Neutropenic Mice Can Be Protected from Fungus Infection by Ex Vivo Expanded Allogeneic Myeloid Progenitors.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3038-3038
Author(s):  
Jos Domen ◽  
Mastura Wahedi ◽  
Esther Danenberg ◽  
Stephanie Smith-Berdan ◽  
Julie Christensen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Hematopoietic Cell Transplantation ◽  
Cellular Therapy ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Myeloid Progenitor ◽  
Large Numbers ◽  
Myeloid Progenitor Cells ◽  
Myeloid Progenitors

Abstract Neutropenia induced by chemotherapy, hematopoietic cell transplantation, as well as radiation-induced neutropenia is accompanied by a significant morbidity and mortality due to increased susceptibility to a variety of pathogens. Despite clinical advances, such as the availability of growth factors that can stimulate granulopoiesis, like G-CSF, and new generations of antibiotics and antifungal agents, the problem persists. Direct cellular therapy with mature granulocytes has met with limited success, and would be difficult to implement due to the large number of cells needed, their very short lifespan and the inability to store the cells between harvesting and use. Our long-term goal is to develop a non-HLA-restricted cell-based short-term bridging therapy to reduce susceptibility to infection and enhance recovery from infections in the setting of neutropenia. The recent characterization of surface marker profiles of early myeloid progenitors has allowed the prospective testing of these cells. It has been clearly shown that the use of limited numbers of a combination of Common Myeloid Progenitors (CMP) and Granulocyte-Macrophage-Progenitors (GMP), purified from bone marrow, can protect mice from a normally lethal challenge with fungus or bacteria [Blood100:4660-7(2002)]. We have extended these observations and tested whether myeloid progenitors (MP) with similar properties can be obtained in large numbers from hematopoietic stem cells (HSC) in vitro. Here we report that a one week, serum-free culture can result in a 200-fold expansion of cells with myeloid progenitor activity with respect to the starting CD117+, CD90.1low, Linneg/low and Sca-1+ KTLS-HSC. Furthermore, these myeloid progenitor cells are as effective as Myeloid Progenitors sorted directly from bone marrow in preventing death due to invasive aspergillosis, even when the cells are fully allogeneic and have been cryopreserved prior to use. These results indicate a way in which myeloid progenitor cells, which can be stored frozen prior to use, can be obtained in large numbers. This process, once translated to human cells, would also open the way for the stockpiling of MP for use in nuclear disasters.

scholarly journals Human bone marrow depleted of CD33-positive cells mediates delayed but durable reconstitution of hematopoiesis: clinical trial of MY9 monoclonal antibody-purged autografts for the treatment of acute myeloid leukemia

Blood ◽  
1992 ◽  
Vol 79 (9) ◽  
pp. 2229-2236 ◽  
Author(s):  
MJ Robertson ◽  
RJ Soiffer ◽  
AS Freedman ◽  
SL Rabinowe ◽  
KC Anderson ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Red Blood Cell Transfusion ◽  
Hematopoietic Stem ◽  
Myeloid Progenitor ◽  
Myeloid Progenitor Cells ◽  
Acute Myeloid

Abstract The CD33 antigen, identified by murine monoclonal antibody anti-MY9, is expressed by clonogenic leukemic cells from almost all patients with acute myeloid leukemia; it is also expressed by normal myeloid progenitor cells. Twelve consecutive patients with de novo acute myeloid leukemia received myeloablative therapy followed by infusion of autologous marrow previously treated in vitro with anti-MY9 and complement. Anti-MY9 and complement treatment eliminated virtually all committed myeloid progenitors (colony-forming unit granulocyte- macrophage) from the autografts. Nevertheless, in the absence of early relapse of leukemia, all patients showed durable trilineage engraftment. The median interval post bone marrow transplantation (BMT) required to achieve an absolute neutrophil count greater than 500/microL was 43 days (range, 16 to 75), to achieve a platelet count greater than 20,000/microL without transfusion was 92 days (range, 35 to 679), and to achieve red blood cell transfusion independence was 105 days (range, 37 to 670). At the time of BM harvest, 10 patients were in second remission, one patient was in first remission, and one patient was in third remission. Eight patients relapsed 3 to 18 months after BMT. Four patients transplanted in second remission remain disease-free 34+, 37+, 52+, and 57+ months after BMT. There was no treatment-related mortality. Early engraftment was significantly delayed in patients receiving CD33-purged autografts compared with concurrently treated patients receiving CD9/CD10-purged autografts for acute lymphoblastic leukemia or patients receiving CD6-purged allografts from HLA- compatible sibling donors. In contrast, both groups of autograft patients required a significantly longer time to achieve neutrophil counts greater than 500/microL and greater than 1,000/microL than did patients receiving normal allogeneic marrow. CD33(+)-committed myeloid progenitor cells thus appear to play an important role in the early phase of hematopoietic reconstitution after BMT. However, our results also show that human marrow depleted of CD33+ cells can sustain durable engraftment after myeloablative therapy, and provide further evidence that the CD33 antigen is absent from the human pluripotent hematopoietic stem cell.

Myc Induces Acute Myeloid Leukemia by Conferring Self-Renewal Activity to Committed Myeloid Progenitor Cells That Resist Apoptosis Via Endogenous Mcl-1.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2634-2634
Author(s):  
Hui Luo ◽  
Jennifer A. Cain ◽  
AnnaLynn Molitoris ◽  
Joseph Opferman ◽  
Michael H. Tomasson
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Median Survival ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Ectopic Expression ◽  
Hematopoietic Stem ◽  
Myeloid Progenitor ◽  
Myeloid Progenitor Cells ◽  
Induced Apoptosis

Abstract Ectopic expression of Myc in most primary cell types induces apoptosis, and cancer development typically requires additional, anti-apoptotic mutations. We reported previously that ectopic expression of Myc in unfractionated murine bone marrow cells induced rapid onset acute myeloid leukemia (AML) without detectable anti-apoptotic mutations. We hypothesized that AML developed in our model because a subset of normal primary bone marrow cells were inherently resistant to Myc-induced apoptosis. Consistent with this model, seven days of Myc activation in the bone marrow of mice caused the reduction of B-lineage cells while at the same time inducing the expansion of myeloid lineage cells. We sought to determine the mechanism by which myeloid progenitor cells evaded Myc-induced apoptosis, and found that Myc-induced AML cells exhibited a distinct profile of pro- and anti-apoptotic proteins, including high levels of the anti-apoptotic Bcl-2 family member Mcl-1. To prioritize apoptosis genes, we examined AML patient microarray data and found MCL1 to be uniformly expressed at high levels in human AML (94/94, 100%). We used Mcl1 heterozygous mice (Mcl1F/null) as bone marrow donors for transduction-transplantation experiments and found that, compared with Mcl1 wild-type (median survival=60 days), haploinsufficiency for Mcl1 completely protected mice from Myc-induced AML (median survival not reached). Mice transplanted with Mcl1F/null cells co-expressing Myc and Bcl2 succumbed rapidly to disease (median survival 25 days). In wild-type mice, defined hematopoietic stem and myeloid progenitor cell populations were not significantly increased by Myc activation. However, Myc transduction conferred serial replating ability to sorted hematopoietic stem and progenitor cells including lineage-committed (Lin+Kit+) progenitors cells. These data demonstrate a critical role for Mcl1 in our AML model and suggest that dysregulation of MYC in MCL1-expressing progenitor cells may mediate AML pathogenesis in humans.

scholarly journals Sensitization of Hematopoietic Stem and Progenitor Cells to Trimetrexate Using Nucleoside Transport Inhibitors

Blood ◽  
1997 ◽  
Vol 90 (9) ◽  
pp. 3546-3554 ◽  
Author(s):  
James A. Allay ◽  
H. Trent Spencer ◽  
Sarah L. Wilkinson ◽  
Judith A. Belt ◽  
Raymond L. Blakley ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Nucleoside Transport ◽  
Hematopoietic Stem ◽  
Myeloid Progenitor Cells ◽  
Nucleoside Transport Inhibitors ◽  
Transport Inhibitors ◽  
Myeloid Progenitors

Abstract Antifolates such as methotrexate (MTX) and trimetrexate (TMTX) are widely used in the treatment of cancer and nonmalignant disorders. Transient, yet sometimes severe myelosuppression is an important limitation to the use of these drugs. It has previously been shown that clonogenic myeloid progenitors and colony-forming units-spleen are resistant to antifolates, suggesting that myelotoxicity occurs late in hematopoietic development. The goal of this study was to define the mechanisms by which primitive hematopoietic cells resist the toxic effects of antifolate drugs. To test the hypothesis that myeloid progenitors may salvage extracellular nucleotide precursors to resist TMTX toxicity, a defined liquid culture system was developed to measure TMTX toxicity in expanding progenitor populations. These in vitro experiments showed that both human and murine progenitors can resist TMTX toxicity by importing thymidine and hypoxanthine from the serum. As predicted from these findings, several drugs that block thymidine transport sensitized progenitors to TMTX in vitro, although to differing degrees. These nucleoside transport inhibitors were used to test whether progenitors and hematopoietic stem cells (HSCs) could be sensitized to TMTX in vivo. Treatment of mice with TMTX and nitrobenzylmercaptopurineriboside phosphate (NBMPR-P), a potent transport inhibitor, caused significant depletions of clonogenic progenitors within the bone marrow (20-fold) and spleen (6-fold). Furthermore, NBMPR-P administration dramatically sensitized HSCs to TMTX, with dual-treated mice showing a greater than 90% reduction in bone marrow repopulating activity. These studies demonstrate that both myeloid progenitor cells and HSCs resist TMTX by using nucleotide salvage mechanisms and that these pathways can be pharmacologically blocked in vivo using nucleoside transport inhibitors. These results have important implications regarding the use of transport inhibitors for cancer therapy and for using variants of dihydrofolate reductase for in vivo selection of genetically modified HSCs.

AML1/ETO and PML/RAR α Can Immortalize Committed Myeloid Progenitor Cells In-Vitro but Not Expand Them In-Vivo.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2544-2544
Author(s):  
Naoki Hosen ◽  
Emmanuelle Passegue ◽  
Irving L. Weissman
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Myeloid Progenitor ◽  
Myeloid Progenitor Cells ◽  
Myeloid Progenitors

Abstract For most leukemia the target cells of transforming mutations are still unknown. Here, we studied the developmental origin of t(8;21)-acute myeloid leukemia (AML), t(15;17)-acute promyelocytic leukemia (APL), and t(9;22)-chronic myeloid leukemia (CML). Purified mouse hematopoietic stem cells (HSCs) and various committed myeloid progenitor cells were retrovirally transduced with AML1/ETO, PML/RARα, or p210 BCR/ABL and subjected to in-vitro serial replating assay and in-vivo transplantation. Myeloid progenitors were efficiently immortalized in-vitro by AML1/ETO or PML/RARα as assayed in serial replating assays. However, following transplantation into lethally irradiated mice, neither AML1/ETO- nor PML/RARα-transduced myeloid progenitors were expanded in-vivo, although cells carrying the fusion gene DNA persisted well beyond their non-transduced control progenitors. In addition, 10 months after the transplant with transduced myeloid progenitor cells, PML/RARα but not AML1/ETO mRNA expression was still detected in committed myeloid progenitors, although PML/RARα-expressing cells were still not expanded. This finding demonstrates the ability of PML/RARα to increase the lifespan of committed myeloid progenitor cells both in-vitro and in-vivo and suggest that t(15;17)-APL could possibly evolve from persisting progenitor-derived cells. In contrast, BCR/ABL-expressing myeloid progenitors disappeared within 3 months post transplantation. Analysis of mice transplanted with transduced-HSCs demonstrated that AML1/ETO induced the accumulation of the most immature subset of HSCs (Lin-/c-kit+/Sca-1+/Flk-2-), while in contrast PML/RARα induced HSCs disappearance in most (5 out of 6) cases. In addition, we demonstrate that p210 BCR/ABL could induce the development of a CML-like disease from transduced HSCs (6 out of 16 cases), which is compatible with previous report. Together, these results suggest the existence of novel pre-leukemic stem cells (pre-LSCs) entities. Slowly expanding pre-LSCs could be generated from HSCs transformed by the expression of AML1/ETO. Persisting pre-LSCs could be generated from myeloid progenitors transformed by the expression of PML/RARα. These pre-LSC populations could mediate the early phases of t(8;21)-AML and t(15;17)-APL pathogenesis and could represent novel key targets for anti-leukemia therapies.

scholarly journals ANTIGENIC DIFFERENCES BETWEEN HEMOPOIETIC STEM CELLS AND MYELOID PROGENITORS

1974 ◽  
Vol 139 (6) ◽  
pp. 1621-1627 ◽  
Author(s):  
Gerrit J. Van den Engh ◽  
Edward S. Golub
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Myeloid Cell ◽  
Myeloid Progenitor ◽  
Myeloid Progenitor Cells ◽  
Myeloid Progenitors

Bone marrow contains pluripotent stem cells which give rise to colonies when injected into irradiated syngenic hosts as well as more differentiated progenitor cells of the myeloid cell which are able to form colonies in vitro. Antisera against brain is known to contain antistem cell antibody. The present experiments were designed to determine if the myeloid progenitor cell still expresses the stem cell antigen. Bone marrow cells were treated with antibrain antiserum plus complement and then survival of stem cells was determined by injection into irradiated hosts. Survival of myeloid progenitor cells was determined by culturing the cells in vitro. It was found that stem cells were eliminated by the antiserum but that myeloid progenitors were not. Inefficient in vitro lysis was ruled out as the reason for this difference since in vitro colonies were not reduced when the cells were treated with anti-immunoglobulin or after passage through an irradiated host. In the differentiation from stem cell to myeloid progenitor there is an associated surface antigen change.

scholarly journals Human bone marrow depleted of CD33-positive cells mediates delayed but durable reconstitution of hematopoiesis: clinical trial of MY9 monoclonal antibody-purged autografts for the treatment of acute myeloid leukemia

Blood ◽  
1992 ◽  
Vol 79 (9) ◽  
pp. 2229-2236 ◽  
Author(s):  
MJ Robertson ◽  
RJ Soiffer ◽  
AS Freedman ◽  
SL Rabinowe ◽  
KC Anderson ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Red Blood Cell Transfusion ◽  
Hematopoietic Stem ◽  
Myeloid Progenitor ◽  
Myeloid Progenitor Cells ◽  
Acute Myeloid

The CD33 antigen, identified by murine monoclonal antibody anti-MY9, is expressed by clonogenic leukemic cells from almost all patients with acute myeloid leukemia; it is also expressed by normal myeloid progenitor cells. Twelve consecutive patients with de novo acute myeloid leukemia received myeloablative therapy followed by infusion of autologous marrow previously treated in vitro with anti-MY9 and complement. Anti-MY9 and complement treatment eliminated virtually all committed myeloid progenitors (colony-forming unit granulocyte- macrophage) from the autografts. Nevertheless, in the absence of early relapse of leukemia, all patients showed durable trilineage engraftment. The median interval post bone marrow transplantation (BMT) required to achieve an absolute neutrophil count greater than 500/microL was 43 days (range, 16 to 75), to achieve a platelet count greater than 20,000/microL without transfusion was 92 days (range, 35 to 679), and to achieve red blood cell transfusion independence was 105 days (range, 37 to 670). At the time of BM harvest, 10 patients were in second remission, one patient was in first remission, and one patient was in third remission. Eight patients relapsed 3 to 18 months after BMT. Four patients transplanted in second remission remain disease-free 34+, 37+, 52+, and 57+ months after BMT. There was no treatment-related mortality. Early engraftment was significantly delayed in patients receiving CD33-purged autografts compared with concurrently treated patients receiving CD9/CD10-purged autografts for acute lymphoblastic leukemia or patients receiving CD6-purged allografts from HLA- compatible sibling donors. In contrast, both groups of autograft patients required a significantly longer time to achieve neutrophil counts greater than 500/microL and greater than 1,000/microL than did patients receiving normal allogeneic marrow. CD33(+)-committed myeloid progenitor cells thus appear to play an important role in the early phase of hematopoietic reconstitution after BMT. However, our results also show that human marrow depleted of CD33+ cells can sustain durable engraftment after myeloablative therapy, and provide further evidence that the CD33 antigen is absent from the human pluripotent hematopoietic stem cell.

scholarly journals Bone marrow lymphoid and myeloid progenitor cells are suppressed in 7,12-dimethylbenz(a)anthracene (DMBA) treated mice

Toxicology ◽  
2010 ◽  
Vol 271 (1-2) ◽  
pp. 27-35 ◽  
Author(s):  
A.U. N’jai ◽  
M. Larsen ◽  
L. Shi ◽  
C.R. Jefcoate ◽  
C.J. Czuprynski
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Myeloid Progenitor ◽  
Myeloid Progenitor Cells

scholarly journals Ex Vivo-Generated CD36+ Erythroid Progenitors Are Highly Permissive to Human Parvovirus B19 Replication

2007 ◽  
Vol 82 (5) ◽  
pp. 2470-2476 ◽  
Author(s):  
Susan Wong ◽  
Ning Zhi ◽  
Claudia Filippone ◽  
Keyvan Keyvanfar ◽  
Sachiko Kajigaya ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Real Time ◽  
Cell Lines ◽  
Ex Vivo ◽  
Parvovirus B19 ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
Human Virus ◽  
Erythroid Progenitor Cells ◽  
Large Numbers

ABSTRACT The pathogenic parvovirus B19 (B19V) has an extreme tropism for human erythroid progenitor cells. In vitro, only a few erythroid leukemic cell lines (JK-1 and KU812Ep6) or megakaryoblastoid cell lines (UT7/Epo and UT7/Epo-S1) with erythroid characteristics support B19V replication, but these cells are only semipermissive. By using recent advances in generating large numbers of human erythroid progenitor cells (EPCs) ex vivo from hematopoietic stem cells (HSCs), we produced a pure population of CD36+ EPCs expanded and differentiated from CD34+ HSCs and assessed the CD36+ EPCs for their permissiveness to B19V infection. Over more than 3 weeks, cells grown in serum-free medium expanded more than 800,000-fold, and 87 to 96% of the CD36+ EPCs were positive for globoside, the cellular receptor for B19V. Immunofluorescence (IF) staining showed that about 77% of the CD36+ EPCs were positive for B19V infection, while about 9% of UT7/Epo-S1 cells were B19V positive. Viral DNA detected by real-time PCR increased by more than 3 logs in CD36+ EPCs; the increase was 1 log in UT7/Epo-S1 cells. Due to the extensive permissivity of CD36+ EPCs, we significantly improved the sensitivity of detection of infectious B19V by real-time reverse transcription-PCR and IF staining 100- and 1,000-fold, respectively, which is greater than the sensitivity of UT7/Epo-S1 cell-based methods. This is the first description of an ex vivo method to produce large numbers of EPCs that are highly permissive to B19V infection and replication, offering a cellular system that mimics in vivo infection with this pathogenic human virus.

Abstract 20010: Diet-induced Obesity Stimulates Inflammatory Monopoiesis From Hematopoietic Stem Progenitor Cells Through Activating Histone Methylation

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Milie Fang ◽  
Rita Mirza ◽  
Timothy J Koh ◽  
Norifumi Urao
Keyword(s):  
Progenitor Cells ◽  
Fold Increase ◽  
Control Mechanisms ◽  
Hematopoietic Stem ◽  
Fluorescent Intensity ◽  
Inflammatory Monocytes ◽  
Diet Induced Obesity ◽  
Histone 3 ◽  
Myeloid Progenitor ◽  
Myeloid Progenitor Cells

Obesity-induced monopoiesis has been implicated in the development of obesity-related complications. Monopoiesis is largely dependent on hematopoietic stem progenitor cells (HSPCs) in the bone marrow (BM). However, little is known about hematopoietic control mechanisms in diet-induced obesity. In a mouse model of diet-induced obesity (DIO), we found leukocytosis (9.465 ± 0.7350 K/ul versus 7.277 ± 0.3450 K/ul in healthy controls, n=7 each? , p=0.023) and increased inflammatory Ly6Chi monocytes in circulation (377.2 ± 40.94/ul vs. 224.8 ± 39.18/ul in lean control; n=7; p=0.023), associated with increased myeloid progenitor cells (60% increase in granulocyte-monocyte progenitor cells, n=4) in the BM. By flow cytometry based profiling, we found that active marks for transcription, histone 3 lysine 4 trimethylation (H3K4me3), are upregulated (1.82-fold increase in mean fluorescent intensity (MFI) vs. lean mice, n=5, p<0.05) in Ly6Chi inflammatory monocytes in DIO mice, along with increased inflammatory gene expression in response to TLR (toll-like receptor) 4 stimulation. In the BM, HSPCs but not myeloid progenitor cells have enriched H3K4me3 in DIO mice (2.84-fold in MFI in cKit+Sca1+Lin- cells, n=5, p<0.05). This activated mark in HSPCs is associated with increased monopoiesis from HSPCs (1.55-fold increase in output CD11b+Ly6Chi monocytes from cultured HSPCs, n=3-5, p<0.01) in response to TLR2 and TLR4 stimulations. Of note, HSPCs produce inflammatory cytokines to promote differentiation into inflammatory monocytes. Moreover, HIF-1a, a potential upstream of H3K4me3, is upregulated (2.29-fold in MFI, n=5, p<0.05) in HSPCs in DIO mice. These results suggest that obesity increases HIF-1a-mediated H3K4me3 enrichment in HSPCs, which in turn contributes to inflammatory cytokine expression and to increased inflammatory monopoiesis. Thus, epigenetic marks in HSPCs could be a target for obesity-related complications.

Diversity and Molecular Modeling of Neutrophil Elastase Mutations in Patients with Severe Congenital Neutropenia and Acute Myelogenous Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1454-1454
Author(s):  
Andrew A.G. Aprikyan ◽  
Steve Stein ◽  
Nara A. Markosyan ◽  
Maxim Totrov ◽  
Ruben Abagyan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Neutrophil Elastase ◽  
Apoptotic Cell ◽  
Myelogenous Leukemia ◽  
Congenital Neutropenia ◽  
Deletion Mutations ◽  
Myeloid Progenitor ◽  
Myeloid Progenitor Cells ◽  
Acute Myelogenous

Abstract Severe congenital neutropenia (SCN) is an inheritable hematopoietic disorder that is characterized by extremely low levels of neutrophils in peripheral circulation and maturation arrest of bone marrow myeloid progenitor cells at the promyelocytic stage of differentiation. SCN patients have recurring severe infections and approximately 10% of these patients evolve to develop acute myelogenous leukemia. Recently we reported that an impaired cell survival and cell cycle arrest of bone marrow myeloid progenitor cells was observed in SCN patients compared with controls. We also reported various heterozygous mutations in the neutrophil elastase (NE) gene encoding a serine protease in approximately 80% of SCN patients. We hypothesized that mutations in the NE gene trigger apoptotic cell death of myeloid progenitor cells and subsequent severe neutropenia. Mutational analysis of 15 families with one or more affected family members revealed that mutant NE was present only in affected but not in healthy members of these families suggesting the causative role for mutant NE in pathogenesis of SCN. Sequencing analysis revealed that none of SCN patients negative for NE mutations examined had mutations in the Gfi-1 or WAS gene. Sequencing DNA samples of SCN and SCN/AML patients revealed 40 mutations that are distributed primarily throughout the exons 2 through 5 of the NE gene and result in substitution, deletion, insertion, or truncation mutations. Molecular modeling of the tertiary structure of NE revealed that all these mutations can be grouped into three major categories. The first category includes 19 substitution and insertion mutations that are grouped around the N-glycosylation sites of the neutrophil elastase and may lead to abnormal targeting and subcellular localization of the mutant protease. The second group includes 9 substitution and deletion mutations that alter the side loop of the NE that is necessary for proper oligomerization of neutrophil elastase. The third category includes 12 substitution, truncation, and deletion mutations that either alter or completely eliminate the carboxy-terminus of the mutant protein leading to conformational changes of the binding pocket of the NE, and subsequently to altered substrate specificity and/or an acquired resistance to elastase inhibitors. SCN patients that evolved to develop AML had either substitution, deletion, or truncation mutations from each of the three categories described above. Most mutations are clearly non-conservative, have destabilizing effect on oligomeric structure of mutant protein, and alter dramatically the affinity of mutant NE to various factors participating in its processing and intracellular transport. Flow cytometry analysis of annexin V-labeled cells revealed that expression of representative mutant but not normal NE from each of the three categories of NE mutations in human promyelocytic HL-60 cells triggered apoptotic cell death similar to that observed in bone marrow progenitor cells in SCN patients. These data indicate that impaired cell survival and block of differentiation in SCN is due to heterozygous mutations in the neutrophil elastase gene. Current studies focused on design and screen of specific protease inhibitors capable of blocking the pro-apoptotic effect of mutant neutrophil elastase.

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