bluuebunny/crossref_metadata_2025 huggingface dataset:
Low Protein Z Levels and the Risk of Venous Thrombosis.
Authors: Martinelli Ida , Battaglioli Tullia , Razzari Cristina , Biguzzi Eugenia , Mannucci Pier Mannuccio
Published: April 2020

Background. Protein Z (PZ) is a vitamin-K dependent protein that serves as cofactor for PZ-dependent protease inhibitor, inactivating factor Xa. Therefore, deficiency of PZ could led to a prothrombotic phenotype. Contrasting data are available on the relationship between PZ and venous thromboembolism (VTE).

Patients and Methods. We carried out a case-control study on 443 patients with deep vein thrombosis of the lower limbs and/or pulmonary embolism who discontinued oral anticoagulant therapy and 394 controls, similar for age and sex. PZ was measured with an enzyme immuno-assay (Diagnostica Stago, France). A thrombophilia screening including DNA testing for factor V Leiden and G20210A prothrombin mutation and measurements of plasma antithrombin, protein C, protein S, and homocysteine (fasting and post-methionine load) was performed. Pregnancy, oral contraceptive use, liver or renal disease were exclusion criteria.

Results. 443 patients and 394 healthy controls who underwent thrombophilia screening were investigated. Median PZ levels (range) were 1.95 μg/mL (0.22–6.26 μg/mL) in patients and 1.79 μg/mL (0.25–7.82 μg/mL) in controls (p=0.07). The odds ratios (adjusted for age, sex and the presence of thrombophilia) for VTE in patients with PZ levels in the lowest quartile was 0.8 (95% CI 0.5–1.1), and remained statistically non significant considering PZ levels below the 10th percentile. However, low PZ levels (lowest quartile) increased the risk of VTE associated with factor V Leiden (odds ratio 14.6, 95% CI 1.9–113), prothrombin G20210A (odds ratio 3.2, 95% CI 1.0–10.3), and hyperhomocysteinemia (odds ratio 4.7, 95% CI 1.7–17.0). These interactions remained, apart for prothrombin G20210A, when PZ levels below the 10th percentile were considered.

Conclusion. Low PZ levels are not an independent risk factor for VTE, but may enhance the relative risk due to already established risk factors, particularly factor V Leiden.
Regulated Shedding of sema4D from the Platelet Surface Produces a Bioactive Second Messenger in Thrombotic Disorders.
Authors: Zhu Li , Bergmeier Wolfgang , Wu Jie , Jiang Hong , Yeboah Nana , Fan Ran , Cieslak Marcin , Milla Marcos E. , Brass Lawrence F.
Published: April 2020

Proteins that are expressed on the platelet surface can participate in contact-dependent signaling events which modulate thrombus formation or, after being shed from the platelet surface, serve as bioactive messengers that affect the function of nearby cells. Here we show for the first time that platelets express the class IV semaphorin known as sema4D or CD100, and that platelet activation causes the regulated shedding of the sema4D extracellular domain in a biologically-active form. Sema4D is a glycosylated 150 kDa disulfide-linked homodimer that has previously been implicated in interactions between T-cells and B-cells. Platelet activation by collagen, thrombin or PMA causes a transient increase in sema4D surface expression peaking at 15 min, followed by a complete loss of expression over 30–60 minutes. These events are accompanied by the release of the sema4D exodomain as a 130 kDa fragment, leaving a 25–30 kDa transmembrane and cytoplasmic domain fragment that is retained by the platelets. The cleavage event required to produce these fragments is inhibited by metalloprotease inhibitors and abolished in platelets from chimeric mice lacking the metalloprotease known as ADAM17 or TACE (TNF alpha converting enzyme). Incubation of recombinant sema4D with ADAM17 identified a single cleavage site just outside the predicted transmembrane domain. Western blots show that human platelets express ADAM17 in both its immature (zymogen) and mature (active) forms, and indicate that at least some of the ADAM17 is located on the platelet surface. ADAM17-dependent cleavage of sema4D does not require platelet aggregation, but the rate is accelerated when aggregation is allowed to occur and slowed when aggregation is prevented. Under both sets of conditions, cleavage of sema4D occurs to a greater extent and more rapidly than the ADAM17-dependent cleavage of GP Ib alpha, suggesting that there is a hierarchy of proteolytic events when platelets are activated. In terms of biological impact, the shedding of sema4D following platelet activation raises the possibility that the soluble extracellular domain of sema4D serves as a bioactive messenger. Two receptors for sema4D have been identified previously: CD72, which is present on lymphocytes where it regulates the activity of the tyrosine phosphatase, SHP-1, and plexin-B1, which is expressed in endothelial cells. Western blots suggest that both of these receptors are expressed on human platelets and show that SHP-1 is associated with CD72 in resting, but not activated platelets. Taken together, these results demonstrate that sema4D undergoes regulated ADAM17-dependent shedding when platelets are activated, and suggest that this results in the production of a bioactive form of the molecule that can affect responses in nearby platelets, lymphocytes and endothelial cells at sites of thrombosis.
Megakaryocytes Derived from Human Embryonic Stem Cells: A Genetically Tractable System To Study Megakaryocytopoiesis and Integrin Function
Authors: Gaur Meenakshi , Kamata Tamihiro , Wang Shaohui , Moran Barry , Shattil Sanford J. , Leavitt Andrew D.
Published: April 2020

The platelet fibrinogen receptor, a heterodimer consisting of integrin subunits αIIb and β3, is required for platelet aggregation, spreading and hemostasis. Platelet agonists such as thrombin and ADP lead to activation of αIIbβ3, thereby enhancing its affinity and avidity for binding fibrinogen (inside-out signaling). Furthermore, fibrinogen binding to αIIbβ3 triggers cytoskeletal changes and granule release (outside-in signaling). Genetic approaches to characterize the molecular pathways involved in αIIbβ3 signaling are not possible with anucleate blood platelets. Therefore we have established an OP9 stromal cell co-culture system to generate megakaryocytes from human embryonic stem cells (hESCs). αIIbβ3 activation, measured by soluble fibrinogen binding to hESC-derived megakaryocytes, αIIb+/GPIbα+ cells, is readily detectable following stimulation with known platelet agonists. Dose-response curves for peptide agonists specific for the two platelet thrombin receptors, protease-activated receptor 1 (PAR1) and PAR4, show a relative responsiveness that mirrors that of human platelets, and sub-maximal ADP responses are augmented by epinephrine. Moreover, hESC-derived megakaryocytes undergo lamellipodia formation, actin filament assembly, and vinculin localization at focal adhesions when plated on a fibrinogen-coated surface, characteristic of αIIbβ3 outside-in signaling. Undifferentiated hESCs genetically modified by lentiviral infection can be cloned and maintained in an undifferentiated state and then differentiated into megakaryocytes capable of αIIbβ3 activation. Thus, using hESCs, we have developed a renewable source of human megakaryocytes, and a genetically tractable system for studying megakaryocytopoiesis and αIIbβ3 signaling in the native cellular environment.
The Mechanism of Cold-Induced Platelet Hyperfunction.
Authors: Han Qiang , Chen Junmei , Peng Yuandong , Bergeron Angela L. , Liu Li , Dong Jing-fei , Lopez Jose A.
Published: April 2020

Refrigerated platelets function better when transfused than do platelets stored at room temperature, although chilled and rewarmed platelets do not survive long in the circulation. Here, we describe the one possible mechanism of this increase in the hemostatic function of platelets upon cold treatment. Cold exposure has been demonstrated to cluster GP Ib-IX complexes on platelets, enabling platelet recognition and phagocytosis by macrophages. We find that, in addition to increasing the binding to the leukocyte integrin Mac-1 (aMb2), clustering of GP Ib-IX complexes also augments von Willebrand factor (VWF)-related functions. Before the functional studies, platelets were chilled to 4°C for 2 hr and rewarmed to 37°C for 30 min. Chilled, rewarmed platelets, whether in platelet-rich plasma (PRP), whole blood, or reconstituted whole blood (all anticoagulated with Cirtate Sodium) perfused through a parallel-plate flow chamber (36 dynes/cm2) attached in greater numbers to VWF-coated surfaces than did platelets from the same donor kept at 37°C or 22°C. The increased function was also reflected in increased thrombus number and size on a surface of type III collagen. The surface coverage, an indicator of thrombus formation, increased from 10% to 25% upon cold. Chilled, rewarmed platelets also responded to lower doses of a VWF-specific agonist, ristocetin Chilled, rewarmed platelets showed 80% aggregation while control platelet only had 10% aggregation at low concentration of ristocetin (0.5mg/ml) treatment, and displayed a decreased threshold for shear-induced aggregation, aggregating at lower shear rates than control platelets at both 22°C and 37°C. Finally, when tested in a platelet function analyzer (PFA-100), chilled, rewarmed platelets demonstrated significantly shorter closure times than control platelets using both epinephrine/collagen and ADP/collagen cartridges. The epinephrine/collagen cartridges clotting time of chilled platelets decreased from normal 180 s to 162 s and ADP/collagen cartridges clotting time decreased from 118s to 99s. To exclude that the observed functional increases were not due to changes in GP Iba expression or prior platelet activation, we evaluated by flow cytometry the surface levels of GP Iba, P-selectin, and activated αIIbβ3.(antibody PAC-1 binding). We found no differences in any of these parameters between chilled, rewarmed platelets and control platelets. These results suggest that, if their rapid clearance can be prevented, cold-stored platelets may have enhanced hemostatic function of potential benefit in the bleeding patient. The results also caution, however, that trials will be necessary to exclude potential thrombotic consequences of infusing hyperfunctional, refrigerated platelets.
The Marginal Microtubule Coil in the Resting Blood Platelet Is a Dynamic Bipolar Array.
Authors: Italiano Joseph E. , Richardson Jennifer L. , Schulze Harald , Drabek Ksenija , Bulinski Chloe , Galjart Niels , Shivdasani Ramesh A. , Hartwig John H. , Patel Sunita R.
Published: April 2020

The discoid shape of the resting blood platelet is maintained by its marginal microtubule band. Structural studies have concluded that this band is composed of a single microtubule coiled 8-12 times around the cell periphery. To understand the dynamics of the microtubule coil, we took advantage of EB1 and EB3, proteins that highlight the ends of growing microtubules. Immunofluorescence microscopy with anti-EB1 revealed clear staining of numerous (8.7 +/− 2.0, range 4–12) comet-like dashes in the microtubule coil, suggesting the presence of several microtubule plus ends. Consistent with this observation, rhodamine-tubulin added to permeabilized platelets incorporates at multiple (7.9 +/−1.9) points throughout the microtubule coil. To visualize microtubule dynamics in platelets, we retrovirally directed megakaryocytes to express the microtubule plus-end marker EB3-GFP and isolated platelets released in these cultures. Fluorescence time-lapse microscopy of EB3-GFP-expressing resting platelets revealed multiple microtubule plus ends that grew in both clockwise and counterclockwise directions. Antibodies that recognize tyrosinated tubulin, which preferentially label newly assembled microtubules and not stable microtubules, stain the microtubule coil. These results indicate that resting platelets contain a bipolar array of microtubules that undergoes continuous assembly. When EB3-GFP-expressing platelets are activated with thrombin, the number of polymerizing microtubules increases dramatically and the microtubules grow into filopodia. Collectively, these results suggest that the marginal band of the resting blood platelet is highly dynamic, bipolar, and contains multiple microtubule plus ends. These ends are amplified in platelet activation and point towards the active edges of the cells and the tips of filopodia.
Evolutionarily Conserved Coupling of Transcription and Alternative Splicing in the Protein 4.1R and 4.1B Genes Regulates N-Terminal Protein Structure.
Authors: Tan Jeff , Parra Marilyn K. , Mohandas Narla , Conboy John G.
Published: April 2020

The protein 4.1R gene is regulated by complex pre-mRNA processing events that facilitate the synthesis of protein isoforms with different structure, function, and subcellular localization in red cells and various nucleated cell types. One of these events involves the stage-specific activation of exon 16 inclusion in erythroblasts, which mechanically stabilizes the membrane skeleton by increasing the protein’s affinity for spectrin and actin. Some of the splicing factor proteins and RNA regulatory elements responsible for this tissue-specific alternative splicing event have been defined. Here we focus on another RNA processing event, in the 5′ end of the transcript that can affect the structure and function of the membrane binding domain of protein 4.1R. We have shown that 4.1R transcripts originating at three far upstream alternative promoters/first exons splice differentially to alternative acceptor sites in exon 2′/2 in a manner that suggests strict coupling between transcription and alternative splicing events. A precisely analogous gene organization and RNA processing pattern has also been shown to occur in the paralogous 4.1B gene. Now we demonstrate that this coupling is evolutionarily conserved among several vertebrate classes from fish to mammals. The 4.1R and 4.1B genes from fish, bird, amphibian, and mammal genomes exhibit shared features including alternative first exons and differential splice acceptors in exon 2. In all cases, the 5′-most exon (exon 1A) splices exclusively to a weaker internal acceptor site in exon 2, skipping a short sequence designated as exon 2′ (17-33nt). Conversely, alternative first exons 1B and/or 1C always splice to the stronger first acceptor site, retaining exon 2′. These correlations are independent of tissue type or species of origin. Since exon 2′ contains a translation initiation site, this regulated splicing event generate protein isoforms with distinct N-termini. We propose that these 4.1 genes represent a physiologically relevant model system for mechanistic analysis of transcription-coupled alternative splicing. We have recently constructed a 9kb “minigene” that successfully reproduces the differential splicing patterns of exons 1A and 1B to exon 2′/2 in transfected cells. This minigene will facilitate identification of the determinants that guide coupling. Current experiments are testing the importance for proper splicing of the transcriptional promoter, first exon sequences, length and sequence of the intron, and sequence of a conserved element within exon 2′. Ultimately these studies should provide new insights into the mechanisms of coupling between far upstream, transcription-related processes and downstream alternative splicing.
Differentiation of Systematically Injected Mesenchymal Stem Cells into Retina Cells in the Rat Eye.
Authors: Park Sung-Kyu , Won Jong-Ho , Bae Sang-Byung , Kim Chan-Kyu , Lee Nam-Su , Lee Kyu-Taeg , Hong Dae-Sik , Park Hee-Sook
Published: April 2020

Presently, no effective treatment are available for retinal degeneration but there are a number of lines of investigation which suggest potential approaches. A number of investigations have shown the multilineage potential of mesenchymal stem cells (MSCs) into neural lineages, which prompted us to attempt their induction into photoreceptors. We evaluated whether systematically injected bone marrow-derived MSCs can be incorporated into the neuroretinal tissues and play an important role in healing retinal wound in the laser-induced retinal trauma model. After Nd;YAG laser-induced retinotomies, MSCs prelabeled with GFP were injected via tail vein. The fundus photographs were taken serially, and eyeballs were enucleated for histological studies. In ophthalmic examination, MSCs injected retinas were showed no hemorrhage, retinal detachment, and vitreous opacity at 4 weeks after MSCs injection. At same time, GFP(+) pigmented cells were detected at the damaged sites and observed more plentiful at the base of the laser-induced break sites. In time-dependent manner, more GFP(+) cells were detected around the damaged retina and incorporated into the neuroretinal tissues through the systemic route. We performed immunohistochemical analyses to evaluate the differential potential of MSCs into neuron. Although retinas were rarely stained within 4 weeks, MSCs injected retinas were consistently and strongly stained after 4 weeks in proportion to the amount of MSCs to engraft into retinas. To test whether MSCs treatment could also induced functional rescue of vision in injured rats, we performed electroretinograms on rat 4 weeks after MSCs injection. Detectable, albeit subnormal, electroretinogram recordings are observed in MSCs injected eyes. However, in all cases electroretinogram from the control eyes was non-detectable. Overall, these results are suggestive of some degree of functional rescue in MSCs injected eyes. Conclusively, we demonstrated the successful transplantation of ex vivo expanded MSCs with minimal side effects when injected intravenously into injured rat retina; we also showed the in vivo survival of these cells, their partial integration, and differentiation into the host retina.
Exploring the Molecular Mechanisms for Enhancing MSC Homing and Lodgement within Sites of Liver Damage/Fibrosis.
Authors: Herrbrich Phillip E. , Meyerrose Todd E. , Nolta Jan A.
Published: April 2020

Exogenous stem cells such as human mesenchymal stem cells (MSC) and hematopoietic stem cells (HSC) have been reported to be applicable for tissue repair, as in the case of chronic liver injury. However, one of the limitations in achieving high enough level of stem cell engraftment at these damaged sites is the low level of recruitment of exogenous stem cells. Our goals in the current studies are to enhance recruitment of human MSC to sites of tissue damage and to equip the MSC with high enzymatic activity to digest through areas of fibrosis surrounding of the damaged tissue, in this case, an immune deficient mouse model of CCl4-mediated liver damage as we have previously reported (Wang et al, 2003). Pretreatment of HepG2 cells with hypoxia significantly increased the levels of the hepatocyte growth factor (HGF) receptor, c-met. Since HGF is both a growth factor and a chemotactic agent found at high levels within sites of hypoxic liver injury, we hypothesized that pre-treatment of MSC with hypoxia would increase the expression of c-met and thus, would enhance the sensitivity of MSC to the chemotactic effect of HGF. First, we demonstrated that in the absence of serum and any exogenous growth factor, a 16-hr hypoxic treatment of MSC increased the total cellular protein level of c-met, as well as the total cellular phosphotyrosine activity following HGF stimulation. 3T3 fibroblasts engineered to secrete human HGF were plated in the bottom well of the transmigration assay and human PKH26-labeled MSC pre-incubated in hypoxic vs. normoxic conditions were added to the upper transwell. Transmigrated cells were enumerated after 3 hours to measure the functional role of c-met induction. Preliminary results show enhanced directional transmigration of hypoxia pre- treated MSC toward an HGF gradient, when compared to normoxic treated MSC. Next, we addressed the issue of liver fibrosis as a possible physical barrier that could block the entry of exogenously recruited MSC into the sites of liver damage. We demonstrated that the combination of hypoxia and HGF modulated the degree of matrix metalloproteinase (mmp2 and mmp9) activity in MSC. In addition, recent studies identified the presence of urokinase plasminogen activator receptor (uPAR) on hematopoietic stem cells. We show here for the first time, that uPAR can also be found on MSC. Ongoing studies are assessing the changes in uPAR and uPA activity following hypoxic culture. We hypothesize that changes in MMP and uPA activity can affect the ability of MSC to engraft/lodge within the fibrotic tissue. In conclusion, we propose that the pre-treatment of MSC with hypoxia and HGF might not only enhance the migration of exogenous stem cells to the site of liver damage but also enhance their ability to degrade the accumulated fibrosis at sites of injury for entry into the regions of damaged tissue.
Use of the NOD/SCID/γcnull Mouse Model To Assess the Hepatocyte-Producing Ability of Human Hematopoietic Cells.
Authors: Fujino Hisanori , Hiramatsu Hidefumi , Tsuchiya Atsunori , Noma Haruyoshi , Shiota Mitsutaka , Umeda Katsutsugu , Yoshimoto Momoko , Heike Toshio , Ito Mamoru , Nakahata Tatsutoshi
Published: April 2020

Hematopoietic cells have been shown to generate nonhematopoietic cells, although the true plasticity of stem cells has been questioned. Here we used the NOD/SCID/γcnull mouse model, which permits efficient engraftment of human hematopoietic stem cells and their multi-lineage differentiation including T cells, to investigate whether human hematopoietic stem cells can differentiate into human hepatocytes. Freshly collected cord blood was depleted of phagocytes with Silica® followed by CD34 positive selection using auto MACS®. These cells were intravenously transplanted into irradiated mice, after which the liver was either undamaged or damaged by chemicals. The livers of these mice contained hepatocyte-specific (albumin, CYP family, TAT, alpha1AT, CPSI, prealbumin, transferrin and RBP4), cholangiocyte-specific (CK19) and vascular endothelial cell-specific (eNOS) human mRNAs. Immunohistochemistry detected the human hepatocyte specific antigens, albumin and alpha-1-antitrypsin-positive hepatocytes, cholangiocytes and CD68+ Kupffer cells. We also found human albumin in the murine bloodstream. Human albumin levels in the peripheral blood of transplanted mice correlate with the degree of PB chimerism and increase with time after transplantation. Furthermore, after obtaining liver cells by collagenase perfusion, flow cytometry revealed the presence of human albumin-positive cells that bear both human and murine MHC molecules, suggesting cell fusion occurs. All of the above phenomena were found in both liver-damaged and undamaged mice. In addition, we found human CD34+ cells are recruited from the murine bone marrow to the liver only in the case of acute liver injury but do not acquire hepatic stem/progenitor characteristics. Our observation suggests there are two pathways that yield hepatic cells from hematopoietic stem cells. The first requires liver damage that recruits CD34+ cells from the bone marrow via the circulation while the second pathway does not involve liver damage and appears to represent a constitutive default pathway of hematopoietic to nonhematopoietic transition. Our model is thus a versatile tool for investigating the development of functional human hepatic cells from hematopoietic cells and the feasibility of using hematopoietic cells in clinical situations.
Hepatogenic Potential of Stem Cells and Lymphoid Precursors.
Authors: Yamada Yasuhiro , Miyake Hirosada , Nishimoto Eishi , Mitsuya Hiroaki , Yonemura Yuji
Published: April 2020

Several research groups have recently reported that certain bone marrow cells (BMCs) differentiate into hepatocytes in vitro as well as in vivo in rodents through both transdifferentiation and cell fusion. Hematopoietic myelomonocytic cells are thought to be the major source of hepatocyte fusion partners in the tyrosinaemia type I mouce transplantation model. Transdifferentiation of murine BMCs can be induced with various cytokines and extracellular matrix. We previously reported that when Sca-1+ BMCs were co-cultured with fetal liver cells (FLCs) on laminin-coated dishes, alpha-fetoprotein (AFP)-expressing BMCs became completely adherent by day 4 and expressed albumin as assessed with immunochemistry and RNA-PCR (Yamada et al., Exp Hematol. in press). In the present study, we attempted to further delineate the characteristics of BMCs that differentiate into hepatic-like cells. It was found that AFP-expressing cells were in CD5+ or B220+ lymphoid lineage, mostly Sca-1+CD5+ lineage and that CD5+CD4−CD8− and CD5+CD4+CD8+ thymocytes expressed AFP. When cKit+Sca-1+ lineage BMCs (KSLs) which did not express AFP, CD5+ BMCs, and CD5+ thymocytes, all from green fluorescence protein (GFP)-expressing transgenic mice, were co-cultured with FLCs from ROSA26 mice (X-gal+ FLCs), fractionated cells gave rise to adherent hepatic-like cells, which expressed albumin and cytokeratin 8 (CK 8) as assessed with immunochemistry and AFP, albumin, transthyretin and dipeptidylpeptidase IV as examined with RNA-PCR. The hepatic-like cells from KSLs, CD5+ BMCs and CD5+ thymocytes emerged at the frequency of 1 in 50, 1x103 and 3.5x105 by titration assay. These data suggest that AFP-expressing cells in BMCs were derived from hematopoietic stem cells and that lymphoid precursors differentiated into hepatic-like cells and their hepatogenic ability could diminish over lymphoid maturation.
The Effect of GSK3β Inhibitor on Murine Multipotent Adult Progenitor Cells (mMAPCs).
Authors: Oki Masayuki , Jiang Yuehua , Verfaillie Catherine M.
Published: April 2020

The Wnt/β-catenin pathway is important for maintenance of the undifferentiated state of human embryonic stem cells (ESCs), hematopoietic stem cells (HSCs) and other stem cells. We here tested if β-catenin activation, by inhibiting GSK3 β with BIO (6-Bromoindirubin-3′-oxime), affects the differentiation status of murine multipotent adult progenitor cells (mMAPCs). mMAPCs were cultured with or without 0.1–2 μ M BIO, LIF, EGF and PDGF-BB, at low density (plating density of 100/cm2 and cells passed every 2 days) or at high confluency (like ESCs). Quantitative RT-PCR (Q-RT-PCR) was done weekly for the ESC-specific pluripotency transcripts, Oct4, Rex1, Nanog, UTF1 and E-Ras, and lineage commitment genes, vWF, VE-cadherin, Sox1, Nestin, CK19, Albumin, HNF3β and AFP. After 2–4 weeks, cells were analyzed by FACS for ESC/MAPC specific antigens, and by immunohistochemsitry for Oct4, β-catenin and E-cadherin protein. Furthermore, cells were differentiated into endothelial cells and hepatocytes by using VEGF-A and HGF/FGF4, respectively for 2 weeks. Q-RT-PCR was used to demonstrate lineage differentiation.

BIO caused a dose-dependent clustering of mMAPCs from 4 days after treatment at both cell densities. FACS phenotype of low- and high-density mMAPCs was not affected by BIO. Nanog mRNA was not detected in any of the cell populations. However, Oct4, Rex1, vWF, and CK19 mRNA levels decreased in a BIO dose-dependent manner in mMAPCs maintained at low density, while they acquired higher levels of Sox1 mRNA (P=NS). At high-density, Oct4 and Rex1 mRNA level decreased in a BIO dose-dependent manner for the first week. On the other hand, 0.1 μ M BIO-treatment induced higher levels of Oct4 and Rex1 in cells maintained at high density. β-catenin and E-cadherin protein was highly expressed in clusters of low-density mMAPCs induced by BIO as well as BIO-treated mMAPCs maintained at ESC densities. In both cell densities, only mMAPCs treated with 1 or 2 μM BIO contained large dense mMAPCs clusters that expressed very high levels of β-catenin and E-cadherin. There was no statistical difference of differentiation potential between BIO conditions and cell densities.

BIO may not allow culture of mMAPCs without loss of Oct4 as it did for ESCs, but induces clusters like ESCs in a BIO-dose dependent manner. High concentrations of BIO enhances β-catenin and E-cadherin protein expression. BIO may induce lineage commitment of mMAPCs. Despite loss of Oct4 mRNA and protein, MAPCs treated with BIO continue to have the ability to differentiate. Ongoing studies are testing whether BIO treated cells maintained at high density also maintain in vivo engraftment and differentiation potential.
Identification of Novel Regulators of Hematopoietic Stem Cell Mobilization.
Authors: Voermans Carlijn , Lento William E. , Uqoezwa Mweia , DiMascio Leah N. , Chhotani Ali , Rattis Frederique M. , Reya Tannishtha
Published: April 2020

In an effort to identify the molecular changes during hematopoietic stem cell (HSC) mobilization we have analyzed genome wide changes in gene expression in HSCs in response to the chemotherapeutic agent cyclophosphamide (Cy) and the growth factor granulocyte colony stimulating factor (G). Cy/G treatment leads to an initial loss of proliferating precursors, followed by a rapid expansion of stem and progenitor cells and their migration to the peripheral blood. While this approach has been capitalized on for harvesting hematopoietic stem cells in clinical therapy, the molecular regulation of this process remains less well understood. To analyze changes in gene expression that occur in vivo as HSCs proliferate and prepare to migrate out of the bone marrow, we enriched for HSC fractions by isolating cells that express c-kit (K), Sca-1(S) and low levels of Thy1.1(T) but do not express lineage (L) markers (KTLS) from untreated mice and mice treated with Cy, G or Cy/G. The gene expression profile of these cells was examined by microarray analysis, relative to HSCs isolated from untreated mice. Interestingly, among the genes upregulated by Cy/G, a large majority (>60%) were a consequence of synergistic activity between Cy and G, while the rest of the upregulated genes could be attributed to activation by Cy or G alone. To test whether this screen allowed identification of novel candidates that regulate HSC function we analyzed the role of transforming growth factor beta induced gene (Big-h3), a gene highly upregulated after Cy/G treatment. Big-h3 is an extracellular matrix protein that mediates the adhesion and migration of various cell types, however its role in HSCs is unknown. Our experiments indicate that overexpression of Big-h3 in HSCs leads to enhanced adhesion to fibronectin as well as upregulation of specific integrins. These experiments suggest that changes in adhesion are an integral component of HSC mobilization, and that Big-h3 is one regulator of such processes. Further functional analysis of other candidates will likely lead to identification of other novel regulators of HSC development and mobilization.
Utility of Quantum Dots for Labeling and Tracking Leukemic Cell Lines, Human Bone Marrow and CD 34+ Umbilical Cord Blood.
Authors: Garon Edward B. , Crooks Gay M. , Tcherniantchouk Oxana , Luong Quang T. , Papaioannou Thanassis , Marcu Laura , Koeffler H. Phillip
Published: April 2020

Quantum dots (QDs) are nanometer scale fluorescent semiconductors that have recently garnered much interest as agents for imaging. QDs have several advantages over conventional fluorescent imaging probes, including high quantum yield, broad absorption spectra and minimal photobleaching. QDs have been used little in hematology.

We studied hematologic cells to determine whether they could be labeled with QDs, whether QDs could be seen after multiple cell divisions, whether QDs remained visible as cells differentiate and whether QDs could target specific antigens on the surface of cells. Using QDs attached to an endocytosed protein, we could intracellularly label all hematologic cells tested, including multiple leukemic cell lines (HL-60 and KG-1), normal human and murine bone marrow, leukemic human bone marrow (AML and CML) and normal human CD34+ cells. The QDs are easily detected by FACS and microscopy.

By counting cell divisions using the cell membrane dye PKH26, we show that QDs can be seen in leukemic cell lines and primary cells through more than four cell divisions. Some cells from leukemic cell lines retain QDs for up to two weeks. We are also able to see QDs in cells through differentiation. We labeled HL-60 cells and cultured them with either 1,25 dihydroxy vitamin D or DMSO. QDs were seen in monocytic-like and neutrophil-like progeny of the labeled HL-60 cells. After labeling CD34+ cells and culturing them in appropriate cytokines, QDs were observed in the resultant monocytes.

We were also able to target QDs specifically to cells expressing certain cell surface antigens. QDs complexed to streptavidin and a biotinylated anti-CD33 antibody homed to myelogenous leukemic cell lines but not lymphomatous cell lines. In summary, we show for the first time that QDs effectively label and track hematolgic cells, adding a new option when addressing cell fate questions in hematology.
GABP (GA Binding Protein) Is Required for Formation of a Retinoic Acid-Induced Myeloid Enhanceosome on the CD18 Promoter.
Authors: Resendes Karen K. , Rosmarin Alan G.
Published: April 2020

CD18, the β chain of the leukocyte integrins, mediates cell-cell and cell-matrix interactions of white blood cells and is required for normal innate immunity. Retinoic acid (RA) induces maturation of myeloid cells; mutations in retinoic acid receptors (RARs) account for acute promyelocytic leukemia. CD18 is transcriptionally regulated by RA in myeloid cells. The proximal promoter of CD18 is transcriptionally regulated by RA but, strikingly, this region is not bound or activated by retinoic acid receptors. Rather, the RA-responsiveness of the CD18 proximal promoter is dependent on ets sites that are bound by the ets transcription factor, GA-binding protein (GABP). The transcriptional co-activator, p300, further increases the RA-responsiveness of CD18. We show that p300 and GABPα, the ets DNA binding subunit of GABP, physically interact in myeloid cells. This interaction uses the cysteine/histidine regions of p300, and the pointed domain (PNT) of GABPα. This demonstrates p300 as the first known interaction partner of the GABPα PNT domain. We prepared a GABPα PNT domain construct for expression in mammalian cells and showed that this PNT construct inhibited the GABPα:p300 interaction and dramatically decreased the RA-responsiveness of the CD18 proximal promoter. We used Chromatin immunoprecipitation (ChIP) to demonstrate that RA induces formation of an enhanceosome on the CD18 promoter; thus, GABPα and p300 on the CD18 proximal promoter recruit RAR/RXR bound to a distal enhancer in the presence of RA. We prepared a dominant negative p300 construct and showed that its expression blocks the formation of the CD18 enhanceosome. These results demonstrate that GABP is required for formation of an enhanceosome that mediates responsiveness of CD18 to RA in myeloid cells. Dominant negative forms of GABPα and p300 interfere with RA responsiveness in myeloid cells and disrupt this enhanceosome. The CD18 enhanceosome is the first known RA-induced enhanceosome and demonstrates its role in mediating retinoid responsiveness in myeloid cells.
Regulation of Ldb1 Stability and Transcriptional Complex Assembly by Single-Stranded DNA-Binding Protein SSDP2.
Authors: Xu Zhixiong , Meng Xianzhang , Cai Ying , Nagarajan Lalitha , Brandt Stephen J.
Published: April 2020

The LIM domain-binding protein Ldb1 is known to form higher order complexes with LIM-homeodomain and LIM-only (LMO) proteins to regulate diverse developmental programs, including hematopoiesis. The level of Ldb1 is critical for its cellular roles, and its turnover is regulated by the E3 ubiquitin ligase RLIM. Single-stranded DNA-binding protein (SSDP), an Ldb1-interacting partner, is an essential gene for embryonic development and has been shown to regulate axis formation in Xenopus and wing development in Drosophila through Ldb1; however, the mechanisms by which SSDPs regulate these and other developmental programs are still obscure. We previously reported that a DNA-binding complex containing the basic helix-loop-helix protein TAL1/SCL, its DNA-binding partner E47, zinc finger protein GATA-1, LIM domain protein LMO2, and Ldb1 stimulates Protein 4.2 (P4.2) transcription in erythroid progenitors through tandem E box-GATA elements in the gene’s proximal promoter. We have now established that SSDP2 is associated with this complex (by supershift analysis) and occupies the promoter of this gene (by chromatin immunoprecipitation analysis) in murine erythroleukemia (MEL) cells. Further, enforced expression of SSDP2 in these cells stimulated P4.2 reporter activity and accumulation of P4.2 and beta-globin mRNAs, and cotransfection of SSDP2 with the five originally identified components of this complex further increased promoter activity in reporter analysis. Importantly, overexpression of SSDP2 in MEL cells significantly increased Ldb1 protein half-life and steady-state levels of Ldb1 and LMO2 protein. This effect on Ldb1 stability required the Ldb1-interacting domain of SSDP2, consisting of its first 94 amino acids (SSDP2(1–94)), and was also observed in Cos7L and CHO cells. We showed, in addition, that SSDP2 or SSDP2(1–94), but not an Ldb1 interaction-defective mutant, prevented RLIM-mediated degradation of both Ldb1 and LMO2 in transfected cells, that SSDP2 protection of LMO2 degradation required Ldb1, and that SSDP2 directly inhibited RLIM-mediated ubiquitination of Ldb1. Immunoprecipitation analysis revealed that overexpression of SSDP2 or SSDP2(1–94) significantly decreased interaction between Ldb1 and RLIM. Finally, SSDP2 protein expression in differentiating MEL cells paralleled this multi-protein DNA-binding activity and overexpression of SSDP2 in these cells dramatically increased E box-GATA DNA-binding activity, with maximal formation of the ternary complex requiring coexpression of SSDP2, Ldb1, and LMO2. Together, these studies reveal a positive role for SSDP2 in erythroid gene expression and identify a biochemical function for SSDP2 in regulating Ldb1 stability and transcriptional complex assembly. The mechanism mediating Ldb1 stabilization appears to involve competitive inhibition of RLIM interaction with Ldb1.
Thrombotic Thrombocytopenic Purpura after Allogeneic Hematopoietic Stem Cell Transplantation: A Complication Still Frequent and Responsible of Severe Outcome.
Authors: Uderzo Cornelio , Sangalli Haidi , Bonanomi Sonia , Morreale Giuseppe , Lanino Edoardo , Annaloro Claudio , Volpe Aldo Della , Alessandrino Paolo , Lazzarino Mario , Renoldi Mila , Ferrari Paolo , Iacobelli Massimo , Busca Alessandro , Locatelli Franco , Balduzzi Adriana , Rovelli Attilio
Published: April 2020

Introduction

Thrombotic thrombocytopenic purpura (TTP) might costitute a severe complication after hematopoietic stem cell transplantation (HSCT) with a variable incidence depending on different diagnostic criteria adopted in the past.

Patients and Methods

The current study enrolled 551 consecutive patients (pts) (314 males, median age 29 years) undergoing HSCT from January 2000 to April 2005 from HLA match (m) or mismatch (mm) family donor (350/551) and unrelated donor (201/551) for malignant (501/551) or non malignant diseases (50/551). The diagnosis of TTP was performed on the basis of homogeneous five clinical and seven laboratory criteria. Univariate (by chi square test and Fisher’s exact test) or multivariate (by logistic regression method) analyses were performed to evaluate the effect of some candidate risk factors on both TTP occurrence and outcome.

Results

Sixty four of 551 pts presented TTP (11,6%) at a median time of 47 days post-HSCT; 59/64 were affected by malignant and 5/64 by non malignant diseases. Multivariate analysis showed GVHD >II° (p=0,0001), stem cell donors (m/mm unrelated or mm related) (p=0,024), female gender (p=0,0186),TBI-based conditioning regimen (p=0,03) and status of hematological remission (p=0,03) as factors influencing the occurrence of TTP. Only three predicting factors for TTP outcome have been demonstrated statistically significative by multivariate analysis: age (p=0,0186), stem cell donors (m/mm unrelated or mm related) (p=0,01) and TTP index (p=0,018). The TTP mortality rate was 40% while the outcome after TTP diagnosis was partially influenced by TTP treatment. In particular, Defibrotide seemed to be a promising drug in univariate analysis compared with alternative therapies for 34 alive and good responders pts out of 64 TTP pts.

Conclusions

The study underlines the possibility to identify those patients who are more prone to develop post-HSCT TTP, complication still frequent (11,6%) when homogeneous diagnostic criteria are adopted. TTP outcome seems to be conditioned by some peculiar risk factors as adult age, m/mm unrelated or mm related donors HSCTs. Prospective randomized therapeutic trials, focusing on the possible advantage of Defibrotide versus other treatments, should be encouraged.
Efficacy of Soluble TNFα Inhibitor (Etanercept) for Treatment of Steroid Refractory Acute Graft Versus Host Disease (GVHD) Post Allogeneic Stem Cell Transplantation (SCT).
Authors: Kennedy Glen A. , Butler Jason , Morton James , Hill Geoff R. , Western Robyn , Durrant Simon
Published: April 2020

Aims: To review the efficacy of the soluble TNFα inhibitor etanercept in treatment of steroid refractory acute GVHD.

Methods: All patients who received etanercept for treatment of biopsy proven steroid refractory acute GVHD at our institution were retrospectively reviewed. GVHD was graded as per Seattle criteria. Standard GVHD prophylaxis post SCT was cyclosporine plus either methotrexate (D1–11) or mycophenolate mofetil (MMF). Standard 1st line therapy used for grade 2–4 GVHD was methylprednisolone 2mg/kg/day; standard 2nd line therapy for steroid refractory GVHD was anti-thymocyte globulin (ATG) + tacrolimus +/− MMF. Etanercept was used either after no-response (NR) >1mth after administration of both 1st line and 2nd line therapies (Group 1), or included as part of standard 2nd line therapy, starting within approximately 2wks of commencing ATG (Group 2). Etanercept dose was 0.4mg/kg (max 25mg) SC twice per week for 4wks, then once per week for 4wks. GVHD response was assessed at end of etanercept therapy. Standard antibiotic prophylaxis included valaciclovir (for HSV / VZV), bactrim / pentamidine (for PCP) and fluconazole (for fungal infection).

Results: In total 17 patients had received etanercept therapy, including 3 with grade 2, 7 with grade 3 and 7 with grade 4 GVHD. Predominant organ affected by GVHD was gut in 9 cases, skin in 5 and combination gut and skin in 3. Of the 3 patients treated in group 1, no responses and no survivors occurred. Group 2 included 14 patients, in whom responses were 10 CR (72%), 1 PR (7%) and 3 NR (21%), with 7 patients (50%) alive at median FU of 6mths (range 3–30mths) post commencing etanercept. For the entire cohort, OS at 12mths is 35%. Causes of death included progressive GVHD in 4 patients and infective complications in 6. Overall infective complications observed included bacterial sepsis (6/17), CMV reactivation (8/17, including CMV pneumonitis in 1), other viral infections (3/17), new, possible or proven aspergillosis (3/17) and progression of previously documented fungal infection (2/17).

Conclusions: Our experience suggests that when used in combination with ATG, etanercept has significant activity in the treatment of steroid refractory acute GVHD, with high response rates and significant survival observed. Better strategies to reduce infectious complications in this setting are needed.
Chronic Graft-Versus-Host Disease after Allogenaic Bone Marrow Transplantation in Japan: Identification of Risk Factors and Prognostication.
Authors: Atsuta Yoshiko , Suzuki Ritsuro , Yamamoto Kazuhito , Terakura Seitaro , Iida Hiroatsu , Kohno Akio , Naoe Tomoki , Sao Hiroshi , Kodera Yoshihisa , Morishima Yasuo , Hamajima Nobuyuki , Morishita Yoshihisa
Published: April 2020

Objective: Chronic graft-versus-host disease (cGVHD) is an important complication of allogeneic hematopoietic stem cell transplantation (HSCT) involving various organs such as skin, eyes, mouth, liver, lung, and gastrointestinal tract, which is associated with major disability and diminished quality of life. There have been several studies to explore risk and prognostic factors in Western countries. However, because the genetic homogeneity is different among the ethnics, such models are not assured to be applicable for Japanese patients. To identify the incidence and risk factors of cGVHD, and prognostic factors for patients who developed cGVHD, we analyzed a series of patients.

Methods: The incidence and prognostic factors for cGVHD were evaluated for 255 patients who survived more than 100 days after bone marrow transplantation (BMT) in Nagoya BMT Group.

Results: One hundred and nineteen (47%) developed cGVHD. Prior acute GVHD (grade 2–4) and use of unrelated donor were significantly associated with the onset of cGVHD. Systemic immunosuppressive therapies were administered to 86 patients (78%), 42 (38%) of whom required secondary therapies. Presence of cGVHD did not have an impact on mortality (hazard ratio [HR]=0.89; 95% confidence interval [CI], 0.59–1.3). Proposed prognostic models from Western countries, Akpek’s score and Lee’s grading system did not successfully classify our patients. Three factors at diagnosis were associated with cGVHD-specific survival; i.e. presence of infection (HR=4.1; 95%CI, 1.6–10.3), ongoing use of corticosteroid at the onset of cGVHD (HR=3.9; 95%CI, 1.7–9.1), and a Karnofsky performance score<80 (HR=4.7; 95%CI, 2.0–11.3). The probability of cGVHD-specific survival at 4 years was 79% (95%CI, 70–86%).

Conclusion: The severity and death rate of Japanese patients with cGVHD was lower than those for populations in Western countries, which might be the result of greater genetic homogeneity of Japanese ethnics. For prognostication, a different model was needed to identify high-risk patients.