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Collisions with buildings cause up to 1 billion bird fatalities annually in the United States and Canada. However, efforts to reduce collisions would benefit from studies conducted at large spatial scales across multiple study sites with standardized methods and consideration of species- and life-history-related variation and correlates of collisions. We addressed these research needs through coordinated collection of data on bird collisions with buildings at sites in the United States (35), Canada (3), and Mexico (2). We collected all carcasses and identified species. After removing records for unidentified carcasses, species lacking distribution-wide population estimates, and species with distributions overlapping fewer than 10 sites, we retained 269 carcasses of 64 species for analysis. We estimated collision vulnerability for 40 bird species with ≥2 fatalities based on their North American population abundance, distribution overlap in study sites, and sampling effort. Of 10 species we identified as most vulnerable to collisions, some have been identified previously (e.g., Black-throated Blue Warbler [Setophaga caerulescens]), whereas others emerged for the first time (e.g., White-breasted Nuthatch [Sitta carolinensis]), possibly because we used a more standardized sampling approach than past studies. Building size and glass area were positively associated with number of collisions for 5 of 8 species with enough observations to analyze independently. Vegetation around buildings influenced collisions for only 1 of those 8 species (Swainson's Thrush [Catharus ustulatus]). Life history predicted collisions; numbers of collisions were greatest for migratory, insectivorous, and woodland-inhabiting species. Our results provide new insight into the species most vulnerable to building collisions, making them potentially in greatest need of conservation attention to reduce collisions and into species- and life-history-related variation and correlates of building collisions, information that can help refine collision management.Correlaciones de las Colisiones de Aves contra Edificios en Tres Países de América del Norte Resumen Las colisiones contra los edificios causan hasta mil millones de fatalidades de aves al año en los Estados Unidos y en Canadá. Sin embargo, los esfuerzos por reducir estas colisiones se beneficiarían con estudios realizados a grandes escalas espaciales en varios sitios de estudio con métodos estandarizados y considerando las variaciones relacionadas a la historia de vida y a la especie y las correlaciones de las colisiones. Abordamos estas necesidades de investigación por medio de una recolección coordinada de datos sobre las colisiones de aves contra edificios en los Estados Unidos (35), Canadá (3) y México (2). Recolectamos todos los cadáveres y los identificamos hasta especie. Después de retirar los registros de cadáveres no identificados, las especies sin estimaciones poblacionales a nivel distribución y las especies con distribuciones traslapadas en menos de diez sitios, nos quedamos con 269 cadáveres de 64 especies para el análisis. Estimamos la vulnerabilidad a colisiones para 40 especies con ≥2 fatalidades con base en la abundancia poblacional para América del Norte, el traslape de su distribución entre los sitios de estudio y el esfuerzo de muestreo. De las diez especies que identificamos como las más vulnerables a las colisiones, algunas han sido identificadas previamente (Setophaga caerulescens), y otras aparecieron por primera vez (Sitta carolinensis), posiblemente debido a que usamos una estrategia de muestreo más estandarizada que en los estudios previos. El tamaño del edificio y el área del vidrio estuvieron asociados positivamente con el número de colisiones para cinco de ocho especies con suficientes observaciones para ser analizadas independientemente. La vegetación alrededor de los edificios influyó sobre las colisiones solamente para una de esas ocho especies Catharus ustulatus). Las historias de vida pronosticaron las colisiones; el número de colisiones fue mayor para las especies migratorias, insectívoras y aquellas que habitan en las zonas boscosas. Nuestros resultados proporcionan una nueva perspectiva hacia las especies más vulnerables a las colisiones contra edificios, lo que las pone en una necesidad potencialmente mayor de atención conservacionista para reducir estas colisiones y de estudio de las variaciones relacionadas con la especie y la historia de vida y las correlaciones de las colisiones contra edificios, información que puede ayudar a refinar el manejo de colisiones.在美国和加拿大, 每年有多达10亿只鸟类死于撞击建筑物。在大空间尺度上跨多个研究地点、采用标准化方法, 并考虑鸟类物种及生活史变异和其它撞击相关因素的研究, 将有助于减少鸟类与建筑物的碰撞。本研究通过整理收集美国、加拿大和墨西哥多个位点 (数量分别为 35 个、3 个和 2 个) 鸟类撞击建筑物的数据来满足以上的研究需求。我们首先收集了所有鸟类尸体并进行物种鉴定。在排除掉不能辨认的尸体、缺少分布范围内种群估计数据的物种、分布区与少于 10 个位点有重叠的物种后, 我们保留了 64 个物种的 269 个死亡事件进行分析。我们根据鸟类的北美种群数量、分布区与研究位点的重叠情况和抽样工作量, 估计了死亡个体≥2 只的 40 种鸟类的撞击脆弱性。本研究确定了最易发生撞击的 10 个物种, 其中包括已经被以往研究提及的物种 (如黑喉蓝林莺 [Setophaga caerulescens]) , 也有首次报道的物种 (如白胸鳾 [Sitta carolinensis]), 这可能是因为我们使用了比以往研究更注重标准化的抽样方法。在有足够观察数据以支持独立分析的 8 个物种中, 有 5 个物种的撞击次数与建筑大小和玻璃面积呈正相关;只有1个物种 (斯氏夜鸫 [Catharus ustulatus]) 的撞击受到建筑物周围植被的影响。生活史也可以预测撞击情况:迁徙鸟类、食虫鸟类和林栖鸟类的撞击次数最多。我们的研究结果为最易发生建筑物撞击伤害的鸟类物种提供了新的见解, 这些物种可能最需要保护以减少撞击, 本研究还深入探究了鸟类物种及生活史变异和其它撞击相关因素, 这些信息有助于改进鸟类撞击管理。【翻译: 胡怡思; 审校: 聂永刚】.

Landscape-scale alterations that accompany urbanization may negatively affect the population structure of wildlife species such as freshwater turtles. Changes to nesting sites and higher mortality rates due to vehicular collisions and increased predator populations may particularly affect immature turtles and mature female turtles. We hypothesized that the proportions of adult female and immature turtles in a population will negatively correlate with landscape urbanization. As a collaborative effort of the Ecological Research as Education Network (EREN), we sampled freshwater turtle populations in 11 states across the central and eastern United States. Contrary to expectations, we found a significant positive relationship between proportions of mature female painted turtles (Chrysemys picta) and urbanization. We did not detect a relationship between urbanization and proportions of immature turtles. Urbanization may alter the thermal environment of nesting sites such that more females are produced as urbanization increases. Our approach of creating a collaborative network of scientists and students at undergraduate institutions proved valuable in terms of testing our hypothesis over a large spatial scale while also allowing students to gain hands-on experience in conservation science.

The spread of malaria is thought to have followed human expansion out of Africa some 60 – 80 thousand years ago. With its prevalence in pantropical countries of the world and epicenter localized in Africa, malaria is now considered an unnecessary burden to overworked and under-resourced healthcare structures. Plants have long afforded a fertile hunting ground for the search and identification of structurally diverse antimalarial agents, such as quinine and artemisinin. This survey examines the antiparasitic properties of the family Amaryllidaceae via the antiplasmodial activities demonstrated for its lycorane alkaloid principles. Of these, 24 were natural compounds identified in 20 species from 11 genera of the Amaryllidaceae family, whilst the remaining 28 were synthetically derived entities based on the lycorane skeleton. These were screened against ten different strains of the malarial parasite Plasmodium falciparum, wherein the parent compound lycorine was shown to be the most potent with an IC50 of 0.029 µg/mL in the FCR-3 strain seen to be the best. Structure-activity relationship studies revealed that good activities were detectable across both the natural compounds as well as the synthetically accessed derivatives. Such studies also highlighted that there are several inherent structural features that define the lycorane alkaloid antiplasmodial pharmacophore, such as the nature of its ring systems and properties of its substituents. Mechanistically, a limited number of studies confirmed that lycorane alkaloids manifest their action by targeting enzymes associated with the plasmodial FAS-II biosynthetic pathways. Overall, these alkaloids have provided useful, convenient, and accessible scaffolds for antimalarial-based drug discovery.

Ethnopharmacological relevance Plants of the family Amaryllidaceae are used widely in traditional medicine in South Africa. Several of these, including representatives of the genus Cyrtanthus find use in the treatment of mental illness and age-related dementia. Aim of the study Based on the distribution of central nervous system-activating alkaloidal constituents within the genus Cyrtanthus , Cyrtanthus contractus was here explored for such compounds which could interact with acetylcholinesterase (AChE), of significance in the progression of neurodegeneration associated with Alzheimer's disease. Materials and methods The known phenanthridone alkaloid narciprimine was isolated via column chromatography of the ethanolic extract of bulbs of Cyrtanthus contractus . The structure of the compound was determined by high field 2D NMR and mass spectroscopic techniques. The classical method of Ellman et al. was used in the determination of AChE inhibitory activity. Results The isolation of narciprimine from Cyrtanthus contractus is a landmark find since it has previously only been identified in Zephyranthes , Narcissus and Lycoris , genera endemic to the Americas, Europe and Asia, respectively. Narciprimine exhibited micromolar inhibitory activity (IC 50 78.9) against the enzyme acetylcholinesterase. Conclusion This work represents the first isolation of narciprimine from an African Amaryllidaceae species, which may be of chemotaxonomic significance. The AChE inhibitory activity of narciprimine, taken together with activities of other structurally similar inhibitors within the series affords further insight to the structural details of the lycorine alkaloid acetylcholinesterase inhibitory pharmacophore.

The Amaryllidaceae occupies a privileged status amongst medicinal plants in having delivered the Alzheimer's drug galanthamine to the clinical market. Following its resounding success, there have been several positive indicators for the emergence of an anticancer drug from the family due to the potent antiproliferative activities manifested by several of its alkaloid constituents. Of these, the phenanthridones such as pancratistatin hold most promise as potential chemotherapeutics having succumbed to various phases of clinical trials. Other cytotoxic targets of the Amaryllidaceae are to be found within the lycorane and crinane groups, as exemplified by crinine and lycorine. Although the molecular targets of these alkaloids still remain elusive, much effort has gone into understanding their mode of action in cancer cells. Recent findings have shown that the apoptotic pathway may be a key factor in cancer cell death instigated by Amaryllidaceae alkaloids. As such, this review seeks to: (a) examine the apoptotic effects of Amaryllidaceae alkaloids in cancer cells; (b) explore the molecular basis to these effects; and (c) provide a pharmacophoric rationale in support of these activities.

Cells cultured from Alzheimer disease leptomeninges or skin were grafted into the cortex of adult thymectomized rats. At 3 days post-implant, plaque-like aggregates were found in the cortex, corpus callosum, septum and caudate nucleus. These structures were immunopositive for human amyloid precursor protein (APP), human amyloid beta peptide (Aβ), cathepsin D, apolipoprotein E and ubiquitin. Aberrant tau + neurites, reactive astrocytes and microglia were associated with many aggregates. Although birefringent amyloid occupied the central area of most aggregates, these structures had disappeared by l month post-implant. Aβ and APP produced by grafted non-neural human cells can penetrate rat brain and form plaque-like structures, which can be effectively cleared by the rat.

The plant family Amaryllidaceae is renowned for its unique alkaloid constituents which possess a significant array of structural diversity. Several of these alkaloids are known for their interesting biological properties, of which galanthamine and pancratistatin have acquired a privileged status due to their relevance in the pharmaceutical arena. In particular, galanthamine represents the first prescription drug emanating from the Amaryllidaceae after its approval by the FDA in 2001 for the treatment of Alzheimer's disease. Following on this commercial success there have been sustained projections for the emergence of an anticancer agent related to pancratistatin due to the potency, selectivity, low toxicity and high tolerability typifying targets of this series of alkaloids. The lycorine series of alkaloids have also garnered widespread interest as cytotoxic agents and were amongst the earliest of the Amaryllidaceae constituents to exhibit such activity. To date over 100 of such naturally-occurring or synthetically-derived alkaloids have been screened for cytotoxic effects against a number of cancer cell lines. This survey examines the cytotoxic properties of lycorine alkaloids, highlights the outcomes of structure-activity relationship orientated studies and affords plausible insights to the mechanistic rationale behind these effects.

The plant family Amaryllidaceae is known for its horticultural and ornamental appeal as well as its medicinal value. In relation to these characteristics, trade in Amaryllid flower varieties (especially daffodils) is a multi-million dollar revenue generator for the floriculture industry. Of greater significance are the medicinal attributes of the family, which has already spawned the Alzheimer's prescription drug galanthamine, a potent and selective inhibitor of the enzyme acetylcholinesterase, of significance in the progression of neurodegeneration associated with motor neuron diseases, with annual global sales of around $150 million. Furthermore, it is anticipated that an anticancer drug target related to the Amaryllidaceae alkaloid pancratistatin, presently under advanced clinical evaluation, will enter commercial circulation within the next decade. Members of the Amaryllidaceae are distributed through both tropical and subtropical regions of the globe, but are of prominence within three distinct geographical locations, including Andean South America, the Mediterranean basin, and southern Africa. The southern African zone is known to harbor at least a third of the worldwide complement of around 1000 species, many of which are widely utilized in the traditional medicinal practices of the indigenous people of the region. Given its therapeutic and economic value, its natural abundance in the southern African region, coupled to its widespread usage in ethnic medicine, the family Amaryllidaceae provides a diverse and accessible platform for phytochemical based drug discovery. A consolidation of its traditional usage as well as its chemical and pharmacological profiles will thus guide efforts aimed at maximizing this potential. In undertaking this survey of the Amaryllidaceae of southern African, we aimed to achieve these goals.

We determined whether the expression of matrix metalloproteinases (MMP) and tissue inhibitors of MMPs (TIMP) in murine macrophages is regulated by the novel synthetic bacterial lipopeptide JBT 3002. Multilamellar liposomes (MLV) encapsulating JBT 3002 (MLV-JBT 3002) stimulated the production of 72-kDa and 92-kDa (gelatinase A and B) type IV collagenase and inhibited the production of murine metalloelastase (MME) in a dose-dependent manner in murine peritoneal macrophages. MLV-JBT 3002 also induced production of TIMP-1. MLV-JBT 3002 did not induce collagenase production in tumor cells. Priming murine macrophages with interferon-gamma (IFN-gamma) inhibited JBT 3002-stimulated production of both MMP-9 and MMP-2 and further inhibited production of MME by a mechanism involving nitric oxide (NO). This conclusion is based on data showing that IFN-gamma failed to inhibit production of MMP in the presence of L-methyl arginine or in macrophages from inducible nitric oxide synthase knockout mice. These data suggest that JBT 3002 differentially regulates the production of various MMPs and TIMP in macrophages.

We determined whether the angiogenesis and growth of murine colon carcinomas growing in the wall of the cecum is dependent on infiltrating leukocytes. Syngeneic BALB/c or SCID mice were treated with a myelosuppressive, maximally tolerated dose of doxorubicin. Parental or multidrug resistant CT-26 colon carcinoma cells were implanted into the cecal wall 3 days after the second intravenous injection of doxorubicin. Control mice developed large, well-vascularized tumors, whereas doxorubicin-pretreated mice did not. Intravenous injection of spleen cells from normal BALB/c or SCID mice one day prior to tumor cell implantation reversed the decreased vascularity and tumorigenicity. The production of proangiogenic molecules and microvessel density in tumors directly correlated with the number of infiltrating leukocytes, suggesting that tumor-infiltrating leukocytes are essential to angiogenesis of murine colon carcinomas.

We studied activation to the tumoricidal state of murine peritoneal macrophages by liposomes containing a new synthetic analogue, JBT3002, of a lipoprotein from the outer wall of a gram-negative bacterium. The liposomes containing JBT3002 or CGP31362 were superior to liposomes containing muramyl tripeptide phosphatidylethanolamine (MTP-PE) for tumoricidal activation in three ways. First, efficient macrophage activation required lower concentrations of JBT3002 or CGP31362 than MTP-PE. Second, macrophage activation by JBT3002 was less dependent on priming by interferon-gamma. Third, MLV-JBT3002 activated tumoricidal properties in both lipopolysaccharide (LPS)-responsive and LPS-nonresponsive macrophages. The activation of tumoricidal properties by MLV-JBP3002 depended on protein tyrosine kinase (PTK) activity associated with phosphorylation of tyrosine. The major mechanism for tumoricidal activity in macrophages incubated with MLV-JBT3002 was due to increased activity of inducible nitric oxide synthase (iNOS) and, hence, production of nitric oxide (NO). We base this conclusion on the results of several experiments. First, MLV-JBT3002 was not directly toxic to tumor target cells. Second, the specific iNOS inhibitor NG-monomethyl-L-arginine abrogated tumor cell lysis by MLV-JBT3002-treated macrophages. Third, macrophages from iNOS knockout mice did not lyse tumor cells, even after incubation with high concentrations of MLV-JBT3002. These data suggest that liposomes containing the synthetic bacterial lipopeptide JBT3002 are potent activators of macrophage tumoricidal properties.

Although humoral responses to Borrelia burgdorferi (Bb) have been shown to be protective in some animal models of Lyme disease, the role of T cells in this disease is less well understood. This work describes three Bb-specific T cell lines that prevent disease progression in syngeneic mice. The T cell lines were generated in C3H mice immunized with Bb in complete Freund's adjuvant. All lines were Bb-specific, CD4+, TCRαβ+, and they proliferated and produced interferon-γ and interleukin-2 on stimulation with Bb. Injection of the cell lines into naive C3H recipients significantly reduced the number of organisms recoverable from the blood and tissues of infected mice and protected them from developing Bb-induced periarthritis. These studies demonstrated that Th1 cells can confer resistance to Bb infection in susceptible mice and suggested that the timing of this T cell response may be critical for determining disease outcome.

The following series of experiments explores the post-xenografting differentiation of a naturally occurring, non-neuronal cell cultured from the leptomeninges of an 84-year-old woman. In culture, flat process-bearing human cells from the leptomeninges were positive for GFAP and 200 kDa neurofilament protein (negative for 68, 160 kDa neurofilament protein). The C3 spinal cord was exposed in 30 adult athymic rats. The hindlimb dorsal columns were transected at C3 and the nerve fibers aspirated to form a pocket, into which 10(6) fast blue-labeled, human leptomeningeal-derived cells were placed. The C3 spinal cord was studied immunohistochemically over 60 days. Three days later the dorsal horn contained fast blue-GFAP-positive astrocyte-like cells that were negative for neurofilament protein. By 7 days, large, process-bearing, fast blue-GFAP-positive (neurofilament protein-negative), astrocyte-like cells joined the native astrocytes of the pia-glia membrane and were in the gray matter of the spinal cord. Some of these astrocyte-like cells were also positive for the human specific histocompatibility complex, HLADR. These data extend the age, species and tissue of origin for pluripotential cells for CNS transplantation.

Astrocytes appear to be excluded from blood vessels. In order to test this observation, pial blood vessels were seeded upon cultures of purified E14 fetal astrocytes. One to seven days later co-cultures were immunohistochemically stained for laminin and fibronectin. Confocal microscopy revealed that laminin-positive cultured astrocytes migrated from the coverslip to and on to the surface of the blood vessels. The fetal astrocytes migrated along the basement membrane but did not enter the blood vessel. Viable (live) basement membrane is a barrier to astrocyte intravasation and is a pathway for astrocyte migration. Published by Elsevier Science Ltd.

Glioblastoma is very rarely found outside the central nervous system. The ability of rat C6 glioblastoma cells to intravasate into central nervous system and pial blood vessels is tested using a rat homografting model and two in vitro models. In vivo, scanning electron microscopy demonstrates that upon grafting C6 cells into implantation pockets in rat cortex, blood vessels can be spared in large digestion cysts formed in host brain parenchyma. Immunocytochemistry of the grafted rat cortex reveals that the glioblastoma cells are upon the blood vessel basement membrane, surrounded by the extracellular matrix material, fibronectin. The endothelial cells of the blood vessel are inside the laminin and fibronectin, and there were areas of endothelial cell hyperplasia. C6 cells are not observed inside blood vessels. In vitro, C6 cell cultures seeded with blood vessels from fresh rat pia exhibit the same relationship of the C6 glioblastoma cells to the blood vessel as those in the other models. The C6 cells migrate upon the pial blood vessel basement membrane but do not intravasate into the blood vessel. To ascertain whether structure and components of the blood vessel basement membrane are important factors in glioblastoma cell exclusion from blood vessels, C6 cells are seeded upon artificial basement membrane hydrated gel wafers. C6 cells migrate into the artificial basement membrane gel wafer by 1 day after seeding. These data indicate that glioblastoma cells are confined to the central nervous system by an inability to pass through vital basement membrane.

The plant family Amaryllidaceae occupies a privileged status within the botanical hierarchy due to its horticultural and ornamental appeal, as well as its widespread usage in the traditional medicinal practices of indigenous peoples across the globe. Of greater significance are the unique, structurally-diverse alkaloid constituents produced by members of the family, which has spawned several biologically significant molecules. In this regard, the Alzheimer's drug galanthamine has gained much prominence due to its selective and reversible inhibitory interaction with the enzyme acetylcholinesterase (AChE), of significance in the progression of neurodegeneration associated with Alzheimer's disease (AD). The lycorine series of compounds within the family have recently emerged as novel inhibitors of AChE, in some instances with higher levels of activity compared with the commercial drug galanthamine, making them attractive targets for natural product and synthetically-driven structure-activity relationship studies. This brief survey traces the emergence of lycorine compounds over the past decade as promising leads in the therapeutic approach towards AD and their possible future advancement onto the clinical stage.

The therapy of cancer is, in reality, the design of therapeutic strategies for therapy of metastatic disease. Metastases consist of unique subpopulations of tumor cells that are derived from the primary tumor, colonize distant target organs, and are able to subvert host immune responses, establish necessary angiogenesis, and obtain a sufficient nutrient supply while evolving to become autonomous from homeostatic mechanisms that function within normal, differentiated tissues. Attempts at eradication of metastases by conventional therapies have generally been unsuccessful due to genetic instability and heterogeneity of metastatic tumors; these properties lead to the emergence of tumor cells that are resistant to most conventional treatments. It may be possible to circumvent this heterogeneity by the activation of tissue macrophages to the tumoricidal state. Activated macrophages are able to kill tumor cells while sparing normal tissues, and efficient activation can be achieved by encapsulation of synthetic muramyl tripeptide analogues into multilamellar vesicles composed of phospholipids. Systemic administration of these liposome-encapsulated compounds leads to tumoricidal activation of alveolar and peritoneal macrophages and eradication of established tumor metastasis in numerous animal tumor models, and this form of therapy is enhanced by combination with parenteral administration of cytokines. Phase III clinical trials of recurrent osteosarcoma are currently in progress. Modulation of the tumor microenvironment by activated macrophages may prove to be an additional modality in treatment strategies that combine the use of biological response modifiers with conventional therapies.

A fluorimetric acetylcholinesterase (AChE) assay was developed and characterized both in solution and with the enzyme entrapped in sol-gel-derived silica. The assay is based on a disulfide-thiol interchange reaction between the intramolecularly quenched dimeric dye BODIPY FL l-cystine and thiocholine generated by the AChE-catalyzed hydrolysis of acetylthiocholine (ATCh), which results in a brightly fluorescent monomeric product owing to the cleavage of the disulfide-coupled form of the dye. The new assay was validated by comparison with the Ellman assay performed under parallel conditions and was used in both kinetic and end point assays. The assay was extended to the fabrication of functional AChE microarrays using contact pin-printing of sol-gel-derived silica. A total of 392 sol-gel formulations were screened for gelation times and 192 of these were further evaluated for array fabrication on four different surfaces using a factor analysis approach. Of these, 66 sol-gel/surface combinations produced robust microarrays, while 26 sol-gel/surface combinations were identified that could produce highly active AChE microarrays. The Z' factor for the on-array assay using an optimal sol-gel/surface combination, which considers both signal variability and difference in signals between positive and negative controls, was determined to be 0.60, which is above the minimum level required for applicability to screening. By overprinting nanoliter volumes of solutions containing the dye, ATCh, and potential inhibitors, these microarrays could be used to screen two libraries of small molecules, one composed of newly synthesized alkaloids and another consisting of ∼1000 known bioactive compounds, both as discrete compounds and mixtures thereof, for activity against AChE. IC(50) values were obtained on microarrays for compounds showing significant inhibitory activity, demonstrating the utility of arrays for quantitative inhibition assays.

We determined whether the intravenous administration of multilamellar vesicle liposomes (MLV) containing a lipopeptide analogue of a fragment from the cell wall of gram-negative bacteria (CGP 31 362) can render BALB/c mouse alveolar macrophages tumoricidal in situ and reduce the incidence of spontaneous lung metastasis of syngeneic renal carcinoma (RENCA) cells. Alveolar macrophages (a) incubated in vitro with MLV containing CGP 31 362 (MLV-31 362) and (b) harvested from mice injected i.v. with MLV-31 362 were rendered cytotoxic against the RENCA cells. Maximum cytotoxic activity of the macrophages was induced by injecting 5 µmol MLV consisting of 250 mg phospholipids and 0.5 mg CGP 31 362. The single i.v. injection of 5 µmol MLV-31 362 produced activation of macrophages that lasted for up to 4 days. Repeated i. v. injections of MLV-31 362 produced a continuous antitumor activity in alveolar macrophages. To study the lipopeptide's effects on metastasis, we injected the left kidneys of BALB/c mice with RENCA cells. The kidney with growing tumor was resected 10 days later and, after a further 2 days, groups of mice were injected i.v. with MLV-31 362 or with MLV-HBSS (twice weekly for 3 weeks). Treatment with MLV-31 362 significantly decreased the median number of spontaneous lung metastases. These data demonstrate that the systemic administration of MLV-31 362 can activate murine lung macrophages in situ and reduce the incidence of spontaneous RENCA lung metastases.

Cultured C6 glioma cells were prelabeled with the plant lectin Phaseolus vulgaris leuco-agglutinin (PHAL) and grafted as a cell suspension (10(6) cells in 5.0 microliters) into freshly made cortical implantation pockets in adult host rats. Animals were killed 1-21 days post-implantation (DPI). The brains were removed, dehydrated, embedded in paraffin and sectioned at 8 microns. Paraffin sections were processed for light level immunofluorescent double labeling for PHAL, a marker for graft derived cells, and glial fibrillary acidic protein (GFAP), a specific marker for C6 glioma cells and astrocytes. Cells positive for both PHAL and GFAP were graft-derived C6 cells. By 7 DPI a large mass developed which extended above the surface of the brain and invaded (displacement of host tissue by a cell mass) the host parenchyma. This mass increased in size over the next 14 days. The invading tumor mass contained double labeled cells at all time periods examined. In addition to the invasion process, grafted C6 cells spread through the host parenchyma by migration (movement of single cells). Individual graft-derived C6 (GFAP/PHAL positive) cells migrated into host cortex surrounding the implantation pocket, corpus callosum ventral to the implantation pocket, ipsilateral internal capsule and bilaterally in the habenula.

Current therapy of human superficial bladder cancer includes the intravesical administration of antitumor drugs and immunomodulators. The purpose of these studies was to determine whether phospholipid liposomes that bind to human bladder cancer cells can improve the delivery of interferon alpha (IFN-alpha) to neoplastic urothelium. The antiproliferative activity of free IFN-alpha and IFN-alpha encapsulated in liposomes was assessed in vitro against the human transitional cell carcinoma line 253J. The cells were exposed to free and liposome-encapsulated IFN-alpha for short periods ranging from 30 minutes to four hours, and inhibition of cell growth was determined three days later. The production of greater than 25 percent cytostasis of 253J cells by free IFN-alpha required four hours of continuous exposure. In contrast, IFN-alpha encapsulated in liposomes produced 35 percent and 60 percent cytostasis after a 30-minute and four-hour exposure, respectively. Liposome-encapsulated IFN-alpha was also effective (50 percent cytostasis) against a subline of 253J cells selected for resistance against free IFN-alpha. Liposomes containing IFN-alpha were stable in the presence of human urine. In vivo studies in mice showed that intravesical administration of radiolabeled IFN-alpha or radiolabeled liposomes did not yield significant systemic absorption and deposition in distant organs. Collectively, these results suggest that the encapsulation of IFN-alpha within multilamellar liposomes may augment its antiproliferative activity, overcome some forms of tumor cell resistance to IFN-alpha, and prove useful for intravesical therapy of superficial bladder cancer.

The selective apoptosis-inducing activity of Amaryllidaceae alkaloids belonging to the crinane-type is reported. A mini-library of natural and synthetic crinane alkaloids was assembled. Biological screening indicated crinamine 4 and haemanthamine 9 to be potent inducers of apoptosis in tumour cells at micromolar concentrations. Structure-activity relationships demonstrated the requirement for both an alpha-C2 bridge and a free hydroxyl at the C-11 position as pharmacophoric requirements for this activity.

Human leptomeningeal (arachnoid and pia mater) cells in culture have been demonstrated in replicated studies to express typical neuronal proteins such as neurofilament protein and neuron-specific enolase. In addition, they can express glial fibrillary acidic protein. The present study examines the possibility that neuronal and glial proteins might be present in rat and human leptomeningeal cells in situ. The neuronal proteins 160 kDa and 200 kDa neurofilaments, neuron-specific enolase and microtubule-associated protein 2 were, however, not immunolocalized in either the pia mater or arachnoid. Glial fibrillary acidic protein and galactocerebroside were also not detected, while fibronectin and vimentin immunoreactivities were robust in all layers of the leptomeninges. Together with the previously reported expression of some neuronal and astroglial markers in cultured human leptomeninges, these observations suggest that culture alters the properties of leptomeningeal cells.

Once prostate cancer metastasizes to bone, conventional chemotherapy is largely ineffective. We hypothesized that inhibition of phosphorylation of the epidermal growth factor receptor (EGF-R) and platelet-derived growth factor receptor (PDGF-R) expressed on tumor cells and tumor-associated endothelial cells, which is associated with tumor progression, in combination with paclitaxel would inhibit experimental prostate cancer bone metastasis and preserve bone structure. We tested this hypothesis in nude mice, using human PC-3MM2 prostate cancer cells. PC-3MM2 cells growing adjacent to bone tissue and endothelial cells within these lesions expressed phosphorylated EGF-R and PDGF-Rα and -β on their surfaces. The percentage of positive endothelial cells and the intensity of receptor expression directly correlated with proximity to bone tissue. Oral administration of PKI166 inhibited the phosphorylation of EGF-R but not PDGF-R, whereas oral administration of STI571 inhibited the phosphorylation of PDGF-R but not EGF-R. Combination therapy using oral PKI166 and STI571 with i.p. injections of paclitaxel induced a high level of apoptosis in tumor vascular endothelial cells and tumor cells in parallel with inhibition of tumor growth in the bone, preservation of bone structure, and reduction of lymph node metastasis. Collectively, these data demonstrate that blockade of phosphorylation of EGF-R and PDGF-R coupled with administration of paclitaxel significantly suppresses experimental human prostate cancer bone metastasis.

Purpose: We sought to determine whether blockade of platelet-derived growth factor receptor (PDGF-R) activation by oral administration of a PDGF-R tyrosine kinase inhibitor (STI571) alone or in combination with i.p. paclitaxel can inhibit the progression of tumors caused by human ovarian carcinoma cells growing in the peritoneal cavity of female nude mice. Experimental Design: In several different experiments, paclitaxel-sensitive and paclitaxel-resistant metastatic human ovarian carcinoma cells were injected into the peritoneal cavity of nude mice. Seven days later, groups (n = 10) of mice began receiving a control treatment, STI571 alone, paclitaxel alone, or a combination of STI571 and paclitaxel. The mice were necropsied after 45 days of treatment. Results: Treatment with combination therapy significantly reduced tumor weight (relative to control or single-agent therapy) in all three human ovarian cancer cell lines. Immunohistochemical analyses revealed that PDGF-R activation was blocked by STI571 administered alone or in combination with paclitaxel. Tumor-associated endothelial cells expressed both PDGF-R and phosphorylated PDGF-R. In mice receiving combination therapy, tumor-associated endothelial cells underwent apoptosis, leading to decreases in microvessel density and tumor cell proliferation relative to control and single-agent therapy. Conclusions: These results show that administration of a PDGF-R tyrosine kinase inhibitor in combination with paclitaxel impairs the progression of ovarian cancer in the peritoneal cavity of nude mice, in part, by blockade of PDGF, an endothelial cell survival factor, which results in the increased apoptosis of tumor-associated endothelial cells.

We evaluated the expression of platelet-derived growth factor (PDGF) ligands and receptors in clinical specimens of human pancreatic adenocarcinomas and determined the therapeutic effect of STI571 (Gleevec), a protein tyrosine kinase inhibitor of PDGF receptor (PDGFR), on human pancreatic carcinoma cells growing in the pancreas and liver of nude mice.Immunohistochemical staining for PDGF-AA and -BB ligands, PDGFR-alpha and -beta, and phosphorylated PDGFR-alpha and -beta was performed on 31 specimens of human pancreatic cancer and L3.6pl human pancreatic adenocarcinoma cell line. To determine the in vivo effects of STI571, nude mice with L3.6pl cells injected into the pancreas were randomized 7 days later to receive one of the following treatments: sterile water p.o. (control), STI571, gemcitabine, or a combination of STI571 and gemcitabine.In 29 of 31 clinical specimens of human pancreatic adenocarcinoma, both tumor cells and tumor-associated endothelial cells expressed phosphorylated PDGFR-alpha and -beta. L3.6pl cells growing in culture expressed moderate amounts of PDGF-AA and little to no PDGFR-alpha or -beta, whereas L3.6pl cells growing in the pancreas of nude mice expressed a high level of PDGF and receptors. Colocalization immunohistochemical analysis demonstrated expression of activated PDGFR-beta by tumor-associated endothelial cells in both the pancreas and in liver metastases. Tumors of mice treated for 4 weeks with STI571 (50 mg/kg or 100 mg/kg p.o. daily) were slightly smaller than controls. Tumors treated with gemcitabine and STI571 (50 mg/kg) were70% smaller than tumors in control mice and 36% smaller than those in mice treated with gemcitabine only (P0.0002 and P0.04, respectively). Combination therapy also inhibited spontaneous metastasis to the liver. Tumors from mice treated with both STI571 and gemcitabine had decreased expression of activated (phosphorylated) PDGFR-alpha and -beta, decreased mean vessel density, decreased cell proliferation, and increased apoptosis of tumor cells.Collectively, these data show that activated PDGFR on tumor cells and tumor-endothelial cells can be a novel target for therapy of pancreatic carcinoma.

Objectives . We determined the expression of platelet-derived growth factor (PDGF), PDGF-receptor (PDGF-R), and phosphorylated PDGF-R (p-PDGF-R) on tumor cells and tumor-associated endothelial cells in clinical specimens of human ovarian carcinoma and human ovarian cancer cells growing in culture and in the peritoneal cavity of nude mice. Methods . Ten specimens of high-grade serous ovarian carcinoma were analyzed using immunohistochemistry (IHC). IHC was used to detect ligand and receptor expression in the human ovarian cancer cells from Hey A8 and SKOV3ip1 growing in culture. Cells from these lines were also implanted orthotopically into the peritoneal cavity of nude mice. IHC was used to determine ligand and receptor expression in tumors that formed in the peritoneal cavity. Results . All 10 evaluable samples expressed both PDGF AA and BB on tumor cells. Tumor cells were positive for PDGF-Rα in 10/10 samples, PDGF-Rβ in 8/10 samples, p-PDGF-Rα in 6/10 samples, and p-PDGF-Rβ in 4/10 samples. p-PDGF-Rα was positive in 4/10 tumor-associated endothelial cell samples and p-PDGF-Rβ was positive in 3/10 samples. Human ovarian cancer cells expressed PDGF, PDGF-R, and p-PDGF-R when growing in culture or in the peritoneal cavity of nude mice. PDGF-R and p-PDGF-R were also present on tumor-associated endothelial cells as demonstrated by simultaneous staining with CD31 antibody. Conclusions . PDGF and the corresponding receptors were expressed in autochthonous human ovarian cancer lesions on both tumor cells and tumor-associated endothelial cells. The ligand and receptor were also present on Hey A8 and SKOV3ip1 human ovarian cancer cells growing in vitro and in the peritoneal cavity of nude mice.

We determined whether chronic administration of IFN-alpha at optimal biological dose inhibits angiogenesis of human pancreatic carcinoma growing in the pancreas of nude mice.Cells of the human pancreatic cancer cell line L3.6pl were implanted into the pancreas of nude mice. Seven days later, groups of mice received s.c. injection with IFN-alpha alone (50,000 units biweekly or 10,000 units daily), i.p. injection with gemcitabine alone (125 mg/kg biweekly), or injection with both daily IFN-alpha and biweekly gemcitabine for 35 days. In a survival study, the mice were treated until they became moribund.Biweekly treatments with 50,000 units of IFN-alpha alone were ineffective. In contrast, daily injections of IFN-alpha (10,000 units/day) alone, biweekly injections of gemcitabine alone, or the combination of IFN-alpha and gemcitabine reduced tumor volume by 53%, 70%, and 87%, respectively. Immunohistochemical analysis revealed that treatment with IFN-alpha alone or with IFN-alpha plus gemcitabine inhibited expression of the proangiogenic molecules basic fibroblast growth factor and matrix metalloproteinase 9 more than did treatment with gemcitabine alone. These treatments also decreased the staining of proliferating cell nuclear antigen within the tumor and induced apoptosis in tumor-associated mouse endothelial cells (staining with CD31/terminal deoxynucleotidyl transferase-mediated nick end labeling), leading to a decrease in microvessel density.These data show that administration of IFN-alpha at optimal biological dose and schedule in combination with gemcitabine induced apoptosis in tumor-associated endothelial cells and decreased growth of human pancreatic cancer cells in the pancreas, leading to a significant increase in survival.

BACKGROUND Expression of platelet-derived growth factor (PDGF) and activation (by autophosphorylation) of its receptor (PDGF-R), a tyrosine kinase, are associated with the growth of metastatic prostate tumor cells in the bone parenchyma. The tyrosine kinase inhibitor STI571 blocks the PDGF signaling pathway by inhibiting PDGF-R autophosphorylation. We examined the effects of STI571, given alone or with paclitaxel (Taxol), on tumor growth in a mouse model of prostate cancer metastasis. METHODS Human prostate cancer PC-3MM2 cells were injected into the tibias of male nude mice. Three days later the mice (20 per group) were randomly assigned to 5 weeks of treatment with oral and injected water (control), daily oral STI571, weekly injected paclitaxel, or STI571 plus paclitaxel. Lesions in bone and the surrounding muscles were then harvested and analyzed by histology, western blotting (for PDGF-R phosphorylation), immunohistochemistry (for expression of proangiogenic molecules), and double immunofluorescence (to identify endothelial cells and apoptotic tumor cells). Growth of bone lesions was monitored by digital radiography. Bone lesions from control mice were used to establish short-term cell cultures for analysis of PDGF-R phosphorylation. All statistical tests were two-sided. RESULTS PC-3MM2 cells cultured from bone lesions and treated in vitro with STI571 had less phosphorylated PDGF-R than untreated cells. In control mice, bone lesions expressed high levels of PDGF and activated (i.e., phosphorylated) PDGF-R, whereas lesions in the adjacent musculature did not. Activated PDGF-R was present on the surface of endothelial cells within the bone lesions but not in endothelial cells of uninjected bone. Mice treated with STI571 or STI571 plus paclitaxel had a lower tumor incidence, smaller tumors, and less bone lysis and lymph node metastasis than mice treated with water or paclitaxel alone (P

We determined whether blockade of the epidermal growth factor receptor (EGF-R) signaling pathway by oral administration of the EGF-R tyrosine kinase inhibitor (PKI 166) alone or in combination with injectable Taxol inhibits the growth of PC-3MM2 human prostate cancer cells in the bone of nude mice.Male nude mice implanted with PC-3MM2 cells in the tibia were treated with oral administrations of PKI 166 or PKI 166 plus injectable Taxol beginning 3 days after implantation. The incidence and size of bone tumors and destruction of bone were determined by digitalized radiography. Expression of epidermal growth factor (EGF), EGF-R, and activated EGF-R in tumor cells and tumor-associated endothelial cells was determined by immunohistochemistry.Oral administration of PKI 166 or PKI 166 plus injectable Taxol reduced the incidence and size of bone tumors and destruction of bone. Immunohistochemical analysis revealed that PC-3MM2 cells growing adjacent to the bone expressed high levels of EGF and activated EGF-R, whereas tumor cells in the adjacent musculature did not. Moreover, endothelial cells within the bone tumor lesions, but not in uninvolved bone or tumors in the muscle, expressed high levels of activated EGF-R. Treatment with PKI 166 and more so with PKI 166 plus Taxol significantly inhibited phosphorylation of EGF-R on tumor and endothelial cells and induced significant apoptosis and endothelial cells within tumor lesions.These data indicate that endothelial cells exposed to EGF produced by tumor cells express activated EGF-R and that targeting EGF-R can produce significant therapeutic effects against prostate cancer bone metastasis.

We determined whether blockade of the epidermal growth factor-receptor (EGF-R) signaling pathway by oral administration of the EGF-R tyrosine kinase inhibitor PKI166 can inhibit angiogenesis and growth of SN12PM6 human renal cell carcinoma (HRCC) in the kidney of nude mice and whether gemcitabine can potentiate these effects. In vitro treatment of HRCC cells with PKI166 inhibited EGF-R autophosphorylation, which correlated with a decrease in expression of Bcl-xl protein and phosphorylation of signal transducers and activators of transcription, particularly signal transducers and activators of transcription 3. PKI166 also decreased expression of vascular endothelial growth factor and basic fibroblast growth factor in a dose-dependent manner. Oral administration of PKI166 or PKI166 and injected gemcitabine or gemcitabine alone beginning 7 days after implantation of SN12PM6 cells into the kidney of athymic nude mice reduced the volume of tumors by 26, 61, and 23%, respectively. In another experiment 28 days after the orthotopic implantation of SN12PM6 cells, nephrectomy was performed followed by 4 weeks of treatment. Treatment with PKI166 and, more so, PKI166 plus gemcitabine significantly inhibited lung metastasis, corresponding to a significant increase in overall length of survival. EGF-R activation was significantly blocked by therapy with PKI166 and was associated with a significant reduction in expression of vascular endothelial growth factor and interleukin-8, decreased microvessel density, decreased staining of proliferating cell nuclear antigen, and increased tumor cell apoptosis. Collectively, the data indicate that targeting activation of EGF-R on HRCC produces significant therapeutic benefits.

We evaluated whether treatment of orthotopic human prostate cancer in nude mice with pegylated IFN-alpha-2b (PEG-IFN-alpha-2b) and docetaxel could represent a two-compartment targeting of primary tumor (tumor cells and tumor-associated endothelial cells) and inhibition of regional lymph node metastasis. The antiangiogenic properties of IFN were combined with the cytotoxic properties of docetaxel, resulting in apoptosis of both tumor cells and endothelium and hence significant inhibition of primary tumor growth. We first determined the optimal biological dose of PEG-IFN-alpha-2b (70,000 IU/week) necessary to down-regulate the expression of basic fibroblast growth factor, matrix metalloprotease-9, and matrix metalloprotease-2. The therapeutic dose of docetaxel (10 mg/kg/week) was determined by efficacy and minimal body weight loss. Therapy beginning 3 days after orthotopic implantation of PC3-MM2 prostate cancer cells reduced tumor weight by 37% in mice treated with PEG-IFN-alpha-2b, by 60% in mice treated with docetaxel, and by 83% in those given both drugs. PEG-IFN-alpha-2b also induced apoptosis of tumor-associated endothelial cells and hence a significant decrease in microvessel density. Our data indicate that the combination of PEG-IFN-alpha and docetaxel inhibits neoplastic angiogenesis by inducing a decrease in the local production of proangiogenic molecules by tumor cells, resulting in increased apoptosis of tumor-associated endothelial cells.

The purpose of this study was to optimize the antitumor andantiangiogenic activities of pegylated IFN-alpha (PEG-IFN-alpha)alone or in combination with paclitaxel against SKOV3ip1 human ovarian cancer cells growing orthotopically in female nude mice. Seven days after the i.p. implantation of tumor cells, groups of mice (n = 10) were injected s.c. once per week (for 4 weeks) with different doses of PEG-IFN-alpha (3,500, 7,000, 35,000, and 350,000 units). PEG-IFN-alpha at 7,000 units significantly decreased tumor incidence and volume. At doses exceeding 7,000 units, PEG-IFN-alpha was less efficacious. In another set of studies conducted 7 days after the i.p. implantation of SKOV3ip1 cells, groups of mice (n = 10) received (once per week for 4 weeks) either s.c. administrations of PEG-IFN-alpha (7,000 units), i.p. injections of paclitaxel (100 microg/wk), or a combination of PEG-IFN-alpha and paclitaxel. The mice were killed 7 days after the last treatment, and tumor burden was assessed. Administration of PEG-IFN-alpha at the optimal biological dose (7,000 units) in combination with paclitaxel significantly decreased angiogenesis and progressive growth of human ovarian carcinoma cells in a synergistic fashion. The combination therapy produced the most significant inhibition in expression of the proangiogenic molecules basic fibroblast growth factor and matrix metalloproteinase-9. Decreased microvessel density, decreased proliferating cell nuclear antigen staining, and increased endothelial cell apoptosis also correlated with therapeutic success. Collectively, the data suggest that combining the optimal biological dose of PEG-IFN-alpha with paclitaxel may provide a novel and effective approach to the treatment of human ovarian carcinoma.

Since vascular endothelial growth factor (VEGF) plays a major role in tumor angiogenesis, we determined whether blockage of VEGF receptor signaling using a novel tyrosine kinase inhibitor (PTK 787) decreases the growth and metastasis of human pancreatic carcinoma growing orthotopically in nude mice. Human pancreatic L3.6pl cells were injected into the pancreas of nude mice. Seven days later, groups of mice were given daily oral administrations of PTK 787 alone, twice weekly i.p. injections of gemcitabine, or combination therapy. The mice were necropsied when control mice became moribund (day 35). Therapy with PTK 787 alone, gemcitabine alone, or the combination of both agents produced respectively 60%, 70%, and 81% inhibition in the volume of pancreatic cancers. The combination therapy significantly decreased the incidence of lymph node and liver metastasis, leading to a significant increase in survival. Microvessel density (MVD) was significantly decreased in tumors treated with either PTK 787 alone or PTK 787 plus gemcitabine. MVD directly correlated with tumor cell proliferation and inversely correlated with apoptosis of tumor cells and associated endothelial cells. Collectively, our results demonstrate that blockade of VEGF-R signaling may provide an additional approach to the therapy of pancreatic cancer.

The alcoholic extract of the fresh bulbs of Cyrtanthus elatus yielded zephyranthine (1) and 1,2-O-diacetylzephyranthine (2), together with three other known alkaloids. Complete assignment of 1H and 13C NMR spectra of compounds 1 and 2 was done by employment of two-dimensional NMR techniques.

The authors recently reported that tumoricidal activation of macrophages by a new synthetic bacterial lipopeptide, JBT 3002, can augment chemotherapy-mediated tumor-cell killing. The aim of this study was to identify the mechanism responsible for the destruction of metastatic cells. Three daily oral doses of JBT 3002 before once-weekly intraperitoneal injections of 100 mg/kg irinotecan for 3 weeks significantly increased the eradication of established CT-26 murine colon cancer liver metastases compared with treatment with irinotecan alone. Immunohistochemical analyses revealed that the hepatic metastases in mice given combination therapy contained infiltrating CD8+ lymphocytes and a dense infiltrate of macrophages expressing both inducible nitric oxide synthase (iNOS) and interleukin-15. In vitro treatment of peritoneal macrophages with JBT 3002 plus interferon-gamma induced the expression of iNOS and the production of nitric oxide. In the presence of a low (subtoxic) dose of irinotecan, these activated macrophages produced significant lysis of CT-26 cells. The high level of cytotoxicity was inhibited by the specific inducible nitric oxide synthase inhibitor, NG-methyl-L-arginine. In contrast, irinotecan-mediated lysis of normal intestinal epithelial IEC-6 cells was not increased by activated macrophages. Scanning electron microscopy revealed that activated macrophages bound to CT-26 tumor cells but not to normal IEC-6 cells, confirming that nitric oxide-mediated cytotoxicity is specific for tumor cells. Collectively, the results suggest that augmentation of the therapeutic efficacy of irinotecan against colon cancer liver metastases by immunomodulation with JBT 3002 may be associated with elevated inducible nitric oxide synthase and endogenous interleukin-15 in tumor-infiltrating macrophages.

A phytochemical investigation of the bulbs of Brunsvigia radulosa yielded the new alkaloid 1-O-acetylnorpluviine, together with the known structures 1-epideacetylbowdensine, crinamine, crinine, hamayne, lycorine, anhydrolycorin-6-one and sternbergine. All structures were established by spectroscopic evidence. Some of the 13C assignments which were reported for crinamine and hamayne were corrected by means of 2D NMR techniques. In order to provide a further structure for biological testing, crinamine was converted to apohaemanthamine. The alkaloids were tested for activity against two strains of cultured Plasmodium falciparum and for cytotoxicity with BL6 mouse melanoma cells.

Astrocytic tumours of the central nervous system express cell adhesion receptors of the integrin superfamily, CD44 and adhesion receptors of the immunoglobulin superfamily. Glioma cells utilize these receptors to adhere to and migrate along components of the extracellular matrix (ECM), which is uniquely distributed and regulated within the brain and the spinal cord. For penetration into healthy brain tissue a number of proteases are expressed, which degrade proteins of the extracellular matrix. Thus, glioma cell invasion into the adjacent brain tissue is dependent on the interaction of glioma cells with the extracellular matrix and the subsequent destruction of matrix barriers. There is a critical balance between expression of various adhesion receptors and proteases. The tight regulation of critical levels of proteases and receptors expressed by glioma cells or other cells is necessary for the "physiological" behaviour of glioma cells. Shifts in the balance of protein expression determine glioma cell behaviour in their micro-environment and can initiate or influence the complex process of glioma cell invasion. The complex receptor-ECM interaction in glioma cell invasion is discussed focussing upon the role of integrin receptors and matrix-metalloproteinases. Influencing these molecules or their regulation may lead to novel therapeutic approaches in the treatment of malignant glioma.

Since previous studies have shown that cells cultured from human leptomeninges can express neuronal and glial antigens under appropriate culture conditions [DeGiorgio L. A. et al. (1994) J. Neurol. Sci. 124, 141 148; Bernstein J. J. et al. (1996) Int. J. Derl Neurosci. 14(5), 681 687], we have studied the developmental characteristics of these cells further by grafting them into young adult rat brains. Cells were labeled in culture with Fast Blue and were identified unequivocally by hybridization with nick-translated human DNA. Intensely Fast Blue positive human leptomeningeal cells were concentrated in the implant pocket and adjacent rat leptomeninges al one and two weeks postimplant. Human and rat leptomeningeal cells were similar morphologically and were equally immunopositive for vimentin and fibronectin. Implanted human cells did not express the neuronal and glial proteins they had in vitro. Cells which hybridized with human DNA corresponded to the intensely Fast Blue positive cells. Small groups of human DNA hybridizing cells were also observed in the choroid plexus. Less intensely Fast Blue positive neurons and glia were found in the brain but these hybridized with rat DNA. A minority of human leptomeningeal cells implanted into rat brain are subsequently found in host leptomeninges where they demonstrate properties characteristic of leptomeningeal fibroblasts. Small numbers of implanted cells can survive for two weeks.

Cultured human leptomeningeal fibroblasts grafted into rat frontal cortex were localized to the implant pocket and to adjacent host leptomeninges. Immunohistochemical studies using a panel of human-specific and domain-specific APP antibodies revealed that all grafted cells expressed both APP and A β in situ. Remarkably, these antibodies also labeled rat pial and ependymal cells as well as reactive astrocytes adjacent to vessels. In addition, apical projections and cell bodies of many cortical pyramidal neurons contained human-specific APP immunoreactive material. Groups of subcortical neurons, particularly those of the amygdala, hippocampal formation and suprachiasmatic nuclei, were similarly labeled. The presence of human APP in host brains was confirmed by immunoblotting. Birefringent Congo Red staining was observed in the cortical neuropil and in leptomeningeal vessels. These data indicate that grafted leptomeningeal fibroblasts hyperexpress APP and A β which can diffuse into parenchyma and be taken up by specific rat cells.

In the alkaloid galanthamine, the plant family Amaryllidaceae has endowed the pharmaceutical community with a potent and selective inhibitor of the enzyme acetylcholinestersae (AChE), of prominence in the chemotherapeutic approach towards motor neuron diseases. Following on the commercial success of this prescription drug in the treatment of Alzheimer's disease, it is anticipated that other drug candidates will in future emerge from the family. In this regard, the phenanthridones, exemplified by narciclasine and pancratistatin, of the lycorine series of Amaryllidaceae alkaloids have shown much promise as remarkably potent and selective anticancer agents, with a drug target of the series destined for the clinical market within the next decade. Given these interesting biological properties and their natural abundance, plants of the Amaryllidaceae have provided a diverse and accessible platform for phytochemical-based drug discovery. The crinane series of Amaryllidaceae alkaloids are also enriched with a significant array of biological properties. As a consequence of their close structural similarity to the anticancer agents of the lycorine series, the cytotoxic potential of crinane alkaloids has been realized through structure-activity relationship (SAR) studies involving targets of both semi-synthetic and natural origin, which has identified several members as leads with promising antiproliferative profiles. As the first of its kind, this review seeks to collate such information from the past few decades in advancing the crinane group as a viable platform for anticancer drug discovery.

Transection of the hindlimb dorsal column fibers leads to a partial deafferentation of the neurons of the nucleus gracilis, the first relay of the ascending dorsal column-medial lemniscal (DC-ML) neural network. In response to the deafferentation, a synaptic renewal cycle is initiated and the neurons of the nucleus gracilis atrophy. The present study examines the molecular changes that occur in synaptic relays of the ascending DC-ML following a hindlimb dorsal column transection. Rats were sacrificed 0.5, 1, 24, or 72 hours postlesion. Steady-state levels of mRNA coding for c-fos were significantly elevated only at 24 hours postlesion in caudal dorsal medulla, which contains the nucleus gracilis. The increased c-fos is neuronal in origin since there is an increased level of c-fos immunoreactivity in both the cluster neurons and the interneurons of the nucleus gracilis. In the third relay of the DC-ML system, the somatomotor cortex, levels of c-fos mRNA were significantly decreased 72 hours postlesion. These data indicate that lesions of the hindlimb dorsal column fibers have transneuronal effects on gene expression that extend to at least the third synaptic relay in the DC-ML system.

Fresh suspensions of human glioblastoma multiforme were preincubated in the plant lectin Phaseolus vulgaris leucoagglutinin (PHAL) and implanted into cortical pockets in adult rat brain. Brains were investigated periodically over 30 postoperative days and the migration of the human glioblastoma cells was traced with anti-PHAL immunofluorescence or the overexpression of human specific p185c-neu a specific marker of a class of human malignant astrocytoma cells. The principal pathway of migration of the implanted human cells in the rat brain was ventrally through cortical gray matter and into the corpus callosum, with rapid lateral distribution in this and other parallel and intersecting white matter fascicles. Human glioblastoma cells also migrated on basement membrane lined blood vessels, pia-glia membrane and spaces of Virchow-Robin, as well as the subependymal space of the ventricles. These paths of migration of human glioblastoma cells in the rat brain are consistent with the pathways of spread of glioblastoma in the human brain as described by Scherer over 50 years ago, indicating that multifocal malignant astrocytomas have common migratory pathways in mature mammalian brain.

Alterations in the immunogenic properties of tumor cells frequently accompany selection for multiple-drug-resistant (MDR) variants. Therefore, studies were performed to examine the hypothesis that overexpression of membrane P-glycoprotein, commonly observed in MDR tumor cells, is associated with enhanced immunogenic properties. Immunogenicity was determined by (a) the ability of drug-sensitive parental UV2237M fibrosarcoma cells and drug-resistant UV2237M variant cells to immunize normal mice against rechallenge with parental tumor cells and (b) the ability of normal syngeneic mice to reject cell inocula that caused progressive tumor growth in immunocompromised mice. Variant UV2237M cell lines included subpopulations selected for a six- to ten-fold increase in mRNA for P-glycoprotein and expression of the MDR phenotype (resistance to doxorubicin) and cells sensitive to doxorubicin (and no expression of MDR properties) but resistant to ouabain. All UV2237M drug-resistant cells were highly immunogenic in immunocompetent mice, regardless of their MDR phenotype. Additional studies showed that CT-26 murine adenocarcinoma cells, sensitive or resistant to doxorubicin (expressing high levels of P-glycoprotein), injected into normal syngeneic Balb/c mice produced rapidly growing tumors. The data do not demonstrate a correlation between the immunogenic properties of drug-resistant tumor cells and the expression of P-glycoprotein.

The immunoglobulin (IgG, IgM, IgA) content of normal, reactive and migrated rat astrocytes was studied in lesioned adult rat cortex and after fetal cortex grafts. Implantation pockets were aspirated in the somatomotor cortex (under bregma) of adult Sprague-Dawley rats. A fetal (E14) rat hemicortex incubated in the plant lectin Phaseolus vulgaris leucoagglutinin (graft premarker) was placed in the aspiration pocket or the pocket left empty (control). Sections of brain were immunohistochemically double labeled for immunoglobulins-GFAP or Phaseolus vulgaris leucoagglutinin-GFAP 3–60 days postoperative. Mature or fetal astrocytes in situ did not contain immunoglobulins. In pocket-only animals, individual reactive astrocytes lining and subjacent to the pocket were positive for rat IgG, IgM and IgA (3–60 days). In grafted animals, graft derived astrocytes (Phaseolus vulgaris leucoagglutinin-GFAP positive) were intermingled with host reactive astrocytes (Phaseolus vulgaris leucoagglutinin negative) lining and subjacent to the pocket. Both classes of astrocytes contained immunoglobulins IgG, IgM and IgA. Immunoglobulin positive graft derived astrocytes migrated into the corpus callosum, cingulum and habenula. These results demonstrate that astrocytes sequester immunoglobulins and probably other serum proteins as a critical function in restoring homeostasis to the injured or diseased nervous system.

✓ Overexpression of human-specific c-neu proto-oncogene transmembrane tyrosine kinase receptor protein (p185) is an index of cell transformation and of poor patient survival in several malignancies. The authors studied this protein in low- and high-grade human malignant astrocytomas before and after xenografting into aspiration pockets in rat cortex. Human-specific p185c-neu-positive cells were found in tumor specimens from all grades of astrocytoma. Significantly fewer p185c-neu-positive cells were observed in the low-grade versus the high-grade astrocytomas examined (p < 0.05). Human specific p185c-neu-positive cells were also positive for the human major histocompatibility complex, human leukocyte antigen (HLA)-DR, as well as glial fibrillary acidic protein and S-100 protein. Fresh suspensions of tumor tissue were prelabeled with the plant lectin Phaseolus vulgaris leukoagglutinin and xenografted into pockets in rat cortex. A class of human p185c-neu-positive cells found in tissue samples from all grades of astrocytoma migrated in the rat brain along parallel and intersecting nerve fiber bundles and basement membrane-lined surfaces. Migrated p185c-neu-positive cells were also positive for HLA-DR, Phaseolus vulgaris leukoagglutinin, glial fibrillary acidic protein, and S-100 protein, suggesting that they were in fact human astrocytoma cells. Simultaneous expression of human p185c-neu, HLA-DR, and glial fibrillary acidic protein was observed in a class of human malignant astrocytoma cells in both tumor tissue and xenografted cells that migrated into rat brain. These molecules are signature proteins for the study of the spread of human malignant astrocytomas in an animal model, and indicate that transformed human malignant astrocytoma cells can migrate within the parenchyma of the central nervous system and could play a role in the development of multifocal tumors.

Background Understanding the mechanism of prostate cancer metastasis is essential to the design of a more effective therapy. An effective therapy for this disease will depend on the development of a clinically relevant in vivo model. Purpose We describe the development of such a model by using orthotopic implantation of human prostate cells in BALB/c nude mice. Method We compared the tumorigenicity of and the incidence of metastasis of human prostate cancer PC-3M and LNCaP-FGC (LNCaP) cell lines subsequent to prostatic (orthotopic) or subcutaneous (ectopic) implantations in male nude mice. Results LNCaP cells produced tumors only in the prostate. Enhanced tumorigenicity at the orthotopic site was found for PC-3M cells. Lymph node metastases were observed in practically all mice given an injection of PC-3M cells in the prostate, but they were uncommon with subcutaneous injection of these cells. Bilateral orchiectomy did not alter the tumorigenicity of either PC-3M or LNCaP cells or the incidence of lymph node metastasis by PC-3M cells. LNCaP tumors in the mouse prostate (but not PC-3M tumors) elaborated detectable levels of human prostate-specific antigen (PSA) in the serum, even when tumors were small (1.5 mm in diameter). Immunohistochemistry analysis revealed the presence of the PSA marker in tissue sections of LNCaP but not of PC-3M tumors. Conclusions The implantation of human prostate cancer cells in an ectopic environment does not permit expression of metastatic potential. In contrast, intraprostatic implantation does. Implications These data suggest that the orthotopic injection of human prostate cancer cells into the nude mouse may provide a valuable model to study the biology and therapy of human prostate cancer.

Fetal basal ganglia astrocytes and C6 glioma cells were plated on the surface of 1.5 cm thick hydrated collagen I wafers. Both cell types migrated through the entire thickness of the wafer within 1 day after plating. The collagen in the wafer was digested and the fine collagen I fibrils were clumped into large strands. By 2-3 days, the collagen strands were digested from the wafers and replaced by a mass of fetal astrocytes or C6 cells joined by their processes. The collagen I digestion and cell migration suggested protease production. In a second series of experiments, cultured C6 cells and E14 fetal astrocytes were immunohistochemically stained for the presence of plasminogen activators as an index of protease production. Both tissue (tPA) and urokinase (uPA) types were observed. Fetal astrocytes and C6 cells were also positive for guanidinobenzoatase, a serine protease associated with migrating cells. These data demonstrate that rapid migration of the cells on and through collagen I fibrils is concomitant with expression of plasminogen activators and proteases which can either activate or function as collagenases and release the cells from the substrate.