Mosaicism for a specific somatic mitochondrial DNA mutation in adult human brain.

The levels of a specific mitochondrial DNA deletion (mtDNA4977) measured in 12 brain regions of 6 normal adults 39 to 82 years old exhibited striking variation among anatomical locations. Comparisons of the same region among individuals showed an increase of mtDNA4977 with age. The three regions with the highest levels, caudate, putamen and substantia nigra, are characterized by a high dopamine metabolism. The breakdown of dopamine by mitochondrial MAO produces H2O2 which can lead to oxygen radical formation. We suggest that mtDNA4977 may be the "tip of the iceberg" of the spectrum of somatic mutations produced by oxidative damage.

Tumor necrosis factor identified in multiple sclerosis brain.

Frozen brain specimens from patients with multiple sclerosis (MS) and other neurologic diseases were analyzed using immunocytochemical techniques for the presence of TNF. In brain lesions in MS, and subacute sclerosing panencephalitis, TNF+ cells were demonstrated. At the lesion site in MS, TNF+ staining is associated with both astrocytes and macrophages. These observations were not made in Alzheimer's disease or normal brain tissue. The presence of TNF in MS lesions suggests a significant role for cytokines and the immune response in disease progression.

Transgenic interleukin 10 prevents induction of experimental autoimmune encephalomyelitis.

The effectiveness of interleukin 10 (IL-10) in the treatment of autoimmune-mediated central nervous system inflammation is controversial. Studies of the model system, experimental autoimmune encephalomyelitis (EAE), using various routes, regimens, and delivery methods of IL-10 suggest that these variables may affect its immunoregulatory function. To study the influence of these factors on IL-10 regulation of EAE pathogenesis, we have analyzed transgenic mice expressing human IL-10 (hIL-10) transgene under the control of a class II major histocompatibility complex (MHC) promoter. The hIL-10 transgenic mice are highly resistant to EAE induced by active immunization, and this resistance appears to be mediated by suppression of autoreactive T cell function. Myelin-reactive T helper 1 cells are induced but nonpathogenic in the IL-10 transgenic mice. Antibody depletion confirmed that EAE resistance is dependent on the presence of the transgenic IL-10. Mice expressing the hIL-10 transgene but not the endogenous murine IL-10 gene demonstrated that transgenic IL-10 from MHC class II-expressing cells is sufficient to block induction of EAE. This study demonstrates that IL-10 can prevent EAE completely if present at appropriate levels and times during disease induction.

The unfolded protein response regulator GRP78/BiP is required for endoplasmic reticulum integrity and stress-induced autophagy in mammalian cells.

In mammalian cells, endoplasmic reticulum (ER) stress has recently been shown to induce autophagy and the induction requires the unfolded protein response (UPR) signaling pathways. However, little is known whether autophagy regulates UPR pathways and how specific UPR targets might control autophagy. Here, we demonstrated that although ER stress-induced autophagy was suppressed by class III phosphatidylinositol-3'-kinase (PI3KC3) inhibitor 3-methyladenine (3-MA), wortmannin and knockdown of Beclin1 using small interfering RNA (siRNA), only 3-MA suppressed UPR activation. We discovered that the UPR regulator and ER chaperone GRP78/BiP is required for stress-induced autophagy. In cells in which GRP78 expression was knocked down by siRNA, despite spontaneous activation of UPR pathways and LC3 conversion, autophagosome formation induced by ER stress as well as by nutrition starvation was inhibited. GRP78 knockdown did not disrupt PI3KC3-Beclin1 association. However, electron microscopic analysis of the intracellular organelle structure reveals that the ER, a putative membrane source for generating autophagosomal double membrane, was massively expanded and disorganized in cells in which GRP78 was knocked down. ER expansion is known to be dependent on the UPR transcription factor XBP-1. Simultaneous knockdown of GRP78 and XBP-1 recovered normal levels of stress-induced autophagosome formation. Thus, these studies uncover 3-MA as an inhibitor of UPR activation and establish GRP78 as a novel obligatory component of autophagy in mammalian cells.

Inhibition of metastatic tumor growth in nude mice by portal vein infusions of matrix-targeted retroviral vectors bearing a cytocidal cyclin G1 construct.

Tumor invasion and associated angiogenesis evoke a remodeling of extracellular matrix components. Retroviral vectors bearing auxiliary matrix-targeting motifs (ie., collagen-binding polypeptides) accumulate at sites of newly exposed collagen, thus promoting tumor site-specific gene delivery. In this study, we assessed the antitumor effects of serial portal vein infusions of matrix-targeted vectors bearing a mutant cyclin G1 (dnG1) construct in a nude mouse model of liver metastasis. The size of tumor foci was dramatically reduced in dnG1 vector-treated mice compared with that in control vector- or PBS-treated animals (P = 0.0002). These findings represent a definitive advance in the development of targeted injectable vectors for metastatic cancer.

Treatment of recurrent malignant gliomas with chronic oral high-dose tamoxifen.

The present clinical trial was undertaken to assess the clinical safety and possible efficacy of administering tamoxifen to patients with recurrent malignant glial tumors at dosages calculated to achieve levels sufficient to inhibit protein kinase C within the tumor cells. Chronic p.o. tamoxifen was administered in very high dosages to 32 patients (20 males and 12 females; age range, 26-75 years; mean, 49 years) with histologically verified malignant glioma [anaplastic astrocytoma (12 patients) or glioblastoma multiforme (20 patients)] who had demonstrated clinical and radiographical progression or recurrence following external beam radiation therapy (and additional chemotherapy in 11; immunotherapy in 2). The dosage of tamoxifen administered was 200 mg/day to males and 160 mg/day to females given in a twice daily schedule. Clinical and radiographical (defined as a greater than 50% decrease in volume of the enhancing lesion volume on magnetic resonance imaging and a decrease in metabolic activity on serial positron emission tomographic scans) response was noted in 8 patients (25%; 4/12 with anaplastic astrocytoma and 4/20 glioblastoma multiforme), with an additional 6 patients (19%) exhibiting stabilization of disease with minimal side effects. Median survival from the time of diagnosis for the entire cohort was 24 months (104 weeks), for the anaplastic astrocytoma group 42.5 months (185 weeks), and for the glioblastoma group 17.4 months (75.5 weeks). From the initiation of tamoxifen, median survival for the entire cohort was 10.1 months (44 weeks), for the anaplastic astrocytoma group 16 months (69 weeks), and for the glioblastoma group 7.2 months (31 weeks). The mean length of follow-up of all patients after initiating tamoxifen was 16 months (69 weeks), while the mean length of follow-up of alive patients is 22.6 months (98 weeks) (range up to 51 months). These data suggest that a subgroup of patients with malignant gliomas respond or stabilize with chronic high-dose tamoxifen therapy. This therapy may represent an alternative or adjuvant to existing chemotherapies for these tumors; further clinical trials are warranted.

Enhancement of radiosensitivity in human malignant glioma cells by hypericin in vitro.

Hypericin, an antidepressant and antiviral agent being evaluated in phase I and II trials for patients with HIV infection, is known to be a potent protein kinase C inhibitor. We have investigated its effects on cellular response to radiation via a tetrazolium-formazan cell growth rate assay using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and clonogenic assay in three human glioblastoma cell lines, U87-MG, A-172, and T98G, and a low-passage malignant glioma culture, 93-492. At a concentration of 5 microM, hypericin inhibited these cells slightly but caused significant radiosensitization (e.g., the cell survival rate after the radiation treatment was 50.2 and 26.0% in cells treated with 6 Gy and 6 Gy plus 5 microM hypericin in U87-MG cells, respectively; P = 0.0285). Hypericin also enhanced the radiosensitivity significantly in the low-passage glioma 93-492 cells. These findings suggest that hypericin represents a potential new agent in combination with radiation therapy of malignant gliomas.

Retroviral vector-mediated transfer of an antisense cyclin G1 construct inhibits osteosarcoma tumor growth in nude mice.

Metastatic osteosarcoma is a potential target for gene therapy, because conventional therapies are only palliative and metastatic disease is invariably fatal. Overexpression of the cyclin G1 (CYCG1) gene is frequently observed in human osteosarcoma cells, and its continued expression is found to be essential for their survival. Previously, we reported that down-regulation of cyclin G1 protein expression induced cytostatic and cytocidal effects in human MG-63 osteosarcoma cells (Skotzko et al., Cancer Research, 1995). Here, we extend these findings in a tumorigenic MNNG/HOS cell line and report on the effective inhibition of tumor growth in vivo by an antisense cyclin G1 retroviral vector when delivered as concentrated high titer vector supernatants directly into rapidly growing subcutaneous tumors in athymic nude mice. Histologic sections from the antisense cyclin G1 vector-treated tumors showed decreased mitotic indices and increased stroma formation within the residual tumors. Furthermore, in situ analysis of the cell-cycle kinetics of residual tumor cells revealed a decrease in the number of cells in S and G2/M phases of the cell cycle concomittant with an accumulation of cells in the G1 phase. Taken together, these studies demonstrate in vivo efficacy of a high-titer antisense cyclin G1 retroviral vector in an animal model of osteosarcoma.

Retrovirus-mediated suicide gene transduction in the vitreous cavity of the eye: feasibility in prevention of proliferative vitreoretinopathy.

In proliferative vitreoretinopathy (PVR), retinal pigment epithelial cells, fibroblasts, or other proliferating cells form contractile membranes in the vitreous cavity of the eye, resulting in traction retinal detachment. Retroviral vector-mediated transfer is a suitable method to transduce the herpes simplex virus thymidine kinase (HSV-tk) gene into proliferating cells in PVR, allowing for the selective killing of these cells. To determine the potential of gene transduction in the environment of the vitreous cavity, we evaluated the effect of vitreous humor on retroviral vector-mediated gene transduction of rabbit dermal fibroblasts in vitro and studied in vivo transduction in rabbit experimental PVR with retroviral vector G1BgSvNa. In addition, we studied the bystander effect in vitro and in vivo in a rabbit model of PVR, with low percentages of HSV-tk-positive cells. Finally, we evaluated the efficacy of intravitreal administration of HSV-tk retroviral vector G1TkSvNa followed by ganciclovir (GCV) in the prevention of experimental PVR. Vitreous humor reduced gene transfer efficiency in vitro in a dose-dependent manner. LacZ expression was found in cells of preretinal or intravitreal membranes of animals of both in vivo and in vitro transduction groups; however, in vivo transduction resulted in a decreased number of transduced cells, with a relative transduction efficiency of approximately 2%. Transduction of HSV-tk was associated with a powerful bystander effect both in vitro and in vivo with significant effects even when HSV-tk-positive cells represented only 1% of the population. In vivo transduction with G1TkSvNa followed by GCV significantly inhibited the development of PVR (p < 0.05). These results suggest that retroviral vector-mediated transfer of HSV-tk into the proliferating cells in PVR may be feasible and may provide a novel therapeutic strategy for this disease.

Systemically expressed soluble Tie2 inhibits intraocular neovascularization.

Retinal and choroidal neovascularization are the most frequent causes of severe and progressive vision loss. Studies have demonstrated that Tie2, an endothelial-specific receptor tyrosine kinase, plays a key role in angiogenesis. In this study, we determined whether adenovirus-mediated gene delivery of extracellular domain of the Tie2 receptor (ExTek) could inhibit experimental retinal and choroidal neovascularization. Immunofluorescence histochemistry with a monoclonal antibody to human Tie2 showed that Tie2 expression is prominent around and within the base of newly formed blood vessels of retinal and choroidal neovascular lesions. A single intramuscular injection of adenovirus expressing ExTek genes achieved plasma levels of ExTek exceeding 500 microg/ml in mice for 10 days (in neonates) and 7 days (in adults). This treatment inhibited retinal neovascularization by 47% (p < 0.05) in a murine model of ischemia-induced retinopathy. The same treatment reduced the incidence and extent of sodium fluorescein leakage from choroidal neovascular lesions by 52% (p < 0.05) and 36% (p < 0.01), respectively, in a laser-induced murine choroidal neovascularization model. The same mice showed a 45% (p < 0.001) reduction of integrated area of the choroidal neovascularization. These findings indicate that Tie2 signaling is a common component of the angiogenic pathway in both retinal and choroidal neovascularization, providing a potentially useful target in the treatment of intraocular neovascular diseases.

Systemic administration of a matrix-targeted retroviral vector is efficacious for cancer gene therapy in mice.

Targeting cytocidal vectors to tumors and associated vasculature in vivo is a long-standing goal of human gene therapy. In the present study, we demonstrated that intravenous infusion of a matrix (i.e., collagen)-targeted retroviral vector provided efficacious gene delivery of a cytocidal mutant cyclin G1 construct (designated Mx-dnG1) in human cancer xenografts in nude mice. A nontargeted CAE-dnG1 vector (p = 0.014), a control matrix-targeted vector bearing a marker gene (Mx-nBg; p = 0.004), and PBS served as controls (p = 0.001). Enhanced vector penetration and transduction of tumor nodules (35.7 +/- 1.4%, mean +/- SD) correlated with therapeutic efficacy without associated toxicity. Kaplan-Meier survival studies were conducted in mice treated with PBS placebo, the nontargeted CAE-dnG1 vector, and the matrix-targeted Mx-dnG1 vector. Using the Tarone log-rank test, the overall p value for comparing all three groups simultaneously was 0.003, with a trend that was significant to a level of 0.004, indicating that the probability of long-term control of tumor growth was significantly greater with the matrix-targeted Mx-dnG1 vector than with the nontargeted CAE-dnG1 vector or PBS placebo. The present study demonstrates that a matrix-targeted retroviral vector deployed by peripheral vein injection (1) accumulated in angiogenic tumor vasculature within 1 hr, (2) transduced tumor cells with high-level efficiency, and (3) enhanced therapeutic gene delivery and long-term efficacy without eliciting appreciable toxicity.

Primary rhabdomyosarcoma of the cerebellum--a light, electron microscopic, and immunohistochemical study.

A primary cerebellar rhabdomyosarcoma (RMS) in a six and a half year old boy is reported. Microscopy of the surgical material revealed lobules of closely packed cells with a high mitotic rate, pleomorphic hyperchromatic nuclei and scant cytoplasm. At their periphery, the lobules merged with rounded cells with similar nuclei but more abundant cytoplasm. These areas were surrounded by interlacing fascicles of strap cells, which were occasionally multinucleated and showed cross striations. Electron microscopy (EM) revealed the primitive nature of the closely packed cells; however, occasional intermediate size filaments were present within their cytoplasm and focal basement membrane accumulation was observed. Cells with more abundant cytoplasm had large accumulations of thick and thin filaments while strap cells showed well-developed cross striations. Immunohistochemical studies (peroxidase-antiperoxidase technique) showed vimentin in the primitive cells and desmin, myoglobin and adenosine triphosphatase as the tumor cells appeared more differentiated. Immunoreaction with antibodies against glial fibrillary acidic protein, S-100 protein and neurofilament protein were negative. Electron microscopic and immunohistochemical studies in this case demonstrated that this was an exclusively mesenchymal tumor with rhabdomyoblastic differentiation and that the pattern of differentiation follows that seen in normal myogenesis.

Soluble TNF-alpha receptors are constitutively shed and downregulate adhesion molecule expression in malignant gliomas.

The regulation of adhesion molecule expression in malignant gliomas by tumor necrosis factor-alpha (TNF) and soluble TNF receptors (TNFR) was examined in the malignant glioma cell line A-172 and in 2 primary glioblastoma cell cultures (LA-492 and LA-567). A-172 cells expressed only the p55 TNF receptor transcripts and protein. The 2 primary cell cultures expressed both the p55 and p75 TNF receptors. In A-172 cells and in 1 of 2 primary glioma cell cultures, TNF upregulated the expression of ICAM-1 and VCAM-1, A-172 and both primary glioma cultures also shed their TNF receptors in the absence of activation by stimulating agents. Soluble p55 (sp55) receptors, but not soluble p75 (sp75) receptors, were found to reduce the TNF induced VCAM-1 and ICAM-1 expression in both the glioma cell line and the primary cell culture. Immunostaining of malignant glioma sections confirmed the presence of soluble TNFR and adhesion molecule expression in glioma cells in situ. These data suggest that soluble TNF receptors may play a role in the mechanism by which malignant gliomas downregulate the effects of infiltrating immune-competent cells.

Viral induced demyelination.

Viral induced demyelination, in both humans and rodent models, has provided unique insights into the cell biology of oligodendroglia, their complex cell-cell interactions and mechanisms of myelin destruction. They illustrate mechanisms of viral persistence, including latent infections in which no infectious virus is readily evident, virus reactivation and viral-induced tissue damage. These studies have also provided excellent paradigms to study the interactions between the immune system and the central nervous system (CNS). Although of interest in their own right, an understanding of the diverse mechanisms used by viruses to induce demyelination may shed light into the etiology and pathogenesis of the common demyelinating disorder multiple sclerosis (MS). This notion is supported by the persistent view that a viral infection acquired during adolescence might initiate MS after a long period of quiescence. Demyelination in both humans and rodents can be initiated by infection with a diverse group of enveloped and non-enveloped RNA and DNA viruses (Table 1). The mechanisms that ultimately result in the loss of CNS myelin appear to be equally diverse as the etiological agents capable of causing diseases which result in demyelination. Although demyelination can be a secondary result of axonal loss, in many examples of viral induced demyelination, myelin loss is primary and associated with axonal sparing. This suggests that demyelination induced by viral infections can result from: 1) a direct viral infection of oligodendroglia resulting in cell death with degeneration of myelin and its subsequent removal; 2) a persistent viral infection, in the presence or absence of infectious virus, resulting in the loss of normal cellular homeostasis and subsequent oligodendroglial death; 3) a vigorous virus-specific inflammatory response wherein the virus replicates in a cell type other than oligodendroglia, but cytokines and other immune mediators directly damage the oligodendroglia or the myelin sheath; or 4) infection initiates activation of an immune response specific for either oligodendroglia or myelin components. Virus-induced inflammation may be associated with the processing of myelin or oligodendroglial components and their presentation to the host's own T cell compartment. Alternatively, antigenic epitopes derived from the viral proteins may exhibit sufficient homology to host components that the immune response to the virus activates autoreactive T cells, i.e. molecular mimicry. Although it is not clear that each of these potential mechanisms participates in the pathogenesis of human demyelinating disease, analysis of the diverse demyelinating viral infections of both humans and rodents provides examples of many of these potential mechanisms.

Mitochondrial DNA deletions are rare in the free radical-rich retinal environment.

We measured the levels of a somatic, 4977 bp deletion of mitochondrial DNA (mtDNA4977) in paired neural retinal and optic nerve tissues from 14 adults and 1 infant using a quantitative PCR assay. MtDNA is prone to free radical damage, and areas in the brain that are exposed to high levels of free radicals are observed to accumulate higher levels of the mtDNA4977 deletion. The levels of mtDNA deletions also increase with age in many tissues. Despite the presence of a free radical rich environment, mtDNA from the neural retina possessed extremely low mtDNA4977 levels (0.0001-0.001%). Deletion levels were always lower than those in the optic nerve from the same eye and do not appear to increase with age. Our results suggest that antioxidant defenses in the neural retina are effective in protecting mtDNA against oxidative damage.

Retinal pathology in Alzheimer's disease. I. Ganglion cell loss in foveal/parafoveal retina.

Morphometric analysis of the numbers of neurons in the ganglion cell layer (GCL) of the central retina (fovea/foveola/parafoveal retina) in eyes from 9 Alzheimer's disease (AD) and 11 age-matched control cases revealed an overall decrease of 25% in total numbers of neurons in AD as compared with control eyes. Detailed analyses of GCL neurons at various eccentricities from the foveola showed that the greatest decrease in neuronal density (43% decrease) occurred in the central 0-0.5 mm (foveal region), while at 0.5-1 mm and at 1-1.5 mm eccentricities, neuronal loss amounted to 24 and 26%, respectively. The temporal region of the central retina appeared most severely affected, with up to 52% decrease in neuronal density near the foveola (central 0-0.5 mm eccentricity). There was close agreement between fellow eyes analyzed separately for three AD and three control cases. Analysis of neuronal sizes showed that all sizes of neurons were similarly affected in AD. In the GCL of control retinas, neurons decreased with age (coefficient of correlation = -0.67), while in AD retinas no such relationship was evident. Since in the central 0-2 mm region of the retina 97% of neurons in the GCL are ganglion cells (while the remaining 3% consist of displaced amacrine cells), these results demonstrate extensive ganglion cell loss in the central retina in AD.

Retinal pathology in Alzheimer's disease. II. Regional neuron loss and glial changes in GCL.

Detailed analyses of neuronal and astrocyte cell numbers in the ganglion cell layer (GCL) of whole-mounted peripheral retinas from 16 Alzheimer's disease (AD) and 11 control eyes (11 and 9 cases, respectively) demonstrate extensive neuronal loss throughout the entire retina in AD as compared to control eyes. The observed neuronal loss is most pronounced in the superior and inferior quadrants, ranging between 40 and 49% throughout the midperipheral regions, and reaching 50-59% in the far peripheral inferior retina, while the overall neuronal loss throughout the entire retina amounts to 36.4% (p < 0.004). Although the 16% increase in astrocyte numbers is not significant, the ratio of astrocytes to neurons is significantly higher (82%; p < 0.0008) in AD as compared to normal retina (0.238 +/- 0.070 vs. 0.131 +/- 0.042). These results are strengthened by the close agreement (within +/- 15% of respective means) found between fellow eyes. Analysis of glial fibrillary acidic protein immunoreactivity (GFAP-ir) in sections of retinas from an additional 12 AD and 19 control cases show increased GFAP-ir with more extensive labeling of astrocytes in the GCL as well as increased labeling of Müller cell end-feet and radial processes in AD as compared to control retinas. The extensive loss of neurons documented in these retinas, accompanied by an increased astrocyte/neuron ratio, provides further support for the substantial involvement of the retina in AD.

Protein kinase C inhibitors induce apoptosis in human malignant glioma cell lines.

Previous work has demonstrated the importance of the protein kinase C (PKC) system in regulating glioma growth, and has led to clinical trials utilizing PKC inhibitors as adjuncts in the therapy of patients harboring malignant gliomas. This study was performed to explore the possibility that inhibition of PKC in gliomas was triggering an apoptosis signal. Glioma cell lines were treated with PKC inhibitors staurosporine (10 nM), and tamoxifen (10 microM). DNA from cells treated with each of these drugs exhibited a 'ladder' pattern of oligonucleosome-sized fragments characteristic of apoptosis, thus suggesting that in glioma cells, these drugs may be cytocidal in action.

Monoclonal antibodies react with neuronal subpopulations in the human nervous system.

Monoclonal antibody probes were used to identify antigenic cross reactivities among neuronal subpopulations and to dissect the human nervous system at several levels of organization. Six monoclonal antibodies, prepared with immunogens from Drosophila melanogaster or human nervous tissue, were used to localize antigens immunocytochemically in normal adult human neocortex, hippocampus, cerebellum, spinal cord, and retina. Four of the six antibodies were neural specific in their reactivity and each stained a unique combination of neurons. The antibodies reacted with at least three subpopulations of cerebral cortical neurons, including discrete populations of pyramidal and nonpyramidal cells. Components of a widely distributed functional system within the spinal cord and cerebellum were labelled by one antibody, which reacted with neurons in the nucleus dorsalis of Clarke, deep cerebellar nuclei, and Purkinje cells. At the single-cell level, three of the monoclonals differentially labelled the photoreceptor cell outer segment, inner segment, and perikaryon. Three of the six antibodies were reactive with specific protein bands on immunoblots of tissue homogenates. This monoclonal antibody panel provides a novel and potentially useful method of analysis of the organization of the normal and diseased human nervous system.

Heterotopic neurons in spinal cord of patients with ALS.

Monoclonal antibody 44.1, an immunocytochemical marker for neurons, identified heterotopically located, multipolar neurons deep within the spinal cord white matter of patients with amyotrophic lateral sclerosis. Displaced neurons were most numerous in the ventral outflow and lateral corticospinal tract regions of all cord levels. These changes may be the result of aberrant neuronal migration during spinal cord development.