Abstract: The present invention provides a genetic method for tethering polypeptides to the yeast cell wall in a form accessible for binding to macromolecules. Combining this method with fluorescence-activated cell sorting provides a means of selecting proteins with increased or decreased affinity for another molecule, altered specificity, or conditional binding. Also provided is a method for genetic fusion of the N terminus of a polypeptide of interest to the C-terminus of the yeast Aga2p cell wall protein. Attaching an scFv antibody fragment to the Aga2p agglutinin effectively mimics the cell surface display of antibodies by B cells in the immune system for affinity maturation in vivo. T cell receptor mutants that are efficiently displayed on the yeast cell surface can be isolated by this method, providing a means of altering T cell receptor binding affinity and specificity by library screening. The selection method also identifies proteins with enhanced phenotypic characteristics, proteins that are displayed at higher levels, proteins that are secreted at higher efficiency and proteins that are more stable.

Abstract: Ets family transcription factors control the expression of a large number of genes in hematopoietic cells. Here we show strikingly precise differential expression of a subset of these genes marking critical, early stages of mouse lymphocyte cell-type specification. Initially, the Ets family member factor Erg was identified during an arrayed cDNA library screen for genes encoding transcription factors expressed specifically during T cell lineage commitment. Multiparameter fluorescence-activated cell sorting for over a dozen cell surface markers was used to isolate 18 distinct primary-cell populations representing discrete T cell and B cell developmental stages, pluripotent lymphoid precursors, immature NK-like cells and myeloid hematopoietic cells. These populations were monitored for mRNA expression of the Erg, Ets-1, Ets-2, Fli-1, Tel, Elf-1, GABPalpha, PU.1 and Spi-B genes. The earliest stages in T cell differentiation show particularly dynamic Ets family gene regulation, with sharp transitions in expression correlating with specification and commitment events. Ets, Spi-B and PU.1 are expressed in these stages but not by later T-lineage cells. Erg is induced during T-lineage specification and then silenced permanently, after commitment, at the beta-selection checkpoint. Spi-B is transiently upregulated during commitment and then silenced at the same stage as Erg. T-lineage commitment itself is marked by repression of PU.1, a factor that regulates B-cell and myeloid genes. These results show that the set of Ets factors mobilized during T-lineage specification and commitment is different from the set that maintains T cell gene expression during thymocyte repertoire selection and in all classes of mature T cells.

Abstract: Primitive subsets of leukemic cells isolated by using fluorescence-activated cell sorting from patients with newly diagnosed Ph+/BCR–ABL+ chronic myeloid leukemia display an abnormal ability to proliferate in vitro in the absence of added growth factors. We now show from analyses of growth-factor gene expression, protein production, and antibody inhibition studies that this deregulated growth can be explained, at least in part, by a novel differentiation-controlled autocrine mechanism. This mechanism involves the consistent and selective activation of IL-3 and granulocyte colony-stimulating factor (G-CSF) production and a stimulation of STAT5 phosphorylation in CD34+ leukemic cells. When these cells differentiate into CD34− cells in vivo, IL-3 and G-CSF production declines, and the cells concomitantly lose their capacity for autonomous growth in vitro despite their continued expression of BCR–ABL. Based on previous studies of normal cells, excessive exposure of the most primitive chronic myeloid leukemia cells to IL-3 and G-CSF through an autocrine mechanism could explain their paradoxically decreased self-renewal in vitro and slow accumulation in vivo, in spite of an increased cycling activity and selective expansion of later compartments.

Abstract: Interaction between the epithelium and the mesenchyme is an essential feature of organogenesis, including hair follicle formation. The dermal papilla (DP), a dense aggregate of specialized dermis-derived stromal cells located at the bottom of the follicle, is a major component of hair that signals the follicular epithelial cells to prolong the hair growth process. However, little is known about DP-specific gene activation with regard to hair induction. In this study we demonstrate that a short fragment (839 bp) of the human versican (a core protein of one of the matrix chondroitin sulfate proteoglycans) promoter is sufficient to activate lacZ reporter gene expression in the DP of postnatal transgenic mice and also in the condensed mesenchyme (the origin of the DP) beneath the hair placode during hair follicle embryogenesis. Using the same versican promoter with green fluorescent protein (GFP), large numbers of fresh pelage DP cells were isolated from newborn transgenic skin by high-speed cell sorting. These GFP-positive DP cells showed abundant versican mRNA, confirming that the reporter molecules reflected endogenous versican gene expression. These sorted GFP-positive cells showed DP-like morphology in culture, but both GFP and versican expression was lost during primary culture. In vivo hair growth assays showed that GFP-positive cells could induce hair when grafted with epithelial cells, whereas GFP-negative cells grafted with epithelium or GFP-positive cells alone did not. These results suggest that versican may play an essential role both in mesenchymal condensation and in hair induction.

Abstract: The Ecballium elaterium trypsin inhibitor II (EETI-II), a member of the squash family of protease inhibitors, is composed of 28 amino acid residues and is a potent inhibitor of trypsin. Its compact structure is defined by a triple-stranded antiparallel beta-sheet, which is held together by three intramolecular disulfide bonds forming a cystine knot. In order to explore the potential of the EETI-II peptide to serve as a structural scaffold for the presentation of randomized oligopeptides, we constructed two EETI-II derivatives, where the six-residue inhibitor loop was replaced by a 13-residue epitope of Sendai virus L-protein and by a 17-residue epitope from human bone Gla-protein. EETI-II and derived variants were produced via fusion to maltose binding protein MalE. By secretion of the fusion into the periplasmic space, fully oxidized and correctly folded EETI-II was obtained in high yield. EETI-II and derived variants could be presented on the Escherichia coli outer membrane by fusion to truncated Lpp'-OmpA', which comprises the first nine residues of mature lipoprotein plus the membrane spanning beta-strand from residues 46-66 of OmpA protein. Gene expression was under control of the strong and tightly regulated tetA promoter/operator. Cell viability was found to be drastically reduced by high level expression of Lpp'-OmpA'-EETI-II fusion protein. To restore cell viability, net accumulation of fusion protein in the outer membrane was reduced to a tolerable level by introduction of an amber codon at position 9 of the lpp' sequence and utilizing an amber suppressor strain as expression host. Cells expressing EETI-II variants containing an epitope were shown to be surface labeled with the respective monoclonal antibody by indirect immunofluorescence corroborating the cell surface exposure of the epitope sequences embedded in the EETI-II cystine knot scaffold. Cells displaying a particular epitope sequence could be enriched 10(7)-fold by combining magnetic cell sorting with fluorescence-activated cell sorting. These results demonstrate that E.coli cell surface display of conformationally constrained peptides tethered to the EETI-II cystine knot scaffold has the potential to become an effective technique for the rapid isolation of small peptide molecules from combinatorial libraries that bind with high affinity to acceptor molecules.

Abstract: The cytokines ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) signal through a receptor complex formed between two transmembrane proteins, gp130 and LIFRbeta. In addition, CNTF also uses a ligand-binding component which is anchored to the cell membrane. In the case of cardiotrophin-1 (CT-1), LIFRbeta is also required in cardiomyocytes, but this has not been proven in neurons, and published data suggest that motoneurons may use a different receptor complex. We used Lifrbeta knockout mice to assess the requirement for this receptor component in the signal transduction of CT-1 in motoneurons. To study purified motoneurons from such mutants, we have developed a method allowing for isolation of highly purified mouse motoneurons. This protocol is based on the immunoaffinity purification of motoneurons using antibodies against the extracellular domain of the neurotrophin receptor, p75, followed by cell sorting using magnetic microbeads. We show that CNTF, LIF, and CT-1 are unable to promote the survival of motoneurons derived from homozygous Lifrbeta-/- mutant embryos. Thus, LIFRbeta is absolutely required to transduce the CT-1 survival signal in motoneurons.

Abstract: While Salmonella infects macrophages, this cell population may not be the only one important for disseminating intracellular bacteria from mucosal sites. Dendritic cells (DC) are present in the Peyer's patches and are mobilized following stimulation. Such characteristics would seem to be ideal for the dissemination of an intracellular, mucosal pathogen. However, it has been difficult to obtain sufficient numbers of DC to assess their ability to harbor Salmonella or to monitor DC in vivo. In the present study, this problem has been addressed by expanding DC in vivo using flt3 ligand, followed by the purification of CD11c+ cells using antibody-coated magnetic beads or by fluorescence-activated cell sorting. Salmonella dublin were found to be efficiently internalized, and to survive and replicate within purified CD11c+ DC, and also in CD11c+, CD8alpha+ or CD11c+, CD11b+ DC subpopulations. The ability of Salmonella to enter DC is of similar magnitude to that reported for macrophages, suggesting that this cell population could be an important host cell for dissemination of this pathogen from mucosal sites. Furthermore, infected DC responded to Salmonella by secretion of IL-1, IL-6 and IL-12. As such, these cells may be important sources of these cytokines during the host response against Salmonella infection.

Abstract: MCF-7 cells migrate through vitronectin-coated filters in response to insulin-like growth factor I (IGF-I); migration is inhibited by the matrix metalloproteinase (MMP) inhibitor BB-94, but not by the serine proteinase inhibitor aprotinin. MMP-9 was identified in the conditioned medium of MCF-7 cells; in addition, fluorescence-activated cell sorting analysis revealed its presence on the cell surface, where MMP-9 activity was also found using a specific fluorogenic peptide. Furthermore, the messenger RNA encoding MMP-9 was detected in MCF-7 cells by PCR. The IGF-I concentration leading to maximal MCF-7 invasion produces an increase in cell surface proteolytic activity after short incubation periods. At 18 h, however, preincubation of MCF-7 cells with IGF-I produces at 18 h a dose-dependent decrease in cell-associated MMP-9 activity and an increase in soluble MMP-9. MCF-7 invasion is dependent on the αvβ5 integrin, a vitronectin receptor. The levels of αv- andβ 5-subunits expressed in MCF-7 cells depend on ...

Abstract: Germ cell transplantation, which offers promising new approaches for research and clinical applications, has focused interest on spermatogonia. This paper describes a procedure that permits the isolation of large quantities of viable spermatogonia. The immunomagnetic isolation procedure was applied to testicular cell suspensions from photoinhibited and photostimulated Djungarian hamsters, mice, and marmoset monkeys. The cells were incubated with a polyclonal rabbit anti-c-kit IgG, binding of which was characterized by immunohistochemical staining. For magnetic labeling, a secondary anti-rabbit IgG conjugated to ferromagnetic microbeads was used. Separation columns allowed the retention of magnetically labeled cells within the matrix. The magnetic fractions were eluted after removal of the column from the magnetic field. All fractions were analyzed for cellular morphology and by flow cytometry. The final enrichment of c-kit-positive cells in the magnetic fraction using fully active testes was in the range of 25-55% with a viability rate of 80-90%. The magnetic fractions of all three species were characterized by high numbers of diploid cells. Cytological analysis revealed a strong enrichment of spermatogonia. No haploid cells were retained in the magnetic fraction. In comparison to conventional procedures, magnetic cell separation is an efficient and fast approach for isolation of spermatogonia.

Abstract: A new procedure to investigate the relationship between bacterial cell size and activity at the cellular level has been developed; it is based on the coupling of radioactive labeling of bacterial cells and cell sorting by flow cytometry after SYTO 13 staining. Before sorting, bacterial cells were incubated in the presence of tritiated leucine using a procedure similar to that used for measuring bacterial production by leucine incorporation and then stained with SYTO 13. Subpopulations of bacterial cells were sorted according to their average right-angle light scatter (RALS) and fluorescence. Average RALS was shown to be significantly related to the average biovolume. Experiments were performed on samples collected at different times in a Mediterranean seawater mesocosm enriched with nitrogen and phosphorus. At four sampling times, bacteria were sorted in two subpopulations (cells smaller and larger than 0.25 μm3). The results indicate that, at each sampling time, the growth rate of larger cells was higher than that of smaller cells. In order to confirm this tendency, cell sorting was performed on six subpopulations differing in average biovolume during the mesocosm follow-up. A clear increase of the bacterial growth rates was observed with increasing cell size for the conditions met in this enriched mesocosm.

Abstract: Background: Over the past decade, cell separation technology has become an important tool in various fields of cell biology allowing for the analysis or subsequent cultivation of specific cell subsets. The objective of the present study was to evaluate if the established sorting techniques fluorescence-activated (FACS) and magnetic cell separation (MACS) affect cell membrane physiology in order to define the most non-perturbing application for the separation of tumor and stromal cells. Materials and Methods: Membrane physiology was monitored in single cell suspensions of adherently grown BT474 breast tumor cells and N1 normal skin fibroblasts using flow cytometry. Cell membrane integrity was evaluated by propidium iodide (PI) staining. Microviscosity within the lipophilic membrane layer was determined by a monomer/excimer method utilizing pyrene decanoic acid, membrane potential measurements were carried out using the fluorescence indicator DiBAC4(3), and Annexin-V-staining reflected transversal membrane asymmetry and an altered phospholipid distribution. Results: Not only the number of preparative cycles prior to cell separation but also the sort conditions during FACS resulted in loss of membrane integrity of a certain cell fraction. If these PI-positive cells were excluded from further analysis, neither MACS nor FACS affected membrane microviscosity while a clear hyperpolarization in both cell types after MACS resulting from exposure to the ferromagnetic matrix of the depletion column and the inhomogeneous magnetic field was shown. In addition, cell sorting of BT474 tumor cells by MACS and FACS was accompanied by the generation of an Annexin-V-positive/PI-negative cell fraction with altered phospholipid distribution. Data were discussed with regard to the sort-induced “stress” conditions such as exposure to hydrodynamic forces or magnetic fields. Conclusions: Both separation procedures modify cell membrane with neither technique being physiologically preferable for subsequent analysis or recultivation of the sorted cells. Cytometry 36:102–111, 1999. © 1999 Wiley-Liss, Inc.

Abstract: To investigate the mechanism by which presenilin (PS) overexpression induces apoptosis, we studied the effects of these proteins on cell cycle progression. Transiently transfected HeLa cells were bromodeoxyuridine (BrdU) labeled to visualize DNA synthesis by immunofluorescence and stained with propidium iodide to measure DNA content by fluorescence-activated cell sorting (FACS). BrdU labeling was decreased in cells expressing presenilin-1 (PS1), presenilin-2 (PS2), an Alzheimer's disease-associated missense mutation PS2(N141I), and the carboxyl-terminally deleted PS2 construct PS2(166aa), compared with mock and neurofilament-light (NF-L) transfected cells. Analysis of BrdU incorporation in mitotically synchronized HeLa cells suggested that cells were arresting in the G1 phase of the cell cycle, and this was confirmed by FACS analysis. Interestingly, cell cycle progression was more inhibited by the expression of PS2(N141I) compared with wild-type PS2. In addition, ATM, the gene product mutated in ataxia-telangiectasia, does not appear to be a downstream effector of PS-induced cell cycle arrest as transfection of PS constructs into an ataxia-telangiectasia cell line also resulted in cell cycle inhibition. Quantitative immunoblotting of whole-cell lysates from PS-transfected cells did not reveal increases or decreases in the steady-state levels of p21, p27, p53, pRb, or c-myc, suggesting that the presenilins mediate cell cycle arrest by mechanisms other than simple changes in the steady-state levels of these cell-cycle-related proteins.

Abstract: Human islet amyloid polypeptide (hIAPP) is co-secreted with insulin from pancreatic islet β cells. This peptide spontaneously aggregates in the form of fibrils, and amyloid deposits are associated with dead or degenerating β cells, a hallmark of noninsulin-dependent diabetes mellitus. We demonstrated that COS-1 cells transfected with vectors expressing hIAPP exhibited intracellular amyloid deposits that were associated with cell death (O'Brien, Butler, Kreutter, Kane, Eberhardt, Am J Pathol 1995, 147:609–616). To establish the mechanism of cell death, we transfected COS-1 cells with vectors expressing amyloidogenic hIAPP or nonamyloidogenic rat IAPP and mutant hIAPP constructs and assayed them for markers characteristic of apoptosis and necrosis by fluorescence-activated cell sorting analysis. Amyloidogenic hIAPP-transfected COS cells contained up to threefold more apoptotic cells present at 96 hours after transfection compared with the nonamyloidogenic vector controls. The hIAPP-induced apoptosis was negligible at 24 and 48 hours after transfection and was maximal at 96 hours which parallels the time course of amyloidogenesis. Immunohistochemical staining and confocal microscopy showed that hIAPP is localized with distinct clustering in the endoplasmic reticulum and Golgi apparatus with no discernable extracellular staining. These experiments provide direct evidence that intracellular hIAPP amyloid causes cell death by triggering apoptotic pathways.

Abstract: The ability of herpes simplex virus type 1 thymidine kinase (HSV-TK)-expressing cells incubated with ganciclovir (GCV) to induce cytotoxicity in neighboring HSV-TK-negative (bystander) cells has been well documented. Although it has been suggested that this bystander cell killing occurs through the transfer of phosphorylated GCV, there is little direct proof that bystander cells can accumulate GCV nucleotides. We have studied the ability of U251 human glioblastoma cells expressing HSV-TK (U251tk cells) to induce cytotoxicity in neighboring U251 bystander cells that lack the viral kinase (U251βgal cells) and evaluated whether this bystander cell killing is mediated by GCV nucleotides. The cytotoxicity studies demonstrated that the ratio of HSV-TK-expressing cells:bystander cells was important in determining the sensitivity of both cell types to GCV. U251tk cells cocultured with an equal number of U251βgal cells (a 50:50 ratio) exhibited a sensitivity to GCV similar to that observed in the absence of bystander cells, with >99.8% cell kill at 1 μm GCV. However, in cultures with 10% U251tk cells and 90% bystander cells (a 10:90 ratio), 1 μm GCV decreased the survival of U251tk cells by only 54%. Strong bystander cell killing was observed at both ratios. In a 50:50 coculture of U251tk and U251βgal cells, the survival of bystander cells was decreased by >99.5% with 3 μm GCV, whereas 30 μm GCV was required to effect a similar decrease in bystander cell survival when 90% of the culture consisted of U251βgal cells. To determine whether this bystander cell killing may be mediated by GCV nucleotides, we developed a technique to separate the two cell populations after coculture. A U251 bystander cell line was developed from the parental cell line by transfection with the cDNA coding for green fluorescent protein (U251gfp cells), which permitted the separation of U251gfp cells from nonfluorescing U251tk cells by flow cytometry with cell sorting. With this technique, bystander cells were isolated in a viable state with >97% purity within 1 h after harvest, permitting analysis of the nucleotide pools for the presence of phosphorylated GCV. The results demonstrated that significant levels of the triphosphate of GCV (GCVTP) accumulated in bystander cells within 4 h of coculture, and this accumulation was dependent upon the percentage of HSV-TK-expressing cells as well as the concentration of GCV and the length of incubation. The proportion of GCVTP in bystander cells was consistently 50–80% of that in HSV-TK-expressing cells in the 50:50 or 10:90 cocultures, suggesting a facile transfer of phosphorylated GCV. However, the actual amount of GCVTP was as much as 8-fold lower in both the U251tk and U251βgal cells cocultured at a ratio of 10:90 compared to those cocultured at a ratio of 50:50, which is consistent with the lesser effect on cell survival. When U251tk and U251gfp cells were cultured with 1-β-d-arabinofuranosylthymine (araT), the 5′-triphosphate of araT accumulated in the bystander cells, demonstrating that the transfer of phosphorylated compounds between these cell types is not restricted to GCV nucleotides. However, the proportion of araT-5′-triphosphate in bystander cells compared to that in HSV-TK-expressing cells was lower than that for GCVTP, and the amount was not sufficient to decrease survival in the bystander population.

Abstract: A human single-chain Fv (scFv) library as fusion to phage was constructed from donors with a high titer of autoantibodies. The library was subjected to three rounds of positive selection on human melanoma cells and negative selection on human peripheral blood mononuclear cells. Two scFv clones, B3 and B4, were isolated that bound melanoma cells in cell ELISA and fluorescence-activated cell sorting. The scFvs were characterized further by immunohistochemistry on a large number of normal human tissues. No cross-reactivity with normal tissues was observed. On the other hand, the target antigens were expressed in sections from several different melanoma patients and in some breast cancer and basal cell carcinoma sections. The unusually high tumor specificity of the B3 and B4 antigens makes them attractive targets for the specific therapy of melanoma. The selection strategy used should be generally applicable to the identification of novel cell surface antigens by antibody phage display.

Abstract: BACKGROUND Interactions of cancer cells with endothelium are a crucial step in metastatic invasion. RGD-recognizing integrins play a definitive role in these interactions. METHODS Fluorescence-activated cell sorting (FACS) analysis of RGD-sensitive integrins in prostate epithelial cells was performed. Attachment inhibition assay was used to characterize functionality of particular integrins. Potential partners for RGD-binding integrins in human umbilical vein endothelial cells (HUVEC) were identified by Western blotting and attachment inhibition assay. To determine the RGD-flanking amino acids optimal for interactions with prostate cell integrins, these cells were biopanned with a phage library. RESULTS Different expressions of RGD-recognizing integrins and distinctions in RGD-dependent adhesion of nonmalignant and cancer cells were observed. Cancer but not control cells were detached from culture plastic by incubation with RGD peptide. Adhesion of carcinoma cells to HUVEC was RGD-sensitive, in contrast to nonmalignant cells. Antibodies against alpha3, alpha5, beta1, and alpha(v)beta3 inhibited interactions of carcinoma cells with HUVEC. Potential ligands for alpha5beta1, alpha3beta1, and alphaVbeta3 integrins, fibronectin, and vitronectin, were detected on the HUVEC surface. Several phages which preferentially bound to the surface of particular prostate cells were selected. CONCLUSIONS Interactions of prostate carcinoma with endothelium are mediated in part via alpha5beta1, alpha3beta1, and alpha(v)beta3 integrins. Because these interactions are RGD-sensitive, synthetic RGD peptides with optimized flanking amino acids can potentially be used as antimetastatic agents.

Abstract: Studies on the effects of time and passage on porcine primary muscle cell cultures and methods to purify myoblasts were conducted using flow cytometry and fluorescence-activated cell sorting (FACS) Primary muscle cells cultured on single plates revealed a small cell ( or = 10 mm diameter) population containing desmin-positive myoblasts and nonmyoblasts The small myoblasts were detectable up to 28 days but after cell sorting and passage, they became indistinguishable from the large myoblast population This indicates that pig muscle contains small self-renewing myoblasts similar to humans, that become larger when induced to proliferate A human myoblast-specific monoclonal antibody allows FACS of both large and small myoblasts from primary cells within 2 days of culture and independent of passage These characteristics of porcine myoblasts indicate that the pig may be a suitable large animal model for myoblast-mediated gene transfer

Abstract: Publisher Summary The use of green fluorescent proteins (GFP) as a reporter in flow cytometry and cell sorting is in its infancy. A major benefit provided by GFP is that it can be measured noninvasively. Alterations to the primary structure of the molecule have provided variants that are readily detectable in both prokaryotic and eukaryotic cells and within subcellular organelles. Further spectral variants that can be combined for multiparametric analyses will find considerable future utility in flow cytometry. In animal cell systems, GFP is particularly suited as an indicator of viral infection. When expressed in viruses, either as a gene fusion or when substituted for a nonessential viral gene, GFP fluorescence provides a sensitive and accurate measure of the proportions of infected cells and the progress of viral infection. Coupling this approach for viral detection with concerted mutagenesis of virus provides a screen for the roles of the various viral components within the infection process. Flow cytometric detection of GFP also finds practical application in gene therapy. By marking the viral vectors containing therapeutic gene products with GFP, the members expressing these products within a population of cells can be identified, selected, and used exclusively for the reconstitution of organisms.

Abstract: High endothelial venules (HEV) allow rapid and selective lymphocyte trafficking from the blood into secondary lymphoid tissues. Here we report the expression profile of active genes in mouse high endothelial cells (HEC). HEC were first purified from mouse lymph nodes (LN) by magnetic cell sorting with MECA-79 mAb and a 3'-directed cDNA library that faithfully represents the composition of mRNA was constructed. A total of 1495 cDNA sequences were obtained from randomly selected clones. Based on their sequence identity, they were grouped into 754 different species [gene signatures (GS)] of which 335 GS were identified in GenBank. Among the previously identified genes, expression of several endothelial cell surface molecules including endoglin and ICAM-1 was detected in HEC. Comparison of the gene expression profile with that of purified CD31(+) flat endothelial cells identified several molecules, such as KC chemokine and Duffy antigen/receptor for chemokines, that are known to be selectively expressed in activated endothelial cells or post-capillary venules. Interestingly, mac25/TAF, which is known to be expressed specifically in tumor vessels and implicated in the regulation of cell adhesion, was highly and selectively expressed in HEC in mouse LN, suggesting that it may participate in regulating HEC-specific functions. Comparison with the expression profiles obtained from 35 different cell types showed at least 22 GS that were apparently specific to HEC. Our results illustrate the expression differences between HEC and CD31(+) flat endothelial cells, and will be useful for the identification and characterization of genes specific for HEC.

Abstract: When Dictyostelium cells are induced to develop between a coverslip and a layer of agarose, they aggregate normally into groups containing up to a thousand cells but are then constrained to form disks only a few cells thick that appear to be equivalent to the three-dimensional mounds formed on top of agarose. Such vertically restricted aggregates frequently develop into elongated motile structures, the flattened equivalent of three-dimensional slugs. The advantage of using this system is that the restricted z-dimension enables direct microscopic visualization of most of the cells in the developing structure. We have used time lapse digital fluorescence microscopy of Dictyostelium strains expressing green fluorescent protein (GFP) under the control of either prestalk or prespore specific promoters to follow cell sorting in these flattened mounds. We find that prestalk and prespore cells expressing GFP arise randomly in early aggregates and then rotate rapidly around the disk mixed with the other cell type. After a few hours, the cell types sort out by a process which involves striking changes in relative cell movement. Once sorted, the cell types move independently of each other showing very little heterotypic adhesion. When a group of prestalk cells reaches the edge of the disk, it moves out and is followed by the prespore cell mass. We suggest that sorting may result from cell type specific changes in adhesion and the consequent disruption of movement in the files of cells that are held together by end-to-end adhesion.

Abstract: Objective: Nef was shown to be the predominant viral protein expressed in HIV-1-infected astrocytes in vivo and in vitro suggesting a distinct role of Nef in this cell type. Nef-induced activation of T cells is well described, whereas the functional activities of Nef in astrocytes are unknown. Our aim was to examine the effect of Nef on growth properties and activation of astrocytes. Design: Human Nef-expressing astrocytic cell lines were established by stable transfection with different wild-type and mutant nef genes derived from laboratory isolates and brain tissue. Methods: Nef-expressing astrocytes were characterized in terms of growth properties (proliferation, growth in soft agar, focus formation and morphology. Apoptotic cell death and expression of activation markers were determined by fluorescent antibody cell sorting. Results: Astrocytic cell lines revealed persistent Nef expression - detectable at the levels of mRNA and protein - and showed altered growth properties and morphology. Elevated expression of activation markers such as glial fibrillary acidic protein and CD88 (complement receptor C5a) was observed; these are regarded as markers for inflammatory processes in the brain. This effect was independent of the nef type or the expression level of the Nef protein. In contrast with previous reports no evidence for increased apoptotic cell death was found in astrocytes expressing Nef stably. Conclusions: Our findings suggest that Nef changes the cellular properties of astrocytes, thus contributing to astrocyte activation and induction of astrogliosis in the central nervous system of individuals with AIDS.

Abstract: The purpose of this study was to isolate pure populations of round spermatids from mouse testis by flow cytometry followed by cell sorting. Cell suspensions from mouse testis were enriched in germ cells by centrifugation on a discontinuous Percoll gradient, then analysed using a FACScalibur flow cytometer measuring the cell size and density. A large and well-delimited population of cells (R1) expected to contain round spermatids was observed on the dot plot diagram. Sorted R1 cells were very homogeneous in size (approximately 11 microns) and displayed the characteristic cytological aspect of round spermatids. Spermatid-specific gene expression was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of R1 cells using primers for protamine 2 gene (PRM2) and SP-10. A positive signal for SP-10 was obtained with a single cell using nested primers. The 5.5 kb transcript of c-kit, which is not expressed in spermatids, was not detected by nested RT-PCR, excluding a contamination with spermatogonia. Our results clearly established that flow cytometry followed by cell sorting allows the isolation of a highly homogeneous population of round spermatids from the testis.

Abstract: Mesenchymal cells from different stages of chick limb buds sort out in monolayer culture, suggesting the presence of different cell affinities dependent on their positions along the proximodistal axis. However, it is still not clear which molecules are responsible for the sorting-out. Here, we propose that N-cadherin, a cell-adhesion molecule, is involved in the sorting-out and is likely to be a component of the mechanism of proximodistal patterning in the developing limb. N-cadherin proteins accumulate in the distal region of the chick limb bud as limb development proceeds. In monolayer culture of distal mesenchymal cells, the stage-dependent levels of N-cadherin proteins are maintained during cell sorting. The results of this study have also demonstrated that an anti-N-cadherin monoclonal antibody, NCD-2, clearly inhibits the cell sorting. Moreover, removal of the apical ectodermal ridge or retinoic-acid treatment of distal cells, which results in a change in the pattern of sorting-out, inhibits the accumulation of N-cadherin proteins, suggesting that the distribution of these proteins is related to the positional identity that gives rise to the different shape and number of cartilage elements along the proximodistal axis.

Abstract: Glioma invasiveness is a complex process involving recognition and attachment of tumor cells to particular extracellular matrix (ECM) molecules prior to migrating into proteolytically modified matrix and inducing angiogenesis. CD44 is a group of transmembrane adhesion molecules found on a wide variety of cells including gliomas that has been suggested as the principal mediator of migration/invasion. The aim of the present study was to demonstrate whether antibody specific for the standard form of CD44 (CD44s, 85–90 kDa) might prevent invasion, thus blocking growth of the 9L gliosarcoma in vivo. High expression of CD44s on the surface of 9L cells and brain tumors was demonstrated by immunochemistry. Fluorescence-activated cell sorting (FACS) demonstrated binding saturation of anti-CD44s monoclonal antibody (mAb) to the receptor at 1 μg/5 × 105 cells. Blocking of CD44s in vitro resulted in a dose-dependent progressive, up to 95% ± 2.5% detachment of 9L cells from ECM-coated culture surfaces. Blocking of CD44s in vivo resulted in significantly reduced 9L brain tumors (2.5% ± 0.4%) – measured as the quotient: tumor surface (mm2)/brain surface (mm2) × 100 – as compared to untreated (16.1% ± 2.2%) or sham-treated rats (16% ± 3.7% to 16.1% ± 3%). We conclude that CD44s-targeted treatment with specific mAb may be an effective means for preventing glioma progression.