The Usefulness of Improved, Newly Developed Polyolefin Container of Platelet, PO-100, with Higher Oxygen Permeability and Content

Abstract The demand for platelet concentrate (PC) has been growing because of the increase of more intensive chemotherapy and hematopoietic stem cell transplantation, and highly invasive surgical operations of several specific areas (i.e. cardiovascular surgery, etc.), which has brought about considerable impact on its decent availability. Furthermore, the short storage period for room temperature stored PC, four days in Japan, five days in worldwide and seven days in the U.S. if they meet several requirements by FDA for the control of the risk of bacterial contamination, has put many PCs become outdated. In response to these requirements, measures have been taken to maximize platelet use, including development of polyolefin containers for platelets that elongate storage time and formulations of higher units. Here, we have developed a polyolefin container, PO-100, which has a 40%/m2 higher oxygen permeability than PO-80, a former, and standard platelet storage bags across the world. Furthermore, PO-100 has two types, one is PO-100/1000 with 1000mL capacity for 4-5 x 1011 platelets content and the other is PO-100/1500 with 1500mL capacity for over 6 x 1011 platelets content. In this study, we evaluated the influence of storage of higher content of platelet in PO-100 on the condition of platelet in the bag. The IRB of Nagasaki University approved this study. After informed consent was obtained from healthy volunteers, single-donor apheresis was performed. Then, platelets were pooled, separated equally into two types of bags, PO-100/1000 (n=6) or PO-100/1500 (n=6), and stored in plasma for up to day 7. We monitored various parameters, which related the quality of PC, in bags over the storage period. The result showed that the in vitro characteristics of platelets; platelet counts, mean platelet volume (MPV), and adenosine diphosphate (ADP)/collagen combined-induced platelet aggregation did not significantly change through 7 days storage period both in PO-100/1000 and PO-100/1500. Furthermore, MPV (fL) (8.63±0.35 vs 8.85±0.58, respectively, p=0.463) and platelet aggregation (%) (81.58±4.45 vs 84.67±1.21, respectively, p=0.118) were equivalent between these two conditions. Although pH gradually declined, it was maintained around 7.0 at day 7 without significant difference in both conditions (7.00±0.10 vs 6.92±0.08, respectively, p=0.063). On the other hand, lactate concentration elevated gradually, but there was no significant difference between two bags at day 7 (133.07±23.70 vs 104.52±15.56, respectively, p=0.097) and the pO2 declined by 30 to 40% from day 0 through day 1 in both conditions, suggesting aerobic metabolism was well maintained with efficient O2 consumption and less lactate accumulation in PO-110 bags regardless of its capacity. Intriguingly, our previous investigation demonstrated that pH and ADP-induced platelet aggregation in PO-80 bags decline steeply, notably after day 3, and values at day 7 were 6.67±0.24 and 56.67±11.34%, respectively, which suggested that higher oxygen permeability might contribute to keep platelets in better condition in PO-100 than PO-80. In conclusion, the present study demonstrated that PO-100 had significantly high utility as a noble container for PC. It was considered that better maintenance of the pH of the medium in PC within the appropriate range through improved higher oxygen permeability and efficient aerobic metabolism resulted in platelets in a better functional state even after 7 days storage. Furthermore, the results in this study might open the possibility of practical application of higher unit products, so that the supply chain of PCs could be conducted favorably. Disclosures Nagai: Kawasumi Laboratories Inc.: Research Funding; Kaneka Corporation: Research Funding; Ono Pharmaceutical Co.Ltd.: Consultancy. Yakushiji:Kawasumi Laboratories Inc.: Employment. Kino:Kawasumi Laboratories Inc.: Employment. Tokunaga:Kawasumi Laboratories Inc.: Employment. Yamaoka:Kawasumi Laboratories Inc.: Employment.

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Enhancement of ADP-induced Platelet Aggregation by Adrenaline in Vivo and Its Prevention

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647788 ◽

1973 ◽

Vol 29 (02) ◽

pp. 490-498 ◽

Cited By ~ 6

Author(s):

Hiroh Yamazaki ◽

Itsuro Kobayashi ◽

Tadahiro Sano ◽

Takio Shimamoto

Keyword(s):

Platelet Count ◽

Platelet Aggregation ◽

Platelet Rich Plasma ◽

The Other ◽

Endothelial Surface ◽

Important Interaction ◽

Blood Coagulability ◽

The Difference

SummaryThe authors previously reported a transient decrease in adhesive platelet count and an enhancement of blood coagulability after administration of a small amount of adrenaline (0.1-1 µg per Kg, i. v.) in man and rabbit. In such circumstances, the sensitivity of platelets to aggregation induced by ADP was studied by an optical density method. Five minutes after i. v. injection of 1 µg per Kg of adrenaline in 10 rabbits, intensity of platelet aggregation increased to 115.1 ± 4.9% (mean ± S. E.) by 10∼5 molar, 121.8 ± 7.8% by 3 × 10-6 molar and 129.4 ± 12.8% of the value before the injection by 10”6 molar ADP. The difference was statistically significant (P<0.01-0.05). The above change was not observed in each group of rabbits injected with saline, 1 µg per Kg of 1-noradrenaline or 0.1 and 10 µg per Kg of adrenaline. Also, it was prevented by oral administration of 10 mg per Kg of phenoxybenzamine or propranolol or aspirin or pyridinolcarbamate 3 hours before the challenge. On the other hand, the enhancement of ADP-induced platelet aggregation was not observed in vitro, when 10-5 or 3 × 10-6 molar and 129.4 ± 12.8% of the value before 10∼6 molar ADP was added to citrated platelet rich plasma (CPRP) of rabbit after incubation at 37°C for 30 second with 0.01, 0.1, 1, 10 or 100 µg per ml of adrenaline or noradrenaline. These results suggest an important interaction between endothelial surface and platelets in connection with the enhancement of ADP-induced platelet aggregation by adrenaline in vivo.

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Studies investigating platelet aggregation and release initiated by sera from patients with thrombotic thrombocytopenic purpura

Blood ◽

10.1182/blood.v69.3.924.bloodjournal693924 ◽

1987 ◽

Vol 69 (3) ◽

pp. 924-928 ◽

Cited By ~ 1

Author(s):

JG Kelton ◽

JC Moore ◽

WG Murphy

Keyword(s):

Platelet Aggregation ◽

Thrombotic Thrombocytopenic Purpura ◽

Disease Process ◽

The Other ◽

Platelet Glycoprotein ◽

Glycoprotein Ib ◽

Thrombocytopenic Purpura ◽

Induced Aggregation

Many patients with thrombotic thrombocytopenic purpura (TTP) have a platelet aggregating factor in their serum that may be pathologically linked with the disease process. To help characterize the type of platelet aggregation and platelet release induced by the sera from seven TTP patients, we measured the ability of a variety of inhibitors of platelet function as well as the ability of monoclonal antibodies (MoAbs) against platelet glycoproteins to inhibit TTP sera-induced platelet aggregation and release. These results were compared with the ability of the same inhibitors to block platelet aggregation induced by ristocetin, collagen, ADP, thrombin, and IgG-immune complexes. Monoclonal antibody directed against platelet glycoprotein Ib totally inhibited ristocetin-induced aggregation and release but had no effect on aggregation and release induced by the TTP sera or by any of the other platelet agonists. However, the MoAb against glycoproteins IIb/IIIa inhibited aggregation and release caused by TTP sera as well as by collagen, thrombin, and ADP but had no effect on aggregation and release induced by ristocetin. The aggregating activity could be abolished by heparin but not by the serine protease inhibitor PMSF (1 mmol/L). And although monomeric human IgG and purified Fc fragments of IgG inhibited IgG-immune complex-induced aggregation and release, they had no effect on TTP sera-induced aggregation and release nor on aggregation and release induced by any of the other agonists. Consistent with these in vitro studies showing no effect of IgG were the in vivo observations that intravenous (IV) IgG was without effect when administered to three patients with TTP. This study indicates that although a von Willebrand factor (vWF)-rich preparation of cryoprecipitate enhances the in vitro platelet aggregation and release caused by sera from the seven TTP patients we studied, the pathway of aggregation and release is not via platelet glycoprotein Ib. Also the aggregating factor of TTP sera is not neutralized in vitro or in vivo by IgG.

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Unraveling the Mechanism of Platelet Aggregation Suppression by Monoterpenoids

Bioengineering ◽

10.3390/bioengineering9010024 ◽

2022 ◽

Vol 9 (1) ◽

pp. 24

Author(s):

Liliya E. Nikitina ◽

Roman S. Pavelyev ◽

Ilmir R. Gilfanov ◽

Sergei V. Kiselev ◽

Zulfiya R. Azizova ◽

...

Keyword(s):

Platelet Aggregation ◽

Cellular Membrane ◽

The Other ◽

P2y12 Receptor ◽

Sulphur Atom ◽

Membrane Stabilization ◽

Nmr Studies ◽

Binding Force ◽

Promising Agent

Platelet aggregation causes various diseases and therefore challenges the development of novel antiaggregatory drugs. In this study, we report the possible mechanism of platelet aggregation suppression by newly synthesized myrtenol-derived monoterpenoids carrying different heteroatoms (sulphur, oxygen, or nitrogen). Despite all tested compounds suppressed the platelet aggregation in vitro, the most significant effect was observed for the S-containing compounds. The molecular docking confirmed the putative interaction of all tested compounds with the platelet’s P2Y12 receptor suggesting that the anti-aggregation properties of monoterpenoids are implemented by blocking the P2Y12 function. The calculated binding force depended on heteroatom in monoterpenoids and significantly decreased with the exchanging of the sulphur atom with oxygen or nitrogen. On the other hand, in NMR studies on dodecyl phosphocholine (DPC) as a membrane model, only S-containing compound was found to be bound with DPC micelles surface. Meanwhile, no stable complexes between DPC micelles with either O- or N-containing compounds were observed. The binding of S-containing compound with cellular membrane reinforces the mechanical properties of the latter, thereby preventing its destabilization and subsequent clot formation on the phospholipid surface. Taken together, our data demonstrate that S-containing myrtenol-derived monoterpenoid suppresses the platelet aggregation in vitro via both membrane stabilization and blocking the P2Y12 receptor and, thus, appears as a promising agent for hemostasis control.

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Use of Medium Potency Statins Is Associated with Improved Outcomes after Frontline RCHOP in Patients with Diffuse Large B-Cell Lymphoma (DLBCL)

Blood ◽

10.1182/blood-2020-136010 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 45-45

Author(s):

Sushanth Gouni ◽

Paolo Strati ◽

Jason Westin ◽

Loretta J. Nastoupil ◽

Raphael E Steiner ◽

...

Keyword(s):

Cell Lines ◽

Research Funding ◽

Response Assessment ◽

Cholesterol Content ◽

In Vivo Studies ◽

High Cholesterol ◽

Improved Outcomes ◽

Significant Difference

Background: Pre-clinical studies show that statins may improve the efficacy of chemoimmunotherapy in patients with DLBCL, through interference with cell membrane-initiated signaling pathways. Clinical retrospective studies, however, yield conflicting data, due to heterogeneous properties of statins, including potency and hydrophilicity. Methods: This is a retrospective analysis of patients with previously untreated, advanced stage DLBCL, non-double hit, treated with frontline R-CHOP between 01/01/2000 and 09/01/2019 (data cut-off 04/15/2020) at MD Anderson Cancer Center, and for whom data regarding statin use at time of initiation of treatment were available. Lugano 2014 response criteria were applied retrospectively for response assessment. Cellular cholesterol levels were analyzed in 6 DLBCL cell lines using an Amplex red fluorometric assay. A doxorubicin (DXR)-resistant cell line was generated exposing SUDHL4 cells to escalating doses of DXR; a DXR-resistant DLBCL patient-derived xenograft (PDX) model was established through serial transplantation and exposure to DXR. Results: 271 patients were included in the analysis, 182 (67%) were older than 60 years, 134 (49%) were male, 212 (72%) had stage IV disease, and 217 (80%) had an IPI score > 3; upon pathological review, 38 (36%) cases were non-GCB type, and 18 (28%) were double-expressors; 214 (79%) were able to complete all planned 6 cycles of RCHOP. Seventy-nine (29%) patients received statins at time of initiation of chemoimmunotherapy: 15 patients received low potency statin, 51 medium and 13 high; 18 patients received hydrophilic statins and 61 lipophilic. Patients receiving statins were significantly older as compared to patients who did not (p<0.001); no other significant difference in baseline characteristics was observed when comparing the 2 groups. Overall, 265 out of 271 patients were evaluable for response, as 6 stopped treatment because of toxicity before first response assessment. Among these, ORR was 95% (252/265) and CR rate was 62% (165/265). ORR rate was identical in patients who were treated with statin and those who did not (95% both, p=1). After a median follow-up of 77 months (95% CI, 70-84 months), 119 patients progressed/died, median PFS was not reached and 6-year PFS was 57%. 6-year PFS rate according to statin intensity was: 48% (low), 72% (medium), 57% (high). PFS. 6-year PFS rate was 64% for hydrophilic and 72% for lipophilic statins. Patients treated with statins had a trend for longer PFS (p=0.06), significantly longer for patients receiving medium potency statins (p=0.04). No significant difference in PFS was observed when comparing patients treated with lipophilic statins to all others (not reached vs 84 months, p=0.22). To confirm these clinical data, in-vitro and in-vivo studies were performed. Six cell lines were tested: 4 with high cholesterol content (SUDHL4, HBL1, HT, and U2932; 5.0-8.0 µg/mg protein), and 2 with low cholesterol content (DOHH2 and OCI-LY19; 1.5-2.0 µg/mg protein); the latter showed the highest sensitivity to DXR-mediated killing. The combination of lovastatin and DXR (10nM) was tested in all 4 cell lines with high cholesterol content, resulting in more cell death than either treatment alone. Lovastatin (at the nanomolar range) resensitized DXR-resistant SUDHL4 cells to DXR. Finally, in a DXR-resistant PDX model, the combination of lovastatin and DXR resulted in delayed tumor growth as compared to chemotherapy alone. Conclusions: Use of medium potency statins is associated with improved outcomes after frontline RCHOP in patients with DLBCL. This was further confirmed in functional in-vitro and in-vivo studies. Future interventional studies, aimed at improving outcomes in these patients using this novel combination, are warranted. Disclosures Westin: Amgen: Consultancy; 47: Research Funding; Kite: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Morphosys: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Curis: Consultancy, Research Funding; Astra Zeneca: Consultancy, Research Funding. Nastoupil:Gamida Cell: Honoraria; Merck: Research Funding; TG Therapeutics: Honoraria, Research Funding; Karus Therapeutics: Research Funding; Janssen: Honoraria, Research Funding; LAM Therapeutics: Research Funding; Novartis: Honoraria, Research Funding; Bayer: Honoraria; Celgene: Honoraria, Research Funding; Genentech, Inc.: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Gilead/KITE: Honoraria. Neelapu:Bristol-Myers Squibb: Other: personal fees, Research Funding; Merck: Other: personal fees, Research Funding; Kite, a Gilead Company: Other: personal fees, Research Funding; Pfizer: Other: personal fees; Celgene: Other: personal fees, Research Funding; Novartis: Other: personal fees; Karus Therapeutics: Research Funding; N/A: Other; Takeda Pharmaceuticals: Patents & Royalties; Acerta: Research Funding; Cellectis: Research Funding; Poseida: Research Funding; Precision Biosciences: Other: personal fees, Research Funding; Legend Biotech: Other; Adicet Bio: Other; Allogene Therapeutics: Other: personal fees, Research Funding; Cell Medica/Kuur: Other: personal fees; Calibr: Other; Incyte: Other: personal fees; Unum Therapeutics: Other, Research Funding. Landgraf:NCI/NIH: Research Funding. Vega:NCI: Research Funding.

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IN VITRO ANTI-PLATELET AGGREGATION ACTIVITY OF HYDROXYPROPYL CELLULOSE–CYSTEAMINE BASED NANOPARTICLES CONTAINING CRUDE BROMELAIN

International Journal of Applied Pharmaceutics ◽

10.22159/ijap.2020v12i3.35053 ◽

2020 ◽

pp. 95-98

Author(s):

DENI RAHMAT ◽

LILIEK NURHIDAYATI ◽

MARCELLA MARCELLA ◽

ROS SUMARNY ◽

DIAN RATIH LAKSMITAWATI

Keyword(s):

Particle Size ◽

Platelet Aggregation ◽

Zeta Potential ◽

Light Transmission ◽

Hydroxypropyl Cellulose ◽

Platelet Activity ◽

Two Samples ◽

Significant Difference ◽

Aggregation Activity

Objective: The aim of the present study was to formulate bromelain into nanoparticles in order to improve its stability and activity. Methods: Crude bromelain was prepared by protein precipitation from the pineapple stem juice using ammonium sulphate at the concentration of 60% (w/v). Nanoparticles containing crude bromelain were generated using the ionic gelation method with hydroxypropyl cellulose–cysteamine (HPC-cysteamine) conjugate as a matrix. Crude bromelain was then added to the HPC-cysteamine solution for ionic interaction to construct the nanoparticles, which were then analyzed for their particle size and zeta potential. The resulting nanoparticles were mixed with adenosine diphosphate (ADP) to perform anti-platelet aggregation. Results: The nanoparticle had 928.3 nm in particle size and-7.25 mV in zeta potential. Anti-platelet activity of crude bromelain and the nanoparticles were determined with modification of light transmission aggregometry (LTA), in which ADP was used to induce an aggregation while a spectrophotometer UV-Vis was used to measure the absorbance at the wavelength of 600 nm. The result showed that crude bromelain and the nanoparticles rendered percentage inhibition of 8.00±1.17% and 48.56±11.19%, respectively. Conclusion: Based on the result of a one-way analysis of variance (ANOVA), it was concluded that there was a significant difference in percentage inhibition between the two samples. The nanoparticles demonstrated a better anti-platelet aggregation activity compared to crude bromelain.

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Effect of Different Intermediary Bases on Microleakage of a Restorative Material in Class II Box Cavities of Primary Teeth

The International Journal of Artificial Organs ◽

10.5301/ijao.5000566 ◽

2017 ◽

Vol 40 (2) ◽

pp. 82-87 ◽

Cited By ~ 1

Author(s):

Faika Y. Abdelmegid ◽

Fouad S. Salama ◽

Waleed M. Al-Mutairi ◽

Saud K. Al-Mutairi ◽

Sultan O. Baghazal

Keyword(s):

Primary Teeth ◽

In Vitro Study ◽

Class Ii ◽

Positive Control ◽

Negative Control ◽

The Other ◽

Control Groups ◽

Significant Difference ◽

The Mean

Introduction The aim of this in vitro study was to assess and compare the effect of different intermediary bases on microleakage between tooth and a nanocomposite interface in Class II box cavities in primary teeth. Methods Standard Class II box cavities were prepared in 52 primary molars and randomly divided into 9 groups according to the intermediary base used (Multicore Flow, Fuji II LC, SDR, Smart Dentin Replacement, and Biodentine). All specimens were subjected to thermocycling and prepared for microleakage testing and evaluation. Results There was significant difference in the mean ranks of microleakage between the 9 groups, which was observed in the gingival side (p<0.0001) and the occlusal side (p<0.0001). The mean ranks microleakage was significantly higher with experimental SDR, experimental Multicore Flow, and positive control materials when compared with the other 6 groups. The microleakage mean ranks were statistically significantly lower in experimental Fuji II LC, experimental Biodentine, and all negative control groups when compared with the other 3 groups. Conclusions Microleakage is affected by the application of intermediate material. Experimental Biodentine and Fuji II LC showed the lowest microleakage while experimental SDR and experimental Multicore Flow showed the highest microleakage.

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Dimensional stability of distances between posterior teeth in maxillary complete dentures

Brazilian Oral Research ◽

10.1590/s1806-83242006000300011 ◽

2006 ◽

Vol 20 (3) ◽

pp. 241-246 ◽

Cited By ~ 4

Author(s):

Rafael Leonardo Xediek Consani ◽

Marcelo Ferraz Mesquita ◽

Lourenço Correr-Sobrinho ◽

Maurício Tanji

Keyword(s):

Dimensional Stability ◽

Storage Period ◽

Acrylic Resin ◽

The Other ◽

Complete Dentures ◽

Posterior Teeth ◽

Anterior Teeth ◽

Significant Difference ◽

Cooling Methods ◽

Made In

The aim of this study was to assess the displacement of posterior teeth in maxillary complete dentures stored in water at 37°C. Twenty acrylic resin-based maxillary complete dentures were constructed with the anterior teeth arranged in normal overlap and the posterior teeth in Angle class I. Metallic pins were placed on the labial cusp of the first premolars (PM), and on the mesiolabial cusp of the second molars (M). The final acrylic resin pressing was made in a metallic flask with aid of the RS tension system, and polymerized in a moist-hot cycle at 74°C for 9 hours. The dentures were deflasked after cooling in their own polymerizing water or after cooling in polymerizing water plus bench storage for 3 hours, and stored in water at 37°C for periods of 7, 30, and 90 days. Following deflasking and after each storage period tested, the PM-PM (premolar to premolar), M-M (molar to molar), LPM-LM (left premolar to left molar), and RPM-RM (right premolar to right molar) distances were measured with an STM Olympus microscope, with an accuracy of 0.0005 mm. Collected data were submitted to ANOVA and Tukey's test (5%). There was no statistically significant difference for the PM-PM, M-M, and LPM-LM distances after all storage periods when the flask cooling methods were considered. With exception of the RPM-RM distance after the 30-days water plus bench storage period, the other distances remained statistically stable.

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Studies investigating platelet aggregation and release initiated by sera from patients with thrombotic thrombocytopenic purpura

Blood ◽

10.1182/blood.v69.3.924.924 ◽

1987 ◽

Vol 69 (3) ◽

pp. 924-928 ◽

Cited By ~ 35

Author(s):

JG Kelton ◽

JC Moore ◽

WG Murphy

Keyword(s):

Platelet Aggregation ◽

Thrombotic Thrombocytopenic Purpura ◽

Disease Process ◽

The Other ◽

Platelet Glycoprotein ◽

Glycoprotein Ib ◽

Thrombocytopenic Purpura ◽

Induced Aggregation

Abstract Many patients with thrombotic thrombocytopenic purpura (TTP) have a platelet aggregating factor in their serum that may be pathologically linked with the disease process. To help characterize the type of platelet aggregation and platelet release induced by the sera from seven TTP patients, we measured the ability of a variety of inhibitors of platelet function as well as the ability of monoclonal antibodies (MoAbs) against platelet glycoproteins to inhibit TTP sera-induced platelet aggregation and release. These results were compared with the ability of the same inhibitors to block platelet aggregation induced by ristocetin, collagen, ADP, thrombin, and IgG-immune complexes. Monoclonal antibody directed against platelet glycoprotein Ib totally inhibited ristocetin-induced aggregation and release but had no effect on aggregation and release induced by the TTP sera or by any of the other platelet agonists. However, the MoAb against glycoproteins IIb/IIIa inhibited aggregation and release caused by TTP sera as well as by collagen, thrombin, and ADP but had no effect on aggregation and release induced by ristocetin. The aggregating activity could be abolished by heparin but not by the serine protease inhibitor PMSF (1 mmol/L). And although monomeric human IgG and purified Fc fragments of IgG inhibited IgG-immune complex-induced aggregation and release, they had no effect on TTP sera-induced aggregation and release nor on aggregation and release induced by any of the other agonists. Consistent with these in vitro studies showing no effect of IgG were the in vivo observations that intravenous (IV) IgG was without effect when administered to three patients with TTP. This study indicates that although a von Willebrand factor (vWF)-rich preparation of cryoprecipitate enhances the in vitro platelet aggregation and release caused by sera from the seven TTP patients we studied, the pathway of aggregation and release is not via platelet glycoprotein Ib. Also the aggregating factor of TTP sera is not neutralized in vitro or in vivo by IgG.

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Durable and Robust Fetal Globin Induction without Anemia in Rhesus Monkeys Following Autologous Hematopoietic Stem Cell Transplant with BCL11A Erythroid Enhancer Editing

Blood ◽

10.1182/blood-2019-129394 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4632-4632 ◽

Cited By ~ 2

Author(s):

Selami Demirci ◽

Jing Zeng ◽

Yuxuan Wu ◽

Naoya Uchida ◽

Jackson Gamer ◽

...

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Mononuclear Cells ◽

Hematologic Toxicity ◽

Cell Transplant ◽

Post Transplantation ◽

Hematopoietic Stem ◽

Significant Difference ◽

Hbf Induction

Elevated fetal hemoglobin (HbF, α2γ2) levels are clinically beneficial for patients with β-hemoglobinopathies. Editing of the erythroid-specific BCL11A enhancer induces HbF, inhibiting sickling and restoring globin chain balance in erythroid cells derived from hematopoietic stem and progenitor cells (HSPCs) from SCD and β-thalassemia patients respectively, without detectable genotoxicity or adverse effects on hematopoietic stem cell (HSC) function (Wu Y, Nat Med, 2019). Here, we sought to evaluate engraftment and HbF induction potential of erythroid-specific BCL11A enhancer edited CD34+ HSPCs in a non-human primate transplantation model in which hemoglobin switching is conserved. We targeted the erythroid-specific +58 DNAse I hypersensitive site of BCL11A, which has identical human and rhesus sequences at the spacer and protospacer adjacent motif (PAM) of the potent #1617 sgRNA. Ribonucleoprotein complex (RNP) composed of 3x-NLS SpCas9 protein and either BCL11A enhancer targeting (#1617) or AAVS1 targeting sgRNA was electroporated into rhesus CD34+ HSPCs (n=3). Following erythroid differentiation, substantial γ-globin expression (54-77%, p<0.01) was observed in BCL11A edited cells (81-85% indels) as compared to 19-25% and 15-24% for non-electroporated and AAVS1 edited cells, respectively, with no significant difference in red blood cell (RBC) enucleation efficiency (44-47%) among groups. We tested BCL11A enhancer editing with autologous HSC transplant in two cohorts, with two macaques per cohort. For cohort 1, we performed competitive engraftment of BCL11A enhancer and AAVS1 edited HSPCs to test long-term reconstitution. For cohort 2, we evaluated BCL11A enhancer editing alone to evaluate HbF induction and hematopoietic reconstitution. For each cohort, purified CD34+ HSPCs were electroporated with RNP one day after G-CSF and plerixafor mobilization and cultured for two days prior to cryopreservation. HSPCs were thawed and infused following 2×5 Gy total body irradiation. For cohort 1 (n=2, ZL25 and ZL22, 1.34-1.39×106 CD34+ HSPCs/kg), we observed reduced indel frequencies (8-41%) at early post-infusion time points compared to cell products (18-49%), suggesting indels in unfractionated HSPCs may overestimate those in engrafting cells and/or hematopoietic ablation was incomplete. From weeks 6 to 83, stable indel frequencies were detected in both BCL11A (~3-18%) and AAVS1 (~10-45%), suggesting no selective advantage for BCL11A enhancer edited, AAVS1 edited, or non-edited HSCs. For cohort 2 (BCL11A enhancer editing alone (n=2, ZM17 and ZM26, 1.78-6.06×106 CD34+ cells/kg), cell products showed improved editing with ~95% indels and ~65-78% γ-globin protein after in vitro erythroid culture. Animals engrafted with typical kinetics and displayed stable indel ratios up to 28 weeks post-transplantation. A significant correlation was detected between γ-globin level and indel frequency comparing all 4 transplanted animals and unedited controls (R2=0.76, p<0.01). In both edited and unedited animals γ-globin levels peaked in the first two months after transplantation and subsequently declined and plateaued. In ZM17 (~70% BCL11A enhancer indels at ~24 weeks), ~12% γ-globin was observed in peripheral blood (PB) at last measurement (compared to 0.5% γ-globin in RBC prior to transplant). In the same animal, editing ranged from 78-81% across all PB and bone marrow (BM) lineages (excluding CD3+ T-cells with 63% indels), including B-lymphoid, myeloid, erythroid, and HSPCs (in particular including 78% indels in CD71+ CD45- erythroblasts). Hemoglobin, hematocrit, and reticulocyte counts and peripheral smear appearance were all normal, suggesting no erythroid toxicity. Colony-forming ability of BM-derived mononuclear cells was similar in edited and control animals. In summary, we evaluated the clinical potential of autologous BCL11A erythroid enhancer editing in rhesus macaques. BCL11A enhancer edited HSCs can persist for at least 83 weeks post-transplant and provide therapeutic levels of HbF in peripheral RBCs without anemia or other apparent hematologic toxicity. Furthermore, these results emphasize input CD34+ HSPC dose and conditioning intensity as critical variables that influence gene editing following autologous HSCT. Overall, these findings support BCL11A erythroid enhancer genome editing as a promising strategy for therapeutic HbF induction. Disclosures Weiss: GlaxoSmithKline: Consultancy; Cellarity INC: Consultancy; Esperian: Consultancy; Beam Therapeutics: Consultancy; Rubius INC: Consultancy.

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A Experimental Study and Clinical Result of Prophylaxis aGVHD with Humanized Anti-CD25 Monoclonal Antibody in Haploidentical BMT.

Blood ◽

10.1182/blood.v104.11.1251.1251 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1251-1251

Author(s):

Shu-Quan Ji ◽

Hui-Ren Chen ◽

Heng-Xiang Wang ◽

Hong-Min Yan ◽

Mei Xue ◽

...

Keyword(s):

Median Time ◽

Conditioning Regimen ◽

Control Group ◽

High Dose ◽

Marrow Graft ◽

Hla Antigen ◽

Hematopoietic Stem ◽

Gvhd Prophylaxis ◽

Significant Difference

Abstract Between February 1999 and March 2004, eighty-seven patients with high risk leukemia, age 3–50 (median 19 year), who needed urgent transplant but no HLA-matched or single HLA-antigen mismatched donors available, received unmanipulated HLA haploidentical BMT. The 87 patients were classified as follows AML 27 (CR1 in 7, CR2 in 15 and 5 in relapse), All 38 (CR1 in 4, CR2 in 30 and 4 in relapse) , CML 22 ( 4 in CP, 12 in AP and 6 in BP). All donors were HLA-haploidentical relatives who had at least two major histocompatibility complex antigen mismatched with the recipients. 87 patients underwent haplo-BMT with G-CSF primed BM as stem cells. All patients received a same conditioning regimen including high dose Ara-C, Cyclophosphamide, antithymocyte globulin and total body irradiation to provide both immunosuppression and myeloablation. GVHD prophylaxis consisted of anti-thymocyte globulin, cyclosporin A, methotrexate and mycofenolate mofitel. 72 patients underwent the transplants with the addition of CD25 mAb (Basiliximab Novartis) for GVHD prophylaxis designated as CD25 mAb group. Basiliximab 20mg each by 30min intravenous infusion on 2 hours before transplantion and day 4 after transplantaion. The other 15 patients without Basiliximab for GVHD prophylaxis were as the control group. The two group of patients were comparable in disease status, HLA-disparity and median age of patients. Immunophenotyping, limited dilution assay and colony forming assays were used to measure the effect of Basiliximab on the subsets of lymphocytes, cytotoxic T lymphocyte precursors (CTLp) and hematopoietic cells. All donors were primed with G-CSF at 3-5ug/kg/d for 7 days and the marrow cells were harvested on the eighth day. G-CSF donor priming significantly increased CD34+ and colony forming progenitors in the marrow grafts. More importantly, it significantly reduced lymphocytes and reversed CD4+/CD8+ lymphocyte ratio in the grafts. Both of group who were treated with and without Basiliximab had similar marrow graft contents. All patients established trilineage engraftments.The median time to achieve an absolute neutrophil count 0.5x109/L was 19 days (range, 13 to 24 days). The median time to achieve platelets above 20x109/L was 22 days (range, 16 to 32 days). Between the two groups were no differences in engraftment. Incidence of grades II–IV acute GVHD were 13.9% with GVHD-related deaths 6.9% in Basiliximab group and 33.3% with 20% GVHD-related deaths in control group. There were a significant difference between the anti-CD25 mAb treated Vs non-treated group.Forty-nine patients who survived over 12 months were eligible for the evaluation of cGVHD. 12 patients developed extensive cGVHD, one in control group and eleven in Basiliximab group. 49 were alive in CR during a median follow-up of 30 months (range3–64 months), 42 in Basiliximab group and 7 in control group. Basiliximab significantly decreased alloreactive CTLp by 10–100 fold in limiting dilution assays. It had no effect on hematopoietic stem and progenitor cells as determined by in vitro colony-forming assays.The addition of basiliximab as aGVHD prophylaxis effectively reduced severe lethal aGVHD in haplo-BMT. It is possible to selectively eliminate or reduce the number of alloreactive T cells with anti CD25 antibody, which results in prevention of or a reduction in the severity of GVHD.

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