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BMS346 - Epithelial Physiology > L6 - Alternative Treatments > Flashcards

L6 - Alternative Treatments Flashcards

1
Q

What was originally thought was going to be the magic bullet for CF treatment

A

Using gene therapy to introduce WT anf fucntional CFTR back into cells

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2
Q

Describe the progression of gene therapy

A

Started with adenoviruses but progressed to using liposome complexes

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3
Q

Describe the conditions of the non viral CFTR trial

A

140 patients tested, 9 doses every 28 days
62 placebo, 78 with p/GM169/GL67A
(ombination of cationic liposome (GL67A) and plasmid DNA expressing CFTR (pGM169)
Placebo was a 0.9% saline

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4
Q

Describe the change in airway function for the non viral CFTR gene therapy

A

In placebo see a steady decline in lung function

In the gene therapy group - there is no improvement in lung function but lung function DOES NOT GET ANY WORSE

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5
Q

What determined how effective the gene therapy was

A

Determined by individuals - some responded well - others didnt

Could be due to differences in the genetic background of these patients

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6
Q

Was is gas trapping

Describe what effect would be seen on gas trapping if this treatment was working

A

Secondary obstruction of the small airways - likely to occur in infants with CF

If treatment is working would expect to see a reduced incidence of gas trapping

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7
Q

What was used a direct measure of how well the gene therapy was being taken up by cells

A

Looking at the change in bronchial DNA

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8
Q

describe the change in bronchial DNA for the placebo group

A

No change - this is expected they have not been exposed to the foreign DNA

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9
Q

Describe the change in bronchial DNA for the gene therapy group

A

Had a range of success

In two of the patients it was completely uneffective

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10
Q

Describe how the chrloride response was measured

A

Injection of a chloride free ISOPROTENEROL solution

This low EC Cl gives a huge driving force for Cl secretion across the apical membrane can mesaure thisby measure the nasal potential

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11
Q

Describe the chloride response in the treatment group

A

Those who had had the gene therapy see the most chloride secretion

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12
Q

What does all of the data presented in the non viral CFTR gene therapy suggest

A

That there is the provision lung function stabilisation

However the population was heterogeneous - some responded well others not so well

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13
Q

What is one critisism of the non viral gene therapy study

A

Saline is not the best control

Should (if in vitro) have used scrambelled CFTr and liposomes - this is for ethical reasons

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14
Q

What is the function of ENaC in CFTR patients

A

Massively enhanced

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15
Q

In the upper airway what it the relationship/interaction between CFTR and ENaC

A

direct inhibition

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16
Q

If CFTR is mutant what is the knockon effect on ENaC

A

There is no longer any inhibition of ENaC
Lose Cl secretion and enhance Na absorption
Thus height of the ASL is EVEN LOWER than expected

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17
Q

If we know CFTR function is enhanced then what would one potential alternative treatment be?

A

Block ENaC - could this lead to an increase in the height of the ASl

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18
Q

Most obvious first experiment to block ENaC

What were the reuslts of this?

A

Using amiloride

This was not effective at all

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19
Q

if amiloride was ineffective what could be tried

A

SPLUNC1

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20
Q

What is SPLUNC1

A

A naturally occuring ENaC antagonist

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21
Q

What does SPLUNC1 stand for

A

Secreted protein short plalate lung and epithelial clone 1

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22
Q

Describe what normally happens to ENaC in the absence of SPLUNC1

A

Normally enzymes such as trypsin etc. catalyse a proteolytic cleavage of ENaC which increases the Po and thus Na absorption is increased

23
Q

What then is the function of SPLUNC1

A

Binds to ENaC preventing cleavage

Suppresses Na absorption across the apical membrane

24
Q

Describe what was seen adding trypsin in the absence of splunc1

A

Currents enhanced - SPLUNC1 cleaves ENaC leading to an increase in Po

25
Q

What was the effect of adding SPLUNC1 wihtout any trypsin

A

Currents recorded were significantly lower

26
Q

Why were currents decreased when SPLUNC1 added on its own WITHOUT ANY TRYPSIN

A

Blocks the endogenous cleavage by other enzymes in the cell

27
Q

Describe what was seen when adding SPLUNC1 and trypsin

A

Currents were smaller than when without any SPLUNC1

28
Q

What was used to take forward the SPLUNC1 experiments …

A

S18

29
Q

What is S18

What was the first question that needed to be adressed when looking at its function

A

It is the ENaC inhibitory domain of SPLUNC1

Need to check that it still inhibits ENaC when it is without the rest of the protein

30
Q

Describe the premise of the experiments used to ensure that S18 binds to and inhibits ENaC

A

Using competitive binding experiments with an alexa fluor 488 SPLUNC1

When SPLUNC1 binds ENaC fluroescence can be detected -

31
Q

Describe the results of the competitive binding experiment at pH 6

A

Not alot is happening here …

Shows ACIDIFICATION INHIBITS THE BINDING

32
Q

What were the results of the SPLUNC1/S18 competitive binding experiment

A

Where s18 and SPLUNC1 are both present there is less relative fluorescence than when just SPLUNC1 alone

This is because s18 competes for ENaC binding and flourecne is only seen if SPLUNC1 binds … so with compeitive inhibition there is less SPLUNC1/ENaC binding

33
Q

Conclusions that can be drawn from the SPLUNC1/S18 compeitive binding experiment?

What is the next question that needs to be addressed

A

That S18 binds to ENaC at the same place

Does this binding prevent cleavage in the same way as SPLUNC1 does

34
Q

With drug experiments what should be used as a control

A

the vehicle only control

E.g. the substance the drug was dissolved in (for insoluble compounds this is usually DMSO)

35
Q

Describe the affect on the ASL of the exposing HBE cells to following

Vehicle only

Amiloride

S18 (why)

A

Height low

No change - dont really know why

Height of the ASL has increased - Na absorption blocked les driving force for the movement of the water out of the ASL between cells

36
Q

Describe the effect on the ASL height when started above optimum height ~30um of the following

Vehicle

Amiloride

S18

A

Height starts too high and ends up too low for both vehicle control and the amiloride

For s18 the height of the ASL stabilises at a higher level

Bringing the height of the ASL down is mediated through the absorption of Na through ENaC - with this inhibited ENaC isnt able to bring the level down as much

37
Q

Looking at amiloride sensitive transepithelial potential and short circuit currents what does a big gap mean

A

Big gap = big function of ENaC
How much ENaC is contributing to the potential (the more this value the more ENaC is providing to the transepithelial potential)

38
Q

what was the effect of S18 on both the transepithelial potential and the short circuit current

A

With S18 ENaC was only contributing 1mV to the Vte (75% inhibition)
with s18 also reduced Isc

39
Q

Sum up the effect of S18 in the HBE cells

A

S18 inhibits the proteolytic cleavage of ENaC

This decrease the Po of the channel and thus the currents are reduced

40
Q

How does the cell model for the alveolar cell differ from the upper airway cells

A

Here both CFTR and ENaC absorb

CFTR is involved in Cl absoprtion and NOT Cl secretion

41
Q

What will the effect of an ENaC blocker in the alveolar cells mean for the height of the ASL

A

Increase the height of the ASL since Na reabsorption is blocked here

42
Q

Describe the effect of the following on alveolar surface liquid height

Vehicle

Amiloride

S18

A

Low

No change

Height of the layer increases

43
Q

What is one of the benifits of using S18

A

There are no systemic effects

44
Q

What is a systemic effect

A

An off target effect at another organ

45
Q

Where would it be expected that S18 would have an off target effect? why is this

A

At the kidney

Amiloride (another ENaC blocker has effects at the kidney

46
Q

Effect of S18 and amiloride on URINE FLOW RATE

A

S18 has no impact on urine flow rate

Amiloride increase since less Na reabsorbed

47
Q

Effect of S18 and amiloride on fractional extretion of Na

A

s18 has a minimal impac

Amiloride has an impact here since ENaC is blocked less na can be reabsorbed and thus fractional excretion will increase

48
Q

Effect of S18 and amiloride on fractional excretion of K

A

S18 - no significant difference
amiloride - decreased fractional excretion of K since Na absorption drives K secretion by the principal cells of the collecting duct

49
Q

Effect of S18 and amiloride on the plasma [K]

A

S18 -no significant difference
Amiloride - over time increases the levels of plasma K, chages the excitability of nerves etc. people would be at risk of cardiac arrhytmias and sudden cardiac death

50
Q

Why don’t mice make the best models for cystic fibrosis

A

Since mice also have Ca activated Cl channels which can take over from CFTr when this is mutated

51
Q

What is the effect of S18 on the height of the ASL in WT mice

A

Increases the height of the ASL

52
Q

What is the effect of S18 in mice models of cystic fibrosis

A

Incrased height of the ASl

53
Q

Since mice arent ideal models of CF was model organism was used to tes S18

How was this model made

What was measured

A

sheep

Pharmacological model - CFTR was blocked

Tracheal mucous velocity was measured

54
Q

Results from the Sheep CF model

A

Tracheal mucous velocity initially decreases as a CFTR inhibitor is added
When treated w/ S18 shows a sustained recover in increase of TMvelocity
Same effect is seen when amiloride is added but these effects are TRANSIENT AND SHORT LIVED

BMS346 - Epithelial Physiology (10 decks)

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