Cystic Fibrosis Cell Bio

I. Introduction

Cystic fibrosis (CF) is the most lethal and common inherited disorder for Caucasians in the United States, particularly of Northern or Central European descent.[1,2] Although the incidence of CF is highest in Caucasians, all races are affected by this disease.[3] There are approximately 70,000 individuals with CF worldwide with about 30,000 living in the United States.[4] Cystic fibrosis is an autosomal recessive disorder in which both parents must be carriers of the gene to pass it on to their child.[5] Approximately 1 in 29 Caucasian Americans carry the CF gene.[2] Most cases are diagnosed by the age of 2 with approximately 1,000 new cases in the United States each year. In the past, the life expectancy for people with CF was very short. Most children did not live to start elementary school.[4] However, due to advances in medicine, people with CF can live into their 30s or 40s and sometimes beyond with approximately 45% of the population with CF being older than 18.[4] Figure 1 illustrates the inheritance process by which a child acquires CF.

Figure 1. Inheritance in CF
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Cystic_Fibrosis-1.gif

Cystic fibrosis affects multiple organ systems in the body with the pulmonary and digestive systems being primarily affected.[2],[4] People with CF have production of unusually thick secretions of mucus that can obstruct the lungs and pancreas.[2],[4] Common symptoms include persistent cough with phlegm, frequent infections of the lungs, wheezing, shortness of breath, salty tasting skin, poor weight gain despite proper diet, and greasy/bulky clay colored stools.[2],[4] The primary cause of morbidity and mortality in people with CF is lung disease from altered pulmonary system functioning.[5]

Cystic fibrosis is primarily diagnosed by a sweat test that analyzes the amount of chloride produced in the child’s sweat.[2],[5] Chloride levels higher than 60 mEq/L indicate CF.[5] Genetic testing is also possible to diagnose CF.[5] However, only 70-75% of gene mutations responsible for CF have been discovered.[5] Therefore, genetic testing is unable to diagnose every case of CF.[5]

The key mechanism through which CF affects the body is abnormal ion transport across epithelial tissues.[6] It is well established that the main epithelial transport problem in CF is a result of a defect in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.[6],[7],[8] In CF, this CFTR gene is mutated leading to disease sequelae affecting many cellular mechanisms. These cellular mechanisms, along with the body systems affected and medical management of CF will be discussed in the sections to follow.

II. Airway Surface Liquid

Figure 5. ASL http://pats.atsjournals.org/content/1/1/2/F3.large.jpg[12]

F3.large.jpg

As stated in the introduction, lung disease from altered pulmonary function is the main cause of morbidity and mortality in patients with CF.[5] Therefore, much research has been performed to determine the pathogenesis of CF that leads to lung disease. With poor ion regulation in epithelial cells being a key component in CF,[6] lack of proper regulation of airway surface liquid (ASL) has been hypothesized to be a major component in CF pathogenesis.[40] ASL covers the lumen in the lungs, specifically the bronchial pathways, and it is important in the immune response as it is responsible for facilitating the clearance of mucus.[13] It is composed of two layers: the periciliary layer (PCL) and the superficial mucus layer.[13] The PCL lies between the mucus layer and the respiratory epithelial cells and has low viscosity, while the mucus layer is much thicker secondary to the presence of mucins that allow for the formation of gel.[13] The optimal height for the PCL is 7 µm, which allows cilia to beat under a low viscosity to clear bacteria and other unwanted particles.[13] Therefore, maintaining the medium of the PCL is very important to allow appropriate mucus clearance by the cilia.[13] The cellular mechanisms of the regulation of ASL and the implications in CF will be described after each component of normal ion regulation the lung has been discussed.

III. Transepithelial Potential Difference

The transepithelial potential difference (TPD) is the balance of charges between the lumen in the lungs/nasal pathways and the interstitium.[11] The Na+/K+-ATPase pump is located on the basolateral membrane of epithelial cells and pumps out 3 Na+ for each 2 K+ absorbed.[13],[14] Therefore, this pump is responsible for creating the electrochemical gradient that is necessary for the passive absorption of Na+ into the cell through ENaC.[13],[14] This passive reabsorption of Na+ generates a negative electrical potential that decreases the force that promotes Cl- secretion.[13],[15]

In healthy individuals, the Na+ is reabsorbed into the cell and Cl- is secreted from the cell to balance the charges and maintain adequate hydration of the ASL.[11] In patients with CF, too much Na+ is reabsorbed into the cell which creates an ionic gradient as Cl- ions cannot be secreted secondary to dysfunctioning CFTR.[11] This increase in Na+ reabsorption into the cell and decrease in Cl- secretion out of the cell causes an increase (more negative) in the resting TPD in patients with CF when compared to normal individuals.[13]

IV. Cellular Mechanisms

A. Normal Ion Regulation in the Lung Epithelial Cells

Because abnormal ion transport across epithelial cells is a hallmark symptom of CF[6], it is imperative to first understand the ion regulation in healthy epithelial cells before investigating the disease process of CF. In normal ion regulation in the lungs, an outside stimulus such as a catecholamine (Epinephrine or Norepinephrine) stimulates the β-adrenergic receptor (ADBR2). This binding allows the release of a G subunit, activating adenylate cyclase.[9] Adenylate cyclase helps convert adenine triphosphate (ATP) to cAMP, and this cAMP activates protein kinase A (PKA). [9] PKA then activates the regulatory (R) domain of CFTR (which allows for the efflux of Cl-) and the sodium epithelium channel (ENaC) (which mediates the reabsorption of Na+).[9] PKA also influences the Na+/K+ ATPase pump, causing Na+ efflux and K+ influx.[9] Refer to [Figure 3].

In addition to regulation by CFTR, Cl- efflux is controlled by Ca2+ activated chloride channels (CaCC).[9] The activation of these channels can be traced back to ATP being released from the airway epithelial cells with an increase in ventilation rate.[9] ATP and its metabolite adenosine (ADO) stimulate purinergic (P2Y2) receptors.[9] P2Y2 then splits phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol triphosphate (IP3) and diacylglycerol (DAG), which facilitates the release of Ca2+ stores that activate CaCC.[10] P2Y2 also helps to mediate ENaC activity by either activating it or inhibiting based on the amount of PIP2.[9] Refer to [Figure 3].

In summary, stimulation of ADBR2 by catecholamines and P2Y2 by ATP cause a cascade of events that ultimately activates several channels and receptors that are responsible for regulating the Na+, Cl-, and K+ coming in and out of the cell. The next few sections will describe the proteins and channels that participate in ion regulation in more depth.

1. Important Components in Ion Regulation and Cystic Fibrosis

The following sections will describe the important channels, proteins and other components involved in normal ion regulation in epithelial cells. Additionally, the differences observed in CF will be discussed. The section ends with [Figure 3], which provides a visual of the role of these channels and proteins in normal ion regulation in epithelial cells.

i. Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

Based on the above explanation of normal ion regulation, one can conclude that CFTR plays a vital role in regulating ions. CFTR is an ATP-gated, chloride (Cl-) ion channel that is regulated by a cAMP/PKA-dependent protein.[16]”-Chromosome 7 contains the gene that is responsible for making the protein CFTR. Patients with CF have two copies of this mutated gene, which has a detrimental effect on the gene’s function.[17] There are 1700 known mutations that affect CFTR with about 75% of CF alleles contain the ΔF508 mutation.[16] This mutation causes a missing phenylalanine at position 508 in the CFTR protein, and the mutated CFTR is usually destroyed by the proteasome after it fails to be transported to the correct location in the cell.[16] There is a small amount of CFTR that arrives at the correct location in the cell, but this protein functions poorly.[16] The function of CFTR is very important as it is responsible for transporting Cl- into and out of cell membranes that regulate mucus, sweat, saliva, tears, and digestive enzymes. These Cl- ions help control the movement of water, which is imperative for fluid mucus production. This protein is also important in regulating the transport of Na+ through channels including ENaC, which will be described in the section below.[17] [Figure 2] demonstrates the effect of CFTR dysfunction on CL- and Na+ regulation.

ii. ENaC

Sodium epithelium channel (ENaC) is a protein in the apical membrane of epithelial cells in the kidney, airway, colon, and ducts of some glands that is composed of three different subunits (alpha, beta, and gamma).[18],[19],[20] It is regulated by CFTR. For example, ENaC is inhibited with CFTR activation in the airways and activated with CFTR activation in the sweat glands.[21],[22],[23],[24],[25],[26] As shown in [Figure 3], ENaC participates in ion regulation by providing an entry channel for Na+ into the epithelial cells. The Na+ is then actively transported into the extracellular fluid through the Na+-K+-ATPase located in the basolateral membrane.[18],[19],[20] Therefore, ENaC plays an integral role in the transport of Na+ in and out of epithelial cells.

Figure 2. CFTR and ENaC Regulation in Sweat Gland and Airway Cells

Normal CFTR/ENaC Regulation in Sweat Glands CFTR/ENaC Dysfunction in Sweat Glands
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In the sweat glands of normal tissue, ENaC is activated with CFTR to maintain balance between electrical charges of both Na+ and Cl- with CFTR allowing Cl- into the cell (opposite of airway cells) and ENaC promoting Na+ into the cell.[27],[28] In a patient with CF, the defective CFTR does not allow for adequate Cl- reabsorption in the sweat gland duct cells.[27],[28] Therefore, Na+ reabsorption via ENaC is inhibited. As a result, the Na+ and Cl- are not reabsorbed into the duct cells and are instead transported out to the skin, causing salty skin.[27],[28]
Normal CFTR/ENaC Regulation in Airways CFTR/ENaC Dysfunction in Airways
http://www.cftrscience.com/img/understanding_cftr/normal_cftr_function_airway.jpg normal_cftr_function_airway.jpg http://www.cftrscience.com/img/understanding_cftr/cftr_dysfunction_airway.jpgcftr_dysfunction_airway.jpg
In the lung tissue of normal individuals, the amount and osmolality of airway surface liquid (ASL) is maintained by ENaC and CFTR. Airway surface liquid is maintained for pulmonary defense as it keeps the lungs moist.[9] Having an appropriate ASL depth of about 7 μm, allows for effective ciliary beating to clear mucus which can limit gas transfer.[9] ENaC moves Na+ into the cell where is it pumped through the Na+-K+-ATPase in the basolateral membrane.[27],[28] For electrical charges to be balanced, Cl- will be secreted out of the cell. This normal balance helps to maintain the right amount of airway surface liquid.[27],[28] In CF patients, there is excess Na+ transport into the cell via ENaC, and because water follows Na+, there is a deficient amount of airway surface liquid, leading to increased problems within the lungs.[27],[28]

iii. CaCC

Ca2+-activated Cl- channels (CaCC) are found in the apical membrane of epithelial cells and are activated by extracellular nucleotides such as ATP (adenosine triphosphate) and UTP (uridine triphosphate) to regulate the flow of Cl- through the cell.[29] Activation of the CaCC channels can occur with an increase in IP3, which stimulates Ca2+ to be released and regulate the efflux of Cl-.[30]

In patients with CF, the channel of CaCC that mediates Cl- secretion is not defective, which may play a role in bypassing the defective Cl- secretion in the mutated CFTR channel.[9] It has been found that both murine and human airways affected by CF have increased CaCC activity to compensate for the defective CFTR pathway and help regulate Cl- secretion in an attempt to decrease further lung pathology.[29]

iv. P2Y2

Purinergic (P2Y2) receptors are located in airway epithelial cells and are activated by ATP, UTP, and adenosine (ADO).[33] Activation of these receptors increase Cl- permeability via stimulation of a Ca2+-dependent outward rectifying chloride channels (ORCC) and CaCC.[32],[33],[35] In addition to CFTR, the ORCC and CaCC serve as distinct alternative pathways for Cl- secretion, making P2Y2 the regulator of Cl- secretion via ORCC activation in individuals with CF.[32],[33],[34],[35], Therefore, P2Y2 agonists have become of interest in developing pharmocological interventions for CF.[33]

Like stated in the normal ion regulation section, P2Y2 also indirectly regulates Na+ reabsorption by influencing ENaC activity.[9][36] When ATP interacts with P2Y2 receptors, there is a depletion in PIP2, which is necessary for ENaC activation that is mediated by protein kinase C.[9] Therefore, a decrease in PIP2 with P2Y2 activation inhibits ENaC stimulation.[9] ENaC inhibition decreases Na+ reabsorption and increases the water concentration in the mucus, making P2Y2 stimulation an important mechanism in patients with CF.[37]

Lastly, P2Y2 receptor activation helps regulate Cl- efflux.[9] This occurs via the cleavage of PIP2 into IP3 and DAG, which causes Ca2+ release from the endoplasmic reticulum and activation of CaCC, leading to Cl- efflux.[9] This pathway is independent of CFTR function, making this another important function of P2Y2 in patients with CF.[38]

Figure 3. Normal Ion Regulation in Lung Epithelial Cells [9]
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745452-fig1.jpg

Legend of Terms

CFTR: cystic fibrosis transmembrane conductance regulator

ENaC: epithelium sodium channel

CaCC: Ca2+activated chloride channel

PLC: phospholipase C

P2Y2: purinergic receptor

ATP: adenosine triphosphate

UTP: uridine triphosphate

ADO: adenosine

AC: adenylate cyclase

ADBR2: beta2-adrenergic receptor

Ab2: ADO-2b

cAMP: cyclic adenosine monophosphate

PKA: protein kinase A

PIP2: phosphatidylinositol 4,5-biphosphate

IP3: inositol triphosphate

DAG:diacylglycerol

v. Protein Kinases

Protein kinases, particularly A and C, play an important role in the activation of CFTR. The activation of protein kinase A (PKA) begins with the stimulation of a G protein receptor (a signaling protein) by catecholamine neurotransmitters, neuropeptides, larger protein hormones, lipids, nucleotides, and other biological molecules.[9] This G protein then activates enzymes such as adenylate cyclase, which synthesizes cAMP from ATP and activates PKA. PKA then activates ENaC, the Na+/K+ ATPase, and CFTR to allow for the regulation of Na+ and Cl-.[9] In addition to PKA, protein kinase C (PKC) has been found to have a positive effect on CFTR by phosphorylating it.[39],[48] According to Seavilleklein et al., PKC helped to increase the responsiveness of CFTR channels to the phosphorylation of PKA specifically within the R domain.[39]The R domain divides the CFTR channel into halves and regulates the opening and closing of the channel via phosporylation by PKA and PKC.[39]

Figure 4. CFTR Protein and Influence of Protein Kinases
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CFTR.jpg

vi. Ion Regulation and ASL

Now that a more comprehensive explanation of ion regulation has been provided, the regulation of ASL using the aforementioned cellular mechanisms can be understood. In healthy cells, the ASL volume, or hydration status, is controlled by the airway epithelial cells switching between absorptive and secretory roles.[13] This is regulated by the movement of ions (Na+ and Cl-) across the basolateral and apical membranes through the interaction of the cellular components mentioned above.[13],[45] Based on the principles of osmosis, the active transport of sodium (Na+) and chloride (Cl-) across the membrane is followed by water to maintain the appropriate hydration of the airway epithelia.[9] The volume of ASL is increased (secretory role) by Cl- secretion through CFTR and calcium activated Cl- channels (CaCC) and is decreased (absorptive role) by Na+ reabsorption through the epithelial sodium channel (ENaC).[13] Therefore, CFTR can increase and decrease ASL volume by secreting Cl- while concurrently inhibiting ENaC to decrease Na+ reabsorption.[13],[45]

Because an osmotic gradient is not controlled in the epithelial cells of the airways, the the flow of ions from the interaction between CFTR, CaCC, and ENaC regulate the force that drives fluid excretion or reabsorption.[13],[46],[47] Like mentioned in the TPD section, the Na+/K+-ATPase pump creates the electrochemical gradient required for ENaC to allow passive transport of Na+ into the cell, creating a negative electrical potential.[13],[15] This negative electrical potential decreases the force that drives secretion of Cl- and subsequent water flux into the lumen, decreasing the volume of ASL.[13],[14],[15] In healthy cells, this Na+ reabsorption and ASL fluid dehydration is reciprocally regulated by adensoine binding to adenoreceptors to cause the cascade of events that ends with the activation of CFTR (increase in Cl- secretion) and inhibition of ENaC (decrease in Na+ reabsorption), resulting in increased ASL volume and hydration.[13] With the gradient for Cl- being nearly 4 times greater outside the cell in the ASL, the reciprocal regulation is important because it creates the electrical driving force to overcome this concentration gradient that favors Cl- absorption into the cell, allowing for Cl- secretion and hydration of the ASL.[13],[41],[42]

In patients with CF, the mucus layer is thickened because of poor ion regulation from CFTR dysfunction.[13]. Lack of CFTR function results in increased Na+ reabsorption and decreased Cl- secretion, creating a more negative transepithelial potential difference (TPD) and a hypopolarized membrane that causes dehydrated or reduced ASL.[13],[14] [43] The weight of the mucus layer can cause the cilia to collapse due to the decrease in PCL height.[13] The collapse of the cilia results in the failure of mucus clearance, the development of adhesions and plaques on the epithelial surface, increased inflammation and infection, and irreversible damage to the bronchioles.[13],[44]

In conclusion, proper ion regulation is important in controlling the volume of ASL to promote appropriate clearance of bacteria and mucus from the lungs.[13] Without functioning CFTR, patients with CF have decreased PCL of the ASL, resulting in cilia collapse and frequent infection.[13]

B. Mitochondria and Cystic Fibrosis

Refer to Mitochondria for normal function.

In patients with cystic fibrosis, it has been found that mitochondria may undergo oxidative stress with the mutation of the CFTR, which can contribute to pulmonary pathology.[49] In normal mitochondria, glutathione (GSH) is an extracellular antioxidant that protects cells from the effects of reactive oxygen species (ROS), which create damaging oxidants.[49] Studies have shown that CFTR may regulate GSH and, therefore, help to neutralize the levels of ROS.[49] In CF, deficient or mutated CFTR may contribute to an increase in oxidative stress within the cell due to lack of regulation of GSH.[49] A study by Velsor et al., reported that mitochondrial GSH in deficient CFTR human lung epithelial cells were 32% lower than in CFTR sufficient cells.[49] Additionally, there was a statistically significant increase in ROS levels and subsequently H2O2 (oxidant) levels in the cells.[49] Therefore, a cell deficient in CFTR may have increased oxidative stress in the mitochondria.

Reduced mitochondrial GSH and resultant increase in ROS levels have been found to cause mitochondrial dysfunction in CF.[50],[51] A study by Kelly-Aubert et al.[50] found that electron transport chain Complex I function is reduced in CF due to oxidative modifications to NADH-ubiquinone oxidoreductase Fe-S protein 1, a Complex I subunit. The changes in Complex I were found to be associated with mitochondrial membrane depolarization.[50] Another study by Antigny et al.[51] found that the mitochondrial membrane depolarization in CF causes reduced mitochondrial Ca2+ uptake. It is thought that these mitochondrial dysfunctions could negatively affect the cell through their influence on ATP production and apoptosis.[50],[51] Therefore, the effect of CFTR deficiency on the mitochondria could contribute to systemic pathologies associated with the disease.

C. Inflammatory Cytokines and Cystic Fibrosis

In comparison to typically developing people, children and adults with CF have higher concentrations of inflammatory cytokines circulating in their blood stream.[52],[53],[54],[55] Therefore, people with CF experience a state of chronic inflammation. This inflammatory state is further exacerbated with respiratory symptom flare ups.[53],[56],[57] Furthermore, there are reports from cross-sectional studies that increased levels of inflammatory mediators circulating in the blood cause increased bone catabolism, resulting in low bone mineral density.[53],[56],[58],[59] The cellular mechanisms contributing to this inflammation are described below.

1. IL-6

Interleukin-6 (IL-6) is produced from T-cells and macrophages and acts as a pro-inflammatory cytokine in CF.[52] It is often released in response to muscle and tendon activity[60],[61] and is one of the mediators of chronic inflammation.[54] Increased levels of IL-6 are observed in people with CF.[53],[54] Furthermore, a longitudinal study by Haworth et al.[62] found that chronically high levels of IL-6 in adults with CF was related to low bone mineral density and urinary excretion of bone resorption marker. Additionally, IL-6 has been associated with increased protein catabolism and muscle wasting, which is observed in CF.[53],[63],[64]

2. TNF-α

Tumor necrosis factor-alpha (TNF-α) is another pro-inflammatory cytokine produced by macrophages that plays a role in chronic inflammation in CF.[52],[65] TNF-α upregulates the activity of other pro-inflammtory cytokines.[65] When left unchecked, this cytokine can have serious adverse consequences such as systemic inflammation and septic shock.[65] In a healthy person, a rapid increase in TNF-α will be counterbalanced by IL-10,[65] however, in CF this balance is disrupted. Like IL-6, TNF-α has also been associated with increased protein catabolism and muscle wasting.[52],[53],[63],[64] TNF-α inhibits the growth and regeneration of skeletal muscle through its ability to reduce the concentrations of Growth Hormone (GH) and insulin-like growth factor 1 (IGF-1).[52] TNF-α causes GH resistance and inhibits the production of IGF-1 in skeletal muscle.[52],[66],[67],[68] As a result of chronic inflammation, the levels of TNF-α are higher in people with CF, and thus, the rates of muscle protein degradation are higher in CF than in healthy people. [52] In order to promote muscle growth, increasing levels of TNF-α should be avoided in people with CF.[52]

3. IL-10

Interleukin-10 (IL-10) is an anti-inflammatory cytokine that can inhibit the activity of pro-inflammatory cytokines that are elevated in CF such as IL-6 and TNF-α.[55] However, IL-10 has been found to be deficient in the lungs in people with CF.[55] This deficiency results in the excessive inflammatory response observed in CF.[55] One of the major causes of morbidity and mortality in CF is chronic Pseudomonas aeruginosa lung infection with excessive inflammation.[55] It has been found that the pulmonary secretions of people with CF contain large concentrations of pro-inflammatory cytokines (such as IL-6 and TNF-α) but reduced concentrations of IL-10.[69],[70],[71],[72],[73][74] Additionally, it has been demonstrated that IL-10 knockout mice and CFTR knockout mice have increased levels of inflammation, weight loss, and mortality from acute and chronic Pseudomonas aeruginosa lung infections.[55],[75] Studies have all suggested that lung epithelial cells are an important source of IL-10 in healthy people and mice,[70],[73] but due to disruptions in lung epithelial cells as a result of CF, IL-10 levels are diminished.

D. Growth Hormone and Cystic Fibrosis

Growth Hormone (GH) mediates the growth of tissues such as muscle and bone and leads to the production of insulin-like growth factor 1 (IGF-1).[52] IGF-1 activates growth pathways and prevents muscle breakdown.[52] GH typically is given to children for catch-up height. However, GH is becoming better understood and is thought to have broader functional effects such as increasing strength and muscle mass.[52] When GH is exogenously given to children with CF, it has a direct effect on protein balance and thus has an anabolic effect on muscle.[52] GH levels are important when considering muscle wasting in people with CF.

V. In Vivo Models

A. Overview

Cystic fibrosis animal models have been created and studied for the following purposes: to understand the pathophysiology of the disease, to identify the genes that alter the severity of the CF phenotype in humans, to assess environmental contributors to the disease severity, to test pharmacological management, and to test gene therapy protocols to correct CF ion transport defects.[6] To date, three CF animal models have been created and studied: a murine (mouse), porcine (pig), and ferret model, making CF the first human genetic disease to be studied in three different animals.[76],[80] Murine models of CF have been studied since the early 1990s.[76] There are currently at least 14 different murine models of CF that display a variety of phenotypes observed with CF, such as failure to thrive and hyper-inflammatory responses in the lungs.[76] However, murine models lack spontaneous lung disease that is seen in human phenotypes of CF.[76] Additionally, models of gut obstruction in weaning mice are clinically different than those of newborn infants, suggesting that there may be biological differences in chloride movement in the gut as regulated by CFTR between humans and mice.[76] Therefore, other animal models of CF were needed to address this gap in the literature.[76]

Two larger animal models were developed using recombinant adeno-associated virus (rAAV)-mediated gene targeting of exon 10.[76] From this development, pig and ferret models have been widely used in current CF research.[76] Promising results have emerged from these models. For example, pig models have been able to develop a lung phenotype similar to that of CF[77] and ferret models have been able to develop lung infections both early and late in life.[76],[78],[79] However, these two models are not without flaws. One barrier to the widespread use of these models are that both animals have severe intestinal problems at birth, which requires surgery in the pig model.[76] Furthermore, the intestinal problems are lethal to 75% of ferrets.[76] Due to these problems, both the pig and ferret models of CF are extremely costly and difficult to use in research.[76] Because each animal model has flaws, it is important to study the pathophysiology of CF in all three animals to provide new insights into CF in humans.[80]

B. Murine Model

The CFTR gene was first cloned in 1989, and the first CF murine model was published just three years later.[81] Because there is a wide spectrum of disease manifestations in humans with CF, it has been difficult to create an animal model that represents the human disease.[81] However, numerous attempts have been generated with 14 murine models reported to date.[31] Mice models have several advantages over the other animal models, which include an increased production rate and decreased maintenance and cost.[81]

Wilke et al. organized the 14 models into the following 5 categories: null mutations, hypomorphic mutations, ΔF508 mutations, other point mutations, and transgenes.[31] [Table 1] is reproduced from Wilke et al.[31] with some modifications mostly related to the columns included. Please refer to this review article for the complete table.[31] The null mutation models and the hypomorphic mutation models are knockout models created by using a selectable marker to interrupt the mouse Cftr coding region resulting in a loss of function.[31] The ΔF508 mutation models and the other point mutation models are created by introducing the mutation (G551D, G480C, R117H) into the endogenous mouse Cftr gene.[31] The Tg(FABPCFTR) transgene model was created to overcome the limitations of severe intestinal disease found in many of the knockout models.[31],[82] This model has a transgene expressing CFTR from a promoter for the intestinal epithelium specifically.[31] [Table 2] describes the phenotypes observed in the CF mutant mice.

Table 1: CF Murine Models This table is reproduced by Wilke et al.[31] with some modifications.

Mouse Mutation Survival to Maturity References
Null Mutations
Cftrtm1Unc S489X Ex10 R <5% [84],[85]
Cftrtm1Cam R487X Ex10 R <5% [86]
Cftrtm1Hsc M1X Ex1 R 25% [87]
Cftrtm3Bay Ex2 R 40% [88]
Cftrtm3Uth Y122X Ex4 R 25% [89],[90]
Hypomorphic Mutations
Cftrtm1Hgu Ex10 I 90% [89]
Cftrtm1Bay Ex3 I 40% [31]
ΔF508
Cftrtm2Cam F508del R <5% [92]
Cftrtm1Kth F508del R 40% [93]
Cftrtm1Eur F508del (H & R) 90% [94]
Other Point Mutations
Cftrtm2Hgu G551D R 65% [95]
Cftrtm3Hgu G480C (H& R) Normal [96]
Cftrtm2Uth R117H R 95% [89],[90]
Transgene Mouse Transgene Phenotype References
Tg(FABPCFTR) CFTR This model promotes the expression of the airway epithelium to account for the limitations in the mice with very severe intestinal disease. [82]
Key: R=replacement, I=insertion, H & R= hit and run, this model deletes the selectable marker from the region of CFTR coding, which causes only the F508del mutation and allows normal transcription rates [94],[83]

Table 2: CF Pathology in Mutant Mice

Body Structure Phenotype
Intestines The hallmark of all but four CF murine models (CFTRtm1HGU, CFTRtmEUR, CFTRtm3HGU, Tg(FABPCFTR)) include an intestinal phenotype that causes severe pathology.[31],[81] The models without this intestinal phenotype have a 90% or greater survival into maturity.[31],[81] The models with intestinal disease express dilatation of the crypt, failure to thrive, accumulation of mucin in the crypts of Lieberkuhn, obstruction of the intestines resulting in perforation, peritonitis, and death.[81] The intestinal obstruction is similar to that seen in humans.[81]
Pancreas Overall, pancreatic pathology in the murine models is much milder than in patients with CF.[81] Two possible factors that are responsible for improved pancreatic function include a Ca2+-mediated Cl- conductance channel (which serves as an alternative channel to CFTR in the pancreas) and some murine models express enough residual CFTR action to create a normal Cl- secretory pathway.[81][97],[98]
Liver CF associated liver disease (CFLD) currently affects more than 50% of adults with CF, making CFLD an increasing clinical problem.[31]. In the liver, CFTR is involved in fluid homeostatsis in the biliary duct system's epithelial cells (cholangiocytes).[31],[99] Unfortunately, there is no major liver pathology noted in most of the CF models.[31] However, liver problems typically manifest in later years in humans, and these mice were studied at a young age.[31]
Gall Bladder The gallbladder in these models appear to have greater abnormalities than the liver.[31] These abnormalities include storage of black bile and neutrophil infiltration in the Cftrtm1Unc and Cftrtm2Hgu models and distended gallbladders in these two models and Cftrtm1Bay.[31],[6],[100] The frequent gallbladder malformations and gallstone formation in humans with CF may benefit from the understanding of this pathology in mice.[31]
Kidney Although no major kidney pathology is present in CF mutant mice, abnormalities in urinary protein secretion were observed in a few of the knockout models.[31],[101],[102] This suggests that CFTR may be involved in the proximal tubules of the kidney, specifically in brush border maturation and membrane trafficking.[31]
Smooth Muscle Cells CFTR is expressed in smooth muscle cells of arteries and airways and myotubes and the sarcoplasmic reticulum of skeletal muscles.[31],[103],[104],[105] Divangahi et al. (2009) observed degeneration of diaphragmatic muscle in Cftrtm1Unc mice infected with Pseudomonas aeroginosa, a chronic infectious agent in patients with CF.[31],[106] The authors hypothesized that the reduced muscle strength, especially of the diaphragm, consistent in patients with CF may influence increased susceptibility to lung disease.[31],[106]
Cartilage Although no cartilage abnormalities have been expressed in human patients with CF, developmental abnormalities in tracheal cartilage have been found in CF mutant mice models.[31] The incomplete formation of rings of tracheal cartilage at birth in Cftrtm1Unc and Cftrtm1Eur murine models was first observed by Bonvin et al.[107] This is the first characteristic to be verified in other CFTR-deficient species,[31] including CFTR knockout ferrets [78] and pigs.[108],[109] The explanation for this developmental abnormality is not yet known.[31]
Bone Because osteopenia and osteoporosis are major problems in patients with CF, study of CF-related bone disease in murine models could lead to new treatment strategies.[31] It has been reported that there are decreased levels of CFTR protein and CFTR mRNA found in human and murine odontoblasts and osteoblasts.[31],[110] In addition, decreased bone density has been described in several studies with CFTR mutant murines.[31],[111],[112],[113] However, it is unknown if this is the result of a CFTR-related defect in the osteoblasts that build bone or the osteoclasts that breaks down bone.( .[31] Although the exact cause of decreased bone density observed in patients with CF and CFTR-deficient mice is unknown, it is hypothesized that CFTR dysfunction in the membrane of bone cells may relate to the changes in bone metabolism leading to osteopenia.[31]
Immune System Abnormal immune cells have been found in CF mutant mice.[31] For example, Bruscia et al. (2009) observed abnormal after abnormal secretion of cytokines that promote inflammation in macrophages from the bone marrow and lung in CF mice after Pseudomonas aeroginosa exposure.[31],[114] Additionally, a pro-inflammatory response with increased secretion of IL-1B and decreased secretion of IL-10 in the alveolar and peritoneal macrophages of Cftrtm1Eur mice after stimulation has been shown.[31],[115],[116] More research is needed to determine if these defects are cell-autonomous in origin versus adaptive in order to conclude that CF is also a disease of the immune system.[31]
Nasal and Trachea The mucus in the nasal cavity found in CF murine models accurately demonstrates the Cl- transport defect and hyper-absorption of Na+.[31],[117] Additionally, all models except for Cftrtm1Hgu and Cftrtm1Eur demonstrate an upregulation of the Ca2+-mediated Cl- secretory response (CaCC) and a decrease in the cAMP-mediated Cl-response (CFTR), similar to what is seen in human CF tissue.[31],[118]
Submucosal Glands Because mice have similar submucousal glands to humans, CF mice models are appropriate to study CF pathogenesis related to the submucosal glands.[31] Abnormal submucosal glands are hypothesized to play an integral role in the development in CF airway disease that contributes to abnormal consistency of mucus in the airways and decreased fluid secretion.[31],[119],[120] Fluid secretion from submucosal glands is dependent on the CFTR Cl- and the K+ channels, which are regulated by cAMP and intracellular Ca2+ signaling pathways.[31] Because the cAMP-stimulated portions of these pathways are absent in CF mutant mice, it has been hypothesized that these pathways are dependent on CFTR.[31],[119],[120] Therefore, CF mutant mice are a valid model to study the function of CFTR and to test drugs to target this protein.[31]
Lungs Like stated in the overview, lack of spontaneous lung inflammation and chronic bacterial infections and inflammation is a limitation in the murine models.[31] However, there are some murine models with lung pathology. The congenic strain B6-Cftrtm1Unc/Cftrtm1Unc is the only murine model to develop spontaneous and progressive lung diseases when placed in a specific-pathogen free environment.[31],[81],[109],[121] The characteristics of the lung disease included hyperinflation of alveoli after the bronchioles, poor mucociliary clearance, fibrosis, recruitment of inflammatory cells, and thickening of parenchymal interstitial tissue.[31],[81] The other models with lung pathology include the Cftrtm1Hgu, the Cftrtm2Hgu, and the Cftrtm1Kth models.[31] The Cftrtm1Hgu model displays cytokine abnormalities, [31],[122] changes in submucosal glands, and damaged mucociliary transport of static particles.[31],[123],[124][125],[126] In the Cftrtm2Hgu models, the inflammation in the lungs was not properly controlled in up to one third of the mice and increased vulnerability to P. aeroginosa and weakened pulmonary clearance was reported..[31],[81],[127],[128] A final phenotype related to the lungs in CF mice is decreased pulmonary levels of iNOS, an implication of CF pathogenesis, in 2 models (Cftrtm1Kth and Cftrtm1Unc).[31],[129],[130]

1. Limitations and Implications

With lung infections being the major cause of morbidity and mortality in patients with CF, the main limitation with the mice models is that the mice do not obtain the spontaneous and chronic bacterial infections that are seen in patients with CF.[81],[80],[131] A second limitation with the mice models are that they do not develop the pancreatic disease that is seen in individuals with CF.[76],[80]

Despite these limitations, the mice models have provided a better understanding of the pathology of CF and have allowed for the identification of promising new therapies.[31] Additionally, mice models will continue to be used secondary to their affordability and versatility.[31]

C. Porcine Model

Limitations in the murine model led to the development of a porcine model of CF.[132] The porcine model was chosen because of pigs' anatomical and physiological similarities to humans.[132] The similarities between humans and pigs in size, biochemistry, genetics, and life span also support the use of this model.[133] Two porcine models of CF have been developed.[108] The CFTR-null model is a knockout model in which the CFTR gene is disrupted, preventing the production of CFTR protein.[108] The CFTR-ΔF508 model is a knock-in model in which the CFTR-ΔF508 mutation is inserted into the DNA, causing disruption of CFTR processing.[108]

1. CFTR-null Model

The CFTR-null model utilizes a recombinant adeno-associated virus vector (rAVV) to target CFTR exon 10 in porcine fetal fibroblasts containing the CFTR allele, creating homologous recombination.[108],[133] The nuclear material from the fibroblasts is then transferred to porcine oocytes using somatic cell nuclear transfer, which results in the production of heterozygote CFTR+/- male piglets.[132],[133] These piglets are bred after they reach sexual maturity, producing male and female heterzygote offspring.[133] The male and female heterozygotes are then bred, producing both heterozygote, CFTR+/-, and homozygote, CFTR+/+ and CFTR-/-, piglets.[133]

i. Phenotype of CFTR-/- Pigs

The newborn CFTR-/- piglets have characteristics that resemble human infants with CF in many organs of the body, including the gastrointestinal tract, pancreas, liver, and gallbladder.[133] In general, the disease is more severe in the CFTR-/- pigs than in newborn humans with CF.[134] The overarching features found in the CFTR-/- pigs' affected organs include prominent epithelial mucus producing cells, mucus accumulation, tissue remodeling, and inflammation.[134] The following provides a detailed description of CFTR-/- pigs phenotype based on organ/system:

  • In the gastrointestinal (GI) tract, 100% of the CFTR-/- pigs develop a neonatal intestinal obstruction called meconium ileus.[133] Meconium ileus occurs in 15% of human infants with CF.[133] The piglets require ileostomy or cecostomy surgeries to bypass the meconium ileus in order to survive.[77] The CFTR-/- pigs’ intestinal tracts have mucinous hyperplasia in the epithelial cells distal to the obstruction but not proximal to the obstruction.[134] Other features of the intestinal tracts include the presence of intestinal atresia, diverticulosis, hypertrophied tunica muscularis, and possible perforation.[134] These conditions can also occur in human infants with CF.[80]
  • In the pancreas, CFTR-/- pigs have many lesions that affect exocrine tissue but spare endocrine tissue.[134] One characteristic of the CFTR-/- pancreas is the filling of acinar and ductular lumens with zymogen material, creating thinning of the acinar and ductular epithelium.[134] The CFTR-/- pancreas also demonstrates duct proliferation, mucus metaplasia of ducts, and inflammation that increases with increased disease severity.[134] While the nature of pancreatic involvement of the CFTR-/- pigs is similar to that of human infants with CF, the severity of damage is greater in the pigs.[80]
  • In the liver, CFTR-/- pigs demonstrate biliary proliferation, which is one of three characteristics of focal biliary cirrhosis (FBC).[134] The other two characteristics of FBC, fibrosis and inflammation, were absent or mild in the CFTR-/- pigs. FBC is also common in people with CF.[80]
  • In the gallbladder, 100% of CFTR-/- pigs demonstrate microgallbladder, compared to 20-30% of human infants with CF.[134] Diffuse epithelial mucinous change, as well as luminal obstruction by mucus and altered bile are also found.[134] Mucinous change with obstruction and stenosis is also observed in the cystic duct.[134]
  • In the pulmonary system, the airway epithelium of these newborn piglets lacks evidence of cellular inflammation or infection.[133] However, abnormalities in electrolyte transport have been discovered in the nasal epithelia, indicating a lack of CFTR activity.[133] Although no abnormalities in CFTR-/- lungs are observed immediately after birth, lung disease spontaneously develops within two or more months after birth.[77] At this stage, CFTR-/- pigs develop airway inflammation, remodeling, mucus accumulation, and infection.[77]

ii. Implications

The similar phenotypes of CFTR-/- pigs and human infants with CF provides hope that the porcine model will help researchers better understand the disease and develop new treatment methods in the future.[133] For now, the model has already provided information regarding the development of lung disease in CF.[77] The causal relationship between infection and inflammation in the lungs has been poorly understood in humans with CF, but research with CFTR-/- pigs has unlocked information about the timing of infection and inflammation.[77] A study by Stoltz et al.[77] found that while CFTR-/- lungs lack inflammation shortly after birth, they have impaired bacterial elimination. These results suggest that impaired bacterial elimination leads to the development of inflammation, which then contributes to lung pathology.[77] This information could have implications for the treatment of human infants with CF, providing support for the use of preventative antibacterial strategies early in life.[77]

2. CFTR-ΔF508 Model

CFTR-ΔF508 Model was developed to model the most common genetic mutation found in CF.[108] It uses the same gene targeting strategy as the CFTR-null model with the exception that the ΔF508 mutation is inserted at exon 10 by the rAAV.[108] The CFTRΔF508/ΔF508 pigs have a phenotype that closely resembles CFTR-/- pigs.[135] Like CFTR-/- pigs, 100% of CFTRΔF508/ΔF508 pigs have meconium ileus that requires surgery in order to survive.[135] They also have similar phenotypic characteristics in the liver, gallbladder, and lungs.[135] However, the CFTRΔF508/ΔF508 pigs have slightly less pancreatic destruction than CFTR-/- pigs.[135] A study by Ostegaard et al. [135] found that CFTRΔF508/ΔF508 pigs retained some residual CFTR function. While this residual function decreased severity of pancreatic involvement, it was insufficient to prevent disease.[135] The results of this study indicate that the disease processes in CF are a result of a lack of CFTR function rather than misprocessing due to genetic mutation.[135]

D. Ferret Model

Due to the inability of mice models to spontaneously develop lung disease, other animals that can prove as better CF models have been researched.[6],[136] Mustela Putorius Furos, which is the domestic ferret, has been discovered as another option in CF for various reasons.[78],[79],[137]

To begin, there are similarities in lung physiology, cell types, and airway morphology between humans and ferrets.[138],[139],[140],[141],[142],[143] Expression of CFTR is identical in humans and ferrets in both the airway epithelium and the submucosal glands, where CFTR concentration is the highest.[78],[144],[145]] In addition to equally high levels of CFTR, ferret and human submucosal glands are abundant in the tracheobronchial airways unlike in that of mice.[144],[145],[146] Humans and ferrets share the same primary secretory cell type in the proximal cartilaginous airways, the goblet cell.[147] Additionally, the CFTR chloride channels present in the airway epithelium of humans and ferrets have similarities.[148] These similarities relate to the pharmacologic and bioelectric properties of the channel.[148]

Ferret models have previously been used in research for other human lung infections, both bacterial and viral,[151],[152],[153],[154],[155],[156] including the influenza virus[157] and SARS[158]. Time to sexual maturity for ferrets is about 5-6 months and gestation period lasts 42 days allowing fairly rapid reproduction time.[79],[137],[159]

1. CFTR-null Model

Multiple methods including somatic cell nuclear transfer[137] and a combination of recombinant adeno-associated virus-mediated gene targeting of CFTR in fibroblasts then nuclear transfer[78] have been trialed in the cloning of ferret models. The CFTR-null model is a knockout model produced utilizing the same method as the CFTR-null porcine model.

i. Phenotype of CFTR-/- Ferrets

  • In regards to the pulmonary system, early in life there is evidence of spontaneous lung infection, similar to humans, in CFTR-/- ferrets that requires antibiotics.[79] A decreased ability to kill bacteria effectively has been observed in newborn CF ferrets.[80] Psedomonas aeruginoa, a pathogen common in a human CF lung, is not present in those of ferret models.[80] Submucosal glands in ferret airways typically have highly expressed CFTR,[79],[144],[145] however, CF ferrets have defective submucosal gland secretion.[79] More research is indicated at this point to determine how these secretions affect innate immune defects related to airway infection.[79]
  • In the GI system, conditions present in CF infants, such as diverticulosis, intestinal atresias, and microcolon, have been seen in ferret models as well.[79] A more common GI condition called meconium ileus is expressed as a similar phenotype in about 75% of CFTR knockout ferrets,[79] which is an increased rate compared to approximately 15% of newborn infants with CF.[131],[160] This increased frequency rate could indicate an increased role of CFTR in hydration of the in-utero intestine of ferrets compared to humans.[80]
  • As opposed to the dissimilarities of pancreatic disease between mice and human models, pathology present in exocrine pancreas of all knockout ferret models mimics that seen in the majority of infants with CF.[160],[161] Specific characteristics of pancreas pathology in ferrets include thickened eosinophilic enzyme precursor secretions as well as enlarged ductules and acini.[79] Although ferret pancreatic disease is not as severe as that present in the pig models, it requires pancreatic enzymes due to exocrine pancreas insufficiency during the first few months of life.[79]
  • The livers of wild type ferrets are indistinguishable from those of CF ferrets through tissue examination.[79] Despite this fact, possible liver disease is suggested in the knockout ferret models by increased levels of bilirubin and plasma alanine aminotransferase.[79] Elevated enzymes without liver pathology is also evident in children with CF. Liver function has been normalized with the oral administration of a bile acid in infants,[162] which has also proven effective in ferrets.[79]
  • Unfortunately, the knockout ferret does not demonstrate any gallbladder disease that can be evident in up to a third of older human patients with CF.[79],[131],[161],[163]
  • Each type of animal model demonstrates growth impairment, however, the knockout ferret model shows the greatest degree of impairment possibly due to decreased gastrointestinal pH.[79],[164],[165] Unlike other species, ferrets also have a shorter intestinal transit time and do not have a cecum.[79],[166],[167]
  • Degeneration of the vas deferens present at birth in the CF ferrets serves as an excellent model due to the sterility caused by deterioration of vas deferens present in approximately 97% of human males with CF[79],[168] and is the only animal model to replicate this occurrence.[79]

ii. Implications

Although there are numerous similarities in pathologies between humans with cystic fibrosis and ferret models, current limitations of ferret model use exist because of meconium ileus or malabsorption of the gastrointestinal tract.[79] Newer knockout models are being developed to correct the gut defect present to improve the use of ferrets in CF research. [79]

VI. Body Systems Affected

Figure 5 demonstrates the effects of CF on multiple organs of the body. This section discusses the changes in the body systems related to the cellular impairments from CFTR dysfunction.

Figure 6: Body Systems Affected by CF
http://www.hgen.pitt.edu/counseling/public_health/images/cystic-fibrosis.gif

cystic-fibrosis.gif

A. Pulmonary System

The pulmonary system is severely affected in individuals with CF with approximately 80% of deaths being the result of lung pathology.[3] In healthy lungs, the airway surface liquid (ALS) is regulated by interactions between CFTR and CaCC. Refer to [Figure 2] for details. However, in human CF tissue, the CFTR is not functional and CaCC is sometimes upregulated to compensate.[31],[171],[101],[102] Despite this compensation, the CFTR deficient lung in CF causes Cl- hyposecretion and Na+ hyperabsorption, leading to increased mucus viscosity.[3] Because of the increased viscosity, the individual is unable to clear the lungs and this retained mucus is an excellent harbor for bacterial growth, leading to increased infections.[3] Pseudomonas aeruginosa is an example of an opportunistic pathogen that frequently causes life-threatening chronic lung infection in patients with CF.[31] CF pathology progresses from initial mucus plugging of small airways to bronchitis to pneumonia and fibrosis involving all bronchi.[3] These changes lead to reduced carbon-dioxide exchange, causing cyanosis, clubbing, hypercapnia, and respiratory acidosis.[3] Eventually, this lung sequelae often leads to respiratory failure and death.[3]

B. Digestive System

Pancreatic enzyme insufficiency and mucus gland secretions with increased viscosity are related to the defective CFTR chloride channel in epithelial cells.[3] This insufficiency leads to blocking of the pancreatic ducts by the thickened secretions and occurs in approximately 90% of patients with CF.[3] This blocking of the ducts causes a chain of events that leads to impaired nutrient absorption due to the inability of pancreatic enzymes to reach the first section of the small intestine, the duodenum.[3] Vitamins A, D, E, K, proteins, and fats are examples of nutrients that are difficult for people with CF to absorb, thereby affecting digestion.[169] The small lobes of the pancreas also become enlarged which causes degeneration and fibrosis.[3]

In addition to impaired nutrient absorption, damage to the pancreas can eventually lead to glucose intolerance and diabetes due to negative effects on beta cells.[3],[169] This type of diabetes develops in approximately 35% of patients with CF by their 20s and 43% after the age of 30.[169] Other clinical presentations of pancreatic insufficiency include malnutrition, recurrent pancreatitis, iron deficiency anemia, bloody diarrhea, stomach pain, and delayed puberty.[3],[169] The stools of patients are foul-smelling as well as frothy and bulky due to a lack of tryptase and amylase.[3]

Gastrointestinal complications in people with CF include meconium ileus and prolapse of the rectum.[3] Meconium ileus, which affects about 10-15% of newborns with CF, results from thickened and dried stools that become impacted and cause obstructions in the small intestine.[3] This earliest sign of CF can present by vomiting, rapid development of dehydration, constipation, abdominal pain or distention, and anemia.[3] Although meconium ileus is the first complication seen, rectal prolapse is the most common.[3] Additional clinical manifestations of GI complications include weight loss, anorexia, thin extremities, failure to thrive, yellowish skin, voracious appetite early on, intussusception of the intestines, and acute gastroesophageal reflux.[3] Comorbidities of the GI system associated with CF include ischemic bowel disease and Crohn’s disease.[3] An excess of pancreatic enzyme supplementation could also lead to fibrosing colonopathy.[3]

C. Reproductive System

In men, CF often leads to the absence of the vas deferens needed to produce sperm causing infertility.[3] If the vas deferens are present in men with CF, there is a blockage or fibrosis which causes azoospermia where at the sperm is prevented from entering the semen.[3]

In women, there is often decreased fertility secondary to the thick mucusal lining of the cervix which prevents conception. However many women may have in vitro fertilization to be able to bear children.[3] Additionally it has recently been found that when women have heightened levels of estrogen such as prior to ovulation, estrogen can in fact inhibit Cl- secretion via the CaCC.[170] During this time, this puts women at an increased risk for infection because the ASL volume is decreased.[170]

D. Hepatobiliary System

Liver disease affects approximately 30-50% of people with CF, and it is the third leading cause of death for people with CF behind pulmonary disease and transplant complications.[31][177] Types of liver disease found in people with CF include focal biliary cirrhosis, multilobular biliary cirrhosis, and portal hypertension.[177] These conditions can eventually progress to liver failure.[177] Gallbladder conditions found in people with CF include microgallbladder, cholelithiasis, neonatal cholestasis, and bile duct obstructions.[177] These liver and gallbladder conditions result from a lack of CFTR function in the epithelial cells, leading to thickened secretions.[177] Steatosis, or fatty liver, is also common in people with CF, but it is developed as a secondary complication of other system disease rather than lack of CFTR function in the liver.[177] It is unclear why some people with CF develop hepatobiliary diseases while other do not, but it is believed that the development of these diseases may be influenced by genetic modifiers.[3],[177]

E. Musculoskeletal System

One common musculoskeletal impairment found in people with CF is low bone mineral density (BMD).[59],[3],[178] A study by Elkin et al.[178] found that increased disease severity is associated with low BMD. The study examined specific risk factors for low BMD in the population of adults with CF and found that risk factors included lower forced expiratory volume in one second (FEV1), higher number of intravenous antibiotic treatments in the past five years, and lower activity level.[178] Glucocorticoid usage and low body mass index were associated with low BMD in the femoral neck.[178] As a result of low BMD, people with CF are at greater risk for fractures, especially in the vertebrae and ribs.[59],[178] Low BMD has been observed in the murine model, but the specific cause of the impairment is unclear.[31] It may be a result of CFTR malfunction in the osteoblasts or osteoclasts, or it could be a secondary effect of increased inflammation or abnormal lipid metabolism.[31]

A study by Aris et al.[59] also found that adults with CF have greater thoracic kyphosis angles than the general population. Other postural abnormalities commonly observed in people with CF include increased scapular elevation and protraction, increased lumbar lordosis, decreased mobility in the spine and ribs, and chronic back pain.[179],[180] A study by Hodges et al.[181] found that, during periods of increased respiratory demand, postural support from the trunk muscles is reduced. The results of this study suggest that the high respiratory demand in CF may contribute to postural abnormalities by reducing postural support from the trunk muscles.[179]

VII. Medical Management

A. Pharmacotherapy

Pharmacotherapy used in the treatment of people with CF includes a variety of medications to manage pulmonary and pancreatic disease.[3] Antibiotic medications are used to prevent and treat lung infections, and they may be administered by oral, inhaled, or intra-venous (IV) methods.[3],[182] Specific antibiotic medications that have been shown to provide added benefits are Macrolides, which may also suppress inflammation, and Azithromycin, which may also help to improve lung function.[3],[182] Inhaled and non-inhaled anti-inflammatory medications are used to decrease inflammation in the airway.[3] The anti-inflammatory medication ibuprofen may also be used to slow decline in lung function.[3] Mucolytic medications, such as Dornase alfa, are used to help thin mucus secretions and improve mucus clearance.[3] Inhaled bronchodilators are used to open the airway.[3] A hypertonic saline solution may be used following administration of a bronchodilator to replace salt in the airway and improve airway clearance.[3] Pancreatic enzyme replacement supplements and vitamin supplements are used to manage digestive system disease by aiding in digestion and improving nutrition.[5] Oral bile acid therapy may be used to improve bile secretion in liver disease.[3] Also, recent studies suggest that using antioxidant supplements may help to decrease inflammation and slow decline in lung function.[50]

B. Airway Clearance Techniques

Airway clearance techniques (ACT) are used by people with CF to help loosen and clear secretions in the lungs.[182] When prescribed, bronchodilators should be administered prior to performing ACT in order to open the airways.[183] The different types of ACT include techniques that use devices such as Oscillating Positive Expiratory Pressure, High Frequency Chest Wall Oscillation, and Positive Expiratory Pressure Therapy, as well as manual and breathing techniques such as Postural Drainage and Percussion, Active Cycle of Breathing Technique, and Autogenic Drainage.[183] The following describes the method and purpose of each technique:

  • Oscillating Positive Expiratory Pressure: The patient breathes through an oscillating device to help loosen secretions.[183]
  • High Frequency Chest Wall Oscillation: An inflatable vest is worn that vibrates to loosen secretions.[183]
  • Positive Expiratory Pressure Therapy: The patient breathes through a mask or mouthpiece that provides resistance during exhalation in order to hold airways open.[183]
  • Postural Drainage and Percussion: The patient uses various positions in combination with chest percussion to drain mucus from all parts of the lungs.[183]
  • Active Cycle of Breathing Technique: Includes several breathing techniques that people with cystic fibrosis can learn to clear mucus and improve spasms.[183]
  • Autogenic Drainage: Involves using varied level of airflow during breathing to first dislodge, then collect, and finally clear mucus.[183]

C. Transplantation

In severe cases, people with CF may require a lung transplant.[5] Liver transplants may also be indicated in cases of liver failure or severe portal hypertension.[3] A study by Egan et al.[184] found that survival rates following lung transplant for CF were 81% at one year, 59% at five years, and 38% at ten years following transplant. Increased CF symptoms in other organs of the body due to immunosuppressive treatments could be a possible complication following transplant.[3]

D. Gene Therapy

Since 1993, attempts have been made to correct the genetic defect in CF through gene therapy.[3] Since lung disease is one of the main causes of morbidity and mortality in CF, research on gene therapy for CF has mostly focused on the lung.[185] Clinical trials using viral vectors, such as adenovirus and adeno-associated virus, only produced transient gene expression with inefficient repeated administration.[185] While these studies failed to produce effective gene therapy treatments, their results support the principle of gene therapy and continued research in this area.[185]

The difficulties with viral vectors led to a focus in research on non-viral vectors.[185] Clinical trials using non-viral vectors have found some improvement in chloride transport in the lungs.[185] However, these trials were unable to demonstrate whether non-viral vectors could produce clinically relevant improvements in lung disease.[185] Therefore, a study is currently taking place in the UK to determine if the non-viral vector cationic lipid GL67A can improve lung disease in CF.[185]

Studies using lentiviral vectors are also showing promise because these viruses may evade the immune system.[185] Research with lentiviral vectors has not yet progressed to the clinical trial stage.[185] Research in gene therapy for CF may be aided by the new development of CFTR-null pigs and ferrets, which could provide new opportunities for research.[185] Therefore, while gene therapy is not yet a treatment for CF, current research shows promise for the future.

VIII. Conclusion

Cystic fibrosis is a common autosomal recessive genetic disorder that affects approximately 70,000 people worldwide.[1],[2],[4] While CF affects multiple body systems, it primarily affects the pulmonary and digestive systems.[2],[4] There are several cellular mechanisms involved in the disease process of CF. In this disease, there is a point mutation on the CFTR gene that results in abnormal ion transport across epithelial cells, which causes the classic signs and symptoms associated with CF,[6] including persistent cough with phlegm, frequent infections of the lungs, wheezing, shortness of breath, and salty sweat.[2],[4] In summary, the mutation on the CFTR negatively affects the Cl- secretion allowing for an increase in Na+ reabsorption via the ENaC.[9] This imbalance in ion regulation causes a decrease in the fluidity of the mucus which makes fluid secretion and mucus clearance very difficult. In many patients with CF, there is an increase in CaCC activity which can help bypass the defective CFTR channel to allow for an increase in Cl- secretion helping to ameliorate the ion dysregulation and increase the fluidity of the mucus.[9] While further research is required, this is an important component in maintaining the negative effects of the disease. Several animal models have been used (mouse, pig, and ferret) to understand the underlying cellular mechanisms of CF and to inform future medical management. There is currently no cure for CF, so future research in human and animal models is crucial to determine best medical management to improve the health, longevity, and quality of life of people with this disease.

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