The Notch signalling pathway in hair growth

The Notch signalling pathway is an important mediator of cell fate selection whose involvement in epidermal appendage formation is now becoming recognised. Hair follicle development and hair formation involve the co-ordinated differentiation of several different cell types in which Notch appears to have a role. We report intricate expression patterns for the Notch-1 receptor and three ligands, Delta-1, Jagged-1 and Jagged-2 in the hair follicle. Notch-1 is expressed in ectodermal-derived cells of the follicle, in the inner cells of the embryonic placode and the follicle bulb, and in the suprabasal cells of the mature outer root sheath. Delta-1 is only expressed during embryonic follicle development and is exclusive to the mesenchymal cells of the pre-papilla located beneath the follicle placode. Expression of Jagged-1 or Jagged-2 overlaps Notch-1 expression at all stages. In mature follicles, Jagged-1 and Jagged-2 are expressed in complementary patterns in the follicle bulb and outer root sheath, Jagged-1 in suprabasal cells and Jagged-2 predominantly in basal cells. In the follicle bulb, Jagged-2 is localised to the inner (basal) bulb cells next to the dermal papilla which do not express Notch-1, whereas Jagged-1 expression in the upper follicle bulb overlaps Notch-1 expression and correlates with bulb cell differentiation into hair shaft cortical and cuticle keratinocytes.     

Negative regulation by interleukin-3 (IL-3) of mouse early B-cell progenitors and stem cells in culture: transduction of the negative signals by betac and betaIL-3 proteins of IL-3 receptor and absence of negative regulation by granulocyte-macrophage colony-stimulating factor

The receptors for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5 share a common signaling subunit betac. However, in the mouse, there is an additional IL-3 signaling protein, betaIL-3, which is specific for IL-3. We have previously reported that IL-3 abrogates the lymphoid potentials of murine lymphohematopoietic progenitors and the reconstituting ability of hematopoietic stem cells. We used bone marrow cells from betac- and betaIL-3-knock-out mice to examine the relative contributions of the receptor proteins to the negative regulation by IL-3. First, we tested the effects of IL-3 on lymphohematopoietic progenitors by using lineage-negative (Lin-) marrow cells of 5-fluorouracil (5-FU)-treated mice in the two-step methylcellulose culture we reported previously. Addition of IL-3 to the combination of steel factor (SF, c-kit ligand) and IL-11 abrogated the B-lymphoid potential of the marrow cells of both types of knock-out mice as well as wild-type mice. Next, we investigated the effects of IL-3 on in vitro expansion of the hematopoietic stem cells. We cultured Lin-Sca-1-positive, c-kit-positive marrow cells from 5-FU-treated mice in suspension in the presence of SF and IL-11 with or without IL-3 for 7 days and tested the reconstituting ability of the cultured cells by transplanting the cells into lethally irradiated Ly-5 congenic mice together with "compromised" marrow cells. Presence of IL-3 in culture abrogated the reconstituting ability of the cells from both types of knock-out mice and the wild-type mice. In contrast, addition of GM-CSF to the suspension culture abrogated neither B-cell potential nor reconstituting abilities of the cultured cells of wild-type mice. These observations may have implications in the choice of cytokines for use in in vitro expansion of human hematopoietic stem cells and progenitors.     

O-glucuronidation, a newly identified metabolic pathway for topotecan and N-desmethyl topotecan

BACKGROUND AND OBJECTIVE: CD34+ hematopoietic progenitor cells (HPCs) constitute a heterogeneous population both in size and in immunological features. Lack of CD38, HLA-DR and lineage committed antigens as well as the co-expression of Thy-1 (CDw90) and c-kit receptor (CD117), are able to identify the so-called stem cells. A flow cytometric study was carried out to investigate the co-expression of Thy-1 and c-kit receptors, both members of Ig superfamily adhesion molecules, involved in cell to cell and cell to stroma interactions, on bone marrow (BM), mobilized peripheral blood (PB) and human umbilical cord blood (HUCB) CD34+ HPCs. DESIGN AND METHODS: Lysed whole blood from 15 BM, 25 mobilized PB and 25 HUCB samples were used to perform a five-dimensional flow cytometric evaluation of both CDw90 and CD117 on CD34+ cells. RESULTS: Few CD34+ cells co-expressed Thy-1 antigen in all three compartments (BM: 11.2 +/- 7.2%; PB: 6.2 +/- 3.6%; HUCB: 6 +/- 2.9%; BM vs PB < 0.04; BM vs HUCB < 0.008; PB vs HUCB ns). c-kit receptor was detected on the majority of CD34+ HPCs, particularly in HUCB (HUCB: 80.7 +/- 8.2%; BM: 72.3 +/- 13.1%; PB: 64.2 +/- 17%; HUCB vs BM < 0.03; HUCB vs PB < 0.0001; BM vs PB ns). CD34+Thy-1+ and CD34+c-kit+ HPCs generally displayed HLA-DR antigen, as expression of early cell commitment. However, the most immature CD34+Thy-1+HLA-DR- (HUCB: 1 +/- 0.6%; BM: 0.4 +/- 03%; PB: 0.7 +/- 0.5%; HUCB vs BM < 0.0001; BM vs PB < 0.04; HUCB vs PB ns) and CD34+c-kit+HLA-DR- HPCs (HUCB: 6.5 +/- 4.4%; BM: 6.3 +/- 4.8%; PB: 2.2 +/- 1.8%; HUCB vs BM ns; BM vs PB < 0.0001; HUCB vs PB < 0.0001) were mainly detected in HUCB. Finally, the greatest percentage of CD34+Thy-1+c-kit+ cells was found in BM (6.9 +/- 4.1%) followed by leukapheretic samples (4.4% +/- 2.7) and then by HUCB (3.7 +/- 1.2%; BM vs PB ns; BM vs HUCB < 0.001; HUCB vs PB ns). INTERPRETATION AND CONCLUSIONS: Since the blood release or HPCs is probably due to a perturbation of the adhesive interactions between these cells and the marrow stroma, the different pattern of Thy-1 and c-kit receptor expression on CD34+ HPCs found in the three hemopoietic compartments evaluated can lead to new knowledge about the mobilization kinetics in which the Ig superfamily adhesion molecules are involved.     

Flt3high and Flt3low CD34+ progenitor cells isolated from human bone marrow are functionally distinct

We generated monoclonal antibodies against the human Flt3 receptor and used them to study the characteristics of normal human bone marrow cells resolved based on Flt3 expression. Human CD34+ or CD34+lin- marrow cells were sorted into two populations: cells expressing high levels of Flt3 receptor (Flt3high) and cells with little or no expression of Flt3 receptor (Flt3low). Flt3 receptor was detected on a subset of CD34+CD38- marrow cells, as well as on CD34+CD19+ B lymphoid progenitors and CD34+CD14+CD64+ monocytic precursors. Flt3 receptor was also present on more mature CD34-CD14+ monocytes. In colony-forming assays, Flt3high cells gave rise mainly to colony-forming unit-granulocyte-macrophage (CFU-GM) colonies, whereas Flt3low cells produced mostly burst-forming unit-erythroid colonies. There was no difference in the number of multilineage CFU-Mix colonies between the two cell fractions. Cell cycle analysis showed that a large number of the Flt3low cells were in the G0 phase of the cell cycle, whereas Flt3high cells were predominantly in G1. Cell numbers in the suspension cultures initiated with Flt3high cells were maintained in the presence of Flt3 ligand (FL) alone, and increased in response to FL plus kit ligand (KL). In contrast, cell numbers in the suspension cultures started with Flt3low cells did not increase in the presence of FL, or FL plus KL. Upregulation of Flt3 receptor on Flt3low cells was not detected during suspension culture. CD14+ monocytes were the major cell type generated from CD34+lin-Flt3high cells in liquid suspension culture, whereas cells generated from CD34+lin-Flt3low cells were mainly CD71+GlycA+ erythroid cells. These results show clear functional differences between CD34+Flt3high and CD34+Flt3low cells and may have implications concerning the in vitro expansion of human hematopoietic progenitor cells.     

CD19+ pro-B cells can give rise to dendritic cells in vitro

Dendritic cells (DC) have the specific capacity of initiating primary T cell responses and ultimately derive from precursors in bone marrow. DC were originally thought to be only of myeloid origin, and myeloid precursor cells could be induced to differentiate into functional DC in response to granulocyte-macrophage (GM)-CSF. However, early CD4low precursor cells from the thymus can also develop into DC when cultured in IL-1beta, IL-3, IL-7, TNF-alpha, stem cell factor, and Flt-3L. In that case, GM-CSF was not required. We now show that CD19+ pro-B cells develop into DC with T cell stimulatory properties when cultured under similar conditions. These pro-B cells acquired the DC-related markers CD11c and NLDC145/DEC205, along with CD80/B7-1, CD86/B7-2, and a high density of MHC class II Ags. The marrow-derived DC did not express CD4 or CD8alpha, which are markers related to thymic DC. These findings are consistent with a new pathway through which DC are generated from B lymphoid precursors.     

Characterization of a B cell progenitor present in neonatal bone marrow and spleen but not in adult bone marrow and spleen

The neonatal period marks an important time in mammalian immunologic development, yet it is often ignored in studies of lymphocyte development. We identified a cell population with the phenotype heat stable Ag (HSA)low lin- CD43low that contained B cell progenitors at a high frequency in the neonatal bone marrow and spleen. Although cells with a similar phenotype can be identified in the bone marrow and spleen of adult animals, these populations showed a greatly reduced frequency of B cell progenitors. B lineage cells were detected after 7 days in culture at a frequency of 1:15 when HSAlow lin- CD43low cells from neonatal bone marrow were cultured on stromal cells and IL-7 under limiting dilution conditions. Under similar conditions, the equivalent population in adult bone marrow had a frequency of B cell progenitors that was less than 1:2000. The expression of terminal deoxynucleotidyl transferase in freshly sorted neonatal HSAlow lin- CD43low cells suggested that cells committed to the lymphocyte lineage were present in this population. These data suggested that the HSAlow lin- CD43low population of cells represents a pool of B lineage precursors that may be responsible for filling the immune compartment early in neonatal life.     

Differential use of very late antigen-4 and -5 integrins by hematopoietic precursors and myeloma cells to adhere to transforming growth factor-beta1-treated bone marrow stroma

The very late antigen (VLA)-4 and VLA-5 integrins mediate hematopoietic progenitor cell attachment to bone marrow (BM) stroma. Transforming growth factor-beta1 (TGF-beta1) is a cytokine present in the BM microenvironment that has been shown to regulate the synthesis of adhesion elements in several cell types. We have investigated whether TGF-beta1 action on human BM stromal cells affected the adhesion of progenitor cells involving integrins VLA-4 and VLA-5. Two precursor cell lines, pre-B Nalm-6 and the multipotential UT-7, attached to untreated primary stroma and to the human BM stromal cell line Str-5 preferentially using VLA-4. However, treatment of the stroma with TGF-beta1 resulted in a significant reduction in the participation of VLA-4 in mediating precursor cell adhesion to stroma and a concomitant increase in the utilization of VLA-5. This effect was not exclusive of normal BM stroma. Treatment with TGF-beta1 of stroma from multiple myeloma BM samples produced a substantial increase in VLA-5 use by the myeloma cell line NCI-H929 to adhere to this stroma. The differential use of VLA-4 and VLA-5 correlated with an increase in fibronectin surface expression by stromal cells in response to TGF-beta1. Adhesion assays to purified fibronectin using Nalm-6 cells showed a predominant utilization of VLA-4 at low concentrations of this ligand, whereas higher concentrations resulted in a preferential use of VLA-5. These results indicate that regulation of fibronectin expression on BM stromal cells by TGF-beta1 results in a modulation of the pattern of integrins used by the precursor and myeloma cells to adhere to BM stroma, which could have important consequences on the proliferation and differentiation of hematopoietic precursor cells as well as on the localization and growth of myeloma cells.     

Reliability for a walk/run test to estimate aerobic capacity in a brain-injured population

We investigated the effect of alpha-phenyl N-tert-butylnitrone (PBN), a spin trap reagent, on the proliferation of murine hematopoietic progenitor cells in vitro. During the addition of PBN to the liquid cultures of murine bone marrow cells containing a combination of interleukin-3, interleukin-6 and the c-kit ligand/stem cell factor, colony-forming cells in vitro (CFC) and the colony-forming unit in the spleen (CFU-S) increased about 1.6-fold and 2.0-fold, respectively, higher than the control culture. These effects were not observed when using dimethyl sulfoxide, which has the ability to scavenge radicals, and 5,5-dimethyl-1-pyrroline N-oxide, another spin trap reagent. Analysis of cultured cells from a 7-day liquid culture with PBN revealed that the ratio of the intracellular glutathione (GSH) and GSH/GSSG (oxidized GSH) content was higher than the control. Adding thiol N-acetylcysteine, a thiol reagent and a precursor of intracellular GSH, also showed similar effects on the liquid culture of murine hematopoietic progenitor cells and the level of intracellular GSH. In contrast, adding DL-buthionine-[S,R]-sulfoximine, a gamma-glutamylcysteine synthetase inhibitor, decreased the intracellular GSH level and did not increase the number of CFC and CFU-S. These results suggest that PBN regulates the content of intracellular thiol molecules, and the possibility of a relationship between the intracellular redox state and the proliferation and differentiation of hematopoietic stem cells.     

Major histocompatibility complex class II molecules, hemopoiesis and the marrow microenvironment

Currently available data indicate that the earliest identifiable hemopoietic progenitor in normal marrow is CD34+ MHC class II-; subsequent expression of MHC class II antigens is maturation and lineage dependent. Studies on embryonal cells suggest that CD34+DR- cells are actually the common precursors for stromal and hemopoietic elements, with the earliest hemopoietic precursor being CD34+DR+. DQ antigens are apparently not expressed in cells of hemopoietic potential and the expression of DQ appears to be regulated differentially from DR and DP. MHC class II antigens are also expressed on some stromal cells, especially those with endothelial and macrophage features. MHC class II molecules are involved in hemopoietic cell/stroma interaction. The presence of anti-MHC class II monoclonal antibodies (MABs) at early stages of stem cell proliferation/differentiation, at least under conditions of marrow stress, induces signals which may result in final, especially granulocytic, differentiation of later precursors. These may interfere with the survival of those cells which are required for long-term hemopoietic reconstitution. Observations in allogeneic marrow transplant recipients support a role of MHC molecules as expected in allogeneic interactions. Results in autologous models point towards a role of MHC class II molecules other than that of a histocompatibility marker insofar as these molecules or signals transmitted by them appear to be involved in the regulation of hemopoiesis.     

Iodination of mouse EGF with chloramine T at 4 degrees C: characterization of the iodinated peptide and comparison with other labelling methods

The BP-1/6C3 molecule expressed by early B lineage cells and some stromal cells has been identified as aminopeptidase A (APA). We have previously demonstrated that IL-7 selectively induces BP-1/APA expression by pre-B cells coincident with their growth. Here we directly demonstrate that BP-1 is preferentially expressed by the proliferating subpopulation of normal B cell progenitors. Furthermore, when non-adherent BALB/c bone marrow cells were incubated with IL-7 in the presence of purified BP-1 antibody, B cell precursor proliferation was markedly inhibited. Modulation of the BP-1/6C3 antigen did not occur in the presence of the BP-1 antibody, but APA enzymatic activity was significantly inhibited. The 6C3 antibody, which recognizes a different epitope on the APA molecule, had no effect on either B cell precursor proliferation or APA enzyme activity. We hypothesized that neutralization of APA by the BP-1 antibody results in inhibition of IL-7 driven B cell precursor proliferation. However, when isolated 14.8+ bone marrow cells were cultured with IL-7 in the presence of the BP-1 antibody, no inhibition of proliferation occurred. This data suggested that the effect of the BP-1 antibody might be related to the action of APA on peptides in the marrow microenvironment which are not present in cultures of isolated B cell precursors. The addition of irradiated non-adherent bone marrow cells to the 14.8+ cell cultures restored the inhibitory effect of the BP-1 antibody. Based on these observations, we propose that APA cleaves a small peptide which serves as a natural inhibitor of B cell precursor proliferation.     

Frequency analysis of the T cell precursors in the thymus

A frequency determination of the T cell precursors in murine adult and fetal thymuses as well as in the bone marrow and fetal liver was made. Cells were serially diluted and injected into deoxyguanosine-treated fetal thymus lobes with a microinjector, and the lobes were cultured for 12 to 21 days. The lobes in which T cell development did not occur were discriminated from those in which T cells developed, and the precursor frequency was determined by Poisson probability distribution analysis. The precursor cell frequencies in adult bone marrow cells (2.4 x 10(-5)) and fetal liver cells (1.7 x 10(-4)) were comparable to those determined previously in in vivo intrathymus transfer experiments. The present study further shows that only a small fraction of fetal thymus cells (0.9-5.0 x 10(-2)), CD4-8- adult thymocytes (1.6 x 10(-2)), and Thy-1 low positive adult thymocytes (3.3 x 10-4)) retain the precursor activity.     

The effects of Flk-2/flt3 ligand as compared with c-kit ligand on short-term and long-term proliferation of CD34+ hematopoietic progenitors elicited from human fetal liver, umbilical cord blood, bone marrow, and mobilized peripheral blood

The Flk-2/flt3 ligand (FL) was evaluated and compared with c-kit ligand (KL) for its in vitro proliferative effects on CD34+ cells from human fetal liver, umbilical cord blood, bone marrow, and mobilized peripheral blood. Using a 7-day liquid culture system, FL in combination with interleukin-3 (IL-3), interleukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF) was comparable with KL in combination with IL-3, IL-6, and G-CSF for the expansion of hematopoietic progenitors. When FL-containing cultures were assayed after 21 or 28 days, a greater number of progenitors were generated as compared with KL-containing cultures. Using bone marrow microvascular endothelial cells as support stroma, cultures supplemented with FL generated a greater number of progenitors in both the nonadherent and adherent layers at day 35. These data suggest that FL ligand, in combination with other cytokines, can be used for short-term ex vivo expansion of hematopoietic progenitors and facilitates the preservation and possible expansion of primitive cells capable of long-term generation of progenitors.     

Kinetics of release of serotonin from isolated secretory granules. I. Amperometric detection of serotonin from electroporated granules

In the CD4+ T cell lineage, two well-defined differentiated populations are the Th1 and Th2 cells, which stem from a common naive T helper precursor (Thp). In this study, we begin to dissect the signaling pathways selectively used by Th1 or Th2 cells as they mature from a common naive precursor in vitro. We show that the maturing Th1 cells mount a vigorous and specific Ca2+ transient upon contact with immunogenic ligand, which is enhanced over that of the naive progenitor cells. As the cells differentiate toward a Th2 phenotype, they quickly lose the ability to engage this pathway, indicating a developmental segregation of intracellular signaling utilization. Moreover, altered peptide ligand stimulation of the Th1 line stimulates a similar Ca2+ transient as native ligand stimulation of the naive precursors, consistent with a quantitative difference in intracellular signaling by these two peptides. These data provide a direct and sequential assessment of a signaling pathway utilization in peripheral T cells as they differentiate to their final functional states.     

Flt3 ligand plus IL-7 supports the expansion of murine thymic B cell progenitors that can mature intrathymically

Flt3 ligand (flt3L) has potent effects on hemopoietic progenitors, dendritic cells, and B lymphopoiesis. We have investigated the effects of flt3L on intrathymic precursors. The addition of flt3L + IL-7 to lobe submersion cultures of murine fetal thymic lobes resulted in the expansion of an immature population of Thy-1(low), CD44(high), HSA(high) cells. This population contained cells with precursor activity, as determined by their capacity to repopulate deoxyguanosine-treated fetal thymic lobes. Upon reentry to the thymic lobe, flt3L + IL-7-cultured Thy-1(low), CD44(high), HSA(high) cells underwent expansion and differentiation into B cells. Two weeks after fetal thymic organ culture following thymic lobe reconstitution, intrathymic cells were Thy-1-, B220+, and a subset was sIgM+. The intrathymic B cells shared features of adult thymic B cells, including CD5 expression and proliferative responses to IL-4 + IL-5 + CD40 ligand, but not to LPS or soluble anti-IgM. Ig production was noted upon stimulation with IL-4 + IL-5 + LPS and IL-4 + IL-5 + CD40 ligand. In conclusion, we have demonstrated that flt3L + IL-7 supports the expansion of a subset of progenitors present in the fetal thymus. The cultured progenitors can repopulate a fetal thymic lobe and develop into mature functional B cells, demonstrating that the fetal thymus is able to support B cell as well as T cell development.     

Cardiac risk in surgery. A review and guidelines for risk evaluation and reduction of cardiac risk in general surgery

We report herein the phenotypic and functional analysis of human bone marrow and thymus derived early T cells. Commitment to T cell lineage is acquired during CD7 antigen expression by CD34+ precursors in human bone marrow and before thymus colonization. Early thymocytes show similar phenotypic characteristics as bone marrow T cells. They rapidly acquire CD4 before the dual expression of CD4 and CD8. Their expansion and differentiation is regulated by two major factors: thymic stroma and cytokines produced by these stroma cells or by thymocytes themselves. Among cytokines, IL1 and sCD23 produced by thymic epithelial cells support in vitro early T cell development.     

Immunophenotypic characterization of stromal cells in aspirated human bone marrow samples

The presence of stromal cells was investigated in aspirated bone marrow prepared by the same method as that used for the initiation of human long-term bone marrow culture (hLTBMC). In previous studies, we performed immunocytochemical staining of cytocentrifuge cell preparations using a panel of antibodies with which we characterized stromal cell populations in hLTBMC. This approach allowed morphological as well as immunophenotypic assessment of cells of interest. Morphologically distinctive cell populations expressing vascular cell adhesion molecule-1 and low-affinity nerve growth factor receptor (NGFR) were observed to be present, but no cells expressing alpha-smooth muscle actin were found. Few macrophages were present, consistent with the origin of hLTBMC stroma-adherent macrophages from monocytes and their precursor cells rather than from mature macrophages among the culture-initiating cells. In the absence of double immunostaining, it was not possible to deduce whether CD34+ cells, which were present in varying numbers in the cytocentrifuge preparations, included stromal as well as primitive hematopoietic cells. In addition to single cells, multicellular tissue fragments containing a variety of stromal cell types were detected in many samples. Their presence raises the possibility that at least some components of hLTBMC stroma may arise by explant growth from complex tissue fragments containing vascular and fibroblastic elements. Overall, our results indicate that demonstration of a variety of stroma-associated antigens, in particular NGFR, provides a useful new tool for identifying stromal elements in aspirated bone marrow.     

Fetal liver contains committed NK progenitors, but is not a site for development of CD34+ cells into T cells

The presence of T and NK cells in the human fetal liver and the fact that fetal liver hemopoietic progenitor cells develop into T and NK cells suggest a role for the fetal liver compartment in T and NK cell development. In this work, we show that the capacity of fetal liver progenitors to develop into T cells, in a human/mouse fetal thymic organ culture system, is restricted to an immature subset of CD34+ CD38- cells. No T cell-committed precursors are contained within the more differentiated CD34+ CD38+ population. This conclusion is supported by the observations that no TCR-delta gene rearrangements and no pre-TCR-alpha expression can be detected in this population. However, NK cells were derived from CD34+ CD38- and CD34+ CD38+ fetal liver cells cultured in the presence of IL-15, IL-7, and Flt-3 ligand. Eighty to ninety percent of cells arising from the CD34+ CD38+ population expressed the NK cell-associated markers CD56, CD16, CD94, and NKR-P1A. Several subpopulations of NK cell precursors were identified by differential expression of these receptors. Based on the detection of populations with a similar antigenic profile in freshly isolated fetal liver cells, we propose a model of NK cell differentiation. Collectively, our findings suggest that CD34+ cells differentiate into NK cells, but not into mature T cells, in the human fetal liver.