Cystic Fibrosis

Question 1

what are the characteristics of mouse models of cystic fibrosis in terms of:

1. intestinal disease
2. pancreatic disease
3. lung disease
4. nasal and tracheal profiles
5. fertility
6. body weight

Answer 1

1. hallmark of most CF mouse models; severe pathologies seen (failure to thrive, intestinal obstruction and abnormal electrophysiology); mirrors human disease closely
2. lack of pancreatic pathology or only mild pancreatic pathology seen in most models. Mouse pancreas may have an alternative compensatory Cl^- channel
3. milder phenotype than human condition. only 1 model displays spontaneous inflammation. possible compensatory Cl channel
4. accurately replicates human profile - hyperabsorption of Na and defective Cl secretion
5. normal fertility in males; females took longer to conceive than WT littermates, which may be due to a slight impaired fertility
6. lower than normal body weight, mirroring the human disease.

Question 2

1. describe the overall lung pathophysiology of cystic fibrosis (2 points)
2. name 3 experiments which have highlighted one of these impacts

Answer 2

1. depletion of PCL, therefore unable to clear bacteria and mucous, making infection likely

CFTR is also found in immune cells, and mutattions cause them to be overstimulated leading to excessive inflammation

* decrease in lung function caused by infection and inflammation*

2. fluorescent dye was used to highlight the height of the PCL following the addition of excell water. Normal cells return the height of the PCL to 7.5μm (associated with fully extended cilia); CF cells return the height to 2.5μm

electron microscopy show that the cilia in CF cells are bent

Vte was studied - in normal cells, this falls over time. this doesnt change in CF cells.
at 0 hours, amiloride had a bigger effect than bumetanide in normal cells; in CF cells, there was very little response to bumetanide, but a very large response to amiloride

at 48 hours, in normal cells, the response to bumetanide was the same as time zero, but the response to amiloride is smaller. In CF cells, there is a slight but non significant increase in response to bumetanide (are other CF channels involved?

Question 3

1. why is work in static cultures not representative of normal physiological conditions?
2. what was performed to ensure physiological conditions?
3. what were the results of adding excess water under these physiological conditions?
4. what was the response to amiloride and bumetanide at 0 and 48 hours under these physiological conditions?
5. what were the effects of DIDs and CFTRinh172 on the height of PCL under these physiological conditions? What does this imply?

Answer 3

1. because breathing places a phasic force (shear stress) on the PCL (0.4-2dynes/cm²)
2. cells were set up on a moving platform to place shear stress on the PCL
3. normal cells reduced the height of the PCL to 15μM

CF cells reduced the height of the PCL to 7.5μL. This still allows cilia to beat to some extent, hence why some mucous movement is seen in CF patients

4. at 0 hours, normal cells have a greater response to A than B; CF cells showed a much greater response to A than B

at 48 hours, normal cells have a greater response to B than A (larger than the static model). In CF cells, there is a reduced response to A, and an increase in B (compensatory Cl channel?)

5. in normal cells, CFTRinh172 reduces the height of the PCL; DIDs further reduced the height of the PCL, suggesting at least one other Cl channel is involved in the maintenance of the PCL

In CF cells, CFTRinh172 has little effect on the height of the PCL. DIDs reduced the height of the PCL to a greater extent than normal cells, suggesting that CF cells are more dependent on/upregulate the other Cl channel.

Question 4

1. what is the concentration of ATP in the BL solution?
2. what causes the release of ATP into the apical solution?
3. What type of receptors are P2X and P2Y? What are they stimulated by?
4. what does P2Y activation lead to?
5. what does P2X activation lead to?
6. what does stimulation of P2Y₂ lead to in terms of Na absorption and Cl secretion?
7. What are P1 receptors stimulated by? How does their activation affect Cl secretion?

Answer 4

1. 0
2. shear stress
3. purine GPCRs. stimulated by ATP
4. increase in [Ca]_i by causing the release from IC stores
5. increase in [Ca]_i by activating PM Ca channels
6. decrease Na reabsorption and increase Cl secretion
7. adenosine. stimulates Cl secretion

Question 5

1. what does apyrase inhibit and how?
2. what does 8-SPT inhibit and how?
3. what were the effects of apyrase and 8-SPT on normal airway cells?
4. what were the effects of apyrase and 8-SPT on CF cells?
5. why are CF cells more dependent on ATP?
6. why is mucous clearance lower than expected in CF cells?
7. How was this (6) investigated?

Answer 5

1. P2Y - hydrolyses ATP
2. P1 - inhibits adenosine
3. apyrase had little effect on PCL height; 8-SPT reduced PCL height - suggests that P1 receptors and adenosine are important for Cl secretion in normal cells under phasic motion. Addition of both adenosine and 8-SPT caused a greater reduction in PCL height thus implicating a small role for ATP
4. apyrase caused a decrease in PCL height; 8-SPT had no effect
5. requred to release Ca that activates compensatory Ca activated Cl channels
6. CF lungs always tend to have a level of infection. RSV, along with other viruses and bacteria break down ATP
7. cells cultured under phasic motion were infected with RSV and the height of the PCL measured.

RSV caused a three fold decrease in the levels of ATP

in normal cells, RSV infection caused the PCL to reduced, but it was still maintained at physiologically relevant levels (9um)

in CF cells, RSV infection caused PCL to be 4.5um - this level fails to sustain mucous clearance.

Question 6

name 8 current therapies of cystic fibrosis

* what is the problem with these therapies?

Answer 6

1. inhaled bronchodilators
2. nebulised/oral antibiotics such as tobramycin, (an aminoglycoside)
3. physiotherapy
4. steriouds
5. fat soluble vitamins
6. pancreatic enzyme suppliments
7. nebulised hypertonic saline
8. mucolytics such as pulmozyne.

* PROBLEM: all treat the symptoms and not the cause

Question 7

1. Name the type of mutation, class of mutation (what is the defect?), incidence and severity of symptoms for:
   a) G551D
   b) ΔF508
2. How were drugs to treat the G551D mutation screened for?
3. How were drugs to treat the ΔF508 mutation screened for?

Answer 7

1a) glycine to aspartate. Class III mutation (affects channel gating); incidence of 1-3%. Produces severe symptoms
1b) phenylalanine deletion; class II mutation (impaired trafficking); indicidence of 90%; produces severe symptoms

2. cells were loaded with a fluorescent compound which changes colour with Vm
   228000 compounds were screened for their ability to change Vm that indicates CFTR activation
3. an immunoblot assay was performed looking at the level of mature CFTR.
   164000 compounds were screened for their ability to increase the amount of mature CFTR

Question 8

1. what mutant does VTX-770 treat?
2. how were the effects of VTX-770 on SSC in rat thyorid cells examined? What were the results?
3. How were the effects of SSC in human bronchial epithelial cells examined? What were the results?
4. When studying PCL volume and cilliary beat frequency:
   a) what was used to stimulate CFTR?
   b) what were the effects of VTX-770 on PCL volume?
   c) what were the effects of VTX-770 on cilliary beat frequency
5. What was the effect of VTX-770 in clinical trials?

Answer 8

1. G551D
2. forskolin was used to stimulate CFTR and SSC measured.
   In normal cells, forskolin caused an increase in SSC that corresponds to CFTR opening
   in G551D cells, SSC does not change in response to forskolin. Addition of VTX-770 causes an increase in SSC
3. performed in G551D/ΔF508 heterozygotes. Amiloride was used to remove the contribution of ENaC to SSC

WT response = 56μA. Mutant response was 5% of WT
VTX-770 cause the response of the mutant to be 50% of WT (same as asymptomatic carriers)

4a) VIP
4b) VTX-770 brings PCL volume to ~ halfway between that of mutant and WT
4c) VTX-770 causes ciliary beat frequency of mutant cells to be the same as WT cells.

5. significant positive result of FEV1 (as a percentage of predicted FEV1)
   fewer negative events such as infections
   salt sweat was still high but below threshold for CF diagnosis
   isoprotenerol, which was used to drive Cl secretion caused a large increase in Cl secretion in the presence of VTX-770.

Question 9

1. what mutation does VTX-809 treat?
2. how were the effects of VTX-809 on the level of mature CFTR analysed? What were the results?
3. how were pulse chase experiments used to examine the levels of immature and mature CFTR in response to VTX-809 treatment? What were the results?
4. how were the effects of VTX-809 on patient cells examined?
5. what were the results of VTX-809 clinical trials?
6. Name a disadvantage of VTX-770 and VTX-809 treatments

Answer 9

1. ΔF508
2. western blotting, which examined the level of mature CFTR as a ratio of immature CFTR

VTX-809 increased the level of mature CFTR

3. human embryonic kindeny cells were exposed to ³⁵S methionine/cysteine, therefore radiolabelling CFTR
   cells were exposed to the radioactivity for different periods of time, and then the proteins were run on gel
   In WT cells, the immature band decreased over time, and the mature band increased over time
   in mutant cells, the immature band decreased over time, however the mature band doesn’t appear
   VTX-809 treatment of mutant cells causes more of the mature band to appear
4. again, amiloride was used to inhibit ENaC, and SSC was measured
   VTX-809 caused the current to increase; a CFTR inhibited these currents, meaning that these currents were mediated by CFTR
5. VTX-809 had little effect on lung function or reducing salt sweat. However, using VTX-809 in combo with VTX-770 caused a dramatic effect on lung function.
6. very expensive

Question 10

1. What is atypical CF characterised by?
2. why must modifier genes be involved?
3. What type of mutations in which protein channel are thought to cause atypical CF and why?

Answer 10

1. mild symptoms, with one or more mutations in CFTR
2. because carriers of CF are asymptomatic, therefore just one mutant CFTR copy is not the cause of atypical CF
3. GOF mutations in ENaC
   this would enhance Na absorption, leading to depletion of PCL
   in combination with 1 mutated CF allele, this would be sufficent to cause CF like symptoms.

Question 11

1. How many mutations in ENaC were found in atypical CF patients?
2. What assumption does this mutation screen make?

Answer 11

1. 11 in alpha subunit; 7 in beta subunit and 8 in gamma subunit
2. that these mutations change protein funcion therefore can cause the disease

Question 12

1. Where is the W493R mutation found?
2. what was the repsonse of normal cells, classical cells and W493R cells in response to amiloride or changing [Cl]
3. what does this say about the W493R mutant?
4. How was the effect of this mutation on Na feedback inhibition measured? What were the effects?
5. How was the effect of this mutation on channel cleaveage measured? what were the effects?
6. How were the effects of this mutation on Na self inhibition measured? What were the effects?
7. Based on these results, what regulatory mechanism is lost in W493R mutants?

Answer 12

1. in the EC loop of the alpha subunit of ENaC
2. normal cells show a bigger response to the addition of amiloride rather than changing Cl
   classical CF cells showed a very large amiloride response (enhanced ENaC function in classical CF)
   W493R cells had a very large amiloride response, and a much large effect of changing Cl than classical CF
3. Cells have normal CFTR function, but enhanced ENaC currents compared to WT
4. EC [Na] was measured in high and low [Na]. The ratio of the 2 indicates ENaC endocytosis
   the ratio was the same in both WT and W493R mutants
5. investigated the current in response to chymotrypsin.
   In WT cells, chymotrypsin caused a large response (cleavage of channel enhances channel activity)
   chymotrypsin caused no effect in W493R cells - was the channel already cleaved?

if channels were already cleaved, you would expect that the single channel trace of W493R cells to be the same as WT channels

6. measured current
   W493R channels had lost Na self inhibition (no decrease in I_Na
7. It is Na self inhibition that is lost leading to enhanced ENaC currents

Question 13

1. Where is the V348M mutation?
2. What is MTSET and what was it used to measure?
3. What were the results of the above experiments?
4. What does this say about the GOF of the V348M mutation?

Answer 13

1. in the beta subunit
2. a sulfhydryl agent which binds to cysteins and stabilises channel with a Po of 1. By taking the ratio of amiloride sensitive currents before and after MTSET treatment, the Po of the channel can be determined
3. WT Po = 0.24; V348M mutant Po = 0.33
4. mutant ENaC is open for longer.

Question 14

1. How was the mouse model of Atypical CF created?
2. describe the phenotype of atypical CF mouse models (4 points)

Answer 14

1. overexpression of the beta subunit of ENaC to mimick GOF
2. reduced PCL height
   reduced mucous clearance
   reduced survival
   reduced clearance of bacteria injected into trachea.