Tangential extreme ultraviolet and soft x-ray diagnostic system for time-resolved temperature measurement on the High Beta Tokamak-Extended Pulse.

The High Beta Tokamak-Extended Pulse has recently incorporated a tangential multi-energy extreme ultraviolet and soft x-ray diagnostic system. This system enables measurements of the electron temperature and the examination of mode dynamics within the tokamak. While other systems have been built for poloidal views over similar temperature ranges, this is the first multi-energy tangential-view system designed to work in a temperature range below 200 eV in a tokamak. To facilitate these measurements, a filter wheel comprising five distinct groups of dual-filters has been developed and implemented. By employing a combination of 0.1 µm aluminum and 0.2 µm titanium filters, the system allows estimation of electron temperature profiles through reconstruction of the emission profile using the standard "double-foil" technique. The influence of impurities and filter oxide layers on measurement outcomes is examined. Results reveal that, while the absolute electron temperature values may exhibit some deviations, key characteristics like the electron temperature profile shape and inversion radius during sawtooth events remain consistent. This consistency confirms the system's suitability for core plasma studies. This system has proven effective in detecting and analyzing internal magnetohydrodynamic phenomena, such as sawteeth.

Mode rotation control in a tokamak with a feedback-driven biased electrode.

Rotation of the plasma and MHD modes in tokamaks has been shown to stabilize resistive wall and tearing modes as well as improve confinement through suppression of edge turbulence. In this work, we control mode rotation with a biased electrode inserted into the plasma of the High Beta Tokamak-Extended Pulse's facility in conjunction with its active GPU (Graphical Processing Unit) feedback system. We first characterize a negative linear relationship between the electrode voltage and mode rotation. Using this relationship, we design, simulate, and implement a proof-of-concept, GPU-based active-control system, which shows consistent success in controlling mode rotation in both feedforward and feedback operation. Controllability is limited by operating conditions, the electrode's voltage range, and by the electrode's proximity to the vessel's walls. The final control system has a 15 µs cycle time, but the addition of various signal filters results in a full cycle latency of 200 µs.

Design and installation of a ferromagnetic wall in tokamak geometry.

Low-activation ferritic steels are leading material candidates for use in next-generation fusion development experiments such as a prospective component test facility and DEMO power reactor. Understanding the interaction of plasmas with a ferromagnetic wall will provide crucial physics for these facilities. In order to study ferromagnetic effects in toroidal geometry, a ferritic wall upgrade was designed and installed in the High Beta Tokamak-Extended Pulse (HBT-EP). Several material options were investigated based on conductivity, magnetic permeability, vacuum compatibility, and other criteria, and the material of choice (high-cobalt steel) is characterized. Installation was accomplished quickly, with minimal impact on existing diagnostics and overall machine performance, and initial results demonstrate the effects of the ferritic wall on plasma stability.

Hypoxia inducible factor (HIF)-2α accelerates disease progression in mouse models of leukemia and lymphoma but is not a poor prognosis factor in human AML.

Hypoxia-inducible factor (HIF)-1α accumulation promotes hematopoietic stem cells' quiescence and is necessary to maintain their self-renewal. However, the role of HIF-2α in hematopoietic cells is less clear. We investigated the role of HIF-2α in leukemia and lymphoma cells. HIF-2α expression was high in subsets of human and mouse leukemia and lymphoma cells, whereas it was low in normal bone marrow leukocytes. To investigate the role of HIF-2α, we transduced human HIF-2α cDNA in mouse syngeneic models of myeloid preleukemia and a transgenic model of B lymphoma. Ectopic expression of HIF-2α accelerated leukemia cell proliferation in vitro. Mice transplanted with cells transduced with HIF-2α died significantly faster of leukemia or B lymphoma than control mice transplanted with empty vector-transduced cells. Conversely, HIF-2α knockdown in human myeloid leukemia HL60 cells decreased proliferation in vitro and significantly prolonged animal survival following transplantation. In human acute myeloid leukemia (AML), HIF-2α mRNA was significantly elevated in several subsets such as the t(15;17), inv(16), complex karyotype and favorable cytogenetic groups. However, patients with high HIF-2α expression had a trend to higher disease-free survival in univariate analysis. The different effects of HIF-2α overexpression in mouse models of leukemia and human AML illustrates the complexity of this mutliclonal disease.

HIF-1α is required for hematopoietic stem cell mobilization and 4-prolyl hydroxylase inhibitors enhance mobilization by stabilizing HIF-1α.

Many patients with hematological neoplasms fail to mobilize sufficient numbers of hematopoietic stem cells (HSCs) in response to granulocyte colony-stimulating factor (G-CSF) precluding subsequent autologous HSC transplantation. Plerixafor, a specific antagonist of the chemokine receptor CXCR4, can rescue some but not all patients who failed to mobilize with G-CSF alone. These refractory poor mobilizers cannot currently benefit from autologous transplantation. To discover alternative targetable pathways to enhance HSC mobilization, we studied the role of hypoxia-inducible factor-1α (HIF-1α) and the effect of HIF-1α pharmacological stabilization on HSC mobilization in mice. We demonstrate in mice with HSC-specific conditional deletion of the Hif1a gene that the oxygen-labile transcription factor HIF-1α is essential for HSC mobilization in response to G-CSF and Plerixafor. Conversely, pharmacological stabilization of HIF-1α with the 4-prolyl hydroxylase inhibitor FG-4497 synergizes with G-CSF and Plerixafor increasing mobilization of reconstituting HSCs 20-fold compared with G-CSF plus Plerixafor, currently the most potent mobilizing combination used in the clinic.

Fast, multi-channel real-time processing of signals with microsecond latency using graphics processing units.

Fast, digital signal processing (DSP) has many applications. Typical hardware options for performing DSP are field-programmable gate arrays (FPGAs), application-specific integrated DSP chips, or general purpose personal computer systems. This paper presents a novel DSP platform that has been developed for feedback control on the HBT-EP tokamak device. The system runs all signal processing exclusively on a Graphics Processing Unit (GPU) to achieve real-time performance with latencies below 8 µs. Signals are transferred into and out of the GPU using PCI Express peer-to-peer direct-memory-access transfers without involvement of the central processing unit or host memory. Tests were performed on the feedback control system of the HBT-EP tokamak using forty 16-bit floating point inputs and outputs each and a sampling rate of up to 250 kHz. Signals were digitized by a D-TACQ ACQ196 module, processing done on an NVIDIA GTX 580 GPU programmed in CUDA, and analog output was generated by D-TACQ AO32CPCI modules.

The novel CXCR4 antagonist POL5551 mobilizes hematopoietic stem and progenitor cells with greater efficiency than Plerixafor.

Mobilized blood has supplanted bone marrow (BM) as the primary source of hematopoietic stem cells for autologous and allogeneic stem cell transplantation. Pharmacologically enforced egress of hematopoietic stem cells from BM, or mobilization, has been achieved by directly or indirectly targeting the CXCL12/CXCR4 axis. Shortcomings of the standard mobilizing agent, granulocyte colony-stimulating factor (G-CSF), administered alone or in combination with the only approved CXCR4 antagonist, Plerixafor, continue to fuel the quest for new mobilizing agents. Using Protein Epitope Mimetics technology, a novel peptidic CXCR4 antagonist, POL5551, was developed. In vitro data presented herein indicate high affinity to and specificity for CXCR4. POL5551 exhibited rapid mobilization kinetics and unprecedented efficiency in C57BL/6 mice, exceeding that of Plerixafor and at higher doses also of G-CSF. POL5551-mobilized stem cells demonstrated adequate transplantation properties. In contrast to G-CSF, POL5551 did not induce major morphological changes in the BM of mice. Moreover, we provide evidence of direct POL5551 binding to hematopoietic stem and progenitor cells (HSPCs) in vivo, strengthening the hypothesis that CXCR4 antagonists mediate mobilization by direct targeting of HSPCs. In summary, POL5551 is a potent mobilizing agent for HSPCs in mice with promising therapeutic potential if these data can be corroborated in humans.

In situ "artificial plasma" calibration of tokamak magnetic sensors.

A unique in situ calibration technique has been used to spatially calibrate and characterize the extensive new magnetic diagnostic set and close-fitting conducting wall of the High Beta Tokamak-Extended Pulse (HBT-EP) experiment. A new set of 216 Mirnov coils has recently been installed inside the vacuum chamber of the device for high-resolution measurements of magnetohydrodynamic phenomena including the effects of eddy currents in the nearby conducting wall. The spatial positions of these sensors are calibrated by energizing several large in situ calibration coils in turn, and using measurements of the magnetic fields produced by the various coils to solve for each sensor's position. Since the calibration coils are built near the nominal location of the plasma current centroid, the technique is referred to as an "artificial plasma" calibration. The fitting procedure for the sensor positions is described, and results of the spatial calibration are compared with those based on metrology. The time response of the sensors is compared with the evolution of the artificial plasma current to deduce the eddy current contribution to each signal. This is compared with simulations using the VALEN electromagnetic code, and the modeled copper thickness profiles of the HBT-EP conducting wall are adjusted to better match experimental measurements of the eddy current decay. Finally, the multiple coils of the artificial plasma system are also used to directly calibrate a non-uniformly wound Fourier Rogowski coil on HBT-EP.

Hematopoietic stem cell mobilizing agents G-CSF, cyclophosphamide or AMD3100 have distinct mechanisms of action on bone marrow HSC niches and bone formation.

The CXCR4 antagonist AMD3100 is progressively replacing cyclophosphamide (CYP) as adjuvant to granulocyte colony-stimulating factor (G-CSF) to mobilize hematopoietic stem cells (HSC) for autologous transplants in patients who failed prior mobilization with G-CSF alone. It has recently emerged that G-CSF mediates HSC mobilization and inhibits bone formation via specific bone marrow (BM) macrophages. We compared the effect of these three mobilizing agents on BM macrophages, bone formation, osteoblasts, HSC niches and HSC reconstitution potential. Both G-CSF and CYP suppressed niche-supportive macrophages and osteoblasts, and inhibited expression of endosteal cytokines resulting in major impairment of HSC reconstitution potential remaining in the mobilized BM. In sharp contrast, although AMD3100 was effective at mobilizing HSC, it did not suppress osteoblasts, endosteal cytokine expression or reconstitution potential of HSC remaining in the mobilized BM. In conclusion, although G-CSF, CYP and AMD3100 efficiently mobilize HSC into the blood, their effects on HSC niches and bone formation are distinct with both G-CSF and CYP targeting HSC niche function and bone formation, whereas AMD3100 directly targets HSC without altering niche function or bone formation.

How we mobilize haemopoietic stem cells.

Mobilization and collection of haemopoietic stem and progenitor cells (HSPC) is the cornerstone of autologous and allogeneic stem cell transplantation for a wide variety of haematological and some non-haematological malignancies. Centres providing this service face the challenge of optimizing the likelihood of successful collection of transplantable doses of cells, while maximizing the efficiency of the apheresis unit and minimizing the risk of toxicity as well as mobilization failure. Recent developments in the understanding of the molecular mechanisms of mobilization have led to the emergence of novel strategies for HSPC mobilization, which may assist in meeting these imperatives. The task for clinicians is how to incorporate the use of these strategies into practice, in the light of emerging evidence for efficacy and safety of these agents. Herein, the literature is reviewed, and a proposed algorithm for HSPC mobilization is presented.

A high-power spatial filter for Thomson scattering stray light reduction.

The Thomson scattering diagnostic on the High Beta Tokamak-Extended Pulse (HBT-EP) is routinely used to measure electron temperature and density during plasma discharges. Avalanche photodiodes in a five-channel interference filter polychromator measure scattered light from a 6 ns, 800 mJ, 1064 nm Nd:YAG laser pulse. A low cost, high-power spatial filter was designed, tested, and added to the laser beamline in order to reduce stray laser light to levels which are acceptable for accurate Rayleigh calibration. A detailed analysis of the spatial filter design and performance is given. The spatial filter can be easily implemented in an existing Thomson scattering system without the need to disturb the vacuum chamber or significantly change the beamline. Although apertures in the spatial filter suffer substantial damage from the focused beam, with proper design they can last long enough to permit absolute calibration.

The endosteal 'osteoblastic' niche and its role in hematopoietic stem cell homing and mobilization.

The concept of hematopoietic stem cell (HSC) niche was formulated in 1978, but HSC niches remained unidentified for the following two decades largely owing to technical limitations. Sophisticated live microscopy techniques and genetic manipulations have identified the endosteal region of the bone marrow (BM) as a preferential site of residence for the most potent HSC - able to reconstitute in serial transplants - with osteoblasts and their progenitors as critical cellular elements of these endosteal niches. This article reviews the path to the discovery of these endosteal niches (often called 'osteoblastic' niches) for HSC, what cell types contribute to these niches with their known physical and biochemical features. In the past decade, a first wave of research uncovered many mechanisms responsible for HSC homing to, and mobilization from, the whole BM tissue. However, the recent discovery of endosteal HSC niches has initiated a second wave of research focusing on the mechanisms by which most primitive HSC lodge into and migrate out of their endosteal niches. The second part of this article reviews the current knowledge of the mechanisms of HSC lodgment into, retention in and mobilization from osteoblastic niches.

Mobilization of bone marrow-derived progenitors.

Bone marrow (BM) is a source of various stem and progenitor cells in the adult, and it is able to regenerate a variety of tissues following transplantation. In the 1970s the first BM stem cells identified were hematopoietic stem cells (HSCs). HSCs have the potential to differentiate into all myeloid (including erythroid) and lymphoid cell lineages in vitro and reconstitute the entire hematopoietic and immune systems following transplantation in vivo. More recently, nonhematopoietic stem and progenitor cells have been identified that can differentiate into other cell types such as endothelial progenitor cells (EPCs), contributing to the neovascularization of tumors as well as ischemic tissues, and mesenchymal stem cells (MSCs), which are able to differentiate into many cells of ectodermal, endodermal, and mesodermal origins in vitro as well as in vivo. Following adequate stimulation, stem and progenitor cells can be forced out of the BM to circulate into the peripheral blood, a phenomenon called "mobilization." This chapter reviews the molecular mechanisms behind mobilization and how these have led to the various strategies employed to mobilize BM-derived stem and progenitor cells in experimental and clinical settings. Mobilization of HSCs will be reviewed first, as it has been best-explored--being used extensively in clinics to transplant large numbers of HSCs to rescue cancer patients requiring hematopoietic reconstitution--and provides a paradigm that can be generalized to the mobilization of other types of BM-derived stem and progenitor cells in order to repair other tissues.

Epitope recognition of antibodies that define the sialomucin, endolyn (CD164), a negative regulator of haematopoiesis.

Endolyn (CD164) is a sialomucin that functions as an adhesion molecule and a negative regulator of CD34+ CD38- human haematopoietic precursor cell proliferation. The 105A5 and 103B2/9E10 CD164 monoclonal antibodies (mAbs), which act as surrogate ligands, recognize distinct glycosylation-dependent classes I and II epitopes located on domain I of the native and recombinant CD164 proteins. Here, we document five new CD164 mAbs, the 96 series, that rely on conformational integrity, but not glycosylation, of exons 2- and 3-encoded CD164 domains, thereby resembling the class III mAbs, N6B6 and 67D2. Although all the 96 series class III mAbs labelled both the 105A5+ and 103B2/9E10+ cells, cross-competition and immunoblotting studies allow them to be categorized into two distinct class III subgroups, i.e. the N6B6-like subgroup that only recognizes 80-100 kDa proteins and the 67D2-like subgroup that also recognizes a higher molecular weight (>220 kDa) form. To more closely define the reactivity patterns of mAbs to the classes I and II epitopes, the global glycosylation patterns of the soluble human (h) CD164 proteins were determined using lectin binding, high-performance liquid chromatography (HPLC) and mass spectrometry. hCD164 recombinant proteins bound to the lectins, Galanthus nivalis agglutinin, Datura stramonium agglutinin, Sambucus nigra agglutinin, Maackia amurensis agglutinin and peanut agglutinin, indicating the presence of high mannose and complex N-glycans, in addition to core 1 O-glycans (the Tn antigen) and alpha2-3 and alpha2-6 sialic acid moieties. Our HPLC and mass spectrometry results revealed both high mannose and complex N-glycosylation with various numbers of branches increasing the complexity of the glycosylation pattern. Most O-glycans were small, core 1 or 2 based. High levels of sialylation in alpha2-3 and alpha2-6 linkages, without sialyl-Lewis X, indicate that the majority of these hCD164 recombinant proteins are unable to bind to selectins in our assay system, but may interact with Siglec molecules.

Vascular cell adhesion molecule-1 (CD106) is cleaved by neutrophil proteases in the bone marrow following hematopoietic progenitor cell mobilization by granulocyte colony-stimulating factor.

Mobilized progenitor cells currently represent the most commonly used source of hematopoietic progenitor cells (HPCs) to effect hematopoietic reconstitution following myeloablative chemotherapies. Despite their widespread use, the molecular mechanisms responsible for the enforced egress of HPCs from the bone marrow (BM) into the circulation in response to mobilizing agents such as cytokines remain to be determined. Results of this study indicate that expression of vascular cell adhesion molecule-1 (VCAM-1) is strongly reduced in vivo in the BM during HPC mobilization by granulocyte colony-stimulating factor (G-CSF) and stem cell factor. Two serine proteases, namely, neutrophil elastase and cathepsin G, were identified, which cleave VCAM-1 and are released by neutrophils accumulating in the BM during the course of immobilization induced by G-CSF. The proposal is made that an essential step contributing to the mobilization of HPCs is the proteolytic cleavage of VCAM-1 expressed by BM stromal cells, an event triggered by the degranulation of neutrophils accumulating in the BM in response to the administration of G-CSF.

Mucin-like molecules as modulators of the survival and proliferation of primitive hematopoietic cells.

Current data suggest that interplay between two classes of molecules contributes to the regulation of hematopoiesis: hematopoietic growth factors, which regulate the survival, proliferation, and development of primitive hematopoietic cells and cell adhesion molecules (CAMs), which are responsible for the localization of hematopoiesis to the bone marrow (BM) and for mediating physical association between developing hematopoietic cells and marrow stromal tissue. A range of cell surface molecules representing several CAM superfamilies including integrins, selectins, the immunoglobulin gene superfamily and an emerging family of mucin-like molecules (the sialomucins) are involved in supporting cell-cell and cell-extracellular matrix (ECM) interactions between primitive hematopoietic cells and the stromal cell-mediated hematopoietic microenvironment (HM) of the bone marrow. There is abundant evidence in non-hematopoietic tissues that CAMs are signalling molecules which participate in a range of signal transduction events important not only for regulating cell adhesion and motility, but also for cell growth and survival. Although the signalling functions of CAMs have not been studied extensively in primitive hematopoietic progenitors (HPCs), extrapolation from burgeoning data in other systems is consistent with the hypothesis that hematopoiesis within the BM is regulated by interaction between signals generated locally by CAMs and those elicited by cytokines. Evidence in support of this notion was initially provided by studies on normal HPCs demonstrating cross-talk between members of the integrin superfamily and cytokine receptors. In this article we review recent reports that mucin-like molecules are also signalling molecules on primitive hematopoietic cells and that the signals they deliver potently inhibit hematopoiesis.

CD164 monoclonal antibodies that block hemopoietic progenitor cell adhesion and proliferation interact with the first mucin domain of the CD164 receptor.

The novel sialomucin, CD164, functions as both an adhesion receptor on human CD34+ cell subsets in bone marrow and as a potent negative regulator of CD34+ hemopoietic progenitor cell proliferation. These diverse effects are mediated by at least two functional epitopes defined by the mAbs, 103B2/9E10 and 105A5. We report here the precise epitope mapping of these mAbs together with that of two other CD164 mAbs, N6B6 and 67D2. Using newly defined CD164 splice variants and a set of soluble recombinant chimeric proteins encoded by exons 1-6 of the CD164 gene, we demonstrate that the 105A5 and 103B2/9E10 functional epitopes map to distinct glycosylated regions within the first mucin domain of CD164. The N6B6 and 67D2 mAbs, in contrast, recognize closely associated and complex epitopes that rely on the conformational integrity of the CD164 molecule and encompass the cysteine-rich regions encoded by exons 2 and 3. On the basis of their sensitivities to reducing agents and to sialidase, O-sialoglycoprotease, and N-glycanase treatments, we have characterized CD164 epitopes and grouped them into three classes by analogy with CD34 epitope classification. The class I 105A5 epitope is sialidase, O-glycosidase, and O-sialoglycoprotease sensitive; the class II 103B2/9E10 epitope is N-glycanase, O-glycosidase, and O-sialoglycoprotease sensitive; and the class III N6B6 and 67D2 epitopes are not removed by such enzyme treatments. Collectively, this study indicates that the previously observed differential expression of CD164 epitopes in adult tissues is linked with cell type specific post-translational modifications and suggests a role for epitope-associated carbohydrate structures in CD164 function.

Functionally defined CD164 epitopes are expressed on CD34(+) cells throughout ontogeny but display distinct distribution patterns in adult hematopoietic and nonhematopoietic tissues.

Three distinct classes of epitopes on human CD164 have been identified. Two of these, recognized by the monoclonal antibodies 105A5 and 103B2/9E10, are the CD164 class I and class II functionally defined epitopes, which cooperate to regulate adhesion and proliferation of CD34(+) cell subsets. In this article, we demonstrate that these 2 CD164 epitopes are expressed on CD34(+) cells throughout ontogeny, in particular on CD34(+ )cell clusters associated with the ventral floor of the dorsal aorta in the developing embryo and on CD34(+) hematopoietic precursor cells in fetal liver, cord blood, and adult bone marrow. While higher levels of expression of these CD164 epitopes occur on the more primitive AC133(hi)CD34(hi)CD38(lo/-) cell population, they also occur on most cord blood Lin(-)CD34(lo/-)CD38(lo/- )cells, which are potential precursors for the AC133(hi)CD34(hi)CD38(lo/-) subset. In direct contrast to these common patterns of expression on hematopoietic precursor cells, notable differences in expression of the CD164 epitopes were observed in postnatal lymphoid and nonhematopoietic tissues, with the class I and class II CD164 epitopes generally exhibiting differential and often reciprocal cellular distribution patterns. This is particularly striking in the colon, where infiltrating lymphoid cells are CD164 class I-positive but class II-negative, while epithelia are weakly CD164 class II-positive. Similarly, in certain lymphoid tissues, high endothelial venules and basal and subcapsular epithelia are CD164 class II-positive, while lymphoid cells are CD164 class I-positive. It therefore seems highly likely that these CD164 class I and II epitopes will mediate reciprocal homing functions in these tissue types.

PSGL-1-mediated adhesion of human hematopoietic progenitors to P-selectin results in suppression of hematopoiesis.

Cellular interactions are critical for the regulation of hematopoiesis. The sialomucin PSGL-1/CD162 mediates the attachment of mature leukocytes to P-selectin. We now show that PSGL-1 also functions as the sole receptor for P-selectin on primitive human CD34+ hematopoietic progenitor cells (HPC). More importantly, ligation of PSGL-1 by immobilized or soluble ligand or anti-PSGL-1 antibody results in a profound suppression of HPC proliferation stimulated by potent combinations of early acting hematopoietic growth factors. These data demonstrate an unanticipated but extremely marked growth-inhibitory effect of P-selectin on hematopoiesis and provide direct evidence that PSGL-1, in addition to its well-documented role as an adhesion molecule on mature leukocytes, is a potent negative regulator of human hematopoietic progenitors.