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This article addresses the debate about the correct application of Green-Kubo expressions for transport coefficients from dissipative particle dynamics simulations. We demonstrate that the Green-Kubo expressions are valid provided that (i) the dynamic model conserves the physical property, whose transport is studied, and (ii) the fluctuations satisfy detailed balance. As a result, the traditional expressions used in molecular dynamics can also be applied to dissipative particle dynamics simulations. However, taking the calculation of the shear viscosity as a paradigmatic example, a random contribution, whose strength scales as 1/δt1/2, with δt the time-step, can cause difficulties if the stress tensor is not separated into the different contributions. We compare our expression to that of Ernst and Brito (M. H. Ernst and R. Brito, Europhys. Lett., 2006, 73, 183-189), which arises from a diametrically different perspective. We demonstrate that the two expressions are completely equivalent and find exactly the same result both analytically and numerically. We show that the differences are not due to the lack of time-reversibility but instead from a pre-averaging of the random contributions. Despite the overall validity of Green-Kubo expressions, we find that the Einstein-Helfand relations (D. C. Malaspina et al. Phys. Chem. Chem. Phys., 2023, 25, 12025-12040) do not suffer from the need to decompose the stress tensor and can readily be used with a high degree of accuracy. Consequently, Einstein-Helfand relations should be seen as the preferred method to calculate transport coefficients from dissipative particle dynamics simulations.

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In this article we demonstrate that contrary to general belief, the standard Einstein-Helfand (EH) formulas are valid for the evaluation of transport coefficients of systems containing dissipative and random forces provided that for these mesoscopic systems: (i) the corresponding conservation laws are satisfied, and (ii) the transition probabilities satisfy detailed balance. Dissipative particle dynamics (DPD) and energy-conserving DPD methods (DPDE), for instance, are archetypical of such mesoscopic approaches satisfying these properties. To verify this statement, we have derived a mesoscopic heat flux form for the DPDE method, suitable for the calculation of the thermal conductivity from an EH expression. We have compared EH measurements against non-equilibrium simulation values for different scenarios, including many-body potentials, and have found excellent agreement in all cases. The expressions are valid notably for systems with density- and temperature-dependent potentials, such as the recently developed generalised DPDE method (GenDPDE) [Avalos et al., Phys. Chem. Chem. Phys., 2019, 21, 24891]. We thus demonstrate that traditional EH formulas in equilibrium simulations can be widely used to obtain transport coefficients, provided that the appropriate expression for the associated flux is used.

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To find a parameter to predict the quality of collected mobilized CD34+ blood as hemopoietic reconstituting cells, the ratio of CFU-GM to CD34+ cells was examined. One hundred six consecutive patients who underwent blood stem cell transplantation at the University of Rochester from 01/01/99 to 12/31/99 were examined retrospectively for the number of days to reach an absolute neutrophil count of 500 or 1000 cells/microl and an absolute platelet count of 20,000 or 50,000 cells/microl without transfusion support as measures of engraftment. Linear regression analyses were conducted to determine factors influencing engraftment. The number of CD34+ cells/kg and CFU-GM/kg correlated highly with the number of nucleated blood cells/kg. In this population, in which 90% of patients received >2 x 10(6) CD34+ cells/kg, neither the number of CD34+ cells/kg nor the number of CFU-GM/kg correlated with the time to engraftment as judged by neutrophil or platelet levels. In contrast, the lower the ratio of CFU-GM to CD34+ cells, the more rapid the reconstitution of platelets to 20,000/microl (P = 0.03) and 50,000/microl (P = 0.02). Thus, a lower ratio of the CFU-GM/CD34+ appended to reflect a greater number of hematopoietic reconstituting cells in the blood cell collection. The CFU-GM/CD34+ ratio is an apparent predictor of earlier platelet engraftment, suggesting that the ratio reflects the engraftment potential of mobilized donor progenitor cells.

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Thrombotic thrombocytopenic purpura pathologically consists of a thrombotic microangiopathy that classically spares lung tissues. We describe a case of TTP that presented as a pulmonary-renal syndrome. In reviewing the international literature, pulmonary involvement is not as rare as once was thought, and the diagnosis of TTP should be considered in the differential diagnosis of pulmonary-renal syndromes.

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The highly packed cell density and the three-dimensional structure in the hematopoietic compartment of bone marrow facilitate cell-to-cell and cell-to-matrix interactions known to be important for hematopoietic activities. To provide a similar environment in vitro, we developed a long-term bone marrow culture (LTBMC) system, continuously perfused with Dexter's medium, employing packed, highly porous bovine collagen microspheres as the matrix support for marrow cell growth. Using murine bone marrow as a model, we found that the culture system differed from the conventional flask culture in the following ways: 1) as revealed by the electron microscopy, the bone marrow cells in the culture system grew in a three-dimensional configuration, similar to that in vivo, 2) the cell output from the culture system at 37 degrees C was virtually the same as that at 33 degrees C, and 3) in the absence of exogenous growth factors, except those in the serum, the culture system produced lymphoid cells and all stages of committed cells (i.e., erythrocytes, granulocytes, macrophages, and megakaryocytes), thus indicating multilineal differentiation of the hematopoietic stem cells. Furthermore, cell clusters resembling erythroblastic islands were observed in the absence of exogenous erythropoietin (Epo). The culture system appears to provide a different microenvironment than that of the flask culture and may be used as an alternative model for hematopoiesis.

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The pleiotropic nature of malignant fibrous histiocytomas (MFH) is manifested as mixed cellular infiltrates consisting of myofibroblasts, histiomonocytes, and neutrophils. We detail in this report the phenotypic characteristics of the human fibrous histiocytoma giant cell tumor (GCT) cell line that establish its mesenchymal origin. The latter is underscored by the ability of GCT cells to express mRNA for transforming growth factor beta (TGF-beta) as well as both A and B chains of platelet-derived growth factor (PDGF). GCT cells also support the binding of CD34+ cells, but less efficiently than do normal marrow stromal cells. Since cytokines elaborated by MFH may mediate in part the recruitment of monocytes and neutrophils into tumor-infiltrated tissues, we have determined the cytokine repertoire of the GCT cell line, already known for its ability to elaborate colony-stimulating factors (CSFs) and interleukin-1 (IL-1). GCT cells express IL-1 alpha, IL-1 beta, IL-6, macrophage colony-stimulating factor (M-CSF or CSF-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and IL-8. No detectable mRNA for IL-3, IL-4, IL-7, and tumor necrosis factor-alpha (TNF-alpha) was detected in GCT cells by polymerase chain reaction (PCR). Expression of cytokine mRNAs was responsive to agents such as dexamethasone (dex), 12-O-tetradecanoyl phorbol 13-acetate (phorbol diester or TPA), and TNF-alpha. Thus, this cell line provides a useful model for understanding the pathobiology of MFH and hematopoietic progenitor interactions with mesenchymal/stromal cells.

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We have found that GM-CSF and DMSO have antagonistic effects on the proliferation but not maturation of asynchronously growing HL-60 cells such that growth in the presence of both more closely resembles normal hematopoiesis (Brennan et al., J. Cell Physiol. 132:246, 1987). Studies were undertaken to determine whether or not the agents affected the same mitogenic pathway and locus in the cell cycle. HL-60 populations containing at least 90% G1 cells were obtained by centrifugal elutriation, exposed to 100 u/ml recombinant human GM-CSF and/or 0-1.25% DMSO, and phosphoprotein changes quantified on autoradiograms of [32P]-orthophosphate-labeled cell proteins separated by giant 2-D gel electrophoresis. Results were correlated with 1) intracellular pH, determined by measurement of BCECF fluorescence; 2) [32P]-orthophosphate uptake; 3) cell cycle progression, determined by flow quantitation of DNA content in mithramycin or propidium iodide-stained cells; and 4) growth, determined by cell volume and concentration. GM-CSF stimulated and DMSO inhibited the GM-CSF-stimulated phosphorylation of 1 protein (approximately 65 kDa, p.i. 5.6) within 2 min of exposure. These effects were sustained through G1, not associated with changes in intracellular pH, and preceded similar antagonistic effects on phosphate uptake (15-30 minutes), cell volume change (16-24 hr), and cell concentration increase (28-32 hr). GM-CSF accelerated and DMSO inhibited G1 to S transit with the most marked antagonism observed in the second cycle following synchronization (28 to 40 hrs). Cell maturation (morphology, NBT reduction) was dominated by DMSO and not antagonized by GM-CSF. We have identified p65 as the nuclear intermediate filament protein, lamin B, on the basis of its locus on gels and its binding of a monoclonal antibody to intermediate filaments and antiserum to human lamin B on immunoblots. These studies suggest that at least part of the GM-CSF-DMSO antagonism is exerted through the same mitogenic pathway, that a major locus of cytokinetic effect is on G1 to S transit, and that nuclear envelope protein phosphorylation is an important early event.

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Adhesive interactions between CD34+ myeloid progenitors, cytomatrix components, and marrow fibroblast and stromal monolayers are described and compared to the binding interactions of the CD34+ myeloid leukemic cell lines KG1a and KG1. Both normal precursors and their leukemic counterparts showed adhesion to marrow stroma and fibroblasts. CD34+ myeloid progenitors bound to the extracellular matrices of marrow stromal cell and fibroblast monolayers and to laminin and fibronectin to a lesser extent than to cellular stromal layers. These adhesive interactions were not inhibited by polyclonal antibodies to laminin or fibronectin, nor by 1 mM Arg-Gly-Asp-Ser (RGDS)-containing peptides. Also, although both normal and leukemic cells expressed the CD18 antigen, binding of these cells to stroma was not inhibited by blocking anti-CD18 monoclonal antibodies. Finally, KG1a adhesion was not blocked in the presence of anti-CD54 (ICAM) antibody, nor was it blocked when galactosyl or mannosyl pyranosides were added. KG1a binding was trypsin sensitive and enhanced in the presence of neuraminidase. These studies serve to characterize adhesive properties of normal and leukemic myeloid progenitors and begin to establish interactions important for the lodgement of early progenitor cells in human marrow.

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The effects of macrophage colony-stimulating factor (M-CSF or CSF-1) on the survival, proliferation, maturation and activation of human blood monocytes were examined. M-CSF (100-1,000 U/ml) doubled the number of monocytes surviving after eight days in culture and accelerated the usual increase in cell volume. Antiserum to M-CSF abolished both of these effects. There was no sizable increase in 3H-thymidine incorporation in monocytes over this time period. Of various factors tested, including gamma-interferon (gamma-IFN), interleukin (IL) 1 alpha, granulocyte CSF (G-CSF), platelet-derived growth factor (PDGF), and lipopolysaccharide (LPS), only granulocyte-macrophage CSF (GM-CSF) could also enhance survival and augment cell volume. While antiserum to human M-CSF eliminated the increase in survival induced by GM-CSF, it could not ablate the GM-CSF-stimulated increase in monocyte cell volume. Monocyte cell surface markers that increase with maturation (i.e., Fc gamma RIII) or with activation (i.e., Fc gamma RI) were unaffected by incubation with M-CSF.

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Adherent cell layers and their associated extracellular matrices form when human marrow is incubated in cultures containing hydrocortisone and horse serum. These stromal layers contain cells positive for alkaline phosphatase; secrete collagens types I and III and fibronectin, bind the anti-actin monoclonal antibodies (MoAbs) HHF and CGA-7; stain with oil red O, and express the acetylated LDL receptor. Highly purified CD34 (My10)-positive progenitor cells attach to these stromal layers, and a 16-fold enrichment of CFU-GM in both stromal attachment and semisolid agar assays was observed. Granulopoiesis persisted up to 40 days (mean duration 25 days) after passaged stroma were recharged with stromal cell-depleted target cells in a two-stage liquid marrow culture system. Although equal to marrow fibroblasts in their ability to bind CD34+ myeloid progenitors, stromal layers were better at supporting granulopoiesis. This system provides an in vitro model to characterize the components of stroma and stroma-cytomatrix that enhance marrow progenitor cell localization and maintenance.

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The growth of eosinophil progenitors (CFU-Eo) and its modulation by hydrocortisone (HC), mononuclear cells, interleukin 1 (IL-1), and interleukin 2 (IL-2) in three cases of eosinophilia are reported in this paper. One microM HC decreased the proportion of CFU-Eo in each of these three patients, and in each case, the addition of autologous mononuclear cells at a 1:1 ratio abrogated the effect of HC on CFU-Eo. As studied in two of the three cases, IL-1 and IL-2 were able to prevent the effect of HC. Further studies showed no effect of HC when monocyte T cell-depleted marrow cells were used as the target population. These results suggest that CFU-Eo production in eosinophilic states is subject to modulation by HC and T lymphocytes.

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Three classes of FcR have been defined on human myeloid cells by their reactivity with mAb; FcRI (mAb 32); FcRII (mAb IV3); and FcRIII (mAb 3G8). We have quantitated the expression of each FcR on human myeloid leukemia cells and cell lines (KG-1, HL-60, U937, and K562). Detailed analysis of FcR surface expression is provided for the U937 cell line after exposure to CSF and cytokines. Increased expression of FcRI and FcRII occurred at 72 h in cells exposed to GCT or Mo cell line-conditioned medium as well as to medium from PHA-treated mononuclear cells. The augmentation of FcRII required protein synthesis and was diminished by a neutralizing antibody to granulocyte-macrophage CSF. We also show that fractions containing natural granulocyte CSF or granulocyte-macrophage CSF as well as r-granulocyte and r-granulocyte-macrophage CSF are capable of inducing FcRII on these cells, whereas other cytokines such as IL-1 and IL-2, TNF-alpha, INF-gamma and macrophages CSF failed to do so.

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We have purified human-active colony-stimulating factors from the human cell line, GCT, using sequential ultrafiltration; cation exchange, gel permeation, and reverse-phase high performance liquid chromatography (RHPLC); and ion-exchange HPLC. Activity eluted from sodium dodecyl sulfate-polyacrylamide gels with a peak at 17,500 daltons. Similar results were obtained by processing 35S-methionine-labeled conditioned medium, which showed a labeled band in the same region of activity. This purified HPLC fraction, which had a specific activity of greater than 1 x 10(7) colonies/mg protein, stimulated neutrophil colonies at day 7 and neutrophil, neutrophil-macrophage, and eosinophil colonies at day 14 of culture, suggesting that it contained both granulocyte (G-CSF) and granulocyte-macrophage (GM-CSF) colony-stimulating factors. It also promoted the growth of erythroid progenitors, and the GM-CSF fraction purified by hydrophobic chromatography had erythroid-enhancing activity. Separation of G-CSF from GM-CSF was accomplished by the addition of trifluoroacetic acid to the mobile phase at the reverse-phase HPLC step.

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Following a report that nerve growth factor preparations have granulocyte-colony-stimulating activity, we investigated the presence of colony-stimulating factors in 7s mouse submaxillary nerve growth factor and its subunits. Macrophage colonies were formed in mouse bone marrow cultures after exposure to preparations of 7s nerve growth factor, the gamma subunit, and, to a small extent, the alpha subunit; the beta subunit, which is responsible for the nerve growth function, did not stimulate colony growth. Furthermore, the esteropeptidase activity of the gamma subunit was not detected in preparations of macrophage colony-stimulating factor purified from the giant cell tumor (GCT) cell line. Immunoprecipitation of radiolabeled gamma subunit with a polyclonal antibody to L-cell macrophage colony-stimulating factor showed a protein band that could represent the gamma subunit of nerve growth factor. Separation of the macrophage activity from the esteropeptidase activity of the gamma subunit was accomplished on the basis of molecular size. Thus, macrophage colony-stimulating factor was a contaminant of nerve growth factor produced by the mouse submaxillary gland and copurified with the gamma subunit.

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We have studied the interactions of dimethyl sulfoxide (DMSO), Giant Cell Tumor (GCT) cell-conditioned medium (GCT CM), and highly purified granulocyte-macrophage colony-stimulating factors (GM-CSF) on the growth and maturation of a highly passaged population of HL-60 cells. DMSO produced dose-dependent inhibition of HL-60 growth in liquid and semisolid media. Growth was partially to completely restored by the addition of GCT CM to cultures. Experiments in which cell volume, cell cycle kinetics, tritiated thymidine (3HTdr) incorporation, cell number, and nitroblue tetrazolium (NBT) reduction were compared during culture indicated that DMSO inhibited the spontaneous increase in cell volume and flow of cells through the cell cycle which occurred in the first day of culture, the increase in 3HTdr incorporation which was detectable by day 2; and the increment in cell counts which occurred by day 3. These effects were opposed by GCT CM. In contrast, the DMSO-induced increase in NBT reduction which occurred by day 6 was not influenced by GCT CM. The major principle opposing DMSO was GM-CSF, since (1) highly purified GM-CSF from GCT cells and recombinant GM-CSF from COS cells transfected with the Mo cell GM-CSF gene overcame greater than 50% of DMSO inhibition; and (2) conditioned media from cells not producing CSF, G-CSF from GCT cells, and recombinant G-CSF from Escherichia coli transfected with the G-CSF gene from 5,637 cells were inactive. DMSO had little or no effect on the elaboration of autostimulatory activity by HL-60 cells. DMSO is a useful agent for inhibiting the spontaneous growth of HL-60 cells and restoring their dependence on GM-CSF, a property which may be mediated through the effects of DMSO on cell cycle kinetics and/or maturation.

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The murine IgG2a monoclonal antibody 6-19 binds to a wide variety of nonhematopoietic cells including human marrow-derived stromal cells but does not bind to marrow or peripheral blood cells. We studied the effects of this antibody and rabbit complement on marrow cells. Fibroblast colonies were eliminated from light density marrow cells by a single incubation with monoclonal antibody 6-19 and complement. The growth and composition of granulocytic and erythroid colonies were unaffected. Specific complement mediated cytotoxicity of the antibody was confirmed on passaged human fibroblasts derived from marrow (more than 99.6% of fibroblasts are killed by a single treatment). Similar results were obtained with human umbilical cord endothelial cells. In addition, such treatment abolished the initiation of Dexter culture stroma. Incubation of bone marrow cell suspensions with this antibody and complement will allow the study of stroma-free marrow cells in long-term liquid cultures.

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The growth of human eosinophil progenitors (CFU-Eo) and the modulation of growth by hydrocortisone were studied as functions of the presence of lymphocytes and monocytes in marrow cells under study; and the source of colony-stimulating factors, specifically, media conditioned by macrophage-like cell line, GCT; phytohemagglutinin-stimulated mononuclear cells (PHA-LCM); or the T cell line, MO. CFU-Eo growth was greatest in marrow containing accessory cells as compared to marrow depleted of accessory cells; and in marrow treated with phytohemagglutinin-stimulated leukocyte conditioned media (PHA-LCM) or MO (T cell line)-conditioned medium (MO-CM) as compared with GCT cell-conditioned medium (GCT-CM). Hydrocortisone reproducibly inhibited eosinophil progenitor growth in unfractionated marrow stimulated by GCT-CM. This effect was abrogated by admixing irradiated mononuclear cells or T lymphocytes with the target marrow or by adding interleukin 1 or interleukin 2 (IL-1, IL-2). Inhibition by hydrocortisone did not occur when monocyte and T lymphocyte depleted marrow was studied. Unlike GCT-CM, MO-CM and PHA-LCM stimulated equal proportions of eosinophil progenitors in nondepleted and accessory cell-depleted marrow and demonstrated less hydrocortisone inhibition. However, both GCT-CM and PHA-LCM produced in the presence of hydrocortisone stimulated significantly fewer CFU-Eos in both unfractionated and accessory cell-depleted marrow target populations. These results indicate that the growth of CFU-Eo and inhibition of growth by hydrocortisone is a direct function of a monocyte-T cell interaction and probably is mediated through effects on the production/release of eosinophil colony stimulating factor (Eo-CSF).

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Lymphomas are an uncommon complication of solid organ transplantation and rarely occur after marrow transplantation. When post-marrow transplant lymphomas have occurred, they have been of donor cell origin and when sought, Epstein-Barr virus DNA has been found in the tumor. A 21-year-old woman developed a poorly differentiated lymphocytic lymphoma 6 months after bone marrow transplantation for acute myeloid leukemia in remission. Cyclosporin A had been used as an immunosuppressant. A chromosomal polymorphism demonstrated that the tumor was of host origin and contained a monoclonal tumor marker, 46,XX INV 4 (p16q12). The tumor did not contain the DNA of the Epstein-Barr virus.

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