6/6/2019 - Shelli Farhadian, MD, PhD

Transcript

• 00:06Good morning, everyone.
• 00:11Good morning, welcome medical grand rounds today is a very special grand rounds. The 2019 physician scientist training award medical grounds in honor of even the stonich given this year to Shelly Farhadian. The title of our talk is more inflammation. During chronic HIV infection. Terms of announcements next week will be our 3rd annual stories of Yale Internal Medison Grand rounds, they'll be 66 stories will be.
• 00:42Recited during the grand rounds and I would based on the previous 2 years. I would suggest you get here early if you'd like to get a seat.
• 00:51The following week June 20th medical grounds will be cancelled because of the start of the new intern year.
• 00:58There's no commercial support for around then there's no conflicts of interest. So I'd like to ask doctor Peter Aaronson to come up and introduce the award.
• 01:13Well, good morning, everyone.
• 01:15Welcome again to a very special medical grand rounds in which we celebrate the life and Legacy Aviva to Stanage, who came to Wales and member of our residency program.
• 01:24On behalf of the Department. I'd like to welcome you his parents dragon, and Predrag, who came here today to share in this special event. Eva's parents had become devoted friends to avail and we're very grateful that they've established and out fundeni. His name to support physician scientist career development in Ark apartment and do their generous donations. This endowment has grown very rapidly and will provide support for future generations of physician scientists in the L Department of Madison, so please join me in thanking them.
• 02:06So today March, the 8th time you're presenting an award leave his name to an outstanding physician scientist training in our Department. The first recipient was even herself who received the award in the 2000 eleven 2012 academic year. Initially, the recipient is Shelly Farhadian will be introduced in a few minutes.
• 02:23And the purpose of named awards and lectures is to highlight individuals whose careers represent the highest ideals of the medical profession.
• 02:31But who was even to stanwich? Why did our Department created award and lecture in the name of this young woman? What ideals were exemplified by Eva's life.
• 02:42Eva was born and raised in Belgrade, Yugoslavia, she had early exposure to family role models in science in Madison. Her mother was an academic pulmonary specialist. Even spent much time with her. God parents, who were PhD chemist. These experiences lead to an early love for chemistry and Madison.
• 03:01But the family is wonderful life in Yugoslavia was disrupted by the ethnic and religious wars that followed the dissolution of the country.
• 03:08Eva and her family became refugees and fled, 1st to Vienna and then to Athens.
• 03:14Even then came to the United States to attend college.
• 03:20At Manhattanville College, even strong interest in chemistry continued as you can see, she received multiple awards for excellence in chemistry.
• 03:28But she was a well rounded student who also received the prize for German studies. She received an award as the top student planning to pursue graduate studies and indeed even applied for PhD graduate programs in biochemistry and molecular biology.
• 03:43Even pursued graduate work in the laboratory of Jerry Lou Rael, who was at the time the chairman of genetics at the University of Cincinnati.
• 03:50Even used were then new methods of mouse genetics to introduce mutations, causing Waban resistance of the sodium pump to uncover rolls for endogenous cardiac glycosides and regulating pump activity, she definitively demonstrated what had long been postulated but never before proved that endogenous cardiac glycosides play a key role in several forms of hypertension.
• 04:11Eva was incredibly productive with 5 first authored papers that averaged over 100 citations. Each many of us on the faculty don't have 5 neighbors with 100 citations? Which.
• 04:22But even easily could have stopped there stop for training and obtained a basic science faculty position with this record of productivity. But she wanted to do translation research and even more importantly, she wanted to care for patients so she decided to go to medical school to become a physician.
• 04:40Even was admitted to the Cleveland Clinic Learner College of Madison, now is a young girl even had always loved her mother's white coat and all that it symbolized so it was a wonderful occasion, when Eva got her own white coat appan entering medical school at Cleveland Clinic.
• 04:57At the time the Cleveland Clinic was a very new medical school that admitted only 30 students per year for education is position scientists.
• 05:03Even had several research experiences during medical school, but our primary research advisor was the outstanding pulmonary physician Scientist Circle Earth's room.
• 05:12Even worked on translation studies on the problem of pulmonary hypertension again. She was very productive. Authoring 3 more papers. But she was not only a brilliant young scientist, but also exceptionally outstanding on her clinical rotations. In fact, even was chosen to receive an award at graduation be cause of her clinical excellence.
• 05:29Even decided to pursue the research pathway of internal medicine residency training combined with fellowship training and pulmonary disease.
• 05:37Evil with later tell her mother that the happiest day of her life was match Day 2011, when she matched the internal Medison program here at Yale.
• 05:46In fact, we were extremely delighted if not surprised that she ranked USTA match with her.
• 05:53But just before graduation evil was found to have ovarian cancer requiring extensive surgery and chemotherapy.
• 06:00But she had a good response and fulfilled her dream to come to Yale.
• 06:04She decided to defer internship until she got a bit stronger, so even became a research fellow with Patty Lee, who was then in our pulmonary section.
• 06:12Even absolutely loved her research work. Iva's tremendous passion and energy is shown by her typical daily activities rising at dawn going running working in the lab late into the night. All this after major surgery and while still receiving chemotherapy.
• 06:28Even worked on signaling mechanisms by which hypoxia causes pulmonary vascular dysregulation.
• 06:34Eva was always full of ideas, Patty Lee, some years ago, had shared her notes from her interactions with Eva. These were the ideas that Patty had given to even when even entered the layout. The very next day. Even came back and gave Patty filled out filled out set of ideas and note the legend at the bottom.
• 07:00So, in fact, in the few months that even work with padding, she made substantial contributions to the project that she was second, author of the major paper that was published describing their work together.
• 07:11But even was hardly a stereotypic science nerd when she wanted to dress down and be silly. She wore the Harvard T shirt. She had given given when one of the hardware. Hospitals was trying recruiter. I actually think she was kind of proud of herself for having the wisdom to pick yellow over Harvard.
• 07:27Indeed, leave abroad passion, Enerji elegance playfulness humor joy to all aspects of her life.
• 07:37But Unfortunately ibiz malignancy progressed, leading to her hospitalization at Yale in December of 2011.
• 07:44Even during the last phase of her illness. Even dismissed or physical ailments and her face. Literally glowed with Joy describing her exciting research ideas. Many of us visit her in the hospital at that time, she had tubes everywhere. But her face. We just light up telling us about her research that she had been conducting with Patty Lee.
• 08:01Eva's passion and love for Science in Madison. Despite the most adverse circumstances possible were truly awe, inspiring indeed. Eva's passion commitment and optimism despite all odds represented the very ideal of what it means to be a physician scientist.
• 08:20Our Department therefore established an annual award in lecture any of his name to recognize the physician scientist trainee in our Department who demonstrates the qualities that Eva so perfectly exemplified and Shelly Farhadian. This year's awardee is a truly outstanding training with many of these same qualities.
• 08:39But In addition to recognizing an outstanding training, you like Shelly. We have another goal and telling even story on this occasion.
• 08:46So for all the students residents and trainees. You folks in the back there, on the audience today. We wish to emphasize that in creating this award be not ask you to strive to be another even to Stanich's even's accomplishments. Even before starting her residency were uniquely exceptional, she was a true force of nature. What we do. Hope is that you will be inspired by Eva's love for Science in Madison and especially by the enormous joy, she found in her work right up to the very last days of her life.
• 09:14And we hope that imbued with evil spirit, you each will find joy and fulfillment as you apply your own special gifts and talents to advance the science or practice or teaching a medison. Thank you very much.
• 09:42And that's my pleasure to introduce Serenas Buddhic from.
• 09:46Neurology is professor of neurology who is the mental ravara ordet today.
• 09:54It's an amazing honor and with great pride that I'm going to introduce doctor Schelling Farhadian to you.
• 10:01So Shelly is obviously the recipient of this award because she has made really, really important.
• 10:09Findings and progress in investigating an area that she has developed a great passion about and I think that one of the things that I've learned listening to even story is that there's a commonality and these young women in the sense that Shelly is somebody who has fixated on questions that she thinks are important, and she's very, very interested in answering questions that are relevant to patients.
• 10:33So I'm at Shelley when she was a beginning, Infectious Disease Fellow. She was attending a lecture that I was giving talking about the brain and HIV and as I write in all of her letters. She interrupted my talk about 10 times asking really great questions and it was clear that things were clicking in her mind about things. She had seen in patients things that she had wondered about things that she was bringing from her own scientific background, which was already quite well developed at that time and thinking about how she could potentially.
• 11:04Understand and answer some of the things that I was saying. Where are the unknowns in her field so?
• 11:10With with with her interest and enthusiasm. I was just absolutely delighted when she sent me her CV and asked me whether she could work in my lab as a fellow doing research with our program because it was clear that she was somebody who is going to contribute enormously to the things that I was interested in and that would be one of those rare people that you just get to sort of launch of and get out of their way because they're going to really do such important things with their lives, so with that. I'll just tell you about Shelly's background so she is. It was an undergraduate at MIT where she majored in math.
• 11:42And then she went to New York, where she pursued an MD at Kurt Weill Cornell Medical School and at the same time, she pursued a PH D Rockefeller University. She worked in the Department of genetics and Leslie Vosshall's lab and completed her thesis.
• 11:57After that, she came to Yale and since then, she's been completed. All of her training at Yale so she did internal Medison residency here. She completed her clinical ID fellowship, and then she completed a fellowship. Enduro infectious disease, where she did research with me and also pursued additional clinical training in new ro ID.
• 12:14In that time, she's really pretty really important science. She's worked on projects that are quite basic and translation, which will talk about today and she's also pursued projects with our team that are more clinical and I hope.
• 12:28In the process of this, she's produced a really important work, she produced a paper last year, which was really the first time that anybody has used the kind of techniques that Shelly's used to look at neurologic diseases with a certain type of specimen again with spinal fluid that will talk about and this work was not only published easily in an important Journal and we got a lot of questions from others about it, but actually I heard it at the end of the year, the granting agency that funded the work the director of the granting agency had highlighted her paper.
• 13:00As one of the major new developments and exciting areas in their program so that's really enormous honor. I heard that from numbers of people who attended that meeting so Shelly. Not surprisingly, has received a lot of awards. She's received a lot of grants. One of these people. That puts her mind to applying for things and she applied for a lot of things and got 1 after the other almost couldn't know what to do with her funding and that includes a career development award from the national student mental health to work in her area of interest.
• 13:30That will support her for the next few years and hopefully again, launcher so we can just all get out of her way so with that. I'd like to introduce Shelly and congratulations. I'm immensely proud of you.
• 13:54Can you hear me OK?
• 13:56Thank you so much. It's such an honor and pleasure to be monks. Friends and mentors and colleagues and new friends and I'm really so grateful to be given this award an really especially grateful to the dystonic family. It was such a pleasure to get to know you better last night. I mean, it really feels like a privilege for me to play even a small part in honoring deepest memory today, so thank you.
• 14:22No.
• 14:25I wanted to start by talking about a clinical case that I saw in our narrow HIV clinic this past fall. So the patient is a 56 year old gentleman who's had longstanding well controlled HIV disease and he was referred to us for evaluation of memory problems, so in terms of these HIV history. He was diagnosed back in 1989. He did have a CNS lymphoma at that time, but otherwise never had any opportunistic infections started antiretroviral therapy pretty early on in the mid 1990s.
• 14:57And says that he really has missed a dose since then, an as a testament to that. He's had an undetectable HIV viral load for as long as he can remember but he was referred to our clinic because for about the last 5 years or so, he'd been having symptoms of worsening memory an gate so his friend, said that during that time, he was acting strangely. He was hoarding things. He was speaking repetitively and he had suffered frequent falls.
• 15:24Previously he was able to work in a high capacity was using computers working for the state government. But because of these cognitive issues had not been able to work for the last few years.
• 15:36When we saw him an exam. It was most notable for a really lay bile mood sort of alternating tearful and happy brisk reflexes, slow and unsteady gait his neurological exam revealed that he had deficits in attention memory and executive function and in fact, he underwent formal neuropsychological testing that really showed the same.
• 16:00His peripheral blood labs showed a CD 4 count of 4:50, so that's pretty good. He has decent immune reconstitution an his HIV viral load was as expected undetectable.
• 16:12Because of his neurological complaints, he underwent a lumbar puncture and his final fluid was pretty bland. So there was just one white blood cell. No elevated protein or glucose an HIV was again not detected this time in his spinal fluid?
• 16:27So he underwent a brain MRI and you can see it's completely abnormal. So forgetting about the old lesion in the frontal lobe. I think what's most striking is really enlarged ventricles in large spaces in the Sol size, so this man has suffered cerebral atrophy. Remember, he's only in his 50s.
• 16:46And the second thing that's really abnormal are these lucencies in the white matter, so these are T2 hyperintense lesions that are primarily Perry ventricular, but diffuse an bilateral.
• 16:58So the question that I'm interested in is assuming that his presentation is somehow related to his HIV disease and we're still not 100% sure about that. But if so, how is it that a well controlled viral infection? Can cause an going neurological damage? Is it that he suffered some sort of early founder effect damage to his brain when he was first infected with HIV and we're just seeing the sick well. I have that now or is there some sort of on going insult to his central nervous system.
• 17:29And if So what is the nature of that insult.
• 17:34So to get these questions. I want to spend the next 40 minutes or so talking about the problem of I'm going neuroinflammation in neuronal injury during HIV in people who are an effective antiretroviral therapy. So just as a brief outline. I'll start with some of the background. What we know about the problem. But we know about the mechanisms of this problem, primarily from autopsy studies and I hope I can make the case for you, that spinal fluid is a really powerful modality for investigating the brain.
• 18:05In living people with HIV so that we don't have to wait for autopsy studies.
• 18:10Next I'll switch gears and talk about some of the research. I've worked on with doctors viewed it over the last few years first clinical project and then something more translational and I hope to summarize what we know and what are still some of the big unanswered questions in the field that hopefully I can contribute to over the next many years.
• 18:30So the case that I told you about is obviously a very extreme and really rare presentation of a neurological problem of HIV in our current era, but prior to the widespread adoption of affective antiretroviral therapy. This presentation was less rare. I sure there are clinicians in the audience who have seen and treated many patients like this back in the 80s and 90s to estimated that about 15 to 30% of people with HIV during that time had some sort of advanced neurological complication.
• 19:02And in fact, this syndrome was the defining presentation of aids for 5% of affected patients at that time, so the syndrome is this combination of cognitive motor and behavioral deficits at the time referred to as aids dementia complex or the aids sub acute encephalopathies. Now we call it HIV associated dementia.
• 19:25This image here is from one of the earliest descriptions. I could find in the literature of what they call the sub acute encephalitis is back in 1983. Obviously, they weren't using MRI at that time. But even on the cat scan. You can see a similarity between this and the brain of our patient that I talked about earlier so stop enlarged ventricles and these lucencies in the white matter, it's pretty diffuse.
• 19:50Early study suggests that in some of these individuals, maybe about half the time. CSF was abnormal with a mild pleocytosis, but in the other half the time it was completely bland.
• 20:03It talked about some of the image. Ng findings and then many of these patients were able to undergo autopsy after their death and the most prominent findings were two one is that they had these extensive nodular lesions of microglial cells that are sort of clumped together, but interesting Lee are not surrounded by inflammatory infiltrates and in that way really distinguishes it from what you would typically see in a viral encephalitis, the second important finding on pathology was of course demyelination.
• 20:34And we still don't really understand the pathogenesis of that.
• 20:38What we do know now is probably this presentation before a RT was the consequences of the fact that HIV establishes infection in the central nervous system and in the absence of drug therapy can cause this widespread encephalitis, an ultimately clinical decline.
• 20:58So since that time. Fortunately, the HIV epidemic has undergone 2 very important epidemiological changes the first, which I'm showing you on the left has to do with the nature of the patients. We see today. So it's estimated from 2016 that about one point 1,000,000 adults. In the United States are living with HIV infection. And amazingly out of that one point 1,000,000 people is estimated that 55% of them so more than half of them are living with a suppressed viral load so their HIV is completely undetectable in blood.
• 21:32The second important change in the epidemic is that HIV is now largely more and more problem of older adults so again from 2016 data half of new diagnosis are made and adults above the age of 50 and almost 50% of adults living with HIV are over the age of 50.
• 21:55So I want to point this out because I think it's important that our research questions reflect this reality so that we're paying attention to the concept of each consequences of HIV infection in people with suppressed viral loads and also that we're thinking about the clinical problems that are encountered maybe disproportionately by older adults with HIV.
• 22:18So as I showed you before prior to the widespread use of antiretroviral therapy. HIV associated events was severe and not uncommon. Now it's an exceedingly uncommon thing to see but we're still seeing very high rates of other sometimes more subtle forms of neurological disease related to HIV infection and I'm sort of listed some of them here. Some of these are probably due to direct action of the virus itself and some of them are probably a consequence of.
• 22:49Chronic immune activation, which we see in the presence of HIV infection despite antiretrovirals therapy.
• 22:56And specifically with regards to nuro cognitive impairment. Several studies from HIV cohorts around the world have demonstrated the scope of this problem, so again, although Frank dementia is rare somewhere between 20 and 70% of people living with HIV on suppressive ART have a mild to moderate to severe form of cognitive impairment.
• 23:23What I find to be one of the most compelling aspects of this problem is that it seems that even in neurologically asymptomatic patients.
• 23:33Inflammation persists in the central nervous system of those with HIV infection.
• 23:38So as an example of one of the ways in which we know this. This is a pet study that was done using a pet lie again specific for activating microglial cells. So a specific immune cell type in the brain and with the authors did was it took patients who had HIV infection or an suppressive antiretroviral therapy and compare their pet imaging to HIV uninfected individuals again. Everyone in the study is neurologically asymptomatic, but what's striking is that?
• 24:08Those with HIV infection despite being asymptomatic despite having a suppressed plasma viral load continue to have stereotypical regions of the brain that show immune activation different from HIV uninfected individuals.
• 24:25So what then is driving this neuroinflammation and neuronal damage during chronic HIV infection.
• 24:32Clearly, it's a multifactorial problem. I'm not going to get into all of this today, but I did want to highlight a few of the factors that contribute to this problem, so people always ask about what about the drugs.
• 24:45That's an important thing to consider an I don't think it's an easy answer. We know that some drugs are more linked to cognitive outcomes, while others are clearly serving a protective role by preventing viral replication in the brain. I want to point out that there are some incredible investigators at Yale working on other aspects of this problem, so bring the emu and Amy Justice looking at the question of whether HIV accelerates the aging process and does that effect, cognitive outcomes. Jenn Edelman, doing really interesting work on substance use disorder and its relationship to geriatric syndromes in HIV.
• 25:20And, of course after spewed it has done so much of the seminal work looking at the impact of HIV infection in the brain during the early days of infection, but I want to focus on today.
• 25:31Are these 2 so the role of low level viral replication in the brain during suppressive ART and the role of chronic immune activation in promoting neuronal damage.
• 25:46So let's take a step back and talk a little bit about what we know about how and when HIV gets into the brain so this is a schematic. I'm showing you the blood brain barrier here in Brown above. It is the blood compartment below it. The brain so we know from doctor speech is working from others that sometime in the first one to 2 weeks after a person is infected with the virus infected CD 4T cells cross the blood brain barrier an answer the central nervous system.
• 26:17What we don't know and which is I think an important unanswered question in the field is whether other cell types might also be carrying the virus into the brain and specifically whether monocytes might be infected and bringing the virus into the central nervous system.
• 26:33Once the virus enter that's the brain, it can replicate and cause damage to nearby neurons through.
• 26:40The release of viral proteins like GP 1:20 and tat that are directly toxic to neighboring neurons.
• 26:48What we don't know is once the virus enters the brain? What is the cell that continues to harbor the virus in the brain and cause bio replication in that compartment? Is it just that their CD. 4T cells that are long lived in the CNS that are doing this, or is it that there are other cell types. Maybe central nervous system specific cell types that may be harboring the virus and that would obviously be an important place for us to intervene. If we're to cure HIV infection and be trying to make a dent in the neurological damage. We see in these individuals.
• 27:22So it's been postulated based on animal studies and other research that one potential site of HIV infection in the central nervous system might be my group glial cells. So I've shown them here is these big green cells. There deep seeded cells in the brain impossible to access and living adults. These are essentially the garbage cleaners of your brain so they sense any sort of debris like bacterial pathogens or.
• 27:53Amyloid deposits or anything else that doesn't quite belong when microglial cells encountered them, they become activated. They engulf and the clear the brain of that debris.
• 28:05Because these cells have been really difficult to access in humans. It's hard to know whether they may be a site of infection in a site that we need to intervene on.
• 28:16What we do know is that in living adults with HIV infection we can measure inflammatory cytokines that are released by activated microglial cells. I've shown some of them here in Green. Some may look familiar like trim 2, which we know has a marker for Alzheimer's disease. For example, and I've listed some of the other ones. I can opt are in that we can again measure their elevated people with HIV and suggest elevated microglial activity in these individuals.
• 28:46Likewise, we can measure elevations in markers of T cell activation again showing that.
• 28:53HIV infection does cause increase in immune activation in the central nervous system in those non ART and many of these markers remain elevated despite antiretroviral therapy.
• 29:07So a great deal of what we know about the state of the brain in people with HIV disease on therapy has come from autopsy studies and I wanted to really emphasize 2 takeaways from the autopsy studies. The 1st is the presence of chronic immune activation in these individuals and I've alluded to this before, and we looked at the pet image in that showed a similar finding but here looking specifically at the brains of individuals who died with HIV disease on a RT but neurologically.
• 29:38Asymptomatic what we find is that these brains are not the same as those of people without HIV so using a marker here that seems for activating microglial cells and looking specifically in the Basil Ganglia. We see a difference in the brains of those who have HIV versus those who are uninfected.
• 30:00The second big tech take away from me from autopsy studies is that it demonstrates to us that HIV virus itself persists in the brain. Despite antiretroviral therapy so this is one of my favourites studies that was published recently. It comes out of the national neuro aids to tissue consortium. It's an incredible organization. It's multisite, but run out of New York City, where people who are living with HIV disease make the commitment while they're still alive to donate their brains for study after they pass away and so these individuals.
• 30:34Are followed clinically during their lifetime and then undergo autopsy you later. So looking at some of these individuals who died from other causes trauma, etc, but had HIV at the time of their death on suppressed antiretroviral therapy. The authors examine let's look specifically at the brain 87. Such brains and found that over half of them contained HIV DNA within the brain tissue. Despite the fact that these individuals have.
• 31:04Undetectable plasma viral loads are on antiretroviral therapy so really further proof that HIV persists in the brain despite what we think of as viral suppression.
• 31:17So the studies have shown you so far have primarily relied on brain autopsy to look at HIV persistence and microglial activation. But we and others have been interested in studying this question in living adults. And so to do so. We're interested in using spinal fluid as a window into the brain.
• 31:37So CSF is produced within the brain by the quarry 8. Plexes cells that reside in the ventricles of the brain while CSF is not the same as the brain. It bathes the brain. It contains proteins and cells that are secreted by the brain and we know from work in multiple sclerosis. Another neuron inflammatory conditions that many of the immune cells that are isolated from CSF reflect the immune cell composition.
• 32:03Of cells and infiltrate the brain so for those reasons, we focused on seeing whether we could UCSF to better understand immune activation and HIV replication in the CNS in individuals who are living on ART.
• 32:18SO2 examples of the power of CSF Studies, one on the left, I'm showing you a biomarker called neurofilament light so this is a protein particle of released by damage neurons or accents and it could be measured in spinal fluid and when we look at the levels of neurofilament light in CSF in individuals with HIV compared to without the levels are much higher in those with HIV and when we look specifically at those who have HIV on ART levels of nerve filament.
• 32:49Are approximately equal to those of an HIV individual? Who is 4 years younger so sort of a nice reflection of that neuronal damage that we normally be taking place during the aging process may be accelerated in those with HIV infection.
• 33:07On the right, I mentioned the octarine earlier as a marker of microglial activation. And here we can measure it in spinal fluid so the four bars here show individuals with HIV with various amounts of HIV RNA detectable in their CSF in all cases, the knee. Octarine levels are above those of uninfected individuals and their highest than those with HIV replication in their spinal fluid?
• 33:34So again, suggesting that immune activation persist this by Artie and that we can measure it in the spinal fluid?
• 33:43And finally studies of CSF cells themselves.
• 33:48Show that despite plasma suppression the virus persists in the brain So what I showed you before it came from biomarker studies in a cellular portion of spinal fluid here. Doctors beautician colleagues took this cells from CSF and found that they were able to detect HIV DNA and more than half of these individuals in those final fluid cells and we don't know did these cells come from peripheral blood and crossover the blood brain barrier where they succeeded from the brain? Which of those cells is it CD 4T cells or something else that are containing the virus?
• 34:23An but again, reflective of the power of using CSF studies to examine the state of the CNS.
• 34:32And finally CSF studies that use a highly sensitive research only single copy HIV. RNA detection assay so these are incredibly powerful assets that can detect even one copy of HIV RNA in CSF.
• 34:47Suggest that
• 34:49If we look at people with plasma HIV RNA suppression. So they have an undetectable viral load. About 20% of those people will have CSF HIV RNA levels that are higher than that in plasma.
• 35:06So to summarize what we know so far, about the scope and the mechanism of the problem. High rates of clinical and sub clinical neurological injury persists in people living with HIV despite plasma viral suppression.
• 35:20Autopsy and CSF Studies demonstrate high markers at T cell, an micro glial cell activation again despite plasma viral suppression.
• 35:29An HIV virus itself remains detectable in the CNS in about half of individuals with HIV despite being on plasma viral suppression.
• 35:40So I want to switch gears now and talk a little bit about some of the research. I've done in the speech lab over the last few years and I'll start by focusing on the question of whether low level viral replication in the CNS may contribute this clinical neurological injury.
• 35:55This question is really motivated by a clinical conundrum that many of us have faced when taking care of patients with HIV so many of our patients will undergo a lumbar puncture for a variety of reasons, including being research. Volunteers in our studies and often times with the lab will come back and report to us that the patient has HIV RNA detectable in spinal fluid again. It's an asymptomatic individual more or less with knowing what is the significance of that clinically?
• 36:25And specifically I'm talking about patients who are reported to have CSF HIV RNA that's detected below the limit of quantification of the clinical acid. So it's a very low level amount of virus, but is that normal to have in your brain and what are the consequences?
• 36:42To answer this question, we perform a retrospective cross sectional analysis of all the research participants. We've seen over the last 3 years here at yell.
• 36:52And I was 42 participants. Most of whom are neurologically asymptomatic everyone underwent LP blood draw a neuropsych testing.
• 37:01Everyone had their CSF send for HIV RNA detection and we split up the participants into 3 groups based on whether and how much HIV. RNA was detected in their CSF so those that had no HIV detected those ahead. It detected below the limit of quantification and those that surprisingly despite being asymptomatic had high levels of HIV RNA in their CSF.
• 37:26Everyone underwent examination for different markers of CNS injury so.
• 37:32We looked at spinal fluid markers of blood brain barrier disruption anoor inflammation markers of immune activation an we also tested for cognitive impairment.
• 37:44And for the purposes of today, I'm going to be comparing these first two groups those that had no HIV RNA detected in CSF and those that had an extremely low level amount of RNA detected in CSF.
• 37:57Just some information about the demographics of the participants shown here, you can see this is a primarily male cohort primarily older African-American, an with moderate rates of comorbid substance use disorder.
• 38:13In terms of their HIV characteristics. I really want to impress that who were seeing in our research studies and who were taking care of in New Haven reflects the National Statistics. So these are folks who have had HIV for a median of 20 years. They are on suppressive antiretroviral therapy their blood CD 4 counts now are above 500 across the board and when we look at their Nater CD 4, so the lowest see before they ever have is not that low so it's above 200. These are not individuals for the most part wherever.
• 38:45Extremely sick from their HIV disease. These are people who are living with longstanding well controlled infection.
• 38:53So when we compare those 2 groups for standard. CSF clinical markers, we find no difference in white blood cell count, but we do, find an increase in CSF protein in those who have HIV RNA detected in CSF, suggesting some amount of neuroinflammation in that group and we also find increased levels of CSF albumen, so albumin is produced in your body and in order for it to get into your brain. It has to cross the blood brain barrier when the ratio of CSF to plasma albumin goes up.
• 39:26That indicates a loss of integrity of the blood brain barrier. So what we're seeing is that this marker of blood brain barrier. Integrity is worse in those who have a small amount of HIV RNA detected versus those who have no HIV RNA detected in CSF.
• 39:44When we looked at a subset of markers of immune activation in spinal fluid we found no difference between the 2:00 groups and when we looked in plasma, which I'm not showing you the data here again. There is no difference.
• 39:58We really wanted to look for more clinical outcome, though, and so we all of our participants underwent detailed neuropsychological testing. The results of which are reported as a Z score. So just to Orient. You a Z score of 0 means that a participant is performing equal to the population average adjusted for race. Educational attainment sex, etc. Z score of minus one or plus. One means that that person performed one standard deviation worse or one standard deviation.
• 40:29Better than the population average.
• 40:32The first thing I'll point out to you is that all of our participants do extremely poorly so if 0 is the population average.
• 40:40On average, all of our participants with HIV infection are doing at least 1 standard deviation worse than the population on their neuro psychological testing.
• 40:52But when we compare these 2 groups to each other for this combines iscor that takes into account performance across 11 or psychological domains. There's no difference between the 2:00 groups.
• 41:03If we look individually at the individual domains that we measured in these patients again for the most part. There's not much of a difference except for a small difference that we detect in executive function, which actually correlate with some of the image. Ng findings that we find also the parts of the brain that might be involved in performing executive function tasks. But for the most part my take home from this is that they're performing really equally to each other.
• 41:31So to conclude on that when comparing adults with HIV RNA detected, but not quantifiable in CSF versus those with HIV RNA not detected we found that the former group may have a decrease in blood brain barrier integrity and may have a decrease in executive function, but not another nurse psychological domains.
• 41:50I think these results are largely reassuring for our patients, but of course, we would need to study them in a larger cohort.
• 41:57So with the time that we have left. I want to switch gears to talk about a different project. One where we try to use really novel techniques to gain a better understanding of chronic immune activation in the central nervous system during a RT.
• 42:12Here are question is what are the immune cell pathways that drive immune activation in chronic HIV infection and to get at this we decided to take a single cell approach so just to make sure we're on the same page here. I want to spend a minute talking about the technique. So imagine you have some sort of heterogeneous tissue like your brain which consists of neurons and glial cells and or maybe your pancreas with Alpha cells and beta cells in traditional RNA sequencing that a piece of that entire tissue is taken.
• 42:42Homogenized and then gene expression is measured in that homogenized tissue so at the end of the day. You can say this gene is highly expressed this gene is lowly expressed within the pancreas as a whole. Let's say, but you wouldn't know anything about the specific cell types within that tissue in single cell analysis. In contrast, each individual cell is mechanically separated from every other cell either by putting them in tiny. Nana Wells or in little oil droplets whatever it is.
• 43:14You're able to get each cell individually and measure RNA expression within each individual cell.
• 43:21After you do that a computer program could then essentially perform a cluster analysis to find groups of cells that have similar gene expression profiles to each other and now you can say something about the individual cell types that make up that heterogeneous tissue.
• 43:37So for our experiments, we collected blood and CSF from HIV positive participants an performed RNA sequencing analysis on them.
• 43:47We took the fresh tissue. We sequence, the individual single cell transcriptomes and then we perform the cluster analysis to look at the immune cell types that we might find it spinal fluid an in blood in these individuals.
• 44:02What we were really interested in was trying to find cell types that are only found in spinal fluid and not find found in peripheral blood because our reasoning was that these might play an important role in promoting neuroinflammation.
• 44:17So for example, in the schematic Here each dot represents a cell and the red are cells that came from blood and blue ourselves. The key from CSF were interested in finding clusters like this cells that have a gene expression profile that makes them unique and that are only found in spinal fluid?
• 44:37So I'm showing you our results here that came from 2. HIV infected research participants. We took blood and CSF from both participants and Ram single cell RNA sequencing.
• 44:49Here, I'm showing you a plot where every dot represents a single cell. The cells are grouped together an colored, according to having a similar gene expression profile.
• 45:00I then went in an assigned identities to those clusters.
• 45:04Using information about gene expression of Canonical marker jeans so for example, you could see in this cluster here. There's really high expression of the CD 20 gene that's this cluster here and so I called those B cells. In doing this we found about 5 different distinct clusters of myeloid cells that we weren't able to give any known identity to just be on their gene expression.
• 45:31If we then look at the same plot and now instead of coloring each cell, according to its cluster, but now instead. We color it, according to that issue. It came from. Now we get a different picture. So that's here and now I take it. Each cell an colored it red or pink if it came from one of the blood samples or dark blue light blue if it came from one of the spinal fluid samples.
• 45:56You can see, there are some clusters like this one, which are the NK cells that came only from blood.
• 46:02There are other clusters like the T cells, up here.
• 46:06These cell types are found in both blood and CSF.
• 46:10What we were excited to find was a cluster like this? A small cluster that only comprises cells that originate it from spinal fluid so this cell type is not present in blood.
• 46:23And this specific cluster that we found here it comprises about 3% of all CSF cells.
• 46:30So what are these cells and this is just a heat map looking at some of the top jeans that are most highly and most differentially expressed in that cluster compared to all others. And I've listed here. Some of those jeans that are highly expressed in that specific cluster that I showed you before.
• 46:50We knew that by comparing that gene list to some published gene expression profile lists that those are jeans that are highly expressed in the human brain in my crew, glial cells. So here I'm actually comparing the jeans that are highly expressed in our little CSF unique. My light cell cluster compared to gene lists that have come out of Micro Glea Studies from animal models of neuro inflammatory diseases and we find significant overlap so we were really excited because.
• 47:21It suggests that we may have identified a new population of cells in spinal fluid that our microglia like at least according to their gene expression profile.
• 47:34We found that these cells are more commonly found in CSF that comes from HIV infected individuals. Then from uninfected individuals and that they are not found in blood samples.
• 47:45And finally we were really interested to see can, we sort these cells out of spinal fluid isolate them so that now we can move on and ask the really important question, which is do they harbor HIV? Are they functionally different in people with HIV compared to those without so to do that. We went back to our original gene list and notice that one of the jeans in codes? What's predicted to be a cell surface protein for which we could find an answer by Tianzhu flow cytometry and that's what we did here so we took spinal fluid cells?
• 48:18All spinal fluid cells from a large volume LP from one individual took the cells that were CD 3 negative so we know they're not T cells.
• 48:28Took those that are see 20 negative so we know there not be cells. And now used our new marker that we only knew about from RNA sequencing analysis and we were in fact, able to isolate a group of these cells from spinal fluid and actually the number comes out to about 3% again? So hopefully, the same population that we identified through single cell RNA seq.
• 48:49When we do the same experiment on peripheral blood immune cells. This population is nowhere to be found so again really reassuring that we're finding something that's specific to CSF.
• 49:01So to conclude on that we use single cell RNA sequencing to identify rare immune subsets in CSF.
• 49:10We found that there are microglia like cells circulating in CSF in patients with HIV and again exciting to us because so many of those open research questions about HIV in the brain has to do with the role of microglia.
• 49:23And we are hopeful that we can isolate these CSF unique cells for hopefully further interrogation.
• 49:31So just to summarize what I've told you despite great advances in HIV treatment rates of neuroinflammation and clinical neurological injury remains stubbornly high and people living with HIV.
• 49:43CSF studies can shed light on neuroinflammation and this includes biomarker or cellular studies of spinal fluid a subset of people living with HIV continue to have detectable HIV in spinal fluid, but the clinical significance of this remains unclear and microglia, which might be a sight of HIV viral persistence now potentially accessible to us through spinal fluid studies and we hope to follow up on this.
• 50:11So these are some of the experiments or questions. I want to start to work on in the short term, including again looking for persistence in these cells everything I've shown you today's cross sectional. I would like to be able to lanja tude only follow people with HIV infection and ask how the cellular profile changes. Overtime and overtreatment whether there's an effective aging or Co infections or treatment on the cellular landscape in these individuals.
• 50:40And sort of big picture questions that I hope to get it in the longer term.
• 50:46One of course, is regarding the reservoir and others about timing at what point is irreversible, neurological damage to seen can we intervene before that, as our patients get older clinically becomes more and more important to us to be able to distinguish cognitive impairment that student and aging process versus cognitive impairment that sued HIV and we still don't have a good way to do that.
• 51:11So that I want to thank you. All for paying attention today and for being here. You know there are so many people that yell who have helped me get to this point, Mike and I came here. About 7 years ago to start our internship in the traditional program. We could not have been luckier than to have doctor. Siegel, who really showed me that he values physician scientists and knowing that that was important to him made it continue to be important to me, even when I was excited by Clinical Medison.
• 51:42Really drawn to that as well.
• 51:44During residency doctor, Q and doctor done. We're excited about my excitement for ID and that helped me so much to stay in the field.
• 51:53An and all that brings me to my time in fellowship with doctor scutage. Just an incredible role model in every way. The perf transitional scientist someone who is really emphasized to me and I hope I can emphasize to the House death also that your clinical experiences in clinical training are not separate from your research in your research interests and although it can seem like this is my clinical time. This is my research time, she's really encouraged me to use my clinical time in my clinical experiences.
• 52:24To identify the difficult problems or the challenging patients and see if we can formulate research questions that have to do with those.
• 52:33I am so grateful to have an amazing research team led by Gen Churella Wonderful Scientific and clinical mentors in ID. I've received outstanding support from the program on Aging and my geriatrics. Families here today from so many other collaborators at yell and elsewhere so thank you very much.
• 53:09OK, we do have time for some questions if possible. I'd like to invite one of our students or residents to ask the 1st question.
• 53:20Yes.
• 53:22Make.
• 53:25I was wondering if you had been able to link some of the any localization of the damage that you see in those pet studies with the potential for where those cells that you identified might actually be located in a normal and abnormal brain. Yeah, it's a great question and we haven't so the spinal fluid doesn't tell you anything about localization. There are some really neat knew RNA seq technologies that actually account for where the cell came from.
• 53:56In a tissue and we would need brain tissue for that and we haven't. But it's important to try to link the location. I agree some of the neuropsych testing does correlate well with the lesions of the brain that are affected non pet, but that's as far as I know.
• 54:14Pretty outstanding talk, I wonder if it because it is a retrovirus.
• 54:19And you see persistent DNA and RNA in these patients if it actually integrated itself in the host genome.
• 54:26And why asking that question they were actually integrated itself within their region that regulates jeans that regulate cognitive function specifically affori was actually excited when I saw that because if we employee.
• 54:42Airport was exciting because actually employee polymorphism is associated with Alzheimer disease. I put for home aside and I was wondering if you would be interested. To actually sequence, the genome and see if you actually integrated that DNA host in the regulatory region and identify those jeans are actually regulated. I love that question. It's about integration sites of HIV DNA in spinal fluid cells or in brain cells. So far, so we have a local expert here at Yale Iaci, who does.
• 55:13Some of the most important work on integration sites, so she would be the best person for us to work with on that.
• 55:19We know something about where HIV integrates in plasma CD 4 sorry in blood. CD 4 cells. So it is a lot of interesting work on that some of it is an onco jeans. For whatever that's worth in the brain that answer is completely unknown. So it probably does integrate. I think detecting HIV DNA means you've detected integrated DNA, but where sub sort of part of what we're doing now is collecting these CSF cells from people with detectable virus.
• 55:50Anne working with colleagues who can just start by sequencing that virus that's an incredibly challenging problem. It's such a minute quantity of virus. That's integrated that even doing the sequencing analysis is going to be a challenge. But for sure. The next step is to see for sale is it a full length integration and where I love that question so definitely.
• 56:14Text Friesen.
• 56:18Thank you. That was wonderful as expected, and very proud of you wanted to comment first that chili and I worked together in the Nathan Smith Clinic and she was a wonderful wonderful clinician caring for very challenging. Patients many of whom still miss you.
• 56:36I have a question on an area that is still debated an wonder whether you research might give us some insight and that is that is the role of antiretrovirals in terms of influencing whether in fact, there is ongoing replication.
• 56:53An particularly the pharmacology of penetration of antiretrovirals into the brain and whether in fact that's very different than in other compartments and might result in less effectiveness of antiretrovirals or even resistance discern antiretrovirals are now that you have virus from these different compartments. Whether it's possible actually to answer that question so the question is about Artie penetration into the brain compartment and related issues. So you know a lot of this literature as well as I do, but 1st.
• 57:26Every time a new drug is brought to market it's not routinely tested for penetrance into CSF. An I think a real loss for us that we don't necessarily know for example, viktar VR new go to no idea what the CSF penetration is all we can do is extrapolate what we know from similar drugs and that are used to treat HIV. But in those that have been studied for the most part parties do penetrate well. We know we can detect them at high concentrations in CSF. We know that in people who have CSF escape.
• 57:59Or HIV detectable in their CSF if we intensify treatment, meaning add certain agents. We will get rid of that virus so suggesting that a RT is useful to further stop viral replication in CNS and people who are off a RT they have sky high levels of HIV in their CSF so unbalanced. It's good to get drugs into the brain and prevent replication there. But in terms of which agents specifically I mean, you know the data on produce inhibitors is worse but.
• 58:29I know I'm answering your question, but I think a lot of it is that from where I'm standing. I don't think it's been a high research priority for clinical trial groups really look at penetration and pharmacokinetics and CSF and I wish that that would change.
• 58:47Yeah, so those are almost impossible to conduct Nancy Angoff and Serena published a paper on that that individual had. I think thousands of copies of our nature. VR name their CSF? What we're finding in our asymptomatic guys were on ART.
• 59:04At the most maybe 300 copies. That's not enough to do standard lab resistance testing so we just don't know.
• 59:15Jeff and Shelly congratulations. This is important work and you're great deserve deserve an individual for this award for sure. I wondered if you looked at aisle 17 and the reason I imagine you have the reason I ask that is that you mentioned demyelination. Anandi myelinating diseases. I owe 17, which is a very pro inflammatory cytokine as you know is supposed to repress.
• 59:45While ago, Denver side precursors that otherwise would really myelinate in areas of demyelination. So I wondered if you looked at I'll 70 I haven't I haven't seen.
• 59:57Any CSF studies in HIV that have although it's possible. I'm missing that but I would love to talk to you about that and see if there's like an easy cited by nasty that we can run on CSF would be interesting.
• 01:00:10OK, in the last minute, I'd like to invite Eva's parents to come up and to present the award plaque to doctor Hayden S Doctor Erickson as well. Thank you all.