Dr. Prathima Nandivada - Hirschsprung Disease: From Swenson to Stem Cells

Good morning, everybody. Dr. Nandavada is a graduate of MIT with a degree in chemistry, followed by medical school at SUNY Stony Brook. Since then, she's been a staple of the Boston surgical community, completing her residency training at the Beth Israel, a research fellowship at Boston Children's under the mentorship of Mark Puder, and continuing on with her pediatric surgery fellowship here at Boston Children's. She's now an assistant professor of surgery at Harvard Medical School and a key member of our colorectal program here. She currently has her own lab and pilot grant for starting her front disease. Dr. Nandavada is well trained, well published, and has the CV you would expect from a premier early career surgeon scientist. What her CV also fails to capture all the things that make her exceptional. She's kind and thoughtful for patients adore her, and she's a role model for how to be an empathetic surgeon. And finally, I can't stress enough how critical she is to us as fellows. I spent countless hours in her office for advice, for support, and to process her experience. My fellowship would not be the same without her support. So please help me to welcome our associate program director and my friend Dr. Nandavada. Thank you, Patrick, for that extremely kind introduction. It's a real special thing to be giving grandparents today. The last time I spoke at this podium was almost 10 years ago when I was finishing my research fellowship with Dr. Puder, and you're probably wondering what about your fellowship, but let's not forget some of them, and I finished in the peak of the COVID-19 pandemic. So we gave our talk from a conference room over Zoom. So it's a special thing. It has given me the opportunity to really reflect on where I've come from, how it's brought me to where I am today, and what I really want as I move forward. And that's very much the theme of this talk. I'd like to talk to you a little bit about how we've come to understand herch-brung disease as we treat it now, and where it's headed, and in particular how surgeons have contributed to that understanding throughout. So Harold Herch-brung wasn't a surgeon, but the story often begins with him. He was a pathologist who really is credited with understanding the disease as an entity. He described it at a childhood disease conference in Berlin in 1886. And it wasn't the first time the idea of a newborn with a megal colon was described, but it was the first time where he had put a couple of cases together. He described the common features, and most notably he suggested that this was congenital in origin. But it really wouldn't be until the 1940s that we came to understand herch-brung disease at a level that allowed us to offer meaningful treatment. And that is in large part thanks to Dr. Orvar Swenson. And so I'd like to talk a little bit about how he came to explore herch-brung disease and help us understand it. And I think there's a lot of interesting lessons that I came across as I read about his story that I think would be relevant for anyone in academic pursuits. So Dr. Swenson was born in 1909 in Sweden. He came over to Independence, Missouri with his family in hopes of better education. At the time, he had to be very wealthy and sweet and to be educated. Very humble beginnings. And in fact, he has a remarkable childhood. We don't have time to talk about. But he and his brother essentially support themselves and put themselves through school by starting a fire starter business. And then later, a major company that makes foes in America at that time. There weren't many companies doing that. A lot of this was inspired by their Boy Scout troop leader. And this Boy Scout ultimately becomes a doctor, goes off to Harvard Medical School and tells the boys you got to go to medical school. So they apply to Harvard Medical School. And Orvar's older brother immediately gets in, but Orvar is wait-listed. And this is very distressing because these brothers do everything together. So naturally, the older brother writes to the dean of medical school and says, look, we're a couple of poor brothers. We don't do anything apart. And it would really save us a lot of money if we could go to the same school. So naturally, the dean of the medical school says, quote, sure, bring your little brother. So that's how Orvar Swenson ends up at Harvard Medical School. And while he gets in, he's not exactly a star student. And at the time, all the premier surgical residencies were in Boston. Still are, in my opinion. But the to get in required an invitation and residents were often picked really early. And Swenson was clearly not going to make the cut. So he ends up in Ohio State where he's an intern. He gets hit by a rheumatic fever, Mrs. Hath has an intern year. And spends the other half in the lab of Dr. Curtis messing around with some equipment to make parastaltic measurements. And it's clear his career is kind of going nowhere. So one of the urologists over in Ohio says, you know, I have some friends at Peter Ben for them. Let me see if I can get you a chance to be a resident there. He takes the exam. He's accepted to the intern class. But there's a 10 month gap to start. So now he has to find something to do. And we've all been there. The thing we want doesn't work out. The lay's disappointments. But he ends up having a phenomenal opportunity. And that is to serve under Dr. Sidney Farber, who's pictured here as a pathology intern. So he spends about 10 months with Farber doing our top season really coming to an understanding of the body in a way that many of us don't get the chance. He ends up excelling at the break. It's a pyramidal program. It's hard to become chief resident, but he does. He gets a two year research fellowship where he studies angina. And he's kind of a classic surgical investigator who's done a little stint in a million different areas that doesn't really have longitudinal exposure the way the NIH likes us to have now. And he does well. And so he gets that magical tone call from Dr. Lad, offering him a job to come to children's hospital and establish a surgical investigative lab. Huge opportunity. He's thrilled to come. He doesn't know what he's going to study. So he starts looking around the wards and finds there are a number of these children with a giant belly, not progressing, ultimately dying. This group of patients with congenital mega colon. And he starts talking to Lad about, well, what can we do for these children? And so Lad tells him about the wealth of literature that's been published since first one described the disease. Papers from the Mayo Clinic about just taking out the dilated colon. Papers about methyl bromide as a treatment. Papers hundreds of papers and hundreds of procedures performed on children. Getting the autonomic nerves to the bowel to get it to recover. And despite everyone saying how great the results are, he tells Hirschfrung, don't believe it. Nothing works. I've tried it all. It doesn't work. So Hirschfrung is inspired. And a reminder, not to believe everything you read. So he becomes kind of friends or interested in Dr. Janeway who at that time is a young pediatrician taking care of these patients with mega colon. And he gets a referral for one of these patients. And Dr. Lad and Janeway say this patient needs a colostomy. It's meant to perform the colostomy. So he does. And he puts it in the dilated bowel. And remarkably, the colostomy works. The child decompresses. Kids do and great. And he starts to get curious. Well, this is interesting. Now I have access to this dilated segment. Let me measure the peristalsis. So going back to that wasted time in Ohio, he knows how to do these experiments because of this time with Curtis performs the peristaltec studies. And remarkably, the peristalsis is normal, despite his hypothesis that it would be apple. So then he gets curious and says, well, maybe I ought to measure it in the distal bowel. No one at this time thought the distal bowel was the problem. Everyone was focused on the dilated segment. And so he does his peristaltec measurements and there's no peristals. And he's excited. He goes to his colleagues. There's no peristalsis in the distal segment. And they look at him and they say, dummy, you're diverted. Approximately. Of course, there's no peristalsis. It doesn't mean anything. So he's a little bit discouraged. But one of the things I love when reading about Smentson is he's very persistently curious. And so he goes and makes a new friend, Dr. Newhauser, this picture here. Same Newhauser of the reading room when you're looking for results from a scan overnight. And he asks Dr. Newhauser, can I look back at all the x-rays and patients with congenital medical ones? And so they review a number of these x-rays and come to the same conclusion, which is it's really hard to see the distal bowel on these films. So they decide together to try something where they put a little bit of barium up through the rectum to try to outline it and get a better picture of what's happening with the distal bowel. And at this point, this is a novel procedure. This is the birth of a barium enema, which eventually becomes our contrast enema that we use ubiquitously. And when they do, they discover, well, actually the distal bowel isn't descending normally. It's smaller than the proximal bowel. And this is the initial kind of impetus to start to wonder, OK, the distal bowel is the problem. So while he's thinking about this, he gets his marching orders from Lad and Janeway to close the stoma. Kids doing better, let's close the stoma. He's hesitant to do this because in his gut, he feels like the distal bowel is the problem, but he does it. And naturally, the child relapses back to having obstructive symptoms, big and bloated, gets a new transfer of his colostomy to get through it. Based off of this, observations clinically and kind of some of the measurements he'd done, he's really interested in removing the distal bowel. He's convinced that's the problem. And he pitches this to his colleagues who think he's nuts. Now keep in mind, there's no laparoscopy, deep pelvic surgery is dangerous in children at this time. So it's not without reason that his colleagues are concerned about this plan, but that's what he wants us to do. And this criticism is hard. And in particular, Dr. Gross is really not into this plan. But our patients are the ones who know us best, trust us most. And this particular patient's mother is absolutely not interested in a long-term colostomy wants to try it. So, this went to books the case. And at the time, the way operations work is the operating schedule comes out. Dr. Gross sits down and he chooses what case he wants to do. It doesn't matter who met the patient or who planned the operation, he gets to do the operation he wants to do. And so he assigns himself to that case. And the resident informs to when Senator First Wenson is pretty deflated at this point because he was excited to see this through and can't. But there's one person who absolutely won't have it and that's the patient's mother who refuses to sign the consent unless Dr. Swenson's the one doing the operation. And so Gross yields and Swenson gets to do the first resection of the distal bowel for this patient. And ultimately, the patient does great. So a reminder, not to be discouraged when you have good ideas and our patients are definitely the ones who give us the greatest opportunities. This patient does well. Swenson's really excited to publish the results. He goes to Gross and says, I really want to describe this. This could be the solution for herch-pronged disease. And he's met with a lot of criticism. This probably isn't herch-pronged disease. It only worked once. And so the original publication of this study is like indescibable, this title. They choose this title that makes it impossible to say for sure that this is a treatment for herch-pronged disease. And this is at the advice of Dr. Gross. And so Swenson and Bill, who performed the first operation, call it a resection of the rectum and rectus sigmoid with preservation of the sphincter for benign spastic lesions producing mega-colon. So I don't know how someone ultimately figured out that this is how herch-pronged disease was treated, but they did, but that title. Jane Ways thrilled with the results. He starts sending Swenson a lot of patients. Patients are doing well, except for one. So he had the patient, the operationist performed kids still having obstructive symptoms. And he gets the idea to use a barium enema to investigate why the filter is not working, which is also, again, novel. Things we do without even thinking about it now, but he applied this early radiologic technique and finds that there's a retained decompressed distal bowel. And this gives him the idea of wouldn't it be nice to have biopsies to tell us where the normal bowel begins? And so he again pitches this to Gross and says, you know, I'd like to do biopsies to figure out where the normal ganglinated bowel is present and Gross tells him he's not. You're going to make a bunch of septic patients on the floor. And again, keep in mind, at the time, this is risky business. And so again, it's not to just credit Dr. Gross, but he's not excited about it and forbid Swenson to do it. And so he doesn't. But things get pretty hard for Swenson after that. Gross isn't happy with him. His access to the hospital and ability to do these cases slowly starts disappearing. And he realizes he's got to go. And so he heads to the floating children's hospital, more recently known as Tufts, now no longer a pediatric center as it was, but that's where he goes to continue his work. And he actually does refine the process for doing biopsies there. He publishes along with a pathologist at Tufts and really continues to publish widely on her spurn disease for the remainder of his career. Now, he had the opportunity to leave Boston at that juncture. He thought of him very highly. He had the opportunity to go out to the Midwest to major surgical academic centers. Floating children's hospital was pretty small in comparison to the experience academically. He was getting at children's. But he says the major reason I didn't leave Boston was that I wanted to follow my kids with mega colon that I had operated on. And I love this is my favorite thing about him is he really took personal responsibility for the fact that he had come up with this operation. And was absolutely committed to making sure that these patients were safe, not just in the 30 day period, but in the long term. And one of the things that he was really worried about, the criticism of his operation from the start was that they were going to destroy the pelvic nerves and cause urinary incontinence and sexual dysfunction. And there was really no way to know that in children. That's one of the hardest things about what we do are results affect them lifelong, but we don't necessarily follow them lifelong. But this was the paper that he put out, I think he was in his late 70s or early 80s, multi-institutional, multi-national where they reported the outcomes of this men's imp procedure. And for his patients, they traveled to meetings all over the country and his patients would come and give updates on how they were doing. So again, a testament to the impression he left on his patients and that really came from really long-term follow-up. And what he showed is the mortality was very low and not even lower over the last 20 years before this was published. The perioperative complications were low. We'll talk about the anoroclidus in soiling. But most importantly, he wasn't seeing huge numbers of erectile dysfunction and incontinence. And so I think he was relatively at peace that his operation wasn't causing the harm that was initially thought. So Swenson really breaks open how to take care of herchemone disease. And what often happens with good ideas is that they build on each other. Despite Swenson's operation being successful, it's still really hard. You have to remember this is a trans-addominal procedure deep in the pelvis, mostly done sharply. It's not hard to imagine that complications were happening from trying this procedure. And people found it to be really difficult. And so in 1960, this is Dr. Duamel. He offers an alternative to the procedure to make it a little safer. Let's not get lower than the peritoneal reflection. Let's resect above that, bring down the ganglionated bowel, plug it into the backside of this egg anglionic rectal pouch, and they should be able to empty. And like most operations with hercheprung, when done well, it works very well. It works beautifully. And the Duamel operation is still widely done, especially in Europe and Canada. And if you keep this pouch small and you avoid spurs, they empty very well. And so Duamel's procedure took off to some degree because it made the operational adhesive. In 1963, Amanda by the name of Suave and Italian surgeon, describes an alternative approach. So when you get low in the rectum instead of not doing that dissection, take out the, get in the endorectal plane. Now you're in a safe place, you're not hitting pelvic nerves and vessels, and you're able to resect down low and then pull your ganglionic segment through the muscular cuff. So Suave's original cuff was quite long. And this worked reasonably well. I think he made his anastomosis actually by hanging the bowel outside of the child, like literally pulling it through and letting it anastomose and then cut it away. And important modifications to that that really makes the Suave procedure what we think of now came from bullying marks. In 1964, I said, well, why don't we just make an anastomosis, which was a nicer way to do it, I think. And then marks came along and said, well, let's cut that posterior cuff because that egging lionic cuff was definitely still a flow restrictor. And so with those two modifications, this operation now becomes the favored way to take care of her schwarm disease surgically. Still transidomal. Some recent information came up in the last few years about potentially who described the first and the rectal dissection for congenital mega-colon. And what was found is that this surgeon, Dr. A. C. Anci, had actually published this procedure 10 years earlier in 1953 in a little known journal because at the time black American surgeons were not allowed to publish in the same journals that the white American surgeons were. And the operation was pretty much exactly what Suave described. And this isn't to say that Suave stole it from him, but rather that it wasn't the first to count. So there's been a consistent effort to now rename the procedure as the Anci Suave to acknowledge some of the inequities that have come from the lack of diversity and academic surgery in the past and something we still grapple with. So bringing us into the 90s, which, according to my son, was a really long time ago, there are now the advent of minimally invasive procedures. And so Wilkin and Jorgison describe a laparoscopic approach, laparoscopy, as we know, or MIS in general makes pelvic surgery actually a lot easier. And so all of a sudden this detection is much more clear. You're able to see the planes better. And then Langer and Deletoura describe a completely transanal approach. So no abdominal scars. If you know your transition is low, why not start transanally, bring the battle out, do your biopsies as you go and make an anastomosis. And both of these are widely used today for the surgical approach to herch-prinus disease, usually a hybrid between these two procedures. So where have we come from there? Well, I think what these surgeons did over the span of about 50 years is they turned a very high risk operation into something that was safe and reproducible. And so in this study, in 2009, that surveyed our APSA membership about 40% were doing laparoscopically, a similar amount doing a transanally, still some open. Those people were doing a divided cuff suave, or yancy suave. I think if you were to reserve people, the enthusiasm about transanal only has diminished in part because of some surprise, long segment disease. And most people are doing really a combination of laparoscopic and transanal. And the Swenson has also become more popular than in the past and is the way we do it here. Interestingly, as the surgery became, as the operations became safer and easier to do over time, people really started comparing the different operations as well as whether or not to stage it in terms of how the patients did. And there have been a number of studies that have looked at this. And at the end of the day, whether you do this in one stage or two, whether you do a do-amel, a yancy suave, a suenson, it doesn't really make a huge difference in terms of your perioperative complications, intercollitis, or soiling and incontinence over time. And so the modern kind of area of really enthusiastic investigation has been, okay, well, we can get the kids through the surgery. And the surgery clearly makes kids better, but we aren't completely curing whatever herch-prung disease is. That the proximal ganglionic bowel doesn't behave normally for all children. The experience of destructive symptoms, the soil, or have frankincontinence. And most worrisome is their risk of ongoing herch-prung associated intercollitis that can cause recurrent hospitalization, intensive care, and in rare cases death. And so in response to how to take care of these children post-operatively, surgeons play a big role in these multidisciplinary colorectal centers that start popping up in the early 2000s, the first of which formed in Cincinnati. And here, historically, this was done in collaboration between the motility center within the GI department and the individual surgeons who care for these patients. This program matured into what we know now is the colorectal and pelvic malformation center with Dr. Jike's arrival in 2016. And it's now a comprehensive program where we offer really impressive bowel management with the use of laxatives, retrograde animas, anti-grade animas. Botox comes into the picture in the late 90s, early 2000s as a solution for obstructive symptoms. And while there was some enthusiasm about the prophylaxic use of Botox, some of the randomized trial set of what's about really don't see a benefit, but it's absolutely a powerful tool in taking care of obstructive symptoms and sphincter management in these patients in the long term. Anominometry matures allows us to diagnose patients who were missed, perhaps, in the earlier periods, and have long standing constipation without performing invasive biopsies, and also helps us manage these patients post-operatively, particularly when they're stigtheraic alesia. And then even more recently here, children's our pelvic floor physiotherapy program has matured into a robust and active program. And our nurse practitioners actually recently put together a poster and are presenting some exciting results on how this has improved the quality of life for our patients. And so if you build it, they will come. And Dr. Jike built it and it has grown in terms of people, the expertise that's offered. And of course, the CPMC takes care of a lot more than herch-pronged disease, but all of those resources are available to our patients with herch-pronged. The office visits and surgical volume have grown, and in fact, 2024 looks like our best year yet, we've done what we did, you know, we're at the halfway point before the first quarter this year. And so it's impressive to be able to say that since the 1950s where this was thought of as a lethal disease and infancy, we're now at a place where this is a relatively routine operation. A complication is a surprise, and we have multidisciplinary care to give these children as excellent quality of life as we can moving forward. And I think that's a lot to be proud of, and it's largely driven by some very excellent surgeons over this time period. But the question kind of still remains, you know, if herch-pronged, if herch-pronged identified the disease, and Swenson showed us it was the distal bowel, and a lot of really amazing E&S science happens after that that helps us really understand that there's a problem with that distal bowel. And we remove that distal bowel and bring down gangling in the bowel. Why do these children continue to have so many problems? And this has been the question that's been really the motivation for the work I'm doing now and hope to do throughout my career. We often think of herch-pronged disease as a disease of the enteric nervous system. The nerves aren't there. The neural crest cells didn't migrate where they needed to go. The gangling are absent. The bowel can't function like it normally does. But we've removed that segment. So what is it about the proximal bowel that's causing problems? And part of it is actually the E&S and the proximal gangling-aided bowel isn't completely normal either. There's differences in the neuronal cell densities. There's differences in the types of neurons that are present, whether they are cold and ergic or nitrogen-ergic. Dr. Goldstein's group has shown there's an abundance of nitrogen neurons. They release a lot of nitric oxide. This can cause smooth muscle relaxation and the dilation we tend to see in these children. We also know that the E&S does a lot more than helps the bowel move. It participates in epithelial transport. It's involved in mucosal immunity. It changes mucosal blood flow. And it actually helps release not only neurotransmitters, but gut peptides that influence the way the intestine works. And so we've come to understand that herch-pronged disease is about a lot more than just the absence of gangling in cells. There's a wider E&S defect that we don't totally understand and probably plays a role into why this proximal bowel doesn't behave as it should. It's also about a lot more than the E&S. People started to become more curious about the epithelium itself and how it interfaces with the E&S, but also about how it functions as a barrier. And this becomes relevant when we think about N-oroclidus. If you think about the last time you probably describe N-oroclidus to one of your patient families, you kind of make a handway the answer about bacteria translocating and causing infection. And the truth is, we really don't know why N-oroclidus happens, but the barrier probably plays a role. In almost any review, you read, we'll talk about reduced barrier function. And some of the data that supports that is different mucins are present. The goblet cells are different, also work from Dr. Bolstein, Dr. Gosan has shown that some of the IgAs are different. And so there's clearly an epithelial component to this that's independent of the E&S that may either be cause or effect, but is part of why this proximal bowel doesn't behave as it should. And so I became really interested in this idea of how the epithelial barrier plays a role in herch-prung disease with respect to N-oroclidus, but also with respect to motility in general. And I came across the work of Dr. Jerry Turner, who's a pathologist at Brigham and Women's, who's an expert in the proteins that comprise these junctions between cells that are really the regulated part of how the epithelial barrier works. And what was particularly exciting to me about what he was doing is he had discovered ways to target these proteins for small molecules therapeutics and actually alter gut permeability using small molecules. So really exciting works that really hadn't been done before and the idea that you could drug the epithelial barrier. So I spent a couple of years in Dr. Turner's lab and learned a lot about tight junctions in particular about these proteins and how they associate with this cytoskeleton of the nearby cells, how transcriptional regulation affects which proteins show up when and how that alters the permeability of the GI tract in response to different stimuli. And one of the things I found really interesting is when we think about permeability, we think about it as a blanket statement, but there are actually these different distinct pathways through which there's regulated barrier permeability. And there are distinct proteins that control these things and tight control of how that barrier is permeable or impermeable in response to different stimuli. There is of course an important to acknowledge there's an unrestricted portion of gut barrier permeability, which is when the epithelial cells die and the barrier literally has a whole limit that's not regulated, things can get across in a way that they shouldn't. So we're really talking about the part where it's a more chronic process, not acute cell loss. And so the first question we asked was, well, if these tight junction proteins are what regulate the barrier in a chronic disease state, are there differences in these proteins between children with first-pronged disease and those without? And this is work that was done by Dr. Lorraine at Rincconcruz, who is a resident from Cleveland Clinic, who was with me for two years, and we have fully his current medical student at BMC. And so we pulled all our patients from 2015 to 2021 who had had a pull through. We excluded patients who didn't have a clear diagnosis of Hirschbrung or who had had their pull through performed at another hospital. Excuse me. And we found 28 patients for whom we had the paraphernal blocks to look at. Our controls for this study were patients who had had either digital colon or rectal biopsies, and they had to be age matched to the months for our cases. And so we found 15, sorry, 16 patients who met that criteria for whom we had paraphernal blocks. And Dr. Jeffrey Goldsmith was really amazing in helping us find all these archived specimens as well as the H&E slides so we could create these tissue microarrays. And what a tissue microarray is is you take a donor block that's the paraphernal block of the tissue that's left after the pathology department has analyzed within you to analyze. You use a punch biopsy to take a two-millimeter section of that specimen, and then you transfer it to a recipient block. And so what you end up with once you're done processing that paraphernal block is a slide that has 20 to 28 patients on one slide, and you're able to stain them and image them in tandem to try to reduce bias over time, and also saves your resources. We stain for a whole panel of tight junction proteins. This is the most that's ever really been looked at in this disease. And we use some of the technology. It's really commonplace in Dr. Turner's lab, including how to automate and whole slide scan these tissue samples so that you end up with these kind of parent slides with all of these different specimens that have high-resolution images that you can look at more carefully. Hayley Leigh and Lorraine are experts now at this post-image processing, which involves annotating all of these images, creating tissue masks, and then running them through a program called Cell Profiler that was developed by the Broad Institute at MIT. And what we get out is some really interesting quantitative data about these images. And so looking at the Poor Pathway Junctional Proteins, which is the Claude and Family, this is a pathway that's very size selective, has to be tiny, less than eight angstroms, and is charge selective in that it favors cation. So we're really talking about solutes and water that are controlled in this carouselular pathway. And what we find in the control, what we see, these little red dots are in the middle of the crypt, this kind of blue cells are the crypt itself. That's where the junction stands. And we found that in children with herch-brung disease, the ganglionic and egg-anglionic portions of the bowel had reduced expression of clot into compared to controls. And this was also true for clotting 15. This is interesting because clotting to 15 are actually sister proteins. They're differentially expressed in development. Clotting to is expressed very highly in the neonatal period and then comes down when we wean from mother's milk and clotting 15 comes up. They have the same function. When you knock out both clotting to and 15 in a mouse model, it's embryonic lethal. When you knock out them individually, they tend to compensate for each other. So it's really interesting that both were down in the herch-brung patients. The other thing that's interesting clotting to you is probably the most widely studied clotting, especially in inflammatory bowel disease. And we think probably its expression is adaptive. It gets upregulated when we need to clear pathogens. When you upregulate clotting too, you make more pores in the membrane. More water can get through and you literally get diarrhea to clear out the pathogens. And it's sort of a primitive response for pathogen clearance. But it can get hijacked in chronic diseases like inflammatory bowel disease where clotting too is abnormally upregulated. It shouldn't be high in an adult. But it is and it can cause a chronic diarrhea state that's not actually clearing any pathogens and is hurting the patients. So clotting too is something that's heavily studied in IBD. And so I, both Dr. Turner and I thought this is really interesting that clotting too is down given that we're talking about an obstructive disease rather than a diarrhea disease. But I have no idea if this is cause or effect or what exactly it means. I can just say these two proteins seem down at the time of reconstruction and it was definitely interesting to continue to look at. The remaining clottings we looked at in this pathway were not different between the controls and disease cases. Looking at our leak pathway, this is more of an inflammatory response pathway regulated by TNF alpha, the dominant proteins or protein called MLCK1 as well as a clutin and the Zona, a clutin's proteins bigger size, about 100 angstroms. This is where you're going to get some of the bacterial byproducts, small molecules, as well as water and solace. And we found that a clutin expression was really decreased in our hersherung and in our hersherung specimens, both in the aganglionic and ganglionic specimens relative to controls. A clutin is not super well understood. It's probably also plays an adaptive role. This is work that was published in Gastro last year by Dr. Turner's group where they showed that when a clutin comes out of the junction, it's internalized into the cell. It probably plays more of a role with how it interacts with apoptotic signals in response to the apoptotic stimuli versus an inflammatory stimulus. And so probably an inflammatory disease is what's happening is that a clutin comes out of the junction. Its expression gets decreased. This actually suppresses apoptotic signals to allow for a return to homeostasis. Again, no idea how this is going to play out in hersherung disease, but it's super interesting and may just be an inflammatory adaptive response. The remaining proteins we looked at for the week halfway were not different between the two groups. So this initial study in human tissues showed us that in fact, there are some differences in the complement of tight junction proteins in the barrier at the time of surgical reconstruction. And we don't know if this is something that develops an adaptation to the disease, whether it's inherent to first-born disease in present even prenaturally, whether it's maladaptive or actually helpful, but those are some of the kind of questions that are coming up for us as we continue this work. We wanted to try to understand a little better what happens to these proteins as the disease progresses. But the human samples were really just looking at the time they have a surgical reception. We don't have access to tissue, a meaningful amount, the suction biopsies don't leave enough behind to ask this question in a time-dependent fashion in humans. So we moved to a mouth's model to ask what happens to tight junction protein expression as the mice get sicker with hersherung disease and an arachyloidus. This was worked on by Lorraine and Leah, and is being continued by Haley Eskivist, who's a resident from Yukon, who's working with me now. The mouth's model we used is a genetic model. It's a knockout of the EDNRB gene. This is the second most common gene that's affected in humans with hersherung disease. We typically think of Rhett. There's not a great Rhett model. And this is a widely studied mouth's model of hersherung disease. And the phenotype in these mice is they develop megacolons. They have distal egg anglianosis, and they also have a melanocyte defect, and so their coat turns white. And so this model, they develop an arachyloidus by week three and are typically either die on their own or need to be used in our eyes by that point because of illness. Tough model to work with as Haley has learned. We collected samples at different time points weekly throughout the progression of the disease, the goal was to measure these tight junction proteins along with some other markers. Created tissue migrares, just like we did in the humans, and stained for a number of these tight junction proteins. We also looked at H&E samples to try and get a sense of when this colitis really start in this model. And so the score in the system we use is one that was developed by Dr. Frickman from working with this model, but is based off of a lot of tidal bombs work. And what we find actually is there's colitis really early in this model, it really starts within the first week. And so some of the challenge here is finding a model that lets us see a little bit more before these myskypsick and these myskypsick pretty early. Some preliminary data from this, we're still really hacking through a lot of the images at this point, but what we can see so far in the proximal colon. So this is the normal ganglionated colon. Because we do see a cluedon drop off over time, which matches what we saw in the humans. And interestingly we're seeing cloud and two come up. So I don't know where we are in the curve when we see it in humans, but it's potentially also an adaptive response that's showing up. We need to compare these to controls and look at all the other specimens, but there's some interesting work coming out of this. And Hayley is working hard on this now. We simultaneously started recognizing that there's a lot of power in the human specimens that we have, but looking retrospectively at our archives while was a powerful tool to begin asking these questions, won't be sufficient in the long term. And so we built and are building a prospective clinical database along with a tissue biorepository. And Dr. Scott Snapper from GI has been immensely helpful in this. As he has a very mature biorepository for IBD and we were able to piggyback on that and leverage his resources. And so Arsum Haroon who's also working with me currently, he's a medical, finished medical school in Pakistan and just matched in general surgery in the United States and some thrills and proud of him and Leah and Hayley are working on this. And so you may notice this if you're in the operating room with Dr. Dick Uri where the team will come by, they'll grab ganglionic and egg ganglionic tissue during the operation. This keeps the tissue relatively perfused. And we're not dealing with as much ischemic effects and tissue is saved for each of these, is processed in each of these ways and stored away for analysis when we get enough samples. And one of the things that is newer that we're doing is putting some of these tissue specimens into a cryo media so that we can develop patient-derived organoids and colonoids as another model for studying the disease. And so this is work that's being done in collaboration with Dr. Daniel Beave who works with Dr. Brolt and his research assistant Emma Mollman. And we've now got about six controls, the controls for this study are actually pea sharps because we get a little bit more tissue from them and we're able to get them also during surgery. And we have about four short segment hersprung and two long segment hersprung. And we're almost there. It takes about two months to get these colonoids into a stable passage, passageable kind of version so that we can study them. We're almost there. Once we have our most recent patient in a state where we can start working with the organoids, we're going to start our first cohort for each group. And the goal is to stay in for these tight junctions and see if there's differences in the human colonoids. Look at single cell analysis to try to understand what cell population show up and if there's a difference between hersprung and not. And then ultimately to plate these as a model layer and measure transepatheliological resistance, which is a measurement of the permeability of the epithelium. And the idea behind these organoids is when you take these tissue samples and you put them through everything that's necessary to make organoids, the only things that actually survive are the stem cells. But the idea is that these stem cells retain phenotype of the original tissue. And this sort of model has been used in other GI diseases. These are colonoids in the sense they only have epithelium. There's no E and S in these organoids. But they've been studied for diseases like IBD and other epithelial based studies. And so I think there's potentially an opportunity to study these for hersprung. In addition to the translational work we're working on, we're still trying to improve the surgical care for our patients. And these are two of the ongoing studies that I'm really excited about. A lot of things are informed from clinical practice. And when we transition from predominantly doing these patients in the newborn period, they're pulled through to our current practice, which is for the most part to establish good irrigations, teach the family, send them home on irrigations and bring them back to their pull through, the question became, well, what's better? And if some of the things that we see at the tissue level are adaptive responses, then you can make different arguments for the benefit of resolving obstruction early versus later and how that impacts the long-term outcomes. There have been some really tiny single center studies as well as one consortium study that was also small and underpowered to answer these questions. But those studies show that there was no difference whether you did it in the newborn period or in the infant period, you know, two or three months after birth. A big systematic review that came out of the MGH did show when you use international data too, that waiting until they're over two months old as associated with decreased soiling and stricter. But heterogeneous data hard to know. And so we pitched this study idea to the Eastern Pediatric Surgical Network and we just had our IRB approved and are going to launch in the next couple of months. And so we're expecting about 10 centers to participate, we're hoping to include over 300 patients. And I think it's going to be a really phenomenal opportunity to look at a disease that at any single center only has so many patients, even though we have a big volume here and ask some really interesting questions. Not only about the optimal timing of performing this operation, but a number of other clinical questions that we could look at once this database is built. And Hayley's leading that project. The second study we're currently working on also just kind of came out of clinical practice. I had a weird run of several patients with gen genital transitions, a Hirschhorn disease. And when you have to go out to the family and tell them this is the kind of Hirschhorn disease, they have it definitely affects you. And I've gotten to know these families very well and have seen how hard this disease is to manage. And as I ravenously read about outcomes in this population to try to educate these families, I found pretty dismal reports, but mostly old reports from places that don't manage children the way our intestinal rehabilitation center does and may not use the same kinds of adjuncts like Omega-Ven that our center does. And so we wanted to look at this population in the current era in the setting of our very excellent care program. And so this is work that Hayley did in collaboration with Priyanka Chu from the care team. We started by just asking what's happening to our small bowel Hirschhorn patients in the care team. And so we had 46 kids, which is the biggest, you know, it seems like a tiny number, but that's a huge number of small intestinal Hirschhorn when you look at the studies that have been published, who had small intestinal Hirschhorn disease followed with 84 months, just the tremendous amount of data. The residual bowel length was 76 centimeters, of course, 95% of these kids require home 2 p.m. on the order of years. There is a high incidence of intestinal failure associated with liver disease in these patients, but none of them died or required transplant. Hayek happened, even in the distal bowel that's not in continuity of 44%, and 76% of these patients required operations to deal with the inflamed distal bowel. The inflamed distal through was attempted in about 28% of these patients, but almost half needed their stomach back. So this is a tough, tough disease for these families to manage. The most interesting thing that came out of here, though, was that bowel length actually wasn't associated with whether or not these children were entirely autonomous at the end of the study period. And this goes against kind of everything we learn about when we think about absorbs in surface area and intestinal rehabilitation, what I learned working with care team and Dr. Puder when I was a research fellow. And so I got really curious about it. And for those of us that take care of these patients, lots of reasons for this kind of come up like, oh, well, the colon's not in line and, you know, they have dysmotility. And yes, those are important things that probably do play a role. But the published study is all say the outcome for herch-pronged disease in the small intestine basically is dependent on small bowel length. And so we found something different. And that made us curious look at this more closely. So this is hot off suppresses. Let me just finish this week. Haley went back and she matched every single one of these small bowel and small intestinal herch-pronged patients with two non-herch-prong-related short-cut care patients. And she matched them on bowel length. And we used percent did predicted bowel length. So this also controls for sex and gestational age. And the group was well balanced. There was no difference in intestinal failure associated liver disease or clapsy, which are some of the factors that can interfere with the success of intestinal rehabilitation. There was no difference between the two groups. And what we found is that small intestinal herch-pronged disease patients, centimeter for centimeters, still had almost twice the duration of PN compared to their short-cut peers of other etiologies. And if you look at entral autonomy in each of the categories when they're matched for bowel length, the herch-pronged kids do worse than patients without herch-pronged disease. And for those of you who care, this makes perfect sense. But to the colorectal folks, it might be interesting, certainly, west to me. But one of the big things that's missing in this story is the colon. And we don't think of the colon as an absorptive service area for nutrients in terms of growth, et cetera, although it probably does do some. But it is absolutely plays a critical role in how we can do intestinal rehabilitation in these patients. And so when we looked at whether any colon was in continuity in these patients, of course, in herch-pronged disease, it was a very, very low percentage, probably patients with dole-mills. And almost 70% of the non-herch-pronged patients had some colon. And if you control for colon and continuity, this difference disappears. So the herch-pronged patients do just as well as the care patients if you take into account whether they have a colon or not. So I think what I'm learning from this is, and in talk, it's been really fun talking with Dr. Jackson, and Daniel Stam, and the care clinic about these patients, is there are some additional challenges when it comes to intestinal rehabilitation in the herch-pronged patients. And we might have to approach it in new ways to try to deal with the fact that they don't have a colon. Diarrhea is going to be a huge problem as we try to advance speeds in these patients. And we have to manage an additional inflammatory niides beyond intestinal failure associated with her disease in Clopsy in terms of this entity and herch-pronged. And how do you play this game knowing that stem cell therapies are down the piax, small bowel transplantation may be an option in the future and keeping that bowel in retains domain to set them up for that. How do you balance that with leaving bowel that is potentially creating a low grade inflammatory state? So lots of interesting questions to ask about that. There's a lot of exciting stuff that's happening with surgeons who are investigating this disease. People are looking at dysbiosis, how the microbiome affects and interacts with the epithelium that's work of Dr. Gosangs out in Colorado. Dr. Gosangs team at MGH is looking at using autologous stem cells to repopulate egg anglionic bowel or potentially augment dysfunctional bowel. Lily Chang out in Virginia is looking at the contractility of this bowel and trying to understand some of the mechanical forces so we can treat dysmotility more effectively. And so I think there's a really exciting horizon for these patients. And I hope I've convinced you that surgeons have been essential to our understanding of this disease from the start. And I think are going to be key players moving forward, even though it's gotten quite frankly a lot harder to do this kind of work. These were my two favorite quotes from Dr. Swenson's interview that he did with ACS where he says quote, we never lacked money and never had to apply for grants. We just started projects and money was there. Dr. Lads somehow always raised money or quote, at the time the NIH was expanding and you could get money for the damn distinct. This is not the case anymore. And so it's an increasingly challenging landscape for certain to navigate. But I don't intend on giving up and I hope you guys don't either. A few of knowledgements. So I want to thank Dr. Turner for giving you the vocabulary, the resources, the generosity of his knowledge. And in particular his postdoc Shabnam Abtahi who's just been a tremendously brilliant resource for us. Dr. Goldstein for the mice and for the encouragement. Dr. Peter for the foundation and support. Dr. Dickie for the opportunity and for teaching me how to take good care of these patients. To our collaborators, to Dr. Fishman and Dr. Jackson for the resources and the space to do this work for my funding resources. And definitely for the amazing trainees that I've had the chance to work with to do this work. Working with you, seeing you get excited about the weird things I get excited about is just the best part of this whole process for me. And then lastly, of course, and most importantly, thank you to my family, my husband Jesse and Karen who put up with the late nights and weekends. And despite not really understanding what I'm doing, our extremely supportive of it. I'd be happy to take your question. Well, I will start by saying for those of you on Zoom and there are a lot, well, a few minutes for questions, so go ahead and type into the Q&A and we'll get what we can. But before we start with Q&A, I want to mention that I have a habit of looking to see who's on Zoom. And there's a whole lot of special guests here this morning with us. One of the huge advantages of hybrid conferences is that sometimes people can attend that might not normally be in the room. So you know who you are. Some of you might have the same last name as Dr. Nandivata and might be relatives on as some other special scientific guests. So thank you for joining us. And because of that, some of you might have understood the science and some might not have. I think all of them could understand Dr. Dela Plains' introduction, which I'm sure if he had time, he could have gone on as good all of us. More about Dr. Nandivata's personhood and role in the department and history here. So if the rest of the talk wasn't that interesting to you, please know that that part was heartfelt and sure and all of our faculty and fellow that would agree. Dr. Nandivata talked about the history of Dr. Swenson in particular and some of the difficulties sort of in finding a path. So we hope it's a little easier these days. Maybe just not by writing a letter and getting in to Harvard Medical School, but hopefully by having it career to do the investigation. So if there's such a thing as a modern triple threat, I think we're looking at one. And we're proud. I want to comment that probably people learned some history about Dr. Swenson that they didn't know. He really a fascinating individual. I had the incredible opportunity years ago when the surgical section in the American Academy of Pediatrics used to have a big dinner. And I think it was this Saturday night. And sometimes we had to take a bus to go from finishing the conference in the hotel to the dinner side, the banquet was a banquet, kind of like the apps of banquet. And I sat down in the bus and then the bus and Dr. Swenson gets on the bus and he stands next to me. He was quite old at this time. And he said, is this he taking what I said here? And it was like a 45 minute ride. So I had the incredible opportunity to spend 45 minutes talking to him once. That's something I'll never forget. I want to open this up to questions for this spectacular talk from Dr. Anipa. A comment. I promise I would look at the zoom and I didn't. I have a question actually for Dr. Schamburger. And I was reading this interview with Dr. Swenson. He talks about how in his late 80s, now he's writing this review on Hirschfung disease for he submits it to pediatrics, the journal pediatrics, not the journal pediatrics, to pediatrics. And the reviewers send him back like this is like a light of consciousness. We can't publish this. This doesn't make any sense. And he writes that there was a very kind and smart chair of pediatrics surgery at Boston Children's Hospital. This is 2002 who offered to quote, pound it into shame so that it was publishable. And it was publishable. And there's no acknowledgement of that individual or in that person that listed as an author. But I assume that was you. No? I don't know that I pounded his paper into a publishable form. He did come to remember when he came to Children's and he was looking for additional individual patients that he could add to his cohort. And so I helped get a bunch of charts and information on the patients that have been treated to Children's. I think ultimately he was disappointed that we didn't have enough additional patients that we'd had the Swanson procedure that he was able to get the number he needed to make it statistically significant. But I do remember him come in at that time. And actually I got to meet Dr. Swanson a couple of other times because ended up his Godson was a classmate of mine in medical school. So I got to meet him a couple of times when he was visiting the Myers. And his doctor fisherman said he was remarkable individual. And he came from Missouri. Yes, he does. I didn't know he came from Missouri. Now in typical modest fashion, there are many of us in this room who have had an manuscript pounded into publishable form by Dr. Schamberger, including myself who he then proceeded to take his name off of the manuscript even though he was the one who got me all the specimens from National Wums to study group at the time. So there are some similarities between Dr. Swanson and Dr. Schamberger. So thank you for putting that out, Pratina. We are grateful for you for this history. I think that there are multiple other forms where this should be presented and I'm sure will and we are certain that your work will proceed forward. And I saw the rain that is also on the zoom. So we thank you for your work and for those present who are still working on it. Thanks to all for joining us and for this tremendous work that you're doing.