 collaborators, Kerry Kenning in the vision of rheumatology, Yvonne and Jerry Kaplan over in pathology. I've also received a lot of help in our Switzerland that have been sending these samples. So the results I'm going to show you appear to apply not to just giant slaughtered arthritis patients in Salt Lake City, but giant slaughtered arthritis patients from across the US, and at least in Switzerland. And actually, we also have some collaborators in Spain as well. And so these are some of the people that have been sending these samples to the resident, Jason Jones. So this is Barry Marshall and Robin Warren. They won the Nobel Prize for identifying helicobacter pylori is a cause for gastric ulcers. Remember, that was systemic diseases, autoimmune diseases, diseases that don't appear to be infectious that may indeed be due to either bacterial or viral causes, or maybe even para-infectious. Maybe a result is a body's immune reaction to an infectious agent. MS might be one of those, actually. But then there's also, these are pictures of chlamydia in atherosclerosis. And that sort of, I don't know that it's been a flash in the pan. There's some people that still believe that chlamydia has something to do with atherosclerosis. But it certainly hasn't panned out like helicobacter has. Now, the University of Utah is home to the artificial heart. It's home to the knockout mouse. But it's also home to cold fusion. So I hope that Birkeld area turns out to be more like the artificial heart and less like cold fusion. So Lynn Gordon, one of my collaborators, actually had a paper out about 12 years ago where she showed some non-human DNA in biopsy specimens, temporality biopsy specimens from patients who had giant sardaritis. And that kind of got me thinking about giant sardaritis being an infectious disease. But based on those studies that she did, she wasn't able to ever prove a specific agent was causing the disease or able to culture an agent out of the arteries or anything like that. But that article always intrigued me. And then when we got a gift from a grateful patient to study giant sardaritis, that seems to be a topic that we could take up. And so I finally was able to convince some collaborators next door to help me with this project. And if you're interested in trying to find a bacterium in a specimen, but you don't know what bacteria you're looking for, it turns out a good way to find it is to look for the 16S subunit of the ribosome. So bacteria, just like human cells or other animal cells, have ribosomes to make proteins. But there's this 16S subunit that's unique to bacteria that's not found in animal cells. And so if you can search for 16S ribosomal RNA in a sample, because ribosomes, if there are bacteria there, ribosomes are very abundant. There's going to be lots of ribosomal RNA that you can look for. And this little diagram here with the black and white bars, these show variable and conserved regions in the gene, the bacterial gene for the 16S RNA. So you can PCR for the conserved regions, and you will pick up all the bacteria in your specimen, not just the particular bacteria. So it's a great screening tool to look for that subunit if you're interested in bacteria. So I've presented before about some of the preliminary work we've done with giant sardaritis and mortality, the accumulation of blood and tissue specimens from my collaborators, binding hepsidon in these specimens, microarray analysis of these specimens. And I'm not going to talk about any of that today in the interest of time. I'm going to go straight to the cool stuff. So we just took eight of these specimens, two control specimens, patients who were biopsied, but whose biopsies turned out to be negative. Those are our controls. And then six positive specimens. So these were taken from five patients. One of them had a bilateral biopsy. So what you do is you take the specimens which are preserved in this stuff called RNA later. You extract their DNA, PCR against the conserved regions of that 16S subunit. You clone the resulting fragments into a vector that then multiplies, gives you a bunch of the 16S RNA. You can sequence it. And then you blast it against known bacterial sequences. So there are these huge databases available online, the sequence in their 16S subunit. And you can blast your sequence that you found against these known sequences and see if there is any homology. And there were several sequences that we came up with that had homology with other bacterial species. But the only species where we had 100% homology was this genus Burkholderia, which I'll describe to you in a minute because probably most of you like me had not heard of it before. But to confirm this experiment, we took these same specimens, did the same DNA extraction. But this time, we PCR'd against the genes that were specific for Burkholderia. So these are DNA sequences that are unique to Burkholderia. They're not found in any of the other bacteria that have been characterized. These were directly sequenced. And we confirmed Burkholderia sequences in four out of our six specimens and neither of the two control specimens. So the two positive specimens where we couldn't confirm Burkholderia sequences came from the same patient. That was the patient who had bilateral biopsies. If it's possible, there's bacteria everywhere, right? And it's possible that there's just dead bacteria somewhere in our operating rooms. Or it's very difficult to keep something completely dense yourself that these are actually live, not just some dead DNA laying around, is to extract RNA and then do a reverse PCR, again, using genes that are specific to Burkholderia. And again, we were able, this time, to identify Burkholderia transcripts, so RNA that had been made the same four out of six specimens and none of the two control specimens. So this seemed to be pretty good evidence that our temporal artery biopsies have this bacteria in them. So Burkholderia are aerobic gram-negative rods. They're obligate intracellular pathogens. Most of them cause plant and animal diseases, but there are only three that are associated with human disease. Some of you might have heard of Burkholderia sapatia because it causes pneumonia and CF patients. But the disease is caused by Pseudomaliae and Malian and one thing that's interesting about this genus is that, in general, they tend to be very antibiotic-resistant and evasive of the host immune system. So patients that have these infections get a double the time. This brings up another point. So the idea that giant sardaritis might be caused by an infectious disease is not something new even within the last 10 years. There's people 50 years ago thought that giant sardaritis could be infectious. And so they would try to gram stain specimens. They would try to culture specimens. They would do electron microscopy. And no one's ever been able to show an infectious organism in these specimens. And that's why the subject was sort of dropped until Lynn Gordon did the study about 12 years ago. And one of the characteristics of this bacteria, I think, explains those initial negative results. Because it's an intracellular pathogen, it's not particularly easy to culture. It's not particularly easy to see, like on gram stain. And I think that may explain why these are these investigations. So the species that we were able to subtype using multi-locus sequencing is bercalderia pseudomalii. It's actually endemic in South Asia and Australia. It's best associated with this disease in that part of the world called neologosis, which is characterized by fever, pneumonia, and it could cause other diseases, which are characterized by cutaneous vascularitis, which is interesting, because that's kind of like giant sardaritis. In some cases, it can cause a piogranulomatous vascularitis and a CNS vascularitis. So these are all characteristics that are sort of like, those are sort of at least reminiscent of giant sardaritis. And this is a very fastidious organism. It has to be grown in selective media to prevent the overgrowth of other flora, which I think explains previous researchers weren't able to culture anything out of these arteries. Because if you don't have your media just right, I mean, it's hard to culture an intracellular pathogen. But mouse model of bercalderia that we've been playing with a little bit over in the, if you give them too much bercalderia, it causes an overwhelming substance of death. If you give them a little bit less, they get this chronic. Bercalderia pseudomaliae is also considered a potential bioterrorist because of these characteristics, because it's, because your body doesn't seem to be able to bite it off, because it's antibiotic resistant. But luckily, we were able to send our specimen. The DOD has a lab in Fort Collins, Colorado State. And we were able to send our specimen over there. And they confirmed that our specimen are the species that we've, there is a commercially available monoclonal antibody against the lipopolysaccharide and the cell wall of bercalderia. And here's one, you know, illustrative example of a control artery and an affected artery. And then there's the immunofluorescence on the right. You can see what the control artery there is. We also use the same commercially available antibody to detect bercalderia LPS in the serum of affected patients. So all of our patients, when they get a biopsy in the operating room, we also get a serum sample. And you can then react their serum against this antibody using an ELISA. And so what our 16 control patients, so these are patients whose biopsies turned out to be negative. We have 29 patients collected from Utah and Spain. All have these very high levels of bercalderia lipopolysaccharide in their serum. This is just for fun. We took one patient who'd been on steroids and their serum was still positive for bercalderia, but it was a little bit, it was on the low side compared to the people who had just been freshly biopsied. And then just to compare it to some other rheumatoid's diseases, we had six rheumatoid arthritis patients, five patients that were in the hospital for other unrelated, 12 patients with takiasus arthritis, which is a disease in younger people that some people think looks a lot like giant arthritis, so we were testing them for fun too. And they seem to be negative for this organism. So the data that we have so far is we've identified bercalderia DNA in these specimens, RNA in our specimens, and we've seen bercalderia protein in the specimens and in the serum of affected patients. We've since been able to culture the organism out of effective arteries, again, by using selected media. It's one of those things that if you know what organism, you want to culture and you make the right conditions in your media, you can culture this organism out of the arteries. We've also taken our species of bercalderia that's sort of like pseudomalyli, keeper cells outside the adventitia of the artery, and dendritic cell activation is a critical step in the process of giant arthritis. So just from people that know about giant arthritis pathogenesis, they know that dendritic cell activation is an important component of that process, and we've been able to show that this bacteria can activate those cells. And also in vitro, we've shown that bercalderia organisms will cause giant cell formation in a bit. We still don't know what bercalderia's role is in giant stardritis. We can't really fulfill Koch's postulates. I'm not gonna drink a beaker full of bercalderia. And a lot of questions remain. I mean, presumably this, like we don't know the epidemiology of this organism yet, that's all yet to be determined, but presumably it's out in the environment everywhere and all of us are exposed to it, and none of us seem to get sick from it. And so why is that? Why is it that there's just a very small number of elderly patients that seem to be susceptible to infection to this organism? Is there some hole in their humoral immunity? Is there some sort of age-related change in the immune system that allows this organism to be cleared and otherwise younger, healthy people, but in certain older people they lose their ability to look, giant stardritis are sick. And they have, if you do an autopsy on these people, they have giant cells all over their body, not just, and yet they're not systemically sick usually when they're treated. And so, but it seems like their immune system is somehow walling off the infection, but just not able to clear it. So after we accumulated these data, patients with giant stardritis on antibiotics, and so so far, just here at the U, we've enrolled six of our, most of them freshly diagnosed, but a few of them, we've given them between one to three months of either doxycycline or minocycline. The organism we've cultured, at least in vitro, appears to be susceptible to these antibiotics because it lacks some of the type three secretion factors that like the biotovar patients have, some patients have been on this protocol. So far we haven't had any. I can tell you the guy that's been on antibiotics the longest is a patient, Dr. Warners. Two or three years ago, he has poor velocity process. He has five compressions, and he got tapered below that amount of steroid. He's doxycycline initially, because after the first month his blood was still positive, home substantially has been like, exactly sure what's gonna happen with these patients. I don't know if they need to be on. What I envision happening is a multi-center placebo-controlled trial of antibiotics plus steroids. You can't just treat these patients with antibiotics. That would be safe or ethical. And so what I envision people getting randomized to steroids plus placebo or steroids plus, it'll have to be multi-center, because we just don't see enough giant sardaritis patients at any single institution to power a study. And then we will look at the relapses and the steroid tapers in the two groups. Yeah, there is. Anastas does have this organization called Nordic Concerns, because you lose a certain amount of control. There's also similar organizations in rheumatology. That's, you know, Curry has tested some of his, just in a small sample, they've all been netted. You know, some people at PMR do go on to develop GCA and cohort of those patients. In the same protocol, not the same bacteria, but that same, you know, you're looking at the 16S ribosomal RNA. That could be used in other diseases like wagons, granulomatosis, or sarcoid, or some of these, some of the unitities. Yeah, I mean, I guess that's sort of a chicken and an egg thing, like maybe the arterioscarosis is caused by the inflammation in the artery. Or maybe arterioscarosis makes it easier for the bacteria to wall itself off and protect itself from the immune system. Okay, so these data were, these data and some additional data, which I haven't presented, were submitted to Science over the summer, and they said it wasn't novel enough, sent it back. And so then it was submitted to Nature Medicine, and they sent back this huge review. And, you know, they had several concerns. They wanted to see some more numbers. They wanted to know if the investigators who actually did the ELISAs and the immunohistochemistry were masked to the disease status of the patient, of the specimen. Those were the, and they wanted to see more with the mouse model. Those were the big, those were the big concerns. We did put together an R01 that will be reviewed at the NEI this month, you know, so I don't have any. Also, the Hepsidon data, which I did not present today, but what I presented to this group previously, those were in the paper and they didn't like that. They wanted that chopped out. So we're gonna try to, they didn't reject the paper. So we're gonna try to address their concerns and resubmit. Well, the ELISAs are reduced. So like, they don't get reduced to the point where they're like in the normal range, but they're reduced, you know, maybe 50%, 60% compared to their baseline. So they still presumably are infected, you know. You can't get rid of the bug with antibiotics. Yeah, there's clearly, well, you know, Greg wants to study that, you know. Yeah, you know, that's absolutely true. The only thing we have is we do have a biomarker. We do have a serum ELISA. And if we can't show that the amount of LPS goes down with antibiotic treatment consistently in treated patients, that shows that the antibiotic effect is at least in part responsible for the therapeutic effect. But you're right, we can't say that it's entirely responsible. I was proposing our R01 submission, you know, to look at, to see if that has anything to do with the whole immune system. But I think that's gonna turn out to be like a related factor. Like I'll bet that HLA locus is somewhere near another locus that actually has to do with disease susceptibility. I don't know that that HLA subtype actually is what causes the disease, but I'll bet you it's, I'm sure it has something to do with disease pathogenesis, but we don't know what it is. You know, that's another thing that I hope to do with this multi-center trial is to prove that this... All right, thanks very much.