Comparison of Nasal Potential Difference and Intestinal Current Measurements as Surrogate Markers for CFTR Function.

OBJECTIVES: Nasal potential difference (NPD) measurement is part of the diagnostic criteria for cystic fibrosis (CF) and now used routinely as an endpoint in clinical trials of correcting the basic defect in CF. Intestinal current measurement (ICM), measured ex vivo on a rectal biopsy, has been used to study cystic fibrosis transmembrane conductance regulator (CFTR) function but has not been compared to NPD in the same subject in adults and children. The aim of the study is to evaluate the potential usefulness of ICM as a marker of CFTR function for treatment studies compared NPD in patients with CF and in healthy control subjects. METHODS: ICM and NPD were performed on healthy controls and patients with CF. The healthy adults were individuals undergoing routine screening colonoscopy at the Beth Israel Deaconess Medical Center. The healthy children were undergoing colonoscopy for suspicion of inflammation in Hadassah Hebrew University Medical Center. The CF adults were recruited from Boston Children's Hospital CF Center and CF Center Worcester Mass, the children with CF from Hadassah CF Center. RESULTS: ICM measurements in healthy control subjects (n = 16) demonstrated a mean (±SE) carbachol response of 16.0 (2.2) µA/cm, histamine response of 13.2 (2.1) µA/cm and a forskolin response of 6.3 (2.0) µA/cm. Basal NPD of -15.9 (1.9) and response to Cl free + isoproterenol of -13.8 (2.0). These responses were inverted in CF subjects (n = 12) for ICM parameters with carbachol response of -3.0 (0.5) µA/cm, histamine -1.0 (0.8) µA/cm and a forskolin response of 0.5 (0.3) and also for NPD parameters; basal NPD of -42.2 (4.3) and response to Cl free + isoproterenol of 4.3 (0.7). Pearson correlation test showed the comparability of ICM and NPD in assessing CFTR function. CONCLUSIONS: ICM is equivalent to NPD in the ability to distinguish patients with CF from controls and could be used as surrogate markers of CFTR activity in treatment protocols.

Genetic and electrophysiological characteristics of recurrent acute pancreatitis.

OBJECTIVES: The aim was to present the workup of patients with acute recurrent pancreatitis (ARP) for genetic analysis and electrophysiological testing. METHODS: Patients with ARP with unknown etiology were referred for genetic testing and evaluation of cystic fibrosis transmembrane conductor regulator (CFTR) function by nasal potential difference (NPD) testing. RESULTS: A total of 67 patients were evaluated. The mean age was 23 ±â€Š17 years (median 17.0 years, range 1.5-72 years); 90% were Jewish and 10% Arab. Ten (15%) patients carried PRSS1 gene mutation (K23R(7), R122H(2), and D21A(1)). One patient had K172E/- (chymotrypsin C [CTRC]) mutation, 1 had I42M (serine protease inhibitor Kazal type 1 [SPINK1])/V235I (CTRC) together with ΔF508/5T, 1 patient had R67H (SPINK1)/V235I (CTRC), and 1 patient had V235I (CTRC)/-. Ten of 67 (15%) patients submitted for CFTR gene testing carried mutations (ΔF508/L997F, ΔF508/5T(11TG), W1282/5T(12TG), W1282X/Y1014C, ΔF508/R31C, R117H/-, R117H/Y1014C, D1152H/-, 5T(11TG)/-, and L997F/-). Fifty-four (80%) patients underwent sweat testing. Of these, 5 had sweat chloride ≥60 mEq/L, and 22 patients had sweat chloride from 40 to 60 mEq/L. Of the 56 (83%) patients had nasal potential difference testing, 4 (6%) with abnormal results. CONCLUSIONS: One-third (34%) of patients with ARP carry mutations for hereditary pancreatitis including rare mutations (K23R), and 12.5% have evidence of cftr mutations and 10% had CFTR dysfunction underscoring the importance of genetic and functional workup of these patients.

Influence of perfusate temperature on nasal potential difference.

Nasal potential difference (NPD) quantifies abnormal ion transport in cystic fibrosis. It has gained acceptance as an outcome measure for the investigation of new therapies. To quantify the effect of solution temperature on NPD, we first examined the effect of switching from room temperature (20-25°C) to warmed (32-37°C) solutions and vice versa during each perfusion step. Secondly, standard protocols were repeated at both temperatures in the same subjects. Changing solution temperature did not alter NPD during perfusion with Ringer's solution (<1 mV) (p>0.1). During perfusion with zero chloride solution, changing from room temperature to warmed solutions tended to decrease absolute NPD (i.e. it became less negative) by 0.9 mV (p>0.1); changing from warmed to room temperature increased NPD by 2.1 mV (p<0.05). During isoprenaline perfusion, changing from room temperature to warmed solutions increased NPD by 1.5 mV (p<0.01) and from warmed to room temperature decreased NPD by 1.4 mV (p<0.05). For full protocols at room temperature or warmed in the same subjects, mean values were similar (n = 24). During warmed perfusion, group results for total chloride response had a larger standard deviation. As this increased variability will probably decrease the power of trials, this study suggests that solutions at room temperature should be recommended for the measurement of NPD.

Nasal potential difference in suspected cystic fibrosis patients with 5T polymorphism.

BACKGROUND: 5T polymorphism is a CFTR mutation with unclear clinical consequences: the phenotype varies from healthy individuals to Cystic Fibrosis (CF). The aim of this study was to evaluate if nasal potential difference (NPD) and sweat testing correlate with symptoms and CF diagnosis in 5T patients. METHODS: 86 patients with 5T who had undergone NPD measurement, were included (6 homozygous (5T/5T), 41 with a PI-CF causing mutation in trans (5T/PI-CF), 11 with a PS-CF causing mutation in trans (5T/PS-CF) and 28 without a known mutation in trans (5T/?). Data including age, phenotype, sweat chloride and follow up were collected. RESULTS: 33% of the 5T/5T patients had abnormal NPD results, compared to 70% in 5T/PI-CF; 33% in 5T/PS-CF and 29% in 5T/?. The percentage of high or borderline sweat chloride was highest in 5T/PI-CF, and 5T/?, compared to 5T/5T and 5T/PS-CF (91, 96, 80, and 63%, respectively). TGm (number of TG repeats in intron 8) analysis was performed in 21 5T/PI-CF patients. TG11 was associated with lower sweat chloride, lower percentage of abnormal NPD and less progression of symptoms compared to TG12 and TG13. CONCLUSION: There is much variation in clinical status among 5T patients. All patients in this study with 5T/PS CF, all patients with both normal NPD and sweat test, and most patients with TG11 were stable or improving over time. Therefore, NPD measurement and TGm status aid to assess if a patient is at high risk for developing CF or CFTR-related disease and if specific follow up in a CF center is required.

Effectiveness of PTC124 treatment of cystic fibrosis caused by nonsense mutations: a prospective phase II trial.

BACKGROUND: In about 10% of patients worldwide and more than 50% of patients in Israel, cystic fibrosis results from nonsense mutations (premature stop codons) in the messenger RNA (mRNA) for the cystic fibrosis transmembrane conductance regulator (CFTR). PTC124 is an orally bioavailable small molecule that is designed to induce ribosomes to selectively read through premature stop codons during mRNA translation, to produce functional CFTR. METHODS: This phase II prospective trial recruited adults with cystic fibrosis who had at least one nonsense mutation in the CFTR gene. Patients were assessed in two 28-day cycles. During the first cycle, patients received PTC124 at 16 mg/kg per day in three doses every day for 14 days, followed by 14 days without treatment; in the second cycle, patients received 40 mg/kg of PTC124 in three doses every day for 14 days, followed by 14 days without treatment. The primary outcome had three components: change in CFTR-mediated total chloride transport; proportion of patients who responded to treatment; and normalisation of chloride transport, as assessed by transepithelial nasal potential difference (PD) at baseline, at the end of each 14-day treatment course, and after 14 days without treatment. The trial was registered with who.int/ictrp, and with clinicaltrials.gov, number NCT00237380. FINDINGS: Transepithelial nasal PD was evaluated in 23 patients in the first cycle and in 21 patients in the second cycle. Mean total chloride transport increased in the first treatment phase, with a change of -7.1 (SD 7.0) mV (p<0.0001), and in the second, with a change of -3.7 (SD 7.3) mV (p=0.032). We recorded a response in total chloride transport (defined as a change in nasal PD of -5 mV or more) in 16 of the 23 patients in the first cycle's treatment phase (p<0.0001) and in eight of the 21 patients in the second cycle (p<0.0001). Total chloride transport entered the normal range for 13 of 23 patients in the first cycle's treatment phase (p=0.0003) and for nine of 21 in the second cycle (p=0.02). Two patients given PTC124 had constipation without intestinal obstruction, and four had mild dysuria. No drug-related serious adverse events were recorded. INTERPRETATION: In patients with cystic fibrosis who have a premature stop codon in the CFTR gene, oral administration of PTC124 to suppress nonsense mutations reduces the epithelial electrophysiological abnormalities caused by CFTR dysfunction.

Cystic fibrosis transmembrane conductance regulator ion channel function testing in recurrent acute pancreatitis.

GOALS: To understand the relationship between acute recurrent pancreatitis and cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction. BACKGROUND: An emerging number of patients present with a nonclassic phenotype of cystic fibrosis (CF) with partial features or single-organ disease only. The association between the phenotype of recurrent pancreatitis CFTR dysfunction is unclear. METHODS: Patients with idiopathic recurrent pancreatitis were referred for electrophysiologic investigation. RESULTS: Thirty-three patients (18 males) aged 20+/-12 years with recurrent pancreatitis were studied. Three patients had mild asthma and 1 patient had mild ulcerative colitis. There was no family history of CF. All patients had normal imaging of the pancreatic duct by endoscopic retrograde cholangiopancreatography or magnetic resonance cholangiopancreatography. No patient was pancreatic insufficient. Mean sweat chloride values were 41+/-14 meq/L (range: 18 to 64). Nasal potential difference (NPD) measurement was pathologic in 7 patients. Mean basal potential difference in these 7 patients was -33+/-13 mV and there was an abnormal response to chloride-free and isoproterenol solutions. There was no difference in sweat chloride concentration between the 2 groups. Mutation analysis revealed W1282X/5T, D1152H/5T, and W1282X/- in 3 patients with abnormal NPD and 1 W1282X allele was found in 1 patient with normal NPD. CONCLUSIONS: In this series, 21% of patients with recurrent pancreatitis have abnormalities of CFTR function. Patients presenting with recurrent, "idiopathic" pancreatitis require CFTR function testing.

Primary sclerosing cholangitis is associated with abnormalities in CFTR.

BACKGROUND: The etiology of primary sclerosing cholangitis (PSC) is unknown. PSC and Cystic Fibrosis related liver disease have common features: chronic inflammation, biliary damage and similar cholangiographic findings. It is unknown whether or not PSC is related to cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction. We hypothesize that a sub-group of PSC patients may be a "single-organ" presentation of CF. METHODS: Patients with PSC underwent nasal potential difference (NPD) measurement, sweat chloride measurement and complete CFTR sequencing by new generation sequencing. RESULTS: 6/32 patients aged 46 ±â€¯13 yrs. had CFTR causing mutations on one allele and 19 had CFTR polymorphisms; 6/23 tested had abnormal and 21 had intermediate sweat tests; 4/32 patients had abnormal NPD. One patient had chronic pancreatitis and was infertile. CONCLUSIONS: 19% of PSC patients had features of CFTR related disorder, 19% carry CFTR mutations and 50% had CFTR polymorphisms. In some patients, PSC may be a single organ presentation of CF or a CFTR-related disorder.

Reproducibility of nasal potential difference measurements in cystic fibrosis.

BACKGROUND: Nasal potential difference (NPD) measurement has been advocated as a diagnostic tool for cystic fibrosis (CF) patients and as a method for assessing the response to new therapies. The purpose of this study was to examine the reproducibility of NPD measurements performed in a single center. METHODS: A total of 68 CF patients with a mean (+/- SD) age of 16 +/- 8 years (age range, 6 to 52 years) underwent NPD measurements on at least two occasions. RESULTS: A total of 25 patients with classic CF (mean age, 21 +/- 8 years) and 43 patients with nonclassic CF (mean age, 14 +/- 8 years) underwent sweat tests and NPD measurements. The mean sweat chloride values were 102 +/- 18 and 54 +/- 14 mEq/L, respectively, for classic CF and nonclassic CF groups. All patients underwent repeat NPD measurements. The basal NPD and the response to amiloride (DeltaAmil) and response to Cl(-) free and isoproterenol (DeltaCl(-) free + iso) were very similar in both measurements. In the classic CF group, the basal potential difference values were -40 +/- 12 vs -39 +/- 11 mV (p = 0.57), respectively, for the first and second measurements; 27 +/- 9 vs 26 +/- 10 mV (p = 0.55), respectively, for DeltaAmil; and 2.1 +/- 3.8 vs 0.4 +/- 2.9 mV (p = 0.07), respectively, for DeltaCl(-) free + iso. In the nonclassic CF group, the values were -32 +/- 13 vs -28 +/- 10 mV (p = 0.008), respectively; 19 +/- 10 vs 17 +/- 8 mV (p = 0.388), respectively; and -3.2 +/- 4.6 vs -3.3 +/- 4.4 mV (p = 0.876), respectively. CONCLUSION: When performed in a single center, NPD is a reproducible test for CF patients and thus may be a useful outcome measurement for assessment of the efficacy of new treatments.

Preconditioning allows engraftment of mouse and human embryonic lung cells, enabling lung repair in mice.

Repair of injured lungs represents a longstanding therapeutic challenge. We show that human and mouse embryonic lung tissue from the canalicular stage of development (20-22 weeks of gestation for humans, and embryonic day 15-16 (E15-E16) for mouse) are enriched with progenitors residing in distinct niches. On the basis of the marked analogy to progenitor niches in bone marrow (BM), we attempted strategies similar to BM transplantation, employing sublethal radiation to vacate lung progenitor niches and to reduce stem cell competition. Intravenous infusion of a single cell suspension of canalicular lung tissue from GFP-marked mice or human fetal donors into naphthalene-injured and irradiated syngeneic or SCID mice, respectively, induced marked long-term lung chimerism. Donor type structures or 'patches' contained epithelial, mesenchymal and endothelial cells. Transplantation of differentially labeled E16 mouse lung cells indicated that these patches were probably of clonal origin from the donor. Recipients of the single cell suspension transplant exhibited marked improvement in lung compliance and tissue damping reflecting the energy dissipation in the lung tissues. Our study provides proof of concept for lung reconstitution by canalicular-stage human lung cells after preconditioning of the pulmonary niche.

Evaluation of the intestinal current measurement method as a diagnostic test for cystic fibrosis.

BACKGROUND: The sweat test and nasal potential difference measurement are now established tools in the diagnostic work up of cystic fibrosis (CF). Intestinal current measurement (ICM) is under consideration as an aid in the diagnosis of CF especially in young children. The aim of this study is to evaluate the diagnostic reliability of ICM. METHODS: Rectal biopsies were obtained from three groups: CF patients, controls, and patients who were suspected for CF. ICMs were performed by mounting the rectal biopsy in an Ussing chamber and sequentially adding secretagogues while recording current changes. RESULTS: Twenty-one CF patients (aged 3.0 ± 3.8 years) and 16 controls (aged 15.6 ± 15.1 years) were examined and have remarkably different results (presented as µA/cm(2) ): carbachol 16.3 ± 6.9, histamine 13.2 ± 8.9, and cAMP/forskolin 4.8 ± 4.0 for control group and carbachol -1.5 ± 5.3 (P < 0.0001), histamine -1.5 ± 3.1 (P < 0.0001), and cAMP/forskolin 0.36 ± 0.67 (P < 0.0001) for the CF group. Our suggested reference values are: +5.40, +3.52, +2.19 for carbachol, histamine, and cAMP/forskolin, respectively. The combination parameter (the arithmetic sum of carbachol, histamine, and cAMP/forskolin) of +7.19 differentiates normal from abnormal (ROC curve analysis, area under the curve = 1.00, both sensitivity and specificity are 100%). This statistical model was applied to 71 patients suspected for CF and revealed that 66 patients had normal ICM results (combination >7.19) and five patients had abnormal ICM results (combination <7.19). CONCLUSION: We have shown that ICM tests may be useful to differentiate between patients suspected to have CF. These results require confirmation so that ICM may be included in diagnostic algorithms.

Nasal potential difference in non-classic cystic fibrosis-long term follow up.

BACKGROUND: Nasal potential difference (NPD) measurement is an electrophysiological test that assesses cystic fibrosis transmembrane conductance regulator (CFTR) activity and is a recognized diagnostic tool in CF. The aim of this study is to assess in the long term the role of NPD in patients whose diagnosis is questionable. METHODS: We performed follow up study on 70 patients with questionable CF (QCF) who were divided previously into two groups according to their NPD results: patients who likely have CF (QCF-CF) (n = 24), and those who likely do not have CF (QCF-non-CF) (n = 46). RESULTS: Sixty out of 70 patients were available for study. Sixteen patients in the QCF-CF group were being followed up at CF Centers as opposed to 1 in the QCF-non-CF group (P < 0.01). Seven patients from the QCF-CF group developed sinusitis during the follow up years compared to none from the QCF-non-CF group. During the years of the follow up, 17 QCF-non-CF patients were diagnosed with other medical conditions that could explain their previous symptoms. On repeated NPD measurement in the QCF-CF group, the results were similar to the original test. CONCLUSIONS: This study supports the diagnostic role of NPD measurement. Larger cohort studies are required for confirmation.