bluuebunny/crossref_metadata_2025 huggingface dataset:
Association of the 1580 (C/T) Polymorphism in the Surfactant Protein B Gene with the Severity of Respiratory Failure after Allogeneic Transplantation.
Authors: Illmer Thomas , Theuser Catrin , Thiede Christian , Schaich Markus , Trautmann Simone , Hoeffken Gert , Ehninger Gerhard , Bornhaeuser Martin
Published: October 2019

Respiratory failure is a serious early complication confounding the clinical effect of allogeneic stem cell transplantation and early detection may be crucial for efficient treatment. We introduced a system of intensive oxigenation monitoring in patients transplanted in our center between November 2001 and December 2003. All allogeneic transplanted patients were followed by a twice daily measurement of oxi-index (paO2/FiO2) during the transplantation period (untill day + 25). Patients that fulfilled criteria of acute lung injury (oxi-index <300) were treated by O2 application vs. non-invasive ventilation (NIV). Failure to increase oxi-index above the threshold of 300 led to referral of the patient to ICU were intensified NIV was applied and/or mechanical ventilation (MV) was initiated based on a defined scoring system. 165 patients were followed for the development of ALI. 48 (29.1%) developed oxiindices <300 (ALI). Development of ALI predicted for a significantly higher rate of ICU admissions (37.5% vs. 6.8% in non-ALI patients; p<0.001) and MV (20.8 % vs. 1.7 in non-ALI patients; p<0.001). ALI was most commonly diagnosed during the engraftment period but was at diagnosis not associated with other criteria like respiration rate, CRP and tachycardia. Since members of the pulmonary innate immune system may be associated with the development of ALI and ARDS we screened the 165 patients for the presence of alternative alleles in the Surfactant Protein B (SP-B) gene. A polymorphism at position +1580 (T/C variation) of SP-B has been previously found to be associated with the development of ARDS. Polymorphism of SP-B were not associated with the development of ALI and did not predict for the need of intensified ICU treatment. However, the rate of MV varied considerably. Nine patients with the T/T genotype (9 out of 42, 21.4%) required intubation and mechanical ventilation whereas none of the patients with C/C had to receive MV (0 out of 30; p=0.01).The data indicate that measurement of the oxiindex is an early sensitive parameter of respiratory failure in allogeneic transplantation and that severity of respiratory damage may be predicted by the T allele at the SP-B +1580 site
Frequent but Reversible and Manageable Cardiac Complications after Successful Haploidentical HLA-Mismatched Hematopoietic Stem Cell Transplantation (HSCT) without Ex Vivo Graft Manipulation.
Authors: Oshima Kumi , Kanda Yoshinobu , Sakata-Yanagimoto Mamiko , Asano-Mori Yuki , Izutsu Koji , Ogawa Seishi , Motokura Toru , Chiba Shigeru , Hirai Hisamaru
Published: October 2019

Alemtuzumab, a humanized monoclonal antibody directed against human CD52, has a strong lympholytic effect. We performed a study to evaluate the safety of unmanipulated HSCT from 2 or 3 loci-mismatched related donors using in vivo alemtuzumab. A total body irradiation-based regimen was used in young patients, while those 50 years old or older received fludarabine-based but myeloablative conditioning. Alemtuzumab was added to these regimens by intravenous infusion at 0.2 mg/kg/day for 6 days (days −8 to −3). We have treated 14 patients thus far. All achieved neutrophil engraftment with complete donor-type chimerism. Only 1 developed grade III-IV acute GVHD. There was no infection-related death. However, 8 patients (57.1%) developed grade II-III cardiac complications, mainly with congestion, according to Bearman’s criteria. We thus retrospectively analyzed the records of 143 adult patients who underwent allogeneic HSCT from 1995 to 2004 to evaluate whether the use of alemtuzumab was an independent risk factor for cardiac complications. On univariate analyses, the cumulative dose of anthracyclines (P=0.001), smoking history (P=0.036), serum ferritin level (P=0.033), left ventricular ejection fraction (P=0.070), HLA-mismatch (P=0.054), and the use of in vivo alemtuzumab (P=0.0002) affected the incidence of grade II to IV cardiac complications with at least borderline significance. Of these, multivariate analysis with backward stepwise selection revealed that the cumulative dose of anthracyclines and the use of in vivo alemtuzumab were independent significant risk factors for cardiac complications (P=0.0016 and P=0.0001, respectively). The duration from transplantation to the onset of cardiac complications was significantly longer in the alemtuzumab group (P=0.02). Fortunately, all the cardiac complications in the alemtuzumab group were successfully treated by the use of diuretics and/or catecholamines. In conclusion, in vivo alemtuzumab has an excellent efficacy to prevent severe GVHD even in HLA-mismatched HSCT. However, the use of in vivo alemtuzumab along with myeloablative conditioning for HLA-mismatched transplantation may affect the incidence of cardiac complications after transplantation. Considering the low incidence of cardiac complications in HLA-matched HSCT with reduced-intensity conditioning, frequent cardiac complications in our series might have resulted not only from alemtuzumab but also from intensive conditioning and/or cytokines excretion associated with the engraftment of HLA-mismatched donor cells. Close monitoring of the cardiac function may be important in such transplantation, especially in patients who had received a high cumulative dose of anthracyclines, because cardiac dysfunction could be manageable by an early detection and early treatment.
Aspirin Down-Regulation of Platelet Reactivity in Patients with Acute Coronary Syndrome: Effects of Erythrocytes.
Authors: Santos Maria T. , Valles Juana , Fuset Maria P. , Perez Francisca , Moscardo Antonio , Aznar Justo , Ruano Miguel
Published: October 2019

Platelet-erythrocyte interactions enhance platelet reactivity (Santos et al.J Clin Invest1991;87:571;Valles et al. Blood2002; 99:3978) and reduce the effect of aspirin (ASA) as antithrombotic modality (Santos et al. Circulation1997;95:63; Valles et al.Circulation1998;97:350). These effects were studied using an experimental approach which independently evaluates platelet activation (TXA2, 5HT release) and recruitment (proaggregatory activity of cell-free releasates) during cell-cell interactions between erythrocytes (RBCs) and collagen-stimulated platelets (PTLs) or in whole blood (WB). In patients with chronic vascular disease treated with ASA, with blunted TXA2, three response groups were found: in Group A (39% of patients), recruitment was blocked in PTLs alone, PTL+RBCs and in WB; in Group B (45%), recruitment was abolished in PTLs alone but continued in the presence of RBCs and in WB; in Group C (16%), recruitment was detected in PTLs alone and was markedly enhanced by RBCs (Valles et al.Circulation1998; 97:350). Using the same experimental approach, 80 patients with acute coronary syndrome (ACS) treated with aspirin (100–300 mg/d) as the only antiplatelet therapy were studied. Other medications were administered according to guidelines. ASA-free (n=80) normal subjects were also evaluated as controls. Results: In ACS patients blockade of TXA2 was observed in only 76% of patients, while partial inhibition (44.7%) was achieved in 24%. In the latter patients platelet recruitment in WB was high, equal to that in ASA-free controls (ACS X=94.58 vs. controls 87.59). When the three response groups described above were studied in the 61 ACS patients with blocked TXA2 synthesis, the proportions differed markedly from chronic patients: Group A (3% vs. 30%), Group B (18% vs. 45%) and Group C (79% vs. 16%). Since TXA2 was blunted in these patients, the results indicate that ACS patients are highly susceptible to stimulation by COX-1 independent mechanisms. In 18% (Group B) this could be attributable to the ASA-insensitive prothrombotic effect of RBCs, while in 79% (Group C) platelet hyperactivity was intrinsic to PTLs and markedly enhanced by RBCs. Conclusions: Evaluation of TXA2 synthesis in ASA-treated ACS patients is advisable, since partial TXA2 inhibition results in full platelet recruitment. COX-1-independent mechanisms of platelet reactivity are prominent in ACS patients and increased by RBCs. Since platelet recruitment is essential in thrombus growth its inhibition by ASA alone or in combination with other antiplatelet drugs could reduce occlusive thrombus formation. (Grant FIS 03/0270).
AMD3100 Plus G-CSF Use in Proven Poor Mobilizers in a Compassionate Use Protocol (CUP).
Authors: McGuirk Joseph , McCarty John , Gibb Kaaren , Badel Karin , Bridger N Gary , Calandra Gary
Published: October 2019

AMD3100 (A), an inhibitor of SDF-1∞ binding to CXCR4, has been used alone and with G-CSF (G) to mobilize CD34+ cells from healthy volunteers and donors and from cancer patients undergoing autologous transplantation. In several studies it enhanced mobilization of poor mobilizers. Therefore, a CUP was submitted to the FDA; more than 100 patients (most commonly NHL; N=42) have been entered. Results from the first 70 patients enrolled are reported here. Three patients underwent a second mobilization with A following initial failure (2) and disease relapse (1); data on the first mobilization is in this report. Entry requirements include documented inability to mobilize 2 x 106 CD34+ cells/kg. Patients must have acceptable cardiac and pulmonary status, no active infection, and to have WBC >3.0 x 109/L, ANC > 1.5 x109/L, PLT >100,000 x 109/L, LFT within 2 x ULN, and creatinine clearance >60 ml/min. as well as other acceptable laboratory values. After signing consent and undergoing history, physical, laboratory, CXR, and ECG evaluation, patients are given G (10 mg/kg) for 4 days. At about 10 p.m. on day 4 they receive 240 mg/kg of A subcutaneously. The next a.m. they receive G and undergo apheresis. The evening A, the a.m. G, and the apheresis are repeated until enough cells are collected or until it is clear insufficient cells could be collected. There were 38 males and 32 females. Five were 18 years or younger (10, 12, 13, 16 and 18 years). The rest ranged in age from 21 to 81 years. Most common diseases were NHL (31), MM (14), AML (8), and Hodgkin’s disease (HD) (10). The 70 patients had undergone 93 possible mobilizations and 16 patients failed multiple prior regimens. Nearly all (91/93) prior mobilization regimens contained G. The most common regimens were G alone (33), cyclophosphamide plus G (17), and cyclophosphamide plus G plus other agents (30). In a subset of patients (N=27) the median time between the prior failed mobilization and the CUP mobilization was 34 days. A was generally well tolerated with AEs being similar to that previously reported. Only one SAE was felt related - patient with worsening of GI symptoms. The percent of patients collecting >2 x 106 cells/kg for the more common cancers were 58 for NHL, 64 for MM, 50 for AML, and 70 for HD. For those collecting >2 x 106 cells/kg (N=40) the median collection was 4.72 106 cells/kg in a median of 3.5 aphereses. The number of these patients transplanted was 36 of 40 with 31 successful engraftments, 3 pending, and two deaths post successful PMN, but not PLT engraftment. Of all the patients transplanted 4 additional patients have died. Durability has been good with at least 24 patients at 6 months or beyond. The success of mobilization compares very favorably with prior published data. A offers a generally safe and effective way to enhance mobilization of patients who have previously failed mobilizations.
Adult Human Blood Leukocytes as an Efficient Source for Tissue-Committed Neural Progenitors.
Authors: Porat Yael , Fulga Valentin , Belkin Danny , Porozov Svetlana , Fisher Yehudit , Belkin Michael , Silverman Willam F.
Published: October 2019

In the last few years, significant progress has been made in the isolation and characterization of bone marrow stem cell populations and their potential to differentiate into a variety of cellular lineages. We hypothesized that peripheral blood can also be used as a source for precursor cells that can become committed progenitors for a variety of tissues. We report here the generation and characterization in vitro of neural progenitor cells from a newly discovered blood-derived multipotent cell population, named synergetic cell population (SCP). Human blood samples were obtained from the Israeli blood bank and SCP cells were purified based on cellular density. Neural progenitors were generated by culturing SCP cells in medium supplemented with autologous serum, followed by activation in a defined serum-free medium containing the specific differentiation-inducing factors F12, B27, bFGF, BDNF, and NGF.

An average of 13.5x106 neural progenitor cells was generated from 450 ml blood. These cells developed irregular perikarya, from which filamentous extensions spread contacting neighboring cells and forming net-like structures. Immunostaining revealed that some of the cells express the early neuronal progenitor markers nestin and b-tubulin and Neu-N, a nuclear protein present in mature neurons. Other cells expressed glial-specific antigens, such as O4 (a marker of oligodendrocytes) and GFAP (a marker of astrocytes). Flow cytometry analysis showed that 44.4% and 34% of the cells were positive for nestin and b-tubulin, respectively. In addition to exhibiting phenotypic evidence of markers specific for the neural lineage, these progenitor cells also responded to the neurotransmitters glutamate and GABA, as detected by calcium influx through voltage-gated calcium channels, demonstrating functional differentiation.

In this study we show that generation of neural progenitors from peripheral blood is feasible and efficient. Blood-derived angiogenic progenitors produced in our system are already safely and efficiently administrated to severe angina pectoris patients in a clinical trial we are conducting in Thailand (reported in a separate abstract by our group). The newly discovered source of these progenitors, the blood-derived multipotent population which we termed SCP, contains both hematopoietic stem cells as well as supportive cells that enable differentiation into various lineages. The therapeutic potential of these neural progenitors will be further characterized and evaluated in vivo using animal models.
Zebrafish KLF4 Is Essential for Primitive Haematopoiesis.
Authors: Perkins Andrew C. , Gardiner Melissa R.
Published: October 2019

Gene knockout studies of Krüppel-like factors (KLFs) in mice have demonstrated key roles in organogenesis. EKLF (Klf1) plays an essential role in definitive erythropoiesis and b-globin gene expression, but primitive (yolk sac) erythropoiesis and embryonic globin gene expression are essentially normal. Since expression of embryonic globin genes is dependent upon proximal CACC box elements, additional KLFs must function during the embryonic wave of erythropoiesis. A number of KLFs have been described in zebrafish. One of these, zKLF4, the homologue of neptune, a Xenopus Laevis KLF expressed in the ventral blood island, cement and hatching glands, is an early marker of lateral plate mesoderm (LPM). From 1 somite and prior to expression of scl and flk1, there is a continuous rim of expression of zKLF4 2–3 cells wide within the LPM extending from the postero-lateral boundary of the polster to the tail. Expression in the posterior lateral plate persists as it migrates medially in a zipper-like movement to generate the intermediate cell mass (ICM), the site of embryonic haematopoiesis. zKLF4 is also expressed in a patch of dorsal mesendodermal cells from 70% epiboly which are fated to become the polster. Morpholino knockdown of zKLF4 results in moderate anaemia which resolves as definitive erythropoiesis replaces primitive erythropoiesis at 48–72 hpf. zKLF4 morphants show a down-regulation in the expression of GATA-1 (>100 fold) and embryonic globin (>10 fold) by real time RT-PCR. Both are expressed during the primitive wave of haematopoiesis. In addition there is a dramatic up-regulation (>100 fold) in the level of cmyb expression in zKLF4 morphant compared to control embryos at 72 hpf, which is consistent with expansion of the secondary definitive wave of erythropoiesis, possibly in response to anemia. These results suggest mammalian KLF orthologs of zKLF4, either KLF4 or KLF2, may play roles in primitive erythropoiesis and embryonic globin gene expression in mice or man.
Development of a Novel Assay Using Bioluminescent Reporter Bacteria To Determine the Sensitivity of AML Cells to Cytosine Arabinoside.
Authors: Smith Graham , Angell Joanna E. , Alloush Habib M. , Smith Margaret A. , Hill Phil J. , Salisbury Vyv C.
Published: October 2019

The nucleoside analogue cytosine arabinoside(Ara-C) has been a cornerstone of remission induction therapy for patients with AML for three decades. Even today, approximately 70% of adults fail to achieve durable complete remission with regimens including Ara-C. This project involves the development of a simple, rapid, economical test to determine Ara-C sensitivity of patient cells to enable clinicians to consider the efficacy of this drug prior to patient treatment.

Bacteria expressing lux genes have been successfully used to study real time pharmacodynamics of antimicrobial agents. We have extended this elegant reporter gene technology to determine cellular penetration of Ara-C, into AML cells. AraC has no effect on E. coli since it does not express deoxycytidine kinase (dCK) and has the ability to deaminate AraC into AraU through the activity of cytidine deaminase (cdd). To render E. coli sensitive to AraC, a cdd-deficient strain was used as the host to express the human dCK gene carried on pTrc99-A plasmid under the control of the lac promoter. The expression of dCK, induced by the addition of IPTG, made E. coli sensitive to AraC. E. coli was then made self-bioluminescent by introducing the pAL2 plasmid carrying the lux operon (luxABCDE)

AML cell lines were incubated with therapeutic levels of Ara-C (100 μM),washed, and re-suspended in fresh media containing bioluminescent modified E. coli and 0.1% saponin, to lyse the AML cells. Light levels and optical density were monitored using a microtitre plate reader (Fluorstar Optima, BMG Labtec).

Cell lines minus drug were used so as to generate control lysates and supernantants.

It was possible to detect significant increases in light levels from the lysate and supernatant of an AML cell-line known to be Ara-C sensitive (KG-1a). The lysate from a cell-line (THP-1) known to deaminate Ara-C through the over production of cytidine deaminase showed no increase in light production in the presence of Ara-C. In addition no difference in light levels was observed from the supernatant of these resistant cells. The control supernatant from both cell lines showed no change in light output. These findings indicate that this assay could be used to evaluate the uptake and metabolism of Ara-C by leukaemia cells. With refinement, the assay could be used to specify at which point in Ara-C metabolism AML cells acquire resistance. Furthermore, this technology could be modified to examine the sensitivity of AML cells to other established chemotherapeutic agents such as anthracyclines thus giving a genuinely helpful chemosensitivity profile to clinicians involved in patient care and customising treatment for individual patients.
A Quick Method for Identifying the Molecular Targets of Potentially Diagnostic New Monoclonal Antibodies Using Protein Array Technology.
Authors: Barry Richard , Murphy Derek , Ip Cho Simon , Cordell Jacqueline , Jones Margaret , Roberton Helen , Cahill Dolores , Mason David
Published: October 2019

During the course of raising monoclonal antibodies, reagents are often produced that are not directed against the immunising antigen. These may pass unnoticed unless a screening step based on immunostaining of human tissue is included. Many of these reagents are auto-antibodies, often directed against intracellular targets (e.g. nuclear components), and the process of hybridoma production serves to “rescue” these self-reactive B cell clones. Such unexpected antibodies of this sort may prove of interest if their distribution is analysed on normal tissues and within the spectrum of leukaemia/lymphoma samples showing specific patterns of staining. However, before they can be used diagnostically or therapeutically, their target molecule needs to be identified, and this can be technically demanding. We describe a novel approach that can be used to define the targets of new monoclonal antibodies to intracellular molecules. The technique involves screening against protein arrays comprising thousands of recombinant human proteins expressed in bacteria. In our initial experiments we showed that a control monoclonal antibody against nucleophosmin identified 22 clones encoding this ubiquitous protein in a human foetal brain cDNA library. Monoclonal antibody JC1 (first published in 1992) was known to recognise an unidentified proliferation-associated nuclear molecule, and screening on the recombinant protein array defined nuclear factor I/X (NFIX) as its probable target. A second antibody (JJ166), reactive with cell nuclei, was shown by the same technique to recognise the mitotic spindle-associated molecule NUMA1 (the gene for which is involved in rare chromosomal translocations). To confirm these putative specificities, the antibodies were shown to react with Cos-1 cells that had been transiently transfected with the corresponding clones from the array (after transfer into a mammalian vector pcDNA4HisMaxC, incorporating an Xpress tag). This new method represents an effective and quick strategy for defining the protein targets of new monoclonal antibodies identified as having diagnostic or other potential value on the basis of their immunostaining patterns.
Proplatelet Formation Is Regulated by the Rho/Rock Pathway.
Authors: Chang Yunhua , Aurade Frederic , Larbret Frederic , Le Couedic Jean Pierre , Momeux Laurence , Zhang Yan Yan , Larghero Jerome , Louache Fawzia , Bertoglio Jacques , Cramer Elisabeth , Vainchenker William , Debili Najet
Published: October 2019

Megakaryopoiesis is a highly specialized cellular process which sustains platelet production. At the end of megakaryopoiesis, megakaryocyte (MKs) fragments into platelets via long and thin cytoplasmic extensions called proplatelets. Proplatelet formation (PPF) is associated essentially with cytoskeleton changes, including actin dynamics. The Rho/Rock pathway is a well characterized regulator of the actin reorganization. In the present study, we have tried to understand the precise role of the Rho/Rock pathway in PPF from human CD34+ derived MKs. Our results show that Rho is expressed in MKs and that its expression and activity remain stable during megakaryopoiesis. Overexpression of a RhoA dominant negatif (RhoA N19) in MKs leads to an increase in PPF. Conversely overexpression of a RhoA spontaneous active (RhoA V14) in MKs leads to a decrease in PPF. These results indicate that Rho activation could inhibit PPF in vitro. It is known that Rho/ROCK promotes actin cytoskeleton dynamics by regulating myosin light chain 2 (MLC2) phosphorylation. To demonstrate that Rho/Rock inhibits PPF through MLC2 phosphorylation, we added MLC kinase inhibitor (P18), Rho inhibitor (TatC3) and ROCK inhibitor (Y27362) in MKs culture just before PPF. Western blot analysis shows that MLC2 phosphorylation was inhibited by these 3 compounds, in contrast, PPF was significantly increased. Moreover, the platelet produced have an identical size and ultrastructure as control platelets and could be normally activated. These results suggest that Rho/ROCK could inhibit PPF through MLC phosphorylation during megakaryopoiesis.
Differential Mapping of the Amino Acids Mediating Agonist and Antagonist Coordination with the Human Thromboxane A2 Receptor Protein.
Authors: Khasawneh Fadi T. , Huang Jin-Sheng , Turek Joseph W. , Le Breton Guy C.
Published: October 2019

Despite the well-documented involvement of thromboxane A2 receptor (TPR) signaling in the pathogenesis of thrombotic diseases, there are currently no rationally-designed antagonists available for clinical use. To a large extent this derives from a lack of knowledge regarding the topography of the TPR ligand binding pocket. On this basis, the purpose of the current study was to identify the specific amino acid residues in the TPR protein which regulate ligand coordination and binding. The sites selected for mutation reside within or in close proximity to a region we previously defined as a TPR ligand binding site, i.e., the C-terminus of the second extracellular loop and the leading edge of the fifth transmembrane domain. Mutation of these residues caused varying effects on the TPR-ligand coordination process. Specifically, the D193A mutant lacked both SQ29,548 (antagonist) binding and U46619 (agonist)-induced calcium mobilization. Three other mutants, F184Y, T186A and S191T, discriminated between SQ29,548 binding and the U46619-mediated functional response. Furthermore, these mutants also revealed a divergence in the binding of two structurally different antagonists, SQ29,548 and BM13.505. Conversely, two separate mutants which exhibited SQ29,548 binding activity yielded either a normal (F196Y) or reduced (S201T) U46619 response. Finally, mutation of other residues directly adjacent to those described above, e.g., E190A and F200A, produced no detectable effects on either SQ29,548 binding or the U46619-induced functional response. In summary, these results identify key amino acids involved in TPR ligand coordination and demonstrate that TPR-specific ligands do not necessarily interact with the same residues in the ligand-binding pocket.
“Maturational” Globin Switching in Primary Primitive Erythroid Cells.
Authors: Palis James , Malik Jeff , Emerson Rachael L. , Bushnell Tim P. , McGrath Kathleen E. , Bulger Michael , Kingsley Paul D.
Published: October 2019

Mammals have two distinct erythroid lineages. The “definitive” erythroid lineage generates small, enucleated erythrocytes that constitute the predominant cell type in the fetal and postnatal circulation. It is preceded by the “primitive” erythroid lineage, which originates in the yolk sac and generates a semi-synchronous wave of large erythroblasts that terminally differentiate in the bloodstream. This feature provides a unique opportunity to investigate changes in gene expression during erythroid maturation. Here, we have examined expression of the various α- and β-globin genes in purified populations of primary primitive erythroid cells isolated from progressive developmental time-points of mouse embryogenesis. Our studies, using both in situ hybridization and quantitative PCR, indicate that βH1 is the predominant β-globin transcript in the early yolk sac. Thus, unlike the human, the murine β-globin genes are not up-regulated in the order of their chromosomal arrangement. As primitive erythroblasts mature from proerythroblasts to reticulocytes, they undergo a βH1- to εy-globin switch, up-regulate low levels of the adult β1- and β2-globins, and down-regulate ζ-globin. These changes in transcript levels correlate with changes in RNA polymerase II density at their promoters and transcribed regions as assayed by ChIP assays. Furthermore, we found that the εy- and βH1-globin genes in primitive erythroblasts reside within a single large hyperacetylated domain. Taken together, these results are consistent with the notion that this βH1- to εy-globin “maturational” switch is dynamically regulated at the transcriptional level. We conclude that the embryonic to adult globin switch that occurs during murine ontogeny is due not only to the sequential appearance of primitive and definitive erythroid lineages but also to striking changes in globin gene expression that occur as primitive erythroblasts mature in the bloodstream.
CD47 Glycoprotein Interacts with p4.1R and p55 in the Erythrocyte.
Authors: Avent Neil D. , Plummer Zoe E. , Head David J.
Published: October 2019

CD47 is a 47–50kDa membrane glycoprotein with 5 known isoforms. The role of CD47 within the erythrocyte membrane remains the subject of much research and debate though we recently provided evidence that CD47 may function as an inducer of eryptosis (Head et al, 2005). As both a cytoskeletal linked fraction and a smaller membrane diffuse fraction of CD47 exists, it is most likely that there are a number of protein species that are able to bind to CD47 at its cytoplasmic face. Our research has focused on a study of the molecular interactions of erythrocyte CD47 with erythrocyte membrane skeletal proteins protein 4.1R (p4.1R), protein 4.2 (p4.2) and p55. Here we demonstrate the ubiquitous expression of all CD47 isoforms in haemopoietic cells and tissues using basic and real-time PCR. Via immunoprecipitation of CD47 from mature erythrocyte membranes using the anti-CD47 mAb BRIC-126, yeast two-hybrid analysis and in vitro co-immunoprecipitation of 35[S] labelled peptides in a cell-free translation procedure, a novel ternary complex involving CD47, p55 and p4.1R has been indicated. More specifically, the potential interaction between p55/p4.1R and the cytoplasmic face of CD47 has been localised to the PDZ and FERM domain of these proteins respectively. Though research suggests p4.2 provides the major cytoskeletal attachment of CD47 (Bruce et al, 2002; Mouro-Chanteloup et al, 2003), a direct interaction between CD47 and p4.2 was not suggested by our study and remains undemonstrated. We continue to further the evidence for a functional role of CD47 in eryptosis. We propose p4.1R links CD47 to the apoptotic machinery of the cell and suggest a mechanism whereby cytoskeletal rearrangement and PS exposure occurs. 4.1null cells have been obtained and are currently being investigated. Further characterisation of the eryptotic pathway may offer insight into potential therapies for erythroleukaemia characterised by resistance of the erythroid lineage to apoptosis. This work has equal significance in the stabilization of red cell preparations in the blood transfusion setting.
Gaucher Disease Psychosine Mis-Traffics Endocytosed Cargo to a Non-Functional, Pre-Lysosomal Compartment.
Authors: Roos Jonathan C. , Wang Susan Z. , Cox Timothy M.
Published: October 2019

Gaucher’s disease is a rare metabolic disorder resulting from a deficiency in glucocerebrosidase activity. It is characterized by the formation of large multinucleate macrophages which accumulate enzyme substrate - principally glucosylsphingosine (psychosine) and glucosylceramide - as well as unrelated products such as cholesterol, lipofuscin, glycopeptides and oligosaccharides. The mechanism of this storage remains obscure. Psychosines have been shown to inhibit cytokinesis (Kanazawa et al., 2000) in cells expressing a G-protein linked receptor: GPR65 (Im et al., 2001). Signalling via GPR65 was shown to affect phosphorylation and calcium release as well as result in the formation of intracellular actin clots. Could such psychosine-triggered signalling account for the secondary accumulation of lipids that are not substrates for glucocerebrosidase in Gaucher disease? We investigated the effects of psychosines and other lysosphingolipids on human macrophages and monocyte-related cell lines expressing GPR65. Trafficking was evaluated by pulse-chase experiments using fluorescent lipids and markers of pH. Changes induced by lysosphingolipids were visualized by electron microscopy and compared with Gaucher cells from affected spleen. Confocal fluorescence and electron microscopy were used to identify sub-cellular compartments. GPR65 expression in Gaucher spleen and other cells was confirmed by RT- PCR.

At sub-pathophysiological concentrations (1-5μM), psychosines but not ceramides disrupt endocytosis: mis-trafficked membrane and ingested materials accumulate in a large vesicular compartment. This multivesicular body bears most lysosomal markers including LAMP1 and LAMP2 but has a neutral pH and does not contain acid phosphatase or Cathepsin D -consistent instead with late endosomes. The ultrastructure of the psychosine-treated monocytic cells closely resembles naturally occurring splenic Gaucher cells and both are also of course multinucleate. This suggests that psychosines dysregulate intracellular trafficking in Gaucher disease. Recycling is perturbed and membrane lipids and other cargo accumulate in a pre-lysosomal compartment where, with glucosylceramide, they may be preferentially concentrated. Since related lysolipid metabolites are present in several other sphingolipidoses, we propose that a common trafficking mechanism accounts for the cellular pathology seen in this class of diseases.

In addition, an active role for psychosines in pathogenesis may also begin to suggest mechanisms to explain why Gaucher patients carry a high relative risk for developing myeloma and other B-cell dyscrasias (Rosenbloom et al., 2005). The majority of these malignancies are associated with mutations at position 14q32. Receptor GPR65 is also located at that position and its regulation may predispose to mutation and eventual transformation.
Intracellular HMGB1 Transactivates the Human IL1B Gene Promoter through Association with an ETS Transcription Factor PU.1.
Authors: Tsukada Junichi , Mouri Fumihiko , Mizobe Takamitsu , Higashi Takehiro , Izumi Hiroto , Kohno Kimitoshi , Tanaka Yoshiya
Published: October 2019

High mobility group box chromosomal protein 1 (HMGB1) was recently identified as a late proinflammatory mediator of endotoxin lethality, which amplifies and sustains the inflammatory processes via macrophage/monocyte activation by extracellular release of HMGB1. However, it was originally described as a nonhistone nuclear protein to bind and distort the structure of DNA and to regulate transcription through association with several transcription factors. Here we investigated the effect of intracellular HMGB1 on transcriptional gene regulation of an immediate early proinflammatory cytokine IL-1β in transient transfection studies using HMGB1 expression vector (pcDNA3HMGB1) and murine macrophage cells RAW264.7. When pcDNA3HMGB1 was introduced into RAW264.7 cells with pGL3HT reporter containing the -131 to +12 minimal IL-1β gene promoter, IL-1β promoter activity was significantly induced by expression of HMGB1. The gene promoter possesses two important transcription factor binding motifs, one for PU.1, a myeloid and B cell-specific transcription factor that belongs to the ETS family, and the other a binding site for NF-IL6. To verify the functional role of intracellular HMGB1 in transactivation of the IL-1β promoter, a PU.1 expression vector and/or pcDNA3HMGB1 were cotransfected into PU.1-deficient murine thymocytes EL4 cells along with pGL3HT reporter. As a result, HMGB1 synergized with PU.1 to transactivate the IL-1β promoter, but not HMGB1 alone. This argument was supported by our GST-pulldown data, which demonstrated direct physical interaction of HMGB1 with PU.1. In addition, deletion of the PU.1 winged helix-turn-helix DNA-binding domain significantly inhibited the association of PU.1 with HMGB1. To determine whether HMGB1 could affect PU.1 DNA-binding affinity, we performed electrophoretic mobility shift assay using a radiolabeled IL-1β -59 to +12 promoter element (DT), recombinant PU.1 (rPU.1) and GST-HMGB1. Two complexes with slower and faster mobilities were generated by the addition of HMGB1 to a mixture of rPU.1 and DT probe. The two complexes were abrogated by preincubation with anti-PU.1 Ab, while anti-HMGB1 Ab reacted only with the complex with a slower mobility, indicating that the complex with a slower mobility formed by addition of HMGB1 contained both HMGB1 and PU.1, while the band with a faster mobility contained only PU.1. From the present study, we propose that intracellular HMGB1 might function as a coactivator in PU.1-mediated transcriptional activation, which facilitate access of PU.1 to specific DNA targets. The fact that PU.1 is a transcription factor essential to macrophages/monocyte-specific proinflammatory cytokine genes further raise the possibility that interaction of PU.1 with HMGB1 may play an important role in the inflammation cascade in macrophages/monocytes.
Thrombopoietin (TPO) Enhances Heamatopoietic Differentiation of Human Embryonic Stem Cells.
Authors: Srivastava Anand S. , Mishra Rangrath , Carrier Ewa
Published: October 2019

Recently, it was demonstrated that TPO enhances hematopoietic differentiation of primate ES cells, but its role in differentiating human ES cells is unknown. We sought to investigate the regulatory mechanism of TPO induced signals mediated by the c-mpl cytoplasmic domain during human embryonic stem (hES) cells hematopoietic commitment. We hypothesize that in human embryonic stem cells, binding of TPO to its c-mpl receptor causes three-dimensional alterations which bring the c-mpl cytoplasmic domain and Janus Kinase into close-proximity and thus induces the phosphorylation and dimerization of STAT5 molecule. Dimerized STAT5 molecules detach from the receptors and migrate to the nucleus where they bind GAS site and induce transcription of a set of target, hematopoiesis-related genes. NIH human ES cell lines (WI01) were used in this experiment. In brief, to induce EB formation, cells were incubated in differentiation medium, which consisted of knockout DMEM medium (GIBCO/BRL, Carlsbad, USA), supplemented with 20% non-heat-inactivated fetal bovine serum (FBS, Hyclone, USA), 1% nonessential amino acids, 1 mM L-glutamine, and 0.1 mM β-mercaptoethanol. Subsequently, DMEM was replaced by IMDM (GIBCO/BRL, USA) with the same supplements and additional two cytokines (100 ng/mL SCF and 100 ng/mL Flt-3 ligand (Flt-3L)) (control group). To investigate the role of TPO and VEGF, cells were additionally treated with 100 ng/mL TPO alone or in combination with 100 ng/mL rhVEGF. All cytokines were from the R&D systems (USA). Significant increase in the numbers of embryoid bodies (EBs) formation in TPO (125/105), TPO/VEGF (150/105 cells) when compared to controls (10/105 planted ES cells) was documented. This corresponded with the increase in CFU-C and the number of CD31/CD34 positive and CD34-positive progenitors. Analysis of gene expression during hematopoietic development demonstrated that TPO/VEGF combination increased mRNA expression of the TPO receptor (TPO-R) and VEGF (VEGF-R) receptors in hematopoietic progenitors obtained from human ES cells. We are in the process of determining the role of JAK/STAT pathway in this process; functional studies involve blocking of TPO/c-mpl using TPO-R-specific antibodies and determining its impact on human ES-derived hematopoiesis.
The Detection and Estimation of Fetal Hemoglobin by Means of the Alkali Denaturation Test
Authors: JONXIS J. H. P. , HUISMAN T. H. J.
Published: October 2019

This study illustrates that the value of both alkali denaturation methods is somewhat limited. At high percentages of fetal hemoglobin, the results obtained with the method of Singer and coworkers, are about 10 per cent too low. The Brinkman and Jonxis method is not accurate when the percentage of fetal hemoglobin is lower than 10 per cent. The alkali resistant fraction found with the method of Singer and associates in adult blood, is a mixture containing fetal hemoglobin. The presence of COHb-A will interfere with the determination of fetal hemoglobin, especially when the optional alkali denaturation method is used.
Polycythemia Associated with Disturbed Function of the Respiratory Center
Authors: PARE PETER , LOWENSTEIN LOUIS
Published: October 2019

1. An unusual case of absolute polycythemia associated with hypoxemia, probably resulting from abnormal function of the respiratory center has been presented.

2. The mechanism of development of this type of polycythemia has been discussed.

3. A classification of polycythemia on the basis of blood volume and arterial oxygen saturation has been suggested.
The Effect of Chloramphenicol and Other Antibiotics on Leukocyte Respiration
Authors: FOLLETTE JAMES H. , SHUGARMAN PETER M. , REYNOLDS JOHN , VALENTINE WILLIAM N. , LAWRENCE JOHN S.
Published: October 2019

1. Leukocyte homogenates show active, reproducible respiratory activity in a fortified system.

2. Chloramphenicol inhibits leukocyte respiration in vitro when used in concentrations similar to other commonly used inhibitors.

3. Chlortetracycline, oxytetracycline, penicillin and streptomycin have no significant effect on leukocyte respiration.

4. Urethane and atabrine produce inhibition of leukocyte respiration, but the latter is more active.

5. The possible sites of action of chloramphenicol in cellular metabolism are discussed.
Studies on Urinary Excretion of Vitamin B12Co60 in Pernicious Anemia for Determining Effective Dosage of Intrinsic Factor Concentrates
Authors: BEST WILLIAM R. , WHITE WILFRID F. , ROBBINS KENNETH C. , LANDMANN WENDELL A. , STEELMAN SANFORD L.
Published: October 2019

Studies were made in pernicious anemia patients on the urinary excretion of B12Co60 after a small oral dose followed by a large parenteral injection of nonradioactive vitamin B12.

(1) Increasing doses of intrinsic factor concentrates give increasing excretions of radioactivity at low doses; little additional increase at moderate doses; and at times a subsequent diminution at excessive doses. Data on 34 tests of a particular intrinsic factor concentrate in 18 pernicious anemia patients tend to support an excretion proportional to the logarithm of intrinsic factor dosage at low to moderate levels, but do not exclude the possibility of a linear approach to a plateau.

(2) Assay by hematologic response was compared with the urinary excretion tests in 13 pernicious anemia patients. This data shows a relation between the two tests though the correlation is far from complete.

(3) Methods are outlined for testing all intrinsic factor preparations with the same amount of tracer vitamin B12 as will be incorporated commercially. More sensitive comparative tests of similar intrinsic factor preparations may be made using smaller amounts of B12Co60.

(4) The literature is reviewed to determine which variations in technic might lead to the most reliable quantitation of intrinsic factor activity.
Experimental Research on the Proliferative and Differentiative Activity of the Erythroblast in Chronic Erythremia, the Chronic Form of Di Guglielmo’s Disease
Authors: BERNARDELLI E. , MELE V. , BANI M.
Published: October 2019

Using the method of culture in vitro, the authors have studied the proliferative and differentiative activities of the erythroblastic tissue in chronic erythremic myelosis.

The results of the research allowed the authors to reach the following conclusions:

1. the erythroblastic cells of chronic erythremic myelosis reveal indisputable differentiative activity in vitro;

2. the rate of maturation of the erythremic erythroblasts is slower than normal;

3. the proliferation of the erythroblastic tissue in chronic erythremic myelosis is normal;

4. in the genesis of the erythroblastic hyperplasia in chronic erythremic myelosis, the most evident factor is a defect of maturation of the erythroblasts.
Final Update of Phase I-II Study of the Farnesyltransferase Inhibitor Tipifarnib in Combination with Idarubicin and Cytarabine for Patients with Newly Diagnosed Acute Myeloid Leukemia or High-Risk Myelodysplastic Syndrome.
Authors: Delmonte John , Kantarjian Hagop M. , Garcia-Manero Guillermo , Estrov Zeev , Ravandi Farhad , Verstovsek Srdan , O’Brien Susan , Koller Charles A. , Faderl Stefan , Thomas Deborah A. , Wright John J. , Cortes Jorge
Published: October 2019

Background: Tipifarnib (Zarnestra, Z) is a non-peptidomimetic farnesyltransferase inhibitor (FTI) with clinical activity in hematologic malignancies, including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and chronic myeloid leukemia. Preclinical data suggest that tipifarnib may be synergistic with some chemotherapeutic agents.

Methods: We designed a phase I/II study in patients (pts) age 15–70 years, with previously untreated AML or high-risk MDS (blasts ≥ 10%), who received induction with idarubicin (Ida) 12 mg/m2/day on days 1–3, cytarabine (ara-C) 1.5 g/m2 IV over 24 hours daily on days 1–4 (days 1–3 only if age ≥ 60 years) and Z, with first cohort (n=6) receiving 200mg PO BID and all others 300 mg PO BID x 21 days every 28 days. Pts achieving a complete remission (CR) received consolidation (5 courses) with Ida 8 mg/m2/day on days 1–2, ara-C 0.75 g/m2/day on days 1–3, and Z 300 mg PO BID x 14 days every 4–6 weeks. Maintenance was with Z 300 mg PO BID x 21 days every 4–6 weeks for 6 months.

Results: We treated 95 pts, median age 50 yrs (range, 17–61 yrs), and all are evaluable for response with a median followup of 61 weeks (range, 35–138). Seventy pts (73%) responded: 61 (64%) achieved a CR and 9 (9%) a CRp. Median CR duration was 72 weeks, (range, 4–121) with median OS 70 weeks (range, 1–138). Response by cytogenetics was: 34/41 (83%) for diploid, 13/19 (69%) with −5/−7, 1/2 (50%) with t(8;21), and 21/32 (65%) with other abnormalities. Response by age was 31/43 (72%) for < 50 yrs, 12/18 (67%) for ≥ 50 yrs with diploid karyotype, and 18/34 (53%) for ≥ 50 yrs with abnormal karyotype. Response by FLT3 status was 12/16 (75%) for mutated, 51/66 (78%) for unmutated. Overall response rate (CR+CRp) in a similar historical population treated with the same chemotherapy regimen, idarubicin/cytarabine (IA), without Z was 72% (p=0.847), median CR duration 52 weeks (range, 2–319) (p=0.493), median OS 65 weeks (range, 3–322) (p=0.698). The most common grade ≥ 3 adverse events have included diarrhea (39%), hypokalemia (27%), rash (21%), and hepatic dysfunction (18%). Fifty-three (56%) pts have required treatment dose reductions during induction, 21 pts (40%) during consolidation, and 3 pts (18%) during maintenance.

Conclusion: We conclude that Z combined with IA induces a high rate of CR in high-risk MDS or AML, but the outcome may not be superior to what is obtained with IA alone.