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Functional comparison of bone marrow-derived liver stem cells: selection strategy for cell-based therapy.

Several distinct subpopulations of bone marrow-derived liver progenitor cells were recently described. However, there is inadequate information comparing these subpopulations from a liver-function point of view. This study was undertaken to compare two subpopulations of liver progenitors: beta(2)-microglobulin (beta(2)m)-negative/Thy-1-positive cells, and liver progenitors obtained from the non-adherent cell fraction after a panning procedure. The cells were cultured under several conditions including high- and low-dose hepatocyte growth factor, various cellular densities, and different media. Growth characteristics, liver-specific metabolic capacity, and liver regeneration-associated gene expression were studied. Both isolation procedures yielded cells that produced albumin and metabolized ammonia into urea. The study demonstrated that the beta(2)m-negative/Thy-1-positive cell fraction metabolized ammonia into urea more efficiently and produced a superior amount of albumin compared with the panned cell fraction. The beta(2)m-negative/Thy-1-positive cell fraction could be optimal for the development of novel cell-based treatment strategies for congenital or acquired liver diseases.

Animals↗

Monoclonal antibody-directed effector cells selectively lyse human melanoma cells in vitro and in vivo.

Monoclonal antibody 9.2.27 (mAb 9.2.27) directed to a chondroitin sulfate proteoglycan on human melanoma cells was able to suppress tumor growth in athymic (nu/nu) mice more effectively when bound with polyethylene glycol to murine effector cells than when injected alone. These "armed" effector cells also proved more effective than the monoclonal antibody in eliciting antibody-dependent cellular cytotoxicity against human melanoma target cells in vitro.

Animals↗

The immune privilege of the eye: human retinal pigment epithelial cells selectively modulate T-cell activation in vitro.

PURPOSE: To examine the effect of human retinal pigment epithelial (RPE) cells on phytohemagglutinin (PHA) activation of T cells. METHODS: Resting peripheral blood lymphocytes (PBLs) were stimulated with PHA with or without the presence of gamma-irradiated RPE cells. Proliferation and the cell cycle profile were thereafter investigated by 3H-thymidine incorporation and flow cytometric analysis. In addition, the PBLs expression of CD69, major histocompatibility complex (MHC) class I and II, CD3, as well as the IL-2 receptor chains were evaluated by flow cytometry, and the content of IL-2 in cell culture supernatant was measured by ELISA. RESULTS: Human RPE cells were found to suppress PHA-induced proliferation, cyclin A, IL-2R-alpha and -gamma, and CD71 expression and decrease the production of IL-2; but RPE cells do not inhibit the PHA-induced expression of early activation markers CD69, MHC class I and II, and of cyclin D of the PBLs. CONCLUSIONS: These results are the first to indicate that RPE cells impede generation of activated T cells by interfering with the induction of high-affinity IL-2R-alphabetagamma, IL-2 production, and the expression of CD71 and cyclin A.

Antigens, CD↗

In vivo primed mouse T cells selectively express T cell-specific serine proteinase-1 and the proteinase-like molecules granzyme B and C.

Previous studies have shown that mouse CD8+ T lymphocyte clones (TLC) produce T cell-specific serine proteinase-1 (MTSP-1) as well as a family of six homologous molecules, termed granzymes B - G, which are structurally related to serine proteinases. Of these proteins, only MTSP-1 has been studied in detail. It has been shown to occur in the majority of CD8+ and a fraction of CD4+ T effector cells in vivo and in vitro and has demonstrable enzyme activity in these cells. The presence of the other serine proteinase-like molecules in T cells is less well defined. We have now analyzed the expression of mRNA species specific for granzymes B - G in activated T cell populations using the sensitive polymerase chain reaction which allows the detection of mRNA species from as little as 2 pg of total cytoplasmic RNA. We demonstrate that MTSP-1 and all six serine proteinase-like transcripts are expressed in a panel of four CD8+ and six CD4+ long-term-cultured TLC, though at greatly differing concentrations. In contrast, in vivo primed T cells of both phenotypes, CD4+ and CD8+, and in vitro activated T cells derived from short-term cultures only express mRNA species specific for MTSP-1 and CCP1 and little of those for CCP2, but no transcripts for granzymes D - G. These findings argue against the participation of granzymes D - G in T cell-mediated functions in vivo.

Animals↗

Alloxan-induced free radical production in isolated cells. Selective effect on islet cells.

Chemiluminescence, as a direct measure of oxygen free radical production, induced in isolated cells, hepatocytes, and red cells by the action of alloxan has been measured. The assay system used luminol, 3 microM, for signal amplification. The buffer used was Krebs-Ringer bicarbonate with 16 mM Hepes, pH 7.4. This buffer did not react with alloxan in the absence of cells. Some chemiluminescence was noted from all cells in the absence of alloxan. In the presence of alloxan, reactions occurred within seconds and islet cells were significantly more reactive to alloxan than either red cells or hepatocytes as defined by alloxan dose-response curves with fixed cell numbers or fixed surface areas. These data indicate a cell specificity for an early action of alloxan perhaps mediated at the cell membrane.

Alloxan↗

Interferon-induced increase in sensitivity of ovarian cancer targets to lysis by lymphokine-activated killer cells: selective effects on HER2/neu-overexpressing cells.

Overexpression of the HER2/neu oncogene in ovarian tumor cells is associated with relative resistance to lymphokine-activated killer (LAK) cell cytotoxicity. Treatment with gamma-interferon (IFN-gamma) (200-2000 units/ml) for 3 days markedly enhanced the sensitivity of HER2/neu-overexpressing ovarian tumor cells to LAK cells but had no effect on the sensitivity of nonexpressing ovarian targets. Increased sensitivity to lysis was associated with an increase in effector-target conjugate formation, the induction of target cell intercellular adhesion molecule 1 (ICAM-1) expression, and the down-regulation of HER2/neu expression. Anti-ICAM-1 antibody blocked the enhanced lysis, indicating that ICAM-1 is important in the increased sensitivity to LAK cells. However, induction of ICAM-1 expression did not correlate well with enhanced sensitivity to lysis; it was maximal after 24 h of exposure to IFN-gamma and still present 24 h after removing IFN-gamma. In contrast, enhanced lysis required 3 days of exposure to IFN-gamma and was reversed within 24 h after removal of IFN-gamma. These data indicate that, although ICAM-1 is necessary, it is not sufficient for the IFN-gamma-induced enhancement of sensitivity to LAK lysis.

Cell Line↗

Cell-based tissue engineering for the intervertebral disc: in vitro studies of human disc cell gene expression and matrix production within selected cell carriers.

BACKGROUND CONTEXT: Little is known about how disc cells attach, proliferate and form extracellular matrix (ECM) within carrier materials. Such information is needed to help formulate criteria for successful cell-carrier interactions in tissue engineering. PURPOSE: To compare proliferation, ECM production and gene expression in annulus cells cultured in a variety of cell carrier materials with potential application in tissue engineering of the disc. STUDY DESIGN: Human intervertebral disc cells from the annulus were used in a prospective study of proliferation, ECM production and gene expression within selected cell carriers. METHODS: Annulus cells from discs of 29 individuals were tested in collagen sponge, collagen gel, agarose, alginate or fibrin gel formulations. In situ hybridization assessed ECM gene expression of Types I and II collagen, aggrecan and chondroitin-6 sulfotransferase. Cell proliferation, cell shape, attachment and ECM production were evaluated. RESULTS: Collagen sponges provided the best microenvironment for disc cell ECM production and gene expression. Although collagen gels often could support good cell growth, such constructs did not result in either abundant ECM production or ECM gene expression, as shown by in situ hybridization. Growth and ECM production and gene expression in alginate, agarose and fibrin microenvironments were inferior. CONCLUSIONS: Tissue engineering techniques open new therapeutic possibilities for use of autologous disc cells, but fundamental questions on how these cells interact with cell carriers are unexplored. Results provide novel data on disc cell gene expression within diverse microenvironments. The collagen sponge proved to be a superior microenvironment.

Absorbable Implants↗

Effects of L- or D-Pro incorporation into hydrophobic or hydrophilic helix face of amphipathic alpha-helical model peptide on structure and cell selectivity.

A synthetic amphipathic alpha-helical model peptide, KLW, displays non-cell selective cytotoxicity. To investigate the effects of L- or D-Pro kink incorporation into hydrophobic or hydrophilic helix face of KLW on structure, cell selectivity, and membrane-binding affinity, we designed a series of four peptides, in which Leu(9) and Lys(11) in the hydrophobic and hydrophilic helix face of KLW, respectively, are substituted with L- or D-Pro. A L- or D-Pro substitution (KLW-L9P or KLW-L9p) of Leu(9) at the hydrophobic helix face of KLW induced a more significant reduction in hemolytic activity with improved antibacterial activity than that (KLW-K11P or KLW-K11p) of Lys(11) in the hydrophilic helix face. In addition, D-Pro-containing peptides (KLW-L9p and KLW-K11p) displayed less hemolytic activity than L-Pro-containing peptides (KLW-L9P and KLW-K11P). Tryptophan fluorescence studies revealed that bacterial cell selectivity of KLW-L9P, KLW-L9p, and KLW-K11p is closely related to selective interactions with negatively charged phospholipids. CD analysis revealed that L- or D-Pro incorporation into KLW reduces the alpha-helicity of the peptide and D-Pro incorporation induces more significant disruption in alpha-helical structure than L-Pro incorporation. Our results collectively suggest that D-Pro incorporation into the hydrophobic helix face of non-cell selective amphipathic alpha-helical peptides may be useful for the design of novel antimicrobial peptides possessing high bacterial cell selectivity without hemolytic activity.

Acrylamide↗

The distribution of wavelength and orientation selective cells in different areas of monkey visual cortex.

The distribution of orientation and wavelength selective cells in visual areas V1, the V4 complex and V5 of macaque monkey cortex was studied by recording from regions representing equivalent eccentricities in each and characterizing cells for their orientational and wavelength preferences. The results showed: (a) that in V1 wavelength selective cells are concentrated in the region of foveal representation (central 0-2 degrees) and decline at increasing eccentricities; (b) that in the V4 complex the concentration of such cells does not vary with eccentricity; rather, there are regional variations that are not eccentricity dependent but follow other, and unknown, rules; (c) that in V5 there are virtually no wavelength selective cells even at central eccentricities; (d) that in V1 fewer than 50% of cells with receptive fields in the central 0-2 degrees are orientation selective and that the proportion of such cells increases to reach a high of about 80% at eccentricities of between 10 and 30 degrees; (e) that in the V4 complex and in V5 the concentration of orientation selective cells decreases at increasing eccentricities; (f) that very few wavelength selective cells in V1 and in the V4 complex are orientation selective. The implications of these findings for a comparison between visual areas of the monkey cortex are discussed.

Animals↗

Primary intestinal epithelial cells selectively transfer R5 HIV-1 to CCR5+ cells.

The upper gastrointestinal tract is a principal route of HIV-1 entry in vertical transmission and after oral-genital contact. The phenotype of the newly acquired virus is predominantly R5 (CCR5-tropic) and not X4 (CXCR4-tropic), although both R5 and X4 viruses are frequently inoculated onto the mucosa. Here we show that primary intestinal (jejunal) epithelial cells express galactosylceramide, an alternative primary receptor for HIV-1, and CCR5 but not CXCR4. Moreover, we show that intestinal epithelial cells transfer R5, but not X4, viruses to CCR5+ indicator cells, which can efficiently replicate and amplify virus expression. Transfer was remarkably efficient and was not inhibited by the fusion blocker T-20, but was substantially reduced by colchicine and low (4 degrees C) temperature, suggesting endocytotic uptake and microtubule-dependent transcytosis of HIV-1. Our finding that CCR5+ intestinal epithelial cells select and transfer exclusively R5 viruses indicates a mechanism for the selective transmission of R5 HIV-1 in primary infection acquired through the upper gastrointestinal tract.

Amino Acid Sequence↗

Impaired stem cell function of CD34+ cells selected by two different immunomagnetic beads systems.

We have been investigating the hematopoietic stem cell (HSC) activity of peripheral blood-derived CD34(+) cells selected by two different laboratory immunomagnetic beads systems (MiniMACS and Isolex 50). In this study, the quality of purified CD34(+) cells was directly compared using clonal cell culture, a cobblestone area-forming cell (CAFC) assay, and an in vivo severe combined immunodeficiency (SCID)-repopulating cell (SRC) assay. It was found that CD34(+) cells selected by these two immunomagnetic methods showed a reduced yield of colony-forming cells and CAFCs compared with cells enriched by the StemSep device (a negative selection method). However, these CD34(+) cells still showed significant SRC activity, including multilineage lymphomyeloid reconstitution. The percentage of human CD45(+) cells in murine bone marrow after transplanting 5 x 10(5) CD34(+) cells selected by the Isolex 50 was significantly lower than after transplanting cells selected by the MiniMACS or the StemSep. Our findings clearly demonstrated that CD34(+) cells selected by the MiniMACS system had superior HSC functions, including SRC activity, compared with cells separated by the Isolex 50 system. More detailed functional analysis of immunomagnetically separated CD34(+) cells may provide useful knowledge for basic research on HSCs as well as for clinical HSC transplantation.

Adult↗

A preliminary analysis of the balance between Th1 and Th2 cells after CD34+ cell-selected autologous PBSC transplantation.

BACKGROUND: CD34+ cell-selected autologous PBSC transplantation (CD34+ APBSCT) is a procedure used for the treatment of patients with malignant disease that is intended to eliminate residual tumor cells from autologous grafts. However, frequent infectious complications after CD34+ APBSCT can occur. A delay of recovery of the absolute number of CD4+ T cells after transplantation was reported to be one disadvantageous factor. As data on T-cell function after CD34+ APBSCT are scanty, we analyzed changes in T-helper cell 1 (Th1) and T-helper cell 2 (Th2) after CD34+ APBSCT to evaluate immune reconstitution. METHODS: Twelve patients underwent APBSCT (CD34+APBSCT group, n=4, and unselected APBSCT, n=8). Peripheral blood (PB) samples were obtained at 2, 4, 8, 12 and 16 weeks after the transplantation. The dynamics of the Th1 and Th2 were analyzed at a single-cell level, using flow cytometry. RESULTS: In the CD34+ APBSCT group, not only the absolute count of CD4+ T cells but also the proportion of Th1 cells in CD4+ T cells and the ratio of Th1 to Th2 after transplantation were significantly decreased at 2 and 4 weeks after transplantation compared with findings in the unselected APBSCT group. DISCUSSION: We suggest that higher rates of infectious complications after CD34+ APBSCT may be due to the inability of residual T cells from the CD34+ cell selection to generate mature T cells that function adequately against infection. Although further study would be required, our preliminary data provide some information on the immune reconstitution after CD34+ APBSCT and differentiation of T lymphocytes into Th1 and Th2 in vivo.

Adult↗

A new method for comparative light and electron microscopic studies of individual cells, selected in the living state.

A method is described which permits comparative light and electronmicroscopic studies of cell cultures, cell spreads or single selected cells which have been kept in the Plastic Film Dish (PFD). The PFD is a versatile large surface tissue culture chamber which, for electron microscopy, is mounted with a transparent FEP-Teflon film bottom. Cells are observed, selected and marked on the PFD-bottom with a high power inverted light microscope. The cells are fixed and dehydrated with a semi-automatic device while they are still in situ in the PFD. During the preparation steps for electron microscopy the topographical relationship between individual cells and between cells and cell support is accurately retained. After embedding and polymerization the Teflon film is easily peeled off the polymerized Epon, leaving a replica of the mark around the selected cell. This permits relocation of the selected cells for ultrathin sectioning in a plane plan-parallel to the original cell support. To enable orientated sectioning of selected cells in a plane perpendicular to the cell support, cells are tagged with Letraset-letters after original embedding and polymerization. Subsequently the re-embedded polymerized specimens are orientated in the microtome in a position which permits controlled thin sectioning of the tagged cells in the previously selected plane.

Cells, Cultured↗

Nonobese diabetic-severe combined immunodeficient mice transplantation of volume-reduced and thawed umbilical cord blood transplants following closed-system immunomagnetic cell selection.

BACKGROUND: Protocols for the expansion of human umbilical cord blood (UCB) progenitors begin with the selection of CD34+ cells from stored frozen and thawed units. Use of an immunomagnetic selection procedure within a closed blood bag system for volume-reduced UCB transplants was evaluated, and the influence of CD34 cell selection on in vivo engraftment potential was studied. STUDY DESIGN AND METHODS: Eleven thawed buffy coat-processed UCB units were processed within a standard blood bag with a washing solution. In six independent experiments, the same dosage of 2 x 104 CD34+ cells from paired selected and nonselected samples was transplanted into NOD-SCID mice. In two experiments, cells from the negative fraction were also transplanted. RESULTS: The purity of CD34+ cells after selection was correlated with the removal of supernatant after the first washing step and therefore with adequate removal of damaged or dead cells (r=0.86, p < 0.01). Mice transplanted with unselected UCB cells had more human cells within their marrow than animals transplanted with selected cells (8.6 +/- 5.9% selected group vs. 19.8 +/- 14.2% unselected group; p=0.04), whereas no engraftment could be observed transplanting cells from the two negative fractions. A higher percentage of human CD45+ cells in the unselected group were found to be positive for CD38, CD14, CD33, and CD19, indicating a higher potential for these unselected progenitors to differentiate into myeloid cells and B cells. CONCLUSIONS: Processing of volume-reduced and thawed UCB transplants within a closed-bag system before immunomagnetic CD34+ cell selection allows for the preparation of CD34+ cells of significant purity at technically useful cell recoveries. However, these experiments indicate a potential impairment of engraftment capacity for the CD34+ cell-enriched fraction.

Animals↗

Bone grafts prepared with selective cell retention technology heal canine segmental defects as effectively as autograft.

Using a canine critical-size segmental defect model, a two-phased study was undertaken to evaluate the healing efficacy of demineralized bone and cancellous chips (DBM-CC) enriched with osteoprogenitor cells using a Selective Cell Retention (SCR) technology. The goals of this study were: 1) to determine the bone-healing efficacy of SCR-enriched grafts versus autograft, and 2) to assess the value of clotting SCR-enriched grafts with platelet-rich plasma (PRP). Thirty dogs were included in Phase I: 18 dogs were treated with an SCR-enriched DBM-CC graft clotted with autologous bone marrow, and were compared to 12 autograft controls. In Phase II, 24 animals were divided into 4 groups of 6 animals, each treated with a different bone graft material: 1) iliac crest autograft, 2) DBM-CC alone, 3) DBM-CC saturated with marrow, and 4) SCR-enriched DBM-CC clotted with PRP. All grafts were placed unilaterally in a 21-mm long osteoperiosteal femoral, instrumented, critical-size defect. Radiographs were obtained for all animals postoperatively and every 4-16 weeks; animals were then sacrificed. All femurs were prepared for histology. Femurs in the Phase II study were also analyzed by micro-CT. At 16 weeks, healing--defined by bridging bone across the defects--was observed in 50% of the DBM-CC alone group and 67% of the DBM-CC saturated with marrow group; 100% of the autograft and SCR-enriched DBM-CC groups were healed. Histologically, grafts clotted with PRP showed more mature bone than those implanted with autologous bone, which in turn were similar to those implanted with bone marrow clotted SCR-enriched grafts. These results demonstrated that: 1) SCR-enriched DBM-CC was equivalent to autograft to repair critical-size defects, and 2) while not statistically significant, PRP may have accelerated bone maturation when used to clot osteoprogenitor-enriched DBM-CC grafts--as compared to cell-enriched, DBM-CC grafts without PRP--in large animal models.

Animals↗

Somatic-cell selection is a major determinant of the blood-cell phenotype in heterozygotes for glucose-6-phosphate dehydrogenase mutations causing severe enzyme deficiency.

X-chromosome inactivation in mammals is regarded as an essentially random process, but the resulting somatic-cell mosaicism creates the opportunity for cell selection. In most people with red-blood-cell glucose-6-phosphate dehydrogenase (G6PD) deficiency, the enzyme-deficient phenotype is only moderately expressed in nucleated cells. However, in a small subset of hemizygous males who suffer from chronic nonspherocytic hemolytic anemia, the underlying mutations (designated class I) cause more-severe G6PD deficiency, and this might provide an opportunity for selection in heterozygous females during development. In order to test this possibility we have analyzed four heterozygotes for class I G6PD mutations: two with G6PD Portici (1178G-->A) and two with G6PD Bari (1187C-->T). We found that in fractionated blood cell types (including erythroid, myeloid, and lymphoid cell lineages) there was a significant excess of G6PD-normal cells. The significant concordance that we have observed in the degree of imbalance in the different blood-cell lineages indicates that a selective mechanism is likely to operate at the level of pluripotent blood stem cells. Thus, it appears that severe G6PD deficiency affects adversely the proliferation or the survival of nucleated blood cells and that this phenotypic characteristic is critical during hematopoiesis.

Autoradiography↗