Low
serotonin-receptor
activity
Thursday May 23,
2013
Research Brief
A
new study on
chronic fatigue syndrome (ME/CFS)
suggests that an autoimmune reaction
to the neurotransmitter serotonin
damages serotonin-sensitive brain
cells. Researchers also concluded
that high levels of bacteria move
through intestinal membrane in
people with ME/CFS, which is known
to play a role in autoimmunity.
Researchers
compared serotonin antibodies in
people with ME/CFS, those with
chronic fatigue who don't meet ME/CFS
criteria, and healthy controls. They
found that autoimmune activity
against serotonin was more than four
times what it was in chronic
fatigue, and twelve times higher
than in healthy people.
Serotonin
autoimmunity was linked to more
severe hyperalgesia (pain
amplification,) fatigue,
brain fog, autonomic symptoms,
sadness, and flu-like symptoms.
Researchers
say serotonin autoimmunity could be
part of the underlying pathology of
the condition, and their results
provide support for ME/CFS being a
neuro-immune disorder.
What
it Means
In
autoimmunity, your immune system
basically gets confused and
identifies a healthy, normal part of
your body as a foreign invader that
should be destroyed. It then treats
it like a virus or bacteria,
creating specialized cells that seek
it out and try to get rid of it.
This response leads to inflammation
and a host of other problems.
Serotonin has
long been believed to play a role in
ME/CFS. In your brain, it's a
neurotransmitter, which means it
transports certain messages from one
brain cell to the next. In the rest
of your body, it's a hormone. The
highest concentration of serotonin
is in your digestive system, where
it plays important roles.
Another known
factor of ME/CFS is chronic immune
system activity. Bascially, it's in
high gear all the time, as if it's
fighting an active illness.
Autoimmunity could help explain
this.
Learn more:
Interessant aan deze studie is
de richting die overeenkomt met eerdere recente bevindingen.
Voor mij persoonlijk
komt het ook sterk overeen met mijn eigen bevindingen.
Metingen geven aan dat ik niet goed tot rust kom.
Onrust is wat bij mij steeds
terugkomt. Mijn lichaam komt niet tot rust. Ik herstel niet met
rust.
Ontspanning is een werking die van serotonine uitgaat. En nu
door de beschadigde serotonine receptor niet goed gebeurd.
Voor mij wordt de puzzel
steeds duidelijker.
Nu wordt het interessant om
uit te vinden of de trigger voor de immuunreactie,
waarschijnlijk een virus, te vernietigen is en er geen
immuunreactie meer komt. Of dat er een andere oplossing komt om
de serotonine receptoren naar behoren te kunnen gaan laten
funtioneren.
Er is ook een
genetische
aanleg mogelijk met daardoor een verandering in het
serotonine systeem en onderactiviteit van
de hypothalamus hypofyse
bijnier as. Er worden een vijftigtal genetische afwijkingen
gevonden bij cvs/me.
Toch
wordt het genetische component bij cvs/me door Dr Kennie de
Meirleir als gering beschouwd als gekeken wordt naar familie die
het ook heeft. Dat is inmiddels niet meer zo. Er zit een
genetische aanleg in ook volgens Meirleir, zeker met betrekking
tot Lyme.
DRACO aankomend
middel tegen dubbelstrengs RNA virussen
Draco is een medicatie waaraan op dit moment gewerkt wordt.
Het kan virussen die dubbel strengs RNA (ds-RNA) produceren een
stevig halt toe roepen.
Hieronder vallen o.a. de groepen adenovirussen, herpes virussen
en poxvirussen maar ook vele andere virussen.
Deze groepen virussen produceren in hun reprodutie proces
dubbelstengs RNA.
De werking berust op het gegeven dat Draco de aanwezigheid
van ds-RNA in een cel herkent. Het middel bindt zich aan het ds-RNA.
Het tweede bestandeel van het middel
(caspase)
zet vervolgens de apoptose, ze
zelfdoding, van de cel in werking en dood daarmee ook het virus.
Resistentie, zoals bij antibiotica's tegen bacteria, zal zich
tegen dit middel niet snel ontwikkelen.
Het is nog in de ontwikkelingsfase maar het middel is
veelbelovend en een aangetoond werkend middel en wordt gezien als
even belangrijk als penicilline was tegen
bacteria.
plosone.org/draco
Rituximab een immuunsteem
(B-cellen) onderdrukkend medicijn werkt bij een
groot deel van mensen met CVS/ME:
VIDEO
Rituximab is zelf een antilichaam wat de B-cellen
werking stil legt. De B-cellen werking is binnen twee weken na de behandeling
met Rituximab stil gelegd maar autoantilichamen overleven twee tot drie maanden
en dat verklaart de vertraagde werking van dit medicijn. De werking zit hem dan
ook waarschijnlijk niet in de B cel reductie maar in de verminderde auto
antilichaam werking. Of mogelijk door de vermindering van het herpusvirus.
Onderzoekers Fluge en
Mella van het Haukeland Universitair
Ziekenhuis in Bergen te Noorwegen hebben
in navolging op een eerdere kleine
studie (3 patiënten) een dubbelblinde
Fase II studie uitgevoerd met het middel
Rituximab bij 30 ME/CVS-patiënten.
Daarbij komen ze tot de vaststelling dat
dit medicijn, dat B-cellen gedurende
enige tijd uitschakelt, met een
vertraging van 2 tot 7 maanden, leidt
tot tot een afname van het gehele
klachtenpatroon bij twee derde van de
patiënten voorlopig al gedurende
meerdere maanden.
Ze concluderen dat de vertraagde reactie erop
wijst dat ME/CVS mogelijk een auto-immuunziekte
is, met andere woorden een ziekte
waarbij het immuunsysteem het lichaam
aanvalt.
De onderzoekers
vonden het eerste bewijs voor de
werkzaamheid van het medicijn per toeval
in 2004, toen ze een patiënte
behandelden die zowel aan de ziekte van
Hodgkin als aan ME/CVS leed. Na
behandeling voor Hodgkin met Rituximab,
bleken de ME/CVS-symptomen de volgende
vijf maanden te verbeteren.
Aan deze recentste
studie namen dertig ME/CVS-patiënten
deel waarvan de helft Rituximab
toegediend kreeg en de andere helft een
placebo. 10 van de 15 patiënten of 67%
van degenen die het medicijn hadden
gekregen, gaven aan dat hun symptomen
verbeterden. In de placebo-groep ging
dit slechts om 2 patiënten (13%).
De theorie van de
leidende onderzoekers - oncologen, is
dat een type witte bloedcel, lymfocyten,
een antilichaam produceert dat het
lichaam aanvalt. Rituximab vernietigt de
lymfocyten, waardoor in sommige gevallen
het immuunsysteem wordt "gereset". In
andere gevallen kwamen de
vermoeidheidssymptomen weer terug
wanneer meer lymfocyten werden
aangemaakt.
De Amerikaanse Dr.
Bell is zeer onder de indruk van de
onderzoeksresultaten en heeft op het
Noorse TV2 verklaard dat hij in de 25
jaar dat hij betrokken is bij ME/CVS,
nog nooit zulke overtuigende en
hoopgevende onderzoeksresultaten heeft
gezien (zie de reportage met Nederlandse
ondertiteling).
Ook medisch
raadgever van de Britse ME Association,
Charles Shepherd, verklaart dat dit
onderzoek heel bemoedigend is voor
ME/CVS-patiënten: "Ten eerste bevestigen
ze een belangrijke abnormaliteit in het
immuunsysteem bij deze ziekte. Ten
tweede geven ze aan dat het aanpassen
van de manier waarop het immuunsysteem
reageert, een effectieve manier kan zijn
om bepaalde ME/CVS-patiënten te helpen."
In Noorwegen zijn
ze reeds gestart met vervolgonderzoeken
die er toe moeten leiden dat het
medicijn op termijn kan worden
voorgeschreven aan ME/CVS-patiënten.
Rituximab en herpusvirussen
Interessante connectie met het
herpesvirus: Herpesvirussen infecteren de B-cellen tijdens de B-cel
transformatie.
Door de B-cellen te vernietigen voordat deze verder transformeren kan mogelijk
het herpus virus verminderd, tijdelijk stil gelegd, of zelfs geheel gestopt
worden.
Ik heb al een jaar geleden (april 2011) gedacht en geschreven dat cvs volgens mij een te
sterke immuunreactie is. Zie Virus en
immuunsysteem. Hierin werd ik niet ondersteund door anderen. Wat ik
constateerde in een gepubliseerd onderzoek was dat het immuunsysteem reageert op eiwitten van virussen en dat
lichaamseigen eiwitten zo zeer lijken op deze eiwitten dat deze ook aangevallen
worden. Met name eiwitten die in verband staan met de B vitamine opname. En
juist deze vitamines zijn belangrijk in de mitochondrien.
Ik denk dat het een combinatie van verschillende virussen is, met name herpus
virussen, die continue deze reactie uitlokken. Op de korte termijn is dit niet
zo schadelijk maar op de lange termijn wel.
Dan gaan de mitochondrien minder goed werken met alle vermoeidheids gevolgen van
dien.
Unieke eiwitten in ruggemergvocht
gevonden bij mensen met chronisch vermoeidheids syndroom
3 groepen mensen zijn onderzocht:
43 mensen met cvs
25 mensen met de ziekte van Lyme
11 gezonde mensen ter controlle
In iedere groep werden meer dan 2500 verschillende
eiwitten gevonden.*
In de cvs groep werden 738 eiwitten gevonden die niet in
beide andere groepen voorkwamen en 1582 eiwitten werden
niet gevonden bij cvs maar wel bij de Lyme en controlle
groep.
Bij de ziekte van Lyme groep werden 692 eiwitten
gevonden die niet in beide andere groepen voorkwamen.
Hiermee is een verschil aangetoond
tussen cvs en Lyme waarin tot nog toe grote
overeenkomsten werden
verondersteld. En ook is nu aagetoond dat het centrale
zenuwstelsel betrokken is bij beide aandoeningen.
Met deze nieuwe onderzoekmethode komt er weer beter
inzicht in cvs en kan er gerichter gewerkt worden aan
diagnose en behandelingen.
*(
Gezond 2630 eiwitten,
cvs 2783
eiwitten en Lyme 2768 eiwitten gemiddeld )
Onderzoekers zijn gestuit op een mogelijke diagnostische methode
om een subgroep patiënten te identificeren met ME/cvs.
In
een proefstudie van zes patiënten hebben wetenschappers
specifieke anti-lichamen ontdekt die worden gelinkt aan de
reactivatie van een latent Epstein-Barr virus. Deze werden
gevonden in bloedstalen van mensen die de klassieke ME/cvs
symptomen hadden en reageerden op behandeling met antivirale
middelen. In controle bloedmonsters van twintig gezonde mensen
werden zulke antilichamen niet gevonden.
Het onderzoeksteam, onder leiding van wetenschappers van de Ohio
State University en de Oakland University William Beaumont
School of Medicine, erkent dat het aantal patiënten klein is.
Maar de onderzoekers zeggen dat de kracht van het onderzoek ligt
in het feit dat ze de beschikking hadden over zestien maanden
lang verzamelde bloedmonsters van elke patiënt.
De onderzoekers zijn van plan om verder te gaan met de
ontwikkeling van een klinische laboratoriumtest die deze anti-lichamen
in bloedmonsters kan detecteren. De studie is gepubliceerd in
het tijdschrift PLOS ONE van 14 november 2012.
Het Epstein-Barr virus is een humaan herpesvirus dat
besmettelijke mononucleosis en meerdere soorten tumoren kan
veroorzaken. Volgens het Centers for Disease Control and
Prevention (CDC), is ongeveer 95% van de Amerikanen er als
volwassene mee geïnfecteerd. Alleen is minder dan de helft van
hen er ook echt ziek van geworden. Als iemand eenmaal
geïnfecteerd is, blijft het virus sluimerend aanwezig in het
lichaam, en kan het opnieuw geactiveerd worden zonder
ziektesymptomen te veroorzaken.
DNA polymerase en dUTPase
Uit de studie bleek, dat bij deze zes patiënten een latent
Epstein-Barr virus opnieuw actief werd. Maar het opnieuw
ontwaakte virus kon zijn volledige kracht om zijn gastcellen
over te nemen niet ontplooien. Toch volstond deze gedeeltelijke
reactivering om op zijn minst twee virale eiwitten voort te
brengen: DNA polymerase en dUTPase. Deze patiënten maakten
antilichamen aan die specifiek ontworpen waren om de twee
eiwitten langer dan een jaar te kunnen herkennen en te
neutraliseren.
De wetenschappers kwamen tot de theorie dat zelfs bij
afwezigheid van een volledig actieve infectie deze virale
eiwitten toch zulke infectieuze chemische signalen kunnen
veroorzaken, dat het immuunsysteem ontregeld wordt en ME/cvs
optreedt. Het hoofdsymptoom van deze aandoening is een ernstige
vermoeidheid gedurende tenminste zes maanden die niet overgaat
door rust, en gepaard gaat met problemen zoals slapte, spierpijn,
geheugenproblemen en depressie. Omdat de ziekte zoveel lijkt op
veel andere aandoeningen, is het moeilijk een diagnose te
stellen. Ongeveer een miljoen Amerikanen hebben ME/cvs, terwijl
volgens experts maar 20% is gediagnostiseerd.
De meest ervaren onderzoekers van de studie waren het eens dat
die bij meer patiënten herhaald moet worden. "Om de waarnemingen
te bevestigen", zei viroloog Ron Glaser, directeur van het
Institute for Behavioral Medicine Research in Ohio State en een
co-auteur van de studie. "Maar na meer dan 20 jaar is dit
tenminste eindelijk iets waar we verder mee kunnen."
Glaser’s eerste medewerkers in deze studie waren Marshall
Williams, professor moleculaire virologie, immunologie en
medische genetica aan de Ohio State, en A. Martin Lerner,
professor interne geneeskunde aan de Oakland University William
Beaumont School of Medicine. Glaser en Williams publiceerden
voor het eerst een artikel in 1988 dat suggereerde dat deze twee
virale eiwitten, geassocieerd met een gedeeltelijke
gereactiveerd Epstein-Barr virus, als biomarkers kunnen
functioneren voor bepaalde aandoeningen, zoals ME/cvs.
Ondertussen werd Lerner in 1986 ernstig ziek en vocht tien jaar
tegen de symptomen van ME/cvs totdat zijn gezondheid drastisch
verbeterde door behandeling met anti-virale middelen.
Lerner, een specialist op het gebied van infectieziekten, heeft
een privé ME/cvs praktijk in Michigan. Dat hij op lange termijn
de kenmerken van van ME/cvs patiënten en de respons op de
behandeling volgde, maakte deze follow-up studie mogelijk.
Dat ME/cvs patiënten verschillende symptomen en meerdere soorten
virale en bacteriële infecties ondergaan, heeft er voor gezorgd
dat onderzoekers geloven dat ME/cvs mogelijk talrijke oorzaken
heeft. Dat gebrek aan eenduidigheid bemoeilijkt ook de diagnose
en de ontwikkeling van behandelingen.
"Een deel van het probleem om een middel of biomarkers voor ME/cvs
te identificeren, is de extreme verscheidenheid onder mensen die
zeggen ME/cvs te hebben. Het aanbrengen van een schifting daarin
heeft het onderzoeksveld vele jaren opgehouden" zei Glaser, die
het
Epstein- Barr virus (EBV)
al decennia lang onderzoekt.
Lerner heeft een hele tijd terug 142 van zijn patiënten in twee
groepen opgesplitst: zij die positief testten op verschillende
antilichamen tegen drie soorten herpesvirussen en reageerden op
maandenlange behandeling met een van twee soorten antivirale
middelen. En een kleinere groep die virale en allerlei co-infecties
had, maar minimaal reageerde op een behandeling met antivirale
middelen. Om dit te traceren, verzamelde hij onder andere langer
dan een jaar van elke patiënt meerdere monsters bloedserum. Voor
dit onderzoek selecteerde hij bloedmonsters van zes van die
patiënten. Vijf vielen onder een subgroep met het Epstein Barr
virus, de zesde had een Epstein Barr virus en een bacteriële co-infectie.
Ter vergelijking verzamelden de onderzoekers monsters van
twintig gezonde personen die qua leeftijd en geslacht
overeenkwamen met de zes ME/cvs patiënten.
Ook Lerner kwam los van dit alles tot de hypothese dat ME/cvs
patiënten mogelijk een gedeeltelijke reactivatie van een virus
ondergingen. Al testten patiënten wellicht negatief op de meest
actieve antilichamen om een virus te bestrijden, toch wisten ze
te herstellen van ME/cvs door een langdurige behandeling met
antiviralen. Van één antiviraal middel dat hij gebruikt, is
bekend dat het de DNA polymerase kan afremmen, die de
reactivatie van het Epstein-Barr virus beheerst.
Williams gebruikte voor de beschikbare bloedstalen van ME/cvs
patiënten en controlepersonen een zeer gevoelige
laboratoriummethode om er achter te komen of die antilichamen
bevatten tegen de twee te bestrijden Epstein Barr virale
ei-witten DNA polymerase en dUTPase, die vroeg in het proces van
de virale reactivatie worden geproduceerd.
In totaal testte 78.8 % van de serum-monsters van de zes ME/cvs
patiënten positief op antilichamen tegen DNA polymerase, en 44.2
% voor antilichamen tegen dUTPase. In de 20 controlemonsters
werden geen antilichamen tegen deze twee eiwitten gevonden.
"Alle zes hadden antilichamen tegen DNA polymerase of EBV
dUTPase, en die antilichamen bleven zo’n 408 dagen be-staan.,"
zei Lerner. "En de aantallen anti-lichamen waren uitzonderlijk
hoog." Hoge aantallen antilichamen die in het bloed circuleren,
duiden op een langdurige immuun-activering tegen deze eiwitten.
Williams merkte op dat de aantallen minder opvallend kunnen zijn
dan de antilichamen die er in eerste instantie waren.
"Kijk naar de meeste gezonde mensen. Er is voor hen geen enkele
reden om een antilichaam tegen een van beide eiwitten te hebben,"
zei hij, "De antilichamen zelf zijn een goede manier om
onderscheid te kunnen maken."
Deze studie
werd mede ondersteund door het NIH (National Institutes of
Health).
Verdere co-auteurs waren o.a. Maria Ariza van de afdeling
moleculaire virologie, immunologie en medische genetica, en
Stanley Lemeshow, decaan van het College of Public Health,
beiden in de staat Ohio, Leonard Jason van de DePaul University,
Safedin Beqaj van Pathology Inc. in Torrance, Californië, en
James Fitzgerald van de University of Michigan School of
Medicine.
Spinal Fluid Proteins Distinguish Lyme Disease from
Chronic Fatigue Syndrome
NEWARK, NJ - Patients who suffer from Neurologic Post
Treatment Lyme disease (nPTLS) and those with the
Chronic Fatigue Syndrome report similar symptoms.
However unique proteins discovered in spinal fluid can
distinguish those two groups from one another and also
from people in normal health, according to new research
conducted by a team led by Steven E. Schutzer, MD, of
the University of Medicine and Dentistry of New Jersey –
New Jersey Medical
School, and Richard D. Smith, Ph.D., of Pacific
Northwest National Laboratory. This finding, published
in the journal PLoS ONE (February 23, 2011), also
suggests that both conditions involve the central
nervous system and that protein abnormalities in the
central nervous system are causes and/or effects of both
conditions.
The investigators analyzed spinal fluid from three
groups of people. One group consisted of 43 patients who
fulfilled the clinical criteria for Chronic Fatigue
Syndrome (CFS). The second group consisted of 25
patients who had been diagnosed with, and treated for,
Lyme disease but did not completely recover. The third
group consisted of 11 healthy control subjects. “Spinal
fluid is like a liquid window to the brain,” says Dr.
Schutzer. By studying the spinal fluid, the research
team hoped to find abnormalities that could be used as
markers of each condition and could lead to improvements
in diagnosis and treatment.
Taking advantage of previously unavailable methods for
detailed analysis of spinal fluid, the investigators
analyzed the fluid by means of high powered mass
spectrometry and special protein separation techniques.
They found that each group had more than 2,500
detectable proteins. The research team discovered that
there were 1) 738 proteins that were identified only in
CFS but not in either healthy normal controls or
patients with nPTLS; 2) 692 proteins found only in the
nPTLS patients. Previously there had been no available
candidate biomarkers to distinguish between the two
syndromes, nor even strong evidence that the central
nervous system is involved in those conditions.
This research represents the most comprehensive analysis
of the complete spinal fluid proteome (collection of
proteins) to date for both Chronic Fatigue Syndrome and
Neurologic Post Treatment Lyme disease (nPTLS). Prior to
this study, many scientists believed that CFS was an
umbrella category that included nPTLS. However these
results call those previous suppositions into question
According to Dr. Schutzer, spinal fluid proteins can
likely be used as a marker of disease, and this study
provides a starting point for research in that area.
“One next step will be to find the best biomarkers that
will give conclusive diagnostic results,” he says. “In
addition, if a protein pathway is found to influence
either disease, scientists could then develop treatments
to target that particular pathway.”
“Newer techniques that are being developed by the team
will allow researchers to dig even deeper and get more
information for these and other neurologic diseases,
says Dr. Smith. "These exciting findings are the tip of
our research iceberg”
Onderzoek
problemen met resistente bacteria
Abstract Top
Background
Neurologic Post Treatment Lyme
disease (nPTLS) and Chronic
Fatigue (CFS) are syndromes
of unknown etiology. They share
features of fatigue and cognitive
dysfunction, making it difficult to
differentiate them. Unresolved is
whether nPTLS is a subset
of CFS.
Methods
and Principal Findings
Pooled cerebrospinal fluid (CSF)
samples from nPTLS patients,
CFS patients, and healthy
volunteers were comprehensively
analyzed using high-resolution mass
spectrometry (MS), coupled with
immunoaffinity depletion methods to
reduce protein-masking by abundant
proteins. Individual patient and
healthy control CSF samples were
analyzed directly employing a
MS-based label-free quantitative
proteomics approach. We found that
both groups, and individuals within
the groups, could be distinguished
from each other and normals based on
their specific CSF proteins
(p<0.01). CFS (n = 43) had
2,783 non-redundant proteins,
nPTLS (n = 25) contained 2,768
proteins, and healthy normals had
2,630 proteins. Preliminary pathway
analysis demonstrated that the data
could be useful for hypothesis
generation on the pathogenetic
mechanisms underlying these two
related syndromes.
Conclusions
nPTLS and CFS have
distinguishing CSF protein
complements. Each condition has a
number of CSF proteins that can be
useful in providing candidates for
future validation studies and
insights on the respective
mechanisms of pathogenesis.
Distinguishing nPTLS and
CFS permits more focused
study of each condition, and can
lead to novel diagnostics and
therapeutic interventions.
Citation: Schutzer
SE, Angel TE, Liu T, Schepmoes AA,
Clauss TR, et al. (2011) Distinct
Cerebrospinal Fluid Proteomes
Differentiate Post-Treatment Lyme
Disease from Chronic Fatigue
Syndrome. PLoS ONE 6(2): e17287.
doi:10.1371/journal.pone.0017287
Editor: Howard
Gendelman, University of Nebraska,
United States of America
Received:
November 29, 2010; Accepted:
January 26, 2011; Published:
February 23, 2011
Copyright: ©
2011 Schutzer et al. This is an
open-access article distributed
under the terms of the Creative
Commons Attribution License, which
permits unrestricted use,
distribution, and reproduction in
any medium, provided the original
author and source are credited.
Funding:
National Institutes of Health,
through NIAID (grant AI088765), NIDA
(grant DA021071), NINDS (grant
NS38636), the National Center for
Research Resources (RR018522), the
Swedish Research Council
(621-2008-3592), Uppsala Berzelii
Technology Center for
Neurodiagnostics, SciLifeLab-Uppsala,
Time for Lyme, Lyme Disease
Association, and the Tami Fund for
support of portions of the research.
Pacific Northwest National
Laboratory units are located in the
Environmental Molecular Sciences
Laboratory, a national scientific
user facility, sponsored by the
Department of Energy (DOE), operated
by Battelle Memorial Institute for
the DOE under Contract DE-AC05-76RL0
1830. The content is solely the
responsibility of the authors and
does not necessarily represent the
official views of the National
Institutes of Health. The funders
had no role in study design, data
collection and analysis, decision to
publish, or preparation of the
manuscript.
Competing interests:
The authors have declared that no
competing interests exist.
* E-mail:
schutzer@umdnj.edu
# These authors contributed
equally to this work.
Introduction Top
Prime objectives in studying
neurologic and psychiatric disorders
are to develop discriminating
markers and generate data that can
provide insight into disease
pathogenesis. This can lead to novel
treatment strategies. Chronic
Fatigue Syndrome (CFS) and
Lyme disease, particularly
Neurologic Post Treatment Lyme
disease syndrome (nPTLS),
represent two conditions that share
common symptoms of fatigue and
cognitive dysfunction
[1]–[7].
Despite extensive research CFS
and nPTLS remain medically
unexplained. There are no biological
markers to distinguish these
syndromes, creating diagnostic
dilemmas and impeding research into
understanding each individual
syndrome.
Cerebrospinal fluid (CSF) is an
ideal body fluid to examine for
signature protein profiles
informative for diagnosis or
etiology of central nervous system
(CNS)-related symptoms and
dysfunction. Not only is the CSF an
accessible liquid extension of the
brain, but recent data suggests CSF
may provide more relevant data than
brain parenchyma itself in certain
neurologic diseases
[8]. Specific abnormalities
found in CSF relating to CFS
and nPTLS would suggest CNS
involvement, and could facilitate
their mechanistic understanding.
Liquid chromatography coupled to
mass spectrometry (LC-MS) is
becoming the method of choice for
examining complex biological
specimens, that contain hundreds to
thousands of proteins
[9], such as CSF
[10]. This is particularly the
case in the initial discovery phase.
This discovery phase may be
viewed as casting a wide net to
maximize identification of as many
proteins as possible in a sample.
This initial list of identified
proteins has value by itself for
qualitative or semi-quantitative
comparisons between diseases. Recent
studies demonstrated the reliability
and reproducibility of LC-MS
results, with different mass
spectrometers across different
laboratories, when performed by
experienced individuals
[9],
[11].
In a discovery phase investigation,
the MS technique is unbiased and
does not require prior knowledge of
what proteins may be in a sample.
This is in contrast to subsequent
validation studies where
targeted approaches are used and
which do require knowledge of target
proteins. In searching for a disease
biomarker, the discovery phase
should provide a list of proteins
and serve as a precursor phase for
targeted approaches. These
subsequent targeted approaches,
whether they use other MS techniques
or are immuno-based, are designed to
validate the use of the biomarker
protein(s) to distinguish one
disease from another.
In practice tailored strategies are
often needed to achieve a balance
between ideal and real world
constraints – especially where
sample volumes and numbers are
limited such as with CSF. In an
ideal situation it is desirable to
have numerous samples from
individuals with a particular
disease. It is further desirable to
have sufficient total protein
content in each sample so that a
variety of protein separation and
fractionation methods can be used
prior to MS analysis. This will
minimize abundant proteins from
masking the detection of less
abundant ones, and will permit full
qualitative and quantitative
analyses. Limited sample numbers and
quantities do not preclude
employment of tailored strategies to
get meaningful results. It should be
remembered that in the example of a
biomarker search, the protein(s)
will be confirmed or dismissed in
future targeted validation studies,
but failure to identify them in the
broad discovery list would preclude
them from examination for
validation.
Until recently, technical hurdles
impeded the use of CSF to
distinguish conditions such as
CFS and nPTLS.
Advances in sample preparation,
separations and MS platform
capabilities enabled us to recently
establish a comprehensive reference
normal CSF proteome
[10]. This provides the basis
for comparative proteome analyses
with other diseases, which should
provide greater insight into their
underlying pathogenesis.
To address the possibility that
CFS and nPTLS could be
distinguished from one another and
healthy subjects, we searched for
distinguishing protein marker
profiles by applying our advanced
proteomics strategy
[10] to characterize the CSF
proteomes from well described
CFS and nPTLS patients
(detailed in Methods). We performed
comparative whole CSF proteome
analyses between CFS,
nPTLS, and healthy normal
controls, and complemented these
findings with label-free
quantitative analysis of individual
subject samples. In addition, we
performed a preliminary pathway
analysis
[12] using these data, to
examine the feasibility of this type
of tool for future investigations to
probe for clues to the pathogenetic
mechanisms behind these diseases.
Materials and Methods Top
Ethics Statement
Approval for the conduct of this
study was obtained from the
Institutional Review Board of New
Jersey Medical School and the
Institutional Review Board of
Pacific Northwest National
Laboratory (Exempt status and
consent not required, using
previously banked de-identified
samples in accordance with federal
regulations).
Overview and Rationale
We performed analysis of pooled CSF
samples allowing for a broad and
deep view as well as qualitative
comparison of each disease-related
and control CSF proteome. To
determine if these two syndromes
could be quantitatively
differentiated we performed a
label-free quantitative analysis of
protein abundances for individual
subject CSF samples. Pooling samples
provided sufficient protein mass for
effective downstream proteomics
analysis following immunoaffinity
depletion of the 14 most abundant
proteins present (representing
approximately 95% of the total
protein mass in CSF), reducing the
dynamic range of protein
concentrations present in CSF, where
proteins with highest concentrations
mask proteins at lower
concentrations from detection.
Coupling immunoaffinity depletion
with strong cation exchange (SCX)
fractionation further reduces sample
complexity, and allowed for the
in-depth analysis of the CSF
proteomes. These comprehensive CSF
proteomics datasets were then used
to create an accurate mass and time
(AMT) tag database for subsequent
label-free quantitative analysis of
individual subject CSF samples. Due
to the limit in sample volume, the
CSF samples used in individual LC-MS
analyses were not immunoaffinity
depleted and fractionated, and
therefore had much lower proteome
coverage compared to the pooled
samples. Nevertheless, the
label-free quantitative analysis of
single subject samples provided a
means for statistical evaluation of
the quantified protein abundances
for many subjects suffering from
CFS and nPTLS as well
as normal healthy volunteers.
Together these analyses represent
the discovery phase of our studies
on CFS and nPTLS,
generating targets for follow up
verification and validation in the
later stages of the biomarker
discovery workflow
[13].
Cerebrospinal Fluid (CSF) specimens
CFS Subjects.
Both pooled and individuals CSF
samples were analyzed. Equal
aliquots from individual CSF samples
were pooled to provide sufficient
volume for extensive fractionation
and two-dimensional LC coupled to
tandem MS (2D-LC-MS/MS) analysis
with immunoaffinity depletion from
30 women and 13 men (n = 43) who
fulfilled the 1994 case definition
for CFS
[1].
All subjects were 18–54 years old
(median = 43) and underwent a
careful history and physical
examination by an expert experienced
in evaluating patients with
medically unexplained fatigue and
pain. Patients had blood tests to
rule out common causes of severe
fatigue such as anemia, liver
disease, hypothyroidism, systemic
lupus erythematosus, and Lyme
disease
[14]. All subjects then
underwent a psychiatric diagnostic
interview designed to identify major
psychiatric diagnoses for exclusion
in this study. Eleven of the
patients were not taking medicines.
Subjects then underwent lumbar
puncture. CSF was sent to the
laboratory for white blood cell (wbc)
count and total protein
[10]. A majority of CFS
patients had normal CSF protein and
cell counts (protein less than 45
mg/dl and wbc less than or equal to
5/mm3). Ten of the
patients had increased protein
values ranging from 46–93 mg/dl
(with a median of 59 mg/dl) and 3
patients had minimally elevated wbc
counts of 6, 7, and 9 respectively.
Individual CSF samples from 14 of
the 43 CFS subjects (aged
33–48 years with a median age of 43
years, 7 female and 7 male) were
also used in direct LC-MS analysis
(i.e., no MS/MS was performed)
without immunoaffinity depletion.
Twelve of the 14 patients had normal
CSF protein levels and all had
normal cell counts. All subjects
provided written informed consent
approved by the Institutional Review
Board.
nPTLS Subjects.
Both pooled and individuals CSF
samples were analyzed. Equal
aliquots from individual CSF samples
were pooled to provide sufficient
volume for extensive fractionation
and 2D-LC-MS/MS analysis with
immunoaffinity depletion from 15
females and 10 males (n = 25) with
nPTLS. All were documented
to have had prior Lyme disease which
met CDC surveillance case definition
criteria
[15],
persistent neurologic features,
including cognitive impairment and
fatigue, despite appropriate
antibiotic treatment
[16],
[17].
Subjects were 17–64 years old
(median = 48). All were seropositive
for antibodies to B. burgdorferi
(the etiologic agent of Lyme
disease). Patients, enrolled in an
NIH funded study, met the following
criteria
[17]:
(1) current positive IgG Western
blot using CDC surveillance criteria
assessed using a single reference
laboratory (University Hospital of
Stony Brook); (2) treatment for Lyme
disease with at least 3 weeks of
intravenous ceftriaxone or
cefotaxime that was completed at
least 4 months before study entry;
and (3) objective evidence of memory
impairment as documented by the
Wechsler Memory Scale-III compared
to age-, sex-and education-adjusted
population norms. nPTLS
subjects were excluded if history or
testing revealed a medical condition
that could cause cognitive
impairment or confound
neuropsychological assessment (e.g.,
neurological disease, autoimmune
disease, unstable thyroid disease,
learning disability, substance
abuse, B12 deficiency). Patients
with cephalosporin allergy or a
history of significant psychiatric
disorder prior to onset of Lyme
disease were also excluded. All
patients had a comprehensive battery
of neurocognitive testing and a
full-physical exam with detailed
rheumatologic and neurologic
assessments. nPTLS patients
then had a lumbar puncture and CSF
was evaluated for cell count, total
protein, glucose, total
gammaglobulin, oligoclonal bands and
evidence of B. burgdorferi
(ELISA, Bb DNA by PCR, and culture
using BSKII medium). None had
evidence of another active
tick-borne disease. A majority of
nPTLS patients included in
the pooled sample had normal CSF
protein and cell counts (protein
less than 45 mg/dl and wbc less than
or equal to 5/mm3),
except for 3 patients who had
elevated protein values of 58, 69,
and 71 mg/dl respectively and 1
patient with elevated wbc count of
6. Individual CSF samples from a
group of 14 of the 25 nPTLS
subjects (aged 25–58 years with a
median age of 48 years, 6 female and
8 male) were also used in direct
LC-MS analysis without
immunoaffinity depletion. Two of the
14 patients had increased CSF
protein levels of 69 and 71 mg/dl
and 1 had a slightly elevated wbc of
6. All subjects provided written
informed consent approved by the
Institutional Review Board.
Normal Controls.
We used the 2D-LC-MS/MS data
obtained previously from pooled CSF
of 11 healthy control subjects
[10]. Briefly, there were 8
women and 3 men, aged 24–55 years
with a median age of 28 years.
Individual CSF samples from another
set of 10 healthy volunteers, age
37–44 years (median = 40) and 5
women and 5 men, were analyzed by
LC-MS analysis without
immunoaffinity depletion.
Immunoaffinity depletion of 14 high
abundance CSF proteins
We had previously shown that this
technique could increase our protein
identification yield by 70%
[10]. Pooled CSF samples from
CFS or nPTLS
patients (total volume of 18 mL
each), were fractionated using a
12.7×79.0 mm Seppro® IgY14 LC10
affinity LC column (Sigma, St Louis,
MO) as previously described
[18].
Pooling was done to compensate for
lack of sufficient volume (and
consequent protein content)
available for immunoaffinity
depletion of individual patient
samples. Both the flow-through
(lower abundance proteins) and bound
fractions from both pooled CSF
samples were collected and processed
identically until LC-MS/MS analysis.
These analyses resulted in an
in-depth characterization of the CSF
proteome and the combined results of
abundant protein and less abundant
protein fractions allowed the
creation of an AMT tag database
[19]
for high-throughput analysis of a
larger number of individual subject
samples using LC-MS.
Protein digestion
CSF proteins (from the
immunoaffinity depletion processed
pooled samples and the individual
samples without immunoaffinity
depletion processing) were digested
with trypsin and cleaned up with SPE
C18 columns as previously described
[10]. Final peptide
concentration was determined by BCA
assay (Pierce, Rockford, IL). All
tryptic digests were snap frozen in
liquid nitrogen and stored at −80°C
until further processing and
analysis.
Strong cation exchange (SCX)
fractionation
A total of 300 µg of tryptic
peptides from both the IgY14 bound
and flow-through fractions from the
pooled CFS and nPTLS
CSF samples were fractionated by SCX
chromatography as described
[20].
Thirty SCX fractions were collected
for each sample and 20% of each
fraction was injected for
reversed-phase LC-MS/MS analysis.
Reversed-phase capillary LC-MS/MS
for CSF pooled fraction analysis
SCX fractions of the IgY14 bound
fraction samples were analyzed on an
LTQ (ThermoFisher, San Jose, CA)
linear ion trap, and SCX fractions
of the IgY14 flow-through fraction
samples were analyzed on an
LTQ-Orbitrap Velos (ThermoFisher)
instrument, operated in
data-dependent mode with the same LC
conditions as previously described
[10].
Reversed-phase capillary LC-MS for
label-free quantification of
unfractionated CSF samples
For label-free quantification
analyzing unfractionated CSF samples
(individual patient samples with
insufficient volume (protein
content) for immunoaffinity
depletion and SCX fractionation),
the LTQ-Orbitrap Velos mass
spectrometer was operated in the
data-dependent mode with full scan
MS spectra (m/z 400–2000) acquired
in the LTQ-Orbitrap Velos with
resolution of 60,000 at m/z 400
(accumulation target: 1,000,000).
MS/MS data acquired here were not
used for the quantitative analysis.
Data analysis
The LTQ raw data from the pooled
samples was extracted using
Extract_MSn (version 3.0;
ThermoFisher) and analyzed with the
SEQUEST algorithm (V27 revision 12;
ThermoFisher) searching the MS/MS
data against the human IPI database
(Version 3.40). Mass tolerances of 3
Daltons for precursor ions and 1
Dalton for fragment ions without an
enzyme defined, as well as static
carboxyamidomethylation of cysteine
and dynamic oxidation of methionine
were used for the database search.
The LTQ-Orbitrap Velos MS/MS data
were first processed by in-house
software DeconMSn
[21] accurately determining the
monoisotopic mass and charge state
of parent ions, followed by SEQUEST
search against the IPI database in
the same fashion as described above,
with the exception that a 0.1-Dalton
mass tolerance for precursor ions
and 1-Dalton mass tolerance for
fragment ions were used. Data
filtering criteria based on the
cross correlation score (Xcorr) and
delta correlation (ΔCn) values along
with tryptic cleavage and charge
states were developed using the
decoy database approach and applied
for filtering the raw data to limit
false positive identifications to
<1% at the peptide level
[22]–[24].
For the LTQ-Orbitrap Velos data, the
distribution of mass deviation (from
the theoretical masses) was first
determined as having a standard
deviation (σ) of 2.05 part per
million (ppm), and a mass error of
smaller than 3σ was used in
combination with Xcorr and ΔCn to
determine the filtering criteria
that resulted in <1% false positive
peptide identifications.
The AMT tag strategy
[19]
was used for label-free
quantification of MS features
observed in the LTQ-Orbitrap Velos
analysis of the individual CSF
samples from normal, CFS
and nPTLS conditions. The
filtered MS/MS peptide
identifications obtained from the
2D-LC-MS/MS analyses of all pooled
CSF samples were included in an AMT
tag database with their theoretical
mass and normalized elution time
(NET; from 0 to 1) recorded. LC-MS
datasets were then analyzed by
in-house software VIPER
[25]
that detects features in mass–NET
space and assigned them to peptides
in the AMT tag database
[26].
The data was further filtered by
requiring that all peptides must be
detected in at least 30% of the
datasets in each of the three
conditions. The false discovery rate
of the AMT tag analysis was
estimated using an 11-Da shift
strategy as previously described
[27].
A false positive rate of <4% was
estimated for each of the LC-MS data
sets. The resulting lists of
peptides from 2D-LC-MS/MS or direct
LC-MS analysis were further
processed by ProteinProphet software
[28] to remove redundancy in
protein identification.
Data normalization and
quantification of the changes in
protein abundance between the
normal, CFS and nPTLS
CSF samples were performed and
visualized using in-house software
DAnTE
[29]. Briefly, peptide
intensities from the LC-MS analyses
of the individual samples (volume
limited) were log2 transformed and
normalized using a mean central
tendency procedure. Peptide
abundances from the individual
samples were then “rolled up” to the
protein level employing the R-rollup
method (based on trends at peptide
level) implemented in DAnTE. ANOVA,
principal component analysis (PCA)
and clustering analyses were also
performed using DAnTE.
Pathway Analysis of the data was
performed with Ingenuity Pathways
Analysis (Ingenuity Systems,
www.ingenuity.com). Canonical
pathway analysis identified the
pathways from the Ingenuity Pathways
Analysis library of canonical
pathways that were most significant
to the CFS and nPTLS
proteins identified. The
significance of the associations
were assessed with the Fisher's
exact test.
Results Top
We first performed pooled sample
analysis, then individual sample
analysis, and then pathway analysis
using the observed proteins. These
analyses represent a discovery phase
of our studies on CFS and
nPTLS, generating targets
which can be followed up in future
verification and validation stages
studies
[13].
Proteomic analysis of pooled CSF
samples
In the pooled analysis, we examined
individual sets of CSF samples from
CFS patients (n = 43) and
nPTLS patients (n = 25),
respectively. We used the proteomic
strategy described in Methods to
assure that the maximum number of
proteins would be analyzed and the
more abundant proteins did not
obscure the less abundant ones
having biomarker potential. The
bound fraction of abundant proteins
from the immunoaffinity depleted
flow through fraction was analyzed
separately and included in the
subsequent analysis. Combining
immunoaffinity-based partitioning,
SCX fractionation and LC-MS/MS, we
identified
1approximately 30,000 peptides
for each pooled sample
corresponding to 2,783 nonredundant
proteins in CFS patient
samples and 2,768 proteins in
nPTLS patient samples, compared
to the 2,630 proteins present in the
CSF of healthy normal control
subjects. These can be graphically
seen in
Figure 1 which shows the number
of proteins identified solely in
each group, and shared or not shared
between the groups (see
Table
S1).
Figure
1 also shows that the nPTLS
and CFS groups shared
significantly more proteins (n =
305) than each disease group shared
with healthy controls (n's = 135 and
166, respectively). (Note that, as
with any assay, when we indicate
that a protein was “not found” or
“not identified” that is defined as
within the limits of detection).
Proteomic analysis of individual CSF
samples
Quantitative analyses were performed
on individual CSF samples from 14
CFS patients and 14
nPTLS patients. They were
compared to 10 normal healthy
volunteers (samples chosen at
random) to provide insights on the
variation among individuals within
and between different groups.
Limited volumes of the individual
samples reduced the sample
preparation options (i.e.,
immunoaffinity depletion and SCX
fractionation), and hence resulted
in less depth of proteome coverage
than possible with the pooled
samples, where approximately 20 ml
were available for depletion and
fractionation. Nevertheless, we
identified 4,522 peptides across all
individual samples, representative
of 474 non-redundant proteins
identified and quantified in the
individual sample analysis (Table
S2).
Unsupervised hierarchical clustering
and PCA were employed to determine
if the observed quantitative
differences in protein abundances
were sufficient to distinguish these
two patient groups (this was de
facto blinded – as samples were
run in a random order and uncoded as
to disease group afterwards). The
proteins considered in the
unsupervised hierarchical clustering
analysis were quantified in
individual samples and found to be
significantly different in abundance
by analysis of variance (ANOVA p ≤
0.01,
Table S3); while PCA analysis
considered all proteins quantified
in each individual sample. The CSF
proteome of the two disease states
were markedly different from each
other (Fig.
2A and B). Individual patients
also showed consistent patterns of
protein abundances discriminating
CFS from nPTLS (Fig.
2A). These results demonstrated
that it is unlikely that any single
subject's CSF sample in the pooled
analysis contributed
disproportionately to the
differential proteome distributions
observed between the disease groups.
Moreover, the individual analyses
also highlighted the potential for
diagnostic marker confirmation upon
extension to larger sample sets in
validation studies.
Illustrative pathway analyses of
protein results from CSF samples
We utilized pathway analysis as an
exploratory tool to assess the value
of our data, beyond distinguishing
the two syndromes from each other,
to see if the data was amenable to
analysis that would help generate
hypotheses of pathogenesis. We chose
representative pathways to analyze
for illustration based in part on
their quantitative ranking (Table
S4) and in part by the potential
relevance of the pathway involved.
Even this limited investigation
demonstrated that there is a wealth
of proteome information that can be
leveraged for hypotheses generation.
Example of proteins in common and
elevated in abundance in the two
disease conditions, compared to
normal, but at different levels.
An illustration, where the same
proteins are elevated in abundance
in both conditions, but at different
magnitudes, is provided by
inspection of proteins in the
complement system. This is of
interest because both syndromes may
be triggered by infections (nPTLS
in all cases by B. burgdorferi;
many CFS cases by one or
more microbes yet to be identified).
We found that the complement cascade
related proteins were identified and
significantly enriched in both
CFS and nPTLS pooled
CSF proteomes by the Fisher Exact
test (p = 0.005) implemented in
Ingenuity Pathways Analysis (Figure
S1A). In individual patient
samples analyzed, we identified and
quantified 4 components (C1S, C4B,
C1QB, C1QC) which are seen with
activation of the complement cascade
and which were differentially
increased in abundance consistently
across the nPTLS patients
compared to CFS (Figure
S1B and C). This represents the
type of data that can be useful in
the formulation of pathogenetic
hypotheses because the role of
complement in these disorders is
under-explored.
Example of proteins solely
identified in one condition.
Analysis of the highly fractionated
pooled patient samples led to the
identification of proteins solely
identified in each of the disease
states. To investigate if these
disease specific proteins have
common annotated functional
properties, we performed pathway
analysis (Tables
S5 and
S6).
As an example, the CDK5 signaling
pathway, was found to be
significantly enriched (p = 0.00009)
for proteins identified only in the
pooled CFS proteome. This
signaling pathway has been linked to
Parkinson's
[30]
and Alzheimer's diseases
[31].
Example of proteins in common and
decreased in abundance in the two
disease conditions, compared to
normal, but at different levels.
In certain cases, proteins were
found to be decreased in both
CFS and nPTLS compared
to healthy normal controls. However,
quantitative distinguishing
differences could still be found
between the two conditions. A
specific example relates to networks
relevant to neurological function
such as axonal guidance (Figures
S2A and B), where the proteins
in CFS were further
decreased relative to nPTLS.
These findings highlight
quantifiable differences between
CFS and nPTLS that may
be found, with respect to certain
proteins such as those that are
known to effect the dynamic changes
in CNS cellular architecture, such
as axon, neurite, and dendritic
spine growth and organization.
Discussion Top
Our results support the concept that
CFS and nPTLS are
distinguishable disorders with
distinct CSF proteomes, where one
can be separated from the other. The
results also demonstrate that each
condition has a multitude of
candidate diagnostic biomarkers for
future validation and optimization
studies. The discovery of many of
the same proteins in each proteome
is important because it allows
comparative pathway analysis, so
that useful hypotheses of
pathogenesis can be formulated and
tested.
Our results represent the most
comprehensive analysis of the whole
CSF proteome to date for both
CFS and nPTLS. These
two disorders have similar symptoms
that have created diagnostic
dilemmas. It has been speculated
that one (nPTLS) is a
subset of the other, but our results
do not support that notion. Our
findings alone do not describe why
CFS or nPTLS
occur, but are provided to
illustrate that CSF proteome
analysis may provide important and
meaningful insights into the
biological processes modulated as a
function of disease and facilitate
the identification of protein
candidates for further
investigation. Analytical strategies
need to be developed for application
to those proteins and their pathways
that may not have been described
yet. Nevertheless, in toto,
these results are encouraging
because there is an abundance of
data now that can be analyzed with
existing tools and future methods to
develop hypotheses on pathogenesis
[9],
[32].
We regard the proteins that were
identified only in one group or
differentially abundant between
groups, as possible or candidate
biomarkers that can be subjected to
further analysis in validation and
verification studies. The clinical
significance of the proteins
identified in each pooled sample is
difficult to determine in the
current discovery phase. As with
most technologic methods, we expect
multiple replicate analyses of the
highly fractionated samples would
result in a reduction of the number
of seemingly unique proteins
identified for each disease group
[33].
An important strategy that can be
used post-discovery towards
validation, is the use of targeted
approaches that are either MS-based,
immuno-based, or a combination of
these approaches
[12],
[34].
One approach, selected reaction
monitoring (SRM) MS, allows for much
higher sensitivity and specificity,
more accurate quantification, and
much higher throughput to be
achieved for simultaneously
measuring many biomarker candidates
in large clinical cohorts
[35]–[37].
This approach also compensates for
any theoretical over-representation
of proteins in pooled samples by a
single or small number of
individuals. This is a strategy that
we plan to use not only for these
diseases, but in the investigation
of other diseases with
neuropsychiatric features. SRM-MS
analysis will permit us to directly
use small-sized samples, such as the
individual CSF samples, enable
verification of marker candidates
that currently do not have available
antibodies (hence not amenable to
conventional analyses such as ELISA
or Western blots), and provide
robust statistical analyses on
individual candidate markers or
combinations of them to determine
which would make the best
biomarker(s) for a particular
disease condition. Immunobased
assays such as ELISA or Western
blots may also be used for targeted
approaches, but will likely have
more utility during a clinical
validation phase where much larger
sample cohorts are used. Some may
choose to apply these methods for
additional orthogonal confirmation
of a result. However, its greater
value may lie in its widespread use
as a common diagnostic platform.
Regardless of the method chosen,
identification of diagnostic CSF
biomarkers may be the necessary
prelude to a search for the same
markers in the highly complex blood,
because it permits targeted searches
for markers that might otherwise be
obscured or have uncertain
relevance.
With respect to biomarkers, we
believe our proteomic strategy
[10], that did not require prior
knowledge of which proteins might be
present in the CSF, will accelerate
the transition from a discovery
phase of candidate biomarkers, as
described in this study, to full
validation for clinical application.
We and others have cited important
elements that should be considered
when an assay or biomarker is being
developed for preliminary or full
validation
[38]–[40].
Distinguishing CFS and
nPTLS will have etiologic
implications which could lead to
novel diagnostics and therapeutic
interventions. On a broader level
the strategy we employed may prove
useful in providing investigative
foundations in other poorly
understood neurological conditions.
Supporting Information Top
Figure S1.
Illustrative example of
pathway analysis with respect to
complement pathways.
Protein network and pathway analysis
was performed employing Ingenuity
Pathways Analysis tools (v8.6-
www.ingenuity.com). A) Proteins
that participate in complement
signaling were significantly
enriched (p = 6×10−20) in
the CSF proteomes for pooled
disease-specific samples. A
comparison of protein abundance
determined by spectral counts
reveals difference between disease
states and normal healthy control
CSF. Proteins with an increased
abundance are colored red and those
that decrease in abundance relative
to normal healthy control are
colored green. B) Proteins annotated
as participating in complement that
were detected in individual patient
analysis are shown the heatmap.
Protein abundances measured by ion
intensity transformed to Z scores
clearly show differences between
CFS and nPTLS
patients. C) Receiver operator
characteristic (ROC) curves
demonstrate the discriminating power
of the select set of proteins that
were detected as having statistical
differences by ANOVA (p<0.05) in
abundance in the analysis of
individual patient samples.
(TIF)
Figure S2.
Illustrative example of
pathway analysis with respect to
axonal guidance pathways.
Protein network and pathway analysis
were performed employing Ingenuity
Pathways Analysis tools (v8.6-
www.ingenuity.com). A) Proteins
that associated with axonal guidance
and signaling were significantly
enriched (p = 6×10−20) in
the CSF proteomes for all pooled
samples. A comparison of protein
abundances determined by spectral
counts revealed differences between
disease states and normal healthy
control CSF. Proteins with an
increased abundance are colored red
and proteins with decreased
abundance relative to
normal/controls are colored green.
B) Normalized protein abundance
clearly differs between CFS
and nPTLS patients. C)
Receiver operator characteristic
(ROC) curves demonstrate the
discriminating power of the select
set of proteins that were detected
in individual CSF samples as well as
in the pooled proteome.
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