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Thanks, everyone.
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So today I'm going to talk about in vitro models and sort of how human in vitro systems are really important for translatability, something that I think everyone in the room would agree.
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So just a few background slides on who we are.
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I promise they're short.
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So we're a preclinical specialist immunology CRO.
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All of our assays are built using primary human immune cell subsets from both healthy and diseased donors.
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And we run validated immune cell assays.
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But the reality is that we customise most of them to fit with our clients target of interest and the biology of interest.
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And we try and work in all the therapeutic areas where the immune system is involved.
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And as we all know, it seems to be involved everywhere these days.
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So the list is forever growing, alright.
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So if we want to push drugs through into the clinic and translate them well, then we need to have good in vitro models to measure immunomodulation.
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And for inflammation and autoimmunity, normally we're looking at assays where we can see immunosuppression and for those where we're looking at immunostimulatory molecules, normally within the field of immuno oncology and infectious disease.
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And below I've listed all the other sort of therapeutic areas where the immune system gets involved.
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And the reality is that a lot of the assays that you set up for inflammation, for autoimmunity, for immuno oncology can be translated into these other therapeutic areas as well.
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So I think when we're thinking about the biology that we want to model, I think we need to ask the question just not does this occur if we culture cells together in a tissue culture dish?
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It's sort of thinking about how that happens in vitro, in vivo, sorry.
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So thinking about the anatomical location and where cell interactions happen.
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And if we think about that, then we sort of understand the kind of biology, biological assays that we need to be developing in vitro.
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So if we can think of this in sort of a disease agnostic manner, then we can think of those interactions that happen in the secondary lymphoid organs.
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So priming and activation of T cells and B cells.
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And how do we model that effectively and how do we bring in the other immune cell subsets that form this very specialised structure that's necessary for these events?
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And then if we move over to the other side again, sort of in this disease agnostic manner, then the effector site, we can think of this as either the tumour or within autoimmunity, the different tissues where that autoimmune reaction's happening.
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How do we best model that?
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We know we've got lots of subsets of cells present here.
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We've got epithelium, fibroblasts, specialised tissue specific cells and then a whole host of resident immune cells as well as those that migrate into the tissue.
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And of course these two things are linked by the blood and the lymphatics.
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So again, that could be another area where you're looking to therapeutically intervene, for example, with migration.
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So again, assays to look at that.
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So if we start off thinking about the interactions that happened in the secondary lymphoid structures, then we can sort of building complexity in the kind of assays that we're running.
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So the real work horse I think for looking at a lot of these interactions is the MLR.
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It's not as sexy as some of the other assays, but it gives us a lot of information and it's relevant if a therapeutic is targeting those cell-cell molecular interactions, T cell signalling pathways for example, or cytokine pathways.
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What it doesn't tell us is if you've got a therapy that's modulating antigen presenting cell function and then we need to move on into the middle of the road sort of family car type assay where we've got antigen specific T cell biology and autologous DCs.
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And then we can start looking at things like DC intrinsic signalling.
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If you're therapeutically targeting antigen loading and presentation, then again, we're building in that.
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And then right out of the other end is sort of your Formula One type assay if you like.
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So that's your tonsil reaggregate culture.
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So now we've taken secondary lymphoid tissue, disaggregated it, reaggregated it, and you've got all the cell types in there that would be within that secondary lymphoid structure, like your T follicular health cells and your B cells, resident dendritic cells.
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And of course, you're looking at antibody production as well.
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And you can look and see how your therapeutic intervenes in that pathway.
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I'm not going to talk about that today, but if you'd like any more information, please just pop by the booth.
4:57
All right, so what kind of molecules can we look at?
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So within autoimmunity, I'm going to give some example data looking at inhibition of positive signalling and the impact that has on activation, proliferation and effect cytokine production.
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But the other areas where you could look sort of for example is blocking co-stimulation.
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So something like a CTLA 4 protein to block there.
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And we heard a nice talk earlier on about how PD 1 can be engaged with agonist and again tone down T cell activation using that kind of approach.
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On the flip side, with immuno oncology, you can look at agonising co-stimulation or blocking negative regulators and I'll give an example of that in the next slide.
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All right.
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So if we look over on the left, we've cultured dendritic cells and allogeneic T cells together and we're looking at interferon gamma production and we can see if we inhibit T cell signalling pathways directly or through rapamycin through the mTOR pathway, then what you see is a suppression of interferon gamma production with these.
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So it's very effective.
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And then if you come down to the bottom and look at CellTrace Violet as a measure of proliferation versus CD25 as an activation marker, you see these molecules block proliferative and activation responses.
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No surprise, this assay works really nicely for these kind of immunosuppressant type molecules.
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On the flip side, if we add a PD1 blocker, in this case pembrolizumab, what you see is again this assay works really nicely and reading out those molecules that are looking to enhance the immune response but in a slightly different way.
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So now we're seeing increase in the interferon gamma production, but you don't impact on proliferation.
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And I think that just highlights that your readout should really be tailored to the molecule that you're targeting.
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It's not a one-size-fits-all these assays.
6:56
So if we then skip on to building in that complexity, so perhaps now you're sort of looking to understand the antigen presenting cell function.
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I will describe how we set up this so we isolate monocytes, differentiate them to dendritic cells and then co-culture them depending on what the question is with tumour cell lysates or potentially with auto antigens if you're interested in autoimmunity.
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They then undergo several rounds of restimulation and you have your functional readout.
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So your ELISpot for example, for interferon gamma producing cells, flow cytometry where cell numbers allow and tumour killing as well if that's the relevant readout.
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So you might be asking, well, that's nice, but what are the questions that I can ask in this system?
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And the reality is you can ask quite a lot in this system.
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So if you have a therapeutic targeting dendritic cell function itself, I'm going to describe a little bit of data there, just using R-848 as a tool to sort of see how you can modulate DC function and then therapeutics targeting the tumour antigens themselves.
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So for example, this could be something where you're trying to generate novel antigens within those tumour cells before you load them into your DCs or an ICD inducer for example, where you're looking to induce immunogenic cell death in those tumour cells.
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Also for therapeutics that are targeting T cell expansion such as cytokine type molecules, I think there's been some talks on that kind of approach at this conference and also the co-inhibitory co-stimulatory pathways that we're all so familiar with now and there's so many of those that can be targeted.
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And I think the nice thing about this system as well that it's very amenable to sort of bispecific approaches where you're looking to engage something on the antigen presenting cell as well as on the T cell itself.
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And then, of course, lastly, sort of therapeutics targeting effective function.
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I'll just give 2 examples of some data, so I'll talk really quickly through this one.
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So if we start down the bottom, it's really annoying not having a pointer.
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But what you see is that we've taken dendritic cells and then we're using just R-848 as a tool to look at maturation.
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And what you can see when we use this, then we have subtle increases in CD40 expression, CD80 expression in terms of MFIs and an increase in the percentage of CD86 expression as well.
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So it's just an example of how you can look at that dendritic cell biology up at the top.
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More relevant for if you're targeting tumour cells themselves, you can measure efferocytosis or phagocytosis.
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So again, isolated dendritic cells, our positive control is Frodo Green labelled bioparticles, those only fluoresce once they go into the acidic compartments within either a macrophage or ADC.
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So it's a nice way of looking to see if you've got internalisation.
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You can see that's very efficient.
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The tumour example that I show here is the Malme-3M.
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We were interested in Mark 1 antigen specific T cell interactions in this context.
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And you can see if you just feed the dendritic cells live Malme-3M cells, not many of them are phagocytosed within this time frame.
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If you heat shock them to induce a bit of ICD like death, then they start to be taken up nicely and those could then go on and be put in co-culture with T cells.
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And this is what happens if you do that.
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So just an example with three different tumour cell lines, so the A549s, Caco-2, DLD-1s.
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So in this particular experiment, we loaded the DCs, they underwent 3 restimulations and then at the end we ran an ELISpot to look at the frequency of antigen responsive cells.
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In the middle you have the controls.
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So those were only stimulated on day 21.
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So the PMA Ionomycin is the positive control.
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You can see that cells are healthy, which is key.
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And then for the A549 1 stimulation you don't really see much at all.
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It's not sufficient to drive that T cell activation.
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But now when you look over the third restimulation with these loaded DCs, then you start to see spots in the A549, the Caco-2, but not the DLD-1.
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And the graphs below just depict the difference between expanded CD4s and CD8s.
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They were separate cultures and you can see how this could be utilised for therapeutics that are targeting sort of that effector cell function and also expansion as well.
11:40
So if we jump on into modelling interactions that occur at tumour or peripheral tissue sites, then I think, you know, the overall goal is probably quite simple for both immuno oncology and autoimmunity.
11:54
Immuno oncology, we want a situation where someone's tumour free if the therapeutics involving T cells, we probably want a memory T cell response in case the tumour re pops up again within autoimmunity inflammation.
12:09
I think this concept of immune resets been around for a long time, but people are sort of increasingly using those terms and talking about immune reset.
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And really the goal here is for lasting tolerance.
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There's sort of many ways where you could potentially drive that by targeting the effector cells themselves or depleting effector T cells.
12:32
So I'm just going to give one quick example of the tumour microenvironment.
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I'm looking at some co-cultures, and then the example that I've chosen for sort of autoimmunity would be interactions that are relevant within psoriasis.
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There's lots of markers there and it looks complicated, but the data that I'm going to show is pretty straightforward to understand.
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All right, so 3D modelling of the tumour.
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So I am just showing here an NK mediated ADCC assay.
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So this would be a depletion type approach.
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I'm using NKs as your effectors.
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And I think pictures are of like normally nicer than graphs.
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So if we just look over on the left at the red blobs.
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So we established the spheroid SkoV3, they express HER2.
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If we culture them with an isotype, then the spheroid size stays fairly constant.
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Now if you add trastuzumab in, you see nice shrinking of the spheroid over time.
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And likewise we're the factor cytokine type approaches.
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That's our positive control.
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Then again, you're driving that shrinking of the tumour and you can measure that over time.
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And over on the right, the graph just depicts that you can get significance across multiple donors with this kind of approach.
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So the utility here is with novel ADCC type antibodies that you want to test.
14:00
All right, so if we come back to the autoimmune setting, sorry, I'm flip flopping between the two, but I think a lot of approaches translate from immuno oncology and to autoimmunity and vice versa.
14:12
I'm going to focus on some TH17 biology and also on neutrophils, just to be controversial and talk to something else other than T cells.
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So in skin, IL17 is a key driver for psoriasis for people that aren't sort of familiar with the disease.
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And the reality with an autoimmune disease is that normally you're working with patients that have that sort of chronic ongoing disease.
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So you want to be looking probably at memory cell subsets in terms of effectors.
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So what we do is sort out firm peripheral blood on TH17 memory cells based on CCR6 expression.
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We can then stimulate them with CD3/28 and a cytokine cocktail over a period of days and adds therapeutic in at this phase if that's the appropriate point for intervention.
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And then look at the percentage of cells which are producing IL-17 or even better.
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If you're pushing cells through into a T regulatory phenotype, then you start to have an advantageous biology, not just blocking the effector sort of subsets.
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And then those cells can either be co-cultured with keratinocytes or with fibroblasts or the supernatants can be taken from the T cells and put on the keratinocytes and fibroblasts.
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And again, that's another opportunity to test therapeutic intervention.
15:37
And the readouts there typically tend to be cytokines and chemokine production with the aim of dampening down those sort of inflammatory mediators that are produced as a consequence of the disease process.
15:51
So the kind of molecules you can test in here really most of them to be honest with you.
15:56
So antibodies blocking soluble factors or blocking receptors could be depleting antibody strategies and all your good checkpoint type molecules.
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But again, I'm just going to talk about therapy targeting signalling pathways.
16:15
So here's the data and I think sort of this frequency of IL17 producing cells is really nice because it can be very challenging in human cells to get good IL17 production.
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If people work with mice, it's much easier than the humans.
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So we've got 50% of cells producing IL17 following this culture.
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You also see the polyfunctional cells, so they interferon gamma 17 co-producers and then a few of the cells that produce gamma alone.
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If we add FK506 into these cultures, as you'd really predict, it's going to inhibit proliferation.
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But the nice thing is it also inhibits cytokine production and that's reflected as well on the graph on the right.
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So it's just an example of the utility of these kind of assays.
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And then last not least, I've got a couple of people in my team that are real neutrophil aficionados.
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So I had to include a neutrophil slide to keep them happy.
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So they tell me neutrophils are the most important cell and who am I to argue?
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So neutrophils contribute to inflammation via NETs, reactive oxygen species, proteolytic enzymes and they're also a driver of auto antigen generation for those of us that like T cells.
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And the data that I'm showing below is from a NETosis type assay.
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So we've purified out neutrophils, we've stimulated them for four hours and you can measure NET production by staining for extracellular DNA.
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You can see it's really quick and that most of those cells will sort of undergo this NETosis process.
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Each of those green blobs is an individual cell.
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And then if you measure that over time, if you look at the blue line, that's our vehicle control.
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And then we've come in with the DPI and you can show that we can inhibit NET formation arm in a dose dependent manner.
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So again, this will be a nice system for testing therapies that aimed at regulating neutrophil biology.
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And of course, sort of one of the challenges with neutrophils is that you don't want to probably regulate them everywhere because then we'd all be in big trouble.
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So I think people that have strategies to specifically dampen down inflammation at the relevant sites, that's where this gets interesting.
18:37
So I just want to quickly acknowledge this lovely lot who produced all the data and much more which haven't shown today.
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And lastly, there's four of us here.
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So please do stop any of us just to say hello or to ask any questions about any of the data that I've presented today.
18:56
Be happy to speak to you.
18:58
Thank you.
