Dear Editor: The blood brain barrier is formed by endothelial cells of cerebral capillaries and is highly selective by preventing specific substances, cells, and pathogens from passing and allowing other needed com-pounds through (3, 16). Primary cultures have been used to study pathophysiology of the blood brain barrier in vitro (5, 6, 8, 12, 24, 25). Isolation and characterization of primary cultures of brain endothelial cells are, however, time consuming; the resulting cultures differ in quality, contaminating cells, and their culture requirements are more fastidious than systemic and/or macrovascular endothelial cells. Previously, we reported the isolation, cultivation, and character-ization of endothelial cells derived from bovine cerebral microvessels (23). These cerebral endothelial cells were relatively pure at early passages (eg, Passage 3) and exhibited specific endothelial and brain characteristics. However, the exhibition of specific brain char-acteristics decreased with passage as also shown by other investigators (5, 8, 10, 12, 21). To facilitate the use of brain microvascular endothelial cells for studies of pathogenesis of CNS disease, we wanted to develop an in vitro model by extending the life span of brain microvascular endothelial cells. In this paper, we report the transfection and immortalization of bovine brain endothelial cells (BBEC) with SV40 large T (7) and demonstrate that these cells maintain original morphologic and func-tional characteristics of brain endothelial cells. Cell culture and transfection. Bovine brain capillaries were iso-lated and cultured as described previously (23). SV40-large T, a pBR 322 based construct (kindly provided by A. Srinivasan, The Wistar Institute, Philadelphia, PA)(7) was transfected into Passage 3 or 4 of the brain microvessel cultures. BBEC monolayers were incubated with pSVT DNA (10-15 gtg/dish at approximately 40-50% confluence along with pSV2neo plasmid (2-4 jsg, obtained from Dr. A. Srinivasan) for co-transfection, and thereafter selected using G418. Islands of transfected cells showing cobblestone appearance were transferred using cloning cylinders, expanded, and characterized as previously described (23) by indirect immunoperoxidase techniques. As shown in Fig. 1, the transfected cells (TBBEC) were positive for Factor VIII related antigen (F-VIII-Rag) and took up AcLDL-DiI, both markers for endothelial cells. Uptake of AcLDL is considered a hallmark for endothelial cells from various organs and species (13, 18), including human (Dr. W. Carley, personal communication) and bovine brain (6, 15, unpublished result). Moreover, the presence of carbonic anhydrase IV (CAIV)(9) and gamma glutamyl transpep-tidase (GGTP) as histochemically assessed using the method of DeBault and Cancilla (4) supported the endothelial character of the isolated cells. No nonendothelial cells were present such as pericytes and glial cells, as determined by immunocytochemistry, respectively, for smooth muscle actin (24) and glial fibrillary acidic protein. Presence of large T antigen in nuclei of transfected cells was confirmed using monoclonal antibody 101 (1)(not shown). These characteristics were maintained up to Passage 60 and cultures are still ongoing. Selected endothelial cell cultures were used in further experiments. Cell adhesion molecules in Tumor Necrosis Factor alpha (TNF) a activated brain endothelial cells. In order to examine responses to cytokines, we incubated confluent cultures of primary (= nontrans-fected)(Passages 4-10) and transfected brain endothelial cells (Passages 18-22) in 24-well plates with TNFo (10 ng/ml) for 4 and 24 h and assessed expression of ELAM and VCAM on cytospin slides …