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Autoimmunity to Serotonin in Chronic Fatigue Syndrome

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.

Lyme RA nieuws
Aankomende nieuwe Lymebehandeling.

Ik ben in regelmatig contact met de toponderzoekers op Lymebied. Zo ook met professor Eva Sapi. Zij werkt voor de Universiteit van New Haven New York. Samen met 70 studenten en Alan MacDonald doet zij dagelijks niets anders dan testen hoe goed middelen werken tegen Borrelia. In een gesprek wat ik enige tijd geleden met haar had kon zij haar enthousiasme niet onderdrukken over een nieuwe medicatie combinatie en de effectiviteit van de werking ervan. Het bestaat uit 3 ingredienten. Het zouden vrij onschuldige middelen zijn. Maar omdat het ter publicatie aan PlosOne is afgegeven mocht ze me niet vertellen wat het precies was. Ze heeft het rond 15 juni 2015 afgegeven en de gemiddelde publicatietijd bij PlosOne is 103 dagen. Maar dat is een gemiddelde. Waarschijnlijk duurt het iets langer. We wachten in spanning af. Waarschijnlijk is Stevia een element, Bee Venom en Lactoferrin en mogelijk Banderol. Maar misschen heeft ze iets nog beters ontdekt of juist een exacte combinatie. Het is afwachten.

Waarschijnlijk maakt Curza er ook deel van uit. http://www.curza.com/About

video de meirleir 1         Is ME een ziekte?
video de meirleir 2         Diagnose
video de meirleir 3         Erfelijk?    (d)

video de meirleir 4         Slaapproblemen

video de meirleir 5         Pijn

video de meirleir 6         Hormonen

video de meirleir 7         Virussen

video de meirleir 8         Bloedsomloop

video de meirleir 9         Maag/darm problemen

video de meirleir 10       ME en andere ziektes

video de meirleir 11       Bestaan en oorzaken

video de meirleir 12       Exacte diagnose

video de meirleir 13       Slaap, pijn en nachtzweten

video de meirleir 14       Behandeling

video de meirleir 15       Remedies en hoop

video de meirleir 16       Behandeling LDN

video de meirleir 17       Hersenen

video de meirleir 18       Witte bloedcellen

video de meirleir 19       Indeling verschillende groepen

video de meirleir             Ontstekingen (Engels)

 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 patiŽnten) een dubbelblinde Fase II studie uitgevoerd met het middel Rituximab bij 30 ME/CVS-patiŽnten. 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 patiŽnten 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 patiŽnte 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-patiŽnten deel waarvan de helft Rituximab toegediend kreeg en de andere helft een placebo. 10 van de 15 patiŽnten of 67% van degenen die het medicijn hadden gekregen, gaven aan dat hun symptomen verbeterden. In de placebo-groep ging dit slechts om 2 patiŽnten (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-patiŽnten: "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-patiŽnten 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-patiŽnten.

 

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 patiŽnten te identificeren met ME/cvs.

In een proefstudie van zes patiŽnten 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 patiŽnten 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 patiŽnt.


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 geÔnfecteerd. Alleen is minder dan de helft van hen er ook echt ziek van geworden. Als iemand eenmaal geÔnfecteerd 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 patiŽnten 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 patiŽnten 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 patiŽnten 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."

Glaserís 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 privť ME/cvs praktijk in Michigan. Dat hij op lange termijn de kenmerken van van ME/cvs patiŽnten en de respons op de behandeling volgde, maakte deze follow-up studie mogelijk.

Dat ME/cvs patiŽnten verschillende symptomen en meerdere soorten virale en bacteriŽle 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 patiŽnten 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 patiŽnt meerdere monsters bloedserum. Voor dit onderzoek selecteerde hij bloedmonsters van zes van die patiŽnten. Vijf vielen onder een subgroep met het Epstein Barr virus, de zesde had een Epstein Barr virus en een bacteriŽle co-infectie. Ter vergelijking verzamelden de onderzoekers monsters van twintig gezonde personen die qua leeftijd en geslacht overeenkwamen met de zes ME/cvs patiŽnten.

Ook Lerner kwam los van dit alles tot de hypothese dat ME/cvs patiŽnten mogelijk een gedeeltelijke reactivatie van een virus ondergingen. Al testten patiŽnten 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 ťťn 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 patiŽnten 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 patiŽnten 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 zoín 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, CaliforniŽ, 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 Ė 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. ďSpinal fluid is like a liquid window to the brain,Ē 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. ďOne next step will be to find the best biomarkers that will give conclusive diagnostic results,Ē he says. ďIn addition, if a protein pathway is found to influence either disease, scientists could then develop treatments to target that particular pathway.Ē

ďNewer 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 icebergĒ


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.

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]Ė[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 Ė 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 18Ė54 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 46Ė93 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 33Ė48 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 17Ė64 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 25Ė58 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 24Ė55 years with a median age of 28 years. Individual CSF samples from another set of 10 healthy volunteers, age 37Ė44 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 Sepproģ 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 −80įC until further processing and analysis.

Strong cation exchange (SCX) fractionation

A total of 300 Ķg 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 400Ė2000) 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]Ė[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 massĖNET 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 ďrolled upĒ 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 ďnot foundĒ or ďnot identifiedĒ that is defined as within the limits of detection).

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Figure 1. Characterization of the proteome from pooled and individual CSF samples.

A) Venn diagram of the qualitative distribution of proteins identified in the pooled, immunodepleted, and fractionated cerebrospinal fluid (CSF) from normal healthy control subjects, Chronic Fatigue Syndrome (CFS), and Neurologic Post Treatment Lyme Syndrome (nPTLS). The numbers of proteins for each of these three categories separately is shown outside the circles below the category (2,630 for true normal controls, 2,783 for CFS, and 2,768 for nPTLS). The subsets of intersections between these categories are shown within the circles. There were 1) 738 proteins that were identified in CFS, but not in either healthy normal controls or nPTLS; 2) 1,582 proteins that were not identified in CFS, but were in either nPTLS disease or healthy normal controls; 3) 692 proteins that were identified in the nPTLS patients, but not in healthy normal controls or CFS; and 4) 1,597 proteins that were not identified in nPTLS, but were identified in either healthy normal controls or CFS. This figure also shows that the nPTLS and CFS groups shared significantly more proteins (n = 305) than each disease group shared with controls (n's = 135 and 166). The specific lists of these subsets are presented in additional Table S1.

doi:10.1371/journal.pone.0017287.g001
 

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 Ė 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.

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Figure 2. Comparative analysis of individual CFS and nPTLS CSF proteomes.

A) Unsupervised hierarchical clustering of 59 proteins (see Table S3) that are differentially abundant as determined by ANOVA (p<0.01) clearly separates these two disease states with the exception of one nPTLS sample clustering with CFS patient samples. B) Principal Component Analysis (PCA) of CFS and nPTLS samples demonstrates that the CSF proteomes, and by extension of the CNS status, differ between CFS and nPTLS.

doi:10.1371/journal.pone.0017287.g002
 

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]Ė[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]Ė[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.

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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.