Download HIV-1 infected macrophage

Macrophage Delivery of Nanoformulated
Antiretroviral Drug to the Brain in a
Murine Model of NeuroAIDS
Huanyu Dou, Cassi B. Grotepas, JoEllyn M. McMillan, Christopher J. Destache, Mahesh
Chaubal, Jane Werling, James Kipp, Barrett Rabinow, and Howard E. Gendelman
The Journal of Immunology
Macrophage


Derived form mononuclear phagocyte (單核吞噬細胞)
Triggered by a range of stimuli including
damaged cells, pathogens and cytokines released by
macrophages already at the site
HIV-associated neurocognitive disorders
HIV Infects primarily vital cells in the human immune system
----- helper T cells, macrophages, and dendritic cells

NeuroAIDS
----- Loss of neurons
----- Astrogliosis (星細胞增生)
----- Neuroinflammation & Encephalitis (腦炎)
----- Microgliosis (小膠質細胞增生)
----- Infiltration of macrophage
----- Formation of multinucleated giant cell (MGC,多核巨細胞)


Cognitive, motor and behavioral impairments
What’s ART?
ART= Antiretroviral Therapy (抗逆轉錄病毒療法)
----- reduce HIV-associated neurocognitive disorder
severity

Current ART limitations:
----- inabilities to combat viral mutation
----- inabilities to achieve continuous, effective drug levels
in virus target tissues
----- HIV-1 levels can rapidly rebound to pretreatment
concentrations if ART is discontinued

Solution:
----- Optimal ART transport across tissue barriers (BBB)

Aim of this study

As the vehicle for virus carriage into the nervous
system, BMM (bone marrow macrophage) could also be
an carrier of antiretroviral drug (Indinavir, HIV蛋白酶抑制劑)
nanoART
----- BMM pharmacological nanoparticle (NP) delivery
system

Keywords:
----- Bone marrow macrophage (BMM)
----- Indinavir nanoparticles (IDV-NP)
----- SCID model of HIV-1 encephalitis (HIVE)
(腦炎)

Murine HIVE model


HIV-1 infected macrophages were
cultured (human MDM)
4~6-wk-old SCID mice
Anesthetized (麻醉) and placed in a stereotaxic
apparatus(立體定位儀) for intracranial injection(顱內注射)
---- sham operated (5 μl saline in left hemisphere)
---- HIV-1 infected macrophage (5x105 cell suspension)

Nanoparticle (NP) preparation
Surfactant coating : Lipoid E80
----- PC, PE
 Size distribution: 1.6 μm (99%<8.4 μm)
 Concentration: 10-2 M
 Marker: rhodamine DHPE (rDHPE; Invitrogen)
----- rDHPE-IDV-NP (red fluorescence)

BMM isolation and cultivation

BMM (bone-marrow macrophage) donors :
4~5-wk-old male BALB/c mice
---- remove femur(股骨)
---- dissociate the bone marrow cells into single-cell
suspensions
---- culture for 10 days with 1000 U/ml M-CSF
IDV treatment & measurement
rDHPE-IDV-NP + BMM
----- concentration :5x10-4 M ,12h
----- i.v. through tail vein
 Evaluate blood and brain tissue IDV levels at
1,3,7,14 days by reverse phase HPLC (RP-HPLC)

Confocal examination

rDHPE-IDV-NP-BMM-targeted migration to the regions of viral
infection
Different antibodies for fluorescence evaluation:
----- Vim: Ab to human specific vimentin-intermediate filaments
(detection of human macrophages in the mouse brain)
----- HIV-1 p24: Ab to HIV-1 p24 Ag
(determine the number of HIV-1-infected macrophage)
----- Iba-1: Ab to ionized calcium-binding adaptor molecule 1
(identify both MDM and murine microglia(小膠質細胞))
----- GFAP: Ab to glial fibrillary acidic protein
(detect astrocytes(星細胞))
----- NF: Ab to H chain (200-kDa) neurofilament Ag
(detect neurons(神經纖維))

IDV-NP-BMM ： Indinavir-nanoparticle-loaded Bone Marrow Macrophage
Uptake & Release of IDV by BMM
BMM
IDV-NP
Test by HPLC
Test by HPLC
Migration of BMM to Infected Brain
infected
sham
HIV-1 p24+
SPIO-BMM
GFAP
Astrogliosis
(Inflammation)
Caused by trauma
BMM Migration
relates to
astrogliosis
Caused by HIVE
Human MDM
Microglia + Human MDM
BMM Migration
relates to
microgliosis
Native and Injected BMM
Elicited by trauma
Drug enters
brain tissue
because of
BMM
Caused by HIVE
IDV-NP-BMM ： Indinavir-nanoparticle-loaded Bone Marrow Macrophage
IDV Concentration Tests
In Blood
In Brain Tissue
Five mouse per group
Vim
p24
IDV-NP-BMM ： Indinavir-nanoparticle-loaded Bone Marrow Macrophage
NF Tangle (p-NF)
NF Loss
P-NF : phosphorylated Neuron Filament
Conclusions

Viral infection and inflammatory responses induced bloodborne BMM migration across BBB.

IDV-NP-BMM treatment led to robust IDV levels up to 14
days and reduced HIV-1 replication in HIVE brain regions.

IDV-NP-BMM treatment would not cause inflammation and
neuron injury.

nanoART targeting to diseased brain through macrophage
carriage is possible and can be considered in
developmental therapeutics for HIV-associated
neurological disease.
Thanks for your attention