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_ The increasing availability of affordable finite element modeling software and computational fluid dynamics codes have given today’s sailing yacht designers tools that, in the past, were only available to designers of yachts competing in the most high-profile events. This paper provides a case study on the structural design of the International America’s Cup Class Yacht Stars & Stripes USA-34, which won the U.S. defender series, the Citizen Cup, leading up to the 29th America’s Cup competition in 1995. The authors have recently been able to publicly share this information, and feel that the problem-solving approach is of direct use to many of today’s designers. The design approach is twofold, the first being the use of Finite Element Analysis to minimize drag-inducing local deformations. Over 80 finite element models were analyzed with varying configurations, starting with an initial baseline structure based on the previous boat, USA-11. These structural improvements reduced local hull deflection from5 to 6 mm per meter on the previous design to less than 1 mm per meter on the USA-34 design. The second part of the design approach isolates the hydrodynamic effects of global bending under rig tension. Illustrative tank tests are provided to demonstrate how the global sagging of the hull, caused by rig tension, can often increase the resistance of a yacht (especially one built for racing). As part of a series of steps taken to mitigate the effect of rig tension, a novel design was developed, with soft patches and flexible structure to permit low-cost modification of the hull in the boatyard, giving approximately a 225mm increase in waterline length and 75 mm reduction in beam if needed—a modification that was made to USA-34 prior to winning the finals of the Citizen Cup, and at a cost of under $500. The reduction of drag-inducing local hull form deformations, and the development of the cost-saving hull modification concept, demonstrate how a creative designer can take advantage of the finite element modeling tools that are now widely available.

Recent advances in single-cell molecular analytical methods and clonal growth assays are enabling more refined models of human hematopoietic lineage restriction processes to be conceptualized. Here, we report the results of integrating single-cell proteome measurements with clonally determined lymphoid, neutrophilic/monocytic, and/or erythroid progeny outputs from >1000 index-sorted CD34+ human cord blood cells in short-term cultures with and without stromal cells. Surface phenotypes of functionally examined cells were individually mapped onto a molecular landscape of the entire CD34+ compartment constructed from single-cell mass cytometric measurements of 14 cell surface markers, 20 signaling/cell cycle proteins, and 6 transcription factors in ∼300 000 cells. This analysis showed that conventionally defined subsets of CD34+ cord blood cells are heterogeneous in their functional properties, transcription factor content, and signaling activities. Importantly, this molecular heterogeneity was reduced but not eliminated in phenotypes that were found to display highly restricted lineage outputs. Integration of the complete proteomic and functional data sets obtained revealed a continuous probabilistic topology of change that includes a multiplicity of lineage restriction trajectories. Each of these reflects progressive but variable changes in the levels of specific signaling intermediates and transcription factors but shared features of decreasing quiescence. Taken together, our results suggest a model in which increasingly narrowed hematopoietic output capabilities in neonatal CD34+ cord blood cells are determined by a history of external stimulation in combination with innately programmed cell state changes.

Elucidation of the identity and diversity of mechanisms that sustain long-term human blood cell production remains an important challenge. Previous studies indicate that, in adult mice, this property is vested in cells identified uniquely by their ability to clonally regenerate detectable, albeit highly variable levels and types, of mature blood cells in serially transplanted recipients. From a multi-parameter analysis of the molecular features of very primitive human cord blood cells that display long-term cell outputs in vitro and in immunodeficient mice, we identified a prospectively separable CD33+CD34+CD38-CD45RA-CD90+CD49f+ phenotype with serially transplantable, but diverse, cell output profiles. Single-cell measurements of the mitogenic response, and the transcriptional, DNA methylation and 40-protein content of this and closely related phenotypes revealed subtle but consistent differences both within and between each subset. These results suggest that multiple regulatory mechanisms combine to maintain different cell output activities of human blood cell precursors with high regenerative potential.

Xenograft models are transforming our understanding of the output capabilities of primitive human hematopoietic cells in vivo . However, many variables that affect posttransplantation reconstitution dynamics remain poorly understood. Here, we show that an equivalent level of human chimerism can be regenerated from human CD34 + cord blood cells transplanted intravenously either with or without additional radiation-inactivated cells into 2- to 6-month-old NOD-Rag1 –/– -IL2Rγc –/– (NRG) mice given a more radioprotective conditioning regimen than is possible in conventionally used, repair-deficient NOD- Prkdc scid/scid - IL2Rγc –/– (NSG) hosts. Comparison of sublethally irradiated and non-irradiated NRG mice and W 41 /W 41 derivatives showed superior chimerism in the W 41 -deficient recipients, with some differential effects on different lineage outputs. Consistently superior outputs were observed in female recipients regardless of their genotype, age, or pretransplantation conditioning, with greater differences apparent later after transplantation. These results define key parameters for optimizing the sensitivity and minimizing the intraexperimental variability of human hematopoietic xenografts generated in increasingly supportive immunodeficient host mice.

SummaryNucleosome position, density, and post-translational modification are widely accepted components of mechanisms regulating DNA transcription but still incompletely understood. We present a modified native ChIP-seq method combined with an analytical framework that allows MNase accessibility to be integrated with histone modification profiles. Application of this methodology to the primitive (CD34+) subset of normal human cord blood cells enabled genomic regions enriched in one versus two nucleosomes marked by histone 3 lysine 4 trimethylation (H3K4me3) and/or histone 3 lysine 27 trimethylation (H3K27me3) to be associated with their transcriptional and DNA methylation states. From this analysis, we defined four classes of promoter-specific profiles and demonstrated that a majority of bivalent marked promoters are heterogeneously marked at a single-cell level in this primitive cell type. Interestingly, extension of this approach to human embryonic stem cells revealed an altered relationship between chromatin modification state and nucleosome content at promoters, suggesting developmental stage-specific organization of histone methylation states.

Plerixafor (P) together with granulocyte colony–stimulating factor (G) is now recognized as an important strategy for mobilizing hematopoietic cells for use in patients given myelosuppressive therapies. However, quantitative comparisons of their ability to mobilize human cells with different hematopoietic activities in vitro or in vivo (in immunodeficient mice) and their interrelationships have not been investigated. To address these questions, we collected samples from 5 normal adult volunteers before and after administering P alone and from another 5 before and after a 4-day course of G and again after a subsequent injection of P. Measurements of their blood content of CD34+ cells, in vitro myeloid colony–forming cells, 3- and 6-week long-term culture (LTC) cell outputs, and levels of circulating human platelets, as well as myeloid and lymphoid cells obtained in immunodeficient mice that received transplants, showed all activities were maximal 4 hours after P preceded by G, and 3-week LTC outputs showed the highest concordance with the 3-week circulating human neutrophil levels obtained in mice that received transplants. Thus, human cells capable of producing neutrophils rapidly in vivo were optimally mobilized by the G + P protocol, and the 3-week LTC assay appears to offer a more specific predictor of their levels than conventional CD34+ cell or colony-forming cell counts.

Investigation of human hematopoietic stem cells (HSCs) has been revolutionized by advances in cell separation and clonal tracking of cells produced in transplanted patients and immunodeficient mice. However, methods able to expand them significantly remain elusive. iPSCs are of continuing interest as an alternative source, although robust conditions that support iPSC generation of human HSC either in vitro or in vivo have also not yet been identified. To determine whether the use of a more compatible in vivo host environment might offer an improvement, we compared the outputs of teratomas generated from human iPSC transplanted (± irradiated mouse fibroblasts engineered to produce human FLT3-L, SCF and IL3) into immunodeficient NOD-Rag1-null- IL2Rgc-null mice with a c-kit deficiency with or without a transgenic source of human IL3, GM-CSF and SCF (NRG-W41± 3GS mice). 6-8 week teratomas generated in the NRG-W41-3GS mice contained 40-fold more human CD34+CD45+ cells than those produced in NRG-W41 mice with yields up to 7 × 106 CD45+ cells. Co-injection of human growth factor-producing mouse fibroblasts further increased the production of human CD34+CD45+ ∼6-fold in the NRG-W41-3GS mice that extended to the phenotypically early GPI80+ and CD49f+ cells within the CD45+CD34+CD38-CD45RA-CD90+ subset. However, despite their display of short-term granulopoietic and erythroid differentiation potential in vitro, no evidence of in vivo engrafting potential was detected. These experiments suggest a persisting failure of conditions in the teratomas to support the acquisition of certain functional properties that primitive human cells require to engraft and set the stage for their future identification.

Recent advances in single-cell molecular analytical methods and clonal growth assays are enabling more refined models of human hematopoietic lineage restriction processes to be conceptualized. Here, we report the results of integrating single-cell proteome measurements with clonally determined lymphoid, neutrophilic/monocytic, and/or erythroid progeny outputs from >1000 index-sorted CD34(+) human cord blood cells in short-term cultures with and without stromal cells. Surface phenotypes of functionally examined cells were individually mapped onto a molecular landscape of the entire CD34(+) compartment constructed from single-cell mass cytometric measurements of 14 cell surface markers, 20 signaling/cell cycle proteins, and 6 transcription factors in ∼300 000 cells. This analysis showed that conventionally defined subsets of CD34(+) cord blood cells are heterogeneous in their functional properties, transcription factor content, and signaling activities. Importantly, this molecular heterogeneity was reduced but not eliminated in phenotypes that were found to display highly restricted lineage outputs. Integration of the complete proteomic and functional data sets obtained revealed a continuous probabilistic topology of change that includes a multiplicity of lineage restriction trajectories. Each of these reflects progressive but variable changes in the levels of specific signaling intermediates and transcription factors but shared features of decreasing quiescence. Taken together, our results suggest a model in which increasingly narrowed hematopoietic output capabilities in neonatal CD34(+) cord blood cells are determined by a history of external stimulation in combination with innately programmed cell state changes.

Without effective therapy, chronic-phase chronic myeloid leukemia (CP-CML) evolves into an acute leukemia (blast crisis [BC]) that displays either myeloid or B-lymphoid characteristics. This transition is often preceded by a clinically recognized, but biologically poorly characterized, accelerated phase (AP). Here, we report that IKAROS protein is absent or reduced in bone marrow blasts from most CML patients with advanced myeloid disease (AP or BC). This contrasts with primitive CP-CML cells and BCR-ABL1-negative acute myeloid leukemia blasts, which express readily detectable IKAROS. To investigate whether loss of IKAROS contributes to myeloid disease progression in CP-CML, we examined the effects of forced expression of a dominant-negative isoform of IKAROS (IK6) in CP-CML patients' CD34(+) cells. We confirmed that IK6 disrupts IKAROS activity in transduced CP-CML cells and showed that it confers on them features of AP-CML, including a prolonged increased output in vitro and in xenografted mice of primitive cells with an enhanced ability to differentiate into basophils. Expression of IK6 in CD34(+) CP-CML cells also led to activation of signal transducer and activator of transcription 5 and transcriptional repression of its negative regulators. These findings implicate loss of IKAROS as a frequent step and potential diagnostic harbinger of progressive myeloid disease in CML patients.

Elucidation of the identity and diversity of mechanisms that sustain long-term human blood cell production remains an important challenge. Previous studies indicate that, in adult mice, this property is vested in cells identified uniquely by their ability to clonally regenerate detectable, albeit highly variable levels and types, of mature blood cells in serially transplanted recipients. From a multi-parameter analysis of the molecular features of very primitive human cord blood cells that display long-term cell outputs in vitro and in immunodeficient mice, we identified a prospectively separable CD33

Human adult stem cell expansion Transfused blood saves lives. Despite the widespread use of this critical resource, it is difficult to increase blood cell numbers outside of the body. By screening thousands of small compounds, Fares et al. identify a molecule that expands human stem cell numbers in cord blood. The researchers generate many variations of that molecule and show that one such compound provides even greater human blood cell expansion. If researchers can provide increased numbers of stem cells and progenitor cells, cord blood should find even greater use in the clinic. Science , this issue p. 1509

Human cord blood (CB) offers an attractive source of cells for clinical transplants because of its rich content of cells with sustained repopulating ability in spite of an apparent deficiency of cells with rapid reconstituting ability. Nevertheless, the clonal dynamics of nonlimiting CB transplants remain poorly understood. To begin to address this question, we exposed CD34+ CB cells to a library of barcoded lentiviruses and used massively parallel sequencing to quantify the clonal distributions of lymphoid and myeloid cells subsequently detected in sequential marrow aspirates obtained from 2 primary NOD/SCID-IL2Rγ(-/-) mice, each transplanted with ∼10(5) of these cells, and for another 6 months in 2 secondary recipients. Of the 196 clones identified, 68 were detected at 4 weeks posttransplant and were often lympho-myeloid. The rest were detected later, after variable periods up to 13 months posttransplant, but with generally increasing stability throughout time, and they included clones in which different lineages were detected. However, definitive evidence of individual cells capable of generating T-, B-, and myeloid cells, for over a year, and self-renewal of this potential was also obtained. These findings highlight the caveats and utility of this model to analyze human hematopoietic stem cell control in vivo.

PURPOSE OF REVIEW Transplantation of hematopoietic cells is now a well established clinical procedure, although optimal outcomes are not always obtained. This reflects insufficient knowledge of the different subsets of primitive cells required to achieve a rapid and permanent recovery of mature blood cell production. Here we review recent findings that extend our understanding of these cells and their regulation, and implications for the ex-vivo expansion of these cells. RECENT FINDINGS Separate subsets of platelet and neutrophil lineage-restricted human hematopoietic cells with rapid but transient repopulating activities have been identified, thus adding to previous evidence of short-term repopulating cells that generate both of these lineages. New studies also suggest intrinsically determined heterogeneity in differentiation potentialities that are sustained at the stem cell level, and have revealed new ways their self-renewal can be influenced. SUMMARY Hematopoietic repopulation posttransplant is highly complex both in terms of the differing numbers and types of cells required for optimal hematopoietic recoveries and the factors that will determine the composition and behavior of a given inoculum. Successful ex-vivo expansion protocols will, thus, need to incorporate conditions that will produce adequate numbers of all cell types required with retention of their full functionality.

Better methods to characterize normal human hematopoietic cells with short-term repopulating activity cells (STRCs) are needed to facilitate improving recovery rates in transplanted patients.We now show that 5-fold more human myeloid cells are produced in sublethally irradiated NOD/SCID-IL-2Receptor-γchain-null (NSG) mice engineered to constitutively produce human interleukin-3, granulocyte-macrophage colony-stimulating factor and Steel factor (NSG-3GS mice) than in regular NSG mice 3 weeks after an intravenous injection of CD34 human cord blood cells. Importantly, the NSG-3GS mice also show a concomitant and matched increase in circulating mature human neutrophils. Imaging NSG-3GS recipients of lenti-luciferase-transduced cells showed that human cells being produced 3 weeks posttransplant were heterogeneously distributed, validating the blood as a more representative measure of transplanted STRC activity. Limiting dilution transplants further demonstrated that the early increase in human granulopoiesis in NSG-3GS mice reflects an expanded output of differentiated cells per STRC rather than an increase in STRC detection.NSG-3GS mice support enhanced clonal outputs from human short-term repopulating cells (STRCs) without affecting their engrafting efficiency. Increased human STRC clone sizes enable their more precise and efficient measurement by peripheral blood monitoring.

Several growth factors (GFs) that together promote quiescent human hematopoietic stem cell (HSC) expansion ex vivo have been identified; however, the molecular mechanisms by which these GFs regulate the survival, proliferation. and differentiation of human HSCs remain poorly understood. We now describe experiments in which we used mass cytometry to simultaneously measure multiple surface markers, transcription factors, active signaling intermediates, viability, and cell-cycle indicators in single CD34+ cord blood cells before and up to 2 hours after their stimulation with stem cell factor, Fms-like tyrosine kinase 3 ligand, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor (5 GFs) either alone or combined. Cells with a CD34+CD38-CD45RA-CD90+CD49f+ (CD49f+) phenotype (∼10% HSCs with >6-month repopulating activity in immunodeficient mice) displayed rapid increases in activated STAT1/3/5, extracellular signal-regulated kinase 1/2, AKT, CREB, and S6 by 1 or more of these GFs, and β-catenin only when the 5 GFs were combined. Certain minority subsets within the CD49f+ compartment were poorly GF-responsive and, among the more GF-responsive subsets of CD49f+ cells, different signaling intermediates correlated with the levels of the myeloid- and lymphoid-associated transcription factors measured. Phenotypically similar, but CD90-CD49f- cells (MPPs) contained lower baseline levels of multiple signaling intermediates than the CD90+CD49f+ cells, but showed similar response amplitudes to the same GFs. Importantly, we found activation or inhibition of AKT and β-catenin directly altered immediate CD49f+ cell survival and proliferation. These findings identify rapid signaling events that 5 GFs elicit directly in the most primitive human hematopoietic cell types to promote their survival and proliferation.

Background: Assessment of the growth and differentiation of human hematopoietic cells transplanted into immunodeficient mice has become an important method for evaluating the numbers and properties of cells with engraftment potential and the progeny they can generate in an in vivo setting. Such xenograft models have undergone numerous modifications and have also been applied with increasing success to primary sources of many types of malignant as well as normal human hematopoietic cells. These advances include the incorporation of cDNAs encoding various human-specific growth factor genes into the genomes of the widely used and long-lived NOD-scid-IL2Rγcnull (NSG) mouse, the creation of regenerated human bone ossicles within the mice to more closely mimic the human marrow microenvironment, recognition of the superior supportive activity of female mice, and more recently of NSG mice additionally compromised by their genetic acquisition of a c-kit deficiency. Exploitation of these modifications to recipients of human hematopoietic cells has greatly increased our understanding of the complex composition of both normal and leukemic populations and their responses to various treatments. Nevertheless, many examples remain of both acute and chronic human myeloid leukemias that do not engraft or engraft very poorly, even in the most permissive mice thus far tested. These include samples of many cases of chronic phase chronic myeloid leukemia (CP-CML), myelodysplastic syndromes (MDS), and approximately one third of all cases of acute myeloid leukemia (AML). We have now created 2 new strains of immunodeficient mice that have the B, T, NK immunodeficiency determined by a SirpαNOD-Rag1-/--IL2Rγc-/- genotype (which is equivalent to that of NSG mice) and a c-kit deficiency obtained by replacement of the wild-type c-kit gene with a homozygous W41/W41genotype (SRG-W41 mice). The second strain has the same SRG-W41 genotype but is also transgenic for human IL3, GM-CSF, and SCF production (SRG-W41-3GS). NRG/SRG mice were chosen as the parental strain because they support similar levels of chimerism as NSG mice when given a radiobiologically equivalent dose of radiation, but can benefit from split or low dose rate irradiation protocols. Results: We first showed that leukemic cells from patients with AML and CP-CML produce progeny more rapidly in sub-lethally irradiated parental SRG-3GS mice than in SRG mice, and that SRG-W41 mice can support higher levels of human myeloid and B-lymphoid chimerism by normal human CD34+ cord blood (CB) and adult bone marrow (BM) cells than similarly suppressed or non-irradiated SRG controls. We then compared the chimerism obtained in different strains of mice transplanted each with 6x106 CD34+ cells from 1 CP-CML patient or ~106 CD34+ cells from 5 different AML patients pre-selected for their previously demonstrated poor or inability to engraft NSG-3GS mice. Within a few weeks, the level of the predominantly myeloid chimerism in 2 non-irradiated male SRG-W41-3GS mice and 2 sub-lethally irradiated female SRG-3GS recipients became equivalent and this equivalence was sustained for over a year. Only 3 of 5 AML samples tested engrafted either sex of sub-lethally irradiated SRG-W41-3GS or SRG-3GS mice, and in each case, the speed and level of engraftment was greatly enhanced in the SRG-W41-3GS mice as compared to sex-matched SRG-3GS hosts (with 20-30-fold and 2-3-fold higher levels at week 6 and 10 respectively in female as compared to male recipients of either strain). All the engrafted SRG-W41-3GS female hosts became moribund within 20 weeks post-transplant with a predominance of human leukemic cells in their BM and enlarged spleens filled with leukemic cells. In contrast, the SRG-3GS recipients of the same AML cells survived more than 20 weeks with a more delayed appearance of leukemic cells. Sublethally irradiated female SRG-W41-3GS recipients of 0.1-0.2x106 CD34+ cells from 3 of 3 chronic myelomonocytic leukemia (CMML) patients tested to date have also shown engraftment at either week 3 or 11 and, in one case, up to 17 weeks post-transplant. Conclusion: The introduction of a c-kit deficiency into highly immunodeficient SRG mice producing human IL3, GM-CSF and SCF transgenically offers new opportunities for examining the growth potential and sensitivity to treatment of a broader spectrum of primary human leukemias and neoplastic conditions than has been previously possible. Disclosures Deininger: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; CTI BioPharma Corp.: Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; BMS: Consultancy, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Delayed recovery of mature blood cells poses a serious, expensive, and often life-threatening problem for many stem cell transplantation recipients, particularly if heavily pretreated and serving as their own donor, or having a CB transplantation as the only therapeutic option. Importantly, the different cells required to ensure a rapid, as well as a permanent, hematopoietic recovery in these patients remain poorly defined. We now show that human CB and mobilized peripheral blood (mPB) collections contain cells that produce platelets and neutrophils within 3 weeks after being transplanted into sublethally irradiated NOD/scid-IL-2Rgammac-null mice. The cells responsible for these 2 outputs are similarly distributed between the aldehyde dehydrogenase-positive and -negative subsets of lineage marker-negative CB and mPB cells, but their overall frequencies vary independently in individual samples. In addition, their total numbers can be seen to be much (> 30-fold) lower in a single 'average' CB transplantation compared with a single 'average' mPB transplantation (normalized for a similar weight of the recipient), consistent with the published differential performance in adult patients of these 2 transplantation products. Experimental testing confirmed the clinical relevance of the surrogate xenotransplantation assay for quantifying cells with rapid platelet regenerative activity, underscoring its potential for future applications., link_to_OA_fulltext

The unique features of the International Sailing Canoe have intrigued naval architects and sailors for years. This paper highlights the historical design development, describes current trends, and presents results from finite element analyses of three popular construction methods: cold-molded cedar, fiberglass, and carbon/epoxy. Comparisons are presented of the bending and torsional stiffness, pitch gyradius, and factors of safety. In general, the carbon/epoxy exhibited the best characteristics, followed by the cold-molded cedar and the fiberglass. The Tsai-Wu quadratic failure criterion developed for laminated plates was used in the factor-of-safety calculations. Factors of safety correlated closely to empirical development.

Background: Assessment of the growth and differentiation of human hematopoietic cells in immunodeficient mice has become vital to much basic and translational research. Historically, the primary focus has been to quantify different types of repopulating cells, based on the diversity and longevity of their clonal outputs in transplanted mice, assuming the results would be relevant to clinical transplants in humans. With the development of mice that lack B, T and NK cells on a variety of backgrounds and that have normal life expectancies but differences in cellular DNA repair ability, an increasing application is to use mice repopulated with human cells to interrogate and perturb mechanisms controlling normal, genetically modified and malignant cell behavior. We have previously shown that C57Bl6 mice homozygous for the W41 mutation of c-kit are fertile with a normal lifespan but have a functionally compromised hematopoietic stem cell (HSC) population. This enables single transplanted syngeneic HSCs to be detected at high frequency in these mice when they have been given a sublethal irradiation dose. Importantly, the HSC-derived clones produced in these mice display the same growth, self-renewal and differentiation abilities as in myeloablated recipients that require a co-transplant of normal mouse bone marrow (BM) cells to support their survival. We now report the development and improved repopulation by human cord blood (CB) CD34+ cells of mice that have the same genetically determined B, T, NK immunodeficiency as NOD/Rag1-/--IL2Rγc-/- (NRG) into which a homozygous W41gene has been introduced. This was achieved by crossing and backcrossing the progeny of NRG x C57Bl6-W41/W41 matings and selecting mice that were homozygous for the Sirpα allele of the NOD mouse, the null Rag1 and null IL2Rgamma chain, genes of the NRG mouse and the W41 gene to obtain all of these on an otherwise mixed NODxC57Bl/6 background (NRG-W41 mice). The NRG mouse was chosen because the Rag1 KO has no effect on the radiosensitivity of other tissues as is the case with the scid (S) gene in the NSG mouse. As a result, use of the NRG mouse allows exploitation of the radioprotective effect of a reduced irradiation dose rate and hence delivery of a selectively higher dose to the HSCs of the host. Results: Initial studies showed that parental NRG mice given 900 cGy split or spread continuously over 3 hrs show similar repopulation by human CD34+ CB cells as NSG mice given 315 cGy, but are more robust with consistent longterm survival. We then performed a pilot experiment using the same transplant design (2x104 CD34+ CB cells/mouse) to compare chimerism obtained in NRG-W41 mice given an estimated “equivalent” radiosensitizing regimen of 150 cGy. The levels of multiple lineages of human cells measured in the BM and spleen 20 weeks post-transplant revealed these were greatly increased in the NRG-W41 mice (>95% human CD45+ cells in the BM vs 40% in NRG mice). Kinetic analysis of human cells in the blood also showed an enhanced output of human myeloid and B-lymphoid cells over time (5-fold higher in the NRG-W41 mice after >3 weeks). Particularly notable was the selectively increased (20-fold) and sustained output of human glycophorin A+ (GPA+) erythroid cells in the NRG-W41 mice (5% human GPA+ cells in the BM of 20-week NRG-W41 mice given 150 cGy and 5x104 CD34+ CB cells/mouse vs 0.25% in the BM of the matched NRG mice given 900 cGy). A similar marked increase (20-fold) was seen on the level of circulating human platelets (SSClowCD41+ CD61+ cells) in comparable groups of transplanted NRG-W41 and NRG mice. We then investigated the extent of repopulation achievable in untreated NRG-W41 recipients. We therefore transplanted mice of both strains with 5x104 CD34+CB cells each and have now followed the levels of human cells in their circulation and BM for up to 20 weeks. Human cells were barely detectable at 3 weeks post-transplant in either strain, but then in the unirradiated NRG-W41 mice only, their levels (all lineages) increased to close to those attained in NRG mice given 900 cGy. Conclusion: NRG-W41 mice support robustly enhanced and long term generation in vivo of a wide range of human hematopoietic cell types including erythrocytes and platelets, with high levels of chimerism achieved even in unirradiated primary recipients transplanted with relatively low numbers of human CD34+ CB cells. Disclosures No relevant conflicts of interest to declare.

Background: Allografting of adult patients remains a widely utilized strategy but rates of treatment-related morbidity and mortality are still high. Granulocyte colony stimulating factor (G-CSF)-stimulated peripheral blood (G-PB) is now the most common source of clinically used hematopoietic allografts. G-PB grafts provide faster rates of neutrophil and platelet recovery compared to unstimulated bone marrow (BM), but G-PB is also associated with an increased risk of graft versus host disease (GVHD). More recently plerixafor has been demonstrated to be an effective hematopoietic cell mobilizing agent, and plerixafor is also now widely used alongside G-CSF to procure PB cells for clinical allografting. However, little is known as to whether plerixafor treatment alone or in combination with G-CSF might have an effect (positive or negative) on the repopulating activity of a BM harvest. Methods: In this study we have evaluated the affect over time of plerixafor ± G-CSF on the hematopoietic activity of the BM and PB of 10 normal adult donors. Donors received one treatment of plerixafor alone (240 μg/kg) vs plerixafor after 4 days of prior G-CSF (5 μg/kg/day). BM and PB samples were taken pre-treatment, post-G-CSF/pre-plerixafor, 4 hrs post-plerixafor, and 24 hours post plerixafor/48 hours post G-CSF. These samples were then analyzed for total nucleated cells (TNCs), CD34+ cells, in vitro colony-forming cells (CFCs), and cells capable of regenerating mature (TNCs) and CD34+ cells in 3-week stroma-containing cultures (LTC-ICs-3 wks) and in short-term (3-week) repopulating cell (STRC) assays in immunodeficient mice. The STRC assessment took advantage of a modified assay with improved sensitivity recently developed by our group, which utilizes NOD/Rag1-/--IL2Rγc-/- mice that constitutively produce human IL-3, GM-CSF and Steel factor (NRG-3GS mice) as the transplant recipients. Results: The G-CSF+plerixafor treatment protocol produced the largest increase in hematopoietic cells in the PB of the donors, as compared to either G-CSF or plerixafor alone. All counts reached peak values 4 hours after administration of plerixafor and 24 hrs after the last administration of G-CSF resulting in maximum increases at that time of 6-fold above baseline for TNCs, 15 for CD34+ cells and 25 for LTC-ICs-3 wks based on absolute measurements of each cell type per µL of PB. In the BM, where corresponding absolute values are not possible, there was a relative decrease in each of these parameters compared to BM TNC values. To assess in vivo STRC activity, collection time-points and sample types were pooled, depleted of CD3+ cells (using diptheria-conjugated OKT3), and 3x106 total cells transplanted IV into each of 2-3 replicate NRG-3GS mice. Four days of G-CSF reduced the STRC activity in donor BM asprates below baseline levels which was rescued by 4 hours after administration of plerixafor to the same donors. This restored human STRC activity included progenitor (CD34+) cells as well as maturing erythroid (glycophorin A+), granulopoietic (CD33+) cells and circulating SSClow human CD41+/CD61+ platelets. Conclusions: A time course study of plerixafor ± G-CSF administration in normal volunteers aligns with previous findings showing that G-CSF+plerixafor treatment induces a transiently higher output of multiple primitive hematopoietic cell types compared to G-CSF or plerixafor alone. These findings suggest an optimal time for harvesting PB and BM for a future clinical trial to determine which may serve as the superior allograft in patients. Disclosures No relevant conflicts of interest to declare.

The growing use of marine composite materials has led to many technical challenges. One is predicting lifetime durability. This step has a large uncertainty as the methods used to design metal ships have never been verified at full-scale for composite vessels. This project systematically extended the isotropic method to the application of fiberglass vessels in the marine environment. The research compared two identical fiberglass vessels having significantly different fatigue histories with undamaged laminates representing a new vessel. Analytical models based on classical lamination theory, finite element analysis, ship motions, probability and wind and wave mechanics were used to predict hull laminate strains. Material properties used in the finite element analysis were based on tested wet and dry conditions. Fatigue tests were used to determine S-N residual stiffness properties. These were compared against strains measured while underway and good correlation was achieved. Fatigue damage indicators were identified which could be used in vessel inspection procedures. Endurance limits were found to be near 25% of static load, indicating a fatigue design factor of 4 is required for these materials. This exceeds ABS and US Navy design guides. Comparisons were made of standard moisture experiments using boiling water versus long-term exposure. Results indicated the boiling water test yielded significantly conservative values and was not a reliable means of predicting long-term effects. Panel tests were compared to a combined coupon and finite element procedure. Results indicated the proposed procedure was a viable substitute, at least for the materials studied. A rational explanation for using thicker outer laminates in marine composites was verified through single-sided moisture flex tests. These showed that the reduced strength and stiffness due to moisture of

Accelerated phase (AP) CML is marked by a rising leucocyte count in the face of maintenance treatment, exaggerated basophilia and accumulating blasts. However, AP is typically short-lived and rapidly succeeded by blast crisis. Genetic alterations resulting in loss of IKAROS activity are associated with the development of both BCR-ABL1+ myeloid and lymphoid leukemias, as well as the related BCR-ABL1-negative MPNs. However, how loss of IKAROS functionality contributes to the malignant features of these leukemias is not clear. Here we demonstrate that direct suppression of IKAROS activity in CD34+ cells from chronic phase (CP) CML patients leads to their rapid acquisition of features of AP without causing progression to acute leukemia. To suppress IKAROS activity in CD34+ CP CML cells, we transduced samples from 4 patients with a lentiviral GFP vector encoding a dominant-negative isoform of IKAROS (dnIK6), or an empty control YFP vector, and compared their progeny generated in cultures containing mouse stromal cells engineered to produce human SCF, IL-3, FLT3L and G-CSF. In all cases, we found dnIK6 transduction caused a rapid (evident within 2 weeks) and sustained (for at least 6 weeks) increase in total cell output (4-20x higher than YFP+ controls at 4-6 weeks, p To interrogate the molecular mechanisms underlying the dnIK6 effects on cell output, we compared the levels of activated signaling proteins by intracellular flow cytometry. This showed increased activation of STAT5 (80±14% higher median fluorescent intensity (MFI), p In summary, IKAROS disruption in patients’ CP CD34+ CML cells recapitulates cardinal features of AP disease, including an increased cell output associated with an enhanced production of basophils and an eventual accumulation of primitive cells. We further identify dnIK6-mediated suppression of SOCS2 and the β-catenin destruction complex, with enhanced activation of STAT5 and β-catenin in progenitor cells, as a likely key mechanism contributing to the altered biology of AP cells. Disclosures: No relevant conflicts of interest to declare.

Abstract 2341 Background: Growth factors (GFs) that stimulate the proliferation of human hematopoietic stem cells (HSCs) in vitro have been identified, but maintenance of the original stem cell potential has remained a challenge. We have confirmed the observation that UG26–1B6 cells produce factors that enhance the maximum yield in vitro of HSCs achievable with SCF, FLT3-L, IL-3, IL-6, and G-CSF (5 GFs) alone (∼5-fold increase in lympho-myeloid repopulating cells by limiting dilution transplant analysis in NOD/SCID-IL-2Rγc null mice assessed 20 weeks post-transplant). It was therefore of interest to determine whether these different outcomes could be correlated with specific signalling events. Methods: CD34+cell-enriched human cord blood (CB) cells were stimulated with 5 GFs ± UG26 conditioned medium (CM) for defined periods, and then fixed in paraformaldehyde, stained for surface markers, alcohol permeabilized and stained with antibodies specific for multiple intracellular signalling intermediates. Samples were then analyzed using either a CyTOF mass cytometer (34 unique parameters) or a LSR Fortessa cell analyzer (11 parameters). Data analysis was performed using a combination of Cytobank, SPADE, and R using the 'flowCore', 'flowType', 'lattice', 'SamSpectral', and 'gplots' packages. Results: Analysis by CyTOF mass cytometry of 106 CD34+ CB cells after 15 minutes of their stimulation ± 5 GFs ± CM showed that the HSC-enriched subset (Lin−CD34+CD38−CD90+CD49f+cells) exhibited similar responses to the 5 GFs and 5 GFs+CM cocktails (average shifts in median intensity [Δasinh (stimulated-unstimulated)] of 0.32 [-0.14 to 1.96] and 0.37 [-0.02 to 1.78]) with increased phosphorylation of S6, STAT3, STAT5, CREB, IκBa (total protein), AKT, Syk/ZAP70, and ERK1/2. However, the addition of CM did selectively reduce the level of pS6 and pAKT seen with 5 GFs alone, and also caused an increase in pCREB. In addition, activation of pSyk/ZAP70 was detected only in HSCs stimulated with 5 GFs + CM. Phosphoflow analysis of the same subset of cells from a second CB pool confirmed the CyTOF results for pAKT, pSTAT5, pCREB and pERK1/2 (variably) following a 15 minute stimulation but not pSyk/ZAP70. After 2 hours of stimulation, the activation patterns were sustained for most targets for cells exposed to 5 GFs + CM but were reduced in 5 GFs alone - particularly pCREB which was decreased to levels in unstimulated cells. After 24 hours, pAKT and pSTAT5 showed continued activation in response to 5 GFs ± CM, whereas pCREB and pERK1/2 signal were reduced to below starting levels. Hartigans' Dip Test for Unimodality revealed significant deviations from unimodality for the mass cytometry HSC data. Principal component analysis (PCA) of the HSC data followed by subsequent clustering also revealed a number of differentially responsive sub-populations including a broadly cytokine non-responsive, non-apoptotic (cleaved PARP−) population, as well as a minority population with high levels of pSrc and pPLCγ, but reduced activation of other pathways. Further analysis using SPADE revealed decreased cytokine responsiveness with higher CD49f intensity. Weak but significant negative correlations between CD49f and pSTAT3, pCREB, and pS6 and positive correlations with pSrc and pPLCγ (holm-adjusted p values In the CyTOF experiment, multipotent progenitors (MPP; Lin−CD34+CD38−CD90−CD49f– cells) showed broadly similar patterns and levels of activation as HSCs when CM was added (p=0.44), but the median activation was attenuated in 5 GFs alone (p=0.048). MPPs also showed lower levels of multiple phosphorylation events, despite both HSC and MPP populations being Ki67−. This was consistent regardless of cell stimulation status, time point or measurement platform. Conclusions: We have discovered heterogeneity in the signal responses of highly purified fractions of very primitive subsets of human CB cells to culture conditions that affect their self-renewal differentially, that distinguish HSCs from MPPs, and that further subdivide the HSC subset. Among the events surveyed, differences in the timing and intensity of signal activation down common pathways were identified rather than any evidence of selective pathway utilization. These findings hold promise for the identification of how external factors modulate the fates of normal and transformed human HSCs when they are stimulated to divide. Disclosures: No relevant conflicts of interest to declare.

Abstract 28 Hematopoietic stem cells (HSC) exhibit heterogeneity in self-renewal and differentiation activity, but the extent to which this is intrinsically determined and extrinsically regulated is still poorly understood. In the mouse, purities of HSCs can now be achieved to allow such questions to be addressed directly. Interestingly, tracking the outputs of large numbers of serial transplantable clones produced from single-cell transplants, or the clonal progenies of vector-marked/barcoded cells indicate the existence in mice of 2 subsets of HSCs with durable self-renewal ability. These 2 subsets are characterized by distinct lineage output programs that are maintained through the many HSC self-renewal divisions required to serially propagate a clone in vivo. To begin to ask whether similar subsets of human HSCs exist, we have created a diverse lentiviral library encoding an estimated >105 different barcode sequences and GFP, and then used this library to track the in vivo clonal outputs of transduced human CD34+ cord blood cells in xenografted mice. For this experiment, CD34+ cells isolated immunomagnetically to a purity of >80% were exposed to virus for 6 hours in the presence of growth factors and then immediately injected intravenously into 2 sublethally irradiated NOD/SCID-IL2Rγ−/− mice (1.2 × 105 cells per mouse; 30% GFP+ cells after 3 days in vitro). Different subsets of human cells were then isolated by FACS from immunostained bone marrow cells aspirated sequentially from the femurs of the mice at intervals from 4–27 weeks post-transplant and the identity, number and size of clones in each established by next generation sequencing of barcoded amplicons derived from each sample. To identify barcodes arising from PCR and sequencing errors and calibrate clone sizes, we included 3 controls of 20, 100 and 500 cells with a known barcode at each datapoint. The data from these controls allowed a threshold of 20 cells per clone to be established with >95% confidence. We then compared the representation of clones among all samples from each mouse to derive the number and size of all clones detected, assuming a mouse contains 2×108 bone marrow cells. This analysis revealed a total of 154 uniquely barcoded clones containing up to 2×108 human hematopoietic cells in the 2 mice (8–30 × 106 in one and 4–165 × 106 in the other at any single time point). Analysis of the representation of each clone over time showed successive waves of repopulation from different clones with lineage output profiles consistent with those obtained by transplanting separate fractions of CD34+ cord blood cells distinguished by their surface phenotypes. Specifically, we detected 50 clones (32% of all clones) that were not sustained at detectable levels beyond 9 weeks post-transplantation and were characterized by robust myeloid differentiation with variable B cell outputs at 4 weeks. Another 30 clones (19%) showed significant but also transient outputs of either or both the myeloid and B cell lineage, disappearing between week 9 and 16 post-transplant. Mature cell output was detected from a total of 74 clones (48%) at the 27 week time point, among which 36 (23%) were not evident during the first 4 months post-transplant. These late-appearing clones were mostly small (contributing up to 3 × 105 total hematopoietic cells at week 27) and made a significantly higher contribution to the total human myeloid population than to the total human B cell population. Notably, the 12 long term clones that showed robust mature cell output detectable in all 3 sites sampled at week 27 when the mice were sacrificed (left leg vs right leg vs pelvis) contained both myeloid and lymphoid cells but with large (>100-fold) variations in their representation in the 3 different sites. This latter finding suggests less trafficking of human cells between sites than expected from parabiotic mouse experiments or substantial differences in the differentiation control exerted in different locations. Additionally, from one of the mice, we obtained the first direct evidence of a large output of human T cells (>9 × 106) that was part of a long term multi-lineage clone detectable at 27 weeks post-transplant. This first use of a barcoding strategy to analyze the clonal dynamics of normal human CD34+ cells with in vivo repopulating activity demonstrates the power of this approach to analyze their lineage outputs and sets the stage for novel applications to expanded and transformed populations. Disclosures: No relevant conflicts of interest to declare.

Abstract 1270 In vivo expansion of hematopoietic stem cells (HSCs) involves local interactions with stimuli generated from non-hematopoietic niche environments, but the full spectrum of molecular mechanisms responsible have remained elusive. Initial experiments in mice showed that highly purified HSCs from adult mouse bone marrow are consistently expanded 3–5-fold with full maintenance of their long term (≥6 months), serially transplantable, multi-lineage repopulating ability when cultured for 7 days in serum-free UG26 stromal-cell conditioned medium (CM) supplemented with 100 ng/ml mouse Steel Factor (SF) and 20 ng/ml mouse IL-11. To explore the potential effects of this CM on HSCs in human cord blood, we conducted an initial experiment in which CD34+CD38− cells were cultured for 7 days in UG26 CM supplemented with 100 ng/ml human Flt3-ligand, 100 ng/ml human SF, and 20 ng/ml each of human IL-3, IL-6 and G-CSF. The results of limiting dilution transplants of the cultured cells in intravenously injected NSG mice showed retention of input numbers of cells with equivalent robust 6-month lympho-myeloid repopulating activity. To characterize the initial target cells and determine whether their proliferative responses might be predictive of their self-renewal behavior, we set up single cell cultures with the CD49f+ subset of CD34+CD38−CD45RA−CD90+Rho−cells with the 5 growth factors in the presence or absence of CM. Under both conditions, 7/13 and 4/13 input cells, respectively, died within the first 72 hours in culture. The subsequent rate of proliferation of the survivors was similar with all completing a first division after 96 hours and a second division 24–48 hours later. By day 8, clones of variable sizes were noted (6–1100 and 4–200 cells/clone, respectively). Clones generated under the same conditions were pooled and injected intravenously into 2 NSG mice each. We then looked for the presence of human cells in the mice by analysis of serial bone marrow aspirates starting 3 weeks post-transplant. Human cells were detected in only one of each of the 2 pairs of mice and, interestingly, in both cases, no evidence of human cells was detectable until 3 months post-transplant. In the positive mouse injected with cells generated in the absence of CM, this repopulation was transient, peaking at ∼0.1% of the mouse bone marrow compartment at 4 months post-transplant and undetectable a month later. In contrast, in the positive recipient of cells from the cultures that contained CM, both lymphoid and myeloid human cells reached much higher levels (together making up ∼20% of the mouse bone marrow compartment) which were maintained for another 3 months when the mouse was sacrificed. Transplants of cells obtained at this time from the marrow gave positive repopulation of secondary mice. In a subsequent experiment, in which similar cultures were initiated with CD34+ cord blood cells, evidence of a late continuing effect of the CM was obtained with a net absolute expansion of CD34+CD45RA−CD90+ cell numbers during the interval between 12 and 21 days in vitro. These findings highlight the important potential of as yet unidentified secreted stromal cell factors to stabilize the stem cell state in HSCs stimulated to proliferate in vitro by growth factors that favor their self-renewal. Disclosures: No relevant conflicts of interest to declare.

Abstract 1892 Cord blood (CB) is becoming an increasingly utilized source of cells for cancer patients who are eligible for therapies that require a transplant to rescue them from toxic side effects on their own hematopoietic cells but lack a suitable HLA-matched donor. This strategy is now routinely used in children, but delayed neutrophil and platelet recovery remain unsolved problems and these problems are exacerbated in adults. To address this issue, we first surveyed the variability in 8 individual CB harvests of parameters routinely used to predict the utility of CB units as transplants (i.e., CD34+ and in vitro myeloid clonogenic progenitor cell frequencies). In addition, we compared their 3-week outputs of CD33/15/66+ cells (neutrophils and monocytes) in the marrow and CD41a+ platelets in the blood of sublethally irradiated NSG mice after the IV transplantation of ∼104 CD34+ cells. These latter assessments were based on ongoing experiments in our lab demonstrating that, at this transplant dose, the outputs measured are linearly related to the number of CD34+ cells injected and detect transplantable progenitor cell types that are biologically distinct from cells with longer term repopulating activity. The results showed variation between CBs in all parameters, a marked lack of correlation between %CD34+ cells or % total CFCs in initial cells and %CD41a+ cells regenerated at 3 weeks/104 CD34+ cells transplanted (R=-0.28 and 0.35, respectively), and a weak correlation between the %CD33/15/66+ cells regenerated at 3 weeks/104 CD34+ cells transplanted and %CD34+ cells or % total CFCs in the initial CB cells (R values of 0.46–0.64). However, although engraftment of primitive human cells in NSG mice appears highly efficient, terminal differentiation of the myeloid lineages in these mice is poor. One possible explanation for this deficiency in mature cell output is that several of the murine growth factors responsible for regulating the production and release of these cells into the circulation in mice are not cross-reactive on human cells. We therefore hypothesized that engineering NSG mice to produce the human counterparts might significantly improve the detection of short term repopulating human cells whose maximum clone size might be limiting in NSG mice. Three potential relevant factors are IL-3, GM-CSF and Steel factor. We therefore backcrossed a line of transgenic NS mice we had created to express human IL-3, GM-CSF and Steel factor onto the NSG strain to produce homozygous NSG mice expressing all 3 of these human factors (NSG-3GS mice). We then compared these NSG-3GS mice with NSG mice in terms of their ability to stimulate the production within 3 weeks of human neutrophil-monocytes and platelets from intravenously transplanted CD34+ cells isolated from pooled CB harvests. The results showed that the levels of neutrophils and monocytes generated in the marrow of the NSG-3GS mice were elevated to levels of >50% of the marrow in 90% of the mice, even at the lowest number of CD34+ cells transplanted. Human neutrophils and monocytes were also elevated in the blood of the NSG-3GS mice where, despite the observed “saturation” of the marrow, there was a linear dose-response in the number of human neutrophils and monocytes present in the blood with increasing CD34+ cells infused. These findings are consistent with the reported activities of these molecules in vitro and in patients suggesting their physiological relevance in this murine xenograft model. We next utilized this assay to characterize the cells responsible for the neutrophil/monocyte and platelet repopulating activities detected in NSG-3GS mice. Preliminary assessment of the CD34+CD45RA- population on the basis of CD123 (IL-3 receptor alpha chain) expression indicates that the CD123+ fraction is enriched for short term (3-week) neutrophil/monocyte repopulating activity, while the CD123- fraction is enriched for short term (3-week) platelet repopulating activity. In summary, NSG-3GS mice significantly enhance the output of human cells with short term human myeloid repopulating ability thereby enabling neutrophil/monocyte outputs as well as platelet outputs to be assessed by analysis of peripheral blood samples. We have also used this tool to obtain evidence that these two outputs are derived from distinct cell types. Direct quantification of these may add to future predictions of graft quality. Disclosures: No relevant conflicts of interest to declare.

A technic to simplify hemodynamic evaluation during atrial fibrillation by right ventricular pacing is described. The hemodynamic data obtained at cardiac catheterization during atrial fibrillation with and without right ventricular pacing were compared in 3 cases. Evaluation of data is simplified and valvular gradients can be measured with greater precision when the wide variability in duration of cardiac cycle and intracardiac pressures usually present during atrial fibrillation are abolished by ventricular pacing.

A new index of selection, α, is presented. It measures the degree to which a predator is more likely to take one kind of prey rather than another, and thus provides a direct estimation of predator behavior. It is unaffected by sampling in a finite population, this being the advantage over other methods of estimating selectivity. If selection is independent of the frequency of prey types, α is constant. Under frequency-dependent predation α changes with frequency. Measures of the frequency-independent and the frequency-dependent components may be obtained when both kinds of selection act together. The method of analysis has been applied to data on the behavior of Japanese quail, Coturnix coturnix japonica, presented with red and blue pastry food under controlled conditions. Variability in response between individual pairs of birds is large. A strong red preference is nevertheless exhibited, and also a distinct tendency for the birds to eat more than a proportional amount of whichever color was the most com...

The prolongation of A-V transmission time that occurs with increasing rates of right atrial pacing has been used to study the effect of ouabain and of ouabain plus atropine on A-V nodal conduction in 14 patients with normal A-V nodes. Drugs were injected into the superior vena cava or pulmonary artery over a 1- to 2-min period. Atropine in a dose of 0.02 mg/kg shortened A-V conduction time by an average of 0.112 sec; ouabain alone in a dose of 0.01 mg/kg lengthened A-V conduction time by 0.053 sec. Ouabain given after large doses of atropine did not prolong A-V conduction at any paced rate. Thus, in these resting patients with normal A-V nodal function, usual digitalizing dosages of ouabain did not have an extravagal or antiadrenergic effect on A-V nodal conduction.

The critical human cells that produce neutrophils and platelets within 3 weeks in recipients of hematopoietic transplants are thought to produce these mature blood cells with the same kinetics in sublethally irradiated immunodeficient mice. Quantification of their numbers indicates their relative underrepresentation in cord blood (CB), likely explaining the clinical inadequacy of single CB units in rescuing hematopoiesis in myelosuppressed adult patients. We here describe that exposure of CD34(+) CB cells ex vivo to growth factors that markedly expand their numbers and colony-forming cell content also rapidly (within 24 hours) produce a significant and sustained net loss of their original short-term repopulating activity. This loss of short-term in vivo repopulating activity affects early platelet production faster than early neutrophil output, consistent with their origin from distinct input populations. Moreover, this growth factor-mediated loss is not abrogated by published strategies to increase progenitor homing despite evidence that the effect on rapid neutrophil production is paralleled in time and amount by a loss of the homing of their committed clonogenic precursors to the bone marrow. These results highlight the inability of in vitro or phenotype assessments to reliably predict clinical engraftment kinetics of cultured CB cells.

1. In an automatic echo detection and classification system wherein sonar equipment transmits pulses toward a target in predetermined and equal time intervals and receives background, dopplered and undopplered echoes which are produced as video output and audio output, the improvement which comprises means for establishing a non-directional background reference signal voltage from the video output, means for separating the audio output into dopplered signal voltage outside the reverberation band and undopplered signal voltage within the reverberation band, means for algebraically adding the background reference signal voltage to both the dopplered and undopplered signal voltages, a first amplifier for the resultant dopplered signal voltage, a target indicator alarm, a first energizing circuit therefor operative by the amplified dopplered signal voltage to indicate that a target has been located, means for obtaining from the resultant undopplered signal voltage a selected signal voltage above a predetermined level, a first gate normally in closed position, a first multivibrator therefor and operable by the selected undopplered signal voltage to move to open position for a predetermined interval to pass a first echo of a predetermined duration, a second gate normally in open position for passing the first echo after it has passed the first gate, a second multivibrator for the second gate operative to close the second gate for a predetermined period, an amplifier for the undopplered signal voltage of the first echo after it has passed through both the first and second gates, a second energizing circuit for the target alarm, said second circuit including the second multivibrator, and being operative to energize the second multivibrator by the amplified undopplered signal voltage of the first echo after it has passed through the second gate and after the predetermined period for which the second gate is closed and a second echo appears, which is substantially within the same range as the first echo, the second gate will be open for passing of the second echo to operate the target indicator alarm.

To the Editor.— The following report is presented to emphasize the increased risk for fire that exists within the Cardiac Care Unit. Report of a Case.— A 73-year-old woman with arteriosclerotic heart disease was admitted with a four-day history of increasing dyspnea. On the seventh hospital day, cardiac arrest occurred. Cardiopulmonary resuscitation was successful and she was sent immediately to the Cardiac Care Unit. Oxygen was administered by face mask and the electrocardiographic monitor showed atrial fibrillation, followed in rapid sequence by supraventricular tachycardia, ventricular tachycardia, and ventricular fibrillation. Defibrillation was promptly attempted with the use of a 400 w-sec discharge delivered via paddles applied to the anterior chest wall. When the first defibrillator discharge did not succeed in restoring a satisfactory rhythm, an intravenous injection of lidocaine, 100 mg, was given and a second 400 w-sec defibrillator discharge was delivered in similar fashion. There was a spark from one