Huanyu Dou

Macrophage Delivery of Nanoformulated Antiretroviral Drug to the Brain in a Murine Model of NeuroAIDS

Antiretroviral therapy (ART) shows variable blood-brain barrier penetration. This may affect the development of neurological complications of HIV infection. In attempts to attenuate viral growth for the nervous system, cell-based nanoformulations were developed with the focus on improving drug pharmacokinetics. We reasoned that ART carriage could be facilitated within blood-borne macrophages traveling across the blood-brain barrier. To test this idea, an HIV-1 encephalitis (HIVE) rodent model was used where HIV-1-infected human monocyte-derived macrophages were stereotactically injected into the subcortex of severe combined immunodeficient mice. ART was prepared using indinavir (IDV) nanoparticles (NP, nanoART) loaded into murine bone marrow macrophages (BMM, IDV-NP-BMM) after ex vivo cultivation. IDV-NP-BMM was administered i.v. to mice resulting in continuous IDV release for 14 days. Rhodamine-labeled IDV-NP was readily observed in areas of HIVE and specifically in brain subregions with active astrogliosis, microgliosis, and neuronal loss. IDV-NP-BMM treatment led to robust IDV levels and reduced HIV-1 replication in HIVE brain regions. We conclude that nanoART targeting to diseased brain through macrophage carriage is possible and can be considered in developmental therapeutics for HIV-associated neurological disease.

Nanotechnology: A Focus on Nanoparticles as a Drug Delivery System

This review will provide an in-depth discussion on the previous development of nanoparticle-based drug delivery systems (DDS) and discuss original research data that includes the therapeutic enhancement of antiretroviral therapy. The use of nanoparticle DDS will allow practitioners to use drugs to target specific areas of the body. In the treatment of malignancies, the use of nanoparticles as a DDS is making measurable treatment impact. Medical imaging will also utilize DDS to illuminate tumors, the brain, or other cellular functions in the body. The utility of nanoparticle DDS to improve human health is potentially enormous.

Neuroprotective Activities of Sodium Valproate in a Murine Model of Human Immunodeficiency Virus-1 Encephalitis

Human immunodeficiency virus-1 (HIV-1) infection of the nervous system can result in neuroinflammatory events leading first to neuronal dysfunction then to cognitive and behavioral impairments in infected people. The multifaceted nature of the disease process, commonly called HIV-1-associated dementia (HAD), provides a number of adjunctive therapeutic opportunities. One proposed adjunctive therapy is sodium valproate (VPA), an anticonvulsant known to promote neurite outgrowth and increase β-catenin through inhibiting glycogen synthase kinase 3β activity and tau phosphorylation. We now show that VPA treatment of rat cortical neurons exposed to HIV-1 gp120 prevents resultant neurotoxic activities. This includes the induction of significant neurite outgrowth and microtubule-associated protein 2 (MAP-2) and neuron-specific nuclear protein (NeuN) antigens in affected neuronal cell bodies and processes. Similarly, VPA protects severe combined immunodeficient (SCID) mice against the neurodegeneration of HIV-1ADA infected monocyte-derived macrophages (MDMs). In SCID mice with HIV-1 MDM-induced encephalitis, VPA treatment significantly reduced neuronal phosphorylatedβ-catenin and tau without affecting HIV-1 replication or glial activation. We conclude that VPA protects neurons against HIV-1 infected MDM neurotoxicity, possibly through its effects on the phosphorylation of tau and β-catenin. The use of VPA as an adjuvant in treatment of human HAD is being pursued.

NanoART synthesis, characterization, uptake, release and toxicology for human monocyte-macrophage drug delivery.

BACKGROUND Factors limiting the efficacy of conventional antiretroviral therapy for HIV-1 infection include treatment adherence, pharmacokinetics and penetration into viral sanctuaries. These affect the rate of viral mutation and drug resistance. In attempts to bypass such limitations, nanoparticles containing ritonavir, indinavir and efavirenz (described as nanoART) were manufactured to assess macrophage-based drug delivery. METHODS NanoART were made by high-pressure homogenization of crystalline drug with various surfactants. Size, charge and shape of the nanoparticles were assessed. Monocyte-derived macrophage nanoART uptake, drug release, migration and cytotoxicity were determined. Drug levels were measured by reverse-phase high-performance liquid chromatography. RESULTS Efficient monocyte-derived macrophage cytoplasmic vesicle uptake in less than 30 min based on size, charge and coating was observed. Antiretroviral drugs were released over 14 days and showed dose-dependent reduction in progeny virion production and HIV-1 p24 antigen. Cytotoxicities resulting from nanoART carriage were limited. CONCLUSION These results support the continued development of macrophage-mediated nanoART carriage for HIV-1 disease.

Neuroprotective Mechanisms of Lithium in Murine Human Immunodeficiency Virus-1 Encephalitis

Lithium (Li) has garnered considerable interest as a neuroprotective drug for a broad range of nervous system disorders. Its neuroprotective activities occur as a consequence of glycogen synthase kinase-3β (GSK-3β) inhibition leading to downstream blockade of β-catenin and Tau phosphorylation. In the present study, we investigated Li-mediated neuroprotective mechanisms in laboratory and murine human immunodeficiency virus-1 (HIV-1) encephalitis (HIVE) models. In laboratory tests, Li protected neurons from neurotoxic secretions of HIV-1-infected monocyte-derived macrophages (MDMs). This neuroprotection was mediated, in part, through the phosphatidyl inositol 3-kinase/Akt and GSK-3β pathways. To examine the effects of Li treatment in vivo, MDMs were injected into the basal ganglia of severe combined immunodeficient mice and then Li was administered (60 mg/kg/d). Seven days after MDM injection, mice were killed and CNS tissue was collected and subjected to immunocytochemical and Western blot assays for leukocyte and neural antigens, GSK-3β, and key kinase substrates such asβ-catenin and Tau. Numbers of HIV-1 p24 antigen-positive MDMs were unaltered by Li treatment of HIVE mice. Similarly, the greatly increased extent of astrocyte and microglia activation in HIVE mice (10-fold and 16-fold, respectively, compared with unmanipulated controls) was also unaltered by Li. In contrast, Li restored HIVE-associated loss of microtubule-associated protein-2-positive neurites and synaptic density while reducing levels or activity of phospho-Tau Ser202, phospho-β-catenin, and GSK-3β. Electrophysiological recordings showed diminished long-term potentiation in hippocampal slices of HIVE mice that were restored by Li. Based on these data, the use of Li as an adjuvant for HIV-1-associated dementia is now being pursued.

HIV‐1‐infected and/or immune‐activated macrophage‐secreted TNF‐α affects human fetal cortical neural progenitor cell proliferation and differentiation

Neurogenesis, tied to the proliferation, migration and differentiation of neural progenitor cells (NPC) is affected during neurodegenerative diseases, but how neurogenesis is affected during HIV‐1 associated dementia (HAD) has not been fully addressed. Here we test the hypothesis that HIV‐1‐infected and/or immune‐activated brain macrophages affect NPC proliferation and differentiation through the regulation of cytokines. We showed that human monocyte‐derived macrophages (MDM) conditioned medium (MCM) induces a dose dependant increase in NPC proliferation. Conditioned media from lipopolysaccharide (LPS)‐activated MDM (LPS‐MCM) or HIV‐infected MCM (HIV‐MCM) induced a profound increase in NPC proliferation. HIV‐infected and LPS‐activated MCM (HIV+LPS‐MCM) induced the most robust increase in NPC proliferation. Moreover, LPS‐MCM and HIV+LPS‐MCM decreased β‐III‐tubulin and increased GFAP expression, demonstrating an induction of gliogenesis and inhibition of neurogenesis. The increase of NPC proliferation and gliogenesis correlated with increases in production of TNF‐α by infected/activated MDM. Although both IL‐1β and TNF‐α induced NPC proliferation and gliogenesis, these effects were only partially abrogated by soluble TNF‐α receptors R1 and R2 (TNF‐R1R2), but not by the IL‐1 receptor antagonist (IL‐1ra). This indicated that the HIV‐1‐infected/LPS‐activated MCM‐mediated effects were, in part, through TNF‐α. These observations were confirmed in severe combined immunodeficient (SCID) mice with HIV‐1 encephalitis (HIVE). In these HIVE mice, NPC injected with HIV‐infected MDM showed more astrocyte differentiation and less neuronal differentiation compared to NPC injection alone. These observations demonstrated that HIV‐1‐infected and immune‐activated MDM could affect neurogenesis through induction of NPC proliferation, inhibition of neurogenesis, and activation of gliogenesis.

Quantitative magnetic resonance and SPECT imaging for macrophage tissue migration and nanoformulated drug delivery

We posit that the same mononuclear phagocytes (MP) [bone marrow (BM) and blood monocytes, tissue macrophages, microglia, and dendritic cells] which serve as targets, reservoirs, and vehicles for HIV dissemination, can be used as vehicles for antiretroviral therapy (ART). Toward this end, BM macrophages (BMM) were used as carriers for nanoparticle‐formulated indinavir (NP‐IDV), and the cell distribution was monitored by single photon emission computed tomography (SPECT), transverse relation time (T2)∗ weighted magnetic resonance imaging (MRI), histology, and γ‐scintillation spectrometry. BMM labeled with super paramagnetic iron oxide and/or 111 indium oxine were infused i.v. into naïve mice. During the first 7 h, greater than 86% of cell label was recorded within the lungs. On Days 1, 3, 5, and 7, less than 10% of BMM were in lungs, and 74–81% and 13–18% were in liver and spleen, respectively. On a tissue volume basis, as determined by SPECT and MRI, BMM densities in spleen and liver were significantly greater than other tissues. Migration into the lymph nodes on Days 1 and 7 accounted for 1.5–2% of the total BMM. Adoptive transfer of BMM loaded with NP‐IDV produced drug levels in lymphoid and nonlymphoid tissues that exceeded reported therapeutic concentrations by 200‐ to 350‐fold on Day 1 and remained in excess of 100‐ to 300‐fold on Day 14. These data show real‐time kinetics and destinations of macrophage trafficking and demonstrate the feasibility of monitoring macrophage‐based, nanoformulated ART.

Neuroregulatory Events Follow Adaptive Immune-Mediated Elimination of HIV-1-Infected Macrophages: Studies in a Murine Model of Viral Encephalitis

HIV-1-specific cellular immunity serves to eliminate infected cells and disease. However, how this process specifically affects the CNS is poorly understood. To mirror the regulatory events that occur in human brain after HIV-1 infection, a murine model of viral encephalitis was used to study relationships, over time, among lymphocyte-mediated infected cell elimination, innate immune responses, and neuropathology. Nonobese diabetic SCID mice were reconstituted with human PBL and a focal encephalitis induced by intracranial injection of autologous HIV-1-infected, monocyte-derived macrophages (MDM). On days 7, 14, and 21 after MDM injection into the basal ganglia, the numbers of human lymphocytes and mouse monocytes, virus-infected MDM, glial (astrocyte and microglial) responses, cytokines, inducible NO (iNOS), neurotrophic factors, and neuronal Ags were determined in brain by immunohistochemistry, real-time PCR, and Western blot assays. Microglia activation, astrocytosis, proinflammatory cytokines, and iNOS expression accompanied the loss of neuronal Ags. This followed entry of human lymphocytes and mouse monocytes into the brain on days 7 and 14. Elimination of virus-infected human MDM, expression of IL-10, neurotropins, and a down-regulation of iNOS coincided with brain tissue restoration. Our results demonstrate that the degree of tissue damage and repair parallels the presence of infected macrophages and effectors of innate and adaptive immunity. This murine model of HIV-1 encephalitis can be useful in elucidating the role played by innate and adaptive immunity in disease progression and resolution.

Neuroprotective Activities of CEP-1347 in Models of NeuroAIDS

When the nervous system is infected with HIV-1, it commonly results in neuroinflammation leading to overt neuronal dysfunction and subsequent cognitive and behavioral impairments. The multifaceted disease process, now referred to as HIV-1–associated neurocognitive disorders (HAND), provides a range of molecular targets for adjunctive therapies. One is CEP-1347, an inhibitor of mixed lineage kinases that elicits neuroprotective and anti-inflammatory responses in models of neurodegenerative diseases. Since HAND is associated with inflammatory encephalopathy induced by virus infection and mononuclear phagocytes (perivascular macrophages and microglia) immune activation, we investigated whether CEP-1347 could ameliorate disease in laboratory models of HAND. We now demonstrate that CEP-1347 reduces the levels of secreted proinflammatory cytokines and chemokines in HIV-1–infected human macrophages and attenuates dose-dependent neurotoxicity in rodent cortical neurons. CEP-1347–treated mice readily achieve therapeutic drug levels in peripheral blood. HIV-1 encephalitis (HIVE) mice, where human virus-infected monocyte-derived macrophages are stereotactically injected into the basal ganglia of CB17 severe combined immunodeficient mice, received daily intraperitoneal injections of CEP-1347. Here, CEP-1347 treatment of HIVE mice showed a dose-dependent reduction in microgliosis. Dendritic integrity and neuronal loss were sustained and prevented, respectively. These results demonstrate that CEP-1347 elicits anti-inflammatory and neuroprotective responses in an HIVE model of human disease and as such warrants further study as an adjunctive therapy for human disease.

Copolymer-1 Induces Adaptive Immune Anti-inflammatory Glial and Neuroprotective Responses in a Murine Model of HIV-1 Encephalitis

Copolymer-1 (COP-1) elicits neuroprotective activities in a wide range of neurodegenerative disorders. This occurs, in part, by adaptive immune-mediated suppression of microglial inflammatory responses. Because HIV infection and immune activation of perivascular macrophages and microglia drive a metabolic encephalopathy, we reasoned that COP-1 could be developed as an adjunctive therapy for disease. To test this, we developed a novel animal model system that reflects HIV-1 encephalitis in rodents with both innate and adaptive arms of the immune system. Bone marrow-derived macrophages were infected with HIV-1/vesicular stomatitis-pseudotyped virus and stereotactically injected into the basal ganglia of syngeneic mice. HIV-1 pseudotyped with vesicular stomatitis virus envelope-infected bone marrow-derived macrophages induced significant neuroinflammation, including astrogliosis and microglial activation with subsequent neuronal damage. Importantly, COP-1 immunization reduced astro- and microgliosis while diminishing neurodegeneration. Hippocampal neurogenesis was, in part, restored. This paralleled reductions in proinflammatory cytokines, including TNF-α and IL-1β, and inducible NO synthase, and increases in brain-derived neurotrophic factor. Ingress of Foxp3- and IL-4-expressing lymphocytes into brains of COP-1-immunized animals was observed. We conclude that COP-1 may warrant therapeutic consideration for HIV-1-associated cognitive impairments.