scholarly journals Adamts13-Cultured Red Blood Cells to Treat Thrombotic Thrombocytopenic Purpura

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 89-89
Author(s):  
Kai Wang ◽  
Khulan Batbayar ◽  
Karl Roberts ◽  
Emmanuel Olivier ◽  
Eric E. Bouhassira
Keyword(s):  
Red Blood Cells ◽  
Thrombotic Thrombocytopenic Purpura ◽  
Blood Cells ◽  
Vascular System ◽  
Conflicts Of Interest ◽  
Direct Injection ◽  
Culture Method ◽  
Thrombocytopenic Purpura ◽  
Membrane Bound

Red Blood Cells (RBCs) have long been considered a potentially useful means of delivering drugs to the circulation because delivery through therapeutic RBCs as compared to direct injection in the plasma can lengthen the half-life of the therapeutic agent in the circulation, spatially restrict the drugs to the lumen of the cardio-vascular system, and shield the drug from the immune system. Despite some progress, loading the cells with therapeutically useful cargo remains technically challenging. We have recently developed PSC-RED, a chemically-defined scalable method to differentiate induced pluripotent stem cells (iPSCs) into unlimited numbers of enucleated cultured RBCs. This provides an ideal method to produce therapeutic RBCs since iPSCs can be genetically manipulated with powerful CRISPR-based technologies. ADAMTS13, whose deficiency is responsible for congenital and acquire Thrombotic Thrombocytopenic Purpura (TTP) is a good target as a therapeutic that could be delivered through drug-carrying RBCs because large amounts of plasma concentrate, or more recently recombinant proteins, are necessary to treat TTP. We report here we have produced engineered erythroid cells that contains globin-LCR driven ADAMTS13 fusion transgenes inserted at safe harbor AAVS1, and that these cells express a membrane bound version of an inhibitor-resistant version of ADAMTS13. We show using flow cytometry that the fusion protein is express at high levels, and using a FRET assay that detect cleavage of the von Willebrand cognate site, that the membrane bound ADAMTS13 is enzymatically active. Comparison of enzymatic activity with plasma concentrate suggests that about 50 billion engineered ADAMTS13-cRBCs would be sufficient to deliver an amount of ADAMTS13 equivalent to 2 liters of plasma concentrate. This suggests that a transfusion of about 10 mL of ADAMTS13-RBCs could be therapeutic for congenital and acquired TTP. The number of cRBCs necessary to treat even a few patients is very large. This has been considered a major obstacle to the development of treatment based on in vitro produced RBCs because of the volume of culture that is necessary to produce the cells. We also report that we have developed a culture method based on holo-fiber bioreactors that allows the production of cRBCs at a density of 5.108 cell/mL which is sufficient to produce enough cells to performed small clinical trials. Disclosures No relevant conflicts of interest to declare.

scholarly journals Thrombotic Thrombocytopenic Purpura and Sickle Cell Crisis

2008 ◽  
Vol 16 (2) ◽  
pp. 224-227 ◽  
Author(s):  
Suresh G. Shelat
Keyword(s):  
Red Blood Cells ◽  
Thrombotic Thrombocytopenic Purpura ◽  
Sickle Cell ◽  
Blood Cells ◽  
Signs And Symptoms ◽  
Activity Level ◽  
Acute Chest Syndrome ◽  
Thrombocytopenic Purpura ◽  
Nucleated Red Blood Cells ◽  
Elevated Lactate

Described is a case of acute chest syndrome in a sickle-cell patient (hemoglobin SS) who also developed signs and symptoms of thrombotic thrombocytopenic purpura, including thrombocytopenia and hemolysis (anemia, elevated lactate dehydrogenase, presence of schistocytes, dark-colored plasma, and elevations in nucleated red blood cells). The ADAMTS13 activity level was normal. Discussed are the diagnosis and therapeutic management issues and the challenges of differentiating the vasoocclusive and hemolytic complications of sickling red blood cells from the thrombotic microangiopathy of thrombotic thrombocytopenic purpura.

Analyzing the Stepwise Developmental Pathway From ES/IPS Cells to Functional Mature Erythrocytes.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2534-2534
Author(s):  
Akira Niwa ◽  
Tomoki Fukatsu ◽  
Katsutsugu Umeda ◽  
Itaru Kato ◽  
Hiromi Sakai ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Specific Antigen ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Oxygen Carriers ◽  
Developmental Pathway ◽  
Ips Cell

Abstract Abstract 2534 Poster Board II-511 Induced pluripotent stem (iPS) cells, reprogrammed somatic cells with embryonic stem (ES) cell–like characteristics, are generated by the introduction of combinations of specific transcription factors. Despite the controversy surrounding the gene manipulation, it is expected that iPS cells should contribute to regenerative medicine, disease investigation, drug screening, toxicology, and drug development in future. In the fields of hematology, iPS cells could become used as a new feasible source for transplantation therapy without immunological barrier and for the investigation of various kinds of hematological defects. Previous studies on ES / iPS cells have already demonstrated that they can develop into various lineages of hematopoietic cells including erythrocytes following the similar processes occurred in embryo and fetus. However, it is important to establish the more effective system for developing functional blood cells. Here we present the methods for selectively inducing mature red blood cells from ES / iPS cells in vitro, and show the functional equality of them to natural blood cells. First, Flk1+ mesodermal progenitors were derived from ES / iPS cells on OP9 stromal cells at an efficacy of more than 50% and collected by fluorescence activated cell sorter. Then, those sorted cells were cultured in the presence of exogenous erythropoietin and stem cell factor. They highly selectively developed into erythroid lineages including enucleated red blood cells. Sequential FACS analysis using the antibodies against transferrin receptor CD71 and erythroid specific antigen Ter119 in combination with DNA staining dye Hoechst 33342 demonstrated that ES / iPS cell-derived erythropoiesis in our system follow the normal erythroid developmental pathway occurred in vivo. RT-PCR and Western blot analyses proved the expression of heme biosynthesis enzymes on the produced erythrocytes. Finally, the oxygen dissociation curve showed that ES / iPS cell-derived erythroid cells are functionally virtually equivalent to natural red blood cells as oxygen carriers. Taken together, our system can present the effective methods of investigating the mechanisms of normal erythropoiesis and the deficits in syndromes with disrupted red blood cell production. Disclosures: No relevant conflicts of interest to declare.

Identification of secreted proteins that can promote erythroid progenitor expansion

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4864-4864
Author(s):  
Bing Xu ◽  
Xiangmeng Wang ◽  
Peng Li ◽  
Yiren Xiao ◽  
Huijuan Dong ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Secreted Proteins ◽  
Erythroid Progenitor ◽  
In Vitro Expansion ◽  
Stromal Cell Line ◽  
Expansion System

Abstract Erythroid blasts are progenitors that can differentiate to mature red blood cells (RBC). Identification of these growth factors and understanding their downstream pathways can help us to optimize RBC production ex vivo. We derived a stromal cell line (PL16) from mouse embryonic fetal livers that promotes proliferation of mouse and human erythroid blasts in vitro. Among specifically highly expressed secreted proteins in PL16, we identified a new growth factor (FA) that is capable of stimulating human erythroid blast expansion in vitro significantly. Furthermore, erythroid blasts from our in vitro expansion system can differentiate into functional mature red blood cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Thrombotic Thrombocytopenic Purpura in Childhood

Blood ◽  
1962 ◽  
Vol 19 (2) ◽  
pp. 181-199 ◽  
Author(s):  
JAMES B. MACWHINNEY ◽  
JAMES T. PACKER ◽  
GERALD MILLER ◽  
ROBERT M. GREENDYKE
Keyword(s):  
Red Blood Cells ◽  
Thrombotic Thrombocytopenic Purpura ◽  
Clinical Features ◽  
Blood Cells ◽  
Patient Case ◽  
Thrombocytopenic Purpura ◽  
Renal Vessels ◽  
Kidney Involvement ◽  
Histologic Changes

Abstract (1) Two cases of thrombotic thrombocytopenic purpura (TTP) occurring in childhood are described. Case 1 is unique in that the patient survived for 12 years. (2) The clinical features of 19 reported cases of TTP in children are reviewed. (3) The presence of morphologic abnormalities of red blood cells and the regular occurrence of kidney involvement in this disorder is emphasized. In one patient (Case 2), histologic changes of the disease were limited to the kidney. (4) Certain hematologic and histologic features shared by TTP and eclampsia are described. (5) Unusual histologic lesions of renal vessels are described.

scholarly journals An Essential Role for the RNA Editor-Exonuclease Axis in Terminal Erythroid Differentiation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 3-3
Author(s):  
Areum Han ◽  
Alena V. Yermalovich ◽  
Vanessa Lundin ◽  
Daniel S. Pearson ◽  
Mariam Hachimi ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Mammalian Cells ◽  
Conflicts Of Interest ◽  
Human Peripheral Blood ◽  
Rna Degradation ◽  
Erythropoietin Receptor ◽  
Conditional Knockout ◽  
Erythroid Progenitors

Erythropoiesis is an intricate process by which lineage-committed erythroid progenitors become mature red blood cells. Reticulocytes are terminal-staged, immature red blood cells with residual RNA after enucleation. In the absence of pathology, reticulocytes are efficiently processed into mature red blood cells and typically represent a small percentage of cells in human peripheral blood. In contrast, when differentiated in vitro from pluripotent stem cells or CD34+ progenitor cells, red cells tend to arrest at the reticulocyte stage. Recent studies have highlighted that uridylation by Terminal Uridylyl Transferases (TUTases) occurs on a broad spectrum of RNA classes in mammalian cells. Oligo-uridylated RNA is recognized by exoribonucleases and targeted for decay. We posited that the machinery behind RNA degradation that accompanies terminal erythropoiesis might involve RNA tail editors coupled to exonuclease activity. Utilizing constitutional murine knockout models, we observed that blood from the TUTase Zcchc6 RNA editor knockout embryos exhibited reticulocytosis and a terminal maturation defect, as documented by FACS, histology, and hematological profiling. Murine strains deficient in the downstream exonuclease Dis3l2 phenocopied the RNA decay defect of the Zcchc6 KO. Conditional knockout murine models of the TUTase-Dis3l2 axis driven by the red cell specific Erythropoietin Receptor-Cre exhibited comparable phenotypes, suggesting a cell intrinsic and niche-independent role for the TUTase-Dis3l2 axis in promoting red blood cell maturation. We are modulating the expression of this axis by various methods to optimize modeling of hemoglobinopathies such as sickle cell anemia. Disclosures No relevant conflicts of interest to declare.

Ultrastructural observations on the in vitro cytoadherence of Plasmodium falciparum infected red blood cells

1990 ◽  
Vol 48 (3) ◽  
pp. 914-915
Author(s):  
D.J.P. Ferguson ◽  
A.R. Berendt ◽  
J. Tansey ◽  
K. Marsh ◽  
C.I. Newbold
Keyword(s):  
Plasmodium Falciparum ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Umbilical Vein ◽  
Cell Types ◽  
Amelanotic Melanoma ◽  
Cytoplasmic Process ◽  
Umbilical Vein Endothelial Cells

In human malaria, the most serious clinical manifestation is cerebral malaria (CM) due to infection with Plasmodium falciparum. The pathology of CM is thought to relate to the fact that red blood cells containing mature forms of the parasite (PRBC) cytoadhere or sequester to post capillary venules of various tissues including the brain. This in vivo phenomenon has been studied in vitro by examining the cytoadherence of PRBCs to various cell types and purified proteins. To date, three Ijiost receptor molecules have been identified; CD36, ICAM-1 and thrombospondin. The specific changes in the PRBC membrane which mediate cytoadherence are less well understood, but they include the sub-membranous deposition of electron-dense material resulting in surface deformations called knobs. Knobs were thought to be essential for cytoadherence, lput recent work has shown that certain knob-negative (K-) lines can cytoadhere. In the present study, we have used electron microscopy to re-examine the interactions between K+ PRBCs and both C32 amelanotic melanoma cells and human umbilical vein endothelial cells (HUVEC).We confirm previous data demonstrating that C32 cells possess numerous microvilli which adhere to the PRBC, mainly via the knobs (Fig. 1). In contrast, the HUVEC were relatively smooth and the PRBCs appeared partially flattened onto the cell surface (Fig. 2). Furthermore, many of the PRBCs exhibited an invagination of the limiting membrane in the attachment zone, often containing a cytoplasmic process from the endothelial cell (Fig. 2).

Effect of liposomal curcumin on red blood cells in vitro

2013 ◽  
Vol 1 (Suppl. 1) ◽  
pp. A4.1
Author(s):  
Angela Storka
Keyword(s):  
Red Blood Cells ◽  
Blood Cells

scholarly journals Calpain activity in patients with thrombotic thrombocytopenic purpura is associated with platelet microparticles

Blood ◽  
1992 ◽  
Vol 80 (9) ◽  
pp. 2246-2251 ◽  
Author(s):  
JG Kelton ◽  
TE Warkentin ◽  
CP Hayward ◽  
WG Murphy ◽  
JC Moore
Keyword(s):  
Thrombotic Thrombocytopenic Purpura ◽  
Membrane Association ◽  
Membrane Glycoproteins ◽  
Calpain Activity ◽  
Thrombocytopenic Purpura ◽  
Calcium Dependent ◽  
Active Protease ◽  
Triton X ◽  
Platelet Microparticles

Abstract Thrombotic thrombocytopenic purpura (TTP) is characterized by thrombocytopenia and disseminated platelet thrombi throughout the microvasculature. Studies by our group have demonstrated calcium- dependent proteolytic activity (calpain) that is no longer detectable in the serum of patients with acute TTP after their recovery. The purpose of this study was to investigate if the protease activity of TTP was detectable in plasma and, therefore, not an in vitro phenomenon secondary to the formation of serum. Additionally, we looked for evidence of membrane association of the active protease in the patients' samples, which would explain the persistence of its activity in the presence of plasma inhibitors. Acute TTP samples, both serum and plasma, were collected from 10 patients with TTP. Calpain was measured using bioassays for enzyme activity and also by detection of the protein using immunoblotting with an anticalpain monoclonal antibody (MoAb). In all instances, calpain could be detected both functionally and antigenically in the acute TTP sera and plasma. No calpain activity could be detected in any of the controls, although antigenic calpain was detectable in one sample from a patient who had undergone cardiopulmonary bypass surgery. To investigate whether the calpain was associated with microparticles in the plasma, the TTP plasma samples were ultrafiltered and ultracentrifuged. Activity was not lost by passage across a 0.2-micron filter but was detectable only in the pellet following ultracentrifugation. Membrane association of the calpain in the microparticles also was demonstrated using solubilization with Triton X-100. Immunoprecipitation studies demonstrated that the calpain activity could be removed by MoAbs against platelet membrane glycoproteins (IX and IIb/IIa) but not by a MoAb against red blood cell membrane glycophorin. These studies indicate that active calpain is associated with platelet microparticles in plasma from patients with TTP.

scholarly journals THE SYNTHESIS OF PROTOPORPHYRIN IN VITRO BY RED BLOOD CELLS OF THE DUCK

1950 ◽  
Vol 183 (2) ◽  
pp. 757-765 ◽  
Author(s):  
David Shemin ◽  
Irving M. London ◽  
D. Rittenberg
Keyword(s):  
Red Blood Cells ◽  
Blood Cells
Sign in / Sign up

Export Citation Format

Share Document