YESTERDAY, TODAY, & TOMORROW
“I think it's absolutely critical...what this team that you're compiling will provide to your customers is not just technology, but also a level of consultation regarding how to integrate the product within their needs within their institution.”
Dr. Betty R. Theriault, DVM, DACLAM
Recently, Allentown sat down with Dr. Betty R. Theriault, Acting President of the Association for Gnotobiotics, and President-Elect of the
International Association for Gnotobiology, for a conversation with us about one of her favorite topics – the past, present and future of Gnotobiotic Research.
The complete transcript of this interview is below.
Why don't we start with the definition of gnotobiotic; what does gnotobiotic mean?
I think it's a good place to start, because that's what I try and do anytime I talk about it, because people may have
misunderstandings or different perceptions about what it is they're talking about. In its most basic form, two Greek
words were put together, "gnoto", meaning "known", and bio, as in "life". When you put "known life" together, the idea
was that the organism that was being discussed had a very defined composite of non-self or associated organisms; that
whatever was not self - to the best of our ability - was defined.
A gnotobiotic animal is an animal for which the organisms that share the environment of that host have been defined to
the best of our ability to detect them based on current technologies. We can use the mouse as an example. We can know
that a particular mouse has a particular population of bacteria, and that it is free of parasites internal and external
or does not have any viral entities associated with it to the extent that we are able to detect.
Then under the "known life" moniker, would that include animals for which there is no additional microbiota, or would that be considered axenic?
Exactly. Underneath the gnotobiotic is the axenic. It's the most extreme example, in that again to the best of our ability to detect, these are
animals that have no other organisms associated with them. To the best of our ability to know, they're free of virus, and I should clarify that.
If there are retroviruses that have been incorporated into their genomic material, we can't do anything about that.
What's a retrovirus?
A retrovirus, more specifically, an endogenous retrovirus is a small RNA-type virus or viral element that is able to encode into the genetic
fabric of the organism. It's difficult to detect, and that is not something that we would be readily able to identify.
Would that potentially have impact on the study?
Only if there becomes a situation where it could have epigenetic influence.. I'm not a virologist by training, but these are viral entities that
lay silent within the organism. Is it something that could be demonstrated in the phenotype? Absolutely, but is it something that, outside of the
known phenotype, could behave differently? Only if it alters gene function. These elements may actually be part of what influences evolutionary processes.
Okay, so that's a great baseline understanding of gnotobiotic. Now that we have established this, what are gnotobiotic studies used for?
I believe that the biggest reason we are using these models once again, because they had a very strong heyday in the past that quieted down,
is the NIH putting out a road map in 2007, which was the Human Microbiome Project.
Tell us about that.
The Human Microbiome Project was piggybacked on, and a springboard off of, what had happened just prior to that. Just prior to that, we had a
convergence of things that happened in science. We had the completion of the human genome being mapped, we had the mouse genome mapped, and we
were also advancing in genetically engineered mouse models. All of these converged together, and in addition there was an understanding that
disease processes were becoming almost epidemic in a proportion that genetics alone couldn't explain.
The idea was to look for what could be epigenetic factors. There had already been some studies that were looking at environmental factors,
endocrine disruptors, these sort of things, but the idea that there were microorganisms associated with the human, and we knew this, and that
perhaps those organisms were playing a role in health and disease. This ended up being an area of interest for the NIH, and one that they
focused financial support towards. I believe that's what exploded this internationally almost overnight.
So, resulting from that increased interest in gnotobiotics, what are people turning this technology loose on? What are the big fish on the horizon?
The big fish, I think, are translational medicines. I think that we're in an environment where there are shrinking or static research dollars,
placed against international human health needs. To combat this we are seeing investments by the Melinda and Bill Gates Foundation, some of these
other private funding sources, and they're going to become bigger players, I believe, as government dollars are maybe static or receding. They
proceed on an outcome-driven investment. I think that, again, if we look at translational medicine, we don't have to look far for conditions
applicable to gnotobiotics. We have type two diabetes. We have inflammatory bowel disease. We have allergies. We have cognitive disorders on the
autism spectrum. These are things that did not exist, or did not exist to the extreme that we're experiencing, and we're all, as humans, looking
for ways to intervene, ways to not only prevent or treat, but to treat at different phases within the disease spectrum. I think that's where our
researchers are focusing their attention, and these germ-free and gnotobiotic models are critical in playing a role in these studies.
I think hidden in there is the fact that researchers still want to know mechanisms. They still want to understand how something works, or to
understand the immune system better, or understanding different types of biological systems better, and hidden in there also is the opportunity
to discover new mechanisms and new pathways, and add to our knowledge base. But the driving force is going to be interventional opportunities.
Why are gnotobiotic studies uniquely tailored to these efforts, as opposed to other non-gnotobiotic studies?
I think that these efforts are not going to be the answer to every study, but what the model system permits us is the ability to modify or identify
the genetic background. That's where we were before the Human Microbiome Project started. We already had genetically engineered mice, and there are
many other organisms, zebrafish, even some early attempts at pigs. There were genetically engineered organisms. It could be a plant, too, it doesn't
necessarily have to be an animal, but we're in the animal realm, so we'll stay here. You can ask in your research model, what's the genetic effect?
Then, what you have is the luxury of saying, "If the animal is sterile, if it's absent of organisms that we have the current capacity to detect, does
the genetic component alone stay true to the model phenotype, or does it change? Does it get worse, or does it get better?"
Then, once you identify if it gets worse or if it gets better, you can strategically add back in either molecules, or organisms, or groups of organisms
and ask, "Is that sufficient enough to either make it better or worse on top of the genetic component?"
As we move forward with gnotobiotics, what are the challenges that researchers are facing when they're trying to apply this type of research?
I think that there are several. I think that we're all challenged by technology. When this road map hit, we had to go back to technology that
was 50 years old. There were very few places that were still using this technology, and there were very few groups that knew how to use it,
but it was the gold standard. So it's where we all went to.
But we know that today's current technology for mouse work – IVCs – impart protection, but also allow for high throughput, for automation.
Yet in the face of this new technology we've still gone back to the most labor-intensive and costly system of the past. What we're faced with
is one particular animal model that, in its current use, is in a conventional or a specific pathogen-free type of environment. What we're
looking to learn is how do we adapt this research model into a gnotobiotic or a germ-free environment, and then how we do this in the most
cost-efficient and timely manner.
I don't think that we have the perfect answer yet. I think we're still searching. And this leads to another point, and that is there's no
perfect fit. There's so much diversity in what's being looked at that we need to be nimble to be able to take current technology, and
maybe not yet identified technology, and bring it to these animal models.
So technology is one challenge. I think that another one is the need for the expertise to interact with these models, because I think that
again, to this day there are still very few groups using germ free animal models.. They are just incredibly complicated and difficult to
maintain that level of sterility, and I think there's limited understanding of how difficult it can be to execute these studies. So that's
a challenge: educating the community about how to do this stuff. So again, I think its technology. I think its expertise and education.
Then I think a challenge is refining the models so that there's a clear understanding of what the research objective is, and then what the limitations are.
So, if I could distill that down just a little bit for my understanding, what you're saying is that there's no silver bullet. You can't say,
"Here's this one piece of technology that's going to solve all the problems." It's a combination of technology, plus expertise, plus education,
plus changing the paradigms and the way we do things. It's a 360-degree approach. They all play their part, but they have to work in tandem to
facilitate this research. Is that about right?
That's a very good summary, yes.
Thank you. So how is the LAS industry meeting these challenges?
I think that industry leaders, Allentown being one, are helping to meet that. They have seen that there's an opportunity for these positive
pressure bio exclusion racks. It brings us back to high throughput. It brings us back into more of the automated advancements that we we're
accustomed to. But the equipment alone, without the education and without working within the system, is not sufficient. That's where I think that
there's a need for a community effort and a community conversation back and forth regarding what are the needs, how might we modify things to meet
those needs, and then not just resting on our laurels, but saying, "Okay, now what's next?" Because guaranteed, that's what's happening in the
research community. They're doing it faster than we're keeping up with them.
So in response to these needs that you've outlined, Allentown has developed our Gnotobiotic Housing Services Team approach. This includes our
technology component – the Sentry SPP positive pressure IVC, plus our team of consultants and experts, plus our service and support structure.
Do you believe that this sort of team approach could have value in helping to foster successful gnoto studies moving forward?
I think it's critical. I think it's absolutely critical. Even when we went back to gold standard flexible film isolators, we had manufacturers,
and they were willing to ship it to you. They might even help you build it…but how you worked with it, and what you did with it, on this you were
entirely left to your own means. Even with today's new technology, working with it is not necessarily intuitive. How do you work with it, what are
some of the pitfalls, what are the risks? What are you going to have to modify at your own institution? You can't take an SOP from one institution,
take that back to your institution, and think that you can just read down a list of steps and it's going to work for you. It will not. It's going
to require some modification.
So what this team that you're compiling will provide to your customers is not just technology, but also a level of consultation regarding
how to integrate the product within their needs within their institution.
It's not a one size fits all...
It can't be. The other thing that it also mirrors, in spirit, exactly what's happening and what's being supported and encouraged in the
research community. It's an effort to break down those individual silos of expertise and get experts together around a particular problem,
and see if not only they can solve that particular problem, but can they potentially see something new that as a group, they can recognize,
but as individuals within the group, cannot be seen. That's the collaborative environment that our researchers find themselves in, and I think
that's the environment that the laboratory animal community needs to find themselves in.
That's great, thank you. Let's talk a little bit about your revival of the Association of Gnotobiotics of North America. Can you tell me about that; what it is and what the plan is for it?
It's not just me. It's too big for little old me. But back when gnotobiotic technology was big, the heyday of the late 50's through the early 90's,
there was a robust community that was using flexible film isolators, maintaining germ-free animals, and conducting germ-free and gnotobiotic research.
There was a robust community of support individuals as well as scientists that were engaged in this association, the Association for Gnotobiotics.
But then the technology and the research shifted and the whole thing attritioned. The association went somewhat dormant. The last elected board held
onto the association and did just enough to maintain it as an active, incorporated, non-profit organization, but without membership, because
everybody retired, everybody attritioned. So it existed but there was no activity.
When we began the development of our gnotobiology program, there was an announcement for a celebration of Philip Drexler's 100th birthday at Notre Dame.
Myself and a handful of my staff went to this symposium and banquet. This was the most fortuitous thing I've done in this whole trajectory, because the
people that were hosting were the dormant board of directors for the association. I said at that time, "We need to revive. This is coming. It's here,
and we need help. We need knowledge. We need all of their input."
They weren't quite sure. They weren't sure because they had their lifetime invested in that organization. They wanted to make sure that they handed
the baton over cautiously, but from that meeting, I got invited to the international association and symposium, which gave me a bigger look.
What helped move the ball forward from that point was when Trenton Schoeb down at UAB got together the gnotobiotic listserv. That enabled people
to start communicating. A new voice got onboard with that, Julia Krout, and she had the energy, and she had the interest in trying to continue to
mobilize to get the organization restarted. With her efforts, plus the platform of that gnotobiotic listserv, we were able to get together a handful -
bigger than a handful - of dedicated people that have been internally functioning as an elected board. We're driven to see this come through to an
active society again, and I fully believe that we're going to be able to have it active and receiving new membership for this coming national AALAS meeting.
If someone is interested or potentially interested in joining this burgeoning association, what should he or she do? Whom should they contact?
They can email me at email@example.com.
We don't currently have a sign-up mechanism for membership, but we can keep a queue to invite interested individuals to join the association once we do.
All right. Last question, then I'll let you go. We've talked about the past and the present as relates to gnotobiotic research.
What's the future look like? What's gnotobiotic research look like five years from now?
The first thing I think, the first word I'll use, is robust. I think this is here to say. It's not going away. I think that there are three things
that we have to keep in focus. The first one, as it relates to the laboratory animal community, is Craig Franklin has a review article in the most
recent Lab Animal. The title of that is "Microbiota and Reproducibility of Rodent Models". We're going to encounter more and more of a need to have
gnotobiotic colonies or various levels of segregation of our animal colonies. That's going to be very challenging to the lab animal community, but many
of us are already facing it. Craig Franklin is way out in the lead in his vision on this.
The second horizon out there is therapeutic intervention - personalized medicine. That's where there's an opportunity for bioceutical and biologic
types of interventions, which are a different approaches to treatment. That's where I think that gnotobiotic and axenic models are going to play a
very important role in those discoveries, because you can very intricately alter the model, and you can add in, and you can take away, and you can
make assessments. You can try to tease out mechanisms in a more powerful way than any other broad group of models will allow.
Then, if all proceeds as I believe it will, there's the real potential for an impact on the 3Rs with the potential development of new technologies such as organoids.
Organoids? Is that a real word?
Yep, it's a real word. It's well understood in the medical community. It was rolled out to national AALAS two years ago, where for certain types of
functional models, you can create in the laboratory, in vitro, a somewhat analogous structure to an organ. It's an organoid.
What's also being looked at is having organs on a chip. This was also presented as a keynote talk at national AALAS a few years ago. They
projected that 10 years out, there might be multiple organs on a slice, and that maybe 20 years out, they might be able to recapitulate interacting systems.
It's exciting stuff. I think that all of these are going to be technologies that converge together, and that's going to continue to refine the animal
models that we use and allow us to look at things in very novel ways that we haven't thought about before.
That's amazing. Dr. Theriault, thank you so much for this time. It's been incredibly interesting and informative.
You're very welcome.
Dr. Betty R. Theriault, DVM, DACLAM, is the Gnotobiotic Research Animal Facility Director, Clinical Veterinarian, Animal Resources Center, Associate Professor, Department of Surgery, at The University of Chicago.