Non-viral ex vivo transduction of human hepatocyte cells to express factor VIII using a human ribosomal DNA-targeting vector

2007 ◽  
Vol 5 (2) ◽  
pp. 347-351 ◽  
Author(s):  
X. LIU ◽  
M. LIU ◽  
Z. XUE ◽  
Q. PAN ◽  
L. WU ◽  
...  
Keyword(s):  
Factor Viii ◽  
Ribosomal Dna ◽  
Ex Vivo ◽  
Human Hepatocyte ◽  
Dna Targeting ◽  
Hepatocyte Cells ◽  
Targeting Vector

In vitro Efficacy of mda-7 Gene for Hepatocellular Carcinoma Gene Therapy Mediated by Human Ribosomal DNA Targeting Vector*

2009 ◽  
Vol 36 (11) ◽  
pp. 1429-1435 ◽  
Author(s):  
Jin-Feng XUE ◽  
Xiong-Hao LIU ◽  
Qiang HE ◽  
Zhi-Gang XUE ◽  
You-Jin HU ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Gene Therapy ◽  
Ribosomal Dna ◽  
Dna Targeting ◽  
Targeting Vector

Synthesis of Factor VIII in Human Hepatocytes in Culture

1988 ◽  
Vol 60 (03) ◽  
pp. 387-391 ◽  
Author(s):  
J Ingerslev ◽  
B Sloth Christiansen ◽  
L Heickendorff ◽  
C Munck Petersen
Keyword(s):  
Factor Viii ◽  
Light Chain ◽  
Gradient Centrifugation ◽  
Human Fibroblasts ◽  
Human Hepatocytes ◽  
Human Hepatocyte ◽  
Blood Monocytes ◽  
Hep G2 ◽  
Human Hepatoma Cells ◽  
Sds Page

SummaryAlthough several investigators have attempted to identify the site of synthesis of factor VIII (FVIII), the cellular species responsible for maintenance of plasma FVIII has not been clearly defined. Indications point at hepatocytes and certain endothelial cells. The present study investigated the FVIII coagulant antigen (VIII : Ag) of hepatocytes obtained by two-step collagenase digests of human liver pieces. Following Percoll gradient centrifugation, less than 1% of cells harvested were non-parenchymal. Lysates of freshly isolated and purified hepatocytes contained 165–250 mU of VIII: Ag/106 cells as defined by a two-site ELISA employing a haemophilic antibody against human FVIII. This material contained a single peak of VIII: Ag polypeptides as jugded from the VIII: Ag ELISA profile of Mono-Q fast protein liquid chromatography fractions. A haemophilic antibody specific for epitopes of the light chain of FVIII, employed in immunoisolation of VIII : Ag in lysate of human hepatocytes, extracted a polypeptide pattern that was studied in a reduced SDS-PAGE electrophoresis gel and compared to that of immunoisolate from normal plasma. After electroblotting onto nitrocellulose and reaction with a monoclonal antibody towards the light chain of FVIII, the appearance of a doublet at 78–79 kDa in both these materials indicated the presence of the light chain of FVIII in human hepatocyte lysate. During culture, human hepatocytes secreted 20–80 mU of VIII: Ag per 1 × 106 cells per 24 hours. Further, a significant secretion of VIII: Ag was found in media of cultured human hepatoma cells, Hep-G2, whereas human blood monocytes and human fibroblasts did not secrete detectable VIII: Ag. In all of these cell cultures, vWf : Ag was indetectable or present as trace. Our results suggest that the human hepatocyte is a production site of FVIII.

Effect of Dextran 70 on the Platelet Distribution of Ex Vivo Thrombi

1979 ◽  
Author(s):  
M. åberg ◽  
A. Rausing ◽  
U. Hedner ◽  
S.-E. Bergent
Keyword(s):  
Light Microscopy ◽  
Factor Viii ◽  
Healthy Volunteers ◽  
Platelet Function ◽  
Small Dose ◽  
Ex Vivo ◽  
Morphological Changes ◽  
Dextran 70 ◽  
Platelet Aggregates

Infusion of dextran 70 impairs the function of factor VIII and increases the lysability of ex vivo thrombi. To find out whether this increased lysability is accompanied by any morphological changes of the thrombi dextran was infused into four healthy volunteers and one patient with von Wiilebrand’s disease. Dextran was also given to four dogs with 51Cr labelled platelets and 1-1abelled fibrinogen. The thrombi were studied in light microscopy and the distribution of isotopes measured. Dextran caused marked changes in the morphology of the thrombi. The platelet aggregates constituting the head were not formed and the platelets were more evenly distributed in the thrombi. The changes were most pronounced 2-4 hours after the infusion. In the patient with von Wiilebrand’s. disease the structure of the thrombus was abnormal already before the infusion of dextran. Even a small dose of dextran given to this patient prevented the formation of a platelet head. When factor VIII concentrate was given the platelet aggregates constituting the head of thrombus again formed. The findings indicate that factor VIII is of importance for the formation and coherence of the platelet aggregates in ex vivo thrombi. Dextran given intravenously reduces platelet aggregabiIity by impairing the function of factor VIII. The alteration of platelet function, accompanied bv profound changes in the morphology of ex vivo thrombi, may explain the increase in lysability which has been previously shown by us.

scholarly journals AdipoR2 is transcriptionally regulated by ER stress-inducible ATF3 in HepG2 human hepatocyte cells

FEBS Journal ◽  
2010 ◽  
Vol 277 (10) ◽  
pp. 2304-2317 ◽  
Author(s):  
In-uk Koh ◽  
Joo H. Lim ◽  
Myung K. Joe ◽  
Won H. Kim ◽  
Myeong H. Jung ◽  
...  
Keyword(s):  
Er Stress ◽  
Human Hepatocyte ◽  
Hepatocyte Cells

scholarly journals The Potential Role of Emicizuamb Prophylaxis in Severe Von Willebrand Disease

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 20-20
Author(s):  
Assaf Arie Barg ◽  
Gili Kenet ◽  
Tami Livnat ◽  
Einat Avishai ◽  
Ivan Budnik ◽  
...  
Keyword(s):  
Factor Viii ◽  
Potential Role ◽  
Prophylactic Treatment ◽  
Ex Vivo ◽  
Peak Height ◽  
Von Willebrand Disease ◽  
Type 3 ◽  
Von Willebrand

Severe Von Willebrand's disease (VWD) may be associated with chronic joint damage and may require prophylactic therapy. In severe VWD, factor VIII (FVIII) levels are low due to rapid clearness. Emicizumab is a humanized bispecific antibody which mimics the function of coagulation factor VIII (FVIII). It has been approved for prophylaxis in hemophilia A. This is the first study assessing the potential role of emicizumab as an alternative prophylactic treatment in a cohort of patients with severe VWD. We present a TG model evaluating patients' hemostasis following ex vivo spiking of their plasma samples with emicizumab. We also report 24 weeks of successful emicizumab prophylaxis in a child with severe VWD and repeated hemarthroses. A cohort of twenty-four VWD patients were included in the study. Fifty-four percent of our patients were males and the cohort consisted of 14 children (≤18 years) and 10 adults. The majority of patients (96%) were of Caucasian origin. Hemarthrosis was encountered in most type 3 VWD patients, whereas none of the type 2 VWD patients had any joint bleeds. Prophylactic treatment was administered in the majority of type 3 VWD patients, whereas type 2 VWD patients largely required only intermittent on demand therapy applied for bleeding episodes or any surgical interventions. Thrombin generation analysis was carried out blindly in plasma obtained from thirteen type 3 VWD patients and eleven type 2 VWD patients. Seventeen healthy volunteers served as a control group. In plasma from type 3 VWD patients, TG was substantially lower than in plasma from type 2 VWD patients, with ETP of 765 nM×min (596-962) vs. 1954 nM×min (1483-2008) (P = 0•001) and peak height of 47 nM (36-65) vs. 262 nM (142-318) (P = 0•002) In order to examine the potential use of emicizumab as an alternative treatment option for type 3 VWD patients, an ex vivo spiking analysis comparing the effect of Haemate P and emicizumab on TG was performed. An improvement in peak height was demonstrated following spiking with both Haemate P concentrations (P = 0•001 for both) and with the higher emicizumab concentration (P = 0•011). Notably, whereas spiking with both Haemate P concentrations increased peak height to near-normal level, spiking with higher emicizumab concentration increased it to a lesser extent (the median was still lower than in normal controls (P = 0•005). Following the decision to treat our impetus patient with emicizumab prophylaxis, TG analyses were performed in the patient's plasma before and during emicizumab loading and maintenance (Figure 3). As expected, patient's initial TG was extremely low and improved following the first administration of emicizumab loading dose (at week 2 after therapy initiation), at which time emicizumab level was 21 µg/mL. Further significant improvement of TG was noted following loading period completion while emicizumab level was 62 µg/mL. Our patient has been treated with emicizumab for more than six months altogether and did not encounter any joint bleeds since the commencement of therapy. During this period, a single dose of Haemate P was administered following tooth exfoliation. Our study contributes towards a better understanding of TG as a surrogate marker of VWD patients' hemostasis. Our data suggests that some severe VWD patients could be safely and efficiently treated with emicizumab. The successful prophylaxis of our patient and our ex vivo laboratory findings should set the ground for further collaborative multicenter studies to examine the efficacy and safety of emicizumab prophylaxis in type 3 VWD patients. Disclosures Barg: roshe: Honoraria, Speakers Bureau. Kenet:PI Healthcare, CSL Behring: Honoraria; Bayer, Pfizer, Takeda, BioMarin, Novo Nordisk: Speakers Bureau; Bayer, Pfizer, Roche, Alnylam (Sanofi), Shire: Research Funding; Bayer, Pfizer, BioMarin, Takeda, Roche, Novo Nordisk, Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: The use of Emicizumab in sever Von Willebrand disease

Effects of Chronic Factor VIII Substitution on Immune Parameters in HIV Seronegative Haemophiliacs: a Comparison between Cryoprecipitate and Factor VIII Concentrate

1996 ◽  
Vol 75 (02) ◽  
pp. 261-266 ◽  
Author(s):  
D P Allersmaa ◽  
W M Smld ◽  
J A van der Does ◽  
J van der Meer ◽  
E Briët
Keyword(s):  
Factor Viii ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Activated T Cells ◽  
Immune Parameters ◽  
Hla Dr ◽  
Factor Viii Concentrates ◽  
Patient Groups ◽  
Previously Treated ◽  
Hiv Negative

SummaryChronic substitution therapy of HIV-negative haemophiliacs with factor VIII products can result in abnormalities of ex-vivo measured immune parameters. To assess a possible relation between these abnormalities and product purity, we analyzed two groups of HIV-negative HCV-positive haemophiliacs, one treated with cryoprecipitate exclusively, the other with more purified factor VIII concentrates. Compared to age matched non-transfused male controls, increased numbers of white cells, granulocytes, IgG and IgM levels and decreased CD4+/CD8+ ratios were found in both patient groups. In the concentrate receivers, the numbers of mononuclear cells, CD4+, CD8+ and CD3+/HLA-DR+ cells indicating activated T-cells, were higher than in the cryoprecipitate group. In conclusion, both cryoprecipitate and intermediate/high purity concentrate recipients showed immune parameter abnormalities. These abnormalities tended to be somewhat more pronounced in patients treated with concentrates. By now there is no indication of the clinical relevance of the abnormalities in previously treated HIV seronegative haemophiliacs.

Plasmatic tissue factor pathway inhibitor is a major determinant of clotting in factor VIII inhibited plasma or blood

2013 ◽  
Vol 109 (03) ◽  
pp. 450-457 ◽  
Author(s):  
Sabine Knappe ◽  
Bernd Jilma ◽  
Ulla Derhaschnig ◽  
Rudolf Hartmann ◽  
Michael Palige ◽  
...  
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Ex Vivo ◽  
Major Determinant ◽  
Prospective Clinical Study ◽  
Acquired Haemophilia ◽  
C Terminus ◽  
Kunitz Domain ◽  
Tissue Factor Pathway

SummaryTissue factor pathway inhibitor (TFPI) is a major inhibitor of coagulation. We therefore hypothesised that high plasmatic TFPI levels are associated with impaired ex vivo clotting in a model of acquired haemophilia. Blood samples were collected in a prospective clinical study from 30 healthy volunteers. Coagulation in normal or factor VIII (FVIII)-inhibited human blood or plasma was measured by the calibrated automated thrombogram (CAT) and rotational thromboelastometry (ROTEM). Both methods are global haemostatic assays that provide insight into the whole coagulation process. Monoclonal mouse antibodies raised against either the C-terminus or the Kunitz domain 2 of TFPI were used to determine full-length (fl-) and total TFPI by an enzyme-immunoassay. Clotting times and parameters of thrombin generation correlated with TFPI levels. Subjects with low fl-TFPI levels had significantly shorter clotting times and a higher endogenous thrombin potential (ETP) compared to those with high fl-TFPI levels (p≤0.005 for all). An even stronger effect was seen in FVIII-inhibited blood/plasma: ROTEM clotting time was 26% shorter (p=0.01) and the ETP assessed by CAT was >2-fold higher in subjects with low fl-TFPI levels (p≤0.0001). Plasmatic TFPI is a major determinant of coagulation in global haemostatic tests particularly when FVIII is missing. Thus, inhibition of TFPI might be a promising novel treatment approach, especially in haemophilia patients with FVIII inhibitors.

High-level expression of porcine factor VIII from genetically modified bone marrow–derived stem cells

Blood ◽  
2006 ◽  
Vol 107 (10) ◽  
pp. 3859-3864 ◽  
Author(s):  
Bagirath Gangadharan ◽  
Ernest T. Parker ◽  
Lucienne M. Ide ◽  
H. Trent Spencer ◽  
Christopher B. Doering
Keyword(s):  
Bone Marrow ◽  
Factor Viii ◽  
Neutralizing Antibodies ◽  
Hemophilia A ◽  
Ex Vivo ◽  
Clinical Success ◽  
Coagulation Factor ◽  
High Level Expression ◽  
Hematopoietic Stem ◽  
High Level

Clinical success for gene therapy of hemophilia A will be judged by achievement of sustained, therapeutic levels of coagulation factor VIII (fVIII). Previous clinical trials have suffered from transient, subtherapeutic expression of human fVIII transgenes. Porcine fVIII contains sequence elements that enable more efficient biosynthesis than human fVIII due to enhanced posttranslational transit through the secretory pathway. In this study, we evaluated ex vivo retroviral gene transfer of a high-expression porcine fVIII transgene into bone marrow–derived stromal and hematopoietic stem/progenitor cells (MSCs and HSCs, respectively) and transplantation into genetically immunocompetent hemophilia A mice. Both MSCs and HSCs demonstrated high-level expression of porcine fVIII in vivo. However, following transplantation of gene-modified MSCs, fVIII activity levels rapidly returned to baseline due to the formation of anti–porcine fVIII–neutralizing antibodies. Alternatively, transplantation of HSCs into myeloablated and nonmyeloablated hemophilia A mice resulted in high-level fVIII expression despite low-level hematopoietic reconstitution by gene-modified cells. FVIII expression was sustained beyond 10 months, indicating that immunologic tolerance to porcine fVIII was achieved. Furthermore, transplantation of bone marrow from primary recipients into naive secondary recipients resulted in sustained, high-level fVIII expression demonstrating successful genetic modification and engraftment of HSCs.

Sign in / Sign up

Export Citation Format

Share Document