The orphan G protein-coupled receptor GPR124/TEM5 is highly expressed in central nervous system (CNS) endothelium. Here, complete null or endothelial-specific GPR124 deletion produced embryonic lethality from CNS-specific angiogenesis arrest in forebrain and neural tube. Conversely, GPR124 overexpression throughout all adult vascular beds produced CNS-specific hyperproliferative vascular malformations. In vivo, GPR124 functioned cell-autonomously in endothelium to regulate sprouting, migration, and developmental expression of the blood-brain barrier marker Glut1, while in vitro, GPR124 mediated Cdc42-dependent directional migration to forebrain-derived, VEGF-independent cues. Our results demonstrate CNS-specific angiogenesis regulation by an endothelial receptor, and illuminate functions of the poorly understood adhesion GPCR subfamily. Further, the striking functional tropism of GPR124 marks this receptor as a therapeutic target for CNS-related vascular pathologies.

Human prominin-1/CD133 has been reported to be expressed in neural and hematopoietic stem/progenitor cells and in embryonic, but not adult, epithelia. This lack of detection of human prominin-1, as defined by its glycosylation-dependent AC133 epitope, is surprising given the expression of the murine ortholog in adult epithelia. Here, we demonstrate, by using a novel prominin-1 antiserum (alphahE2), that the decrease of AC133 immunoreactivity observed during differentiation of the colonic adenocarcinoma-derived Caco-2 cells is not paralleled by a down-regulation of prominin-1. We have also shown that alphahE2 immunoreactivity, but not AC133 immunoreactivity, is present in several adult human tissues, such as kidney proximal tubules and the parietal layer of Bowman's capsule of juxtamedullary nephrons, and in lactiferous ducts of the mammary gland. These observations suggest that only the AC133 epitope is down-regulated upon cell differentiation. Furthermore, alphahE2 immunoreactivity has been detected in several kidney carcinomas derived from proximal tubules, independent of their grading. Interestingly, in one particular case, the AC133 epitope, which is restricted to stem cells in normal adult tissue, was up-regulated in the vicinity of the tumor. Our data thus show that (1) in adults, the expression of human prominin-1 is not limited to stem and progenitor cells, and (2) the epitopes of prominin-1 might be useful for investigating solid cancers.

Summary:Monitoring the engraftment of donor cells after allogeneic blood stem cell transplantation (BSCT) may be important for the early diagnosis of graft failure or relapse of disease. Several techniques have been reported for this purpose. PCR-based assays analyzing polymorphic short tandem repeat (STR) markers are attractive because they are sensitive and can be performed rapidly. The intent of the present study was to test a novel approach for the quantification of mixed chimerism using a commercial multiplex STR assay with fluorescence-based detection for forensic purposes. The feasibility of this assay and the accuracy of quantitative results was tested using serial cell mixtures of unrelated individuals. Sample preparation was optimized to obtain information from minute amounts of starting material, eg from patients with aplasia or from sorted cell populations. Using the STR-PCR, discrimination between donor and recipient was possible in all patients analyzed (n = 25). Cell dilution experiments showed a linear correlation between the cell numbers added and the proportions found, with the limit of detection for a minor cell population being 5%. Comparison of values obtained with standard FISH analysis in patients transplanted from sex-mismatched donors showed an excellent correlation with the STR-PCR results. Taken together, this procedure allows the rapid, versatile and accurate quantification of mixed chimerism, even with minuscule numbers of cells. Keywords: chimerism; short-tandem repeats (STR); quantification; PCR; fluorescence detection Allogeneic blood stem cell transplantation is frequently performed in patients with nonmalignant and malignant hematological diseases such as severe aplastic anemia (SAA), severe combined immunodeficiency (SCID), acute and chronic leukemia and lymphoma. Detection of the degree of chimerism after transplantation is an important method for monitoring the engraftment of donor cells and allows early detection of graft failure. This seems to be especially important in patients at risk for graft failure, ie patients Correspondence: C Thiede, Medizinische Klinik und Poliklinik I, Universitätsklinikum Carl Gustav Carus der Technischen Universität, Fetscherstrasse 74, 01307 Dresden, Germany Received 12 September 1998; accepted 4 January 1999 receiving T cell-depleted stem cell grafts from unrelated donors.1 In addition, novel therapeutic approaches like nonmyeloablative stem cell transplantation 2,3 are dependent on rapid information on the degree of mixed chimerism to schedule therapeutic interventions, eg donor lymphocyte infusion (DLI).Several approaches have been published for the detection of chimerism. In sex-mismatched transplantation settings, information on the ratio between donor and recipient can be obtained efficiently and rapidly by using fluorescent in situ hybridization (FISH) with probes specific for X-and Y-chromosome.4,5 PCR-based amplification of a single variable number of tandem repeat (VNTR) or short tandem repeat (STR) markers is another frequently perf...

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Prominin/CD133 is a 115/120-kDa integral membrane glycoprotein specifically associated with plasma membrane protrusions in epithelial and non-epithelial cells including neuroepithelial and hematopoietic stem cells. Here we report the identification as well as molecular and cell biological characterization of mouse, rat, and human prominin-2, a 112-kDa glycoprotein structurally related to prominin (referred to as prominin-1). Although the amino acid identity between prominin-2 and prominin-1 is low (<30%), their genomic organization is strikingly similar, suggesting an early gene duplication event. Like prominin-1, prominin-2 exhibits a characteristic membrane topology with five transmembrane segments and two large glycosylated extracellular loops. Upon its ectopic expression in Chinese hamster ovary cells as a green fluorescent protein fusion chimera, prominin-2 was also found to be associated with plasma membrane protrusions, as revealed by its co-localization with prominin-1, suggesting a related role. Consistent with this, prominin-2 shows a similar tissue distribution to prominin-1, being highly expressed in the adult kidney and detected all along the digestive tract as well as in various other epithelial tissues. However, in contrast to prominin-1, prominin-2 was not detected in the eye, which perhaps explains why a loss-of function mutation in the human prominin-1 gene causes retinal degeneration but no other obvious pathological signs. Finally, we present evidence for the existence of a family of pentaspan membrane proteins, the prominins, which are conserved in evolution.