bluuebunny/crossref_metadata_2025 huggingface dataset:
Growth Characteristics of Acute Myelogenous Leukemia Progenitors That Initiate Malignant Hematopoiesis in Nonobese Diabetic/Severe Combined Immunodeficient Mice
Authors: Ailles Laurie E. , Gerhard Brigitte , Kawagoe Hiroyuki , Hogge Donna E.
Published: October 2019

The use of immunodeficient mice, particularly of the nonobese diabetic/severe combined immunodeficient (NOD/SCID) strain, has allowed detection of very primitive malignant progenitors from patients with acute myelogenous leukemia (AML). To define the sensitivity and reproducibility with which the engraftment of different AML cells can be detected, 61 different samples from patients with newly diagnosed AML representing a variety of cytogenetic and French-American-British (FAB) subtypes were injected into NOD/SCID mice. Eight weeks after intravenous injection of 107 AML cells, the average percent of human cells in mouse bone marrow was 13.3%, with 70% of samples showing easily detectable engraftment of CD45+ cells. AML samples with cytogenetic changes associated with a poor clinical prognosis tended to engraft to higher levels than those with changes associated with a good prognosis. Cells with FAB subtypes M3 and, to a lesser extent, M2, engrafted more poorly (P = .002 and .06, respectively) than those from other subtypes. Intraperitoneal injection of human interleukin-3 and Steel factor thrice weekly for 4 weeks did not enhance the levels of AML cell engraftment. However, AML samples that showed cytokine-independent colony growth in methylcellulose assay or expressed growth-factor mRNA in malignant blasts achieved significantly higher levels of engraftment than those which were cytokine dependent in culture or failed to express cytokine message (P < .03 and P < .02, respectively). In 6 patient samples, the frequency of NOD/SCID leukemia-initiating cells (NOD/SL-IC) varied from 0.7 to 45 per 107 cells, which was 200- to 800-fold lower than the frequency of AML long-term culture-initiating cells (AML LTC-IC) in the same samples. Each NOD/SL-IC will produce more than 106leukemic blasts as well as many AML-CFC and AML LTC-IC as detected 8 weeks postinjection into mice. Serial transplant experiments showed the ability of NOD/SL-IC to maintain their own numbers over at least 3 to 4 weeks in vivo. The ability of these progenitors to self-renew combined with their potential to differentiate to produce large numbers of more mature progenitors and leukemic blasts suggests that the NOD/SL-IC assay identifies leukemic ‘stem cells’ that may maintain the malignant clone in human patients. The further use of this assay should facilitate studies of AML stem cell biology and the evolution of novel therapeutic strategies.
Children With Acute Lymphoblastic Leukemia Who Receive T-Cell–Depleted HLA Mismatched Marrow Allografts From Unrelated Donors Have an Increased Incidence of Primary Graft Failure but a Similar Overall Transplant Outcome
Authors: Green Ann , Clarke Emer , Hunt Linda , Canterbury Andrew , Lankester Alan , Hale Geoff , Waldmann Herman , Goodman Sally , Cornish Jacqueline M. , Marks David I. , Steward Colin G. , Oakhill Anthony , Pamphilon Derwood H.
Published: October 2019

Disparity for HLA in unrelated donor bone marrow transplantation (BMT) increases the risk of graft rejection and graft-versus-host disease (GVHD) and may compromise transplant outcome. We have compared the outcome of matched and mismatched transplants from unrelated donors in 137 children with acute lymphoblastic leukemia (ALL). Their disease status was complete remission (CR)-1, 24 patients; CR-2, 88 patients; CR-3, 18 patients; CR-4, 2 patients; and relapse, 5 patients. CAMPATH monoclonal antibodies were used for T-cell depletion and cyclosporin A was given to 134 children together with short-course methotrexate in 43, mainly when there was HLA disparity. Fifty-two donor/recipient pairs were HLA-mismatched, 41 at HLA-A and -B and 11 at HLA-DR and -DQ loci. Overall graft failure was increased in recipients of marrow mismatched at either HLA-A, -B, -DR, or -DQ (15.7% v 4.8%; P = .057) mainly because there was a higher proportion of children with primary graft failure (11.8% v 1.2%; P = .012). The presence of an HLA-C locus mismatch did not independently increase the likelihood of graft failure. There was no significant difference in the incidence of acute GVHD ≥ grade 2 between the matched and mismatched groups (P = .849). For patients in CR-2, the risk of relapse post-BMT was significantly lower if leukemic relapse occurred off-treatment (P = .005). The Kaplan-Meier overall and leukemia-free survival (LFS) estimates for recipients of matched and mismatched BMT, respectively, at 36 months were 49% versus 42% (P = .380) and 45% versus 40% (P = .654). Although HLA mismatching results in an increased occurrence of primary graft failure with T-cell–depleted allografts, it allows more donors to be identified rapidly for children with ALL without compromising overall transplant outcome.
Efficient and Durable Gene Marking of Hematopoietic Progenitor Cells in Nonhuman Primates After Nonablative Conditioning
Authors: Rosenzweig M. , MacVittie T.J. , Harper D. , Hempel D. , Glickman R.L. , Johnson R.P. , Farese A.M. , Whiting-Theobald N. , Linton G.F. , Yamasaki G. , Jordan C.T. , Malech H.L.
Published: October 2019

Optimization of mobilization, harvest, and transduction of hematopoietic stem cells is critical to successful stem cell gene therapy. We evaluated the utility of a novel protocol involving Flt3-ligand (Flt3-L) and granulocyte colony-stimulating factor (G-CSF) mobilization of peripheral blood stem cells and retrovirus transduction using hematopoietic growth factors to introduce a reporter gene, murine CD24 (mCD24), into hematopoietic stem cells in nonhuman primates. Rhesus macaques were treated with Flt3-L (200 μg/kg) and G-CSF (20 μg/kg) for 7 days and autologous CD34+ peripheral blood stem cells harvested by leukapheresis. CD34+ cells were transduced with an MFGS-based retrovirus vector encoding mCD24 using 4 daily transductions with centrifugations in the presence of Flt3-L (100 ng/mL), human stem cell factor (50 ng/mL), and PIXY321 (50 ng/mL) in serum-free medium. An important and novel feature of this study is that enhanced in vivo engraftment of transduced stem cells was achieved by conditioning the animals with a low-morbidity regimen of sublethal irradiation (320 to 400 cGy) on the day of transplantation. Engraftment was monitored sequentially in the bone marrow and blood using both multiparameter flow cytometry and semi-quantitative DNA polymerase chain reaction (PCR). Our data show successful and persistent engraftment of transduced primitive progenitors capable of giving rise to marked cells of multiple hematopoietic lineages, including granulocytes, monocytes, and B and T lymphocytes. At 4 to 6 weeks posttransplantation, 47% ± 32% (n = 4) of granulocytes expressed mCD24 antigen at the cell surface. Peak in vivo levels of genetically modified peripheral blood lymphocytes approached 35% ± 22% (n = 4) as assessed both by flow cytometry and PCR 6 to 10 weeks posttransplantation. In addition, naı̈ve (CD45RA+and CD62L+) CD4+ and CD8+cells were the predominant phenotype of the marked CD3+ T cells detected at early time points. A high level of marking persisted at between 10% and 15% of peripheral blood leukocytes for 4 months and at lower levels past 6 months in some animals. A cytotoxic T-lymphocyte response against mCD24 was detected in only 1 animal. This degree of persistent long-lived, high-level gene marking of multiple hematopoietic lineages, including naı̈ve T cells, using a nonablative marrow conditioning regimen represents an important step toward the ultimate goal of high-level permanent transduced gene expression in stem cells.
The Use of Granulocyte Colony-Stimulating Factor During Retroviral Transduction on Fibronectin Fragment CH-296 Enhances Gene Transfer Into Hematopoietic Repopulating Cells in Dogs
Authors: Goerner Martin , Bruno Benedetto , McSweeney Peter A. , Buron Greg , Storb Rainer , Kiem Hans-Peter
Published: October 2019

A competitive repopulation assay in the dog was used to develop improved gene transfer protocols for hematopoietic stem cell gene therapy. Using this assay, we previously showed improved gene transfer into canine hematopoietic repopulating cells when CD34-enriched marrow cells were cocultivated on gibbon ape leukemia virus (GALV)–based retrovirus vector-producing cells. In the present study, we have investigated the use of fibronectin fragment CH-296 and 2 growth factor combinations to further improve gene transfer efficiency. CD34-enriched marrow cells from each dog were prestimulated for 24 hours and then divided into 3 equal fractions. Two fractions were placed into flasks coated with either CH-296 or bovine serum albumin (BSA) and virus-containing medium supplemented with growth factors, and protamine sulfate was replaced 4 times over a 48-hour period. One fraction was cocultivated on irradiated PG13 (GALV-pseudotype) packaging cells for 48 hours. In 2 animals, cells of the different fractions were transduced in the presence of human FLT-3 ligand (FLT3L), canine stem cell factor (cSCF), and human megakaryocyte growth and development factor (MGDF), and in 2 other dogs, transduction was performed in the presence of FLT3L, cSCF, and canine granulocyte-colony stimulating factor (cG-CSF). The vectors used contained small sequence differences, allowing differentiation of cells genetically marked by the different vectors. After transduction, nonadherent and adherent cells from all 3 fractions were pooled and infused into lethally irradiated dogs. Polymerase chain reaction and Southern blot analysis were used to determine the persistence of the transferred vectors in the peripheral blood and marrow cells after transplantation. The highest levels of gene transfer were obtained when cells were transduced in the presence of FLT3L, cSCF, and cG-CSF (gene transfer levels of more than 10% for more than 8 months so far). Compared with the 2 animals that received cells transduced with FLT3L, cSCF, and MGDF, gene transfer levels were significantly higher when dogs received cells that were transduced in the presence of cG-CSF. Transduction on CH-296 resulted in gene transfer levels that were at least as high as transduction by cocultivation. In summary, the overall levels of gene transfer obtained with these conditions should be sufficiently high to allow stem cell gene therapy studies aimed at correcting genetic diseases in dogs as a model for human gene therapy.
PU.1 and the Granulocyte- and Macrophage Colony-Stimulating Factor Receptors Play Distinct Roles in Late-Stage Myeloid Cell Differentiation
Authors: Anderson Karen L. , Smith Kent A. , Perkin Hugh , Hermanson Gary , Anderson Carol-Gay , Jolly Douglas J. , Maki Richard A. , Torbett Bruce E.
Published: October 2019

PU.1 is a hematopoietic cell–specific ets family transcription factor. Gene disruption of PU.1 results in a cell autonomous defect in hematopoietic progenitor cells that manifests as abnormal myeloid and B-lymphoid development. Of the myeloid lineages, no mature macrophages develop, and the neutrophils that develop are aberrantly and incompletely matured. One of the documented abnormalities of PU.1 null (deficient) hematopoietic cells is a failure to express receptors for granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage (GM)-CSF, and M-CSF. To elucidate the roles of the myeloid growth factor receptors in myeloid cell differentiation, and to distinguish their role from that of PU.1, we have restored expression of the G- and M-CSF receptors in PU.1-deficient cells using retroviral vectors. We have similarly expressed PU.1 in these cells. Whereas expression of growth factor receptors merely allows a PU.1-deficient cell line to survive and grow in the relevant growth factor, expression of PU.1 enables the development of F4/80+, Mac-1+/CD11b+ macrophages, expression of gp91phox and generation of superoxide, and expression of secondary granule genes for neutrophil collagenase and gelatinase. These studies reinforce the idea that availability of PU.1 is crucial for normal myeloid development and clarify some of the molecular events in developing neutrophils and macrophages that are critically dependent on PU.1.
Eosinophil Apoptosis Is Mediated by Stimulators of Cellular Oxidative Metabolisms and Inhibited by Antioxidants: Involvement of a Thiol-Sensitive Redox Regulation in Eosinophil Cell Death
Authors: Wedi Bettina , Straede Julia , Wieland Britta , Kapp Alexander
Published: October 2019

The mechanisms for induction of eosinophil apoptosis remain uncertain. The role of oxidative stress has not been investigated. The present study was undertaken to determine the role of reactive oxygen species (ROS) and selective antioxidants in eosinophil apoptosis. Eosinophils were cultured with sodium arsenite (SA) known to induce intracellular oxidative metabolites. There was a significant increase in the rate of eosinophil apoptosis with low concentrations of arsenite, whereas high concentrations showed rates of apoptosis similar to control medium. Investigating the role of intracellular oxidants by flow cytometry, we found that while inducing apoptosis, SA more than anti-Fas resulted in a significant dose-dependent production of intracellular H2O2. In contrast, the extracellular release of superoxide decreased after stimulation with SA or anti-Fas as assessed by lucigenin-dependent chemiluminescence. Coincubation experiments demonstrated that arsenite-induced apoptosis can be nearly completely prevented by selective antioxidants such as glutathione (GSH) and N-acetyl-cysteine (NAC), but not dimethyl sulfoxide (DMSO) or taurine (TAUR). Moreover, GSH and NAC significantly reduced eosinophil apoptosis mediated by a monoclonal antibody directed to Fas antigen. Next it was shown that GSH and NAC, but not DMSO or TAUR, were able to significantly delay spontaneous apoptosis in unstimulated eosinophils. Taken together, these data point to an important role of oxygen-dependent mechanisms in the regulation of eosinophil survival and apoptosis. We propose that eosinophil apoptosis may be related to the ability of the cell to maintain an appropriate oxidant-antioxidant balance.
ABL1 Methylation Is a Distinct Molecular Event Associated With Clonal Evolution of Chronic Myeloid Leukemia
Authors: Asimakopoulos Fotis A. , Shteper Pesach J. , Krichevsky Svetlana , Fibach Eitan , Polliack Aaron , Rachmilewitz Eliezer , Ben-Neriah Yinon , Ben-Yehuda Dina
Published: October 2019

Methylation of the proximal promoter of the ABL1 oncogene is a common epigenetic alteration associated with clinical progression of chronic myeloid leukemia (CML). In this study we queried whether both the Ph′-associated and normal ABL1 alleles undergo methylation; what may be the proportion of hematopoietic progenitors bearing methylated ABL1 promoters in chronic versus acute phase disease; whether methylation affects the promoter uniformly or in patches with discrete clinical relevance; and, finally, whether methylation of ABL1 reflects a generalized process or is gene-specific. To address these issues, we adapted the techniques of methylation-specific PCR and bisulfite-sequencing to study the regulatory regions of ABL1 and other genes with a role in DNA repair or genotoxic stress response. In cell lines established from CML blast crisis, which only carry a single ABL1 allele nested within the BCR-ABL fusion gene, ABL1 promoters were universally methylated. By contrast, in clinical samples from patients at advanced stages of disease, both methylated and unmethylated promoter alleles were detectable. To distinguish between allele-specific methylation and a mixed cell population pattern, we studied the methylation status of ABL1 in colonies derived from single hematopoietic progenitors. Our results showed that both methylated and unmethylated promoter alleles coexisted in the same colony. Furthermore, ABL1 methylation was noted in the vast majority of colonies from blast crisis, but not chronic-phase CML. Both cell lines and clinical samples from acute-phase CML showed nearly uniform hypermethylation along the promoter region. Finally, we showed that ABL1 methylation does not reflect a generalized process and may be unique among DNA repair/genotoxic stress response genes. Our data suggest that specific methylation of the Ph′-associatedABL1 allele accompanies clonal evolution in CML.
In Vitro and In Vivo Effects of a Farnesyltransferase Inhibitor onNf1-Deficient Hematopoietic Cells
Authors: Mahgoub Nidal , Taylor Brigit R. , Gratiot Mary , Kohl Nancy E. , Gibbs Jackson B. , Jacks Tyler , Shannon Kevin M.
Published: October 2019

Oncogenic RAS alleles encode proteins that accumulate in the guanosine triphosphate (GTP)-bound state. Because post-translational processing of Ras by farnesyltransferase is essential for biologic function, inhibitors of this enzyme have been developed as rational cancer therapeutics. We have investigated farnesyltransferase inhibitor (FTI) L-744,832 in an in vivo murine model of myeloid leukemia that is associated with inactivation of the Nf1 tumor suppressor gene.Nf1 encodes a GTPase activating protein for Ras, andNf1-deficient (Nf1−/−) hematopoietic cells show hyperactive Ras signaling through the mitogen-activated protein (MAP) kinase pathway. L-744,832 inhibited H-Ras prenylation in cell lines and in primary hematopoietic cells and abrogated the in vitro growth of myeloid progenitor colonies in response to granulocyte-macrophage colony-stimulating factor (GM-CSF). This FTI also partially blocked GM-CSF–induced MAP kinase activation, but did not reduce constitutively elevated levels of MAP kinase activity in primaryNf1−/− cells. Injection of a single dose of 40 or 80 mg/kg of L-744,832 increased the amount of unprocessed H-Ras in bone marrow cells, but had no detectable effect on N-Ras. Adoptive transfer ofNf1−/− hematopoietic cells into irradiated mice induces a myeloproliferative disorder that did not respond to L-744,832 treatment. We speculate that the lack of efficacy in this model is due to the resistance of N-Ras and K-Ras processing to inhibition by this FTI.
The primacy of the gastrointestinal tract as a target organ of acute graft-versus-host disease: rationale for the use of cytokine shields in allogeneic bone marrow transplantation
Authors: Hill Geoffrey R. , Ferrara James L. M.
Published: October 2019

Acute graft-versus-host disease (GVHD), the major complication of allogeneic bone marrow transplantation (BMT), limits the application of this curative but toxic therapy. Studies of inflammatory pathways involved in GVHD in animals have shown that the gastrointestinal (GI) tract plays a major role in the amplification of systemic disease. Damage to the GI tract increases the translocation of inflammatory stimuli such as endotoxin, which promotes further inflammation and additional GI tract damage. The GI tract is therefore critical to the propagation of the “cytokine storm” characteristic of acute GVHD. Experimental approaches to the prevention of GVHD include reducing the damage to the GI tract by fortification of the GI mucosal barrier through novel “cytokine shields” such as IL-11 or keratinocyte growth factor. Such strategies have reduced GVHD while preserving a graft-versus-leukemia effect in animal models, and they now deserve formal testing in carefully designed clinical trials.
Shortage of circulating naive CD8+ T cells provides new insights on immunodeficiency in aging
Authors: Fagnoni Francesco F. , Vescovini Rosanna , Passeri Giovanni , Bologna Giovanni , Pedrazzoni Mario , Lavagetto Giampaolo , Casti Amos , Franceschi Claudio , Passeri Mario , Sansoni Paolo
Published: October 2019

Clinical observations indicate that elderly people are prone to severe, often lethal infectious diseases induced by novel pathogens. Since the ability to mount primary immune responses relies on the availability of naive T cells, the circulating naive T-cell reservoir was evaluated throughout the human life span. Naive T cells were identified as CD95− T lymphocytes for their phenotypic and functional features. Indeed, the lack of CD95 marker is sufficient to identify a population of naive T cells, as defined by coincidence with previously characterized CD45RA+ CD62L+ T cells. Naive CD95− T cells, as expected, require a costimulatory signal, such as CD28, to optimally proliferate after anti-CD3 stimulation. Cytofluorimetric analysis of circulating T lymphocytes from 120 healthy subjects ranging in age from 18 to 105 years revealed that naive T cells decreased sharply with age. The younger subjects had a naive T-lymphocyte count of 825 ± 48 cells/μL, and the centenarians had a naive T-lymphocyte count of 177 ± 28 cells/μL. Surprisingly, the naive T-cell count was lower in CD8+than in CD4+ subsets at any age, and the oldest individuals were almost completely depleted of circulating naive CD8+ T cells (13 ± 4 cells/μL). Concomitantly, a progressive expansion of CD28− T cells occurs with age, which can be interpreted as a compensatory mechanism. These data provide new insights into age-related T-cell–mediated immunodeficiency and reveal some analogies of T-cell dynamics between advanced aging and human immunodeficiency virus (HIV) infection. In conclusion, the exhaustion of the naive CD8+ T-cell reservoir, which has never been reported before, suggests that this T-cell pool is a major target of the aging process and may define a parameter possibly related to the life span of humans.
Killer inhibitory receptor (CD158b) modulates the lytic activity of tumor-specific T lymphocytes infiltrating renal cell carcinomas
Authors: Guerra Nadia , Guillard Maryvonne , Angevin Eric , Echchakir Hamid , Escudier Bernard , Moretta Alessandro , Chouaib Salem , Caignard Anne
Published: October 2019

In this study, we showed that renal tumors contain substantial subsets of CD8+ p58+ T cells. From 1 of these tumors, T cells were amplified in mixed lymphocytes–tumor cell cultures and p58+ T cells were selected immunologically. After expansion, phenotypic and functional features of p58+ and p58− T cells were examined. The p58+ T cells expressed p58.2 receptor and corresponded to CD3+, CD8+, T-cell receptor (TCR) /β+ T cells that were CD56+ and CD28−. Functionally, p58+ T cells showed a low level of lytic activity against autologous tumor cells that was dramatically and specifically increased by anti-p58.2 monoclonal antibody. On the other hand, p58− CD8+ T cells did not lyse autologous tumor cells and had non-major histocompatibility complex–restricted cytotoxicity against K562 and Daudi cells. A p58+ cytotoxic T lymphocyte (CTL) clone (4C7) with the same characteristics as the p58+ T-cell line was derived. This CTL clone did not lyse autologous normal B cells but lysed several HLA-A1+ renal tumor cell lines. Analysis of TCR repertoire diversity showed that the p58+T-cell line contained 3 TCR rearrangements, whereas the TCR repertoire of p58− T cells was polyclonal. Interestingly, TCR transcripts of p58+ T cells and of CTL clone 4C7 were detected as prominent ex vivo in tumor cells but not in peripheral blood mononuclear cells, suggesting that these cells are antigen specific and amplified at the tumor site.
Differential expression and regulation of GTPases (RhoA and Rac2) and GDIs (LyGDI and RhoGDI) in neutrophils from patients with severe congenital neutropenia
Authors: Kasper Brigitte , Tidow Nicola , Grothues Dirk , Welte Karl
Published: October 2019

Severe congenital neutropenia (SCN) or Kostmann syndrome is a disorder of myelopoiesis characterized by a maturation arrest at the stage of promyelocytes or myelocytes in bone marrow and absolute neutrophil counts less than 200/μL in peripheral blood. Treatment of these patients with granulocyte colony-stimulating factor (G-CSF) leads to a significant increase in circulating neutrophils and a reduction in infection-related events in more than 95% of the patients. To date, little is known regarding the underlying pathomechanism of SCN. G-CSF-induced neutrophils of patients with SCN are functionally defective (eg, chemotaxis, superoxide anion generation, Ca++mobilization). Two guanosine triphosphatases (GTPases), Rac2 and RhoA, were described to be involved in many neutrophil functions. The expression of these GTPases and their regulation in patients' neutrophils were of interest. This study determined that the guanosine diphosphate (GDP)-dissociation inhibitor RhoGDI is overexpressed at the protein level in patients' neutrophils and that overexpression is a result of G-CSF treatment. RhoA and LyGDI are expressed at similar levels, whereas Rac2 shows a decreased expression. In addition, association of Rac2 and RhoGDI or LyGDI is abrogated or not detectable based on the low Rac2 expression in patients' neutrophils.
Effects of erythropoietin on platelet reactivity and thrombopoiesis in humans
Authors: Stohlawetz Petra Jilma , Dzirlo Larisa , Hergovich Nicole , Lackner Edith , Mensik Christa , Eichler Hans Georg , Kabrna Eva , Geissler Klaus , Jilma Bernd
Published: October 2019

A recent study in dogs suggested that erythropoietin (EPO) not only promotes the synthesis of increased numbers of reticulated platelets but that these newly produced platelets are hyperreactive compared with controls. Because of the increasing use of EPO in the perioperative setting, we characterized the effects of EPO on platelet reactivity in healthy human volunteers. In a randomized, controlled trial, we studied the effects of EPO on platelet reactivity, thrombopoiesis, and endothelial activation in circumstances similar to those of autologous blood donation. Thirty healthy male volunteers received placebo or EPO (100 or 500 U/kg of body weight given intravenously) three times a week for 2 weeks and underwent phlebotomy on days 8 and 15. Thrombin receptor–activating peptide induced expression of P-selectin, and CD63 increased 2- to 3-fold during EPO treatment. The enhanced platelet reactivity was also reflected by a 50% increase in soluble P-selectin in plasma. Plasma E-selectin levels increased in a dose-dependent fashion by more than 100% during EPO treatment, indicating substantial activation of endothelial cells. A 10% to 20% increase in platelet counts was observed in both EPO groups on day 5. In the placebo group, platelets increased only several days after the first phlebotomy. The increase in platelet counts was not reflected by changes in the amounts of reticulated platelets or circulating progenitor cells. In summary, we found that EPO markedly enhances endothelial activation and platelet reactivity, which may adversely affect patients at cardiovascular risk. However, the increased platelet reactivity could be exploited in patients with platelet dysfunction.
Quantitative measure of c-abl andp15 methylation in chronic myelogenous leukemia: biological implications
Authors: Nguyen TuDung T. , Mohrbacher Ann F. , Tsai Yvonne C. , Groffen John , Heisterkamp Nora , Nichols Peter W. , Yu Mimi C. , Lübbert Michael , Jones Peter A.
Published: October 2019

We used a sensitive, quantitative bisulfite PCR assay, methylation sensitive single nucleotide primer extension (Ms-SNuPE), to measure methylation of the 5′ CpG islands of c-abl andp15 in chronic myelogenous leukemia (CML) patients during progression. We found that the Pa promoter of c-abl was methylated in 81% (17/21) of the white blood cells (WBCs) of CML patients, which correlates with previous reports. In contrast, WBCs from healthy donors, acute myelogenous leukemias, acute lymphocytic leukemias, and myelodysplastic syndromes were unmethylated at thec-abl Pa promoter locus. We also observed p15hypermethylation in 24% (8/34) of CML cases. Methylation of thep15 but not c-abl Pa promoters was associated with CML progression (P = 0.047 vs 0.46), and the two events were independently acquired. We conclude that de novo methylation ofc-abl and p15 both occur in CML, and analysis of DNA methylation changes using the bisulfite-based MS-SNuPE assay allows both a sensitive and quantitative assessment of these molecular events compared to other methods currently utilized.
Inhibition of in vitro spontaneous apoptosis by IL-7 correlates with Bcl-2 up-regulation, cortical/mature immunophenotype, and better early cytoreduction of childhood T-cell acute lymphoblastic leukemia
Authors: Karawajew Leonid , Ruppert Velia , Wuchter Christian , Kösser Annett , Schrappe Martin , Dörken Bernd , Ludwig Wolf-Dieter
Published: October 2019

In normal T-cell development, IL-7 plays a nonredundant role as an antiapoptic factor by regulating Bcl-2 expression in pro-T cells. In the current study, we addressed the roles of IL-7 and related cytokines as apoptosis-modulating factors in precursor T-cell acute lymphoblastic leukemia (T-ALL). To this end, leukemic blasts from pediatric patients with T-ALL were prospectively investigated as to their responsiveness to IL-7, IL-4, and IL-2 (in terms of modulation of spontaneous apoptosis, assessed by flow cytometry), cytokine receptor expression profiles, and expression levels of Bcl-2 and Bax proteins. IL-7, in contrast to IL-4 and IL-2, was highly efficient in apoptosis inhibition , and this effect correlated with the expression levels of IL-7R chain and with the up-regulation of Bcl-2 protein expression (P< .0001). Subclassification of T-ALL samples (n = 130) according to their in vitro IL-7 responses revealed that IL-7 refractory samples were more frequently positive for CD34 (P< .0001) and the myeloid-associated antigen CD33 (P= .01), whereas IL-7 responsiveness was associated with an expression of more mature differentiation-associated T-cell antigens (CD1a, surface CD3, CD4/8; P < .05). Furthermore, the extent of apoptosis inhibition by IL-7 in vitro quantitatively correlated with early cytoreduction as determined by the prednisone peripheral blood response on day 8 and cytoreduction in the marrow on day 15 (n = 87;P < .05). Multivariate analysis of the apoptosis-related parameters investigated, including spontaneous apoptosis, its inhibition by IL-7, and expression levels of Bcl-2 and Bax, showed that only IL-7 responsiveness has an independent impact on early cytoreduction (P < .05), thus indicating a potential prognostic relevance of IL-7 sensitivity in T-ALL.
Severity of iron overload in patients with sickle cell disease receiving chronic red blood cell transfusion therapy
Authors: Harmatz Paul , Butensky Ellen , Quirolo Keith , Williams Roger , Ferrell Linda , Moyer Thomas , Golden Daniel , Neumayr Lynne , Vichinsky Elliott
Published: October 2019

Chronic transfusion therapy is being used more frequently to prevent and treat the complications of sickle cell disease. Previous studies have shown that the iron overload that results from such therapy in other patient populations is associated with significant morbidity and mortality. In this study we examined the extent of iron overload as well as the presence of liver injury and the predictive value of ferritin in estimating iron overload in children with sickle cell disease who receive chronic red blood cell transfusions. A poor correlation was observed between serum ferritin and the quantitative iron on liver biopsy (mean 13.68 ± 6.64 mg/g dry weight;R = 0.350, P = .142). Quantitative iron was highly correlated with the months of transfusion (R = 0.795, P < .001), but serum ferritin at biopsy did not correlate with months of transfusion (R = 0.308, P = .200). Sixteen patients had abnormal biopsies showing mild to moderate changes on evaluation of inflammation or fibrosis. Liver iron was correlated with fibrosis score (R = 0.50, P = .042). No complications were associated with the liver biopsy. Our data suggest that, in patients with sickle cell disease, ferritin is a poor marker for accurately assessing iron overload and should not be used to direct long-term chelation therapy. Despite high levels of liver iron, the associated liver injury was not severe.
Therapeutic levels of human factor VIII and IX using HIV-1–based lentiviral vectors in mouse liver
Authors: Park Frank , Ohashi Kazuo , Kay Mark A.
Published: October 2019

Lentiviral vectors have the potential to play an important role in hemophilia gene therapy. The present study used human immunodeficiency virus (HIV)-based lentiviral vectors containing an EF1 enhancer/promoter driving human factors VIII (hFVIII) or IX (hFIX) complementary DNA expression for portal vein injection into C57Bl/6 mice. Increasing doses of hFIX-expressing lentivirus resulted in a dose-dependent, sustained increase in serum hFIX levels up to approximately 50-60 ng/mL. Partial hepatectomy resulted in a 4- to 6-fold increase (P < 0.005) in serum hFIX of up to 350 ng/mL compared with the nonhepatectomized counterparts. The expression of plasma hFVIII reached 30 ng/mL (15% of normal) but was transient as the plasma levels fell concomitant with the formation of anti-hFVIII antibodies. However, hFVIII levels were persistent in immunodeficient C57Bl/6 scid mice, suggesting humoral immunity-limited gene expression in immunocompetent mice. This study demonstrates that lentiviral vectors can produce therapeutic levels of coagulation factors in vivo, which can be enhanced with hepatocellular proliferation.
Constitutive elevation of serum alpha-fetoprotein in Fanconi anemia
Authors: Cassinat Bruno , Guardiola Philippe , Chevret Sylvie , Schlageter Marie-Hélène , Toubert Marie-Elisabeth , Rain Jean-Didier , Gluckman Eliane
Published: October 2019

The diagnosis of Fanconi anemia (FA) is based on the association of congenital malformations, bone marrow failure syndrome, and hypersensitivity to chromosomal breaks induced by cross-linking agents. In the absence of typical features, the diagnosis is not easy to establish because there is no simple and cost-effective test; thus, investigators must rely on specialized analyses of chromosomal breaks. Because we observed elevated serum alpha-fetoprotein (sAFP) levels in FA patients, we investigated this parameter as a possible diagnostic tool. Serum AFP levels from 61 FA patients and 27 controls with acquired aplastic anemia or other inherited bone marrow failure syndromes were analyzed using a fluoroimmunoassay based on the TRACE technology. Serum AFP levels were significantly more elevated (P < .0001) in FA than in non-FA aplastic patients. In the detection of FA patients among patients with bone marrow failure syndromes, this assay had a sensitivity of 93% and a specificity of 100%. This elevation was not explained by liver abnormalities. Levels of sAFP were unchanged during at least 4 years of follow-up, and allogeneic bone marrow transplantation did not modify sAFP levels. Three of 4 FA patients with mosaicism as well as 5 of 6 FA patients with myelodysplastic syndrome were detected by this test. Heterozygous parents of FA patients had normal sAFP levels. Measurement of sAFP levels with this automated, cost-effective, and reproducible fluoroimmunoassay could be proposed for the preliminary diagnosis of FA whenever this disorder is suspected.
Graphene Oxide in a Composite with Silver Nanoparticles Reduces the Fibroblast and Endothelial Cell Cytotoxicity of an Antibacterial Nanoplatform
Authors: Wierzbicki Mateusz , Jaworski Sławomir , Sawosz Ewa , Jung Anna , Gielerak Grzegorz , Jaremek Henryk , Łojkowski Witold , Woźniak Bartosz , Stobiński Leszek , Małolepszy Artur , Chwalibog André
Published: October 2019

Antibacterial surfaces coated with nanomaterials, including silver nanoparticles, are considered effective alternative antimicrobial agents that can be used instead of antibiotics and chemical agents. However, reports of the potential toxicity of these materials raise questions about the safety of their use in biomedical applications. The objective of this research was to reduce the human cell cytotoxicity of silver nanoparticle-coated polyurethane foils by complexing silver nanoparticles with graphene oxide. The antimicrobial activity of nanoplatforms coated with silver nanoparticles, graphene oxide and the composite of silver nanoparticles and graphene oxide was assessed with Salmonella enteritidis. Cytotoxicity was analysed by an analysis of the viability and morphology of human fibroblasts, human umbilical vein endothelial cells (HUVECs) and chicken embryo chorioallantoic membrane. Additionally, the synthesis level of inflammatory proteins was examined for fibroblasts cultured on different nanoplatforms. The nanoplatform coated with the silver nanoparticles and graphene oxide composite showed strongest antibacterial properties, although nanoplatforms coated with only silver nanoparticles or graphene oxide also resulted in decreased S. enteritidis growth. Furthermore, a nanoplatform coated with silver nanoparticles and graphene oxide composite showed limited immunological stimulation and significantly reduced cytotoxicity towards fibroblasts, HUVECs and chicken embryo chorioallantoic membrane in comparison to the nanoplatform coated only with silver nanoparticles, due to the higher stability of the nanomaterials in the nanocomposite.
Genetic diversity and structure of Chinese grass shrimp, Palaemonetes sinensis, inferred from transcriptome-derived microsatellite markers
Authors: Zhao Yingying , Zhu Xiaochen , Li Zhi , Xu Weibin , Dong Jing , Wei Hua , Li Yingdong , Li Xiaodong
Published: October 2019

Background

The Chinese grass shrimp,Palaemonetes sinensis, is an economically important freshwater shrimp in China, and the study of genetic diversity and structure can positively contribute to the exploration of germplasm resources and assist in the understanding ofP. sinensisaquaculture. Microsatellite markers are widely used in research of genetic backgrounds since it is considered an important molecular marker for the analyses of genetic diversity and structure. Hence, the aim of this study was to evaluate the genetic diversity and structure of wildP. sinensispopulations in China using the polymorphic microsatellite makers from the transcriptome.

Results

Sixteen polymorphic microsatellite markers were developed forP. sinensisfrom transcriptome, and analyzed for differences in genetic diversity and structure in multiple wildP. sinensispopulations in China. Totally of 319 individual shrimps from seven different populations were genotyped to find that allelic polymorphisms varied in two to thirteen alleles seen in the entire loci. Compared to other populations analyzed, the two populations including LD and SJ showed lower genetic diversity. Both the genetic distance (D) and Wrights fixation index (FST) comparing any two populations also indicated that LD and SJ populations differed from the other five populations. An UPGMA tree analysis showed three main clusters containing SJ, LD and other populations which were also confirmed using STRUCTURE analysis.

Conclusion

This is the first study where polymorphic microsatellite markers from the transcriptome were used to analyze genetic diversity and structures of different wildP. sinensispopulations. All the polymorphic microsatellite makers are believed useful for evaluating the extent of the genetic diversity and population structure ofP. sinensis. Compared to the other five populations, the LD and SJ populations exhibited lower genetic diversity, and the genetic structure was differed from the other five populations. Therefore, they needed to be protected against further declines in genetic diversity. The other five populations, LP, LA, LSL, LSY and LSH, are all belonging to Liaohe River Drainage with a relatively high genetic diversity, and hence can be considered as hot spots for in-situ conservation ofP. sinensisas well as sources of desirable alleles for breeding values.
Ipilimumab-induced renal granulomatous arteritis: a case report
Authors: Lemoine Mathilde , Dilly Baptiste , Curie Alexandre , Hébert Vivien , Laurent Charlotte , Hanoy Mélanie , Grangé Steven , Guerrot Dominique , François Arnaud , Bertrand Dominique
Published: October 2019

Background

Immune Checkpoint Inhibitors (ICPIs) are promising new drugs in treatment of advanced tumours targeting cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed cell death protein-1 (PD1) or its ligand (PDL-1). Ipilimumab is a monoclonal antibody targeting the CTLA-4 receptor used in treatment of metastatic melanoma. By increasing activity of the immune system, ICPIs lead to immune-related adverse events, such as dermatitis, colitis or hepatitis. ICPIs-related kidney adverse events are rare and acute tubulointerstitial nephritis with or without granuloma have mainly been reported.

Case presentation

We report a case of acute kidney injury in a patient with melanoma treated by ipilimumab. Kidney biopsy revealed acute interlobular and juxtaglomerular granulomatous arteritis, which has not yet been reported in patients treated by ICPIs. Kidney function partially recovered after ipilimumab discontinuation and oral prednisone. Unfortunately, the patient died a few months later from progression of his melanoma.

Conclusion

This case highlights a new mechanism of acute kidney injury related to ICPIs and supports the interest of kidney biopsy in case of ICPIs related acute renal failure.
Describing mortality trends for major cancer sites in 133 intermediate regions of Brazil and an ecological study of its causes
Authors: Bigoni Alessandro , Ferreira Antunes José Leopoldo , Weiderpass Elisabete , Kjærheim Kristina
Published: October 2019

Background

In Brazil, 211 thousand (16.14%) of all death certificates in 2016 identified cancer as the underlying cause of death, and it is expected that around 320 thousand will receive a cancer diagnosis in 2019. We aimed to describe trends of cancer mortality from 1996 to 2016, in 133 intermediate regions of Brazil, and to discuss macro-regional differences of trends by human development and healthcare provision.

Methods

This ecological study assessed georeferenced official data on population and mortality, health spending, and healthcare provision from Brazilian governmental agencies. The regional office of the United Nations Development Program provided data on the Human Development Index in Brazil. Deaths by misclassified or unspecified causes (garbage codes) were redistributed proportionally to known causes. Age-standardized mortality rates used the world population as reference. Prais-Winsten autoregression allowed calculating trends for each region, sex and cancer type.

Results

Trends were predominantly on the increase in the North and Northeast, whereas they were mainly decreasing or stationary in the South, Southeast, and Center-West. Also, the variation of trends within intermediate regions was more pronounced in the North and Northeast. Intermediate regions with higher human development, government health spending, and hospital beds had more favorable trends for all cancers and many specific cancer types.

Conclusions

Patterns of cancer trends in the country reflect differences in human development and the provision of health resources across the regions. Increasing trends of cancer mortality in low-income Brazilian regions can overburden their already fragile health infrastructure. Improving the healthcare provision and reducing socioeconomic disparities can prevent increasing trends of mortality by all cancers and specific cancer types in Brazilian more impoverished regions.
Comparison of vascular parameters between normal cynomolgus macaques and healthy humans by optical coherence tomography angiography
Authors: Peng Jingyi , Zhong Liuxueying , Ma Li , Jin Jiayi , Zheng Yongxin , Jin Chenjin
Published: October 2019

Background

The metabolic activity of retina is higher than other human tissues and is crucial to the vision. Cynomolgus macaques is widely used in ophthalmic disease research. The evaluation and comparison of macular and optic disc vascular circulation parameters between normal adult cynomolgus macaques and healthy adult humans using OCT-A can promote better use of nonhuman primate models in studies of ophthalmic vascular disease.

Methods

Twelve normal adult cynomolgus macaques with a mean age of 4.91 ± 0.43 years were studied for data collection. The macula of 28 adult healthy humans (14 males and 14 females), with a mean age of 25.11 ± 6.21 years and the optic discs of 9 adult healthy humans (4 males and 5 females) with a mean age of 28.56 ± 6.78 years were measured. The vessel density (VD) was measured using an RTVue XR with AngioVue. The scan sizes of the macular and optic discs were 3 × 3 mm and 4.5 × 4.5 mm, respectively.

Results

OCT-A can image the superficial and deep capillary plexuses and radial peripapillary capillary network. In RPC layer of the optic disc, the VD in the nasal quadrant was lower than the VD in the inferior temporal quadrant. Similarities and significant differences in VD between healthy humans and cynomolgus macaques were obtained using OCT-A.

Conclusions

This study provides normal vascular parameters for adult cynomolgus macaques using OCT-A to help establish an optical parameter database for cynomolgus macaques and compare VD between healthy humans and cynomolgus macaques to promote choroid-retinopathy research.

Trial registration

Current Controlled TrialsNCT03692169, retrospectively registered on 26 sept 2018.
College students’ interpretations of food security questions: results from cognitive interviews
Authors: Nikolaus Cassandra J. , Ellison Brenna , Nickols-Richardson Sharon M.
Published: October 2019

Background

Food insecurity (FI) – the lack of sufficient access to food to maintain a healthy lifestyle – among college (i.e. post-secondary or higher education institution) students has become a prominent issue in the U.S. However, it is not clear if high rates of FI among students are due to the modern experience in higher education institutions or due to underlying issues in common surveying methods. To understand if there were underlying content validity issues, the present study had two primary research questions: 1) How do students interpret the U.S. Department of Agriculture (USDA) Food Security Survey Module (FSSM) questionnaire items, and 2) How do responses of students experiencing FI compare with the theorized experiences and coping responses?

Methods

Thirty-three undergraduate students, aged 18- to 24-years old and fluent in English were recruited from a single 4-year university. During a 60-min session, participants completed the 10-item Adult FSSM and then were cognitively interviewed about their responses using the think-aloud method. Interview transcripts were analysed by two researchers using a collaborative process and basic interpretative approach.

Results

Students were on average 19.5 years old (± 1.2 years), the majority were in their freshman or sophomore (i.e., first or second) year, and 67% (n = 22) experienced FI. Results indicated that students’ interpretations of key terms – such as “money for more,” “balanced meals,” and “real hunger” – diverge from expectations. Furthermore, students categorized as food insecure reported experiences and responses to FI that varied from theoretical dimensions of the process.

Conclusions

Though limited by sample size and representativeness, the present results indicate that the content validity of the FSSM may be compromised in this population and the managed process of FI may present differently among college students. Further psychometric research on modifications to the FSSM or with new FI assessment tools should be conducted with college students.