Transcript Joanne Weinberg

WEINBERG CIFASD DEVELOPMENTAL PROJECT
With Tina Chambers and the CIFASD team
Co-I: Dr. Charlis Raineki
PhD student/Post-doc: Tamara Bodnar
Identifying an immune signature characteristic
of FASD: Implications for possible immunebased intervention strategies
CIFASD MEETING, AUSTIN, TX
MARCH 8-9, 2016
Specific Aim 1

Based on our animal model data, our Aim was to probe for a unique immune signature
(early pro-inflammatory bias?) in children with FASD that may be linked to maternal
immune function
◦ Year 1:
◦ Assay plasma samples from pregnant women for cytokine, C-reactive protein (CRP),
cortisol expression
◦ Assay plasma levels of placental corticotropin releasing hormone (pCRH) – increases
20-40 fold over gestation; critical role in gestation, fetal development, and parturition
- For mom, pCRH may mediate association between early life adversity and
postpartum state (eg., postpartum depression)
- For fetus, critical for development/ fetal programming: CRH levels provide objective
evidence that fetus has been exposed and is responding to maternal stress signals;
role in organization of fetal nervous system and developmental trajectories/outcomes
◦ Year 2:
◦ Assess both plasma (cytokines, CRP, cortisol) and salivary (cortisol, CRP) markers in the
children in this cohort
◦ Parallel measures allow us to determine if there are alcohol-specific alterations in saliva that
can be used as a proxy for those in blood and also provide an index of endocrine-immune
interaction
◦ Investigate whether nutritional supplementation can modulate the expected proinflammatory/endocrine profiles in both mothers and children
Graded increase in rate of pCRH production over the
second half of gestation in women exposed to childhood
trauma vs no trauma – the greater the trauma, the
steeper the rise in CRH
Moog et al., Biol
Psychiatry, 2015
Deliverables for Aim 1, Year 1
Aim 1/ Year 1
Maternal Plasma
Status
Done
Cytokines (40 analytes)
✔
CRP
✔
To do
Cortisol
By 5/31/16
pCRH
By 5/31/16
Data analysis
In progress
Manuscripts based on these data
one on cytokines and stress markers; one
with Rajesh, combined data
Goal: SummerFall, 2016
Abstract submitted to RSA 2016:
Bodnar T., Raineki C., Wertelecki W., Yevtushok L., Zymak-Zakutnya N., Weinberg
J., Chambers C.D., and the CIFASD. Alcohol consumption during pregnancy alters
maternal immune parameters: Implications for the offspring
Deliverables for Aim 1, Year 2
Aim 1/ Year 2
Child Plasma
Cytokines (40 analytes)
CRP
Cortisol
Status
Done
To do
Will begin to run assays as soon
as we have plasma samples from
38 children (1 plate) –
(103 samples recently received
at Davis; aliquots to be sent to
us)
Data analysis
Goals:
Abstract for RSA 2017
Manuscript based on these data
January 2017
Summer-Fall,
2017
Data to date
Maternal plasma: 4 major patterns/findings

Alcohol-consuming moms with affected children different
from alcohol-consuming moms whose children are not
affected, and low-no alcohol consumption group (A vs B+C)

Effect of alcohol consumption per se: alcohol-consuming
moms, regardless of whether child is affected, different from
moms with low to no alcohol consumption (A+B vs C)

Alcohol-consuming mom with unaffected children different
from alcohol-consuming moms with affected children and
moms with low-no alcohol consumption (B vs A+C)

Patterns change over the course of gestation in all groups

A=35; B=22, C=94 @ intake (~second trimester) and 3rd
trimester
Unique immune (cytokine) signatures in
maternal plasma during pregnancy, which are
associated with child outcome
CIFASD second trimester - average
1
Eotaxin.3
IL.12p70
SAA
CRP
Eotaxin
IL.1b
MIP.1a
IL.4
IL.6
IL.5
bFGF
IL.2
IFN.g
TNF.b
TARC
IP.10
sICAM.1
MDC
MIP.1b
MCP.1
IL.10
IL.15
VEGF
VEGF.D
PIGF
IL.8
MCP.4
IL.17A
TNF.a
sVCAM.1
VEGF.C
IL.16
IL.1a
GM.CSF
IL.7
sFlt.1
IL.13
Tie.2
IL.12p40
e
1
A: Alcoholaffected
2
B: Alcoholunaffected
3
C1: No alcoholControl
4
C2: No alcoholAffected
How this project has interacted with
other projects/investigators in CIFASD

Collaboration with Tina Chambers to analyze samples from
mothers and children in CIFASD Ukraine cohorts

Link with Rajesh Miranda to enhance the understanding/reach
/ impact of our findings
• Sample overlap for moms: Group A, 13; Group B, 16; Group C, 18

Links with other data sets based on the Ukraine cohorts
(epigenetics) and other studies (Eberhart) to further enhance
the understanding/reach/impact of our findings
Linking data sets markedly broadens the data base and increases
our ability to achieve the goal of utilizing biological variables,
linked to critical clinical, social, behavioral, etc measures, in the
development of biomarkers of alcohol exposure/child outcomes
One example of the power of integration across
projects within CIFASD – discovery of possible
biomarkers of alcohol consumption/exposure/effects
Rajesh: Pathway Overrepresentation
Analysis: Top 6 predictive miRNAs
KEGG pathway
p-value
#genes
#miRNAs
ECM-receptor interaction
9.77E-10
14
4
Signaling pathways regulating pluripotency of stem cells
8.38E-06
32
6
Mucin type O-Glycan biosynthesis
0.001841561
6
3
PI3K-Akt signaling pathway
0.015396328
54
6
Glycosaminoglycan biosynthesis - heparan sulfate / heparin
0.016306392
6
2
mTOR signaling pathway
0.018986944
16
6
Wnt signaling pathway
0.038492015
23
5
p53 signaling pathway
0.038492015
17
6
Amyotrophic lateral sclerosis (ALS)
0.038492015
13
6
MAPK signaling pathway
0.044578177
40
6
hsa-miR-503-5p
MIMAT0002874
hsa-miR-33a-5p
MIMAT0000091
hsa-let-7a-5p
MIMAT0000062
hsa-miR-17-5p
MIMAT0000070
hsa-miR-10b-5p
MIMAT0000254
hsa-miR-376b-3p MIMAT0002172
mirPath v.3/MicroT-CDS
mTOR pathway integrates signals from neurotransmitters, growth
factors, hormones, cytokines, and stress mediators
- involved in immune cell metabolism, differentiation and function, and more broadly, in brain
development, synaptic pruning
Estes & McAllister, Nature
Reviews Neuroscience, 2015
Chambers et al., Alcohol Clin Exp Res 39:43A, 2015:
mTOR signaling was identified among the 255 differentially
methylated genes in A vs B+C (children with FASD vs no FASD)
•
•
Human gene symbols
– ALDH2
– FANCD2
– ADH1B2
– ADH1B3
– MARS
– CDON
– AGRN
– SHH
– INSR
– PDGFRA
– HINFP
– PLK1
– VANGL2
– FOXD1
– PIK3
– PTEN
– AKT
– mTOR
Differentially methylated genes – AB
–
–
–
•
ALDH8A1
ADH5
FOXK1
Differentially methylated genes – FAS/noFAS
–
–
–
–
–
–
–
–
–
–
–
–
–
ALDH18A1
ALDH8A1
ADH1C
ADH5
FANCI
AGRN (body; hypomethylated in FAS)
INSR (body; hypomethylated in FAS)
PLK1S1
VANGL2 (body; hypomethylated in FAS)
FOXN3, FOXP1, FOXA3, FOXN4, FOXE3, FOXR1
PIK3R5, PIK3AP1, PIK3C2B, PIK3R5, PIK3AP1, PIK3C2B
AKT3 (3’UTR; hypomethylated in FAS)
MTOR (body; hypomethylated in FAS)
McCarthy & Eberhart, Cell Mol Life Sci 71, 2014: Geneethanol interactions underlying FASD: Ethanol
interacts with growth factor signaling via inhibition of
PI3K/AKT/mTOR signaling, which can lead to cell death
What did we gain from Consortium
structure?

This project could not be done as an individual
project without the Consortium
◦ Access to the Consortium has enabled us to translate
questions/hypotheses/findings from our animal model
to unique, well-phenotyped clinical cohorts
◦ Ability to link immune/inflammation findings to clinical,
social, environmental variables in mothers, and
subsequently, outcomes in children (Helen’ talk)
◦ Integration across projects increases ability to identify
critical pathways/mechanisms/markers
◦ Synergies among projects happen naturally within
CIFASD
Novel, innovative aims for Phase
IV of CIFASD
Background

IMPLICATIONS OF A DOHAD FRAMEWORK FAR BEYOND
IMMUNE/HEALTH OUTCOMES
◦
◦
◦
◦
◦

Research on the health outcomes of children with FASD is a neglected area
DOHAD perspective emerging as critical for understanding not only health
and well-being, but also functioning of children, adolescents and young
adults in their daily lives
Immune activation/inflammation/cytokines play a key role in brain
development
Increasing evidence demonstrates that altered immune activation may
underlie altered cognition, attention, behavior, self-regulation, adaptive
functioning
We add a new dimension to CIFASD that increases our understanding of
alcohol’s impacts, provides novel markers that distinguish groups from each
other
DATA WILL PAVE THE WAY FOR THE DEVELOPMENT OF NOVEL IMMUNEBASED INTERVENTIONS
◦
Opportunity for us to extend our work from the bench to the bedside –
critical findings in our animal model provide the basis for the proposed
research and opportunity for preclinical testing of novel interventions
ASD risk factors during pregnancy converge on
FASD
maternal immune system activation
Alcohol ± stress ± …..
Estes & McAllister, Nature
Reviews Neuroscience, 2015
Aim 1: Replication/Validation Cohorts

Replication/validation cohorts with matched mother infant pairs:
Validate predictive value of biomarkers related to neuroimmune profile (cytokines, CRP,
◦
cortisol)


◦
Replicate under different cultural settings/ SES/ environmental stress/ alcohol intake levels/
ethnic make-up – how much do environmental variables modify/modulate outcomes for
mothers and children
◦
Does the immune signature hold in a different environment?
◦
If yes, this speaks to the robustness of the findings.
◦
If no, insight into environmental factors, beyond alcohol, that contribute to the outcomes observed
Ukraine
◦
180 new moms enrolled – have blood at 2 times during pregnancy
◦
Feeder group of kids, now ~ 2 yrs old – can be called back to give blood, saliva, follow up
testing
◦
Measure: cortisol in blood spots at birth; cytokines, CRP, CBG in blood; cortisol, CRP in saliva.
San Diego – Biobank
◦
30 children already diagnosed by Ken Jones currently in registry
◦
Have newborn blood spots (cortisol, cytokines); can be called back for current blood draw,
saliva collection – measures as above – developmental trajectory
◦
Have mid-gestational blood sample on ~80% of mothers
Aim 2: Immune and Stress Markers
in Breast Milk: Implications for Development

Analyze inflammatory markers in breast milk and link to
microbiome in children – further link mother to child outcomes

Enhanced or reduced levels of maternal cytokines may be transmitted to
nursing offspring through the milk, and could thus adversely impact
neurobiological development of the offspring.
Measure immune and stress markers in milk, eg., immunoglobulins,
cortisol, cytokines
Nutritional panel; Include breast fed and formula fed infants (Chambers)
Insight into mechanisms underlying immune signature in alcohol-exposed
children
Role of inflammatory pathways in growth trajectories
Digestive tract of newborn colonized with microorganisms from the
mother and surrounding environment, and markedly influenced by breast
feeding.
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This Aim could integrate/synergize with investigation of child microbiome –
fecal and skin samples (in collaboration with Dr. Rob Knight, UCSD)
Aim 3: Intervention


Goal: Develop novel immune-based interventions that
could be used as adjunctive treatments to improve child
functional/neurobehavioral/health outcomes
To be developed following analysis of blood from alcoholexposed children

Preclinical testing - proof of principle, to provide evidence
for launch of clinical trial
Boy, I don’t know what
they’ve injected me with,
but I feel great!
Aim 3: Intervention (cont’d)

Clinically approved OTC drugs already in use; benefits with minimal risk
◦
Minocycline – broad based antibiotic/microglial inhibitor: In use for > 30 yrs; nonantibiotic biological effects in experimental models of: TBI, ineuropathic pain,
diseases with pro-inflammatory bias (autoimmune [RA, inflammatory bowel disease],
dermatitis, periodontitis)
◦
◦
Ibudilast – Type IV-phosphodiesterase inhibitor, anti-inflammatory; crosses BBB and
modulates glial cell activity; suppresses release of cytokines and other inflammatory
signals, and inhibits the production of reactive oxygen species; neuroprotective
◦
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Open label pilot trial in 25 children with Angelman Syndrome, clinical and neuropsychological measures
suggest drug is well-tolerated, improved adaptive behavior
Maternal immune activation (MIA) models of autism, schizophrenia: postnatal treatment of MIA offspring
prevented synaptic (decr number, turnover dendritic spines) and behavioural (repetitive behaviours)
impairments
Shown to decrease alcohol drinking (alcohol-preferring rat model) – supports viability of as possible
treatment for alcohol dependence
Indomethacin – NSAID, inhibits prostaglandins: indicated as analgesic in certain
conditions; anti-inflammatory (juvenile arthritis, bursitis, arthritic gout, rheumatoid
arthritis)
Administration during pregnancy (early brain development), lactation (third
trimester equivalent thru toddler/young childhood), early and late adolescence
Measure immune, cognitive, behavioral, adaptive outcomes:
◦
◦
◦
Response to LPS (inflammatory challenge)
Learning and memory (eg., Morris water maze, T-maze [working memory)
Vulnerability to depression/anxiety (EPM, FST, novelty-suppressed feeding)
Additional areas of interest

Placenta, cord blood from new Ukraine cohort
◦ Pilot studies ongoing on placenta, cord blood collection, storage,
shipping from Khmelnytsky
◦ We could contribute to proposed research


◦
Link mother to child in this cohort
◦
Measure 11β-HSD1, 11β-HSD2, and glucocorticoid receptors (GR) – role in fetal
programming
◦
Cytokines in cord blood
Of particular interest, partner with other CIFASD investigators who
may be interested in analyzing placental tissue to broaden the
analysis.
Link our findings to genetic/epigenetic analyses from Division of
Genome Information Sciences at UCSD – stress and inflammatory
pathway genes in placenta and cord blood cells - relate to
outcomes/vulnerability/resilience
3 Priority Areas in FASD Research

Early diagnosis:
◦ The earlier an individual is diagnosed, the earlier we can intervene to improve
outcome. We need to develop better tools for early assessment and diagnosis.
These could be behavioral, neurobiological, genetic/epigenetic, etc or involve a
combination of approaches. What have we learned from all of the current
CIFASD approaches (face/brain/environment) that can help move us forward.

Along these same lines, a particular need is ability to distinguish ARND
from other neurodevelopmental disorders.
◦

Can we develop a more specific profile for ARND, encompassing a variety of
domains, that will help with early diagnosis.
Early intervention:
◦ Progress already being made on this front, but more needed.
◦ How can we intervene to improve outcomes for affected children
◦ A combination of approaches (neurobiological, behavioral, environmental, etc)
is probably the most powerful way to go.