Increased differentiation of dermal mast cells in mice lacking the Mpl gene.

Thrombopoietin interactions with its receptor, Mpl, play an important role in the regulation of hematopoietic stem/progenitor cell proliferation and differentiation. In this study, we report that the mast cell restricted progenitor cells (MCP) and the mast cell precursors in the bone marrow of wild-type mice express Mpl on their surface. Furthermore, targeted deletion of the Mpl gene in mice decreases the number of MCP while increasing the number of mast cell precursors present in the marrow and spleen. It also increases the number of mast cells present in the dermis, in the peritoneal cavity, and in the gut of the mice. In addition, serosal mast cells from Mpl(null) mice have a distinctive differentiation profile similar to that expressed by wild-type dermal mast cells. These results suggest that not only does ligation of thrombopoietin with the Mpl receptor exert an effect at the mast cell restricted progenitor cell level, but also plays an unexpected yet important role in mast cell maturation.

Thrombopoietin inhibits murine mast cell differentiation.

We have recently shown that Mpl, the thrombopoietin receptor, is expressed on murine mast cells and on their precursors and that targeted deletion of the Mpl gene increases mast cell differentiation in mice. Here we report that treatment of mice with thrombopoietin or addition of this growth factor to bone marrow-derived mast cell cultures severely hampers the generation of mature cells from their precursors by inducing apoptosis. Analysis of the expression profiling of mast cells obtained in the presence of thrombopoietin suggests that thrombopoietin induces apoptosis of mast cells by reducing expression of the transcription factor Mitf and its target antiapoptotic gene Bcl2.

A pathobiologic pathway linking thrombopoietin, GATA-1, and TGF-beta1 in the development of myelofibrosis.

Idiopathic myelofibrosis (IM) is a disease characterized by marrow fibrosis, abnormal stem/progenitor cell trafficking, and extramedullary hematopoiesis frequently associated with alterations in megakaryocytes (Mks). Mice harboring genetic alterations in either the extrinsic (ectopic thrombopoietin expression, TPO(high) mice) or intrinsic (hypomorphic GATA-1 mutation, GATA-1(low) mice) control of Mk differentiation develop myelofibrosis, a syndrome similar to IM. The relationship, if any, between the pathobiologic mechanism leading to the development of myelofibrosis in the 2 animal models is not understood. Here we show that plasma from GATA-1(low) mice contained normal levels of TPO. On the other hand, Mks from TPO-treated wild-type animals (TPO(high) mice), as those from GATA-1(low) animals, had similar morphologic abnormalities and contained low GATA-1. In both animal models, development of myelofibrosis was associated with high transforming growth factor beta1 (TGF-beta1) content in extracellular fluids of marrow and spleen. Surprisingly, TPO treatment of GATA-1(low) mice restored the GATA-1 content in Mks and halted both defective thrombocytopoiesis and fibrosis. These data indicate that the TPO(high) and GATA-1(low) alterations are linked in an upstream-downstream relationship along a pathobiologic pathway leading to development of myelofibrosis in mice and, possibly, of IM in humans.

Animal experimentation in Italy. Legislation and the authorization of research protocols.

In Italy, the European Directive 86/609/EEC ("The Council Directive on the approximation of laws, regulations and administrative provisions of the Member States regarding the protection of animals used for experimental and other scientific purposes") has been given effect with Legislative Decree 116/92, whose main objective is to guarantee the welfare of animals used in research and to prevent unnecessary experiments on animals from being carried out. The regulatory authority for controlling the use of laboratory animals in Italy is the Ministry of Health, which requires that researchers requesting authorization to perform experiments on animals submit not only a copy of the experimental protocol but also a detailed application form that focuses on how the animals will be used. In the evaluation process, the Istituto Superiore di Sanità (Italy's National Institute of Health), in particular, the Service for Biotechnology and Animal Welfare, plays a key decision-making role. The evaluation is conducted by experts in the given area of research and by a veterinarian specifically trained in evaluating experimental protocols involving animal use. In the present work, the evaluation process is explained and a point-by-point description of the application form is provided.

Robust levels of long-term multilineage reconstitution in the absence of stem cell self-replication in W/Wv mice transplanted with purified stem cells.

Isolation of primitive blood stem cells by different methods results in cell populations with distinct biological activities. This study was aimed at resolving differences in the frequency of multilineage reconstituting cells (MRC) and their precursors (pMRC) in cell populations isolated by positive selection for Sca-1 compared to those isolated by negative selection for 15-1.1. Separation of wheat germ agglutinin-positive mouse bone marrow cells into 15-1.1neg or Sca-1+ subsets was performed by flow cytometry. The isolated cells were transplanted into W/Wv or normal irradiated recipient mice and reconstitution was evaluated over time. Sca-1+ cells were less frequent and contained more MRC than 15-1.1neg cells, while pMRC were found mainly among 15-1.1neg cells. MRC activity was exclusively contained within the Sca-1+ subpopulation of 15-1.1neg cells, but marrow from 7 robustly engrafted W/Wv mice did not contain donor-derived MRC, indicating that 15-1.1negSca-1+ cells contain low numbers of pMRC. Functional differences between 15-1.1neg and Sca-1+ cells were further confirmed by reverse transcriptase (RT)-PCR gene expression analysis. Early hematopoiesis-specific transcription factors (Scl, Gata-2, and Gata-1) were amplified from cDNA prepared from Sca-1+ but not 15-1.1neg cells. This study indicates that cell populations isolated as Sca-1+ are functionally distinct from those isolated as 15-1.1neg in that few pMRC are included among Sca-1+ cells and that MRC and pMRC are two distinct and separable cell populations.

Role of endogenous reverse transcriptase in murine early embryo development.

We report that a reverse transcriptase (RT) activity is present in early cleavage stage embryos as determined by a Polymerase chain reaction (PCR)-based detection assay. In an attempt to establish whether this activity plays a role in early embryonic development, we have blocked the endogenous RT by two independent approaches: (1) embryos were exposed to nevirapine, a highly specific nonnucleoside inhibitor of RT activity; (2) anti-RT antibody was microinjected into the nucleus of one blastomere of 2-cell embryos. When embryos were exposed to nevirapine in the developmental window between late 1-cell and 4-cell stages, development was arrested before the blastocyst stage. In contrast, development was not affected when embryos were exposed to nevirapine after the eight-cell stage. Developmental arrest was also induced when anti-RT antibody was microinjected in one blastomere of 2-cell embryos. Analysis of gene expression by RT-PCR in nevirapine-arrested 2-cell embryos revealed an extensive and specific reprogramming of gene expression, involving both developmentally regulated and constitutively expressed genes, compared to control embryos. These results support the conclusion that an endogenous RT activity is required in mouse early embryogenesis specifically between the late 1-cell and the 4-cell stage.

Mouse early embryos obtained by natural breeding or in vitro fertilization display a differential sensitivity to extremely low-frequency electromagnetic fields.

We have investigated the sensitivity of pre-implantation embryos obtained by natural breeding (NB) or in vitro fertilization (IVF) to extremely low-frequency magnetic fields (ELF-MF). Fertilized eggs obtained by NB were removed from mothers 12h after mating and cultured in vitro for 5 days under continuous ELF-MF exposure (constant strength of 50Hz and various intensities, i.e. 60, 120 and 220 microT). Alternatively, zygotes obtained by IVF were subjected to ELF-MF exposure (50Hz, 60 microT), starting 12h after IVF for 5 days. We found that ELF-MF exposure causes a small yet significant (P<0.05) decrease in the survival rate of NB-derived embryos at the latest stages of pre-implantation development, i.e. the eight cell-to-blastocyst transition. In embryos exposed to the highest field intensity (220 microT), the effect became apparent somewhat earlier. When IVF-derived embryos were exposed to ELF-MF, the reduction in the rate of embryo survival was more pronounced and the difference from controls was more significant (P<0.01). Moreover, the decreased survival rate in IVF embryos became apparent as early as the first cleavage and persisted throughout pre-implantation. These results suggest that IVF-derived embryos are more sensitive than NB-generated embryos to ELF-MF, and that this sensitivity occurs earlier in development.

Exposure of normal and transformed cells to nevirapine, a reverse transcriptase inhibitor, reduces cell growth and promotes differentiation.

Endogenous, nontelomeric reverse transcriptase (RT) is encoded by two classes of repeated elements: retrotransposons and endogenous retroviruses. Expression of RT-coding genes is generally repressed in differentiated nonpathological tissues, yet is active in the mammalian germ line, embryonic tissues and tumor cells. Nevirapine is a non-nucleoside RT inhibitor with a well-characterized inhibitory activity on RT enzymes of retroviral origin. Here, we show that nevirapine is also an effective inhibitor of the endogenous RT in murine and human cell lines. In addition, progenitor and transformed cells undergo a significant reduction in the rate of cell growth upon exposure to nevirapine. This is accompanied by the onset of differentiation, as depicted in F9 and C2C7 progenitor cells cultures in which nevirapine triggers the expression of differentiation-specific markers. Consistent with this, an extensive reprogramming of cell cycle gene expression was depicted in nevirapine-treated F9 cultures. Furthermore, nevirapine exposure rescued the differentiation block present in acute myeloid leukemia (AML) cell lines and primary blasts from two AML patients, as indicated by morphological, functional and immunophenotypic assays. The finding that an RT inhibitor can modulate cell proliferation and differentiation suggests that RT may represent a novel target in the development of therapeutical approaches to neoplasia.

GATA-1 as a regulator of mast cell differentiation revealed by the phenotype of the GATA-1low mouse mutant.

Here it is shown that the phenotype of adult mice lacking the first enhancer (DNA hypersensitive site I) and the distal promoter of the GATA-1 gene (neo Delta HS or GATA-1(low) mutants) reveals defects in mast cell development. These include the presence of morphologically abnormal alcian blue(+) mast cells and apoptotic metachromatic(-) mast cell precursors in connective tissues and peritoneal lavage and numerous (60-70% of all the progenitors) "unique" trilineage cells committed to erythroid, megakaryocytic, and mast pathways in the bone marrow and spleen. These abnormalities, which were mirrored by impaired mast differentiation in vitro, were reversed by retroviral-mediated expression of GATA-1 cDNA. These data indicate an essential role for GATA-1 in mast cell differentiation.

Development of myelofibrosis in mice genetically impaired for GATA-1 expression (GATA-1(low) mice).

The phenotype induced by the GATA-1(low) (neodeltaHS) mutation is here further characterized by analyzing the hemopoietic system during the aging (up to 20 months) of a GATA-1(low) colony (135 mutants and 40 normal littermates). Mutants expressed normal hematocrit values (Hct = 45.9 +/- 4.0) until 12 months but became anemic from 15 months on (Hct = 30.9 +/- 3.9; P <.05). Anemia was associated with several markers of myelofibrosis such as the presence of tear-drop poikilocytes and progenitor cells in the blood, collagen fibers in the marrow and in the spleen, and hemopoietic foci in the liver. Semiquantitative reverse transcription-polymerase chain reaction showed that growth factor genes implicated in the development of myelofibrosis (such as osteocalcin, transforming growth factor-beta1, platelet-derived growth factor, and vascular endothelial growth factor) were all expressed in the marrow from the mutants at higher levels than in corresponding normal tissues. The GATA-1(low) mutants experienced a slow progression of the disease because the final exitus was not observed until at least 15 months with a probability of survival more favorable than that of W/Wv mice concurrently kept in the animal facility (P <.001, by Kaplan-Meier analysis). In conclusion, impaired GATA-1 expression may contribute to the development of myelofibrosis, and the GATA-1(low) mutants may represent a suitable animal model for the human disease that may shed light on its pathogenesis.