Nan Gong

Effects of Pluronic and Doxorubicin on Drug Uptake, Cellular Metabolism, Apoptosis and Tumor Inhibition in Animal Models of MDR Cancers

Cancer chemotherapy is believed to be impeded by multidrug resistance (MDR). Pluronic (triblock copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), PEO-b-PPO-b-PEO) were previously shown to sensitize MDR tumors to antineoplastic agents. This study uses animal models of Lewis lung carcinoma (3LL-M27) and T-lymphocytic leukemia (P388/ADR and P388) derived solid tumors to delineate mechanisms of sensitization of MDR tumors by Pluronic P85 (P85) in vivo. First, non-invasive single photon emission computed tomography (SPECT) and tumor tissue radioactivity sampling demonstrate that intravenous co-administration of P85 with a Pgp substrate, 99Tc-sestamibi, greatly increases the tumor uptake of this substrate in the MDR tumors. Second, 31P magnetic resonance spectroscopy (31P-MRS) in live animals and tumor tissue sampling for ATP suggest that P85 and doxorubicin (Dox) formulations induce pronounced ATP depletion in MDR tumors. Third, these formulations are shown to increase tumor apoptosis in vivo by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and reverse transcription polymerase chain reaction (RT-PCR) for caspases 8 and 9. Altogether, formulation of Dox with P85 results in increased inhibition of the growth solid tumors in mice and represents novel and promising strategy for therapy of drug resistant cancers.

Neuromodulatory Activities of CD4+CD25+ Regulatory T Cells in a Murine Model of HIV-1-Associated Neurodegeneration

HIV-1-associated neurocognitive impairments are intrinsically linked to microglial immune activation, persistent viral infection, and inflammation. In the era of antiretroviral therapy, more subtle cognitive impairments occur without adaptive immune compromise. We posit that adaptive immunity is neuroprotective, serving in both the elimination of infected cells through CD8+ cytotoxic T cell activities and the regulation of neuroinflammatory responses of activated microglia. For the latter, little is known. Thus, we studied the neuromodulatory effects of CD4+ regulatory T cells (Treg; CD4+CD25+) or effector T cells in HIV-1-associated neurodegeneration. A newly developed HIV-1 encephalitis mouse model was used wherein murine bone marrow-derived macrophages are infected with a full-length HIV-1YU2/vesicular stomatitis viral pseudotype and injected into basal ganglia of syngeneic immunocompetent mice. Adoptive transfer of CD3-activated Treg attenuated astrogliosis and microglia inflammation with concomitant neuroprotection. Moreover, Treg-mediated anti-inflammatory activities and neuroprotection were associated with up-regulation of brain-derived neurotrophic factor and glial cell-derived neurotrophic factor expression and down-regulation of proinflammatory cytokines, oxidative stress, and viral replication. Effector T cells showed contrary effects. These results, taken together, demonstrate the importance of Treg in disease control and raise the possibility of their utility for therapeutic strategies.

Genomic and proteomic microglial profiling: pathways for neuroprotective inflammatory responses following nerve fragment clearance and activation

Microglia, a primary immune effector cell of the central nervous system (CNS) affects homeostatic, neuroprotective, regenerative and degenerative outcomes in health and disease. Despite these broad neuroimmune activities linked to specific environmental cues, a precise cellular genetic profile for microglia in the context of disease and repair has not been elucidated. To this end we used nucleic acid microarrays, proteomics, immunochemical and histochemical tests to profile microglia in neuroprotective immune responses. Optic and sciatic nerve (ON and SN) fragments were used to stimulate microglia in order to reflect immune consequences of nervous system injury. Lipopolysaccharide and latex beads‐induced microglial activation served as positive controls. Cytosolic and secreted proteins were profiled by surface enhanced laser desorption ionization‐time of flight (SELDI‐TOF) ProteinChip®, 1D and 2D difference gel electrophoresis. Proteins were identified by peptide sequencing with tandem mass spectrometry, ELISA and western blot tests. Temporal expression of pro‐inflammatory cytokines, antioxidants, neurotrophins, and lysosomal enzyme expression provided, for the first time, a unique profile of secreted microglia proteins with neuroregulatory functions. Most importantly, this molecular and biochemical signature supports a broad range of microglial functions for debris clearance and promotion of neural repair after injury.

Proteomic Modeling for HIV-1 Infected Microglia-Astrocyte Crosstalk

Background HIV-1-infected and immune competent brain mononuclear phagocytes (MP; macrophages and microglia) secrete cellular and viral toxins that affect neuronal damage during advanced disease. In contrast, astrocytes can affect disease by modulating the nervous systems microenvironment. Interestingly, little is known how astrocytes communicate with MP to influence disease. Methods and Findings MP-astrocyte crosstalk was investigated by a proteomic platform analysis using vesicular stomatitis virus pseudotyped HIV infected murine microglia. The microglial-astrocyte dialogue was significant and affected microglial cytoskeleton by modulation of cell death and migratory pathways. These were mediated, in part, through F-actin polymerization and filament formation. Astrocyte secretions attenuated HIV-1 infected microglia neurotoxicity and viral growth linked to the regulation of reactive oxygen species. Conclusions These observations provide unique insights into glial crosstalk during disease by supporting astrocyte-mediated regulation of microglial function and its influence on the onset and progression of neuroAIDS. The results open new insights into previously undisclosed pathogenic mechanisms and open the potential for biomarker discovery and therapeutics that may influence the course of HIV-1-mediated neurodegeneration.

HIV-1-Infected Astrocytes and the Microglial Proteome

The human immunodeficiency virus (HIV) invades the central nervous system early after viral exposure but causes progressive cognitive, behavior, and motor impairments years later with the onset of immune deficiency. Although in the brain, HIV preferentially replicates productively in cells of mononuclear phagocyte (MP; blood borne macrophage and microglia), astrocytes also can be infected, at low and variable frequency, particularly in patients with encephalitis. Among their many functions, astrocytes network with microglia to provide the first line of defense against microbial infection; however, very little is known about astrocytes’ consequences on MP. Here, we addressed this question using co-culture systems of HIV-infected mouse astrocytes and microglia. Pseudotyped vesicular stomatis virus/HIV was used to circumvent the absence of viral receptors and ensure cell genotypic uniformity for studies of intercellular communication. The study demonstrated that infected astrocytes show modest changes in protein elements compared to uninfected cells. In contrast, infected astrocytes induce robust changes in the proteome of HIV-1-infected microglia. Accelerated cell death and redox proteins, among others, were produced in abundance. The observations confirmed the potential of astrocytes to influence the neuropathogenesis of HIV-1 infection by specifically altering the neurotoxic potential of infected microglia and regulating viral maturation.

Monocyte Chemotactic Protein-1 Regulates Voltage-Gated K+ Channels and Macrophage Transmigration

Progressive human immunodeficiency virus (HIV)-1 infection and virus-induced neuroinflammatory responses effectuate monocyte-macrophage transmigration across the blood–brain barrier (BBB). A key factor in mediating these events is monocyte chemotactic protein-1 (MCP-1). Upregulated glial-derived MCP-1 in HIV-1-infected brain tissues generates a gradient for monocyte recruitment into the nervous system. We posit that the inter-relationships between MCP-1, voltage-gated ion channels, cell shape and volume, and cell mobility underlie monocyte transmigration across the BBB. In this regard, MCP-1 serves both as a chemoattractant and an inducer of monocyte-macrophage ion flux affecting cell shape and mobility. To address this hypothesis, MCP-1-treated bone marrow-derived macrophages (BMM) were analyzed for gene and protein expression, electrophysiology, and capacity to migrate across a laboratory constructed BBB. MCP-1 enhanced K+ channel gene (KCNA3) and channel protein expression. Electrophysiological studies revealed that MCP-1 increased outward K+ currents in a dose-dependent manner. In vitro studies demonstrated that MCP-1 increased BMM migration across an artificial BBB, and the MCP-1-induced BMM migration was blocked by tetraethylammonium, a voltage-gated K+ channel blocker. Together these data demonstrated that MCP-1 affects macrophage migratory movement through regulation of voltage-gated K+ channels and, as such, provides a novel therapeutic strategy for neuroAIDS.

Brain ingress of regulatory T cells in a murine model of HIV-1 encephalitis

CD4+CD25+ regulatory T cells (Treg) transform the HIV-1 infected macrophage from a neurotoxic to a neuroprotective phenotype. This was demonstrated previously in a murine model of HIV-1 encephalitis induced by intracranial injection of HIV-1/vesicular stomatitis virus-infected bone marrow macrophages. In this report, relationships between Treg ingress of end organ tissues, notably the brain, and neuroprotection were investigated. Treg from EGFP-transgenic donor mice were expanded, labeled with indium-111, and adoptively transferred. Treg distribution was assayed by single photon emission computed tomography and immunohistochemistry. Treg readily migrated across the blood brain barrier and were retained within virus-induced neuroinflammatory sites. In non-inflamed peripheral tissues (liver and spleen) Treg were depleted. These observations demonstrate that Treg migrate to sites of inflammation where they modulate immune responses.

Genomic and proteomic microglial profiling: pathways for neuroprotective inflammatory responses following nerve fragment clearance and activation: Proteomic fingerprint of nerve-activated microglia

Microglia, a primary immune effector cell of the central nervous system (CNS) affects homeostatic, neuroprotective, regenerative and degenerative outcomes in health and disease. Despite these broad neuroimmune activities linked to specific environmental cues, a precise cellular genetic profile for microglia in the context of disease and repair has not been elucidated. To this end we used nucleic acid microarrays, proteomics, immunochemical and histochemical tests to profile microglia in neuroprotective immune responses. Optic and sciatic nerve (ON and SN) fragments were used to stimulate microglia in order to reflect immune consequences of nervous system injury. Lipopolysaccharide and latex beads‐induced microglial activation served as positive controls. Cytosolic and secreted proteins were profiled by surface enhanced laser desorption ionization‐time of flight (SELDI‐TOF) ProteinChip®, 1D and 2D difference gel electrophoresis. Proteins were identified by peptide sequencing with tandem mass spectrometry, ELISA and western blot tests. Temporal expression of pro‐inflammatory cytokines, antioxidants, neurotrophins, and lysosomal enzyme expression provided, for the first time, a unique profile of secreted microglia proteins with neuroregulatory functions. Most importantly, this molecular and biochemical signature supports a broad range of microglial functions for debris clearance and promotion of neural repair after injury.

Genomic and proteomic microglial profiling

Microglia, a primary immune effector cell of the central nervous system (CNS) affects homeostatic, neuroprotective, regenerative and degenerative outcomes in health and disease. Despite these broad neuroimmune activities linked to specific environmental cues, a precise cellular genetic profile for microglia in the context of disease and repair has not been elucidated. To this end we used nucleic acid microarrays, proteomics, immunochemical and histochemical tests to profile microglia in neuroprotective immune responses. Optic and sciatic nerve (ON and SN) fragments were used to stimulate microglia in order to reflect immune consequences of nervous system injury. Lipopolysaccharide and latex beads‐induced microglial activation served as positive controls. Cytosolic and secreted proteins were profiled by surface enhanced laser desorption ionization‐time of flight (SELDI‐TOF) ProteinChip®, 1D and 2D difference gel electrophoresis. Proteins were identified by peptide sequencing with tandem mass spectrometry, ELISA and western blot tests. Temporal expression of pro‐inflammatory cytokines, antioxidants, neurotrophins, and lysosomal enzyme expression provided, for the first time, a unique profile of secreted microglia proteins with neuroregulatory functions. Most importantly, this molecular and biochemical signature supports a broad range of microglial functions for debris clearance and promotion of neural repair after injury.