Brainmap Seminar October 13 2010 - Center for Biomedical...

transcript

Eva‐Maria Ratai

Department of Radiology / Neuroradiology

Athinoula

A. Martinos

Center for Biomedical Imaging

MGH / Harvard Medical School

Brainmap

Seminar ‐

October 13th

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

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

et al. Int

Rev Psychiatry 2008; McArthur et al. J Neuroimmunol

2004;Sacktor

et al. J Neurovirol

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

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

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

Days Post Infection

MRS changes in frontal cortex (FC)Plasma Viral Loads

0 5 10 15 20 25 30

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

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.

-5 0 5 10 15 20 25 30

MI/Cr In vivoCho/Cr In vivo GFAP

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

Days Post Infection

Plasma VL

Within 10 weeks of infection –AIDS

Histopathological examination revealed severe SIVE

SIV infection + CD8 depletion results in rapid neuronal injury (NAA/Cr)

0 20 40 60 801.00

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

Days Post Infection

CD14+ CD16+ CD14lo CD16+**

SIVnef

Immunohistochemistry

Perivascular macrophages are activated and CD16+

SIV+/CD8- Uninfected control

1) reduce the virus in the plasma using antiretroviral therapy (ART)

Combination ART in Accelerated AIDS Model

4 animals underwent combination ART at 4 weeks pi

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

Days Post Infection

Untreated cART tretaed

0 10 20 30 40 50 600

+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

Days Post Infection

NAA/Cr (FC) Untreated NAA/Cr (FC) Treated with CART

FC WM BG0.6

p = 0.015

p = 0.04

Untreated Treated

NAA/Cr levels in the frontal cortex NAA/Cr in treated and untreated cohorts at last scans before sacrifice

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

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.

1) reduce the virus in the plasma using antiretroviral therapy (ART),

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

7SIV+CD8-

Study Design

CD8 T lymphocyte Depletion and Cohort Designation

-10 0 10 20 30 40 50 60

5.0x104

1.0x105

1.5x105

2.0x105

2.5x105

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

700CD14+CD16- Monocytes

Days Post Infection

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

350Activated CD14+CD16+ Monocytes

Days Post Infection

MN0 10 20 30 40 50 60 70

100Activated CD14loCD16+ Monocytes

Days Post Infection

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

6P=0.0057

P=0.050

VL Brain (FC)

Untreated MN(short-term)

MN(persist.)

4.0 4.5 5.0 5.5 6.070

Untreat 8wk Untreat 6wk MN pers depl MN short-term depl

Log VL Brain (FC) (copies/g)

P=0.0008R

ρ=-0.77

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

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

High Resolution MRSI at 7T

VOI = 4 cm

VOI =3.5 cm

1.5 cmFOV = 6 cm, ×16 CSI

FOV = 6 cm, ×16 CSI

cm, ×

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

Cr6 cm

2.6 cm

cm0.05 cm3

Resulting in these spectra that still show good SNR at resolution of 0.05

cm3 or 50 μL

axial metabolic maps

Higher spectral resolution

STEAM: TE/TR=15/3000; NA: 256

MRS@7.0T

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

GonenSongtao

Brainmap Seminar October 13 2010 - Center for Biomedical...

Documents