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PURPOSE Heterogeneity of the extent of angiogenesis induced by exogenous growth factors may be determined by genetic influences. Because angiogenesis is the formation of new vessels from preexisting ones, strain-related influences on naive resting limbal vessel phenotype and gene expression were determined in mice having divergently low and high angiogenic responses. METHODS Resting limbal vessel surface area and density and extent of bFGF-induced corneal angiogenesis were determined in C57BL/6J, BALB/cJ, F1 intercross identical with C57BL/6J X 129S3/SvIM, and 129S3/SvIM mouse strains by quantitative three-dimensional reconstruction confocal microscopy. Strain-related influences on pro- and antiangiogenic gene expression in naive cornea were determined by quantitative real-time RT-PCR. RESULTS The strain-dependent rank order of resting limbal vessel surface area and resting vessel density paralleled bFGF-induced neovascularization: 129S3/SvIM > BALB/cJ, F1 > C57BL/6J (P < 0.0006). Pigment epithelium-derived factor (PEDF) was increased more than 67-fold compared to Ang-2 in resting cornea of both C57BL/6J and 129S3/SvIM strains (P < 0.0001; P < 0.0001), suggesting a strongly antiangiogenic environment. The corneas of the C57BL/6J mice demonstrated 1.8-, 1.5-, and 1.7-fold increased mRNA levels for Flt-1, VEGF, and bFGF, respectively (P < 0.02; P < 0.04; P < 0.02); however, TSP-1 expression was increased 2.4-fold compared with 129S3/SvIM (P < 0.0004). CONCLUSIONS Strain-dependent differences in the resting limbal vessel surface area and density correlated with heterogeneity in the extent of bFGF-induced angiogenesis. Differences in pro- and antiangiogenic gene expression levels in resting cornea may influence vascular limbal phenotype during quiescence and may predict susceptibility to angiogenesis-dependent diseases.

PURPOSE. Microphthalmia is a relatively common ocular malformation. Molecular mechanisms that lead to this dire condition are largely unknown. Msx genes have been shown to be expressed in the developing eye. In the Msx1;Msx2, double mutant mouse, eye development arrests early in embryogenesis. To investigate possible functions of Msx2 in early ocular development, we created transgenic animals that overexpress Msx2. METHODS. Msx2 transgenic embryos and nontransgenic littermates were examined histopathologically. The effect of Msx2 overexpression on retinal cell proliferation was assayed by bromodeoxyuridine (BrdU) incorporation and immunohistochemical staining. Apoptosis was determined by TUNEL label-ing. Expression of retina and retinal pigmented epithelium (RPE)-specific genes was investigated by performing in situ hybridization or immunohistochemical staining. RESULTS. Forced expression of the Msx2 gene resulted in optic nerve aplasia and microphthalmia in all transgenic animals. In developing retinas of Msx2 transgenic animals, proliferation was significantly reduced and increased numbers of retinal cells underwent apoptosis. Marker analysis showed suppression of Bmp4 and induction of Bmp7 gene expression in the optic vesicle. Ectopic concurrent expression of the RPE cell markers Cx43 and Trp-2 in the neural retinal layer suggests cell fate respecification. CONCLUSION. These results indicate that forced expression of Msx2 perturbs BMP signaling in the developing eye and is accompanied by an increase in retinal cell death and a reduction in cell proliferation. Thus, deregulated Msx2 gene expression may be a plausible genetic mechanism by which the autosomal dominant form of congenital microphthalmia may arise.

CD8+ T cells infiltrating the CNS control infection by the neurotropic JHM strain of mouse hepatitis virus. Differential susceptibility of infected cell types to clearance by perforin or IFN-γ uncovered distinct, nonredundant roles for these antiviral mechanisms. To separately evaluate each effector function specifically in the context of CD8+ T cells, pathogenesis was analyzed in mice deficient in both perforin and IFN-γ (PKO/GKO) or selectively reconstituted for each function by transfer of CD8+ T cells. Untreated PKO/GKO mice were unable to control the infection and died of lethal encephalomyelitis within 16 days, despite substantially higher CD8+ T cell accumulation in the CNS compared with controls. Uncontrolled infection was associated with limited MHC class I up-regulation and an absence of class II expression on microglia, coinciding with decreased CD4+ T cells in CNS infiltrates. CD8+ T cells from perforin-deficient and wild-type donors reduced virus replication in PKO/GKO recipients. By contrast, IFN-γ-deficient donor CD8+ T cells did not affect virus replication. The inability of perforin-mediated mechanisms to control virus in the absence of IFN-γ coincided with reduced class I expression. These data not only confirm direct antiviral activity of IFN-γ within the CNS but also demonstrate IFN-γ-dependent MHC surface expression to guarantee local T cell effector function in tissues inherently low in MHC expression. The data further imply that IFN-γ plays a crucial role in pathogenesis by regulating the balance between virus replication in oligodendrocytes, CD8+ T cell effector function, and demyelination.

The relationship(s) between viral virulence and matrix metalloproteinase (MMP) expression in the central nervous system (CNS) of mice undergoing lethal and sublethal infections with neurotropic mouse hepatitis virus was investigated. Lethal infection induced increased levels of MMP-3 and MMP-12 mRNAs as well as that of tissue inhibitor of matrix metalloproteinases 1 (TIMP-1) compared to sublethal infection. Increased induction of MMP, TIMP, and chemokine expression correlated with increased virus replication but not with inflammatory cell infiltration. Infection of immunosuppressed mice suggested that expression of most MMP, TIMP, and chemokine mRNA was induced primarily in CNS-resident cells. By contrast, MMP-9 protein activity was associated with the infiltration of neutrophils into the CNS. These data indicate an association between the magnitude of inflammatory gene expression within the CNS and viral virulence.

To evaluate the effect of hepatocyte growth factor (HGF) on the integrity and function of tight junctions and adherens junctions in the retinal pigment epithelial (RPE) monolayer.Fresh bovine eyes were dissected to obtain 2- to 3-mm(2) explants of intact RPE with underlying choroid and sclera. Explants were cultured with or without HGF (20 ng/mL) for various periods (20 minutes to 72 hours). Junction integrity was assessed by transmission and scanning electron microscopy; localization, expression, and phosphorylation of junction proteins; and measurement of transepithelial resistance (TER), diffusion of fluorescent labeling in the plasma membrane, and the migration of RPE cells from the monolayer.Untreated explants consisted of polarized cells with apical microvilli and well-developed tight and adherens junctions. After HGF treatment, the explants showed loss of tight and adherens junctions ultrastructurally, diffusion of fluorescent label from apical to lateral membrane domains, and increased chemotactic migration of RPE cells from the monolayer. Primary cultures of confluent RPE cells showed a progressive decrease in TER. Western blot analysis showed rapid tyrosine phosphorylation of ZO-1, occludin, and beta-catenin within 20 minutes of stimulation. There was a marked loss of ZO-1 protein within 1 hour of HGF treatment. After 6 hours of treatment with HGF, occludin, claudin-1, and beta-catenin were redistributed from the membrane to the cytoplasm.Treatment of RPE explants with HGF results in rapid disassembly of tight and adherens junctions associated with loss or redistribution of junctional proteins, decreased TER, and increased migration of RPE cells from the monolayer.

Cells deficient in a major DNA double-strand break repair pathway (nonhomologous DNA end joining [NHEJ]) have increased spontaneous chromosome breaks [1–3]; however, the source of these chromosome breaks has remained undefined. Here, we show that the observed spontaneous chromosome breaks are partially suppressed by reducing the cellular oxygen tension. Conversely, elevating the level of reactive oxygen species by overexpressing the antioxidant enzyme superoxide dismutase 1 (SOD1), in a transgenic mouse, increases chromosome breakage. The effect of SOD1 can also be modulated by cellular oxygen tension. The elevated chromosome breakage correlates histologically with a significant increase in the amount of neuronal cell death in Ku86−/− SOD1 transgenic embryos over that seen in Ku86−/− embryos. Therefore, oxygen metabolism is a major source of the genomic instability observed in NHEJ-deficient cells and, presumably, in all cells.

The authors prospectively correlated results of magnetic resonance (MR) imaging and histologic examination of the dura in 17 patients with craniofacial and calvarial neoplasms and possible intracranial extension. Contrast material-enhanced MR imaging revealed dural enhancement in 14 patients and no enhancement in three. In four cases, the dura deep to the tumor appeared as a continuous band of enhancement and there was a thin unbroken hypointense zone between the dura and the tumor. Histologic examination in these four patients revealed only dural inflammation without neoplastic spread. In 10 patients, there was a break in the continuity of the enhancement of the underlying dura or a portion of the dura and overlying enhancing tumor could not be separated on MR images. The hypointense zone was focally absent in nine of these cases, and in all 10 there was invasion of the dura by adjacent tumor. Among the other three patients, in whom dural enhancement was not discernible, one had dural invasion by tumor while the other two did not.

Age-related macular degeneration (AMD) is a leading cause of blindness in the developed world. The retinal pigment epithelium (RPE) is a critical site of pathology in AMD and αB crystallin expression is increased in RPE and associated drusen in AMD. The purpose of this study was to investigate the role of αB crystallin in sodium iodate (NaIO3)-induced retinal degeneration, a model of AMD in which the primary site of pathology is the RPE. Dose dependent effects of intravenous NaIO3 (20-70 mg/kg) on development of retinal degeneration (fundus photography) and RPE and retinal neuronal loss (histology) were determined in wild type and αB crystallin knockout mice. Absence of αB crystallin augmented retinal degeneration in low dose (20 mg/kg) NaIO3-treated mice and increased retinal cell apoptosis which was mainly localized to the RPE layer. Generation of reactive oxygen species (ROS) was observed with NaIO3 in mouse and human RPE which increased further after αB crystallin knockout or siRNA knockdown, respectively. NaIO3 upregulated AKT phosphorylation and peroxisome proliferator-activator receptor-γ (PPARγ) which was suppressed after αB crystallin siRNA knockdown. Further, PPARγ ligand inhibited NaIO3-induced ROS generation. Our data suggest that αB crystallin plays a critical role in protection of NaIO3-induced oxidative stress and retinal degeneration in part through upregulation of AKT phosphorylation and PPARγ expression.

Studies on the selective retinal degeneration induced by sodium iodate (NaIO3) date back to 1941; Sorsby (1941) described the effect of intravenously injected NaIO3 solution on the rabbit retina. Since then, NaIO3-induced retinal degeneration has been described in different mammalian species including sheep, rabbit, rat and mouse with varying doses and routes of administration. At the present time, the murine NaIO3 model is the most widely used because it results in reproducible, patchy retinal degeneration (Figure 1) and can be studied in a wide variety of wild type and genetic knockout strains. Many studies reporting the pathologic changes in retinal structure at specific time points and electrophysiological changes in retinal function over time have followed (Franco et al., 2009; Redfern et al., 2011). More recently, the model has gained a resurgence of interest because of the ability to utilize high resolution in vivo imaging to study structural changes in the retina in live animals. Techniques such as spectral domain optical coherence tomography (OCT) and confocal scanning laser ophthalmoscopy (cSLO) allow longitudinal evaluation of retinal structure (Machalinska et al., 2014; Yang et al., 2014); thus making this model amenable to evaluation of novel therapeutic approaches to retinal degeneration such as cellular therapies. Figure 1 Fundus photograph of murine retina 3 weeks after intravenous injection of NaIO3 (129S6/SvTac strain). The sequence of events leading to retinal damage after NaIO3 administration has been extensively evaluated; however, comparisons of different studies is at times difficult because of differences in the source of NaIO3, methods of administration, animals (type, strain, and age) and time points analyzed after injection. A classic study using high dose NaIO3 (100 mg/kg) reported rapid and selective necrotic cell death of the retinal pigment epithelium (RPE; the layer of pigmented epithelial cells that support the function and survival of the photoreceptors) beginning 6 hours after injection (Kiuchi et al., 2002). Others have evaluated lower doses of NaIO3 treatment and found progressively increasing RPE damage from 15–35 mg/kg (Franco et al., 2009; Machalinska et al., 2014) but no discernible change at 10 mg/kg (Wang et al., 2014). The death of RPE is followed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cell death of photoreceptors, presumably by apoptosis (Kiuchi et al., 2002). Recently, the mechanism of photoreceptor dysfunction and death has been questioned. Carido et al. (2014) demonstrated increased calpain activity, without any increase in caspase 3 activation, 3 days after NaIO3 injection, and suggested that NaIO3 may be triggering a TUNEL-positive, non-apoptotic cell death mechanism in photoreceptors. As well, others suggest a direct effect of NaIO3 on photoreceptors, with visual dysfunction and rapid suppression of photoreceptor genes noted with 20–30 mg/kg intravenous dose in adult C57BL/6J mice (Wang et al., 2014). The molecular mechanism of RPE damage that occurs after NaIO3 administration is thought to be mediated through induction of oxidative stress. We have shown that murine RPE cultured in the presence of NaIO3 show accumulation of reactive oxygen species (ROS) that co-localizes with mitochondria (Zhou et al., 2014). These results suggest that future studies focused on therapy with antioxidants such as superoxide dismutase could be of considerable interest. With respect to signaling mechanisms, it is known that a rapid activation of AKT and reduction of RPE65 (a RPE-specific protein) occurs in mice subjected to oxidative stress. NaIO3-induced oxidative stress caused inactivation of PTEN and loss of its interactions with junctional proteins suggesting an important role for PTEN in normal RPE cell function as well in response to oxidative stimuli. Another target that has been recently suggested is Nrf2. Sachdeva et al. (2014) showed that Nrf2 signaling is impaired in the aging RPE when an oxidative insult with NaIO3 was imposed. Not only did the RPE of older mice (15 months) show impaired induction of Nrf2 as compared to young (2 months old) mice, they exhibited higher levels of superoxide anion and malondialdehyde. When Keap1, the negative regulator of Nrf2 was conditionally knocked down, the Nrf2 signaling and protection of RPE was restored. Our laboratory has presented evidence that the small heat shock protein αB crystallin protects the retina from NaIO3 induced retinal degeneration (Zhou et al., 2014). Low dose NaIO3 (20 mg/kg) injection in αB crystallin knockout mice augmented RPE cell death and subsequent retinal degeneration when compared to wild type control mice. Further, using cultured human RPE cells we showed that NaIO3 induced increased AKT phosphorylation and PPARγ expression in RPE cells which could be partially blocked by αB crystallin siRNA knockdown. Our findings suggest that αB crystallin plays a significant role in protection of NaIO3-induced oxidative stress and retinal degeneration in part through upregulation of AKT phosphorylation and PPARγ expression. We are currently evaluating the potential of αB crystallin or αB crystallin peptide nanoparticles as a therapeutic approach for treatment of retinal degeneration. The NaIO3 model has emerged as a valuable model to study RPE regeneration and cellular therapies in the retina. Machalinska et al. (2014) have shown that regeneration of the RPE occurs 3 months after induction of low dose sodium iodate induced retinal degeneration. Carido et al. (2014) transplanted human embryonic stem cell (hESC)-derived RPE cells 1 week after a single intravenous injection of NaIO3 (70 mg/kg) to mice. While NaIO3 injection caused severe RPE cell loss, photoreceptor degeneration and altered gene and protein expression in the inner nuclear layer (INL) and outer nucler layer (ONL) of the retina, transplantation of donor hESC-derived RPE cells formed extensive monolayers showing mature RPE cell morphology, organization and function. Furthermore, the transplanted RPE within these monolayers showed RPE-specific functions such as phagocytosis of host photoreceptor outer segments. In addition, hESC-derived RPE produced considerable collagen IV which is a component of the underlying Bruch membrane. Although most studies deal with hESC transplantation, the beneficial role of stem-cell derived neural progenitor cells to restore retinal degeneration in the NaIO3 model has also been reported recently (Amirpour et al., 2012). As well, after transplantation of rat mesenchymal stem cells (with and without erythropoietin gene modification) into the subretinal space of NaIO3 treated rats, the transplanted cells adopted RPE morphology and function 8 weeks after transplantation (Guan et al., 2013). Many investigators see considerable potential of the NaIO3 model for the study of novel therapies for geographic atrophy, a blinding complication of age-related macular degeneration (AMD) for which there is currently no effective treatment. While no animal model fully recapitulates each of the pathologic features found in the disorder, further refinement of the NaIO3 model to better mimic the defined geographic degeneration seen in AMD is underway in our lab and should represent a valuable advance. Finally, geographic atrophy is a slowly progressive disease occurring in an elderly population. The NaIO3 model is typically performed in young mice; it will be important to evaluate whether aging alters NaIO3 toxicity and sensitivity using older mice in various strains. The advent of high resolution imaging will allow longitudinal studies over extended periods of time and provides impetus for use of imaging endpoints for evaluation of preclinical efficacy of novel therapeutics for geographic atrophy. This work is supported in part by grants EY01545 and by core grant EY03040, the Arnold and Mabel Beckman Foundation, and an unrestricted grant to the Department of Ophthalmology from Research to Prevent Blindness Inc., New York, NY. We thank Dr. Parameswaran G. Sreekumar for the fundus images in Figure 1.

Background: The aim of this study is to see the effect of interferon β (IFN-β) on cell proliferation and the protein kinase C (PKC) signaling pathway. Methods: Proliferation of cultured human retinal pigment epithelium (RPE) cells, with various concentrations of IFN-β, and with or without 3% fetal calf serum (FCS), was assessed by cell counting. Effects of short (3 h) or prolonged (48 h) exposure of RPE cells to natural human IFN-β were assessed by 3H-thymidine uptake. Cytosolic and membranous PKC activity over time in cells treated with IFN-β and calphostin C was also measured.Results: IFN-β inhibited the increased proliferation by FCS in the prolonged-exposure assay. The PKC inhibitor calphostin C also showed an inhibitory effect on RPE cell growth and 3H-thymidine uptake in the chronic exposure with FCS. Short treatment with IFN-β had no inhibitory or stimulatory effect on 3H-thymidine uptake. Cytosolic and membranous PKC activity was strongly upregulated after short IFN-β exposure but returned to original levels after 1 h. PKC activity was downregulated both in the cytosol and membrane after 24 or 48 h. Conclusion: IFN-β inhibited RPE proliferation in vitro and the effect is mediated by upregulation of the PKC pathway.

Angiogenesis is required for the progression of tumors from a benign to a malignant phenotype and for metastasis. Malignant tumor cells secrete factors such as vascular endothelial growth factor (VEGF), which bind to their cognate receptors on endothelial cells to induce angiogenesis. Here it is shown that several tumor types express VEGF receptors (VEGFRs) and that inhibition of VEGF (VEGF antisense oligonucleotide AS-3) or VEGFRs (neutralizing antibodies) inhibited the proliferation of these cell lines in vitro. Furthermore, this effect was abrogated by exogenous VEGF. Thus, VEGF is an autocrine growth factor for tumor cell lines that express VEGFRs. A modified form of VEGF AS-3 (AS-3m), in which flanking 4 nucleotides were substituted with 2-O-methylnucleosides (mixed backbone oligonucleotides), retained specificity and was active when given orally or systemically in vitro and in murine tumor models. In VEGFR-2–expressing tumors, VEGF inhibition may have dual functions: direct inhibition of tumor cell growth and inhibition of angiogenesis.

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.

CD8+ T cells control infection by the neurotropic JHM strain of mouse hepatitis virus (JHMV) by eliminating infectious virus and reducing CNS pathology (Stohlman et al. 1995). Virus-specific CTL in BALB/c mice (H-2d) respond almost exclusively to a single immunodominant epitope in the nucleocapsid (N) protein (Bergmann et al. 1993). During acute JHMV infection, CD8+ T cells account for up to 40% of CNS mononuclear cells (MNC) (Bergmann et al. 1999). Following viral clearance, virus specific CD8+ T cells remain in CNS for several weeks (Bergmann et al. 1999).

The central nervous system (CNS) is refractive to many aspects of the immune system primarily due to its limited ability to repair damage induced by the cytopathic mechanisms deployed by most immune cells. A primary obstacle to CNS inflammation is the blood-brain-barrier (BBB), which limits entry of immune cells into the CNS. To pass the BBB, inflammatory cells release matrix metalloproteinases (MMPs); a growing family of proteases with overlapping substrate specificities for components of the extracellular matrix (reveiwed by Kieseier et al. 1999). MMPs break down the extracellular matrix surrounding the endothelial layer of BBB thereby permitting peripheral immune cells to traverse the BBB and migrate through the parenchyma of the CNS in response to inflammatory signals. To limit potential damage resulting from infiltration of inflammatory cells, MMP activity is tightly regulated at both the level of gene expression and proenzyme activation as well as by expression of a second gene family, the tissue inhibitors of MMPs (TIMPs). TIMPs act as competitive inhibitors for the active sites of MMPs and thus limit inflammatory infiltrates.

Replication of mouse hepatitis virus strain JHM (JHMV) in the central nervous system (CNS) is controlled by CD8+ T cells. However, persistent infection and subsequent chronic demyelination are established. Understanding effector mechanisms during acute infection may help understand viral persistence. CD8+ T cells respond to viral infections via two different cellular mechanisms, lysis of infected cells and secretion of anti-viral cytokines (Kaki et al 1995, Ruby et al 1991, Young et al 1995). JHMV replication in astrocytes and microglia is controlled by perforin-dependent cytolysis (Lin et al 1997) whereas replication in oligodendrocytes is controlled via IFN-γ (Parra et al 1999). The contributions of IFN-γ and perforin-dependent CD8+ T cell function in viral clearance were examined in SCID mice following adoptive transfer of CD8+ T cells deficient in IFN-γ secretion.

Neurological disease induced by neurotropic coronaviruses has received considerable attention not only as a model for virus induced demyelination, but also of immune regulation and viral persistence within the central nervous system (CNS). The neurotropic JHM strain (JHMV) of mouse hepatitis virus (MHV) produces an acute CNS infection characterized by encephalomyelitis and demyelination. Several strains and variants have been derived from the lethal, parental JHMV to better study the demyelinating process as a subacute disease resembling the human CNS disease, Multiple Sclerosis. A concise overview of historical aspects, derivation of neurotropic Coronavirus strains and variants, together with types of neurological disease and immune responses associated with these viruses is provided in the previous volume of this series (Perlman, 1998) and in several other recent reviews (Houtman and Fleming 1996, Lane and Buchmeier 1997, Stohlman et al 1999). Advances in molecular immunology techniques, combined with the use of selective exclusion of immune components, either using antibody (Ab) mediated depletion or genetic disruption, has resulted in significant progress in dissecting the interactions between virus, CNS cells, and immune responses and their role in demyelination. This chapter therefore focuses on advances of the past 3-4 years in elucidating interactions between CNS infection and immune responses leading to persistence. As MHV variants differ vastly in tropism, spread, clinical symptoms and mortality, this review focuses largely on a murine model of infection using the neuroattenuated 2.2v-l variant of JHMV (Fleming et al 1986). This mAb selected variant establishes a persistent infection associated with extensive, subacute, ongoing CNS demyelination in the absence of infectious virus. During acute infection this virus replicates primarily in microglia, astrocytes and oligodendrocytes. Although the immune response of immunocompetent hosts generally eliminates infectious virus within 14 days post infection (p.i.), survivors remain persistently infected as evidenced by the detection of viral RNA (vRNA). Despite suffering from varying degrees of encephalitis and paralysis during acute infection, mice generally recover from clinical symptoms and remain asymptomatic during viral CNS persistence.

Background: A new model of choroidal neovascularization (CNV) has been developed in the primate by implanting vascular endothelial growth factor (VEGF)-impregnated microspheres in the subretinal space. Methods: CNV was induced in Macaca mulatta monkeys by implanting VEGF-impregnated gelatin microspheres in the subretinal space. Progression of CNV was followed for 24 weeks after surgery using fluorescein angiography. Eyes were enucleated at various time points, and lesions were evaluated for evidence of CNV by light microscopy and by immunohistochemical staining. Results: CNV developed in 12 (92%) of 13 eyes. Fluorescein leakage was first observed in the 2nd postoperative week and was apparent for the following 12 weeks. CD31 staining for endothelial cells was first observed at day 7 and was evident for the following 8 weeks. Glial fibrillary acidic protein staining revealed a glial adhesion between the proliferative membrane and the retina at 6 weeks after implantation. Smooth muscle actin-positive cells were found a + 2 weeks and remained prominent for at least the next 6 weeks. Cytokeratin-positive retinal pigment epithelial (RPE) cells, first identified in the proliferative membrane at day 3, predominated throughout the growth of the membrane. Macrophages (RAM-II positive) were present at day 3 but were no longer observed after day 7. Conclusion: In monkeys, subretinal implantation of VEGF-impregnated gelatin microspheres leads to the development of CNV. Early, disciform and reparative stages of CNV were observed, similar to those seen in humans. This model will be useful for studying the pathogenesis of CNV and for evaluating potential treatment strategies.

Hypericin, a polycyclic dione used as an antidepressant, has been shown to inhibit the protein kinase C (PKC) pathway. Many of the pathologic responses found in proliferative vitreoretinopathy (PVR) are dependent upon PKC. Therefore, we studied the effect of hypericin on the treatment of experimental PVR.PVR was induced in pigmented rabbits by intravitreal injection of 50,000 rabbit conjunctival fibroblasts after vitrectomy. Subsequently, the eyes received an intravitreal injection of either balanced salt solution (BSS, 0.1 mL) (group A, control) or hypericin (0.1 mL) in doses of 1 muM (group B), 10 muM (group C), and 100 muM (group D). The eyes were examined ophthalmoscopically on days 1, 3, 7, 14, and 28 after surgery and the stage of PVR was evaluated (0 to V). The effect of hypericin on retinal morphology and function was also determined for the eyes injected with 100 muM hypericin with no fibroblasts by light microscopy and electroretinogram (ERG).In the control eyes, the retina was detached after 5 days, membranes had formed on and beneath it, and the PVR had progressed to higher stages over time. In the eyes injected with hypericin, the PVR also progressed; however, the severity of PVR on each day was lower than that in control eyes on that day. PVR was significantly inhibited in groups C and D as compared with the control eyes after day 5 (P0.05). Histological examination of the hypericin-treated control eyes disclosed no morphological change, and ERG analysis revealed no significant functional change.Intravitreal injection of hypericin is a safe and effective means of reducing experimental PVR.

The pattern of interferon-gamma-induced major histocompatibility complex Class II antigen expression was evaluated on the retinal pigment epithelium. Experiments were performed in vitro using explant cultures of aged and fetal human eyes and in vivo in albino rabbits. The human explants were stimulated with 50 U ml-1 interferon-gamma for 3 days prior to immunostaining for Class II. The rabbit eyes were subretinally injected in vivo with 50 microl of interferon-gamma (500 U ml-1) and analyzed immunohistochemically 3 days later. A heterogeneous pattern of Class II expression was present in the interferon-gamma-stimulated retinal pigment epithelial cells, in both the in vivo and the in vitro experiments. In aged human eyes the percent of Class-II positive cells was higher in the periphery than in the posterior pole (macular region) after interferon-gamma stimulation (P0.01). No such difference was found in the fetal eyes. These data demonstrate that retinal pigment epithelial cells are heterogeneous in their response to interferon-gamma. The results are supportive of previous studies demonstrating the structural and proliferative heterogeneity of the retinal pigment epithelium. Together, these studies provide support for the possibility of functional retinal pigment epithelial heterogeneity.

Objective To investigate the localization of Nϵ -(carboxymethyl)lysine (CML), a component and major immunologic epitope of advanced glycation end products, in aged eyes and choroidal neovascular membranes (CNVMs) surgically excised from eyes with age-related macular degeneration. Methods Immunohistochemistry for CML was performed using 8 snap-frozen, surgically excised CNVMs. Twelve eyes from patients aged 69 to 82 years and 2 donor eyes, 1 each from a 23-week-old fetus and 21-year-old patient, without age-related macular degeneration or diabetic retinopathy were also examined. To determine if retinal pigment epithelial cells in CNVMs accumulate advanced glycation end products, cytokeratin and CML were stained in paired serial sections. Results Soft, macular drusen and/or basal laminar and basal linear deposits were observed in 8 of 12 aged eyes. Each case showed CML accumulation, while overlying retinal pigment epithelial cells showed no accumulation in all 12 eyes. In CNVMs, however, retinal pigment epithelial cells showed CML accumulation in their cytoplasm. Conclusion The additional accumulation of advanced glycation end products in soft, macular drusen and/or retinal pigment epithelial cells may play a role in the pathogenesis of CNVM formation in age-related macular degeneration. Clinical Relevance Recently, advanced glycation end products have been found to play a role both in aging changes and neovascularization. Localization of advanced glycation end products in the above-mentioned tissue may lead to a better understanding of the pathogenesis of age-related macular degeneration.

✓ The authors report the case of a 48-year-old woman who presented with a nonprolactin-secreting adenoma and a preoperative prolactin level of 662 ng/ml. The patient's neoplasm subsequently enlarged despite normalization of her prolactin level with dopamine agonist therapy. Hyperprolactinemia, with levels of prolactin as high as 150 ng/ml, is commonly associated with sellar tumors and is attributed to disruption of the normal delivery of dopamine to the adenohypophysis. The prolactin level found in this patient represents the highest level attributed to the stalk-section effect reported in the literature and underscores the need for repeated radiographic assessment of patients who are undergoing treatment with bromocriptine and have prolactin levels in the 25 to 1000 ng/ml range.

Diabetic retinopathy is a major cause of acquired blindness due to the development of retinal neovascularization and associated traction retinal detachment. It is commonly treated with retinal photocoagulation therapy; however, progression to blindness remains a significant problem. To determine the feasibility of adjunctive anti-angiogenic gene therapy, we evaluated the capability of retroviral vectors, which transfer exogenous genes only into dividing cells, to transfer and express a β-galactosidase gene selectively into photocoagulation sites. Thirty-five rabbits received 30 retinal photocoagulation burns in the right eye followed 2 days later by β-galactosidase (G1nBgSvNa) or control (G1XSvNa) vector injection into the subretinal space. β-galactosidase expression was observed in the photocoagulation sites from 5 days after vector administration (31.7 ± 7.0 %) to 12 weeks (6.7 ± 3.4 %). Immunohistochemical studies of the treated retinas using antibody Ber-MAC3 and anti-cytokeratin antibodies revealed that transduced cells were macrophages and retinal pigment epithelial cells. To determine feasibility in a primate, two monkeys received 10 laser burns in the macula superior to the fovea followed 2 days later by G1nBgSvNa vector. β-galactosidase expression was found in photocoagulation sites and foveal retina was well preserved. We conclude that gene transfer to retinal photocoagulation sites provides stable expression of the transduced gene with relatively high efficiency. This feasibility study suggests the possibility of transferring genes encoding for anti-angiogenic factors into photocoagulation sites to improve the efficacy of laser photocoagulation therapy. [Diabetologia (1998) 41: 500–506]

Little is known about the molecular mechanisms of tumor progression in the pituitary. However, animal studies suggest that the Rb gene may be involved in the development of pituitary carcinoma. Pathologic examination of a pituitary tumor that included both benign and malignant components provided insight into this mechanism. Both benign and malignant tumors were immunoreactive for ACTH. The benign adenoma showed strong nuclear immunoreactivity for Rb, however, both the adjacent sellar carcinoma and its metastases were Rb-negative. This study suggests that loss of Rb may in some cases be important in the progression of pituitary adenoma to carcinoma.

Growth factor-directed migration is a critical component of the wound healing response although little is known about the signaling pathways involved. We examined the effect of inhibiting the mitogen-activated protein kinase (MAPK) pathway on platelet-derived growth factor (PDGF) and fibronectin-induced cell migration of human retinal pigment epithelial (RPE) cells. Using transwell cell-culture chambers, the effect of PDGF-BB (10–50 ng/ml) and fibronectin on components of migration was measured with or without the MAPK pathway inhibitor PD98059 (10–30 μM). MAPK activation of serum-starved cells by PDGF-BB was demonstrated by an immunoprecipitation/kinase assay and by immunohistochemistry using antibody specific for phosphorylated MAPK. PDGF-BB (10 ng/ml) stimulated MAPK activity in RPE (10 min) and its nuclear localization (1 h). PD98059 inhibited the activation of MAPK by PDGF-BB or serum. PDGF-BB stimulated RPE chemokinesis, chemotaxis, and haptotaxis; chemokinesis was additively increased and chemotaxis synergistically increased by the presence of a fibronectin substratum. PD98059 potently inhibited fibronectin-induced haptotaxis and PDGF-BB-induced chemotaxis but inhibited chemokinesis only at higher PDGF-BB (50 ng/ml) concentrations in the presence of fibronectin substratum. These results demonstrate that MAPK is critically involved in multiple components of RPE migrationin vitroand suggest the potential of targeting MAPK to inhibit RPE migrationin vivo.

Background Choroidal neovascular membranes (CNVMs) in age-related macular degeneration show progressive histologic changes from active, cellular, highly vascularized membranes to inactive paucicellular scars. The purpose of this study was to determine whether apoptosis was involved in the evolution of these changes, what cell types are involved, and whether there was an association with the Fas antigen (Fas or CD95) and Fas ligand (FasL). Methods Serial frozen sections from 10 surgically excised CNVMs were stained by the TUNEL (terminal deoxynucleotidyl transferase–mediated biotin-deoxyuridine 5-triphosphate nick-end labeling) method for detection of DNA strand breaks and by propidium iodide staining for morphologic detection of apoptosis. Immunoperoxidase staining was used for detection of Fas, FasL, and cell-type specific antigens. Results Highly vascularized membranes contained cells with TUNEL-positive nuclei, particularly in the regions of neovascularization, while fibrotic membranes showed few, if any, TUNEL-positive cells. Many of the TUNEL-positive cells were stromal retinal pigment epithelial cells, although smaller numbers were identified as endothelial cells and macrophages. Confocal microscopy of propidium idoide–stained sections confirmed the presence of apoptotic nuclei. The extent of Fas antigen expression correlated with extent of apoptosis. FasL expression was found in all specimens but was most intense in the highly vascularized membranes. Conclusions Highly vascularized CNVMs related to age-related macular degeneration show apoptosis in stromal retinal pigment epithelial cells, endothelial cells, and occasional macrophages. Apoptosis is associated with prominent Fas and FasL expression.

The role of CD4+ T cells in altering the activity of cytotoxic T lymphocytes (CTL) during infection of the central nervous system (CNS) by the neuroptropic JHMV strain of mouse hepatitis virus was examined. Adoptive transfer of in vitro activated CTL into CD4-depleted and control recipients showed that CTL were not effective in reducing JHMV replication within the CNS. The distribution of CD4+ and CD8+ T cells within the CNS during JHMV infection showed that the CD4+ T cells remained in perivascular and subarachnoid spaces and few entered the parenchyma. By contrast approximately half of the CD8+ T cells entered the parenchyma. In CD4-depleted mice the trafficking of CD8+ T cells was not inhibited; however, the majority of the cells were found to be apoptotic. These data suggested that CD4+ T cells were not required for CTL induction but were required for the maintenance of CTL viability. The limited role of CD4+ T cells in CTL induction was confirmed by comparison of CTL activity from CD4-depleted and control mice.

We have developed a defective-interfering (DI) RNA of mouse hepatitis virus (MHV) as a vector for expressing a variety of cellular and viral genes including the chloramphenicol acetyltransferase (CAT), hemagglutinin’esterase (HE), and gamma interferon. Here, we used the HE-expressing DI RNA for examining the role of HE protein in viral pathogenesis. The pseudorecombinant virus containing an expressed HE protein was generated by infecting cells with MHV-A59, which does not express HE, and transfecting the in vitro-transcribed DI RNA containing the HE gene. These pseudorecombinant viruses (DE-HE A59) were then inoculated intracerebrally into mice. Viruses recovered from cells infected with A59 and transfected with DI RNA expressing the CAT gene (DE-CAT A59) were used as a control. At various time points after inoculation, mice were observed for clinical symptoms. Tissues (brains and livers) were obtained for determining the replication of DI RNA by RT-PCR, virus replication by plaque assay, antigen expression by immunohistochemistry, and pathological changes. Results showed that all mice infected with DE-CAT A59 succumbed to infection by 9 days postinfection (d p.i). These data are identical to the pathogenesis of the parental A59 virus, demonstrating that inclusion of the DI RNA did not by itself alter pathogenesis. In contrast, only 40% of mice infected with DE-HE A59 succumbed to infection. The subgenomic mRNAs transcribed from the DI vector were detected at 1 and 2 d p.i. but not at subsequent time points, indicating that the genes in the DI vector were expressed only at an early stage of viral infection. No significant difference in virus replication in the brains was detected between these two groups of mice, suggesting that virus replication in brains was not affected by the expression of the HE. Histopathological examination showed only a small increase in the extent of inflammatory cell infiltration and reduced viral antigen in the mice infected with DE-HE A59. There was no difference in virus replication in the livers at 2 and 4 d p.i., but a 3 log10 reduction was detected in the livers of mice infected with DE-HE A59 at 6 d p.i. Histological examination showed a significant reduction in viral antigen, inflammation and necrosis in mice infected with DE-HE A59. These results indicate that the expression of HE from the DI vector altered the viral pathogenesis. This study thus demonstrates the usefulness of this system in studying the role of viral or cellular genes expressed locally at the sites of viral infection in viral pathogenesis.

The roles of CD4+ T cells, IFN-γ and TNF-α in viral clearance from the central nervous system (CNS) were examined in perforin gene deficient (PKO) mice. Depletion of CD4+ T cells from the PKO mice resulted in a significant 1 log10 PFU/gm increase in viral titer over control-treated PKO mice. PKO mice treated with anti-IFN-α mAb also had a significant 1 log10 increase in infectious virus whereas inhibition of TNF-a did not alter viral clearance or clinical disease in the PKO mice. These data suggest, in addition to perforin-mediated cytolysis, CD4+ T cells and IFN-γ, but not TNF-α could contribute to JHMV clearance from the CNS.

To evaluate the effect of hypericin on bovine choroidal endothelial cell proliferation and cord formation and on protein kinase C activity.The effect of hypericin (0.1-5 microM) on bovine choroidal endothelial cell proliferation was determined by cell number counting and a 3H-thymidine uptake assay in media containing 1, 5 or 10% serum. For the cord formation assay, bovine choroidal endothelial cells were seeded on basement membrane matrix, and the lengths of the capillary-like structures (cords) formed were quantified by image analysis. The effect of hypericin on cord formation was evaluated in the presence of serum or vascular endothelial growth factor. The effect of hypericin on protein kinase C activity was also measured in the presence or absence of light.Hypericin inhibited bovine choroidal endothelial cell proliferation in a dose-dependent manner in the presence of light but not in the dark. Serum dose-dependently masked the inhibition of DNA synthesis by hypericin. Cord formation by bovine choroidal endothelial cells was stimulated by serum or vascular endothelial growth factor and inhibited by hypericin in the presence of light. Protein kinase C activity was completely inhibited by hypericin in the presence of light but only mildly inhibited in the absence of light.Hypericin inhibits bovine choroidal endothelial cell proliferation and cord formation and choroidal endothelial cell protein kinase C activity. These results suggest that hypericin should be further investigated in animal models for its potential to inhibit subretinal neovascularization.

The potential role(s) of cytokines in the reduction of infectious virus and persistent viral infection in the central nervous system was examined by determining the kinetics of cytokine mRNA expression following infection with the neurotropic JHM strain of mouse hepatitis virus. Mice were infected with an antibody escape variant which produces a nonlethal encephalomyelitis and compared to a clonal virus population which produces a fulminant fatal encephalomyelitis. Infection with both viruses induced the accumulation of mRNAs associated with Th1- and Th2-type cytokines, including IFN-gamma, IL-4, and IL-10. Peak mRNA accumulations were coincident with the clearance of virus and there was no obvious differences between lethally and nonlethally infected mice. TNF-alpha mRNA was induced more rapidly in lethally infected mice compared to mice undergoing a nonfatal encephalomyelitis. Rapid transient increases in the mRNAs encoding IL-12, iNOS, IL-1alpha, IL-1beta, and IL-6 occurred following infection. Nonlethal infections were associated with increased IL-12, IL-1beta, and earlier expression of IL-6, while lethal infections were associated with increased iNOS and IL-1alpha mRNA. These data suggest a rapid but differential response within the central nervous system cells to infection by different JHMV variants. However, neither the accumulation nor kinetics of induction provide evidence to distinguish lethal infections from nonlethal infections leading to a persistent infection. Accumulation of both Th1 and Th2 cytokines in the central nervous system of JHMV-infected mice is consistent with the participation of both cytokines and cell immune effectors during resolution of acute viral-induced encephalomyelitis.

Human retinal pigment epithelial (HRPE) cells are a major cell component in proliferative vitreoretinopathy (PVR) membranes. We investigated the feasibility of killing HRPE cells by retroviral-mediated transfer of the herpes simplex virus-thymidine kinase (HSV-tk) gene, also known as the suicide gene, into HRPE cells followed by ganciclovir treatment, and to study the so-called bystander effect. Such a treatment plan might serve as a possible therapy for PVR.Transduction efficiency was determined using retroviral vectors encoding the beta-galactosidase reporter gene. To evaluate the efficacy of suicide gene therapy. HRPE cells were transduced with retroviral vectors encoding the HSV-tk gene (G1TkSvNa), with empty vectors or without vectors, and were treated with 5 micrograms/ml ganciclovir. Sensitivity of HSV-tk positive HRPE cells to various concentrations of ganciclovir was evaluated. To demonstrate the bystander effect, HSV-tk positive cells were cultured with HSV-tk negative cells at varying proportions.Transduction efficiency in vitro was 15.1 +/- 4.8%. Cell growth was significantly inhibited after transduction with G1TkSvNa followed by ganciclovir treatment (P0.01). Ganciclovir showed dose- and time-dependent cytotoxicity only on HSV-tk positive cells. The concentration that resulted in 50% inhibited was 0.1 micrograms/ml. In terms of the bystander effect, after ganciclovir treatment the viability of co-cultured cells decreased with increasing populations of HSV-tk positive cells.HRPE cells were successfully transduced with the HSV-tk gene via retroviral vectors and displayed a strong bystander effect after treatment with ganciclovir. These results suggest that retrovirus-mediated suicide gene therapy might be a feasible treatment strategy for PVR.

Transforming growth factor beta-2 (TGF-B2) is secreted by glioma cells and is known to decrease leukocyte–endothelium interaction. TGF-B2 alone and in conjunction with soluble tumor necrosis factor (TNF) p55 receptor, was found to decrease the expression of TNF induced VCAM-1 on the malignant glioma cell line A-172 and human cerebral microvessel endothelial (CNS-EC) cells. Co-culture of A-172 glioma cells led to a decrease in VCAM-1 expression; this effect on CNS-EC in co-culture could be simulated by glioma supernatant alone. These results suggest that malignant gliomas, by secreting TGF-B2 and releasing soluble TNF receptors, modulate adhesion molecules.

Prolactin (PRL)-producing pituitary adenomas are in some cases associated with deposition of abundant spherical amyloid; however, the origin of the amyloid has not been established. In this report, a PRL-producing pituitary adenoma composed almost entirely of spherical amyloid was analyzed biochemically. The tumor was removed surgically from a 56-year-old man. Immunohistochemical analysis revealed that residual tumor cells were strongly positive for PRL, while the spherical amyloid was not. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a band of approximately 4 kDa associated with the amyloid, which was not present in a non-amyloid producing prolactinoma. The 4-kDa band is similar in size to other known amyloidogenic peptides. Immunoblot analysis of the tumor material using polyclonal anti-human PRL antibodies revealed a small amount of normal-sized PRL; however, the abundant 4-kDa band was nonimmunoreactive. Amino acid sequencing showed that this peptide represents the first 34 amino acids of the intact PRL protein with a predicted size of 4313 Da. The presence of a small amount of normal-sized PRL in this tumor, as well as elevated circulating levels of PRL implies that intact PRL is being abnormally processed in the formation of spherical amyloid.

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.

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% (p0.004). Although the 16% increase in astrocyte numbers is not significant, the ratio of astrocytes to neurons is significantly higher (82%; p0.0008) 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.

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 (p0.05). 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.

The present study was undertaken to explore the role of the Protein Kinase C (PKC) signal transduction system in growth regulation of pituitary adenomas. Primary tumor cultures were plated from fresh surgical tumor specimens. The PKC inhibitors Staurosporine and Tamoxifen were added at varying dosages to the cell cultures. Measurements of cell proliferation were performed by [3H]-thymidine uptake and the [3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H tetrazolium bromide] (MTT) assay. After a 48 h treatment period, both [3H]-thymidine uptake and absorbance on the MTT assay decreased in a dose-related manner in both the staurosporine and tamoxifen-treated cultures (IC50 of 10 nM and 30 µM respectively). Direct measurement of PKC activity using an in vitro assay revealed very high activity (range of 1465-5708 pmol/min/ mg protein; within the range previously published for malignant glioma specimens) in 12 frozen specimens of pituitary adenomas (9 nonfunctional adenomas, 1 prolactinoma, 1 gonadotrophin-secreting and 1 corticotroph-secreting adenoma). In contrast, PKC activity measured in normal adenohypophysis was comparatively very low. These data indicate that pituitary adenoma cells display high PKC activity and are sensitive to growth inhibition by PKC inhibitors. These data suggest a role for the PKC system in regulating pituitary tumor growth, which may have implications for future therapy of these tumors.

Summary: We report a patient with medically refractory complex partial seizures (CPS) caused by a cerebral neurocytoma located near the amygdala. Neurocytoma represents an important addition to the differential diagnosis and, in particular, must be differentiated from oligoden-droglioma and dysembryoplastic neuroepithelial tumor. Accurate pathological differentiation has therapeutic and prognostic implications.

Neutralizing anti-tumor necrosis factor alpha (TNF-alpha) antibody treatment of mice infected with the neurotropic JHMV strain of mouse hepatitis virus showed no reduction of either virus-induced encephalomyelitis or central nervous system demyelination. TNF-alpha-positive cells were present in the central nervous system during infection; however, TNF-alpha could not be colocalized with JHMV-infected cells. In vitro, TNF-alpha mRNA rapidly accumulated following JHMV infection; however, no TNF-alpha was secreted because of inhibition of translation. Both live and UV-inactivated virus inhibited TNF-alpha secretion induced by lipopolysaccharide. These data show that TNF-alpha is not secreted from infected cells and indicate that if contributes to either JHMV-induced acute encephalomyelitis nor primary demyelination.

This study was initiated to evaluate a role for gamma interferon (IFN-gamma) in herpes simplex virus type 1 (HSV-1) infection. At the acute stage of infection in mice, HSV-1 replication in trigeminal ganglia and brain stem tissue was modestly but consistently enhanced in mice from which IFN-gamma was by ablated monoclonal antibody treatment and in mice genetically lacking the IFN-gamma receptor (Rgko mice). As determined by reverse transcriptase PCR, IFN-gamma and tumor necrosis factor alpha transcripts were present in trigeminal ganglia during both acute and latent HSV-1 infection. CD4+ and CD8+ T cells were detected initially in trigeminal ganglia at day 5 after HSV-1 inoculation, and these cells persisted for 6 months into latency. The T cells were focused around morphologically normal neurons that showed no signs of active infection, but many of which expressed HSV-1 latency-associated transcripts. Secreted IFN-gamma was present up to 6 months into latency in areas of the T-cell infiltration. By 9 months into latency, both the T-cell infiltrate and IFN-gamma expression had cleared, although there remained a slight increase in macrophage levels in trigeminal ganglia. In HSV-1-infected brain stem tissue, T cells and IFN-gamma expression were present at 1 month but were gone by 6 months after infection. Our hypothesis is that the persistence of T cells and the sustained IFN-gamma expression occur in response to an HSV-1 antigen(s) in the nervous system. This hypothesis is consistent with a new model of HSV-1 latency which suggests that limited HSV-1 antigen expression occurs during latency (M. Kosz-Vnenchak, J. Jacobson, D.M. Coen, and D.M. Knipe, J. Virol. 67:5383-5393, 1993). We speculate that prolonged secretion of IFN-gamma during latency may modulate a reactivated HSV-1 infection.

Retinal pigment epithelial (RPE) cells produce and respond to a variety of cytokines; however, molecular and biochemical studies are restricted by the limited access to large numbers of pure cells and the variability associated with different donor sources. Despite success in establishing primary human RPE (HRPE) cell cultures, the inability to sustain consistent proliferation rates and morphology over several passages remains a concern. This problem was approached by using an immortalized line of simian virus (SV)40 transformed fetal HRPE cells (SVRPE). Cytokine production, receptor expression and responsiveness in the SVRPE cell line was analyzed to determine the usefulness of this model for studying HRPE-cytokine interactions. Using reverse transcriptase polymerase chain reaction (RT-PCR), HRPE and SVRPE cells demonstrated an identical pattern of interleukin-1 receptor (IL-1R), IL-2R (alpha sub-unit), IL-6R, interferon (IFN)-gamma R and tumor necrosis factor-alpha (TNF)R p55 expression. No amplification products for TNFR p75 or granulocyte/macrophage colony stimulating factor (GM-CSF)R were demonstrated in either population. IFN-gamma stimulation induced surface human leukocyte antigen (HLA)-DR in both SVRPE and HRPE, while TNF treatment induced surface expression of intercellular adhesion molecule (ICAM)-1 on SVRPE and upregulated ICAM from basal levels on HRPE. Both cell types showed amplification products for interleukin (IL)-1 beta, IL-6 and transforming growth factor (TGF)-beta 1 using RT-PCR. The bioassays demonstrated that both populations of unstimulated cells constitutively secrete very low levels of TGF-beta and no IL-6.(ABSTRACT TRUNCATED AT 250 WORDS)

Summary: The incidence of seizures related to primary brain tumors is 20–80%. High-dose tamoxifen was recently reported as a novel treatment for patients with malignant gliomas who have failed standard therapies. Tamoxifen inhibits protein kinase C (PKC) in vitro and thus may regulate glioma cell growth by modulating intracellular signal transduction. We report a patient with a recurrent supratentorial pilocytic astrocytoma who had an untoward interaction between high-dose tamoxifen therapy and phenytoin (PHT), drugs that share a common enzyme for metabolism, therefore emphasizing the need to monitor concomitant antiepileptic drug (AED) levels when high-dose tamoxifen therapy is instituted.

To describe observations of adenohypophysitis on magnetic resonance (MR) images.Clinical, enhanced MR imaging, surgical, and histologic findings were retrospectively studied in four female patients and one male patient with adenohypophysitis who presented with headaches, pituitary insufficiency, or hyperprolactinemia. Results were compared with MR imaging, surgical, and pathologic findings in 128 consecutive cases of newly diagnosed pituitary adenomas.In two of the four female patients, disease onset was not associated with a recent history of pregnancy. Imaging findings included a slightly lobulated, intensely enhancing pituitary mass (n = 5); enhancement along the infundibulum (n = 4); and adjacent dural enhancement (n = 4). Four patients had extrapituitary involvement. Some of these findings were noted in patients with pituitary adenomas complicated by infarction, hemorrhage, or necrosis.The observed clinical and MR imaging findings are suggestive of adenohypophysitis; the latter, however, are not specific and may be seen in some complicated cases of pituitary adenoma and other rare forms of pituitary inflammation. Biopsy may be needed to establish the correct diagnosis if a trial of steroid therapy fails.

Objective: To elucidate the mechanism of bovine choroidal endothelial (BCE) cell angiogenesis and, in particular, the role of retinal pigment epithelial (RPE) cells by use of an in vitro coculture assay system. Methods: The BCE cells were isolated from choroidal tissues and cultured. They were embedded in type I collagen gel and incubated. The gel-embedded BCE cells were then covered with a monolayer of RPE cells, pericytes, choroidal fibroblasts, or additional BCE cells on culture day 0 or day 14. The BCE cells in culture formed a meshwork of tubelike structures. The length of the tubelike structures (micrometers per field) was quantified by image analysis, as an indicator of angiogenesis. The effect of RPE cells on normal and growth-arrested BCE cell tube formation was evaluated in this assay system. The mechanism of RPEinduced angiogenesis was studied by parallel experiments with the use of neutralizing antibodies against specific growth factors (basic fibroblast growth factor, vascular endothelial growth factor, and transforming growth factor β). Results: Ultrastructural analysis revealed that the tubelike structures had features typical of choroidal endothelial cells. Cocultures initiated on day 0 revealed that BCE cell angiogenesis was promoted by overlying RPE cells and, to a lesser extent, by pericytes, choroidal fibroblasts, and additional BCE cells when compared with BCE cells without covering cells. In cocultures initiated after BCE tube formation (day 14), there was inhibition of BCE angiogenesis by overlying RPE cells when compared with cultures without overlay or with an overlay of BCE cells. The RPE cells stimulated tube formation of growth-arrested BCE cells less effectively than did normal BCE cells. Neutralizing antibody for basic fibroblast growth factor and vascular endothelial growth factor, but not transforming growth factor β, inhibited control and RPE-induced tube formation by BCE cells. Conclusions: Overlying RPE cells stimulate the formation of tubelike structures by choroidal endothelial cells more effectively than do fibroblasts or pericytes but inhibit BCE tube formation in older cultures. The effect involves endothelial proliferation and differentiation. The stimulatory effect of overlying RPE cells can be inhibited by neutralizing antibodies to vascular endothelial growth factor and basic fibroblast growth factor, which suggests that these growth factors play an important role in this phenomenon.

Acute infection of the central nervous system by the neurotropic JHM strain of mouse hepatitis virus (JHMV) induces nucleocapsid protein specific cytotoxic T lymphocytes (CTL) not found in the periphery (S. Stohlman, S. Kyuwa, J. Polo, D. Brady, M. Lai, and C. Bergmann, J. Virol. 67:7050-7059, 1993). Peripheral induction of CTL specific for the nucleocapsid protein of JHMV by vaccination with recombinant vaccinia viruses was unable to provide significant protection to a subsequent lethal virus challenge. By contrast, the transfer of nucleoprotein-specific CTL protected mice from a subsequent lethal challenge by reducing virus replication within the central nervous system, demonstrating the importance of the CTL response to this epitope in JHMV infection. Transfer of these CTL directly into the central nervous system was at least 10-fold more effective than peripheral transfer. Histological analysis indicated that the CTL reduced virus replication in ependymal cells, astrocytes, and microglia. Although the CTL were relatively ineffective at reducing virus replication in oligodendroglia, survivors showed minimal evidence of virus persistence within the central nervous system and no evidence of chronic ongoing demyelination.

Infection with JHMV results in the transcriptional activation of two host cell genes encoding proinflammatory cytokines, tumor necrosis factor (TNF)-a and interleukin (IL)-ip. Analysis of irradiated mice showed that IL-1β mRNA accumulation in the central nervous system was predominantly derived from the mononuclear infiltrate. By contrast, accumulation of TNF-α mRNA was unaffected by immunosuppression, suggesting that resident cells were the source of this cytokine. Infected mice were treated with anti-TNF antibody to determine if TNF-α contributed to either the encephalomyelitis or demyelination associated with JHMV infection. Surprisingly, neither the cellular infiltrate nor demyelination were affected. In vitro analysis showed that IL-1β but not TNF was secreted from JHMV infected macrophages. The absence of TNF secretion is due to a block in translation of the TNF mRNA which accumulates during infection.