Endocrine parameters of cystic fibrosis: Back to basics

Dramatic changes in the life expectancy of cystic fibrosis (CF) patients are occurring, creating a cohort of aging individuals experiencing long‐term complications of this chronic disease. The two most common of these complications include CF‐related diabetes and CF bone disease. The clinical implications of each have become better understood, as have potential therapies. However, data obtained from the basic science studies of both diseases have not been widely recognized. In this review, we focus on the known and hypothesized pathogenesis of these two disorders. Additionally, the molecular underpinnings of CF will be explained along with the potential interactions with endocrine disease phenotypes. J. Cell. Biochem. 108: 353–361, 2009. © 2009 Wiley‐Liss, Inc.     

CFTR haplotypes in northern Iranian population

Background

Cystic fibrosis (CF) is a multiorganic autosomal recessive disorder, caused by mutation in cystic fibrosis transmembrane conductance regulator (CFTR). CF is highly heterogeneous in Iranian population and molecular diagnosis based on direct identification of mutations is not completely efficient. The use of polymorphic intragenic markers not only can facilitate phenotype prediction in prenatal diagnosis by gene tracking, but also can lead to the demonstration of possible associations between haplotypes and specific mutations.

Methods

60 CF patients and 53 fertile normal subjects originating from North of Iran were analyzed for F508del mutation and c.1210-12T(5_9), c.1408A>G and c.744-33GATT(6_8) polymorphisms.

Results

c.1210-12T[7] is the most prevalent allele in normal individuals and CF non-F508del patients with 87.7%and 86.7% frequencies respectively. c.1408A>G survey showed that frequency of allele G and A is nearly equal in both non-F508del CF patients and normal individuals. c.744-33GATT(6_8) study showed that 7 repeat is the most prevalent allele in normal individuals and non-F508del CF patients with 80.2% and 82.1% frequencies respectively. The [c.1408A; c.1210-12T[9]; c.744-33GATT[6]] haplotype was only associated with mutant alleles including F508del.

Conclusions

The allelic distribution and heterozygosity results suggest that c.1408A>G, c.1210-12T(5_9) and c.744-33GATT(6_8) can contribute to carrier detection and prenatal diagnosis of CF in Iranian families with previous history of the disease.     

CFTR型氯离子通道研究进展

囊性纤维化跨膜传导调节因子（CFTR）是一种重要的氯离子通道,突变易引起囊性纤维化病变,故得名。一系列研究表明,CFTR由5个结构域组成：两个跨膜结构域形成氯离子通道;两个核苷酸结合结构域调节通道的开闭;一个调节结构域主要影响氯通道的活动。这些结构域通过协同作用共同控制了氯离子的跨膜流动,而一些突变可以影响细胞功能而导致囊性纤维化的发生。本文通过介绍CFTR基本结构、调节机制、与囊性纤维化病变的关系及针对CFTR的治疗而对CFTR型氯离子通道有一个的全面的理解。     

黏液性/浆液性胰腺囊性肿瘤囊液蛋白质糖基化差异表达研究

随着影像学技术的进步,胰腺囊性病变(恶性胰腺囊性病变包括黏液性囊腺瘤(mucinous cystic neoplasm,MCN)和导管内乳头状黏液瘤(intraductal papillary mucinous neoplasm,IPMN)以及黏液性囊腺癌(mucinous cystic adenocarcinoma,MCA)的检出率有所提高,但是区分囊性病变的良恶性仍然是一个难题.本研究根据外科手术病理结果及囊液液基细胞结果,从120例经CT或MRI方法诊断为胰腺囊性肿瘤患者中选取35例样本,其中胰腺黏液性囊腺瘤(MCN)组17例与浆液性囊腺瘤(serous cystic neoplasm,SCN)组18例,通过超声内镜下细针穿刺(endoscopic ultrasonography-guided fine needle aspiration,EUS-FNA)方法吸取囊液,采用凝集素芯片分析蛋白质糖链谱差异.经t检验结果表明,MCN组与SCN组相比,6种凝集素,STL、WGA、BPL、DBA、PTL-Ⅰ及MAL-Ⅰ,识别的糖链结构二者之间存在明显差异(P0.05),其中凝集素BPL、DBA、WGA、STL识别的糖链结构在MCN囊液中呈高表达(R2.0),例如DBA特异识别的Tn抗原表达的增多,可能与肿瘤上皮细胞分泌的黏蛋白增多密切相关.而凝集素PTL-Ⅰ、MAL-Ⅰ特异识别Galβ-1、4Glc NAc结构以及Gal NAcα-1、3Gal结构在MCN囊液中表达降低(R0.5).通过凝集素印记法检测了STL和BPL分别于MCN、SCN囊液中蛋白的结合情况,结果表明STL和BPL与囊液中的糖蛋白结合明显强于SCN组.本文通过比较MCN和SCN糖蛋白糖谱表达差异,寻找胰腺囊性肿瘤诊断和肿瘤良恶性评估的新方法,同时为探索胰腺囊性肿瘤发生发展机制和治疗的潜在靶点奠定基础.     

Traffic pattern of cystic fibrosis transmembrane regulator through the early exocytic pathway

The pathway of transport of the cystic fibrosis transmembrane regulator (CFTR) through the early exocytic pathway has not been examined. In contrast to most membrane proteins that are concentrated during export from the ER and therefore readily detectable at elevated levels in pre-Golgi intermediates and Golgi compartments, wild-type CFTR could not be detected in these compartments using deconvolution immunofluorescence microscopy. To determine the basis for this unusual feature, we analyzed CFTR localization using quantitative immunoelectron microscopy (IEM). We found that wild-type CFTR is present in pre-Golgi compartments and peripheral tubular elements associated with the cis and trans faces of the Golgi stack, albeit at a concentration 2-fold lower than that found in the endoplasmic reticulum (ER). delta F508 CFTR, a mutant form that is not efficiently delivered to the cell surface and the most common mutation in cystic fibrosis, could also be detected at a reduced concentration in pre-Golgi intermediates and peripheral cis Golgi elements, but not in post-Golgi compartments. Our results suggest that the low level of wild-type CFTR in the Golgi region reflects a limiting step in selective recruitment by the ER export machinery, an event that is largely deficient in delta F508. We raise the possibility that novel modes of selective anterograde and retrograde traffic between the ER and the Golgi may serve to regulate CFTR function in the early secretory compartments.     

Membrane function in cystic fibrosis. I. Putrescine transport in normal and cystic fibrosis fibroblasts

Putrescine transport was examined in normal and cystic fibrosis fibroblasts. No differences were observed in accumulation pattern, kinetics of uptake, or efflux between CF and normal cells. In both growing and growth-arrested CF and normal fibroblasts, exogenously supplied putrescine remained unchanged for at least 60 min. Some differences were observed in the response of CF and normal cells to environmental (media) changes.This research was supported by a grant from the Cystic Fibrosis Foundation and by a grant from the National Institutes of Health, Training Grant (GM01316 11 GNC).     

Bicarbonate and chloride secretion in Calu-3 human airway epithelial cells

Serous cells are the predominant site of cystic fibrosis transmembrane conductance regulator expression in the airways, and they make a significant contribution to the volume, composition, and consistency of the submucosal gland secretions. We have employed the human airway serous cell line Calu-3 as a model system to investigate the mechanisms of serous cell anion secretion. Forskolin-stimulated Calu-3 cells secrete HCO-3 by a Cl-offdependent, serosal Na+-dependent, serosal bumetanide-insensitive, and serosal 4,4'-dinitrostilben-2,2'-disulfonic acid (DNDS)-sensitive, electrogenic mechanism as judged by transepithelial currents, isotopic fluxes, and the results of ion substitution, pharmacology, and pH studies. Similar studies revealed that stimulation of Calu-3 cells with 1-ethyl-2-benzimidazolinone (1-EBIO), an activator of basolateral membrane Ca2+-activated K+ channels, reduced HCO-3 secretion and caused the secretion of Cl- by a bumetanide-sensitive, electrogenic mechanism. Nystatin permeabilization of Calu-3 monolayers demonstrated 1-EBIO activated a charybdotoxin- and clotrimazole- inhibited basolateral membrane K+ current. Patch-clamp studies confirmed the presence of an intermediate conductance inwardly rectified K+ channel with this pharmacological profile. We propose that hyperpolarization of the basolateral membrane voltage elicits a switch from HCO-3 secretion to Cl- secretion because the uptake of HCO-3 across the basolateral membrane is mediated by a 4,4 '-dinitrostilben-2,2'-disulfonic acid (DNDS)-sensitive Na+:HCO-3 cotransporter. Since the stoichiometry reported for Na+:HCO-3 cotransport is 1:2 or 1:3, hyperpolarization of the basolateral membrane potential by 1-EBIO would inhibit HCO-3 entry and favor the secretion of Cl-. Therefore, differential regulation of the basolateral membrane K+ conductance by secretory agonists could provide a means of stimulating HCO-3 and Cl- secretion. In this context, cystic fibrosis transmembrane conductance regulator could serve as both a HCO-3 and a Cl- channel, mediating the apical membrane exit of either anion depending on basolateral membrane anion entry mechanisms and the driving forces that prevail. If these results with Calu-3 cells accurately reflect the transport properties of native submucosal gland serous cells, then HCO-3 secretion in the human airways warrants greater attention.     

Ultrastructural features of spermatozoa and their phylogenetic application in Zaprionus (Diptera,Drosophilidae)

The genus Zaprionus consists of approximately 60 species of drosophilids that are native to the Afrotropical region. The phylogenetic position of Zaprionus within the Drosophilidae family is still unresolved. In the present study, ultrastructural features of spermatozoa of 6 species of Zaprionus as well as the species Drosophila willistoni and Scaptodrosophila latifasciaeformis were analyzed. The ultrastructure revealed that the species have the same flagellar ultrastructure. Two mitochondrial derivatives, one larger than the other, close to the axoneme were present, primarily in D. willistoni (subgenus Sophophora). Except for Z. davidi and Z. tuberculatus, the analyzed species had paracrystalline material in both mitochondrial derivatives. Moreover, the testes showed 64 spermatozoa per bundle in all of the species. In the cluster analysis, 6 Zaprionus species were grouped closely, but there were some incongruent positions in the cladogram. The results indicated that sperm ultrastructure is an important tool for elucidating the phylogeny and taxonomy of insects.     

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR): CLOSED AND OPEN STATE CHANNEL MODELS*

The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily. CFTR controls the flow of anions through the apical membrane of epithelia. Dysfunctional CFTR causes the common lethal genetic disease cystic fibrosis. Transitions between open and closed states of CFTR are regulated by ATP binding and hydrolysis on the cytosolic nucleotide binding domains, which are coupled with the transmembrane (TM) domains forming the pathway for anion permeation. Lack of structural data hampers a global understanding of CFTR and thus the development of “rational” approaches directly targeting defective CFTR. In this work, we explored possible conformational states of the CFTR gating cycle by means of homology modeling. As templates, we used structures of homologous ABC transporters, namely TM(287–288), ABC-B10, McjD, and Sav1866. In the light of published experimental results, structural analysis of the transmembrane cavity suggests that the TM(287–288)-based CFTR model could correspond to a commonly occupied closed state, whereas the McjD-based model could represent an open state. The models capture the important role played by Phe-337 as a filter/gating residue and provide structural information on the conformational transition from closed to open channel.     

Glycosylation of VSV glycoprotein is similar in cystic fibrosis,heterozygous carrier,and normal human fibroblasts

The single envelope glycoprotein of vesicular stomatitis virus was used as a specific probe of glycosyltransferase activities in fibroblasts from two cystic fibrosis patients, an obligate heterozygous carrier and a normal individual. Gel filtration of pronasedigested glycopeptides from both purified virions and infected cell-associated VSV glycoprotein which had been labeled with [³H] glucosamine did not reveal any significant differences in the glycosylation patterns between the different cell cultures. All 4 cell lines were apparently able to synthesize the mannose- and glucosamine-containing core structure and branch chains terminating in sialic acid which are characteristic of asparagine-linked carbohydrate side chains in cellular glycoproteins. Analysis of tryptic glycopeptides by anion-exchange chromotography indicated that the same 2 major sites on the virus polypeptide were recognized and glycosylated in all 4 VSV-infected cell cultures. These studies suggest that the basic biochemical defect(s) in cystic fibrosis is not an absence or deficiency in enzymes responsible for the biosynthesis of complex carbohydrate side chains.     

American Association of Clinical Endocrinology Consensus Statement: Comprehensive Type 2 Diabetes Management Algorithm – 2023 Update

ObjectiveThis consensus statement provides (1) visual guidance in concise graphic algorithms to assist with clinical decision-making of health care professionals in the management of persons with type 2 diabetes mellitus to improve patient care and (2) a summary of details to support the visual guidance found in each algorithm.MethodsThe American Association of Clinical Endocrinology (AACE) selected a task force of medical experts who updated the 2020 AACE Comprehensive Type 2 Diabetes Management Algorithm based on the 2022 AACE Clinical Practice Guideline: Developing a Diabetes Mellitus Comprehensive Care Plan and consensus of task force authors.ResultsThis algorithm for management of persons with type 2 diabetes includes 11 distinct sections: (1) Principles for the Management of Type 2 Diabetes; (2) Complications-Centric Model for the Care of Persons with Overweight/Obesity; (3) Prediabetes Algorithm; (4) Atherosclerotic Cardiovascular Disease Risk Reduction Algorithm: Dyslipidemia; (5) Atherosclerotic Cardiovascular Disease Risk Reduction Algorithm: Hypertension; (6) Complications-Centric Algorithm for Glycemic Control; (7) Glucose-Centric Algorithm for Glycemic Control; (8) Algorithm for Adding/Intensifying Insulin; (9) Profiles of Antihyperglycemic Medications; (10) Profiles of Weight-Loss Medications (new); and (11) Vaccine Recommendations for Persons with Diabetes Mellitus (new), which summarizes recommendations from the Advisory Committee on Immunization Practices of the U.S. Centers for Disease Control and Prevention.ConclusionsAligning with the 2022 AACE diabetes guideline update, this 2023 diabetes algorithm update emphasizes lifestyle modification and treatment of overweight/obesity as key pillars in the management of prediabetes and diabetes mellitus and highlights the importance of appropriate management of atherosclerotic risk factors of dyslipidemia and hypertension. One notable new theme is an emphasis on a complication-centric approach, beyond glucose levels, to frame decisions regarding first-line pharmacologic choices for the treatment of persons with diabetes. The algorithm also includes access/cost of medications as factors related to health equity to consider in clinical decision-making.     

Extracellular zinc and ATP-gated P2X receptor calcium entry channels: New zinc receptors as physiological sensors and therapeutic targets

In this review, we focus on two attributes of P2X receptor channel function, one essential and one novel. First, we propose that P2X receptors are extracellular sensors as well as receptors and ion channels. In particular, the large extracellular domain (that comprises 70% of the molecular mass of the receptor channel protein) lends itself to be a cellular sensor. Moreover, its exquisite sensitivity to extracellular pH, ionic strength, and multiple ligands evokes the function of a sensor. Second, we propose that P2X receptors are extracellular zinc receptors as well as receptors for nucleotides. We provide novel data in multiple publications and illustrative data in this invited review to suggest that zinc triggers ATP-independent activation of P2X receptor channel function. In this light, P2X receptors are the cellular site of integration between autocrine and paracrine zinc signaling and autocrine and paracrine purinergic signaling. P2X receptors may sense changes in these ligands as well as in extracellular pH and ionic strength and transduce these sensations via calcium and/or sodium entry and changes in membrane potential.     

Calculating expected lung deposition of aerosolized administration of AAV vector in human clinical studies

BACKGROUND: Cystic fibrosis is an autosomal recessive disease affecting approximately 1 in 2500 live births. Introducing the cDNA that codes for normal cystic fibrosis transmembrane conductance regulator (CFTR) to the small airways of the lung could result in restoring the CFTR function. A number of vectors for lung gene therapy have been tried and adeno-associated virus (AAV) vectors offer promise. The vector is delivered to the lung using a breath-actuated jet nebulizer. The purpose of this project was to determine the aerosolized AAV (tgAAVCF) particle size distribution (PSD) in order to calculate target doses for lung delivery. METHODS: A tgAAVCF solution was nebulized using the Pari LC Plus (n = 3), and the PSD was determined by coupling laser diffraction and inertial impaction (NGI) techniques. The NGI allowed for quantification of the tgAAVCF at each stage of impaction, ensuring that rAAV-CFTR vector is present and not empty particles. Applying the results to mathematical algorithms allowed for the calculation of expected pulmonary deposition. RESULTS: The mass median diameter (MMD) for the tgAAVCF was 2.78 +/- 0.43 microm. If the system works ideally and the patient only receives aerosol on inspiration, the patient would receive 47 +/- 0% of the initial dose placed in the nebulizer, with 72 +/- 0.73% of this being deposited beyond the vocal cords. CONCLUSIONS: This technology for categorizing the pulmonary delivery system for lung gene therapy vectors can be adapted for advanced aerosol delivery systems or other vectors.     

Binding screen for cystic fibrosis transmembrane conductance regulator correctors finds new chemical matter and yields insights into cystic fibrosis therapeutic strategy

The most common mutation in cystic fibrosis (CF) patients is deletion of F508 (ΔF508) in the first nucleotide binding domain (NBD1) of the CF transmembrane conductance regulator (CFTR). ΔF508 causes a decrease in the trafficking of CFTR to the cell surface and reduces the thermal stability of isolated NBD1; it is well established that both of these effects can be rescued by additional revertant mutations in NBD1. The current paradigm in CF small molecule drug discovery is that, like revertant mutations, a path may exist to ΔF508 CFTR correction through a small molecule chaperone binding to NBD1. We, therefore, set out to find small molecule binders of NBD1 and test whether it is possible to develop these molecules into potent binders that increase CFTR trafficking in CF‐patient‐derived human bronchial epithelial cells. Several fragments were identified that bind NBD1 at either the CFFT‐001 site or the BIA site. However, repeated attempts to improve the affinity of these fragments resulted in only modest gains. Although these results cannot prove that there is no possibility of finding a high‐affinity small molecule binder of NBD1, they are discouraging and lead us to hypothesize that the nature of these two binding sites, and isolated NBD1 itself, may not contain the features needed to build high‐affinity interactions. Future work in this area may, therefore, require constructs including other domains of CFTR in addition to NBD1, if high‐affinity small molecule binding is to be achieved.     

A Joint Location-Scale Test Improves Power to Detect Associated SNPs,Gene Sets,and Pathways

Gene-based, pathway, and other multivariate association methods are motivated by the possibility of GxG and GxE interactions; however, accounting for such interactions is limited by the challenges associated with adequate modeling information. Here we propose an easy-to-implement joint location-scale (JLS) association testing framework for single-variant and multivariate analysis that accounts for interactions without explicitly modeling them. We apply the JLS method to a gene-set analysis of cystic fibrosis (CF) lung disease, which is influenced by multiple environmental and genetic factors. We identify and replicate an association between the constituents of the apical plasma membrane and CF lung disease (p = 0.0099 and p = 0.0180, respectively) and highlight a role for the SLC9A3-SLC9A3R1/2-EZR complex in contributing to CF lung disease. Many association studies could benefit from re-analysis with the JLS method that leverages complex genetic architecture for SNP, gene, and pathway identification. Analytical verification, simulation, and additional proof-of-principle applications support our approach.     

The effects of proteins secreted by fibroblasts from patients with cystic fibrosis on hamster tracheal explants

Summary Hamster tracheal explants have been used to assay for mucosecretory activity in media taken from cultures of fibroblasts isolated from patients with cystic fibrosis (CF). Cystic fibrosis and normal sera were first used to establish optimal conditions for mucus release in the hamster tracheal ring assay. Unless protein levels were maintained at 5% serum concentration or greater there was loss of cilia, nonspecific mucus accumulation, and extensive epithelial damage to the luminal surface. Likewise, it was shown that exposure of the explants to unconcentrated conditioned media from CF (GM 770, 768, 1348, 142) or normal (GM 3349, 38) cultured fibroblasts for 1, 6, or 12 h resulted in the same type of damage and this was due to low protein levels. When the protein concentration of the conditioned media was increased with fetal bovine serum, the morphological integrity of the explants was maintained, demonstrating that there was no apparent difference between CF and normal fibroblast-secreted proteins in ability to induce mucus release. The ciliary inhibitory capacity of CF serum-derived or fibroblast-derived factor had been reported to require IgG for activity. However, addition of IgG to high molecular weight (VoP10) or low molecular weight (VeP10) secreted proteins had no apparent effect on stimulating secretion. In conclusion, it is possible that CF fibroblasts do not secrete a protein that has the mucostimulatory effect and thus these cells may not be suitable for studying the CF-related activity.     

Spatial distribution of microbial communities in the cystic fibrosis lung

Cystic fibrosis (CF) is a common fatal genetic disorder with mortality most often resulting from microbial infections of the lungs. Culture-independent studies of CF-associated microbial communities have indicated that microbial diversity in the CF airways is much higher than suggested by culturing alone. However, these studies have relied on indirect methods to sample the CF lung such as expectorated sputum and bronchoalveolar lavage (BAL). Here, we characterize the diversity of microbial communities in tissue sections from anatomically distinct regions of the CF lung using barcoded 16S amplicon pyrosequencing. Microbial communities differed significantly between different areas of the lungs, and few taxa were common to microbial communities in all anatomical regions surveyed. Our results indicate that CF lung infections are not only polymicrobial, but also spatially heterogeneous suggesting that treatment regimes tailored to dominant populations in sputum or BAL samples may be ineffective against infections in some areas of the lung.     

Plasma chromium concentrations in normal infants and cystic fibrosis patients

Plasma Cr concentrations have been studied in normal children aged 0–14 yr. Levels ranged from 0.65 to 0.88 μg/1 and did not change with age. Plasma concentrations of CF patients given 0.5–0.75 μg Cr/kg/d in addition to their diet were similar to normal values. There was no correlation between these plasma values and growth retardation.