What do gunshot wounds, burns, heart attacks, arthritis,
asthma, and cancer all share in common? Apart from inflicting
misery, these conditions--and others too--involve inflammation,
an immune response to injury and infection that normally
protects, but sometimes endangers or kills patients. Caused
by immune cells accumulating at a site of injury, inflammation
typically guards against infection and speeds recovery; it
is a critical process and, per se, does not cause disease.
But unchecked inflammation that spreads or fails to subside
poses chronic and acute health risks for millions of people.
Asthma patients, for instance, can’t breathe because inflammatory
compounds cause airway linings to swell and mucus to spread
in the lungs. Inflammation also exacerbates cancer, scientists
believe, by facilitating the proliferation of abnormal cells.
An acute condition called sepsis--caused when infection or
inflammation spills into the bloodstream--produces organ
failure and shock in critically ill patients. Up to 215,000
Americans die from sepsis every year, according to the National
Institute of General Medical Sciences. Worldwide, sepsis
is estimated to kill 1,400 people each day, according to
a consensus document published in the June 1992 issue of
Chest.
In light of its implications, inflammation has become
one of the hottest areas in biomedical research. J. Perren
Cobb, a professor of surgery and genetics at Washington
University in St. Louis, says a wide array of medical specialties
stand to benefit from these investigations. “Inflammation
is a major unifying syndrome, the investigation of which
provides opportunities for multidisciplinary convergence,” he
explains. “Studies of inflammation cut across all the domains
at the NIH; it’s a fundamental process in human biology
that ties everything together.”
|
image: Bryson/Custom Medical Stock Photo |
Growing evidence suggests that genetic factors drive
key aspects of an individual’s inflammatory outcome. Scientists
studying inflammation are trying to identify the genes
that drive inflammation as well as biomarkers from throughout
the course of inflammation. Stephen Chanock, who heads
the Section on Genomic Variation in the Pediatric Oncology
Branch at the National Cancer Institute, emphasizes that
the current critical care orientation of this research
has broad multidisciplinary implications that extend to
environmental health. “Injuries represent the ultimate
gene-environment interactions,” he explains. “Usually environmental
health focuses on chronic exposures, but in this case we’re
studying environmental insults that are more dangerous
and intense. So, the ‘environment’ in environmental health
isn’t just about pollution, it’s also experiential. We’re
developing practical methods for looking at inflammation
that will ultimately be applied to larger public health
issues.”
Toward Better Knowledge of Inflammation
Today, genomics defines the cutting edge of inflammation
research. Genomic studies, in addition to their proteomic
and metabolomic cousins, aim to resolve an age-old mystery:
namely, why some patients recover readily from inflammation
while others suffer and die from it. The current research
emphasis focuses on critical care, particularly of trauma
and burn patients, who face the lethal dangers of septic
complications. Ideally, new gene-based discoveries will
provide diagnostic biomarkers to predict who among these
patients will react poorly to inflammation and why. If
doctors could reliably predict this outcome in advance,
they might tailor antibiotics and other treatment options
to a patient’s own inflammatory system, potentially saving
lives.
Better knowledge of inflammation biology could also spawn
new treatment options, Cobb says. The newest drug for sepsis--an
Eli Lilly and Company product called Xigris that came on
the market in 2001--helps some patients, but its cost is
exorbitant: nearly $7,000 per course of treatment. What’s
more, the drug reduces the risk of death by just 6% and
can produce side effects such as excessive bleeding.
Among the numerous programs moving inflammation research
forward is an effort funded by a National Institute of
General Medical Sciences “glue grant,” so named because
it “glues together” multidisciplinary efforts to tackle
biomedical questions beyond the means of any one research
group. This program, called Inflammation and the Host Response
to Injury, strives to determine why patients can have dramatically
different outcomes after traumatic injuries and burns.
Headed by Ronald Tompkins, a professor of surgery at Harvard
Medical School and chief of Massachusetts General Hospital’s
Burn Service, the program uses genomic and proteomic methods
to study inflammation at 22 clinical centers located throughout
the country. A total of $37 million was made available
for the program’s first five years.
|
image: Digital Vision |
When the Inflammation and Host Response to Injury program
was launched in 2001, its leaders decided to create a broad
research infrastructure with uniform protocols as a first
priority. “One of our first challenges was to develop guidelines,
not just for the sample collection and analysis, but also
for patient management,” says Lyle Moldawer, a glue grant
recipient and professor of surgery at the University of
Florida College of Medicine. “We recognized that all the
funded centers have different protocols for the immediate
care of trauma and burn patients, and we were concerned
that those differences in early management might contribute
to gene expression changes.”
Tompkins says creating a uniform infrastructure for the
program was like building a highway. “We needed the gas
stations, the on-ramps, the off-ramps,” he says. “No one
had ever tried to introduce this technology into critical
care medicine before.” With standard operating procedures
in place and the program now in its fourth year, scientists
have begun to address a subsequent challenge: extracting
useful knowledge from the reams of genomic data flowing
out of the program’s 22 clinics.
At the same time the glue grant program was gearing up,
Cobb, senior investigator Anthony Suffredini of the NIH
Critical Care Medicine Department, and Robert Danner, who
heads the Infectious Diseases Section in the same department,
created the Consortium for Expression Profile Studies in
Sepsis specifically to identify the needs of those applying
genomic methods to critical care. The consortium hosted
four meetings throughout the country before evolving into
the NIH Functional Genomics of Critical Illness and Injury
Symposia series, which now provides a forum where glue
grant recipients and others discuss research progress and
results. The most recent symposium, hosted by the NIH at
its Bethesda campus on 21-22 April 2005, was attended by
scientists from 10 countries, all seeking to advance genomics
in inflammation research.
An Inflammation Primer
Once triggered, inflammation proceeds similarly whether
caused by pollutants, pathogens, trauma, radiation, or
burns. Localized mast cells in affected tissues produce
histamine, a chemical mediator that dilates blood vessels
at the site of injury, producing redness and heat. Histamine
also renders blood vessels permeable, so leukocytes (white
blood cells) can reach the injury. Leukocytes are attracted
to the injury site by chemotactic proteins known as chemokines,
which are secreted by endothelial cells of the blood vessels.
Leukocytes originate in bone marrow and include diverse
cell types, such as neutrophils, eosinophils, basophils,
monocytes, lymphocytes, and macrophages. Neutrophils arrive
at the affected area first. These remarkable cells roam
the body and kill pathogens on demand with a toxic blend
of free radicals and protein-chewing enzymes that destroy
bacterial cell walls. Monocytes engulf cellular debris
and mature into macrophages, which are larger leukocytes
that consume entire bacteria. These cells also secrete
a variety of cytokines that recruit and activate other
cell types. Lymphocytes are divided in two broad classes--B
cells and T cells--each with different roles. B cells,
once activated, make antibodies that attack foreign substances,
while T cells kill infected cells directly.
Chemical mediators released by leukocytes during inflammation
come in many varieties. Cytokines, for instance, help to
regulate inflammation, whereas interleukins regulate T
cell activity and produce systemic effects such as fever.
Normally, the whole inflammation process is self-limited
and short-lived; leukocytes disperse after dispensing with
infectious agents, and inflammation dies down within hours
or days. Problems crop up when the response persists or
spreads systemically, damaging and killing normal tissues
in the process. Chronic inflammation can persist for years,
causing illnesses that end with the suffix “-itis,” such
as bronchitis, arthritis, and bursitis. Systemic inflammation--sepsis
being one variety--occurs when cytokines reach the bloodstream
and spread through the body, damaging organs far from the
initial injury’s source.
Candidate Genes
No one knows precisely what happens when inflammation
goes awry. Years of immunology research have implicated
hundreds of genes in abnormal inflammation, but the evidence
linking them to particular outcomes is weak. Of these genes,
the one coding for C-reactive protein (CRP), an acute-phase
molecule whose levels shoot up during systemic inflammation,
is perhaps the best known. High CRP levels are prognosticators
for heart disease and stroke (which are both linked to
inflammation), but its role in these conditions remains
unclear. Another well-known gene--tumor necrosis factor-alpha
(TNF-)--codes
for a pro-inflammatory cytokine that normally regulates
leukocyte and endothelial cell activity, in addition to
other functions.
|
image: Photodisc |
By the 1990s, however, candidate gene studies had yet
to produce clinical benefits for inflammation. Suffredini
says scientists at the time were extremely frustrated with
the lack of progress. “People were throwing up their hands
and feeling [painted] into corners,” he says.
A turning point emerged at the turn of the millennium,
when a rough draft of the human genome and the advent of
microarrays made it possible to assess the expression of
thousands of genes simultaneously. “The analogy is that
for years, we’d been working on the ground to see how candidate
genes interact,” Cobb explains. “But microarrays allowed
us to look down at the genome from twenty thousand feet,
so to speak, and that has enabled us to model much broader
interactions.”
With these tools, scientists could search for entirely
new genes and molecular pathways involved in disease processes.
Cancer researchers were among the first to exploit the
technology for clinical aims, Suffredini says, inspiring
their counterparts in critical care to do the same. Thus,
inflammation research entered a new phase of gene discovery
that drives much of the progress in the field today. Scientists
are now investigating a variation in the promoter region
of TNF- (the
region that initiates protein production after binding
transcription factors) that might contribute to sepsis.
While cancer genomics inspired similar efforts in critical
care, both specialties operate under vastly different research
settings. For one thing, cancer patients typically have
the time and awareness to provide informed consent for
blood and tissue sampling. In addition, the cohorts tend
to be large and matched for age, sex, treatment history,
and other parameters that can influence genomic profiles.
Trauma and burn patients, on the other hand, are rushed--often
unconscious--into the emergency room or intensive care
unit, where live-saving treatment is the first priority.
In this frenetic environment, informed consent is difficult
to secure, and research sampling becomes a secondary concern.
Moreover, cancer and trauma induce totally different
types of gene expression--whereas tumors typically produce
localized, stable expression profiles corresponding to
small portions of the genome, critical injuries trigger
enormous genomic changes that affect all tissues and shift
rapidly over time. Temporal factors are extremely important
in critical care sampling because they have a tremendous
influence on the gene profile; a sample taken 15 minutes
after injury will be vastly different than one taken several
hours later.
Into the Data
According to Tompkins, investigators with the glue grant
program chose to investigate normal and abnormal inflammation
trajectories sequentially, each in five-year increments.
Genomic and proteomic data for the normal trajectory--compiled
using samples from trauma and burn patients who recovered
uneventfully--are now being analyzed.
At the same time, program scientists augmented the clinical
research with additional genomewide expression studies
of leukocytes sampled from healthy volunteers dosed intravenously
with bacterial endotoxin. These studies--which induced
low-level systemic inflammation that permitted validation
of sample processing protocols--enabled scientists to compare
baseline and inflammatory genomic changes at varying time
points. Patients weren’t harmed by the experiments, and
all responses returned to normal within 24 hours.
The results, published in the 31 August 2005 issue of Nature,
showed how complex inflammatory networks really are--between
3,000 and 5,000 genes, up to 20% of the entire genome,
were activated, according to Moldawer, one of the study’s
authors. “The research revealed that the magnitude of the
changes was much larger than we anticipated,” he says. “We
expected to see up-regulation of stress-related genes during
the acute phase, but much to our surprise, the diversity
of the changes was much greater than we thought it would
be.”
|
image: Digital Vision |
Many of those changes, Moldawer adds, were seen in genes
involved in mitochondrial energy transfer, protein synthesis,
and antigen recognition--in short, biological processes
that enable leukocytes to become more efficient antimicrobial
agents, he says. Preliminary analyses suggested the magnitude
and nature of the endotoxin response shared some similarities
with the response seen in real patients. At press time,
the clinical data from actual patient cohorts were still
being assessed.
Although the amounts of genomic data may be computationally
daunting, recent evidence from another study suggests efforts
to distinguish good inflammatory outcomes from bad might
have promise. This study, published in the 29 March 2005 Proceedings
of the National Academy of Sciences, made several key
discoveries. First, hospitalization and repeated sampling
had only a modest effect on gene expression in healthy
volunteers. Thus, the experience of being hospitalized
(with its enforced bed rest and defined nutritional intake)
is unlikely to influence gene expression in ways that undermine
the detection of signature profiles for specific inflammatory
outcomes. Second, the researchers showed that gene expression
differences in whole-blood leukocytes drawn from severe
trauma patients could be divided into injury-specific patterns.
Taken together, says coauthor Tompkins, the findings indicate
that expression profiling may yield “low-hanging fruit” in
the form of highly correlated data.
Linking Sepsis-Related Genes to Biology
Meanwhile, researchers in Germany have shown that subsets
of genes can be linked directly to sepsis. Among these
researchers is Trinad Chakraborty, who directs the Institute
of Medical Microbiology at Justus-Liebig University. Chakraborty
is completing a study of genomic factors contributing to
sepsis in patients with multiple trauma or pneumonia. The
study--part of a broader effort to understand why patient
outcomes differ after similar injuries and illnesses--involved
screening up to 20,000 genes in peripheral blood during
a 14-day post-injury period. The effort, conducted in 185
patients, found 690 genes whose expression appears to correlate
with sepsis. In future research, Chakraborty plans to look
for single-nucleotide polymorphisms within candidate genes
that predispose the sepsis phenotype, and to identify protein-based
biomarkers for diagnostic use.
But Chakraborty adds that computational challenges are
a serious holdup. “When we started the research, getting
the microarrays to be sufficiently robust was the bottleneck,” he
says. “Now we’ve resolved that problem, and bioinformatics
is the bottleneck.” He and his colleagues hope to trim
the 690 genes to a lesser population of 25 or so. “Then
we could develop an algorithm that recognizes a profile
within that smaller set of genes to indicate whether you
have a likelihood of sepsis or not.”
U.S. scientists have also correlated genes with sepsis
and used these findings to suggest a preliminary mechanism
for its lethality. Led by Hector Wong, who directs the
Division of Critical Care Medicine at Cincinnati Children’s
Hospital Medical Center, the scientists used microarrays
to compare gene profiles between children who survived
sepsis and those who died from it. Children respond uniquely
to sepsis in that their fatality rates are much lower than
those of adults--roughly 10% compared to 30% among the
latter, says Chanock.
|
Small advances. Fatality rates
from sepsis are much lower in children than adults,
so much may be learned from how children’s bodies
deal with inflammation.
image: Photodisc |
Wong suspects that children respond better to sepsis in
part because they have fewer comorbidities such as diabetes
and heart disease (a status that is changing somewhat with
rising childhood obesity). But he further suspects genetic
factors underlie important biological differences that
improve their outcomes, though at this point he can’t say
how.
In recent studies presented at the April symposium, Wong
found that among nonsurviving children, six genes coding
for metallothionein--a protein that binds zinc and removes
it from the bloodstream--appeared to be highly expressed.
These findings led him to a hypothesis: if severely septic
children had high blood metallothionein levels, he proposed,
then their blood zinc levels might be correspondingly low. “And
in fact, that turned out to be true,” he says.
Another interesting finding was that the profiles showed
altered expression patterns for a host of proteins that
either depend on zinc or take part in zinc homeostasis. “So
there’s a lot of biology there to look at,” Wong says. “We
don’t know how or whether zinc is involved; there’s very
little information out there about the effects of acute
zinc deficiency. I find it hard to believe the foundation
for sepsis is zinc, but . . . I think it can be tested.” After
considering this position further, Wong adds that this
is how high-throughput investigations are useful: they
suggest biological mechanisms that scientists can explore
further in the laboratory.
Future Needs
Today, a genomic research culture is slowly seeping into
the front lines of care for the critically ill and injured.
But establishing that culture isn’t easy--emergency room
and intensive care unit settings challenge researchers
in many ways. Issues like informed consent for study participation
and repeated intrusive blood sampling to assess temporal
changes in the genome are difficult to manage, Tompkins
says. Ideally, new technologies will reduce sample volume
requirements, lessen the amount of time required for microarray
analysis (which now averages 24 hours), and reduce microarray
costs to the extent that they can be used routinely in
the clinic.
Chakraborty adds that microarray platforms need to accommodate
sample degradation too. As it stands now, he says, RNA
in blood samples drawn in the emergency room has a higher
degradation potential than RNA in samples drawn from the
more controlled environment of a research laboratory. “The
platforms need to become more robust,” Chakraborty says. “That
way, if the quality of the RNA drops to fifty or seventy
percent rather than a hundred percent, we would still be
able to get meaningful results.” Researchers with the glue
grant program are also seeking to set up guidelines for
standardized research procedures that will help lessen
the potential for sample degradation.
Inflammation genomics also poses enormous computational
challenges. Studies that lack sufficient statistical rigor
are a persistent problem, Cobb says--emergency room and
intensive care unit cohorts tend to be smaller than optimal,
and patients come in after the trauma has occurred so they
can’t serve as their own controls. At the same time, lists
of inflammation-specific genes identified during microarray
experiments need to be incorporated into biological models
that describe their molecular interactions. Bioinformatic
research and associated databases are continually advancing
to meet these needs, however, and collaborations among
research groups both within the United States and abroad
are helping to drive the science forward.
Cobb emphasizes that despite its broad public health
impact, inflammation research has yet to achieve the same
public awareness as that of cancer or heart disease. “We
need to do a better job of educating people about the importance
of this process,” he says. This means reaching other scientists
as well as the public, whose concerns often drive research
funding.
In the meantime, genomic methods have generated incremental
advances in our understanding of inflammation. Scientists
have barely scratched the surface of its vast complexity,
but perhaps in the not-too-distant future, patients will
reap the benefits of their efforts.
Charles W. Schmidt