Stanford investigators used high-throughput analysis to link inflammation to chronic fatigue syndrome, a difficult-to-diagnose disease with no known cure.

Jose Montoya and his colleagues have found evidence inflammation may be the culprit behind chronic fatigue syndrome, a disease with no known cure.

Steve Fisch

Researchers at the Stanford University School of Medicine have linked chronic fatigue syndrome to variations in 17 immune-system signaling proteins, or cytokines, whose concentrations in the blood correlate with the disease’s severity.

The findings provide evidence that inflammation is a powerful driver of this mysterious condition, whose underpinnings have eluded researchers for 35 years.

The findings, described in a study published online July 31 in the Proceedings of the National Academy of Sciences, could lead to further understanding of this condition and be used to improve the diagnosis and treatment of the disorder, which has been notably difficult.

More than 1 million people in the United States suffer from chronic fatigue syndrome, also known as myalgic encephomyelitis and designated by the acronym ME/CFS. It is a disease with no known cure or even reliably effective treatments. Three of every four ME/CFS patients are women, for reasons that are not understood. It characteristically arises in two major waves: among adolescents between the ages of 15 and 20, and in adults between 30 and 35. The condition typically persists for decades.

“Chronic fatigue syndrome can turn a life of productive activity into one of dependency and desolation,” said Jose Montoya, MD, professor of infectious diseases, who is the study’s lead author. Some spontaneous recoveries occur during the first year, he said, but rarely after the condition has persisted more than five years.

The study’s senior author is Mark Davis, PhD, professor of immunology and microbiology and director of Stanford’s Institute for Immunity, Transplantation and Infection. More 

‘Solid basis for a diagnostic blood test’

“There’s been a great deal of controversy and confusion surrounding ME/CFS — even whether it is an actual disease,” said Davis. “Our findings show clearly that it’s an inflammatory disease and provide a solid basis for a diagnostic blood test.” 

Many, but not all, ME/CFS patients experience flulike symptoms common in inflammation-driven diseases, Montoya said. But because its symptoms are so diffuse —sometimes manifesting as heart problems, sometimes as mental impairment nicknamed “brain fog,” other times as indigestion, diarrhea, constipation, muscle pain, tender lymph nodes and so forth — it often goes undiagnosed, even among patients who’ve visited a half-dozen or more different specialists in an effort to determine what’s wrong with them.

Mark Davis

Montoya, who oversees the Stanford ME/CFS Initiative, came across his first ME/CFS patient in 2004, an experience he said he’s never forgotten.

 “I have seen the horrors of this disease, multiplied by hundreds of patients,” he said. “It’s been observed and talked about for 35 years now, sometimes with the onus of being described as a psychological condition. But chronic fatigue syndrome is by no means a figment of the imagination. This is real.”

Antivirals, anti-inflammatories and immune-modulating drugs have led to symptomatic improvement in some cases, Montoya said. But no single pathogenic agent that can be fingered as the ultimate ME/CFS trigger has yet been isolated, while previous efforts to identify immunological abnormalities behind the disease have met with conflicting and confusing results.

Still, the sporadic effectiveness of antiviral and anti-inflammatory drugs has spurred Montoya to undertake a systematic study to see if the inflammation that’s been a will-o’-the-wisp in those previous searches could be definitively pinned down.

To attack this problem, he called on Davis, who helped create the Human Immune Monitoring Center. Since its inception a decade ago, the center has served as an engine for large-scale, data-intensive immunological analysis of human blood and tissue samples. Directed by study co-author Holden Maecker, PhD, a professor of microbiology and immunology, the center is equipped to rapidly assess gene variations and activity levels, frequencies of numerous immune cell types, blood concentrations of scores of immune proteins, activation states of intercellular signaling models, and more on a massive scale.

Finding patterns

This approach is akin to being able to look for and find larger patterns — analogous to whole words or sentences — in order to locate a desired paragraph in a lengthy manuscript, rather than just try to locate it by counting the number of times in which the letter A appears in every paragraph.

The scientists analyzed blood samples from 192 of Montoya’s patients, as well as from 392 healthy control subjects. The average age of patients and controls was about 50. Patients’ average duration of symptoms was somewhat more than 10 years.

Importantly, the study design took into account patients’ disease severity and duration. The scientists found that some cytokine levels were lower in patients with mild forms of ME/CFS than in the control subjects, but elevated in ME/CFS patients with relatively severe manifestations. Averaging the results for patients versus controls with respect to these measures would have obscured this phenomenon, which Montoya said he thinks may reflect different genetic predispositions, among patients, to progress to mild versus severe disease.

I have seen the horrors of this disease, multiplied by hundreds of patients.

I have seen the horrors of this disease, multiplied by hundreds of patients.

When comparing patients versus control subjects, the researchers found that only two of the 51 cytokines they measured were different. Tumor growth factor beta was higher and resistin was lower in ME/CFS patients. However, the investigators found that the concentrations of 17 of the cytokines tracked disease severity. Thirteen of those 17 cytokines are pro-inflammatory.

TGF-beta is often thought of as an anti-inflammatory rather than a pro-inflammatory cytokine. But it’s known to take on a pro-inflammatory character in some cases, including certain cancers. ME/CFS patients have a higher than normal incidence of lymphoma, and Montoya speculated that TGF-beta’s elevation in ME/CFS patients could turn out to be a link.

One of the cytokines whose levels corresponded to disease severity, leptin, is secreted by fat tissue. Best known as a satiety reporter that tells the brain when somebody’s stomach is full, leptin is also an active pro-inflammatory substance. Generally, leptin is more abundant in women’s blood than in men’s, which could throw light on why more women than men have ME/CFS.

More generally speaking, the study’s results hold implications for the design of future studies of disease, including clinical trials testing immunomodulatory drugs’ potential as ME/CFS therapies.

“For decades, the ‘case vs. healthy controls’ study design has served well to advance our understanding of many diseases,” Montoya said. “However, it’s possible that for certain pathologies in humans, analysis by disease severity or duration would be likely to provide further insights.”

Other Stanford co-authors of the study are clinical research coordinator Jill Anderson; Tyson Holmes, PhD, senior research engineer at the Institute for Immunity, Transplantation and Infection; Yael Rosenberg-Hasson, PhD, immunoassay and technical director at the institute; Cristina Tato, PhD, MPH, research and science analyst at the institute; former study coordinator Ian Valencia; and Lily Chu, MSHS, a board member of the Stanford University ME/CFS Initiative.

The study was funded by the National Institutes of Health (grant U19AI057229), the Stanford ME/CFS Initiative Fund and an anonymous donor.

Stanford’s departments of Medicine and of Microbiology and Immunology also supported the work.    

By Bruce Goldman via Stanford Medicine News Center

 Less 

Society Citation Dean L. Winslow, MD, FIDSA, a compassionate, committed clinician, teacher, and military veteran who has been a champion for patients with HIV and victims of war and disaster, is the recipient of a 2017 IDSA Society Citation. First awarded in 1977, this is a discretionary award given in recognition of exemplary contribution to IDSA, an outstanding discovery in the field of infectious diseases, or a lifetime of outstanding achievement.

Dr. Winslow is vice chair of the Department of Medicine and a professor of medicine in the Division of Hospital Medicine and the Division of Infectious Diseases and Geographic Medicine at Stanford University, where he has been on the faculty since 1998. He is also academic physician-in-chief at Stanford/ValleyCare, a community teaching hospital. His professional career began in private practice in Delaware, where he started the state’s first multidisciplinary clinic for HIV-infected patients in 1985. During his later time in industry, he worked on studies of HIV drug resistance as a bench scientist and designed the clinical trials leading to the Food and Drug Administration’s approval of efavirenz. He also helped direct the clinical studies of nelfinavir and led the group responsible for regulatory approval of the first pharmacogenomics diagnostic device for HIV-1 drug resistance.

Before returning to Stanford fulltime in 2013, Dr. Winslow was chief of the Division of AIDS Medicine and chair of the Department of Medicine at Santa Clara Valley Medical Center, the institution’s largest academic and clinical unit. In addition to caring for adults, Dr. Winslow regularly attended on the pediatric infectious diseases consult service at the county hospital. From 2003 to 2011, Dr. Winslow also deployed to Iraq and Afghanistan six times as a flight surgeon in the Air National Guard in support of combat operations there, including serving as the commander of an Air Force combat hospital unit in Baghdad in 2008. Closer to home, he coordinated military public health and force protection in Louisiana after Hurricane Katrina. He has been awarded numerous military decorations for his service.

In addition to his care for wounded service personnel in the Middle East, Dr. Winslow also treated many local civilians. A passionate supporter of human rights and health, he has, since 2006, arranged for medical care, transportation, and housing in the U.S. for more than 20 Iraqi children and adults with complicated medical conditions for which surgical care was not available in Iraq. He has been recognized by the Iraqi Army for his humanitarian service to the Iraqi people and was named a Paul Harris Fellow by Rotary International.

After earning his medical degree from Jefferson Medical College in Philadelphia, Dr. Winslow completed his internship and residency in internal medicine at the Medical Center of Delaware, followed by a fellowship in infectious diseases at the Ochsner Clinic in New Orleans. A highly regarded attending physician on the ID consultation services at Stanford and the Palo Alto Veterans Administration Medical Center, Dr. Winslow has received multiple teaching awards. His wide-ranging knowledge and experience, combined with his passion for teaching and patient care, are legendary among ID fellows and residents. The author of more than 60 peer- reviewed publications and more than 90 presentations at national and international meetings, he is the current chair of IDSA’s Standards and Practice Guidelines Committee.

Dr. Winslow’s extensive knowledge, deep compassion, and wide-ranging experience over more than four decades have greatly impacted patients around the world, his colleagues, and the next generation of ID physicians. The Society is delighted to add a 2017 Society Citation to his long list of accomplishments

Source: Department of Medicine News

Harrington Discovery Institute has selected Paul Bollyky, MD, (assistant professor, infectious diseases) as one of its 2017 Scholar-Innovators. Chosen for his work on a novel drug for Type I Diabetes, Bollyky is one of 11 awardees this year.

The Institute grants each recipient a minimum of $100,000 (along with its drug development expertise and project management support) for breakthrough discoveries defined by innovation, creativity and potential clinical impact.

Bollyky says the award is “designed to accelerate breakthrough discoveries into new medicines” and adds, “In our case, this work will fund development of a novel therapeutic to prevent autoimmune diabetes.” Noting how innovative the research is, Upi Singh, MD, (chief, infectious diseases) says, “An infectious disease (ID) doc doing diabetes research! As I always say, everything is really ID. [This is] a nice example that science boundaries really are blurring and the best science is done at the interface of multiple disciplines.”

Throughout his career, Bollyky has earned a number of awards and grants, including a Catalyst Award from the Dr. Ralph and Marian Falk Medical Research Trust, the Outstanding Faculty Mentor Award, a SPARK Innovation Grant, a seed grant from Stanford Institute for Immunity, Transplantation and Infection and a Career Development Award from the Juvenile Diabetes Research Foundation.

Bollyky attended college at Columbia University in New York City.  He did his Ph.D. at Oxford University and went to Harvard Medical School for his M.D. He completed his residency at Brigham and Women’s Hospital, an infectious disease fellowship at the University of Washington and a post-doctoral fellowship at the Benaroya Research Institute.  He was appointed to the Stanford faculty in 2013. 

A number of faculty members, students, trainees, staff and affiliated staff won awards for dedication to and excellence in graduate and medical education, patient care and teaching.

Dr. Dora Ho has been recognized for her exemplary clinical and humanistic skills by being awarded the Alwin C. Rambar-James B.D. Mark Award for Excellence in Patient Care. 

The annual award recognizes a Stanford physician for compassion in working with patients and their families, excellence in providing medical treatment and effectiveness and pleasantness in interactions with patient-care staff. The Rambar award was established in 1985 to honor the late Alwin Rambar, a Chicago pediatrician long associated with the medical school. It was renamed in 1997 to include James Mark, a former chief of staff, Stanford thoracic surgeon and professor emeritus who was also Rambar’s son-in-law.

Source: Department of Medicine News

Recently, Paul Bollyky, MD, DPhil (assistant professor, Infectious Diseases), learned that he was to receive a Catalyst Award from the Dr. Ralph and Marian Falk Medical Research Trust. The one-year grant of almost one-half million dollars aims to fund preliminary steps (such as planning, project and team development, purchase of tools, hiring of personnel) that will, according to the Trust, enable awardees to undertake a high risk/high reward project “that addresses critical scientific and therapeutic roadblocks.”

Bollyky’s project aims to solve the problem of antibiotic resistance by the bacteriaPseudomonas aeruginosa, a major cause of pneumonia, infected wounds, and hospital-acquired infections. Antibiotic-resistant infections are not a new phenomenon, but they are a frustration to infectious diseases physicians whose patients have to fight them with fewer options all the time. “These Pseudomonas infections have evolved resistance to most, and in some cases, all of the antibiotics that we have to use against them,” explains Bollyky.

“As an infectious diseases doctor on the wards, I often see patients with these infections. And I have had patients where we’ve amputated limbs because we don’t have any antibiotics that work against their infection. If antibiotic choices are like bullets in a gun, I often find that I’m out of bullets to use against Pseudomonas infections. If we’re lucky we may come up with another solution for treating these bugs.”

Bollyky’s target is a bacteriophage, or a virus, that infects bacteria like Pseudomonas.  While most bacteriophages parasitize bacteria, Bollyky and his collaborators discovered that certain bacteriophages actually partner with their bacterial hosts. In a recent article in Cell Host & Microbe (2015;18:549-59), they showed that these bacteriophages play a pivotal role in the formation of biofilm, which is an adherent coating that allows bacteria to colonize surfaces like wounds and catheters. Both bacteriophage and bacteria thereby work together to cause infections. “What that means,” Bollyky explains, “is that we have a target that we can go after to treat antibiotic-resistant Pseudomonas infections.”

He continues: “I think the reason why the Falk Trust gave us this funding is because we have this novel target that puts another bullet in the gun, so to speak. In particular, the Catalyst Award is funding a pair of strategies to develop therapies that target this family of bacteriophages, and we’re optimistic that one and maybe both strategies will give us some novel therapies to treat multi-drug resistant Pseudomonas.”

In order to jump-start this project, Bollyky plans to use the Catalyst Award monies to hire a dedicated microbiologist, add needed equipment in his lab, and invest in reagents and materials to develop his technology. “What I’m most excited about,” he says, “is getting a slug of resources and, to be honest, a vote of confidence from a handful of reviewers. This gives us both the momentum and the resources to take this to the next level. It propels the whole lab in a new and pretty exciting direction.”

This breadth of science has allowed him to rapidly integrate and contribute greatly to the Stanford academic/intellectual environment

Ideally, a successful, milestone-driven Catalyst Award will lead to another grant, a two-year Transformational Award from the Falk Trust, which offers $1 million of support to approximately half of Catalyst awardees following a second round of competitive applications.

Upinder Singh, MD (chief, Infectious Diseases and Geographic Medicine) cites two features that make Dr. Bollyky such a successful scientist: “One is his ability to think outside the box, and the second is the overall breadth of his research program (ranging from autoimmunity, inflammation, to antibiotic resistance [this grant] to biofilm formation). This breadth of science has allowed him to rapidly integrate and contribute greatly to the Stanford academic/intellectual environment. He has already collaborated (and in some cases published) with a number of faculty (including in Bone Marrow Transplant, Neurology, Immunology, Pediatrics).

Asked how he feels about having received this first award from the Falk Trust, Bollyky says: “To use an outer-space metaphor, the Catalyst Award is the first stage that allows this project — and optimistically my lab — to get off the ground. And then if we make the second stage of the Falk funding, the Transformational Award, then that’s the booster that will really put us into orbit.”

We hope to report on the orbiting Bollyky lab in about a year.

Stanford Researchers Uncover New Bacterial Diversity Inside U.S. Navy Dolphins

• Steve Fyffe, from Stanford CISAC

Chairman of Joint Chiefs of Staff Gen. Martin E. Dempsey watches a dolphin jump out of the water during a tour of the U.S. Navy Marine Mammal Program in San Diego on March 12, 2012.

Photo credit: 

U.S. Navy

Stanford researchers working with the U.S. Navy’s Marine Mammal Program in San Diego have discovered a startling variety of newly-recognized bacteria living inside the highly trained dolphins that the Navy uses to protect its ships and submarines, find submerged sea mines and detect underwater intruders. They found similar types of bacteria in wild dolphins as well.

“About three quarters of the bacterial species we found in the dolphins’ mouths are completely new to us,” said David Relman, Stanford professor of microbiology and medicine, and co-author of a paper published in the journal Nature Communications on Wednesday.

These previously unknown bacteria represent “whole new realms of life,” according to Relman.

“Bacteria are among the most well-studied microbes, so it was surprising to discover the degree to which the kinds of bacteria we found were types that have never been described,” he said. “What novelty means is not just new names of species, families, classes or phyla…there’s reason to believe that along with this taxonomic novelty, there’s functional novelty.”

The U.S. Navy has been training dolphins and sea lions to carry out defensive military missions from their bases in San Diego and elsewhere since the early 1960s.

Over the years, it has also funded scientific research and become the single largest contributor to the scientific literature on marine mammals, producing more than 800 publications, according to the Navy.

Relman started working with the Navy more than 15 years ago to identify bacteria suspected of causing stomach ulcers in their dolphins.

His latest project to catalog the bacterial communities (or microbiota) living inside the dolphins began when the Navy asked him to help develop a probiotic bacterial strain that could keep their dolphins healthy, or help sick dolphins get better.

Navy trainers took regular swabs from the dolphins’ mouths and rectal areas, using what looked like a Q-tip, and shipped the samples to Stanford on dry ice for analysis.

They also collected samples of the air the dolphins exhaled from their blowholes (known as “chuff”) onto sterile filter paper, as well as samples of their gastric juices using a tube that the dolphins would swallow on command, and for comparison, bacteria from the surrounding water.

The study found a similar amount of diversity and novelty in bacterial samples taken from wild dolphins living in Sarasota Bay off the west coast of Florida, although there were slight differences in the bacteria from the dolphins’ mouths.

Relman said he hoped to develop a profile of the normal microbial communities in healthy dolphins and other marine mammals, so that scientists could detect any early change that might signify an imminent disease, or health problems caused by climate change and ocean warming.

“There’s a lot of concern about the changing conditions of the oceans and what the impact could be on the health of wild marine mammals,” Relman said. “We would love to be able to develop a diagnostic test that would tell us when marine mammals are beginning to suffer from the ill effects of a change in their environment.”

The research could help solve other mysteries, such as how dolphins digest their food, even though they swallow fish whole without chewing them.

The key could be a unique bacterial group that’s also been identified in an endangered species of freshwater dolphins living in China’s Yangtze River, said Elisabeth Bik, a research associate at the Stanford Department of Medicine and lead author on the paper.

“It’s a very intriguing bacterial group that nobody has seen before in any other terrestrial animal group,” said Bik. “I would really love to know more about those bacteria and sequence their genomes to understand more about their functional capacity.”

The study also examined oral, gastric and rectal samples from the Navy’s trained sea lions.

“The sea lions and dolphins are kept at the same facility, they’re fed exactly the same fish, and they’re swimming in the same water…but they’re very, very different in terms of microbiota,” Bik said.

Unlike dolphins, sea lions share many common types of bacteria with their terrestrial cousins.

“Sea lions weren’t that different from other carnivores like dogs and cats,” Bik said. “They’re evolutionarily related to them, and their microbiota looks very similar to those animals. But dolphins don’t really have a terrestrial mammal that’s closely related, and their microbiota looks very different from anything else that people have seen.”

Relman said his team was planning on expanding their study to include other marine mammals such as sea otters, killer whales, baleen whales, grey whales, harbor seals, elephant seals and manatees. Their purpose, in part, is to understand how life in the sea, over the millions of years since the return of mammals, may have shaped the structure of their microbial communities and the roles they play in marine mammal health.

They’re already working to analyze more than 80 samples of killer whale stool that the U.S. National Oceanic and Atmospheric Administration has gathered with the help of specially trained sniffer dogs, which stand on the bow of their boats and point to fresh killer whale feces before it sinks.

The California Department of Fish and Wildlife is contributing samples from the sea otters and seals it studies as part of its conservation, ecological, and monitoring programs.

And the Marine Mammal Center in Sausalito, which is the West Coast’s largest rescue and rehabilitation facility for marine mammals, is sending samples from the seals in its care.

Relman said the research could help scientists begin to answer fundamental questions about life in the ocean.

“Marine mammals remain one of the more poorly understood habitats for studying microbial life, and there would be lots of reasons for thinking that these are important environments to study, in part because of the relevance for the health of these marine mammals, but also because they represent a view into what it means to live in the sea and the nature of our relationship with this vast aspect of our environment,” Relman said.

Collaborators and co-authors on this study included Stephanie Venn-Watson and Kevin Carlin from the National Marine Mammal Foundation, and Eric Jensen from the Space and Naval Warfare Systems Center Pacific, in San Diego.

By Bruce Goldman, Department of Medicine News

High diversity among certain cells that help fight viruses and tumors is strongly associated with the likelihood of subsequent infection by HIV, Stanford University School of Medicine researchers have found.

Natural killer cells, or NK cells, are lymphocytes, a type of white blood cell. NK cells’ increased diversity, the scientists learned, may stem from prior exposures to viruses.

The findings, described in a study published July 22 in Science Translational Medicine, could spur the development of blood tests capable of flagging individuals’ susceptibility to viral infection. The study also offers insights into the workings of NK cells, a somewhat poorly understood but crucial group of immune cells. 

“This puts NK-cell diversity on the map as a metric of immune function,” said Catherine Blish, MD, PhD, assistant professor of infectious diseases and geographical medicine and the study’s senior author. “But it was a first foray. Before we can say definitively that NK-cell diversity predicts a person’s susceptibility to infection, we need to validate these findings by looking at large numbers of individuals in a different population.

“NK cells are particularly suited to detecting and demolishing virally infected or cancerous cells,” Blish added. “They arrive on the scene quickly, and they act quickly. An NK cell can kill an infected cell in 10 minutes.”

Unexpected finding

Unexpectedly, it was higher, rather than lower, diversity in this immune-cell population that turned out to be associated with increased HIV susceptibility in the study. The investigators had figured that, as is the case with B cells and T cells — the two other, better-known types of lymphocytes — diversity in NK cells would be a strength, not a detriment, Blish said.

“Our hypothesis was wrong,” she said. “We didn’t think NK-cell diversity would be a bad thing, or that NK cells’ diversification would occur to the extent that it does with viral exposure.”

Using a cutting-edge, single-cell analytic technology called mass cytometry, Blish and her colleagues, including the study’s lead author, graduate student Dara Strauss-Albee, showed that overall diversity in people’s NK-cell repertoires is low at birth and steadily accumulates over the course of a lifetime.

An individual T cell has surface receptors that recognize unique protein snippets, called peptides, on other cells’ surfaces. The structures of these receptors, which can discern “healthy” versus “suspect” peptides, differs from T cell to T cell. So a healthy person’s legion of T cells can surveil and sort out hundreds of millions of different peptides representing possible invaders.

Unlike their T cell cousins, NK cells don’t have surface receptors that recognize unique peptides. Instead, these lymphocytes harbor various combinations of generic receptors. Some receptors recognize signs of other cells’ normalcy, and others recognize signs that a cell is stressed — due, say, to viral infection or cancerous mutation. On recognizing their targeted features on other cells’ surfaces, an NK cell’s “normalcy” receptors tend to inhibit it, while its stress-recognizing receptors activate it.

All told, NK cells can have many thousands of different combinations of these receptors on their surfaces, with each combination yielding a slightly different overall activation threshold. An NK cell’s surface features also vary depending on its degree of maturation.

Mass cytometry analysis of NK cells exposed in a dish to HIV — as well as to West Nile virus, which differs substantially from HIV in its makeup and its modus operandi — showed that exposure to virus-infected cells leads to differentiation of NK cells and to an increased diversity among them. But diversification in the NK-cell population, the experiments indicated, was associated with a diminished ability of these cells’ ability to replicate and kill.

The researchers also showed that while healthy human adults differ considerably from one another in the diversity of their NK-cell populations, a given adult’s NK-cell population remains quite stable, changing little over periods of many months. An examination of NK cells extracted from umbilical-cord blood showed that newborns’ NK-cell population is much less diverse.

Blish said she believes that viral exposure during one’s lifetime is the driving force behind the maturation, differentiation and diversification of NK cells.

Blish said she believes that viral exposure during one’s lifetime is the driving force behind the maturation, differentiation and diversification of NK cells.

HIV link

In order to assess the impact of NK-cell diversity on adult humans’ viral susceptibility, Blish and her associates turned to blood samples that had been drawn during the Mama Salama Study, a longitudinal study of just over 1,300 healthy pregnant or postpartum Kenyan women. In that study, 25 of the women were found to have HIV. For 13 of these women, blood drawn both before and after infection was available.

This puts NK-cell diversity on the map as a metric of immune function.

Using mass cytometry, the researchers carried out a precise analysis of NK cells in the women’s blood and observed a strong positive correlation between the diversity of a woman’s NK cell population and her likelihood of becoming infected with HIV. This correlation held up when the scientists controlled for age, marital status, knowledge of sexual partners’ HIV status and history of trading sex for money or goods. The two groups of women were also statistically indistinguishable with respect to their sexually transmitted disease status or their reported frequency of recent unprotected sex.

The NK-diversity-dependent difference in these women’s likelihood of HIV infection was huge. Those with the most NK-cell diversity were 10 times as likely as those with the least diversity to become infected.

A 10-fold risk increase based solely on NK-cell diversity is far from negligible, said Blish. “By way of comparison, having syphilis increases the risk of contracting HIV two- to four-fold, while circumcised men’s HIV risk is reduced by a factor of 2.5 or 3,” she said.

The observations could have clinical potential, most immediately by spotlighting people who need to be closely monitored for possible viral infections and, perhaps, prophylactically treated. But Blish cautioned that the study remains preliminary.

Other Stanford co-authors are professor of statistics Susan Holmes, PhD; statistics graduate student Julia Fukuyama; research assistant Emily Liang (now a medical student at UCLA); and immunology graduate student Justin Jarrell.

The study was funded by a Beckman Young Investigator Award, a National Institutes of Health New Innovator Award (grant DP2AI11219301) and a National Science Foundation training grant.

Zika infection causes developing cranial cells to secrete neurotoxic levels of immune molecules

New research shows that cranial neural crest cells can be infected by the Zika virus, causing them to secrete high levels of cytokines that can affect neurons in the developing brain.

SEP 29 2016

Babies born to women infected with the Zika virus can suffer from a birth defect called microcephaly, or abnormally small heads. A new study from Stanford found that the infection can affect the cells that give rise to the bones and cartilage of the skull and face.
Alila Medical Media/Shutterstock

Infection by the Zika virus causes a population of cells in the cranium of a developing embryo to secrete neurotoxic levels of immune signaling molecules called cytokines, according to a new study by researchers at the Stanford University School of Medicine.

Although the study was conducted solely in cells grown in a laboratory dish, it may provide one possible explanation for why babies born to women infected with the virus can suffer from a birth defect called microcephaly, or abnormally small heads.

“Affected babies have small brains and small skulls,” said assistant professor of medicine Catherine Blish, MD, PhD. “Cells in the cranial neural crest give rise to the bones and cartilage of the skull and face, and they also form an important supportive niche for the developing brain. We wondered if the Zika virus could infect cranial neural crest cells, perhaps giving rise to deficits in skull formation and altered neural development.”

The study was published online Sept. 29 in Cell Host & Microbe. Blish is the senior author. Graduate students Nicholas Bayless and Rachel Greenberg share lead authorship of the study.

A reservoir for the virus

Cranial neural crest cells are cells that arise in humans within about five to six weeks of conception. Although they first appear along what eventually becomes the spinal cord, the neural crest cells migrate over time to affect facial morphology and differentiate into bone, cartilage and connective tissue of the head and face. They also provide critical molecular signals that support nearby developing neurons in the brain. 

Catherine Blish

Bayless and Greenberg used a technique developed in the laboratory of study co-author Joanna Wysocka, PhD, a professor of chemical and systems biology and of developmental biology, to convert human embryonic stem cells into cranial neural crest precursors in the laboratory. Wysocka’s research focuses on understanding how these cells affect the embryonic development of facial features, including those of humans and chimpanzees.

Recent research has focused on the effect of Zika virus infection of the neural precursor cells that give rise to neurons in the developing brain. But Bayless and Greenberg found that not only can cranial neural crest cells also be infected by the Zika virus, they respond differently than their neighboring neural precursor cells to the infection. Rather than rapidly dying, as the neural precursors do, the cranial neural crest cells act as a reservoir for the virus by allowing it to replicate repeatedly. In addition, they begin to secrete high levels of cytokines, including leukemia inhibitory factor and vascular endothelial growth factor, known to affect neural development.

“The magnitude of altered cytokine secretion caught us by surprise,” said Blish. “These molecules are important for neurogenesis, and the infected cells are secreting them at high levels.”

Abnormally shaped cells

When Bayless and Greenberg incubated neural precursor cells together with infected neural crest cells, the neural precursors appeared abnormal and were more likely to initiate a program of cellular suicide.

Joanna Wysocka

They next exposed the neural precursor cells to leukemia inhibitory factor and vascular endothelial growth factor at levels equivalent to those secreted by the infected cells. After three days, they observed an increase in structures associated with cellular migration and growth, as well as a significant increase in the frequency of cell death.

“At least in vitro, these elevated levels of cytokines appear to induce premature differentiation and migration,” said Wysocka. “This abnormal developmental program then leads to cell death.”

The Zika virus, which is spread by the Aedes genus of mosquito, has sprung into the public eye over the past year as it has become increasingly apparent that pregnant women infected with the virus can pass it to the fetus, causing devastating birth defects. Outbreaks of the virus are currently occurring in multiple countries and in three American territories: American Samoa, Puerto Rico and the U.S. Virgin Islands. Local mosquito-borne transmission has also been identified in two areas of Miami, and the World Health Organization has designated the disease a public health emergency of international significance.

“Our study brings attention to the possibility that other infected embryonic cell types in the developing head can influence Zika-associated birth defects, including microcephaly, perhaps through signaling to neighboring cells or by serving as a viral replication reservoir,” said Wysocka. “Formation of the cranial neural crest cells and a cross-talk between brain and craniofacial development occurs during the first three months of human fetal development, which is when epidemiological studies have suggested that Zika infection correlates with poor birth outcomes.”

The researchers said that although the findings are intriguing and merit further study,  their studies were conducted only on cells grown in a laboratory and it is possible that other factors related to Zika infection may affect brain size and outcomes in affected infants.

Tomek Swigut, PhD, a senior scientist in Wysocka’s lab, is also a co-author of the study.

The research was supported by the National Institutes of Health (grants 1DP2AI112193 and DE024430), the Tashia and John Morgridge Faculty Scholar Program from the Stanford Child Health Research Institute, the March of Dimes Birth Defect Foundation, the Howard Hughes Medical Institute, a seed grant from the Stanford Center for Systems Biology and two Ruth L. Kirschstein awards.

Stanford’s Department of Medicine also supported the work.

• PRESS RELEASES 
• 

  By KRISTA CONGER

  Krista Conger is a science writer for the medical school's Office of Communication & Public Affairs. Email her at kristac@stanford.edu.