It was 2001, immunologist Barbara Vilen's first full year as an assistant professor at the University of North Carolina (UNC), Chapel Hill's School of Medicine and she had already earned a seed grant from the Lupus Research Institute (LRI), a patient-funded research foundation. Each year, LRI grant recipients gather to report results and exchange ideas not only with other scientists but also with patients. At Vilen's first such meeting, she noticed one lupus patient listening intently to each talk. At the end of the meeting, the patient presented a check to the foundation--a tangible result of a year of door-to-door fundraising in her community.
"How can you study basic science mechanisms [in humans] when you don't have enough cells? The only way I can see to do that is to take what we have in the mouse and ask, Does it happen in humans? ... If we're right, we're golden; if we're wrong, you get a bad track record. We're taking a risk. We might be wrong, but at this point we need to at least try." --Barbara Vilen, UNC
"She's etched in my mind," Vilen says. "It was so important to her that we figure something out. How could you leave there and not want to figure something out? It put a face to the disease."
Vilen planned to continue the work she had started during her postdoctoral research in John Cambier's lab at the National Jewish Medical and Research Center in Denver studying the basic signal transduction mechanisms of B cells, the cells that produce antibodies to attack foreign antigens--or in the case of lupus, the body's own cell parts. She had already invested years of training in learning about basic molecular mechanisms and how to study them.
But Vilen couldn't shake the image of that patient seeking treatments for a disease that had defied all attempts at targeted treatments and cures. In time, that encounter with a patient would inspire her to shift away from basic molecular mechanisms and toward work that could be applied to human patients. It's a risky shift and not an easy one, given that Vilen has training and a track record in mouse studies and neither in human research. She's in the midst of that transition, which has presented challenges and satisfactions she never imagined as a basic scientist.
The complexity of lupus makes the disease difficult to study. In lupus patients, so-called self-antigens--nucleic acids and proteins found in every cell's nucleus--trigger the creation of antibodies that attack otherwise healthy tissue. But that relatively straightforward picture is clouded by the substantial patient-to-patient variations. For example, patients with antibodies to the so-called Smith Antigen, a ribonucleoprotein complex, tend to have more severe disease symptoms than patients without these antibodies. Adding to the difficulty is the fact that the symptoms are intermittent: Patients might present at the clinic one week feeling fine, but the next week have a "flare"--a sudden worsening that can include swollen, painful joints, fever, rashes, and hair loss.
Scientists know that faulty B cells are the central culprit in the disease, but why and how these cells lose their tolerance to the body's own cells and begin to produce self-antibodies is not well understood. Most immunologists focused on lupus had been exploring what was happening inside the B cell; numerous labs were dissecting its signal transduction pathways. Vilen decided instead to examine what B cells are doing in the secondary lymphoid organs--the lymph nodes and spleen. It is known that B cells spend a lot of their time in specific compartments within these organs. Vilen wanted to know what was happening there.
Vilen's lab focused on B cell interactions with macrophages and myeloid dendritic cells, work it did initially in a mouse model. Macrophages and myeloid dendritic cells are among the body's cellular first responders, part of the body's innate immune response to infection. They act in parallel to the body's adaptive immune system, which produces antibodies for long-lasting immune protection, to control the spread of infection. Macrophages and dendritic cells react to antigens by releasing inflammatory cytokines such at interleukin-6 and tumor necrosis factor alpha. Vilen's group showed that, in addition to producing inflammation, these cytokines can shut down the B cells' ability to transform into antibody-secreting cells, which is their key immunological function.
Furthermore, by comparing healthy mice with mice that contain the mouse equivalent of lupus, they discovered that in a normal immune system this B cell-regulatory pathway occurred only when the immune response was activated by self-antigens--and not when it was stimulated by bacterial or viral antigens. They had found a new pathway that stopped autoimmunity cold. In lupus patients, Vilen hypothesized, something must have gone wrong with this normal shutdown mechanism.
"That was a day of joy in the lab," Vilen says. "We showed that you could have an innate immune response, and the naive B cells go on to make neutralizing antibody, and the autoreactive B cells at the same time are selectively shut off so you don't get autoimmunity."
With her new interest in application, Vilen wasn't content just to write up her results for a scientific journal; she also applied for patents. She has already received one patent, for using the ideas generated in this research to supress B cell activation in lupus, and has another patent pending. The idea is to override the malfunctioning signals that allow self-antigens to activate B cells.
In addition, as a basic scientist, Vilen might have continued to pursue this research in mice. But her desire to apply her findings to human lupus patients inspired her to test how her lab's findings applied to people by studying the behavior of lupus patients' B cells and their interactions with myeloid dendritic cells in culture.
But the move into humans has proven to be a complex endeavor and, for Vilen, unfamiliar territory. Questions about whom to study became a major issue. "Do we want newly diagnosed patients who've never been on meds? Do we want active patients on meds who are having a flare? Do we want patients who are in remission and are on meds?" she says. "This is when we realized we need a clinical collaborator."
So in 2008, Vilen teamed with UNC rheumatologist Robert Roubey to seek his help in answering such questions--and also to get access to patient blood samples. At about that time, Ph.D. student Shannon Jones also joined Vilen's team. Jones had recently started a then-new program at UNC Chapel Hill to introduce Ph.D. students to clinical medicine, funded by the Howard Hughes Medical Institute's Med into Grad initiative.
Jones had direct experience with the disease. "My aunt has lupus," Jones says. "I saw what she went through. Now she's been on corticosteroids [nonspecific anti-inflammatory drugs] for years, and it's taking a toll. We need something else."
One of the first lessons the scientists learned was that patients don't like having their blood drawn--especially patients who aren't feeling well to begin with. "Sick people don't really like to be poked at," Vilen says. "And I agree, I wouldn't want to be poked at either." But to study B cells circulating in the blood stream, they needed at least 60 to 100 ml of blood to retrieve enough B cells to determine whether lupus patients have disruptions to the newly discovered signaling pathway.
Patient samples, they realized, are a precious resource and not easy to obtain. So before they started asking patients to submit to a regular blood draw, they decided to take a step back, to think more carefully about the biology of the disease and how the B cell behavior might be affected by variables such as the stage of a patient's disease and what medications the patients are taking. Those discussions are currently under way.
Along with defining the patient population it wants to study, the group has been doing parallel work in mice to better define the significant components of B cell self-antigen recognition.
For example, one of the challenges of trying to move from mouse studies to human studies, Vilen learned, is that the molecular players are different. For instance, a literature search revealed that the compelling mouse data would not translate directly to humans because the B cell receptor the researchers studied in mice, toll-like receptor (TLR) 4, is not very active in human B cells. Instead, human B cells in the lymphoid organs signal mainly through TLR7 and TLR9.
To work out the cast of characters relevant to human macrophage B cell regulation, a second translational research graduate student, Amanda Wisz, joined the group to study TLR7 and TLR9 in mouse models and then in normal human volunteers. "I'm trying to find out if those cell-mediated mechanisms will work on seven and nine and what will the factors be; will they be soluble, like with TLR 4?," Wisz wonders.
Wisz is also shadowing Roubey in the clinic. "He focuses a lot on trying to teach me about clinical presentation and diagnosis," Wisz says. "That I think will be helpful in classifying what kind of patients we'll need" for the clinical phase of their research. Eventually, Vilen and Wisz want to see if TLR7 and TLR9 are dysregulated in lupus patients.
"There's a lot of phenomenology, characterization, and observational studies going on [in human lupus research], and that's important," Vilen says. "But we need to now get down to something nitty-gritty, and how are we going to do that? How can you study basic science mechanisms [in humans] when you don't have enough cells? The only way I can see to do that is to take what we have in the mouse and ask, Does it happen in humans? ... If we're right, we're golden; if we're wrong, you get a bad track record. We're taking a risk. We might be wrong, but at this point we need to at least try."
The patients are counting on it.
Karyn Hede is a freelance writer in Chapel Hill, North Carolina.