Angiostatin blocks tumor angiogenesis in vivo, almost certainly through its demonstrated ability to block endothelial cell migration and proliferation. Although the mechanism of angiostatin action remains unknown, identification of F 1-FO ATP synthase as the major angiostatin-binding site on the endothelial cell surface suggests that ATP metabolism may play a role in the angiostatin response. Previous studies noting the presence of F 1 ATP synthase subunits on endothelial cells and certain cancer cells did not determine whether this enzyme was functional in ATP synthesis. We now demonstrate that all components of the F 1 ATP synthase catalytic core are present on the endothelial cell surface, where they colocalize into discrete punctate structures. The surfaceassociated enzyme is active in ATP synthesis as shown by dual-label TLC and bioluminescence assays. Both ATP synthase and ATPase activities of the enzyme are inhibited by angiostatin as well as by antibodies directed against the ␣-and ␤-subunits of ATP synthase in cell-based and biochemical assays. Our data suggest that angiostatin inhibits vascularization by suppression of endothelialsurface ATP metabolism, which, in turn, may regulate vascular physiology by established mechanisms. We now have shown that antibodies directed against subunits of ATP synthase exhibit endothelial cell-inhibitory activities comparable to that of angiostatin, indicating that these antibodies function as angiostatin mimetics.

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Whether astrocytes utilize B7:CD28 co-stimulation to activate T cells mediating CNS inflammatory disease is controversial. In this report, primary astrocytes and murine astrocyte lines, generated by immortalization at two different times, day 7 or 45 of culture, were examined for their capability to express B7 co-stimulatory molecules and to participate in B7:CD28 co-stimulation. Following exposure to IFN-gamma, primary astrocytes and astrocyte lines up-regulated MHC class II and B7-2 (CD86) molecules. However, B7-1 (CD80) expression was not inducible on primary astrocytes examined after IFN-gamma stimulation beginning on day 7 or on astrocyte lines immortalized on day 7. B7-1 expression was inducible on primary astrocytes examined later and could be up-regulated on astrocyte lines immortalized later. Unlike B7-1, temporal discordant expression of other co-stimulatory/adhesion molecules was not observed. Both B7-1(-)/B7-2(+) and B7-1(+)/B7-2(+) astrocyte lines were capable of stimulating proliferation of encephalitogenic Th1 cells, utilizing B7-2 for B7:CD28 co-stimulation. However, lines derived from immortalization later (B7-1(+)/B7-2(+)) were more effective in stimulating proliferation of naive myelin basic protein-specific CD4(+) T cells. Astrocyte lines that expressed both B7-1 and B7-2 also stimulated Thp cells to secrete proinflammatory Th1 cytokines, whereas lines that expressed B7-2 only stimulated Thp cells to produce a Th2 cytokine pattern. Thus, we demonstrate for the first time that individual astrocytes can differentially express B7-1 molecules, which may correlate with their ability to stimulate proinflammatory and regulatory patterns of cytokine production. These results suggest that astrocytes have potential for both promoting and down-regulating T cell responses, and that temporal differences in expression of B7 molecules should be considered when evaluating immune regulation by astrocytes.

This study examined the impact of otitis media with effusion (OME) and associated hearing loss between 6 and 48 months of age on attention dimensions (i.e., selective/focus, sustained) during the elementary school years. A prospective cohort design in which 74 African American infants were recruited between ages 6 and 12 months. Ear examinations were done repeatedly using both otoscopy and tympanometry, and hearing was assessed using standard audiometric procedures between 6 and 48 months. Multiple measures of attention (i.e., direct assessment, behavioral observations, parent/teacher ratings) were administered from kindergarten through second grade to assess two theoretical dimensions of attention: selective/focused and sustained. The home environment was assessed annually. Results indicated that neither early childhood OME nor hearing loss showed significant correlations with any of the longitudinal or cross-sectional measures of selective/focused attention and sustained attention. In contrast, children with mothers who had fewer years of education and who lived in less responsive and supportive home environments scored higher on both parent and teacher ratings of sustained attention (i.e., hyperactivity) through the second grade of elementary school. For NEPSY Auditory Attention in second grade, a significant interaction between the Home Observation for Measurement of the Environment and hearing loss was uncovered. This interaction showed that children with hearing loss from poor home environments experienced greater difficulties on the NEPSY Auditory Attention task than those with hearing loss from good home environments. These findings do not support a direct linkage of a history of OME and associated hearing loss to difficulties in selective/focused attention or sustained attention in early elementary school children. Relationships between sociodemographic variables and attention-related functions appear stronger and should be considered as mediators in any examination of the linkages between early OME and subsequent attention functions.

Astrocytes are nonprofessional APCs that may participate in Ag presentation and activation of pathogenic CD4+ T cells involved in central nervous system (CNS) inflammatory diseases. Using immortalized pure astrocytes as a complement to the study of primary astrocytes, we investigated whether these astrocytes express elements involved in the class II endocytic pathway and if they are capable of processing native myelin basic protein (MBP), a step that could be necessary for initiating or perpetuating T cell recognition of this self-Ag in vivo. Upon IFN-γ-stimulation, primary and immortalized astrocytes up-regulate class II transactivator (CIITA), invariant chain (Ii) (p31 and p41), H-2Ma, and H-2Mb. Analysis of CIITA cDNA sequences demonstrated that CIITA transcription in astrocytes is directed by a promoter (type IV) that mediates IFN-γ-inducible CIITA expression and encodes a CIITA protein that differs in its N-terminal sequence from CIITA reported in professional APC. Comparing live and fixed APC for Ag presentation, we show that Ag processing by APC is required for presentation of native MBP to autopathogenic T cells specific for the major MBP epitope, Ac1-11. We have observed that primary astrocytes and some, but not all, astrocyte lines in the absence of contaminating microglia are capable of processing and presenting native MBP, suggesting that there may be heterogeneity. Our study provides definitive evidence that astrocytes are capable of processing CNS autoantigen, indicating that astrocytes have potential for processing and presentation of CNS autoantigen to proinflammatory T cells in CNS autoimmune disease.

NK T cells are a T cell subset in the human that express an invariant α-chain (Vα24invt T cells). Because of the well-described immunomodulation by glucocorticoids on activation-induced cell death (AICD), the effects of dexamethasone and anti-CD3 stimulation on Vα24invt T cell clones and CD4+ T cell clones were investigated. Dexamethasone significantly enhanced anti-CD3-mediated proliferation of Vα24invt T cells, whereas CD4+ T cells were inhibited. Addition of neutralizing IL-2 Ab partially abrogated dexamethasone-induced potentiation of Vα24invt T cell proliferation, indicating a role for autocrine IL-2 production in corticosteroid-mediated proliferative augmentation. Dexamethasone treatment of anti-CD3-stimulated Vα24invt T cells did not synergize with anti-Fas blockade in enhancing proliferation or preventing AICD. The Vα24invt T cell response to dexamethasone was dependent on the TCR signal strength. In the presence of dexamethasone, lower doses of anti-CD3 inhibited proliferation of Vα24invt T cells and CD4+ T cells; at higher doses of anti-CD3, which caused inhibition of CD4+ T cells, the Vα24invt T cell clones proliferated and were rescued from AICD. These results demonstrate significant differences in TCR signal strength required between Vα24invt T cells and CD4+ cells, and suggest important immunomodulatory consequences for endogenous and exogenous corticosteroids in immune responses.

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