All right. Good afternoon, everybody. We're going to get started with our session today. Thank you for attending I'm honored to be one of the moderators today I'm Betsy Verna from Columbia University in New York and I have here with me jazz Bajaj from VCU who is also going to be our first speaker for this session and Jazz is going to talk about microbiota and hepatic encephalopathy, and it's just a quick reminder We're going to save all questions for the end of the session when all the speakers will be invited up for a panel discussion Can we get started with the slides, please Or not we have time I Do have a good memory, but not that Interpretive dance, maybe you can tell some jokes Okay, here we go Thank you. Thank you. Dr. Werner. Thank you Dr. Biggins dr. Rocky and to all of you and to the portal hypertension sig. I'm at dr Andrea got out D got RD could not be here. So I'm going to try and Combine two Not so distant concepts in one talk as much as possible So why is the gut a major target for HE portal hypertension and cirrhosis? How can the gut microbiota be used as prognostic and diagnostic markers in HE? Current treatments that focus on the gut and potential issues and targets for future HE related therapy. We've tried very hard Thanks to our organizers not to overlap with each other But certain things which I think are worthy of overlap. Maybe you may see the same concept over and over So let's talk about the factors that impact gut microbiota composition and function lest we might all think that this is a static thing It's not it's multiple these things at least in cirrhosis that can affect this the prominent things could be disease etiology active alcohol use disease stage Medicines and organ failures and of course the law if you see the biggest font is for diet Diet is really a very critical thing. And in addition most of these studies have been done on bacteria But fungi archaea protists and viruses are also really important parts of this Sphere of microbial analysis that we want to talk about. So this is the gut liver axis You have the intestinal barrier. You have the intestine portal vein itself And then you have the sinusoids and there are multiple bacterial products as well as bacteria themselves that can translocate through this go directly to the liver or go through the lymphatic Circulation and then affect the liver systemically So there are depending on what string of bacteria that you pull out you may have multiple different mechanisms Not only from one bacteria, but several other working together So to give an example of alcohol-related liver disease There are of course you have brain dysfunction depression that can lead to alcohol misuse in some people and many people don't have Alcohol-related liver disease, but many people do with alcohol-related liver disease. You can have steatosis Compensated decompensated cirrhosis alcohol-related hepatitis and death and liver transplant in addition This all can also take hepatic encephalopathy nutritional deficiencies and withdrawal. All of these can affect the brain These are where all in human studies Microbiota change structure and or function have been described to be a problem So I can tell you right from the beginning as to who develops problems because of alcohol Who develops problems as a result of alcohol use and who actually lives or dies Has been associated with the gut microbiota of that patient in addition Etiology versus cirrhosis related is very important because when we see a patient we see the entire patient You can't peel apart what was happening before they actually started to drink alcohol What drove them to drink the alcohol what is related to the cirrhosis? So I'm going to also tell you whether you can use the microbiota to differentiate What kind of brain dysfunction this person actually has This is a very complicated schematic and that is the reason because it is very complex There's a change in the microbiota as well as the gut liver axis right here in the gut bacteria Sorry gut barrier. The there's a structural barrier change and there's also an immunological barrier failure in Not me in patients with cirrhosis Just to keep you on your toes then you have Inflammatory and immune changes not just in the circulating situation Circulation but in also the reticular endothelial system that results in Bacterial translocation as well as organ failure. So microbiota are one small part of this This is one small part of whatever is going wrong with your patient patient So you have to remember microbiota are important, but they're not the only thing that may go wrong with your patient So why is the gut important and this is directly towards a chi? We know that people wake up comatose people wake up after bowel movements So we know it works The question is how and why and should everyone have 20 bowel movements or could we actually do something better for these patients? So this is not one of those things We have to start through some scratch that this does not work and let's figure out why it does not work now We need to figure out why it does work and how best to use it So the important question is can you develop encephalopathy hepatic encephalopathy in the absence of gut microbiota? The answer at least in germ-free mice is no if you have a germ-free mouse that you actually made cirrhotic and you have a Conventional mouse that you made cirrhotic here. You can see not only are the changes in the inflammation in the cerebellum and the cortex Significantly higher in the mouse that was conventionally cirrhotic, but not in germ-free. In addition. There was also a change in the microglial activation as well as activation of the the The G FAP which is a astroglial marker So it tells you in patient in mice who has cirrhosis in the germ-free condition. They do not have brain inflammation What's important these mice have high ammonia? Why is that as many other people have shown there's a glutaminase enzyme in the small bowel that actually can split Ammonia from the glutamine in the diet and actually this can cause hyper aminemia But that hyper aminemia in the absence of microbes does not result in brain inflammation Moreover mice are very discerning the germ-free mice if you take mice who got FMT from hepatic encephalopathy patients There's a huge Increase in the micro in the brain inflammation and then when you give them post FMT this actually quenches down So it is very important. It's not only the presence of microbiota, but which microbiota are in there so microbiota from a healthy person does not induce brain inflammation microbiota for a patient with the HE and Antibiotics induces a lot of inflammation and when you give the healthy person FMT from a healthy donor and you put that in the mouse that is quenched So it tells you microbiota are quantitatively and qualitatively related to the development of encephalopathy Of course, this cannot be done in humans, but that's the best we have right now What about portal hypertension itself portal hypertension the story is a little more complicated without microbes the portal pressure in this Specific thing because you can't do while bile duct ligation because if you do bile duct ligation You basically switch off the bile to the microbiome to the microbiome which in itself is a huge variation But when you do the partial portal vein ligation, you can study both the the bile duct is still intact But you can actually study this in this nice study done by Dr. Gortardi Gortardi's group the portal pressure was normal in the germ-free condition But when you try to induce it with the port partial portion pressure It did not work and moreover the this things are a little more complicated When you look at the cells called the panet cells These are cells found in the small intestine that produce defensins and can also at least be in lymphangiogenic and angiogenic Which we know are mediators for the development of portal hypertension So what they found is in the absence of microbes the panet cells did not produce the lymphangiocyte a lip and just it were not involved in angiogenesis and lymphanogenesis But when this was actually the microbes were introduced this did so it's kind of a mixed bag. It's not a hundred percent Yes, you need the microbes, but it tells you in the right situation any person any microbes can actually do this And it is reflected in the human studies. I'm not going to go over the details But this is a mixed bag some studies show that it is associated microbiome some studies show No, and this study are done from Latvia Lithuania shows that there is the circulating microbiome measured with the HV PG There is absolutely no correlation at all when you talk about the microbiome itself However, the proof of concept like I told you that that comatose people wake up with lactulose Is there a proof of concept of actually using high-dose antibiotics and the answer is yes But it's not hundred percent whether it's purely a microbial effect like what we see in potentially in hepatic encephalopathy So hepatic encephalopathy there is much more clear-cut Microbial origin of at least association of the symptoms whereas in portal hypertension alone It is not as clear-cut either in animal models or in human experiences So, let's go a little bit further we talked about the potential pathogenesis and the need for having bacteria let's talk about using these microbiota as prognosticators and biosensors so this is the first study that we had done in which we actually did a very crude analysis of patients cognitive function and Stool family and in what we found I'm not going to go over the details But the inflammation was highly correlated with microbiota that are associated with negative outcomes, which is porphyromon adacy We're going to talk a little bit about more about this later These are oral bacteria that are if you remember porphyromonas gingivalis is the number one cause of gingivitis or gum disease in the world And this is one of the most common inflammatory diseases present worldwide that is infectious and you have alkali genesis. These are both These is gram negative and they were associated with poor cognitive function Then you can say okay stool is not something the body keeps on interacting with what about the other stuff? this is the microbiota present in the colonic mucosa, which is clearly interacting with the mouse with the with the body and The cognitive function was very very different in those who had good bacteria of short-chain fatty acid producing bacteria such as lachnospiraceae Ruminococci and their constituents, so it tells you that the brain function at least is related to this But when you take a patient with cirrhosis and hepatic encephalopathy, there are many processes that go on there's inflammatory markers there's in there's inflammatory change and there's hyper aminemia when ammonia is taken up by the brain by the Astrocytes the astrocytes swell up and the myo-nos at all and choline goes down goes goes down and glutamate and glutamine go up So what we found is there are specific bacteria that are associated with protection From hyper aminemia, whereas the other ones are associated with negative connotations So enterobacteriaceae, which is gram-negative rods, which are associated with poor outcomes were associated with hyper aminemia associated changes in the brain Whereas lachnospiraceae and ruminococci, which I just talked about which produce short-chain fatty acids and some parts of which Change the primary to secondary bile acids were associated from protection from the brain being swollen up because of hyper aminemia But what was very interesting is this was the gray mat This was the astrocytic change was the change in the white matter, which we assumed was because of inflammation the oral bacteria Remember porphyromonad AC we were talking about that is porphyromonad gingivalis that was directly correlated Specifically with the brain function of the white matter So your patient comes to you it come the patient comes with the whole things together You have a patient who has neuronal dysfunction and you have a patient who has glial and astroglial dysfunction there are different groups of bacteria that Actually are associated with all of these and if you do not take care of the mouth and the gut and the liver at the Same time the patient will have a problem which will be residual or self-perpetuating So, how can we actually make this so that patients can actually be? These these can actually be used in patients this study shows in in patients with encephalopathy You could predict with bacteria at the time of admission who is going to have a recurrence of then Encephalopathy after discharge and that this study we found that in single and multi-center studies Bacteria were able to tell you who is going to live or die during that admission and that is very scary It's not like these patients are ignored while they're in the hospital But it tells you the time to act on the patient's bacteria Maybe when they're out patients and not wait till they're already in patients because the horse may have left the barn at that time So, how do we operationalize this knowledge for hepatic encephalopathy? One study we did across the Naxal consortium is we took 602 patients who came in without Grade 3 and 4 encephalopathy and we followed them until they developed that and what we found that many serum metabolites including some producing the micro bacteria Microbiota and the thyroxin was able to actually predict who develops encephalopathy and this was validated with a clinical lab at least with thyroxin Developing grade 3 and 4 encephalopathy is a huge thing And especially if you miss it and the patient develops aspiration pneumonia This is a big problem And if the patient continues to be comatose you do not know what their wishes were you do not they're not good transplant candidates so this is a product this is a thing that you want to predict better than what you were doing and The challenging thing is actually not only including who has covert or minimal hepatic encephalopathy Which patients do very badly as far as cognitive tests. This is associated with poor outcomes This is associated poor quality of life. So our patients we want to not only make sure that they are Improving their quantity of life. They're also improving the quality of life the cognitive tests that we do for this are impossible to do in clinic No one even has time to do a one-minute animal naming test So the question is can we operationalize this thing to find out who does not have covert HE? So you can at least say this person does not need anything else and at least the proof of concept across these three Different tests we found that regardless of whether they were whichever test was given there were certain bacteria Remember the same lachnospirases which make short-chain fatty acids were higher in those who were free of HE Whereas in saliva it was the reverse the streptococcus AC which often produced urease that caused the ammonia to go higher was higher in those who had HE So at least a proof of concept we know in humans that this can be used for this What about differentiating about the patient remember that first thing that I talked about alcohol patients can have Whatever caused them to drink in the first place the alcohol related change in the brain itself the addiction physiology itself The cirrhosis itself and finally the cirrhosis related hepatic encephalopathy It's very difficult for us as clinicians with our 15 minutes appointments to peel all of these apart Can microbes guide us and the answer is yes There are different microbes that are highly expressed in people who have predominant addiction behavior Rather than those related to the consequences of cirrhosis because there are two separate cognitive tests that we can do In these patients that separate the two one is the inhibitory control test That is more patients just cannot stop doing what they know is wrong And that is more a dose is associated with addiction behavior Whereas PHES is much it's much more a compendium of visual spatial changes that is associated with the encephalopathy and cirrhosis And they were clear-cut bacterial differences showing that there's proof of concept again with this What about post-traumatic stress disorder? I work in the Veterans Hospital in which Alcohol post-traumatic stress disorder they come kind of as a package deal and we were able to find out that patients with cirrhosis with Post-traumatic stress disorder had a very different microbial profile than those who didn't and last but not the least is the aging population Patients with cirrhosis typically do not have memory disorders memory disorders are much more associated with dementia And as the patients with cirrhosis are growing older the overlap between dementia and encephalopathy is getting closer and closer and closer And you have patients who have both amnestic means amnesia non-amnestic means MHE and Microbiota were able to differentiate who actually had Amnestic disorder and who didn't so again these have gone out of the textbooks potentially not in clinic just yet But going towards the factor that we can knowing the microbiota composition is may actually be helpful in the distant future near future So what are the challenges with gut therapies and microbiota is target for future therapies a lot of this will be touched with my Colleagues that are coming, but I had a sneak peek at their Talks so some of these things are not covered So the current number one treatment is lactulose if you see in this condition Patients with hepatic one episode of encephalopathy who were given lactulose versus placebo They had much more likelihood of not getting an encephalopathy episode, but there's a problem with lactulose like I said multiple bowel movements Dehydration it's cheap as far as a medication concern But it's very expensive as far as human resources are concerned you to spend a lot of time explaining to the patient what to do What not to do and patients suddenly now have to come clean about their bowel movements to people whom they don't know so it's a big problem and I'm not going to go over the details but we actually gave scenarios because all the Indian studies with lactulose was so positive and all the American and the Western studies were so negative that we Gave them an association liquor how much how many bowel movements will you tolerate? Before you get hospitalized and all Americans said we'd rather be hospitalized than have more bowel movements. That's exactly what you're dealing with So this is a problem with our current therapies, but in India it was not the case So it was it's a big cultural change and it's a big cultural difference when you try to have one one therapy across different cultures Rifaximin clearly approves the blood Gut liver brain axis in this study the the driving impairment was improved and this nice study done by dr Shawcross that she'll speak a little bit more about later. There were changes not only in the gut microbiota But in some clinical outcomes, but Rifaximin needs bile to solubilize for optimal bioavailability And what is lacking in patients with cirrhosis is bile reaching the intestine So if the current formulation of Rifaximin needs is so hydrophobic that it needs bile It's not really going to work optimally in the patients who require it the most which are patients with decompensated cirrhosis It is expensive in certain countries It's really expensive in the United States and it's not available in certain countries, which is also a barrier So therefore SSD is the soluble Solid dispersion which is as the name suggests Soluble in water was started and the proof-of-concept studies were done both in inpatients as well as outpatients The outpatient studies were in patients with controlled ascites to prevent the second episode of encephalopathy Encephalopathy or ascites or SBP and this was one formulation Which is 40 mg QD IR was associated with this in inpatients It was more related to patients who are not waking up after 12 hours Then you randomize them to lactose plus these formulations versus lactose alone and the same IR Formulation was associated with a shorter time to waking up. So this is now actually being studied in a larger Concept. So in summary microbial change is linked with HE. It's necessary to develop. It's related to disease severity It is very specific for HE even in a patient who has multiple reasons to have cognitive impairment and it can predict Therapeutic response and the cult are conclusions. The gut alterations are important in the pathogenesis progression and prognostication The evidence regarding portal hypertension is more challenging to interpret Specify several medications for HE directly and indirectly affect the milieu. There is scope for improvement Newer formulation refax min are shown promise likely due to the water solubility and larger trials include has been started Deeper microbial modulation using FMT are shown promise and will be discussed later in this symposium These are my acknowledgements, especially to dr. D got RD and thank you for your time Thank you for that excellent overview and opening to this session next it's my pleasure to introduce dr Shawcross from King's College in London who's going to speak to us today about the use of antibiotics for GI bleeding and ascites complications Worth the risk to the microbiome Thank you very much, I just need to wait for some slides hopefully So thank you jazz and Betsy and and Don and Scott for asking me to speak in this fantastic symposium today And what I've been asked to talk to you about is really the use of antibiotics for gastrointestinal bleeding and ascites complications Is it worth the risk to the gut microbiome? That's The wrong slide deck that's currently Shawcross, please. Yeah, what's that originally? This one, yeah I'll try again. Good, right. Hopefully that Oh How does it go forward is it just okay fine just playing up these are my disclosures as well It's not working You just doing that one, okay Okay, then let's start again and so over the next I was asked to speak for about 18 minutes I might try and make it 15 if I can just in it to keep in the interest of time and lots of conversation Is really I'm going to talk to you about why patients with cirrhosis and portal hypertension is susceptible to infection We're going to talk a lot about bacterial translocation and how that's a driver of cirrhosis associated immune dysfunction and then why infection and inflammation are such important precipitants of Decompensation in patients with cirrhosis and the development of a CLF. So a little bit of overlap there and I'm going to talk about focus particularly on variceal bleeding and ascites Whereas jazz is obviously just talked about He and then I'm just going to follow that by just talking a little bit now then about the impact that antibiotics actually have It's good and bad There's huge concerns that antibiotics themselves worse than dysbiosis and we're going to talk a little bit about antimicrobial resistance just to finish and So just just just to start off with really if you focus on the panel on the left hand side, this is hopefully all of us in the room, a healthy individual, and we have a robust healthy gut barrier. And this gut barrier actually prevents bacteria and bacterial degradation products translocating across the gut into the portal vein into the liver. And even if things do pass across, and actually even in normal health you do get some translocation, it's rapidly dealt with by the liver with very little fallout. In our patients with cirrhosis on the right hand side, and particularly once you develop portal hypertension, you get significant defects in the gut barrier and you get the gut dysbiosis, which Jazz has very eloquently talked about. You get translocation of bacteria and bacterial degradation products across the gut wall into the liver, and then the liver's full of macrophages, Kupfer cells, of which actually carry a receptor for toll-like receptor 4, which is a receptor for endotoxin. And they detect all of the endotoxin passing across and initiate a really robust inflammatory reaction where you get a localized hepatitis within the liver, but then you get infiltration of monocytes and neutrophils which kick out all sorts of pro-inflammatory cytokines, reactive oxygen species, and so on. And that initiates a pro-inflammatory response which activates all of your immune cells. And Jazz has already showed you this slide, but I really wanted just to suggest that this is really, really probably the most important part that actually initiates cirrhosis-associated immune dysfunction. And then the fact that our cirrhotic patients then have reduced protein synthesis, they make less complement, they make less acute phase proteins and so on, they're sarcopenic and so on, they're malnourished, that really impacts on the cirrhosis-associated immune dysfunction and the cells eventually become exhausted and can't fight infection. Jazz also mentioned the gut barrier, but I also wanted to really reinforce that not only do we get translocation because we get leakiness in the gut, we get reduced mucus at the gut barrier and also reduced IgA secretion as well. These all contribute. And this finally culminates really in this pro-inflammatory response that leads or predisposes our patients with cirrhosis to decompensation, to tissue injury, to acute on chronic liver failure, and worse still really. And there's a very nice study that's been published in Europe from the PREDICT study, this enrolled 1,273 patients, and really what this showed is that in patients with acute decompensation and ACLF, infection is still the most important driver of the development of decompensation and the development of acute on chronic liver failure, and that's any kind of decompensating event, it's not one specific one. And what's really important to look at is that in patients with compensated cirrhosis versus acute decompensation versus ACLF, you get this kind of stepwise progression in a rise in inflammatory markers. So you get an increase in white cell count, you get an increase in CRP, you get an increase in any pro-inflammatory marker here, we've got TNF-alpha and interleukin A. So these are all really important events. And what that does is that actually increases your likelihood of developing infection, both bacterial and fungal, over the following 28 days. And this is a study from Javier Fernandez, another easel-based study, which basically shows that patients with acute decompensation over the following 28 days have about a 20% risk of developing an infection. If you've got ACLF-1 it's about 40%, if you've got ACLF-3 it's about 80%. So this really is something we need to take seriously. And the probability of 90-day transplant-free survival in these patients when they have an infection is really poor compared to when they don't have an infection, and this is some data showing patients with acute decompensation are obviously less vulnerable than patients with ACLF. This is quite an old study but it's a beautiful study. This is a study from Sheila Sherlock's old unit at the Royal Free in London that was led by Andy Burrows, and really this looked at the role of bacterial infection in patients presenting with acute variceal bleeding. And one of the things that they were able to show in this study is that they looked at a group of patients who had controlled variceal bleeding and they looked at a group who had uncontrolled variceal bleeding, and it was very clear in this study, I'm not sure it necessarily shows here, that the patients who had uncontrolled variceal bleeding had acute bacterial infections often that were poorly controlled despite antibiotic prophylaxis. So if there's anything that we've learned from this is that bacterial infection is a huge driver for re-bleeding. And if we actually fast-forward the clock on now 25 years, this is another study where actually we are routinely giving patients antimicrobial prophylaxis when they present with acute variceal bleed, we still don't have a huge amount of impact. And in this study, bacterial infections still occur after the patient presents with a variceal bleed in about 20% of patients despite antibiotic prophylaxis. And the majority of these patients get respiratory bacterial infections, and that's possibly not surprising because they are having airway interventions and manipulations and aspiration pneumonia is very high in this group. But the other thing that's quite scary is that about 50% of these patients have a multi-drug resistant organisms isolated as well, particularly in this study in third generation Kevlar sporins. So this is showing us that there's good and there's bad. So we have this kind of trade-off really between giving antibiotics to reduce infections and save lives versus actually the risk of inducing multi-drug resistant organisms. And I think this study here that's very recently been published just a year ago actually sort of shows that beautifully. And this was in a parcel study where they looked at giving antimicrobial prophylaxis to patients presenting with ACLF. And they randomized patients to either norfloxacin or placebo. And what they were able to show is that at day 30 and at day 90 those patients randomized to norfloxacin had markedly reduced infection rates here. It didn't actually equate to transplant free survival ultimately, but certainly there reduced infection rates. But scarily once again lots of multi-drug resistant organisms and in the patients treated with norfloxacin they also developed candiduria. So we may be predisposing patients to getting fungal infections as well as bacterial. And I think we're becoming aware now that actually multi-drug resistant bacterial infections are a huge problem in cirrhosis. This is data once again from Javier Fernandez from Europe that actually looks at two huge patient populations. It looks at the canonic study which was over a thousand patients and it looked at a second series of patients with more than 800 patients from Eastern and Southern and Western Europe. And what they showed is that the prevalence of multi-drug resistant organisms arose from 29% to 38% between 2011 and 2017. And actually the incidence of multi-drug resistant organisms in the decompensated patients in my hospital is 35% currently and it's going up. It's frightening. And just to give you another example, this again is a very recently published study which has looked at rectal colonisation of resistant bacteria. Again two European cohorts, Barcelona and Frankfurt, and they basically show that if you can, if with a rectal swab you can show colonisation of a particular multi-drug resistant strain then that's likely to lead to subsequent infection with that strain. Interestingly in the Barcelona cohort they had lots of ESBL, in the Frankfurt one they had lots of VRE. So this is all really scary stuff. And you know WHO have really become aware of this as well and this is more of a public health slide than anything else but they're predicting that there will be 10 million deaths, excess deaths, by 2050 due to antimicrobial resistance costing the world about 66 trillion pounds if we don't develop further antibiotics or do something about it. And this is another Lancet review which shows that drug resistant infections are now the leading cause of deaths worldwide. I mean I think COVID probably did try to compete with this subsequently but you know it kills more people than HIV and AIDS and breast cancer and malaria. So Jazz has already talked about gut dysbiosis being really really important in encephalopathy and I'm just going to sort of carry this theme on forwards but also to suggest to you that actually antibiotics also worsen dysbiosis. And this is some of my own data but they're on many many published studies showing very very similar data sets that actually store microbial diversity is reduced in patients with cirrhosis and you get a reduction in things like Firmicutes in the green here and an increase in things like bacteroidetes. And this really has importance in things that we're talking about. Now this is this is not a criticism of Jazz's study but actually the study that Jazz first did looking at FMT in 20 patients with cirrhosis, a very seminal study, actually was groundbreaking because it showed impact on encephalopathy but it also beautifully showed us what antibiotics do to your gut microbiome. And in this study patients were treated with amoxicillin and ciprofloxacin and metronidazole prior to being given FMT and you can see that that dysbiosis worsens before it gets better after the FMT. And I think there's a lot of learning here that we need to take on board. And actually this is a study of mine which is we're just in the process of getting published now which again was another FMT study and I'm not talking about FMT specifically but one of the things that we noticed is in the patients where FMT didn't work is because we gave them antibiotics about seven to thirty days later and it takes away all the good we've done with the FMT. So antibiotics are good as well as bad. And really that's the problem I think we really have to think about now is this there's this challenge between addressing the susceptibility susceptibility that patients with cirrhosis have to infection and the increased risk of dying versus antimicrobial resistance. And I put this little cartoon together if you imagine this chap in the middle is your patient with cirrhosis and portal hypertension it's difficult to know which way he goes. It's bad news both ways and this is going to be a really difficult thing for us to navigate through. So one of the things that people have suggested is maybe this is just systemic antibiotics maybe if we use non-absorbable antibiotics maybe this might be a better thing. And one of the things that that people have been looking at is whether rifaximin for example will have an impact on outcomes in patients with cirrhosis. And this is a retrospective study from my unit at King's and it is retrospective it's not prospective but we looked at patients listed for transplantation and looked at patients who were on rifaximin and the outcomes of patients on rifaximin versus the patients who weren't. And on multivariate analysis there were significant reductions in variceal hemorrhage, SBP and actually all cause admissions in patients on rifaximin to compare to those. It also reduced when people were hospitalized it reduced their mean length of stay and actually increased the time to being re-hospitalized. So there may be something there and in fact there are multiple studies now systematic reviews and meta-analyses that have looked at the impact of rifaximin on the incidence of SBP. I've just picked one in the interest of time that suggests that there is a huge benefit of rifaximin in reducing SBP. So Jaz mentioned the RIFSYS trial and one of the things that we wanted to look at when we undertook the RIFSYS trial is that we were interested to understand mechanisms. Mechanisms of how it affects the gut microbiome but mechanisms also about how it affects the systemic immune system and immune functioning. And one of the things that we noticed in this double blind randomized placebo controlled trial is that patients who were randomized to rifaximin but not to placebo actually were less likely to develop infection over just 90 days. So clearly there is an impact there. Interestingly also there is a significant reduction in pro-inflammatory markers in the blood stream and here is an example of TNF-alpha the one that I've talked about a lot today. But also when you look at the expression of tolite receptor 4 on immune cells and in this case neutrophils you can see that in the rifaximin treated patients there is a reduction in tolite receptor 4 expression suggesting that there is less gut derived systemic driver for inflammation. And one of the other things that we wanted to do in this study was also to see how that reduction in systemic inflammation relates to the gut microbiome. And one of the things that, as Jaz mentioned, patients with cirrhosis have is they have bugs that you normally find in your mouth and your buccal mucosa translocating down into your gut and that's also more prevalent in patients also who are on proton pump inhibitors. And some of those bacteria things like streptococci and vionella actually have a silodase tail here and this silodase tail actually breaks down gut mucus. So if those bacteria are in the wrong place, it's beneficial in the mouth, it helps you to digest your food, if it happens in your gut that actually breaks down your gut barrier and that's a bad thing. So rifaximin may have a benefit there in bolstering the gut barrier but does it actually have any impact on portal hypertension? And Jaz also mentioned this study in amongst several others but this is just one that I've picked out just because it was randomized and double-blinded placebo control. This was a NENA chemo study that looked at a small number of patients that were randomized to rifaximin or placebo and they didn't see any impact in portal pressure changes with patients on rifaximin or changes in cardiac output or GFR. So I guess I've shown you some data that suggests rifaximin could be helpful here in preventing infections in patients with cirrhosis but is this also going to cause antimicrobial resistance? I guess we don't really know. This is some of Jaz's group's recently published data looking at antibiotic resistance genes in sequencing data and although it only included a small number of patients treated with rifaximin, just 19, they didn't really see much in an increase in resistance genes but I think we need to do bigger studies and I think this will be important that we all think about doing that. So this is my last slide and really what I just wanted to summarize here is that patients with cirrhosis and portal hypertension, as Jaz alluded to, have gut dysbiosis. They have a lot of things going wrong at the gut barrier leading to intestinal inflammation and translocation of bacteria into the portal vein and into the liver. Interestingly intestinal inflammation may also be a driver for antimicrobial resistance and I think what we need to do is all of us think about targeting the gut and the liver axis with therapies that might reduce that intestinal inflammation and that translocation and paradoxically it might not be antibiotics that might be the right thing to do. It might be that we need to think about antimicrobial therapies that kind of bolster that gut barrier and reduce the inflammation and I hope that's going to give a great introduction now to Patty. So that's where I'm going to end so thank you very much everyone for listening. Thank you Dr. Shawcross. Our next speaker, already spoiler alert given by Dr. Shawcross, is Dr. Patricia Bloom from University of Michigan who's going to talk to us about fecal microbiota transplant for cirrhosis and portal hypertension. Is it ready for prime time? Okay so it's the left click. Thank you. So thank you so much the organizers for inviting me. I'm truly honored to be invited to give this talk today. Here are my disclosures. I'll just note that I have a research grant from Vedanta Biosciences who makes VE303 which is an FMT alternative that I'll talk about later. So is fecal microbiota transplant or FMT ready for prime time in cirrhosis and portal hypertension? I hope no one has an epilepsy condition. We have a little bit of a flicker here. The short answer is no but I'll spend the next 18 minutes explaining why and hopefully sharing a little optimism about the future of FMT. So I'd like to set the stage for this talk with a framework. Oh pause. The wheel. Okay apparently I'm not supposed to keep clicking. Okay so here's a framework for the talk. Let's think about what are the criteria that we need to have to know that a drug is safe and effective to give our patients. We need to know that there is a clear and clinically meaningful indication for the therapy. We need to have data from large randomized trials and the FDA often asks for not one but two phase three trials. We need to have sufficient data that there is efficacy that outweighs potential safety risks. We need to understand the pharmacokinetics of that drug and make sure that those pharmacokinetics are acceptable. And so I'll walk through each of these as it relates to FMT in cirrhosis. So the first criteria is we need to have a clear and clinically meaningful indication and if we prove that FMT is able to do any of these things I have on the screen it will be well on its way for approval. These are clear and clinically meaningful endpoints for the drug. As Jazz and Debbie already eloquently reviewed we know that there's an altered gastrointestinal microbiota composition in cirrhosis but this can't be the primary outcome for FMT. It's likely on the pathway to relevant clinical outcomes but it cannot be the primary indication. Number two we need more large clinical trials and I'm just here comparing our data on FMT to other approved drugs in hepatology. There have been five small clinical trials using FMT for different indications in cirrhosis and as you can see here the number of patients with cirrhosis who've received FMT is far smaller than for example the 228 patients that received rifaximin in clinical trials for approval and pales in comparison to the number of patients who received GP to cure hepatitis C virus. So my point here is there's a very good reason for enthusiasm for FMT in cirrhosis but we are still early in our experience currently. So let's talk about the evidence for efficacy and there is some promising evidence for efficacy of using FMT for multiple conditions within cirrhosis. So there have been three trials thus far looking at if FMT improves cognitive outcomes in patients with a history of overt encephalopathy and this figure I've created here is a composite of the two trials that used oral FMT capsules and there was an overall improvement of 2.8 psychometric HE score points after FMT and 2.8 may seem like a small number but that's a very clinically meaningful improvement for the FEZ score. And then also FMT has been trialed in a group of patients with alcohol-related cirrhosis and FMT was found to decrease cravings for alcohol and even decrease biomarkers of alcohol use in the urine. So even though these are small studies there's a reason FMT has been given a talk at the at the liver meeting there is some reason for for enthusiasm and some early signs of clinical efficacy. And thankfully more data is on the way. In the coming years hundreds of patients with cirrhosis will be receiving FMT. There are five trials in different countries being conducted these are all placebo controlled trials they are administering FMT through different routes it's typically oral FMT capsules but there is one study using nasal jejunal administration and another with enemas and each of these trials has a different subgroup of patients within cirrhosis it's targeting a different primary endpoint and then also which may be obvious because these studies are being conducted in different countries they're using different FMT donors. So in the coming years we're going to have a lot more data about the efficacy and safety of FMT and maybe be able to answer some more nuanced questions like which FMT donor is best and what is the best administration strategy. I'll be brief here this is my one mechanism slide just because Jas and Debbie covered this so in so much detail but FMT what it's doing is it's delivering a package of potentially beneficial bacteria into the gut of a recipient and those bacteria can do many beneficial functions they can ferment fibers to produce short-chain fatty acids they can metabolize bile acids to make secondary bile acids they can increase tight junction proteins and all of those mechanisms combined may really improve gut barrier function and help correct this leaky gut we hear so much about in cirrhosis. Those beneficial bacteria may also nudge potentially harmful bacteria out of that ecosystem and as such decrease endotoxin production and perhaps ammonia production as well. So the the sum total of these different mechanisms could be, number one, improving gut barrier function, number two, decreasing systemic inflammation, and then number three, decreasing neuroinflammation in our patients with cirrhosis and portal hypertension. And I just want to make a little note here that C. difficile is not the same thing as cirrhosis, which may be obvious to the group. But I bring up C. difficile just because that's obviously the classic condition that we're using FMT for. But what's happening in FMT for C. diff is that FMT is establishing colonization resistance to a very specific bacteria, toxigenic C. difficile, which is not what's happening in cirrhosis. In cirrhosis, we don't have this singular pathogen that we're using FMT to establish resistance to. So we can't conflate these two indications. Now, moving on to another criteria for making FMT prime time, let's talk about safety. At this point, many thousands of patients have received FMT for C. difficile. And time and time again, we see very encouraging safety data. And I think largely, experts agree that FMT really and truly is overall safe. There have been a few cases, notable cases, of severe adverse events. My group published these two cases of extended spectrum beta-lactamase E. coli transmitted via FMT to two patients, one of whom had cirrhosis. And I want to just clarify, too, that this was not some kind of back alley, do-it-yourself FMT, if such a thing exists. This was an academic stool biobank following detailed FDA protocols for screening their donors. And subsequently, recently, OpenBiome has published another series of cases where shiga toxin-producing E. coli was transmitted, again, despite really extensive donor screening. And now, living in the COVID pandemic, we know that SARS-CoV-2, as well as monkeypox, can be potentially transmitted through stool. So there will always be unknown pathogens, and also pathogens that are challenging to test for. Many of the stool tests we currently use have low sensitivity for pathogens at low abundance in stool. And so this risk will always be there, even though overall, we are seeing that FMT is safe. And FMT has been used for conditions outside of gastrointestinal and infectious indications. It's been used for all of these domains that you see here on the screen. So we just need to keep in mind that we should be looking for potential off-target beneficial, as well as adverse events in each of these areas as we administer FMT to patients with cirrhosis. There are unique pharmacology challenges associated with FMT, which, of course, then translate to regulatory challenges. The first is that it is dependent on a human donor. This means that every batch will be different. It also means that the supply chain is dependent on human behavior. If your donor goes on vacation internationally, or eats some new food, or gets a UTI, or gets a UTI and then uses antibiotics. Number two, the active ingredient in FMT for cirrhosis is unknown. And so we can't be entirely sure that the active ingredient for our patients is the same with each dosing of FMT. I've already talked about how pathogen testing is complicated. We also don't know exactly what the best preparation of FMT is for cirrhosis. Fresh or frozen, anaerobically prepared versus aerobically prepared. If we lose a lot of the anaerobes in preparation, we may be potentially losing some of the beneficial microbes. We need to ensure that the bacteria actually get from the donor through the processing, through the storage, through the recipient's stomach, and into the intestine, which is a unique challenge of FMT. And we need to keep in mind that it's not just the bacteria we are transmitting with FMT, right? It's all the metabolic products and immune products that come from those bacteria. And finally, there are many peritreatment factors that likely modulate the effect of FMT. So with all of these unique aspects to the pharmacology of FMT, it has been a challenging regulatory environment. Now, the FDA has been allowing providers to prescribe FMT even though it is still investigational. It is not currently approved. And they are allowed to do so under enforcement discretion. And so this means that a provider can consent the patient to have FMT for C. difficile, and they just need to be aware that it's an investigational product and that there are risks associated with it. And there's no current sign that the FDA is about to approve FMT the way we currently conceive of it through stool biobanks. However, I will just note that an FMT similar product made by rebiotics may be nearing approval. And I'll talk about that a little later. The European Union has the same challenges with regulating this. And they just put out a document in June basically describing the vast heterogeneity between European Union states and how they regulate and make FMT, which may be leading to heterogeneity and efficacy and safety. So now I'll move into a couple open interesting questions about FMT and cirrhosis in my last few minutes here. The first question is, which FMT donor is best? If we know what the magic sauce, the special active ingredient is that is in FMT that works for cirrhosis, perhaps we could choose donors that are super pooper, super donors who are enriched for those particular microbial functions. This is a real book, by the way, that you can buy. This is a study looking at 2,000 patients with C. difficile who obtained FMT from 28 different donors. And what you can see here is that there actually was not a lot of variability in the clinical efficacy of FMT between donors. So we really don't believe there is a donor effect in C. difficile. However, there is some evidence of a donor effect in non-C. difficile conditions. So this was a trial of FMT and ulcerative colitis, and they found that donor B, whoever that was, seemed to have a higher clinical response rate than other donors. But here's the catch. How many people do you need to enroll in your trial to adequately assess for a donor effect? It's at least 100. So we currently don't have that data available for cirrhosis but with the upcoming trials, perhaps we'll get more data on which donor is best. And Jazz did a really logical thing in some of his studies of FMT, which is we know that patients with hepatic encephalopathy, they have low abundance of lacnospiraceae and ruminococciaceae. And these are two families that tend to have high short-chain fatty acid production, which we've already discussed has a good impact on barrier function. And so what he did is he selected for a donor that was rich in these families and then used that in his FMT studies, and there was a positive clinical effect, as we already discussed. Interestingly, the lacnospiraceae actually didn't significantly engraft in the recipient. So there wasn't actually a significant increase in lacnospiraceae, but there was still a clinical effect. So interesting that it doesn't always, if you enrich a donor for something, it doesn't always mean that it engrafts in the recipient. So we've talked now about donor effect. What about a recipient effect? Are there certain recipients who do better with FMT? And this has been seen in C. difficile as well as non-C. difficile indications. And we have to keep in mind, this FMT is not going into a void, okay? There's an existing microbiota ecosystem in the recipient that the FMT is entering into. And it's quite possible, including in cirrhosis, that there are certain people who are going to have better efficacy with FMT than others. My group recently showed that there are different enterotypes in cirrhosis. So enterotype just means a cohesive gut microbial profile. So we took duodenal mucosa and found that there were two different clusters or two different enterotypes of patients with cirrhosis. So I have no data to support what I'm about to say, but it'd be an interesting thing to look at. I wonder if there's an enterotype effect with FMT. So in other words, patients who are in cluster one, if they have a better outcome with FMT, for example, than patients in cluster two. And it's not just FMT that is out there now. As you go down this figure, you can narrow the composition of FMT, remove certain elements, and keep others. And as you narrow the composition, you might be enhancing safety and consistency and become more targeted. There's a trade-off, though. You may also be losing efficacy, especially if you're getting rid of whatever the active ingredient is for FMT. And here are some of the other microbiome therapeutics that are being tried in C. difficile. And I'll just highlight RBX2660. This was recently recommended by an FDA advisory panel for approval. You may have seen that in the news. So it isn't fully approved yet, but it's been recommended for approval. And this product is fairly similar to FMT. It's derived from a healthy donor, and they use good manufacturing practices to make it a fairly consistent product. But it is still based on humans. And each of these products has had good safety and efficacy data. As you go down the table, you get more and more refined. And VE303 is actually a product that is not derived from humans. It's clonally produced. And I'll just note here, again, I have a research grant from Vedanta Biosciences that makes VE303. And we are trying that in patients with cirrhosis and hepatic encephalopathy in a trial. So my last couple of thoughts here, suggestions for future research. I think we should perhaps start considering pre-enrollment microbiome analysis, and either restricting enrollment or stratifying enrollment by the baseline microbiota characteristics. And we might still need more data yet as to figure out what those characteristics will be, will stratify by. We should also be assessing for strain engraftment so that we have a hope of figuring out which strains are associated with the clinical outcomes we're looking for. While there is a lot of enthusiasm and should be for FMT, we should also be considering these other more defined consortium products as it might have a more likely pathway towards regulatory approval. And then also rigorous safety monitoring, even outside those infectious and GI adverse events. So I'll just wonder out loud here, I wonder if this is going to be another example of personalized medicine, now personalized microbiome medicine, where perhaps we can isolate the specific microbiome problem a patient with cirrhosis and portal hypertension has, and then treat that problem. So give them whatever their microbiome is missing. We're still a ways off from that. So to conclude, there is a lot of reason for enthusiasm around FMT in cirrhosis and portal hypertension in some small trials. It has improved cognitive outcomes, decreased alcohol use. But there remains some weaknesses. There's a concern about infections and unknown contents of FMT. There's some challenges as we keep adding more testing to FMT. It becomes more costly and higher burden. And in the future, we'll think more about which donors are best, which recipients are best to have FMT, the best mode and method of delivery also. So thank you for your attention. Thank you for that excellent talk. And we're now gonna move on to Bern Schnabel from University of California in San Diego, who's gonna talk to us about non-FMT microbial-based therapy for cirrhosis and portal hypertension. Thank you so much for the invitation and the SIG for inviting me. Okay, these are my disclosures. I wanna highlight that I will talk about a research collaboration with Prodigy, and I'm the founder of Enterica Bio, which is pursuing clinical trials with phages. So if we wanna look at the microbiome-centered therapies that we can use for patients with liver disease, we can essentially separate the therapies into two big pockets. One is the untargeted therapies, and then we have the targeted therapies. For the untargeted therapies, we had already heard about a lot with FMT from Patty. We have also heard about the antibiotics from Debbie, and then we also have probiotics. I'm not gonna focus on these untargeted therapies today. What I wanna highlight is more these targeted therapies, and I wanna show you how we can very precisely now target bacteria, and especially start editing the gut microbiota. However, if you wanna really do a targeted, very precise editing of the gut microbiota, we obviously need to have pathogens and pathobionts as target present and available. And I wanna show you, I will focus today firstly, mostly on alcoholic hepatitis. My alcoholic hepatitis will arise in most of the patients with an underlying liver cirrhosis, has a very high mortality, no effective therapy. And we know, and Debbie already pointed this out, that in patients with alcoholic hepatitis and also cirrhosis, this is just one example, unpublished study from us, but there are many studies now out that have shown very similar findings, that in these patients with alcoholic hepatitis and cirrhosis you have now this overproduction or overrepresentation of virulence factors in the gut microbiome as compared to patients who have less liver disease or not advanced liver disease or controls. You have a whole variety of bacteria that are encoding these virulence factors, and you can look that about 2 3rds of all these patients in this cohort have, with alcoholic hepatitis, actually have virulence factors in their gut microbiome. So you can describe this more or less, during homeostasis you have with a person, healthy person with no liver disease, you have a very nice gut ecology. And then once you have this dysregulated microbiota, and that's what we currently consider in the cirrhotic patients, as we have heard before, you have pathobionts that have virulence factors inside, where they can actually also allow or secrete these virulence factors to the environment. And what I want to focus on in the next is one specific virulence factor that we have discovered several years ago, and based on this virulence factor I want to highlight like three different targeted therapies. This virulence factor is called cytolysin, which is coming from enterococcus faecalis specifically. It's a secreted product, and you can see that about 30% of the patients with alcoholic hepatitis are cytolysin positive in the stool. If a patient is cytolysin positive, there is a very high chance that these patients will die, and we have also confirmed this now in an independent cohort that is currently still unpublished. What is cytolysin doing? Cytolysin is a toxin, and in this case we isolated primary mouse hepatocytes and just put the recombinant cytolysin on these hepatocytes, and you can see you have a very nice dose-dependent effect where the cytolysin kills these primary mouse hepatocytes. You get at one point the saturation in this killing, and you can also appreciate on the right side that this toxic effect is happening very rapidly, so within two hours you get the maximum effect of cytotoxicity, and I'm just highlighting or just summarizing what we currently believe about this, what the cytolysin is doing. In these patients with alcoholic hepatitis, we have this increase in the cytolytic enterococcus faecalis in the intestine that can translocate, as we have heard, via the portal vein to the liver. Here they can either bind to pathogen recognition receptors to exert and induce inflammation, but they can also now directly kill the hepatocytes. So what therapeutic strategies can we now use? And I'm highlighting you three strategies, and in the following I want to explain these in a little bit more details, how we can specifically target now these bacteria or their secreted products. The first is we can use phages, and I brought a small short movie and hopefully with an audio. Can you, should I start this, or? Brace structure consists of a head, a sheath, and tail fibers. The tail fibers are what mediate attachment to the bacterial cell. The DNA stored in the head will then travel down the sheath and be injected inside the cell. Once inside the cell, the phage will hijack the cellular machinery to make many copies of itself. Lastly, the newly assembled phages burst forth from the bacterium, which resets their phage life cycle and kills the bacteria in the process. And this is the most important principle of phages. They can recognize very specific bacteria. They replicate inside, then they release the progeny to the outside one, and the bacterium is killed. And the progeny, again, they can replicate, or they can infect new bacteria again. They can replicate inside, and you have this very nice perpetuating system, ongoing system, as long as you have the pathobion or the pathogen around. Once the pathogen or the bacteria are gone, the phage will also be, the phages are also gone. So we have done this in a preclinical model. I'm just showing you also just one slide. You have germ-free mice. We colonized them with stool from patients with alcoholic hepatitis who were acetyllysine positive. And then we used a phage cocktail against a bacterium that is not present in mouse or humans. And then we used our specific Enterococcus faecalis cocktail phages that now specifically target the cytolytic Enterococcus faecalis. And then we subjected these mice to chronic alcohol feeding. And you can see here that in the mice that were treated with this specific Enterococcus faecalis phage cocktail, you get an improvement in liver disease as compared to the control phage-treated mice. You see an improvement in liver steatosis. But most importantly, we can also now decrease the cytolysin in the liver of these mice. So this is one, and this is also just a recent published study from us, which also highlights that once we give these phages to the mice orally, these phages actually do not only stay in the intestine, but we can also detect them in the systemic circulation. As you can see here, obviously, the most highest abundance of these phages, when you give them by oral gavage to these mice, are in the feces, but we can also find them in the liver spleen and the serum. And this is very important also for our cirrhotic patients because they have these translocated pathobionts. So potentially, these phages, after oral gavage or after oral administration, can also target now these translocated systemic pathobionts. The most interesting thing in this, in our mouse model, is after 24 hours, these phages are gone from the systemic circulation, so meaning that these pathobionts have been probably cleared from the mouse. There are still many clinical challenges out with phage therapy, and we can also discuss this and address this more in the Q&A session. Obviously, is phage-based therapy safe for clinical practice? All what we know right now, yes, even if we give it intravenously. Can it replace antibiotic treatment? If not, when to choose which option, or both simultaneously? We do not know yet. How to decide the best administration route and the dose for each phage therapy, orally via IV administration? Which is better, a single phage or a phage cocktail? And what we also do not know, what are the long-term effects of phage-based therapy on the overall human microbiota, and in general, on the health of the patients? So this is one targeted therapy that I wanted to mention to you, and show you how we can now precisely edit the gut microbiome and target the gut microbiome with bacteriophages. In the next example, I wanna show you another concept, again, how we can use decoy receptors, again, to target these enterococcus faecalis that secrete the cytolysin. And in this, I just wanna introduce you a little bit to the decoy receptor. We have chosen a receptor that is called complement receptor of immunoglobulin superfamily CRIG. In the new nomenclature, it's also called VSIG4. And by RNA-seq analysis, this is a secondary analysis, we have also shown that in these patients, even in early stage of alcoholic liver disease, but then mostly in the patients with alcoholic hepatitis, CRIG in the liver is downregulated. CRIG is predominantly expressed or exclusively expressed on the macrophages and the Kupfer cells in the liver of these, in our liver or in patients with alcoholic hepatitis. And you can see this dramatic downregulation of CRIG on these Kupfer cells in patients with alcoholic hepatitis and underlying cirrhosis. So what is CRIG doing? This is also recently published, and I'm also just giving you a summary. So what we believe, and this is in a healthy state, enterococcus faecalis, you have a very low abundance in a regular human being who does not have any chronic disease. If there is a translocation, these gram-positive bacteria would bind now to CRIG on the Kupfer cells, and these are phagocytosis receptors that are specifically recognizing gram-positive bacteria. So they would catch these bacteria once they are translocated. They are endocytosing them into the lysosome. These bacteria, pathogens, or pathobionts, are now killed. And this is a classical concept that has been brought forward by Andrew McPherson. Initially he said the liver is essentially a second fireball. So once the gut barrier is broken, you still have your Kupfer cells in the liver, and they can now rescue the body from this translocating bacteria. Now what happens during disease, what we currently believe in, as I've shown you before, enterococcus faecalis is now increased. They can translocate now, and under these conditions that I have shown you, alcoholic hepatitis, you have a dramatic downregulation of this CRIG, of this phagocytosis receptor in the liver. Less of this bacteria can now be phagocytosed. More can stick around in the liver. They can, first of all, increase inflammation by binding to pathogen recognition receptors, but they can also now be released into the systemic circulation. So what we have done, we have used this CRIG, and this is showing you the structure from the CRIG. You have an extracellular domain, and this binds the gram-positive bacteria, and then you have the intracellular transmembrane that anchors it into the cell membrane from Kupfer cells. So we have engineered, in collaboration with Genentech, this soluble extracellular domain, which we also call CRIG-IG. And this is a soluble protein, so you can essentially give it intravenously to this, at least we have given it to mice, not to patients yet. And we also show, by fluorescently labeling this CRIG-IG, that it specifically binds gram-positive bacteria. So we have incubated this with the enterococcus faecalis, and by FACS analysis, you can see that these gram-positive bacteria bind to the CRIG-IG by shifting the fluorescence to the right, while this is not the case with the E. coli or gram-negative bacteria. So we have used this now in an animal model, and we have again used an alcohol feeding model, and you can very nicely see that we have with treating these mice with an intravenous application of this CRIG-IG, as compared with the control-IG, you get a significant improvement in liver injury, liver steatosis, and liver inflammation. So this gives you a second example how we can now design a molecule targeting very specifically gram-positive bacteria, pathobionts, which we can now use for editing, in this case, the systemic microbiota rather than the gut microbiota. And in my last example, I want to just show you what we have recently done, which is still unpublished, where we have used, in fact, antimicrobial antibodies to target not only the bacteria themselves, but also their secreted products. The D. coli receptors and the phages would only target the entire bacterium. In this case, now we can target the cytolysin that is secreted. And what we have done, we have immunized now chickens with the antigen. We used either cytolysin or enterococcus faecalis alone or in combination. And then the chicken IgY, so these antibodies, they can be found in the yolk from the egg in a very high concentration. And this has a very dramatic advantage. First of all, the chickens stay immunized for up to 81 days. They have their daily eggs, and you can extract the IgY. You can purify the IgY from these eggs, or you can also use the egg powder. And it's incredible, you can get up to 1.5 grams of chicken IgY per month. Very cheap, very efficient method to target now pathogens or anything else. And this has been also used in clinical trials, so you can also give it to a patient. And the advantage is here also that they are resistant to the gastric acid. So they are resistant up to a pH of 3.5. And that has shown some effects in human clinical trials. So that's what we have done now. So we have immunized chickens now either with cytolysin or with the cytolysin and the enterococcus faecalis. And this is coming back to our primary mouse C parasites. When we induce cell death with the cytolysin and then we treat them with the control IgY, you can see that we still do not see a difference between not treated and IgY control treated. But now if we use the IgY that is specifically recognizing the cytolysin, you can very nicely see that at least in vitro we can neutralize cytolysin and we can get a decrease in this cytotoxic death from this primary mouse hepatocytes. So you can say, sure that works in vitro, but really show me the data that really works for us in vivo. And I was very skeptical in the beginning, would this really work in our mouse model? And again, the advantage here is that we can give it to the mouse continuously via the liquid diet, which contains the alcohol, and we can just add the IgY to this liquid diet. And that's what we did. And to my surprise and thanks to the persistence of the postdoc, you can see that, just let me walk you through this, we have the ethanol-fed mice, we have the vehicly treated mice, you have the chicken IgY control antibody treated mice, and then you have a combination of enterococcus faecalis plus cytolysin, and then this is the cytolysin IgY alone. So you can very nicely see that we can get a significant decrease in liver injury, but just using this chicken IgY against the cytolysin in this mouse model, we get a decrease in liver steatosis and a decrease in the cytolysin in the liver. So these are my key takeaways. Cirrhosis, hopefully we have all been able to convince you that it's associated with changes in the gut microbiota. Intestinal dysbiosis, I think, represents a very attractive target for therapy, and hopefully I have been able to convince you that targeted therapeutics are now also can be used, at least in preclinical models, and hopefully we can translate them into clinical trials. Thank you for your attention. Great. Thanks, everybody, for your attention. And we're now going to take a few questions. If people want to come up to the microphone, please introduce yourself and then articulate your question. Thanks. Don Rocky. I'm from MUSC. So thanks to all the speakers for great talks. So you all have convinced me that there is dysbiosis with cirrhosis, no question. My question is, is it a cause or effect? I guess, Bernd, the chicken or the egg question. So, and Bernd, I think you suggested that it may be a cause, right? So you have abnormal bacteria and E. faecalis, whatever, and that causes liver injury. But my question is, could it be that cirrhosis itself occurs first and then that somehow leads to the dysbiosis? So I'm just curious. What are all of y'all's thoughts about this? Don, thank you for the question. I mean, this is a very important question that you're asking. I just answer it and then I give it to the other panel. Specifically for this enterococcus faecalis at the cytolysin positive, we did not find them in patients with preclinical disease or pre-cirrhotic disease, not preclinical disease. So in patients with alcohol abuse, and let's say it's just early stages of liver fibrosis, we did not see this. So I think this is definitely something which eventually this bug finds the niche in a cirrhotic patient. We just have not identified how is the niche defined. Is it because we have less bile acids or is it like some other factors that we do not understand yet? So to answer your question regarding putting human feces or feces from mice that have cirrhosis into germ-free mice or something like that, that in itself does not cause liver fibrosis. It causes liver injury because these mice have not been associated with any bugs before. However, if you have, say suppose, a mouse from a person who is much more advanced in their liver disease and then expose them to the liver disease inducing agent, these microbiota will then induce a much worse liver disease. So they are complicit in my mind. They're not causative. You certainly want to reduce it, but as I said, this is not the be-all and end-all of liver disease. The liver itself damage also has to be fixed with the microbiota. And I think this is, to me, the more and more I learn about this, the more I realize that part one that I know nothing and part two that it is actually, it's not enough for us to just fix the microbiome and hope that the liver will get better. I don't know what my other colleagues think. That's an excellent question, isn't it? And I don't think anyone's really shown the answer. I always used to think it was cause and I think actually the only way we can really do this is to do epidemiological studies where we look at people who are normal and look at people who develop liver disease down the line and see how the microbiome changes. But one thing that I have learned from the FMT studies that I've done is that FMT will reverse dysbiosis, but it only lasts for about 90 days before it wears off and you have to retreat people. So the question then is, is that the liver disease driving the change or is it actually something the patient's doing in terms of their diet, which is something that Jazz raised as well. Many patients with liver disease have really bad diets as well. So it's difficult to know and I think we probably need to do some epidemiological studies. Agree. To be brief, I think it's both the chicken and the egg. Thank you guys again. I'm Dr. Akash from India. I was just asking to take it from another perspective from the study that Judge quoted about differences in acceptance of lactulose between Indian patients and United States. I think we're getting to see there's differences in India also depending whether there's an urban and a rural divide. So is it possible to identify, there is possibly a group of patients who had a single episode of precipitated encephalopathy who are Child A, now non-sarcopenic, who have been just put on lactulose for over and over again. So is it possible to predict those groups either by clinical parameters or by microbial signatures whom we can take off lactulose as a viral axis? So this was my original thing published in Metabolic Brain Disease because no one else would publish it in 2009 because I had these people condemned to a life of lactulose. I said, okay, let me withdraw the lactulose and see the microbiome, what happens to them. And to my horror, the microbiome actually started getting worse a little bit and some of them actually went back. Now these were not the perfect patients that you're describing, but you do not this is, it's a little difficult to do this because when you take the lactulose off or take the medications off and they develop another encephalopathy episode, then it's very hard to justify why you did that in that patient. In my practice, and I don't know what other people have thought, many of these people who are very stable that you put on lactulose, they automatically have stopped it by the time they come to see you. And the other things, the nuance that we have now gotten to know that we have actually tried to follow the Bristol stool scale rather than the number of bowel movements, at least in the United States patients, which makes it much more acceptable. So we don't insist on having three or four large bowel movements, which put people off with a lot of gas, but it's actually small amounts of lactulose, as long as the stools go Bristol 5. So that acceptance of that is a little bit more. I don't know what other people think. Obviously, I'll probably come from a UK perspective, but at the end of the day, lactulose is a prebiotic. So actually, it's going to have some positive effect on your microbiome. There's no doubt about that. But I think we prescribe too much lactulose and that's what spoils it. Actually, you can use quite small doses to have quite a profound effect. And actually, I think your study showed it doesn't really make any difference on the dosing. And I think actually, rather than stop it, we should just use smaller doses that patients will tolerate. Yeah, I agree with what's been said and I would say that we do need more data on when it's safe to stop lactulose and maybe there is a microbiome signature that helps tell us that. I will say there's been so much research into rifaximin recently and what that does to the microbiome, but I recently just did a literature search on what do we know about lactulose and what it does to the microbiome. And most of those studies are from the 1960s through the 1980s and there was a lot of good research done then. But we haven't quite looked at that question with modern techniques, so it's worth another look. Thank you, esteemed panel. I am Nipun Verma from PGI Chandigarh, India. Dr. Schnabel, really fascinating work. The question is, what is the relative contribution of candida virus and bacteria in causing the portal hypertension-related complications in cirrhosis and then etiology-specific derivation like ALD has a different and NAFLD has a different thing. Candida lysine is one of the beautifully shown in different studies. Question number one, this is. And the question number two is, are there any ways to improvise the colonization resistance? Like say for an example, these cirrhotic patients are at high risk of MDRO colonization and we recently conducted a study found 60% of these patients in India are at least colonized with MDRO. So is there a way to improvise the colonization resistance using microbial therapies? I take question one. It seems to be easier. So, candida lysine you mentioned, so candida definitely, I mean we see in many of these patients especially with alcoholic hepatitis, but also in other cirrhotic patients like Dr. Bashash published that we have candida overgrowth. And this is not only proportionally, but we also see this if we just culture the stool from these patients, you have many more colony-forming units of this candida, especially candida albicans in these patients with cirrhosis and especially alcoholic hepatitis. The way I see it, you have this multi-microbial synergy which probably all contribute to the disease outcome and potentially to liver failure and liver injury. From a clinical perspective, cytolysine at least compared to the candida lysine has a much higher impact on disease outcome and correlation with MELD or with mortality and survival as compared to the candida lysine. Debbie will answer the horrible question because she had that talk, not because we hate her, but to get to your point, we just finished a study which is an oral presentation in two days about the transkingdom, everything. You put shotgun metagenomics, again it's not actually viral particles, etc., and it turns out it's really the bacteria that are driving this, the second are the phages, and then archaea, which none of us talked about, which are the methanogens, which can slow down the transit and potentially can worsen constipation, increase encephalopathy, were associated a little bit but not a whole lot, and fungi, basically if you take the whole thing, because they're so sparse, were not really that when you take the whole microbiota, unless you look for them specifically. So ultimately it is bacteria and bacterial modifiers that do end up being, if the phages also obviously act on the bacteria primarily, they do end up being the major kingdom that everything else works on or works with, and once that is decimated with antibiotics then the fungi come into a little more, and now to Debbie for the solving world AMR problem. Multidrug resistant organisms was the second part of your question, wasn't it? And I think the most important thing to say is that most multidrug resistant organisms start off life in the gut microbiome. That's where they begin, and they proliferate, and I showed you the data from the study, just doing a rectal swab, and you can show that they're there. We've recently finished a study in 32 patients where we looked at the impact of FMT on patients with multidrug resistant organisms, and 25% of those patients had VRE. And FMT cures AMR basically, so all of the patients that had VRE were cured by FMT, and literally the manuscript has been written, I just have to send it, but hopefully we'll be able to show you some more data to support that as a good way, in much the same way as you treat C. diff, but actually you may be able to treat multiple MDROs. But if you look at your experience, a little MDRO goes a long way Indeed. And we're seeing that more of the potential healthy FMT donors are actually carrying multidrug resistant organisms. Maybe some people in this room, you might be carrying multidrug resistant organisms and not know. Anything apart from FMT which can improvise this colonization resistance, so you don't carry that MDRO organism. There are some non-pharmacological ways such as pectins have been shown to improvise the gut microbiome and reduce the attachment of MDRO bacteria. Inulin, definitely. A recent study that came out also showed the same thing. It improves bile acids which would actually help the clearance, but none of them have actually reached the threshold of us prescribing them because many of these are food supplements, and a lot of them can be actually eaten in food also right now. And there was a company that is sadly no more which was making oligosaccharides, which one I'm talking about. And that had shown some resistance, but the problem is if the patient got one more dose of antibiotic, everything was wiped out and you have to start from fresh again. So that's the challenge. Thank you. I think we have time for one last question. Alex here from Washington University in St. Louis. Dr. Bloom mentioned enterotypes and I was thinking a little bit about this in terms of an engraftment issue. In a lot of grafts we try to do induction first. I was wondering if there's any data on pretreatment of patients before FMT, either non-specifically with antibiotics versus targeted therapies along with what Dr. Schnabel presented in terms of trying to alter the conditions before you even start the FMT. Yeah, and so in, again, the primary indication for FMT is C. difficile infection and actually all of those patients have pretreatment with antibiotics. So this is the way we've been delivering FMT broadly. And there was something just recently published on what if you don't give the vancomycin first and you just jump right in. And I believe that giving vancomycin first had better efficacy in terms of engraftment. I know some of the non-FMT microbiome products have found enhanced engraftment when there is pre-antibiotic treatment. So I think in cirrhosis it's certainly an open question. There's a risk, obviously, as we heard from Debbie, of giving people antibiotics. So we're, you know, between a rock and a hard place. I don't think there's the right answer yet. Yeah, in our study where we were forced to give those multi-antibiotics, it did not work. It actually made things worse for everything. And the FMT, when we put it in, it barely brought it back to what it was before the antibiotics were started. And therefore, our second trial we did without antibiotics. So in cirrhosis, it's already reached a little too far. Thank you. Okay, everybody. I'd like to thank this fantastic panel of speakers and all of you in the audience for your engagement. I'd like to thank the leadership of the Portal Hypertension SIG, Scott Biggins and Don Rocky, who organized this. And I want to remind everybody that our SIG, the Portal Hypertension SIG, has an in-person meeting tomorrow from 1030 to 1130 at the Marriott Marquis. And we would love to see you all there to get you engaged. Thank you.

Fecal microbiota transplant

Cirrhosis

Portal hypertension

Liver diseases

Gut microbiome

Imbalance of gut bacteria

Enterococcus faecalis

Toxin

Targeted therapies

Bacteriophages

Antimicrobial antibodies

Research

Clinical trials