Thank you, and good morning. I have no financial conflicts of interest. I am currently funded by NHLBI, and much of the work that I will show here has been funded by NIGMS, NHLBI, and NICHD. So we've heard a lot recently about the cytokine storm. And following on the theme from a couple of presentations ago, certainly much of what we see in the ICU is due to hyper inflammation, whether it be capillary leak, fever, leukocytosis, organ dysfunction. We know that many of our patients are inflamed. And certainly, severe hyper inflammation and inflation, when very high and or prolonged, is associated with adverse outcomes. But we are also increasingly recognizing that concurrently, or at the same time, under the surface, many of our patients are also immune suppressed. And this immune suppression can itself also be severe and or prolonged. And it's harder to detect at the bedside. It's harder to know which of our patients with multiple organ dysfunction are suppressed. Now, there are certain things that we can do in the laboratory to test this. And I'm going to talk briefly to you about one of these assays that is particularly well suited to study in children. And that is the ex vivo LPS induced TNF alpha production capacity assay. Basically, we take whole blood. And we need as little as 50 microliters of whole blood. Stimulate that with a standardized solution of lipopolysaccharide, or LPS. Incubate that for four hours at 37 degrees. And then quantify the amount of TNF alpha that is produced. Now, TNF alpha is an inflammatory cytokine. And it's important to note that this assay is very different from measuring plasma cytokines. If I were to measure TNF alpha in the plasma of a healthy person, it should be very, very low. If I take whole blood from a healthy person, stimulate it with LPS, the resultant TNF alpha production should be high. And so when we look at this in healthy children, for instance, the TNF alpha production capacity tends to be about 1,000 picograms per ml. When we do this in children in the ICU, particularly those with multiple organ dysfunction syndrome and or sepsis, those results are typically lower. Now, we can also, in the same blood sample, look at the plasma circulating inflammatory mediators, including cytokines and others, so that we can, at the same time, also have a snapshot or a window into the inflammatory state of the patient. And this assay has a couple of different advantages for pediatric patients. One is blood volume. So we can do this with relatively small volumes of blood, which is important for our small patients, who really don't have much in the way of circulating blood volume available for study, particularly when we're talking about infants or neonates. The other advantage to this assay is that it can be standardized for multi-center study with rather simple on-site processing that can be accomplished by clinical research coordinators. So this is very important also for pediatric study because of patient numbers requiring that really we do expand our studies to multiple centers. And so at the Immune Surveillance Laboratory at Nationwide Children's Hospital, we have developed a standardized approach to this assay, where we manufacture and quality control these LPS stimulation kits and ship those to centers. The blood is drawn, and the stimulation happens on site. And then the supernatant is shipped back to us, either for batch processing or for real-time immune function phenotyping with a turnaround time that can be as little as 24 hours or sometimes even less from the time of blood draw to getting results back to the center. And using this method, we've shown that this severe immune suppression is associated with adverse outcomes across a variety of critical illness and injury, not just sepsis. And so this low LPS-induced TNF alpha production capacity has been associated with the subsequent development of nosocomial infection in children following traumatic injury in the ICU. It's been associated with mortality and critical viral illness, including influenza, and is independently associated with duration of organ dysfunction in children with severe sepsis or septic shock, even when accounting for baseline severity of organ dysfunction. So clearly, critically ill and injured children are immune suppressed, and we need to turn the immune system back on, right? Well, it's not so simple, because again, hyperinflammation is also present and is also bad for you. And so for each of the studies that I just showed you, elevated inflammatory mediators were also associated with development of subsequent nosocomial infection in children with critical trauma, mortality and influenza, and duration of organ dysfunction in sepsis. And so clearly, we need to be thinking about both sides of hyperinflammation as well as immune suppression. And it's important to note that each individual child is going to be in a different place along that spectrum of both immune suppression as well as inflammation. And this was further highlighted by the recent phenome study conducted by the Capcorn Network that was nine centers at the time, enrolled 401 children with severe sepsis, and measured biomarkers for four different phenotypes of MODS. And I'm going to call your attention to the bottom two here, immunoparalysis, which you've heard about before, which was measured by this standardized LPS-induced TNF alpha production assay, as well as macrophage activation syndrome-like sepsis, or a hyperinflammatory sepsis, which was indicated by the presence of hepatic dysfunction, hematologic dysfunction, and an elevated ferritin. Within the phenome study, there were 289 children with MODS, with an overall mortality of 11%. About 30% of children with MODS had immunoparalysis. And this is a number that's fairly consistent across our studies of children with multiple organ dysfunction. 24% of the children had immunoparalysis without MAS. And for those children, the mortality was 16%, or about one in six children who died. A smaller proportion of children had this hyperinflammatory MAS-like sepsis syndrome, but with a mortality rate that was nearly 50%, which is quite high for pediatric sepsis. And there was this interesting group in between that had both and had a mortality rate of about 40%. And so clearly, I think the path forward, as has already been talked about this morning, is that we need to establish immunologic balance. And that is probably unlikely to happen with a one-size-fits-all approach, because again, each patient is in a different place in terms of both immune suppression and inflammation. And what we want, in the end, is an immune system whereby your immune cells are capable of clearing existing infection, of promoting organ recovery, and of fighting new infection, but without damaging systemic inflammation. And I would posit, again, that this is going to take a personalized or precision approach. And so to this end, the Capcorn Network is now in the midst of conducting the personalized immune modulation in sepsis-induced MODS, or PRECISE trial. The PRECISE trial is essentially an immune phenotype guided RCT, whereby based on these immune biomarkers, children are either enrolled in the GRACE-2 trial of GM-CSF for reversal of immunoparalysis, or the TRIPS trial targeting that hyperinflammatory sepsis syndrome. There's a planned enrollment of 1,000 children across 24 sites of the Capcorn Network, with a primary outcome variable of cumulative PILOD2 score, PILOD2 score being a pediatric-specific organ dysfunction score, through day 28, with death being given the highest possible score. The 24 centers of the Capcorn Network are as you see here, and really represent a broad swath of centers across the country. The way that this works, essentially, is children with sepsis-induced MODS, or multiple organ dysfunction syndrome, are enrolled and are phenotyped by the ex vivo LPS-induced TNF-alpha production capacity on the immune suppression side to identify immunoparalysis, and by ferritin and CRP to identify hyperinflammatory sepsis. For those children who have immunoparalysis, or severe immune suppression, without significant elevations of ferritin or CRP, such that they may have MAS, so these are the children with immunoparalysis without MAS-like sepsis, are randomized to GM-CSF or placebo. Those children who have a CRP and or ferritin level indicative of potential MAS-like sepsis syndrome, or this hyperinflammatory sepsis, are randomized to the IL-1 receptor antagonist, anakinra or placebo. Children who have neither are observed, followed over time for both immune profiling, as well as outcomes. And those children have a very high ferritin, indicative of a potential for hemophagocytic lymphohistiocytosis, say that six times fast, or HLH, or autoimmune-type MAS, are not a part of the trial per se, they are observed, and the clinical teams are identified of the need for potential workup and management per local standard of care. So enrollment in this trial is ongoing. We hope to have more information coming out of this, and really see this as a step forward toward that end goal of personalized, precision-targeted immunomodulation for critical illness, recognizing that slide that we saw earlier this morning of the failures of anti-inflammatory therapies could probably equally be matched by failure of therapies to turn the immune system back on if you apply it to everyone. What we really need to know is how to get the right drug to the right patient at the right time, and we believe this is one step forward. And so in summary, immune phenotypes characterized by severe immune suppression and by hyperinflammation are both present in critically ill children. They can be identified by small-volume assays that are amenable to multicenter study. This, I think, represents a great opportunity to move forward toward truly targeted immunomodulatory therapies in children to really finally, at some point, we hope, achieve the goal of restoration of immunologic balance and improved outcomes for these highly vulnerable children with multiple organ dysfunction syndrome. And with that, I'd say thank you, and I'm happy to take questions. Thank you.

cytokine storm

critically ill children

immune suppression

ex vivo LPS induced TNF alpha production capacity assay

personalized immunomodulation strategies