Efficient c-kit receptor-targeted gene transfer to primary human CD34-selected hematopoietic stem cells.

We have previously reported effective gene transfer with a targeted molecular conjugate adenovirus vector through the c-kit receptor in hematopoietic progenitor cell lines. However, a c-kit-targeted recombinant retroviral vector failed to transduce cells, indicating the existence of significant differences for c-kit target gene transfer between these two viruses. Here we demonstrate that conjugation of an adenovirus to a c-kit-retargeted retrovirus vector enables retroviral transduction. This finding suggests the requirement of endosomalysis for successful c-kit-targeted gene transfer. Furthermore, we show efficient gene transfer to, and high transgene expression (66%) in, CD34-selected, c-kit(+) human peripheral blood stem cells using a c-kit-targeted adenovirus vector. These findings may have important implications for future vector development in c-kit-targeted stem cell gene transfer.

ABL1 promoter methylation can exist independently of BCR-ABL transcription in chronic myeloid leukemia hematopoietic progenitors.

Formation of the hybrid BCR-ABL gene is responsible for >95% of chronic myeloid leukemia (CML). The alternative, downstream ABL promoter (Pa), which is usually retained in this chimeric oncogene, was reported to be methylated in many CML patients, but there has been controversy as to whether this methylation is a frequent change in bone marrow (BM) in early chronic phase (CP) or only past this stage. Also, the relevance of Pa promoter methylation to BCR-ABL expression in CML is unclear. We examined methylation of the ABL Pa promoter in uncultured BM samples and in colonies derived from their hematopoietic precursor cells by bisulfite and PCR-based assays (combined bisulfite restriction analysis and methylation-specific PCR). BM from seven CP CML patients at diagnosis had about 20-60% of the copies of the ABL Pa promoter methylated. No Pa methylation was detected in normal BMs or colonies derived from them. In contrast, most colonies from CP CML patients had Pa methylation. Surprisingly, 18-49% of the CML-derived colonies with this methylation reproducibly had no detectable BCR-ABL RNA on nested reverse transcription-PCR. Furthermore, the percentage of BCR-ABL RNA-positive colonies was almost same among the colonies not displaying Pa methylation as among the colonies in which this methylation was found. We conclude that ABL Pa methylation is often an early marker of CML in hematopoietic precursors and in total mononuclear BM cells but that it is not associated with an increased frequency of BCR-ABL RNA-positive cells. This methylation might be emblematic of cancer-associated hypermethylation elsewhere in the genome with the consequent silencing of tumor suppressor genes seen in many malignancies.

Il-17 mobilizes peripheral blood stem cells with short- and long-term repopulating ability in mice.

Autologous and allogeneic bone marrow transplantations have evolved as important cancer therapy modalities. For both indications, peripheral blood has been shown to have distinct advantages over bone marrow as the stem cell source. Cytokine combinations for mobilization have enhanced stem cell yield and accelerated engraftment. However, novel mobilizing agents and strategies are needed to further improve clinical outcomes. Within the donor graft, the dynamic equilibrium between T cells and stem cells critically influences engraftment and transplantation results. IL-17 is a cytokine produced almost exclusively from activated T cells. IL-17 was expressed in vivo with adenovirus technology. Here, proof-of-principle studies demonstrate that IL-17 effectively mobilizes hemopoietic precursor cells (CFU-granulocyte-erythrocyte-macrophage-monocyte, CFU-high proliferative potential) and primitive hemopoietic stem cells (Lin(-/low)c-kit(+)Sca1(+)). Moreover, mouse IL-17 adenovirus-mobilized peripheral blood stem cells rescued lethally irradiated mice. Bone marrow was found to be 45-75% of donor origin at 1 year. In secondary recipients, donor-derived bone marrow cells ranged from 45 to 95%. These data show that IL-17 mobilizes stem cells in mice with short- and long-term reconstituting capacity. Additional comparative studies are needed as well as studies in tumor models to refine distinct potential clinical applications for IL-17-mobilized peripheral blood stem cells.

IL-17 stimulates granulopoiesis in mice: use of an alternate, novel gene therapy-derived method for in vivo evaluation of cytokines.

IL-17 is a novel cytokine secreted principally by CD4+ T cells. It has been shown to support the growth of hemopoietic progenitors in vitro; however, its in vivo effects are presently unknown. Adenovirus-mediated gene transfer of the murine IL-17 cDNA targeted to the liver (5 x 10(9) plaque-forming units (PFU) intravenous) resulted in a transiently transgenic phenotype, with dramatic effects on in vivo granulopoiesis. Initially, there was a significant increase (fivefold) in the peripheral white blood count (WBC), including a 10-fold rise in the absolute neutrophil count. This was associated with a doubling in the spleen size over 7-14 days after gene transfer, which returned to near baseline by day 21, although the white blood cell count remained elevated. There was a profound stimulation of splenic hemopoiesis as demonstrated by an increase in total cellularity by 50% 7 days after gene transfer and an increase in hemopoietic colony formation. A maximal increase in frequency of high proliferative potential colonies (HPPC) (11-fold) and CFU-granulocyte-macrophage (GM) and CFU-granulocyte-erythrocyte-megakaryocyte-monocyte (GEMM) (CFU) (6-fold) was seen on day 3 after IL-17 gene transfer. Both CFU and HPPC remained significantly elevated in the spleen throughout day 21, but at reduced levels compared with day 3. Bone marrow CFU and HPPC were elevated on day 3 only by 75% and 25%, respectively, without changes in total cellularity. Thus, murine IL-17 is a cytokine that can stimulate granulopoiesis in vivo. Since IL-17 is principally produced by CD4+ T cells, this cytokine could have therapeutic implications in AIDS-related bone marrow failure and opportunistic infections.

Durable remission of locally advanced breast cancer with multimodality management.

We treated 20 women with locally advanced breast cancer between January 1991 and September 1996. The treatment regimen included 4 cycles of intensive doxorubicin (30 mg/m2/d on 3 consecutive days every 2 weeks with G-CSF support), followed by appropriate surgery, followed by high dose therapy with cyclophosphamide, carboplatin and thiotepa (STAMP V, CTCb). Of the 20 patients, seven presented with inflammatory breast cancer, three with Stage IIIB, seven with stage IIIA, one with multifocal Stage IIB and two with Stage IV M1 (ipsilateral supraclavicular lymph node involvement) (including one who had an inflammatory primary) disease. Six patients had not undergone mastectomy at the time of entering the protocol. These six received the doxorubicin in a neoadjuvant fashion and were thus evaluable for tumor response. The remaining 14 received doxorubicin as adjuvant therapy prior to intensification and transplantation. All patients underwent local-regional radiation therapy and were placed on oral tamoxifen. Doxorubicin was well tolerated in this schedule with all but three patients receiving all their cycles on schedule. Both BM and PBPC were easily collected after this regimen and, when reinfused, resulted in the prompt recovery of granulocytes (median 11 days to 500 absolute granulocyte count) and platelets (median 13 days to 20,000 platelets). The six patients who received doxorubicin prior to mastectomy all had major clinical responses, but were found to have microscopic focii of breast cancer in the mastectomy specimens. The overall treatment was well tolerated with the exception of one treatment-related death (5%). The overall and relapse free survival are 70% and 58% respectively with a median follow-up of 40 months (range 12-74 months). When the Stage IV patients are censored, the relapse-free survival rate is 69%. In the bone marrow transplant phase of treatment, the major non-hematologic toxicities were stomatitis (70%) and anorexia requiring parental nutrition (75%).

Providing a Microenvironment for the Development of Human CD34+ Hematopoietic Cells in SCID Mice.

In order to develop a convenient small-animal model that can support the differentiation of human bone-marrow-derived CD34+ cells, we transplanted SCID mice with an immortalized human stromal cell line, Lof(11-10). The Lof(11-10) cell line has been characterized to produce human cytokines capable of supporting primitive human hematopoietic cell proliferation in vitro. Intraperitoneal injection of Lof(11-10) cells into irradiated SCID mice by itself resulted in a dose-dependent survival of the mice from lethal irradiation. The radioprotective survival was reflected by an increase in the growth and number of mouse bone-marrow-derived committed hematopoietic progenitors. The Lof(11-10) cells localized to the spleen, but not to the bone marrow of these animals and resulted in detectable levels of circulating human IL-6 in their plasma. Secondary intravenous injections of either human or simian CD34+ cells into the Lof(11-10)-transplanted SCID mice resulted in engraftment of injected cells within the bone marrow of these mice. The utility of this small-animal model that allows the growth and differentiation of human CD34+ cells and its potential use in clinical gene therapy protocols are discussed. Copyright 1997 S. Karger AG, Basel

Efficiency of human HLA-mismatched CD34+ cells from unrelated donors in establishing in vitro hematopoiesis in allogeneic long-term marrow cultures.

We have examined the capacity of highly purified human CD34+ marrow cell isolates from unrelated, HLA-mismatched donors to establish in vitro hematopoiesis on recipient marrow stromal cells in 2-stage hematopoietic long-term marrow cultures (H-LTMC). HLA-typing of both peripheral blood mononuclear cells and CD34+ marrow cells was performed for both HLA class I and HLA class II antigens for eight healthy individuals. Significant antigenic mismatches for these molecules ranged from three to six antigens for each recipient-donor pair. Comparison of MHC antigen expression by peripheral blood cells and CD34+ marrow cell isolates confirmed the presence of identical HLA-A, -B, and -C, and -DR specificities on the surface of these cells. Typing of -DQ specificities, however, was not consistently reactive on CD34+ cells. The > or = 20% plating efficiency of purified CD34+ cells for BFU-E, CFU-GM, and CFU-MIX allowed us to use inoculum doses of 10(3), 10(4), and 10(5) cells to determine the efficiency of allogeneic CD34+ cells in achieving in vitro engraftment and the establishment of hematopoiesis in H-LTMC. Engraftment of adherent BFU-E, CFU-GM, and CFU-MIX was equally efficient for autologous and allogeneic CD34+ cells. In vitro hematopoiesis reflected by the cumulative recoveries of progenitor cells over time was also equivalent for allogeneic and autologous CD34+ cells. These results demonstrate that highly purified, HLA-mismatched CD34+ marrow cells proliferate and establish in vitro hematopoiesis as efficiently as autologous cells in marrow derived stromal cell cultures and confirm that interactions between stromal cells and highly purified CD34+, DR-, and CD34+, DR+ marrow cell isolates are not MHC-restricted.

Exposure of human CD34+ cells to human immunodeficiency virus type 1 does not influence their expansion and proliferation of hematopoietic progenitors in vitro.

The susceptibility of highly purified human CD34+ cells to monocytotropic (Ba-L) and lymphotropic (A018-post) strains of human immunodeficiency virus-1 (HIV-1) was examined. Liquid cultures initiated with fresh immunomagnetically purified CD34+ cells using the K6.1 CD34 monoclonal antibody (MoAb) (K6.1/CD34+) were positive for HIV expression 2 weeks after exposure to HIV-1 Ba-L. These cells were initially greater than 90% CD34+ and had undetectable monocyte contamination by flow-cytometric staining and side-scatter analyses, respectively, and undetectable T-cell contamination by CD3 polymerase chain reaction (PCR) analysis. However, secondary CD34+ liquid cultures reselected from the primary liquid cultures 24 hours after HIV exposure by panning with the ICH3 CD34 MoAb (ICH3/CD34+) and maintained for an additional 14 days were negative for HIV expression. The ICH3-unbound cells were positive for both spliced and unspliced HIV RNA when exposed to HIV-1 Ba-L, and were DNA PCR positive when exposed to either monocytotropic or lymphotropic HIV-1. To further test that CD34+ cells were not infectible by HIV-1, we exposed K6.1/CD34+ cells continuously to HIV-1 in a culture system capable of maintaining and expanding primitive CD34+ cells. HIV-exposed K6.1/CD34+ cells proliferated and expanded as efficiently as uninfected cultures. However, when reselected magnetically using the K6.1 CD34 MoAb after expansion for 7 days, bound K6.1/CD34+ cells were again negative for HIV-1 expression, whereas unbound cells were positive for HIV-1 expression. These findings suggest that a sequential CD34+ cell-selection process, in which the two selections are separated by a brief culture period, can yield a population of CD34+ cells that are not infected with HIV-1. This process may be useful in the design of stem or progenitor cell-based transplantation therapies for HIV infection.

Dengue-2 virus infection of human bone marrow: characterization of dengue-2 antigen-positive stromal cells.

Dengue is often associated with neutropenia and thrombocytopenia, suggesting that cells of the bone marrow may be targets of dengue viral infections. In this study we infected long-term marrow cultures with dengue type-2 (DEN-2) virus and characterized the viral antigen-positive cells. Using immunofluorescence microscopy and immunohistochemical staining, we demonstrated two types of stromal cells that were positive for DEN-2 virus antigens. The first was a population of relatively small (approximately 25 microns) CD11b/CD18 (MAC-1)-positive cells. When stained with anti-DEN-2 polyclonal antibody, these cells showed viral antigen-positive inclusions and, when stained with anti-tubulin or anti-vimentin antibodies, they showed a diffuse pattern of fluorescence, consistent with mobile dendritic cells with phagocytic functions. The second population of DEN-2 antigen-positive cells comprised a smaller proportion of the total cells. It was made up of larger cells (> 100 microns) that had a well-formed cytoskeletal system as demonstrated by intense staining with anti-actin, anti-tubulin, and anti-vimentin antibodies. When stained with anti-DEN-2 antibody, these cells showed a more diffuse pattern of fluorescence in the perinuclear and Golgi regions, consistent with ongoing virus replication. These large, strongly adherent cells were positive for nerve growth factor receptor, consistent with their identification as adventitial reticular cells. The molecule that mediates the virus interaction with susceptible cells has not previously been identified. Using plasma membrane proteins isolated from K562 cells, virus-binding studies suggest that an approximately 100-kD membrane protein may be involved in the initial virus-cell interaction.

Characterization of a Human Stromal Cell Line Supporting Hematopoietic Progenitor Cell Proliferation: Effect of HIV Expression.

Our objective was to determine the role that bone marrow-derived stromal cells have on human hematopoiesis in HIV infection. In particular, we dissected the heterogeneous bone marrow microenvironment to study the effect HIV expression might have on the cell population capable of producing the cytokines which will support human CD34+ cell differentiation. A stromal cell line, Lof(11-10), was established from human bone marrow by transfecting a plasmid containing the SV40 large T-antigen and isolating foci exhibiting a transformed phenotype. The Lof(11-10) cell line was characterized to determine its susceptibility to HIV infection, to identify its cytokine production profile, and to test the ability of conditioned media from this line to support CD34+ cell differentiation in the presence and absence of HIV expression. Nine cytokines were detected by RT-PCR and ELISA analysis. Conditioned media obtained from the Lof(11-10) cell line was able to support CD34+ celle differentiation. However, because the Lof(11-10) cells are not infectible by HIV, molecular clones of HIV were introduced into these cells by transfection. There was no qualitative difference in the levels of cytokine production between HIV-expressing and control Lof(11-10) cells. Furthermore, conditioned media derived from HIV-expressing and control Lof(11-10) cells added to bone marrow-derived CD34+ progenitor cells yielded similar colony formation in methylcellulose assays. Our data suggest that HIV infection of the cytokine-producing cells within the bone marrow microenvironment, as represented by the Lof(11-10) cell line, results in both normal cytokine production and hematopoiesis in spite of HIV expression. This report adds to the evidence against stromal cells being a significant target of HIV and establishes a system for comparison with more relevant models. Copyright 1995 S. Karger AG, Basel

Mechanisms of dengue virus-induced bone marrow suppression.

Infection with many flaviviruses is associated with transient suppression of haematopoiesis. Of the flaviviruses of man, none are more accessible to clinical and laboratory study than dengue. Consequently, the clinical syndrome of dengue-associated bone marrow suppression has been well documented. A review of experimental dengue infections of volunteers and histopathological studies of bone marrow from patients with severe dengue virus infection suggests that marrow suppression evolves rapidly through several phases: (1) onset of marrow suppression within 3-4 days of infection; (2) onset of host inflammatory responses in the marrow and of fever shortly thereafter; (3) occurrence of a neutrophil nadir on the fourth to fifth day after onset of fever; (4) almost simultaneously, immune activation sufficient to neutralize viraemia and accelerate elimination of infected cells; (5) remission of symptoms; and (6) resolution of cytopenias. Clinical observations and experimental data bear on possible mechanisms of dengue virus-mediated marrow suppression. Work from the authors' laboratory in which long-term bone marrow cultures were used to investigate interactions between dengue virus and bone marrow cells (stromal elements and haematopoietic progenitors) is also reviewed. Long-term marrow culture (LTMC) was a useful experimental system. In vitro, early blast cells as well as the more differentiated haematopoietic elements were abortively infected, killed and eliminated by phagocytosis by specialized marrow macrophages called dendritic cells. Moreover, the ARC from stroma rather than haematopoietic precursors were productively infected. When ARC were infected, stroma failed to support haematopoiesis. Cytokine production by virus-infected stromal cells was altered. A hypothesis is proposed to account for dengue virus-induced marrow suppression. Down-regulation of haematopoiesis is probably a protective mechanism of the microenvironment that limits injury to the marrow stem/progenitor cell compartment during the subsequent process of elimination of infected cells.

Effects of anti-CD33 blocked ricin immunotoxin on the capacity of CD34+ human marrow cells to establish in vitro hematopoiesis in long-term marrow cultures.

Human marrow cells that express the CD34 antigen but lack CD33 are able to initiate sustained, multilineage in vitro hematopoiesis in long-term Dexter cultures and are believed to include the primitive stem cells responsible for effecting long-term hematopoietic reconstitution in vivo following marrow transplantation. In studies described in this report we investigated the effects of a novel anti-CD33 immunotoxin on the clonogenic potential of normal human CD34+ marrow cells and on the ability of these cells to initiate hematopoiesis in two-stage Dexter cultures (long-term marrow cultures, LTMC). This immunotoxin (anti-CD33-bR), shown previously to kill both clonogenic myelogenous leukemia cells and normal mature myeloid progenitor cells (granulocyte-macrophage colony-forming units, CFU-GM), consists of an anti-CD33 monoclonal antibody conjugated to purified ricin that has been modified by blocking the carbohydrate binding domains of the ricin B-chain to eliminate nonspecific binding. For our studies, normal CD34+ human marrow cells were isolated from the light-density (less than 1.070 g/ml) cells of aspirated marrow by positive selection with immunomagnetic beads linked to the monoclonal antibody K6.1. These cell isolates were highly enriched with both multipotential and lineage-restricted clonogenic, hematopoietic progenitors (mixed lineage colony-forming units, CFU-Mix; CFU-GM; and erythroid burst-forming units, BFU-E) which constituted greater than or equal to 20% of the cells. Recovery of clonogenic progenitors from these CD34+ cell preparations, following treatment with anti-CD33-bR (10 nM), was reduced by greater than or equal to 85% for CFU-GM and 20%-40% for CFU-Mix and BFU-E. However, the capacity of these cells to initiate hematopoietic LTMC was preserved. Indeed, the production of high proliferative potential (HPP) CFU-GM, BFU-E, and CFU-Mix in cultures seeded with 10(5) anti-CD33-bR-treated CD34+ marrow cells was substantially greater than that observed in LTMC seeded with equivalent numbers of untreated CD34+ cells. Moreover, concentrations of long-term culture initiating cells in CD34+ cell isolates, quantified by a limiting dilution technique, were found to be increased following anti-CD33-bR treatment. These findings support the potential usefulness of anti-CD33-bR for in vitro marrow purging or in vivo treatment to eliminate CD33+ leukemic clones, while sparing normal CD34+/CD33- stem cells that support normal hematopoiesis and hematopoietic reconstitution in vivo.

Agranulocytosis associated with "Mexican aspirin" (dipyrone): evidence for an autoimmune mechanism affecting multipotential hematopoietic progenitors.

A case of acute, transient agranulocytosis and thrombocytopenia associated with ingestion of dipyrone is reported. This once widely used analgesic, which is now banned in the United States, was obtained by the patient as "aspirin" while traveling in Mexico. Studies of the effects of this patient's serum on purified CD34+ marrow cells, which were highly enriched for hematopoietic progenitors, showed not only a drug-dependent suppression of the in vitro growth of myeloid progenitors, as has been reported previously, but also a drug-dependent suppression of primitive multipotential progenitors (CFU-Mix) and erythroid progenitors (BFU-E). These findings indicate that autoimmune, antibody-hapten interactions which have been reported to occur in dipyrone- and aminopyrine-induced agranulocytosis are not restricted to the neutrophil lineage.

Effects of neuraminidase on the regulation of erythropoiesis: III: Characterization of carbohydrate moieties on the surface of thymic regulatory cells that interact with erythroid colony-forming cells.

In this study we further define cell surface carbohydrate structures relevant to cellular interactions that regulate erythropoiesis. An analysis of thymocyte cell surface negativity was made using fluoresceinated poly-L-ornithine (FITC poly-L-ornithine) as a probe that binds to negatively charged sites (i.e., sialic acid residues) at the cell surface. Two distinct subpopulations are labeled, comprising both intensely as well as weakly fluorescent subpopulations of thymocytes. Prior treatment of thymocytes with Vibrio cholerae neuraminidase (VCN), which removes cell surface sialic acid residues, markedly reduced the FITC poly-L-ornithine surface labeling of these cells. Distinct enzymatic modifications of regulatory cell functions were also assessed by the ability of thymocytes to function as separate regulatory subpopulations. Confirming our previous observations, treating thymocytes with VCN impaired the enhancement activity but had little effect on thymocyte regulatory ability to suppress erythroid colony growth. In contrast, treatment of thymocytes with galactose oxidase (GAO) or beta-galactosidase (beta-GAL) removed suppressor activity either before or after VCN treatment. A further exposure of GAO-treated thymocytes to sodium borohydride or hydroxylamine, which reduce D-galactose residues, restores their suppressor function and prevents enhancement. These differential enzymatic effects on thymocyte regulatory cell functions suggest that different carbohydrate structures may be involved in helper and suppressor activities for erythroid colony formation. Sialic acid residues may be associated with certain cells that function to enhance erythropoiesis, and D-galactose residues may be associated with the suppressor subpopulation.

Effects of in vivo neuraminidase on the regulation of erythropoiesis. II: Modulation of erythroid colony formation by thymic regulatory cells.

Effects of the enzyme vibrio-cholerae neuraminidase (VCN) on the marrow-derived erythropoietic progenitor CFU-E and thymic regulatory cells were examined in vitro 1 and 24 h after i.v. injection of the enzyme. An in vivo enzymatic modification of bone marrow and thymic helper regulatory cell function occurs within 1 h after i.v. injection of VCN and results in suppression of both CFU-E colony formation and thymic helper cell function. These inhibitory effects of neuraminidase, however, are no longer detectable by 24 h after injection. More importantly, these inhibitory effects can be reversed by adding thymocytes from control animals to cocultures of enzymatically modified marrow or thymic regulatory cells. These findings: 1) suggest that regulatory cells from the bone marrow and thymus may be enzymatically modified in vivo in a reversible manner, suggesting a noncytotoxic effect of the enzyme on accessory cells, and 2) confirm the importance of sialic acid for the helper function but not for the suppressor function of thymocytes and CFU-E colony formation in vitro.

Antigenic analysis of hematopoiesis. V. Characterization of My-10 antigen expression by normal lymphohematopoietic progenitor cells.

The My-10 glycoprotein is an hematopoietic cell surface antigen expressed specifically by undifferentiated (blast) cells, constituting 1%-4% of normal adult bone marrow leukocytes. We used several immunological and in vitro culture methods to analyze the expression of this unique antigen on a variety of lymphohematopoietic progenitor cells. Colony-forming cells (CFC) for granulocyte-monocyte colonies (CFC-GM) and erythroid colonies (BFU-E) were predominantly My-10 positive. CFC with higher proliferative potential were more strongly My-10 positive than CFC with lower proliferative potential, and those for mixed-lineage and blast cell colonies were even more uniformly My-10 positive. Cells maintaining CFC-GM number in short-term marrow culture (pre-CFC) were found to be My-10 positive, as were lymphoid precursors defined by their content of intranuclear terminal deoxynucleotidyl transferase. More mature erythroid precursors (CFU-E) were heterogeneous for antigen expression and lost My-10 antigen progressively, in parallel with advancing maturational stage. The My-10 antigen permits rapid identification and purification of hematopoietic progenitor cells for further study or potential clinical application. The disappearance of the My-10 antigen, moreover, may be a probe for differentiation-linked cellular events.