Dr. James Baraniuk, Assistant Professor of Medicine, Georgetown University
Accompanying Document: CFS Research: Transition from the Qualitative Consensus Tradition to a Quantitative, Translational, Hypothesis-Driven, Systems Biology Strategy
Dr. Baraniuk opened his presentation by saying that he was moved to be giving his presentation in the John Eisenberg Memorial Lecture Room. Dr. Eisenberg was the department chairman at Georgetown University and the professor who gave Dr. Baraniuk his start in clinical research.
One of the things that Dr. Eisenberg encouraged was thinking out of the box. Those in CFS have to make a change from the qualitative approach and consensus tradition toward a hypothesis-based type of investigation and new methods, said Dr. Baraniuk. The bywords now are using a “systems biology” or overall approach as to what is going on in multiple areas within an individual.
He gave the example of a patient evaluation that compares pain symptoms (headache, myalgia, arthralgia, sore throat, tender lymph nodes) and psychoneural symptoms (cognitive memory problems, sleep disturbances, exertional exhaustion) with fatigue.
The hypothesis: The sum for the minor criteria will be proportional to the level of fatigue. Significant fatigue for the diagnosis was defined as moderate (3) or severe (4).
Taking the sum of the minor criteria and comparing it with significant fatigue split the population into normal subjects, those with chronic idiopathic fatigue (CIF), and those with CFS. There was no difference by age or gender. Using the sum of minor criteria was superior to using SF-36 to separate the controls, those with CIS, and those with CFS. The implications for quantification of CFS criterion severities:
Scoring the severity of each CFS criterion provides more information about each individual.
The sum of the minor criteria correlates with fatigue and is a good predictor of CFS.
The next step is to explore whether cluster or multivariate analysis will identify subsets of CFS subjects in order to find out if there are common mechanisms that are creating subsets of CFS patients. Dr. Baraniuk said that he wants to use the current grant to find objective markers that will go along with those potential subsets. The grant (RO1 ES-015382) is funded by the National Institute of Environmental Health Sciences (NIEHS).
Researchers are:
Doing lumbar punctures in 50 CFS and 50 control subjects to analyze the proteomics of the cerebrospinal fluid.
Making psychometric comparisons using a lengthy questionnaire
Making pain comparisons using dolorimetry, isometric hand grip studies, and capsaicin skin tests for neurogenic inflammation.
A problem encountered during the study was spinal headaches, which are very severe and occur after a lumbar puncture. With the idea of making lemons out of lemonade, researchers decided to see what could be learn from spinal headaches about CFS pathology. Researchers discovered that
88 percent of CFS subjects had migraines. In checking the literature, researchers found that 84 percent of the migraine populations report fatigue. In fact,
67 percent of Dr. Baraniuk’s chronic migraine patients have CFS. This statistic suggests that there is more than a chance relationship and that perhaps there is a mechanism that is shared.
Migraines plus CFS suggests:
Autonomic abnormalities
Neurogenic inflammation
very likely contribute to the high level of overlap between migraines and CFS. From a treatment perspective,
if you identify migraines in 88 percent of your CFS people, you should get them started on appropriate therapy.
From this we wanted to look more at the autonomic nervous system.
The hypothesis: Autonomic control during isometric hand grip maneuvers will be dysfunctional in CFS but not in healthy controls.
Subjects squeezed a hand strain gauge as hard as they could.
Researchers measured blood pressure, heart rate, and nasal dimensions (surrogates of sympathetic nervous system vasoconstriction) during a restful “SHAM” contraction.
Subjects squeezed a hand strain gauge at 30 percent of maximum for as long as they could; researchers determined autonomic responses.
There is something called the muscle reflex. When muscles are activated, nerves stimulate the brain stem and the brain stem stimulates the sympathetic nervous system centers, which send out messages that work to increase blood flow to the local contracting muscle. The dynamics of the squeeze and release of the hand grip stimulate multimodal Type C neurons. We used to think that these neurons would have just one sensory receptor and would mediate just one sensation.
Now it’s apparent that they have a wide variety of receptors, especially the multi-modal neurons. It turns out that the acid that’s released in the muscle will activate the acid ion channel that then leads to great up regulation of the sensitivity of the TRPV, which depolarizes the nerves and sends an incredibly strong pain message to the brain. In turn, that leads to sympathetic discharge of a variety of chemicals, one of which is ATP, which if it’s released in the local muscle area, will have a positive feedback effect increasing the pain.
When you stimulate the sympathetic nervous system, you actually squeeze down on the blood vessels in your nose and as a result, you have an increase in nasal patency. This turns out to be a good measure of autonomic function or in the case of CFS, dysfunction.
Isometric Hand Grip in CFS
Controls had slightly higher maximum muscle strength, equivalent duration of muscle contractions, and similar pain scores. Researchers expected that the CFS people would have higher pain scores.
As far as the autonomic component was concerned, the first factor that changes during a contraction is that the diastolic blood pressure begins to increase. That’s also the measure that goes up the highest in the healthy controls. There’s a very significant difference between the controls and the CFS people.
The systolic blood pressure goes up next, but usually very late in the contraction, and similarly, the heart rate goes up fairly late.
As far as the nose goes, the cross-sectional area for air flow also goes up in the controls significantly greater than in the CFS people. In CFS there’s a group that has a swelling of their mucosa when they should be constricting.
Conclusion: Mucosal and systemic sympathetic reflexes are dysfunctional in the pathogenesis of CFS and the nonallergic rhinitis of CFS. The primary defect is apparently autonomic dysregulation in the brain stem.
"It’s all in your head.” That’s now the mission statement for our laboratory,” said Dr. Baraniuk. “It’s all in your head because that’s where most of your nerves are. And that’s where the problem lies, I believe.”
“That led to our approach to look at cerebral spinal fluid. By way of review, you have blood flowing into a central structure in the center of the brain. As that blood flows through that structure, you have a very carefully generated fluid produced—the cerebral spinal fluid. It surrounds the brain, circulates throughout the day, goes down your spinal column, and we can collect it by doing a lumbar puncture.”
Hypothesis: Central nervous system dysfunction is a critical component of CFS and related syndromes such as FM, Persian Gulf War Illness, multiple chemical sensitivity, irritable bowel and bladder syndromes, and other allied conditions. Analysis of the fluid flowing from the brain will identify factors that may indicate the pathology of CFS to direct the creation of new treatments, and serve as diagnostic biomarkers for future testing.
Researchers obtained cerebrospinal fluid from Gulf War Illness (GWI) subjects (most also had CFS), FM subjects (many had CFS), and control subjects for a total of 62 subjects. Researchers compared pooled samples from GWI, pooled samples from CFS, and pooled samples from controls. The identified the proteins that were only present in the GWI and CFS people. They also took individual specimens from the CFS and GWI people and compared them to individual specimens from control subjects.
Dr. Baraniuk: It was mind boggling because we ended up with about 10 proteins that were present only in our CFS group and not present in the control group. The odds of this are about 10 to the minus 15. We’re currently doing the proteomic analysis using a high-end orbitrap mass spectrometer to see if we get the same pattern. The key proteins from the CFS-related proteome, I think, give us some potential clues as to what’s going on (chart from presentation slide):
CFS – related Proteome (set of proteins)
Protease – Antiprotease Imbalance
Macroglobulin
Orosomucoid 1 and 2
Structural Injury
Amyloid APLP1
C4B (C3)
Gelsolin (apoptosis)
Oxidant Injury
Ceruloplasmin
Carnosine dipeptidase 1
Vascular Dysregulation
Autotaxin
Pigment Epithelium Derived Factor (EPDF)
Vasoconstrction (ischemia)
Endothelial proliferation (repair)
Leptomeningeal Activation
Keratins 4, 10, 16, 17
Structural Repair
Brain-enhanced hyaluronan binding (BEHAB)
With the new spectrometer, researchers get about 100 times more ions per run, 100 times more peptides, and about 20 times more proteins per run. We haven’t broken the code on the patient diagnosis in order to stay unbiased, so we don’t know what the mix of controls versus CFS is in the first two-dozen samples. A total of 4,075 were identified in the 24 samples.
The orbitrap system has really dropped down the level of sensitivity so that we’re picking up a lot of proteins that we could not pick up previously. We have over 200 unique proteins from these 24 samples. We had a large number of adhesion markers and it will take awhile to sort through the many different names that appear when you start doing the bioinformatic searches. After the next batch, we’ll probably be able to start doing quantitative analysis to compare the abundance of proteins between our CFS and control group.
We’re making progress—we’re getting there. Although this started out with some people thinking that it would be just a blind hog searching for acorns, I felt that we would find specific illness-related proteins. Now, based upon the distribution the types of proteins that we’ve found, we’ve been able to extend our work into other grants in other areas where we’re currently funded and are starting, for instance, a clinical treatment trial that I hope to adapt to CFS.
Committee Discussion
Dr. Baraniuk: It’s important for all of these that we identify by mass spectrometry to go on and do a quantitative assay. For all the proteins, we have that work to do once we identify which ones are significantly different. Specifically for the cytokines, we’re comparing plasma and cerebral spinal fluid.
Dr. Klimas: Are you looking for any pathogens?
Dr. Baraniuk: In the previous study we searched for all bacteria and all viruses and found one protein. But is was not a viral protein.
