Eva Maria Ratai Department of Radiology / Neuroradiology Athinoula A. Martinos Center for Biomedical Imaging MGH / Harvard Medical School

Transcription

1 Eva Maria Ratai Department of Radiology / Neuroradiology Athinoula A. Martinos Center for Biomedical Imaging MGH / Harvard Medical School Brainmap Seminar October 13 th 2010

2 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 HIV 375,000 people have died from the disease HIV infection/aids is a leading cause of death for young adults in the US

3 Estimated HIV/AIDS prevalence among young adults

4 Estimated number of People living with HIV/AIDS

5 HIV Time Course timecourse.png

6 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

7 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

8 NeuroAIDS in the ART era ART has reduced the incidence of HAD Less 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

9 Neuropathogenesis rojan Horse Mechanism Adapted from Kaul et al. Nature 2001

10 HIV Entry into the Brain and the Role of the Periphery ART has reduced the incidence of CNS disease Controlling 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 HAD Pulliam et al. Lancet 1007

11 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

12 How do we monitor neuronal injury or neuroprotection?

13 Neuroimaging techniques MRI and opportunistic infections Toxoplasmosis Progressive multifocal leukoencephalopathy (PML) Primary CNS Lymphoma

14 MRI and neuroaids Morphological alterations Hyperintensities in white matter (and basal ganglia) on T2 weighted images Cortical atrophy at later stages of the disease HIV encephalitis Functional imaging (MRS, DTI and pmri) can reveal abnormalities before structural atrophy or focal CNS lesions are visible Normal

15 MR Spectroscopy Biochemical information Representative MR spectrum of a normal human 3T NAA Cho Cr MI Glu/Gln Lipids & Macromolecules

16 Detectable metabolites N 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 membranemetabolism 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

17 Cho Before NAA * * Tracey, et al. Neurology 1996

18 Frontal white matter HIV+ moderate dementia HIV+ mild dementia HIV+ minor motor cognitive disorder Healthy control subject Chang & Ernst Chang at al Neurology

19 Animal Models Greater 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

20 SIV Macaque Model of NeuroAIDS Closest relative to HIV Infects CD4+ macrophages, lymphocytes & microglia AIDS similar to humans Same neuropathology SIVE = HIVE Accumulation of viral-laden perivascular macrophages and multinucleated giant cells, astrogliosis, microgliosis, and neuronal injury 1 H MRS changes are very similar to those found in humans with HIV-associated neurocognitive disorders

21 To study the acute phase of infection 15 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 cm 3 using a PRESS sequence (TE/TR = 35/3000) Spectra were processed off line using a SAGE GE

22 SIV Macaque in vivo MRS: Acute Changes Plasma Viral Loads MRS changes in frontal cortex (FC) Plasma Viral Load (Eq./mL) Days Post Infection % changes Metabolite/Cr NAA/Cr Cho/Cr MI/Cr * Days Post Infection (dpi) * * * * Peak viremia at 11 dpi NAA/Cr at 13 dpi Cho/Cr at 11 dpi and at 27 dpi MI/Cr at 11,13 and 25 dpi Greco et al. MRM 2004; Ratai et al. BMC Neuroscience 2009

23 IHC: Acute Infection neuronal dysfunction Synaptophysin (presynaptic integrity) Altered synaptic transmission Cresyl Violet (stains neurons) Control SIV 14 days pi bar represents 25 microns

24 Acute Infection: Correlation between NAA/Cr and SYN 1.1 NAA/Cr Measured Ex-vivo r s = 0.72 p = Lentz et al, Radiology 2005

25 IHC: Acute Infection astroglial activation Changes in GFAP and in vivo Cho/Cr and MI/Cr after SIV infection MI/Cr In vivo Cho/Cr In vivo GFAP Metabolites Measured by In vivo 1H MRS Days Post Infection (DPI) GFAP Glial fibrillary acidic protein (GFAP) are indicative of astrogliosis Peaks of all three measures were attained at days, coincident with peak viremia. Kim et al, AJNR 2005

26 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

27 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

28 Methods Four 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

29 Results CD8 depletion viral loads, and clinical findings All animals were persistently CD8 lymphocytes depleted (> 28days) Plasma Viral Load (Eq./mL) Plasma VL Days Post Infection Within 10 weeks of infection AIDS Histopathological examination revealed severe SIVE

30 SIV infection + CD8 depletion results in rapid neuronal injury (NAA/Cr) Before infection 10 week post infection NAA/Cr Frontal Cortex NAA/Cr in FC NAA/Cr FC * * δ / ppm In vivo MRS pre & 10 wks pi single macaque Days Post Infection ANOVA: p = 0.02 Williams et al, Journal of Clinical investigation 2005

31 Neuronal injury confirmed post mortem Controls SIV+ CD8- C and D: Cresyl violet severe neuronal cortical damage E and F: MAP-2 (marker for post synaptic integrity) decreased MAP-2 expression 4 controls vs. 4 SIV+/CD8- (~10 wks pi)

32 Expansion and Infection of CD14+CD16+ monocyte subsets CD14loCD ** ** ** CD14+ CD16+ CD14lo CD16+ ** % of Monocytes * * * Days Post Infection 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

33 Immunohistochemistry SIV+/CD8- Uninfected control CD68+ CD16+ SIVnef Perivascular macrophages are activated and CD16+

34 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

35 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

36 SIV+, CD8 Macaque Viral Load Without and With CART Plasma Viral Load Activated Monocytes Viral Load (Copy eq./ml) cart Untreated cart tretaed CD14+CD cart Untreated cart treated Days Post Infection Days Post Infection Plasma viral load dropped 1 2 log units following cart therapy on day 28 post infection Reduction in CD14+CD16+ monocytes with CART

37 Neuronal Recovery with cart NAA/Cr levels in the frontal cortex NAA/Cr FC NAA/Cr (FC) Untreated NAA/Cr (FC) Treated with CART CART NAA/Cr in treated and untreated cohorts at last scans before sacrifice NAA/Cr Untreated Treated p = p = Days Post Infection 0.6 FC WM BG CART results in reversal of neuronal injury

38 Immunohistochemistry with cart SIV+/CD8- SIV+/CD8- cart Uninfected control CD68+ CD16+ SIVnef

39 CART decreases virus burden in brain Frontal Cortex Putamen Real time RT-PCR Annamalai et al, Am J Pathol. 2010

40 Neuronal Recovery by CART is Accompanied by Reversal of Astrocytosis Optical Density per mm * * Uninfected CD8- Controls 4 wpi 8 wpi CART * * * * 0 SYN MAP-2 GFAP IHC supports CART s ability to reverse of neuronal injury and astrocytosis Ratai et al., ISNV, Miami 2009

41 Conclusions Activated CD14CD16 monocytes traffic virus into the CNS Even non-cns penetrating drugs control viral infection and prevent activation of monocytes, thereby reducing neuronal injury This provides explanation why AZT (even inflectional therapies) reduced presence of dementia so quickly.

42 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

43 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

44 Objective Minocycline s ability to prevent neuroaids 1. Neuroprotective effect: in vivo MRS NAA/Cr and post mortem IHC Synaptophysin and Microtubuleassociated 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)

45 Minocycline s Neuroprotective effect Inhibiting the activation of microglia, inhibits inflammation

46 Study Design 11 rhesus macaque studied until 8 weeks post infection (wpi) 7 animals minocycline (4 mg /kg orally) starting 4 wpi 4 SIV+/ CD8- Untreated MRS MRS MRS MRS MRS MRS Sacrifice 7 SIV+/ CD8- MN treated 4 wks MN Sacrifice SIV+ CD8-4 wpi 8 wpi MRS, 3 Tesla PRESS (TE/TR=30/2500) in 4 brain regions PC, FC, BG and WM (LC Model) Flow Cytometry

47 CD8 T lymphocyte Depletion and Cohort Designation CD8 lymphocyte count 2.5x x x x x10 4 Anti CD8 M5207 M5407 M7207 M1308 M7307 M1508 M1608 M3408 M7407 M7507 M1408 SIV+CD8- Untreated MN long-term CD8 MN short-term CD DPI

48 Viral burden

49 Ratai et al. PLoS ONE 2010

50 Minocycline treated animals reveal higher NAA/Cr at their last scans

51 Neuroprotection Confirmed by quantitative Neuropathology * *

52 Minocycline normalizes astrogliosis and meliorates microgliosis GFAP: marker of astrogliosis IBA-1: marker for microglial activation * P = 0.07

53 Minocycline reduces Campbell et al. Journal of Infectious disease 2010 submitted activation of monocytes Activated CD14+CD16+ monocyte traffic virus into the CNS Abs. CD14+CD16- Monocytes / ul CD14+CD16- Monocytes MN Days Post Infection

54 Minocycline reduces Campbell et al. Journal of Infectious disease 2010 submitted activation of monocytes Activated CD14+CD16+ monocyte traffic virus into the CNS Abs. CD14+CD16+ Monocytes / ul Activated CD14+CD16+ Monocytes MN Days Post Infection Abs. CD14loCD16+ Monocytes / ul Activated CD14loCD16+ Monocytes MN Days Post Infection

55 Activated monocytes correlate inversely with neuronal marker NAA/Cr For the other one R = p = R = p =

56 Viral burden in the CNS Log VL Brain [copies / g] Untreated VL Brain (FC) P=0.050 MN (persist.) P= MN (short-term) NAA/Cr FC (%) Correlation between NAA/Cr (FC) and Brain VL (FC) Untreat 8wk Untreat 6wk MN pers depl MN short-term depl Log VL Brain (FC) (copies/g) P= R ρ =-0.77

57 Conclusions Minocycline was found to be neuroprotective Possible mechanisms towards MN s neuroprotection: 1) reduction of inflammatory response by downregulation of glial cell activation in the brain 2) reductions of plasma, CSF and brain viral burden 3) a reduction in a subset of monocytes considered to be responsible for viral infection of the CNS by cell trafficking mechanisms

58 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.

59 Future Studies How does MN reduce monocyte activation? Mechanistic Studies Prevent HIV from entering the brain Next experiment: use anti VLA 4 antibody to block monocyte/t cell traffic into CNS Move to high resolution MRSI and (higher field strength)

60 Increased Signal to Noise Ratio at higher Magnetic Field Strengths Signal N V 2 γ B 0 1.5T N V : Number of Spins γ : gyromagnetic ratio B : magnetic field 0 3T Linear increase in signal with field strength if T1 and T2 relaxation times, coil and system losses and RF penetration effects, do not change significantly 7T

61 High Resolution MRSI at 7T a b 4 HSI VOI = 4 cm FOV = 6 cm, 16 CSI 1.5 cm FOV = 6 cm, 16 CSI VOI =4 cm VOI =3.5 cm FOV = 6 cm, 16 CSI 3D MRSI pulse sequences with 2D (CSI) 16 x 16 phase encoding 1D Hadamard spectroscopic imaging (HSI) resulting in 4 slabs in z isotropic spatial resolution at (0.375 cm) 3 =0.05 cm 3 in 25 min. Gonen at al, MRM 2008

62 High Resolution MRSI Cho Cr NAA NAA 0.05 cm cm Cr 2.6 cm 6 cm O 3 Cho ppm axial metabolic maps Resulting in these spectra that still show good SNR at resolution of 0.05 cm 3 or 50 μl

63 Higher spectral resolution STEAM: TE/TR=15/3000; NA: 256

64 Acknowledgements Jeffrey P. Bombardier Robert Fell Robert Fuller Jane Greco Reza Hakimelahi Elkan Halpern Julian He Chan Gyu Joo John Kim Vallent Lee Margaret R. Lentz Katharine Turk R. Gilberto González Lakshman Annamalai Angela Carville Elizabeth Curran Ronald Desrosier Mike O Connell Shawn O Neil Susan V. Westmoreland Jeffrey Lifson Mike Piatak Patrick Autissier Tricia Burdo Jennifer Campbell Kenneth C. Williams Eliezer Masliah Oded Gonen Songtao Liu Keith Reiman R24 RR016001, N01 AI Shannon Luboyeski Elisabeth Moeller Joanne Morris NIH grants R21NS (EMR), R01NS (RGG), R01NS (KW), R01NS37654 (KW), R01MH62962 (EM), MH59754 (EM), MH62512 (EM), and RR00168 (NEPRC), and P41RR14075