Delta-like 4 is indispensable in thymic environment specific for T cell development

The thymic microenvironment is required for T cell development in vivo. However, in vitro studies have shown that when hematopoietic progenitors acquire Notch signaling via Delta-like (Dll)1 or Dll4, they differentiate into the T cell lineage in the absence of a thymic microenvironment. It is not clear, however, whether the thymus supports T cell development specifically by providing Notch signaling. To address this issue, we generated mice with a loxP-flanked allele of Dll4 and induced gene deletion specifically in thymic epithelial cells (TECs). In the thymus of mutant mice, the expression of Dll4 was abrogated on the epithelium, and the proportion of hematopoietic cells bearing the intracellular fragment of Notch1 (ICN1) was markedly decreased. Corresponding to this, CD4 CD8 double-positive or single-positive T cells were not detected in the thymus. Further analysis showed that the double-negative cell fraction was lacking T cell progenitors. The enforced expression of ICN1 in hematopoietic progenitors restored thymic T cell differentiation, even when the TECs were deficient in Dll4. These results indicate that the thymus-specific environment for determining T cell fate indispensably requires Dll4 expression to induce Notch signaling in the thymic immigrant cells

Characterisation of apoptosis in the neuronal cell line, ND7/23

The process of programmed cell death or apoptosis plays a critical role in the development of the nervous system and ensures that the correct number of neurons are finally present (Wyllie et al, 1980). Post-mitotic neurons undergo programmed cell death, the extent of which is determined by the availability of trophic factors derived from the target field (Oppehneim 1991). However, early in development extensive cell death is observed when proliferating neuroblasts cease dividing and differentiate into mature non-dividing neurons and during adulthood when neuronal cell death occurs abnormally as a result of excitotoxicity, anoxia and ischemia (Robert-Lewis et a L, 1993). The neuronal cell line, ND7 (Wood et al., 1991), was used as a model system to analyze the processes involved in neuronal development. ND7 cells can be maintained in continuously growing culture in the presence of serum. When transferred to serum-free medium, the ND7 cells are induced to cease dividing and undergo morphological differentiation, acquiring numerous dendritic processes typical of differentiated neurons (Wheatley et al., 1991). Following transfer to serum-free medium a number of cells die rather than differentiate. Biochemical and morphological analysis confirmed that ND7 cells were dying by apoptosis when serum-starved (Wyllie et al., 1980). However, not all cells died when transferred to serum-free medium, suggesting that cells had the choice to either die or differentiate. Indeed, apoptosis can be regulated in ND7 cells. Addition of RA to differentiating cells enhanced cell death whereas addition of cAMP promoted both survival and differentiation. Furthermore, apoptosis was dependent on de novo protein synthesis and was inhibited by aurintricarboxylic acid which is known to inhibit the nuclease activity responsible for the DNA degradation characteristic of this mode of cell death. The heat shock response and the heat shock proteins (hsps) have been implicated in the protection of cells against a variety of stresses (Lindquist 1986; Lindquist and Craig 1988). Indeed, heat shock conferred ND7 cells protection against apoptosis. An increase in the level of hsp synthesis was paralleled by an increase in cell survival. Furthermore, heat shock protected neonatal rat primary dorsal root ganglia neurons from NGF deprivation, establishing the ND7 cell line as a suitable model to study the effects of heat shock on apoptosis in sensory neurons. To determine the role played by individual heat shock proteins in the protection against apoptosis, the two major inducible hsps, hsp90 and hsp70, were over expressed in ND7 cells. Cell lines over expressing either hsp90 or hsp70 showed no significant difference in survival following transfer to serum-free medium indicating that these hsp alone do not mediate the protective effect of heat stress. However, cell lines over expressing hsp90 and hsp70 were significantly more resistant to heat than the parental ND7 cells, showing for the first time that over expression of hsp70 and hsp90 can confer thermotolerance to neuronal cells

Synergies of VEGF inhibition and photodynamic therapy in the treatment of age-related macular degeneration

Photodynamic therapy (PDT) and the administration of compounds acting against vascular endothelial growth factor (anti-VEGF) are approved for the treatment of choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD). Experimental evidence that the combined use of both treatment options may improve therapeutic outcome is presented

Simultaneous but not prior inhibition of VEGF165 enhances the efficacy of photodynamic therapy in multiple models of ocular neovascularization

PURPOSE: To investigate the effect of the combined treatment of photodynamic therapy and specific VEGF165 inhibition with pegaptanib sodium (Macugen; Eyetech Pharmaceuticals, Lexington, MA) on ocular neovascularization. METHODS: Photodynamic therapy's (PDT's) effects on the integrity of pegaptanib sodium were analyzed by HPLC, a VEGF165-binding assay, and a VEGF165-induced tissue factor gene expression assay. The effects of mono- or combined treatment on vessel growth and regression were determined in a murine corneal neovascularization model. The effects of combined treatment on vessel growth were also determined in a murine choroidal neovascularization model. RESULTS: PDT did not affect the chemical composition of pegaptanib sodium nor the efficacy of pegaptanib sodium in the inhibition of VEGF165 binding to Flt-1 and VEGF165-induced gene expression. In an animal model of effects on existing ocular neovascular lesions (corneal neovascularization), PDT monotherapy yielded an initial regression of these vessels, but there followed a rapid regrowth. In contrast, pegaptanib sodium monotherapy yielded little regression but potently abrogated further vessel growth. The combination of pegaptanib sodium and PDT resulted in the regression of the neovascular lesions, as observed with PDT alone, but also prevented significant vessel regrowth, leading to a significantly greater reduction in lesion size than did each monotherapy. In addition, there was a significantly greater effect of the combination of pegaptanib sodium and PDT on lesion size in choroidal neovascularization than with each monotherapy. Pretreatment with pegaptanib sodium appeared to decrease the efficacy of PDT-induced vessel regression in corneal neovascularization, and as such the enhanced efficacy over monotherapy when the agents were delivered simultaneously was not observed. CONCLUSIONS: Although the combined simultaneous treatment of ocular neovascularization with PDT and pegaptanib sodium may provide a more effective approach for the regression and overall treatment of CNV associated with AMD, the order of addition of these treatments may play a role in achieving optimal efficacy