Endosialin/TEM1 was originally discovered as a human embryonic fibroblast-specific antigen and was later found to be differentially expressed in tumor stroma and endothelium. Endosialin/TEM1 overexpression has been observed in many cancers of various tissue origin, including colon, breast, pancreatic, and lung. The knockout (KO) mouse model showed the absence of endosialin/TEM1 expression reduced growth, invasion, and metastasis of human tumor xenografts. In addition, lack of endosialin/TEM1 led to an increase in small immature blood vessels and decreased numbers of medium and large tumor vessels. This abnormal angiogenic response could be responsible for the reduced tumor growth and invasion observed in endosialin/TEM1 KO mice, suggesting a role for endosialin/TEM1 in controlling the interaction among tumor cells, endothelia, and stromal matrix. Here we report the identification of fibronectin (FN) and collagen types I and IV as specific ligands for endosialin/TEM1. More importantly, cells expressing endosialin/TEM1 exhibit enhanced adhesion to FN as well as enhanced migration through matrigel, although these properties could be blocked by a humanized antibody directed against human endosialin/TEM1. Our results pinpoint to a molecular mechanism by which expression of endosialin/TEM1 in the tumor stroma and endothelium may support tumor progression and invasion.CD248 ͉ fibronectin ͉ stroma ͉ collagen T he process of angiogenesis, which is critical for physiological tissue growth, wound healing, and embryonic development, also is required for the formation of large solid tumors (1). Within the developing capillary, extracellular matrix (ECM) proteins serve as a structural scaffold for proliferating endothelial and tumor tissues and, more important, provide support for the growth of tumor cells. Therefore, anticancer strategies aimed at disrupting these processes could result in effective therapies.Endosialin/TEM1 (CD248) originally discovered as a human embryonic fibroblast-specific antigen was later found to be differentially expressed in tumor stroma and endothelium. The monoclonal antibody FB5, generated through immunization of mice with human embryonic fibroblasts, was found to recognize an antigen, named endosialin, present on tumor stromal cells (2). Subsequently, examination of gene expression patterns in normal and neoplastic tissue indicated up-regulation of endosialin/TEM1 mRNA expression in tumor neovessels (3). Similar endosialin/TEM1 expression levels were noted in human colorectal cancer (4), breast cancer tissues (5), and histiocytomas (6). Moreover, human endosialin expression has been observed in highly invasive glioblastoma, anaplastic astrocytomas, and metastatic carcinomas, including melanomas (7,8).Tem1 knockout (KO) mice develop normally and exhibit normal wound healing, suggesting that endosialin/TEM1 is not required for neovascularization during fetal development or wound repair (9). However, when colorectal cancer cells were implanted in the abdominal sites of Tem1 KO mice, the loss of en...

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The BAR adaptor proteins encoded by the RVS167 and RVS161 genes from Saccharomyces cerevisiae form a complex that regulates actin, endocytosis, and viability following starvation or osmotic stress. In this study, we identified a human homolog of RVS161, termed BIN3 (bridging integrator-3), and a Schizosaccharomyces pombe homolog of RVS161, termed hob3؉ (homolog of Bin3). In human tissues, the BIN3 gene was expressed ubiquitously except for brain. S. pombe cells lacking Hob3p were often multinucleate and characterized by increased amounts of calcofluor-stained material and mislocalized F-actin. For example, while wild-type cells localized F-actin to cell ends during interphase, hob3⌬ mutants had F-actin patches distributed randomly around the cell. In addition, medial F-actin rings were rarely found in hob3⌬ mutants. Notably, in contrast to S. cerevisiae rvs161⌬ mutants, hob3⌬ mutants showed no measurable defects in endocytosis or response to osmotic stress, yet hob3؉ complemented the osmosensitivity of a rvs161⌬ mutant. BIN3 failed to rescue the osmosensitivity of rvs161⌬, but the actin localization defects of hob3⌬ mutants were completely rescued by BIN3 and partially rescued by RVS161. These findings suggest that hob3؉ and BIN3 regulate F-actin localization, like RVS161, but that other roles for this gene have diverged somewhat during evolution.

BackgroundStaphylococcal enterotoxins are considered potential biowarfare agents that can be spread through ingestion or inhalation. Staphylococcal enterotoxin B (SEB) is a widely studied superantigen that can directly stimulate T-cells to release a massive amount of proinflammatory cytokines by bridging the MHC II molecules on an antigen presenting cell (APC) and the Vβ chains of the T-cell receptor (TCR). This potentially can lead to toxic, debilitating and lethal effects. Currently, there are no preventative measures for SEB exposure, only supportive therapies.MethodsTo develop a potential therapeutic candidate to combat SEB exposure, we have generated three human B-cell hybridomas that produce human monoclonal antibodies (HuMAbs) to SEB. These HuMAbs were screened for specificity, affinity and the ability to block SEB activity in vitro as well as its lethal effect in vivo.ResultsThe high-affinity HuMAbs, as determined by BiaCore analysis, were specific to SEB with minimal crossreactivity to related toxins by ELISA. In an immunoblotting experiment, our HuMAbs bound SEB mixed in a cell lysate and did not bind any of the lysate proteins. In an in vitro cell-based assay, these HuMAbs could inhibit SEB-induced secretion of the proinflammatory cytokines (INF-γ and TNF-α) by primary human lymphocytes with high potency. In an in vivo LPS-potentiated mouse model, our lead antibody, HuMAb-154, was capable of neutralizing up to 100 μg of SEB challenge equivalent to 500 times over the reported LD50 (0.2 μg) , protecting mice from death. Extended survival was also observed when HuMAb-154 was administered after SEB challenge.ConclusionWe have generated high-affinity SEB-specific antibodies capable of neutralizing SEB in vitro as well as in vivo in a mouse model. Taken together, these results suggest that our antibodies hold the potential as passive immunotherapies for both prophylactic and therapeutic countermeasures of SEB exposure.