This image of a section through the midface of a mouse embryo illustrates fusion of the tissues that form the secondary palate above the tongue. Green staining illustrates cells expressing a key enzyme that mediates DNA methylation, blue indicates nuclei of all cells, red indicates epithelial cells.

Cleft lip and palate are the most common craniofacial birth defects in humans, affecting more than 175,000 newborns around the world each year. Yet despite decades of research, it’s still not known what causes most cases or what can be done to prevent them. But a recent study from the University of Wisconsin School of Veterinary Medicine (SVM) has uncovered new information about orofacial development in mice that researchers believe could one day help reduce the risk of these birth defects in humans.

Published this week in the Proceedings of the National Academy of Sciences (PNAS) the study provides the first direct evidence of a mechanism called DNA methylation being required for craniofacial development. DNA methylation is a process where a group of molecules are added to DNA that change the expression of genes without actually altering the DNA sequence. It’s also affected by various environmental factors. The researchers discovered that disruption to DNA methylation interferes with development of the lip and palate and causes these birth defects in mice.

Rob Lipinski

Led by Robert Lipinski, associate professor of comparative biosciences at the UW School of Veterinary Medicine, the research is an important step toward developing preventive strategies that could one day lessen the risk of cleft lip and palate, known collectively as orofacial clefts (OFCs), in both animals and humans.

“We knew from past research that genetics and the environment interact to cause these types of birth defects, but our understanding of the environmental component lagged far behind that of genetics.” says Lipinski. “Unlike genetics, we don’t have a permanent record of the prenatal environment that can be examined retrospectively, but connecting OFCs to DNA methylation helps narrow our focus on the particular environmental influences that modify the risk for these types of birth defects.”

His team’s work confirmed the essential role of DNA methylation in regulating orofacial development during embryonic development and demonstrates how disruptions to that process alter the ability of stem cells to form the connective tissue of craniofacial bone and cartilage, resulting in OFCs.

Lipinski and his team arrived at these results by first genetically manipulating DNA methylation in two separate groups of mouse embryos. The experiments resulted in seemingly contradictory results, with OFCs developing in one group of mice, but not the other. To understand why there was a difference between the groups, the team conducted another round of experiments in which they inhibited DNA methylation in mouse embryos at different stages of development. The timing of when DNA methylation occurs was critical to the development of orofacial clefts.

They found that exposure on the 10th gestational day resulted in OFCs but administering the same inhibitor just 48 hours later resulted in normal orofacial development.

Identifying this narrow window of gestational sensitivity is important, Lipinski says, because it not only helps narrow the focus of the next stage of his team’s research but it will also help design future public education initiatives once more is known about the modifiable environmental and behavioral risk factors that impact OFC risk in humans.

The 10th gestational day in mouse embryos corresponds with the beginning of the 5th week of embryonic development in humans–a stage at which many pregnancies may not yet be recognized.

“We know DNA methylation can be influenced by a variety of environmental factors, including maternal stress, diet, and exposure to drugs, toxins and environmental pollutants, and having a better understanding how orofacial development is regulated by environmentally sensitive mechanisms could directly inform birth defect prevention strategies,” he says. “This next phase of our team’s research is focused on identifying specific factors that influence DNA methylation during orofacial development and which could therefore alter OFC risk.”

Lipinski and his team are uniquely positioned to pursue this next stage of research because of another important outcome of the study: a new in vitro model the team developed. The model will allow them to rapidly screen thousands of dietary and environmental factors in a laboratory dish before testing the impact of specific factors on cleft susceptibility in mouse models.

The results in cell and animal models will help the researchers more quickly and accurately identify factors likely to be of consequence to human development.

Orofacial clefts of the upper lip and palate affect approximately 1 in 700 newborns, and individuals with OFCs navigate feeding difficulties as infants that require multiple surgeries, dental procedures, and speech therapy during childhood and adolescence. Studies have shown higher mortality rates at all stages of life for individuals with OFCs.

This study was supported by funding from the National Institutes of Health under award numbers R03DE027162, R56DE030917, RO1DE032710, U01 DK11807, and R01DK099328, and T32ES007015. Additional support was also provided by the University of Wisconsin Hilldale Undergraduate Research Award.

It’s a significant step in efforts to help people with the disease, says UW–Madison Waisman Center senior scientist Tracy Hagemann, who led the study alongside Albee Messing, professor emeritus of comparative biosciences and founder of the Alexander Disease Lab. With University of Alabama at Birmingham colleague Michael Brenner, Messing discovered the gene responsible for Alexander disease more than 20 years ago.

People born with Alexander disease may develop an enlarged brain and head, experience seizures or delayed development, have stiffness in their arms and legs, and have intellectual disabilities. The disease, which involves destruction of the white matter of the brain, is often not diagnosed until symptoms are pronounced, says Hagemann.

Tracy Hagemann

Albee Messing

The new study, published Nov. 17 in Science Translational Medicine, provided preliminary data instrumental for a human clinical trial currently being led by Ionis Pharmaceuticals. Hagemann, Messing, and the Alexander Disease Lab are not directly involved.

However, working with Ionis Pharmaceuticals, the researchers developed a treatment that consists of small pieces of DNA called antisense oligonucleotides, which in their rat model was able to target mRNA in cells and tag the mRNA for destruction, effectively halting it from creating proteins.

One feature of Alexander disease is the formation of abnormal protein aggregates called Rosenthal fibers, caused by mutations in the gene that makes a protein called GFAP. The connection between this abnormal GFAP and the white matter destruction seen in Alexander disease is not yet clear, but changes in the protein are an intrinsic part of the disease in almost all cases.

Studies with a mouse model developed by Hagemann, Messing and their collaborators, and published three years ago, showed that antisense oligonucleotides were able to reduce GFAP and clear Rosenthal fibers. However, mice display only subtle symptoms of Alexander disease and researchers can’t measure important improvements in behavior or quality of life that may result from treatment.

The research team was able to develop a rat model that better represents the white matter damage and physical manifestations seen in humans. The model also provides better opportunities to assess symptom improvement in response to antisense oligonucleotide treatment.

A hallmark of Alexander disease is the buildup of abnormal proteins called Rosenthal fibers, pictured here (red) in brain tissue from a study of a rat model of the disease. The study identified a potential treatment and helped provide early data for a human clinical trial now underway. Tracy Hagemann et al.

“Alexander disease is considered a leukodystrophy, where white matter deficits develop, and we don’t see evidence of that or motor impairment in the mouse model,” says Hagemann. “So, for a preclinical model, the rats are much improved compared to the mice.”

The rats treated with antisense oligonucleotides before they developed major physical symptoms stayed virtually indistinguishable from their healthy littermates. When treatment began after the rats were severely impaired, their symptoms not only drastically improved; they also experienced a reversal in some of the damage to their white matter.

The antisense oligonucleotides, she explains, “clear out the GFAP aggregates (or Rosenthal fibers), and not only can we prevent the disease from happening by treating animals at an early stage before they’re really showing significant clinical signs, we can treat them when they’re at their worst and see reversal of some of the disease phenotypes.”

In people, Hagemann says, “we’ll be happy if we can stop the disease from progressing. But if you can actually see some reversal of symptoms that have already occurred, that would be wonderful.”

In addition to creating a foundation for clinical trials, the rat model has also paved the way to study aspects of the disease that are not yet understood, Hagemann notes, including the first opportunity to study the link between the GFAP mutations and white matter deficits in mammals.

These developments have been possible because of Messing’s extensive work on Alexander disease over the last 25 years, as well as the contributions of colleagues around the world, Hagemann adds. Messing’s “dedication and commitment to Alexander disease research has deepened our understanding of the disorder immensely.”

Study co-authors include Robert F. Berman of the University of California, Davis; Mel B. Feany at Brigham and Women’s Hospital, Harvard Medical School and Boston Children’s Hospital; and Ming-Der Perng of National Tsing Hua University. The study was supported by grants from the National Institutes of Health, including the National Institutes of Health’s Eunice Kennedy Shriver National Institute of Child Health and Human Development (HD076892, HD03352, HD090256, and HD103526) and the National Institute of Neurological Disorders and Stroke. It was also supported by Children Living with Inherited Metabolic Disease and the Juanma Fund. 

Mason Konsitzke, 7, plays in his bedroom at home in Stoughton. Photo: Jeff Miller

Mason Konsitzke is 7. He loves food (especially when he can share it with others) and anything military (both of his grandfathers served). He likes to fly kites and play with his 5-year-old sister, Alexandra. But Mason was born with a disease called neurofibromatosis type 1, or NF1, and each day can present new challenges for him and his family.

NF1 is a genetic disease caused by changes, or mutations, to a single gene in the human DNA library. Roughly one out of 3,000 babies born in the United States have the disease. That’s more than three times as many as have cystic fibrosis. Yet few people have ever heard of NF1.

Mutations in the NF1 gene cause defects in the neurofibromin 1 protein, which acts as a tumor suppressor. Children with NF1 can develop painful tumors along their nerve tracts, including their skin and in their eyes. Sometimes this renders them blind. They are often diagnosed with autism spectrum disorder, though not all children with NF1 are also autistic, and they are sometimes diagnosed with attention deficit hyperactivity disorder. They may have soft bones that bend and break. They are at a higher risk for cancer. And there is no cure.

It was not a disease Mason’s parents, Charles and Malia Konsitzke, had ever heard of. As a newborn, he was healthy. But when Mason was 6 months old, the couple began to suspect something was wrong. Mason developed coffee-and-cream-colored spots all over his body. His father later learned these were a hallmark of NF1. Mason received a genetic diagnosis of the disorder just before his first birthday.

“We were like deer in the headlights,” Malia says. “We were in shock, wondering, what does this mean for us? What does it mean for Mason?”

At 18 months, Mason began to lose his ability to speak. He was falling over, screaming constantly and deliberately banging his head. That’s when an MRI revealed a tumor called a plexiform neurofibroma in a mesh of nerves in the left side of his face. It was growing fast.

A father turns to science

Charles “Chuck” Konsitzke is the associate director of UW-Madison’s Biotechnology Center, a sort of one-stop shop for scientists in need of DNA sequencing, genome editing and other services.

Upon Mason’s diagnosis he began to delve into published NF1 research. He wanted to know where it was happening, who was doing it and how he might be able to help. He sought opinions from experts, wondering how the field could be improved. Many identified the same bottleneck: the lack of a good research model.

In biology, research models are animals, cells, plants, microbes and other living things that allow scientists to study biological processes and recreate diseases in order to better understand them. Good models yield information relevant to humans, but the right model can sometimes be difficult to find.

Seen through a microscope, a researcher guides a micro-needle (at right) to inject DNA into a pig embryo at UW–Madison’s Biotechnology Center. Photo: Jeff Miller

NF1 is especially complex, affects many systems of the body and touches many areas of scientific inquiry, from cancer research to neurobiology. Chuck began to search for a better model and in 2013, when Mason was 3, he settled on pigs. Pigs are similar to humans in many ways that other common research animals, such as mice and flies, are not. That includes their size, which means drugs and devices that work on humans can also be tested on pigs. They have a robust immune system, which rodents lack. And they’re intelligent, so scientists can study changes to their cognition.

Chuck then went on the hunt for researchers who studied swine.

Braving the risks

Dhanansayan “Dhanu” Shanmuganayagam, a nutrition and animal sciences professor in the UW–Madison College of Agricultural and Life Sciences, has spent most of his career using swine to study human diseases, particularly heart disease. In fact, he and colleagues in the animal sciences department created the Wisconsin Miniature Swine, a pig that, like people, can develop heart disease under the right conditions.

Dhanansayan “Dhanu” Shanmuganayagam speaks about his NF1 research at a symposium for patients and families at UW-Madison in May. Photo: Jeff Miller

Dhanu’s office was a few blocks from Chuck’s but they’d never met until a few years ago, when they bumped into one another while helping campaign for a new building on campus. They got to know one another and Chuck asked Dhanu whether he had ever heard NF1. He hadn’t. Chuck told him about Mason, about the need for a better model, about the promise that pigs offered to help understand and treat the disease. Would Dhanu join forces to help create that model, Chuck asked.

Dhanu took some time to think about it. He consulted the members of his laboratory. All would be helping to forge this new path. His risks would be their risks. A pig model could fail, leading them all down a blind alley. That kind of outcome can derail a scientist’s entire career.

Dhanu told Chuck he was in.

The risks remain significant, he says, “but I’ve come to terms with it and it’s fine. I’ve been lucky in my career to work on things that have gone to clinic. If it works it’s going to be impactful.”

Meanwhile, Chuck consulted a legal team to ensure he was clear of conflicts of interest, and took steps to ensure his involvement was ethical and not problematic for his staff at the Biotechnology Center.

There aren’t many places in the world where this kind of work – melding basic science with clinical research and a large animal model like swine – is possible. UW–Madison has large biomedical research centers, the capacity for high-powered basic science, and a 1,500-pig research facility called the Swine Research and Teaching Center (SRTC), based in Arlington, a 35-minute drive from campus.

“It’s a brave new frontier, to move into swine,” says David H. Gutmann, a physician and researcher at the Washington University School of Medicine in St. Louis, considered one of the foremost NF1 experts in the world. “I’m glad they’re doing this work at UW–Madison because the combination of specialized resources and expertise are found in very few places worldwide.”

Like scissors for genes

Dhanu and Chuck determined the course they wanted to chart included gene editing, using a powerful new tool known as CRISPR, which stands for Clustered Regularly Interspaced Short Palindromic Repeats.

The genetic technology is reshaping basic biological research. Like a pair of molecular scissors, CRISPR enables scientists to target a stretch of cellular DNA for alteration. They can cut out pieces of DNA or swap out letters in the genome, changing the message it encodes or shutting off genes entirely.

The two set their sights on creating pigs that carry the NF1 mutations they and other researchers are most interested in studying. “But we had to figure out where to start,” says Dhanu. “It’s like learning to fly a space shuttle.”

With Dhanu’s lab manager and lead scientist Jen Meudt at the helm, the team dove in. But the challenges were many.

They had to learn about swine reproduction, about CRISPR and gene editing, how to perform the necessary surgeries on pigs, how to time events so no step of the process failed and ruined all the efforts before it. Again and again, they hit roadblocks.

It took more than a year, but finally, they came up with a plan: The researchers would use artificial insemination to impregnate a female pig carefully primed to produce more eggs than she naturally would. Shortly after fertilization, they would remove the embryos, whisk them to the Biotechnology Center and inject them with a solution containing the gene-editing CRISPR. This would have to be done quickly, while the embryos were still a single cell. When the single cell divided, all the subsequent cells would contain the NF1 mutation. Inject too late and the pig would develop into a mosaic of cells that contain the mutation and those that do not.

Researchers at UW–Madison created a breed of pig called Wisconsin Miniature Swine to better model and understand human diseases. Photo by: Jeff Miller

Researcher Kathy Krentz uses a microscope and micro-needle to inject DNA into an embryo at the UW–Madison Biotechnology Center. The embryos are then rushed to Arlington where the host mother awaits. Photo by: Jeff Miller

A closeup of the micro-needle. Photo by: Jeff Miller

Dhanu Shamuganayagam and Jen Meudt prepare to transplant the genetically edited pig embryos into the host mother. Photo by: Jeff Miller

Implanting the embryos. Photo by: Jeff Miller

Larry Britzman and his 11-year-old daughter, Mackenzie, talk to advocate Lindsay Geier during a campus event for patients and their families in May. Photo by: Jeff Miller

Siblings Alexandra, 5, and Mason Konsitzke, 7, work on colored drawings while playing at the Konsitzke family home in Stoughton, Wis., on May 26, 2017. Photo by: Jeff Miller

Then it would be off to the surrogate mother, a pig chosen to reproductively match the embryo-donating pig. The researchers would perform surgery to implant the CRISPR embryos into her womb. If all went well, months later she would give birth to piglets, at least some of which would carry the desired NF1 mutations.

A few months passed. On November 7, 2016, Chuck and Dhanu were meeting in Madison with a group from the Neurofibromatosis (NF) Network, which supports research and clinical care for NF1. They were sipping coffee when a text came in from Jen: “The mom carrying NF piglets is delivering right now.”

The piglets – eight in all, and four with the NF1 mutation – were a living embodiment of the team’s hard work. They had proved that they could create pigs genetically-engineered to carry the disease. It was an emotional experience for the scientists, involving tears and prayers. They immediately went out to celebrate.

Why are pigs used in research? Learn more

Then they set to work building on that success. One of the four piglets with the mutation was a male. Mason named him Tank. His job is to sire more piglets with the mutation since the changes conferred by CRISPR were designed to be passed on from generation to generation.

The team took the process they’d developed and applied it to other NF1 mutations, including some related to cancer. And they set an even more ambitious goal: precision medicine. A pig personalized for every child with NF1.

With CRISPR, the researchers believe they can take the genetic fingerprint of an individual child’s NF1 mutation and create a pig with that same mutation. They can then test potential medications and treatments and see if they’ll work. Can tumors, like the one that afflicts Mason, be shrunk?

The promise of precision

By the time Mason reached pre-kindergarten, the tumor in his face had grown into his cranial sinus. His parents were told he could lose his sight and his ability to taste.

Surgery wasn’t an option. It was too risky and could leave Mason in even greater pain, permanently. “He’s literally been in pain his whole life,” Malia says.

Then, for reasons doctors couldn’t explain, the tumor stopped progressing. He regained his speech and no longer screamed or struggled to stay upright. His doctors keep a close watch on the tumor with MRI scans. And they continue to work to determine the best medication regimen for the other symptoms that come with his particular variant of NF1. His treatment must be continuously modified. Because of NF1’s unique manifestations, each child is an experiment unto himself.

Pigs develop faster than children do so they offer the possibility of helping predict how NF1 might affect a particular child, enabling parents, doctors, teachers and others to prepare. Earlier intervention for a child who develops autism could lead to better outcomes. Doctors could start working to find drugs to treat tumors before they grow too large.

“Precision medicine is more than matching the right drug to the right gene. With NF1 it’s more complicated and involves searching for the factors that make each individual with NF1 unique,” says Washington University’s David Gutmann. “This is an amazing opportunity to find the risk factors that put an affected child at risk for developing a brain tumor, a bone defect, or another serious complication of NF1.”

Washington University researcher David Gutmann, center, speaks with UW–Madison’s Dhanu Shanmuganayagam, left, and Neha Patel. Photo: Jeff Miller

Dhanu, Chuck and Jen are not doing this work on their own. The team now includes many talented individuals like Biotechnology Center scientists C. Dustin Rubinstein, Kathy Krentz and Michael Sussman, along with Jamie Reichert and his team at the Swine Research and Teaching Center. And there’s now a broader research group, the UW NF1 Translational Research team, that includes Thomas Crenshaw, an animal sciences professor and department chair, and Marc Wolman, a professor of integrative biology.

They have also enlisted the skill and knowledge of Neha Patel, a pediatrician at the University of Wisconsin School of Medicine and Public Health who treats about 150 children with NF1 in Wisconsin and surrounding regions.

Dhanu hopes to make the NF1 pigs accessible to other researchers around the country, charging only what it costs to produce them. And the team plans to use the pigs to help identify metabolic and cellular pathways common to the variety of NF1 mutations, to help target and develop better drugs.

But to accomplish all of this requires funding.

“We’re at a critical moment,” Dhanu says. “We have to turn our successes into funding opportunities.”

Parents and health advocates Lindsay and Ryan Geier participate in a panel discussion during the May event for patients and families. Their daughter Lauren has NF1. Photo: Jeff Miller

The UW NF1 Translational Research team has bootstrapped most of its work so far, relying primarily on funding and donations from the NF Network. Most of that comes from an annual charity golf tournament the Konsitzkes and four other families help organize and run. Called Links for Lauren, the tournament honors Lauren Geier, an 8-year-old girl in Madison with NF1.

Families can play a surprisingly influential role in the fight against rare diseases.“They often provide critical resources and financial support at the earliest stages of a high-risk project, when funding from federal agencies is not possible,” says David Guttman. “Our families, they inspire us because they ask us to do things that are really meaningful and take risks by taking the roads not frequently traveled. Through their involvement they can move the field forward in ways that no one else can.”

Where there’s research, there’s hope

Larry Britzman had no idea there were pigs at UW–Madison that might one day help children like his 12-year-old daughter Mackenzie. He learned that, and much more, in May when he traveled to campus from La Valle, Wisconsin, for a symposium for patients and families.

“I didn’t realize each child is specific,” he says. “I didn’t realize UW has swine research and there aren’t too many facilities in the country researching NF1.”

The NF1 team hopes to host the symposium each year, to invite families to learn more about the science of NF1, to give them a chance to meet researchers and clinicians, and to ask questions and meet other families living with the disease.

“We’ve gone very far in two years because it hasn’t been just about building a model, it’s also been about creating a community around it,” says Dhanu.

The opportunity to work so closely with and on behalf of the people who may ultimately benefit from his work is not something he’d ever experienced. And it’s been profoundly rewarding.

Not long ago, he invited a family whose college-aged daughter has NF1 into his lab. They’d been donors to NF1 causes for years but had never talked to a researcher. “It meant a lot to them and my first thought was: ‘How can we do more of this?’”

He and his lab members now participate in running events like the Madison Half Marathon, often with The NF Team organization, to raise money for NF1 research and to increase awareness. The runners sport neon yellow performance shirts with bold, black lettering. They also participate in the annual charity golf tournament.

“As scientists, we don’t often see the payoff of what we’re working on,” Dhanu says. “It redefines our research priorities and it also aids discovery. The best people to note observations are the people who live with it.”

To him, success can be measured by individuals. “Even if our research just raises awareness and someone gets treated because of what we do, that alone is big,” he says.

Chuck believes the disease is underdiagnosed because very few people are genetically tested for it and most physicians are not familiar with it. So they may diagnose patients with autism or a behavioral disorder and miss the broader picture.

That has frustrated Danielle Wood, a teacher and mother of two who lives in Reedsburg, Wisconsin. Her daughter, Bernadette, is 2 and was diagnosed with NF1 as an infant. Along with springy blonde curls and an arresting smile, Bernadette has a weak abdominal wall, which causes her pain and may require surgery. She wears braces to support her frail ankles.

Danielle, too, has NF1. Her mother had it and so did her grandmother. Though her condition is mild – she simply wears glasses for poor vision caused by a tumor on her optic nerve – deciding whether to have children was hard. Because it is a dominant mutation, Danielle and her husband had at least a 50 percent chance of giving birth to a baby with the disease. Having grown up with NF1, Danielle felt she had a good idea of what to expect. She now sees herself as an advocate for Bernadette.

“While things never move as fast as we want them to, there’s a tremendous amount of exciting progress in this field and where there’s research, there’s hope,” says David Gutmann. UW–Madison is “in a really great position because (it has) young faculty who are excited and a patient community that is challenging them to improve the lives of people with NF1 through research.”

This is what drives Chuck, Dhanu and the rest of the UW NF1 Translational Research team, which is working to establish a NF1 Center for Excellence at UW–Madison. Not only is this possible, David Gutmann says, it is necessary. “There is no established therapy for NF1 and no magic bullet that works for all kids or adults. The challenge for us is to learn more about this disorder so that personalized and effective treatments emerge.”

Moreover, he says, what NF1 teaches researchers will inform their approaches to other conditions. And he’s excited to see what the future holds.

“All of us in the NF field get up every morning and are excited to get to work. What we learn from our colleagues and our families each day brings us one step closer to that better future for children and adults with NF,” he says. “I can imagine getting up every morning and running to work to see what’s happening with those pigs.”

For Mason, the pigs don’t play much of a role in his daily life today. Rather, he relies on regular visits to therapists and other professionals both in and out of school to help him manage his symptoms. He also benefits from the support of his family, from Chuck and Malia to aunts and uncles who have learned all they can about NF1. And the family dog, Donatella, is his packmate, Malia says.

At 7, Mason can still take all that for granted. He can focus on what he loves best, like sharing the tastiest mini pizzas he can make. You should try the pepperoni.

Mason at home with his parents and sister Alexandra, 5. Photo: Jeff Miller

Mason Konsitzke, 7, plays in his bedroom at home in Stoughton. Photo: Jeff Miller

Mason Konsitzke is 7. He loves food (especially when he can share it with others) and anything military (both of his grandfathers served). He likes to fly kites and play with his 5-year-old sister, Alexandra. But Mason was born with a disease called neurofibromatosis type 1, or NF1, and each day can present new challenges for him and his family.

NF1 is a genetic disease caused by changes, or mutations, to a single gene in the human DNA library. Roughly one out of 3,000 babies born in the United States have the disease. That’s more than three times as many as have cystic fibrosis. Yet few people have ever heard of NF1.

Mutations in the NF1 gene cause defects in the neurofibromin 1 protein, which acts as a tumor suppressor. Children with NF1 can develop painful tumors along their nerve tracts, including their skin and in their eyes. Sometimes this renders them blind. They are often diagnosed with autism spectrum disorder, though not all children with NF1 are also autistic, and they are sometimes diagnosed with attention deficit hyperactivity disorder. They may have soft bones that bend and break. They are at a higher risk for cancer. And there is no cure.

It was not a disease Mason’s parents, Charles and Malia Konsitzke, had ever heard of. As a newborn, he was healthy. But when Mason was 6 months old, the couple began to suspect something was wrong. Mason developed coffee-and-cream-colored spots all over his body. His father later learned these were a hallmark of NF1. Mason received a genetic diagnosis of the disorder just before his first birthday.

“We were like deer in the headlights,” Malia says. “We were in shock, wondering, what does this mean for us? What does it mean for Mason?”

At 18 months, Mason began to lose his ability to speak. He was falling over, screaming constantly and deliberately banging his head. That’s when an MRI revealed a tumor called a plexiform neurofibroma in a mesh of nerves in the left side of his face. It was growing fast.

A father turns to science

Charles “Chuck” Konsitzke is the associate director of UW-Madison’s Biotechnology Center, a sort of one-stop shop for scientists in need of DNA sequencing, genome editing and other services.

Upon Mason’s diagnosis he began to delve into published NF1 research. He wanted to know where it was happening, who was doing it and how he might be able to help. He sought opinions from experts, wondering how the field could be improved. Many identified the same bottleneck: the lack of a good research model.

In biology, research models are animals, cells, plants, microbes and other living things that allow scientists to study biological processes and recreate diseases in order to better understand them. Good models yield information relevant to humans, but the right model can sometimes be difficult to find.

Seen through a microscope, a researcher guides a micro-needle (at right) to inject DNA into a pig embryo at UW–Madison’s Biotechnology Center. Photo: Jeff Miller

NF1 is especially complex, affects many systems of the body and touches many areas of scientific inquiry, from cancer research to neurobiology. Chuck began to search for a better model and in 2013, when Mason was 3, he settled on pigs. Pigs are similar to humans in many ways that other common research animals, such as mice and flies, are not. That includes their size, which means drugs and devices that work on humans can also be tested on pigs. They have a robust immune system, which rodents lack. And they’re intelligent, so scientists can study changes to their cognition.

Chuck then went on the hunt for researchers who studied swine.

Braving the risks

Dhanansayan “Dhanu” Shanmuganayagam, a nutrition and animal sciences professor in the UW–Madison College of Agricultural and Life Sciences, has spent most of his career using swine to study human diseases, particularly heart disease. In fact, he and colleagues in the animal sciences department created the Wisconsin Miniature Swine, a pig that, like people, can develop heart disease under the right conditions.

Dhanansayan “Dhanu” Shanmuganayagam speaks about his NF1 research at a symposium for patients and families at UW-Madison in May. Photo: Jeff Miller

Dhanu’s office was a few blocks from Chuck’s but they’d never met until a few years ago, when they bumped into one another while helping campaign for a new building on campus. They got to know one another and Chuck asked Dhanu whether he had ever heard NF1. He hadn’t. Chuck told him about Mason, about the need for a better model, about the promise that pigs offered to help understand and treat the disease. Would Dhanu join forces to help create that model, Chuck asked.

Dhanu took some time to think about it. He consulted the members of his laboratory. All would be helping to forge this new path. His risks would be their risks. A pig model could fail, leading them all down a blind alley. That kind of outcome can derail a scientist’s entire career.

Dhanu told Chuck he was in.

The risks remain significant, he says, “but I’ve come to terms with it and it’s fine. I’ve been lucky in my career to work on things that have gone to clinic. If it works it’s going to be impactful.”

Meanwhile, Chuck consulted a legal team to ensure he was clear of conflicts of interest, and took steps to ensure his involvement was ethical and not problematic for his staff at the Biotechnology Center.

There aren’t many places in the world where this kind of work – melding basic science with clinical research and a large animal model like swine – is possible. UW–Madison has large biomedical research centers, the capacity for high-powered basic science, and a 1,500-pig research facility called the Swine Research and Teaching Center (SRTC), based in Arlington, a 35-minute drive from campus.

“It’s a brave new frontier, to move into swine,” says David H. Gutmann, a physician and researcher at the Washington University School of Medicine in St. Louis, considered one of the foremost NF1 experts in the world. “I’m glad they’re doing this work at UW–Madison because the combination of specialized resources and expertise are found in very few places worldwide.”

Like scissors for genes

Dhanu and Chuck determined the course they wanted to chart included gene editing, using a powerful new tool known as CRISPR, which stands for Clustered Regularly Interspaced Short Palindromic Repeats.

The genetic technology is reshaping basic biological research. Like a pair of molecular scissors, CRISPR enables scientists to target a stretch of cellular DNA for alteration. They can cut out pieces of DNA or swap out letters in the genome, changing the message it encodes or shutting off genes entirely.

The two set their sights on creating pigs that carry the NF1 mutations they and other researchers are most interested in studying. “But we had to figure out where to start,” says Dhanu. “It’s like learning to fly a space shuttle.”

With Dhanu’s lab manager and lead scientist Jen Meudt at the helm, the team dove in. But the challenges were many.

They had to learn about swine reproduction, about CRISPR and gene editing, how to perform the necessary surgeries on pigs, how to time events so no step of the process failed and ruined all the efforts before it. Again and again, they hit roadblocks.

It took more than a year, but finally, they came up with a plan: The researchers would use artificial insemination to impregnate a female pig carefully primed to produce more eggs than she naturally would. Shortly after fertilization, they would remove the embryos, whisk them to the Biotechnology Center and inject them with a solution containing the gene-editing CRISPR. This would have to be done quickly, while the embryos were still a single cell. When the single cell divided, all the subsequent cells would contain the NF1 mutation. Inject too late and the pig would develop into a mosaic of cells that contain the mutation and those that do not.

Researchers at UW–Madison created a breed of pig called Wisconsin Miniature Swine to better model and understand human diseases. Photo by: Jeff Miller

Researcher Kathy Krentz uses a microscope and micro-needle to inject DNA into an embryo at the UW–Madison Biotechnology Center. The embryos are then rushed to Arlington where the host mother awaits. Photo by: Jeff Miller

A closeup of the micro-needle. Photo by: Jeff Miller

Dhanu Shamuganayagam and Jen Meudt prepare to transplant the genetically edited pig embryos into the host mother. Photo by: Jeff Miller

Implanting the embryos. Photo by: Jeff Miller

Larry Britzman and his 11-year-old daughter, Mackenzie, talk to advocate Lindsay Geier during a campus event for patients and their families in May. Photo by: Jeff Miller

Siblings Alexandra, 5, and Mason Konsitzke, 7, work on colored drawings while playing at the Konsitzke family home in Stoughton, Wis., on May 26, 2017. Photo by: Jeff Miller

Then it would be off to the surrogate mother, a pig chosen to reproductively match the embryo-donating pig. The researchers would perform surgery to implant the CRISPR embryos into her womb. If all went well, months later she would give birth to piglets, at least some of which would carry the desired NF1 mutations.

A few months passed. On November 7, 2016, Chuck and Dhanu were meeting in Madison with a group from the Neurofibromatosis (NF) Network, which supports research and clinical care for NF1. They were sipping coffee when a text came in from Jen: “The mom carrying NF piglets is delivering right now.”

The piglets – eight in all, and four with the NF1 mutation – were a living embodiment of the team’s hard work. They had proved that they could create pigs genetically-engineered to carry the disease. It was an emotional experience for the scientists, involving tears and prayers. They immediately went out to celebrate.

Why are pigs used in research? Learn more

Then they set to work building on that success. One of the four piglets with the mutation was a male. Mason named him Tank. His job is to sire more piglets with the mutation since the changes conferred by CRISPR were designed to be passed on from generation to generation.

The team took the process they’d developed and applied it to other NF1 mutations, including some related to cancer. And they set an even more ambitious goal: precision medicine. A pig personalized for every child with NF1.

With CRISPR, the researchers believe they can take the genetic fingerprint of an individual child’s NF1 mutation and create a pig with that same mutation. They can then test potential medications and treatments and see if they’ll work. Can tumors, like the one that afflicts Mason, be shrunk?

The promise of precision

By the time Mason reached pre-kindergarten, the tumor in his face had grown into his cranial sinus. His parents were told he could lose his sight and his ability to taste.

Surgery wasn’t an option. It was too risky and could leave Mason in even greater pain, permanently. “He’s literally been in pain his whole life,” Malia says.

Then, for reasons doctors couldn’t explain, the tumor stopped progressing. He regained his speech and no longer screamed or struggled to stay upright. His doctors keep a close watch on the tumor with MRI scans. And they continue to work to determine the best medication regimen for the other symptoms that come with his particular variant of NF1. His treatment must be continuously modified. Because of NF1’s unique manifestations, each child is an experiment unto himself.

Pigs develop faster than children do so they offer the possibility of helping predict how NF1 might affect a particular child, enabling parents, doctors, teachers and others to prepare. Earlier intervention for a child who develops autism could lead to better outcomes. Doctors could start working to find drugs to treat tumors before they grow too large.

“Precision medicine is more than matching the right drug to the right gene. With NF1 it’s more complicated and involves searching for the factors that make each individual with NF1 unique,” says Washington University’s David Gutmann. “This is an amazing opportunity to find the risk factors that put an affected child at risk for developing a brain tumor, a bone defect, or another serious complication of NF1.”

Washington University researcher David Gutmann, center, speaks with UW–Madison’s Dhanu Shanmuganayagam, left, and Neha Patel. Photo: Jeff Miller

Dhanu, Chuck and Jen are not doing this work on their own. The team now includes many talented individuals like Biotechnology Center scientists C. Dustin Rubinstein, Kathy Krentz and Michael Sussman, along with Jamie Reichert and his team at the Swine Research and Teaching Center. And there’s now a broader research group, the UW NF1 Translational Research team, that includes Thomas Crenshaw, an animal sciences professor and department chair, and Marc Wolman, a professor of integrative biology.

They have also enlisted the skill and knowledge of Neha Patel, a pediatrician at the University of Wisconsin School of Medicine and Public Health who treats about 150 children with NF1 in Wisconsin and surrounding regions.

Dhanu hopes to make the NF1 pigs accessible to other researchers around the country, charging only what it costs to produce them. And the team plans to use the pigs to help identify metabolic and cellular pathways common to the variety of NF1 mutations, to help target and develop better drugs.

But to accomplish all of this requires funding.

“We’re at a critical moment,” Dhanu says. “We have to turn our successes into funding opportunities.”

Parents and health advocates Lindsay and Ryan Geier participate in a panel discussion during the May event for patients and families. Their daughter Lauren has NF1. Photo: Jeff Miller

The UW NF1 Translational Research team has bootstrapped most of its work so far, relying primarily on funding and donations from the NF Network. Most of that comes from an annual charity golf tournament the Konsitzkes and four other families help organize and run. Called Links for Lauren, the tournament honors Lauren Geier, an 8-year-old girl in Madison with NF1.

Families can play a surprisingly influential role in the fight against rare diseases.“They often provide critical resources and financial support at the earliest stages of a high-risk project, when funding from federal agencies is not possible,” says David Guttman. “Our families, they inspire us because they ask us to do things that are really meaningful and take risks by taking the roads not frequently traveled. Through their involvement they can move the field forward in ways that no one else can.”

Where there’s research, there’s hope

Larry Britzman had no idea there were pigs at UW–Madison that might one day help children like his 12-year-old daughter Mackenzie. He learned that, and much more, in May when he traveled to campus from La Valle, Wisconsin, for a symposium for patients and families.

“I didn’t realize each child is specific,” he says. “I didn’t realize UW has swine research and there aren’t too many facilities in the country researching NF1.”

The NF1 team hopes to host the symposium each year, to invite families to learn more about the science of NF1, to give them a chance to meet researchers and clinicians, and to ask questions and meet other families living with the disease.

“We’ve gone very far in two years because it hasn’t been just about building a model, it’s also been about creating a community around it,” says Dhanu.

The opportunity to work so closely with and on behalf of the people who may ultimately benefit from his work is not something he’d ever experienced. And it’s been profoundly rewarding.

Not long ago, he invited a family whose college-aged daughter has NF1 into his lab. They’d been donors to NF1 causes for years but had never talked to a researcher. “It meant a lot to them and my first thought was: ‘How can we do more of this?’”

He and his lab members now participate in running events like the Madison Half Marathon, often with The NF Team organization, to raise money for NF1 research and to increase awareness. The runners sport neon yellow performance shirts with bold, black lettering. They also participate in the annual charity golf tournament.

“As scientists, we don’t often see the payoff of what we’re working on,” Dhanu says. “It redefines our research priorities and it also aids discovery. The best people to note observations are the people who live with it.”

To him, success can be measured by individuals. “Even if our research just raises awareness and someone gets treated because of what we do, that alone is big,” he says.

Chuck believes the disease is underdiagnosed because very few people are genetically tested for it and most physicians are not familiar with it. So they may diagnose patients with autism or a behavioral disorder and miss the broader picture.

That has frustrated Danielle Wood, a teacher and mother of two who lives in Reedsburg, Wisconsin. Her daughter, Bernadette, is 2 and was diagnosed with NF1 as an infant. Along with springy blonde curls and an arresting smile, Bernadette has a weak abdominal wall, which causes her pain and may require surgery. She wears braces to support her frail ankles.

Danielle, too, has NF1. Her mother had it and so did her grandmother. Though her condition is mild – she simply wears glasses for poor vision caused by a tumor on her optic nerve – deciding whether to have children was hard. Because it is a dominant mutation, Danielle and her husband had at least a 50 percent chance of giving birth to a baby with the disease. Having grown up with NF1, Danielle felt she had a good idea of what to expect. She now sees herself as an advocate for Bernadette.

“While things never move as fast as we want them to, there’s a tremendous amount of exciting progress in this field and where there’s research, there’s hope,” says David Gutmann. UW–Madison is “in a really great position because (it has) young faculty who are excited and a patient community that is challenging them to improve the lives of people with NF1 through research.”

This is what drives Chuck, Dhanu and the rest of the UW NF1 Translational Research team, which is working to establish a NF1 Center for Excellence at UW–Madison. Not only is this possible, David Gutmann says, it is necessary. “There is no established therapy for NF1 and no magic bullet that works for all kids or adults. The challenge for us is to learn more about this disorder so that personalized and effective treatments emerge.”

Moreover, he says, what NF1 teaches researchers will inform their approaches to other conditions. And he’s excited to see what the future holds.

“All of us in the NF field get up every morning and are excited to get to work. What we learn from our colleagues and our families each day brings us one step closer to that better future for children and adults with NF,” he says. “I can imagine getting up every morning and running to work to see what’s happening with those pigs.”

For Mason, the pigs don’t play much of a role in his daily life today. Rather, he relies on regular visits to therapists and other professionals both in and out of school to help him manage his symptoms. He also benefits from the support of his family, from Chuck and Malia to aunts and uncles who have learned all they can about NF1. And the family dog, Donatella, is his packmate, Malia says.

At 7, Mason can still take all that for granted. He can focus on what he loves best, like sharing the tastiest mini pizzas he can make. You should try the pepperoni.

Mason at home with his parents and sister Alexandra, 5. Photo: Jeff Miller

In a UW-Madison lab, a vacuum tube holds a blood-fed strain of Aedes aegypti mosquito, which can carry the Zika virus. New research suggests the virus poses a wider threat in human pregnancies than generally appreciated. Photo: Jeff Miller

Zika virus infection passes efficiently from a pregnant monkey to its fetus, spreading inflammatory damage throughout the tissues that support the fetus and its developing nervous system — suggesting the virus poses a wider threat in human pregnancies than generally appreciated, University of Wisconsin-Madison scientists have found.

The UW–Madison researchers, along with collaborators at Duke University and the University of California, Davis, published their study of Zika-infected pregnancies today in the journal PLOS Pathogens.

Their work, which was funded by the National Institutes of Health, followed the pregnancies from infection in the first or third trimester, regularly assessing maternal infection and fetal development and examining the extent of infection in the fetus when the pregnancies reached term.

The UW researchers infected four pregnant rhesus macaque monkeys at the Wisconsin National Primate Research Center with a Zika virus dose similar to what would be transferred by a mosquito bite, and found evidence that the virus was present in each monkey’s fetus.

Golos

Antony

Dudley

“That is a very high level — 100 percent exposure — of the virus to the fetus along with inflammation and tissue injury in an animal model that mirrors the infection in human pregnancies quite closely,” says Ted Golos, a UW–Madison reproductive physiologist and professor of comparative biosciences and obstetrics and gynecology. “It’s sobering. If microcephaly is the tip of the iceberg for babies infected in pregnancy, the rest of the iceberg may be bigger than we’ve imagined.”

Three of the fetuses involved had small heads, but not quite so small relative to normal that they would meet the human standard for diagnosing microcephaly — the most striking and widely discussed result of Zika infection since Brazilian doctors raised alarm in 2014 of many babies with arrested brain development.

The new study did not find abnormal brain development, but the researchers did discover unusual inflammation in the fetal eyes, in the retinas and optic nerves, in pregnancies infected during the first trimester.

“Our eyes are basically part of our central nervous system. The optic nerve grows right out from the fetal brain during pregnancy,” says Kathleen Antony, a UW–Madison professor of maternal fetal medicine and an author of the study. “So it makes some sense to see this damage in the monkeys and in human pregnancy — problems such as chorioretinal atrophy or microphthalmia in which the whole eye or parts of the eye just don’t grow to the expected size.”

“It’s sobering. If microcephaly is the tip of the iceberg for babies infected in pregnancy, the rest of the iceberg may be bigger than we’ve imagined.”

Ted Golos

The similarities between the monkey pregnancies and reported complications in Zika-affected human pregnancies further establish Zika infection in monkeys as a way to study the progression of the infection and associated health problems in people.

“There are so many things about Zika infection we can’t study as well in pregnant humans — or fast enough to make a difference for a lot of people who may be infected,” says Dawn Dudley, a UW–Madison pathology research scientist and one of the lead authors of the new research with Antony and obstetrics and gynecology graduate student Sydney Nguyen.

An animal model opens the door to studying how Zika infection interacts with other infections (like dengue virus), how the effects of early pregnancy infection might be different from later infection, and, according to Dudley, whether quick treatment with some antiviral therapies could manage the damage of what has come to be known as congenital Zika syndrome.

“The precise pathway that the virus takes from mom’s bloodstream to the fetal bloodstream, across that interface, cannot be studied except in an animal model,” says Golos, whose research group found damage from Zika infection in every part of the interface between mother and fetus — the placenta, amniotic fluid in the womb and the lining of uterus.

While the immediate effects may not be as dramatic as microcephaly, “the results we’re seeing in monkey pregnancies make us think that, as they grow, more human babies might develop Zika-related disease pathology than is currently appreciated,” Golos says.

This work was supported by NIH grants R01 AI107157-01A1, R01AI116382-01A1S1, DP2HD075699 and P51 OD011106. This research was conducted in part at a facility constructed with support from Research Facilities Improvement Program grant numbers RR15459-01 and RR020141-01.

Listeria monocytogenes, the common food-borne bacteria depicted in this illustration based on electron microscope imagery, can cause miscarriage, stillbirth and premature labor in pregnant women. Image: James Archer/Centers for Disease Control

Listeria, a common food-borne bacterium, may pose a greater risk of miscarriage in the early stages of pregnancy than appreciated, according to researchers at the University of Wisconsin–Madison School of Veterinary Medicine studying how pathogens affect fetal development and change the outcome of pregnancy.

“For many years, listeria has been associated with adverse outcomes in pregnancy, but particularly at the end of pregnancy,” says Ted Golos, a UW–Madison reproductive physiologist and professor of comparative biosciences and obstetrics and gynecology. “What wasn’t known with much clarity before this study is that it appears it’s a severe risk factor in early pregnancy.”

Golos and his collaborators published their results Feb. 21, 2017 in the journal mBio.

Listeria — stained green in this image — invades cells in the uterine lining of monkeys in their first trimester of pregnancy. Image: Gregory J. Wiepz/UW-Madison

According to the Centers for Disease Control, listeria makes about 1,600 Americans sick each year — a relatively small number, but a group heavy on newborn babies and older adults with undeveloped or weak immune systems.

“The problem with this organism is not a huge number of cases. It’s that when it is identified, it’s associated with severe outcomes,” says Charles Czuprynski, a UW–Madison professor of pathobiological sciences and director of the UW–Madison Food Research Institute.

Pregnant women are warned to avoid many of the foods — among them unpasteurized milk and soft cheese, raw sprouts, melon and deli meats not carefully handled — that can harbor listeria, because the bacterium is known to cause miscarriage and stillbirth, and spur premature labor. Those severe outcomes have resulted in a zero-tolerance regulatory policy for listeria in ready-to-eat foods. But when it occurs, listeria infection in pregnancy may go unnoticed. The few recognizable symptoms are nearly indistinguishable from the discomfort most newly pregnant women feel.

“It’s striking that mom doesn’t get particularly ill from listeria infection, but it has a profound impact on the fetus,” says Golos, whose work is funded by the National Institutes of Health. “That’s familiar now, because we’ve been talking about the same difference in Zika virus.”

Sophia Kathariou, a North Carolina State University professor of food science and microbiology, provided a strain of listeria that caused miscarriage, stillbirth and premature delivery in at least 11 pregnant women in 2000. Four pregnant rhesus macaques at the Wisconsin National Primate Research Center were fed doses of the listeria comparable to what one might encounter in contaminated food. Bryce Wolfe, a UW–Madison graduate student studying cellular and molecular pathology who is lead author of the study, monitored the speed and progression of listeria’s spread.

Ted Golos

Charles Czuprynski

“What’s particularly striking about the work Bryce did is the detailed information we now have about the organism,” Czuprynski says. “The animal ingested it; she tracked it being shed in feces and showing up in the bloodstream. They did ultrasound analysis of the fetus, and could then show events in terms of where the organism was preceding fetal demise.”

None of the monkeys showed obvious signs of infection before their pregnancies came to abrupt ends. But in tissue samples taken after each monkey experienced intrauterine fetal death, Wolfe found listeria had invaded the placenta — the connection between the mother-to-be and the fetus, which usually prevents transmission of bacteria — as well as the endometrium, the lining of the uterus.

“In that region, there’s a rich population of specialized immune cells, and it is exquisitely regulated,” says Wolfe. “When you introduce a pathogen into the midst of this, it’s not very surprising that it’s going to cause some sort of adverse outcome disrupting this balance.”

The researchers believe the inflammation caused by the maternal immune response to the fast-moving listeria also affects the placenta, keeping it from protecting the fetus.

“There are effective antibiotics available. It is treatable. The issue is that because it’s asymptomatic, the fetus may be infected by the time anyone realizes the mother was infected.”

Bryce Wolfe

“It should be a barrier,” Golos says. “But we’re hypothesizing that the maternal immune system’s attempt to clear the bacteria actually results in collateral damage to the placenta that then allows the bacteria to invade the fetus.”

The results suggest listeria (and perhaps other pathogens) may be the culprit in some miscarriages that usually go without diagnosed cause, but the bacteria’s stealth and speed may still make it hard to control.

“There are effective antibiotics available. It is treatable,” Wolfe says. “The issue is that because it’s asymptomatic, the fetus may be infected by the time anyone realizes the mother was infected.”

Golos and Wolfe plan to continue work with listeria to better define how the bacterium targets the reproductive tract, its incubation time and the problems it causes leading up to miscarriage. Their goal is to provide basic knowledge about the progression of infection and the maternal immune response to intracellular pathogens in pregnancy, which may help other researchers battling similar dangers such as Zika virus.

That is the primary finding of a study published Feb. 14 in the journal Cell Reports by a team led by University of Wisconsin-Madison researchers Alan Attie and Federico Rey. The new report describes experiments in mice showing how genetic variation in a host animal shapes the microbiome — a rich ecosystem of mostly beneficial microorganisms that resides in the gut — and sets the table for the onset of metabolic disease.

“We’re trying to use genetics to find out how bugs affect diabetes and metabolism,” explains Attie, a UW-Madison professor of biochemistry and a corresponding author of the study.

Nacho Vivas, lab manager at the Rey Lab in the Bacteriology Department, checks on a group of germ-free mice inside a sterile lab environment. Photo: Bryce Richter

Peeling back the complex interplay of genes, diet and the trillions of microorganisms that live in the guts of humans and other animals, Rey, Attie and their colleagues are beginning to work out the subtleties of how host genes shape the composition of the microbiome and contribute to an animal’s phenotype and, ultimately, diet-induced metabolic disease.

Metabolic diseases such as diabetes have long been known to be influenced by both genes and diet. Understanding the role of the microbes that live in the gut and help process nutrients not only promises a fuller understanding of the link between genes, diet and disease, but may also be a pathway to pinpointing the genes responsible for conditions like diabetes.

“We’re asking whether or not there is a chain of causality between gut microbiota and (disease) phenotype,” says Attie. “Genetics is the anchor. If something is associated with a gene, it is truly a causal relationship, not just a correlation.”

“We’re trying to use genetics to find out how bugs affect diabetes and metabolism,” explains Alan Attie, a UW-Madison professor of biochemistry. Photo: College of Agricultural and Life Sciences/UW-Madison

To leverage that approach, the new Wisconsin study employed a cohort of eight strains of mice whose genetics collectively mirror the genetic diversity of the human population.

“These mice show tremendous phenotypic diversity,” says Attie. “Some are lean. Some are susceptible to obesity. Some are resistant to obesity. Some of these phenotypes can be partially transmitted by gut microbiota.”

Clues to the influence of genes on the composition of the microbiome emerged from experiments where mice were raised in a germ-free environment and challenged by a diet high in fat and sugar. Through fecal transplants, microbiomes could be effectively traded bewteen strains, helping researchers home in on the interplay between genes and the microbiome.

“Our study suggests that a lot of the genetic variation we see among these eight strains of mice is reflected in their microbiomes,” notes Rey, a UW-Madison professor of bacteriology and a corresponding author of the study. “And we have evidence that the composition of the gut microbiota is controlled by the genomes of the mice. We’re trying to find the genes that control the composition of the gut microbiota and (dictate) host phenotype.”

“Our study suggests that a lot of the genetic variation we see among these eight strains of mice is reflected in their microbiomes,” notes Federico Rey, a UW-Madison professor of bacteriology. Photo: College of Agricultural and Life Sciences/UW-Madison

In response to diet, the Wisconsin group observed a “remarkable variation” in mice whose genetics make them prone to diabetes. They also noticed an accompanying change in the makeup of the animals’ gut microbiomes. Some of the bacteria, according to Rey and Attie, could be linked to metabolic traits such as body weight, and glucose and insulin levels.

The microbiome plays a crucial role in processing nutrients. Food not metabolized directly by a host like a mouse or a human is subsequently processed in the gut by the bacteria of the microbiome. As the microbes metabolize food, they produce an astonishing number of small molecules, chemicals and hormones that circulate in a host and can influence health in an animal.

Among those metabolites, perhaps as many as 20,000 in all, are what are called short-chain fatty acids, which serve as signaling molecules in the intestine and associated organs like the liver and pancreas. In particular, they are key regulators of energy and glucose.

Gut microbes also influence the physiology of the host by modifying bile acids produced by the liver, which are also processed by the microbiome to produce secondary metabolites that can exert an influence on disease and health.

Mice in the study that were put on a rich diet and received microbiome transplants helped the Wisconsin team expose functional differences attributable to two different transplanted microbiomes, including a link between the gut microbiome and insulin secretion.

Now, in a study comparing mice raised in a “germ free” environment and mice raised under more typical lab conditions, scientists have identified yet another key role of the microbes that live within us: mediator of host gene expression through the epigenome, the chemical information that regulates which genes in cells are active.

Writing online Nov. 23 in the journal Molecular Cell, a team of researchers from the University of Wisconsin-Madison describes new research helping tease out the mechanics of how the gut microbiome communicates with the cells of its host to switch genes on and off. The upshot of the study, another indictment of the so-called Western diet (high in saturated fats, sugar and red meat), reveals how the metabolites produced by the bacteria in the stomach chemically communicate with cells, including cells far beyond the colon, to dictate gene expression and health in its host.

Nacho Vivas, lab manager at the Rey Lab in the Bacteriology Department at the University of Wisconsin-Madison, checks on a group of germ-free mice inside a sterile lab environment. Photo: Bryce Richter

“The bugs are somehow driving gene expression in the host through alteration of the epigenome,” explains John Denu, a UW-Madison professor of biomolecular chemistry and a senior researcher at the Wisconsin Institute for Discovery, and a co-author of the new study. “We’re starting to understand the mechanism of how and why diet and the microbiome matter.”

The study, which was led by Kimberly Krautkramer, an MD/Ph.D. student in the UW School of Medicine and Public Health, revealed key differences in gene regulation in conventionally raised mice and mice raised in a germ-free environment. The mice were provided with two distinct diets:  one rich in plant carbohydrates similar to fruits and vegetables humans consume; the other mimicking a Western diet, high in simple sugars and fat.

Kimberly Krautkramer Photo: Patricia Pointer/Wisconsin Institute for Discovery

A plant-based diet, according to Federico Rey, a UW-Madison professor of bacteriology and also a co-corresponding author of the new report, yields a richer microbiome: “A good diet translates to a beautifully complex microbiome,” Rey says.

“And we see that the gut microbiome affects the host epigenome in a diet-dependent manner. A plant-based diet seems to favor host-microbe communication.”

The new Wisconsin study shows that a small set of short-chain fatty acids produced as the gut bacteria consume, metabolize and ferment nutrients from plants are important chemical messengers, communicating with the cells of the host through the epigenome. “One of the findings here is that microbial metabolism or fermentation of plant fiber results in the production of short-chain fatty acids. These molecules, and potentially many others, are partially responsible for the communication” with the epigenome, says Denu.

In the study, the gut microbiota of the animals that were fed a diet rich in sugar and fat have a diminished capacity to communicate with host cells. According to the Wisconsin team, that may be a hint that the template for a healthy human microbiome was set in the distant past, when food from plants made up a larger portion of diet and sugar and fat were less available than in contemporary diets with more meat and processed foods.

John Denu

Federico Rey

“As we move away from plant-based diets, we may be losing some of that communication between microbes and host,” notes Rey. “With a Western-type diet, it seems like the communication between microbes and host gets lost.”

Foods rich in fat and sugar, especially processed foods, are more easily digested by the host, but are not necessarily a good source of food for the flora inhabiting the gut. The result is a less diverse microbiome and less communication to the host, according to the researchers.

A surprising finding in the study is that the chemical communication between the microbiome and host cells is far reaching. In addition to talking to cells in the colon, the microbiome also seems to be communicating with cells in the liver and in fatty tissue far removed from the gut. That, says Denu, is more evidence of the importance of the microbiome to the well-being of its host.

The upshot of the study is another indictment of the so-called Western diet.

The kicker experiment in the study, says Denu, was providing mice raised in a germ-free environment with three different short-chain fatty acids that the study showed to be important messengers to the epigenome. The supplement was enough to promote the kind of healthy interplay between microbiota and host cells seen in mice given a diet high in plant fiber.

“It helps show that the collection of three short-chain fatty acids produced in the plant-based diet are likely major communicators,” adds Denu. “We see that it is not just the microbe. It’s microbial metabolism.”

This research was funded by the National Institutes of Health under grants F30 DK108494 and GM059789-15/P250VA. Additional support was provided by the Clinical and Translational Science Award program through the NIH National Center for Advancing Translational Sciences grants UL1TR000427, KL2TR000428, DK108259, and DK101573.

The dressing is now cleared by the FDA as a class II medical device, for prescription and over-the-counter use.

Like many dressings now used to treat burns and other persistent wounds, Microlyte Ag contains silver to kill bacteria – but in much smaller quantities.

“Silver is an excellent antimicrobial agent,” says Agarwal, a co-founder of the company in the Madison suburb Fitchburg, “as it is active against a broad range of bacteria and yeast. But the large silver loads found in conventional silver dressings can be toxic to skin cells. Our dressing uses as little as 1 percent as much silver as the competition, and yet the tests we submitted to the FDA showed that Microlyte kills more than 99.99 percent of bacteria that it contacts.”

This chronic pressure sore on an Irish wolfhound received conventional treatment for more than three months at the UW-Madison School of Veterinary Medicine.

Fourteen days after application of Microlyte, the sore is closed and well on its way to healing. Photos: Imbed Biosciences

That kill ratio even appeared in tests against some of the nastiest hospital-acquired superbugs, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococcus.

Microlyte overcomes a key problem with existing dressings: stiffness. Under a low-power microscope, a wound has bumps and fissures — hiding places for bacteria. The Microlyte dressing inherently adheres to moist surfaces and is so flexible that it drops into the fissures, leading to the sweet combination of greater destruction of bacteria at much lower doses of silver.

Microlyte has several other advantages, Agarwal says. It retains moisture yet is ultrathin and breathable, allowing oxygen to reach the wound and gases to exit, all factors that promote healing. The slow release of the silver means the dressing can remain in place for at least one day. And because the material is a hydrogel (a water-based gel), it can simply be rinsed off as needed before replacement.

Experience with animals shows that the ultra-thin dressing simply sloughs off as the wound heals. All of these advantages should reduce the need to change dressings, which can be so painful that sedation is needed, especially for children.

“Reducing or eliminating dressing changes reduces the pain that the patient experiences,” says co-founder Michael Schurr, chair of general surgery at the Mountain Area Health Education Center in Asheville, North Carolina, and adjunct professor of surgery at the University of North Carolina. “It also reduces costs in supplies and reduces the burden to the health care system that supplies visiting nurses to do the dressing changes.”

“We are seeing in a limited number of cases that it does provide us with a remarkable new tool for dealing with chronic wounds” in dogs and cats treated at the UW-Madison School of Veterinary Medicine, says Jonathan McAnulty, chair of the Department of Surgical Sciences. “We certainly have no reason to think that this will be different with humans,” adds McAnulty, who is also a company co-founder. “The principles are the same, and a lot of the problems are the same.”

Imbed’s Microlyte dressing is applied to a wound on a pig. The ultra-thin dressing conforms to the wound, bringing the antimicrobial silver into direct contact with bacteria. Photo: Imbed Biosciences

The dramatic closure of wounds that have resisted months of conventional treatment “suggests that chronic bacterial contamination of the wound surface, even when it looks relatively healthy, is a significant factor inhibiting healing in many cases,” McAnulty says. “Once we treat with our dressing, we start to see very dramatic closure of these wounds.”

McAnulty says he’s starting to use Microlyte earlier in treatment. “Certainly it seems appropriate for prevention of infection as well as treatment.”

The ultra-thin dressing material was invented in the lab of Nicholas Abbott, a UW–Madison professor of chemical and biological engineering, when Agarwal was a postdoctoral fellow and where he is now an honorary associate scientist.

The dressing will compete in the $2 billion market sector of “advanced wound dressings,” which are used to treat diabetic ulcers, venous ulcers, burns, bedsores and other difficult wounds.

Imbed has 10 employees. The company is developing other ideas for wound treatment and discussing commercial-scale production of Microlyte. Currently, it plans to reach the market through licensing agreements with hospital suppliers.

Research reported in this press release was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under award number R44AR061913.

“There is incredibly important, potentially lifesaving research that goes on in Wisconsin that relies on fetal material received from federally regulated tissue banks,” said Dr. Robert Golden, dean of the UW–Madison School of Medicine and Public Health and the university’s vice chancellor of medical affairs, at a hearing before the State Assembly’s Committee on Criminal Justice and Public Safety. “On our campus, cancers including lymphoma, stomach cancer and other diseases have NIH-supported labs actively working on promising new treatment. All of this will stop if this bill is passed.”

That doesn’t sit well with Mary Jo Gordon, who relies on research with fetal tissues to keep her heart beating.

Gordon and many members of her family have long QT syndrome, a genetic heart disorder that causes heart arrhythmia and cardiac arrests like the one that claimed her sister.

A long list of common drugs from antibiotics to asthma medications can trigger the attacks, so the Federal Drug Administration now requires every new medication to be tested for safety among those with long QT and similar disorders.

“We couldn’t do this without these tissue samples available to the researchers who are doing this work,” Gordon told legislators. “By halting research, you’re putting at risk my beautiful nieces and nephews. I ask that you not put our health and safety at risk, not take away our hope for treatment and a cure.”

With research on various cancers, Parkinson’s disease, arthritis, new vaccines and more threatened by the bill, Dr. Brad Schwartz, biochemist and CEO of the Morgridge Institute for Research, wants legislators to consider the implications of outlawing efforts to help so many.

“It would be an ethical failing if we did not do everything we could to try to help people with these diseases,” Schwartz says.

The bill would also hamstring a vibrant, growing piece of the state’s economy. UW–Madison labs whose work relies in some part on fetal tissue or cell lines employ more than 1,400 people and account for about $76 million in federal research funding in the most recent fiscal year.

“These scientists will leave as soon as they can, taking with them not only their $76 million, but their collaborating fellow scientists,” says Golden, who believes the legislation would also endanger Wisconsin’s only federally supported comprehensive cancer center — where a small, but vital, portion of research employs fetal tissue.

Such a law could also cost the state its future scientific leaders, innovators and entrepreneurs.

“Talented Wisconsin students have the opportunity to learn skills that will be valuable to the economy of the future,” Schwartz says. “Anything we do to make that research impossible to accomplish will drive those talented young people to other parts of the country.”

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