Human BMVEC’s were isolated from temporal lobe tissues removed during neurosurgical procedures and cultured as previously described [6Liu NQ, Lossinsky AS, Popik W, et al. Human immunodeficiency virus type 1 enters brain microvascular endothelia by macropinocytosis dependent on lipid rafts and the mitogen-activated protein kinase signaling pathway J Virol 2002; 76(13): 6689-700.]. Confluent monolayers of BMVEC’s were prepared by seeding 100,000 cells into each well of an 8- well tissue culture- treated slides (Becton Dickinson Falcon, Bedford, Massachusetts, USA) containing 0.5 ml of DME/F12 (InVitrogen, Carlsbad, California, USA) with 10% fetal calf serum and incubating cell cultures for 4-6 days to confluence. To study cocaine’s signaling, cocaine (Research Triangle Institute, North Carolina, USA) (1:100 dilution of freshly diluted 100x stock in DME/F12) was added to achieve the indicated concentration in the medium.

The National Neurological AIDS Bank at UCLA provided formalin-fixed, paraffin-embedded frontal lobe and hippocampal tissues of five HIV-1-positive patients (four patients had a history of drug abuse including cocaine with additional alcohol and opiate abuse in some; the last patient had no history of drug abuse) and two amyotrophic lateral sclerosis (ALS) brain tissues. The autopsy interval ranged between 5 to 6 h except that of one ALS patient, which was 22 h. The Manhattan HIV Brain Bank provided brain tissue of a patient with minor cognitive impairment who had brain lymphoma and was taking medically prescribed opiates. The UCLA Brain Bank provided HIV-1-negative control tissues (amyotrophic lateral sclerosis) and two brain tissues of HAD patients who died in the late 1980’s. The protocol was reviewed and approved by the UCLA Institutional Review Board.

Tissues were subjected to antigen retrieval solution (DAKO) in a steamer for 20 min and were treated with Dual endogenous enzyme block and Protein block (DAKO, Carpinteria, California, USA). IHC was performed by the Envision technique (with primary rabbit and mouse antibodies) or the LSAB 2 technique (with primary goat antibodies) (DAKO) as previously described [7Fiala M, Liu QN, Sayre J, et al. Cyclooxygenase-2-positive macrophages infiltrate the Alzheimer's disease brain and damage the blood-brain barrier Eur J Clin Invest 2002; 32(5): 360-71.]. The primary antibodies were anti-gp120, anti-p24, anti-Nef (all three, which are described below, were provided by the AIDS Research and Reference Reagent Program, NIH, Bethesda, USA), anti-NeuN (Chemicon, Temecula, California, USA), anti-CD68 and anti-GFAP (DAKO).

Two gp120 monoclonal antibodies were used: ID6 [8Gomez-Roman VR, Cao C, Bai Y, et al. Phage-displayed mimotopes recognizing a biologically active anti-HIV-1 gp120 murine monoclonal antibody J Acquir Immune Defic Syndr 2002; 31(2): 147-53.] and 670-30D [9Zolla-Pazner S, O'Leary J, Burda S, et al. Serotyping of primary human immunodeficiency virus type 1 isolates from diverse geographic locations by flow cytometry J Virol 1995; 69(6): 3807-15.]. The murine ID6 monoclonal antibody was generated after immunization of a mouse with a recombinant gp160 immunogen. Subsequent analysis using screening of phage display libraries with ID6 indicated that the epitope was contained within amino acids 86-100 of the highly conserved C1 region of gp120. One of the phage mimotopes generated by screening with ID6 (designated phage 3.3) bound to ID6 with a high affinity. Two IHC studies were done using ID6 absorbed with the relevant gp120 epitope. In the first, equal volumes of the ID6 antibody (1:100) and recombinant gp120_JR-FL (250 ng/μL) were incubated at 37^o C for 3 hrs and, after ultracentrifugation, the top half of the suspension was used in IHC. Recombinant gp120 was produced in Chinese hamster ovary cells as described [10Mossman SP, Bex F, Berglund P, et al. Protection against lethal simian immunodeficiency virus SIVsmmPBj14 disease by a recombinant Semliki Forest virus gp160 vaccine and by a gp120 subunit vaccine J Virol 1996; 70(3): 1953-60.]. In the second study, ID6 antibody (1:100) was incubated with phage 2.13 (negative phage mimotope) or phage 3.3 (positive phage mimotope) [8Gomez-Roman VR, Cao C, Bai Y, et al. Phage-displayed mimotopes recognizing a biologically active anti-HIV-1 gp120 murine monoclonal antibody J Acquir Immune Defic Syndr 2002; 31(2): 147-53.] (each at 1:800) and processed as above.

Gag was detected using monoclonal anti-p25/24 (ISF2) and anti-p24 (AG 3.0); Nef was stained using anti-HIV-1 IIIB Nef (AE6) and anti-Nef (EH1).

BMVEC cultures grown on cover slips in 24-well plastic dishes were fixed by immersion according to standard protocols that take into consideration our desire to produce excellent ultrastructural preservation of EC cytoskeletal filaments, microtubules, and EC junctional complexes. For this reason, we fixed the BMVEC at room temperature (25EC) to avoid disruption of EC cytoskeletal components such as microtubules. The fixative contained 2.5-3.0% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4 [11Haudenschild CC, Cotran RS, Gimbrone MA Jr, Folkman J. Fine structure of vascular endothelium in culture J Ultrastruct Res 1975; 50(1): 22-32.]. BMVEC were post-fixed with 1% osmium tetroxide, dehydrated in a graded series of ethanol, and en bloc stained with uranyl acetate [12Hayat MA. The production of artifacts Ultrastruct Pathol 1981 Jan-Mar; 2(1): 93. discussion]. The coverslips were infiltrated with liquid epoxy plastic overnight in a desiccator at 25EC. On the next day, in liquid plastic, the coverslips were sliced into several pieces and flat-embedded with the BMVEC positioned upward then polymerized in a 60EC oven overnight. For TEM and HVEM studies, plastic thick- and thin-sections were cut with either the Leica Ultracut UCT and Sorvall (MT-1) Ultramicrotomes and stained with lead and uranyl salts, according to standard protocols (Hayat, 1981). Thin-sections were collected on grids, stained with uranium and lead salts and examined with the FEI Morgagni Electron Microscope.

Confluent monolayers of BMVEC’s were incubated overnight in serum-free DME/F12 medium and treated with cocaine as indicated. Cell extracts were prepared by washing cell monolayers three times with phosphate buffered saline (PBS), scraping the cells into SDS sample buffer (125 mM Tris pH 6.8, 4% SDS, 10% glycerol, 0.006% bromophenol blue, 2% 2-mercaptoethanol), then passing them through a 26-gauge needle. After 5 min of boiling, the protein samples were centrifuged for 2 min and separated by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (SDS-PAGE) [13Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 1970; 227(5259): 680-5.]; the proteins were transferred to nitrocellulose membranes (30 V for 18 h or 90 V for 2 h) [14Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications 1979 Biotechnology 1992; 24: 145-9.] and incubated with specific antibodies. The antibodies of interest included anti-phospho-myosin light chain 2 and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling, Danvers, Massachusetts, USA); anti-phospho-extracellular signal-regulated kinases 1 and 2 (Erk1/2) (New England BioLab, Beverly, Massachusetts, USA); anti-phosphor-specific myristoylated alanine-rich C kinase substrate (MARCKS) (ProteinTech Group, Chicago, Illinois, USA); and anti- myosin light chain (MLC) (Sigma, St. Louis, Missouri, USA). Immunoreactive proteins were detected with enhanced chemiluminescent detection system (ECL) according to the manufacturer's directions (Amersham, Little Chalfont, UK). The experiments were repeated three times. To evaluate the protein loading, the membranes were stained with GAPDH or MLC antibody.

Cocaine concentrations 10^-5 to 10^-7M disrupted endothelial junctions and produced gaps between BMVEC’s (Fig. 1).

Fig. (1) Cocaine disrupts BMVEC junctions. Confluent BMVEC monolayers were treated with the indicated concentration of cocaine for 1h; the cells were then washed, fixed, stained with phalloidin-ALEXA 594 and photographed by Hamamatsu camera on the Olympus Bmax microscope (40x). The arrows indicate gaps between BMVEC’s.

Fig. (2) Cocaine and benztropine induce phosphorylation of Erk 1/2, MARCKS and MLC in BMVEC’s.

Fig. (3) Ultrastructural features of monocyte migration through the BMVEC model of the blood-brain barrier. (A) Endothelial layer becomes thick above the pores through which the monocytes are migrating (1,000x section of the BBB model with migrating monocytes). (B) Monocytes migrate between endothelial cells and through the pore (TEM, 10,000 x).

Fig. (4) Ultrastructural features of monocyte migration through the BMVEC model of the blood-brain barrier. Compared to the untreated cells (a), cocaine-treated BMVEC’s (b) display cytoplasmic vacuolization (TEM, 10,000 x).

Fig. (5) Perivascular infiltration by macrophages in HAD brain tissues. (a) Minimal perivascular macrophage infiltration in patient 1 (IHC with CD68 antibody). (b) Robust macrophage infiltration of vessels in an AIDS patient dying in late 1980’s.

Fig.(6). (gp120). Immunohistochemical demonstration of HIV-1 antigens in frontal lobe neurons of the patient #5 with HAD (the results of staining in patients #1-4 were comparable). (a) Positive neuronal gp120 staining (anti-gp120 (ID6) (brown) and anti-MAP2 (red), 40x). (b) Positive gp120 staining of neurons and negative staining of astrocytes (anti-gp120 (ID6) (brown) and anti-GFAP (red), 40x). (c) Positive gp120 staining of neurons (anti-gp120 (ID6) absorbed with the negative phage 2.13, 100x). (d) Negative gp120 staining of neurons (anti-gp120 (ID6) absorbed with the positive phage 3.3, 100x).

Fig. (7) (p24, Nef) Immunohistochemical demonstration of HIV-1 antigens in frontal lobe neurons of the patient #5 with HAD (the results of staining in patients #1-4 were comparable) (a). Positive neuronal staining with anti p24 (ISF2) (40x, inset 100x). (b) Positive neuronal staining with anti p24 (AG 3.0) (100x). (c) Positive neuronal staining with anti-Nef (AE6). (d) Positive neuronal staining with anti-Nef (EH1).

Cocaine concentrations 10^-8M and below had minimal effects on BMVEC’s. Benztropine is a therapeutic agent that, in addition to binding to the biogenic amine transporters and muscarinic receptors, binds with high affinity to a cocaine-binding site on BMVEC’s [3Fiala M, Eshleman AJ, Cashman J, et al. Cocaine increases human immunodeficiency virus type 1 neuroinvasion through remodeling brain microvascular endothelial cells J Neurovirol 2005; 11: 1-11.]. Similarly as cocaine, 10^-5 to 10^-7M benztropine produced gaps between endothelial cells (not shown). 10^-5 to 10^-7M cocaine treatment of BMVEC’s induced phosphorylation of Erk 1/2, MARSKC and MLC (Fig. 2). Cocaine concentrations10^-8 and below did not consistently induce cell signaling. Benztropine has a higher affinity for the cocaine-binding site on BMVEC’s than cocaine [3Fiala M, Eshleman AJ, Cashman J, et al. Cocaine increases human immunodeficiency virus type 1 neuroinvasion through remodeling brain microvascular endothelial cells J Neurovirol 2005; 11: 1-11.]. Accordingly, stimulation by 10^-6 M benztropine induced more prolonged signaling through Erk1/2 and MLC phosphorylation in comparison to cocaine (Fig. 2).

Confluent BMVEC monolayers were treated with the indicated concentration and time of exposure to cocaine; cell extracts were prepared and analyzed by SDS-PAGE with antibodies to phospho-Erk 1/2, MARCKS and MLC. Staining with antibodies to GAPDH and MLC demonstrated equal loading.

In the BBB model, monocyte migration was stimulated by MCP-1 in the lower chamber. Monocytes migrated between the endothelial cells, which became thick above each pore (Fig. 3A). Monocytes migrated through the pore into the brain chamber (Fig. 3B). The rate of monocyte migration was increased by cocaine, as previously published [2Zhang L, Looney D, Taub D, et al. Cocaine opens the blood-brain barrier to HIV-1 invasion J Neurovirol 1998; 4: 619-26.].

24-h treatment of BMVEC’s with 10^-6M cocaine produced robust vacuolization of the endothelial cell cytoplasm (Fig. 4) and increased monocyte transmigration, as previously published [15Gan X, Zhang L, Taub D, et al. Cocaine enhances endothelial adhesion molecules and leukocyte migration Clin Immunol 1999; 91: 68-76.].

As shown above (Fig. 1), cocaine at the concentrations in the plasma of cocaine’s addicts (10^–6M to 10^–8M) disrupts BBB. We have searched for increased evidence of HIV-1 in brain tissues of drug-abusing patients with HAD.

To detect viral “foot-prints” in brain tissues, we performed IHC with the monoclonal gp120 antibody ID6 with brain tissues of six HAD patients, four with a history of drug abuse, one with a history of opiate use for medical reasons, and one without any history of drug use. All patients were intensively treated until their death with HAART. The neuropathology was remarkable for absence of perivascular infiltration by macrophages and lack of gp120 staining of microglia, which were noted in brain tissues of patients with HAD who died in the eighties (Fig. 5). The brain tissues of the patient without a history of drug abuse were completely negative for gp120 (Table 1).

Table. 1

HIV-1 Positivea and Controlb Patients – Diagnostica and Virologicalb Data

However, the five patients with a history of drug abuse or medical use of morphine had expression of gp120 (brown) in neurons, as shown by double staining with MAP-2 antibody (red) (Fig. 6a), but not in astrocytes (Fig. 6b).

To confirm the specificity of gp120 staining with the ID6 antibody, this antibody was absorbed with the phage 3.3 mimotope (Fig. 6d), which removed the reactivity, whereas absorption with the unrelated phage 2.13 had no effect on the staining (Fig. 6c). Absorption of the ID6 antibody with gp120 also reduced neuronal staining (not shown).

Furthermore, ID6 antibody did not stain neurons of control patients (e.g., ALS case) and unrelated antibody (CD20) did not stain neurons in HAD brain tissues. In addition, neurons in the frontal lobe tissues were positively stained by two different p24 (Fig. 7a,b) and two different Nef antibodies (Fig. 7c,d).