So, for the next 15 minutes, I will talk a little bit about strategies to enhance liver regeneration. These are my disclosures. I will show data from this company, Generon. I don't receive fees from that, but Mayo Clinic does receive fees from Generon. So we'll start at a high level about liver regeneration. So we know that in various types of species that regeneration occurs very naturally, such as lower vertebrates, and in mammals that when you do a partial hepatectomy, you have complete and effective liver regeneration. Now, in the setting of injury, especially chronic liver injury, we know that there's impaired liver regeneration. And this occurs in part due to extracellular matrix deposition. And we know that the regeneration even in response to injury in the absence of fibrosis is replete and can lead to proper regeneration. So in alcoholic hepatitis, we have problems not only with fibrosis, knowing that most of the patients with alcoholic hepatitis have coexistent fibrosis, but we also have steatosis, which we also know impairs liver regeneration. And furthermore, corticosteroids are often administered in these patients, and this may also impair liver regeneration. So this sets the background of why alcoholic hepatitis may be a target for regenerative therapies. So in the talk, I will go over a few different strategies here. I'm going to, again, show a few things that may be more foresight-oriented, not ready for prime time, but things that we really need to be thinking about. I'll show you a little bit of data about scaffolds, bioengineered scaffolds, reprogramming of cells, extracellular vesicles, cell therapies. And then we'll spend most of the time talking more current state about drug therapies that are under evaluation, including GCSF and interleukin-22. So this slide shows a few of the futuristic therapies that may not be so far in the future. This panel here shows you basically a bioprinted liver using, just like printers you may have that print for your paper, you can also have organic compounds that are used in the printer to generate actual organs. The middle shows a study from Scott Nyberg at Mayo that we collaborated with looking at scaffolds. And these scaffolds can be perfused, actually, and this is an MRI which shows perfusion of blood through this scaffold device that is essentially a repopulated organ. So this can provide additional assistance to the native liver, which is impaired. And then finally we know a number of companies and strategies going on looking at humanized pigs and approaches to generate hepatocytes through xenotransplant and then using these cells in a variety of ways, either through injection or through ex vivo gene delivery or again using scaffold systems to try to provide temporary regenerative solutions for patients. And alcoholic hepatitis is probably a very good indication for some of these types of regenerative therapies, given that if you can temporize patients over a period of time and they're able to maintain abstinence, then you may have natural improvements. So in terms of reprogramming, these are some studies from Ramon Batalha's group making the case that hepatic progenitor cells in alcoholic hepatitis are actually going towards a cholangiocyte fate rather than a hepatocyte fate and this is accounting for impaired regeneration. This is shown in this graph from an earlier paper that there's an increase in the laminin fibronectin ratio in tissues in these patients with alcoholic hepatitis with laminin predisposing towards cholangiocyte differentiation of progenitor cells. And this is a more recent paper from Ramon's group with a number of collaborators, including ourselves as well, showing that HNF4 might be a key molecular target that mediates this process by which patients with alcoholic hepatitis have impaired liver regeneration. You can see a down regulation of a number of HNF4 target genes and ChIP-seq studies that complemented this were able to show impaired epigenetic activation of HNF4 both at the histone level as well as at the DNA methylation level. Another approach that is still a little bit futuristic but probably not too far down the horizon are extracellular vesicles. This is a cartoon from Dr. Alpini's group in a nice review that he wrote looking at these vesicles and we know these vesicles are in a number of different body fluids and a number of approaches can be used to categorize these vesicles into different categories. Probably right now it's not so relevant to go into the different categories, but these vesicles can contain a number of proteins, microRNAs, and RNA, mRNA as well. And there may be a number of potential utilities for these vesicles. One is a potential biomarker through the amount of vesicles circulating as well as the cargo in those vesicles. Another is the potential pathogenic role of these vesicles and how they may mediate cell-cell communication. And then finally, you can imagine that these vesicles may actually serve as a delivery vehicle as well if you could load vesicles with various molecules to use them for therapeutic delivery. So the next section will move into cell-based therapies. This is a study from a few years ago looking at cell-based therapies in alcohol-related liver disease. And in this study from Europe, patients with alcohol-associated liver disease were randomized to receive either bone marrow mononuclear cell transplantation or standard medical therapy. And the point being here that this is essentially a negative study showing that bone marrow mononuclear cell transplantation in these patients was not able to achieve a primary endpoint of improved outcomes in these patients with alcohol-associated liver disease. However, there's been new approaches now used to try GCSF, which stimulates liver regeneration and also recruits bone marrow cells. So this is shown in this schematic cartoon here from Richard Moreau and Dr. Ratou, whereby GCSF has these putative effects both on hepatocyte progenitor cells as well as mobilization of CD34 stem cells. I should point out, I'm going to show you some positive data, but there was also a negative study on ACLF from Europe, and I believe another study from Europe will be presented at this meeting on GCSF. So these are a couple of the studies from India. This is from Virendra Singh, and this is from Shiv Sarin, complementary study protocols, but one of them essentially showing GCSF was more effective than standard of care in improving survival in patients with severe alcoholic hepatitis. And then this study from Shiv Sarin showing that in patients who were corticosteroid non-responders who received GCSF, there was an improvement in survival, again, compared to a placebo, providing evidence that GCSF may be effective in patients with alcoholic hepatitis. So in the last five minutes here, I'll talk a little bit about IL-22. You heard a little bit from Bern earlier about IL-22. It's produced from blood cells, promotes liver repair, reduces fibrosis, and has no immunosuppressive effects. Initial work in the liver was done by Bin Gao and Svetlana Radeva many years back, showing that IL-22 could stimulate a number of potentially beneficial effects in hepatocytes, including hepatoprotection, liver regeneration, and preventing steatosis. And some work also suggests potential beneficial effects as an antifibrotic as well. This is a cartoon showing some of the translational studies going on and the link of IL-22 with hepatocyte injury. I should have updated this. I think Bin Gao just has a paper in Journal of Hepatology going into much greater depth about the mechanism by which IL-22 has beneficial effects in acute and chronic liver disease. But just as a capsule, while alcohol can activate caspases and stimulate cell death as well as exosome release, which can serve as a biomarker, as I mentioned, we know that IL-22 can counteract a number of these effects through a number of pathways, which I won't show you here. But as I mentioned, there will be a paper in press in Journal of Hepatology that I would refer you to. So we'll move on now in the last couple minutes to the open label study looking at IL-22. This was done through the TREAT consortium. So F652 is a recombinant fusion protein consisting of human interleukin-22. This was basically a dose escalation study looking at a variety of doses in patients with both moderate and severe liver disease. And this was the dose finding at 10, 30, and 45 micrograms per kilogram. And this is the demographics. Something really important to highlight here, I think, disease stratification was done at MELD score 11 to 20, and then more severe MELD score of 21 to 28. And since this was really a safety and tolerability study as the primary endpoint, this is just to show there were some possible associated adverse effects, but nothing very dramatic. PK data showing nice curves of peaks in response to dosing with reductions in the blood levels. And then when you give a second dose again, you get the elevation. And this is really the take-home point showing changes in MELD score in the total groups and in the subgroups. And then towards improved survival at day 28, improved MELD score day 42, and then improvements in the moderate group, significant at day 28 and day 42. And then in the severe group, significant improvements by day 42. Now I'll point out there's no control arm in this study. So what was done was a propensity score analysis. This is essentially using prior cohorts of patients receiving or natural history or steroid treatments and then looking at little responses and comparing little responses to the little response in patients who received F652. And that's shown here with 82% little response in the study I just showed you with historical cohorts in much lower little responses. These two are very low. And we did add a third cohort in response to reviewer query where the little responses were more in about a 50% range, but still not in the range that we saw with F652. And then some secondary mechanistic endpoints, looking at extracellular vesicles, which are increased in these patients shown by multiple groups, including Dr. Szabo and ourselves and others, showing increased vesicle numbers, which drop over time, as well as increases in cytokine levels that are inflammatory cytokines, which are attenuated in association with dosing of IL-22. So this was now accepted in hepatology, so should be available in hepatology online soon. So with that, I will conclude and thank everyone, especially Doug Simonetto and Patrick Kamath and the rest of the Alkepnet team, as well as the folks from other Mayo Clinic sites, Harry Yang, Hugo, and others who recruited all the patients through the Mayo Clinic Enterprise. Thank you. So you will all agree with me that all the three presentations were excellent, and not only we saw the currently ongoing clinical trials, but also the future promising targets in different areas on pathophysiology of this disease. So now the session is open for questions and discussion or comments. So let me start the ball rolling, Dr. Berndt-Schnabel. You showed that the NEJM paper, there was one patient who died after, if you call it, bug as a drug. So how we can tether out whether this is disease-related or dilly, if we can use the word, in terms of patients of alcoholic hepatitis di. Yeah, I mean, that's a very good point. I mean, the donor, they have tracked this donor back, and the donor is just healthy. So I think in a healthy human being, even if you have like drug-resistant bugs in your liver, intestine will not cause any disease. I think the major point is, you know, the one patient who died, I believe he had a myoablative therapy for bone marrow transplant that they did, this was a clinical trial, and they did the prophylactic FMT. So in such a very severe diseased group, which I agree, our alcoholic hepatitis are also very diseased. You know, they have a very immune-compromised immune system. So I think we have to be very careful, and obviously, I think screening factors now from the FDA will require to screen for such drug-resistant bacteria, especially in such immunocompromised patients. Mark? Yeah, I'd like to pick on Ben's as well, actually. So you make comments about FMT being a little bit crude, which I don't disagree with. But if you give limited colonies of organisms or single organisms as probiotic therapy, how do you know those are going to survive against the challenges of colonization resistance? I completely agree with you, Mark. I think, you know, the probiotics currently, very few are colonizing. So if eventually we go to, you know, a more precise approach, you know, away from probiotics, they have many effects. We honestly don't know probably all of the effects. So if you move more towards precision medicine, you know, what I have shown you with the bioengineered bacteria, I think this might be one of the futures. I think this will be not, most likely, they will not colonize. So you will have to have them, you know, administrative, administrated or given to the patient probably daily. Can I just say congratulations on your phage study? Oh, thank you. Yeah, and microphone at the back. Okay, John Clary, Barcelona. So this question is for Dr. Szabo. So I'm asking about the inflammasome system. So I have the question regarding this. So it is a draggable target. Can you, for example, modulate the sample of the inflammasome or you just reduce the levels of the inducers, like reducing ATP? That's a very good question. So the normal molecule NLRP3 modulators are in development, but they haven't made it to kind of clinical trials at this point. But theoretically, yes, there are pharmaceutical company efforts to essentially develop inhibitors that will potentially prevent the assembly of the inflammasome. So the second question is the relative contribution of the inflammasome. So it is a goofer cell, immune cell related, or hepatocytes also contribute to the role of inflammasome? So you're bringing up a very good question. The inflammasome components actually are expressed pretty much in all cell type in the liver to various extent in terms of the functional assembly of the inflammasome. It happens in every cell types. It has not been systematically studied, but inflammasome assembly and activity happens in hepatocytes. It can happen in stellate cells and I believe in endothelial cells has also been shown. So it's not limited only to the immune cell types. The question is how effective is that and what is the target of that? In alcoholic liver disease, at least in the animal model, we found relatively minimal activity of the inflammasome in hepatocytes compared to immune cells. That's what we kind of know so far. Can I ask a question to Bernd? Bernd, the IL-22 bacteria is very clever. I wondered your opinion on how much of the effect is at the gut level versus a direct liver effect. You know there's other companies besides Generon that have IL-22 recombinant products actually being developed for IBD, but maybe you can just comment about all this. So we designed this essentially not more or less as proof of concept that IL-22 is beneficial for alcoholic liver disease. Dr. Gao has shown this through many years now. So what we did and this was essentially just to bring in a little bit of novelty and also show that IL-22 is very important in the intestinal compartment. So what we did is we titrated in this mouse model the dose and how often we gave this engineered IL-22 producing bacteria to a level where we do not see an increase in systemic IL-22. So if you give this daily in a high dose, you will, and we have shown this, you know, we have not published it, but I'm saying we have tested it, that you can also increase systemic IL-22 levels. But we have dosed it to a level that we cannot at least not detect by ELISA, you know, that systemic IL-22 levels go up. So we believe this is, you know, proof of concept at least if you give it like through a, you know, in the gut compartment and you have like very beneficial effects on the intestinal epithelial cells. And as you know, Vijay, I mean, the, you know, intestinal enterocytes like hepatocytes are one of the few cell types that have the IL-22 receptors. So Bernd, another question for you. We're collaborating with the same people that made your bugs for a different approach. And so I think it's a great idea. The question is, how does the FDA feel about engineered bugs? Do you have any idea about that? I haven't asked them. I have to ask them about the phage therapy. So, I don't know. So I think obviously modifying gut flora is going to be a very important thing. We have tremendous strategies out here and this is going to be an important issue to get the FDA on board on this. Another question for Bernd. You didn't have time to really talk about Canada very much. Do you want to mention that? Yeah. So, Canada, we published now this also like in a series of papers is that independent from the liver disease stage, if you look at patients with alcohol use disorder, essentially you get in those sequencing for fungi, essentially the diversity of fungi in patients with alcohol use disorder, but also alcoholic hepatitis is essentially wiped out. So you have very low diversity of fungi in these patient populations. And what they are replaced to by is essentially Canada and this mostly Canada albicans. So essentially Canada takes over in these patients, which is kind of interesting to us. Because if a patient with alcoholic hepatitis gets a Canada or fungimia infection, essentially that's, probably the patient cannot be rescued. Now the interesting thing is, although they have like this Canada infections, the percentage of infections that we see with fungi, I think compared to bacteria, it's very, very low. I think you would agree to that. And that is important, I think, because the Canada, it's a hundred times bigger than bacteria. So bacteria have like, they are a couple of microns and a Canada is like 100 microns. So I think to get the Canada across the gut barrier, unless you have an alteration, unless you're an IBD patient, it's probably much harder than to get bacteria across the barrier. But obviously, I mean, this would be interesting to see if we can use anti-fungi medications. In animal model, obviously they work, but in humans, I don't think there has been any trial yet. John G., the uric acid data was very interesting. Have you done anything on humans with that? Is there a possibility of just repurposing a drug? So we went as far as designing a trial, but it turns out that to get probenicid, nobody produces that. So it's as simple as that. It's sort of an area that we probably want to get back to, because it has been used very, very widely, actually, in the past, particularly in renal medicine. I think we have a couple more minutes, if I can ask Yongi just a simple, I mean, I don't know, but why was the trial of anakinra, pentoxyphilin, and zinc, the rationale for pentoxyphilin, and why not only anakinra? It was just to... It was very simple... Global anti-inflammation, or... Well, so the concept at that point was that we wanted to combine the three kind of, some approaches to improve gut permeability, to reduce inflammation, and potentially provide hepatocyte protection. This trial was designed before the study from Dr. Tursk came out, identifying the kind of lack of benefit of probenicid. I mean, I'm sorry, of pentoxyphilin. Hi. I'm from Miami. A question, basically, on a practical sense. If I go back, when I go back tomorrow or next day to my hospital, and I see a patient with alcoholic hepatitis, out of what you have found, what can I use today to help my patient with alcoholic hepatitis? I'm going to put him on steroids, 20% chance it's going to improve. Should I give him vitamin C? Should I give him probiotics? Should I give him zinc? Should I give him NAC? What do you guys think I can use today? I'm sorry. I think at this point, I would just stick with what we have a lot of data for. As frustrating as I think is for all of us, the clinical trials are not at the point where we can make firm recommendations. So, we use steroids in patients who have severe alcoholic hepatitis, potentially use the 7-Day Lilies score to stratify. Then, unfortunately, the rest leaves the supportive care, nutrition that cannot be emphasized big enough in this disease, and treatment of the alcohol use disorder. I think we all have to remember that as hepatologists, we may not be well-equipped to deal with the alcohol use disorder treatment. So, bring in your colleagues who have expertise from psychiatry and social work, and work as a team. Well, if there are no more questions, we wrap up the first session. Thank you, all the speakers, for excellent deliberation.

liver regeneration

alcoholic hepatitis

chronic liver injury

regenerative therapies

drug therapies

clinical trials