Excess soluble CD40L contributes to blood brain barrier permeability in vivo: implications for HIV-associated neurocognitive disorders.

Ethics Statement

All experiments involving the use of laboratory animals were carried out in accordance with the Animal Welfare Act and the National Institute of Health (NIH) guidelines, and the animal protocol was approved by the University Committee on Animal Resources of the University of Rochester Medical Center. The facilities and programs of the Vivarium and Division of Laboratory Animal Medicine of the School of Medicine and Dentistry are fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC) and are in compliance with state law, federal statute, and NIH policy. All animals used in these studies, strains C57BL/6 and B6.129s2-CD40lg tm1Imx /J, were purchased from The Jackson Laboratory, Bar Harbor, ME. Reagents and Antibodies HIV Tat 1–72 was obtained from Dr. Avindra Nath (National Institute of Neurological Disorders and Stroke, Baltimore, MD) and Philip Ray (University of Kentucky, Lexington, KY). Production of this protein has been described previously [23] – [25] and the purified protein was found to be >98% pure, with less than 1 pg/mg endotoxin content of Tat protein [23] – [25] . We have also tested the effects of heat inactivated Tat (H.I. Tat) that was prepared in the same manner, but subsequently incubated at 85°C for 30 minutes, as previously described [26] , [27] , as this allows a control for nonspecific contaminants that may arise during production and processing of the protein. We found no significant differences between treatment with H.I. Tat and saline controls, and therefore, in some cases saline controls alone are displayed. It should also be noted that when working with Tat all instruments were siliconized with Sigmacote (Sigma-Aldrich, St. Louis, MO) to avoid loss of the reagent. Fluorescein sodium salt was purchased from Sigma-Aldrich (St. Louis, MO); recombinant mouse CD40L was purchased from R&D Systems (Minneapolis, MN). Antibodies against mouse granulocyte antigen 1 (Gr1; also known as Ly-6C/G) conjugated to Alexa Fluor 488, and Texas Red Dextran were obtained from Invitrogen (Carlsbad, CA), while phycoerythrin-conjugated anti-mouse CCR-2 was purchased from R&D Systems (Minneapolis, MN). Antibodies for platelet depletion, as well as non-immune rat immunoglobulin control antibodies, were purchased from Emfret Analytics (Eibelstadt, Germany). ELISA Ten to twelve-week old wild-type C57BL/6 (WT) mice (n = 5 for each group) were injected retro-orbitally with HIV Tat (100 ng/g body weight). One-hour post-injection, whole blood was collected via cardiac exsanguination and sequentially centrifuged to obtain platelet poor plasma (PPP). Soluble CD40L or platelet factor 4 (PF4) concentrations were measured in PPP samples using either a mouse sCD40L or PF4 ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol. Samples were compared using an unpaired t-test with statistical significance indicated in the figure as **p