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Eva‐Maria Ratai
Department of Radiology / Neuroradiology
Athinoula
A. Martinos
Center for Biomedical Imaging
MGH / Harvard Medical School
Brainmap
Seminar ‐
October 13th
2010
HIV infection/AIDS
Worldwide around 33 million people were living with HIV at the end of 2008
In the US ~1 Million people have been infected with HIV375,000 people have died from the diseaseHIV infection/AIDS is a leading cause of death for young adults in the US
Estimated HIV/AIDS prevalence among young adults
http://en.wikipedia.org/wiki/File:HIV_Epidem.png
Estimated number of People living with HIV/AIDS
http://en.wikipedia.org/wiki/File:People_living_with_HIV_AIDS_world_map.PNG
HIV Time Course
http://en.wikipedia.org/wiki/File:Hiv‐timecourse.png
NeuroAIDS or AIDS Dementia Complex (ADC)
or HIV‐associated Dementia (HAD)
First defined as a clinical complication of AIDS in 1986
The clinical manifestations cognitive impairment
loss of motor function
behavior deficits
and physical symptoms
20% ‐30% of HIV‐infected patients developed mild neurocognitive disorder and 10‐20% developed frank dementia
Navia
et al, Ann Neurol 1996
Pathology
Within days, the virus can be detected in the brain
Pathological findings Monocytes are infected by the virus
infiltration of monocyte‐derived macrophages into the brain
inflammation: HIV encephalitis (HIVE)
microgliosis
reactive astrocytosis
neuronal atrophy and loss
NeuroAIDS in the ART era
ART has reduced the incidence of HADLess severe manifestations of the disease persist Its prevalence is again increasing as patients have a longer life expectancy As the virus gains resistance to ART, the incidence of severe neurodegeneration will increase The problem is exacerbated by the limited penetration of antiretroviral agents into the CNS Development of effective therapies for HIV related CNS disease is hindered by incomplete understanding of the pathogenesis
Nath
et al. Int
Rev Psychiatry 2008; McArthur et al. J Neuroimmunol
2004;Sacktor
et al. J Neurovirol
2002
Neuropathogenesis – Trojan Horse Mechanism
Adapted from Kaul
et al. Nature 2001
HIV Entry into the Brain and the Role of the Periphery
ART has reduced the incidence of CNS diseaseControlling HIV replication in the periphery limits the development of HIV dementia
HIV dementia typically does not present before onset of AIDS
→ A threshold level of virus is required to develop disease
Factors in the periphery are important triggers leading to dementia
CD14+CD16+ monocytes are predictive of HADPulliam et al. Lancet 1007
Potential Routes of Treatment
Based on this mechanism there are three approaches by which AIDS in the brain can be prevented:
1) reduce the virus in the plasma using antiretroviral therapy (ART)
2) prevent HIV from entering the brain, and/or
3) use adjunct therapies that act in the brain to provide neuroprotection
How do we monitor neuronal injury or neuroprotection?
Neuroimaging techniques MRI and opportunistic infections
Toxoplasmosis
Progressive multifocalleukoencephalopathy
(PML) Primary CNS Lymphoma
MRI and neuroAIDS
Morphological alterations Hyperintensities in white matter (and basal ganglia) on T2 weighted images
Cortical atrophy at later stages of the disease
Functional imaging (MRS, DTI and pMRI) can reveal abnormalities before structural atrophy or focal CNS lesions are visible
HIVencephalitis
Normal
MR SpectroscopyBiochemical information
Representative MR spectrum of a normal human brain @ 3T NAA
Glu/Gln
ChoCr
MI Lipids & Macromolecules
Detectable metabolitesN‐Acetyl Aspartate (NAA) at 2 ppm: produced in neurons, majority found in neurons in the adult brain
Marker of neuronal density and viability
Osmolyte, Precursor of NAAG, Source of acetate for myelin lipid synthesis, facilitates energy metabolism in neuronal mitochondria
↓ NAA (NAA/Cr) in patients with advanced neurocognitive symptoms
Choline‐containing compounds (Cho) at 3.2 ppm: related to cell/lipid membrane metabolism
↑ Cho/Cr in HIV‐infected patients is possibly associated with an immune response that includes cerebral inflammation or gliosis
Myo‐inositol (MI) at ~3.6 ppm: osmolyte located primarily in glia, ↑ in MI/Cr ‐ a marker of gliosis or inflammation
Tracey, et al.
Neurology 1996
*
*
Cho
Before NAA
Chang & ErnstChang at al. 1999 Neurology
Frontal white matter
Healthy control subject
HIV+ minor
motor cognitive disorder
HIV+ mild dementia
HIV+ moderate
dementia
Animal ModelsGreater flexibility to explore the questions concerning HIV neuropathogenesis in a controlled manner and to monitor treatment response
FIV infection of cats
Transgenic HIV mice models and SCID mice models [injection of HIV‐1 infected macrophages into the brains of severe combined immunodeficient mice]
SIV macaque models
SIV Macaque Model of NeuroAIDS
Closest relative to HIV
Infects CD4+ macrophages, lymphocytes & microglia
AIDS similar to humans
Same neuropathology SIVE = HIVEAccumulation of viral-laden perivascular macrophages and multinucleated giant cells, astrogliosis, microgliosis, and neuronal injury1H MRS changes are very similar to those found in humans with HIV-associated neurocognitive disorders
To study the acute phase of infection15 animals were inoculated with SIVmac251
MRI and 1H‐MRS were performed on a clinical 1.5 T GE Scanner and monitored for the first month of infection
MR spectra were obtained from the frontal cortex, white matter semiovale and basal ganglia with a voxel size of 3.4 cm3 using a PRESS sequence (TE/TR = 35/3000)
Spectra were processed off‐line using a SAGE‐GE
SIV Macaque in vivo
MRS: Acute Changes
Greco et al. MRM 2004; Ratai et al. BMC Neuroscience 2009
↓
NAA/Cr at 13 dpi↑
Cho/Cr at 11 dpiand ↓
at 27 dpi↑
MI/Cr at 11,13 and 25 dpi
0 5 10 15 20 25 30101
102
103
104
105
106
107
Pla
sma
Vira
l Loa
d (E
q./m
L)
Days Post Infection
MRS changes in frontal cortex (FC)Plasma Viral Loads
0 5 10 15 20 25 30
-0.2
-0.1
0.0
0.1
0.2
0.3 *
*
**
% c
hang
es M
etab
olite
/Cr
Days Post Infection (dpi)
NAA/Cr Cho/Cr MI/Cr
*
Peak viremia
at 11 dpi
Synaptophysin(presynaptic
integrity)
Altered synaptic transmission
Cresyl Violet(stains neurons)
Control SIV 14 days pibar represents 25 microns
IHC: Acute Infection – neuronal dysfunction
160 180 200 220 240 260 280 300 320 3400.5
0.6
0.7
0.8
0.9
1.0
1.1
NA
A/C
r Mea
sure
d Ex
-viv
o
Acute Infection: Correlation between NAA/Cr and SYN
rs = 0.72 p = 0.013
Lentz et al, Radiology 2005
IHC: Acute Infection –
astroglial activation
↑ Glial fibrillary acidic protein (GFAP) are indicative of astrogliosisPeaks of all three measures were attained at 11–12 days, coincident with peak viremia.
0.45
0.5
0.55
0.6
0.65
0.7
0.75
0.8
0.85
120
140
160
180
200
220
240
260
280
-5 0 5 10 15 20 25 30
MI/Cr In vivoCho/Cr In vivo GFAP
Met
abol
ites
Mea
sure
d by
In v
ivo
1H M
RSG
FAP
Days Post Infection (DPI)
Changes in GFAP and in vivo Cho/Cr and MI/Cr
after SIV infection
Kim et al, AJNR 2005
Conclusions: Acute Changes –
traditional Model
↑ Cho and MI may reflect inflammation microglial/astroglial activation
↓ NAA, evidence of neuronal injury Following immunological control of viremia, decline in Cho to levels below baseline
Monitored animals longitudinally (2 years) ‐ variable metabolic response over time course of infection due low incidence of SIVE
The accelerated SIV Model of neuroAIDS
Traditional SIV macaque model:~ 25% develop SIVE
progression to terminal AIDS may take several years
To accelerate AIDS progression, CD8+ T lymphocytes were depleted
permits the virus to replicate
85% of persistently CD8-depleted animals develop SIVE
AIDS and SIVE with months
MethodsFour rhesus macaques were inoculated with SIVmac251
CD8 lymphocytes were depleted by IV administration of cM‐T807 at days 6, 8 and 12 pi
MRI/MRS before infection and ~every 2 weeks thereafter until the endpoint of the study (~ 10 wpi).
Blood samples were drawn for plasma and CSF viral load analyses and for flow cytometry and FACsorting
Post mortem pathology and IHC
Results ‐
CD8 depletion viral loads, and clinical findings
All animals were persistently CD8 lymphocytes depleted (> 28days)
-7 0 7 14 21 28 35 42 49 56 63 70101
102
103
104
105
106
107
108
Pla
sma
Vira
l Loa
d (E
q./m
L)
Days Post Infection
Plasma VL
Within 10 weeks of infection –AIDS
Histopathological examination revealed severe SIVE
δ
/ ppm
SIV infection + CD8 depletion results in rapid neuronal injury (NAA/Cr)
0 20 40 60 801.00
1.05
1.10
1.15
1.20
1.25
1.30
1.35
1.40
**N
AA
/Cr
FC
Days Post Infection
NAA/Cr in FC
Before infection 10 week post infection
ANOVA: p = 0.02
NAA/CrFrontal Cortex
In vivo MRS pre & 10 wks pisingle macaque
Williams et al, Journal of Clinical investigation 2005
Neuronal injury confirmed post mortem
4 controls vs.4 SIV+/CD8-
(~10 wks pi)
C and D: Cresyl
violetsevere neuronal cortical damage
E and F: MAP-2 (marker for post synaptic integrity)decreased MAP-2 expression
Controls SIV+ CD8-
Expansion and Infection of CD14+CD16+ monocyte
subsets
Normally, majority of monocytes are CD14+CD16‐ and 10% CD16+
Following viral infection and inflammation # of activated monocytes(CD14+CD16+ ) increases
These CD14+CD16+ and CD14loCD16+ monocyte subset traffic the virus across the BBB
CD14loCD16+
0 10 20 30 40 50 600
10
20
30
40
50
60
70
80
***
****
**
% o
f Mon
ocyt
es
Days Post Infection
CD14+ CD16+ CD14lo CD16+**
CD68+
CD16+
SIVnef
Immunohistochemistry
Perivascular macrophages are activated and CD16+
SIV+/CD8- Uninfected control
Potential Routes of Treatment
Based on this mechanism there are three approaches by which AIDS in the brain can be prevented:
1) reduce the virus in the plasma using antiretroviral therapy (ART)
2) prevent HIV from entering the brain, and/or
3) use adjunct therapies that act in the brain to provide neuroprotection
Combination ART in Accelerated AIDS Model
•
4 animals underwent combination ART at 4 weeks pi
•
cART
consisted of PMPA and RCV that do NOT
penetrate CNS
•
PMPA: (R)‐9‐(2‐phosphonylmethoxypropyl) adenine
•
RCV: racemic
β‐2′,3′‐dideoxy‐5‐fluoro‐3′‐thiacytidine
SIV+, CD8‐
Macaque Viral Load Without and With CART
Plasma viral load dropped 1‐2 log units following cART therapy on day 28 post infection
-10 0 10 20 30 40 50 60 70101
102
103
104
105
106
107
108
Vira
l Loa
d (C
opy
eq./m
L)
Days Post Infection
Untreated cART tretaed
cART
0 10 20 30 40 50 600
5
10
15
20
25
30
35
cART
CD
14+C
D16
+Days Post Infection
Untreated cART treated
Reduction in CD14+CD16+ monocytes with CART
Plasma Viral Load Activated Monocytes
Neuronal Recovery with cART
CART results in reversal of neuronal injury
0 10 20 30 40 50 601.00
1.05
1.10
1.15
1.20
1.25
1.30
1.35
1.40
CART
NA
A/C
r FC
Days Post Infection
NAA/Cr (FC) Untreated NAA/Cr (FC) Treated with CART
FC WM BG0.6
0.8
1.0
1.2
1.4
1.6
1.8
p = 0.015
p = 0.04
NAA
/Cr
Untreated Treated
NAA/Cr levels in the frontal cortex NAA/Cr in treated and untreated cohorts at last scans before sacrifice
CD68+
CD16+
SIVnef
Immunohistochemistry with cARTSIV+/CD8- Uninfected control
SIV+/CD8-cART
CART decreases virus burden in brain
Frontal Cortex
Real time RT-PCR
Annamalai
et al, Am J Pathol. 2010
Putamen
Neuronal Recovery by CART is Accompanied by Reversal of Astrocytosis
IHC supports CART’s ability to reverse of neuronal injury and astrocytosis
Ratai et al., ISNV, Miami 2009
SYN MAP-2 GFAP0
50
100
150
200
250
300
350
**
**
*
Opt
ical
Den
sity
per
mm
2
Uninfected CD8- Controls SIV+/CD8- @ 4 wpi SIV+/CD8- @ 8 wpi CART
*
Conclusions
Activated CD14CD16 monocytes traffic virus into the CNSEven non-CNS penetrating drugs control viral infection and prevent activation of monocytes, thereby reducing neuronal injuryThis provides explanation why AZT (even inflectional therapies) reduced presence of dementia so quickly.
Potential Routes of Treatment
Based on this mechanism there are three approaches by which AIDS in the brain can be prevented:
1) reduce the virus in the plasma using antiretroviral therapy (ART),
2) prevent HIV from entering the brain, and/or
3) use adjunct therapies that act in the brain to provide neuroprotection
Minocycline
Minocycline (MN), a well-tolerated, inexpensive, anti-inflammatory, tetracycline-type antibiotic crossed BBB
Advantageous effects against inflammation, microglial activation, apoptotic cell death, and viral production in an accelerated macaque model of neuroAIDS
Zink et al, JAMA 2005
ObjectiveMinocycline’s ability to prevent neuroAIDS
1. Neuroprotective effect: in vivo MRS NAA/Cr and post mortem IHC Synaptophysin and Microtubule-associated protein 2 (MAP2)
2. Glial activation: IBA1 (calcium binding adaptor molecule 1) and Glial fibrillary acidic protein (GFAP), markers for microglial and astroglial activation
3. Anti-viral effects in blood, CSF and brain by PCR
4. CD14CD16 monocyctes (responsible for viral trafficking into CNS)
Minocycline’s Neuroprotective effect
Inhibiting the activation of microglia, inhibits inflammation
11 rhesus macaque studied until 8 weeks post infection (wpi) 7 animals minocycline (4 mg /kg orally) starting 4 wpi
MRS, 3 Tesla PRESS (TE/TR=30/2500) in 4 brain regions PC, FC, BG and WM (LC Model)Flow Cytometry
4 wpi 8 wpi
SIV+/ CD8-Untreated
SIV+/ CD8-MN treated 4 wks MN
MRS MRS MRS MRS MRS MRSSacrifice
Sacrifice
4
7SIV+CD8-
Study Design
CD8 T lymphocyte Depletion and Cohort Designation
-10 0 10 20 30 40 50 60
0.0
5.0x104
1.0x105
1.5x105
2.0x105
2.5x105
CD
8 ly
mph
ocyt
e co
unt
DPI
M5207 M5407 M7207 M1308 M7307 M1508 M1608 M3408 M7407 M7507 M1408
SIV+CD8-
Untreated
MN long-term CD8
MN short-term CD8
Anti CD8
Viral burden
Ratai et al. PLoS
ONE 2010
Minocycline treated animals reveal higher NAA/Cr at their last scans
Neuroprotection Confirmed by quantitative Neuropathology
* *
Minocycline normalizes astrogliosis
and ameliorates microgliosis
GFAP: marker of astrogliosis IBA-1: marker for microglial activation
*P = 0.07
Minocycline reduces activation of monocytes
Activated CD14+CD16+ monocyte traffic virus into the CNS
Campbell et al. Journal of Infectious disease 2010 submitted
0 10 20 30 40 50 60 70
0
100
200
300
400
500
600
700CD14+CD16- Monocytes
Abs
. CD
14+C
D16
- Mon
ocyt
es /
uL
Days Post Infection
MN
Minocycline reduces activation of monocytes
Activated CD14+CD16+ monocyte traffic virus into the CNS
Campbell et al. Journal of Infectious disease 2010 submitted
0 10 20 30 40 50 60 70
0
50
100
150
200
250
300
350Activated CD14+CD16+ Monocytes
Abs
. CD
14+C
D16
+ M
onoc
ytes
/ uL
Days Post Infection
MN0 10 20 30 40 50 60 70
0
20
40
60
80
100Activated CD14loCD16+ Monocytes
Abs
. CD
14lo
CD
16+
Mon
ocyt
es /
uL
Days Post Infection
MN
Activated monocytes
correlate inversely with neuronal marker NAA/Cr
R = -0.72p = 0.001
For the other one
R = -0.71p = 0.0006
Viral burden in the CNS
4
5
6P=0.0057
P=0.050
VL Brain (FC)
Untreated MN(short-term)
MN(persist.)
Log
VL
Bra
in[c
opie
s / g
]
4.0 4.5 5.0 5.5 6.070
75
80
85
90
95
100
105
110
115
Untreat 8wk Untreat 6wk MN pers depl MN short-term depl
Log VL Brain (FC) (copies/g)
P=0.0008R
ρ=-0.77
NA
A/C
r FC
(%)
Correlation between NAA/Cr (FC) and Brain VL (FC)
Conclusions
Minocycline was found to be neuroprotectivePossible mechanisms towards MN’s neuroprotection:
1) reduction of inflammatory response by downregulationof glial cell activation in the brain2) reductions of plasma, CSF and brain viral burden3) a reduction in a subset of monocytes considered to be responsible for viral infection of the CNS by cell trafficking mechanisms
Conclusions
Better profile for all neuro-inflammation markers in the short-term depleted animals that had also lower plasma viral load
Best strategy to treat neuroAIDS is by the use of antiretroviral therapy in combination with minocycline.
Future Studies
How does MN reduce monocyte activation?Mechanistic Studies
Prevent HIV from entering the brainNext experiment: use anti‐VLA‐4 antibody to block monocyte/T cell traffic into CNS
Move to high‐resolution MRSI and (higher field strength)
Increased Signal to Noise Ratio at higher Magnetic Field Strengths
fieldmagneticBratioicgyromagnet
SpinsofNumberN
BNSignal
V
V
::
:
0
02
γ
γ ⋅⋅∝
Linear increase in signal with field strengthif T1 and T2 relaxation times,coil and system losses and RF penetration effects, do not change significantly
1.5T
3T
7T
High Resolution MRSI at 7T
VOI = 4 cm
VOI =3.5 cm
VO
I=4
cm
1.5 cmFOV = 6 cm, ×16 CSI
FOV = 6 cm, ×16 CSI
FOV
=6
cm, ×
16 C
SI
×4 H
SI
a b
Gonen at al, MRM 2008
3D MRSI pulse sequences with 2D (CSI) 16 x 16 phase encoding1D Hadamard spectroscopic imaging (HSI) resulting in 4 slabs in zisotropic spatial resolution at (0.375 cm)3=0.05 cm3 in 25 min.
High Resolution MRSI
1.52.02.53.03.5 ppm
NAACho Cr NAA
Cho
Cr6 cm
2.6 cm
3.4
cm0.05 cm3
Resulting in these spectra that still show good SNR at resolution of 0.05
cm3 or 50 μL
axial metabolic maps
O 3
Acknowledgements
Jeffrey P. BombardierRobert FellRobert FullerJane GrecoReza HakimelahiElkan
HalpernJulian HeChan‐Gyu
JooJohn KimVallent
LeeMargaret R. LentzKatharine TurkR. Gilberto González
Lakshman
Annamalai
Angela Carville
Elizabeth Curran
Ronald Desrosier
Mike O’Connell
Shawn O’Neil
Susan V. Westmoreland
Patrick Autissier
Tricia Burdo
Jennifer Campbell
Kenneth C. Williams
Eliezer
Masliah
NIH grants R21NS059331 (EMR), R01NS050041
(RGG), R01NS040237 (KW), R01NS37654 (KW),
R01MH62962 (EM), MH59754 (EM), MH62512 (EM),
and RR00168 (NEPRC), and P41RR14075
Jeffrey LifsonMike Piatak
Keith ReimanR24 RR016001, N01
AI040101
Shannon LuboyeskiElisabeth Moeller Joanne Morris
Oded
GonenSongtao
Liu
