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from: Vivienne Hughes to: GUmail date: Sun, Sep 16, 2018 at 11:03 PMsubject: Re: PP-Baraniukmailed-by: griffith.edu.au
Provocation Testing in CFS/MEJames N. Baraniuk, MDGeorgetown University, Washington DC USA
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Provocation Testing in CFS/ME
James N. Baraniuk, MDGeorgetown University, Washington DC USA
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Provocation Testing in CFS/ME
• Hypothesis:
• Baseline symptoms, cognitive and other functions are relatively similar to normal when CFS/ME subjects are well rested.
• It is necessary to use provocation methods to perturb body systems and induce subjective and objective evidence of dysfunction in CFS/ME compared to control subjects.
Null Hypothesis 1:
There are no differences between CFS/ME and control subjects.
Null Hypothesis 2: There are substantial differences between CFS/ME and control subjects at all times that can be detected without provocations that worsen symptoms or cause exacerbations.
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Georgetown University
• Telephone screening for • 1994 CDC Fukuda criteria CFS/ME subjects
• 2000 “Kansas” criteria Gulf War Illness (GWI)
• Control subjects for sedentary activity status, and
• Exclude major medical and psychiatric diseases.
• All subjects complete online questionnaires to assess:• Baseline Fatigue
• Somatic symptoms• Pain
• Interoception
• Psychological symptoms
• Quality Of Life
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Clinical Research Unit on Day 1
• Scripted history and physical examination
• Routine blood work
• Serial blood work (pre-exercise baseline)
• Dolorimetry – systemic hyperalgesia = tenderness
• Heart Rate Variability• EKG
• Vital signs during recumbent and standing postures
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Chronic
Multisymptom
Illness (CMI) 2
GWI
“Kansas” 3CFS
“Fukuda” 4FM 2010 17 FM 2011 ?? FM 1990 15
Tenderness
Musculoskeletal PainMyalgia Widespread
Pain
Widespread
Pain
Widespread
PainArthralgia
FatigueFatigue,
Sleep
Fatigue Fatigue Fatigue
SleepWaking
unrefreshed
Waking
unrefreshed
Post-exertional
malaise
Cognition,
Mood
Cognition, Mood
Neurological
Cognition Cognition Cognition
Depressed
Headache Headache
GastrointestinalSomatic
symptoms
Abdominal painRespiratory Sore throat
Skin Sore lymph nodes
≥1 chronic symptom
in ≥2 categories≥3 of 6 categories
Fatigue plus
≥4 of 8Severity scores Severity scores
Pain +
Tenderness
Defining CFS and its Differential Diagnosis
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Chronic
Multisymptom
Illness (CMI) 2
GWI
“Kansas” 3CFS
“Fukuda” 4FM 2010 17 FM 2011 ?? FM 1990 15
Tenderness
Musculoskeletal PainMyalgia Widespread
Pain
Widespread
Pain
Widespread
PainArthralgia
FatigueFatigue,
Sleep
Fatigue Fatigue Fatigue
SleepWaking
unrefreshed
Waking
unrefreshed
Post-exertional
malaise
Cognition,
Mood
Cognition, Mood
Neurological
Cognition Cognition Cognition
Depressed
Headache Headache
GastrointestinalSomatic
symptoms
Abdominal painRespiratory Sore throat
Skin Sore lymph nodes
≥1 chronic symptom
in ≥2 categories≥3 of 6 categories
Fatigue plus
≥4 of 8Severity scores Severity scores
Pain +
Tenderness
Defining CFS and its Differential Diagnosis
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Chronic
Multisymptom
Illness (CMI) 2
GWI
“Kansas” 3CFS
“Fukuda” 4FM 2010 17 FM 2011 ?? FM 1990 15
Tenderness
Musculoskeletal PainMyalgia Widespread
Pain
Widespread
Pain
Widespread
PainArthralgia
FatigueFatigue,
Sleep
Fatigue Fatigue Fatigue
SleepWaking
unrefreshed
Waking
unrefreshed
Post-exertional
malaise
Cognition,
Mood
Cognition, Mood
Neurological
Cognition Cognition Cognition
Depressed
Headache Headache
GastrointestinalSomatic
symptoms
Abdominal painRespiratory Sore throat
Skin Sore lymph nodes
≥1 chronic symptom
in ≥2 categories≥3 of 6 categories
Fatigue plus
≥4 of 8Severity scores Severity scores
Pain +
Tenderness
Defining CFS and its Differential Diagnosis
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Center for Epidemiological Studies – Depression (CESD)
20 items scored 0 to 3 by severity in past week
Cut-off score ≥ 16 / 60This threshold has a 30% false positive rate [Vilagut, 2016]Sensitivity = 87% Specificity = 70%
CESD remains the gold standard for epidemiological studies that screen populations and countries for risk of depression
Factor Analysis: Somatic, Depressed, Anhedonia, Interpersonal
Somatic = Fatigue, Sleep, Cognition, Effort, Bother, Talk less, AppetiteThe 4 factor scores have never been evaluated in depression or control groups
There was a “strong relationship between the symptoms of depression as measured by the Center for Epidemiologic Studies-Depression Scale (CES-D) and fatigue, but fatigue was neither sensitive nor specific for the diagnosis of depression.”Fuhrer R, Wessely S. The epidemiology of fatigue and depression: a French primary-care study. PsycholMed. 1995 Sep;25(5):895-905. Review. PubMed PMID: 8588008.
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Center for Epidemiological Studies – Depression (CESD)
4 Factors: Somatic, Depressed, Anhedonia, Interpersonal
24% of US population at risk for depression using total score (black) threshold of 16 / 60(NHANES & sedentary controls)
Median
Traditional threshold for Total CESD Score (black) = DepressionScore of 16 / 60 = 24%
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Center for Epidemiological Studies – Depression (CESD)
4 Factors: Somatic, Depressed, Anhedonia, Interpersonal
94% of Depressed subjects in PROMIS studies ≥ 16 / 60
Total CESD Score (black) threshold of 16 / 60
Depression
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Center for Epidemiological Studies – Depression (CESD)
4 Factors: Somatic, Depressed, Anhedonia, InterpersonalSC CFS GWI
High Somatic Domain Scores cause positive CESD in 54% of CFS
Depressed Depressed
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Center for Epidemiological Studies – Depression (CESD)
• 4 Factors:
• Threshold score is too low • ≥16/60 20% false positive
• Somatic• High somatic scores drive CESD and depression in CFS
• Depressed• Present in CFS but does not drive CESD score
• Anhedonia• Present in CFS but does not drive CESD score
• Interpersonal
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Systemic Hyperalgesia in CFS, GWI and FM Females
0%
10%
20%
30%
40%
0 3 6 9 12
Fre
qu
en
cy
Dolorimetry (bins of 1 kg)
GWI n=68
CFS n=174
FM n=28
SC n=133
4.5 kg
0
0.2
0.4
0.6
0.8
1
0 0.2 0.4 0.6 0.8 1
Sen
siti
vity
1 - Specificity
GWI
CFS
H&P CFS (green, n=174), GWI (red, n=68), Control (black, n=133)Pain + Tenderness Fibromyalgia (1990) (yellow, n=28)
Dolorimetry Method:Apply pressure with dolorimeter at 18 traditional FM tender points Determine pressure that causes pain Average kg Frequency distributions
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MRI - Exercise Study to Model Exertional Exhaustion
HYPOTHESES:
1. CFS have depression
2. CFS have tenderness (systemic hyperalgesia)
3. Submaximal exercise on DAY 1 reduced VO2 on DAY 2
4. Submaximal exercise on DAY 1 postural tachycardia in 1/3rd
5. Exercise worsens orthostatic intolerance
6. MRI before exercise on DAY 1 will show good brain function
MRI after exercise on DAY 2 will show bad brain function
7. Cerebrospinal fluid contains CFS biomarkers
MRI DAY 1 DAY 2 MRI LPQ H&P
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MRI - Exercise Study to Model Exertional Exhaustion
MRI DAY 1 DAY 2 MRI LPQ H&P
Ceiling vs
FloorEffects
FunctionalDisordersMigraine
MRI: 3T Seimens TrimTrio, 32 element head coil, new PRISMABrain anatomy – voxel based morphometryMolecular spectroscopyResting state scan for Resting State NetworksCognitive Task n-Back working memory task
Attention = 0-back = “See a letter. Press a button.”Working memory = 2-back = “See a string of letters…
Submaximal test: Does exercise on Day 1 cause a decrease in exercise on DAY 2?
“Brain” biomarkers
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MRI - Exercise Study to Model Exertional Exhaustion
Background:
GWI Submaximal exercise on DAY 1 postural tachycardia in 1/3rd
Stress Test Activated Reversible Tachycardia = START = 25%
No postural tachycardia before exercise
Postural tachycardia after exercise
≥ 2 time points with ΔHR ≥30 bpm after exercise
ΔHR = Standing – Recumbent
No ΔHR = STOPP = 62% of CFS
POTS = 13% of CFS = GWI = Control = Literature Standing Up
MRI DAY 1 DAY 2 MRI LPQ H&P
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Orthostatic Problems
• Orthostatic Intolerance
• Symptoms
• Dizziness, Lightheaded
• Orthostatic Instability
• Change in heart rate with posture
• Change in autonomic tone• Vagal – Parasympathetic to slow
down the heart• Sympathetic to speed up the
heart
• Results are biased from Heads Up Tilt Table testing• High false positive rate• Not physiological
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Dizziness Before Exercise
Control CFS
Recumbent Standing Recumbent Standing
CFS had significantly more Dizziness than Controls while supine and standing. Dizziness Score > 2/20 reported in 9/25 Controls and 33/39 CFS (FET, p
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Dizziness After Exercise
Postural Orthostatic Tachycardia
Exercise-Induced Postural
Tachycardia
Postural Orthostatic Tachycardia
Exercise-Induced Postural
Tachycardia
Exercise increased Dizziness in 22/39 CFSPOTS did not explain Dizziness in CFS
Exercise caused symptoms that could not be explained by postural tachycardia
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Dizziness After Exercise
Dizziness While Lying Down in 41% CFSExercise increased Dizziness in 22/39 CFS (56%)
POTS did not explain Dizziness before exercisePostural Tachycardia did not explain the increased Dizziness after exercise (85%)Receiver Operating Characteristics defined Dizziness > 2 / 20
No Orthostatic Intolerance (No OI) = 28%Postural Orthostatic Intolerance = Standing = 31% Persistent Orthostatic Intolerance = Recumbent & Standing = 41%
Recumbent Standing
Control CFS
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Recumbent Dizziness in CFS
• Recumbent Dizziness in CFS in 41%• Persistent Orthostatic Intolerance (symptoms)
• Dizziness gets worse in 85% of CFS• Component of Post-Exertional Malaise?
• Dizziness is not related to postural tachycardia• Orthostatic instability (heart rate)
• Postural tachycardia is related to decreased parasympathetic tone • Decreased vagal messages from the brain stem allow the heart to speed up• NULL HYPOTHESIS: Autonomic dysfunction (orthostatic instability) is not the
cause of dizziness and orthostatic intolerance
• HYPOTHESIS: It is all in your head (Vestibular, Brain stem, other?)
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MRI - Exercise Study to Model Exertional Exhaustion
HYPOTHESES:
1. CFS have depression
2. CFS have tenderness (systemic hyperalgesia)
3. Submaximal exercise on DAY 1 reduced VO2 on DAY 2
4. Submaximal exercise on DAY 1 postural tachycardia in 1/3rd of CFS
5. Exercise worsens orthostatic intolerance (symptoms)
6. MRI before exercise on DAY 1 will show good brain function
MRI after exercise on DAY 2 will show bad brain function
MRI DAY 1 DAY 2 MRI LPQ H&P
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MRI - Exercise Study to Model Exertional Exhaustion
HYPOTHESES:
1. CFS have depression
2. CFS have tenderness (systemic hyperalgesia)
3. Submaximal exercise on DAY 1 reduced VO2 on DAY 2
4. Submaximal exercise on DAY 1 postural tachycardia in 1/3rd
5. Exercise worsens orthostatic intolerance
6. MRI before exercise on DAY 1 will show good brain function
MRI after exercise on DAY 2 will show bad brain function
7. Cerebrospinal fluid contains CFS biomarkers
MRI DAY 1 DAY 2 MRI LPQ H&P
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Cerebrospinal Fluid for Cytokines
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Cerebrospinal Fluid for Metabolomics
• Untargeted “Discovery”• 17 positive mode and 22 negative mode peaks between sedentary control,
CFS and GWI groups at Nonexercise and Post-Exercise• None identified in databases or by hand sequencing
• Targeted Biocrates 180 analytes• 2 significantly different
• C5-OH (C3-DC-M)• PC ae C44:4
Pearson Correlations
Positive Mode
Negative Mode
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Unknowns in LC-MS-MS
Positive Mode: Pooled Nonexercise (white) vs. Post-exercise (grey)Acrolein suspected Positive Mode @ 456 sec
18 Negative Mode Peaks at 249 sec
e.g. M460T249nm
Distinguish CFS from GWI in Nonexercise specimens
Mean ± SD
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No exercise Post-exerciseTargeted Metabolomics
Control
CFS
GWI
GWISTART
GWISTOPP
Control
Exercise
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Lumbar Puncture Cerebrospinal fluid miRNA
DNA genome & genes
pre-mRNA
Spliceosome & Processing
Mature mRNA miRNA~22 ntd
Mature mRNAmiRNA
miRNA binds mRNAmRNA destroyedNo proteinTranslate mRNA
into protein
miRNAs fine tuneprotein expression
In general, miRNAs were reduced
after exercise
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sc0>SC SC>sc0 gwi0>cfs0 gwi0>START START>gwi0 gwi0>STOPP cfs0>CFS
33 targets 224 targets targets? 23 targets 36 targets 48 targets 61 targets
miR-328
miR-608
miR-425-3p
miR-30d-5p
miR-204-5p
miR-1180
miR-328
miR-608
miR-200a-5p
miR-93-3p
let-7i-5p
miR-425-3p
miR-328
miR-608
miR-200a-5p
miR-93-3p
let-7i-5p
miR-328
miR-608
miR-200a-5p
miR-93-3p
miR-92a-3p
Lumbar Puncture Cerebrospinal fluid miRNA DIANA for gene targets
No Exercisesc0 = sedentary controls
cfs0 = CFS no exercisegwi0 = GWI with no exercise
Post Exercise – After DAY2 ExericseSC = sedentary controls
CFS = CFSSTART = GWI Stress Test Activated Reverisible TachycardiaSTOPP = GWI Stress Test Originated Phantom Perception
In general, miRNAs were equivalent within No Exercise and Post-Exercise specimensmiRNAs were higher before exercise and (cfs0>CFS, gwi0>START, gwi0>STOPP)
[except for sc0>SC and SC>sc0]
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# GO Category p-value Genes #
miRNA
1 nucleoplasm (GO:0005654) 8.19E-05 8 4
2 ribonucleoprotein complex
(GO:0030529)
0.00067 5 4
3 organelle (GO:0043226) 0.00067 16 4
4 mitotic cell cycle (GO:0000278) 0.0020 4 3
5 cytosol (GO:0005829) 0.0020 9 4
6 cellular component assembly
(GO:0022607)
0.0047 6 4
7 gene expression (GO:0010467) 0.0055 4 4
8 RNA binding (GO:0003723) 0.0056 7 4
9 cellular nitrogen compound
metabolic process (GO:0034641)
0.0056 10 4
10 exoribonuclease activity,
producing 5'-phosphomonoesters
(GO:0016896)
0.0093 1 3
11 cytoplasmic ribonucleoprotein
granule (GO:0036464)
0.0093 2 4
12 DNA replication initiation
(GO:0006270)
0.0095 2 3
13 ATP-dependent chromatin
remodeling (GO:0043044)
0.0095 2 4
# KEGG pathway p-value #
genes
#
miRNA
1 Fatty acid
biosynthesis (hsa00061)
3.12E-36 1 2
2 Fatty acid
metabolism (hsa01212)
2.46E-13 2 2
3 Adherens junction (hsa04520) 0.000256 7 4
4 Lysine degradation (hsa00310) 0.000485 4 3
5 Transcriptional misregulation
in cancer (hsa05202)
0.000515 7 4
6 Viral myocarditis (hsa05416) 0.003162 7 4
7 Cell cycle (hsa04110) 0.005299 11 4
Combinations of miRNAs DIANA Intersection of Pathways Weighted Targets
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# Modules of target proteins with linkers Top pathway for each moduleCy0 ACTB, CDC42, ELK4, FASN, FYN, IGF1R, IQGAP1, PTPRJ, SETD7, ZNF703
(Linkers: CCT8, CSNK2A2, CTNNB1, MAPK3, STAT3)Adherens junction
Cy1 BAG6, DYRK1A, EWSR1, H3F3B, KMT2D, SCD, SIN3A(Linkers: EP300, HIST1H4A, RXRA)
Transcriptional misregulation in cancer
Cy2 DDX5, HNRNPC, NCL, PCBP2, PRPF8, SCARB2, TCF3(Linker: HSPA8)
Processing of Capped Intron-Containing Pre-mRNA (Reactome)
Cy3 ASH1L, DCP2, DYNC1H1, SPOPL, TGFBR1, (Linkers: SUFU, UBC)
Beta5 beta6 beta7 and beta8 integrin cell surface interactions (NCI PID)
Cy4 CCND2, CCNE2, MCM7, RB1 Cell cycle (KEGG)
miRNA DIANA target genes pathways plausible druggable targets and drugs
Ingenuity Pathway Analysis (IPA)
Cytoscapemodules
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MRI - Exercise Study to Model Exertional Exhaustion
HYPOTHESES:
1. CFS have depression
2. CFS have tenderness (systemic hyperalgesia)
3. Submaximal exercise on DAY 1 reduced VO2 on DAY 2
4. Submaximal exercise on DAY 1 postural tachycardia in 1/3rd of CFS
5. Exercise worsens orthostatic intolerance
6. MRI before exercise on DAY 1 will show good brain function
MRI after exercise on DAY 2 will show bad brain function
7. Cerebrospinal fluid will contain CFS biomarkers
MRI DAY 1 DAY 2 MRI LPQ H&P
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Provocation Testing in CFS/ME
• Common Data Elements to share data between sites
• Evidence-based definitions for CFS, ME, SEID, GWI, FM, depression
• Systemic hyperalgesia in CFS and GWI
• Post-Exertional Malaise / Exertional Exhaustion• Model reveals pathophysiological changes induced by exertion• Unclear if VO2max is changed or diagnostic
• Orthostatic Intolerance (symptoms) vs. Orthostatic Instability (HR)• Recumbent Dizziness “Persistent Orthostatic Intolerance”• Exercise makes orthostatic symptoms worse• POTS and START (ΔHR) are not relevant to symptoms in the broad group of CFS
• Cerebrospinal Fluid• Metabolomics analytes are significantly changed but not identified• miRNA Informatics Infer pathways Mechanisms?
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Thank you for the invitation, opportunity,
and fellowship
James N. Baraniuk MD
Georgetown University, Washington DC