Betty Nakamoto

Mutations in proto-oncogene GFI1 cause human neutropenia and target ELA2

Mice lacking the transcriptional repressor oncoprotein Gfi1 are unexpectedly neutropenic. We therefore screened GFI1 as a candidate for association with neutropenia in affected individuals without mutations in ELA2 (encoding neutrophil elastase), the most common cause of severe congenital neutropenia (SCN; ref. 3). We found dominant negative zinc finger mutations that disable transcriptional repressor activity. The phenotype also includes immunodeficient lymphocytes and production of a circulating population of myeloid cells that appear immature. We show by chromatin immunoprecipitation, gel shift, reporter assays and elevated expression of ELA2 in vivo in neutropenic individuals that GFI1 represses ELA2, linking these two genes in a common pathway involved in myeloid differentiation.

Cells with hemopoietic potential residing in muscle are itinerant bone marrow–derived cells

OBJECTIVE The nature of cells residing in muscle giving rise to hemopoietic colonies in vitro or hemopoietic reconstitution in vivo has been unclear. The goal of the present study was to characterize these cells and uncover their potential site of origin. MATERIALS AND METHODS Cells prepared from muscle were characterized for surface antigens (CD45, CD34, c-kit, Sca-1, CD31, VCAM-1), for their in vitro clonogenic capacity and in vivo repopulation potential either as unpurified cells or sorted subsets (CD45(+), CD45(-)). The presence of bone marrow (BM)-derived cells in muscle of mice reconstituted with marked BM cells before and after cytokine-induced mobilization was also examined. RESULTS Our data show: 1) The yield of CD45(+) cells is higher in muscle of neonates and young animals. Their composite phenotype does not favor contamination by blood. 2) The capacity of fresh muscle cell explants to give rise to colonies in vitro and hemopoietic reconstitution in vivo is associated with CD45(+) cells. 3) Irradiated recipients reconstituted with marked BM cells harbor marked BM-derived cells (CD45(+) or CD45(-)) in their muscle several months after transplant. 4) Cytokine-induced mobilization of transplanted animals modestly increases the yield of BM-derived cells recovered from muscle, unlike the yields from spleen, liver, or peripheral blood (PB). CONCLUSIONS Our data suggest a reinterpretation of previously published conclusions: hemopoietic colonies derived from fresh muscle explants do not originate from transdifferentiated muscle cells, but from BM-derived cells residing in muscle; the hemopoietic reconstituting potential of muscle cells is likewise attributed to these cells.

Altered SDF-1/CXCR4 axis in patients with primary myelofibrosis and in the Gata1low mouse model of the disease

OBJECTIVE To assess whether alterations in the stromal cell-derived factor-1 (SDF-1)/CXCR4 occur in patients with primary myelofibrosis (PMF) and in Gata1 low mice, an animal model for myelofibrosis, and whether these abnormalities might account for increased stem/progenitor cell trafficking. MATERIALS AND METHODS In the mouse, SDF-1 mRNA levels were assayed in liver, spleen, and marrow. SDF-1 protein levels were quantified in plasma and marrow and CXCR4 mRNA and protein levels were evaluated on stem/progenitor cells and megakaryocytes purified from the marrow. SDF-1 protein levels were also evaluated in plasma and in marrow biopsy specimens obtained from normal donors and PMF patients. RESULTS In Gata1 low mice, the plasma SDF-1 protein was five times higher than normal in younger animals. Furthermore, SDF-1 immunostaining of marrow sections progressively increased with age. Similar abnormalities were observed in PMF patients. In fact, plasma SDF-1 levels in PMF patients were significantly higher (by twofold) than normal (p < 0.01) and SDF-1 immunostaining of marrow biopsy specimens demonstrated increased SDF-1 deposition in specific areas. In two of the patients, SDF-1 deposition was normalized by curative therapy with allogenic stem cell transplantation. Similar to what already has been reported for PMF patients, the marrow from Gata1 low mice contained fewer CXCR4 pos CD117 pos cells and these cells expressed low levels of CXCR4 mRNA and protein. CONCLUSION Similar abnormalities in the SDF-1/CXCR4 axis are observed in PMF patients and in the Gata1 low mice model of myelofibrosis. We suggest that these abnormalities contribute to the increased stem/progenitor cell trafficking observed in this mouse model as well as patients with PMF.

The role of CS1 moiety of fibronectin in VLA4‐mediated haemopoietic progenitor trafficking

In vitro the integrin VLA4 mediates the adhesion of haemopoietic progenitors to bone marrow stroma through an interaction with its ligands VCAM‐1 and the CS1 moiety of fibronectin. The VLA4/VCAM‐1 pathway has been implicated in haemopoietic trafficking in vivo since antibodies to both VLA4 and VCAM‐1 decrease the homing (lodgement) of transplanted progenitors and mobilize progenitors. However, the role of the CS1 domain of fibronectin in progenitor trafficking in vivo has not been explored. We studied the effect of competitive inhibition of the VLA4/CS1 pathway on progenitor homing and mobilization in mice. Pre‐incubation of bone marrow cells with a CS1 inhibitor did not alter the number of CFU‐C or CFU‐S12 lodged to the bone marrow of lethally irradiated mice 3 h after transplantation. In addition, continuous administration of a CS1 inhibitor did not increase the number of CFU‐C in the peripheral blood. In order to study the role of the VLA4/CS1 pathway in trafficking of more primitive progenitors we studied whether administration of a CS1 inhibitor mobilized radioprotective cells. In contrast to the effect of anti‐VCAM‐1 which mobilized cells capable of rescuing 100% of lethally irradiated mice, administration of a CS1 inhibitor did not increase the number of radioprotective cells in the peripheral blood. Haemopoietic progenitors also bind to the RGD motif of fibronectin through an interaction with VLA5 and we therefore also studied the effect of antibodies to VLA5 on progenitor homing and mobilization. Antibody to VLA5 did not alter bone marrow lodgement at 3 h or increase the number of circulating haemopoietic progenitors. These studies therefore imply that, in contrast to VCAM‐1, the CS1 moiety of fibronectin is not a significant ligand in VLA4 mediated progenitor trafficking in vivo.

Combinatorial and distinct roles of α5 and α4 integrins in stress erythropoiesis in mice

To delineate the role of specific members of β₁ integrins in stress erythropoiesis in the adult, we compared the response to phenylhydrazine stress in 3 genetically deficient models. The survival of β₁-conditionally deficient mice after phenylhydrazine is severely compromised because of their inability to mount a successful life saving splenic erythroid response, a phenotype reproduced in β₁(Δ/Δ) reconstituted animals. The response of bone marrow to phenylhydrazine-induced stress was, unlike that of spleen, appropriate in terms of progenitor cell expansion and mobilization to peripheral blood although late differentiation defects qualitatively similar to those in spleen were present in bone marrow. In contrast to β₁-deficient mice, α₄(Δ/Δ) mice showed only a kinetic delay in recovery and similar to β₁(Δ/Δ), terminal maturation defects in both bone marrow and spleen, which were not present in VCAM-1(Δ/Δ) mice. Convergence of information from these comparative studies lends new insight to the distinct in vivo roles of α₄ and α₅ integrins in erythroid stress, suggesting that the presence of mainly α₅β₁ integrin in all hematopoietic progenitor cells interacting with splenic microenvironmental ligands/cells is instrumental for their survival and accumulation during hemolytic stress, whereas presence of α₄ or of both α₅ and α₄, is important for completion of terminal maturation steps.

Somatic-Cell Mutation Monitoring System Based on Human Hemoglobin Mutants

The system described in this chapter was developed as a means of detecting rare red cells, in genetically normal (HbA/HbA) individuals, that are heterozygous for an abnormal hemoglobin. It is assumed, first, that mutations arise spontaneously in human hemopoietic stem cells, as they do in gametal stem cells, and second, that somatic mutations of globin-chain genes do not diminish the viability of affected stem cells. The latter assumption is a reasonable one, since phenotypic expression of such mutations occurs very late in hemopoietic cell differentiation. It is expected that as a result of these stem cell mutations, lines of stem cells containing the mutant globin genes are established and produce erythrocytes heterozygous for structurally abnormal globin chains. Development of appropriate methods of screening blood samples should then permit detection and enumeration of red cells that contain an abnormal hemoglobin as a result of somatic mutation in a stem cell.

Modeling promising nonmyeloablative conditioning regimens in nonhuman primates.

Minimal conditioning or even no conditioning would be the preferred preparation for most gene therapy applications for nonmalignant diseases. However, reduced intensity conditioning (RIC) regimens in patients with nonhematologic malignancies have not led to long-term engraftment unless a selective advantage was present for the transplanted donor cells. Similar findings have also been observed in a number of large animal studies. Inadequate myelosuppression levels were thought to be responsible for the outcomes. To address this issue several innovative protocols in small animals have been presented with selective hematopoietic myelosuppression and less systemic toxicity. Such protocols promised to curb the transplant-related morbidity and mortality in myeloablative conditioning and provide effective long-term engraftment, especially in patients with gene-corrected autografts. In the present study we have tested some of these promising RIC regimens in nonhuman primates, a clinically relevant large animal model. Our data suggest that transient myelosuppression induced by anti-c-Kit antibody in conjunction with low-dose irradiation may lead to long-term engraftment, albeit at low levels. The animals with busulfan conditioning with or without anti-c-Kit that received gene-modified autologous transplants with green fluorescent protein expression had similar myelosuppression, but failed long-term engraftment and despite immunosuppressive treatment had all the hallmarks seen previously in similar models without immunosuppression. Our preliminary data expand current knowledge of RIC and emphasize the need to explore whether specific and directed myelosuppression alone is adequate in the absence of microenvironmental modulation, or whether innovative combinations are necessary for safe and effective engraftment.