HIV replication was examined by measuring viral RT activity as previously described (41). In these assays, 90,000 monocytes were cultured in 96-well plates for seven days, then infected with HIV-1_ADA at a MOI of 0.01 and washed 24 hr later to remove virus. Media was changed every other day. To estimate HIV-1 replication, RT activity was determined by incubating 10 mL of sample with a reaction mixture consisting of 0.05% Nonidet P-40 (Sigma) and [³H]dTTP (2 Ci/mmol; Amersham, Arlington Heights, IL) in Tris-HCl buffer (pH 7.9) for 24 hr at 37°C on days 3, 5, 7 and 10. Radiolabeled nucleotides were precipitated on paper filters in an automatic cell harvester (Skatron, Sterling, VA), and incorporated activity was measured by liquid scintillation spectroscopy.

Cerebral cultures, containing neurons and glia in similar proportions to that found in the brain, were derived from the cerebral hemisphere of embryonic C57Bl/6 mice (Jackson Laboratories) on day 17 of gestation and cultured as described previously (42) following dissociation in 0.027% trypsin. Neuron-enriched cells were resuspended in neurobasal medium (Invitrogen, Grand Island, NY) with heat-inactivated fetal calf serum supplemented with B-27, 500 μM glutamine, and 25 μM glutamate then seeded at a density of 2.8 × 10⁵ cells/cm² on poly-d-lysine-coated 96 and 24-well plates.

Ninety thousand human MDM were cultured in 96-well plates and treated with 0, 80, 160 or 220 nM CEP-1347 24 hr before, at, or 4 hr after viral infection. Media was changed every other day and appropriate concentrations of CEP-1347 added. Cultures were monitored for 10 days. Cell viability assays were performed on days 1, 3, 5, 7 and 10 by measuring mitochondrial activity by reduction of tetrazolium salt MTT as described previously (43).

One million human MDM were cultured in 24-well plates and treated with/without CEP-1347 as outlined above, cultured for 5 days and then washed with PBS. Media was replaced with neural basal media (Invitrogen) every 24 hr. At that time, a ratio of 1:5 (supernatant:media) fluids were placed onto murine cortical neurons for 24 hrs to assess neurotoxicity by neuronal immunohistochemistry. For the neuronal antigen measurements, cells were blocked with 5% normal goat sera for 1 hr then incubated with antibodies to microtubule-associated protein 2 (MAP-2) (neural cell bodies, axons and dendrites, 1:1000) and neuron-specific nuclear protein (NeuN) (1:100) for 1 hr (Chemicon International, Inc., Temecula, CA). Cells were then washed three times with PBS and then incubated with fluorescent antibodies (1:1000 dilutions) to rabbit and mouse Alexa Fluor® 488 (green) and 594 (red) as a secondary antibody (Molecular Probes Inc., Eugene, OR) for 1 hr. Cells were washed twice with water, and water was allowed to cover the wells. Plates were read at 617 and 519 nM for MAP-2 and NeuN using a fluorescent plate reader, and the data were quantified (4 wells/group). Data were expresses as the changes in the mean of fluorescence intensity (MFI) in treated versus non-treated MCN. Lactate dehydrogenize (LDH) levels in neuronal fluids were determined by a cytotoxicity detection kit (Roche, Indianapolis, IN). In this case, MCN culture plates were read at 490 nM. Data were expressed as percent changes in MCN LDH-release amongst conditioned media treatments as compared to 1nM staurosporin (St)-treated cells (listed as 100%). For characterization of neuronal morphology double-immunofluorescence staining was carried out using Alexa Fluor® 488 (green) and 594 (red) as a secondary antibody (Molecular Probes Inc.). Confocal laser scanning imaging was used, which contains Argon, and Argon/Krypton lasers allowing for up to 4 Acousto-Optical Tunable Filter (AOTF) modulated excitation lines of 488 nm, 514 nm, 568 nm and 633 nm (Melles Griot, Prairie Technologies, Inc., Madison, WI). The lasers are connected to SweptField scanner head (Nikon Instruments, Melville, NY) attached with Cascade 512B back illuminated 12-bit CCD digital camera (Roper Scientific, Duluth, GA), which are connected to the side port of Nikon TE2000U Inverted Microscope with a high-resolution X-Y-Z-motorized stage.

One million human MDM were cultured in 24-well plates and infected with HIV-1_ADA at an MOI of 0.01 for 1 day, then washed with PBS. Infection was allowed to grow for 5 days. On day 5, cells were treated with 220 nM CEP-1347 for 45 min, washed with PBS, and phenol-red free and serum free media were added. Supernatants were harvested and assayed 24 hr later by Panomic antibody array 3.0 (Panomics, Inc., Fremont, CA) for human cytokines. Panomic antibody array 3.0 measured human cytokines: ApoI/Fas, Leptin, RANTES, ICAM-1, IL-2, IL-7, CTLA, MIP1α, TGFβ, VCAM-1, IL-3, IL-8, Eotaxin, MIP1β, IFNγ, VEGF, IL-4, IL-10, GM-CSF, MIP4, TNF-α, IL-1α, IL-5, IL-12(p40), EGF, MIP-5, TNFRI, IL-1β, IL-6, IL-15, IP-10, MMP3, TNFRII, IL-IRα, IL-6R, and IL-17. For the Cytometric Bead Array (CBA) flow cytometric of secreted chemokine analysis, one million human MDM were cultured in 24-well plates and infected with HIV-1_ADA at an MOI of 0.01 for 1 day, then washed with PBS. At time of infection, cells were treated with 80, 160 or 220 nM CEP-1347. Half media exchanges were carried out on day 2 and 4 with respective drug concentrations. On days 3 and 5, 50 μL of supernatant was assayed for each group by CBA Human Inflammation Kit and Chemokine Kit (BD Biosciences, San Diego, CA). Human Inflammation and Chemokine Kit measures IL-8, IL-1β, IL-6, IL-10, TNF-α, IL-12p70 and IL-8, RANTES, MIG, MCP-1, and IP-10, respectively.

MDM were infected and cultured with CEP-1347 for 5 days then lysed using RIPA lysis buffer (Fischer Scientific, Pittsburgh, PA) containing additional protease and phosphatase inhibitors (Calbiochem, San Diego, CA). Protein concentrations of whole cell lysates were estimated using BSA assay and stored at −80°C. Cell lysates were allowed to thaw on ice then processed for Western by boiling with SDS laemmli buffer (Bio-Rad Laboratories, Hercules, CA). Whole cell lysate (40 μg protein) was loaded into each well of the Bio-Rad SDS gradient PAGE ranging from 4–15 % and run at 100 V for 1 hour 30 minutes. Protein was transferred to polyvinylidene fluoride (PVDF) membrane (Millipore Billerica, MA) using Bio-Rad semidry transfer apparatus at 25 V for 1 h. The membrane was then blocked with 5% BSA in TBS. Primary antibodies used were anti-phosphorylated-MLK3 (1:1000, Cell Signaling Technology, Inc., Danvers, MA), anti-MLK3 (1:1000, Cell Signaling Technology, Inc.), anti-JNK (1:1000, Cell Signaling), anti-phosphorylated-JNK (1:2000, Cell Signaling Technology, Inc.), anti-p38 (1:1000, Cell Signaling), anti-phosphorylated-p38 (1:1000, Cell Signaling Technology, Inc.), anti-phosphorylated- nuclear factor-kappa beta (NF-κB) p65 (Ser 468) (1:1000, Cell Signaling) anti-NF-κB p65 (1:1000, Cell Signaling), anti-phosphorylated-NF-κB p105 (Ser933) (1:1000, Cell Signaling), anti- NF-κB p105/50 (1:1000, Cell Signaling) and GAPDH (1:1000, Cell Signaling Technology, Inc.). Primary antibodies were detected with HRP-linked secondary antibodies, anti-mouse or anti-rabbit as per antibody used (1:20,000, Chemicon International) followed by detection by ECL femto-detection reagent (Pierce Biotechnology, Rockford, IL) and subsequent exposure to X-ray films for 5–30 min (for phosphoproteins up to 6h). Blots were quantified by inverting scanned images of the blots using the program ImageJ (Naitonal Institutes of Health, NIH) and measuring intensity. Readings were normalized to respective GAPDH expression levels and compared to uninfected, untreated MDM.

Five-week old male CB17/scid mice and C57Bl/6 mice were purchased from Charles River Laboratory, Wilmington, WA. Animals were administered i.p. injections either 1.5 or 15.0 mg/kg CEP-1347. The C57B1/6 mice received both drug dosages while the CB17/scid received only the lower 1.5 mg/kg dosage. Blood was extracted by cheek puncture at 0, 0.5, 1, 2, 4, 6 or 8 hr. Blood samples were centrifuged at 5000 rpm for 10 min at 4°C. Plasma was collected and stored at −20°C pending analysis. The plasma samples were prepared for bioanalysis by adding ten volumes of acetonitrile containing an internal standard (alprenolol). After the samples were vortexed and centrifuged, the supernatant was transferred to a 96-well plate for analysis by liquid chromatography/mass spectrometry. The amount of CEP-1347 in the sample was quantified using a plasma standard curve made via serial dilution in a concentration range from 10 to 5000 ng/mL.

Four-week old male C.B-17/IcrCrl-SCIDbr (CB17/scid) mice were purchased from Charles River Laboratory. Animals were maintained in sterile microisolator cages under pathogen-free conditions in the Laboratory of Animal Medicine at the University of Nebraska Medical Center in accordance with ethical guidelines for care of laboratory animals set forth by the National Institutes of Health. HIV-1_ADA-infected MDM (1.5 × 10⁵ cells infected at an MOI of 0.1 in 5 μl) were stereotactically injected intracranially (i.c.) after 1 day of viral infection and referred to as HIVE mice (44). The higher multiplicity used in the animal studies as compared to the in vitro experiments reflected the need to infect a larger proportion of cells prior to cell injections. CEP-1347 was then administered i.p. daily for 7 days at doses 0.5, 1.0, 1.5, 5.0 and 15.0 mg/kg/d (n = 4 mice/treatment group). Vehicle only was the control. CB17/scid mice received i.c. injections of media (sham-operated) and served as additional controls. Seventeen animals were included in each group. Animals were treated with vehicle or CEP-1347 starting 1day post-i.c. injection and for 7 days following MDM injections and CEP-1347 treatments.

Brain tissue was collected at necropsy, fixed in 4% phosphate-buffered paraformaldehyde and embedded in paraffin. Paraffin blocks were cut until the injection site of the human MDM was identified. HIV-1 p24 antigen (clone Kal-1; Dako, Carpinteria, CA) was used to test for virus infected human MDM. For each mouse, 30–100 serial (5-μm-thick) sections were cut from the injection site and 3–7 sections (10 sections apart) analyzed. Antibodies to vimentin intermediate filaments (clone VIM 3B4; Boehringer Mannheim, Indianapolis, IN) were used for detection of human cells in mouse brains. Mouse microglia were detected by antibodies to Iba-1 (WAKO, Osaka, Japan), and astrocytes were detected by antibodies for glial fibrillary acidic protein (GFAP; Dako). NeuN, MAP-2 (both from Chemicon International Inc.) and heavy chain (200kDa) neurofilaments (Dako) were used for detection of neurons. Appropriate secondary antibodies and the Vectastain Elite ABC kit (Vector Laboratories, Berlingame, CA) were used to complete the immunohistochemical tests. All sections were counterstained with Mayer’s hematoxylin. The numbers of human MDM and HIV-1 p24 antigen-positive cells were counted with a Nikon Microphot-FXA microscope. All obtained images were imported into Image-Pro Plus, v. 4.0 (Media Cybernetics, Silver Spring, MD) for quantifying area (%) of GFAP, Iba-1, MAP-2 and NeuN positive staining.

The levels of HIV RNA in the injected hemisphere were determined by real time PCR using ABI 7000 prism (Perkin-Elmer, Applied Biosystems, Foster City, CA). Briefly, total RNA was extracted from the brain tissues. The HIV-1gag RNA specific primers and probe were used as previously described (45) and are: forward, 5′-ACA TCA AGC CAT GCA AAT -3′; reverse, 5′-ATC TGG CCT GGT GCA ATA GG -3′; and probe, 5′-CAT CAA TGA GGA AGC TGC AGA ATG GGA TAG A -3′. The reverse primer was used to make cDNA from RNA, which was further amplified using primers and probe at 50°C for 2 min., 95°C for 10 min., and 40 cycles at 95°C for 15 sec. and 60°C for 1 min. Separate GAPDH amplifications were used as an endogenous control to ensure that equal amounts of RNA were used. For GAPDH, Mac-1, TNF, GFAP and IL-10 Taqman gene expression assays were used (Applied Biosystems). Results were expressed as mean copy number ± SEM.

The data presented above suggested that CEP-1347 treatment reduces the neurotoxicity mediated by HIV-1-infected MDM in vitro. To analyze this possibility, we used primary MCN cultures. Supernatants were collected from human MDM that were treated 24 hr before, at, or 4 hr post HIV-1_ADA infection with CEP-1347 concentrations of 0, 80, and 160 nM and cultured for 5 days in the presence of CEP-1347. On day 4, cells were washed, and media replaced with neural basal media (Invitrogen) for 24 hr without CEP-1347. On day 5, supernatants were harvested. These conditioned supernatants were placed on 10-day-old cultures of primary MCN for 24 hr, and neurotoxicity was assessed by measuring MAP-2 and NeuN staining. The results were then verified by conducting LDH release assays. The morphological changes for MCN are shown in Figures 2 and ​and33 include quantitative MFI measurements for MFI for dendrites and neuronal nuclei (MAP-2 at 617 nm and NeuN at 519 nm wavelength, respectively). Conditioned media-treated MCN was subtracted in all evaluations. In untreated MCN cultures, neurons were evenly distributed and connected with each other with a high density of dendritic branchpoints and long neuritic processes; these cells also contained prominent cell bodies (Figure 2A). This morphology was unaltered by treatment with CEP-1347 (data not shown). After exposure to HIV-1 conditioned media, neurons (Figure 2B) displayed a low density of dendritic nodes, shorter neurites, and a loss of connected processes. In contrast, conditioned media from CEP-1347-treated, HIV-1 infected MDM did not elicit these neurotoxic effects (Figure 2C and D, Figure 3A). In this latter case, neurites were retained and dendritic nodes showed long processes in high density, at similar levels to controls (untreated and uninfected cells); prominent cell bodies were also seen. The greatest increase in MAP-2 immunostaining was seen in cultures that were exposed to supernatant fluids from HIV-1 infected MDM treated with 160 nM CEP-1347 (P < 0.0001, compared to cultures exposed to conditioned media from untreated HIV-1 infected MDM). MCN that were exposed to conditioned supernatants from HIV-1 infected MDM treated with 80 nM CEP-1347 also showed increased levels of MAP-2, but this result did not achieve statistical significance (P < 0.087). NeuN immunostaining was also significantly increased in MCN cultures that were exposed to conditioned supernatants from HIV-1 infected MDM that had been treated with 160 nM of CEP-1347, versus untreated HIV-1 infected MDM (P < 0.0005). MCN exposed to conditioned media from HIV-1 infected MDM treated with 80 nM CEP-1347 also showed elevated numbers of NeuN reactive cells, compared to cultures exposed to supernatants from untreated HIV-1 infected MDM, although this result did not achieve statistical significance. Neuronal protection was also assessed by measuring LDH levels in the extracellular milieu. LDH release from MCN subjected to different MDM conditioned medias were compared to 1nM St-mediated neuronal destruction (100%, Figure 3B). LDH release from neurons treated with conditioned media collected from non-treated MDM was subtracted in these evaluations. Conditioned media collected from HIV-1 infected MDM elicited high levels of LDH release in MCN cultures. Levels of LDH release were lower in cultures exposed to conditioned media from HIV-1 infected MDM cultures that were treated with both 80 and 160 nM CEP-1347 (P = 0.05 and P = 0.013, respectively, Figure 3B).

In order to gain insight into the metabolism of CEP-1347 in our SCID mouse model of HIVE, we measured plasma concentrations of CEP-1347 following i.p. administration at dosages of 1.5 mg/kg and 15.0 mg/kg in CB17/scid mice (n = 4 mice/treatment/time point) and collected blood at varying time points as depicted in Figure 4.

Human HIV-1_ADA infected MDM were stereotactically injected into the basal ganglia of CB17/scid mice. Histopathological changes observed in murine brain tissue paralleled those seen for human HIVE. This included HIV-1 infection in perivascular and parenchymal human MDM, the formation of multinucleated giant cells, astrocytosis, and neuronal dropout. Therapeutic efficacy of different dosages of CEP-1347 was evaluated using immunohistochemistry in HIVE scid mice after administration. Human HIV-1_ADA-infected MDM were stereotactically injected in the basal ganglia of CB17/scid mice (n = 4 mice/treatment group). CEP-1347 was then administered i.p. daily for 7 days at doses of 0 mg/kg/d (vehicle only), 0.5 mg/kg/d, 1.0 mg/kg/d, 1.5 mg/kg/d, 5.0 mg/kg/d and 15.0 mg/kg/d. Morphological changes in astrocytes surrounding the lesion site were evaluated using GFAP immunostaining. Quantitation of GFAP expression used three sections from each animal: immediately before, at, and immediately after the lesion site. GFAP expression was quantified by determining GFAP positive area as a percentage of total image area per microscopy field. Astrogliosis was not unaltered in CEP-1347 treated HIVE mice, when compared to vehicle-only treated HIVE mice (Figure 5E to H). Serial brain sections were stained with Iba-1 to determine activation of microglia. Microglia stained area was calculated as a percentage of the area of the entire microscopy field. Microglial activation was significantly decreased in all CEP-1347 treated groups when compared to vehicle-only treated HIVE mice (2.71 ± 0.61). Results of microglial activation for 0.5 mg/kg/d, 1.0 mg/kg/d, 1.5 mg/kg/d, 5.0 mg/kg/d and 15.0 mg/kg/d were 0.91 ± 0.16 (P < 0 .0001 compared to HIVE mice treated with vehicle alone), 1.13 ± 0.26 (P < 0 .01), 0.87 ± 0.40 (P < 0 .001), 0.75 ± 0.17 (P < 0 .0001), and 0.41 ± 0.06 (P < 0 .00001), respectively (Figure 5A to C). Microglial activation decreased with increasing concentrations of CEP-1347 (Figure 5D).