Pilot Intervention Using Food Challenges and Video Technology for Promoting Fruit and Vegetable Consumption.

Objective: To evaluate the feasibility of a Social Cognitive Theory-based intervention on cognitive, affective, and behavioral outcomes in a college nutrition course.

Design: A pre-post quasi-experimental design.

Setting: Large metropolitan university.

Opposing Discourses Surrounding Type 2 Diabetes in Eastern Appalachian Kentucky.

Type 2 diabetes disproportionately affects individuals in Appalachia. This study investigated discourse among friends and family with different diabetes risk levels in Central Appalachia. Eighteen semistructured, in-depth interviews were conducted with Appalachian Kentuckians.

Social Constructions of Stigmatizing Discourse Around Type 2 Diabetes Diagnoses in Appalachian Kentucky.

Type 2 diabetes is a growing problem among Appalachian Kentucky residents. Several issues contribute to diabetes disparities in the region, including lack of access to health care and geographic isolation. Previous studies also indicate that social stigma may be associated with type 2 diabetes.

Secreted histidyl-tRNA synthetase splice variants elaborate major epitopes for autoantibodies in inflammatory myositis.

Inflammatory and debilitating myositis and interstitial lung disease are commonly associated with autoantibodies (anti-Jo-1 antibodies) to cytoplasmic histidyl-tRNA synthetase (HisRS). Anti-Jo-1 antibodies from different disease-afflicted patients react mostly with spatially separated epitopes in the three-dimensional structure of human HisRS. We noted that two HisRS splice variants (SVs) include these spatially separated regions, but each SV lacks the HisRS catalytic domain.

Sweat chloride as a biomarker of CFTR activity: proof of concept and ivacaftor clinical trial data.

Background: We examined data from a Phase 2 trial {NCT00457821} of ivacaftor, a CFTR potentiator, in cystic fibrosis (CF) patients with aG551D mutation to evaluate standardized approaches to sweat chloride measurement and to explore the use of sweat chloride and nasal potential difference (NPD) to estimate CFTR activity.

Methods: Sweat chloride and NPD were secondary endpoints in this placebo-controlled, multicenter trial. Standardization of sweat collection, processing,and analysis was employed for the first time.

Multicenter intestinal current measurements in rectal biopsies from CF and non-CF subjects to monitor CFTR function.

Intestinal current measurements (ICM) from rectal biopsies are a sensitive means to detect cystic fibrosis transmembrane conductance regulator (CFTR) function, but have not been optimized for multicenter use. We piloted multicenter standard operating procedures (SOPs) to detect CFTR activity by ICM and examined key questions for use in clinical trials. SOPs for ICM using human rectal biopsies were developed across three centers and used to characterize ion transport from non-CF and CF subjects (two severe CFTR mutations).

Correction of the F508del-CFTR protein processing defect in vitro by the investigational drug VX-809.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene that impair the function of CFTR, an epithelial chloride channel required for proper function of the lung, pancreas, and other organs. Most patients with CF carry the F508del CFTR mutation, which causes defective CFTR protein folding and processing in the endoplasmic reticulum, resulting in minimal amounts of CFTR at the cell surface. One strategy to treat these patients is to correct the processing of F508del-CFTR with small molecules.

Results of a phase IIa study of VX-809, an investigational CFTR corrector compound, in subjects with cystic fibrosis homozygous for the F508del-CFTR mutation.

Background: VX-809, a cystic fibrosis transmembrane conductance regulator (CFTR) modulator, has been shown to increase the cell surface density of functional F508del-CFTR in vitro.

Methods: A randomised, double-blind, placebo-controlled study evaluated the safety, tolerability and pharmacodynamics of VX-809 in adult patients with cystic fibrosis (n=89) who were homozygous for the F508del-CFTR mutation. Subjects were randomised to one of four VX-809 28 day dose groups (25, 50, 100 and 200 mg) or matching placebo.

Introduction to Section I: the relevance of CF diagnostic tools for measuring restoration of CFTR function after therapeutic interventions in human clinical trials.

The pilocarpine sweat test, and in vivo assessment of CFTR function via nasal potential difference or intestinal current measurement are important tools to confirm the diagnosis of CF in subjects with suggestive symptoms. Since these tests reflect CFTR function and thus relate to the basic disease process in CF, changes in these parameters are also being used to assess the pharmacologic effect of compounds aimed at restoring CFTR function. However, longitudinal data proving that changes in these measurements are associated with meaningful clinical improvements in the course of disease in CF patients are needed.

Therapeutics development for cystic fibrosis: a successful model for a multisystem genetic disease.

Cystic fibrosis (CF) is a progressive genetic disease primarily involving the respiratory and gastrointestinal tracts. Multiple therapies directed at CF symptoms and clinical management strategies have emerged from iterative cycles of therapeutics development, helping to change the face of CF from a fatal childhood affliction to a disease in which nearly 50% of U.S.

Effect of VX-770 in persons with cystic fibrosis and the G551D-CFTR mutation.

Background: A new approach in the treatment of cystic fibrosis involves improving the function of mutant cystic fibrosis transmembrane conductance regulator (CFTR). VX-770, a CFTR potentiator, has been shown to increase the activity of wild-type and defective cell-surface CFTR in vitro.

Methods: We randomly assigned 39 adults with cystic fibrosis and at least one G551D-CFTR allele to receive oral VX-770 every 12 hours at a dose of 25, 75, or 150 mg or placebo for 14 days (in part 1 of the study) or VX-770 every 12 hours at a dose of 150 or 250 mg or placebo for 28 days (in part 2 of the study).

An international randomized multicenter comparison of nasal potential difference techniques.

Background: The transepithelial nasal potential difference (NPD) is used to assess cystic fibrosis transmembrane conductance regulator (CFTR) activity. Unreliability, excessive artifacts, and lack of standardization of current testing systems can compromise its use as a diagnostic test and outcome measure for clinical trials.

Methods: To determine whether a nonperfusing (agar gel) nasal catheter for NPD measurement is more reliable and less susceptible to artifacts than a continuously perfusing nasal catheter, we performed a multicenter, randomized, crossover trial comparing a standardized NPD protocol using an agar nasal catheter with the same protocol using a continuously perfusing catheter.

Rescue of CF airway epithelial cell function in vitro by a CFTR potentiator, VX-770.

Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR), a protein kinase A (PKA)-activated epithelial anion channel involved in salt and fluid transport in multiple organs, including the lung. Most CF mutations either reduce the number of CFTR channels at the cell surface (e.g.

A pipeline of therapies for cystic fibrosis.

Therapeutics development for cystic fibrosis (CF) involves a coordinated effort among many groups, including individuals with CF and their caregivers, clinical research teams, and those in academia and industry who have discovered and developed the therapeutic strategies. In the United States, the Cystic Fibrosis Foundation (CFF) has devoted over $875 million to facilitate and coordinate this process since 1986, resulting in the clinical development and/or assessment of ~50 drug candidates during that time. The more than 30 compounds currently in the pipeline of Foundation-funded therapeutics are used as a platform to discuss why and how therapeutic strategies are brought into clinical development.

Cystic fibrosis foundation: achieving the mission.

The Cystic Fibrosis Foundation is a voluntary, nonprofit, health organization whose mission is "to assure the development of the means to cure and control cystic fibrosis and to improve the quality of life for those with the disease." While substantial progress has been made, as evidenced by a marked increase in the median predicted age of survival, much work remains to be done. Ongoing medical programs and activities of the Cystic Fibrosis Foundation, which span basic science, drug discovery, drug development, clinical care, patient education, and advocacy, will be described in this article.

cis-Acting elements within CFTR 5'-flanking DNA are not sufficient to decrease gene expression in response to phorbol ester.

The cystic fibrosis transmembrane conductance regulator gene (CFTR) is regulated in a tissue-specific and developmental fashion. Although it has been known for some time that phorbol esters decrease CFTR expression in cell lines that have high CFTR mRNA levels, the cis-acting elements that control this down-regulation remain ill-defined. The role of cis-acting elements within the CFTR minimal promoter in modulating responses to phorbol 12-myristate 13-acetate (PMA) and forskolin was assessed using luciferase reporter gene (luc)-containing plasmids transfected into Calu-3 and HT-29 cells.

Positional cloning utilizing genomic DNA microarrays: the Niemann-Pick type C gene as a model system.

A major obstacle in positional cloning is identifying the specific mutated gene from within a large physical contig. Here we describe the application of DNA microarray technology to a defined genomic region (physical map) to identify: (i) exons without a priori sequence data and (ii) the disease gene based on differential gene expression in a recessive disorder. The feasibility was tested using resources from the positional cloning of the Neimann-Pick Type C (NP-C) disease gene, NPC1.

CFTR intron 1 increases luciferase expression driven by CFTR 5'-flanking DNA in a yeast artificial chromosome.

The DNA elements that account for the highly regulated expression of the cystic fibrosis transmembrane conductance regulator gene (CFTR) are poorly understood. The goal of this study was to assess the feasibility of using a yeast artificial chromosome (YAC)-based reporter gene construct to define these elements further. An approximately 350-kb YAC (y5'luc) was constructed by replacing CFTR with a luciferase reporter gene (luc).

Design of modified oligodeoxyribonucleotide probes to detect telomere repeat sequences in FISH assays.

A series of dye-labeled oligonucleotide probes containing base and sugar modifications were tested for the ability to detect telomeric repeat sequences in FISH assays. These modified oligonucleotides, all 18 nt in length, were complementary to either the cytidine-rich (C(3)TA(2))(n)or guanosine-rich (T(2)AG(3))(n)telomere target sequences. Oligonucleotides were modified to either increase target affinity by enhancing duplex stability [2'-OMe ribose sugars and 5-(1-propynyl)pyrimidine residues] or inhibit the formation of inter- or intramolecular structures (7-deazaguanosine and 6-thioguanosine residues), which might interfere with binding to the target.

Fluorescent chloride indicators to assess the efficacy of CFTR cDNA delivery.

Cl(-)-sensitive fluorescent indicators have been used extensively in cell culture systems to measure the Cl(-)-transporting function of the cystic fibrosis transmembrane conductance regulator protein CFTR. These indicators have been used in establishing a surrogate end point to assess the efficacy of CFTR cDNA delivery in human gene therapy trials. The ability to measure Cl- transport with high sensitivity in small and heterogeneous tissue samples makes the use of Cl- indicators potentially attractive in gene delivery studies.

An improved method for routine preparation of intact artificial chromosome DNA (340-1000 kb) for transfection into human cells.

The transfer of high molecular weight (HMW) DNA into mammalian cells is an important strategy for assessing human gene expression and chromosome structure and function. However, using current methods, it is difficult to dependably prepare intact HMW DNA because of the susceptibility of the DNA to degradation and physical shearing. Here we describe a strategy whereby intact artificial chromosome DNA (as large as 1 Mb) can be routinely prepared from yeast.

Isolation of human transcripts expressed in hamster cells from YACs by cDNA representational difference analysis.

Gene isolation methods used during positional cloning rely on physical contigs consisting of bacterial artificial chromosomes, P1, or cosmid clones. However, in most instances, the initial framework for physical mapping consists of contigs of yeast artificial chromosome (YACs), large vectors that are suboptimal substrates for gene isolation. Here we report a strategy to identify gene sequences contained within a YAC by using cDNA representational difference analysis (RDA) to directly isolate transcripts expressed from the YAC in mammalian cells.

Human artificial chromosomes generated by modification of a yeast artificial chromosome containing both human alpha satellite and single-copy DNA sequences.

A human artificial chromosome (HAC) vector was constructed from a 1-Mb yeast artificial chromosome (YAC) that was selected based on its size from among several YACs identified by screening a randomly chosen subset of the Centre d'Etude du Polymorphisme Humain (CEPH) (Paris) YAC library with a degenerate alpha satellite probe. This YAC, which also included non-alpha satellite DNA, was modified to contain human telomeric DNA and a putative origin of replication from the human beta-globin locus. The resultant HAC vector was introduced into human cells by lipid-mediated DNA transfection, and HACs were identified that bound the active kinetochore protein CENP-E and were mitotically stable in the absence of selection for at least 100 generations.

Humanizing the yeast telomerase template.

Saccharomyces cerevisiae contains an irregular telomere sequence (TG1-3)n, which differs from the regular repeat (TTAGGG)n found at the telomeres of higher organisms including humans. We have modified the entire 16-nt template region of the S. cerevisiae telomerase RNA gene (TLC1) to produce (TTAGGG)n repeats, the human telomere sequence.

Transient gene expression from yeast artificial chromosome DNA in mammalian cells is enhanced by adenovirus.

The introduction of high molecular weight DNA into mammalian cells is useful for gene expression studies. However, current transfection strategies are inefficient, necessitating propagation of stable DNA transformants prior to analysis of gene expression. Here we demonstrate that transient lipid-mediated DNA transfection can be used to assess gene expression from yeast artificial chromosomes (YACs) containing the 230 kb cystic fibrosis transmembrane conductance regulator gene ( CFTR ) and Escherichia coli lacZ .