RespoLecoDecko 2 Flashcards
(607 cards)
1
Q
Structural Lung Cell Biology
Desmosomes are between which cells in the epithelium?
A
Between Basal cells and epithelial
2
Q
Structural Lung Cell Biology
Adherens junctions are between which cells?
A
Epithelial cells
3
Q
Structural Lung Cell Biology
Describe Trachea/Bronchus epithelium?
A
Polarised Layer Pseudostratified Apical and Basal surfaces Columnar Basal cells not polarised
4
Q
Structural Lung Cell Biology
What cells are present in the conducting airways?
A
Ciliated cells
Goblet cells
Basal cells
5
Q
Structural Lung Cell Biology
How does the organisation differ with bronchioles (vs Bronchi)?
A
Bronchioles = cuboidal (rather than columnar)
6
Q
Structural Lung Cell Biology
Describe orientation of cilia.
A
All have the same orientation on a cell
Cells have similar orientations to neighbours
Beat towards pharynx
7
Q
Structural Lung Cell Biology
What two exocrine cells are within the airways?
A
Serous = watery secretions
Mucous cells = Mucous
8
Q
Structural Lung Cell Biology
List non-ciliated // secretory cells present in airways (structural)
A
Brush Cells Neuroendocrine cells Basal cells (precursors)
9
Q
Structural Lung Cell Biology
Which cell is the most abundant in the alveoli?
A
Type 2 pneumocytes
10
Q
Structural Lung Cell Biology
What is the function of type 2 pneumocytes?
A
Secretory cells
Differentiate into T1
11
Q
Structural Lung Cell Biology
Which cell contributes to the vast surface area of the alveoli?
A
T1 pneumocytes
97% of SA
12
Q
Structural Lung Cell Biology
Describe structure of AECI
A
Small nucleus Basic organelles 2 plasma membranes Apical glycoprotein coat Cannot regenerate
13
Q
Structural Lung Cell Biology
Describe structure of AECII
A
Cuboidal, thick
Many organelles
Surfactant producers
(A549 developed from adenocarcinoma - cell used in labs)
14
Q
Structural Lung Cell Biology
Describe capillary endothelial cell structure
A
2 plasma membranes
Polarised
Glycocalyx in lumen
Microfilaments+tubules
Microvesicles
Tight junctions - NOT as tight as epithelium
Tightness = not uniform ie less tight in capillary (mediated by VE-cadheren)
Adherens junctions = movement of fluid + solutes
No fenestrations
Basal side attached to SM-like pericytes
15
Q
Structural Lung Cell Biology
What stimulates VE-cadheren to break apart?
A
IL1B, TNF-a
16
Q
Structural Lung Cell Biology
What protein enhances VE-cadheren barriers EVEN in the presence of inflammatory stimuli?
A
SLIT
17
Q
Structural Lung Cell Biology
Name some differences of the endothelium of larger blood vessels in comparison to capillaries
A
Increased thickness Arterial = Elliptical Venous = Polygonal More organelles Unique organelles eg Weibal Palade bodies (make VWF+F8)
18
Q
Structural Lung Cell Biology
Describe functions of fibroblasts?
A
Form fibres eg elastin // collagen
Also contractile
19
Q
Airway Mucus Production and ‘Pharmacotherapy’ of Overproduction
How many “barrels of beer” of air do we breath each day?
A
73 barrels
Equivalent to ~12k litres
20
Q
Airway Mucus Production and ‘Pharmacotherapy’ of Overproduction
What did the Copenhagen Heart study find with regards to Mortality?
A
R:R Increased with CMH (chon muc hyper)
Increased even more if concomitant infection
21
Q
Airway Mucus Production and ‘Pharmacotherapy’ of Overproduction
What did the copenhagen heart study find with regards to LF? How does this differ between sexes?
A
CMH associated with excess decline in LF
~25ml/yr with men
~10ml/yr with women
Appears to affect men more
22
Q
Airway Mucus Production and ‘Pharmacotherapy’ of Overproduction
What did the copenhagen Heart study discover with regards to hospitalisation? How does this differ between sexes?
A
CMH associated with:
- 5x risk of hospitalisation in men
- 5x risk of hospitalisation with women
23
Q
Airway Mucus Production and ‘Pharmacotherapy’ of Overproduction
What happens to plasma exudation (into airway) in asthma?
A
Increased
24
Q
Airway Mucus Production and ‘Pharmacotherapy’ of Overproduction
Why are goblet cells seen in increased numbers in COPD/Asthma?
A
Increased DIFFERENTIATION of other cells to goblet cells
25
Airway Mucus Production and 'Pharmacotherapy' of Overproduction
What happens to submucosal glands in Asth/COPD?
Hypertrophy
26
Airway Mucus Production and 'Pharmacotherapy' of Overproduction
Describe the "cascade" of mucus hypersecretion
Inflammatory stimuli -> inflammation + nerve activation -> Secretagogues -> inc MUC secretion + expression -> Secretory cell hyperplasia -> mucus hypersecretion -> Clinical symptoms
27
Airway Mucus Production and 'Pharmacotherapy' of Overproduction
Using the "cascade" of CMH, discuss the possibilities of targets
Inflammation -> anti inflammatory drugs
Nerve activation -> Nerve inhibitors (must be highly selective)
Secretagogues -> mediator antagonists / anticholinergics
Mucus secretion -> anti-exocytosis
Muc expression -> EGFR TK inhibitors / MAPK/MEK ERK/ hCLCA1 inhibitors
RAR-a antagonists
Secretory cell hyperplasia -> pro-apoptotic factors
Mucus hypersecretion -> mucolytics/hydrators
28
Airway Mucus Production and 'Pharmacotherapy' of Overproduction
What did Jay A Nadel discover in 1999?
EGFR (ERB1) is intimately involved in rat model of Goblet cell hyperplasia + mucin synthesis
Can be inhibited by EGF TK inhibitor BIBX1522
29
Airway Mucus Production and 'Pharmacotherapy' of Overproduction
Why might EGFRs be a good target in CMH?
Upregulation of ErbB receptors in asthmatics/smokers/COPD
Upregulation of ErbB ligands (EGF/TGFb) in COPD/asthma
Upregulated on Neutro/Eosino/Macrophages
30
Airway Mucus Production and 'Pharmacotherapy' of Overproduction
Describe EGF receptor signalling mechanism
ErbB1/2 monomers come together to form heterodimer on ligand binding
ATP phosphorylates tryrosine residues Grb2/SOS/SHC
Intracellular signalling:
- GDP -> GTP
RAS->RAF->MEK->ERK
ERK activates TF SP1
Transcription of MUC5AC
31
Airway Mucus Production and 'Pharmacotherapy' of Overproduction
Which Erb inhibitor caused dose-response inhibition of MUC5AC expression?
ErbB1 inhibitor
| ErbB2 inihibitor DID NOT inhibit MUC5AC expression
32
Airway Mucus Production and 'Pharmacotherapy' of Overproduction
What happened with Erb inhibitor (BIBW2948) when trialled?
FEV1 decline (on visit 5) actually worse in Rx group
Poorly tolerated by Pts
May need antidifferentiation rather than proliferation
33
Airway Mucus Production and 'Pharmacotherapy' of Overproduction
List 3 main MUC genes in respiratory tract?
MUC5AC
MUC5B
MUC2
34
Airway Mucus Production and 'Pharmacotherapy' of Overproduction
Describe structure of mucin monomers
Highly glycosylated
Sulf-hydryl groups allow joining of monomers end-end to form huge mature mucin polymers. Among largest molecules in nature
35
Airway Mucus Production and 'Pharmacotherapy' of Overproduction
Describe mucin exocytosis. Think of the ion that enables this.
When packaged water is removed and calcium is added which promotes folding of large molecules.
When exocytosed, water is added which washes out calcium. Sudden expansion as mucin proteins unfold.
36
Airway Mucus Production and 'Pharmacotherapy' of Overproduction
How does C.botulinum block secretion of neurotransmitters?
Cleaves SNARE proteins using endopeptidase enzymes
| Vesicles fuse to membranes via SNARE complexes
37
Airway Mucus Production and 'Pharmacotherapy' of Overproduction
What was the hypothesis for Botox use in CMH?
Botox could target EGFR
EGFR on epithelial cells
EGFR upregulated in asthma/smokers/COPD
38
Airway Mucus Production and 'Pharmacotherapy' of Overproduction
What three domains are present on botox neuro-tox? BoNT/C
Neuronal binding domain
Translocation domain
Catalytic domain
39
Airway Mucus Production and 'Pharmacotherapy' of Overproduction
How was BoNT/C structure altered for potential use for CMH?
Neuronal binding domain removed + replaced with EGF (able to bind to EGFR)
40
Airway Mucus Production and 'Pharmacotherapy' of Overproduction
What 3 proteins form the SNARE complex for mucin exocytosis?
Cellubrevin
Syntax
SNAP
41
Airway Mucus Production and 'Pharmacotherapy' of Overproduction
Describe the full theoretical mechanism of BoNT/C use for CMH?
Binds to EGFR
EGFR complex endocytosed
H+ concentration increases in endosome
Translocation domain allows BoNT/C to move into cytoplasm
Cleaves Syntaxin domain of SNARE complex (measured by antibody to syntaxin)
Mucin granule not released
42
Airway Mucus Production and 'Pharmacotherapy' of Overproduction
Why did BoNT/C fail?
Inhibited mucus secretion very well - conc dependent.
No affect on cell survival.
BUT CONCENTRATION had to be titrated -> too much resulted in intracellular BUILDUP of mucin
43
Airway Mucus Production and 'Pharmacotherapy' of Overproduction
What similar molecule (mechanistically BoNT/C) failed?
MARCKS antagonist protein (chaperones mucin vesicles)
44
Airway Mucus Production and 'Pharmacotherapy' of Overproduction
CF CMH drugs
Potentiators and correctors
| HS still a front-runner + cheap
45
Pulmonary Vessels
| What is the intima?
Single layer of endothelial cells
46
Pulmonary Vessels
| What does the adventita comprise of?
Fibroblasts
| ECM
47
Pulmonary Vessels
| What does the media contain?
Several layers of SM
48
Pulmonary Vessels
| Where does the external elastic laminum lie?
Between media + adventita
49
Pulmonary Vessels
| Where does the internal elastic laminum lie
Between intima and media
50
Pulmonary Vessels
| Differences between artery and veins
```
A - away
V - towards
A - High press
V - low
A - thicker, muscular
V - thinner but larger diameter
A - more elastic
v - high compliance
...................
```
51
Pulmonary Vessels
| How many generations in the pulmonary artery tree?
17
52
Pulmonary Vessels
| What are the main generations?
Elastic arteries
Muscular Arteries
Arterioles
53
Pulmonary Vessels
| Main function of muscular arteries?
Diversion of blood to improve V/Q
54
Pulmonary Vessels
| Discuss properties of Elastic arteries?
```
Thin walled
Highly elastic -> transmission of energy from heart
Have own blood supply
Few SM cells
Systolic and diastolic BP
```
55
Pulmonary Vessels
| What are arteriole functions?
Control BF to capillaries.
| Main resistance to blood flow -> determine overall pressure.
56
Pulmonary Vessels
| Describe the tri-partite structure
Alveolar cell // capillary // air
| T4 collagen for strength
57
Pulmonary Vessels
| How is pulmonary BP measured?
Swan-ganz catheter. Measures pulmonary wedge pressure -> balloon inflates in the PA.
PAWP = Left atrial Pressure
58
Pulmonary Vessels
| How can PVR be obtained?
```
PVR = (Mean PAP - LAP(PAWP))/CO
Normally = 1
```
59
Pulmonary Vessels
| What pressures cause fluid movement
```
Oncotic pressure - proteins either side (-28/-14)
Hydrostatic pressure (+7/-8)
Net pressure is +1 to interstitium
```
60
Pulmonary Vessels
| What is Laplace's relationship? (tension)
Tension (s) = (Pressure x radius) / thickness
| Increasing thickness reduces stress
61
Pulmonary Vessels
| Where do bronchial vessels arise from?
Aorta -> bronchial artery -> bronchial vein -> azygos vein to RA
62
Pulmonary Vessels
| List vasodilators in pulmonary circulation?
O2
NO
Prostacyclin
63
Pulmonary Vessels
| List vasoconstrictors in pulmonary circulation?
```
Endothelin-1
Low O2
High CO2
TX
TNFa
```
64
Pulmonary Vessels
| What are the effects of lung volume on pulmonary vascular resistance?
Extremely low - ie near RV results in compression of extra-alveolar vessels
Extremely high - near TLC - compresses intra-alveolar vessels
Results in U shaped diagram when combined
65
Pulmonary Vessels
| What is the effect of gravity on BF?
Inc at bottom of lung
| Ventilation - better at top
66
Pulmonary Vessels
| Normal CO.
~6mls/min
67
Pulmonary Vessels
| List 2 metabolic functions of the lung
Conversion of ANG1-2 by ACE in endothelial cells
| Bradykinin and serotonin degradation
68
Extracellular Secreted Proteases
| What is seen with regards to the antiprotease-protease balance in COPD?
Inc EC proteases
Dec antiproteases
Imbalance
Multifaceted -> can be that there are reduced amounts // dec function
Usually MUCH more antiproteases than proteases -> ie a1antitrypsin
69
Extracellular Secreted Proteases
| What are the main classes of protease in the lung?
MMP
Serine
Cysteine
70
Extracellular Secreted Proteases
| How many MMPs are there?
25
71
Extracellular Secreted Proteases
| What metal is at the active site?
Zinc
72
Extracellular Secreted Proteases
| How many membrane bound vs soluble (MMPS)?
6 Membrane bound
| 19 soluble
73
Extracellular Secreted Proteases
| What do MMPs 1,8,13 degrade?
Collagen
74
Extracellular Secreted Proteases
| What do MMPs 2 and 9 degrade?
Gelatin
Elastin
Collagen
75
Extracellular Secreted Proteases
| What do the Stromelysins (MMPs 3,10,11) break down?
Broad spectrum ECM
76
Extracellular Secreted Proteases
| Matrilysin (MMP7) breaks down what?
Broad spectrum
Elastin
Collagen
77
Extracellular Secreted Proteases
| What other purpose do MMPs have?
Modulation of inflammation via processing of cytokines/chemokines/GFs/adhesion molecules/other proteases
78
Extracellular Secreted Proteases
| What are the three cell sources of MMPs?
Neutrophils 8+9
Macrophages 1+2+7+9+12
Epithelium 1+2+9
79
Extracellular Secreted Proteases
| MMPs 3+9 are elevated in which lung disease?
Asthma
80
Extracellular Secreted Proteases
| MMP 9 alone is elevated in which lung disease?
CF
| Bergin et al 2013
81
Extracellular Secreted Proteases
| Which MMPs are elevated in COPD?
1,2,8,9
| Segura-Valdez et al 2000
82
Extracellular Secreted Proteases
| What is the difference between MMPs synthesised in epithelial cells vs neutrophils?
MMPs in epithelial cells = inactive pro-peptide
| Neutrophils = active
83
Extracellular Secreted Proteases
| What was the interesting discover by Senlor and Anthonlsen 1998?
MME deficient mice had reduced emphysema at 6 months in comparison to MME+ve
84
Extracellular Secreted Proteases
| What are the endogenous inhibitors of MMPs?
TIMPs
| Tissue inhibitors of MMPS
85
Extracellular Secreted Proteases
| How many TIMPs are there?
4
| Each capable of reversibly inhibiting all MMPs
86
Extracellular Secreted Proteases
| What other molecule inhibits MMPs 2+9?
a2-macroglobulin
| One of the largest plasma proteins
87
Extracellular Secreted Proteases
| In what disease is a2-macroglobulin found in the lungs?
ARDS
88
Extracellular Secreted Proteases
| Why are TIMPs increased in chronic lung disease?
Synthesised alongside MMPs.
TIMPs increased to control proteolytic damage.
Release + activity of TIMPs cannot restore balance
89
Extracellular Secreted Proteases
| What residues are present in the catalytic domain of serine proteases?
Serine
Histidine
Aspartic acid
90
Extracellular Secreted Proteases
| What are the main serine proteases in the lungs?
Neutrophil elastase
Proteinase 3
Cathepsin-G
Mast cell chymase and trypase
91
Extracellular Secreted Proteases
| What are the cellular sources of serine proteases?
Macrophages
Neutrophils
Mast cells
- Granulocytes
92
Extracellular Secreted Proteases
| What interesting phenomenon occurs with macrophages and NE/Cathepsin G?
Able to release both bound to surface receptors
| Able to engulf both and re-release
93
Extracellular Secreted Proteases
| What did Shapiro et al 2003 discover with regards to NE?
NE +ve mice had increased MLI with smoke exposure in comparison to NE-ve
94
Extracellular Secreted Proteases
Describe levels of NE in CF and COPD
Roghanian et al 2008?
CF actually has significantly more NE than COPD
| also inc in neutrophilic asthma
95
Extracellular Secreted Proteases
| What inhibits serine proteases?
SERPINs
eg a1-antiT
Secretory leukoprotease inhibitor - SLPI
Elafin
96
Extracellular Secreted Proteases
| Where is a1-antitrypsin made?
Liver
| Can be made by alveolar macrophages + epithelial cells
97
Extracellular Secreted Proteases
| What do many a1-antitrypsin deficient babies die from?
Fibrosis of liver
98
Extracellular Secreted Proteases
| a1-antiT, reversible or irreversible?
Irreversible
Distorts active site via methionine residue
J Stoller 2005
99
Extracellular Secreted Proteases
| Where is SLPI and elafin made?
All resp epithelium
Released into lumen of lung
Can be made by macrophages and neutrophils
100
Extracellular Secreted Proteases
| SLPI and Elafin, ir/reversible?
Low deg reversibility
101
Extracellular Secreted Proteases
| a1-antitrypsin is increased in which diseases?
Asthma
| Chronic Bronchitis
102
Extracellular Secreted Proteases
| When NE generates peptides, what does this stimulate?
Collagen synthesis in fibroblasts
- Role in tissue remodelling
103
Extracellular Secreted Proteases
| What cleaves ICAM 1 adhesion molecule?
MMP9 and NE
104
Extracellular Secreted Proteases
| What does proteolytic elastin degredation stimulate? (2)
Neutrophil chemotaxis
PAR1 receptor on fibroblasts stimulating TGF-B release
105
Extracellular Secreted Proteases
| What activates TGF-B itself?
MMPs
NE
Cathepsin-G
....causing fibroblast migration
differentiation (to myofibroBs)
proliferation
106
Extracellular Secreted Proteases
| Growth factors sit in connective tissue and are relatively inactive. What causes their release?
Neutrophil MMP-9 release
107
Extracellular Secreted Proteases
| What cytokine does cathepsin G FURTHER activate?
ENA-78
| Goes from active->very active -> degraded
108
Extracellular Secreted Proteases
| What cytokines do cathepsin G + NE deactivate?
IL-6
IL-8
TNF-a
109
Extracellular Secreted Proteases
| MMP-9 activates + FURTHER activate which cytokine?
IL-8
110
Extracellular Secreted Proteases
| NF-kB is activated by which protease?
NE
111
Extracellular Secreted Proteases
| In what way is NF-kB activation described as a "vicious cycle"
NE release results in NF-KB transcription
This causes MMP release from macrophages
MMPs activated by NE/PR3/CathG
Thus proteases can stimulate further proteases
112
Extracellular Secreted Proteases
In what other ways can proteases potentiate their own effects?
a1-antiT
anti-proteases can be BROKEN DOWN by proteases
EG. TIMPs can be inactivated by NE
EG. a1-antiT can be inactivated by MMPs and NE itself
Degredation of a1-antiT by MMP-9 releases peptide fragments THAT ARE CHEMOTACTIC FOR NEUTROPHILS
113
Extracellular Secreted Proteases
| Which antiproteases are resistant to MMP degredation
SLPI and elafin
114
Extracellular Secreted Proteases
| TIMPs can be broken down by.......
NE
115
Extracellular Secreted Proteases
| a1-antiT can be broken down by......
MMP-9
| NE
116
Extracellular Secreted Proteases
| Which proteases act as mucin secretagogues via EGF receptors on goblet cells?
NE
| Proteinase 3
117
Cytokines
| How is IL1B activated?
Via the inflammasome which cleaves it to its active form
Allows storage of cytokines -> faster release than transcription
118
Cytokines
| What does the NALP 3 inflammasome consist of?
NALP-3
ASC
Caspase
119
Cytokines
| What activates the NALP3 inflammasome?
PAMPS
Bacterial toxins
ROS
Danger signals -> large particles ie asbestos,alum,MSU,CPPD // UV // skin irritants
120
Cytokines
| What cytokines does the inflammasome activate?
IL1B
IL18
IL33
121
Cytokines
| Aside from cytokine activation, what other role does the inflammasome have?
Antigen processing and presentation to T cell
122
Cytokines
| What are the main cells that contain inflammasomes?
APCs
123
Cytokines
| Describe the JAK/STAT pathway?
Intracellular signalling pathway
2 chains on receptor dimerize as ligand binds
This phosphorylates 2 JAK domains (cytoplasmic)
Allows binding of STATS (of which there are 4)
STATS are phosphorylated by activated JAKS
Phosphorylated STAT dimers translocate to nucleus -> act as TF
124
Cytokines
| What influences which genes are transcribed from STATs?
Cell architecture and phenotype
Type of STAT
Cytokine that binds
125
Cytokines
| Which cytokines signal via MyD88?
IL33, IL1a/beta
126
Cytokines
| Describe IL-33 intracellular signalling?
MyD88 associates to cytoplasmic domain upon ligand binding and receptor dimerisation
Activates the TRAF-6 pathway
Activation of TRAF-6 has many downstream effects, one of which is activation of NF-kB
127
Cytokines
| Describe the downstream effects of LPS stimulation of TLR-4 (or equally IL-1/IL1R)
MyD88 signalling activates TRAF-6
| Results in NFkB activation
128
Cytokines
| Which cytokines does NFkB stimulate production/release of? (acute phase cytokines)
TNFa
IL6
IL1
129
Cytokines
| In what way can cytokine receptors regulate themselves?
Has another subset that does not possess cytoplasmic signalling domains, therefore no onward signalling
Also able to secrete a soluble form of the receptor which mops up ligands
130
Cytokines
| Define pleiotropic cytokine
Cytokine that is able to act on multiple cell types
131
Cytokines
| Define multifunctional cytokine
Same cytokine able to regulate different functions
132
Cytokines
| What are the acute phase cytokines?
IL-1/6/TNFa
133
Cytokines
| What do the acute phase cytokines stimulate in the liver?
Acute phase protein production -> C reactive protein, mannose binding lectin
Causes activation of complement opsonisation
134
Cytokines
| What do the acute phase cytokines stimulate in the bone marrow?
Neutrophil mobilisation -> increased phagocytosis
135
Cytokines
| What do the acute phase cytokines stimulate in the hypothalamus
Inc body temp
-> decreased viral and bacterial replication. Increased antigen processing
Increased specific immune response
136
Cytokines
| What do the acute phase cytokines stimulate in fat + muscle?
Protein + energy mobilisation -> allows increased body temperature
-> decreased viral and bacterial replication. Increased antigen processing
Increased specific immune response
137
Cytokines
| What do the acute phase cytokines stimulate in dendritic cells?
TNFa stimulates migration to LNs and maturation -> initiates adaptive immune response
138
Cytokines
| What are the three types of IFN?
```
T1 = a,b
T2 = y
T3 = lamda, theta
```
139
Cytokines
| What are the 3 main functions of IFNs?
Induce resistance to viral replication
Increase MHC class 1 expression and antigen presentation in all cells.
Activation of NK cells to virus infected cells
140
Cytokines
| What cytokines do activated macrophages secrete?
Acute phase: IL1b, IL6, TNF
| Also: IL8/12
141
Cytokines
| What are the local vs systemic effects of IL1B
```
Locally = activates endothelium+lymphocytes
Systemically = Fever, IL-6 production
```
142
Cytokines
| What are the local vs systemic effects of TNFa?
```
Local = inc permeability
Increased IgG + cell entry
Increased fluid drainage to lymphatics
Systemic = fever, mobilisation of metabolites
Shock
```
143
Cytokines
| What are the local vs systemic effects of IL-6?
Local - lymphocyte activity + antibody production
| Systemic - Fever , induces acute phase protein production
144
Cytokines
| Explain naive CD4+ T cell differentiation?
```
Naive T cell has ability to differentiate into:
Treg (IL-2, TGFB)
TH17 (IL1,6,23,TGFB)
TH2 (IL4)
TFH (IL6,21)
TH1 (IL12,18, IFNy)
when exposed to (cytokines)
```
145
Cytokines
| What are the functions of TRegs
Suppression of immune/inflammatory responses
146
Cytokines
| What are the functions of TH17 cells?
Inflammation stimulation -> Neutrophil bacterial/fungal killing
147
Cytokines
| What are the functions of TH2 cells?
Allergic + helminth responses -> goblet cells/eosin/Bcell stimulation
148
Cytokines
| What are the functions of TFH cells?
Aid B cells in germinal centres -> antibody formation
149
Cytokines
| What are the functions of TH1 cells?
Cell mediated immunity, macrophage activation
150
Cytokines
| What are the main cytokines produced by TRegs?
IL10
| TGFB
151
Cytokines
| What are the main cytokines produced by TH17?
IL17A+F
| IL22
152
Cytokines
| What are the main cytokines produced by TH2?
IL4,5,13
153
Cytokines
| What are the main cytokines produced by TFH?
IL4,21
154
Cytokines
| What are the main cytokines produced by TH1 cells?
IFN-y, TNFa
155
Cytokines
| TH1 generation is blocked by which cytokine?
IL4
156
Cytokines
| TH2 generation is blocked by which cytokine?
IFN-y
157
Cytokines
| TH2 cytokine locus encodes which cytokines?
IL4,5,13
| All share same TF gata3. Binds start site of all.
158
Cytokines
| Which STAT is at the start of IL4,13 loci?
STAT6
159
Cytokines
| Which cytokines do TH2 cells secrete?
4/5/13
160
Cytokines
| What STAT do IL4/13 signal through?
STAT6 (phosphorylated dimer)
161
Cytokines
| What STAT does IL5 signal through
STAT5A+B (phosphorylated dimer)
162
Cytokines
| Functions of IL4
Increase VCAM-1
Induces TH2 cell formation (self-stimulates)
Stimulates IgE production and release
163
Cytokines
| Function of IL5
EOSINOPHILIA CYTOKINE
| - Stimulates activation, differentiation and increased survival
164
Cytokines
| IL13 function
Critical mediatior of AHR and mucus secretion
165
Cytokines
| What is the role of IL33 in generation of asthma?
Any cellular damage results in IL33 release (in pro form).
IL33 is cleaved and then stimulates IL5/13 release on mast cells via MYD88 signalling
If cell undergoes apoptosis then IL33 is not released
166
Eicosanoids
| What is broken down to form LTs and prostanoids?
Arachidonic acid
167
Eicosanoids
| Describe the four-character abbreviation rule for eicosanoids, with PGE2.
```
PGE2
PG = prostaglandin
E = (E) class
2 = number of double bonds
```
168
Eicosanoids
| What enzyme breaks down membrane phospholipids to form aracidonic acid?
Phospholipase A2
169
Eicosanoids
| Which two molecules can arachidonic acid be further broken into?
5-HPETE
| Prostaglandin-G2
170
Eicosanoids
| What enzyme breaks arachidonic acid into 5HPETE?
5-LO
| 5-lipo-oxygenase
171
Eicosanoids
| What enzyme breaks down arachidonic acid to PG?
COX1/2
172
Eicosanoids
| What is PGG2 converted into?
PGH2
173
Eicosanoids
| Describe the full pathway of LT synthesis (enzymes in brackets). Finish at LT.
Membrane phospholipid -> (phospholipase-A2) arachidonic acid -> 5HPETE (5-LO) -> Leukotreine synthesis
174
Eicosanoids
| Describe the full pathway for prostanoid synthesis?
Membrane phospholipid -> (phospholipase A2) arachidonic acid -> (COX) PGG2 -> PGH2 -> prostanoid synthesis
175
Eicosanoids
| Which COX is inducible and which is constitutive
COX1 is in all
| COX2 inducible
176
Eicosanoids
| Describe the FURTHER pathway from 5HPETE?
5HPETE -> LTA4->LTC4->LTD4->LTE4
(LTC-E4)-> CysLT1/2 R
OR
5HPETE-> (WITH H2O) LTB4 -> BLT1/2
177
Eicosanoids
| Enzyme for LTA4 -> LTC4?
LTC4 Synthase
178
Eicosanoids
| Enzyme for LTC4->LTD4?
y-GTP
179
Eicosanoids
| Enzyme for LTD4->LTE4?
Dipeptidase
180
Eicosanoids
| Enzyme for LTA4->LTB4
LTA4 hydrolase
181
Eicosanoids
| Cysteinyl LTs signal intracellularly through...
G proteins
182
Eicosanoids
| What are LTs implicated in, in asthma?
Airflow Ob
Inc mucus
Mucosal accumulation
Bronchoconstriction
183
Eicosanoids
| What two classes of drugs target this system?
LT antagonists eg montelukast
| 5-LO inhibitor eg Zileuton
184
Eicosanoids
| Main cells with cyst-LT Receptors?
Mast cells
Eosinophils
Basophils and Macrophages
Also lymphocytes but less so
185
Eicosanoids
What is seen with regards to LTs in asthma?
Wenzel et al 1996
Wenzel et al discovered that LTB4 and cyst LTs in statistically sig higher levels in asthmatics by BAL
HIgher in evenings vs mornings
186
Eicosanoids
| What is seen with regards to LTs in asthma sputum? Aggarwaal et al 2009
Sig increased CystLTs in sputum of mild + mod asthma. However severe asthma = less than mild/mod
187
Eicosanoids
| Describe aspirin sensitive asthma?
Giving aspirin will exacerbate symptoms. Dec prostanoid synthesis results in increased LT production.
188
Eicosanoids
| What proportion of asthmatics have aspirin sensitive asthma?
20%
189
Eicosanoids
As well as increased LTs in asthma, what other component is increased?
Sousa et al 2002
CystLT-Rs
| Sousa et al 2002
190
Eicosanoids
| List 3 CystLT-R antagonist therapies?
Montelukast
Zafirlukast
Pranlukast
191
Eicosanoids
The LT pathway is insensitive to which widely used asthma drug?
Gyllfors et al 2006
Dworeld et al 94
Corticosteroids
192
Eicosanoids
| What trend is seen with montelukast and FEV1? Nayak et al 2004
New baseline FEV1 change of +10% in severe asthma
| Nayak et al 2004
193
Eicosanoids
What happens with combination steroid+montelukast?
Laviolette et al 1998
Improvement in FEV1 is enhanced.
| Laviolette et al 1998
194
Eicosanoids
Briefly describe the heterogeneity of asthma in children?
Szefler et al
Some eosinophilic predominant (worse outcome), some neutrophilic, Some respond to only steroids/montelukast/both
Treating is a fumble in the dark and drug companies hate children
195
Eicosanoids
| Why might the other side of the pathway, LTB4 synthesis be a promising target in severe asthma?
- LTB4 is chemotactic for neutrophils
- Primes dendritic cells
- Activates eosinophils too
- Activate CD4/8 T cells
- Also promotes proliferation and migration of airway SM
- Also stimulates release of proinflammatory mediators
196
Eicosanoids
Neutrophils negatively correlate with....
Green et al 2002
FEV1
197
Eicosanoids
| Why else may LTB4 be a good target in asthma? (excluding the ROLES of LTB4) (3)
Polymorphisms in LTB4 associated with asthma severity
Increased LTB4 results in more enzyme production
LTB4 elevated in urine, sputum, BAL, EBC, blood
- Associated with neutrophilic asthma
198
Eicosanoids
| How could LTB4 be inhibited? (3)
5-LO/FLAP inhibitors
BLT1/2 antagonists (Rs)
LTA4 hydrolase
199
Eicosanoids
| What were the problems with 5LO/FLAP inhibitors?
5LO/FLAP inhibitors
- Also reduce LTA4
- THEREFORE NO PRODUCTION OF lipoxins = antiinflammatory
Poorly tolerated also
200
Eicosanoids
| What are the problems of BLT1/2 antagonists?
Other inflammatory mediators signal through LTB4 receptor
| STRANGELY very potent antagonists also acted as partial agonists?
201
Eicosanoids
| What were the problems of LTA4H inhibitors?
When released from cells LTA4 hydrolase also breaks down proline-glycine-proline which is pro-inflammatory
202
Eicosanoids
| What drug inhibited neutrophil chemotaxis by LTB4?
Coversin
203
Eicosanoids "might come up in future"
Learn the bastards
204
Eicosanoids
| What are the 5 subclasses of PGs?
```
PGD2
PGE2
PGF2a
PGI2 (prostacyclin)
TXA2
```
205
Eicosanoids
| Receptor for PGD2?
DP1/2
206
Eicosanoids
| Receptor for PGE2?
EP1-4
207
Eicosanoids
| Receptor for PGF2a
FP
208
Eicosanoids
| Receptor for PGI2
IP
209
Eicosanoids
| Receptor for TXA2
TP
210
Eicosanoids
| Which two PGs antagonise one another?
TXA2
| PGI2 (prostacyclin)
211
Eicosanoids
| Which PG is anti-inflammatory?
PGI2 - prostacyclin
212
Eicosanoids
| Which 3 PGs are responsible for bronchoconstriction?
TXA2
PGD2
PGF2
213
Eicosanoids
| Which PG other than prostacyclin reverses bronchoconstriction?
PGE2
214
Eicosanoids
| Airway secretion is potentiated by which PGs?
PGD2
PGE2
PGF2
215
Eicosanoids
| Which PGs cause inc chemotaxis?
PGD2>PGE2
216
Eicosanoids
| Prostaglandin D2 can also bind to which receptor?
CRHT2
| as well as D2R/DPR
217
Eicosanoids
| What cells have CRHT2 receptors?
```
Eosinophils
IL-C
Mast cell
Effector T cell
Basophils
```
---- Causes release of many cytokines il5/9/13/33
218
Eicosanoids
Why may PGE2 be a potential therapeutic avenue?
Gauvreau et al 1999
As previously stated = antiinflammatory/broncho/muco
Alleviates allergen induced FEV1 decline
Also decreased eosinophil levels
Gauvreau et al 1999
219
Eicosanoids
| What disease has increased PGE2?
Appears to be increased in COPD
220
Eicosanoids
| What problem did Walters and Davies (1982) discover with PGE2?
Caused cough -> activates nerve cells
Walter and Davies 1982
It can also act via any of the EP1-4 receptors ---> cough response was via EP3 Maher et al 2009
221
Eicosanoids
| Which EP receptor is bronchoprotective + antiinflammatory?
EP4
222
Eicosanoids
| Which EP receptor is responsible for cough?
EP3
223
Genetic Aspects of CF
| How does CFTR affect sodium reabsorption?
CFTR when functioning inhibits ENaC
| Therefore in CF Na absoption (and water) is even greater
224
Genetic Aspects of CF
| What happens to cilia in CF?
Become shorter + squished ->low water volume thins the serous/muc layer
225
Genetic Aspects of CF
| What is the bicarbonate hypothesis?
CFTR also pumps out bicarbonate.
May have reduced pH therefore in CF.
Results in impaired bacterial killing.
Host defense proteins cannot function as well
226
Genetic Aspects of CF
| Inheritance of CF
AR
227
Genetic Aspects of CF
| What ethnicities are affected least by CFTR?
African/american
| Asian
228
Genetic Aspects of CF
| How many mutations are there?
>2000
229
Genetic Aspects of CF
| What is a missense mutation? (42%)
Wrong amino acid incorprated
230
Genetic Aspects of CF
| What is a nonsense mutation?
Stop codon inserted too early
231
Genetic Aspects of CF
| What is the most freq mutation?
Missense
232
Genetic Aspects of CF
| What may be a heterozygous advantage of CF carrier?
Cholera?
233
Genetic Aspects of CF
| What is the most common mutation, and what type of mutation is it?
delta-F508
| Frame shift
234
Genetic Aspects of CF
| What are the 6 classes of molecular consequences?
Normal
No synthesis
Block in processing -> stuck in ER (deltaF)
Reduced freq opening (due to block in regulation)
Channel conductance wrong
Reduced synthesis
Reduced survival
235
Genetic Aspects of CF
| Which patients have severe pancreatic disease vs not? (classes)
Classes 1-3 = severe
| 4-6 = mild
236
Genetic Aspects of CF
| How do potentiators work?
Bind to defective channels - allows opening and closing
237
Genetic Aspects of CF
What did oral administration of VX-770
(kalydeco//Ivacaftor) result in?
Elborn 2011
Dec Sweat chloride (50%)
10% FEV1 improvement
Increased time until 1st exacerbation
3% change in weight
238
Genetic Aspects of CF
| What mutation is Ivacaftor/Kalydeco for?
G551D
| 5% mutations
239
Genetic Aspects of CF
| What method discovered luma+ivacaftor
High throughput screening
240
Genetic Aspects of CF
| What was the result of the phase 2a study on lumacaftor? (corrector for dF508)
Dose dependent change in sweat chloride vs placebo
BUT
NO effect on AEs, LF, patient symptoms
241
Genetic Aspects of CF
| How were the disappointing results of lumacaftor (partially) overcome?
Combination of Iva and lumacaftor
Still only 3% inc in FEV1
Not approved by NICE
242
```
Genetic Aspects of CF
How does ataluren work and what mutation/class is it for?
```
Bind to mRNA and convinces ribosome to carry on translating
For class 1
Stop mutations
By week 48 change vs placebo FEV1 = 6.7% (only -0.2% from week 0) ONLY WITH THOSE NOT ON inhaled ABx WHEN COMPARED TO ABx
243
Genetic Aspects of CF
| What study depicted the importance of subgroup analysis and drug interaction?
Trial of Ataluren
| 6.7% change in FEV1 when compared those on INHALED abx with not
244
Genetic Aspects of CF
| Is the genotype/phenotype correlation good/bad with lung?
Big effect of environment on lungs
Correlation is bad
Two siblings with same mutation vastly different.
With the pancreas genotype/pheno does correlate
245
Genetic Aspects of CF
| What are putative modifiers?
```
Genes that may alter phenotype of CF
ie
Host defense
Other ion channels
Muc genes
B2 receptors
Chaperones etc
```
246
Genetic Aspects of CF
| Why can't knockout mice emulate lung CF?
Alternative cl- channel
247
PCD
| Inheritance of PCD?
AR 1/20k
248
PCD
| Where can cilia be found in human body?
Resp tract
Lining ventricles
Eustacean tubes
Sperm/fallopean
249
PCD
| What are the clinical features of PCD?
```
Respiratory symptoms
- neonatal resp symptoms
Wet prod cough
Recurrent infections
Bronchiectasis
Chronic Rhinits/sinusitis
Recurrent otitis media
55% situs inversus
6% heterotaxy = loadsa probsheart
Other features:
Hydrocephalus
Retinal abnormalities
Abnormal sperm flagella
```
250
PCD
| NNO - high/low PCD?
80-85% lower in PCD
| Breath hold technique
251
PCD
| What are the benefits /negs NNO?
Quick, becoming more portable, noninvasive
Some defects have levels closer to normal
Breath hold technique
252
PCD
| Benefits / Negs nasal brushing?
Able to visualise structure and function.
Can culture cells
Is invasive/uncomfortable
False-positivess --affected by infection?
Must be repeated 3x if abormal though
Can have 2ndary
253
PCD
| What is the structure of cilia?
9+2 microtubules
| Inner and outer dynein arms
254
PCD
| What is the function of the dynein arm?
Motor protein -> causes movement by grabbing and sliding
| Radial spoke structure gives signal
255
PCD
| Most common defect in cilia?
```
Outer dynein = 37%
Outer and inner = 21%
Inner = 18%
Transposition defect = 11% = random arrangement
Normal ultrastructure 13%
```
256
PCD
| Success rate of culture?
52%
257
PCD
| Adv/Dis of immunofluorescence?
Less cells required
Relatively quick
125£ per patient
Panel of antibodies to confirm
258
PCD
| What is the hydin defect?
A tiny regulator,
| Defect cannot be seen in EM, only visualised with Electron-microscopy
259
PCD
| How many gene mutations?
>37
260
PCD
| What is ciliary aplasia?
No cilia at all
261
PCD
| What are the implications of missed diagnosis?
```
Prevention of:
Bronchiectasis
Progressive red in LF
Mismanagement of ENT
Inappropriate ENT surgery
Developmental/speech delay
Cardiac splenic gut abnormalities
IVF couselling
```
262
PCD
| Management
```
Chest physio
Serial sputum samples
H influenza/Paeruginosa
ABx
Serial LF testing
```
263
PCD
| What are preventative methods?
Vaccinations
Early diagnosis
Avoid smoking
264
PCD
| What ENT Rx should be avoided?
Surgery. Wait until older
265
PCD
| What is the prognosis of PCD?
Normal life span possible
| Resolution of ear disease by late childhood
266
PCD?
| How is chron rhinosinusitis Rx?
```
Douching
Corticosteroids
Abx
anti-leukotrienes
anti-histamines
Endoscopic sinus surgery can be performed
```
267
Cell-Based Therapy of the Lung
| Define regenerative medicine?
An emerging medical endeavour aimed at regeneration via small molecules, biological therapies, medical devices or genes and cells.
Aims to replace or repair human cells tissue or organs to replace ordinary functions.
268
Cell-Based Therapy of the Lung
| Difference between progenitor vs stem cells?
Stem cells - self renew to make more stem cells. toti/pluripotent
Progenitor - cannot self renew, differentiate into mature cell types - MULTIPOTENT
269
Cell-Based Therapy of the Lung
| Which cell types are being used more widely now in cellular based therapy?
ESCs
| iPSCs
270
Cell-Based Therapy of the Lung
| Why are ESCs used more?
Able to differentiate into virtually all tissues
271
Cell-Based Therapy of the Lung
| What are the problems of ESC use?
Can be subject to rejection but heterologous - possibility of harvesting when an embryo from self though but STILL A RISK
Difficult to induce differentiation with 100% efficacy - can mean a small percentage of undifferentiated cells might remain -> could form teratomas
272
Cell-Based Therapy of the Lung
| What are the pros of MSCs?
Available from various sources - BM/adipose
| Expand with high efficiency
273
Cell-Based Therapy of the Lung
| Limitations of MSCs
Low numbers -> ex vivo expansion required
Senescece (no further div) after ex-vivo expansion
Genetic instability after ex vivo expansion
274
Cell-Based Therapy of the Lung
| Takahashi and Yamanaka received a nobel prize for the development of what?
Developing the technique to induce adult somatic stem cells to form iPSCs. Behave like ESCs
Via transfer of number of genes
275
Cell-Based Therapy of the Lung
| Main benefit of iPSCs
Will not remove teratoma possibilities but DOES remove the risk of rejection if autologous cells are used
- The possibilities of HLA matched heterologous banks are being explored
276
Cell-Based Therapy of the Lung
| Name 2 other benefits of iPSCs with regards to expansion?
High capacity for expansion [120] (iPSCs MSCs) without losing MSC phenotype
Reduced senescence
(Hopes to overcome aging issues when using BM-MSCs
277
Cell-Based Therapy of the Lung
| What two steps did Yamanaka and Takahashi use to create iPSC-MSCs from somatic cells?
Insertion of TF genes in "reprogramming step"
THEN
"Differentiation stage" -
addition of MSC differentiation growth factors
278
Cell-Based Therapy of the Lung
| What TF genes were inserted into somatic cells when first generations iPSCs-MSCs
Oct4
Sox2
Nanog
Lin28
279
Cell-Based Therapy of the Lung
| What (3) growth factors are used is the "differentiation stage" when first Yamanaka and Takahashi created iPSCs-MSCs.
bFGF
PDGF
EGF
280
Cell-Based Therapy of the Lung
| What are the 3 general mechanisms of action of MSCs?
Replacement of damaged cells via differentiation
Paracrine regulation - immunoregulatory effects through secretion of paracrine factors
Mitochondrial transfer to damaged cells
281
Cell-Based Therapy of the Lung
| What are tunnelling nanotubules?
extracellular projections formed in-vitro between cells allowing transfer of vesicles and organelles such as mitochondria
282
Cell-Based Therapy of the Lung
| Describe the proposed mechanism of TNTs?
Miro-1 loads mitrochondria onto milton accessory protein which attaches itself to KIF-5 protein.
KIF 5 protein has motor on the bottom
Complex then tracks along the actin fibres within microtubules.
Mitochondria transported to epithelial cell from BM-MSC
283
Cell-Based Therapy of the Lung
| Is mitochondria TNT transfer bi or uni directional?
Bi-directional
284
Cell-Based Therapy of the Lung
| What have pre-clinical studies revealed with regards to MSC lifespan in vivo?
MSCs disappear within 1 day of injection
| High localisation to lung
285
Cell-Based Therapy of the Lung
| What have pre-clinical studies rebealed with regards to differentiation of MSCs in vivo?
Consensus that they rarely differentiate into tissue resident cells
286
Cell-Based Therapy of the Lung
| With regards to MSCs, if they have short lifespans and do not differentiate in vivo often, how do they work?
- Downreguation of proteases + proinflammatory mediatiors
- Upregulation of others eg TGF-B
- Induce macrophages to M2 phenotype
- Inhibit M1 phenotype which improves inflammation + elastolysis
287
Cell-Based Therapy of the Lung
| How many clinical trials have been published for COPD?
4 (out of 17)
288
Cell-Based Therapy of the Lung
| What did the first study show?
```
Brazilian phase 1 study
4 patients
Stage IV COPD
BM-MSC injection
No adverse effects, BUT did not fully discuss causes of a death in the subsequent follow up study (1 yr stussek et al)
```
289
Cell-Based Therapy of the Lung
| What did the prochymal study show?
```
Study of 62 patients
4 monthly infusions
mod-severe COPD
No AEs
No inc in LF
Decrease in CRP
```
290
Cell-Based Therapy of the Lung
| What did the Netherlands COPD study find?
```
1st adjuvant study
Prior to LVRS in 7 patients
Stage IV Emphysema
No AEs
Lung tissue showed no fibrotic responses
```
291
Cell-Based Therapy of the Lung
| What did the second adjuvant COPD MSC study find?
```
10 Gold III/IV patients
Adjuvant to 1 way endobronchial valve insertion
No AEs
Safe to do
Significant reduction in CRP
```
292
Cell-Based Therapy of the Lung
| What can affect the ability of a mitochondria to function?
Ageing
Oxidative stress
Inflammation
293
Cell-Based Therapy of the Lung
| What are the main cellular functions of mitochondria
```
Energy production
Oxidative stress
Calcium
Apoptosis / survival
Inflammation
```
294
Cell-Based Therapy of the Lung
| Briefly discuss the mechanism of mitochondria in energy production
Glucose->pyruvate (glycolysis) -> acetyl coA
Acetyl CoA enters mitochondra
Krebs occurs in cytoplasm of mitochondria
Generates NADH2/FADH2
Both donate electrons to electron carriers 1-4
Flow of electrons allows pumping of H+ ions into intramembrane space
Buildup/Gradient
O2 combined with terminal electrons + H+ ions to make water
ATPase makes ATP as H+ ions flow through
Can make ROS at end
295
Cell-Based Therapy of the Lung
| How does oxidative stress disrupt mitochondrial energy production?
Disrupts KREBs and electron carrier chain
Leads to increased ROS production
Self-stimulating
296
Cell-Based Therapy of the Lung
What did Weigman and Michaeloudes discover in 2015 (mito/ox)?
4, STAGE 2 patients
Did endobronchial biopsies
Membrane potential reduced in COPD vs healthy (sig)
Mitochondial ROS levels also significantly higher in COPD
297
Cell-Based Therapy of the Lung
| Weigman and Michaeloudes also developed a mouse model of COPD. How was this acheived? 2015
Ozone exposure
3hr per day
2x per week
1-6 weeks
298
Cell-Based Therapy of the Lung
| What values did the mouse OZONE model of COPD produce?
Sig reduced depolarisation (at 1+6 weeks)
Sig increased ROS 1+6
Sig inc total BAL cells 1+6
Sig increased airway hyper-responsiveness
299
Cell-Based Therapy of the Lung
What were the results of CSE to mitochondria?
10/25/50%
Time/dose dependencies of:
Reduced membrane potential
Increased ROS levels
Increased apoptosis % of apoptotic cells
300
Cell-Based Therapy of the Lung
| What did Pank do in his study of CSE + ASMCs +iPSC-MSCs?
```
Cocultured ASMCs and iPSC-MSCs - 20 hrs
CSE for 4 hours
Measured by flow cytometry
Compared to single culture + no CSE control ASMCs
Measured
MitoROS
Membrane potential
Apoptosis
```
301
Cell-Based Therapy of the Lung
| What were the results of Pank's CSE ASMCs iPSC-MSCs coculture study?
Sig dif:
- ROS levels between control, different CSE concentrations AND between coculture vs ASMCs
- Sig fold change in membrane potential of COvsNon but non sig between CSE concs (coculture only)
- Sig dif apoptosis COvsNON but not between CSE concs
302
Cell-Based Therapy of the Lung
| What did Pank do next with his CSE ASMCs iPSC-MSCs coculture study? What were the results?
Labelled iPSC-MSCs
- Mitochondria
- Actin filaments
Exposed to CSE
Successfully labelled and documented TNTs
TNT formation increased with dose dependant CSE 10/25%
Even in absence of CSE (ox stress) 20% of iPSC-MSC cells formed TNTs
303
Cell-Based Therapy of the Lung
| What did Pank do after the coculture of iPSC-MSCs and ASMCs? What were the results?
Looked at the concept in vivo using ozone mouse model. iPSC-MSCs were administered prophylactically
- No attenuation of membrane potential in MSC ozone group
- No increase in ROS in MSC ozone group (sig dif vs saline ozone)
- Attenuated apoptosis with MSC group (sign dif vs Saline ozone)
- Slight non sig improvement in proliferation
- Sig attenuated AHR to ACh in MSC group
- Sig reduction in total BAL cells vs OzSaline
Cells disappeared within 24hrs
304
Oxidative Stress
| What is O2 role in metabolism?
O2 is the terminal electron acceptor
305
Oxidative Stress
How many electrons does O2 need to form 2
molecules of water with H+ ions?
4
306
Oxidative Stress
| Equation for O2 to water formation? * = free electron
O2->O2*->H2O2->*OH->H2O
One water molecule generated with H2O2->*OH
307
Oxidative Stress
| What did Gerschman et al discover in 1954 about O2?
High O2 killed rats
| Increased *OH formation (extremely oxidative)
308
Oxidative Stress
| What did studies in 68/69/84 discover?
The enzymes that reverse superoxide formations
Xanthine Oxidase
Superoxide dismutase
NADPH oxidase
309
Oxidative Stress
| In '87 Palmer et al discovered what with regards to RNS?
That NO (thought to be a pollutant) is produced within the body
310
Oxidative Stress
| In '90 Bredt discovered what?
The NOsynthase enzyme
311
Oxidative Stress
| What is the formation equation of NO?
L-arginine cleaved to Lcitrulline
| NOS forms NO as NADPH->NADP+
312
Oxidative Stress
| In 1985 Blough discovered what?
NO + superoxide -> peroxynitrite
313
Oxidative Stress
| What us a free radical?
Any species capable of independent existence that contains one or more unpaired electrons
314
Oxidative Stress
| What is ROS?
A collective term incorporating oxygen free radicals and associated oxidants and reductants
315
Oxidative Stress
| What is a RNS?
Nitrogen centred free radicals and associated species
316
Oxidative Stress
| Why is ground state oxygen, the oxygen we breath in, relatively inactive?
Outer electrons are spinning in the same difrection?
317
Oxidative Stress
| Why is singlet oxygen more reactive therefore?
Energy causes an outermost electron to pair with the other unpaired electron resulting in one free ring, and another occupied by two antiparallel spinning electrons
318
Oxidative Stress
| List 3 commonly encountered ROS and RNS involved in signalling?
H2O2
O2*-
*NO
319
Oxidative Stress
| List 3 commonly encountered ROS and RNS involved in damage?
HOCl = hydrochlorous acid
*OH
ONOO- = peroxynitrite
320
Oxidative Stress
| What is "oxidative stress"?
A consequence of aerobic respiration and metabolism.
| Oxygen intermediates can cause damage when repair mechanisms are overwhelmed.
321
Oxidative Stress
| Sources of anti-ox?
Constitutive
Inducible
Dietary
322
Oxidative Stress
| How do anti-ox's work?
Prevent formation of Oxs
Removal of Oxs
Repair mechanisms
Modulation of redox signalling
323
Oxidative Stress
| What is an anti-oxidant?
Any substance that delays or inhibits oxidation of a subtrate, AT LOW CONCENTRATIONS
324
Oxidative Stress
| Name an iron-binding anti-ox?
Transferrin
Normally only 25% iron loaded
Only binds Fe3+
325
Oxidative Stress
| Name an iron oxidising antioxidant?
Caeruloplasmin
Its an acute phase protein
Converts FE2+->Fe3+
(less damaging form)
326
Oxidative Stress
| What contributes to production of ROS?
```
Aerobic Respiration
Respiratory burst (NO) of activated inflammatory cells
Halide peroxidases
NOX and DuOX oxidases in endothelial and epithelial cells
Ischaemia
Reperfusion
Electron transfer reactions
Hyperoxia
```
327
Oxidative Stress
| Thinking back to the electron reduction of O2 to water, why may hyperoxia be a problem?
Results in increased superoxide anion O2*- formation.
| Under normoxia, O2* production is largely beneficial as it acts as signalling
328
Oxidative Stress
| Thinking back to the electron reduction of O2 to water, how does hypoxia cause increased superoxide formation?
Hypoxia causes mitochondrial dysfunction by disrupting key cytochrome enzymes.
Progression down the reduction to water chain is inhibited and O2*- builds up.
Reperfusion worsens this as the damage to cyt is irreversible.
329
Oxidative Stress
| XDH breaks down what?
Adenosine and related products to uric acid
| Uses NADPH as cofactor
330
Oxidative Stress
| What happens to XDH under hyperoxia?
XDH->XOD
| Xanthine oxidase
331
Oxidative Stress
| What does XOD catalyse?
Essentially the same reaction -> breaks down adenosine products to uric acid.
HOWEVER O2 is used as a cofactor
THEREFORE
O2*- and H2O2 are formed
332
Oxidative Stress
| What else can cause XDH->XOD?
Ischaemia
| Re-oxygenation worsens
333
Oxidative Stress
| Why is XDH->XOD evolutionarily useful?
Seemingly antimicrobial but limited evidence
334
Oxidative Stress
| FOCUS ON NEUTROPHILS SUGGESTED
Oxidative Stress
| FOCUS ON NEUTROPHILS SUGGESTED
335
Oxidative Stress
| Principal ROS in neutrophils
O2*-
| HOCl
336
Oxidative Stress
Describe the NADPH oxidase "respiratory burst"
2O2 +NADPH ->
2O2+NADPH-> 2O2*- + NADP+ + H+
337
Oxidative Stress
| Where is NADPH oxidase located?
On the phagosome membrane in neutrophils
338
Oxidative Stress
| What is the function of this "respiratory burst" in phagosomes?
Classically thought to be involved in direct mitochondrial killing BUT its not extremely toxic
May activate proteases (pH drops)
May be involved in redox signalling
May be converted into more damaging species
339
Oxidative Stress
| What do NOX and DuOX enzymes produce?
Purposeful ezymatic production of O2*- and H2O2
340
Oxidative Stress
| What are the functions of NOX and DuOX enzymes?
Host defence
Redox signalling
- Influence cell proliferation, differentiation and gene regulation.
341
Oxidative Stress
| NOX and DuOX, inducible true or false?
True
342
Oxidative Stress
| What are the targets of NOX and DuOX enzymes?
Haem peroxidases
Redox active thiols
Limited reactivity
343
Oxidative Stress
| Where can NOX and DuOX enzymes be expressed?
```
Epithelium
Endothelium
Smooth Muscle
Fibroblasts
Inflammatory cells
```
344
Oxidative Stress
| What is the principle cell of myeloperoxidase?
Neutrophil
345
Oxidative Stress
MPO
Complete equation:
H2O2 + Cl- + H+ ->(MPO)
H2O2 + Cl- + H+ -> HOCl + H2O
HOCl = damaging itself
WHY IS HOCl production EVEN more damaging?
346
Oxidative stress
| Why is HOCl production even more damaging?
Can form hydroxyl radicals:
| HOCl + Fe2+ -> Fe3+ + Cl- + *OH
347
Oxidative Stress
| When MPO activation and production of HOCl occur?
Following phagocytosis of particles opsonized with IgG
348
Oxidative Stress
| iNOS is upregulated on what cells?
Endo+epithelium, macrophages, endothelium, possibly neutrophils
349
Oxidative Stress
| What disruptive molecule is produced if NO* and O2*- are produced in equimolar amounts?
ONOO-
Capable of hydroxylation, S-nitrosylation and Nitration
350
Oxidative Stress
| What are the functions of NETS?
Trap and destroy microbes
351
Oxidative Stress
| What are NETS formed from?
Decondensed chromatin and antimicrobial proteins
352
Oxidative Stress
| What are the two mechanisms of NET formation?
RAPID formation w/o cell death - REQUIRES ROS for priming
ALTERNATIVE formation with cell death
requires NOX-2 activity
NOX = NADPH oxidase
Linked to sepsis + autoimmune disease
353
Oxidative Stress
| Functions of Fe.
Cell proliferation and microbial virulence
Direct iron signalling + redox
Generation of ROS
- Controlled = inflammatory responses, cellular fate, TFs
- Uncontrolled = molecular damage, organ dysfunction
354
Oxidative Stress
| What is the FENTON reaction?
Iron catalysing formation of *OH from H2O2
| Fe2+ + H2O2 -> Fe3+ + OH- + *OH
355
Oxidative Stress
| What propagating reaction can Fe2+ cause?
Lipid oxidation
Fe2+ forms alkoxyl radicals
Fe3+ forms peroxyl radicals
Terminated by chain breaking antioxidants such as vitamin E
356
Oxidative Stress
| What does uncontrolled oxidative stress (ie damage) cause?
Modification of proteins, lipids and DNA
Dysfunction of biological molecules
Accumulation of end-products that may be bioactive/toxic
357
Oxidative Stress
| Name some common oxidations that take place if stress is abhorrent?
```
Thiol oxidation (can be irreversible)
Protein carbonyl formation
Aromatic hydroxylation -> form non biological tyrosines
Chlorination and Nitration of tyrosines
Fe lipid oxygenation
```
358
Oxidative Stress
| What enzyme convert oxidised thiols to normal redox states?
Thioredoxin
Others exist ie:
Glutaredoxin
Nucleoredoxin
GSNO-reductase
359
Oxidative Stress
| Describe how oxidative stress can cause pro OR anti-inflammatory events?
Inc oxidative stress -> increased redox signalling -> transcription of:
Nrf-2 = anti-inflammatory
NFkB, AP-1 = pro-inflammatory
360
Oxidative Stress
| How do extreme oxidising signals cause adverse effects?
TF activation can be compromised -> immunosuppression
Anti-inflammatory and anti-oxidant Nrf-2 is suppressed by extreme oxidising conditions
361
Oxidative Stress
Human studies of antioxidants have been disappointing. Why may this have happened?
What new approaches could be taken?
Heterogenous populations with established disease
Multiple targets + types of antioxidants
Time of administration
At risk populations
Combinational regimes ie chelators and scavenging antioxidants
Duration of administration (may compromise antioxidant protective responses)
362
Adaptive Immunity
| What is the hallmark of adaptive immunity?
Memory
| Greater magnitiude and faster resolution at secondary encounter
363
Adaptive Immunity
| What are the two arms of adaptive immunity?
Humoral response = B cell antibody production
| Cell mediated = T cell killing/cytokines
364
Adaptive Immunity
| Three monomer antibodies?
IgG IgD IgE
365
Adaptive Immunity
| Dimer antibody?
IgA
366
Adaptive Immunity
| Which antibody exists as a pentamer?
IgM
367
Adaptive Immunity
| What molecule connects antibody monomers?
J chain
368
Adaptive Immunity
| What antibody is the B cell receptor?
IgD
Naive B cells
B cells also have IgM
369
Adaptive Immunity
| What receptors recognise the heavy chain (Fc) portion of the antibody?
FcR
370
Adaptive Immunity
| IgE function?
Eosinophils activation via FcEpsilonR
Causes degranulation
IgE is bound to eosinophils
371
Adaptive Immunity
| Which antibodies cause opsonisation/phago?
IgG or IgA
Via
FcGammaR or FcAlphaR
372
Adaptive Immunity
| Most abundant antibody in blood?
IgG
373
Adaptive Immunity
| Where is IgA mainly found?
Mucinous surfaces
374
Adaptive Immunity
| What is the homeostatic mechanism of antibody regulation?
FcR can be found without disease
Bind antibodies + recycle in acidic endosomes
Re-released and dissociates at physiological ph
375
Adaptive Immunity
| Why is IgA good for the airway and gut?
Relatively immunologically inert
| These areas are exposed to pathogens frequently
376
Adaptive Immunity
| What is affinity maturation?
Process whereby as B cells undergo clonal expansion they improve their antibody efficacy by mutation
377
Adaptive Immunity
| What secondary lymphoid tissue do B cells undergo affinity maturation?
Germinal centres
378
Adaptive Immunity
| What are the three possible outcomes of a B cell in the germinal centre?
Infinity improves via FDC (follicular dendritic cell) presentation and TH cell activation
Low affinity = apoptosis
Autoreactive = TH cell mediated apoptosis via fas death receptor + withdrawal of survival cytokines
379
Adaptive Immunity
| Once a B cell has proven capability in the germinal centre, what are the 3 possible differentiation outcomes?
Plasma cell - secretory
Class-switched memory B cell - do not secrete antibody but can rapidly enter germinal centres to redo the process
IgM+ IgD+ memory B cell
380
Adaptive Immunity
| What did Tas et al depict very nicely using the brainbow mouse?
Brainbow mouse has each cell fluorescently marked
| Depicted that all cells same colour in germinal centres post administration of vaccine
381
Adaptive Immunity
| What protein induces mutations that drive affinity maturation?
AID protein
Activation induced cytidine deaminase
Deaminates cytosine -> uracil which is corrected to thymine
C:G = T:A
Tas et al also depicted that deletion of the AIDCA gene in brainbow mice resulted in non-high affinity antibody responses
382
Adaptive Immunity
| Describe process by which IgA is secreted?
Secreted in lamina propria
Binds to IgA receptor
Endocytosed
Endosome fuses with apical membrane alongside secretory component of receptor (left behind)
383
Adaptive Immunity
| What is the most abundantly produced antibody?
IgA
| 9-25g per day
384
Adaptive Immunity
| Do T cells undergo affinity maturation?
No, either have the T cell that can recognise antigen or don't
385
Adaptive Immunity
| When undergoing clonal expansion how are T cells with the highest affinity selected for?
Compete for survival cytokines (IL2)
| Those with most affinity survive
386
Adaptive Immunity
| What are the two main types of CD4+ and CD8+ T cells?
```
Effector = "do the things"
Memory = sit inside lymphoid tissue primed for clonal expansion
```
387
Adaptive Immunity
| What are the functions of effector CD8+ cells?
Kill infected target cells
| Activate macrophages
388
Adaptive Immunity
| What are the functions of effector CD4+ cells?
Activation of B cells and macrophages
389
Adaptive Immunity
| What is the main mechanism of killing in CD8+ (also NK, NKT and some subsets of CD4+ T cells)?
Cytolysis
- Forms close membrane-membrane interaction via receptor molecules such as ICAM 1
- Polarises itself and moves actin and granules to target cell membrane interaction
- Lines up cytoplasmic granules
Either secretes granules into target cell or lack significant stimuli and dissociates
Target cell may secrete survival molecules EG PDL1/2
390
Adaptive Immunity
What is the process by which cytoplasmic granules in CD8+ t cells cause death or target cells?
Granzyme B + Perforin
Granules of Granzyme B and perforin are released
Endocytosed by target cell
Perforin forms pores in vesicles inside target cell
Granzyme B exits and causes mitochondria to release cytC = death by caspase 9 (procaspase 9 and APAF 1)
OR
Granzyme B can directly activate Procaspase 3
Apoptosis
391
Adaptive Immunity
What is the difference between administration of a vaccine intra-nasally vs systemically?
(study compared in mice WU et al influenza)
IN gave more protection than IP (peritonally)
IN mice lost less weight than IP
Suggested there is a degree of localised tissue resident memory
T cells
392
Adaptive Immunity
| What did Jozwick et al discover with tissue resident CD8+ lymphocytes?
Increased number cause reduced viral load and symptom scores
393
Adaptive Immunity
| What did Harker discover with regards to B cells in the lungs?
Resident B cell germinal centres in the lungs - not much known about these.
Even see these in COPD in the alveoli (separate paper)
394
Inflammatory Cells of the Airways
| What is NALT?
Nasal associated lymphoid tissue
Layer of epithelial cells, M cells, goblet cells and intra-epthelial lymphocytes
Mature B cells = IgA
395
Inflammatory Cells of the Airways
| What is BALT? What cells are are?
Bronchus associated lymphoid tissue
- Germinal centres
- Parafollicular T cells
- IELS
396
Inflammatory Cells of the Airways
| Name some challenges to maintaining homeostasis in the lung?
```
Air pressure
Endogenous microbiome in lower airways
Inhaled allergens
Particulate matter
Pollution
```
397
Inflammatory Cells of the Airways
| Lifespan of epithelium?
30-50 days
398
Inflammatory Cells of the Airways
| What protein forms tight junctions?
ZO-1
399
Inflammatory Cells of the Airways
| How do some allergens traverse the epithelium? Eg house dust mites?
Some have proteases
400
Inflammatory Cells of the Airways
| How many BAL cells (%) are alveolar macrophages in the healthy airways?
95% in controls (ie lab mice)
| Most humans have 70-80%
401
Inflammatory Cells of the Airways
| Where are alveolar macrophages found?
Close proximity to AECI+II
402
Inflammatory Cells of the Airways
| Differentiation/origin of macrophage?
Monocyte-> differentiates in tissue to macrophage -> differentitates into alveolar macrophage
403
Inflammatory Cells of the Airways
| When do macrophages colonize the airways?
First few days of birth
404
Inflammatory Cells of the Airways
How do alveolar macrophages replisnish themselves?
Hasthimoto et al
In situ proliferation and self renewal
| Hashimoto et al
405
Inflammatory Cells of the Airways
| What stimulates macrophages to form M1-like phenotype (macrophages do not exist as either 1/2 but more of continuum)?
LPS
Bacteria
IFN-y
GM-CSF
406
Inflammatory Cells of the Airways
| What stimulates M2 tissue healing like phenotype in macrophages?
IL4,10,13
| M-CSF
407
Inflammatory Cells of the Airways
| Lifespan of macrophages
40% turnover in 1 year - long
408
Inflammatory Cells of the Airways
| 2 General critical roles of Alv Macrophages?
Immune surveillance
Homeostasis
- Prevent inflammation with innocuous stimulu
- Induce inflammation with pathogens
- Resolution of inflammation - clearance of self cells and ECM products
409
Inflammatory Cells of the Airways
| Macrophage phagocytosis steps
```
Phagocytosis of cell
Fusion with lysosome
Degredation into fragments
Fragments presented on plasma membrane
Leftover fragments released by exocytosis
```
410
Inflammatory Cells of the Airways
| How are alveolar macrophages different from other macrophages?
Poor antigen presentation
Low ROS
Naturally hyporesponsive under steady state
411
Inflammatory Cells of the Airways
| What cytokines do Alv Macrophages produce in health?
IL-10
TGF-b
Low amounts
412
Inflammatory Cells of the Airways
| What activates alveolar macrophages?
IL-1
IFN-a
IL-8
Phagocytosis
413
Inflammatory Cells of the Airways
| What 4 main cytokines do alveolar macrophages produce when activated?
```
IL-12
TNF
IFN-y
IL-8
Recruitment of DC/NK/Neutrophils
```
414
Inflammatory Cells of the Airways
TIP "IF I WAS TO ASK YOU WHAT THE MAIN MECHANISMS BY WHICH HOMEOSTASIS IS MAINTAINED BY ALVEOLAR MACROPHAGE - EPITHELIAL CELL INTERACTIONS THIS IS WHAT YOU'D ANSWER
Inflammatory Cells of the Airways
TIP "IF I WAS TO ASK YOU WHAT THE MAIN MECHANISMS BY WHICH HOMEOSTASIS IS MAINTAINED BY ALVEOLAR MACROPHAGE - EPITHELIAL CELL INTERACTIONS THIS IS WHAT YOU'D ANSWER
415
Inflammatory Cells of the Airways
TIP "IF I WAS TO ASK YOU WHAT THE MAIN MECHANISMS BY WHICH HOMEOSTASIS IS MAINTAINED BY ALVEOLAR MACROPHAGE - EPITHELIAL CELL INTERACTIONS THIS IS WHAT YOU'D ANSWER
Via IL-10 / IL-10R interaction
- JAK1/STAT3 -> SOCS3 ->inhibition of inflammatory cytokine expression
TLR2/4 -. NFkB
Epithelium expresses CD200, macrophage CD200R (has associated RASA2 and DOK2 -> ERK/p38/JNK signalling -> induce inflammatory transcription
Macrophage has TGFbR -> binds (alongside TGFb) to avB6 integrin
Epithelium expressed SPA and SPD -> bind to SIRPa on macrophage -> Signals via SHP1->RHOA -> inhibits phagocytosis
Macrophage TREM2 receptor inhibits inflammatory transcription
416
Inflammatory Cells of the Airways
| List anti-inflammatory receptors on macrophages.
```
Mannose
IL10
SIRPa
TGFbR
TREM
CD200
```
417
Inflammatory Cells of the Airways
| List the pro-inflammatory receptors
```
IL1R
IFNGR
TNFR
CD-14 TLR2/6
TLR4
Oxidative stress increases TLR expression
```
418
Inflammatory Cells of the Airways
| What study discovered that alv Macros develop from fetal monocytes in the first week of life in mice?
Guillams et al 2013
419
Inflammatory Cells of the Airways
| Where do the signals that stimulate tissue resident macros->alv macros?
Lung microenvironment
- rolk sac macros/fetal liver/adult monocytes can develop into alv macros in Knockout mice with an empty niche - Van der Laar et al 2016
Same with peitoneal macrophages - Lavin et al 2014
420
Inflammatory Cells of the Airways
| Describe the chronology of the macrophage with regards to the lung?
Fetal macrophages peak -> differentiate into tissue resident alv macros in the first week.
As infection/pollution/smoke take their toll, these are replaced by monocyte derived alv macros gradually in adult life
421
Inflammatory Cells of the Airways
| What is they hypothesis with regards to monocyte derived alveolar macrophages in late life?
Although morphologically similar to alveolar macrophages, these are believed to be more pro-inflammatory
Possibly why more pneumonias etc in later life.
422
Chemokines in the lung
More CD8+ T cells, asthma or COPD? Jeffery et al
Deaths vs healthy
COPD 8 fold change from baseline.
| Asthma only 2
423
Chemokines in the lung
Fold change of Macrophages - more in COPD or asthma? Jeffery et al
Deaths vs healthy
```
COPD = 9
Asthma = 0
```
424
Chemokines in the lung
Fold change eosinophils asthma vs COPD Jeffery et al
Deaths vs healthy
COPD 4
| Asthma 93
425
Chemokines in the lung
| 3 types of chemoattractants
Chemokines = small proteins
Lipids eg LTB4 (neutrophil chemo)
Peptides eg fMLP - bacterial cell wall peptide
PGP - matrix breakdown peptide
426
Chemokines in the lung
| How do the majority of chemokines/attractants signal?
G protein coupled receptor signalling
427
Chemokines in the lung
| Describe the process of G protein coupling
Agonist binds to 7TM receptor
G protein (a,b,y subunits) binds to cytoplasmic domain
GTP -> GDP by a subunit
(a subunit is a a GTPase on any G protein coupled receptor)
GDP+a subunit bind to PLC
PLC degrades membrane and forms PIP2
PIP2 broken to IP3 + DAG
IP3 opens Ca2+ on actin cytoskeleton -> actin rearrangment
DAG activates PKC
Remaining b+y subunits activate PI3K which upreguates adhesion molecules
PI3K can also rearrange actin
428
Chemokines in the lung
| What is the a subunit of G protein receptors?
GTPase
429
Chemokines in the lung
| What does IP3 activate in G protein coupled receptors?
IP3 opens Ca2+ on actin cytoskeleton -> actin rearrangment
430
Chemokines in the lung
| What is redundancy?
- More than one chemokine per receptor
- Single chemokine can bind to multiple receptors
- More than one receptor per cell
431
Chemokines in the lung
| Problems with redundancy?
- Different chemokines are not always equipotent at a single receptor
- Different chemokines may have different functions at the same receptor
- The same chemokine receptors on a cell may have different functions
432
Chemokines in the lung
| List 3 ways of measuring chemotaxis?
Boyden chamber
Transwell systems
Videomicroscopy with vector plot
Surrogates -> shape change assays , Ca2+ mobilisation
433
Chemokines in the lung
| What did video microscopy and vector plot depict with regards to chemotaxis?
Even the same cells when exposed to the same conc of chemoattractants do not move in the same direction/at same velocity
434
Chemokines in the lung
| How do Trevor Hansel's surrogate methods for determining chemotaxis work?
Take blood
Measure shape change (Must occur with chemotaxis)
Measure upregulated adhesion molecules such as CD11b
435
Chemokines in the lung
| Describe graph of conc chemokine vs number of cells migrated
```
Bell shaped (upside-down U)
Cell migration decreases after point of maximal migration
```
436
Chemokines in the lung
Briefly explain how cells move with regard to "polarisation"
Wang et al 2002
Receptors polarise to head, which leads migration.
Fast receptor turnover and recycling allows it to sense direction
WANG et al 2002
437
Chemokines in the lung
| As a result, explain why HUGE amounts result in little/no movement?
Cell unable to sense gradient to polarise and move
438
Chemokines in the lung
| Describe how chemokine-chemokine interactions can alter chemotaxis?
Can get homodimers
EG CXCL8 dimers reduce CXCR1 binding
Can get heterodimers
EG CXCL8-CXCL4 increases migration but is anti-proliferative on ECs
439
Chemokines in the lung
Explain why CCR3 antagonists (GW766994) failed in asthmatics?
CCR3 = on eosinophils
Neighbour et al 2014
```
No effect on blood eosinophillia
No effect FEV1
No improvement in PC20
No symptomatic improvement
Neighbour et al 2014
REDUNDANCY
```
440
Chemokines in the lung
| What was discovered with regards to neutrophil / peripheral blood monocyte migration to CXCL1 (GROa)
Sig increased migration in COPD vs smokers/ non COPD
441
Chemokines in the lung
What was discovered with regards to PMBC movement by CXCL9/10/11
in normal/smokers/COPD
Sig increased in COPD
442
Chemokines in the lung
| Describe why differing levels of chemotaxis occur to the same stimuli in different diseases?
Receptor recycling
```
Ligand binds
a subunit = GTP->GDP
Allows binding of ARRESTIN protein
Removed from surface -> endosome
2 fates:
- Broken down
- Recycled
IN COPD WITH GROa the receptors are recycled more quickly
```
443
Chemokines in the lung
| Describe the process of receptor recycling?
```
Ligand binds
a subunit = GTP->GDP
Allows binding of ARRESTIN protein
Removed from surface -> endosome
2 fates:
- Broken down
- Recycled
IN COPD WITH GROa the receptors are recycled more quickly
```
444
Chemokines in the lung
What's the difference between healthy vs COPD directional movement + velocity?
Stockley et al 2013
COPD = loss of direction
COPD = move faster
Stockley et al 2013
445
Chemokines in the lung
What corrects COPD neutrophil loss of direction and fast movement?
Stockley et al 2013
PI3K inhibitor
| Stockley et al 2013
446
Chemokines in the lung
| What is the only chemokine receptor licenced for use? What does it antagonise?
Maraviroc (HIV)
| CCR5
447
Chemokines in the lung
Several CXCR1/2 antagonists from multiple pharmaceutical companies failed clinical trials, despite showing very promising in vitro dose response results. Why is this? What happened with healthy volunteers?
Worked on healthy volunteers at reducing neutrophils in sputum (absolute + percentage). BETTER THAN PRED
```
On COPD patients for 6-18 months
Dec sputum eosinophils
Inc FEV1 by 67ml
Inc FEV1 in smokers by 168ml
BUT
!!!Dec ANC -> neutropaenic sepsis possible!!!
```
Rennard et al 2015
448
Chemokines in the lung
| How could p38 inhibitors, PI3K inhibitors, PDE4 inhibitors, and reservatrol stop enhanced leukocyte recruitment?
Act as actin polymerisation / cytoskeleton inhibitors
449
Chemokines in the lung
| Why did actin/cytoskeleton inhibitors fail?
Multiple side effects or not bioavailable
450
Chemokines in the lung
| How could antibodies be used to stop enhanced leukocyte recruitment?
a/bs to cytokines
a/bs to receptors
But redundancies
451
Chemokines in the lung
| What are the three classes of drugs being investigated to stop enhanced leukocyte recruitment?
Actin polymerisation / cytoskeleton inhibitors
A/bs
Chemokine receptor antagonists
452
Chemokines in the lung
Why may a CCR2/5 antagonist also inhibit the actions of CXCR4?? WHY ARE WE SHITE AT DRUG MAKING
Sohy et al 2009
Because receptors can exist as homodimers, heterodimers and oligodimers
CCR2-CCR5
CCR2-CXCR4
CCR5-CXCR4
Can get negative cooperativity.
453
Chemokines in the lung
| Explain negative cooperativity with regards to CCR2-CCR5?
CCR5 ligand binding inhibits CCR2 ligand binding
454
Clinical Lung Cancer
| 3 types of lung cancer
Small cell
Non Small cell
Carcinoid (nonSC)
455
Clinical Lung Cancer
| Which type of lung carcinoma is traditionally treated with chemotherapy?
Small cell
| Non-small cell rx with other means
456
Clinical Lung Cancer
| Most prevalent lung cancer?
NSCLC 88%
SCLC 11%
Carcinoid 1%
457
Clinical Lung Cancer
| What percentage of smokers get lung cancer?
12%
458
Clinical Lung Cancer
| What stage are most diagnosed in?
```
4 (53%)
Stage 1 (19%)
```
459
Clinical Lung Cancer
| Why do only 15% of stage 1-2 have surgery?
Comorbidities
Choice
Age
460
Clinical Lung Cancer
| Lung cancer 1 yr survival overall?
38%
| 31% in 2010
461
Clinical Lung Cancer
| What is the Will Roger's effect?
Idea that early detection technically prolongs survival but doesn't really
462
Clinical Lung Cancer
What did the national lung screening trial research team discover with regards to regular low dose CT vs chest radiography in individuals over 50 with 30+ pack years?
Risk of dying from lung cancer reduced by 20%
463
Clinical Lung Cancer
| Will low dose CT regular screening take place in the UK?
Unlikey despite the body of evidence (UKLungScreening+NLSTRT)
"we are broke"
464
Clinical Lung Cancer
| What is happening to the incidence on non-smoking lung cancer + smoking related lung cancer?
Smoking related lung cancer incidence is decreasing.
Non-smoking lung cancer incidence has increased by 12% - unknown why (women/asian/adeno/nonsmoking-NO MEANS OF DETECTING)
465
Clinical Lung Cancer
| How are stage 1 LC patients Rx? What is the rationale for this?
Surgery
No studies actually compare surgery vs non surg.
Surgery "seen" as definitive -> no one would enter such a trial
Surgery + comorbidities/age has also never been compared in study
ONE RCT did compare surgery to radiotherapy -> radiotherapy was better but the world shunned the trial
466
Clinical Lung Cancer
| What did the West Brompton study show regarding SCLC and NSCLC resections?
Despite popular opinion being chemo for SC and surgery for NSCLC the survival was actually pretty similar. Nobody "believed"
467
Clinical Lung Cancer
| How many trials are commencing worldwide to determine the extent of resection?
3
468
Clinical Lung Cancer
| What is the new form of radiotherapy that is being installed UK-wide?
SABR
Stereotactic ablative body radiotherapy
Delivers higher doses of radiation that can be targeted better -> as a result less toxicity
469
Clinical Lung Cancer
| Why is SABR already near-outdated? What is the new machine being tested?
MR-Linac
| MRI guided linear accelerator
470
Clinical Lung Cancer
| What did the SABRTOOTH trial 2015 show when comparing SABR vs Surgery in stage 1 NSCLC high risk pts?
Closed due to failure to accrue as people wanted surgery
Pooled analysis with another terminated trial depicted a HAZARD ratio of 0.14 indicating the risk of death is reduced by 86%!!!
471
Clinical Lung Cancer
| What treatment being developed may be EVEN better than MRI-Linac
```
National Proton Beam Therapy Programme
UK gov £250ml to build two facilities
- Reduced radiation dose
- Reduced oesophagitis
- Well tolerated
```
472
Clinical Lung Cancer
| Systemic generalised old chemotherapy success rate?
20%
473
Clinical Lung Cancer
| What was the hazard ratio of standard chemo vs Gefitinib EGFR TK inhibitor?
0.48
| Risk of death reduced by 52% survival increase
474
Clinical Lung Cancer
What fusion gene was first discovered in mice by Japanese scientists that lead to Pfizer developing and running a human trial?
EML4-ALK7
Nearly every person responded to Crizotanib which was fast tracked to FDA approval
Only 4% lung cancers have this fusion gene though
475
Clinical Lung Cancer
| What was the HR reduction of nivolumab vs Docetaxel in NSCLC?
0.59
| = 41% reduced risk of death
476
Clinical Lung Cancer
| What did the PACIFIC trial show with regards to Durvalumab use after chemo-radio in stage 3 NSCLC? (HR)
HR 0.52
| = risk of death reduced by 48%
477
Basic Science Lung Cancer
| What is a driver mutation?
Mutation that causes cancer cell to grow
478
Basic Science Lung Cancer
| What is the soil and seed hypothesis?
Circulating cancer cells die if they do not establish blood supply, or come into contact with WBCs in cirulation
479
Basic Science Lung Cancer
| How does a microarray chip work?
All complementary DNA binds.
Upon binding light is emitted.
Colour and intensity indicates hybridisation.
Only done for abhorrent genes eg EGFR mutation
CHEAP
480
Basic Science Lung Cancer
| How does illumina work?
Maps out EACH nucleotide base sequence using colour emission detected by laser as each binds. Add one by one, then wash out, wait for emission, No emission = wash out and next base tried
ATCG coded with different colours
481
Basic Science Lung Cancer
| How does thermofisher work?
Addition of one base at a time
| When each base binds a proton is released and is detected by a voltage sensor
482
Basic Science Lung Cancer
| What did the Cancer Genome Atlas Research Network discover when sequencing the exxons of lungs adenocarcinoma?
8.9 mutations per megabase
| TP53, KRAS and EGFR accounted for 80% of mutations
483
Basic Science Lung Cancer
| What did the Cancer Genome Atlas Research Network discover when sequencing the exxons of squamous cell lung carcinomas?
81% have TP53 mutations
484
Basic Science Lung Cancer
How does the development of osimertinib (EGFR TK inhibitor) highlight that even with new Rxs there is room for improvement?
When compared to standard EGFR TK inhibitor it showed:
HR for progression 0.46 = 54% reduced risk of progression
HR for death 0.63 = risk of death reduced by 37%
485
Basic Science Lung Cancer
| Name a receptor that T cells use to recognise host cells (vs tumour)
PD1 (recognises PDL1)
486
Basic Science Lung Cancer
| How can a tumour cell evade T cells?
If an oncogenic pathway results in upregulation of PDL1
OR
Tumour cell binds via MHC, INF-y release from T cel to tumour cell results in Stat signalling and upregulation of PDL1
487
Basic Science Lung Cancer
| What percentage of cells require PDL1 expression to benefit from immunotherapy
50%
488
Basic Science Lung Cancer
| What percentage of patients have tissue confirmed lung cancer? National lung cancer audit
75%
489
Basic Science Lung Cancer
| What are the cons of tissue diagnosis?
```
Invasive - complications include risk of death
Time consuming -> multiple specialists
Procedure waiting times
Expensive 2-3k pp
May underdiagnose EGRF mutations
```
490
Basic Science Lung Cancer
| What is the cost of diagnostic assessment overall for lung cancer?
~3-5k£
491
Basic Science Lung Cancer
| List blood based biomarker possibilties (6)
Circulating tumour cells
DNA - methylation, mutations, rearrangements
NA - microRNA
Proteins - altered levels, altered processing, other modifications
Immune response - immune cell profile, autoantibodies, cytokines
Metaboltes - cancer metabolites, altered host metabolism
492
Basic Science Lung Cancer
| What ambitious blood test are illumina (yes the genome mapping company) developing themselves?
PAN-CANCER BLOOD TEST
493
Basic Science Lung Cancer
| What are the methods of detecting circulating cancer cells?
```
CTC Clearbridge chip
Screencell-filter
Marker proteins
Direct visualisation
Circulating RNA and DNA
```
494
Basic Science Lung Cancer
| What are circulating epithelial cells likely to represent?
Cancer
495
Basic Science Lung Cancer
| What is the only FDA approved circulating cancer cell machine?
Cell search
496
Basic Science Lung Cancer
| How does "Cell search" work?
Antibodies to Circulating tumour cell surface markers
Antibody is bound to magnet
Separated by magnetic field
497
Basic Science Lung Cancer
| What is a benefit of cell search?
High recovery capacity 93%
and detection limit
Allows cell enumeration
498
Basic Science Lung Cancer
| Negatives of "cell search"?
Requires non-standard expensive equipement/reagents
Cannot detect EpCAM NEGATIVE -CTCs
Skin cells EpCAM positive
499
Basic Science Lung Cancer
| How does CTC Clearbridge chip work?
Micro chip - smaller than a US 1 cent coin
Blood = flushed through
Normal cells = malleable and small enough to pass through microfluidic filters
Cancer cells = stuck on tiny pillars
Wash with saline
Sent to pathologist
500
Basic Science Lung Cancer
| What problems may be encountered by the pathologist with clearbridge?
- Lack of architecture
| - Cells beaten and squished
501
Basic Science Lung Cancer
| What was the sensitivity of clearbridge overall?
40%
502
Basic Science Lung Cancer
| What were the results of comparison between two different pathologists with clearbridge?
Sensitivity 40 vs 59%
| Specificity of 67 vs 0%
503
Basic Science Lung Cancer
| What is the SPIND acronym for (specificity)?
In a test with high
Specificity
a Positive test rules IN Disease
504
Basic Science Lung Cancer
| What is the SNOUT acronym?
In a test with high sensitivity a negative result rules out disease
505
Basic Science Lung Cancer
| How does screen cell work?
Like a vacutainer with a filter
506
Basic Science Lung Cancer
| What was the intraobserver agreement among pathologists with regards to screen-cell? Why was this performef?
Agreement of 64%
15vs19% confirmatory diagnosis
Not sufficiently concordent
Wanted to test human ability vs test
507
Basic Science Lung Cancer
| What proportion of patients have CTCs in early stage cancer?
50%
508
Basic Science Lung Cancer
| How can centrifugal forces be used to detect CTCs?
CTCs larger -> separated from blood cells
509
Basic Science Lung Cancer
| How does "cell extraction" work with regards to CTC detection?
Involves removal of normal cells eg WBC via CD45
510
Basic Science Lung Cancer
| What was the specificity of 2 groups of compared pathologists cell extraction? CTC
Each had 100%
511
Basic Science Lung Cancer
| Why has cell extraction not been rolled out?
WIDE CIs.
| Need to test on larger N numbers
512
Basic Science Lung Cancer
| What is sample heterogeneity?
Idea that blood tests may miss CTCs
| Mutation frequency varies
513
Basic Science Lung Cancer
| Why is circulating DNA being investigated more?
More evenly distributed
514
Basic Science Lung Cancer
What did Lim et al discover with regards to comparison of FFPE (formalin fixed paraffin embedded tissue) vs CTCs vs Plasma ctDNA [Kras mutation]
COMPARE CTCs to ctDNA
cDNA sensitivty of 96% and specificity of 95%, good CIs
CTC all over the place
Sens - 52%
Spec - 88%
515
Basic Science Lung Cancer
What did Lim et al discover with regards to comparison of FFPE (formalin fixed paraffin embedded tissue) vs CTCs vs Plasma ctDNA
[Kras]
Compare ctDNA to FFPE
FFPE - Mutation identified 61% of time
ctDNA - 68%
Agreement of 85%
516
Basic Science Lung Cancer
| What was the sensitivity of ctDNA KRAS detection ?
68%sensitivity
Specificity 91%
PPV 98%
Confidence intervals approaching clinically sound levels
517
Basic Science Lung Cancer
| What does the tracer X consortium hope to acheive?
A method of detecting the original mutation of the "parent" cancer cell
Targeting this would kill all cancer cells.
Creates a genetic tree by sequencing all cell genomes and looking at frequencies of mutations
Cancer is highly heterogeneous
518
Basic Science Lung Cancer
| What does the a higher number of sub-clonal mutations correlate with
Earlier death
519
Pulmonary Hypertension
| Definition of PHTN?
mPAP > 25mmHg
520
Pulmonary Hypertension
| What is normal pulmonary blood pressure?
<18mmHg
521
Pulmonary Hypertension
| What is the PVR?
PVR = mPAP-PAWP/CO
522
Pulmonary Hypertension
| What does a PAWP >15mmHg mean?
Post capillary PHTN
LA pressure high
LS-heart disease
523
Pulmonary Hypertension
| What does a PAWP <15mmHg represent?
Pre-capillary or pulmonary arterial hypertension
524
Pulmonary Hypertension
| What group are PAWP >15mmHg patients?
2 - PH-LHD
525
Pulmonary Hypertension
| What group are PAWP <15 patients(s)
1,3,4,
Pulmonary arterial hypertension
PH-lung
CTEPH
526
Pulmonary Hypertension
| Describe the pathophysiology of pulmonary arterial hypertension?
Raised PA pressure
Vascular proliferation and arterial wall thickening - LAPLACE
Vasoconstriction and in-situ thrombosis
527
Pulmonary Hypertension
| What do PAH patients die from. What is the untreated mortality?
RVH -> dilation -> failure
| 40%
528
Pulmonary Hypertension
What shape is normal RV?
What shape is a failing RV?
Crescent
Becomes more circular
Wall thickens and septa deviates
Compressed LV
529
Pulmonary Hypertension
| What are the five clinical classifications of PHTN?
1. PAH
2. PHTN ass. LHD
3. PHTN ass. Lung disease
4. PHTN ass. thromboembolix
5. Unclear//multifactorial
530
```
Pulmonary Hypertension
What class of PHTN does IPHTN / heritable causes fall under?
```
1
531
Pulmonary Hypertension
| List geners associated with PAH
BMPR2
ALK-1
SMAD9
CAV1
532
```
Pulmonary Hypertension
List lung diseases associated with class 3?
```
COPD
Sleep disordered breathing
ILD
High altitude
533
Pulmonary Hypertension
| Describe progressive nature of the disease?
```
Symptomless as CO can maintain
As CO goes down symptoms occur
RV begins to fail
Resistance constantly rises until death
PAP may fall as RV fails
```
534
Pulmonary Hypertension
| Symptoms and signs of PH?
```
Non specific
- Breathlessness
Fatigue
Chest pain
Syncope / pre Syncope
```
- RV failure signs
Tachypnoea
535
Pulmonary Hypertension
| Mean survival PPH w/o Rx
2.8yrs
Mortality varies per cause eg with congenital HD much lower mortality -> heart compensates to RV adaptation with remodelling when young
536
Pulmonary Hypertension
| What is the proportion of patiens with idiopathic PHTN? NAPH data
33.6% NAPH data
537
Pulmonary Hypertension
| What percentage is heritable?
10%
538
Pulmonary Hypertension
| M:F
1:2.5
539
Pulmonary Hypertension
| Median age at presentation
40s
540
Pulmonary Hypertension
| Who should be screened for PHTN? "detect study"
Only scleroderma pts
| DETECT study
541
Pulmonary Hypertension
| What are the 3 primary investigations for PH?
Bloods - BNP, TFT, FES
ECG - RVH + strain (t wave inversion chest leads) pattern, prone to AF/flutter
CXR - megaly, dilated PAs, peripheral oligaemia
542
Pulmonary Hypertension
| Rx
```
Warfarin (IPAH)
O2 follows COPD guidelines
Diuretics
Specific subtype Rx
Lung transplantation
Exercise training
PAH - Endothelin antagonists/NO/phosphodiesterase inhibitor/prostacyclin
```
543
Pulmonary Hypertension
| Why is pregnancy bad?
Mortality 30%
| Due to increased circulating blood volume
544
Pulmonary Hypertension
| Problem with endothelin antagonists?
S/Es
| Drug interactions -> warfarin/statins/Cyp450s
545
Pulmonary Hypertension
| PDE5 inhibitors. How do they work?
Inhibit PDE5 degredation of cGMP which increases activity of NO
Sildenafil/tadalafil
546
Pulmonary Hypertension
Which drug has shown an improvement in survival?
What are the problems with this Rx?
IV prostacyclin
25% survival increase
Need a constant Groshling Line port -> kept sterile and maintained/checked
- Terrible side effects but do normalise as patient acclimatises
- Expensive
547
Pulmonary Hypertension
What is CTEPH?
What Rx?
Chronic thrombo emboli -> causes PH -> occludes
| Endarterectomy -> instant relief
548
Common Disease Models
| Advantages of cell line cell models?
Easy to use
Cost effective
Unlimited supply (if freeze early passage)
Tissue Specific
549
Common Disease Models
| Disadvantages of cell line cell models?
Cancer cells
Adaptation in cell culturing
Lack of tissue architecture
No heterogeneous population
550
Common Disease Models
| What are the advantages of a primary cell models, eg NHBE normal human bronchial epithelial cells?
Cells maintain important markers and functions seen in vivo
551
Common Disease Models
| What are the disadvantages of primary cell models, eg NHBE?
Primary cells are extremely sensitive
Require additional nutrients not included in classical mediums
Difficult to maintain
552
Common Disease Models
| What are the advantages of stem cell models?
Better knowledge of growth/differentiation
Key to reversing aging
Cure development defects before they occur
Manipulated to different cell types
Full function unkown
New mechanisms of action described-> could be harnessed
553
Common Disease Models
| What are the disadvantages of stem cell models?
High uncertainties
Unknown S/Es
Playing god + ethical
Perpetuates cloning humans
554
Common Disease Models
| Advantages of Celegans model?
```
Free living nematode
First multicellular organism to have its whole genome sequenced
Six chromosomes 100mb
Easy growing
Good for molecular genetics
```
555
Common Disease Models
| Disadvantages of C elegans model?
Small size
| Evolutionarily far from mammals
556
Common Disease Models
| Drosophila model advantages?
4 chromosomes
Bioengineering
A tool in drug discover - fuck knows
557
Common Disease Models
| Disadvantages of Drosophila model?
No easy measure of complex behaviour
Can only be maintained as living creatures - no permanent conservation possible
Less complex immune system (adaptive)
558
Common Disease Models
| Advantages of zebra fish?
```
Genetic similarity to humans
25 Chromosomes
Easy to house and care
Impact of mutation easy to see
Lots of offspring
Easier to introduce genetic changes
```
559
Common Disease Models
| Disadvantages of Zebra-fish?
Not a mammal
Water system maintenance
Fly genetics is more powerful
560
Common Disease Models
| Primate model adv?
Genotype-environment interactions
Complex pedigrees even more powerful
Hypotheses can be tested prospectively
Essential and invasive experiments can be conducted
561
Common Disease Models
| Primates dis?
```
Cost
Genetic heterogeneity
Availability
Restrictions and regulations
- hard to work on in UK
```
562
Common Disease Models
| Most commonly used lab model?
Mus Musculis - mouse
- inbred + outbred strains
WHOLE genome has been sequenced + annotated
563
Common Disease Models
| Advantages of mouse models?
```
Mammals
10=15 offspring
450 inbred strains
Short generation time
Genetic manipulations and knockouts
```
564
Common Disease Models
| Dis of mouse models?
Alike, yet dif
85% similarity of genomes
Big distinctions
Mouse brain 70% neurons and 30% glia - human=opposite
Short generation time may not be good for aging related diseases
565
Common Disease Models
| What does isogenic mean? Mice models
Genetically identical
566
Common Disease Models
| What does Coisogenic mean?
Mutation/transgene/targetted allele which arose directly on that strain
567
Common Disease Models
| What does congenic mean?
A variant larger than a gene but otherwise isogenic
568
Common Disease Models
| What does consomic mean?
Complete chromosome from one strain of mice in the background of a second strain
569
Common Disease Models
| What is an inbred mouse?
Mice bread to be isogenic
570
Common Disease Models
| How are mouse embryonic cells used?
Can be used to introduce exogenous DNA / manipulate endogenous
571
Common Disease Models
| What is a transgenic mouse?
Mouse engineered to have an introduced gene
Fertilised eggs collected and microinjected with DNA - 20% of the surviving mice have the DNA at one site in a chromosome
572
Common Disease Models
| What is a chimera?
Embryo/foetus/adults that are mixtures of genetically distinct cells
573
Common Disease Models
| What is ENU
A chemical used to introduce A,T changes (87%) or G,C mutations (13%) in DNA
- introduces missense, splice or nonsense mutations
574
Common Disease Models
| What is homologous recombination?
When a fragment of genomic DNA is introduced into a mammalian cell it can be recombined with homologous endogenous sequences
575
Common Disease Models
| List the steps of gene targetting?
Generate the targetting vector
Extrapolate into cells
Culture ES cells in presence of a selectant
Identify cells that have the recombinated DNA
Inject ES cells into blastocysts
Identify chimaeric pups
Generate progeny that carry the target gene
576
Common Disease Models
| What is Cre/loxP // FLP/FRT?
Site specific DNA recombinase
Cleaves DNA and inserts new
Can be used for deletions/insertions/modifications
Can be triggered by stimuli selective for the cells wished to be targetted
577
Common Disease Models
| What is zinc-finger nuclease?
Method of knockout formation/dna engineering
Zinc fingers engineered allowing nucleases to target
DNA binding domain (cutom designed)
Non specific DNA cleavage domain - cleaves next to binding domains
578
Common Disease Models
| Adv of zinc finger nucleases?
Short time course to generate knockouts - 6 months
Targeted gene disruption with high efficiency
ES independence: applied to other species
579
Common Disease Models
| Disadvantages of ZN nuclease
Screening and assembly is technically challenging
Commercial ZFN molecules = expensive
Difficult to perform large fragment replacement
Off target effects require screening of animals
580
Common Disease Models
| What is transcription activator-like effector nuclease? How does it work?
Method of genomic engineering
Present in xanthomonas bacteria
- molecules capable of DNA binding and activation of target genes via mimicry of eukaryotic TFs
581
Common Disease Models
| What is CRISPR? How does it work?
Cas (CRISPR-associated) proteins recognize and cut exogenous DNA
Replaced with DNA associated to CRISPR molecule
582
Common Disease Models
Adv of CRISPR
Dis
- Easy
- Many applications
- Highest efficiency and safety
- Food industry -> personalised medicine
- off targeting problem and screening required
583
ILD
| In general and simply, why does fibrosis occur?
Epithelial injury -> activates myofibroblasts -> Abhorrent wound healing
```
Failure of:
ECM degradation
Scar regression
Re-epithelialisation
Myofibroblast apoptosis
```
584
ILD
| General histology of ILD?
Thickened interstitium - scar tissue, cells, connective tissue
585
ILD
| What is the simplified classifications of the ILDS?
```
Idiopathic
Systemic involvement
Associated with a cause/exposure
Heterogenous
Fibroblastic foci
```
586
ILD
| Important Hx questions?
```
Exposures home/work/hobbies
Drugs - pneumotox
Smoking
Fhx
Systemic symptoms
```
587
ILD
| General Ix for ILD?
P/E
Routine biochem
Full autoimmune screen -> ANA ENA anti-ccp, anti-dsdna, CK, precipitations
HRCT chest
Bronchoscopy with BAL (only if CT is non-confirmatory)
ONLY AFTER ALL THESE HAVE PROVIDED NO CAUSE OR ASSOCIATION CAN IT BE CLASSED ILD
588
ILD
| How many deaths from IPF?
1% of deaths, 5k per yr
| Equal to that of leukaemia but isn't known by many patients
589
ILD
| ILD, how has the survival changed over the last decade?
It hasnt changed in a decade
50% die by 3-4 yrs
20% survival at 5 yrs
590
ILD
| Prevalence IPF?
over 50yrs it increases nearly exponentially
| Rare before 40
591
ILD
| Risk factors IPF?
3-20% familial
Gender M>F
Env - smoking, dust, air pollutions with acute exacerbations, viruses/bacteria
592
ILD
| First mutation discovered in IPF?
SP-C (surfactant protein C)
Protein A2
1-2% Familial IPF
593
ILD
| What are telomeres?
Repetitive pieces of DNA
At each replication these repeats are lost
When telomeres are critically short cells undergo apoptosis/cell cycle arrest
Shorten with age
594
ILD
| What percentage of mutations do telomerase mutations account for?
```
10-15% familial cases
1-3% sporadic cases
Armanios et al 2007
Tsakiri et al 2007
Cronkhite et al 2008
```
595
ILD
| What did Alder et al PNAS 2008 discover with regards to IPF alveolar epithelial cells?
Shorter telomeres than normal
| Both carriers and sporadic IPF
596
ILD
| What gene has the strongest association with IPF, discovered by genome wide association studies?
MUC5B
34% familial
38% sporadic IPF
9% of control population
597
ILD
| What can clinicians NOT predict with IPF?
Unpredicable disease course
- Some respond to exacerbations worse -> accelerates fibrosis and progression
- Some may rapidly progress in general
Unable to differentiate
598
ILD
| What did stuart et al 2014discover with regards to telomere length in IPF?
May be able to be used as an independent marker of poor prognosis in IPF.
Shorter telomeres in more severe disease.
Repeated in 3 different cohorts, dose-effect response
599
ILD
What did the PANTHER trial compare?
60 week doule blind RCT
IPF clinical research network
Compared placebo, Nacetylcysteine monotherapy and triple therapy
Primary endpoint FVC change
600
ILD
| What were the results of the PANTHER trial?
STOPPED EARLY
Hospitalisation probability
20% inc risk of death
601
ILD
| What are the two IPF approved drugs?
Both anti-fibrotic drugs
- Pirfenidone - unknown MoA - anti TGF, oxidant?
- Nintedanib - triple TK inhibitor VEGF, PDGF, FGF
602
ILD
What was pirfenidones mean change in FVC at 1 yr vs placebo?
King et al NEJM 2014
-250 vs -450
| Patients retained ~200mls
603
ILD
| What was nintedanibs mean FVC change at 1 yr when compared to placebo? Richeldi et al 2014 NEJM
-100 vs -200
| ROUGHLY 100MLS of RETAINED
604
ILD
| WHat percentage of patients cannot tolerate IPF drugs?
15-20%
605
ILD
| What is connective tissue ILD survival like in comparison to IPF?
```
60% survival 5 yrs
Must assess for extra thoracic signs -> may be few differentiation symptoms
- raynaulds
- Scleo.
- Prox muscle weakness
- Raynalds
- Gottrons
- Mechanics hands
```
606
ILD
| What is the most common histological pattern seen in CTD-ILD?
NSIP
Inflammation admixed with fibrosis
HOMO-genous
Often see airways within 2cm of lung periphery
607
ILD
| What drug was shown to stabilise disease in SSC-ild SCLERO
Oral cyclophosphamide
| MMF
