With Basic Biology and Clinical Savvy, Scientists Solve A Mystery in The Nose

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In her basic science laboratory at the Johns Hopkins University School of Medicine, Jean Kim, M.D., Ph.D., had just discovered extraordinarily high levels of a protein in the nose. It was 2006, a little over 10 years after Kim began her work at Johns Hopkins as a surgeon scientist specializing in sinus surgeries and studying the root of her patients’ chronic inflammation in sinus tissue. Despite her years of expertise, Kim was puzzled by this protein’s overexpression in the nose. But the new finding sparked hope that she could find a new treatment for her patients.

The protein Kim discovered is produced by the gene DMBT1 (deleted in malignant brain tumors 1), which makes a wide variety of proteins in the body with diverse names and functions, and is present in other parts of the airway.

DMBT1’s appearance in the tissue of nasal polyps was confusing. Why was it present at such high levels, and what was its connection to the highly inflamed tissue in the nose and nasal polyps?

The structure of DMBT1’s protein was also puzzling, with its mosaic of sugars and other molecules on its surface, the purposes of which were largely unknown at the time. Kim would have to wait 15 years and rally a multidisciplinary team of scientists to unlock the mysteries of DMBT1.

Perseverance and Collaboration

Science is all about the relentless pursuit of truths, big and small. On one of the smallest stages of the body, at the levels of proteins and cells, searching for these truths is a huge task, often requiring years of study and dedication. Even then, findings can be elusive.

When breakthroughs do occur, the discoveries create a ripple effect in scientific and clinical communities. Every clinical finding, trial or new treatment is propped up on the foundations of basic science discoveries that reveal how the smallest parts of the body function.

Collaboration among basic scientists and clinicians, as well as access to human tissues, is crucial in research that seeks answers about how the body works. Teamwork with scientists who have different specialties often brings new perspectives to a research project, leading to creative solutions in the face of difficult work.

In the case of Kim’s research on DMBT1, partnerships with colleagues from Johns Hopkins research departments would help solve her protein mystery.

It’s an example of how cross-disciplinary work among basic research scientists and clinicians is vital to find the root of health problems.

Kim’s interest in treating and studying inflammatory airway diseases is long-lived. After receiving doctorates from the University of Pittsburgh School of Medicine, Kim completed a five-year residency in otolaryngology–head and neck surgery at the Johns Hopkins University School of Medicine where she found a niche in clinical and translational research.

She became laser focused on finding the cause of chronic rhinosinusitis and developing better treatments for it.

“It’s important for me as a clinician to give my patients the best treatment I can,” says Kim. “And to improve treatment, I knew I needed to find out how and why these conditions occur.”

Everyone has experienced the minor and temporary discomfort of a stuffy nose, but a long-lasting plugged nose is nothing to sneeze at. When a stuffy nose lasts for months or years at a time, it could be chronic rhinosinusitis. Difficulty breathing and excess mucus drainage are just two of the symptoms that come with this sort of congestion.

It’s estimated that up to 15% of the U.S. adult population has the condition. When accompanied by nasal polyps — a more severe type of chronic rhinosinusitis that occurs in approximately 3% of U.S. adults — the condition can cause more serious health issues such as sleep apnea and asthmalike symptoms.

While chronic rhinosinusitis is far more problematic than a stuffy nose, the inflammation that often accompanies common colds and allergies offers clues to the condition’s origins. The “puffed up,” or inflamed, tissues are often signs that immune cells in our nose and sinus tissue are going haywire. This inflammation causes symptoms such as congestion, runny nose, breathing difficulties and excess mucus.

Immune cells known as eosinophils can make sinus inflammation last longer, turning a simple stuffy nose into chronic rhinosinusitis.

Corticosteroids are often used to try to treat the condition by decreasing inflammation, but these drugs aren’t advised for long-term use.

A Need for Different Research Perspectives

Kim knew from her years of studying chronic rhinosinusitis with nasal polyps that her patients deserved more effective treatments. First, she had to identify possible causes of the disease, and to do that, Kim turned to perspectives from researchers in different fields who were also interested in airway inflammation.

“To succeed in biomedical science, we need to be inherently multidisciplinary,” says Andrew Ewald, Ph. D., director of the Department of Cell Biology at the Johns Hopkins University School of Medicine. “As we try to understand these complex processes, we can benefit from researchers with different skill sets and ideas.”

Ewald, who studies the spread of cancer, has collaborated with clinicians and other basic scientists alike during his years at Johns Hopkins. Many of these collaborations in cancer research are forged through formalized programs based on research interests that span Johns Hopkins departments and schools. For example, the cancer invasion and metastasis program at the Sidney Kimmel Comprehensive Cancer Center — a group that Ewald co-leads — has 42 members from 16 departments across three schools. Ewald says he has seen this model create collaborations and new research projects among clinicians, basic research scientists and public health experts.

“It can be hard to figure out how your research fits into larger bodies of research, like cancer, if you’re working with one molecule,” says Ewald. He admires the cross-collaboration efforts in other Johns Hopkins departments, though he says it would wonderful to see additional initiatives linking researchers together like the cancer invasion and metastasis program.

The Chance Encounter That Began the Collaboration of a Lifetime

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From left to right: Jean Kim, Anabel Alvarenga and Ron Schnaar

Still searching for answers about the tumor-suppressing protein created by DMBT1, Kim attended a seminar at Johns Hopkins on inflammation and eosinophils in the body’s airways. She had no idea she’d meet research collaborators who would bring her closer to answering why the gene’s protein lurked in nasal passageways.

Enter pharmacologist Ron Schnaar, Ph.D., and postdoctoral fellow Anabel Alvarenga, Ph.D., who changed the trajectory of Kim’s – and their own – interest in the proteins involved in airway inflammation.

An expert in investigating proteins linked to inflammation, Schnaar, a professor of pharmacology and molecular sciences at the Johns Hopkins University School of Medicine, has studied how these proteins interact with cells in airways and the nervous system. With decades of expertise in cellular biology, Schnaar’s recent projects focused on Siglec-8, a protein found on the surface of eosinophils, the inflammation-causing immune cells.

At the time of the seminar, Alvarenga was working toward her Ph.D. in glycobiology in Schnaar’s lab and had authored more than a dozen research articles on links between Siglec-8 and eosinophils.

“Our teamwork started so casually — Dr. Kim had a scientific question, we had our own inquiries, and we realized that we could help each other find answers,” says Alvarenga.

At the seminar, the three scientists struck up a conversation on their recent research, converging on inflammation in the airways caused by eosinophils. With different perspectives but shared research interests, Kim, Schnaar and Alvarenga began their collaboration. Schnaar and Alvarenga brought their basic scientist perspective as well as their expertise in protein research, while Kim’s perspective as a clinician provided insights on human physiology and the role of proteins in the body.

The team decided on more studies focusing on the protein Siglec-8, which Schnaar and Alvarenga had previously investigated, and its role in inflammation in the airways. They believed Siglec-8 was binding with another molecule to decrease inflammation in the airways.

At the time, the researchers were using asthma as a disease model in their hunt for information on Siglec-8. In asthma, eosinophils are overactive, causing inflammation in the airways and lungs that makes it difficult to breathe. Where the researchers found eosinophils, they also found Siglec-8.

Using lung and airway tissues from people with asthma who donated their tissues to research when they died, Schnaar and Alvarenga scoured the tissues microscopically, staining them for Siglec-8. Schnaar and Alvarenga had previously found the molecule that Siglec-8 latched onto, but in quantities so small that the molecule could not be isolated and identified. But Siglec-8 and its mysterious molecular companion were nowhere to be found on the surface of the airway tissue they observed.

“There were so many lab meetings where we had to report that the tests we were running just weren’t working,” says Alvarenga. “Our perseverance was tested.”

A Connection with the Clinic

Obtaining human tissue for testing is necessary for many basic scientists such as Schnaar and Alvarenga, but finding and getting the best kind of tissue can be a difficult process that requires collaboration among clinicians and basic scientists.

At the intersection of clinical and basic sciences at the Johns Hopkins University School of Medicine is the Division of Clinical Pharmacology, which helps pharmacology and biomedical researchers locate the right tissues for testing and links scientists to clinicians and other researchers with the same interests.

In this division is the drug development unit, directed by Craig Hendrix, M.D. The unit’s team of clinicians and scientists has helped hundreds of pharmacological research projects determine research methods for experiments and link researchers with human tissues that are approved and available to be studied. One of their major goals is to move research into clinical trials.

“We’re situated in the perfect position to help basic scientists move their research forward,” says Hendrix. “They bring their knowledge of molecules in the body, and we bring them the clinical resources they need to test their ideas and expand their research horizons.”

The drug development unit works closely with Johns Hopkins clinicians to responsibly and safely obtain human tissues from volunteers for experimental purposes in basic science and translational research. The biological samples include blood, urine and biopsied tissues from people who have allowed their samples to be studied.

For scientists in pharmacology who don’t have a background in medicine, the unit helps determine which human tissues are best to study based on a researcher’s unique project.

Namandje Bumpus, Ph.D., says the unit’s help in fostering collaboration among basic scientists and clinicians is important to understand the human body and develop new treatments.

“A lot of important progress is made in my field when you bring together folks with different expertise and get them to think about the same scientific idea,” says Bumpus, professor and director of the Department of Pharmacology and Molecular Sciences. “The clinicians we work with have such a good sense of what human tissues we should study.”

An Unexpected Turn

Kim’s experience working with sinus and airway tissue became critical for her research with Schnaar and Alvarenga. She developed a theory that her patients’ tissues held the key to unlocking the mystery of the molecule bound to the Siglec-8 protein. Because inflammation in the airways is similar to the inflammation of chronic rhinosinusitis with nasal polyps, it was possible that Kim’s patients had high Siglec-8 activity in their nose and sinuses.

“I suspected these patients made this protein like a factory,” she says. “It had to be in their tissues, and I had the ability to get Ron and Anabel the samples that could answer our questions.”

To test the team’s new theory, Kim acquired samples of nasal washes and nasal polyp tissue during pre-surgery care from her patients who had chronic rhinosinusitis with nasal polyps. The team doubled down on its search for Siglec-8 and the mystery molecule.

“Lo and behold, the samples lit up like a huge light bulb, and there it was: Siglec-8 ligand, wrapped up in a massive glycoprotein,” says Kim.

But why was Siglec-8 ligand missing from the lung tissue that Schnaar and Alvarenga previously studied? It turns out that the airway tissue had been prepared for storage in a manner that completely washed away Siglec-8 and the molecule it bound to. Kim says the work highlights the importance of obtaining and studying human tissue in its most natural form, directly from patients being treated for these conditions and free from artifacts caused by tissue processing and handling.

Kim’s nasal polyp tissue and nasal wash samples were not prepared in the same way, since they were fresh tissue.

The Protein Mystery Solved at Last

Using their expertise in protein analysis and isolation techniques, Schnaar and Alvarenga found that the mysterious molecule bound to Siglec-8 was a protein created by a gene that Kim was all too familiar with: DMBT1.

This year, Alvarenga, Kim and Schnaar published their findings that the protein made by DMBT1 and Siglec-8 formed a massive protein complex when bound together: DMBT1S8. It had evaded discovery for years because it looked very similar to, and was often mistaken for, another airway protein.

DMBT1’s protein, with a patchwork of sugars on its surface, has one sugar that acts as a weapon against the inflammation-causing eosinophils. The sugar can specifically target the Siglec-8 receptor on eosinophils.

Kim, Schnaar and Alvarenga discovered that when inflammation in the airways occurs due to eosinophil immune cells, the sugary part of DMBT1’s protein lock onto Siglec-8, binding with it to create DMBT1S8.

The protein complex DMBT1S8 then arrives to intercept the eosinophils and halt inflammation in the airways by setting off a chain of events that ends with the death of eosinophil cells. Of the many proteins throughout the body’s airways, DMBT1’s protein in this system is the only one that carries the proper sugars to shut down eosinophil activity.

The team had some answers at last, and Kim finally had an idea about why the DMBT1 protein showed up in nasal polyps all those years ago. The body responded to the inflammation from chronic rhinosinusitis by linking DMBT1 with Siglec-8 in an attempt to decrease the inflammation.

Kim’s search for possible treatments for her patients with chronic rhinosinusitis with nasal polyps wasn’t over, though.

Once again working with Schnaar and Alvarenga, Kim specifically looked for the combined DMBT1S8 molecule in patients with chronic rhinosinusitis.

By exclusively testing nasal washes and tissues from her patients, the team found that the DMBT1S8 levels were far higher in those who had chronic rhinosinusitis with nasal polyps than those who had chronic rhinosinusitis without nasal polyps.

The researchers also discovered that DMBT1S8 is likely to be found in a layer of the nasal polyp called the submucosal glands.

Looking Toward Future Collaborations

Kim is optimistic that future DMBT1S8 research, including learning how to control the protein complex, could provide long-term treatments that are more effective for patients with chronic rhinosinusitis with nasal polyps.

Kim and Alvarenga say they gained a great deal from their teamwork that they plan to carry forward to future research projects.

“I’ve learned so much from this research project,” says Kim. “Having a perspective of opening yourself up to the opinion and help of your colleagues not necessarily in your field is so important. In the end, we all want to find truth in our science and help people with our work.”