bluuebunny/crossref_metadata_2025 huggingface dataset:
Chemokine SDF-1 enhances circulating CD34+ cell proliferation in synergy with cytokines: possible role in progenitor survival
Authors: Lataillade Jean-Jacques , Clay Denis , Dupuy Catherine , Rigal Sylvain , Jasmin Claude , Bourin Philippe , Bousse-Kerdilès Marie-Caroline Le
Published: October 2019

The chemokine stromal cell-derived factor-1 (SDF-1), and its receptor, CXCR-4, have been implicated in the homing and mobilization of human CD34+ cells. We show here that SDF-1 may also be involved in hematopoiesis, promoting the proliferation of human CD34+ cells purified from normal adult peripheral blood (PB). CXCR-4 was expressed on PB CD34+ cells. The amount of CXCR-4 on PB CD34+ cells was 10 times higher when CD34+ cells were purified following overnight incubation. CXCR-4 overexpression was correlated with a primitive PB CD34+ cell subset defined by a CD34high CD38lowCD71lowc-KitlowThy-1+antigenic profile. The functional significance of CXCR-4 expression was ascertained by assessing the promoting effect of SDF-1 on cell cycle, proliferation, and colony formation. SDF-1 alone increased the percentage of CD34+ cells in the S+G2/M phases and sustained their survival. In synergy with cytokines, SDF-1 increased PB CD34+ and CD34highCD38low cell expansion and colony formation. SDF-1 also stimulated the growth of colonies derived from primitive progenitors released from quiescence by anti–TGF-β treatment. Thus, our results shed new light on the potential role of this chemokine in the stem cell engraftment process, which involves migration, adhesion, and proliferation. Furthermore, both adhesion-induced CXCR-4 overexpression and SDF-1 stimulating activity may be of clinical relevance for improving cell therapy settings in stem cell transplantation.
Adaptor proteins CRK and CRKL associate with the serine/threonine protein kinase GCKR promoting GCKR and SAPK activation
Authors: Shi Chong-Shan , Tuscano Joseph , Kehrl John H.
Published: October 2019

STE20-related kinases play significant regulatory roles in a range of cellular responses to environmental stimuli. GCKR (also referred to as KHS1) is a serine/threonine protein kinase that has an STE20-like protein kinase domain and that stimulates the stress-activated protein kinase (SAPK, also referred to as Jun kinase or JNK) pathway. GCKR has a large C-terminal regulatory domain that provides sites for interactions with other proteins. Adaptor proteins mediate the interactions between signaling molecules. In this study we showed that the adaptor proteins Crk and CrkL associated with GCKR. When Crk-I, Crk-II, or CrkL was transiently expressed in HEK 293T cells along with GCKR, each coimmunoprecipitated with GCKR. Furthermore, in the Bcr-Abl transformed cell line, K562 endogenous GCKR and CrkL coimmunoprecipitated, indicating a constitutive association. Detection of the CrkL-GCKR interaction required the SH3 domains of CrkL and 2 regions in GCKR—1 between amino acids 387 and 395 that contains a consensus SH3 binding motif and the other between amino acids 599 and 696. Crk or CrkL overexpression increased GCKR catalytic activity. A dominant negative form of Ras abolished Crk- or CrkL-induced GCKR activation, suggesting a dependence on Ras activation for their activation of GCKR. Finally, we showed impairment of the known ability of CrkL to activate the SAPK pathway by a catalytically inactive form of GCKR or by a GCKR antisense construct. Thus, GCKR associates with other proteins through interactions mediated by SH2/SH3 adaptor proteins, which can lead to GCKR and SAPK activation.
Quantitative PCR analysis of HbF inducers in primary human adult erythroid cells
Authors: Smith Reginald D. , Li Jin , Noguchi Constance T. , Schechter Alan N.
Published: October 2019

The development and evaluation of drugs to elevate fetal hemoglobin in the treatment of the genetic diseases of hemoglobin would be facilitated by the availability of reliable cell assays. We have used real-time, quantitative polymerase chain reaction (PCR) analyses of globin messenger RNA (mRNA) levels in a biphasic, erythropoietin-dependent primary culture system for human adult erythroid cells in order to assay compounds for their ability to modulate levels of adult (β) and fetal (γ) globin mRNA. Complementary DNA synthesized from total RNA extracted at timed intervals from aliquots of cells were assayed throughout the period that the culture was studied. γ-globin mRNA levels were found to be much lower (less than 1%) than β-globin mRNA levels. At concentrations of agents chosen for minimal effect on cell division, we find that the 3 drugs studied, 5-azacytidine (5μmol/L), hydroxyurea (40μmol/L), and butyric acid (0.5mmol/L), significantly increase γ-globin mRNA levels. Interestingly, hydroxyurea also had a small stimulatory effect on β-globin mRNA levels, while butyric acid caused a twofold inhibition of β-globin mRNA levels, and 5-azacytidine had little effect on β-globin mRNA levels. The net result of all 3 drugs was to increase the γ/(γ + β) mRNA ratios by threefold to fivefold. These data suggest that the mechanism is distinct for each drug. The profile of butyric-acid–induced changes on globin gene expression is also quite distinct from changes produced by trichostatin A, a known histone deacetylase inhibitor. Quantitative PCR analyses of human erythroid cells should prove useful for studying the mechanism(s) of action of known inducers of γ-globin and identifying new drug candidates.
Acquired loss of p53 induces blastic transformation in p210bcr/abl-expressing hematopoietic cells: a transgenic study for blast crisis of human CML
Authors: Honda Hiroaki , Ushijima Toshikazu , Wakazono Kuniko , Oda Hideaki , Tanaka Yuji , Aizawa Shin-ichi , Ishikawa Takatoshi , Yazaki Yoshio , Hirai Hisamaru
Published: October 2019

Chronic myelogenous leukemia (CML) begins with an indolent chronic phase but inevitably progresses to a fatal blast crisis. Although the Philadelphia chromosome, which generates p210bcr/abl, is a unique chromosomal abnormality in the chronic phase, additional chromosomal abnormalities are frequently detected in the blast crisis, suggesting that superimposed genetic events are responsible for disease progression. To investigate whether loss of p53 plays a role in the evolution of CML, we crossmated p210bcr/abl-transgenic (BCR/ABLtg/−) mice with p53-heterozygous (p53+/−) mice and generated p210bcr/abl-transgenic, p53-heterozygous (BCR/ABLtg/−p53+/−) mice, in which a somatic alteration in the residual normal p53 allele directly abrogates p53 function. TheBCR/ABLtg/−p53+/− mice died in a short period compared with their wild-type (BCR/ABL−/−p53+/+), p53 heterozygous (BCR/ABL−/−p53+/−), and p210bcr/abl transgenic (BCR/ABLtg/−p53+/+) litter mates. They had rapid proliferation of blast cells, which was preceded by subclinical or clinical signs of a myeloproliferative disorder resembling human CML. The blast cells were clonal in origin and expressed p210bcr/abl with an increased kinase activity. Interestingly, the residual normal p53 allele was frequently and preferentially lost in the tumor tissues, implying that a certain mechanism facilitating the loss of p53 allele exists in p210bcr/abl-expressing hematopoietic cells. Our study presents in vivo evidence that acquired loss of p53 contributes to the blastic transformation of p210bcr/abl-expressing hematopoietic cells and provides insights into the molecular mechanism for blast crisis of human CML.
Long-term outcome of continuous 24-hour deferoxamine infusion via indwelling intravenous catheters in high-risk β-thalassemia
Authors: Davis Bernard A. , Porter John B.
Published: October 2019

The optimal regimen of intravenous deferoxamine for iron overload in high-risk homozygous β-thalassemia is unknown because only short-term follow-up has been described in small patient groups. We report the outcome over a 16-year period of a continuous 24-hour deferoxamine regimen, with dose adjustment for serum ferritin, delivered via 25 indwelling intravenous lines for 17 patients. Treatment indications were cardiac arrhythmias, left ventricular dysfunction, gross iron overload, and intolerability of subcutaneous deferoxamine. Cardiac arrhythmias were reversed in 6 of 6 patients, and the left ventricular ejection fraction improved in 7 of 9 patients from a mean (± SEM) of 36 ± 2% to 49 ± 3% (P = .002, n = 9). The serum ferritin fell in a biphasic manner from a pretherapy mean of 6281 ± 562 μg/L to 3736 ± 466 μg/L (P = .001), falling rapidly and proportionally to the pretreatment ferritin (r2 = 0.99) for values >3000 μg/L but falling less rapidly below this value (at 133 ± 22 μg/L/mo). The principal catheter-related complications were infection and thromboembolism (1.15 and 0.48 per 1000 catheter days, respectively), rates similar to other patient groups. Only one case of reversible deferoxamine toxicity was observed (retinal) when the therapeutic index was briefly exceeded. An actuarial survival of 61% at 13 years with no treatment-related mortality provides evidence of the value of this protocol.
Inhibition of thrombin generation by the zymogen factor VII: implications for the treatment of hemophilia A by factor VIIa
Authors: van 't Veer Cornelis , Golden Neal J. , Mann Kenneth G.
Published: October 2019

Factor VII circulates as a single chain inactive zymogen (10 nmol/L) and a trace (∼10-100 pmol/L) circulates as the 2-chain form, factor VIIa. Factor VII and factor VIIa were studied in a coagulation model using plasma concentrations of purified coagulation factors with reactions initiated with relipidated tissue factor (TF). Factor VII (10 nmol/L) extended the lag phase of thrombin generation initiated by 100 pmol/L factor VIIa and low TF. With the coagulation inhibitors TFPI and AT-III present, factor VII both extended the lag phase of the reaction and depressed the rate of thrombin generation. The inhibition of factor Xa generation by factor VII is consistent with its competition with factor VIIa for TF. Thrombin generation with TF concentrations >100 pmol/L was not inhibited by factor VII. At low tissue factor concentrations (<25 pmol/L) thrombin generation becomes sensitive to the absence of factor VIII. In the absence of factor VIII, factor VII significantly inhibits TF-initiated thrombin generation by 100 pmol/L factor VIIa. In this hemophilia A model, approximately 2 nmol/L factor VIIa is needed to overcome the inhibition of physiologic (10 nmol/L) factor VII. At 10 nmol/L, factor VIIa provided a thrombin generation response in the hemophilia model (0% factor VIII, 10 nmol/L factor VII) equivalent to that observed with normal plasma, (100% factor VIII, 10 nmol/L factor VII, 100 pmol/L factor VIIa). These results suggest that the therapeutic efficacy of factor VIIa in the medical treatment of hemophiliacs with inhibitors is, in part, based on overcoming the factor VII inhibitory effect.
Thioredoxin prolongs survival of B-type chronic lymphocytic leukemia cells
Authors: Nilsson Joacim , Söderberg Ola , Nilsson Kenneth , Rosén Anders
Published: October 2019

Thioredoxin (Trx) is a ubiquitous protein disulfide oxidoreductase with antioxidant, cytokine, and chemotactic properties. Previously, we showed that Trx, in synergy with interleukin 1 (IL-1), IL-2, IL-4, tumor necrosis factor  (TNF-), and CD40-ligation induced S-phase entry and mitosis in normal B cells and B-type chronic lymphocytic leukemia (B-CLL) cells. The viability of B-CLL cells stimulated by these protocols is high, and it has been hypothesized that the overexpression of Bcl-2 found in B-CLL protects the cells from apoptosis in vitro and in vivo. In this study, we have analyzed the response of cells derived from 12 samples of patients with B-CLL to recombinant human Trx in spontaneous apoptosis, with special reference to the Bcl-2 expression. Long-term cultures of B-CLL clones showed significantly higher viability when supplemented with human Trx (P = .031), also exemplified with clones surviving more than 2 months. Short-term cultures of B-CLL cells exposed to 1 μg/mL of Trx for 1, 5, or 12 days maintained expression or delayed down-regulation of Bcl-2 compared with control cultures containing RPMI 1640 medium and 10% fetal calf serum only (P = .032, .002, .026, respectively). All B-CLL cells expressed constitutive Trx at varying but low levels, in contrast to adult T-cell leukemias, which overexpress Trx, as previously reported. We found that Trx added to B-CLL cells increased in a dose-dependent fashion the release of TNF-, which has been suggested to be an autocrine growth factor for these cells. In conclusion, we have found that human recombinant Trx induced TNF- secretion, maintained Bcl-2, and reduced apoptosis in B-CLL cells.
Hodgkin and Reed-Sternberg cells represent an expansion of a single clone originating from a germinal center B-cell with functional immunoglobulin gene rearrangements but defective immunoglobulin transcription
Authors: Marafioti Theresa , Hummel Michael , Foss Hans-Dieter , Laumen Helmut , Korbjuhn Petra , Anagnostopoulos Ioannis , Lammert Hetty , Demel Gudrun , Theil Jan , Wirth Thomas , Stein Harald
Published: October 2019

Single cell studies aimed at clarifying the nature and clonality of Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin's disease (HD) have so far produced conflicting results. Using an improved single cell procedure, the HRS cells of 25 patients with nodular sclerosing HD lacking B- and T-cell antigens, with and without Epstein-Barr virus infection, were analyzed for the presence of immunoglobulin (Ig) gene rearrangements. One patient with HD developed follicular lymphoma 2 years later. Both lymphomas originated from a common precursor identified as a germinal center B cell. The data show that all but one of the investigated cases harbored rearranged Ig genes, which were clonal in all instances and carried a high load of somatic mutations. The Ig coding capacity was preserved in 18 of the 24 cases (75%) with rearrangements. However, expression of Ig messenger RNA was not detectable in the HRS cells with the exception of Ig kappa light chain expression in some tumor cells of 1 case. The lack of Ig gene transcription in HRS cells was confirmed by analyzing the HD cell lines L428 and KM-H2 in transient transfection experiments. An Ig promoter/enhancer reporter construct showed virtually no activity in these cells compared to 5 control B-cell lines. We conclude that (1) classical HD is a B-cell lymphoma in most instances, (2) HRS cells are clonal without any exception, (3) they are derived from germinal center B-cells that (4) mostly lack crippling mutations but (5) have consistently lost their Ig gene transcription ability, due to functional defects in the Ig gene regulatory elements.
Glucose-6-phosphate dehydrogenase Aveiro: a de novo mutation associated with chronic nonspherocytic hemolytic anemia
Authors: Costa Elı́sio , Cabeda José Manuel , Vieira Emilia , Pinto Rui , Pereira Susana Aires , Ferraz Leonor , Santos Rosário , Barbot José
Published: October 2019

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common X-linked enzyme abnormality. The clinical phenotype is variable but often predictable from the molecular lesion. Class I variants (the most severe forms of the disease) cluster within exon 10, in a region that, at the protein level, is believed to be involved in dimerization. Here we describe a de novo mutation (C269Y) of a new class I variant (G6PD Aveiro) that maps to exon 8. Mutant and normal alleles were found in both hematopoietic and buccal cells, indicating the presence of mosaicism. The available model of the protein predicts that this lesion lies in proximity to the dimer interface of the molecule. A possible mechanism to explain the severity of the defect is proposed.
The Exodus subfamily of CC chemokines inhibits the proliferation of chronic myelogenous leukemia progenitors
Authors: Hromas Robert , Cripe Larry , Hangoc Giao , Cooper Scott , Broxmeyer Hal E.
Published: October 2019

Chemokines are a family of related proteins that regulate leukocyte infiltration into inflamed tissue and play important roles in disease processes. Among the biologic activities of chemokines is inhibition of proliferation of normal hematopoietic progenitors. However, chemokines that inhibit normal progenitors rarely inhibit proliferation of hematopoietic progenitors from patients with chronic myelogenous leukemia (CML). We and others recently cloned a subfamily of CC chemokines that share similar amino-terminal peptide sequences and a remarkable ability to chemoattract T cells. These chemokines, Exodus-1/LARC/MIP-3, Exodus-2/SLC/6Ckine/TCA4, and Exodus-3/CKβ11/MIP-3β, were found to inhibit proliferation of normal human marrow progenitors. The study described here found that these chemokines also inhibited the proliferation of progenitors in every sample of marrow from patients with CML that was tested. This demonstration of consistent inhibition of CML progenitor proliferation makes the 3 Exodus chemokines unique among chemokines.
The GATA-E box-GATA motif in the EKLF promoter is required for in vivo expression
Authors: Anderson Kathleen P. , Crable Scott C. , Lingrel Jerry B.
Published: October 2019

The erythroid Krüppel-like factor (EKLF) is a key regulatory protein in globin gene expression. This zinc finger transcription factor is required for expression of the adult β globin gene, and it has been suggested that it plays an important role in the developmental switch from fetal γ to adult β globin gene expression. We have previously described a sequence element in the distal promoter region of the mouse EKLF gene that is critical for the expression of this transcription factor. The element consists of an E box motif flanked by 2 GATA-1 binding sites. Here we demonstrate that mutation of the E box or the GATA-1 consensus sequences eliminates expression from the EKLF promoter in transgenic mice. These results confirm the importance of this activator element for in vivo expression of the EKLF gene.
Activation of Akt kinase by granulocyte colony-stimulating factor (G-CSF): evidence for the role of a tyrosine kinase activity distinct from the janus kinases
Authors: Dong Fan , Larner Andrew C.
Published: October 2019

Activation of the serine/threonine kinase Akt has been shown to be a critical component for growth factor and cytokine stimulation of cell survival. Although some of the immediate upstream activators of Akt have been defined, the roles of tyrosine kinases in the activation of Akt are not well delineated. Granulocyte colony-stimulating factor (G-CSF) regulates the proliferation, differentiation, and survival of neutrophilic granulocytes. G-CSF exerts its actions by stimulating several signaling cascades after binding its cell surface receptor. Both Jak (Janus) and Src families of tyrosine kinases are stimulated by incubation of cells with G-CSF. In this report, we show that G-CSF stimulation of cells leads to activation of Akt. The membrane-proximal 55 amino acids of the G-CSF receptor cytoplasmic domain are sufficient for mediating Akt activation. However, activation of Akt appears to be downregulated by the receptor's carboxy-terminal region of 98 amino acids, a region that has been shown to be truncated in some patients with acute myeloid leukemia associated with severe congenital neutropenia. Furthermore, we demonstrate that G-CSF–induced activation of Akt requires the activities of Src family kinases but can be clearly dissociated from G-CSF–stimulated activation of Stats (signal transducers and activators of transcripton) by the Jak kinases. Thus, cytokine activation of the Jak/Stat and other signaling cascades can be functionally separated.
Integrin α2β1 (VLA-2) is a principal receptor used by neutrophils for locomotion in extravascular tissue
Authors: Werr Joachim , Johansson Joakim , Eriksson Einar E. , Hedqvist Per , Ruoslahti Erkki , Lindbom Lennart
Published: October 2019

Cell adhesion molecules are critically involved in the multistep process of leukocyte recruitment in inflammation. The specific receptors used by polymorphonuclear leukocytes (PMN) for locomotion in extravascular tissue have as yet not been identified. By means of immunofluorescence flow cytometry and laser scanning confocal microscopy, this study demonstrated that surface expression of the 2β1 (VLA-2) integrin, though absent on blood PMN, is induced in extravasated PMN collected from human skin blister chambers, and rat PMN accumulated in the peritoneal cavity after chemotactic stimulation. Intravital time-lapse videomicroscopy was used to investigate chemoattractant-induced PMN locomotion in the rat mesentery in vivo. Local administration of function-blocking monoclonal antibody or peptide recognizing the 2β1 integrin reduced PMN migration velocity in the extravascular tissue by 73% ± 3% and 70% ± 10%, respectively ( means ± SD). The distance f-met-leu-phe peptide (fMLP)-stimulated human PMN migrated in a collagen gel in vitro was markedly reduced by treatment with anti-2 mAbs or peptide, whereas no effect was observed with antibodies or peptides recognizing the 4β1 or 5β1integrins. Further evidence for a critical role of expression of 2β1 integrin in PMN locomotion in extravascular tissue was obtained in the mouse air pouch model of acute inflammation where chemoattractant-induced PMN recruitment was substantially inhibited by local anti-2 mAb treatment. Thus, expression of 2β1 integrin on extravasated PMN has been identified and a novel role of this receptor in regulating the extravascular phase of leukocyte trafficking in inflammation has been formulated.
Recombinant full-length tissue factor pathway inhibitor fails to bind to the cell surface: implications for catabolism in vitro and in vivo
Authors: Ho Guyu , Narita Masaaki , Broze George J. , Schwartz Alan L.
Published: October 2019

Tissue factor pathway inhibitor (TFPI) plays a key role in the regulation of tissue factor-initiated blood coagulation secondary to loss of the integrity of the blood vessel wall. TFPI is a naturally occurring Kunitz-type protease inhibitor that inhibits coagulation factor Xa and, in a factor Xa-dependent manner, mediates feedback inhibition of the factor VIIa/tissuefactor catalytic complex. In vivo full-length TFPI is thought to be primarily bound to the vascular endothelium and the high affinity binding requires an intact carboxy terminus. Here we describe a full-length TFPI molecule, expressed in mouse C127 cells (TFPIC127), which exhibits virtually no cellular binding yet contains the intact carboxy terminus. This TFPI (TFPIC127) is neither internalized nor degraded via the TFPI endocytic receptor, LDL-receptor–related protein. Pharmacokinetic studies of TFPIC127 in vivo demonstrate a 10-fold prolongation in the plasma half-life, compared with that of bacterial recombinant TFPI.
Characterization of the vasculogenic block in the absence of vascular endothelial growth factor-A
Authors: Bautch Victoria L. , Redick Sambra D. , Scalia Aaron , Harmaty Marco , Carmeliet Peter , Rapoport Rebecca
Published: October 2019

Vascular endothelial growth factor (VEGF) signaling is required for both differentiation and proliferation of vascular endothelium. Analysis of differentiated embryonic stem cells with one or both VEGF-A alleles deleted showed that both the differentiation and the expansion of endothelial cells are blocked during vasculogenesis. Blood island formation was reduced by half in hemizygous mutant VEGF cultures and by 10-fold in homozygous mutant VEGF cultures. Homozygous mutant cultures could be partially rescued by the addition of exogenous VEGF. RNA levels for the endothelial adhesion receptors ICAM-2 and PECAM were reduced in homozygous mutant cultures, but ICAM-2 RNA levels decreased substantially, whereas PECAM RNA levels remained at hemizygous levels. The quantitative data correlated with the antibody staining patterns because cells that were not organized into vessels expressed PECAM but not ICAM-2. These PECAM+ cell clumps accumulated in mutant cultures as vessel density decreased, suggesting that they were endothelial cell precursors blocked from maturation. A subset of PECAM+ cells in clumps expressed stage-specific embryonic antigen-1 (SSEA-1), and all were ICAM-2(−) and CD34(−), whereas vascular endothelial cells incorporated into vessels were PECAM(+), ICAM-2(+), CD34(+), and SSEA-1(−). Analysis of flk-1 expression indicated that a subset of vascular precursor cells coexpressed PECAM and flk-1. These data suggest that VEGF signaling acts in a dose-dependent manner to affect both a specific differentiation step and the subsequent expansion of endothelial cells.
Cyclophosphamide induces type I interferon and augments the number of CD44hi T lymphocytes in mice: implications for strategies of chemoimmunotherapy of cancer
Authors: Schiavoni Giovanna , Mattei Fabrizio , Di Pucchio Tiziana , Santini Stefano M. , Bracci Laura , Belardelli Filippo , Proietti Enrico
Published: October 2019

In a previous study, we reported that a single injection of cyclophosphamide (CTX) in tumor-bearing mice resulted in tumor eradication when the animals were subsequently injected with tumor-sensitized lymphocytes. Notably, CTX acted by inducing bystander effects on T cells, and the response to the combined CTX/adoptive immunotherapy regimen was inhibited in mice treated with antibodies to mouse interferon (IFN)–/β. In the present study, we have investigated whether CTX induced the expression of type I IFN, and we have characterized the CTX effects on the phenotype of T cells in normal mice. CTX injection resulted in an accumulation of type I IFN messenger RNA in the spleen of inoculated mice, at 24 to 48 hours, that was associated with IFN detection in the majority of the animals. CTX also enhanced the expression of the Ly-6C on spleen lymphocytes. This enhancement was inhibited in mice treated with anti–type I IFN antibodies. Moreover, CTX induced a long-lasting increase in in vivo lymphocyte proliferation and in the percentage of CD44hiCD4+ and CD44hiCD8+T lymphocytes. These results demonstrate that CTX is an inducer of type I IFN in vivo and enhances the number of T cells exhibiting the CD44hi memory phenotype. Since type I IFN has been recently recognized as the important cytokine for the in vivo expansion and long-term survival of memory T cells, we suggest that induction of this cytokine may explain at least part of the immunomodulatory effects observed after CTX treatment. Finally, these findings provide a new rationale for combined treatments with CTX and adoptive immunotherapy in cancer patients.
Growth of FasL-bearing tumor cells in syngeneic murine host induces apoptosis and toxicity in Fas+ organs
Authors: Zeytun Ahmet , Nagarkatti Mitzi , Nagarkatti Prakash S.
Published: October 2019

In the current study, we investigated whether the growth of FasL-bearing tumor cells would induce apoptosis and toxicity in organs that express high level of Fas. Sera from C57BL/6 +/+(wild-type) mice injected with syngeneic FasL+ tumors, LSA, or EL-4, showed significantly higher levels of soluble FasL than that from the nontumor-bearing mice. Furthermore, the soluble FasL was functional inasmuch as the sera from tumor-bearing mice were able to induce apoptosis in Fas+ but not Fas−targets. Histopathologic studies and in situ TUNEL assay to detect apoptosis were carried out in C57BL/6 +/+(Fas+) or C57BL/6 lpr/lpr (Fas−) mice injected with syngeneic LSA and EL-4 tumor cells. The morphology of the liver and thymus from tumor bearing C57BL/6 +/+ mice showed marked damage and tissue destruction. In contrast, the liver and thymus from tumor-bearing C57BL/6 lpr/lpr mice showed minimal damage. Furthermore, the tumor-bearing C57BL/6 +/+, but not the C57BL/6 lpr/lpr, mice exhibited significant apoptosis in the liver and thymus. The FasL responsible for toxicity was tumor derived rather than host derived; tumor-bearing C57BL/6 gld/gld(FasL-defective) mice also exhibited significant apoptosis in the liver and thymus. Together, these data suggested that the in vivo growth of FasL-bearing tumor cells can induce significant apoptosis and toxicity in Fas+ tissues of the host. Such toxicity may be mediated by the soluble FasL produced by tumor cells.
Dehydration of mature and immature sickle red blood cells during fast oxygenation/deoxygenation cycles: role of KCl cotransport and extracellular calcium
Authors: McGoron Anthony J. , Joiner Clinton H. , Palascak Mary B. , Claussen William J. , Franco Robert S.
Published: October 2019

Sickle red blood cells (RBC) become dehydrated as a consequence of potassium loss. This process depends at least partly on deoxygenation and may be influenced by the presence of oxygenation/deoxygenation cycles and the frequency of cycling. In this study, sickle RBC were subjected to approximately 180 oxygenation/deoxygenation cycles during 4 hours to evaluate RBC dehydration with cycle periods more similar to in vivo cycles than those in previous studies. A continuous-flow, steady-state apparatus circulated a dilute RBC suspension through gas-permeable silicone tubing with segments that were exposed to either nitrogen or ambient oxygen. The percentage of sickling and partial pressure of oxygen were measured by means of sampling ports in the deoxygenation and oxygenation regions. The density increase (dehydration) of young (transferrin receptor-positive) and mature (transferrin receptor-negative) RBC and the requirements for calcium and chloride were evaluated. Density increase correlated with the percentage of sickled cells at the deoxygenation sampling port and was observed only in the presence of calcium, thereby implicating the calcium-dependent potassium channel (Gardos pathway). Density increase was not dependent on the presence of chloride, making it unlikely that KCl cotransport was an important pathway under these conditions.
Continuous salt stress-induced long non-coding RNAs and DNA methylation patterns in soybean roots
Authors: Chen Rui , Li Ming , Zhang Huiyuan , Duan Lijin , Sun Xianjun , Jiang Qiyan , Zhang Hui , Hu Zheng
Published: October 2019

Background

Environmental stimuli can activate a series of physiological and biochemical responses in plants accompanied by extensive transcriptional reprogramming. Long non-coding RNAs (lncRNAs), as versatile regulators, control gene expression in multiple ways and participate in the adaptation to biotic and abiotic stresses.

Results

In this study, soybean seedlings were continuously cultured for 15 days with high salinity solutions started from seed germination. Strand-specific whole transcriptome sequencing and stringent bioinformatic analysis led to the identification of 3030 long intergenic non-coding RNAs (lincRNAs) and 275 natural antisense transcripts (lncNATs) in soybean roots. In contrast to mRNAs, newly identified lncRNAs exhibited less exons, similar AU content to UTRs, even distribution across the genome and low evolutionary conservation. Remarkably, more than 75% of discovered lncRNAs that were activated or up-regulated by continuous salt stress mainly targeted proteins with binding and catalytic activities. Furthermore, two DNA methylation maps with single-base resolution were generated by using reduced representation bisulfite sequencing, offering a genome-wide perspective and important clues for epigenetic regulation of stress-associated lncRNAs and protein-coding genes.

Conclusions

Taken together, our findings systematically demonstrated the characteristics of continuous salt stress-induced lncRNAs and extended the knowledge of corresponding methylation profiling, providing valuable evidence for a better understanding of how plants cope with long-term salt stress circumstances.
Intensified household contact tracing, prevention and treatment support versus enhanced standard of care for contacts of tuberculosis cases in South Africa: study protocol for a household cluster-randomised trial
Authors: MacPherson Peter , Webb Emily L. , Variava Ebrahim , Lala Sanjay G. , Milovanovic Minja , Ratsela Andrew , Lebina Limakatso , Kinghorn Anthony , Martinson Neil A.
Published: October 2019

Background

Household contact tracing of index TB cases has been advocated as a key part of TB control for many years, but has not been widely implemented in many low-resource setting because of the current dearth of high quality evidence for effectiveness. Innovative strategies for earlier, more effective treatment are particularly important in contexts with hyper-endemic levels of HIV, where levels of TB infection remain extremely high.

Methods

We present the design of a household cluster-randomised controlled trial of interventions aimed at improving TB-free survival and reducing childhood prevalence of Mycobacterium tuberculosis infection among household contacts of index TB cases diagnosed in two provinces of South Africa. Households of index TB cases will be randomly allocated in a 1:1 ratio to receive either an intensified home screening and linkage for TB and HIV intervention, or enhanced standard of care. The primary outcome will compare between groups the TB-free survival of household contacts over 15 months. All participants, or their next-of-kin, will provide written informed consent to participate.

Discussion

Evidence from randomised trials is required to identify cost-effective approaches to TB case-finding that can be applied at scale in sub-Saharan Africa.

Trial registration

ISRCTN16006202 (01/02/2017: retrospectively registered) and NHREC4399 (11/04/2016: prospectively registered). Protocol version: 4.0 (date: 18th January 2018).
Iranian midwives’ attitudes and beliefs toward physiological childbirth: a cross-sectional study
Authors: Sadeghzadeh Narges , Amiri-Farahani Leila , Haghani Shima , Hasanpoor-Azghady Syedeh Batool
Published: October 2019

Background

The significant role of midwives in providing labor and delivery care underscores the necessity of the identification of attitudes and beliefs of these groups of maternity care providers toward physiological childbirth. The purpose of the current study was to describe midwives’ attitudes and beliefs toward physiological childbirth and identify its related factors.

Methods

This cross-sectional study was carried out on 200 midwives working in the labor and delivery wards of selected hospitals in an urban area of Tehran, Iran, through the continuous sampling method from May to July 2018. The data were collected using a two-part demographic characteristic form and Midwives’ Attitudes and Beliefs Questionnaire-Revised toward physiological childbirth. Data analysis was performed in SPSS software (version 25). The significance level was set atP < 0.05.

Results

The mean score of midwives’ attitudes and beliefs toward physiological childbirth were 119.90 with a standard deviation of 9.30. Moreover, of the different domains of Midwives’ Attitudes and Beliefs Questionnaire-Revised, the women’s experience of birth (78.53) and the medical model conflict (51.05) obtained the highest and lowest scores, respectively. According to the multiple linear regression model analysis, the total mean score of midwives’ attitudes and beliefs toward physiological childbirth was significantly correlated with the level of education and interest in the profession (P < 0.05). Midwives with a master degree obtained higher scores (4.32) in terms of attitudes and beliefs toward physiologic childbirth, compared to those with an associate or bachelor degree. Also, there were 0.09 increases in the attitude and belief score of midwives per one score increase in their interest in the profession. It can be concluded that these two variables explained 16% of the variation in the scores of midwives’ attitudes and beliefs to physiological childbirth.

Conclusion

The results of this study showed that midwives with higher levels of education and more interest in their profession had more positive attitudes and beliefs toward physiological childbirth. Therefore, it is necessary to motivate midwives to obtain higher levels of education and increase their interest in the profession to promote physiological childbirth.
Implementing a toolkit for the prevention, management and control of carbapenemase-producing Enterobacteriaceae in English acute hospitals trusts: a qualitative evaluation
Authors: Schneider Annegret , Coope Caroline , Michie Susan , Puleston Richard , Hopkins Susan , Oliver Isabel
Published: October 2019

Background

Antimicrobial resistance is an increasing problem in hospitals world-wide. Following other countries, English hospitals experienced outbreaks of carbapenemase-producing Enterobacteriaceae (CPE), a bacterial infection commonly resistant to last resort antibiotics. One way to improve CPE prevention, management and control is the production of guidelines, such as the CPE toolkit published by Public Health England in December 2013. The aim of this research was to investigate the implementation of the CPE toolkit and to identify barriers and facilitators to inform future policies.

Methods

Acute hospital trusts (N = 12) were purposively sampled based on their self-assessed CPE colonisation rates and time point of introducing local CPE action plans. Following maximum variation sampling, 44 interviews with hospital staff were conducted between April and August 2017 using a semi-structured topic guide based on the Capability, Opportunity, Motivation and Behaviour Model and the Theoretical Domains Framework, covering areas of influences on behaviour. Interviews were audio-recorded, transcribed verbatim and analysed using thematic analysis.

Results

The national CPE toolkit was widely disseminated within infection prevention and control teams (IPCT), but awareness was rare among other hospital staff. Local plans, developed by IPCTs referring to the CPE toolkit while considering local circumstances, were in place in all hospitals. Implementation barriers included: shortage of isolation facilities for CPE patients, time pressures, and competing demands. Facilitators were within hospital and across-hospital collaborations and knowledge sharing, availability of dedicated IPCTs, leadership support and prioritisation of CPE as an important concern. Participants using the CPE toolkit had mixed views, appreciating its readability and clarity about patient management, but voicing concerns about the lack of transparency on the level of evidence and the practicality of implementation. They recommended regular updates, additional clarifications, tailored information and implementation guidance.

Conclusions

There were problems with the awareness and implementation of the CPE toolkit and frontline staff saw room for improvement, identifying implementation barriers and facilitators. An updated CPE toolkit version should provide comprehensive and instructive guidance on evidence-based CPE prevention, management and control procedures and their implementation in a modular format with sections tailored to hospitals’ CPE status and to different staff groups.
Semi-quantitative visual assessment of chest radiography is associated with clinical outcomes in critically ill patients
Authors: Mason Stefanie E. , Dieffenbach Paul B. , Englert Joshua A. , Rogers Angela A. , Massaro Anthony F. , Fredenburgh Laura E. , Higuera Angelica , Pinilla-Vera Mayra , Vilas Marta , San Jose Estepar Raul , Washko George R. , Baron Rebecca M. , Ash Samuel Y.
Published: October 2019

Background

Respiratory pathology is a major driver of mortality in the intensive care unit (ICU), even in the absence of a primary respiratory diagnosis. Prior work has demonstrated that a visual scoring system applied to chest radiographs (CXR) is associated with adverse outcomes in ICU patients with Acute Respiratory Distress Syndrome (ARDS). We hypothesized that a simple, semi-quantitative CXR score would be associated with clinical outcomes for the general ICU population, regardless of underlying diagnosis.

Methods

All individuals enrolled in the Registry of Critical Illness at Brigham and Women’s Hospital between June 2008 and August 2018 who had a CXR within 24 h of admission were included. Each patient’s CXR was assigned an opacification score of 0–4 in each of four quadrants with the total score being the sum of all four quadrants. Multivariable negative binomial, logistic, and Cox regression, adjusted for age, sex, race, immunosuppression, a history of chronic obstructive pulmonary disease, a history of congestive heart failure, and APACHE II scores, were used to assess the total score’s association with ICU length of stay (LOS), duration of mechanical ventilation, in-hospital mortality, 60-day mortality, and overall mortality, respectively.

Results

A total of 560 patients were included. Higher CXR scores were associated with increased mortality; for every one-point increase in score, in-hospital mortality increased 10% (OR 1.10, CI 1.05–1.16, p < 0.001) and 60-day mortality increased by 12% (OR 1.12, CI 1.07–1.17, p < 0.001). CXR scores were also independently associated with both ICU length of stay (rate ratio 1.06, CI 1.04–1.07, p < 0.001) and duration of mechanical ventilation (rate ratio 1.05, CI 1.02–1.07, p < 0.001).

Conclusions

Higher values on a simple visual score of a patient’s CXR on admission to the medical ICU are associated with increased in-hospital mortality, 60-day mortality, overall mortality, length of ICU stay, and duration of mechanical ventilation.
Assessment of spinal cord motion as a new diagnostic MRI-parameter in cervical spinal canal stenosis: study protocol on a prospective longitudinal trial
Authors: Wolf Katharina , Krafft Axel J. , Egger Karl , Klingler Jan-Helge , Hubbe Ulrich , Reisert Marco , Hohenhaus Marc
Published: October 2019

Background

Increased spinal cord motion has been proven to be a relevant finding within spinal canal stenosis disclosed by phase-contrast MRI (PC-MRI). Adapted PC-MRI is a suitable and reliable method within the well deliberated setting. As the decision between conservative and operative treatment can be challenging in some cases, further diagnostic marker would facilitate the diagnostic process. We hypothesize that increased spinal cord motion will correlate to clinical course and functional impairment and will contribute as a new diagnostic marker.

Methods

A monocentric, prospective longitudinal observational trial on cervical spinal canal stenosis will be conducted at the University Medical Center Freiburg. Patients (n = 130) with relevant cervical spinal canal stenosis, being defined by at least contact to the spinal cord, will be included. Also, we will examine a control group of healthy volunteers (n = 20) as proof-of-principle. We will observe two openly assigned branches of participants undergoing conservative and surgical decompressive treatment (based on current German Guidelines) over a time course of 12 month, including a total of 4 visits. We will conduct a broad assessment of clinical parameters, standard scores and gradings, electrophysiological measurements, standard MRI, and adapted functional PC-MRI of spinal cord motion. Primary endpoint is the evaluation of an expected negative correlation of absolute spinal cord displacement to clinical impairment. Secondary endpoints are the evaluation of positive correlation of increased absolute spinal cord displacement to prolonged evoked potentials, prediction of clinical course by absolute spinal cord displacement, and demonstration of normalized spinal cord motion after decompressive surgery.

Discussion

With the use of adapted, non-invasive PC-MRI as a quantitative method for assessment of spinal cord motion, further objective diagnostic information can be gained, that might improve the therapeutic decision-making process. This study will offer the needed data in order to establish PC-MRI on spinal cord motion within the diagnostic work-up of patients suffering from spinal canal stenosis.

Trial registration

German Clinical Trials Register, ID: DRKS00012962, Register date 2018/01/17
Minimal invasive aortic valve replacement: associations of radiological assessments with procedure complexity
Authors: Boti Bruce R. , Hindori Vikash G. , Schade Emilio L. , Kougioumtzoglou Athina M. , Verbeek Eva C. , Driessen-Waaijer Annet , Cocchieri Riccardo , de Mol Bas A. J. M. , Planken Nils R. , Kaya Abdullah , Marquering Henk A.
Published: October 2019

Objectives

Limited aortic annulus exposure during minimal invasive aortic valve replacement (mini-AVR) proves to be challenging and contributes to procedure complexity, resulting in longer procedure times. New innovations like sutureless valves have been introduced to reduce procedure complexity. Additionally, preoperative imaging could also contribute to reducing procedure times. Therefore, we hypothesize that Computed Tomography (CT)-image based measurements are associated with mini-AVR complexity.

Methods

One hundred patients who underwent a mini-sternotomy and had a preoperative CT scan were included. With a CT-based mini-AVR planning tool, we measured access distance, access angle, annulus dimensions, and calcium volume. The associations of these measurements with cardiopulmonary bypass (CPB) time and aortic cross-clamp (AoX) time were assessed using univariable and multivariable regression models. In the multivariable models, these measurements were adjusted for age and suture technique.

Results

In the univariable regression models, calcium volume and annulus dimensions were associated with longer CPB and AoX time. After adjusting for age and suture technique, increasing calcium volume was still associated with longer CPB (adjusted β-coefficient 0.002, 95%-CI (0.005, 0.019), p-value = 0.002) and AoX time (adjusted β-coefficient 0.010, 95%-CI (0.004, 0.016), p-value = 0.002). However, after adjusting for these confounders, the association between annulus dimensions and procedure times lost statistical significance.

Conclusion

Increase in calcium volume are associated with longer CPB and AoX times, with age and sutureless valve implantation as independent confounders. In contrast to previous studies, access angle was not associated with procedure complexity.
Epigenetic down-regulation of the HIST1 locus predicts better prognosis in acute myeloid leukemia with NPM1 mutation
Authors: Garciaz Sylvain , N’guyen Dasi Lia , Finetti Pascal , Chevalier Christine , Vernerey Julien , Poplineau Mathilde , Platet Nadine , Audebert Stéphane , Pophillat Matthieu , Camoin Luc , Bertucci François , Calmels Boris , Récher Christian , Birnbaum Daniel , Chabannon Christian , Vey Norbert , Duprez Estelle
Published: October 2019

Background

The epigenetic machinery is frequently altered in acute myeloid leukemia. Focusing on cytogenetically normal (CN) AML, we previously described an abnormal H3K27me3 enrichment covering 70 kb on theHIST1cluster (6.p22) in CN-AML patient blasts. Here, we further investigate the molecular, functional, and prognosis significance of this epigenetic alteration named H3K27me3HIST1inNPM1-mutated (NPM1mut) CN-AML.

Results

We found that three quarter of theNPM1mut CN-AML patients were H3K27me3HIST1high. H3K27me3HIST1highgroup of patients was associated with a favorable outcome independently of known molecular risk factors. In gene expression profiling, the H3K27me3HIST1highmark was associated with lower expression of the histone genesHIST1H1D,HIST1H2BG,HIST1H2AE, andHIST1H3Fand an upregulation of genes involved in myelomonocytic differentiation. Mass spectrometry analyses confirmed that the linker histone protein H1d, but not the other histone H1 subtypes, was downregulated in the H3K27me3HIST1highgroup of patients. H1d knockdown primed ATRA-mediated differentiation of OCI-AML3 and U937 AML cell lines, as assessed on CD11b/CD11c markers, morphological and gene expression analyses.

Conclusions

Our data suggest thatNPM1mut AML prognosis depends on the epigenetic silencing of theHIST1cluster and that, among the H3K27me3 silenced histone genes,HIST1H1Dplays a role in AML blast differentiation.

Graphical abstract