Perspectives On Medical Research
Volume 4, 1993
The Failure of Animal Models of Cystic Fibrosis
Introduction: The Human Disease
Cystic fibrosis (CF) is a progressive disease characterized by dysfunction of the secretory epithelial cells that line the sweat glands, salivary glands, intestines, pancreas, trachea, and bronchi.(1) CF's pulmonary manifestations-thickened mucus that is difficult to expectorate, recurrent bacterial infection, and destruction of lung tissue--account for most symptoms and deaths caused by the disease. Ultimately CF is fatal.(2)
Inherited as a recessive trait, CF is one of the commonest human genetic diseases, affecting about one in 2,000 children. In the United States, eight million (one in 31) people are believed to carry a CF gene.(1)
CF arises from a defect in the gene that codes for the protein "cystic fibrosis transmembrane regulator" (CFTR).(3) While some of its functions have yet to be elucidated, CFTR is believed to span the width of a cell's membrane and act as a channel through which chloride ions pass into and out of the cell. (Alternatively, CFTR may indirectly control the passage of chloride ions by activating another protein that serves as the chloride-ion channel.) In CF patients, the CFTR present in certain types of secretory epithelial cells fails to respond to biochemical cues that these cells should either absorb or secrete chloride ions; instead the chloride-ion channels remain closed.(1,4) Defective chloride-ion transport leads to CF's symptoms.(2)
In normal individuals, chloride ions transported into the trachea and bronchi draw water into these airways; the water hydrates secretions and keeps them fluid. Because CF patients lack this chloride-ion transport, their respiratory secretions become thickened and difficult to expectorate. The thickened mucus provides a site for chronic bacterial infection. White blood cells, drawn to the lungs by this infection, degenerate and add their DNA to the already-thickened mixture, further increasing viscosity. Eventually airway obstruction impairs respiration, leads to the destruction of lung tissue, and proves fatal.(2)Advances in understanding and treating CF have derived from clinical and in vitro research. CF researchers (including a number of animal experimenters) generally agree that there is no valid animal "model" of CF.(5-8) Nevertheless, researchers continue to propose various animal models, either naturally occurring (genetic) or artificially induced.
Clinical and In Vitro Advances
Improved diagnosis and supportive care, as well as an increased emphasis on therapies that focus on CF's respiratory symptoms, have extended the life expectancy of CF patients--from the pre-teens in the 1940s to the mid- to late-twenties today.(1) Antibiotics have proved beneficial in controlling CF-associated bacterial lung infections.(1) Various other drugs, such as deoxyribonuclease (DNase) and acetylcysteine, have been partially effective in thinning CF's tenacious respiratory secretions and facilitating their clearance; these drugs are administered as aerosols.(1)
The use of aerosolized drugs to loosen pulmonary secretions followed a serendipitous observation in tuberculosis patients. In the 1940s, clinicians such as Joseph B. Miller were treating tuberculosis by administering the antibiotic streptomycin in an aerosol inhalant containing the detergent Triton A-20. These clinicians noted that Triton A-20 had a side-effect of liquefying sputum. Before treatment, the patients had difficulty clearing bronchial secretions. "Almost immediately after beginning treatment with Triton A-20 aerosols," Miller recalls, "their secretions became thin and watery" and were easily expectorated. Triton A-20 was then used to treat other chronic pulmonary diseases, including CF. The "thinning effect was found to be constant and dependable," Miller notes.(9) (After Triton A-20 had been widely and safely used in children and adults for years, Miller and Edward H. Boyer conducted Triton A-20 toxicity tests in rats. Their finding of no pulmonary toxicity was "in complete agreement with the clinical experiences"--and apparently superfluous.)(9)
Following Triton A-20's success, researchers sought other drugs that liquefy respiratory secretions. To better identify agents capable of thinning these secretions, investigators collected sputum from CF patients and analyzed its composition. CF bronchial secretions, they found, have considerably higher DNA concentration than bronchial secretions resulting from other pulmonary diseases.(10,11) In CF, as previously mentioned, white blood cells drawn to the lungs by infection degenerate, releasing DNA that forms a gel and mixes with already-thickened mucus to further increase sputum viscosity.(2,10,11) By mixing sputum samples from CF patients with various agents, researchers screened for drugs that decrease the viscosity of CF secretions. In vitro, bovine pancreatic DNase I (an enzyme that digests DNA) was found to be especially effective.(12,13) Clinical trials showed that bovine DNase was effective in treating CF; in 1958 it was approved for use.(13) In following years, however, clinicians noted occasional adverse respiratory reactions--perhaps allergic reactions to bovine protein in the enzyme, perhaps reactions to cell damage caused by contaminants (other bovine pancreatic enzymes) in the DNase solution.(13) Recently a recombinant human DNase (rhDNase) produced in human embryonic kidney cells was evaluated in vitro using sputum from CF patients; the rhDNase rapidly thinned the sputum.(13) A preliminary study of rhDNase in CF patients has indicated that the enzyme is safe and effective in thinning respiratory secretions.(2)
Like the finding of high DNA levels in CF secretions, the link between CF and defective ion transport was first discovered in CF patients. During a 1948 heat wave in New York City, clinicians W. R. Kessler and Dorothy H. Andersen noted that 5 out of 10 children admitted to Babies Hospital for heat prostration had previously been diagnosed with CF. In a 1951 Pediatrics paper, they speculated that children with CF might be especially prone to heat prostration.(14) Based on this report, pediatrician Paul A. di Sant'Agnese and his colleagues exposed normal children and those with CF to an ambient temperature of 90°F and collected their sweat for study. In the sweat of CF patients, chloride-ion concentrations were triple those of normal controls; sodium-ion levels, too, were greatly elevated. Urine analysis revealed that in CF patients the kidneys conserve chloride and sodium ions, but continued and uncontrolled loss of these ions through sweat eventually leads to heat prostration.(15) These findings confirmed clinical impressions that CF patients are particularly vulnerable to heat stress and indicated that CF affects more of the body's physiology than had previously been realized; they also strongly supported the hypothesis that CF involves a defect in ion transport.(15)
The precise nature of CF's ion-transport defect was determined in vitro. In the 1980s, Paul Quinton demonstrated that chloride ions cannot penetrate the membranes of certain types of cells from CF patients.(1) The major breakthrough in understanding CF's molecular basis came in 1989 when, using in vitro techniques, researchers studying human molecular genetics pinpointed a defective gene (the CFTR gene) in CF patients that they suspected was responsible for causing the disease.(1) Within a year, researchers transferred a normal human CFTR gene, in vitro, into airway(4) and pancreatic(16) epithelial cells from CF patients; the cells acquired the ability to transport chloride ions normally. This in vitro work confirmed that a mutant CFTR gene causes CF's defect in chloride-ion transport and indicated that transfer of a normal CFTR gene might normalize chloride-ion transport in CF patients.(4,16)
Transfer of the Normal Human CFTR Gene to Rats
In work reported in 1992, Melissa A. Rosenfeld et al. transferred a norma human CFTR gene--in vitro--into epithelial cells from CF patients by infecting these cells with Ad-CFTR, replication-deficient adenovirus containing the CFT1 gene. The cells then produced structurally normal human CFTR and showed normal chloride-ion transport.(8)
Rosenfeld and her colleagues followed this in
vitro success with an in vivo demonstration.
Again using Ad-CFTR infection, they transferred a
normal humai CFTR gene into epithelial cells in the
lungs of cotton rats. Like the cells from CF patients,
the cells in the rats’ lungs responded to the
transfer by producing structurally normal human CFTR.
With regard to this result of gene transfer, then,
the rat work merely duplicated previous in vitro work.
The rat work was even less useful with regard to
the transfer's effect on chloride-ion transport:
Unlike the in vitro research, the rat research could not reveal whether transfer of a normal human CFTR gene can correct a defect in chloride-ion transport--because the rats never had such a defect. Rosenfeld et al. acknowledge, "As there is no animal model for cystic fibrosis, it is not possible to demonstrate definitively the function of the Ad-CFTR-directed CFTR protein in vivo."(8)
To date, progress in transfer of the normal human CFFR gene has derived from in vitro work, not animal experimentation.
Animal Models of CF
Two genetic diseases in mice have been studied as
models of CF. One of these diseases, a "cribriform
degeneration" suffered by some DBA/2J mice, involves
degeneration of the central nervous system characterized
by cell death that leaves scattered cavities in the
brain and spinal cord. At about 2½ to 3 weeks
of age, mice with cribriform degeneration begin to
show stunted growth, overall weakness, and incoordination.(17)
Because the sweat of CF patients had shown elevated
levels of sodium and chloride ions,(15) the finding,
in the early 1970s, that mice with cribriform degeneration
have abnormally high sodium-ion levels in their fur
sparked interest in this disease as a model of CF.(17)
However, researchers soon discovered that the abnormally
high sodium-ion concentrations in the fur of the mice
is not accompanied by sweat-gland dysfunction (which
occurs in CF). The only ion-transport dysfunction found
was excessive sodium-ion secretion by the parotid salivary
glands. Through their saliva, the mice were transferring
sodium ions to their fur--by licking themselves during
grooming.(17) In addition, the researchers' focus on
sodium ions would later prove to have been misguided:
Paul Quinton’s in vitro work would reveal
that CF arises from a defect in the transport of chloride,
not sodium, ions.(1)
Another proposed genetic model of CF involves the use of CBA/J mice. Like CF patients, some CBA/J mice suffer from exocrine pancreatic insufficiency (EPI), inadequate secretion of digestive enzymes produced in the pancreas. For this reason, mouse EPI has been suggested as a model of the EPI of CF patients.(18) Although both EPIs result from the destruction of pancreatic cells, the causes of the cell destruction fundamentally differ. (19-21) In CF patients, the destruction results from obstruction of the pancreatic ducts by thickened secretions;(19) in CBA/J mice, it results from digestive enzymes produced in the pancreas being activated within the pancreatic cells themselves (instead of being secreted into the small intestine and activated there, as is normal).(20,21) Because mouse EPI and the EPI of CF patients have different causes, the mouse model is unlikely to reveal ways of halting the pancreatic destruction that accompanies CF.
Other researchers have used artificially induced bacterial infection in nonhuman animals as a model of the pulmonary infection seen in CF patients.(5) For decades, it has been known that the vast majority of teenage CF patients suffer recurrent respiratory infection by the bacterium Pseudomonas aeruginosa--infection associated with increased morbidity and mortality.(5) Among those who do not suffer this chronic infection, many have developed opsonizing antibodies (antibodies that facilitate white blood cells' engulfment and destruction of bacteria) against mucoid exopolysaccharide (MEP), a carbohydrate in the bacterium's outer coat.(5) Following clinical observation of the benefits of opsonizing MEP antibodies, experimenters examined whether these antibodies confer protection against P. aeruginosa in nonhuman animals as well. Gerald B. Pier and colleagues injected rats and mice with MEP and then exposed them, via the trachea, to P. aeruginosa. Those animals who had responded to MEP injection by producing opsonizing MEP antibodies developed significantly fewer P. aeruginosa infections than those who had not produced these antibodies. These findings, however, provided no new information of any practical significance; they merely paralleled findings in CF patients.(5)
The most popular CF animal models involve the use
of drugs to impair the autonomic nervous system (ANS),
which regulates involuntary functions. Drugs such as
isoproterenol or pilocarpine are sometimes used to
block the action of catecholamines (neurotransmitters
active in the ANS) and create symptoms similar to those
seen in CF. Usually, however, reserpine is used to
deplete catecholamine reserves and thereby impair the
In the mid-1970s, researchers introduced reserpine-induced ANS dysfunction as a model of CF.(18) In rats, seven daily reserpine injections cause an accumulation of secretions that blocks the ducts of some exocrine glands.(22,23) This blockage resembles that in CF.(22) Although reserpine-treated rats produce excessive intestinal mucus,(22,23) as do CF patients,(23) the rat mucus differs from the human mucus in biochemical composition.(23) In addition, the cause of exocrine-gland pathology in CF (a defect in chloride-ion transport)(1) fundamentally differs from the cause in reserpine-treated rats (ANS dysfunction).(18,22) Originally the reserpinetreated rat (RR) model arose from the belief that faulty ANS control caused the dysfunction of some exocrine glands in CF.(18) Since then, however, in vitro work has shown that CF exocrine-gland pathology is due to an inherent molecular flaw in the secretory epithelial cells of exocrine glands, not to abnormal ANS innervation.(22)
Studies of the RR model of CF have continued because
reserpine-treated rats possess some secretory epithelial
cells (for example, in the pancreas, salivary glands,
and trachea) that appear to have a chloride-ion transport
defect.(24) Researchers have found, however, that ion
transport in rats treated with reserpine or other compounds
that impair the ANS differs in important ways from
ion transport in CF patients. In the 1980s, human studies
revealed that in CF patients the epithelial cells of
the nose, trachea, and bronchi have an electrical potential
difference, or PD (the difference in charge between
a cell's inside and outside) of approximately -50 mV,
while in control subjects the PD is approximately -20
mV.(6) The PD is more negative in CF patients because
their respiratory epithelial cells can absorb sodium
ions (which are positively charged) but not chloride
ions (negatively charged)--creating excess negativity
outside the cell. Following these clinical findings,
Duncan F. Rogers et al. treated rats with reserpine
and isoproterenol and measured the effects on the PD
of tracheal epithelial cells in vivo. The
treatments did not produce the desired increase in
PD negativity; instead, the tracheal PD of the treated
rats did not significantly differ from that of controls.
Rogers et al. concluded that the imbalance in ion absorption
that produces excessively negative airway PD in CF
patients is, in all likelihood, "not present in
As reported by gastroenterologist H. J. Veeze and colleagues, CF and the RR model also appear to differ in intestinal chloride-ion transport: In CF patients this transport is blocked, but in reserpine-treated rats it is normal. Veeze et al. conducted in vitro work in which they treated cells from the small intestine of CF patients with various drugs and metabolites that stimulate chloride-ion secretion in normal human intestinal cells. As expected, the treated cells failed to secrete chloride ions. In contrast, when Veeze et al. exposed cells from the large intestine of reserpine-treated rats to these same compounds, the cells did secrete chloride ions. The researchers interpreted the results as indicating "normal function and regulation" of intestinal chloride-ion channels in the reserpine-treated rat.(25)
Further, in humans, CFTR-mediated chloride-ion transport (defective in CF patients) is regulated primarily by the metabolite cyclic AMP, with a minor contribution from calcium ions.(24) In rats, however, calcium ions are the major regulator of chloride-ion transport--at least in the pancreas and salivary glands, where chloride-ion transport is impaired by reserpine.(24) Because of these differences, J. Ricardo Martinez and A. M. Martinez have written that studies of pancreatic and salivary cells in reserpine-treated rats are of questionable relevance to CF.(24)
Most recently, animal experimenters have attempted to study CF by means of a transgenic-mouse model. In 1992, J.N. Snouwaert and colleagues announced that they had produced transgenic mice who carry a defective human CFTR gene and, as a consequence, lack functional CFTR.(26) Although researchers working with the transgenic-mouse model are touting it as a breakthrough,(26-28) the pathology in the transgenic mice significantly differs from that in CF. In the mice, the primary pathology is intestinal obstruction due to thickened mucus. Most CF patients, however, do not experience intestinal obstruction.(26) Further, the mice do not manifest the pancreatic, reproductive, or respiratory pathologies found in CF.(26,27) Although they show abnormal chloride transport in nasal epithelial cells and some increase in the number of tracheal cells that produce mucus, the mice do not develop pulmonary obstructive disease--the most life-threatening feature of CF.(26,27) A number of factors could underlie the absence of pulmonary obstruction in the mice. Compared to humans, mice have far fewer mucus-producing glands lining their trachea.(27) Also, unlike humans, mice possess a second ion-transport system in their airway; this second system may compensate, at least partially, for the loss of functional CFTR.(27,28) Given these major differences in pathology, anatomy, and physiology between humans and mice, how can data obtained from the transgenic mice be meaningfully extrapolated to humans? The transgenicmouse model is highly unlikely to provide insights applicable to treatment of CF.
The history of cystic fibrosis research reveals that
progress in understanding and treating the disease
has derived from clinical and in vitro studies.
Researchers generally agree that no valid animal model
of cystic fibrosis exists. The animal models currently
being manipulated or proposed are fundamentally flawed.
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