bluuebunny/crossref_metadata_2025 huggingface dataset:
Acquired Deficiency of von Willebrand Factor-Cleaving Protease in a Patient With Thrombotic Thrombocytopenic Purpura
Authors: Furlan Miha , Robles Rodolfo , Solenthaler Max , Lämmle Bernhard
Published: October 2019

Plasma of patients with thrombotic thrombocytopenic purpura (TTP) has been shown to contain unusually large von Willebrand factor (vWF) multimers that may cause platelet agglutination in vivo. Fresh frozen plasma infusions and plasma exchange represent the most efficient therapy of acute TTP. A specific protease responsible for cleavage of vWF multimers has been recently isolated from normal human plasma and was found to be deficient in four patients with chronic relapsing TTP. We examined the activity of the vWF-cleaving protease in plasma samples collected over a period of 400 days from a further patient with recurrent episodes of TTP who was treated by plasma exchange, plasma infusion, vincristine, corticosteroid therapy, and splenectomy. Complete deficiency of the vWF-cleaving protease was established during the first episode of TTP. The ensuing normalization of the platelet count was associated with the appearance of the protease activity. Three months after remission from the initial TTP event, the vWF-cleaving protease again disappeared and the platelet count gradually decreased. Relapses of severe thrombocytopenia occurred 7 and 11 months after the first acute episode of TTP. Deficient protease activity was associated with the presence in the patient plasma of an inhibitor that was found to be an IgG. Plasma exchange/infusion was followed by a temporary increase in the antibody titer, whereas treatment with vincristine led to a recovery of the platelet count without affecting the inhibitor concentration. Splenectomy and corticosteroid treatment resulted in disappearance of the autoantibody and normalization of the protease activity and of the platelet count. Our data suggest that the thrombocytopenia in this patient with TTP was associated with a lack of the vWF-cleaving protease activity depleted by an autoimmune mechanism. This case, together with our previously reported patients, leads us to conclude that acquired as well as constitutional deficiency of the vWF-cleaving protease may predispose to TTP.
Inhibition of Eosinophil Rolling and Recruitment in P-Selectin– and Intracellular Adhesion Molecule-1–Deficient Mice
Authors: Broide David H. , Humber David , Sriramarao P.
Published: October 2019

To determine the relative in vivo importance of endothelial expressed adhesion molecules to eosinophil rolling, adhesion, and transmigration, we have induced eosinophilic peritonitis using ragweed allergen in P-selectin–deficient, intracellular adhesion molecule-1 (ICAM-1)–deficient and control wild-type mice. Circulating leukocytes visualized by intravital microscopy exhibited reduced rolling and firm adhesion in P-selectin–deficient mice and reduced firm adhesion in ICAM-1–deficient mice. Eosinophils exhibited reduced rolling and firm adhesion to endothelium in P-selectin–deficient mice. Eosinophil recruitment in P-selectin–deficient mice (∼75% inhibition of eosinophil recruitment) and ICAM-1–deficient mice (∼67% inhibition of eosinophil recruitment) was significantly reduced compared with wild-type mice. Eosinophil recruitment was not completely inhibited in P-selectin/ICAM-1 double-mutant mice (eosinophil recruitment inhibited ∼62%). However, pretreatment of P-selectin/ICAM-1–deficient mice with an anti-vascular cell adhesion molecule (VCAM) antibody induced near complete inhibition of eosinophil recruitment. Overall, these studies show that eosinophil rolling and firm adhesion is significantly reduced in P-selectin–deficient mice and that P-selectin, ICAM-1, and VCAM are important to eosinophil peritoneal recruitment after ragweed challenge.
Tumor B Cells From Non-Hodgkin's Lymphoma Are Resistant to CD95 (Fas/Apo-1)–Mediated Apoptosis
Authors: Plumas Joël , Jacob Marie-Christine , Chaperot Laurence , Molens Jean-Paul , Sotto Jean-Jacques , Bensa Jean-Claude
Published: October 2019

Apoptosis mediated by the CD95 (Fas/Apo-1) molecule plays a crucial role in the regulation of the B-cell immune response. In this study, we examined the function of the CD95 antigen in B-cell–derived non-Hodgkin's lymphoma (NHL), a malignant disease of mature B cells. Membrane CD95 molecules were found to be constitutively expressed in a large number of NHL, including mantle cell (MCL, n = 10), lymphocytic (LCL, n = 10), follicular (FL, n = 11), and diffuse large cell lymphoma (DLCL, n = 9) with, however, different levels of intensity. Indeed, the levels of CD95 were low in MCL and LCL as compared with FL and DLCL. However, regardless of the intensity of expression, CD95 triggering with anti-CD95 monoclonal antibody (MoAb) did not induce apoptosis of lymphoma B cells, while these cells underwent apoptosis after irradiation or staurosporine treatment. Further experiments were then performed to address whether apoptosis could be restored by B-cell activation via CD40 cross-linking. We showed that CD40 engagement in the presence of interleukin (IL)-4 was more effective than CD40 engagement alone in upregulating the CD95 antigen and induced CD95-mediated cell death in nontumoral B cells. Concerning malignant B cells, CD40 ligation in the presence of IL-4 strongly increased CD95 expression, but did not markedly increase CD95-induced apoptosis. Furthermore, using cytotoxic T cells, we showed that CD95L was also ineffective in inducing apoptosis in lymphoma B cells, whereas these cells were killed by the perforin pathway. Our findings suggest that the CD95-mediated cell death pathway is altered in malignant cells from the NHL we tested. This could be a mechanism allowing lymphoma B cells to escape from immune regulation.
Abnormal Myelocytic Cell Development in Interleukin-2 (IL-2)–Deficient Mice: Evidence for the Involvement of IL-2 in Myelopoiesis
Authors: Reya Tannishtha , Contractor Nikhat V. , Couzens Matthew S. , Wasik Mariusz A. , Emerson Stephen G. , Carding Simon R.
Published: October 2019

Mice lacking interleukin-2 (IL-2) developed a severe hematopoietic disorder characterized by the abnormal development of myeloid cells and neutropenia. Analysis of the bone marrow of IL-2–deficient (IL-2−/−) mice showed that the number of mature polymorphonuclear cells was decreased by 65% to 75%, and granulocyte/macrophage precursor cells were reduced by 50%. Bone marrow cells from IL-2−/− mice were unable to sustain myelopoiesis in lethally irradiated mice and in long-term bone marrow cultures (LTBMC). The addition of exogenous IL-2 to LTBMC of IL-2−/− cells partially restored hematopoietic progenitor activity. In the bone marrow of wild-type mice, immature (Mac-1lo) myeloid cells, including myeloblasts and promyelocytes, constitutively expressed the β-chain of the IL-2R, and the number of Mac-1loIL-2Rβ+ cells was increased by twofold to threefold in IL-2−/− mice. During culture in the presence of IL-2 and the absence of stromal cells, Mac-1loIL-2Rβ+ immature myeloid cells proliferated and gave rise to mature granulocytes and macrophages. Collectively, these observations indicate that defective myelopoiesis in IL-2−/− mice is at least in part a consequence of their direct dependency on IL-2, and by regulating the growth of immature myeloid cells, IL-2 plays an important role in the homeostatic regulation of myelocytic cell generation.
Bifurcated Dendritic Cell Differentiation In Vitro From Murine Lineage Phenotype-Negative c-kit+ Bone Marrow Hematopoietic Progenitor Cells
Authors: Zhang Yi , Harada Akihisa , Wang Jian-bin , Zhang Yan-yun , Hashimoto Shin-ichi , Naito Makoto , Matsushima Kouji
Published: October 2019

We have recently established the culture system to generate dendritic cells (DCs) from murine Lin−c-kit+ bone marrow hematopoietic progenitor cells (HPCs) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) + stem cell factor (SCF) + tumor necrosis factor-α (TNF-α). We present here the identification of two DC precursor subsets originated from HPCs with the phenotype of CD11b−/dullCD11c+ and CD11b+hiCD11c+ that develop independently at early time points (days 4 to 6) in the same culture conditions. Both of CD11b−/dullCD11c+ and CD11b+hiCD11c+ precursors could differentiate at day 10 to 14 into CD11b−/dullCD11c+ mature DCs with typical morphology, phenotype, and the ability to stimulate allogenic mixed leukocyte reaction (MLR). However, the endocytic capacity of fluorescein isothiocyanate-dextran was markedly reduced during the differentiation. CD11b−/dullCD11c+precursors expressed high levels of Ia, CD86, CD40, and E-cadherin molecules, but not c-fms transcript, and mature DCs derived from this precursor subset continue to express abundant E-cadherin antigen, a discernible marker for Langerhans cells. In contrast, CD11b+hiCD11c+ precursors expressed c-fms mRNA, but low levels of Ia, CD86, and E-cadherin, whereas CD40 was undetectable. CD11b−/dullCD11c+mature DCs differentiated from these precursors displayed abundant c-fms mRNA and nonspecific esterase activity. Interestingly, CD11b+hiCD11c+precursors, but not CD11b−/dullCD11c+precursors, may be bipotent cells that can be induced by M-CSF to differentiate into macrophages. All of these results suggest that CD11b−/dullCD11c+ and CD11b+hiCD11c+ cells are distinct DC precursors derived from Lin−c-kit+ HPCs, which differentiate into mature DCs through bifurcated and independent DC differentiation pathways.
Epstein-Barr Virus Infects and Induces Apoptosis in Human Neutrophils
Authors: Larochelle Bernard , Flamand Louis , Gourde Pierrette , Beauchamp Denis , Gosselin Jean
Published: October 2019

The role of neutrophils during Epstein-Barr virus (EBV) infection is not known. Disruption of the initial and nonspecific immune response may favor the spread of EBV infection. We have previously shown that EBV interacts with human neutrophils and modulates protein expression. In this study we have investigated the ability of EBV to infect neutrophils. Electron microscopy studies showed penetration of virus and its subsequent localization to the nucleus. The presence of viral genomes in isolated nuclei from neutrophils was also shown by polymerase chain reaction (PCR). Expression of viral transcripts like EBNA-2 (Epstein-Barr nuclear antigen-2) and ZEBRA (BamHI Z EBV replication activator) was not detected by reverse transcriptase (RT)-PCR, suggesting that EBV does not seem to establish a latent or a lytic infection in neutrophils. However, at 20 hours post-EBV infection, 77% of cells were apoptotic as compared to 22% in uninfected cell cultures, as evaluated by flow cytometry. This EBV-induced apoptosis was prevented by the addition of granulocyte-macrophage colony-stimulating factor to the cell cultures. Apoptotic cell death seems to implicate the Fas/Fas ligand (L) pathway, as reflected by an increase of Fas/Fas L expression on neutrophils treated with EBV and an increase of soluble Fas L, which may function in an autocrine/paracrine pathway to mediate cell death. Lastly, EBV genome was detected from neutrophils of infectious mononucleosis (IM) patients in contrast to neutrophils obtained from healthy EBV-seropositive donors. Our findings on the interactions of EBV with neutrophils will then provide new insights on the immunosuppressive effects associated with EBV infection.
Sustained Gene Expression in Retrovirally Transduced, Engrafting Human Hematopoietic Stem Cells and Their Lympho-Myeloid Progeny
Authors: Cheng Linzhao , Du Changchun , Lavau Catherine , Chen Shirley , Tong Jie , Chen Benjamin P. , Scollay Roland , Hawley Robert G. , Hill Beth
Published: October 2019

Inefficient retroviral-mediated gene transfer to human hematopoietic stem cells (HSC) and insufficient gene expression in progeny cells derived from transduced HSC are two major problems associated with HSC-based gene therapy. In this study we evaluated the ability of a murine stem cell virus (MSCV)-based retroviral vector carrying the low-affinity human nerve growth factor receptor (NGFR) gene as reporter to maintain gene expression in transduced human hematopoietic cells. CD34+ cells lacking lineage differentiation markers (CD34+Lin−) isolated from human bone marrow and mobilized peripheral blood were transduced using an optimized clinically applicable protocol. Under the conditions used, greater than 75% of the CD34+ cell population retained the Lin− phenotype after 4 days in culture and at least 30% of these expressed a high level of NGFR (NGFR+) as assessed by fluorescence-activated cell sorter analysis. When these CD34+Lin−NGFR+ cells sorted 2 days posttransduction were assayed in vitro in clonogenic and long-term stromal cultures, sustained reporter expression was observed in differentiated erythroid and myeloid cells derived from transduced progenitors, and in differentiated B-lineage cells after 6 weeks. Moreover, when these transduced CD34+Lin−NGFR+ cells were used to repopulate human bone grafts implanted in severe combined immunodeficient mice, MSCV-directed NGFR expression could be detected on 37% ± 6% (n = 5) of the donor-type human cells recovered 9 weeks postinjection. These findings suggest potential utility of the MSCV retroviral vector in the development of effective therapies involving gene-modified HSC.
Protease-Activated Receptor 1 (PAR-1) Is Required and Rate-Limiting for Thrombin-Enhanced Experimental Pulmonary Metastasis
Authors: Nierodzik Mary Lynn , Chen Kui , Takeshita Kenichi , Li Jian-Jun , Huang Yao-Qi , Feng Xue-Sheng , D’Andrea Michael R. , Andrade-Gordon Patricia , Karpatkin Simon
Published: October 2019

Thrombin-treated tumor cells induce a metastatic phenotype in experimental pulmonary murine metastasis. Thrombin binds to a unique protease-activated receptor (PAR-1) that requires N-terminal proteolytic cleavage for activation by its tethered end. A 14-mer thrombin receptor activation peptide (TRAP) of the tethered end induces the same cellular changes as thrombin. Four murine tumor cells (Lewis lung, CT26 colon CA, B16F10 melanoma, and CCL163 fibroblasts) contain PAR-1, as detected by reverse transcriptase-polymerase chain reaction (RT-PCR). B16F10 cells did not contain the two other thrombin receptors, PAR-3 and glycoprotein Ib. TRAP-treated B16F10 tumor cells enhance pulmonary metastasis 41- to 48-fold (n = 17). Thrombin-treated B16F10 cells transfected with full-length murine PAR-1 sense cDNA (S6, S7, S14, and S22) enhanced their adhesion to fibronectin 1.5- to 2.4-fold (n = 5, P < .04), whereas thrombin-treated wild-type cells do not. S6 (adhesion index, 1.5-fold) and S14 (index, 2.4-fold) when examined by RT-PCR and Northern analysis showed minimal expression of PAR-1 for S6 over wild-type and considerable expression for S14. Immunohistochemistry showed greater expression of PAR-1 for S14 compared with wild-type or empty-plasmid transfected cells. In vivo experiments with the thrombin-treated S14 transfectant showed a fivefold to sixfold increase in metastases compared with empty-plasmid transfected thrombin-treated naive cells or S6 cells (n = 20, P = .0001 to .02). Antisense had no effect on thrombin-stimulated tumor mass. Thus, PAR-1 ligation and expression enhances and regulates tumor metastasis.
Cytokine Gene Polymorphisms Associating With Severe Acute Graft-Versus-Host Disease in HLA-Identical Sibling Transplants
Authors: Middleton Peter G. , Taylor Penelope R.A. , Jackson Graham , Proctor Stephen J. , Dickinson Anne M.
Published: October 2019

It is now well known that the initial phase of graft-versus-host disease (GVHD) involves cytokine release during preconditioning of the recipient of an allogeneic bone marrow transplant (BMT). Tumor necrosis factor  (TNF), in particular, has been implicated in pathological damage and is released pretransplant due to irradiation and cytotoxic preconditioning regimens. Interleukin-10 (IL-10), a natural immunosuppressant of TNF , may be involved in downregulation of these responses, which may be an individual patient-specific effect. In this study, we determined the genotype for polymorphisms associated with TNF and IL-10 in 80 potential allo-BMT recipients and correlated the genotype with the severity of GVHD in 49 patients for whom clinical data relating to GVHD was available. The widely studied TNF −308 polymorphism does not show any significant associations, but the d3 homozygous allele of the TNFd microsatellite is preferentially associated with grade III/IV GVHD (7 of 11 patients) compared with its occurrence in 8 of 38 patients with grade 0/II GVHD (P = .006). Alleles of the IL-10 −1064 promoter region microsatellite polymorphism that possess greater numbers of dinucleotide (CA) repeats also significantly associate with more severe GVHD. This region has been demonstrated to be important in the regulation of the IL-10 promoter. Eighteen of 38 patients with grade 0-II GVHD possessed alleles with greater numbers (12 or more) of dinucleotide repeats, compared with 9 of 11 cases with grade III-IV GVHD (P < .02). Of the 38 patients with grade 0-II GVHD, 3 of 38 had a both TNFd3/d3 and IL-10 (12-15) genotype, compared with 6 of 11 patients with grade III-IV GVHD (P < .001). There was no association of either the TNFd or IL-10 microsatellite polymorphisms with mortality (P = .43 and .51, respectively). Our results suggest that patient cytokine gene polymorphism genotypes may influence GVHD outcome by affecting cytokine activation during the pretransplant conditioning regimens, and these results are the first to suggest a genetic predisposition to this important transplant-related complication.
Intravascular Coagulation Activation in a Murine Model of Thrombomodulin Deficiency: Effects of Lesion Size, Age, and Hypoxia on Fibrin Deposition
Authors: Healy Aileen M. , Hancock Wayne W. , Christie Patricia D. , Rayburn Helen B. , Rosenberg Robert D.
Published: October 2019

We consecutively inactivated both alleles of the thrombomodulin (TM) gene in murine embryonic stem (ES) cells and generated TM-deficient (TM−/−) chimeric mice. Quantitation of an ES-cell marker and protein C cofactor activity indicates that up to 50% of pulmonary endothelial cells are ES-cell derived and therefore TM deficient. Infusions of 125I-fibrinogen into mice show a significant increase (fourfold, P < .005) in radiolabeled cross-linked fibrin in TM−/− chimeric mouse lung as compared with wild-type mice. However, only chimeric mice that exhibit at least a 30% reduction in protein C cofactor activity and are at least 15 months old display this phenotype. Immunocytochemical localization of TM in chimeras shows a mosaic pattern of expression in both large and small blood vessels. Colocalization of cross-linked fibrin and neo (used to replace TM) reveals that fibrin is deposited in TM−/− regions. However, the fibrin deposits were largely restricted to pulmonary vessels with a lumenal area greater than 100 μm2. The hypercoagulable phenotype can be induced in younger chimeric mice by exposure to hypoxia, which causes a fivefold increase in β-fibrin levels in lung. Our findings show that TM chimerism results in spontaneous, intravascular fibrin deposition that is dependent on age and the magnitude of the TM deficiency.
Role of Spontaneous and Interleukin-2–Induced Natural Killer Cell Activity in the Cytotoxicity and Rejection of Fas+and Fas− Tumor Cells
Authors: Bradley Mike , Zeytun Ahmet , Rafi-Janajreh Asimah , Nagarkatti Prakash S. , Nagarkatti Mitzi
Published: October 2019

In the current study, we investigated whether the naive, poly I:C or interleukin-2 (IL-2)–induced natural killer (NK)/lymphokine-activated killer (LAK) cells use perforin and/or Fas ligand (FasL) to mediated cytotoxicity. We correlated these findings with the ability of mice to reject syngeneic Fas+ and Fas− tumor cells either spontaneously or after IL-2 treatment. The spontaneous NK-cell–mediated cytotoxicity was primarily perforin based, whereas the poly I:C and IL-2–induced NK/LAK activity was both FasL and perforin dependent. L1210 Fas+ tumor targets were more sensitive than L1210 Fas− targets to poly I:C and IL-2–induced cytotoxicity in wild-type, gld/gld, and perforin knockout mice. When L1210 Fas+ and Fas– tumor cells were injected subcutaneously (sc) or intraperitoneally into syngeneic mice, Fas− tumor cells caused mortality earlier than Fas+ tumor cells. Also, approximately 20% of the mice injected sc with L1210 Fas+ tumor cells survived the challenge(>60 days), whereas all mice injected similarly with L1210 Fas− tumor cells died. When immunotherapy using IL-2 (10,000 U, three times/d for a week, followed by once/d for an additional week) was attempted in mice injected sc with tumor cells, IL-2 treatment was very effective against mice bearing L1210 Fas+ (40% survival) but not L1210 Fas− (0% survival) tumors. These data correlated with the finding that the LAK cells from IL-2–injected mice caused increased cytotoxicity against L1210 Fas+ when compared with L1210 Fas− targets. Also, L1210 Fas+tumor-bearing mice showed increased tumor-specific cytotoxic T lymphocyte (CTL) activity when compared with those bearing L1210 Fas− tumor cells. Together our studies show for the first time that expression of Fas on tumor targets makes them more immunogenic as well as susceptible to CTL- and IL-2–induced LAK activity. The Fas+ tumor cells are also more responsive to immunotherapy with IL-2.
Norethisterone Treatment, a Major Risk-Factor for Veno-Occlusive Disease in the Liver After Allogeneic Bone Marrow Transplantation
Authors: Hägglund Hans , Remberger Mats , Klaesson Sven , Lönnqvist Berit , Ljungman Per , Ringdén Olle
Published: October 2019

In this single-center study, we retrospectively analyzed incidence and risk factors for hepatic veno-occlusive disease (VOD) in 249 consecutive patients who underwent allogeneic hematopoietic stem cell transplantation between January 1990 and June 1995. Twenty-four of the 249 transplanted patients developed VOD. The probabilities of developing VOD were 17% among women and 7% in men (P = .01). In women treated with norethisterone, the incidence was 27% compared with 3% in women without this treatment (P = .007). One-year survival rates were 17% and 73% in patients with (n = 24) or without VOD (n = 225), respectively. The use of heparin prophylaxis (100 IE/kg/24 hours for 1 month) did not alter the incidence or 1-year mortality of VOD. In multivariate analysis, the following risk factors were significant: norethisterone treatment (P < .001), bilirubin >26 μmol/L before bone marrow transplantation (BMT) (P = .002), one HLA-antigen mismatch (P = .003), previous abdominal irradiation (P = .02), and conditioning with busulphan (P = .02). Our conclusion is that norethisterone treatment should not be used in patients undergoing BMT and heparin prophylaxis did not affect the incidence or mortality of VOD.
Improving the Outcome of Bone Marrow Transplantation by Using CD52 Monoclonal Antibodies to Prevent Graft-Versus-Host Disease and Graft Rejection
Authors: Hale Geoff , Zhang Mei-Jie , Bunjes Donald , Prentice H. Grant , Spence David , Horowitz Mary M. , Barrett A. John , Waldmann Herman
Published: October 2019

Graft-versus-host disease (GVHD) is a major cause of mortality and morbidity after allogeneic bone marrow transplantation, but can be avoided by removing T lymphocytes from the donor bone marrow. However, T-cell depletion increases the risk of graft rejection. This study examined the use of CD52 monoclonal antibodies to eliminate T cells from both donor marrow and recipient to prevent both GVHD and rejection. Seventy patients receiving HLA-identical sibling transplants for acute myelogenous leukemia (AML) in first remission were studied. An IgM (CAMPATH-1M) was used for in vitro depletion of the graft and an IgG (CAMPATH-1G) for in vivo depletion of the recipient before graft infusion. No posttransplant immunosuppression was given. Results were compared with two control groups: (1) 50 patients who received bone marrow depleted with CAMPATH-1M, but no CAMPATH-1G in vivo; and (2) 459 patients reported to the International Bone Marrow Transplant Registry (IBMTR) who received nondepleted grafts and conventional GVHD prophylaxis with cyclosporin A (CyA) and methotrexate (MTX). The incidence of acute GVHD was 4% in the treatment group compared with 35% in the CyA/MTX group (P < .001). Chronic GVHD was also exceptionally low in the treatment group (3% v 36%; P< .001). The problem of graft rejection, which had been frequent in the historic CAMPATH-1M group (31%), was largely overcome in the treatment group (6%). Thus, transplant-related mortality of the treatment group (15% at 5 years) was lower than for the CyA/MTX group (26%; P = .04). There was little difference in the risk of leukemia relapse between the treatment group (30% at 5 years) and the CyA/MTX group (29%). Survival of the treatment group at 6 months was better than the CyA/MTX group (92% v 78%), although at 5 years the difference was not significant (62% v 58%) and neither was the difference in leukemia-free survival (60% v52%). We conclude that T-cell depletion is a useful strategy to prevent GVHD, provided that measures are taken to ensure engraftment. Using CAMPATH-1G to deplete residual host lymphocytes is a simple and practical method to do this. At least in AML, the beneficial reduction in GVHD can be achieved without an increased risk of relapse.
Structurally Specific Heparan Sulfates Support Primitive Human Hematopoiesis by Formation of a Multimolecular Stem Cell Niche
Authors: Gupta Pankaj , Oegema Theodore R. , Brazil Joseph J. , Dudek Arkadiusz Z. , Slungaard Arne , Verfaillie Catherine M.
Published: October 2019

Stem cell localization, conservation, and differentiation is believed to occur in niches in the marrow stromal microenvironment. Our recent observation that long-term in vitro human hematopoiesis requires a stromal heparan sulfate proteoglycan (HSPG) led us to hypothesize that such HSPG may orchestrate the formation of the stem cell niche. We compared the structure and function of HS from M2-10B4, a hematopoiesis-supportive cell line, with HS from a nonsupportive cell line, FHS-173-We. Long-term culture-initiating cell (LTC-IC) maintenance was enhanced by PG from supportive cells but not by PG from nonsupportive cells (P < .005). The supportive HS were significantly larger and more highly sulfated than the nonsupportive HS. Specifically, supportive HS contained higher 6-O-sulfation on the glucosamine residues. In agreement with these observations, purified 6-O-sulfated heparin and highly 6-O-sulfated bovine kidney HS similarly maintained LTC-IC. In contrast, completely desulfated heparin, N-sulfated heparin, and unmodified heparin did not support LTC-IC maintenance. Moreover, the supportive HS promoted LTC-IC maintenance but not differentiation of CD34+/HLA-DR−cells into colony-forming cells (CFCs) and mature blood cells. The supportive HS but not the nonsupportive HS bound both cytokines and matrix components critical for hematopoiesis, including interleukin-3 (IL-3), macrophage inflammatory protein-1 (MIP-1), and thrombospondin (TSP). Significantly more CD34+ cells adhered directly to immobilized O-sulfated heparin than to N-sulfated or desulfated heparin. Thus, hematopoiesis-supportive stromal HSPG possessing large, highly 6-O-sulfated HS mediate the juxtaposition of hematopoietic progenitors with stromal cells, specific growth-promoting (IL-3) and growth-inhibitory (MIP-1 and platelet factor 4 [PF4]) cytokines, and extracellular matrix (ECM) proteins such as TSP. We conclude that the structural specificity of stromal HSPG that determines the selective colocalization of cytokines and ECM components leads to the formation of discrete niches, thereby orchestrating the controlled growth and differentiation of stem cells. These findings may have important implications for ex vivo expansion of and gene transfer into primitive hematopoietic progenitors.
Abrogation of the Hematological and Biological Activities of the Interleukin-3/Granulocyte-Macrophage Colony-Stimulating Factor Fusion Protein PIXY321 by Neutralizing Anti-PIXY321 Antibodies in Cancer Patients Receiving High-Dose Carboplatin
Authors: Miller Langdon L. , Korn Edward L. , Stevens Diane S. , Janik John E. , Gause Barry L. , Kopp William C. , Holmlund Jon T. , Curti Brendan D. , Sznol Mario , Smith John W. , Urba Walter J. , Donegan Sarah E. , Watson Thelma M. , Longo Dan L.
Published: October 2019

This dose-escalation study was performed to evaluate the hematologic activity, biological effects, immunogenicity, and toxicity of PIXY321 (an interleukin-3/granulocyte-macrophage colony-stimulating factor fusion protein) administered after high-dose carboplatin (CBDCA) treatment. Patients with advanced cancers received CBDCA at 800 mg/m2 intravenously on day 0 of repeated 28-day cycles. In part A of the study, patients were treated with CBDCA alone during cycle 1 and then received PIXY321 on days 1 through 18 of cycle 2 and later cycles. In part B, patients received 18 days of PIXY321 beginning on day 1 of all CBDCA cycles, including cycle 1. PIXY321 was administered subcutaneously in 2 divided doses. Total doses of 135, 250, 500, 750, and 1,000 μg/m2/d were administered to successive cohorts of 3 to 6 patients in part A. In part B, patient groups received PIXY321 doses of 750, 1,000, and 1,250 μg/m2/d. The hematologic effects of PIXY321 were assessed in the first 2 cycles of therapy. Anti-PIXY321 antibody formation was assessed by enzyme-linked immunosorbent assay (ELISA) and neutralization assay. Of the 49 patients enrolled, 31 were fully evaluable for hematologic efficacy. When comparing the first B cycle (cycle B-1; with PIXY321) with the first A cycle (cycle A-1; without PIXY321), the fusion protein had no significant effect on platelet nadirs or duration of platelets less than 20,000/μL but was able to speed the time of recovery of platelet counts to 100,000/μL (15v 20 days; P = .01). Significant improvements in neutrophil nadir and duration of ANC less than 500 were observed in cycles A-2 and B-1 (with PIXY321) as compared with cycle A-1 (without PIXY321). Initial PIXY321 prophylaxis (cycle A-2 and cycle B-1), enhanced the recovery of ANC to greater than 1,500/μL by an average of at least 8 days as compared with cycle A-1 (without PIXY321;P ≤ .004). However, positive PIXY321 hematologic effects were lost in the second course of PIXY321 among patients treated in part B. ELISA analysis showed that 92% of patients had developed neutralizing anti-PIXY321 antibodies by the completion of 2 PIXY321-containing cycles. The incidental action of PIXY321 to depress serum cholesterol levels was also abrogated during cycle B-2. We conclude that PIXY321 was active in speeding hematologic recovery but that neutralizing anti-PIXY321 antibody formation suppressed the hematologic and biochemical effects by the second cycle of PIXY321 administration. The immunogenicity of this fusion protein provides a cautionary warning that clinical development of bioengineered human molecules requires thorough testing for immune neutralization.
Transcriptional Targeting of Retroviral Vectors to the Erythroblastic Progeny of Transduced Hematopoietic Stem Cells
Authors: Grande Alexis , Piovani Bianca , Aiuti Alessandro , Ottolenghi Sergio , Mavilio Fulvio , Ferrari Giuliana
Published: October 2019

Targeted expression to specific tissues or cell lineages is a necessary feature of a gene therapy vector for many clinical applications, such as correction of hemoglobinopathies or thalassemias by transplantation of genetically modified hematopoietic stem cells. We developed retroviral vectors in which the constitutive viral enhancer in the U3 region of the 3′ LTR is replaced by an autoregulatory enhancer of the erythroid-specific GATA-1 transcription factor gene. The replaced enhancer is propagated to the 5′ LTR upon integration into the target cell genome. The modified vectors were used to transduce human hematopoietic cell lines, cord blood-derived CD34+ stem/progenitor cells, and murine bone marrow repopulating stem cells. The expression of appropriate reporter genes (▵LNGFR, EGFP) was analyzed in the differentiated progeny of transduced stem cells in vitro, in liquid culture as well as in clonogenic assay, and in vivo, after bone marrow transplantation in lethally irradiated mice. The GATA-1 autoregulatory enhancer effectively restricts the expression of the LTR-driven proviral transcription unit to the erythroblastic progeny of both human progenitors and mouse-repopulating stem cells. Packaging of viral particles, integration into the target genome, and stability of the integrated provirus are not affected by the LTR modification. Enhancer replacement is therefore an effective strategy to target expression of a retroviral transgene to a specific progeny of transduced hematopoietic stem cells.
Activation of Hematopoietic Progenitor Kinase-1 by Erythropoietin
Authors: Nagata Yuka , Kiefer Friedemann , Watanabe Takeshi , Todokoro Kazuo
Published: October 2019

Hematopoietic progenitor kinase-1 (HPK1), which is expressed predominantly in hematopoietic cells, was identified as a mammalian Ste20 homologue that, upon transfection, leads to activation of JNK/SAPK in nonhematopoietic cells. The JNK/SAPK pathway is activated by various environmental stresses and proinflammatory and hematopoietic cytokines. Upstream activators of HPK1 currently remain elusive, and its precise role in hematopoiesis has yet to be defined. We therefore examined the possible involvement of HPK1 in erythropoietin (Epo) and environmental stress-induced JNK/SAPK activation in the Epo-dependent FD-EPO cells and Epo-responsive SKT6 cells. We found that Epo, but not environmental stresses, induced rapid and transient activation of HPK1, whereas both induced activation of JNK/SAPK. A screen for HPK1 binding proteins identified the hematopoietic cell-specific protein 1 (HS1) as a potential HPK1 interaction partner. We found HPK1 constitutively associated with HS1 and that HS1 was tyrosine-phosphorylated in response to cellular stresses as well as Epo stimulation. Furthermore, antisense oligonucleotides to HPK1 suppressed Epo-dependent cell growth and Epo-induced erythroid differentiation. We therefore conclude that Epo induces activation of both HPK1 and HS1, whereas cellular stresses activate only HS1, and that the HPK1-JNK/SAPK pathway is involved in Epo-induced growth and differentiation signals.
Monoclonal Lym-1 Antibody-Dependent Cytolysis by Neutrophils Exposed to Granulocyte-Macrophage Colony-Stimulating Factor: Intervention of FcγRII (CD32), CD11b-CD18 Integrins, and CD66b Glycoproteins
Authors: Ottonello L. , Epstein A.L. , Dapino P. , Barbera P. , Morone P. , Dallegri F.
Published: October 2019

Murine monoclonal antibody (MoAb) Lym-1 is an IgG2a able to bind HLA-DR variants on malignant B cells and suitable for serotherapeutic approaches in B-lymphoma patients. We have previously shown that Lym-1 can synergize with granulocyte-macrophage colony-stimulating factor (GM-CSF) to trigger neutrophil cytolysis towards Raji cells used as a model of B-lymphoma targets. Here we provide evidence for the intervention of certain neutrophil receptors or surface molecules in this model of cell-mediated lysis. The lysis was completely inhibited by the anti-FcγRII MoAb IV.3 and unaffected by the anti-FcγRIII MoAb 3G8. This suggests that neutrophil cytolysis involves FcγRII without cooperation of this receptor with FcγRIII. Moreover, the lysis was inhibited by an anti-CD18 MoAb (MEM48) and by a MoAb specific for carcinoembryonic antigen (CEA)-like and glycophosphatidyl inositol (GPI)-linked glycoproteins (CD66b). Using an immunofluorescence staining procedure, cross-linking of CD66b induced the redistribution of CD11b on neutrophils with distinct areas of CD11b clustering via a process susceptible of inhibition by D-mannose. This is consistent with the ability of CD11b-CD18 and CD66b to undergo lectin-like physical interactions on the neutrophil surface. Such a type of interaction is presumably instrumental for neutrophil cytolytic activity in that the lysis was inhibited by D-mannose and enhanced by the MoAb VIM-12, which mimics the cooperation between CD11b and GPI-anchored molecules by specifically interacting with CD11b lectin-like sites. Therefore, the present results prove the absolute requirement for FcγRII in neutrophil GM-CSF/Lym-1–mediated cytolysis and, on the other hand, define the crucial role of CD66b and CD11b/CD18 in the expression of the cell lytic potential.
Transient Association of the Nicotinamide Adenine Dinucleotide Phosphate Oxidase Subunits p47phox and p67phox With Phagosomes in Neutrophils From Patients With X-Linked Chronic Granulomatous Disease
Authors: Allen Lee-Ann H. , DeLeo Frank R. , Gallois Annabelle , Toyoshima Satoshi , Suzuki Kensuke , Nauseef William M.
Published: October 2019

Optimal microbicidal activity of polymorphonuclear leukocytes (PMNs) requires recruitment of a functional nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to the phagosome. In this study, we used a synchronized phagocytosis assay and immunofluorescence microscopy (IFM) to examine the association of cytosolic NADPH oxidase subunits with phagosomes containing opsonized zymosan (OpZ). Ingestion of OpZ began within 30 seconds of particle binding and forming phagosomes were enriched for both F-actin and the actin-binding protein p57. NADPH oxidase subunits p47phox and p67phox were also recruited to forming phagosomes and were retained on mature phagosomes for at least 15 minutes. Colocalization of F-actin, p57, and p47phox on phagosomes was confirmed by immunoblotting. Translocation of p67phox, but not p57, to forming phagosomes was deficient in PMNs lacking p47phox. Surprisingly, we found that in PMNs from six individuals with X-linked chronic granulomatous disease (CGD), p47phox and p67phox accumulated in the periphagosomal area during ingestion of OpZ. However, in marked contrast to normal PMNs, p47phox and p67phox were shed from nascent phagosomes along with F-actin and p57 once OpZ was internalized (≈5 minutes). These data support a model in which flavocytochrome b is required for stable membrane binding of p47phox and p67phox, but not their association with the cytoskeleton or transport to the cell periphery.
Depletion of Alloreactive T Cells by a Specific Anti–Interleukin-2 Receptor p55 Chain Immunotoxin Does Not Impair In Vitro Antileukemia and Antiviral Activity
Authors: Montagna Daniela , Yvon Eric , Calcaterra Valeria , Comoli Patrizia , Locatelli Franco , Maccario Rita , Fisher Alain , Cavazzana-Calvo Marina
Published: October 2019

The success of bone marrow transplantation (BMT) from HLA-disparate donors depends on the development of new strategies able, on one hand, to efficiently prevent graft-versus-host disease (GVHD) and, on the other hand, to protect leukemic patients from relapse and infections. Using an immunotoxin (IT) directed against the  chain (p55) of the human interleukin-2 receptor (RFT5-SMPT-dgA), we previously showed that it is possible to kill mature T cells activated against a specific HLA complex by a one-way mixed lymphocyte culture (MLC). The present study was performed to investigate whether this protocol of allodepletion affects the capacity of residual T cells to display antileukemia and antiviral activity evaluated by limiting dilution assays (LDA), measuring the frequency of cytotoxic T-lymphocyte precursors (CTLp) directed against autologous leukemic blasts (LB) and cytomegalovirus (CMV)- and Epstein-Barr virus (EBV)-infected target cells. Antileukemia activity was evaluated in peripheral blood mononuclear cells (PBMC) of 3 patients treated for acute myeloid leukemia who had developed a high frequency of LB-reactive CTLp after either autologous or allogeneic BMT. Results demonstrate that (1) depletion with RFT5-SMPT-dgA efficiently inhibited MLC; (2) fresh PBMC of patients yielded a high frequency of LB-reactive CTLp comparable to that of the mock-treated PBMC; and (3) effector cells obtained after allodepletion fully retained the capacity to lyse pretransplant LB. By contrast, the frequency of CTLp directed against patient’s pretransplant BM remission cells was always undetectable. Data obtained in 4 healthy donors showed that specifically allodepleted T cells recognized and killed autologous CMV-infected fibroblasts and autologous EBV–B-lymphoblastoid cell lines. In conclusion, our data indicate that allodepletion using RFT5-SMPT-dgA efficiently removed alloreactive cells, while sparing in vitro antileukemic and antiviral cytotoxic responses.
The Rh blood group system: a review
Authors: Avent Neil D. , Reid Marion E.
Published: October 2019

The Rh blood group system is one of the most polymorphic and immunogenic systems known in humans. In the past decade, intense investigation has yielded considerable knowledge of the molecular background of this system. The genes encoding 2 distinct Rh proteins that carry C or c together with either E or e antigens, and the D antigen, have been cloned, and the molecular bases of many of the antigens and of the phenotypes have been determined. A related protein, the Rh glycoprotein is essential for assembly of the Rh protein complex in the erythrocyte membrane and for expression of Rh antigens. The purpose of this review is to provide an overview of several aspects of the Rh blood group system, including the confusing terminology, progress in molecular understanding, and how this developing knowledge can be used in the clinical setting. Extensive documentation is provided to enable the interested reader to obtain further information.
N-RAS gene mutation in patients with aplastic anemia and aplastic anemia/ paroxysmal nocturnal hemoglobinuria during evolution to clonal disease
Authors: Mortazavi Y. , Tooze J. A. , Gordon-Smith E. C. , Rutherford T. R.
Published: October 2019

Long-term survivors of aplastic anemia (AA) have a high incidence of clonal disorders, in particular paroxysmal nocturnal hemoglobinuria (PNH), myelodysplastic syndromes (MDS), and acute nonlymphocytic leukemia. To investigate the potential involvement of N-RAS gene mutations in the predisposition to leukemic evolution, a subset of patients at potentially increased risk for clonal disease was selected based on evidence of existing clonal evolution. Nine patients showed a monoclonal pattern of X-chromosome inactivation, 18 demonstrated a PNH clone, and in 3 MDS developed during the course of this study. No mutations were detected during the aplastic phase of disease; 2 of 3 patients with MDS after AA also showed no mutations. However, in 1 patient in whom the disease transformed from AA/PNH to MDS, a mutation of GGT → GAT at N-RAS codon 13 became detectable, whereas the PNH mutation disappeared. The authors conclude that N-RAS mutations are not an early event preceding transformation of AA or AA/PNH to leukemia. In a subset of patients, RAS mutations may occur at the time of evolution to MDS, but preexisting RAS mutations do not explain the propensity of AA to leukemogenesis. Although PNH is also associated with leukemia, this may arise in the non-PNH cells, indicating that PIG-A gene mutation is not per se oncogenic.
Elevated cytosolic phospholipase A2 expression and activity in human neutrophils during sepsis
Authors: Levy R. , Dana R. , Hazan I. , Levy I. , Weber G. , Smoliakov R. , Pesach I. , Riesenberg K. , Schlaeffer F.
Published: October 2019

Sepsis is defined as the systemic inflammatory response to infection. Phospholipase A2 (PLA2) plays an important role in inflammation processes by initiating the production of inflammatory mediators. The role of cytosolic PLA (cPLA2) has not yet been identified in inflammatory and infectious disease clinical settings. The aim of the present research was to determine whether cPLA2 activity has a role during sepsis. Since neutrophil activation has been documented during sepsis, these cells were chosen as a model to evaluate the function of cPLA2 in this clinical setting. cPLA2 was studied at 3 levels: activity, protein expression, and messenger RNA (mRNA). Neutrophils from 32 septic patients with and without bacteremia were examined. cPLA2 activity was measured using labeled phosphatidyl choline vesicles as a substrate, and total PLA2 was determined by the release of labeled arachidonic acid from prelabeled cells. A significant increase in cPLA2activity, protein expression, and total PLA2 activity in neutrophils was detected during sepsis. mRNA levels, detected by reverse transcriptase–polymerase chain reaction, were significantly higher during sepsis, indicating that the increase in the amount of cPLA2 is regulated on the mRNA level. The significant elevation of cPLA2 activity and expression in neutrophils during sepsis suggests that this enzyme plays a major role in neutrophil function in this clinical setting.
C-C chemokine profile of cord blood mononuclear cells: selective defect in RANTES production
Authors: Hariharan Deepa , Ho Wenzhe , Cutilli Joann , Campbell Donald E. , Douglas Steven D.
Published: October 2019

Three C-C chemokines inhibit human immunodeficiency virus (HIV) entry into macrophages: macrophage inflammatory protein-1 (MIP-1), MIP-1β, and regulated-upon activation, normal T-cell expressed and secreted (RANTES). We studied the ability of placental cord blood mononuclear cells (CBMC) to secrete these C-C chemokines in comparison to adult blood mononuclear cells (ABMC). CBMC had diminished ability to secrete RANTES, as determined by enzyme-linked immunosorbent assay. Secretion of MIP-1 and MIP-1β were similar in CBMC and ABMC. Whereas MIP-1 and MIP-1β secretion were comparable in monocytes and lymphocytes, RANTES was secreted primarily by lymphocytes. Flow cytometric analysis of RANTES expression showed diminished intracellular RANTES expression in cord blood lymphocytes (CBL) compared to adult (peripheral) blood lymphocytes (ABL). A subset analysis of RANTES-producing CBL and ABL demonstrated that RANTES was produced predominantly by CD8+/CD45RO+ cells. CBL had a reduced proportion of CD8+/CD45RO+ cells compared with ABL, which may account for the diminished RANTES secretion by CBMC. These results may be relevant to the pathogenesis of perinatal HIV infection.
A primitive hematopoietic cell is the target for the leukemic transformation in human Philadelphia-positive acute lymphoblastic leukemia
Authors: Cobaleda C. , Gutiérrez-Cianca N. , Pérez-Losada J. , Flores T. , Garcı́a-Sanz R. , González M. , Sánchez-Garcı́a I.
Published: October 2019

BCR-ABL is a chimeric oncogene generated by translocation of sequences from the chromosomal counterpart (c-ABLgene) on chromosome 9 into the BCR gene on chromosome 22. Alternative chimeric proteins, BCR-ABLp190 and BCR-ABLp210, are produced that are characteristic of chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph1-ALL). In CML, the transformation occurs at the level of pluripotent stem cells. However, Ph1-ALL is thought to affect progenitor cells with lymphoid differentiation. Here we demonstrate that the cell capable of initiating human Ph1-ALL in non-obese diabetic mice with severe combined immunodeficiency disease (NOD/SCID), termed SCID leukemia–initiating cell (SL-IC), possesses the differentiative and proliferative capacities and the potential for self-renewal expected of a leukemic stem cell. The SL-ICs from all Ph1-ALL analyzed, regardless of the heterogeneity in maturation characteristics of the leukemic blasts, were exclusively CD34+CD38−, which is similar to the cell-surface phenotype of normal SCID-repopulating cells. This indicates that normal primitive cells, rather than committed progenitor cells, are the target for leukemic transformation in Ph1-ALL.