriptional coactivator in stress signaling cascades. These active intracellular pathways are associated with increased surface expression of critical adaptor molecules, including the growth factor receptor CD114 and the receptor for advanced glycation end-products (RAGE), a CREB inducer and sensor for host-derived damage-associated molecular patterns (DAMPs). Most CF airway fluids lack any detectable soluble RAGE, an inhibitory decoy receptor for DAMPs. Concomitantly, CF airway fluids displayed high and consequently unopposed levels of S100A12; a potent mucosa- and neutrophil-derived DAMP. CF airway neutrophils also show increased surface levels of 2 critical CREB targets, the purine-recycling enzyme CD39 and the multifunctional, mTOR-inducing CXCR4 receptor. This coordinated set of events occurs in all patients, even in the context of minimal airway inflammation and well-preserved lung function. Taken together, our data demonstrate an early and sustained activation of host-responsive metabolic and stress pathways upon neutrophil entry into CF airways, suggesting potential targets for therapeutic modulation.

Profound functional and signaling changes in viable inflammatory neutrophils homing to cystic fibrosis airways.

Blood neutrophils recruited to cystic fibrosis (CF) airways are believed to be rapidly killed by resident bacteria and to passively release elastase and other toxic by-products that promote disease progression. By single-cell analysis, we demonstrate that profound functional and signaling changes readily occur within viable neutrophils recruited to CF airways, compared with their blood counterparts. Airway neutrophils have undergone conventional activation, as shown by decreased intracellular glutathione, increased lipid raft assembly, surface mobilization of CD11b+ and CD66b+ granules, and increased levels of the cytoskeleton-associated phospho-Syk kinase. Unexpectedly, they also mobilize to the surface CD63+ elastase-rich granules, usually confined intracellularly, and lose surface expression of CD16 and CD14, both key receptors in phagocytosis. Furthermore, they express CD80, major histocompatibility complex type II, and the prostaglandin D2 receptor CD294, all normally associated with other lineages, which reflects functional reprogramming. This notion is reinforced by their decreased total phosphotyrosine levels, mirroring a postactivated stage, and increased levels of the phospho-S6 ribosomal protein, a key anabolic switch. Thus, we identified a subset of neutrophils within CF airways with a viable but dysfunctional phenotype. This subset provides a possible therapeutic target and indicates a need to revisit current paradigms of CF airway disease.

Identification of novel and rare mutations in California Hispanic and African American cystic fibrosis patients.

In ethnic heterogeneous California, complete genetic information is currently lacking to build solid population-based cystic fibrosis (CF) screening programs because a large proportion of mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR/ABCC7) are still unknown, especially in non-Caucasian patients. A total of 402 [46 African American+356 Hispanic] Hispanic and African American patients from California CF patient registry were included in this study. Patients with at least one unidentified mutant allele were asked to donate blood samples for further analysis, first by Genzyme Genetics for a panel of 87 known mutations, followed by temporal temperature gradient gel electrophoresis (TTGE) scanning of the entire coding exons of CFTR gene. A total of eight novel mutations; one missense mutation, one splice-site mutation and six frame-shift mutations were identified. In addition to the eight novel mutations, 20 [corrected] distinct rare mutations that are not in the current available commercial mutation panels were identified by TTGE. The overall detection rate was raised to 95.7% for African American and 94.5% for Hispanic. The discovery of recurrent rare and novel mutations improves the diagnosis and care of persons with CF and improves our ability to adequately and equitably provide screening and genetic counseling services to non-Caucasians.

Standardized procedure for measurement of nasal potential difference: an outcome measure in multicenter cystic fibrosis clinical trials.

Patients with cystic fibrosis (CF) can be discriminated from healthy subjects by measurement of the nasal potential difference, which has become a useful outcome measure for therapies directed toward correcting defective electrolyte transport in CF. A standard operating procedure was developed by a CF Foundation clinical trials network, to be followed by all sites performing collaborative studies. Key variables in the measurement included type of voltmeter, exploring probe, reference electrodes, and solutions used to assess both sodium transport and chloride conductance. Eight sites submitted data on 3-8 normal and 4-5 CF subjects. Baseline voltage, an index of sodium transport, was -18.2 +/- 8.3 mV (mean +/- SD) for normals, and -45.3 +/- 11.4 mV for CF patients. There was no CFTR-mediated chloride secretion in CF subjects, as evidenced by the lack of response to perfusion with zero chloride + beta agonist solutions (+3.2 +/- 3.5 mV) vs. that in normals (-23.7 +/- 10.2 mV). The standardized nasal potential difference measurement minimizes variability between operators and study sites. Valid and consistent results can be attained with trained operators and attention to technical details. These data demonstrate the procedure to be sufficient for multicenter studies in the CF Foundation network.