Neurodevelopmental and neuropsychiatric behaviour defects arise from 14-3-3ζ deficiency.

Complex neuropsychiatric disorders are believed to arise from multiple synergistic deficiencies within connected biological networks controlling neuronal migration, axonal pathfinding and synapse formation. Here, we show that deletion of 14-3-3ζ causes neurodevelopmental anomalies similar to those seen in neuropsychiatric disorders such as schizophrenia, autism spectrum disorder and bipolar disorder. 14-3-3ζ-deficient mice displayed striking behavioural and cognitive deficiencies including a reduced capacity to learn and remember, hyperactivity and disrupted sensorimotor gating. These deficits are accompanied by subtle developmental abnormalities of the hippocampus that are underpinned by aberrant neuronal migration. Significantly, 14-3-3ζ-deficient mice exhibited abnormal mossy fibre navigation and glutamatergic synapse formation. The molecular basis of these defects involves the schizophrenia risk factor, DISC1, which interacts isoform specifically with 14-3-3ζ. Our data provide the first evidence of a direct role for 14-3-3ζ deficiency in the aetiology of neurodevelopmental disorders and identifies 14-3-3ζ as a central risk factor in the schizophrenia protein interaction network.

Murine marrow cells expanded in culture with IL-3, IL-6, IL-11, and SCF acquire an engraftment defect in normal hosts.

Stimulatory cytokines may induce murine hematopoietic progenitor cells (HPCs) to survive, self-renew, proliferate, or differentiate. We studied the role of active cell cycling induced by the cytokines interleukin-3 (IL-3), IL-6, IL-11, and Steel factor (SF) on murine progenitor cell frequency and cell cycle status in an in vitro system and on engraftment potential in nonmyeloablated mice. Marrow exposure to IL-3, IL-6, IL-11, and SF in in vitro liquid culture maintained or expanded seven factor-responsive high and low proliferative potential colony-forming cells (HPP-CFC and LPP-CFC). The HPP-CFC and LPP-CFC were dormant at the initiation of culture, as determined by 3H-thymidine suicide. There was an increase in the number and proliferation of HPP-CFC and LPP-CFC at 48 hours; by 48 hours, 62% of HPP-CFC and 56% of LPP-CFC were killed by 3H-TdR exposure. In engraftment studies of cytokine-stimulated marrow cells into normal hosts, female BALB/c mice received the equivalent of 40 x 10(6) starting male marrow cells exposed to cytokines in vitro for 48 hours for 3 consecutive days and were sacrificed 8 weeks after transplantation. Control groups received either 40 x 10(6) male uncultured marrow cells, 40 x 10(6) starting marrow cells cultured in medium without growth factors for 48 hours, or phosphate-buffered saline (PBS) for 3 days. Engraftment of male cytokine-treated cells was analyzed by Southern blot analysis using the Y-chromosome-specific pY2-cDNA probe. There was minimal engraftment (approaching background levels) in marrow, spleen, and thymus of nonmyeloablated female recipients. Transplant recipients that had received uncultured marrow directly after sacrifice showed engraftment levels of 21% (11 mice; range = 8 to 44%) into marrow, of 9% (range = 0 to 22%) into spleen, and 13% (range = 2 to 43%) into thymus. We conclude that active cell cycling of marrow cells induced by cytokine stimulation is associated with an engraftment defect in the normal host.

Monoclonal antibody BB9 raised against bone marrow stromal cells identifies a cell-surface glycoprotein expressed by primitive human hemopoietic progenitors.

OBJECTIVE: The identification of cell-surface antigens whose expression is limited to primitive hematopoietic progenitor cells (HPC) is of major value in the identification, isolation, and characterization of candidate stem cells in human hemopoietic tissues. Based on the observation that bone marrow stromal cells and primitive HPC share several cell-surface antigens, we sought to generate monoclonal antibodies to HPC by immunization with cultured human stromal cells. METHODS: BALB/c mouse were immunized with human bone marrow (BM)-derived stromal cells. Splenocytes isolated from immunized mice were fused with the NS-1 murine myeloma cell line and resulting hybridomas selected in HAT medium, then screened for reactivity against stromal cells, peripheral blood (PB), and BM cells. RESULTS: A monoclonal antibody (MAb), BB9, was identified based on its binding to stromal cells, a minor subpopulation of mononuclear cells in adult human BM, and corresponding lack of reactivity with leukocytes in PB. BB9 bound to a minor subpopulation of BM CD34(+) cells characterized by high-level CD34 antigen and Thy-1 expression, low-absent expression of CD38, low retention of Rhodamine 123, and quiescent cycle status as evidenced by lack of labeling with Ki67. CD34(+)BB9(+) cells, in contrast to CD34(+)BB9(-) cells, demonstrated a capacity to sustain hematopoiesis in pre-CFU culture stimulated by the combination of IL-3, IL-6, G-CSF, and SCF. BB9 also demonstrated binding to CD34(+) cells from mobilized PB. CONCLUSION: Collectively, these data therefore demonstrate that MAb BB9 identifies an antigen, which is selectively expressed by hierarchically primitive human HPC and also by stromal cells.

Stem cell transplantation in the normal nonmyeloablated host: relationship between cell dose, schedule, and engraftment.

In previous studies we have shown high rates of stable engraftment when 40 million male BALB/c cells were infused intravenously daily for 5 days (a total of 200 million cells) to normal nonmyeloablated female hosts. The present studies evaluate engraftment of male BALB/c bone marrow cells in female host marrow, spleen, and thymus 20-25 weeks after transplantation using varying cell dosages within a 5-day schedule. Engraftment in recipient mice was assessed by detection of male specific sequence in recipient DNA from each organ. When 40 million cells were given per daily injection for 1, 2, 3, 4, or 5 days, engraftment percentages in host marrow were 11 +/- 0.83, 20 +/- 2.0, 23 +/- 2.5, 32 +/- 6.3, and 39% +/- 5.7 (+/- standard error of mean), respectively, yielding engraftment percentages per million cells infused of 0.28, 0.25, 0.19, 0.20, and 0.20%, respectively. When levels of 2.5, 5, 10, 20, or 40 million cells were injected 5 times over a 5-day schedule into normal BALB/c female hosts, progressively increasing levels of engraftment from 3 +/- 0.6 to 39% +/- 5.7 were seen in host marrow. Highest levels of engraftment per million cells injected were obtained on days 1 and 2 of a 5-day schedule and with a level of 10 million cells given daily over 5 days. Engraftment profiles varied with spleen and thymus and percent engraftment was generally lower than for marrow. The present work indicates that regardless of cell level infused or number of infusions, rates of engraftment observed in marrow approached or exceeded the highest rates of engraftment estimated by theoretical calculations based on replacing host cells ("replacement model") or adding to host cells ("incremental model"). Engraftment in spleen and thymus was lower, but also at times approached or exceeded theoretical maxima. These data show extraordinary levels of engraftment in normal hosts, suggesting that rates in this competitive model are superior to those seen in irradiated hosts; alternatively, there may be selective repression of host stem cell proliferation and differentiation.

Targeting of acute myeloid leukemia in vitro and in vivo with an anti-CD123 mAb engineered for optimal ADCC.

Acute myeloid leukemia (AML) is a biologically heterogeneous group of related diseases in urgent need of better therapeutic options. Despite this heterogeneity, overexpression of the interleukin (IL)-3 receptor α-chain (IL-3 Rα/CD123) on both the blast and leukemic stem cell (LSC) populations is a common occurrence, a finding that has generated wide interest in devising new therapeutic approaches that target CD123 in AML patients. We report here the development of CSL362, a monoclonal antibody to CD123 that has been humanized, affinity-matured and Fc-engineered for increased affinity for human CD16 (FcγRIIIa). In vitro studies demonstrated that CSL362 potently induces antibody-dependent cell-mediated cytotoxicity of both AML blasts and CD34(+)CD38(-)CD123(+) LSC by NK cells. Importantly, CSL362 was highly effective in vivo reducing leukemic cell growth in AML xenograft mouse models and potently depleting plasmacytoid dendritic cells and basophils in cynomolgus monkeys. Significantly, we demonstrated CSL362-dependent autologous depletion of AML blasts ex vivo, indicating that CSL362 enables the efficient killing of AML cells by the patient's own NK cells. These studies offer a new therapeutic option for AML patients with adequate NK-cell function and warrant the clinical development of CSL362 for the treatment of AML.

CD34+ cells and their derivatives contain mRNA for CD4 and human immunodeficiency virus (HIV) co-receptors and are susceptible to infection with M- and T-tropic HIV.

Highly purified (>98%) CD34+ cells directly after isolation (D0) or 2 weeks in culture (D14) were CD4+ and contained mRNA for the T-tropic HIV co-receptor, CXCR-4, and minor co-receptor, CCR-2B. D14 but not D0 cells were RT-PCR positive for mRNA for the major M-tropic human immunodeficiency virus (HIV) co-receptor, CCR-5, and potential co-receptor, CCR-1. D14 and D0 cells were susceptible to T- (HXB2) and M-tropic HIV (Bal), showing greater virus production with Bal than HXB2, and with higher virus production levels in D14 compared to D0 cells. Seven days post-infection of D0 cells Bal DNA was present in CD14bright and CD14- fractions, suggesting D0 infection of diverse progenitor types. HXB2 DNA was detected in CD14bright cells alone indicating D0 infection of monocyte progenitors only. It is concluded that CD34+ cells and cultured derivatives are susceptible to M- and T-tropic HIV and this correlates in part with co-receptor expression at the mRNA level.

Ex vivo expansion of murine marrow cells with interleukin-3 (IL-3), IL-6, IL-11, and stem cell factor leads to impaired engraftment in irradiated hosts.

In vitro incubation of bone marrow cells with cytokines has been used as an approach to expand stem cells and to facilitate retroviral integration. Expansion of hematopoietic progenitor cells has been monitored by different in vitro assays and in a few instances by in vivo marrow renewal in myeloablated hosts. This is the first report of studies, using two competitive transplant models, in which cytokine-treated cells, obtained from nonpretreated donors (eg, 5-fluorouracil), were competed with normal cells. A basic assumption is that the expansion of progenitors assayed in vitro as high- and low-proliferative potential colony-forming cells (HPP- and LPP-CFCs) indicates an expansion of stem cells which will repopulate in vivo. This study shows that culture of marrow cells with four cytokines (stem cell factor, interleukin-3 [IL-3], IL-6, IL-11) induces significant expansion and proliferation of HPP-CFC and LPP-CFC. Cell-cycle analysis showed that these hematopoietic progenitors were induced to actively cell cycle by culture with these cytokines. In the first competitive transplant model, which uses Ly5.2/Ly5.1 congenic mice, cytokine-cultured Ly5.2 cells competed with noncultured Ly5.1 cells led to 5% +/- 1% engraftment at 12 weeks and to 4% +/- 2% engraftment at 22 weeks posttransplantation for the cytokine exposed cells. Noncultured Ly5.2 cells competed with cultured Ly5.1 cells led to 70% +/- 1% engraftment at 12 weeks and to 93% +/- 2% engraftment at 22 weeks posttransplantation. In the second model, which uses BALB/c marrow of opposite genders, cultured male cells lead to 13% +/- 9% engraftment at 10 weeks and 2% +/- 1% engraftment at 14 weeks posttransplantation; noncultured male cells lead to 70% +/- 2% and 95% +/- 2% engraftment at 10 and 14 weeks posttransplantation, respectively. Data presented here from two different competitive transplant studies show a defect of cytokine expanded marrow related to cell cycle activation which manifests as defective long-term repopulating capability in irradiated host mice. The engraftment defect is more profound at longer time intervals, suggesting that the most striking effect may be on long-term repopulating cells.

New approaches in the treatment of asthma.

Asthma is a common and complex inflammatory disease of the airways that remains incurable. Current forms of therapy are long term and may exhibit associated side-effect problems. Major participants in the development of an asthma phenotype include the triggering stimuli such as the allergens themselves, cells such as T cells, epithelial cells and mast cells that produce a variety of cytokines including IL-5, GM-CSF, IL-3, IL-4 and IL-13 and chemokines such as eotaxin. Significantly, the eosinophil, a specialized blood cell type, is invariably associated with this disease. The eosinophil has long been incriminated in the pathology of asthma due to its ability to release preformed and unique toxic substances as well as newly formed pro-inflammatory mediators. The regulation of eosinophil production and function is carried out by soluble peptides or factors. Of these IL-5, GM-CSF and IL-3 are of paramount importance as they control eosinophil functional activity and are the only known eosinophilopoietic factors. In addition they regulate the eosinophil life span by inhibiting apoptosis. While one therapeutic approach in asthma is directed at inhibiting single eosinophil products such as leukotrienes or single eosinophil regulators such as IL-5, we believe that the simultaneous inhibition of more than one component is preferable. This may be particularly important with eosinophil regulators in that not only IL-5, but also GM-CSF has been repeatedly implicated in clinical studies of asthma. The fact that GM-CSF is produced by many cells in the body and in copious amounts by lung epithelial cells highlights this need further. Our approach takes advantage of the fact that the IL-5 and GM-CSF receptors (as well as IL-3 receptors) utilize a shared subunit to bind, with high affinity, to these cytokines and the same common subunit mediates signal transduction culminating in all the biological activities mentioned. By generating the monoclonal antibody BION-1 to the cytokine binding region of the common subunit (betac) we have shown that the approach of inhibiting IL-5, GM-CSF and IL-3 binding and the resulting stimulation of eosinophil production and function with a single agent is feasible. Furthermore we have used BION-1 as a tool to crystallize and define the structure of the cytokine binding domain of betac. This knowledge and this approach may lead to the generation of novel therapeutics for the treatment of asthma.

High levels of engraftment with a single infusion of bone marrow cells into normal unprepared mice.

Repetitive infusion of 40 million male murine marrow cells (total 200 million cells) into normal unprepared female BALB/c hosts for 5 consecutive days results in high levels of engraftment at 1-25 months postinfusion, as determined by Southern blot analysis using a Y chromosome-specific probe. We investigated the importance of the schedule of injections in this engraftment model. Surprisingly, a single infusion of 200 x 10(6) male BALB/c bone marrow cells analyzed at 7-14 weeks postinfusion resulted in engraftment levels in individual female mice of over 50% with mean values of 25 +/- 2% for 44 individual transplant points. Engraftment levels in spleen and thymus were 14 +/- 1% and 18 +/- 3%, respectively. Including heparin in the infusion increased engraftment in marrow, spleen, and thymus. Administration of the cells over five or 10 separate infusions, rather than in one injection, did not increase engraftment levels. If the infused bone marrow cells seeded equally between host spleen, thymus, and bone marrow, and if all cells engrafted, the bone marrow engraftment seen here approaches the theoretical maximum. This suggests either a large number of available "niches" or the displacement of host marrow cells by infused marrow. The latter possibility is upheld by cell counts per tibia/femur and total seven-factor HPP-CFC/tibia, which were not increased. These data suggest that a single infusion of marrow homes quantitatively to spleen, thymus, and bone marrow, possibly displacing host cells in the process.

Engraftment of bone marrow cells into normal unprepared hosts: effects of 5-fluorouracil and cell cycle status.

Bone marrow from animals treated with 5-fluorouracil (5FU) competes equally with normal marrow when assessed in vivo in an irradiated mouse, but shows markedly defective engraftment when transplanted into noncytoablated hosts. Using Southern Blot analysis and a Y-chromosome specific probe, we determined the level of engraftment of male donor cells in the bone marrow, spleen, and thymus of unprepared female hosts. We have confirmed the defective engraftment of marrow harvested 6 days after 5FU (FU-6) and transplanted into unprepared hosts and shown that this defect is transient; by 35 days after 5FU (FU-35), engraftment has returned to levels seen with normal marrow. FU-6 marrow represents an actively cycling population of stem cells, and we hypothesize that the cycle status of the stem cell may relate to its capacity to engraft in the nonirradiated host. Accordingly, we have evaluated the cycle status of engrafting normal and FU-6 marrow into normal hosts using an in vivo hydroxyurea technique. We have shown that those cells engrafting from normal marrow and over 70% of the cells engrafting from FU-6 marrow were quiescent, demonstrating no killing with hydroxyurea. We have also used fluorescent in situ hybridization (FISH) analysis with a Y-chromosome probe and demonstrated that normal and post-5FU engraftment patterns in peripheral blood were similar to those seen in bone marrow, spleen, and thymus. Altogether these data indicate that cells engrafting in normal, unprepared hosts are dormant, and the defect that occurs after 5FU is concomitant with the induction of these cells to transit the cell cycle.

Cytokine-facilitated transduction leads to low-level engraftment in nonablated hosts.

Using a murine bone marrow transplantation model, we evaluated the long-term engraftment of retrovirally transduced bone marrow cells in nonmyeloablated hosts. Male bone marrow was stimulated in a cocktail of interleukin-3 (IL-3), IL-6, IL-11, and stem cell factor (SCF) for 48 hours, then cocultured on the retroviral producer line MDR18.1 for an additional 24 hours. Functional transduction of hematopoietic progenitors was detected in vitro by reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of multiple drug resistance 1 (MDR1) mRNA from high proliferative potential-colony forming cell (HPP-CFC) colonies. After retroviral transduction, male bone marrow cells were injected into nonablated female mice. Transplant recipients received three TAXOL (Bristol-Myers, Princeton, NJ) injections (10 mg/kg) over a 14-month period. Transplant recipient tissues were analyzed by Southern blot and fluorescence in situ hybridization for Y-chromosome-specific sequences and showed donor cell engraftment of approximately 9%. However, polymerase chain reaction amplification of DNAs from bone marrow, spleen, and peripheral blood showed no evidence of the transduced MDR1 gene. RT-PCR analysis of total bone marrow RNA showed that transcripts from the MDR1 gene were present in a fraction of the engrafted donor cells. These data show functional transfer of the MDR1 gene into nonmyeloablated murine hosts. However, the high rates of in vitro transduction into HPP-CFC, coupled with the low in vivo engraftment rate of donor cells containing the MDR1 gene, suggest that the majority of stem cells that incorporated the retroviral construct did not stably engraft in the host. Based on additional studies that indicate that ex vivo culture of bone marrow induces an engraftment defect concomitantly with progression of cells through S phase, we propose that the cell cycle transit required for proviral integration reduces or impairs the ability of transduced cells to stably engraft.

Increased recruitment of hematopoietic progenitor cells underlies the ex vivo expansion potential of FLT3 ligand.

The ligand for flt-3 (FLT3L) exhibits striking structural homology with stem cell factor (SCF) and monocyte colony-stimulating factor (M-CSF) and also acts in synergy with a range of other hematopoietic growth factors (HGF). In this study, we show that FLT3L responsive hematopoietic progenitor cells (HPC) are CD34+CD38-, rhodamine 123dull, and hydroperoxycyclophosphamide (4-HC) resistant. To investigate the basis for the capacity of FLT3L to augment the de novo generation of myeloid progenitors from CD34+CD38- cells, single bone marrow CD34+CD38- cells were sorted into Terasaki wells containing serum-free medium supplemented with interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor (G-CSF), SCF (4 HGF) +/- FLT3L. Under these conditions, FLT3L recruited approximately twofold more CD34+CD38- cells into division than 4 HGF alone. The enhanced proliferative response to FLT3L was evident by day 3 and was maintained at all subsequent time points examined. In accord with these findings, we also show that transduction of CD34+CD38- cells with the LAPSN retrovirus is enhanced by FLT3L. The results of these experiments therefore indicate that increased recruitment of primitive HPC into cell cycle underlies the ex vivo expansion potential of FLT3L and also its ability to improve retroviral transduction of HPC.