In Vitro Blood-Brain Barrier Modeling adapted for Peripheral Blood Mononuclear Cell Transmigration from HIV-Positive Patients for Clinical Research on Therapeutic Drug Intervention.

OBJECTIVE: HIV-associated cognitive impairment (HACI) continues to persist for HIV-seropositive individuals who are on antiretroviral therapy (ART). HACI develops in part when HIV-infected monocytes (MOs) transmigrate through the blood-brain barrier (BBB) and secrete pro-inflammatory cytokines and chemokines, which leads to neuronal damage. In vitro BBB models are important tools that can elucidate mechanisms of MO transmigration. Previously described in vitro BBB models relied on pathology specimens, resulting in potentially variable and inconsistent results. This project reports on a reliable and consistent alternative in vitro BBB model that has the potential to be used in clinical research intervention studies analyzing the effects of ART on the BBB and on MO transmigration. METHODS: A bilayer BBB model was established with commercially available astrocytes and endothelial cells on a 3µm PET membrane insert to allow the contact of astrocytic foot processes with endothelial cells. Inserts were cultured in growth medium for 7 days before exposure to HIV- or HIV+ peripheral blood mononuclear cells (PBMCs). PBMCs were allowed to transmigrate across the BBB for 24 hours. RESULTS: Confluency and integrity measurements by trans-endothelial electrical resistance (TEER) (136.7 ± 18.3Ω/cm2) and permeability (5.64 ± 2.20%) verified the integrity of the in vitro BBB model. Transmigrated MOs and non-MOs were collected and counted (6.0x104 MOs; 1.1x105 non-MOs). Markers indicative of glial fibrillary acidic protein (GFAP), von Willebrand factor (vWF), and p-glycoprotein (Pgp) were revealed in immunofluorescence staining (IF), indicating BBB phenotype and functionality. CONCLUSION: Potential applications for this model include assessing the HIV DNA copy numbers of transmigrated cells (pre- and post-targeted ART) and understanding the role of oxidative stress related to HIV DNA and HACI.

Different levels of HIV DNA copy numbers in cerebrospinal fluid cellular subsets.

Inequities in the incidence of HIV infection and AIDS with continued persistence of HIV-associated neurocognitive disorders (HAND) exist in populations in Hawaii (HI) and Puerto Rico (PR). We previously reported that peripheral monocyte HIV DNA levels are high in patients in Hawaii with HAND and we now hypothesize that similar findings would be observed in the cerebrospinal fluid (CSF) cellular subsets. Cerebrospinal fluid cells were obtained from patients from PR and HI undergoing neurocognitive testing and sorted into monocytes (CD14+) and lymphocytes (CD14-) and HIV DNA was measured. From six PR subjects (three HAND, three normal cognition, NC) and six HI subjects (three HAND, three NC), HIV DNA burden in CD14+ cells was higher in HAND than NC patients; NC patients had higher HIV DNA burden in CD14-cells versus HAND. Differences in HIV DNA burden in particular CSF cellular subsets suggest that HIV DNA burden may play a role in HAND neuropathogenesis.