PubMed Health⌕ Search

Biomedical subjects

T Lapidot

Publications and source records attributed to T Lapidot.

At least 37 records · Page 2Linked to original sources
PubMed1999

The chemokine SDF-1 stimulates integrin-mediated arrest of CD34(+) cells on vascular endothelium under shear flow.

The chemokine SDF-1 plays a central role in the repopulation of the bone marrow (BM) by circulating CD34(+) progenitors, but the mechanisms of its action remain obscure. To extravasate to target tissue, a blood-borne cell must arrest firmly on vascular endothelium. Murine hematopoietic progenitors were recently shown in vivo to roll along BM microvessels that display selectins and integrins. We now show that SDF-1 is constitutively expressed by human BM endothelium. In vitro, human CD34(+) cells establish efficient rolling on P-selectin, E-selectin, and the CD44 ligand hyaluronic acid under physiological shear flow. ICAM-1 alone did not tether CD34(+) cells under flow, but, in the presence of surface-bound SDF-1, CD34(+) progenitors rolling on endothelial selectin rapidly developed firm adhesion to the endothelial surface, mediated by an interaction between ICAM-1 and its integrin ligand, which coimmobilized with SDF-1. Human CD34(+) cells accumulated efficiently on TNF-activated human umbilical cord endothelial cells in the absence of SDF-1, but they required immobilized SDF-1 to develop firm integrin-mediated adhesion and spreading. In the absence of selectins, SDF-1 also promoted VLA-4-mediated, Gi protein-dependent tethering and firm adhesion to VCAM-1 under shear flow. To our knowledge, this is the first demonstration that SDF-1 expressed on vascular endothelium is crucial for translating rolling adhesion of CD34(+) progenitors into firm adhesion by increasing the adhesiveness of the integrins VLA-4 and LFA-1 to their respective endothelial ligands, VCAM-1 and ICAM-1.

Antigens, CD34↗
PubMed1998

High level engraftment of NOD/SCID mice by primitive normal and leukemic hematopoietic cells from patients with chronic myeloid leukemia in chronic phase.

We have previously shown that intravenously injected peripheral blood (PB) or bone marrow (BM) cells from newly diagnosed chronic myeloid leukemia (CML) patients can engraft the BM of sublethally irradiated severe combined immunodeficient (SCID) mice. We now report engraftment results for chronic phase CML cells in nonobese diabetic (NOD)/SCID recipients which show the superiority of this latter model. Transplantation of NOD/SCID mice with 7 to 10 x 10(7) patient PB or BM cells resulted in the continuing presence of human cells in the BM of the mice for up to 7 months, and primitive human CD34+ cells, including those detectable as colony-forming cells (CFC), as long-term culture-initiating cells, or by their coexpression of Thy-1, were found in a higher proportion of the NOD/SCID recipients analyzed, and at higher levels than were seen previously in SCID recipients. The human CFC and total human cells present in the BM of the NOD/SCID mice transplanted with CML cells also contained higher proportions of leukemic cells than were obtained in the SCID model, and NOD/SCID mice could be repopulated with transplants of enriched CD34+ cells from patients with CML. These results suggest that the NOD/SCID mouse may allow greater engraftment and amplification of both normal and leukemic (Ph+) cells sufficient for the quantitation and characterization of the normal and leukemic stem cells present in patients with CML. In addition, this model should make practical the investigation of mechanisms underlying progression of the disease and the development of more effective in vivo therapies.

Adult↗
PubMed1998

Targeted disruption of the mouse Caspase 8 gene ablates cell death induction by the TNF receptors, Fas/Apo1, and DR3 and is lethal prenatally.

Homozygous targeted disruption of the mouse Caspase 8 (Casp8) gene was found to be lethal in utero. The Caspase 8 null embryos exhibited impaired heart muscle development and congested accumulation of erythrocytes. Recovery of hematopoietic colony-forming cells from the embryos was very low. In fibroblast strains derived from these embryos, the TNF receptors, Fas/Apo1, and DR3 were able to activate the Jun N-terminal kinase and to trigger IkappaB alpha phosphorylation and degradation. They failed, however, to induce cell death, while doing so effectively in wild-type fibroblasts. These findings indicate that Caspase 8 plays a necessary and nonredundant role in death induction by several receptors of the TNF/NGF family and serves a vital role in embryonal development.

Animals↗
PubMed1997

Kinetic evidence of the regeneration of multilineage hematopoiesis from primitive cells in normal human bone marrow transplanted into immunodeficient mice.

Based on initial observations of human CD34+ Thy-1+ cells and long-term culture-initiating cells (LTC-IC) in the bone marrow of some sublethally irradiated severe combined immunodeficient (SCID) mice transplanted intravenously with normal human marrow cells, and the subsequent finding that the NOD/LtSz-scid/scid (NOD/SCID) mouse supports higher levels of human cell engraftment, we undertook a series of time course experiments to examine posttransplant changes in the number, tissue distribution, cycling activity, and in vivo differentiation pattern of various human hematopoietic progenitor cell populations in this latter mouse model. These studies showed typical rapid posttransplant recovery curves for human CD34- CD19+ (B-lineage) cells, CD34+ granulopoietic, erythroid, and multilineage colony-forming cells (CFC), LTC-IC, and CD34+ Thy-1+ cells from a small initial population representing <0.1% of the original transplant. The most primitive human cell populations reached maximum values at 5 weeks posttransplant, after which they declined. More mature cell types peaked after another 5 weeks and then declined. A 2-week course of thrice weekly injections of human Steel factor, interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin (administered just before the mice were killed for analysis) did not alter the pace of regeneration of either primitive or mature human hematopoietic cells, or their predominantly granulopoietic and B-lymphoid pattern of differentiation, although a significant enhancing effect on the level of human cell engraftment sustained after 3 months was noted. Cycling studies showed the human CFC present at 4 to 5 weeks posttransplant to be rapidly proliferating even in mice not given human growth factors. However, by 10 weeks and thereafter, only quiescent human CFC were detected; interestingly, even in mice that were given the 2-week course of growth factor injections. These studies indicate the use of this model for future analysis of the properties and in vivo regulation of primitive human hematopoietic cells that possess in vivo repopulating ability.

Animals↗
PubMed1997

Increased circulating colony-stimulating factor-1 (CSF-1) in SJL/J mice with radiation-induced acute myeloid leukemia (AML) is associated with autocrine regulation of AML cells by CSF-1.

The SJL/J mouse strain has a high spontaneous incidence of a B-cell neoplasm, reticulum cell neoplasm type B (RCN B). In addition, following irradiation, 10% to 30% of these mice develop acute myelomonocytic leukemia (radiation-induced acute myeloid leukemia [RI-AML]), an incidence that can be increased to 50% by treatment of the mice with corticosteroids after irradiation. The role played by the mononuclear phagocyte growth factor, colony-stimulating factor-1 (CSF-1), in the development of RI-AML in SJL/J mice was investigated. Mice dying of RI-AML, but not those dying of RCN B or without disease, possessed elevated concentrations of circulating CSF-1. In addition, in mice developing RI-AML with a more prolonged latency, circulating CSF-1 concentrations were increased before overt expression of RI-AML. First-passage tumors from 14 different RI-AMLs all contained high concentrations of CSF-1, and six of six different first- or second-passage tumors expressed the CSF-1 receptor (CSF-1 R). Furthermore, in vitro colony formation by first- or second-passage tumor cells from 20 of 20 different RI-AMLs was blocked by neutralizing anti-CSF-1 antibody, and four of four of these tumors were inhibited by anti-CSF-1R antibody. The results of these antibody neutralization studies, coupled with the observation of elevated circulating CSF-1 in mice developing RI-AML, show an autocrine role for CSF-1 in RI-AML development in SJL/J mice. Southern blot analysis of tumor DNA from six of six of these tumors failed to reveal any rearrangements in the genes for CSF-1 or the CSF-1R. Studies in humans have shown that patients with AML possess elevated levels of circulating CSF-1 and that AML cells can express CSF-1 and the CSF-1R. Thus, RI-AML in the SJL/J mouse appears to be a useful model for human AML.

Acute Disease↗
PubMed1997

Induction of T cell adhesion to extracellular matrix or endothelial cell ligands by soluble or matrix-bound interleukin-7.

The putative effects of interleukin (IL)-7, operating in the context of extracellular matrix (ECM), on the adhesion of human T cells were examined. Recombinant human, IL-7 was found to bind ECM or fibronectin (FN) with IC50 values of 10-100 nM. Nanogram amounts of both soluble and, especially, FN- or ECM-bound IL-7, which differentially affected the morphologies of FN-adherent T cells, induced the adhesion of resting CD4+ and CD8+ T cells in dose-dependent and beta 1 integrin-dependent manners. Under static and flow conditions, soluble IL-7 also induced the binding of unstimulated T cells to vascular cell adhesion molecule-1, suggesting that this cytokine can also modulate integrin binding to endothelial cell ligands. The effects of affinity modulation by IL-7 of FN-specific beta 1 integrins depend on the presence of soluble FN, which inhibited T cell adhesion to FN induced by FN-bound IL-7 or by an integrin-specific affinity-modulating monoclonal antibody, but not by soluble IL-7 or phorbol 12-myristate 13-acetate. These findings provide an example of a major ECM integrin ligand, FN, which is capable of modulating its adhesive interactions with specific immune cells by associating with and presenting a cytokine in a bio-active state.

Cell Adhesion↗
PubMed1997

Immune-deficient SCID and NOD/SCID mice models as functional assays for studying normal and malignant human hematopoiesis.

Many events and requirements of the developmental program of human hematopoietic stem cells have not yet been discovered. A major impediment has been the lack of an appropriate experimental system. At present the conditions for maintaining human stem cells in vitro are not fully known. As a result within a short period the small stem cell pool is lost due to differentiation, making it difficult to examine the correlation between these cells and their function in vivo. Most of our knowledge of hematopoietic stem cells is from animal models in which purified stem cell canididates are assayed based on their functional ability to rescue lethally conditioned recipients. The permanent correction of many genetic disorders of the hematopoietic system requires efficient methods for introducing genes into stem cells in vitro. However, progress has been hindered by the absence of preclinical models that assay the repopulating capacity of primitive human cells. In addition, the development of therapy for malignant diseases also requires assays to identify the target leukemic stem cells based on their ability to initiate the disease. The recent development of methods to transplant or implant both normal and leukemic cells into immune-deficient mice provides the foundation for human stem cell assays. These models assay the repopulating capacity of primitive human cells and provide an important approach to identify and characterize human stem cells, both normal and leukemic. This review focuses on the development of functional assays for normal and leukemic human stem cells and on the new insights that these models are beginning to provide on the organization of the human stem cell hierarchy.

Animals↗
PubMed1997

Interleukin-6 receptor-interleukin-6 fusion proteins with enhanced interleukin-6 type pleiotropic activities.

An sIL-6R/IL-6 chimera, directly fusing the natural forms of soluble IL-6 receptor and IL-6, as found in human body fluids, was produced in transfected human cells. The secreted p85 glycoprotein was active at a concentration of 120 pM to produce growth-arrest and spindleoid differentiation of murine melanoma F10.9 cells, which do not respond to IL-6 alone. This fusion protein was as active as the yeast-produced p56 fusion protein containing a shortened sIL-6R, linked through a flexible peptide chain to IL-6 (Hyper IL-6). The concentration of Hyper IL-6 needed to arrest the growth of F10.9 cells was much lower than that needed of a combination of IL-6 and sIL-6R, added separately. Hyper IL-6 was also more active than IL-6 in stimulating growth of murine plasmacytoma T1165 cells, the half maximal stimulation being obtained at 2 pM Hyper IL-6 versus 23 pM for IL-6. In order to evaluate the effect of the fused sIL-6R/IL-6 proteins on human hematopoietic primitive progenitor cells, they were added to suspension cultures of CD34+ cells from human cord blood in addition to both flt3/flk2 ligand (FL) and stem cell factor (SCF). Fused sIL-6R/IL-6 produced a marked stimulation of cell expansion and a marked increase in the number of colony forming units when subsequently plated in semi-solid medium with IL-3, GM-CSF, SCF and erythropoietin. Ex-vivo maintenance and expansion of early progenitor cells in bone marrow transplantation protocols may be a potential application for the sIL-6R/IL-6 chimeric glycoproteins.

Animals↗
PubMed1996

Identification of human juvenile chronic myelogenous leukemia stem cells capable of initiating the disease in primary and secondary SCID mice.

Most juvenile chronic myelogenous leukemia (JCML) cells have limited long-term proliferative capacity, and only a minority of immature cells give rise to colonies in semisolid cultures. Clonogenic JCML progenitors cannot be maintained in culture because they differentiate, and within a few weeks the leukemic clone is lost. This makes it difficult to identify the cell that initiates and maintains the disease in patients. To determine the proliferative capacity of JCML cells in vivo, bone marrow (BM), peripheral blood, or spleen cells from eight patients with JCML either at diagnosis or during treatment were transplanted into sublethally irradiated severe combined immune deficient (SCID) mice. JCML cells from all patients homed to the murine BM and proliferated extensively in response to exogenous stimulation with granulocyte-macrophage colony-stimulating factor. Within a few weeks, highly engrafted mice became ill and cachectic due to infiltration of leukemic cells and secretion of tumor necrosis factor-alpha. Murine BM, spleen, and liver were infiltrated with leukemic blasts, and typical JCML colony-forming progenitors could be recovered. Kinetic experiments demonstrated that only a small minority of transplanted cells homed to the murine BM, and that these cells initiated and maintained the disease in vivo by extensive proliferation and differentiation. To characterize the cell-surface phenotype of the JCML initiating cell (JCML-IC), JCML blood or spleen cells were fractionated on the basis of CD34/CD38 marker expression and transplanted into SCID mice. Only immature CD34+ cells could initiate the disease, while mature CD34- cells did not engraft. Within the CD34+ compartment, there was enrichment for JCML-ICs by immature cells with a CD34+/CD38- stem-cell-like phenotype. Mice transplanted with more mature CD34+/CD38+ populations that also contained clonogenic JCML progenitors were poorly engrafted. These results indicate that the JCML-IC is an earlier stage of development than clonogenic JCML progenitors. Additional evidence that the JCML-IC has stem-cell properties comes from secondary transplant experiments that test the self-renewal capacity. The JCML-IC from all three patients tested could successfully reinitiate the disease in secondary murine recipients. Thus, we have developed a functional in vivo model that replicates many aspects of human JCML, and have used this model to identify and characterize JCML-ICs and their stem-cell properties.

Animals↗
PubMed1996

Normal and leukemic SCID-repopulating cells (SRC) coexist in the bone marrow and peripheral blood from CML patients in chronic phase, whereas leukemic SRC are detected in blast crisis.

Progress in understanding the abnormal regulation of hematopoiesis in chronic myelogenous leukemia (CML) would be facilitated if neoplastic cells, at all stages of the disease, could be studied in an animal model. In this report, we show that irradiated severe combined immunodeficient (SCID) mice can be transplanted with both normal (Philadelphia chromosome [Ph]-negative) and neoplastic (Ph+) cells from CML patients with either chronic or blast phase disease. Mice transplanted with peripheral blood (PB) or bone marrow (BM) cells from 9 of 12 chronic phase CML patients were well engrafted with human cells including multilineage colony-forming progenitors and CD34+ cells for at least 90 days posttransplantation. Repeated posttransplant injections of cytokines did not enhance the number of engrafted human cells. Interestingly, approximately 70% of the human progenitors found in the engrafted SCID BM were Ph-, suggesting that the growth of primitive normal cells is favored in this in vivo transplant model. A similar number of normal cells were found in mice transplanted with either PB or BM cells, suggesting that elevated numbers of primitive normal cells are present in CML PB. When cells from patients with CML in either myeloid or lymphoid blast crisis were transplanted into SCID mice, the BM of these mice was more rapidly repopulated and to a higher level than that observed with transplants of chronic phase cells. Moreover, all human colony-forming progenitors present in the BM of mice transplanted with blast crisis cells were Ph+, and the majority of cells showed the same morphological features of the blast crisis cells originally transplanted. These experiments provide a starting point for the creation of an animal model of CML and establish the feasibility of using this model for the future characterization of transplantable CML stem cells during disease progression.

Animals↗
PubMed1996

Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy.

The development of stem-cell gene therapy is hindered by the absence of repopulation assays for primitive human hematopoietic cells. Current methods of gene transfer rely on in vitro colony-forming cell (CFC) and long-term culture-initiating cell (LTC-IC) assays, as well as inference from other mammalian species. We have identified a novel human hematopoietic cell, the SCID-repopulating cell (SRC), a cell more primitive than most LTC-ICs and CFCs. The SRC, exclusively present in the CD4+CD8- fraction, is capable of multilineage repopulation of the bone marrow of nonobese diabetic mice with severe combined immunodeficiency disease (NOD/SCID mice). SRCs were rarely transduced with retroviruses, distinguishing them from most CFCs and LTC-ICs. This observation is consistent with the low level of gene marking seen in human gene therapy trials. An SRC assay may aid in the characterization of hematopoiesis, as well as the improvement of transduction methods.

ADP-ribosyl Cyclase↗
PubMed1995

Engraftment of immune-deficient mice with primitive hematopoietic cells from beta-thalassemia and sickle cell anemia patients: implications for evaluating human gene therapy protocols.

Permanent correction of genetic deficiencies of the hematopoietic system requires gene transfer into stem cells and long-term lineage specific expression after autologous transplantation. However, progress to develop gene therapy protocols has been hampered by the absence of in vivo assays that detect genetically deficient human hematopoietic stem cells and their diseased differentiated progeny. The establishment of systems to transplant human cells into immune-deficient SCID mice provides such an assay. We report that primitive bone marrow cells from beta-thalassemia major and sickle cell anemia patients engraft immune-deficient mice, giving rise to high levels of human erythroid and myeloid cells in response to treatment with human cytokines. The bone marrow of transplanted mice contained the entire erythroid lineage from BFU-E to mature erythrocytes expressing human gamma, beta or beta s-globin. Moreover, human erythroid cells from mice transplanted with sickle cell anemia bone marrow showed characteristic sickling under reducing conditions in an in vitro assay. This model provides a powerful in vivo system that can be used to evaluate the efficiency of globin gene transfer into primitive human hematopoietic cells, lineage-specific expression in mature erythrocytes, and ultimately correction of the cellular defect found in the erythroid lineage.

Anemia, Sickle Cell↗
PubMed1994

Immature human cord blood progenitors engraft and proliferate to high levels in severe combined immunodeficient mice.

Unseparated or Ficoll-Hypaque (Pharmacia, Piscataway, NJ)--fractionated human cord blood cells were transplanted into sublethally irradiated severe combined immunodeficient (SCID) mice. High levels of multilineage engraftment, including myeloid and lymphoid lineages, were obtained with 80% of the donor samples as assessed by DNA analysis, fluorescence-activated cell sorting (FACS), and morphology. In contrast to previous and concurrent studies with adult human bone marrow (BM), treatment with human cytokines was not required to establish high-level human cell engraftment, suggesting that neonatal cells either respond differently to the murine microenvironment or they provide their own cytokines in a paracrine fashion. Committed and multipotential myelo-erythroid progenitors were detected using in vitro colony assays and FACS analysis of the murine BM showed the presence of immature CD34+ cells. In addition, human hematopoiesis was maintained for at least 14 weeks providing further evidence that immature hematopoietic precursors had engrafted the murine BM. This in vivo model for human cord blood-derived hematopoiesis will be useful to gain new insights into the biology of neonatal hematopoietic cells and to evaluate their role in gene therapy. There is growing evidence that there are ontogeny-related changes in immature human hematopoietic cells, and therefore, the animal models we have developed for adult and neonatal human hematopoiesis provide useful tools to evaluate these changes in vivo.

Animals↗
PubMed1994

A cell initiating human acute myeloid leukaemia after transplantation into SCID mice.

Most human acute myeloid leukaemia (AML) cells have limited proliferative capacity, suggesting that the leukaemic clone may be maintained by a rare population of stem cells. This putative leukaemic stem cell has not been characterized because the available in vitro assays can only detect progenitors with limited proliferative and replating potential. We have now identified an AML-initiating cell by transplantation into severe combined immune-deficient (SCID) mice. These cells homed to the bone marrow and proliferated extensively in response to in vivo cytokine treatment, resulting in a pattern of dissemination and leukaemic cell morphology similar to that seen in the original patients. Limiting dilution analysis showed that the frequency of these leukaemia-initiating cells in the peripheral blood of AML patients was one engraftment unit in 250,000 cells. We fractionated AML cells on the basis of cell-surface-marker expression and found that the leukaemia-initiating cells that could engraft SCID mice to produce large numbers of colony-forming progenitors were CD34+ CD38-; however, the CD34+ CD38+ and CD34- fractions contained no cells with these properties. This in vivo model replicates many aspects of human AML and defines a new leukaemia-initiating cell which is less mature than colony-forming cells.

ADP-ribosyl Cyclase↗
PubMed1994

SCID mice as an in vivo model of human cord blood hematopoiesis.

Cord blood is increasingly used as an alternative stem cell source for autologous and allogeneic transplantation, particularly in pediatric patients. We therefore adopted our protocol for transplanting human adult bone marrow cells into severe combined immunodeficient (SCID) mice [1] to develop an in vivo model for cord blood hematopoiesis. Intravenous injection of unfractionated or Ficoll-separated cord blood cells into sublethally irradiated SCID mice led to high levels of human hematopoiesis in the majority of the recipients [2]. Multilineage human hematopoiesis including committed and multipotential myeloerythroid progenitors as well as CD19+ B-lymphoid cells were observed in the murine bone marrow for at least 18 weeks. Together, these data indicate that the SCID mice were engrafted with an immature cell that was able to maintain multiple progenitor lineages in vivo. In contrast to our experiences with adult bone marrow, high levels of human cell engraftment in the mouse could be achieved without exogenous cytokine treatment, suggesting that the cord blood cells respond differently to the murine microenvironment. Alternatively, the cord blood cells might have been able to provide themselves with the necessary growth factors in a paracrine fashion. This model will be useful in gaining new insights into the biology of immature human cord blood progenitors and cord blood transplantation.

Animals↗
PubMed1993

Engraftment of human lymphoid cells into newborn SCID mice leads to graft-versus-host disease.

Although there has been considerable progress in transplanting normal human hematopoietic cells into immune-deficient mice, the establishment of a functional human immune system has proved to be difficult. Current methods of introducing mature human lymphoid cells into adult SCID mice lead to oligoclonal engraftment with restricted dissemination to various organs. We have attempted to improve human lymphoid cell engraftment in mice, both qualitatively and quantitatively, by injecting human bone marrow cells and peripheral blood leukocytes intraperitoneally into newborn SCID mice. Newborn mice were used as recipients because certain immune functions such as natural killer cell activity do not develop until several weeks after birth and the numerous growth factors secreted in young mice may facilitate the engraftment and proliferation of transplanted human cells. At various times after transplantation, the presence of human cells in different organs was determined by Southern blot analysis using a human specific probe. Within 4 weeks, 70% of the mice were engrafted with human cells. Human cell engraftment of the bone marrow, spleen, lungs, kidneys, liver, and thymus exceeded 10% in at least 40% of the transplanted mice; most of these highly engrafted mice were sick. Flow cytometry and immunocytochemistry indicated these organs were heavily infiltrated with mature T and B lymphocytes. Histologic and molecular analysis showed massive human cell infiltrates within the liver, lung and spleen. The presence of human IgG and IgM antibodies against mouse red blood cells provided evidence that the engrafted human cells retained some immune function. Mice transplanted with peripheral blood leukocytes from donors that were allergic to mouse antigens engrafted to the same extent as normal cells but in addition developed the classical symptoms of acute allogeneic graft-versus-host disease (GVHD) including infiltrates of the skin, gut, and liver. The newborn SCID system provides a new in vivo model to study human xenoreactivity and GVHD.

Animals↗