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very much accepting. Um, thanks for giving me the opportunity to discuss just sort of part of my PhD project. So I'm going to talk predominantly about the transcript analysis, transcription analysis I've done of areas of men in jail and Prevacid information in postmortem m s case. So just a bit of a background of my PhD in general. So it's interested in looking at tertiary lymphoid structures in M s, which are collections of immune cells that gather within the meninges. Um, and they're they're sort of interchangeable. Terms for these in the literature is sometimes described as B cell follicles because they're predominantly our do consist of B cells, and so they form these sort of aggregates in the meninges. But they are quite heterogeneous and that you can't see loose collections of B and T cells within the meninges, and they can vary in size. And they're described throughout the cerebral hemispheres, but most typically in the deep, still kind of the cerebral hemispheres and typically overlying some peeled grey matter lesions, and they've also been described in the cerebellum and spinal cord. We know from post mortem studies that patients who have TLS have a more severe disease course. Uh, so this is work done by on how well that showed that cases that have these follicles or TLS and got a diagnosis of M s At younger ages, they had a shorter disease, duration, shorter time to progressive stages of M s and a shorter time from progression to death. We also know again from post mortem studies they have a more severe cortical pathology which is again linked to progressive MS and they have high numbers of grey Matter lesions and greater areas of demyelination with in the gray matter, what isn't totally clear in M s is exactly the exact immune cell composition of the structures. And whether it changes in the different disease stage is it's also very difficult to know which patients have TLS. And so research predominantly relies on post mortem studies. My overall hypothesis that these structures are distinct immunologically in early versus longer duration, M s. And one of the injury with this is is do we can determine whether TLS have a distinct immunologist center and therefore can we better predict which patients have them and which patients therefore may like, more likely get a progressive disease course and my overall Ames's to characterize the TLS cases you're pathologically and then compare their cellular and molecular profiles. But today I'll just be talking about the molecular work. So I'm using for Olympics party embedded tissue from the dementia and tissue collections, and this is held here in Queens. And crucially, this tissue predates introduction of disease modifying therapies. Um, therefore should represent the natural disease history, and in most cases it's a relatively short time interval to postmortem, which is very critical for my transcript stomach work. Initially, I set out to characterize and update the clinical data base and then select the patient or the cases that would go then to my study. And so, within this tissue collections, there's 214 s cases and then I further stratify these by disease duration. Another unique thing about this tissue collections. We have quite a large number of short disease duration cases, but not all were eligible for my studies to others had other meningeal pathology or didn't have the appropriate CNS blocks, so they were excluded, which left 12. So, firstly, then I undertook screening to identify which cases had TLS is the incidence of this isn't known in this tissue collection. So to do this I used histological stain to identify the number of cells and encountered the number of cells running blood vessels and the meningitis in the parenchyma and a substantial scores. So there's more greater than 50 cells. And these are cases that are more likely to have TLS and the images at the bottom of two cases that have these aggregates. Um, that may represent TLS. So it was these cases in that moved forward to my transcript stomach work, whereas interested in looking at the gene expression, particularly differences between the TLS and then the other men in jail and very vascular inflammatory sites. Um, so doctor Michelle Norton, who's a post hoc in our group one, uh, an award to use Nano string platform, which allowed hold transcription analysis of 25 regions of interest per side. Um, so firstly, I validated which cases I could detect R N a N. And then these two of these were then selected to go for the Nano string hold transcription analysis, and these were the two cases that were selected. So these, uh, this is the tissue section at the bottom of these images, and one of the cases had a short disease duration of four months, and the other kids had a longer disease duration of six years and had a progressive phenotype. Then this is a heat map showing the top differential expressed genes in the meningeal areas, um, in the T l s and then the other meningitis areas that didn't have TLS and our highest express genes were immunoglobulins of which the immunoglobulin G subclasses I G four and I G three were the highest expressed by also had kidney cancer markers to see X e l nine cc L5 and c x er four. So I move forward, then to validate these candidates using an RNA scope multiplex assay. So essentially, that s a then allowed me to look at for 12 or in a target within a single tissue section. And I selected similar reasons of interest to that used by the Nano string. Bass said So the tertiary infrastructure in the case, um so this is the same case that went for the nano string? Uh, whole trip to analysis. So the TLS was selected for analysis as we're other regions to other men in jail areas, other grey matter, pre vascular areas that were near to and away from the TLS and then white matter, Perry Vascular areas. So this is looking at the IgM and the, uh, GI expression. And this image here is showing that expression I g and green and I gm and yellow through the through the part of the TLS and each dot represents a single RNA transcript. So you can see here that I, GM and I g are quite highly expressed and it's interspersed throughout the TLS. I also saw that in the neighboring Grey Matter blood vessel that they're also was high expression of my GM um, and I g in the neighboring in the nearby Grey Matter blood vessel. Uh, this was also seen in the White Matter blood vessels. I also saw expression of iga again. This is a representative image of the TLS where we can see the r n a dots here within the cell nuclei and again this was observed in the gray matter blood vessel near the TLS and in white matter blood vessels and then looking at the different chemical in cytokines expressions so expression of see X er four was observed in all regions. This is just again a representative images showing TLS and again you see, that's quite high expression cell nuclei here of see x er four within the TLS c c L5 was again observed and TLS But, um, this was really only one or two dots per sort of cell. Um, and again it was seen in the TLS neighboring grey Grey matter, blood vessels and White Matter blood vessel space. And for see x e l nine. This was overall quite lowly, expressed in all of the region's, um, this is seen here in yellow here again, only 1 to 2 RNA at dots per cell. So currently, what I'm working on is then now to look at the protein expression of the immunoglobulin G subclasses these where our highest express jeans and to determine if they are distinct in the early versus late post mortem cases and then hopefully also then to look at the different protein levels and sort of selected cohort of patients, blood and CSF samples. So I just like to thank everybody who help me along the way, particularly Doctor Michelle Norton who has a lot of the training and supervision of this and also see my Thunder's. I'm happy to take any questions. It's really impressive, Rachel. I'm sure there's questions in a few people and ask some questions. Luckily, it's really nice. I think this works fantastic. I'm really interested in your reasons of interest. So obviously you're looking at the blood vessels in the brain and the white matter Do you know that myelin status of your regions of interest in terms of an answering was demyelinated. It was like normal appearing Grey Matter or, you know, demyelinating grey matter because obviously decided the tertiary infrastructure should get widespread demyelination. So I'm just wondering, like in your different regions where they're different levels of mile and present. Yes. So a mile and stain was done as part of the analysis and regions were selected based on where they were in the location to the TLS, but also in the normal appearing grey matter, mostly normal. Appearing well, there was both both okay. And did you find a difference in the ambulance between the normal appearing the demyelinating? So that is work. I'm hoping. Yeah, yeah, carry forward with yeah, I'm very interested in what? The results of that. So, yeah. So that's something I'm interested in looking at now that validated the targets. No, that's great. Okay, thank you very much. Okay, for your talk. Very exciting what you're doing. And my question is actually about the ls that you're going to find that you're finding in Manages, Uh, I wanted to ask a little bit more precise about the region. Is it possible to say, like in which, uh, matter you're finding them like, is it like, uh, more like leptomeninges? Or maybe your, uh it's not, like so much as possible to say, like a very precisely which matter of many years Yeah, they're they're fine. That was in the left. Um, and so in the in the pill, in the PM And, uh, yeah. Oh, and you know, excellent talk, Rachel. So I suppose it's not to unexpected to find our gg sub classes within the topic because they have the cells. So I just wondered what we're planning on doing any do labeling to see which population could be in there. Yeah. So, again, that's what I'd like to do is combine the IgG subclass work with a marker of the cell. So a plasma cell marker and another one will probably see the 20 to get an overall B cell count. But there are multiple. You think you might look at other kinds of Yes, another part of the Ph DS and looking at the different immune cells subsets within it. So that will form part of that work. Yeah, Very nice. Thank you. Um, I just thought that it's really important about the heterogeneity of this cell composition. And is it? Do you think it's possible to measure D C. K those cells? So it will just depend on what you get the genotype, and then you can infer that from RN A sequencing. Yeah, and I haven't looked at, but yeah, but I talked about the trial that we're using and obviously, which penetrates into the C. N s. And that works through activation by the CK. So it's interesting to see whether these cells are Yeah, there was another handout. I'm sorry. I've forgotten who someone Is there another question around there? You can go ahead and Yeah, I was just wondering what you were showing up for a moment. Yeah, I didn't I didn't know it was 13. So yes. So that wasn't That didn't actually come up in our database. We looked at the data base. Of course, the XL 13. Um, it didn't come up, so But essentially, it wasn't detectable act. If it was there, it wasn't detectable by the banana stream. Yeah, yeah, yeah. And it's a very brief question. And to finish Yeah, so I am. Detect Went used ubiquitin as it's highly, highly expressed protein CNS. So I optimized the protocol to detect. See if I can detect ubiquitous in any in the samples. Yeah, so thank you, too, Rachel.