Successful Xenografts of Second Trimester Human Fetal Brain and Retinal Tissue in the Anterior Chamber of the Eye of Adult Immunosuppressed Rats

Successful xenografting of first trimester human fetal CNS tissue and retina has been reported in the literature. We wished to test the feasibility ofusing the anterior chamber ofthe rat eye to support the development of more mature human fetal xenografts. Here we report on the successful outcome of human brain and retinal transplants. Adult host rats immunosuppressed with cyclosporin A accepted these xenografts and supported their further development. Periodic examination of the host eyes using a direct ophthalmoscope or an ophthalmic slit lamp permitted direct visual monitoring of the health and growth of the transplants. Histologically it was possible to identify neuronal, macroglial, and microglial (macrophage) cell types within the grafts. Mitotic activity and histogenetic differentiation took place. Blood vessels filled with hematic cells were commonly present within the grafts. The walls of these vessels prevented the leakageofhorseradish peroxidase, suggesting the presence of a functional brain-blood barrier in the graft. These results indicate that it is possible to use a small animal model to study normal and pathological phenomena oniate fetal human neural tissues. Our group has already taken advantage of the model to achieve HIV infectivity offetal human brain outside the human body

The role of endothelial chemokine receptor CCR2 in monocyte chemoattractant protein-1-mediated leukocyte migration across the blood-brain barrier

Previous results from this laboratory revealed the presence of high-affinity, saturable binding sites for monocyte chemoattractant protein-1 (MCP-1) along human brain microvessels (Andjelkovic et al., 1999; Andjelkovic and Pachter, 2000), which suggested that CCR2, the recognized receptor for this chemokine, was expressed by the brain microvascular endothelium. To directly test the role of CCR2 in mediating MCP-1 interactions with the brain microvasculature, MCP-1 binding activity was assessed in murine brain microvessels isolated from wildtype mice and CCR2(−/−) mice engineered to lack this receptor. Results demonstrate that MCP-1 binding is greatly attenuated in microvessels prepared from CCR2(−/−) mice as compared to their wildtype controls. Moreover, microvessels from wildtype mice exhibited MCP-1-induced downmodulation following MCP-1 binding, and that the recovery of this binding activity was not dependent upon de novo protein synthesis. Furthermore, it was shown that MCP-1 was internalized within wild-type microvessels, but not within microvessels obtained from CCR2(−/−) mice. This further validated that CCR2 is obligatory for MCP-1 endocytosis. The internalization of MCP-1, but not transferrin, was inhibited by the disruption of caveolae. Internalized MCP-1 also co-localized at some sites with caveolin-1, a major protein of caveolae, implying that this chemokine is endocytosed, in part, via non-clathrin-coated vesicles. These results prompt consideration that MCP-1 signals may be relayed across the blood-brain barrier by highly specialized interactions of this chemokine with its cognate receptor—CCR2—along brain microvascular endothelial cells. ^ The contribution of CCR2 to MCP-1-induced transendothelial migration was evaluated through ‘mix-and-match’ experiments where endothelial or mononuclear cells from either wildtype or CCR2(−/−) were utilized. Specifically, this study assessed if CCR2 expression by brain microvascular endothelial cells (BMEC) facilitates peritoneal macrophage (pMØ) transendothelial migration. Results were that the expression of CCR2 by pMØ is required for these cells to undergo MCP-1-stimulated transmigration. Moreover, it was also demonstrated that CCR2 expression by BMEC was found to be critical for pMØ transmigration. Furthermore, cellular components from both wildtype and CCR2(−/−) were found to support transendothelial migration in response to another chemokine, MIP-1α. These findings underscore the contribution of CCR2 in MCP-1-stimulated transmigration and highlight a novel role for chemokine receptors on endothelia.

Cytokines and arachidonic metabolites produced during human immunodeficiency virus (HIV)-infected macrophage-astroglia interactions: implications for the neuropathogenesis of HIV disease

Human immunodeficiency virus (HIV) infection of brain macrophages and astroglial proliferation are central features of HIV-induced central nervous system (CNS) disorders. These observations suggest that glial cellular interactions participate in disease. In an experimental system to examine this process, we found that cocultures of HIV-infected monocytes and astroglia release high levels of cytokines and arachidonate metabolites leading to neuronotoxicity. HIV-l^D^-infected monocytes cocultured with human glia (astrocytoma, neuroglia, and primary human astrocytes) synthesized tumor necrosis factor (TNF-o 0 and interleukin 1B (IblB) as assayed by coupled reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and biological activity. The cytokine induction was selective, cell specific, and associated with induction of arachidonic acid metabolites. TNF-B, Iblc~, IL-6, interferon c ~ (IFN-c~), and IFN-'y were not produced. Leukotriene B4, leukotriene D4, lipoxin A4, and platelet-activating factor were detected in large amounts after high-performance liquid chromatography separation and correlated with cytokine activity. Specific inhibitors of the arachidonic cascade markedly diminished the cytokine response suggesting regulatory relationships between these factors. Cocultures of HIV-infected monocytes and neuroblastoma or endothelial cells, or HIV-infected monocyte fluids, sucros