August 28, 2024 • 41 mins
Join us in this fascinating episode of Pediatrics Now as we sit down with Dr. Paolo Casali, University of Texas Asheville Smith Professor and Distinguished Research Professor in Microbiology, Immunology, and Molecular Genetics. Dr. Casali and his team have made groundbreaking strides in creating the first fully humanized immune system in mice.
Discover the meticulous process behind this scientific breakthrough, from grafting human hematopoietic stem cells to the hormonal conditioning that ensures a fully functional human immune system in mice. Dr. Casali shares insights into the potential this innovation holds for vaccine development, cancer research, and reducing reliance on non-human primates for testing.
Dr. Casali's journey from basic molecular research to this pioneering work is a testament to the impact of dedication and experience in the field of immunology. Tune in to learn how this advancement could revolutionize medical science and pave the way for more effective therapies and vaccines.
Mark as Played
Transcript
Episode Transcript
Available transcripts are automatically generated. Complete accuracy is not guaranteed.
(00:00):
Music.
(00:09):
I am so honored today because here in the podcast studio with me is Dr. Paolo Casali.
He's the University of Texas Asheville Smith Professor and Distinguished Research
Professor, Microbiology, Immunology, Molecular Genetics.
He's Professor of Medicine at the University of Texas Long School of Medicine.
(00:30):
Dr. Casali, first, thank you so much for being here in the podcast studio today.
My pleasure. So you and your team have accomplished something that is so amazing
and so interesting and will affect medicine for countless years to come.
So you all have created the first humanized immune system in mice.
(00:54):
Well, thank you for your kind words. Actually, this is not the first humanized mouse.
Humanized mice have been attempted to be constructed since the 80s,
the late 80s, when there was an urgent need to model in vivo the human immune response to HIV.
(01:23):
And so those were the first, so to say, humanized mice.
In fact, those precursors of humanized mice were very rudimental, very primitive.
And those mice never quite mounted a good immune response to antigens or viruses, whatever.
(01:54):
Immunogen. And this has been really the shortcoming of the different varieties
of humanized mice throughout the last many years.
And I think it's proper to say that our mice are the first fully humanized mice.
(02:17):
The first mice with essentially a 100% human So you and your team are the first
to create mice with a fully humanized immune system. And fully functional.
And fully functional. It's incredible.
So what does this mean for medicine?
(02:40):
Well, I think it's a simple and complex answer, but the first one was to define
what humanized minds mean.
And what it means is these are mice with a FURI, virtually, or FURI,
(03:03):
as one could argue here, human immune system.
So the mice are mice, but they are immunodeficient mice, virtually without immune system, so to say.
And this mice accept a graft consisting of human hematopoietic stem cells.
(03:28):
And these are the cells that will give rise, will proliferate and differentiate
and give rise to a whole human immune system.
And these stem cells are hematopoietic, meaning they are stem cells that will
(03:50):
give rise to cells of the immune systems,
but of the lymphoid lineage,
such as B and D lymphocytes, myeloid systems, such as granulocytes,
neutrophils, and macrophage.
And then once grafted in immunodeficient mice,
(04:14):
again, then within a number of weeks, you will have those mice fully constituted
in the immune system, functional immune system.
Now, how that is done, it's important to understand what the potential of these humanized mice is.
(04:34):
So it is done by crafting hematopoietic, human hematopoietic stem cells from
core blood, from neonatal core blood.
And so we take cold blood with the help of the OBGYN department.
(04:59):
Fresh cold blood. We purify the hematopoietic stem cells from that cold blood.
And then graft within 24 or 48 hours of birth this sort of special immunodeficient
mice those are pups, they are neonates,
(05:20):
and we graft those mice intracardically with a small insulin needle.
And the grafting is
done in the left ventricle to maximize the dissemination of stem cells throughout
(05:41):
all districts of the bodies so that those stem cells will become resident of
wherever bone marrow will develop.
Now, these are special immunodeficient mice in that they carry a mutation of
(06:02):
the CKIT receptor, and they are referred to as W41.
We did not generate these immune-efficient mice.
These mice have been available for about four years.
The interesting thing is that nobody really took advantage of them to...
(06:24):
To create immunized mice with a fully functional immune system.
More precisely, I should say that the two different groups,
one in Minnesota, the other one in Germany, who created these immune-efficient
(06:44):
mice with the same mutation,
W44 mutation, actually did craft them with human hematopoietic stem cells,
and they did quite full characterization of the developing immune cell elements.
But they didn't really do much of functional studies on the immune response
(07:14):
potential of these valleys. And this is what we have done.
What we have done, which has been different from any other humanized mouse,
has been the intracardiac grafting.
And importantly, the hormonal conditioning with estradiol, with estrogen, of male or female pups.
(07:44):
And this is what really has made a big difference in terms of the development of the immune system.
And the functioning and the differentiation, further differentiating, I should say,
further differentiation of immune cell elements once these mice were challenged,
(08:09):
were immunized with different conjugated optins, and those are main, main molecules.
But how many years in the making has this been? It has been five years.
Is this a dream that you've had for a long time to accomplish this?
Or how, Paolo, can you tell us about how this came about, how you got into this? How I got into this?
(08:32):
I think my age has played a major role in it, in that experience, I should say.
My many years of research in immunology have always been, has certainly always
been with the human immune system and immune response in mind.
(08:54):
But my research has always been very basic, very molecular,
very much into the cell, namely B cells, B lymphocytes, which are the cells
that make antibodies, and very much with a molecular genetic flavor,
very much about regulation of genes that encode antibodies.
(09:19):
Cross-reaching somatic permutation of those genes.
And with my aging, I became, I have become.
Much more determined and, I should say,
(09:41):
much more motivated to do something that will have an immediate relevance to vaccine development,
to the human antibody response,
but particularly antibody response,
because I always worked on B lymphocytes and, as I say, antibody genes.
And I should say that my persuasion is not only mine, has become stronger and
(10:09):
stronger than if you want a good vaccine,
you need to be able to generate a good antibody response, one that is very specific
and neutralizing a virus or a bacterium or whatever else or a toxin.
And so, once we have become more and more familiar with the intimate molecular
(10:37):
mechanism that underpins the generation of a mature antibody response,
again, one that matures, one that is highly specific and can neutralize microbial
agents, and one has certain features.
(11:01):
Local features in the antibodies.
And that is, again, those antibodies are generally IgG or IgA and they're generally,
or they're invariably actually,
somatically mutated in the variable region of the antibody molecule.
(11:22):
And this is what provides a substrate for the selection of high-affinity mutants,
sub-mutants, I should say,
in those B-cell groups that are selected by the antigen driving the response, such as the HA,
the glycoprotein spike of influenza virus,
(11:44):
or the surface antigen on bacteria, fragilin in salmonella, for instance.
And so the idea was, at this point, we need to be able to do.
Experiment in vitro in a 100% immune system.
(12:07):
Because if we can do that, then we are in the position of, finally,
of being able to do vaccine development.
Vaccine testing of drug, if not development, but certainly testing,
(12:29):
particularly, well, in any case, of immune modulators, such as checkpoint inhibitors
in the case of the antibody, the immune response to tumors,
solid tumors, and many other things that one cannot do.
To this day, if you develop a vaccine, the last step the FDA wants you to do
(12:56):
is efficacy before going even to phase one. in non-human primates.
Non-human primates are not exactly human.
They're very similar, very close to us, incredibly close to us,
but in many ways are not exactly human.
And this is perhaps not even the major obstacle in the use of human primates.
(13:23):
One problem with human primates is that they are so close to us that But you don't want,
I certainly don't, wouldn't want to do what has been done with human primates, with those primates,
non-human primates, for many years in the whole story of HIV, for instance.
(13:49):
Certainly, many things have been done with those non-human primates that shouldn't
have been done and which today are not acceptable and not done.
Because of cruelty? Yes.
And unnecessarily, unnecessary experiments.
So this will reduce the amount of testing that needs to be done on non-human primates.
(14:16):
Hopefully, if these mice turned out to be really what they are,
but I'm being very cautious here
but they turn out to be really what they seem to be and I think they are,
then you could do.
A lot of vaccine development because you're dealing with an in vivo human immune system.
(14:39):
And you cannot do that in humans for ethical reasons. Yes.
It will help dramatically pave the way for even better vaccines,
more targeted vaccines.
Yes. And then when it comes to tumors.
It's already been done with a few THX mice.
(15:00):
Targeted therapy? These are our mice. Here, actually, we are collaborating with
giving over those mice, our mice, to a couple of groups working with different tumors, solid tumors.
So this investigator grafted tumor tissue from humans into the mice and then
(15:23):
studied the overall reaction to that tumor.
And that reaction is human, be it inflammatory or immune, exquisitely immune-responsive.
Wow. So some investigators started doing that.
You worked, Paolo, for many years as an immunologist. And so instead of retiring
(15:48):
like a lot of people do, you really revved up your research and the urgency
to make a difference and get this done.
Yes. And the NIH offers experience and knowledge, but it also offers security.
Because I was always well-funded with the NIH. But at this point,
I could afford to do something that I was not funded for. Really?
(16:14):
That's amazing. Like, we're going to do it. I'm going to make this happen, right? That's right.
Can you tell for us a practical example in, say, for pediatrics?
Well, one thing that we did with these mice, again, we refer to them as THX
mice, three true human X mice.
(16:36):
This was not me, this was our development office.
So one thing that we already did, and it's published in this paper that you mentioned,
is we vaccinated these mice with the messenger RNA Pfizer COVID vaccine,
using an immunization schedule which is the same as for humans.
(16:59):
And the mice made a neutralizing, strong antibody response neutralizing to SARS-CoV-2.
And those are human antibodies, obviously. And we analyze in details the mohawk
genetic makeup and mohawk composition of that response.
(17:19):
So again, that's good.
We did the The same, and that's published as well, with purified Fragilin from Salmonella.
We immunized those mice with Fragilin and showed that they mounted a neutralizing
antibody response to Salmonella,
neutralizing as assessed by in vitro the classic colony neutralization in petri dishes.
(17:47):
But then we also show that the THX mice vaccinated with flagellin survived,
fought and survived infectionally like flagellin.
The non-vaccinated ones died.
So the neutralizing response mounted by the vaccinated mice died.
(18:14):
It was a protective response, protecting from infection. Wow. Those mice survived.
I lost a little weight. Wow. They made it. Wow. The other ones, I, all of them died.
And, Paolu, I'm imagining it when all the scientists at NASA and the landing
(18:36):
is happening in space and there's lots of excitement.
When you all realized you had done this, can you describe for me what it was
like or what the feeling was like?
Or is it dramatic like that? Or it's more you're in the lab, you knew it was coming?
I'm trying to imagine the moment when you realized this is, we did it. That's a good question.
(19:00):
The climax or the outcome of the death was tempered by the fact that this is so labor intensive.
They went front and get there. It's okay.
It's so tedious, so labor-intensive. Labor-intensive, yeah. Can you tell me
what you were feeling when you realized?
(19:21):
That's, in many ways, what's expected.
So, you know, science these days, particularly biomedical science,
it's very incremental in progress.
So it's very different from 50 or 80 years ago. You knew the rocket was going to get there. Right.
(19:44):
So do you, what's the next step? What's the plan from here now that? That's a good question.
We are trying to do different things using DHX-Vis.
Certainly the decision has been made on my part to do any experiment from now
(20:07):
on in THX rise so there are different things we are doing we are uh.
We have identified actually a transcription factor that is critical for the
maturation of the antibody response.
It's critical for the expression of AID, activation-induced cytodiaminase,
(20:30):
which acts on DNA and divert
the DNA repair machinery of B lymphocytes to act, to effect class switch DNA recombination,
and somatic hypermutation.
I say divert, I say it diverts, but in fact it's a physiological process.
(20:54):
It diverts the physiological process of DNA repair,
which comes into place, comes into being in a major way when you go to the beach and you're in the sun,
and all the DNA repair over your body.
(21:14):
So that process is physiological, but a process in bilinphocytes that enables
the maturation or the affinity maturation of those antibodies,
meaning enables the generation of submutants with a much higher specificity for the antigen.
(21:37):
Again, be it viral glycoprotein or bacterial components.
Toxin, bacterial toxin. That process is physiological as well,
but it's really a diversity, diverting of the more general process ongoing in your body all the time.
(21:59):
And whenever there is a mutation, it needs to be corrected in the DNA.
So we have been working on that, and we have been working on that in vitro, as well as in mice.
And now, for the last, latest stages, of that overall investigation.
We are using THX mice.
So whatever we will find,
(22:24):
actually whatever we are proving, attempting to prove, will be even more relevant
because it will be in a human system in vivo.
That's for instance one thing. There are other things. We have identified a
transcription factor that seems to be critical for the maturation,
(22:48):
the generation, I should say, of mammary B lymphocytes.
Mammary B cells or B lymphocytes, we refer to them as immunologists as B cells.
Those B cells, the generation of those B cells is critical for the effectiveness of a vaccine.
(23:09):
If a vaccine cannot induce degeneration on memory B cells, then it's not a good vaccine.
It's not a vaccine because those are the cells.
After the vaccination, will persist in your body for months and years,
and they will be recalled, reactivated, when the same antigen comes in again.
(23:34):
For instance, if those B cells are generating response to influenza vaccine,
influenza virus vaccine, and then in a couple of years, you will come across that virus.
That virus will reactivate those cells. Those cells will proliferate and further
differentiate and make tons of antibodies to the virus.
(24:00):
Wow. So understanding, getting
to know what are the critical transcription factors and what are the other factors
that are critical for the generation of memory B cells is critical for vaccine development.
(24:20):
In the future, do you see other humanized organs in mice?
I mean, are we going to have a fully humanized mouse?
Or what is the future? It sounds like science fiction, but it's medical science now.
Well, it's interesting. That's a very interesting question.
In fact, prior to the development of our mice, which because of hormonal conditioning,
(24:48):
And I think actually that deserves a few words.
Because of monoconditioning, they develop a fully human immune system.
Prior to that, different attempts were made to graft,
again, earlier humanized mice with human fragments of human thymus,
(25:10):
for ISA to promote the T-cell differentiation of those mice or graft other.
Other fragments of other organs in the attempt of, again, promoting a better
differentiation of the immune system.
(25:32):
So one could speculate, or it's not a big speculation, that you could graft
actually organs or pieces of organs, fragments of organs, such as liver or something else, human.
So that in this case, you would study as targets of the immune immune system
(25:57):
or the human immune system,
targets of autoantibody response, SIS.
We show that our mice can mount an autoimmune response, which is really to all
effects similar as the autoimmune response in lupus, human lupus.
(26:19):
So one can certainly foresee that. But I want to go back to the hormonal conditioning.
The hormonal conditioning that is critical for the full development of the human
immune system in THX mice is in fact estrogen condition.
So these mice, once they're grafted, they even rise to a certain extent to all immune element,
(26:51):
which happens at the age of 20 weeks, 18, 20 weeks,
then the mice are put on estradiol E2, estradiol is E2, and that's the most
potent and the most abundant.
(27:13):
Humans, particularly in females, and we put E2 in water, so the mice,
the little mice drink water and they get E2.
Now interesting, the blood concentration of E2 that derive, that are generated
(27:33):
by the drinking E2 in water at a very small concentration,
they're actually very physiological for humans.
In female or male THX mice, adult THX mice, the blood concentration we do is
around 60-70 picolams per ml.
(27:54):
In women, depending on the different stages of the cycle,
the blood concentration is from from 30 to 500 picoderms per hour,
or even 1,500 picoderms or more in pregnancy.
So both female and male THX mice,
(28:18):
have the same relative ego by human physiological levels, level of ISTO.
But that makes a huge difference.
But in the overall differentiation of the immune system as well as in the immune
response once the whole immune system is differentiated.
(28:43):
I mean, a lot of people think of mice as pests, but really they could be helping
to save humanity. Oh, they always did.
Hundred years. And we need to think about that. But even more so now with this,
Yes, it sounds incredible.
But the fact is that those mice have a fully immune system, but a human cell
(29:09):
did not make it to the germline.
So they can reproduce, but they reproduce as mice.
It's the same immune-deficient mice.
So they have to be constructed. Humanized mice have to be constructed.
And this is why we have several triangles, meaning one male and two females,
(29:30):
and 10, 12 cages all the time, breeding those immune-deficient mice.
And then we keep crafting new pups.
And so we have quite a few at any time, quite a few THX mice.
Do they look different than other mice? Not really, actually.
(29:52):
And actually interesting, they are very healthy, and they live a normal mouse life.
We have constructed already 500 THX mice,
and we've never seen any pathological reaction or anything, any short life.
(30:17):
So that's good. are, I think, a question that one wants to ask, why estrogen?
Well, and that came from two things.
One, there is a lot of anecdotal literature going back quite a long time.
And no systematic study was ever made.
(30:40):
But it's well accepted that women mount a much better antibody response to viral
and bacterial vaccines than men.
And there are many papers, mostly anecdotal again, in not great journals, but it is accepted.
(31:05):
I certainly embraced it.
And the second point is that we have published three or four or five very basic
papers in the last many years,
particularly two or three in journal biochemistry or chemistry,
one in natural immunology in 2010 or 97.
(31:30):
And those were very basic papers in which we show that a transcription factor, which is all T4,
which is very important in the differentiation, development of the esophagus,
but it's also very important in the differentiation differentiation of B cells, or mammalian B cells,
(31:54):
and the regulation of the expression,
of EID, which at the moment, as I say, is critical.
The generation of mature antibodies, the IFA and the ATP.
And we saw that, we showed that clearly in those couple of papers,
(32:18):
that Oxy-4 is actually under regulation by E2, by estrogen, by estradiol E2.
And that E2 actually, after it binds ER, estrogen receptor alpha,
Alpha binds to the promoter of that gene, of oxy-4 gene, and promotes the expression of oxy-4.
(32:45):
And then oxy-4 promotes the expression of AID.
So that was the molecular underpinning for really going ahead with estradiol conditioning.
The fact that we could put E2 in water of coke made it very easy.
Patients who are vaccine-hesitant. Is that particularly frustrating for you
(33:09):
as well, how there's this, as we all know, people who may not understand,
and there's so much misinformation out there?
I have no words. I mean, that's no words. What can you say?
Yeah, it's just… I mean, to me,
(33:31):
the most troubling thing is that that belief or those beliefs are germane to
all sorts of different people,
well-educated people, wealthy people who have access to all sorts of information.
(33:52):
As much as other people. So that's the troubling, very, that's very troubling.
And it's so inspiring, though, to see what you're doing.
You worked for many years at the University of California. Yes.
And before that, you lived in Italy. That's where you're from. Yes.
(34:13):
I went to medical school in Milano, University of Milano. And you still go back
there? You have a house there?
Yes, I go back, again, not very often, But I'm lucky because this is a house
in the mountains, north of Milan. And I share it with my sister.
What do you love about Italy? Not sure what I love. I know you said you don't
(34:33):
miss it. You love living in Texas and the United States.
What I love about Italy are Ferraris, Maseratis, and not so much Lamborghinis.
But there is a whole history about all that. I, when I went to the,
(34:53):
probably a lot of us have been watching the Olympics and I, I went to the 2002
Olympics and went to a lot of the events and the award ceremonies and the houses of the athletes.
And I got to go to the, the Italian house.
And then I had dinner with some of the athletes, and it was my first time.
(35:14):
I was a lot younger then, but first time saying where I was so surprised when
I said, so do you want to move to America?
And they were just like, why would we want to do that? We love our country.
And I'm like, I thought most people wanted to move to America,
and I felt so embarrassed.
They're like, no, we're having a fun time, but we love our country.
And all the people did move to America. Thank you.
(35:37):
At the end of the 19th century, beginning of the 20th century,
they went to Ellis, Ireland. Yes.
Many Italians in Hong Kong, Ireland. Yes. Everywhere, actually,
in Chicago, California.
Bank of America was founded by two Italian brothers. Oh, really?
(35:58):
And here on Pediatrics Now, we love quotes. I love quotes.
You have a favorite quote that pertains to what we're talking about.
If you think that research is expensive, try disease.
Yes. That's a great one. And that was by? By Mary Lasker. And that's from the?
(36:19):
The same as the Lasker Award. Who is that person? Can you tell us about the person?
Well, actually, it's a very wealthy family.
And the Lasker Award has always been considered the American Nobel Award.
Many Laskar awardees went on to receive the Nobel Prize, and many didn't. Yeah.
(36:45):
Or several men did not.
Paolo, anything else you want to say about this that we didn't cover?
This has been such an honor, and it's so fascinating.
Thank you for your kind words.
I think that what you want to say is well women are better than men that's the
(37:09):
best demonstration it has been in the making.
Is there anything you want to say for the hope of the future,
well I think that a lot of things can be finally done in vivo in humans,
in mice not in humans but it's a human immune system Now, this is not the answer
(37:32):
to everything, but certainly, you know, like vaccine development,
again, checkpoint inhibitors, it's also important.
Many other monoclonal therapeutics.
It's quite incredible to me that my lab in New York was at the cutting edge
(37:55):
in the late 80s, early 90s, in making human monoclonal antibodies.
Today, 80% of the drugs under consideration by the FDA are human monoclonal
antibodies. Right, wow. It's incredible. Yes.
And, you know… That must feel good.
(38:16):
Yes and no, because back then I should have continued making human monogonal antibodies.
But it's interesting, that was also the time when computing power increased
exponentially at the beginning.
(38:37):
We believe that, well, you can model anything three-dimensionally.
Why do we need to go to the trouble of making human aminoclonal antibodies?
Once you know what the antigen is, you can design a molecule that can lock in,
need not be a monoclonal antibody.
(38:58):
And we were wrong. because in spite of
all the modeling and the structural crystallography and resolution you do,
at the end, almost 40 years later, 35 years later,
the truth is that nature does it right and easy.
(39:20):
We are back. We have been back making monochromatic bodies.
The modeling didn't get us anywhere.
Well, with respect, I should say, qualify my statement, didn't get us anywhere
in terms of coming up with molecules that have the precision,
(39:40):
the specificity, specificity and affinity of an antibody in neutralizing something,
in locking onto something, the antigen, again again, be it the whatever phylogenetic
structure or pathologist.
We went back to antibodies.
That's full circle.
(40:00):
I'm talking about the late 80s, early 90s, so 35 years ago.
We thought that, well, we had an incredible progress back then of structural
studies and modeling, and now we are again.
Again, that's incredible.
Monoclonal antibodies are used for, in my generation, we would think making
(40:28):
good vaccines, good antibodies, or good deuterized antibody therapeutics.
But in fact, monoclonal antibodies are used to over your cholesterol.
The most incredible application that one would never have thought of. It's incredible.
(40:49):
Anything else, Paolo, you want to mention about your next step or anything we didn't cover?
Yeah, my next step, I should be retiring at this point. So you are really going to retire? I'm not.
You're not going to? Everybody tells me.
Dr. Paolo Casali with the University of Texas Health Science Center at San Antonio,
(41:14):
thank you so much for being on Pediatrics Now.
Music.
Music.
(00:09):
I am so honored today because here in the podcast studio with me is Dr. Paolo Casali.
He's the University of Texas Asheville Smith Professor and Distinguished Research
Professor, Microbiology, Immunology, Molecular Genetics.
He's Professor of Medicine at the University of Texas Long School of Medicine.
(00:30):
Dr. Casali, first, thank you so much for being here in the podcast studio today.
My pleasure. So you and your team have accomplished something that is so amazing
and so interesting and will affect medicine for countless years to come.
So you all have created the first humanized immune system in mice.
(00:54):
Well, thank you for your kind words. Actually, this is not the first humanized mouse.
Humanized mice have been attempted to be constructed since the 80s,
the late 80s, when there was an urgent need to model in vivo the human immune response to HIV.
(01:23):
And so those were the first, so to say, humanized mice.
In fact, those precursors of humanized mice were very rudimental, very primitive.
And those mice never quite mounted a good immune response to antigens or viruses, whatever.
(01:54):
Immunogen. And this has been really the shortcoming of the different varieties
of humanized mice throughout the last many years.
And I think it's proper to say that our mice are the first fully humanized mice.
(02:17):
The first mice with essentially a 100% human So you and your team are the first
to create mice with a fully humanized immune system. And fully functional.
And fully functional. It's incredible.
So what does this mean for medicine?
(02:40):
Well, I think it's a simple and complex answer, but the first one was to define
what humanized minds mean.
And what it means is these are mice with a FURI, virtually, or FURI,
(03:03):
as one could argue here, human immune system.
So the mice are mice, but they are immunodeficient mice, virtually without immune system, so to say.
And this mice accept a graft consisting of human hematopoietic stem cells.
(03:28):
And these are the cells that will give rise, will proliferate and differentiate
and give rise to a whole human immune system.
And these stem cells are hematopoietic, meaning they are stem cells that will
(03:50):
give rise to cells of the immune systems,
but of the lymphoid lineage,
such as B and D lymphocytes, myeloid systems, such as granulocytes,
neutrophils, and macrophage.
And then once grafted in immunodeficient mice,
(04:14):
again, then within a number of weeks, you will have those mice fully constituted
in the immune system, functional immune system.
Now, how that is done, it's important to understand what the potential of these humanized mice is.
(04:34):
So it is done by crafting hematopoietic, human hematopoietic stem cells from
core blood, from neonatal core blood.
And so we take cold blood with the help of the OBGYN department.
(04:59):
Fresh cold blood. We purify the hematopoietic stem cells from that cold blood.
And then graft within 24 or 48 hours of birth this sort of special immunodeficient
mice those are pups, they are neonates,
(05:20):
and we graft those mice intracardically with a small insulin needle.
And the grafting is
done in the left ventricle to maximize the dissemination of stem cells throughout
(05:41):
all districts of the bodies so that those stem cells will become resident of
wherever bone marrow will develop.
Now, these are special immunodeficient mice in that they carry a mutation of
(06:02):
the CKIT receptor, and they are referred to as W41.
We did not generate these immune-efficient mice.
These mice have been available for about four years.
The interesting thing is that nobody really took advantage of them to...
(06:24):
To create immunized mice with a fully functional immune system.
More precisely, I should say that the two different groups,
one in Minnesota, the other one in Germany, who created these immune-efficient
(06:44):
mice with the same mutation,
W44 mutation, actually did craft them with human hematopoietic stem cells,
and they did quite full characterization of the developing immune cell elements.
But they didn't really do much of functional studies on the immune response
(07:14):
potential of these valleys. And this is what we have done.
What we have done, which has been different from any other humanized mouse,
has been the intracardiac grafting.
And importantly, the hormonal conditioning with estradiol, with estrogen, of male or female pups.
(07:44):
And this is what really has made a big difference in terms of the development of the immune system.
And the functioning and the differentiation, further differentiating, I should say,
further differentiation of immune cell elements once these mice were challenged,
(08:09):
were immunized with different conjugated optins, and those are main, main molecules.
But how many years in the making has this been? It has been five years.
Is this a dream that you've had for a long time to accomplish this?
Or how, Paolo, can you tell us about how this came about, how you got into this? How I got into this?
(08:32):
I think my age has played a major role in it, in that experience, I should say.
My many years of research in immunology have always been, has certainly always
been with the human immune system and immune response in mind.
(08:54):
But my research has always been very basic, very molecular,
very much into the cell, namely B cells, B lymphocytes, which are the cells
that make antibodies, and very much with a molecular genetic flavor,
very much about regulation of genes that encode antibodies.
(09:19):
Cross-reaching somatic permutation of those genes.
And with my aging, I became, I have become.
Much more determined and, I should say,
(09:41):
much more motivated to do something that will have an immediate relevance to vaccine development,
to the human antibody response,
but particularly antibody response,
because I always worked on B lymphocytes and, as I say, antibody genes.
And I should say that my persuasion is not only mine, has become stronger and
(10:09):
stronger than if you want a good vaccine,
you need to be able to generate a good antibody response, one that is very specific
and neutralizing a virus or a bacterium or whatever else or a toxin.
And so, once we have become more and more familiar with the intimate molecular
(10:37):
mechanism that underpins the generation of a mature antibody response,
again, one that matures, one that is highly specific and can neutralize microbial
agents, and one has certain features.
(11:01):
Local features in the antibodies.
And that is, again, those antibodies are generally IgG or IgA and they're generally,
or they're invariably actually,
somatically mutated in the variable region of the antibody molecule.
(11:22):
And this is what provides a substrate for the selection of high-affinity mutants,
sub-mutants, I should say,
in those B-cell groups that are selected by the antigen driving the response, such as the HA,
the glycoprotein spike of influenza virus,
(11:44):
or the surface antigen on bacteria, fragilin in salmonella, for instance.
And so the idea was, at this point, we need to be able to do.
Experiment in vitro in a 100% immune system.
(12:07):
Because if we can do that, then we are in the position of, finally,
of being able to do vaccine development.
Vaccine testing of drug, if not development, but certainly testing,
(12:29):
particularly, well, in any case, of immune modulators, such as checkpoint inhibitors
in the case of the antibody, the immune response to tumors,
solid tumors, and many other things that one cannot do.
To this day, if you develop a vaccine, the last step the FDA wants you to do
(12:56):
is efficacy before going even to phase one. in non-human primates.
Non-human primates are not exactly human.
They're very similar, very close to us, incredibly close to us,
but in many ways are not exactly human.
And this is perhaps not even the major obstacle in the use of human primates.
(13:23):
One problem with human primates is that they are so close to us that But you don't want,
I certainly don't, wouldn't want to do what has been done with human primates, with those primates,
non-human primates, for many years in the whole story of HIV, for instance.
(13:49):
Certainly, many things have been done with those non-human primates that shouldn't
have been done and which today are not acceptable and not done.
Because of cruelty? Yes.
And unnecessarily, unnecessary experiments.
So this will reduce the amount of testing that needs to be done on non-human primates.
(14:16):
Hopefully, if these mice turned out to be really what they are,
but I'm being very cautious here
but they turn out to be really what they seem to be and I think they are,
then you could do.
A lot of vaccine development because you're dealing with an in vivo human immune system.
(14:39):
And you cannot do that in humans for ethical reasons. Yes.
It will help dramatically pave the way for even better vaccines,
more targeted vaccines.
Yes. And then when it comes to tumors.
It's already been done with a few THX mice.
(15:00):
Targeted therapy? These are our mice. Here, actually, we are collaborating with
giving over those mice, our mice, to a couple of groups working with different tumors, solid tumors.
So this investigator grafted tumor tissue from humans into the mice and then
(15:23):
studied the overall reaction to that tumor.
And that reaction is human, be it inflammatory or immune, exquisitely immune-responsive.
Wow. So some investigators started doing that.
You worked, Paolo, for many years as an immunologist. And so instead of retiring
(15:48):
like a lot of people do, you really revved up your research and the urgency
to make a difference and get this done.
Yes. And the NIH offers experience and knowledge, but it also offers security.
Because I was always well-funded with the NIH. But at this point,
I could afford to do something that I was not funded for. Really?
(16:14):
That's amazing. Like, we're going to do it. I'm going to make this happen, right? That's right.
Can you tell for us a practical example in, say, for pediatrics?
Well, one thing that we did with these mice, again, we refer to them as THX
mice, three true human X mice.
(16:36):
This was not me, this was our development office.
So one thing that we already did, and it's published in this paper that you mentioned,
is we vaccinated these mice with the messenger RNA Pfizer COVID vaccine,
using an immunization schedule which is the same as for humans.
(16:59):
And the mice made a neutralizing, strong antibody response neutralizing to SARS-CoV-2.
And those are human antibodies, obviously. And we analyze in details the mohawk
genetic makeup and mohawk composition of that response.
(17:19):
So again, that's good.
We did the The same, and that's published as well, with purified Fragilin from Salmonella.
We immunized those mice with Fragilin and showed that they mounted a neutralizing
antibody response to Salmonella,
neutralizing as assessed by in vitro the classic colony neutralization in petri dishes.
(17:47):
But then we also show that the THX mice vaccinated with flagellin survived,
fought and survived infectionally like flagellin.
The non-vaccinated ones died.
So the neutralizing response mounted by the vaccinated mice died.
(18:14):
It was a protective response, protecting from infection. Wow. Those mice survived.
I lost a little weight. Wow. They made it. Wow. The other ones, I, all of them died.
And, Paolu, I'm imagining it when all the scientists at NASA and the landing
(18:36):
is happening in space and there's lots of excitement.
When you all realized you had done this, can you describe for me what it was
like or what the feeling was like?
Or is it dramatic like that? Or it's more you're in the lab, you knew it was coming?
I'm trying to imagine the moment when you realized this is, we did it. That's a good question.
(19:00):
The climax or the outcome of the death was tempered by the fact that this is so labor intensive.
They went front and get there. It's okay.
It's so tedious, so labor-intensive. Labor-intensive, yeah. Can you tell me
what you were feeling when you realized?
(19:21):
That's, in many ways, what's expected.
So, you know, science these days, particularly biomedical science,
it's very incremental in progress.
So it's very different from 50 or 80 years ago. You knew the rocket was going to get there. Right.
(19:44):
So do you, what's the next step? What's the plan from here now that? That's a good question.
We are trying to do different things using DHX-Vis.
Certainly the decision has been made on my part to do any experiment from now
(20:07):
on in THX rise so there are different things we are doing we are uh.
We have identified actually a transcription factor that is critical for the
maturation of the antibody response.
It's critical for the expression of AID, activation-induced cytodiaminase,
(20:30):
which acts on DNA and divert
the DNA repair machinery of B lymphocytes to act, to effect class switch DNA recombination,
and somatic hypermutation.
I say divert, I say it diverts, but in fact it's a physiological process.
(20:54):
It diverts the physiological process of DNA repair,
which comes into place, comes into being in a major way when you go to the beach and you're in the sun,
and all the DNA repair over your body.
(21:14):
So that process is physiological, but a process in bilinphocytes that enables
the maturation or the affinity maturation of those antibodies,
meaning enables the generation of submutants with a much higher specificity for the antigen.
(21:37):
Again, be it viral glycoprotein or bacterial components.
Toxin, bacterial toxin. That process is physiological as well,
but it's really a diversity, diverting of the more general process ongoing in your body all the time.
(21:59):
And whenever there is a mutation, it needs to be corrected in the DNA.
So we have been working on that, and we have been working on that in vitro, as well as in mice.
And now, for the last, latest stages, of that overall investigation.
We are using THX mice.
So whatever we will find,
(22:24):
actually whatever we are proving, attempting to prove, will be even more relevant
because it will be in a human system in vivo.
That's for instance one thing. There are other things. We have identified a
transcription factor that seems to be critical for the maturation,
(22:48):
the generation, I should say, of mammary B lymphocytes.
Mammary B cells or B lymphocytes, we refer to them as immunologists as B cells.
Those B cells, the generation of those B cells is critical for the effectiveness of a vaccine.
(23:09):
If a vaccine cannot induce degeneration on memory B cells, then it's not a good vaccine.
It's not a vaccine because those are the cells.
After the vaccination, will persist in your body for months and years,
and they will be recalled, reactivated, when the same antigen comes in again.
(23:34):
For instance, if those B cells are generating response to influenza vaccine,
influenza virus vaccine, and then in a couple of years, you will come across that virus.
That virus will reactivate those cells. Those cells will proliferate and further
differentiate and make tons of antibodies to the virus.
(24:00):
Wow. So understanding, getting
to know what are the critical transcription factors and what are the other factors
that are critical for the generation of memory B cells is critical for vaccine development.
(24:20):
In the future, do you see other humanized organs in mice?
I mean, are we going to have a fully humanized mouse?
Or what is the future? It sounds like science fiction, but it's medical science now.
Well, it's interesting. That's a very interesting question.
In fact, prior to the development of our mice, which because of hormonal conditioning,
(24:48):
And I think actually that deserves a few words.
Because of monoconditioning, they develop a fully human immune system.
Prior to that, different attempts were made to graft,
again, earlier humanized mice with human fragments of human thymus,
(25:10):
for ISA to promote the T-cell differentiation of those mice or graft other.
Other fragments of other organs in the attempt of, again, promoting a better
differentiation of the immune system.
(25:32):
So one could speculate, or it's not a big speculation, that you could graft
actually organs or pieces of organs, fragments of organs, such as liver or something else, human.
So that in this case, you would study as targets of the immune immune system
(25:57):
or the human immune system,
targets of autoantibody response, SIS.
We show that our mice can mount an autoimmune response, which is really to all
effects similar as the autoimmune response in lupus, human lupus.
(26:19):
So one can certainly foresee that. But I want to go back to the hormonal conditioning.
The hormonal conditioning that is critical for the full development of the human
immune system in THX mice is in fact estrogen condition.
So these mice, once they're grafted, they even rise to a certain extent to all immune element,
(26:51):
which happens at the age of 20 weeks, 18, 20 weeks,
then the mice are put on estradiol E2, estradiol is E2, and that's the most
potent and the most abundant.
(27:13):
Humans, particularly in females, and we put E2 in water, so the mice,
the little mice drink water and they get E2.
Now interesting, the blood concentration of E2 that derive, that are generated
(27:33):
by the drinking E2 in water at a very small concentration,
they're actually very physiological for humans.
In female or male THX mice, adult THX mice, the blood concentration we do is
around 60-70 picolams per ml.
(27:54):
In women, depending on the different stages of the cycle,
the blood concentration is from from 30 to 500 picoderms per hour,
or even 1,500 picoderms or more in pregnancy.
So both female and male THX mice,
(28:18):
have the same relative ego by human physiological levels, level of ISTO.
But that makes a huge difference.
But in the overall differentiation of the immune system as well as in the immune
response once the whole immune system is differentiated.
(28:43):
I mean, a lot of people think of mice as pests, but really they could be helping
to save humanity. Oh, they always did.
Hundred years. And we need to think about that. But even more so now with this,
Yes, it sounds incredible.
But the fact is that those mice have a fully immune system, but a human cell
(29:09):
did not make it to the germline.
So they can reproduce, but they reproduce as mice.
It's the same immune-deficient mice.
So they have to be constructed. Humanized mice have to be constructed.
And this is why we have several triangles, meaning one male and two females,
(29:30):
and 10, 12 cages all the time, breeding those immune-deficient mice.
And then we keep crafting new pups.
And so we have quite a few at any time, quite a few THX mice.
Do they look different than other mice? Not really, actually.
(29:52):
And actually interesting, they are very healthy, and they live a normal mouse life.
We have constructed already 500 THX mice,
and we've never seen any pathological reaction or anything, any short life.
(30:17):
So that's good. are, I think, a question that one wants to ask, why estrogen?
Well, and that came from two things.
One, there is a lot of anecdotal literature going back quite a long time.
And no systematic study was ever made.
(30:40):
But it's well accepted that women mount a much better antibody response to viral
and bacterial vaccines than men.
And there are many papers, mostly anecdotal again, in not great journals, but it is accepted.
(31:05):
I certainly embraced it.
And the second point is that we have published three or four or five very basic
papers in the last many years,
particularly two or three in journal biochemistry or chemistry,
one in natural immunology in 2010 or 97.
(31:30):
And those were very basic papers in which we show that a transcription factor, which is all T4,
which is very important in the differentiation, development of the esophagus,
but it's also very important in the differentiation differentiation of B cells, or mammalian B cells,
(31:54):
and the regulation of the expression,
of EID, which at the moment, as I say, is critical.
The generation of mature antibodies, the IFA and the ATP.
And we saw that, we showed that clearly in those couple of papers,
(32:18):
that Oxy-4 is actually under regulation by E2, by estrogen, by estradiol E2.
And that E2 actually, after it binds ER, estrogen receptor alpha,
Alpha binds to the promoter of that gene, of oxy-4 gene, and promotes the expression of oxy-4.
(32:45):
And then oxy-4 promotes the expression of AID.
So that was the molecular underpinning for really going ahead with estradiol conditioning.
The fact that we could put E2 in water of coke made it very easy.
Patients who are vaccine-hesitant. Is that particularly frustrating for you
(33:09):
as well, how there's this, as we all know, people who may not understand,
and there's so much misinformation out there?
I have no words. I mean, that's no words. What can you say?
Yeah, it's just… I mean, to me,
(33:31):
the most troubling thing is that that belief or those beliefs are germane to
all sorts of different people,
well-educated people, wealthy people who have access to all sorts of information.
(33:52):
As much as other people. So that's the troubling, very, that's very troubling.
And it's so inspiring, though, to see what you're doing.
You worked for many years at the University of California. Yes.
And before that, you lived in Italy. That's where you're from. Yes.
(34:13):
I went to medical school in Milano, University of Milano. And you still go back
there? You have a house there?
Yes, I go back, again, not very often, But I'm lucky because this is a house
in the mountains, north of Milan. And I share it with my sister.
What do you love about Italy? Not sure what I love. I know you said you don't
(34:33):
miss it. You love living in Texas and the United States.
What I love about Italy are Ferraris, Maseratis, and not so much Lamborghinis.
But there is a whole history about all that. I, when I went to the,
(34:53):
probably a lot of us have been watching the Olympics and I, I went to the 2002
Olympics and went to a lot of the events and the award ceremonies and the houses of the athletes.
And I got to go to the, the Italian house.
And then I had dinner with some of the athletes, and it was my first time.
(35:14):
I was a lot younger then, but first time saying where I was so surprised when
I said, so do you want to move to America?
And they were just like, why would we want to do that? We love our country.
And I'm like, I thought most people wanted to move to America,
and I felt so embarrassed.
They're like, no, we're having a fun time, but we love our country.
And all the people did move to America. Thank you.
(35:37):
At the end of the 19th century, beginning of the 20th century,
they went to Ellis, Ireland. Yes.
Many Italians in Hong Kong, Ireland. Yes. Everywhere, actually,
in Chicago, California.
Bank of America was founded by two Italian brothers. Oh, really?
(35:58):
And here on Pediatrics Now, we love quotes. I love quotes.
You have a favorite quote that pertains to what we're talking about.
If you think that research is expensive, try disease.
Yes. That's a great one. And that was by? By Mary Lasker. And that's from the?
(36:19):
The same as the Lasker Award. Who is that person? Can you tell us about the person?
Well, actually, it's a very wealthy family.
And the Lasker Award has always been considered the American Nobel Award.
Many Laskar awardees went on to receive the Nobel Prize, and many didn't. Yeah.
(36:45):
Or several men did not.
Paolo, anything else you want to say about this that we didn't cover?
This has been such an honor, and it's so fascinating.
Thank you for your kind words.
I think that what you want to say is well women are better than men that's the
(37:09):
best demonstration it has been in the making.
Is there anything you want to say for the hope of the future,
well I think that a lot of things can be finally done in vivo in humans,
in mice not in humans but it's a human immune system Now, this is not the answer
(37:32):
to everything, but certainly, you know, like vaccine development,
again, checkpoint inhibitors, it's also important.
Many other monoclonal therapeutics.
It's quite incredible to me that my lab in New York was at the cutting edge
(37:55):
in the late 80s, early 90s, in making human monoclonal antibodies.
Today, 80% of the drugs under consideration by the FDA are human monoclonal
antibodies. Right, wow. It's incredible. Yes.
And, you know… That must feel good.
(38:16):
Yes and no, because back then I should have continued making human monogonal antibodies.
But it's interesting, that was also the time when computing power increased
exponentially at the beginning.
(38:37):
We believe that, well, you can model anything three-dimensionally.
Why do we need to go to the trouble of making human aminoclonal antibodies?
Once you know what the antigen is, you can design a molecule that can lock in,
need not be a monoclonal antibody.
(38:58):
And we were wrong. because in spite of
all the modeling and the structural crystallography and resolution you do,
at the end, almost 40 years later, 35 years later,
the truth is that nature does it right and easy.
(39:20):
We are back. We have been back making monochromatic bodies.
The modeling didn't get us anywhere.
Well, with respect, I should say, qualify my statement, didn't get us anywhere
in terms of coming up with molecules that have the precision,
(39:40):
the specificity, specificity and affinity of an antibody in neutralizing something,
in locking onto something, the antigen, again again, be it the whatever phylogenetic
structure or pathologist.
We went back to antibodies.
That's full circle.
(40:00):
I'm talking about the late 80s, early 90s, so 35 years ago.
We thought that, well, we had an incredible progress back then of structural
studies and modeling, and now we are again.
Again, that's incredible.
Monoclonal antibodies are used for, in my generation, we would think making
(40:28):
good vaccines, good antibodies, or good deuterized antibody therapeutics.
But in fact, monoclonal antibodies are used to over your cholesterol.
The most incredible application that one would never have thought of. It's incredible.
(40:49):
Anything else, Paolo, you want to mention about your next step or anything we didn't cover?
Yeah, my next step, I should be retiring at this point. So you are really going to retire? I'm not.
You're not going to? Everybody tells me.
Dr. Paolo Casali with the University of Texas Health Science Center at San Antonio,
(41:14):
thank you so much for being on Pediatrics Now.
Music.
Popular Podcasts
On Purpose with Jay Shetty
I’m Jay Shetty host of On Purpose the worlds #1 Mental Health podcast and I’m so grateful you found us. I started this podcast 5 years ago to invite you into conversations and workshops that are designed to help make you happier, healthier and more healed. I believe that when you (yes you) feel seen, heard and understood you’re able to deal with relationship struggles, work challenges and life’s ups and downs with more ease and grace. I interview experts, celebrities, thought leaders and athletes so that we can grow our mindset, build better habits and uncover a side of them we’ve never seen before. New episodes every Monday and Friday. Your support means the world to me and I don’t take it for granted — click the follow button and leave a review to help us spread the love with On Purpose. I can’t wait for you to listen to your first or 500th episode!
Stuff You Should Know
If you've ever wanted to know about champagne, satanism, the Stonewall Uprising, chaos theory, LSD, El Nino, true crime and Rosa Parks, then look no further. Josh and Chuck have you covered.
Dateline NBC
Current and classic episodes, featuring compelling true-crime mysteries, powerful documentaries and in-depth investigations. Follow now to get the latest episodes of Dateline NBC completely free, or subscribe to Dateline Premium for ad-free listening and exclusive bonus content: DatelinePremium.com