bluuebunny/crossref_metadata_2025 huggingface dataset:
LPS Stimulates Platelet Secretion and Promotes Platelet Aggregation Via TLR4/MyD88 and the cGMP-Dependent Protein Kinase Pathway.
Authors: Zhang Guoying , Welch Emily , Malik Asrar B. , Du Xiaoping , Li Zhenyu
Published: April 2020

Bacterial lipopolysaccharide (LPS) induces rapid thrombocytopenia, hypotension and sepsis. Although growing evidence suggests that platelet activation plays a critical role in LPS-induced thrombocytopenia and tissue damage, the mechanism of LPS-mediated platelet activation is unclear. Here we show that LPS stimulated platelet secretion of dense and alpha granules as indicated by ATP release and P-selectin expression, and thus enhanced platelet activation induced by low concentrations of platelet agonists. Platelets express components of the LPS receptor-signaling complex, including Toll-like receptor (TLR4), CD14, MD2, and MyD88. The effect of LPS on platelet activation was abolished by an anti-TLR4 blocking antibody or TLR4 knockout. Furthermore, LPS-induced potentiation of platelet aggregation and FeCl3-induced thrombus formation were abolished in MyD88 knockout mice. Importantly, TLR4 mediates LPS-induced cGMP elevation and the stimulatory effect of LPS on platelet aggregation was also abolished by inhibitors of nitric oxide synthase (NOS) and the cGMP-dependent protein kinase (PKG). Thus, LPS promotes platelet secretion and aggregation through a TLR4/MyD88 and cGMP/PKG-dependent pathway.
Donor-Cell Derived Aplastic Anemia (AA) with a Small Population of CD55−CD59− Blood Cells after Allogeneic Stem Cell Transplantation: Evidence for the Immune System Attack Against Normal Hematopoietic Stem Cells in AA.
Authors: Sugimori Chiharu , Mochizuki Kanako , Yamazaki Hirohito , Nakao Shinji
Published: April 2020

Acquired aplastic anemia (AA) is thought to be caused by the immune system attack against hematopoietic stem cells. However, there is no direct evidence that an immune system attack against normal hematopoietic stem cells leads to development of AA. The immune system attack may be directed toward abnormal stem cells given the fact that some patients with myelodysplastic syndrome respond to immunosuppressive therapy. Although the presence of a small population of CD55−CD59− blood cells represents a reliable marker for the immune pathophysiology of AA, little is known regarding when and how such paroxysmal nocturnal hemoglobinuria (PNH)-type cells appear in patients with AA. The development of AA with a small population of PNH-type cells was recently observed in an allogeneic stem cell transplant (SCT) recipient. This patient, a 59-year-old male, who had been treated with allogeneic peripheral blood stem cell transplantation (PBSCT) from an HLA-compatible sibling for treatment of very severe AA in March 2002, developed severe pancytopenia in December 2005. Late graft failure (LGF) without residual recipient cells was diagnosed based on the results of a chimerism analysis. Sensitive flow cytometry failed to reveal any increase in the proportion of CD55−CD59− PNH-type blood cells. The patient underwent a second PBSCT from the original donor without preconditioning in February 2006. Although his pancytopenia was completely resolved by day 20, his blood counts gradually decreased from day 60 without any apparent complications. Flow cytometry revealed small populations of PNH-type granulocytes in his peripheral blood (Figure 1). Both the PNH-type and normal phenotype granulocytes were of donor origin. PIG-A gene analyses showed the PNH-type granulocytes in the patient to be a clonal stem cell with an insertion of thymine at position 593 (codon 198). Similar results were obtained from the sorted PNH-type granulocytes obtained 6 months later. The patient was treated with horse antithymocyte globulin and cyclosporine. The patient required no further transfusions after 88 days of the therapy and remains well as of August, 2007. The small population of PNH-type cells was not detectable in any of 50 SCT recipients showing stable engraftment or in an AA patient suffering graft rejection after a SCT. These findings suggest that some factors expressed by the patient induced an immune system attack against autologous hematopoietic cells, leading to de novo development of donor-cell derived AA. This is the first evidence that an immune system attack against normal hematopoietic stem cells results in AA associated with a clonal expansion of a PIG-A mutant which may originally be present in the donor bone marrow.

Figure Figure
Disease-Related Symptoms Reported across a Broad Population of Patients with Paroxysmal Nocturnal Hemoglobinuria.
Authors: Meyers Gabrielle , Weitz Ilene , Lamy Thierry , Cahn Jean-Yves , Kroon Henk-André , Severino Beth , Uranga Maria Teresa , Alonso Miguel Sanz , Vela José Antonio García , Hill Anita
Published: April 2020

In paroxysmal nocturnal hemoglobinuria (PNH), lack of the GPI-anchored terminal complement inhibitor CD59 from erythrocytes renders them susceptible to chronic hemolysis, which is central to the signs and symptoms of PNH. Patients are at elevated risk for thrombosis, experience anemia that may require transfusion support, and suffer from fatigue that can be severe. Patients often have a poor quality of life resulting from PNH related symptoms including pain, dyspnea, dysphagia and erectile dysfunction, which negatively impact quality of life. The prevalence and severity of symptoms were explored in the context of a multi-national content validation study, of patients not receiving eculizumab therapy, employing the Functional Assessment of Chronic Illness Therapy (FACIT)-Fatigue and the European Organization for Research and Treatment of Cancer Quality of Life Core 30 (EORTC QLQ-C30) instruments. Symptom questions were asked of 29 PNH patients (19 men, 10 women, mean age 41.2±13.2 years) from the United Kingdom, United States, France and Spain. More than half (52%) had PNH for over 5 years. Most (76%) reported never having had a blood clot, 31% reported not receiving any medication for their PNH, and 59% reported either that they had never been transfused or had not received transfusion within the last year for PNH. Patients viewed overall quality of life, global health, functioning, fatigue, pain, and shortness of breath as important PNH-related signs/symptoms. Both the FACIT-Fatigue and EORTC instruments were relevant and adequate in assessing the level of fatigue and other quality of life measures in PNH. The burden of disease in this multicultural and diverse cohort of patients was significant: 76% were forced to modify their daily activities to manage their PNH and 17% were unemployed due to PNH. Nearly all (96%) complained of fatigue and more than half reported abdominal pain, headache and shortness of breath (Table). Patients also commonly reported dysphagia (41%) and erectile dysfunction (47% in males). Most patients reported these PNH-related symptoms as moderate to very severe, and a substantial majority reported distress associated with the symptoms. Significant disease burden was identified in a diverse population of PNH patients, most of which had minimal or no transfusion requirements and a low incidence of thrombosis. Therapy that controls hemolysis and thereby improves fatigue, pain, shortness of breath, dysphagia and erectile dysfunction may prove beneficial for PNH patients with these disease characteristics.
Impaired CD163 Mediated Hemoglobin-Scavenging and Hemolytic Crisis in Patients Treated with CD33 Targeted Chemotherapy (Gemtuzumab Ozogamicin, Mylotarg™).
Authors: Maniecki Maciej B. , Hasle Henrik , Friis-Hansen Lennart , Lausen Birgitte , Nielsen Ove J. , Bendix Knud , Moestrup Soren K. , Moller Holger J.
Published: April 2020

Hemoglobin liberated to plasma during intravascular hemolyses is rapidly bound to haptoglobin. The hemoglobin-haptoglobin complexes undergo endocytosis through the monocyte/macrophage specific scavenger receptor for hemoglobin (CD163). This mechanism protects against oxidative and NO-scavenging adverse effects of free hemoglobin. In this study, we describe a novel syndrome of severe intravascular hemolysis and serious hemolytic crisis due to impaired hemoglobin scavenging in three acute myeloid leukemia patients following CD33-directed therapy with the immunotoxin gemtuzumab ozogamicin (GO, Mylotarg™). A synchronous high free hemoglobin, haptoglobin, and low bilirubin after septicemia-induced intravascular hemolysis indicated abrogated clearance of haptoglobin-hemoglobin complexes. This was further supported by low levels of plasma soluble CD163 and a concordant low number of CD163-expressing monocytes. We show that CD163 positive monocytes and bone marrow macrophages coexpress CD33 thus suggesting that the GO-induced cellular cytotoxicity of CD33 positive cells eradicates a significant part of the CD163 positive monocytes and macrophages. The patients had severe inflammation and serious organ failure symptoms that may be a direct effect of the persistent high level of free hemoglobin. One of the patients had a fatal outcome whereas, the other two recovered from the hemolytic episode, and the peripheral blood CD163 expression returned to normal. The risk of a serious hemolytic crisis should be considered following CD33-targeted chemotherapy. Furthermore, the cases provide circumstantial evidence of a key role of CD163 plasma hemoglobin clearance in vivo.
Heritability of Commonly Measured Blood Cell Phenotypes in the San Antonio Family Heart Study.
Authors: Viel Kevin R. , Howard Tom , Curran Joanne E. , Almasy Laura , Moses Eric K. , Glahn David C. , Ameri Afshin , John Blangero
Published: April 2020

Heritability (h2) indicates the extent to which genes contribute to the observed inter-individual variation in a phenotype. Traits that have a high h2 may be fruitful targets of a subsequent genome screen to identify quantitative trait loci (QTL) and the genetic variants underlying them. We have estimated the heritabilities of measurements that commonly comprise a complete blood cell count (CBC) for members of the San Antonio Family Heart Study (table). The number of white blood cells per μL of blood (WBC) was not under genetic influence (h2 = 0.160, p = 0.253), however, the individual components of the differential (lymphocyte, monocyte, and granulocyte percentage) did appear to be. Red blood cell count (RBC) had the highest heritability (h2 = 0.638, p < 0.001) with both the mean corpuscular volume (MCV) and the red cell distribution width (RDW) demonstrating moderate heritability. The platelet count (Plt) and the mean platelet volume (MPV) had a mild genetic influence that was nominally significant. Currently, the fourth longitudinal examination of this cohort is in progress and to date we have measurements for 154 Mexican American subjects in 16 nuclear families. If prior recruitment rate is maintained, bivariate analyses assessing pleiotropy between traits may be available for presentation. Some of these traits have been investigated previously, but for many these are the first reported h2 estimates to our knowledge. This investigation is a necessary first step to understanding the contributions of genes to these important clinical measurements and to the elucidation of the biological pathways involved in their regulation, which may lead to improved diagnostics and therapies to treat blood cell-related disorders.

Heritabilities of commonly measured blood cell phenotypes Phenotype h2 (p-value) 1 Inverse normal transformed value WBC 0.163 (0.253) LY (%) 0.599 (<0.001) MO (%) 0.603 (0.011) GR (%) 0.500 (0.004) RBC 0.638 (<0.001) Hgb 0.283 (0.094) Hct 0.432 (0.023) MCV 0.476 (0.005) MCH 0.429 (0.011) MCHC1 0.249 (0.139) RDW1 0.422 (0.014) Plt 0.290 (0.013) MPV 0.298 (0.006)
Treatment Decisions for Young Women with Supra-Diaphragmatic, Limited-Stage Hodgkin Lymphoma: Exploration of Options Using a Markov Decision Analysis.
Authors: Hicks Lisa , Dent Rebecca , Meyer Ralph M. , Naimark David
Published: April 2020

Background: Treatment decisions for patients with limited-stage Hodgkin lymphoma (HL) involve trade-offs between combined modality therapy (CMT), which includes involved field radiation therapy (IFRT), versus ABVD alone. While CMT is associated with superior disease control, these gains must be balanced against late-effects associated with radiation, including a long-term increased risk of solid cancers. This balance may be particularly important for young women in whom IFRT would include portions of the breast, and in whom substantially increased relative risks (RR) of breast cancer have been reported. The use of a Markov decision model (MDM) may assist in evaluating the principles of this trade-off.

Methods: We developed a MDM to explore which strategy, CMT or ABVD alone, maximizes life expectancy among young women with limited-stage HL involving supraclavicular, axillary, and/or mediastinal adenopathy and whose IFRT would therefore include portions of the breast. Three base cases assessing women ages 20, 30 and 40 years at the time of HL diagnosis were evaluated. Published data on the RR of breast and other cancers following HL treatment, the risk of HL relapse with CMT and ABVD alone, expected survival with breast and other cancers, the risk of relapse following salvage HL therapy, the risk of leukemia after salvage HL therapy, and expected survival with secondary leukemia were used to populate the model. The RR of breast and other solid cancers was assumed to be negligible for the first 10 years after treatment and constant thereafter. The risk of HL relapse was assumed to be constant during the first five years post-treatment, and substantially lower thereafter. Uncertainty in critical variables was explored with deterministic sensitivity analysis.

Results: In our model, women age 20, 30, and 40 years at the time of HL diagnosis have a life expectancy of 56.1, 64.1, and 70.1 years with CMT, and 62.5, 66.1, and 69.9 years with chemotherapy alone. For women age 40 years at HL diagnosis, CMT was the favored strategy at all time points. For younger women, ABVD alone maximized life expectancy, but this benefit was manifest only > 20 years after HL diagnosis. Our model was robust in one-way sensitivity analyses across a plausible range of critical variables. In two-way analyses, the model was sensitive to extreme values for the relative risk of breast cancer post-CMT, the relative risk of other cancer post-CMT, and the risk of HL relapse following ABVD alone.

Conclusions: The optimum treatment strategy for young women with limited-stage, supra-diaphragmatic HL is likely influenced by age at diagnosis. Use of a MDM may assist in evaluating the trade-offs associated with current treatment options.
Clincial Evidence That Very Small Embryonic Like (VSEL) Stem Cells Are Mobilizaed into Peripheral Blood in Patents after Stroke.
Authors: Kucia Magda , Paczkowska Edyta , Koziarska Dorota , Halasa Maciej , Safranow Krzysztof , Karbicka Anna , Nowik Marta , Nowacki Przemyslaw , Ratajczak Mariusz Z. , Machalinski Boguslaw
Published: April 2020

Murine bone marrow (BM) contains a mobile population of CXCR4+SSEA-1+Sca-1+lin−CD45− very small embryonic like (VSEL) stem cells (Leukemia2006:20; 857) that are mobilized into peripheral blood (PB) in SDF-1 dependent manner e.g., after pharmacological mobilization with G-CSF or in murine model of stroke (Leukemia2006:20;18). We proposed that VSEL could be potentially employed in regeneration of damaged brain or spinal cord. Since a similar population of CXCR4+CD133+CD34+SSEA-4+Oct-4+lin−CD45− cells resides also in human BM (Leukemia2007:21;297), we asked whether in humans, similarly as in mice, the stroke-related stress may trigger mobilization of these cells from BM into PB. To address this question, we evaluated a number of VSEL in 60 stroke patients and similar number of age-matched controls. The PB samples were harvested during first 24 h, day +3 and day + 7 after stroke, and compared to normal controls. The circulating VSEL in PB were evaluated by employing

RQ-PCR, FACS and direct immunofluorescence analysis.

To perform these studies, first the mRNA was extracted from circulating PB mononuclear cells (MNC) and the expression of pluripotent (e.g., Oct-4, Nanog) and neural specific markers (GFAP, Nestin, β-III-tubulin, Olig1, Olig) was evaluated by RQ-PCR. Next, the circulating PB MNC were analyzed by FACS for the presence of cells that express CXCR4+, CD133+ and CD34+ antigens. Finally, we measured the serum concentration of SDF-1 by ELISA. We found in stroke patients

an increase in total number of circulating CXCR4+D133+CD34+ cells, ∼ × 8 increase in expression of mRNA for Oct-4 and Nanog in circulating PB MNC that was subsequently confirmed by immunofluorescence staining for a presence of CXCR4+Oct-4+ cells, and increase in serum concentration of SDF-1.

Additionally, we found a positive correlation between the extensiveness of a stroke and the number of CXCR4+ VSEL circulating in PB. We conclude that the stroke triggers the mobilization of CXCR4+ VSEL. We hypothesize that these cells could have a prognostic value in stroke patients as well as use for regeneration of neural tissues. Currently, we are testing this hypothesis in the murine model of stroke.
Urokinase (uPA) Induces Endothelial Cell Survival Via the X-Linked Inhibitor of Apoptosis Protein (XIAP).
Authors: Prager Gerald W. , Brunner Patrick M. , Mihaly Judit , Koschelnick Yuri , Binder Bernd R.
Published: April 2020

uPA plays an important role in angiogenesis: Originally, the urokinase system has been implicated to assist the angiogenic process by it’s proteolytic properties. It is now becoming increasingly evident that uPA additionally elicits many pro-angiogenic responses like differentiation, proliferation and cell migration in a non-proteolytic fashion via induction of intracellular signal transduction. In this study we demonstrate that in endothelial cells uPA protects against apoptosis by transcriptional upregulation of inhibitor of apoptosis proteins (IAPs), among them most prominently the X-linked inhibitor of apoptosis protein (XIAP). In contrast to canonical growth factors, like vascular endothelial growth factor (VEGF), uPA elicits anti-apoptosis independently of the PI3-kinase pathway. uPA-induced cell survival is dependent on the type of extracellular matrix used indicating the involvement of integrin adhesion receptors. Thereby, uPA induces phosphorylation of the CDC42 downstream effector p21-activated kinase 1 (PAK1), which leads to IkappaB kinase alpha (IKKa) phosphorylation, a prerequisite for NFkappaB activation. Blocking NFkappaB by using the specific NFkappaB inhibitor BAY 11–7082 or by adenoviral-mediated overexpression of its inhibitor, IkB, inhibits uPA-induced XIAP expression as well as uPA-induced cell survival. Downregulating XIAP expression by small interfering RNA techniques significantly reduces cell survival efficiencies of uPA in endothelial cells. From these data we conclude that uPA activation, which is a main player in endothelial cell migration and invasion, provides an additional, PI3-kinase independent cell survival mechanism.
Cleaved Kininogen Inhibits Endothelial Progenitor Cell Differentiation by Interfering with the avb3 Integrin-MMP-2 Pathway.
Authors: Colman Robert W. , Schmuckler Noah G. , Wu Yi
Published: April 2020

Circulating endothelial progenitor cells (EPCs) are involved in repairing damaged vasculature and in tumor angiogenesis, thus promoting wide interest in their therapeutic potential in ischemic diseases and cancer. Cleaved high molecular weight kininogen (HKa) potently inhibits endothelial cell (EC) functions including in vivo angiogenesis. We have shown that HKa inhibits differentiation of ECs. We now test a new hypothesis that HKa can inhibit EPC differentiation by blocking matrix metalloprotease-2 (MMP-2) activation. EPCs were isolated from human blood and cultured on collagen type I-coated surface. As detected by flow cytometry and Western-blot, they expressed MMP-2 and membrane-type 1-MMP (MT1-MMP), avb3 integrin, uPAR and caveolin-1, but did not express monocyte markers such as HLA-DR and CD14. A clone-forming assay using retroviral infection indicated that an early single EPC exhibited colony forming property, but mature ECs such as human umbilical vein endothelial cells (HUVECs) did not. Upon stimulation by VEGF, EPCs, but not HUVECs, formed vacuoles and differentiated into capillary networks. Gene silencing of either MT1-MMP or MMP-2 by siRNA completely blocked the vacuole and tube formation by EPCs, suggesting that MT1-MMP and MMP-2 are both essential for differentiation of EPCs. HKa inhibited tube formation by EPCs, as well as the conversion of pro-MMP-2 to MMP-2 as a functioning concentration in the conditioned medium. CS-1 cells expressing endogenous av subunit were transfected with human b3 cDNA. We found that HKa inhibition of MMP-2 activation in these cells is dependent on avb3 heterodimer. The binding of avb3 integrin to MMP-2 was via RGD-dependent and RGD-independent mechanisms, both of which, however, were completely blocked by HKa. Further, we found that HKa inhibited the association of MMP-2 with immunoprecipitated avb3 integrin, and prevented MMP-2 localization in caveolae in EPCs. Since HKa inhibited VEGF-stimulated proliferation of EPCs but not HUVECs on collagen-coated surface, we postulate that EPCs are more sensitive than mature ECs to HKa inhibition. Thus, HKa targets EPCs and inhibits their differentiation via blocking avb3 integrin and MMP-2 interaction. These observations provide novel insight into understanding the interactions between the kallikrein-kinin system and biologic functions of EPCs.
A Chemical Genetic Screen Identified Small Molecules Modulating Zebrafish Vascular Development and Angiogenesis.
Authors: Xue Rui , Keating Armand , Wang Youdong , Stewart Duncan , Wen Xiao-Yan
Published: April 2020

The zebrafish has emerged as an important model for studying vascular development and angiogenesis. Advantages over other models include rapid embryonic development and optical clarity of the embryos. Abnormal angiogenesis is linked to over 70 health conditions and inhibition of angiogenesis is an excellent target for cancer therapy (including hematopoietic malignancies) as tumor growth requires new blood vessels. In this study, we performed a chemical genetic screen in developing zebrafish embryos to identify compounds that modulate zebrafish vascular development and angiogenesis. A zebrafish transgenic line with the Flk1 promoter controlling the GFP (green fluorescent protein) reporter was used. The entire vascular network of the Flk1:GFP fish was marked by GFP and could be visualized under fluorescence microscopy. Screens were performed in 96 well plates with three embryos/well. The outer membranes of healthy zebrafish embryos expressing green fluorescent vasculature were removed by enzymatic digestion with protease at the 16–18 somite stages. Small molecules/compounds were then added to the water at concentrations of 0.1 μM, 1 μM, and 5 μM for 72 hrs. After 24, 48 and 72 hrs of exposure to the compound, the embryos were visually inspected for viability, gross morphological defects, heartbeating rate and circulation. The known angiogenic inhibitor, PD173074 was used as control. An initial screening of 780 compounds in a small molecule compound library identified four small molecules with potent activities in inhibiting zebrafish vascular development. Notably, 1–5 μM of Hit #1 inhibited the growth of cranial vessels and disrupted vascular patterning, which resulted in uneven spacing between intersegmental vessels. While Hit #2 and #5 also inhibited the growth of cranial vessels, the vascular patterning remained unaffected, however, the drug-treated embryos had weak or missing intersegmental vessels. Fish embryos treated by Hit #3 had an enlarged heart and thinner vessels. Interestingly, this screen also identified one compound (Hit #4) with pro-angiogenic activity. Embryos treated with Hit #4 had increased numbers of intersegmental vessels. Hit #2 is a known inhibitor of c-Jun N-terminal kinase and angiogenesis (SP600125), was recently reported to inhibit the proliferation and migration of human endothelial cells in vitro as well as inhibit solid tumor growth in mice. This observation lends validity to the zebrafish screen and the potential utility of the other hits. Experiments are currently in progress to investigate the detailed time course of the angiogenic inhibition and the potential molecular mechanism. Studies of these angiogenic inhibitors may lead to the development of potent anti-cancer drugs while the pro-angiogenic compound may prove useful in facilitating tissue/organ regeneration. We conclude that the zebrafish model is likely to yield valuable information regarding vasculogenesis and its manipulation.
The Interaction of Platelet GPIB with the VWF-A1 Domain in Solution or Immobilized Is Regulated by the D3 Domain (A.A. 1238–1260) and the A2A3 Domains
Authors: Cruz Miguel A. , Martin Cecilia , Wu Tao , Zu Cheng
Published: April 2020

We have used recombinant von Willebrand factor (VWF) fragments to investigate the influence of the flanking regions of the A1 domain on the interaction with platelet glycoprotein (GP)Ibα. One fragment, the r1261–1874, represented mainly the A1A2A3 domains and the other, r1238–1874, included the C-terminal region of the D3 domain. When immobilized onto a surface, the two fragments tethered platelets similarly at high shear rates. The two fragments bound identically to immobilized fixed platelets in a ristocetin-dependent and saturable manner. In the absence of ristocetin, the r1261–1874 had a 50% of its GPIb-binding activity with the modulator while the r1238–1874 only had < 5% binding activity. The r1261–1874 fragment effectively inhibited both ristocetin-induced platelet agglutination and shear-induced platelet aggregation. In contrast, the r1238–1874 failed to show an inhibitory capacity in the two assays, indicating a limited interaction with platelet GPIbα in solution. We also analyzed the single bond lifetimes in a range of constant forces of GPIbα with either the single A1 domain (a.a. 1238–1471) or the A1A2A3 fragment using atomic force microscopy (AFM). When the protein was directly adsorbed on a Petri dish, the GPIbα bound with longer lifetimes to the A1A2A3 than the isolated A1 domain, suggesting that a conformational change in the A1 is influenced by the A2A3 domains. In conclusion, the C-terminal region of D3 domain (a.a.1238–1260) and the A2A3 domains negatively regulate the binding of the A1 domain to platelet GPIbα in suspension. On the other hand, the A2A3 domains may have a positive influence on the bond formed between the GPIbα and a surface-bound A1 domain.
Delta Like 4 Expressing Bone Marrow-Derived Endothelial Progenitor Cells Regulate Tumour Angiogenesis
Authors: Real Carla , Caiado Francisco , Igreja Catia , Elias Ana P. , Borges Cristina , Duarte Antonio , Dias Sergio
Published: April 2020

Bone marrow-derived endothelial progenitor cells (BM-EPCs) have been implicated in adult neoangiogenesis and consequently used as therapies for human pathologies with endothelial damage. The administration of these cells in human patients temporally improves endothelial function, although the engraftment of these cells in newly formed vessels is inefficient. Conversely, therapeutic stratagies to block EPC contribution during tumor angiogenesis have been proposed. In this work, we analysed the role of the Notch/Delta signalling pathway in EPC function during tumour neoangiogenesis, by regulating the expression of Notch ligand, delta-like 4 (Dll4) in these cells. Sublethally irradiated NOD-SCID mice received WT, Dll4+/− (Dll4 heterozygous mice) or Dll4 SiRNA-treated BM-EPCs and were subcutaneously inoculated with well established Human or murine tumor xenografts. Tumours growing in Dll4-depleted EPCs transplanted mice presented increased microvessel density when compared with WT EPCs transplanted mice or non-transplanted controls, regardless of VEGF expression. Although with increased vessel number, tumours of Dll4+/− EPC transplanted mice presented increased hypoxia and decreased tumour cell proliferation, suggesting an impairment in vessel function. In addition, these tumours present a diminished expression of PDGF, a vessel stabilizing factor, and increased expression of Ang2, known as a vessel destabilizing factor. We next verified whether the vessel destabilization observed in tumors after Dll4-depleted EPCs transplant might be due to a diferential endothelial differentiation or incorporation of EPCs in the tumour vasculature. In order to answer this question we quantified the incorporation of WT and Dll4-depleted EPCs in tumour vessels. Accordingly to our results, the presence of Dll4-depleted EPCs was reduced compared to WT EPCs, suggesting that Dll4-depleted EPCs might have reduced capacity to adhere to the renewing tumor vasculature, or to the underlying basement membrane. To test this, we used an in vitro endothelial differentiation assay, and observed a defect on the adhesion of of Dll4-depleted EPCs to extracellular matrix, which was correlated with a reduced expression of integrin subunits a3 and b1. These results suggest that the reduction of Dll4 on EPCs reduces integrin expression interfering with their ability to adhere, incorporate and stabilize the tumor vasculature during tumor neoangiogenesis. Therefore, EPCs have a major role in vessel stabilization in active neoangiogenic sites by the regulation of Dll4 expression. We propose that targeting the Notch/Dll4 pathway on EPCs, modulating vessel stability, may have therapeutic potential.
Gamma-Retroviral and Lentiviral Vectors Have Different Preferences for Specific Classes of Genes and Specific Genomic Features in Human Hematopoietic Cells
Authors: Recchia Alessandra , Testa Anna , Cittaro Davide , Felice Barbara , Cattoglio Claudia , Luzi Lucilla , Mavilio Fulvio
Published: April 2020

Integration of gamma-retroviral (RV) and lentiviral (LV) vectors follows non-random patterns in mammalian genomes, with a common preference for active chromatin regions. The molecular basis of the interaction between retroviral pre-integration complexes and the human genome is poorly understood, particularly in the case of gamma-retroviruses. We have mapped a large number of RV and LV integrations in human CD34+ hematopoietic cells, transduced in vitro and analyzed without selection. Recurrent insertion sites (hot spots) account for >20% of the RV integration events, while they are significantly less frequent in the case of LV vectors. RV but not LV hot spots are highly enriched in proto-oncogenes, cancer-associated CIS, and growth-controlling genes. Genes involved in hematopoietic and immune system development are targeted at high frequency and enriched in hot spots, suggesting that the CD34+ gene expression program is instrumental in directing RV integration. To obtain information about the genetic determinants of retroviral integration preferences, we investigated the role of transcriptional regulatory element within the vector as well as the chromatin-specific determinants of the target sites in directing RV and LV integration. Comparative analysis of the “integromes” of vectors carrying different transcriptional regulatory elements provide information on the role of transcriptional complexes bound to genomic and viral sequences in targeting retroviral pre-integration complexes (PICs). Analysis of the chromatin characteristics around integration sites provides information about specific structures, protein components or epigenetic modifications that may favor the binding of retroviral PICs. Genomic sequences flanking the integration sites (±1 kb) were annotated and analyzed using TRANSFAC and JASPAR matrix database and UCSC track. This analysis shows that the content and arrangement of putative transcription factor binding sites around the insertion points is different between RV and LV vectors, and between vectors carrying different LTR modifications. The frequency, coverage and position of the major class of repeats in our datasets were also analyzed. Hyper Geometric Distribution analysis revealed a significant abundance of SINE and LINE repeats around the RV vector integration sites. These data indicate that vector design and the target cell gene expression program have a significant impact in determining the integration characteristics of retroviral vectors.
Quantifying Capsid Peptide:MHC I Complexes Following Adeno-Associated Virus (AAV) Transduction
Authors: Pien Gary C. , Hasbrouck Nicole C. , Maus Marcela V. , Mingozzi Federico , High Katherine A.
Published: April 2020

Recent data from a clinical trial of adeno-associated virus (AAV) 2-mediated hepatic gene transfer of factor IX into hemophilia B subjects demonstrated the presence of AAV2-capsid-specific CD8+ T cells and identified an immunodominant AAV2 epitope. The expansion and contraction of this CD8+ T cell population coincided with the rise and fall of an asymptomatic elevation in liver transaminases, and correlated with the expression and subsequent loss of factor IX expression. These observations suggest that CD8+ T cells directed against AAV capsid antigen clear the transduced hepatocytes. Indeed, others in our laboratory have directly shown the lysis of AAV-transduced hepatocytes by CD8+ T cells in vitro. However, the stoichiometry and kinetics of AAV capsid degradation and processing for MHC class I presentation are presently unknown. We now report the generation of soluble AAV capsid-specific T cell receptor (TCR) tetramers and their use in quantifying capsid peptide:MHC I complexes presented on cell surfaces. CD8+ T cell clones specific for AAV2-derived peptide 74 (VPQYGYLTL) presented in the context of HLA-B*0702 were isolated and used to generate TCR cDNA. This cDNA was transfected into CHO cells to produce soluble TCR α and β chain fusion proteins. The soluble TCR was biotinylated, then tetramerized to streptavidin-conjugated fluorochrome for use in flow cytometric analyses. Cell lines expressing HLA-B*0702 were loaded with cognate AAV2 peptide, a cross-reactive AAV1 peptide (IPQYGYLTL), or irrelevant HLA-B*0702 epitopes derived from HIV-1 gp120 (IPRRIRGGL) or EBV nuclear antigen (RPPIFIRRL). Staining with TCR-tetramers reveals that this reagent binds AAV-derived peptides specifically. In addition, this reagent exhibits HLA-specificity, as cognate peptide loaded onto cells which express alternate HLA alleles was not detected. These results demonstrate that TCR-tetramers retain their antigen and MHC context specificities. Capsid peptide titration experiments using HLA-B*0702-bearing JYA2B7 and SK-MES-1 cell lines reveal that TCR-tetramers exhibit a limit of detection of approximately 105 cell surface peptide:MHC I complexes. Efficiency of detection is estimated at 1 peptide:MHC I complex for every 1010 peptide molecules added to cell culture. Preliminary studies indicate that the number of presenting complexes wanes by 48 hours following peptide loading. In contrast, AAV1-mediated transduction experiments demonstrate that capsid peptide:MHC I complexes are detectable after 48 hrs. In conclusion, these results are in agreement with our central hypothesis that AAV capsid is degraded upon transduction and gains access to MHC Class I antigen presentation pathways. Studies with the TCR-tetramers will allow definition of kinetics and capsid dose-dependence of antigen presentation, defining parameters of cell vulnerability to host immune-mediated clearance of transduced cells.
GABP Transcription Factor Is Required for Myeloid Cell Development In Vivo.
Authors: Yang Zhong-fa , Drumea Karen , Rosmarin Alan G.
Published: April 2020

GABP is an ets transcription factor that regulates genes that are required for innate immunity, including CD18 (β2 leukocyte integrin), lysozyme, and neutrophil elastase. GABP consists of two distinct and unrelated proteins. GABPα binds to DNA through its ets domain and recruits GABPβ, which contains the transactivation domain; together, they form a functional tetrameric transcription factor complex. We recently showed that GABP is required for entry into S phase of the cell cycle through its regulation of genes that are required for DNA synthesis and cyclin dependent kinase inhibitors (Yang, et al. Nature Cell Biol9:339, 2007). Furthermore, GABP is an essential component of a retinoic acid responsive myeloid enhanceosome (Resendes and Rosmarin Mol Cell Biol26:3060, 2006). We cloned Gabpa (the gene that encodes mouse Gabpα) from a mouse genomic BAC library and prepared a targeting vector in which the ets domain is flanked by loxP recombination sites (floxed allele). Deletion of both floxed Gabpa alleles causes an early embryonic lethal defect. In order to define the role of Gabpα in myelopoiesis, we bred floxed Gabpa mice to mice that bear the Mx1-Cre transgene, which drives expression of Cre recombinase in response to injection of the synthetic polynucleotide, poly I-C. Deletion of Gabpa dramatically reduced granulocytes and monocytes in the peripheral blood, spleen, and bone marrow, but myeloid cells recovered within weeks. In vitro colony forming assays indicated that myeloid cells in these mice were derived only from Gabpa replete myeloid precursors (that failed to delete both Gabpa alleles), suggesting strong pressure to retain Gabpα in vivo. We used a novel competitive bone marrow transplantation approach to determine if Gabp is required for myeloid cell development in vivo. Sub-lethally irradiated wild-type recipient mice bearing leukocyte marker CD45.1 received equal proportions of bone marrow from wild type CD45.1 donor mice and floxed-Mx1-Cre donor mice that bear CD45.2. Both the CD45.2 (floxed-Mx1-Cre) and CD45.1 (wild type) bone marrow engrafted well. Mice were then injected with pI-pC to induce Cre-mediated deletion of floxed Gabpa. The mature myeloid and T cell compartments were derived almost entirely from wild type CD45.1 cells. This indicates that the proliferation and/or differentiation of myeloid and T cell lineages requires Gabp. In contrast, B cell development was not impaired. We conclude that Gabpa disruption causes a striking loss of myeloid cells in vivo and corroborates prior in vitro data that GABP plays a crucial role in proliferation of myeloid progenitor cells.
Iron Absorption: Comparison of Oral Heme-Bound Iron with Inorganic Ferrous Sulfate.
Authors: Estrada Dexter T. , Schwartz Kenneth A.
Published: April 2020

Oral iron supplementation is an effective method for treating iron deficiency anemia. However, current preparations of inorganic iron have significant gastrointestinal adverse effects, that sometimes prevent patients from complying with their prescribed iron. Gut absorption of heme bound iron is via a different pathway than inorganic iron. Heme-bound iron may also have fewer gastrointestinal side effects as it is present in red meat and other specially prepared foods. If it can be shown that oral heme-bound iron is absorbed as readily as oral inorganic iron, then patients who cannot tolerate inorganic iron would have a less expensive alternative to parenteral iron which is costly and can cause life-threatening risk of anaphylaxis. The purpose of this study was to determine if there is a significant difference between the oral absorption of heme-bound iron, in the form of cooked bovine blood soup, and inorganic ferrous sulfate. Ten normal adult subjects were recruited for participation in this study and informed consents were obtained. All subjects had normal baseline fasting serum iron studies and ferritin. After blood for a fasting serum iron was obtained, subjects ingested a cooked bovine blood soup, containing 18 mg of elemental heme iron. Repeat iron studies were taken 2 hours after ingestion of the heme iron preparation. After a 2 week washout period, the same 10 subjects had baseline serum iron drawn followed by ingestion of 18 mg of elemental iron in the form of inorganic ferrous sulfate elixir. Serum iron was again measured 2 hours after ingestion of ferrous sulfate. The mean increase in serum iron after ingestion of bovine blood preparation was 18.5 micrograms/dL. In comparison, the mean increase in serum iron after ingestion of ferrous sulfate was 37.9 micrograms/dL. A paired T test analysis did not show any significant difference in the increase of serum iron from baseline to 2 hours between the two iron preparations (P>0.1112). No adverse effects were reported by any of the subjects after ingestion of either preparation. In conclusion, heme iron ingested in the form of a cooked bovine blood soup is well tolerated, produces increases in the concentrations of serum iron that are comparable to those produced by ferrous sulfate, and supports the principle that reasonable amounts of iron can be absorbed from heme.
Prevalence and Trend of Hematopoietic Stem Cell Transplantation for Adult Aplastic Anemia/Pure Red Cell Aplasia in Korea.
Authors: Kim Hawk , Baek Jin Ho , Shin Su Jin , Min Young Joo , Park Jae-Hoo , Lee Je-Hwan , Lee Kyoo-Hyung
Published: April 2020

Aplastic anemia (AA) and pure red cell aplasia (PRCA) seems more frequent in Asian countries including Korea. However, there is no exact data regarding its prevalence and frequency of allogeneic hematopoietic stem cell transplantation (HSCT) in Korea. Here we present the demographic data of AA/PRCA in Korea. Data were prepared by retrieving computerized database of National Health Insurance Corporation and Korea National Statistical Office. HSCT data were collected from all HSCT centers in Korea through Korea Hematopoietic Stem Cell Transplantation Nurses’ Society. The selection criteria of PRCA and AA were D60 and D61 in ICD-10 code. Study years for crude incidence/prevalence of AA/PRCA and trend of HSCT for AA/PRCA were from 2000 to 2005, and from 1997 to 2006, respectively. All patients were 10 or more years old. In terms of prevalence, PRCA was decreasing annually from 2000 to 2005 with 0.97, 1.12, 0.72, 0.61, 0.67, and 0.47 per 100,000 persons, respectively. On the contrary, the prevalence of AA was almost same from 2000 to 2005 with 11.28, 12.93, 12.36, 11.07, 11.09, and 10.54 per 100,000 persons, respectively. Female was more affected to both AA and PRCA. The average female to male ratios in AA and PRCA were 1.38 and 1.82. The peak decades of AA were fourth and fifth decades. On the other hand, the peak decades of PRCA were second and sixth decades. The frequency of HSCT for AA/PRCA was increasing in spite of the decreasing crude incidence during a decade from 1997 to 2006, in that 48, 56, 78, 88, 69, 68, 76, 85, 86, and 92, respectively. The frequency of HSCT was similar in both genders. In spite of the possibility of over-estimation, the prevalence of AA and PRCA was very higher in Korea than in western countries.
The Comparison of the Cytobiological Role of Bone Marrow CD4+ T Cells in Aplastic Anemia Patients and Healthy Subjects.
Authors: Zheng Miao , Liu Wenli , Zhou Jianfeng , Sun Hanying , Zhang Yicheng
Published: April 2020

Recent studies indicate that immune-associated aplastic anemia (AA) resembles such autoimmune diseases as insulin-dependent diabetes and chronic autoimmune thyroiditis that belong to organ-specific autoimmune diseases. Many independent investigation groups have successfully isolated the pathopoiesis-associated T cell clone causing hematopoiesis failure with a CD4 phenotype from AA patients peripheral blood and bone marrow (BM). In the current study, BM CD4+T cells were isolated from both AA patients and healthy subjects with immunomagnetic beads sorting and were compared with regard to proliferation capability, apoptosis features and the impacts of their secreted cytokines on hematopoiesis stem/progenitor cells. With the improved MTT method, BM CD4+T cells of AA group presented 1.6 times more enhanced reproductive activity than those of normal group. Induced by high concentrational CD3 monoclonal antibody for 18h, evident apoptosis cells could be seen under the electron microscope in both normal group and AA group. Quantified by flow cytometry using Annexin-V FITC/PI dual staining method, apoptosis rates in the early and advanced stages of AA group were more than those of normal group (P<0.01). Apoptosis rate in early stage: normal group(7.03±0.86)%, AA group(16.11±1.37)%; apoptosis rate in advanced stage: normal group(2.07±0.42)%, AA group(8.05±0.36)%. The influence of BM CD4+T cell culture supernatant on CFU-GM formation of cord blood CD34+ hematopoiesis stem/progenitor cells: the CFU-GM count in AA group was: (74.50±9.50)/104 cells; the CFU-GM count in normal group was: (124.25±19.80)/104 cells, observing under an inverted microscope. A significant difference was present in the comparison between the two groups (P<0.01). CyclinD3 mRNA and protein expression levels of cord blood CD34+ cells were both down-regulated induced by BM CD4+ T cell culture supernatant of AA patients. These results indicate a strong likelihood of an abnormally proliferative and activated state of BM CD4+T cells among AA patients and its intimate correlation with AA hematopoiesis failure by secreting some soluble humoral agents that are able to inhibit CyclinD3 and consequently suppress hematopoietic stem cell in proliferation.
Ectopic Expression of HDAC9 in Murine Lymphoid System Leads to Altered Lymphocyte Numbers and Proliferation as Well as Predisposition to Tumorigenesis.
Authors: Gil Veronica S. , Howell Louise M.C. , Yeung Jenny , Petrie Kevin R. , Smith Adrian , Sebire Neil , Matutes Estela , Wotherspoon Andy , Enver Tariq , So Eric , Zelent Arthur
Published: April 2020

Reversible acetylation of lysine residues on histone tails is associated with changes to chromatin structure and plays a key role in regulation of gene expression. In this process, histone hypoacetylation is generally associated with gene silencing and pharmacological inhibition of histone deacetylases (HDACs) leads usually to activation of gene expression. Decreased histone acetylation is a hallmark of cancer cells and increased HDAC expression or their mistargetting to specific gene promoters has been associated with a variety of tumors. In the past we have identified and cloned class IIa HDAC9. The HDAC9 gene is located in chromosome 7p21, which is frequently amplified in B-cell tumours such as mantle cell lymphoma (MCL) and in B-cell non-Hodgkin’s lymphoma cell lines. Consistently, initial analysis of patient samples and/or publicly available microarray data highlighted high levels of HDAC9 expression in chronic lymphocytic leukemia, folicullar lymphoma and MCL. Within the normal lymphoid system, HDAC9 is co-expressed with BCL-6 in germinal center B-cells (∼60% of cells). HDAC9 is also expressed in marginal zone B cells and a fraction of CD38 or CD27 positive subepithelial tonsilar cells. In order to examine the role of HDAC9 in the lymphoid development and pathogenesis of lymphoid malignancies we used Ig heavy chain enhancer (Eμ), which drives gene expression from early stages of B-cell development, to ectopically express HDAC9 in transgenic mice. Hemizygous and homozygous mice expressing Flag epitope tagged human HDAC9 (fHDAC9) transgene display throughout their lifespan altered B-cell development. Immunophenotypic analysis of B-cells isolated from bone marrow (BM) revealed an absence of cells expressing the pre-B/immature-B cell markers normally associated with C-E Hardy’s fractions. In vitro functional clonogenic assays for IL-7 responsive BM-derived B-cell progenitors demonstrated an increase (∼50%) in colony numbers in the transgenic BM. Moreover, morphologic and flow cytometric analyses of the transgenic colonies, but not those derived from normal BM, revealed the presence of granulocyte/macrophage colony forming units expressing the HDAC9 transgene, suggesting a lympho-myeloid lineage switch. This correlates with the finding that extramedullary myelopoiesis occurs in a fraction of mice presenting splenomegaly (44%). Furthermore, a subgroup of homozygous Eμ-fHDAC9 mice (n=16) developed tumours (81%) at middle age, and present with enlarged lymph nodes (6%) and abnormal hematopoietic elements in peripheral blood and BM. Taken together these data suggest that HDAC9 plays a role in B-cell maturation and its ectopic expression in early B-cells leads to perturbation of normal B-cell development, possibly predisposing transgenic mice to tumorigenesis.