Nina Dmitrieva

Depletion of Peripheral Macrophages and Brain Microglia Increases Brain Tumor Titers of Oncolytic Viruses

Clinical trials have proven oncolytic virotherapy to be safe but not effective. We have shown that oncolytic viruses (OV) injected into intracranial gliomas established in rodents are rapidly cleared, and this is associated with up-regulation of markers (CD68 and CD163) of cells of monocytic lineage (monocytes/microglia/macrophages). However, it is unclear whether these cells directly impede intratumoral persistence of OV through phagocytosis and whether they infiltrate the tumor from the blood or the brain parenchyma. To investigate this, we depleted phagocytes with clodronate liposomes (CL) in vivo through systemic delivery and ex vivo in brain slice models with gliomas. Interestingly, systemic CL depleted over 80% of peripheral CD163+ macrophages in animal spleen and peripheral blood, thereby decreasing intratumoral infiltration of these cells, but CD68+ cells were unchanged. Intratumoral viral titers increased 5-fold. In contrast, ex vivo CL depleted only CD68+ cells from brain slices, and intratumoral viral titers increased 10-fold. These data indicate that phagocytosis by both peripheral CD163+ and brain-resident CD68+ cells infiltrating tumor directly affects viral clearance from tumor. Thus, improved therapeutic efficacy may require modulation of these innate immune cells. In support of this new therapeutic paradigm, we observed intratumoral up-regulation of CD68+ and CD163+ cells following treatment with OV in a patient with glioblastoma.

Lithium inhibits invasion of glioma cells; possible involvement of glycogen synthase kinase-3

Therapies targeting glioma cells that diffusely infiltrate normal brain are highly sought after. Our aim was to identify novel approaches to this problem using glioma spheroid migration assays. Lithium, a currently approved drug for the treatment of bipolar illnesses, has not been previously examined in the context of glioma migration. We found that lithium treatment potently blocked glioma cell migration in spheroid, wound-healing, and brain slice assays. The effects observed were dose dependent and reversible, and worked using every glioma cell line tested. In addition, there was little effect on cell viability at lithium concentrations that inhibit migration, showing that this is a specific effect. Lithium treatment was associated with a marked change in cell morphology, with cells retracting the long extensions at their leading edge. Examination of known targets of lithium showed that inositol monophosphatase inhibition had no effect on glioma migration, whereas inhibition of glycogen synthase kinase-3 (GSK-3) did. This suggested that the effects of lithium on glioma cell migration could possibly be mediated through GSK-3. Specific pharmacologic GSK-3 inhibitors and siRNA knockdown of GSK-3alpha or GSK-3beta isoforms both reduced cell motility. These data outline previously unidentified pathways and inhibitors that may be useful for the development of novel anti-invasive therapeutics for the treatment of brain tumors.

Effects of the destruction of starburst-cholinergic amacrine cells by the toxin AF64A on rabbit retinal directional selectivity

The effects of intraocular injections of ethylcholine mustard aziridinium ion (AF64A), an irreversible inhibitor of choline uptake, on the rabbit retina were assessed electrophysiologically, pharmacologically, anatomically, and behaviorally. Survival times from 1 day to 30 days were investigated. After 24 h, the shortest time tested, the directional selectivity of On-Off responding ganglion cells having the characteristic morphology of On-Off directionally selective directionally selective (DS) ganglion cells, as revealed by intracellular dye injection, was significantly reduced, both by an apparent decrease of preferred direction responses and an increase in responses to null-direction movement. No toxin-mediated changes in the dendritic trees of these cells were noted. Cells in AF64A-affected retinas having the DS morphology did not respond significantly to GABAergic or cholinergic agents such as picrotoxin and eserine, but did respond to nicotine. Recordings from a small random sample of other ganglion cell classes in the same retinas yielded no obvious changes in response properties. The direct effects on starburst (cholinergic) amacrine cells, which were identified by intraocular injection of the fluorescent dye DAPI with the AF64A, were investigated by intracellular injections of Lucifer yellow, and by immunohistochemical staining with antibodies to choline acetyltransferase (ChAT). Although starburst amacrine cell somas survived the AF64A treatment for at least several days, the dendrites could not be visualized by fluorescent dye injection in affected retinas due to dye leakage of the injected fluorescent dye from either the soma or proximal dendritic region. ChAT staining revealed a sequence in which ChAT-positive cells were undetectable first in the inner nuclear layer, and then in the ganglion cell layer. Cholinergic amacrine cells in the central retina were also affected before those in the periphery. The electrophysiological changes observed typically preceded the loss of ChAT activity. Behavioral tests for optokinetic nystagmus responses also revealed a lack of such responses in the affected eyes.

Synaptic Connections of Starburst Amacrine Cells and Localization of Acetylcholine Receptors in Primate Retinas

Starburst amacrine cells in the macaque retina were studied by electron microscopic immunohistochemistry. We found that these amacrine cells make a type of synapse not described previously; they are presynaptic to axon terminals of bipolar cells. We also confirmed that starburst amacrine cells are presynaptic to ganglion cell dendrites and amacrine cell processes. In order to determine the functions of these synapses, we localized acetylcholine receptors using a monoclonal antibody (mAb210) that recognizes human α3‐ and α5‐containing nicotinic receptors and also antisera against the five known subtypes of muscarinic receptors. The majority of the mAb210‐immunoreactive perikarya were amacrine cells and ganglion cells, but a subpopulation of bipolar cells was also labeled. A subset of bipolar cells and a subset of horizontal cells were labeled with antibodies to M3 muscarinic receptors. A subset of amacrine cells, including those that contain cholecystokinin, were labeled with antibodies to M2 receptors. Taken together, these results suggest that acetylcholine can modulate the activity of retinal ganglion cells by multiple pathways. J. Comp. Neurol. 461:76–90, 2003.

Enhanced antitumor efficacy of vasculostatin (Vstat120) expressing oncolytic HSV-1.

Oncolytic viral (OV) therapy is a promising therapeutic modality for brain tumors. Vasculostatin (Vstat120) is the cleaved and secreted extracellular fragment of brain-specific angiogenesis inhibitor 1 (BAI1), a brain-specific receptor. To date, the therapeutic efficacy of Vstat120 delivery into established tumors has not been investigated. Here we tested the therapeutic efficacy of combining Vstat120 gene delivery in conjunction with OV therapy. We constructed RAMBO (Rapid Antiangiogenesis Mediated By Oncolytic virus), which expresses Vstat120 under the control of the herpes simplex virus (HSV) IE4/5 promoter. Secreted Vstat120 was detected as soon as 4 hours postinfection in vitro and was retained for up to 13 days after OV therapy in subcutaneous tumors. RAMBO-produced Vstat120 efficiently inhibited endothelial cell migration and tube formation in vitro (P = 0.0005 and P = 0.0184, respectively) and inhibited angiogenesis (P = 0.007) in vivo. There was a significant suppression of intracranial and subcutaneous glioma growth in mice treated with RAMBO compared to the control virus, HSVQ (P = 0.0021 and P < 0.05, respectively). Statistically significant reduction in tumor vascular volume fraction (VVF) and microvessel density (MVD) was observed in tumors treated with RAMBO. This is the first study to report the antitumor effects of Vstat120 delivery into established tumors and supports the further development of RAMBO as a possible cancer therapy.

Amacrine, ganglion, and displaced amacrine cells in the rabbit retina express nicotinic acetylcholine receptors.

Acetylcholine (ACh) in the vertebrate retina affects the response properties of many ganglion cells, including those that display directional selectivity. Three beta and eight alpha subunits of neuronal nicotinic acetylcholine receptors (nAChRs) have been purified and antibodies have been raised against many of them. Here we describe biochemical and immunocytochemical studies of nAChRs in the rabbit retina. Radioimmunoassay and Western blot analysis demonstrated that many of the nAChRs recognized by a monoclonal antibody (mAb210) contain beta2 subunits, some of which are in combination with alpha3 and possibly other subunits. MAb210-immunoreactive cells in the inner nuclear layer (INL) were 7-14 microm in diameter and were restricted to the innermost one or two tiers of cells, although occasional cells were found in the middle of the INL. At least 60% of the cells in the ganglion cell layer (GCL) in the visual streak displayed mAb210 immunoreactivity; these neurons ranged from 7-18 microm in diameter. The dendrites of cells in both the INL and GCL could sometimes be followed until they entered one of two dense, poorly defined, bands of processes in the inner plexiform layer (IPL) that overlap the arbors of the cholinergic starburst cells. Parvalbumin and serotonin-positive neurons did not exhibit nAChR immunoreactivity. Although the level of receptor expression appeared to be low, mAb210 immunoreactivity was observed in some of the ChAT-positive (starburst) amacrine cells.

Chondroitinase ABC I-Mediated Enhancement of Oncolytic Virus Spread and Antitumor Efficacy

Purpose: The inhibitory role of secreted chondroitin sulfate proteoglycans on oncolytic viral (OV) therapy was examined. Chondroitinase ABC (Chase-ABC) is a bacterial enzyme that can remove chondroitin sulfate glycosaminoglycans from proteoglycans without any deleterious effects in vivo. We examined the effect of Chase-ABC on OV spread and efficacy. Experimental Design: Three-dimensional glioma spheroids placed on cultured brain slices were utilized to evaluate OV spread. Replication-conditional OV-expressing Chase-ABC (OV-Chase) was engineered using HSQuik technology and tested for spread and efficacy in glioma spheroids. Subcutaneous and intracranial glioma xenografts were utilized to compare antitumor efficacy of OV-Chase, rHsvQ (control), and PBS. Titration of viral particles was performed from OV-treated subcutaneous tumors. Glioma invasion was assessed in collagen-embedded glioma spheroids in vitro and in intracranial tumors. All statistical tests were two sided. Results: Treatment with Chase-ABC in cultured glioma cells significantly enhanced OV spread in glioma spheroids grown on brain slices (P < 0.0001). Inoculation of subcutaneous glioma xenografts with Chase-expressing OV significantly increased viral titer (>10 times, P = 0.0008), inhibited tumor growth, and significantly increased overall animal survival (P < 0.006) compared with treatment with parental rHsvQ virus. Single OV-Chase administration in intracranial xenografts also resulted in longer median survival of animals than rHsvQ treatment (32 vs. 21 days, P < 0.018). Glioma cell migration and invasion were not increased by OV-Chase treatment. Conclusions: We conclude that degradation of glioma extracellular matrix with OV-expressing bacterial Chase-ABC enhanced OV spread and antitumor efficacy. Clin Cancer Res; 17(6); 1362–72. ©2010 AACR.

Expression of Alpha 7 Nicotinic Acetylcholine Receptors by Bipolar, Amacrine, and Ganglion Cells of the Rabbit Retina

Cholinergic agents affect the light responses of many ganglion cells (GCs) in the mammalian retina by activating nicotinic acetylcholine receptors (nAChRs). Whereas retinal neurons that express β2 subunit-containing nAChRs have been characterized in the rabbit retina, expression patterns of other nAChR subtypes remain unclear. Therefore, we evaluated the expression of α7 nAChRs in retinal neurons by means of single-, double-, and triple-label immunohistochemistry. Our data demonstrate that, in the rabbit retina, several types of bipolar cells, amacrine cells, and cells in the GC layer express α7 nAChRs. At least three different populations of cone bipolar cells exhibited α7 labeling, whereas glycine-immunoreactive amacrine cells comprised the majority of α7-positive amacrine cells. Some GABAergic amacrine cells also displayed α7 immunoreactivity; α7 labeling was never detected in rod bipolar cells or rod amacrine cells (All amacrine cells). Our data suggest that activation of α7 nAChRs by acetylcholine (ACh) or choline may affect glutamate release from several types of cone bipolar cells, modulating GC responses. ACh-induced excitation of inhibitory amacrine cells might cause either inhibition or disinhibition of other amacrine and GC circuits. Finally, ACh may act on α7 nAChRs expressed by GCs themselves.

The relationship between GABA-containing cells and the cholinergic circuitry in the rabbit retina

As a part of ongoing efforts to understand the cholinergic circuitry in the mammalian retina, we studied the coexpression of nicotinic acetylcholine receptors (nAChRs) and gamma-aminobutyric acid (GABA), the GABA transporter 1 (GAT-1), or choline acetyltransferase (ChAT) immunoreactivity in the rabbit retina. Double-label experiments with monoclonal antibody 210 (mAb 210) against nAChRs and antibodies against GABA revealed that several populations of GABA-containing amacrine, displaced amacrine, and ganglion cells displayed nAChR immunoreactivity. Some of them also exhibited ChAT immunoreactivity and were identified as the cholinoceptive starburst cells. Other GABAergic amacrine cells positive for mAb 210 were not cholinergic. Simultaneous visualization of mAb 210 and GAT-1 immunoreactivity revealed that 10% of GAT-1 immunoreactive amacrine cells contained nAChRs. Ninety-nine percent of the GAT-1 labeled cells demonstrated GABA immunoreactivity, but only 75% of the GABAergic cells were outlined by GAT-1 staining. Neither population of starburst cells exhibited GAT-1 immunoreactivity. Thus, mAb 210 expressing, GAT-1 positive cells in the rabbit retina constitute a noncholinergic subset of GABAergic amacrine cells. Taken together, our results suggest that some GABAergic amacrine cells are cholinoceptive, raising the possibility that ACh, acting through nAChRs, can modulate the release of GABA in the rabbit retina.

Identification of cholinoceptive glycinergic neurons in the mammalian retina

The light‐evoked release of acetylcholine (ACh) affects the responses of many retinal ganglion cells, in part via nicotinic acetylcholine receptors (nAChRs). nAChRs that contain β2α3 neuronal nicotinic acetylcholine receptors have been identified and localized in the rabbit retina; these nAChRs are recognized by the monoclonal antibody mAb210. We have examined the expression of β2α3 nAChRs by glycinergic amacrine cells in the rabbit retina and have identified different subpopulations of nicotinic cholinoceptive glycinergic cells using double and triple immunohistochemistry with quantitative analysis. Here we demonstrate that about 70% of the cholinoceptive amacrine cells in rabbit retina are glycinergic cells. At least three nonoverlapping subpopulations of mAb210 glycine‐immunoreactive cells can be distinguished with antibodies against calretinin, calbindin, and γ‐aminobutyric acid (GABA)A receptors. The cholinergic cells in rabbit retina are thought to synapse only on other cholinergic cells and ganglion cells. Thus, the expression of β2α3 nAChRs on diverse populations of glycinergic cells is puzzling. To explore this finding, the subcellular localization of β2α3 was studied at the electron microscopic level. mAb210 immunoreactivity was localized on the dendrites of amacrines and ganglion cells throughout the inner plexiform layer, and much of the labeling was not associated with recognizable synapses. Thus, our findings indicate that ACh in the mammalian retina may modulate glycinergic circuits via extrasynaptic β2α3 nAChRs. J. Comp. Neurol. 456:167–175, 2003.