Dr. Laura Crotty Alexander is an associate professor of medicine at the University of California San Diego, and staff physician and researcher at the VA San Diego Healthcare System. Her research interests are primarily focused on E-cigarettes. She studies the impact of E-cigarettes on airway inflammation and innate immune function. We were able to sit down with Dr. Crotty Alexander to learn more about her research and get her thoughts on the field!
A: I bounced around quite a bit. As an undergraduate at Duke University, I did three years in a brain tumour immunology lab, mainly studying dendritic cells, macrophages, and cytokines, so that was my gateway into the world of immunology.
I spent a year at the NIH as part of the Clinical Research Training Program. I switched my focus to T cell receptors and antigen-presenting cells while there; trying to figure out where in the body our immune cells first start presenting antigens during an HIV infection, with the hope of targeting those areas for vaccine development.
I decided to pursue a fellowship in pulmonary and critical care medicine, so I ended up in the lab of Dr. Victor Nizet here at UCSD, focused on host defenses and innate immunity. I started studying bacterial virulence, looking at the roles of neutrophils and macrophages in fighting infections. That brought me into the world of inhalants because I started looking at the effect of cigarette smoke on bacteria.
The vast majority of the work that has been done shows that cigarette smoking is deleterious to human cells and human organs, but we are colonized by bacteria and actually have more bacteria in our body than we do mammalian cells. So, I began looking at the effect of cigarette smoke on the bacteria that colonize us and found that it makes them more pathogenic and aggressive.
At that time, I was seeing pulmonary patients in clinic who had never been able to quit smoking and who kept asking me “should I switch to E cigarettes?” … “Would that be a way to decrease my smoking or quit smoking?” and my response was, “I have no idea, we don’t know anything about these devices”, and then I realized that I was ideally set-up to answer those questions, and determine whether these novel nicotine delivery devices are safe for human use. So that is how I entered the E-cigarette field that now predominates my research.
A: I always struggle with that; I would say my area is the inflammatory effects of inhalants throughout the body.
I don’t like wasting organs, so even though I’m a lung doctor and I mainly focus on the cells of host defense, when we do a harvest on these mice, that we’ve exposed for weeks and months to E-cigarettes, we harvest almost every part of the body. I’ve developed collaborations with brain, cardiac, liver, renal, bone, and GI researchers, so that these mice can be optimized that when they give their lives, we get the maximum amount of information.
My favorite part about this research is that we don’t know what E-cigarettes will do to the body over time, and seeing the catastrophic effects of cigarette smoking that people didn’t realize for decades, it’s just really inspirational to be involved in figuring this out, so that people will be informed as to what the health effects of vaping will be. Also, our results may contribute to the design of safer products, which is also highly motivating.
A: I feel like over the past seven years the field has really developed. Seven years ago, there were just a few of us working in the field, but the number of E-cigarette researchers has expanded year after year.
There is research going on covering every part of potential toxicology, inflammation, organ dysfunction; there is some fascinating work going on about why people use them, why adolescents pick them up. There is all kinds of really cool stuff going on, I’d say it’s a pretty vibrant area research right now. Internationally and in the United States, there are a lot of researchers across the world trying to answer these questions.
A: Yes, I battle with this problem. As a scientist, things are not black and white, so it’s really hard for me to summarize things and say E-cigarettes are….”good” or “bad”.
I would say that when I first entered the field I was probably more like a lay person and really believed that the E-cigarettes were going to be significantly less harmful than conventional tobacco, just because conventional tobacco is so incredibly bad; how could anything come close to matching it? In our original studies it did look like it was going to be less than 10% of the health impact but this data was from cells in a dish and our short-term mouse exposure models.
Pretty much every year I have been convinced more, that E-cigarettes are a lot worse than I ever thought that they could be. The signals coming out are much stronger, in terms of direct adverse impact on human cells and on human organ function. I think that in the end, E-cigarettes are still not going to be as bad as conventional tobacco but unfortunately tobacco is a leading killer across the world. Making that comparison can be problematic, just because it makes them seem safe. So, I think that E-cigarettes are going to kill a significant number of people. They already have killed people, and they’re going to cause all kinds of damage across the body.
One of the things that I’ve been most impressed by, I really felt as a lung doctor that that’s where the major impact was going to be and it turns out that the lungs are pretty capable of responding to a stressful insult. We know that E-cigarette vaping causes a lot of different lung diseases but when we look at the lung under the microscope, pathologically, we see less signs of emphysema, less signs of fibrosis. What has really struck me is that when we look in the heart, the kidney, the brain, and the GI track there are huge changes in these distal organs due to vaping. It really shines a light on the fact that, yes, you are breathing in these chemicals, yes, they’re hitting lung first, but they cross into the bloodstream and impact the rest of your body.
A: It is incredibly hard to study. New devices are coming out every month, the new e-liquids are coming out every day, and you can’t test every single chemical that is in those e-liquids.
A: I started with the flexiVent back in 2009, so 11 years.
A: Absolutely, I would say of the projects that we do, 90% of them rely on using the inExpose and flexiVent systems. As we own two flexiVents and basically two inExpose systems, we have been able to share that with a lot of people; so beyond us, several people use both of those machines to forward their research too.
A: First, we were using the flexiVent for our asthma studies. We look at molecular pathways of pathogenesis in asthma in the hopes of identifying new therapeutic targets. Unfortunately, there are many asthmatics who do not respond to the current medications, so we’re always looking for new therapeutic modalities. The flexiVent really allowed us to look for effects of certain pathways on the airway’s reactivity.
The flexiVent is fantastic because not only does it measure airway resistance and lung compliance at baseline, but we can also do methacholine challenges. We nebulize increasing doses of methacholine over about a 20-minute period and we can determine if these asthmatic mice have more or less airway reactivity and thus understand whether the pathway we’re looking at is affecting asthma pathogenesis. That part of our work helped us identify HIF-1α and HIF-2α as playing a role in certain cell types in asthma.
Then we moved into the E-cigarette exposures. For that we use the inExpose system which allowed us to deliver the E-cigarette aerosols, first in the nose-only way, so that we could try and do a really clean look at what breathing in the E-cigarette aerosols does to the body. Then we then moved into using the whole-body enclosure system to reduce stress on the subjects and expose up to three times a day.
A: At our max we were basically running the system from 8AM to 5PM and we were just swapping out subjects every half an hour, to an hour throughout the day, so that is intense. It is also why we need a team. We usually have about 10 people in the lab, so that we can spread out the exposures to make these kinds of studies, which are sometimes six months long, feasible.
We used the SCIREQ system first for vape pens and then we moved into the box MOD E-cigarette device using the SCIREQ setup, and then we moved into the JUUL device.
The inExpose has allowed us to use all the different types of vaping devices. We design our own E-liquid and we also buy E-liquid, so that we’re testing commercially made products. For pretty much any E-cigarette that we’re testing we always assess lung function, especially for the first experiment that we do. Typically, we’ll do a week exposure, a month exposure, three months exposure, and then we put them on the flexiVent for full lung function mechanics. We also do methacholine challenges because there is a clinical signal that E-cigarettes increase asthma exacerbations and wheeze symptoms. We also assess lung compliance to see if the E-cigarettes are driving a parenchymal process like lung fibrosis or interstitial pneumonias. We use both SCIREQ systems for pretty much every E-cigarette experiment that we do.You use the inExpose for exposures and the flexiVent for lung mechanics, do you see SCIREQ equipment as an important part of your research?
A: On the asthma side we’re working with HIF-1α and HIF-2α knockouts in different cell lines. On the E-cigarette side we are using the KRAS knockout mouse, which spontaneously develops lung cancers. We are using this model to try to determine whether E cigarette aerosol exposure drives tumorigenesis or promotes tumor growth once the cancers are present, which have been two big questions.
The C57BL/6 mouse is so hardy, we haven’t we haven’t had any problems.
A: Luckily the KRAS mouse comes from one of our collaborators, who does lots of different work, so we just tell him when we would like to have mice and they tell us how many they have of that age.
The good thing about the inExpose system is that with the 16 openings for mice we will actually do staggered starts. We might start with one batch of mice one week, start them with controls, and then we add mice the next week. That can make harvest weeks much more manageable.
We tend to run three different experiments at the same time. If we run an exposure for three months then I want to use all 16 spaces. We pick three experiments we want to do and we have those mice at staggered starts but then we use the same exposures for that time.
A: I think when we have answered a question, that is probably the most satisfying thing. That satisfaction comes when we are looking at the data and we combine it all, run the right stats; that moment when you see a result that is significant, it’s a real finding. I think that is probably the greatest sense of joy.
Also, when trainees get excited about research. They do so much work and we’re training them every single day on the research front but when they actually get excited about asking and answering these questions. When they understand why we do this and they have that spark to want to continue doing research themselves, that’s also very satisfying.
A: Be patient. Keep a stiff upper lip. Give it time.
I don’t think anybody can decide whether they want to do research as a career over one year or two years. I would say you would need to give it at least two years. There are so many different types of research. I’ve come across people who worked for years in a bench research lab but then when they moved into clinical research they loved it, or somebody was in one particular bench lab and perhaps it wasn’t a great environment or the work didn’t click with them, but then and then they moved to another lab and really loved it.
So, I would say give it time and try out different areas before saying you’re not into research.
A: Right now we’ve shifted to a 90% focus on COVID. Unfortunately, we have had to stop all the E-cigarette work until we re-open. It’s probably going to be a few months out.
One thing that we’re really focusing on is the E-cigarette aerosol effects in the brain. We are examining some pathways in the brain and then continuing our inflammatory profiling of the heart, kidney, GI tract and lungs. We’re really hopeful that we’ll be able to put together a model of the effects of E-cigarette vapor exposure in the lungs and other organs.
A: We’re taking neutrophils that are circulating in the blood of COVID patients and running functional assays. Neutrophils cause so much collateral damage in the lungs, that I feel like it is likely to be one of the pathogenic mechanisms behind the acute lung injury in COVID.
Patients who are hospitalized with COVID have high inflammatory markers in the circulation. I feel like the circulating neutrophils of those patients are likely to be altered and I’m worried that as soon as they hit the lungs, if endothelial markers drive neutrophils infiltration, that they are going to go in, cause damage through oxidative bursts, and drive the disease.