morning's virtual grand rounds. We continue our series of presentations from our graduating research fellows. Today we will hear from Bethany Farh, Stephanie Laisau and Jamie Nell, who have all done tremendous research during their time here. And just a reminder to those in the audience that this is a webinar setup. And so if you do have any questions or comments, please virtually raise your hand and one of the moderators will grant you permission to speak. Starting us, starting us off this morning will be Bethany Farh. Bethany Hales from Wisconsin did her medical school training in Chicago before heading to Louisiana for her general surgery residency. Throughout her time at Boston Children's, she has done it all. Tremendous amount of call and moonlighting on our clinical services both with the consults and in the ICU. She's also completed the critical care fellowship and has also done an MPH through the Harvard School of Public Health. She's worked with many mentors within the department for her research and trauma and critical care. And today she will share with us some highlights from her work. So thank you, Bethany and take the way. All right. Good morning everyone. Get my slides up here. Okay. Okay. Well, you can all see my slides. Today I'll be reviewing my time here at Boston Children's and presenting some data from our pilot study by impedance spectroscopy for a total body water assessment in pediatric surgical patients. I'll be discussing three aspects of my time at BCH, including clinical development through surgical critical care fellowship, academic development through the MPH program at the Harvard Chan School, and research development through investigations and trauma critical care and nutrition. The surgical critical care fellowship provides exposure to a variety of pediatric illnesses, including trauma and complex surgery, as well as medical and surgical subspecialty cases. On these cases, give amazing clinical experiences, including participation in ECMO, advanced ventilation, resuscitation, as well as opportunities for procedures like intubation, placement of lines, chest tubes, and drains. Additionally, the environment of inquiry and collaboration in ICUs really fosters an excellent teaching and learning experience through informal and formal case discussions, as well as rounds and conferences. Following the SEC fellowship, I began master's program in the School of Public Health that served as a foundation for research. The clinical effectiveness program provides a background in epidemiology, as well as spiosetitistics, and a practice to come to support researchers and project development. The options I had there also led to coursework and research methodologies listed on the slide, as well as opportunities to take classwork in areas of personal interests, which for me included injury prevention and antibiotic resistance. I also participated in an ICU multi-disciplinary nutrition group called ICU Energy. It's comprised of physicians, researchers, and bieticians with weekly meetings to discuss research projects, learn from challenging ICU cases, and review new literature in the field. These activities, the SEC fellowship MPH coursework and the ICU energy group, prepared me for research by solidifying areas of clinical interest, expanding my knowledge of research methodology, and obtaining support through mentorship, with doctors, mooni, samarice towns, and nila shmata. With doctor mooni's mentorship, I conducted multiple studies in trauma and general surgery, and had the opportunity to present at local regional and national conferences. I also participated in several multi-center trauma studies, which I believe is important for entering pediatric trauma research. With the guidance of nila shmata and samarice towns, and I was involved in a variety of critical care and nutrition projects as well, that provided the opportunity for regional national conference presentation. And now I'll take the opportunity to tell you about our initial findings from our study Biomedpedance Spectroscopy for Total Body Water Assessment and Pediatric Certical Patients. This was a prospective pilot study using Biomedpedance Spectroscopy or BIS. BIS is a hand-held put-of-care device that measures body compartment volumes using tissue and pedants to current and multiple frequencies. For this device, it's 256 frequencies. And the output from the device includes measures of total body water, extra cellar water, and intracellular water. Through coal plot modeling, it provides additional measures like fat mass and fat free mass and phase angle, which is thought to be an indicator of cell membrane integrity. This device and similar BIA devices that use fewer frequencies have been used in multiple adult and pediatric studies, looking at both nutrition outcomes and hydration. Cienes of our study were to assess the feasibility of using BIS in surgical patients, and to compare the outcomes of total body water to fluid balance recorded as ins and outs in the chart using plots of both correlation and agreement. We hypothesized that use of BIS in this population would be feasible with at least 50% completion rate and no significant adverse events. The device's low risk and skin irritation from the electrode stickers appeared to be the most likely possible adverse event. When comparing to fluid balance, hypothesis was that there would be moderate correlation before agreement between these two measures. We included pediatric patient scheduled for elective procedures to be able to obtain a baseline measure of total body water that would correlate to an admission fluid balance of zero. We included complex procedures that were expected to have an admission of several days following surgery. The initial protocol was for ICU status only, but several missed patients and slow enrollment prompted a protocol change to floor patient's status as well. The data collected included basic demographics as well as daily post-out total body water measurements from the BIS device and fluid balance from the medical chart as well as other clinical variables. Enrollment of a convenient sample of 20 patients was completed in March of this year. The consenting patients of consenting patients three were excluded, one because the surgery was canceled and two because they were admitted to the flooring's set of ICU prior to our protocol change. Medium patient age was two and a half years and 65% were male. Procedures performed were 50% thoracic mainly trakeopexy and 50% abdominal with a variety of case types. To assess BIS feasibility, we looked at the following parameters. Enrollment overall was 70% and 20 of 23 enrolled patients or 87% completed pre and post-out measures. Some patients declined measurement on specific post-up days which yielded a measurement completion rate of 79% between post-up days 1 and 3. Measurements were quick and there were no adverse events. 49 measures were taken in triplicate without changing steady conditions yielding an interclass correlation of 0.9. These parameters indicate the MIS device is safe and has high measurement completion rate and thus is feasible for total body water measurement in this population. Comparison of total body water from BIS to fluid belts from charting for post-up day 1 is shown here. Data recorded are changed from baseline corrected for patient weight with units of mills per kilo. The spiraling correlation on the left is moderate at 0.59. The bland almond plot of agreement on the right shows a mean bias of 26 mills per kilo but with wide limits of agreement from negative 100 11 to positive 163 mills per kilo and several with means well over 100 mills per kilo change from baseline. Here are shown spiraling correlation in bland almond means and limits of agreement for post-up days 1, 2 and 3 as well as cumulative findings. You can see that the spiraling correlation is moderate on post-up day 1 but declines thereafter and loses statistical significance. The bland almond limits of agreement also widened in subsequent post-up days. It's interesting that the BIS measures total body water at 26 mills per kilo more than fluid balance on post-up day 1 is shown in the mean bias column but then the relationship reverses on post-up day 2 and 3 with fluid balance measuring greater than total body water and this can be explained by the trend scene in each measure over time. So here you can see figures from box plots of the trend in the first three post-up days for total body water on the left and the fluid balance on the right. The total body water measures are highest on post-up day 1 and then trend back toward the baseline but the recorded cumulative fluid balance increases with each post-up day. If the total body water measurement is to believe then the fluid balance recorded as ends and outs may not be accurate over time. The cumulative fluid balance may not reflect true fluid status at any given time point. So from these findings we see that BIS is feasible and there's moderate correlation in early post-up measures that degrade quickly. There are also wide limits in the blend outment plot indicating core agreement between these measures. As such the measures should not be used interchangeably. The outliers seen in means of both measures and variation in trends may be indicative of errors of measurement from either method. cumulative fluid balance as measured by ins and outs may not be accurate over time. This measure should be further investigated. Gathering more data will help to determine the usefulness of BIS for patients with fluid overload. In whom accurate total body water measures could be helpful in guiding dioresis, timing of extubation and other clinical decision making. Measurement against a gold standard would be ideal like a deuterium dilution but it's not practical for situations of rapid fluid change. So as such fluid balance and volume status remain a challenge that will continue to plague clinicians, least for the type being. In addition to these findings conducting this study allowed me a unique opportunity to experience the entire clinical research process in all its facets including study design and refinement of study through my MPH practicum. The IRB and approval processes needed gaining volume from medical staff as well as consenting subjects establishing maintaining a database and learning new techniques and statistical analyses. These skills and experiences will follow me into my future career. Finally I would like to say thank you to my research mentors, doctors, Mooni, Nellish, Madden, Samarice, Townsend as well as to Dr. Walden and my critical care family. She is a clinician and as a professional colleague and with that I wanted to say thank you to the pediatric surgery fellows and my critical care co-fellows as well and thank you to Dr. Schamburger and Dr. Fishman for the amazing opportunities I've had here. Surgery department as well for their teaching and instruction on IC rounds overnight and weekend call and educational conferences and with that I'd be happy to take any questions. Thank you for your hard work and your time spent with us. I'm on this heavy unit facing me around to try and get work out of me and thank you so much for finishing the many things you would accomplish here. Thank you actually. Good luck and it's a big easy. Okay thank you Bethany. We'll move on to our second presenter Jamie Nell. Jamie comes to us from Sunny San Diego, California where she did both her undergraduate and her medical school training. She is currently a resident in the Brigham General Surgery program and has spent the past couple of years doing research in intestinal failure under the mentorship of Dr. Jack Vic and Dr. Modi. Today we will hear from her about her work with the Vermont Oxford Network, specifically in North Developmental Disability and Healthcare needs among survivors with Necrotizing and Herc lightest. Thank you so much Jamie for all your hard work and take it away please. All right I'm going to set up my screen sharing. All right good morning and thank you all for the opportunity to present today. I've been fortunate to work with Dr. Jack Vic and Dr. Modi in the Center for Advanced Detestinal Rehabilitation for the past two years and it's been an incredible experience to work with a wonderful group of people as part of a multi-disciplinary team to get to know the intestinal failure patients and their families and to study the underlying diseases associated comorbidities and long-term outcomes as these patients grow into adulthood. I worked on several projects during my research time at Boston Children's but today I would like to discuss some of the work I've done with the Vermont Oxford Network starting with neurodevelopmental disability and healthcare needs among survivors of surgical neck. As you all know Necrotizing Disease, the primarily affects premature infants up to half of patients ultimately require surgical intervention. Bedside Pairsnial Drainage was first described as a temporizing measure for infants deemed too ill for laparotomy and over time it became clear that 38 to 75% of infants actually survived without the need for further intervention. As a result PPD has gained popularity and is now used in approximately 50% of infants with surgical neck in the US. Two randomized controlled trials have attempted to evaluate mortality differences between PPD and laparotomy. However, an either group was able to identify a statistically significant difference. It's also important to note that surgical neck is associated with worse neurodevelopmental outcomes at follow-up when compared to both medical neck and no neck with nearly 40% of infants demonstrating severe neurodevelopmental disability. Given the lack of evidence of any mortality difference, the increasing utilization of PPD and the no-neurodervalmental disability in this population, we thought to determine if there were any differences in outcomes of follow-up between infants who received laparotomy or PPD. The primary outcome we investigated was severe neurodevelopmental disability and secondary outcomes included healthcare needs after discharge. Our group has had an ongoing partnership with the Vermont Oxford Network, which is a non-profit worldwide collaboration dedicated to dedicated to improving quality safety and value of neonatal intensive care. The VLBW infant database collects prospective data on approximately 90% of the VLBW infants in the US. In addition, Bonn has an ELBW follow-up database, which prospectically collects data on infants at 16 to 26 months' corrective age. We use the ELBW follow-up database to evaluate infants formed between 2006 and 2016 at the 44 US Centers participating in the ELBW follow-up project. Neck was defined by visualization during surgery or autopsy or as one clinical and one radiographic finding which correlates to Bell stage 2 or higher. Severe neurodevelopmental disability included any one of the following, bilateral blindness, hearing loss requiring amplification, inability to walk 10 steps, cerebral palsy, or any daily scales of infant and toddler development to main score less than 70. Healthcare needs after discharge assess for medical re-hospitalizations, home health support, and post-disturge surgery. The surgical groups were analyzed in an intention to treat fashion, with those receiving PPD at any point in their clinical course evaluated as a single group. Such that this group included infants who received PPD alone and PPD plus laparotomy. We identified 1211 infants with surgical neck during the study period, 55% underwent laparotomy alone, and 45% underwent PPD. Follow-up data was available for 181 laparotomy alone infants and 124 PPD infants resulting in follow-up rates of 59 and 53% respectively. We next evaluated characteristics of surviving ELBW infants who underwent laparotomy versus PPD. Median gestational age of laparotomy and PPD infants were similar, however, PPD infants had lower median birth weights and higher rates of neonatal comorbidities, including bacterial sepsis, severe IVH, severe ROP, and chronic lung disease, all of which have been identified as risk factors for neurodevelopmental impairment. We also compared evaluated and not evaluated infants to determine if the follow-up population was representative of ELBW survivors of surgical neck as a whole. Consistent with prior studies using the bond ELBW followed database, evaluated infants for more likely to be inborn and less likely to be Hispanic for Black. Variations between evaluated and not evaluated infants in each surgical group were small and not felt to be clinically significant. Laparotomy and PPD infants had similarly higher rates of severe neurodevelopmental disability, 42 and 41% respectively. Infants who underwent PPD had similar rates of medical re-hospitalization, home support, and post-disturbed surgery. Medical re-hospitalizations were primarily due to respiratory illnesses in both groups, and among infants requiring home support, PPD infants were more likely to require oxygen or cardiac pulmonary monitoring, which was consistent with the higher rates of chronic lung disease in this group. Risk ratios adjusted for birth weight, sex, multiple births, maternal race, and clustering between hospitals were calculated comparing infants to underwent any PPD to those undergoing laparotomy alone. There were no significant differences in any outcome on adjusted analysis. A subgroup analysis of the 124 infants in the PPD group was undertaken to evaluate differences between infants receiving PPD alone, represented 60% of that population, and those who received PPD plus laparotomy, represented 40%. Median birth weights were slightly lower in the group that received PPD plus laparotomy compared to PPD alone, 650 versus 695 grams. In clinical characteristics, immunosciences were similar, although infants who underwent PPD plus laparotomy had longer median rates of stay and higher median rates of predesturbed surgery. Infants who underwent PPD alone had slightly lower rates of all outcomes as seen here. Adjusted risk ratios compared to laparotomy alone showed a significantly lower rate of medical re-hospitalizations and post-dischrored surgery in PPD alone infants. In conclusion, rates of neurodevelopmental disability and post-dischrored healthcare needs are high in infants with surgical neck. However, despite lower median birth weights and higher neonatal risk factors, infants undergoing PPD did not demonstrate worse outcomes. The reasons for this are unclear, but maybe due to different general anesthesia exposure, the risks of transport, different early nutritional status, or different inflammatory responses. The PPD group is also likely a dichotomy's cohort, as PPD has been applied to an increasingly stable population of infants, possibly representing those with stiffer minimine neck. Further studies are needed to identify patient factors that will allow surgeons to tailor intervention for the most optimal outcomes for individual patients. Next, I'd like to discuss the impact of neck on outcomes in VLBW infants with neurologic injury. Interventricular hemorrhage preferentially affects VLBW infants at risk for other comorbidities, including neck. While neck and IVH are individually associated with increased mortality, their combined incidents and effects are poorly defined. An article recently published by a group at Seattle Children's, assessing the clinical practice of neonatologist and surgeons when treating premature infants with neck, found that for an infant with a concurrent diagnosis of severe IVH, physicians were much more likely to recommend comfort care only. We therefore sought to evaluate the incidents and mortality of VLBW infants with neck and IVH to identify any differences when they occur simultaneously. We evaluated VLBW infants form between 2014 and 2018 in the bond VLBW database. IVH was characterized as absent, mild, grade 1 through 2, or severe, grade 3 through 4. Risk ratios are adjusted for birth weight, small for gestationally, intonatal steroids, and clustering of infants within hospitals were calculated. Approximately 187,000 VLBW infants were identified during the stem period. The majority, 75% did not have IVH, 17% had mild IVH, and 8% had severe IVH. As expected, increasing severity of IVH was associated with lower birth weight, more comorbidities, and as shown here, an increasing severity of neck, particularly surgical neck and black. Mortality increased with severity of IVH and severity of neck independently, consistent with prior studies. Infants without IVH had a notable increase in mortality with increasing severity of neck shown here in white. This increase was less marked in infants with mild IVH shown in gray, and was nearly absent in those with severe IVH shown in black. On adjusted analysis, comparing medical and surgical neck to no neck, the higher mortality seen with increased severity of neck was mitigated by increasing severity of IVH, with a minimal increase in those with severe IVH. In conclusion, infants with IVH are more likely to have neck, which is likely related to the underlying prematurity of the population, and mortality increases with severity of neck and severity of IVH independently. The increased mortality is not substantially compounded, and infants with severe IVH, when the two diseases occur together. Further studies evaluating long-term, neurodevelopmental outcomes, and health training, will help further guide the management of these complex patients. I wanted to thank everybody in the Department of Surgery, especially Dr. Jackson and Dr. Modi, as well as my co-fellows, Sam. Thank you to Dr. Fishman, Dr. Schamburger, Dr. Lola Hayan, Dr. Fowze for this opportunity. I also wanted to acknowledge the support of the entire care team who taught me so much, and finally, thank you to my husband, Brent, and my daughter, Ellie, who remain the most important part of my life. I'm happy to take any questions. Jamie, can you hear me? I can. Great. Hey, it's Baron. I'm really glad that Jamie decided to highlight these two papers, because I think they not only are going to be really impactful, as you guys can tell, but they highlight some of her tremendous characteristics. Jamie's really just humble and mature, and you can see from these studies that she's been great at collaborating with our partners at Vermont Oxford, and producing these really great studies. Jamie, we're really proud of you. You have really grown into a great clinical researcher. What most strikes me about Jamie is just how thoughtful she is in designing these studies, and thinking about what outcome measures and variables to look at. So the paper and the study and the end result has been just really tremendous. I think she's going to go on to great things. I'm proud of you as a Brigham Reson. I'm proud of you as our research fellow. I'm really looking forward to great things for you, and I wish you all the best. Thank you. Jamie, can you hear me? Yes. It's Dr. Z. Congratulations on completing your fellowship. I did have a question about neurodevelopmental outcomes using the Bayley scores for surgical neck patients. All the statistical work that you've done, I'm sure you've done a fair amount of that with Steve and the Vermont Oxford Group. Apart from maybe trying like a propensity score matching of lab versus PPD in terms of baseline factors, all the multi-variable analysis that you've done apparently doesn't show much of a difference in those neurodevelopmental outcomes. Although irrespective of the surgical treatment, the incidence of severe neurodevelopmental outcomes is quite high, and I was just wondering from your perspective, tying several studies that you've actually worked on, any insights into any management interventions for surgical neck patients that might somehow be valuable to look at more closely to reduce those high levels of severe neurodevelopmental outcomes, i.e. Bayley scores less than 70 points. Yeah, I think it's a really important question because these kids are very, very severely disabled when you're talking about the factors that we're looking at and affecting that would be very important to their long-term prognosis and eventually their quality of life. I don't think that we have any fantastic answers, but I think a lot of the things that we thought about when we were writing up this paper was how much does operative exposure actually affect these kids? Does multiple early operations really the right way to go? Can we maximize their nutrition, which is something I know our ICU teams and our 14s really, really focus on, but I think that needs to remain in the spotlight. And then I think basic science studies comparing the different levels of inflammation and different surgical options would be important to see if that has an effect on neurodevelopmental. Certainly a very multifaceted situation in terms of intervention management and outcomes, and you've really been doing a great job. Thank you. With that, we'll move on to our last speaker, Stephanie Lazo. Stephanie considers New Jersey home. She attended Dartmouth College and received her MD from Cornell. She is currently a resident in the B.I. General surgery program and has spent her research fellowship working in basic and translational research with Dr. Dario Fousa. She's also done various clinical research projects with the fetal group in Dr. Buck Miller. Stephanie has been recognized for accomplishments at both the regional and national level with various awards. Her main research efforts focus on tranfamiotic stem cell therapy, and today she will give us some insight on expanding applications of fetal gene therapy and stem cell delivery. Thank you, Stephanie, for all your hard work and take it away. Thank you. Okay, I believe I'm sharing my screen. Okay, thank you very much for the opportunity to present. I have been privileged to work on a variety of projects during my two-year fellowship in the Fousa Lab, including in vitro cell-based work and several animal-based translational studies on different disease models and species. I have also been involved in several clinical projects with Dr. Buck Miller and Dr. Chen. In the interest of time, today I will be focusing on four rat-based studies in which we aim to better understand how tranfamiotic stem cell therapy works and to expand its applications. Transmiotic stem cell therapy or trace it is a minimally invasive strategy proposed by her lab to treat congenital anomalies. Trace it involves the procurement and ex-fibo-expansion of fetal derived mizankalmalsem cells or MSCs followed by their injection back into the amiotic cavity in large numbers to augment their native biological role in fetal tissue repair. So far, trace it has demonstrated potential therapeutic benefit in animal models of spina bifida, gastroskeces, and congenital diaphragmatic hernia, with varied effects depending upon the anomaly. Essential goal of my fellowship was to determine whether trace its clinical applications could be further expanded through a variety of potential strategies listed here. Firstly, we looked at whether trace it could be a strategy for fetal gene therapy. While fetal gene therapy has many potential targets in general, we specifically explored the use of genetically modified donor cells to increase trace its effects. Secondly, we explored whether stem cells other than MSCs could be delivered via trace it. Today, we'll present a summary of these first two goals informed by some new relevant mechanistic insights. In order to expand trace it's benefits, we first had to understand how it works. Therefore, we first performed a mechanistic analysis of trace its effects in the setting of spina bifida. For this application of trace it, our lab uses the rodent model, which is based on administration of retinomic acid to the mother through oral gavage under stational day 10 or E10, to induce fetal spina bifida defects. Intraamnotic injections of MSCs are then performed on E17 as seen in this video here, followed by euthanasia just prior to term at E21 for analysis of defect coverage. Using this model, our lab has previously shown that trace it promotes higher rates of coverage of the spina bifida defect by a host derived neoscan compared to control animals. In studies of donor cell homing after trace it in this model, our lab has found that donor cells home robustly to the placenta and fetal bone marrow rather than to the defect site itself, suggesting a possible important role for hematogenous routing of donor cells to the bone marrow in the host response to trace it. In our initial mechanistic screening of trace it in the rodent model of spina bifida, we hypothesized that defect coverage after trace it is a result of a combination of complex processes briefly summarized here. In this first analysis, we specifically focused on changes in bone marrow stem cell clonal populations and pericran activity. Using the same retinomic acid model of spina bifida, rat fetuses were divided into three groups. The first two groups underwent intra-amiotic injection of amniotic fluid derived MSCs or AFMSCs versus volume match saline and a third group underwent no further treatment. After euthanasia, spina bifida defects were isolated and split for correlated reverse transcription quantitative PCR or RTQPCR analysis of pericran activity and histological analysis of defect coverage. Bone marrow samples were also isolated from the lower extremities for RTQPCR. RTQPCR analysis quantified the expression of several pericran factors known to be secreted by MSCs in the setting of tissue repair, both at the defect and bone marrow. The expression of different stem cell markers within the bone marrow was also quantified as a surrogate for cell clonality assessments. At the defect, we found significantly decreased expression of fibroblast growth factor tube or FGF2 and at epidermal growth factor or EGF and histologically covered compared to uncovered defects. This suggests a possible negative feedback loop on FGF2 and EGF expression after coverage has already been achieved. At the bone marrow, fetuses with covered defects were significantly more likely to have below threshold expression of both transforming growth factor beta 1 and CD45, a hematochedic marker. Combined with the previous cell homing findings, these data further suggest that the bone marrow and more specifically its mz. angomal stem cell pool are essential component of their host response to trace it. In conclusion, this first mechanistic study fetal bone marrow activity is central to trace its therapeutic effects and experimental spine of it beta. Local pericain factor activity at the defect site also appears to contribute, specifically involving at least FGF2 and EGF expression. In parallel, sorry, in parallel to that mechanistic study, we also investigated whether trace it could be a vehicle for fetal gene therapy through the use of genetically modified donor cells. In this first proof of concept study, which I will not have time to discuss in detail beyond this slide, we modified donor AFMSE to overexpress a transtine, in this case endothelian 3 or EDN3, and found that trace it was able to promote delivery of that transtine to the fetal bone marrow after intra-amiotic injection compared to controls. While there are numerous foreseeable applications of transtine delivery to fetal tissues via trace it, which the upcoming fellows will explore, we used our initial mechanistic findings suggesting a role for FGF2 in the spine of epiphyda coverage process to hypothesize that the modification of donor cells to overexpress FGF2 could possibly enhance coverage after trace it in the rodent model of cyanobiphyda. To provide some background, FGF2 is also known as basic fibroblast growth factor. It is an important perconfactor that is part of a large FGF family as seen here. It binds specific FGF receptors on the cell surface that service transmembrane tyrosine kinase receptors, leading to the activation of a variety of downstream signaling cascades with a multitude of functions relevant for tissue repair and regeneration. To test our hypothesis, Syngeneic Lewis Rat AFMSC's underwent lentiviral transduction to overexpress FGF2 compared to a control cell population. Preservation of mizinkamol progenitor identity was confirmed by cytometry. RTQPCR and ELISA were performed in vitro to compare FGF2 transgene mRNA expression and protein levels respectively, in the FGF2 overexpressing AFMSC's compared to control cells. In vivo, we use the same retinoic acid-induced model of cyanobiphyda. Two groups of rat fetuses then underwent entramiatic injection on E17 with either FGF2 overexpressing AFMSC's or concentration matched control AFMSC's. A third group underwent no further treatment. Euthanasia was performed at term with histological analysis of defect coverage. On RTQPCR and ELISA analyses in vitro, we observed 10.1-fold higher FGF2 mRNA expression and 3.1-fold higher FGF2 protein levels in the FGF2 overexpressing AFMSC's compared to control cells. Of note, FGF2 is normally expressed at baseline by AFMSC's. In vivo, when comparing defect coverage rates by treatment group, we saw significantly higher rates of defect coverage in both cell-injected groups compared to the untreated group. However, we also saw significantly higher coverage rates in the FGF2 overexpressing AFMSC injected group compared to the control AFMSC injected group. In conclusion, we found that FGF2 overexpressing in donor MSCs increased defect coverage rates in a rodent model of trace it for cyanobiphyda. Therefore, the genetic engineering of donor cells appears to be a promising strategy for the enhancement of this emerging therapy for cyanobiphyda and potentially other congenital anomalies. Next, I wanted to briefly discuss another project in which we aim to further expand trace its clinical applications through the use of a new donor cell. In this study, we introduced the hematopoietic stem cell or HSC, while our previous studies have all used mesenchymal stem cells, given significant interest in the in utero transplantation of HSCs to achieve long-term graph, long-term treatment of hematological conditions with transplant tolerance. We aim to investigate if HSCs can hone to hematopoietic sites after simple intramiotic injection. Until now, these cells have been delivered by highly invasive methods in utero with risk of complications. We used a Syngenetic Lewis Rat model for this study. Lewis Rat HSCs were used with the HSC phenotype, including CD34 positivity confirmed on flow cytometry. In vivo, healthy dams underwent intramiotic injection on E17 with either Luciferase labeled HSCs or a control injection of acelular Luciferase protein to control for cell death and passive release of Luciferase. Euthanasia was then performed at term, with procurement of all fetal organs as listed here, with specific interest in organs involved in hematogen as traficing, or hematopoietas listed in red. The Luciferase assay was then performed to evaluate mnemonessence in each tissue as a surrogate of cell presence. Compared to controls, HSCs were identified in all tissues listed here in red, notably including placenta, umbilical cord, bone marrow, and thymus. From this study, we concluded that donor HSCs can reach the fetal circulation and travel to the fetal bone marrow in a variety of other fetal sites after intramiotic delivery, in large numbers in a syngenetic healthy rat model. Tracit may therefore become a practical and minimally invasive strategy for the prenatal administration of HSCs. In addition, Tracit could also potentially be used to deliver genetically modified HSCs for the prenatal treatment of hemiclovenopathy such as sickle cell disease and many others. Thank you very much for your attention. I am very grateful to Dr. Falza for his mentorship over the past two years, as well as to Ena and my co-fellows, Danny, Sarah, and Alex for their support in completing these projects. I also wanted to thank my clinical research mentors, Dr. Buck Miller and Dr. Chen, as well as Dr. Z and C for their statistical assistance, and lastly, thank you to the Department leadership for supporting our work. Thank you again and I'm happy to answer any questions. Dr. Falza, thank you are unmuted. All right. So I just wanted to say that Stephanie made it look easy. She had to synthesize her presentations with this. These projects carried a fair bit of frustration, particularly in the pilot phase and required a lot of perseverance and skill to bring to the execution phase, which she beautifully did. I think this is original important work. I expect that Stephanie's age index will get a boost from them in the near future. In fact, she has already started to receive national and international recognition for that. Extending my comments from last night, I'm very proud to have been a part of Stephanie's journey. I reiterate my gratitude to her admiration for her work ethos over all competence, and I'm looking forward to following her career. Thank you very very much, Stephanie. I wonder if some of them oftentimes we hold questions. I certainly did. Because we're worried that we may not run our time for subsequent presenters. I wonder if we could reopen the floor, so to speak. The questions for all three of our excellent research presenters. I might start, maybe probably because Stephanie's microphone is still open. I might start in reverse order and ask sort of a 30,000-foot question. That would be with all the elegant science that's been done by you and your colleagues and predecessors, many of the technical details have been worked out for tracing. There are several clinical applicability cases that you and others have made. What do you see as the biggest hurdle to getting us into the first human? What was it going to take? What did that application be? Thank you very much for the question. Dr. Fausa has actually already been in discussions with the FDA to try to push forward trace it for the gastroskeases application. One thing that the FDA has asked for that we are still in the process of trying to obtain data for would be postnatal functional data after trace it for gastroskeases. One thing we've struggled with is to obtain that data because it's very challenging to keep these animals alive postnatally once we've created defects. In the near future, our lab is going to be working on larger animal models, especially a gastroskeases trying to obtain that postnatal functioning data. Thank you. I wonder if for simplicity, some of the microphone opening, if I could just ask each person one question, then we'll see if there's time for others. In reverse order, it's a good journey. You know, neck is this elusive condition that since the day that I trained it was in your shoes, we don't understand very much more about the ideology or pathophysiology of neck, nor do we have a great handle on better therapies. Of course, a few decades ago, Pardial drainage was introduced and there was this big question, is it better or worse or equivalent? It's a source of really the first randomized prospective trial in pH of surgery. And we still have sort of equipoids. The Vermont Autornetwork is a massive dataset that allows us to look at many, many most children across the nation. And it still shows, as you demonstrated, lots of close similarities. But even with the massive dataset, of course, it's collecting what's done. There's nothing randomized about the care of those children. So my 30,000-foot question for you would be, do you think the lack of randomization increases or decreases the likelihood that we would see a difference between treatments, outcomes, whether it be life and death or whether it be neurodevelopmental outcomes, etc. Because you could imagine that some sets of babies are chosen for one therapy versus another for whatever local reason there might be. And sometimes lack of randomization blinds an outcome, but sometimes it actually may increase in this very, do you really, you went to that? Yeah, I think that's a great question. It's something that we've talked about a lot. Randomization is important because as you can see, the patients receiving the different surgical treatments are different groups of patients. It's just something that's really confounded our ability to learn more about outcomes and PPD versus LAPRAW. I think actually having a view of what's happening now gives us a lot of unique information. I think our understanding of this is that there are certain patients who definitely need LAPRAW. For whom PPD is probably not the right initial intervention. And finding the group of patients who would really benefit from that is determined to be determined a lot by those factors that surgeons are already taking into consideration before they are doing their procedures. I think randomization for these kids is really, really hard despite having 10 years of data looking at tens of thousands of patients, we really only came up with several hundred to evaluate. And when you look at the randomized control trials, you see that they had trouble convincing surgeons to randomize their patients. And so I think that all of our randomized data for NAC really has to take that into consideration. I think by having views from both perspectives, we'll be able to learn a little bit more. Terrific. I hope that your work leads towards that clarity. 35 years from now when you're in my shoes. And lastly, I would ask, Bethany also a long distance picture question. I remember being in your shoes and one of the most complex parts of caring for sick surgical patients was fluid balance. And we had different names for things like capillary lexin enrollment. We debated the importance of oncotic pressure and crystalline versus colloid. All these things are still ongoing. And you've demonstrated a tool to demonstrate maybe an improvement in the way that we could look at that total body fluid and fluid balance measurement. I would also ask that the big picture or sort of old arching due is 35 years from now, will we have a, with all of the increased technology that we have and all the fancy monitoring and all the advanced biochemical techniques, will we have a way of actually knowing when our patients are fluid overloaded or fluid, need to be fluid avid and when we turn that switch from giving off a fluid to diuretic. Thank you, Dr. Fishman. And certainly a question that has been an ongoing challenge for a very long time. I think this, this tool could potentially help in the post-resuscitative phase of care where you want to have a better idea of are you, are you recovering from fluid overload? Do you still need to diurease? Is it an appropriate time to extubate, extubate a patient? Questions where we talk a lot about the fluid balance and the ins and outs are the most practical tool we have right now, but there are certainly plenty of reasons to believe that that's not a very accurate measurement, especially over time, the further you get out from the initial surgery. So I think devices like this will continue to improve and I hope that they can help assist us in making those decisions, as well as some more non-invasive tools in that resuscitative phase. And this device doesn't measure intravascular volumes so it can't contribute in that way, but I think I certainly hope there will be more developments to get an accurate picture of fluid status without using invasive measures like swan catheters, etc. So I hope so. I just want to tell, I can't tell, in Zoom only Somala knows if there's other hands up, but before she moves on to others, I just want to take the opportunity, well, my microphone is on is to, again, as last night, comment on what is spectacular, group of presentations you guys have made, what a tremendous amount of learning you've done for yourself, but also for us, and I think you've taught your mentors each something. I think is clear that we're looking at futures of the field and the possibilities for your careers is limitless, and you should all reach for this guy and we will really enjoy watching you moving forward. Thank you so much, and I know that it's Zoom and you can't hear all the clapping, but everybody was clapping at least virtually for each of you after your presentations. So congratulations so much. Thank you. There is one more hand up, Dr. Kim. You are also unmuted if you have any questions or comments to make. Yeah, hi. Thanks to all three of you for your excellent talks and all of your hard work. I do have a specific question for Stephanie since she brought up one of the topics that's in your and your to my heart in your stem cell transplantation. With regard to the inoculum, you mentioned CD34, but I didn't see or maybe I missed it, whether it was just CD34, or they were just user for a pod of cells within the inoculum, or if you're enriched for that. Hi, Dr. Kim. Thank you for the question. Are you asking if we specifically selected for CD34 cells? Yep. So we actually have struggled on our own. It's very challenging to isolate rat HSEs. So we actually purchased these cells. They were commercially available from a company, and we are not actually aware of the process that they used to isolate the cells. It's possible that they did select for CD34. All we received was their flow cytometry data, suggesting that the cells that we injected had at least 90% CD34 positivity. So that's very enriched then. And then the second question is when you looked at the different organs, did you look for evidence of live cells, or was it simply just the cypherase activity? So using a Luciferase assay, we were only able to assess for the Luciferase activity in the tissues. However, we do use a Luciferase protein control, which we believe should control for the possibility of cell death and passive release of Luciferase at those tissues. But I agree we do not know absolutely that the cells are alive in the tissues where we are able to recognize Luciferase. That's all I have. Again, thanks to all three of you for your outstanding work. Don't believe there are any other questions. Thank you again to all three of you for your excellent presentations and thanks to all the research fellows and critical care fellows for their presentations over the past couple of weeks. I would simply echo what Dr. Fishman said with respect to graduation yesterday, being able to step back and hear all the work that you've done outside of the clinical arena was quite impressive. And so thank you so much for everything that you do in the clinical arena, but also outside as well. And thanks to everybody for participating this morning. And that's a wrap.
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