Monday, October 20, 2014

Immune System Disruption: The search for answers

Immune system disruption
The search for answers

By Kris Newby
Illustration by Jeffrey Decoster
Photography by Timothy Archibald

Erin keeps a photo of herself playing soccer in the living room of her
tidy cottage near San Francisco Bay. It captures her image frozen in
time and space, hurtling like a comet between two opponents, her
white-blond ponytail fanned out like flames.


video interview-

Related reading: The Institute for Immunity, Transplantation and Infection

Related reading: Hacking the immune system: The hunt for chronic
fatigue biomarkers

"She was a midfielder with boundless energy, lightning fast," recalls
the coach of her Big Ten college soccer team.

Erin, in her early 30s, always assumed that soccer would be at the
center of her life. As a little girl, her favorite toy was a soccer
ball, a present from a cousin living in Rome. At age 4, she drew a
picture of herself competing in the Olympics. In high school, she was
invited to try out for the national team's talent pool. After college,
she played for the Detroit Jaguars, a semi-professional team.

But her dream of playing competitive soccer abruptly ended after a
trip to Mexico in 2007.

"I was doing social work at an orphanage when I got sick," says Erin
(who asked that her real name not be used). "I passed out and was
hospitalized with a high fever, low blood pressure and swollen lymph
nodes. After that, I was never the same."

Thus began her seven-year journey battling a devastating illness with
no known cause or cure. She was bedridden for all but four hours a
day. She could stand only for 20 minutes without fainting. But the
worst symptom was the brain fog.

"It was like my thoughts were stuck in molasses," says Erin.

No one could figure out what was wrong or how to fix it. She was
labeled with chronic fatigue syndrome. Despair set in as the door to
her old life slowly closed.

Interrogating the immune system

That same year a door opened on the other end of San Francisco Bay, in
a windowless basement of a clinical research building at Stanford
University. Here Mark Davis, PhD, an immunologist with a computer
hacker's mindset, was launching a center that aimed to break open the
black box called the human immune system. This dynamic network of
biological sensors, cells, secretions and genes is like a sixth sense,
able to detect microbial friend from foe in the food we eat, the
things we touch and the air we breathe. The most intelligent facets of
the immune system are still a mystery. How does it differentiate
between the cells that are part of you and the interlopers? What are
the steps involved in launching an army of white blood cells to attack
a microbial invader? How does the system dial down the resulting
tissue-damaging inflammation? How do our traitorous cells — the
cancers — make themselves invisible to our immune system?

Davis, director of Stanford's Institute for Immunology,
Transplantation and Infection, is in the right place at the right time
for this quest, swimming in the primordial soup of creative
disruption, Silicon Valley. At long Stanford cafeteria tables
frequented by geneticists, bioengineers, math geniuses, computer
programmers, surgeons and cancer biologists, ideas just happen. And
sometimes, visionary entrepreneurs throw money at these ideas. Such is
the case with Davis' Human Immune Monitoring Center, which before long
had enough funding to acquire a CyTOF, a time-of-flight mass
spectrometer for high-speed acquisition of multiparametric single-cell
data. (If someone asks you if you want one of these instruments, just
say yes.)

The CyTOF enables researchers to detect 40 different components within
a single cell at the rate of 1,000 cells per second. It not only
measures static levels of proteins, useful in identifying different
types of immune cells, but it also detects minute changes in signaling
proteins within cells in response to various stimuli. Using this

device, a staggering amount of data is generated. And armed with
big-data analytical methods developed during the Human Genome Project,
Davis' multidisciplinary team is trying to bring order and meaning to
the output.

When it's all done, the team hopes to create a map of what a healthy
person's immune system should look like and a flow chart depicting how
immune-system signaling pathways work.

The ultimate goal of Davis' "Human Immunome Project" is to develop
better tools to answer immunological questions no one can answer now.
One of the first: What is wrong with the immune systems of patients
like Erin, and how can we help them get better?

Two years into her illness, Erin was broken. On any given day, she
would cycle through a laundry list of symptoms: brain fog, dizziness,
light sensitivity, a sore throat, nausea, swollen lymph nodes,
crushing fatigue, a racing heart, ear ringing, drenching sweats and

During this time, she had lost some of her most active and athletic
friends, who grew impatient with the waxing and waning symptoms that
prevented her from the leaving the house on most days.

"I had times where I'd shut the blinds, lie down and hope for a better
day," says Erin. "Literally, my escape was through my dreams. I just
couldn't stand to be in my body."

Her life revolved around doctors' appointments. One physician ruled
out infectious diseases. Neurologists examined her for seizure
disorders and brain tumors. A rheumatologist evaluated her for
systemic lupus erythematosus and other inflammatory diseases. An
endocrinologist agreed that the origin of her fatigue was not the
thyroid, the adrenals or any other gland. A cardiologist assured her
it was not her heart.

None of them could settle on a definitive diagnosis, so the physicians
tagged her with the insurance code for chronic fatigue syndrome, a
controversial diagnosis for a set of symptoms also sometimes labeled
as CFIDS — for chronic fatigue and immune dysfunction syndrome — and
ME — for myalgic encephalomyelitis. The dominant moniker today is

No one knows what causes ME/CFS. Some think that an infectious agent
or overactive immune system triggers it. Others blame genetic flaws,
environmental factors or a combination of any of the above.

Roughly 17 million people worldwide (1 million to 4 million in the
United States) have ME/CFS. It strikes people of all ages and racial,
ethnic and socioeconomic groups. It is diagnosed two to four times
more often in women than men.

It's a syndrome that gets little respect in the medical community
because, with no tangible cause and an ever-changing constellation of
symptoms, patients often get labeled as hypochondriacs, malingerers or
seekers of addictive pain medications. The primary diagnostic
criterion for this condition is infuriatingly vague — "six or more
consecutive months of severe fatigue" — virtually unchanged since

As Erin went from specialist to specialist, well-meaning doctors grew
frustrated with their inability to help her. One day Erin blacked out
while driving, almost hitting a streetlight. After another fainting
accident, an emergency room physician told her, "On hot days, women

"I felt objectified, like a slab of meat," says Erin.

Finally, in 2009 Erin was diagnosed with postural orthostatic
tachycardia syndrome — which accounted for her fainting spells. For
treatment, her cardiologist sent her to Stanford Hospital's cardiology
clinic to see one of the nation's few POTS specialists, cardiac
electrophysiologist Karen Friday, MD.

POTS, which often accompanies ME/CFS, is a fainting disorder
associated with an abnormal increase in heart rate and low blood
pressure. The mechanism is unknown, but some people develop it after
contracting viral or bacterial infections like mononucleosis,
pneumonia or Lyme disease. Friday prescribed fludrocortisone to manage
Erin's low blood pressure, but to explore the possibility of an
underlying microbial trigger, she sent Erin to see Stanford professor
of infectious diseases José Montoya, MD.

Montoya, 54, dapper in his white coat and tie and smiling widely,
greeted Erin with a bear hug and told her in his thick Colombian
accent, "I want to make your life beautiful again."

"Dr. Montoya was a shining beacon of hope," says Erin.

Montoya's ethos to reduce patient suffering was shaped by a
hardworking, single mother and the iron-fisted priests at his Catholic
school in Cali, Colombia. He was accepted into medical school at age
18, after receiving the third-highest qualifying exam score in his
native country that year. After medical school he went on to Tulane
University School of Medicine for his residency, then joined the
infectious disease division at Stanford. At Stanford he became a
world-recognized authority on infections affecting heart transplant
recipients and on toxoplasmosis, a common parasitic disease.

Montoya conducted a detailed medical history and physical exam on
Erin, then ordered a battery of tests for viruses, bacteria and fungi.
His wide-net diagnostic approach paid off; he found two blood-borne
microbes — Human Herpesvirus-4 and the coxsackie virus — known to
cause chronic disease and POTS.

Though Montoya wasn't sure if these viruses were at the root of Erin's
illness or merely collateral infections, he started her on a high dose
of the antiviral drug famciclovir. Erin was relieved to finally have a
physician who wasn't going to punt her case to another specialist.

"I wanted to live my life again," says Erin.

Montoya is one of only a handful of clinician-researchers who accept
ME/CFS patients, and he currently has a waiting list of about 150.

Back in 2005, while attending a conference on toxoplasmosis in Paris,
Montoya told his mentor that he wanted to research ME/CFS. His mentor
scoffed at the idea, pointing to a homeless person lying in a Parisian

"That's going to be you if you go into chronic fatigue research," the
mentor told him.

The hard truth is that most medical research labs rely in large part
on U.S. government funding, and the ME/CFS research budget is
insufficient to support a typical university research lab.

The National Institutes of Health, the largest funder of medical
research in the United States, allocated only $5 million for ME/CFS
research in 2013. (To put this in context, the annual NIH research
budget for multiple sclerosis, with 400,000 sufferers, is $112
million.) The reasons behind this underfunding are complicated.

One factor is that the NIH funding process favors well-defined
diseases that fit neatly into medical specialties like cardiology,
cancer and neurology. Most of these medical societies have organized
lobbying efforts, sometimes backed by pharmaceutical or medical
technology companies. Another factor is that collectively ME/CFS
patients are too sick to organize, raise money and lobby for research
dollars. And then there is the stigma associated with the condition;
some NIH grant reviewers are reluctant to fund research because they
believe that ME/CFS is a psychosomatic, "all in the head," disorder.
(To remedy this, the NIH recently created a special emphasis panel so
that researchers familiar with the condition review grant

But none of this deterred Montoya, who was driven to do something for
the suffering patients queuing up for appointments.

Opportunity knocked in 2008 when a wealthy donor met with Montoya to
talk about the ME/CFS problem. He asked if a $5 million donation for
research could make a difference.

Montoya could hardly believe the sum, replying, "Yes, give me five years."

With the freedom of private funding, Montoya was able to take a
multifaceted and rigorous approach to analyzing ME/CFS. Traditionally,
NIH funding is awarded through medical specialty groups that tend to
favor research that tests one narrow hypothesis about a disease. For
example, a researcher might get funded to screen blood samples for one
virus, or treat patients with one drug. This approach takes a long
time, and researchers typically aren't able to share and build on
discoveries for years.

Montoya's game plan was to use a big-picture, big-data strategy to
find out what was wrong with patients like Erin. His first step in
launching the Stanford Initiative on Infection-Associated Chronic
Diseases was to convince a dozen or so academic investigators to
venture out of their comfort zones to research a wildly unpopular
disease using technologies yet to be developed.

Montoya convinced experts in immunology, rheumatology, genetics,
bioengineering, anesthesiology, neuroradiology, cardiology,
psychiatry, infectious diseases and bioinformatics to all work
together. The team members would be searching blood samples for
infectious microbes, inflammation-related molecules and genetic flaws.
They'd do brain scans and physical exams. They'd survey study subjects
for fatigue levels and medical histories. Then they'd compare all this
data with that of healthy people to see what was different. Next, he
launched a Bay Area recruitment campaign for 200 patients who met the
Centers for Disease Control's definition for chronic fatigue syndrome,
including Erin, and 400 age- and sex-matched healthy volunteers, all
of whom agreed to donate eight tubes of blood and be poked, scanned
and surveyed over the next decade.

The most complex part of the ME/CFS initiative was the exploration
into what was happening with the immune system of these patients. For
this role, he needed an expert who didn't care about the ME/CFS stigma
or how things have been done in the past. So he called on Mark Davis.

There will be blood

Davis, in well-worn jeans and running shoes, leans back in his chair,
surrounded by pillar-piles of scientific papers. At first glance one
might assume that he is — in California-speak — a mellow dude.

But looks can be deceiving, because Davis, who discovered how T cells
help a body fight off infections, is all about the fight. [See story,
page 38.]

As if to prove this point, Davis reaches into a random stack of paper
and pulls out a black-and-white photo of a collegiate fencing match.

"This is me," he says, pointing to a man in white flying off the
ground, plunging the tip of a silver foil directly between the eyes of
a masked opponent. "I like to poke people."

He then reaches into another stack of paper and pulls out the Dec. 19,
2008, issue of Immunity. It is a poke-in-the-eye to fellow
immunologists, an essay titled, "A Prescription for Human Immunology."

In this oft-quoted paper, he describes immunology as a field known for
its "impenetrable jargon, byzantine complexity and acrimonious

He also chides many of his colleagues for spending too much time on
mouse studies and not enough on human studies. For immunological
studies, mice are fast and easy. They can be bred with specific
diseases, such as diabetes or Parkinson's, and then dissected to
evaluate the effectiveness of experimental treatments. There are
relatively few regulatory, financial and ethical hurdles to working
with mice. He emphasizes that lab mice live in isolated, disease-free,
temperature-controlled environments, far different from the crowded,
germ-ridden urban habitats of your typical Homo sapiens. (Most humans
are infected with six different herpes viruses, and who knows what
else.) The other problem with "mouse models" is that their common
ancestors are genetically separated from Homo sapiens by some 65
million years.

"Inbred mice have not, in most cases, been a reliable guide for
developing treatments for human immunological diseases," says Davis.

Instead, he would like to shift the focus of immunology research back
to where it can do the most good — to humans and their blood. And
along the way, he'd like to slay a few sacred cows of medicine.

First off, Davis believes it's time to rethink the CBC or complete
blood count, the most commonly ordered medical test on the planet. The
CBC, which has changed little since it was put into mainstream use in
the '50s, provides physicians with relative numbers of a patient's
red, white and platelet blood cells. This test isn't really "complete"
and it doesn't begin to capture the nuances of a working immune

As researchers gain a better understanding of this system, he'd like
to develop a new set of metrics for immune system health that
communicates more of a continuum of health rather than a
black-and-white declaration. If the immune system is underactive, a
person is open to infections, mutations and premature aging. If it is
overactive, a person may suffer from allergies, autoimmune disease and
excessive inflammation. Davis wants to redefine health as an immune
system in balance, then develop better reporting tools to help
clinicians determine if a patient is fighting a virus, a bacteria, an
allergy or environmental toxins.

Davis' field of dreams for this effort is Stanford's Human Immune
Monitoring Center. Launched in 2007, today the center consists of
dozens of instruments that provide standardized, state-of-the-art
immune system analysis at the RNA, protein and cellular levels. Its
gene-sequencing instrumentation is located in a nearby building and
shared with the Stanford Functional Genomics Facility. For researchers
both inside and outside of the university, the center's 15-person
staff provides a one-stop shop for these services. At the start of a
project, the center's director, Holden Maecker, PhD, meets with
investigators to help plan studies and determine needs, such as what
samples to take, how to store those samples and which tests will best
answer their scientific questions. There is also assistance on results
archiving, reporting and data mining.

"We have about 60 different projects under way at the center right
now," says Maecker. These include searches for immune biomarkers for
aging, Alzheimer's, autoimmune disease, cancer, chronic pain,
rejection in organ transplantation and viral infections.

"I believe this is the only facility of its kind anywhere," says Davis.

Montoya's chronic illness initiative is the largest project in the
HIMC at this time, and the complexity of the task ahead is daunting.
The staff is looking for meaningful patterns in the many components of
the 600 blood samples, including dozens of cytokines, 35 cell-surface
proteins, 15 or so types of blood cells, and more than 47,000 genes
and regulatory nucleic acids. The challenge is not only to quantify
the normal ranges for these components, but also to understand
relationships between the components and reverse-engineer the cascade
of biochemical reactions that drive immune system processes. He
anticipates it will take about a year to run all 600 samples through
the processes.

"It's like dumping a hundred different puzzles on the floor and trying
to find two pieces that fit," says Davis.

The workhorses for these tasks are the center's two CyTOFs. Stanford
has seven of these $630,000 instruments, more than any other academic
medical center, thanks to Garry Nolan, PhD, a Stanford professor of
microbiology and immunology. Nolan purchased the first commercially
available CyTOF, and as an early adopter developed protocols for using
it in cancer biology, immunology and cell biology. He now holds equity
in Fluidigm, a company that manufactures the machines.

His enthusiasm for the technology and his willingness to share what
he's learned has catalyzed an active Stanford community of CyTOF

"With seven CyTOFs on campus, Stanford continues to innovate and lead
the way in 'deep-profiling' studies of the immune system," says Nolan

Through this work, the Stanford team hopes to gain a better
understanding of the complex, inner workings of the immune system.
This will ultimately give physicians and their patients the tools to
answer the fundamental question, "How is my immune system doing

Putting the pieces together

This past March, four years after the launch of the ME/CFS initiative,
Montoya held an all-day symposium to present early findings on what's
happening within the hearts, blood, brains and genomes of ME/CFS
patients. The lecture hall was packed with researchers from Australia,
Canada, the United Kingdom and the United States, as well as patients,
all eager for any news on a research agenda that had been stalled for

(These presentations can be watched at

At the end of the day, the biggest news was the identification of a
number of biological markers that indicate ME/CFS patients may be
suffering from out-of-control inflammation.

First up was a neuroinflammation researcher, Jarred Younger, PhD, who
worked with the HIMC to measure daily fluctuations of 74 blood markers
and cytokines. For this study, published in the Journal of
Translational Medicine
(, volunteers
gave blood once a day for 25 days, and reported their fatigue levels
on a hand-held computer twice a day. (Younger moved to the University
of Alabama in Birmingham in August.) Through complex statistical
analysis, the team found 12 cytokines that were consistently elevated
on days that ME/CFS patients felt the most fatigued. One of these
cytokines, leptin, activates microglial cells, the brain's first line
of defense against infections. When microglial cells are primed, they
start pumping out signaling chemicals that generate the flulike
symptoms commonly reported by ME/CFS patients — fatigue, headaches and
brain fog.

Amit Kaushal, a medical resident with a PhD in bioinformatics, did the
first pass on genomic analysis. For his part of the investigation, he
scanned the blood of 200 ME/CFS patients and 400 healthy subjects for
47,000 gene elements, then ran this data through the Nextbio Disease
Atlas, a publically accessible database that catalogs gene markers
associated with specific diseases. After analysis, he found genetic
markers in the blood of ME/CFS patients similar to those in patients
with well-defined chronic inflammatory diseases.

The quarterback for the search for infectious microbes is W. Ian
Lipkin, MD, a renowned microbe hunter and the director of the Center
for Infection and Immunity at Columbia University's Mailman School of
Public Health. He is using high-throughput sequencing platforms that
enable rapid identification and molecular characterization of known
and novel disease agents.

"We decided to go in without any preconceived notions about what we'd
find," says Lipkin. "Our approach is comprehensive, rigorous and quite

In the first analysis, his team found no significant differences in
the types of infectious organisms present in the blood of people with
ME/CFS or their matched normal controls. In the next phase he'll
search inside the blood cells and analyze the gastrointestinal
microbiome for the presence of bacteria or viruses that may trigger
the immunological disturbances that are so disabling in ME/CFS. The
objective of this work is to identify the agents responsible for
initiating and perpetuating disease. This could lead to vaccines,
drugs or probiotic interventions.

While not all of these results have been published or independently
confirmed, the researchers were excited about finding measurable,
physical differences between ME/CFS patients and healthy controls.
(Stanford assistant professor of radiology Michael Zeineh, MD, has
identified structural brain abnormalities in the ME/CFS patients —
findings are slated for publication in the coming months.) More pieces
of the puzzle are coming together, providing other ME/CFS researchers
with ideas to build on. For ME/CFS patients it was validation — their
symptoms are real, with measurable biological markers.

Eight months after seeing Montoya, Erin's recovery from ME/CFS started
with fleeting windows of cognitive clarity. She was on high-dose
antivirals and POTS medications for about five years, and her recovery
was infuriatingly slow and inconsistent. It was like wiping off a
mirror in a steamy bathroom. She saw her former self briefly, then the
image fogged over again.

Montoya doesn't know why these drugs worked for Erin, but he knew from
treating other patients that beating back viral infections sometimes
helps get an immune system back into balance.

Erin also believes that the antiviral and POTS drugs were instrumental
in her recovery, but other factors — family support, meditation and
Montoya's coaching — were also important.

One of Montoya's key messages to ME/CFS patients is this: If you have
one good day, don't try to make up for lost time by overexerting

"Don't burn out your engine," says Montoya, because the resulting
crash can reset the recovery process by months.

And at appointments he would remind Erin, "Take in all the love that
is all around you and use it to heal."

During her illness, Erin's mother became her advocate, managing her
medical issues and driving her to appointments. Her father added her
to his health insurance policy so she could afford the visits to
medical specialists. And her sister, who is her best friend and
housemate, was her wingman.

"My sister was my voice of hope," says Erin. "She'd tell me, 'OK, you
took five steps forward and two back today, but maybe tomorrow you'll
take six steps forward and only one back.'"

It's been seven years since Erin fell into the abyss of a mysterious
illness. This girl interrupted, now a woman, is picking up the pieces
of her life and starting to live again.

Today, she works as a social worker and therapist. She plays soccer in
a local league. She's also started dating again. It gives her hope
that researchers are finally focused on ME/CFS and that others may be
able to benefit from the treatment that has given her life back.

As she packs for a camping trip, she reflects on how this illness has
changed her.

"It's made me a person of more depth and compassion," says Erin.
"Before, I'd been so active, I didn't have the opportunity to sit with
myself in this way and take a deeper inward journey. Adventure had
been the focus of my life. As I sit with clients who are coming in
with devastating situations, with unknown futures, I'm able to share
with them hope and the power of self-fulfilling prophesies. I help
them find those things inside, spiritually, that will help them meet
the adversities in their lives."

At this point her voice becomes soft, almost a whisper, as she says,
"I'll always miss playing soccer at a competitive level, but I've
gained so much. It's helped me reinterpret what success looks like.
It's not everything you achieve and how many games you win. It's the
process of getting there. This is my biggest achievement — recovering
from this illness."

In soccer, a "hat trick" is where a player scores three goals in a
row. Montoya achieved his first goal, the launch of the first major
ME/CFS research initiative, with a little funding luck and the
recruitment of a top-notch research team. With the assistance of Davis
and his immune system hackers, he's close to reaching his second goal:
the identification of biomarkers and causes, which will enable
physicians to provide a definitive diagnosis and treatment options to
patients suffering from this debilitating condition.

The third goal of his hoped-for hat trick will be a whole new way to
look at the human immune system. It's a game changer. It will provide
researchers with a new playbook of research strategies to help them
discover the causes of other confounding conditions, from Lyme disease
to multiple sclerosis to fibromyalgia. It will provide clinicians with
a better set of metrics for assessing patients' health. And then the
patients lying in dark rooms with forgotten diseases, whose numbers
could fill hundreds of soccer stadiums, will have reasons to stand up
and cheer.

Kris Newby is the communications manager for Spectrum, the Stanford
Center for Clinical and Translational Research and Education. Email
her at [email protected]

Email the author- [email protected] , [email protected]

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