ABSTRACT
Progress in AIDS treatment shifted emphasis towards limiting adverse effects of antiviral drugs while improving the treatment of hard-to-reach viral reservoirs. Many therapeutic nucleoside reverse transcriptase inhibitors (NRTI) have a limited access to the central nervous system (CNS). Increased NRTI levels induced various complications during the therapy, including neurotoxicity, due to the NRTI toxicity to mitochondria. Here, we describe an innovative design of biodegradable cationic cholesterol-ε-polylysine nanogel carriers for delivery of triphosphorylated NRTIs that demonstrated high anti-HIV activity along with low neurotoxicity, warranting minimal side effects following systemic administration. Efficient CNS targeting was achieved by nanogel modification with brain-specific peptide vectors. Novel dual and triple-drug nanoformulations, analogous to therapeutic NRTI cocktails, displayed equal or higher antiviral activity in HIV-infected macrophages compared to free drugs. Our results suggest potential alternative approach to HIV-1 treatment focused on the effective nanodrug delivery to viral reservoirs in the CNS and reduced neurotoxicity.
Subject(s)
Brain/drug effects , Cations , Drug Carriers , Nanostructures , Reverse Transcriptase Inhibitors/administration & dosage , Apoptosis/drug effects , Blood-Brain Barrier/drug effects , Cell Line , Cell Survival/drug effects , Cholesterol , Drug Delivery Systems , Gels , HIV Reverse Transcriptase , HIV-1/drug effects , Humans , Mitochondria/drug effects , Neurons/drug effects , Polylysine , Reverse Transcriptase Inhibitors/chemistryABSTRACT
The release of histamine from mast cells and basophils during allergic reactions can regulate functions of T cells and may influence the nature of the immune response to a given antigen. The effects of histamine on T lymphocytes are associated with its binding to H2-receptors linked with adenylate cyclase, elevation of cAMP levels and activation of cAMP-dependent protein kinase (PKA). In this report we explore the role of PKA in histamine-mediated effects on IL-2 mRNA expression and IL-2 protein secretion. Fresh isolated mouse splenocytes (C57Bl/6) were pretreated with histamine (10(-4) M) for 1 h in the presence or absence of Rp-cAMPS (50 microM), an inhibitor of PKA regulatory subunit. The cells were then washed thoroughly and activated with plate-bound anti-CD3 (5 microg/ml), or PHA (1:100) or PMA + ionomycin (10 ng/ml, 1 microg/ml) for 6 h. Pretreatment with histamine inhibited IL-2 mRNA expression and secretion in cells activated with anti-CD3 or PMA, but not in cells activated with PMA + ionomycin. Rp-cAMPS prevented histamine-mediated suppression and did not itself affect IL-2 production. These results provide evidence that histamine affected IL-2 production when the cells were activated via the T cell receptor (TCR)/CD3 complex, but did not interfere with signal transduction pathways downstream of PKC leading to production of IL-2. These effects of histamine on IL-2 secretion and mRNA expression were mediated via PKA.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/physiology , Histamine/physiology , Interleukin-2/biosynthesis , RNA, Messenger/biosynthesis , Spleen/metabolism , Animals , Antibodies/pharmacology , CD3 Complex/immunology , Cyclic AMP/analogs & derivatives , Cyclic AMP/pharmacology , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/metabolism , Enzyme Activation , Enzyme Inhibitors/pharmacology , Enzyme-Linked Immunosorbent Assay , Interleukin-2/metabolism , Ionomycin/pharmacology , Lymphocyte Activation/physiology , Male , Mice , Mice, Inbred C57BL , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Spleen/drug effects , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Thionucleotides/pharmacologyABSTRACT
Histamine and IL-5 are important autacoid mediators involved in the etiology of allergic diseases. IL-5 is the main factor of eosinophilic reactions in allergy. It has been suggested that the protein kinase A-dependent (PKA) pathway of signal transduction may play the main role in histamine-induced elevation of interleukin-5 production. This study was designed to investigate the effects of the inhibitors of regulatory and catalytic subunits of PKA on histamine-mediated elevation of IL-5 production. In our study, histamine at a concentration range of 10(-4)-10(-6) M enhanced IL-5 production in D10.G4.1 cells, a mouse Th2 helper cell line. Pretreatment of this cell line with histamine at a concentration of 10(-4) M for 6-9 h had the maximum stimulatory effects (226-420%) on IL-5 production. Other cAMP-elevating agents including forskolin and Bt2-cAMP produced similar effects. The PKA inhibitors N-[2-(methylaminoethyl]-5-isoquinoline-sulfonamide (H-8) and Rp-diastereomer of adenosine cyclic 3',5'-phosphorothioate (Rp-cAMPS) were used for the inhibition of catalytic and regulatory subunits of PKA, respectively. Pretreatment of D10.G4.1 cells with H-8 at a concentration of 10(-5) M completely prevented the effects of histamine at a concentration range of 10(-6)-10(-4) M. Rp-cAMPS at 10(-5) M also prevented histamine-induced stimulation. Neither inhibitor affected IL-5 production when tested alone. These observations suggest a role for PKA in histamine-mediated increase in IL-5 production.
