Transcript will be available Friday, Oct 2
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Host – Dan Keller
Hello, and welcome to Episode Fifty-Three of Multiple Sclerosis Discovery, the podcast of the MS Discovery Forum. I’m your host, Dan Keller.
This week’s podcast features Dr. Jonathan Kipnis, who discusses his recent discovery of lymphatic vessels in the meninges. But first, here are some new items in the MS Discovery Forum.
According to our curated list of the latest scientific articles related to MS, 34 such articles were published between August 21st and 28th. To see these publications and the articles we selected as Editors Picks, go to msdiscovery.org and click on Papers.
Our Drug-Development Pipeline includes continually updated information on 44 investigational agents for MS. This week, we’ve added 6 pieces of information about alemtuzumab and fingolimod. To find information on all 44 compounds, visit msdiscovery.org and click first on Research Resources and then on Drug-Development Pipeline.
The MSDF team is looking forward to attending next month’s ECTRIMS meeting in Barcelona, Spain. If you, too, will be at the conference and would like to meet with us – or if you’re interested in being interviewed about your research for a future podcast – please email us at editor@msdiscovery.org.
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And now to Part 1 of our interview with Dr. Jonathan Kipnis, Professor of Neuroscience and Director of the Center for Brain Immunology and Glia at the University of Virginia in Charlottesville. His group recently published in Nature their discovery and characterization of lymphatic vessels in the meninges.
Interviewer – Dan Keller
You've described in this paper about meningeal lymphatics, the novel but actually more conventional path for cerebrospinal fluid drainage from the CNS than I guess had been thought of before; it's sort of conventional as revolutionary. Can you tell me what you found and what led you to look?
Interviewee – Jonathan Kipnis
Yes, so we've been interested in the role of meningeal immune system for quite some time, and we've shown that changes in meningeal immunity could impact brain after a CNS injury, or also for normal brain function. So, for example, mice that have impaired meningeal immunity would show cognitive deficits and would show some little bit more prone to stress and other phenotypes.
So we've been very interested in understanding how meningeal immunity is being regulated. So the assumption was at some point that there is no immune cells in the brain, which is true, except for microglial which reside in the brain and compose 10% of the brain cells, but there is no peripheral immune cells within the brain. But in very nearby areas, which is the surroundings of the brain – the choroid plexus, the meninges, and the CSF – that's where actually there are immune cells, and there are all types of immune cells. And so we have been very interested to understand how the cells are getting in and getting out.
Through the use of parabiotic mice, we demonstrated last year, we showed that immune population of the meninges is not static; the cells are being repopulated, and about 50% of T cells, for example, is being exchanged within about 10 days, and major exchange between the CSF or the meninges with the deep cervical lymph nodes. So nothing was really new, we just sort of established things maybe more solid way. Those cells can get in while still nobody understands very well how they get in; we'll assume they get in through the meningeal vasculature, which is probably true.
But then how do they get out, or what happens with cells after they get to the CNS? Well, the assumptions were, well, they either die, magically disappear, or crawl under the nose through the cribriform plate and into the deep cervical lymph nodes through the nasal mucosa. They were okay explanations, but in our systems we did not find any of it to be sufficiently explaining what's going on in this really fast and pretty dramatic exchange of the immune system within the meningeal spaces.
So when we just looking at it a bit closer, and it is very, very well established that there is lymphatic drainage from the CNS, so this needs to be remembered. So people in many labs have shown that if you put stuff in the brain – which stuff I mean proteins – if you put proteins or antigens in the brain, whether it's in the parenchyma or in the meninges or in the CSF, you will find those proteins, and you will find immune response to these proteins in the deep cervical lymph nodes.
The question is, of course, how do they get there? And the path which was described just did not work in our hands, and so I was lucky to get a very, very talented postdoc, Antoine Louveau, at the lab. He realized that for us to understand how things get in and out, the only way to do it is to do live imaging and also to do a whole mount of the entire meninges. And I think that's when it was a breakthrough point. So Antoine laid out the entire meninges and was looking for location of the immune cells. And he said let's see where I see maximum accumulation of the immune cells, and then let's see how these places will change when we expose mouse to, for example, stress, learning, or EAE inflammation, viral infection, or whatever, let's see how these areas of dense immune population will change.
And so he realized that there is a lot of immune activity around the major sinuses in the meninges, and then he saw that there are immune cells which are in the vascular structures which were not blood vessels. And I think that was the turning point. And I said, okay, if the cells are within the vasculature which is not blood vasculature, what would it be? Well, so I went to colleagues here and said what do you label lymphatic vessels with? And they didn't understand why would you want to label for lymphatic vessels, because they don't work with the brain. And so we labeled a lymphatic marker and we saw the vessels, which were lining the major sinuses and going all the way along them. So that's a very long answer to your very simple question.
MSDF
And Antoine Louveau's technique here that was the key to it was doing in situ fixation so he could get the meninges out intact?
Dr. Kipnis
To let's assume the meninges came out intact on the brain, and let's assume we had this beautiful staining. Let's say we did the coronal staining, and let's say we're labeling for lymphatic vessels. So you can imagine that what you'll see is at the border of the brain you will see a dot; you will see maybe three dots because there are three vessels going along the sinuses. And when you see a dot, you never take a dot seriously in immunohistochemistry. Now that we know that this dot represents the vessel, then we can actually go back and do those coronal sections and look at it. But back then only by seeing the whole meninges mounted as one on a slide, and by seeing those vessels there, I mean that's when we knew. And so to us it was obvious this is something that absolutely went under-noticed. And this technique of whole-mount meninges, I think, was absolutely crucial.
MSDF
Did he find these vessels in all layers of the meninges, or any specific ones?
Dr. Kipnis
No, no. Major lymphatic vessels are following the superior sagittal and the transverse sinuses, which is in the dura. So all the blood from the brain is being – at least in mice. In humans it goes a little bit different, but also through the sinuses, although sinuses are located so not all the blood in the human brain goes through the parasagittal sinus, but in the mouse brain all the blood goes through the sinuses in the dura. So major sinuses through which all the blood is being collected from the brain, and then goes out there. And so along those sinuses we find the lymphatic vessels, so they are sitting in the dura.
MSDF
And this system also has been found in humans?
Dr. Kipnis
Well, that's a good question. You know, it's very hard to obtain high-quality human samples from the dura, because nobody really cares about this area. So we were lucky in the triple operation of Bea Lopes, who's a really great neuropathologist here at UV; she was able to give us, I think, nine samples from patients of dura of the sinus; these were all fixed in formalin. So we looked at those, and as you can imagine, the sinus in the human is huge, so obviously compared to a mouse. So in two out of nine, we were able to identify vessels that looked like meningeal vessels, but I think it warrants much deeper and much farther investigation to be able to say, yes, here they are. But if you ask me personally, why wouldn't they be? Why would mice have them and humans won't have them. So I think it's a matter of identifying their location and the best markers to use for them, but I think they should be there again. In two out of the nine samples, we were able to demonstrate that this is something that looks very, very good.
MSDF
And you did immunohistochemistry on these to show the lymphatic properties and not general blood circulatory vasculature properties, either in the mice or human?
Dr. Kipnis
Oh, yes. So in the mice we identified the characteristics of those vessels really, really well. You know, nothing is perfect, that every marker on mouse markers are expressed by different cells, so you need here to provide a series of markers and to demonstrate that this is also indeed the real lymphatic cells. So we stained for LYVE1 and we showed beautiful staining with LYVE1, also with macrophages. And those are vascular structures and they came out to be macrophages. But one of the major transcription factors that will define lymphatic and endothelial cells is a Prox1. So we demonstrated two ways of Prox1; one is transgenic mouse and the other is staining for Prox1. And we also did two other molecules. One is a Podoplanin which is expressed in tissue lymphatics, and these vessels are expressive.
And the other molecule, she is very interesting. It's a receptor for VEGF3, VEGF-C. And this receptor is first on the lymphatic and endothelial cells. In the periphery, lymphatic and endothelial cells will respond to recombinant VEGF-C and will expand. So what we did here, we also injected a recombinant VEGF-C and we showed these vessels expanding. So we know now that the receptor is actually functional in the vessels, but also we now can expand the vessels. Whether it will impact any neurological disease, we don't know, but at least we have the capability to do so.
And then we also identified them by flow cytometry. We took samples from skin and from diaphragm where lymphatics are very, very well defined, and using the exact same antibodies we did also flow cytometry on our meningeal samples. And we show that the cells look exactly like they look from the skin and from the diaphragm; of course, the numbers are much smaller. So I think in terms of their calculation in a mouse, we are very convinced.
Now for humans it's more difficult. Like I said, the sample was in formalin and it's very hard to work with those samples, and, again, the area is huge to go through. So we were able in humans to get two markers to work; one was LYVE1 and the other was Podoplanin. We could not make Prox1 to work, I think it's a problem with the antibody and not with the vessel or potentially with the tissue as well. And these vessels would not label for some other markers, which would be characteristic of, for example, macrophages. So we were able to attack on them two out of four markers that would potentially allow for him to see. But we are now trying to identify those vessels by other means in humans as well, and I think flow cytometry may be the way to go.
MSDF
Now you've shown that these lymphatic vessels drain into the deep cervical lymph nodes, and it looks like you've also been able to rule out drainage through the cribriform plate back into the cervical lymph nodes. Is that true?
Dr. Kipnis
I'm glad you bring this up, this is very important. So if you think of CSF, CSF is composed of several things. So we have the liquid itself, we have the macromolecules within the CSF, and then we have the immune cells within the CSF. So I don't think there is anybody would argue against liquid being drained through the cribriform plate and through the granulation; this is funny to argue. And obviously we are not claiming anything until we're absolutely sure; there is beautiful works from many, many labs showing that. But for the macromolecules and for the immune cells, the path which was proposed through the cribriform plate most probably if it's not a wrong one, it's probably not the major one.
[transition music]
Thank you for listening to Episode Fifty-Three of Multiple Sclerosis Discovery. This podcast was produced by the MS Discovery Forum, MSDF, the premier source of independent news and information on MS research. Msdiscovery.org is part of the non-profit Accelerated Cure Project for Multiple Sclerosis. Robert McBurney is our President and CEO, and Hollie Schmidt is vice president of scientific operations.
Msdiscovery.org aims to focus attention on what is known and not yet known about the causes of MS and related conditions, their pathological mechanisms, and potential ways to intervene. By communicating this information in a way that builds bridges among different disciplines, we hope to open new routes toward significant clinical advances.
We’re interested in your opinions. Please join the discussion on one of our online forums or send comments, criticisms, and suggestions to editor@msdiscovery.org.
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[intro music]
Host – Dan Keller
Hello, and welcome to Episode Fifty-Two of Multiple Sclerosis Discovery, the podcast of the MS Discovery Forum. I’m your host, Dan Keller.
This week’s podcast features Dr. David Tabby, who discusses the incidence of headache in MS. But first, here are some new items in the MS Discovery Forum.
According to our curated list of the latest scientific articles related to MS, 53 such articles were published between August 14 and 21. To see these publications and the articles we selected as Editors Picks, go to msdiscovery.org and click on Papers.
We’ve made some recent updates to our Funding Opportunities and our Meetings and Events listings, both of which can be found under our Professional Resources tab. Be sure to take a look at a newly-posted funding announcement entitled, “National MS Society: Health Care Delivery and Policy Research Contracts.” If you know of any meetings, events, or funding opportunities that are missing from our lists, please email us at editor@msdiscovery.org so that we can include them.
Our Drug-Development Pipeline includes continually updated information on 44 investigational agents for MS. This past week, we’ve added one new trial and 15 other pieces of information. The drugs with important additions are dimethyl fumarate, fingolimod, glatiramer acetate, interferon beta-1a, interferon beta-1b, and ofatumumab. To find information on all 44 compounds, visit msdiscovery.org and click first on Research Resources and then on Drug-Development Pipeline.
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And now to the interview. Dr. David Tabby is an adult general practice neurologist, with a subspecialty in multiple sclerosis, in Bala Cynwyd, Pennsylvania. He was formerly associated with Drexel University College of Medicine in Philadelphia where he carried out the study on headache and MS that we discussed.
Interviewer – Dan Keller
Could you just tell me what the aim was? I think you were looking the variables affecting headache occurrence in MS patients.
Interviewee – David Tabby
Well, to be perfectly frank about that, my business manager had noted that many of my MS patients also had migraine, and she asked what’s going on with that. So we did a cursory review and found that close to 50% of my MS patients also had migraine. So that prompted the survey in a more formal fashion.
MSDF
Whom did you look at, and what were some of the variables you looked at?
Dr. Tabby
Well, we looked at everyone who was willing to answer a survey. And we wanted to include some typical migraine variables like frequency and intensity and triggers and age of onset and duration at that time and which came first, MS or migraine.
MSDF
And what were some of the major findings?
Dr. Tabby
I think our biggest contribution was that it seemed that we could correlate exacerbations with increase in headache, which I don’t think was known before that. Sometimes headache a little bit before the exacerbation, and sometimes exacerbation first, then with headache. But that raised the question about the role of inflammation in both headache and MS exacerbations. You know, I don’t think one is the cause of the other, but they’re clearly related.
MSDF
What about migraine with aura or without? Are there differences in the MS presentations or symptoms or relations in time?
Dr. Tabby
We didn’t have a lot of migraine with aura patients. That’s only about 20% of the migraine population anyway. We didn’t have a big enough number to make any kind of association that would be distinguishing migraine with or the migraine without aura.
MSDF
Do you have a proposed mechanism in mind how these two may be linked – mechanism of migraine, mechanism of MS?
Dr. Tabby
I don’t think there’s a lot of work about what the immune system’s role is in migraine, but stress affects the immune system. My patients tell me that a particularly stressful event can seem to precipitate an exacerbation, and, no big secret, that stress can precipitate a migraine.
MSDF
Fatigue can be a trigger, and obviously, that’s a large proportion of MS patients have it. So do you think there may be a link there, one triggering the other?
Dr. Tabby
90% of MS patients will complain of fatigue at some point, and I think that’s clearly got something to do with it. So anyone with migraine will tell you that when they overdo it and get overtired, they get a migraine.
Interviewer – Dan Keller
I think I saw something in the paper was talking about metalloproteinases in the CNS and leakiness of vessels and allowing either antigens out or T cells in. Does that have any legs or where does that idea come from?
Dr. Tabby
That idea came from a paper that we found in our literature review of the basic pathophysiology of an MS exacerbation that matrix metalloproteinases have to be activated first, to increase vascular permeability of the CNS. Migraine was thought, at one time, to be a primarily vascular issue. It’s not. It’s primarily a neurochemical issue that affects the vasculature, but results in dramatic changes in blood vessel permeability. There’s a kind of leakage of fluid around blood vessels, which helps make them more sensitive.
MSDF
There’s also a component of calcium flux in migraine. Would that have any effect on the immune system? There’s calcineurin inhibitors that are immunosuppressants, so I’m wondering if calcium in itself – could be an imbalance be a stimulant?
Dr. Tabby
The role of calcium in modulating neural function can’t be overstated. I’m not ready to give you an exact mechanism of how that might function.
MSDF
Patients with migraine supposedly have more symptomatic clinical course of MS. Do you have any numbers there? What did you find?
Dr. Tabby
We weren’t a longitudinal study. We’re really just a snapshot kind of picture to determine, you know, a relationship now. That was another one of our hypotheses that the worse headache you had the worse MS you were going to have. Our take-home message from the paper was MS doctors should be very aggressive about addressing migraine symptoms in their patients, because we think that it could have an effect in long-term prognosis in MS. And it’s not just the patient complaining about, oh, I have a really bad headache. There is a relationship. It’s been known for many years. I mean, you might have seen in our bibliography that some of the papers went back to the 1960s about the relationship with headache and MS. So, just to reiterate, migraine prophylactic therapy, migraine abortive therapy is important in the context of treating MS.
MSDF
Can pre-existing migraine before the MS diagnosis give you any clue as to being a risk factor in itself?
Dr. Tabby
That’s an excellent point. We didn’t have a big enough group to come to that conclusion, but 12% of Americans have migraine – 18% of women, 6% of men. Less than 1% of the population has MS. Something else has to be happening.
MSDF
Also, I guess you found that people with stabbing pain had more acute MS exacerbations than if they had other more throbbing kinds of pain.
Dr. Tabby
Well, we were just using that as an indicator of severity of the headache. Stabbing’s not a typical word used to describe migraine. We gave choices. We just didn’t leave it open-ended. We gave some example words for their responses to the survey to tick off. Yes, stabbing was one of them, and there was a correlation between the ones who said stabbing and worse exacerbation.
MSDF
Some of the patients you surveyed did not have pre-existing migraine, only after their MS diagnosis. Do you think that migraine is something that one should investigate whether they actually may be in a prodrome of MS, if they present late - develop migraine late in life – later in life?
Dr. Tabby
In our world, pretty much everyone with a bad headache who sees a doctor about it, at some point is going to get some sort of imaging study of the brain. It might be a CAT scan, in which case there might not be enough information. But if it’s an MRI, there’s a good chance of seeing high-signal lesions scattered through the white matter. They may be real small and not particularly typical of MS. But not uncommonly, they’ll see a pattern that looks really quite like MS. And you examine the patient, and you don’t find anything that’s consistent with MS. You do a history, and you don’t find any symptoms that are consistent with MS. So this is the sort of thing you just file away and see what happens. I don’t think anyone would suggest that treatment for MS should be started at that point.
There’s an entity that’s finally gotten a name, called radiologically isolated syndrome, which people get MRIs of their brain for some reason – head trauma, headache, anything really – and the pattern looks really quite like MS, but there’s no clinical support of MS. So same thing; we don’t forget about these people. You recheck their MRIs at some point in the future. You look for new lesions or enhancing lesions. I think the latest statistic is somewhere around 50% of them are eventually going to present as having multiple sclerosis.
MSDF
Would each entity be treated or each condition be treated as if it existed in isolation? Or do they complicate the management of the other?
Dr. Tabby
My approach has been to treat them independently. Just follow the basic principles of headache treatment for the headache and the basic principles for MS treatment and adjustments of therapy and symptomatic therapy for MS. I would not suggest some interlinked therapy system.
MSDF
But something like interferon and possibly other drugs may cause exacerbation of headache. Would that lead to noncompliance of the MS treatment or would you switch drugs or how do you handle that?
Dr. Tabby
That doesn’t necessarily involve the headache issue. I think someone who has bad headaches on interferon who’s not taking their medicine, you know, you might try to fix it – fix the headache that is. But you’re probably fighting an uphill battle when that patient has negative associations with taking their medicine. I still tend to think of these two things as related but separate.
MSDF
So what’s the big take-home message for a physician?
Dr. Tabby
Really just about treating migraine aggressively in their MS patients, and keeping in mind that we work very hard to preserve function to reduce the accumulation of disability, with drugs and other sorts of interventions. But intervening for migraine may be just one other way to reduce the burden of disability in the future, for your patients.
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MSDF
Thank you for listening to Episode Fifty-Two of Multiple Sclerosis Discovery. This podcast was produced by the MS Discovery Forum, MSDF, the premier source of independent news and information on MS research. Msdiscovery.org is part of the non-profit Accelerated Cure Project for Multiple Sclerosis. Robert McBurney is our President and CEO, and Hollie Schmidt is vice president of scientific operations.
Msdiscovery.org aims to focus attention on what is known and not yet known about the causes of MS and related conditions, their pathological mechanisms, and potential ways to intervene. By communicating this information in a way that builds bridges among different disciplines, we hope to open new routes toward significant clinical advances.
We’re interested in your opinions. Please join the discussion on one of our online forums or send comments, criticisms, and suggestions to editor@msdiscovery.org.
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