scholarly journals Activation-independent, antibody-mediated removal of GPVI from circulating human platelets: development of a novel NOD/SCID mouse model to evaluate the in vivo effectiveness of anti-human platelet agents

Blood ◽  
2006 ◽  
Vol 108 (3) ◽  
pp. 908-914 ◽  
Author(s):  
B. Boylan
Keyword(s):  
Mouse Model ◽  
Scid Mouse ◽  
Human Platelet ◽  
Human Platelets ◽  
Scid Mouse Model

scholarly journals P38 mitogen activated protein kinase inhibitor improves platelet in vitro parameters and in vivo survival in a SCID mouse model of transfusion for platelets stored at cold or temperature cycled conditions for 14 days

PLoS ONE ◽  
2021 ◽  
Vol 16 (5) ◽  
pp. e0250120
Author(s):  
Andrey Skripchenko ◽  
Monique P. Gelderman ◽  
Jaroslav G. Vostal
Keyword(s):  
Mouse Model ◽  
Cold Storage ◽  
Scid Mouse ◽  
Room Temperature ◽  
Human Platelet ◽  
Human Platelets ◽  
Bacterial Proliferation ◽  
Scid Mouse Model

Platelets for transfusion are stored at room temperature (20–24°C) up to 7 days but decline in biochemical and morphological parameters during storage and can support bacterial proliferation. This decline is reduced with p38MAPK inhibitor, VX-702. Storage of platelets in the cold (4–6°C) can reduce bacterial proliferation but platelets get activated and have reduced circulation when transfused. Thermocycling (cold storage with brief periodic warm ups) reduces some of the effects of cold storage. We evaluated in vitro properties and in vivo circulation in SCID mouse model of human platelet transfusion of platelets stored in cold or thermocycled for 14 days with and without VX-702. Apheresis platelet units (N = 15) were each aliquoted into five storage bags and stored under different conditions: room temperature; cold temperature; thermocycled temperature; cold temperature with VX-702; thermocycled temperature with VX-702. Platelet in vitro parameters were evaluated at 1, 7 and 14 days. On day 14, platelets were infused into SCID mice to assess their retention in circulation by flow cytometry. VX-702 reduced negative platelet parameters associated with cold and thermocycled storage such as an increase in expression of activation markers CD62, CD63 and of phosphatidylserine (marker of apoptosis measured by Annexin binding) and lowered the rise in lactate (marker of increase in anaerobic metabolism). However, VX-702 did not inhibit agonist-induced platelet aggregation indicating that it does not interfere with platelet hemostatic function. In vivo, VX-702 improved initial recovery and area under the curve in circulation of human platelets infused into a mouse model that has been previously validated against a human platelet infusion clinical trial. In conclusion, inhibition of p38MAPK during 14-days platelet storage in cold or thermocycling conditions improved in vitro platelet parameters and platelet circulation in the mouse model indicating that VX-702 may improve cell physiology and clinical performance of human platelets stored in cold conditions.

scholarly journals A NOD/SCID mouse model for the assessment of human platelet aggregation in vivo

2012 ◽  
Vol 10 (3) ◽  
pp. 490-492 ◽  
Author(s):  
L. HOLBROOK ◽  
C. MOORE ◽  
D. SANZ-ROSA ◽  
A. SOLOMON ◽  
M. EMERSON
Keyword(s):  
Platelet Aggregation ◽  
Mouse Model ◽  
Scid Mouse ◽  
Human Platelet ◽  
Human Platelet Aggregation ◽  
Scid Mouse Model

Assessment of human platelet survival in the NOD/SCID mouse model: technical considerations

Transfusion ◽  
2016 ◽  
Vol 56 (6) ◽  
pp. 1370-1376 ◽  
Author(s):  
Julia Fuhrmann ◽  
Rabie Jouni ◽  
Jenny Alex ◽  
Heike Zöllner ◽  
Jan Wesche ◽  
...  
Keyword(s):  
Mouse Model ◽  
Scid Mouse ◽  
Human Platelet ◽  
Platelet Survival ◽  
Scid Mouse Model ◽  
Technical Considerations

Role of Il-2, Il-7, and IL-15 in T Cell Mediated Graft-vs-Leukemia Against Human Acute Lymphoblastic Leukemia in a NOD/scid Chimeric Mouse Model.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5256-5256
Author(s):  
Doug Cipkala ◽  
Kelly McQuown ◽  
Lindsay Hendey ◽  
Michael Boyer
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Mouse Model ◽  
Scid Mouse ◽  
In Vitro Cytotoxicity ◽  
Scid Mouse Model

Abstract The use of cytotoxic T-lymphocytes (CTL) has been attempted experimentally with various tumors to achieve disease control. Factors that may influence GVT include CTL cytotoxicity, ability to home to disease sites, and survival of T cells in the host. The objective of our study is to evaluate the GVL effects of human alloreactive CTL against ALL in a chimeric NOD/scid mouse model. CTL were generated from random blood donor PBMCs stimulated with the 697 human ALL cell line and supplemented with IL-2, -7, or -15. CTL were analyzed for in vitro cytotoxicity against 697 cells, phenotype, and in vitro migration on day 14. NOD/scid mice were injected with 107 697 ALL cells followed by 5x106 CTL. Mice were sacrificed seven days following CTL injection and residual leukemia was measured in the bone marrow and spleen via flow cytometry. The ratios of CD8/CD4 positive T cells at the time of injection were 46/21% for IL-2, 52/31% for IL-7, and 45/14% for IL-15 cultured CTL (n=13). Control mice not receiving CTL had a baseline leukemia burden of 2.01% and 0.15% in the bone marrow and spleen, respectively (n=15). Mice treated with IL-15 cultured CTL had a reduction in tumor burden to 0.2% (n=13, p=0.01) and 0.05% (n=13, p=0.01) in bone marrow and spleen, respectively. Those treated with IL-2 or IL-7 cultured CTL showed no significant difference in leukemia burden in either the bone marrow (IL-2 1.28%, Il-7 5.97%) or spleen (IL-2 0.4%, IL-7 0.33%). No residual CTL could be identified in the bone marrow or spleen at the time of sacrifice in any CTL group. CTL grown in each cytokine resulted in similar in vitro cytotoxicity at an effector:target ratio of 10:1 (IL-2 41.3%, IL-7 37.7%, IL-15 45.3%, n=12–15, p>0.05 for all groups) and had statistically similar intracellular perforin and granzyme-B expression. In vitro CTL migration to a human mesenchymal stem cell line was greatest with IL-15 CTL (30.5%, n=4), followed by IL-7 CTL (18.9%, n=4), and least in IL-2 CTL (17.9%, n=4), though the differences were not significant. In vitro CTL migration was analyzed to an SDF-1α gradient as CXCR4/SDF-1α interactions are necessary for hematopoietic progenitor cell homing to the bone marrow. IL-15 cultured CTL showed the highest migration (48.8%, n=8) as compared to IL-2 (21.7%, n=6, p=0.048) or IL-7 CTL (35.9%, n=8, p>0.05). However, surface expression of CXCR4 measured by flow cytometry was significantly higher in IL-7 CTL (89.4%, n=9) compared to IL-2 CTL (52.2%, n=9, p<0.001) and IL-15 CTL (65.4%, n=10, p=0.002). Experiments are currently underway to further evaluate the role of CXCR4/SDF-1α in GVL. Preliminary in vivo experiments do not suggest any significant differences in CTL engraftment when evaluated at 24 hours post injection. Expression of the anti-apoptotic bcl-2 protein was greatest on IL-7 (MFI=5295, n=13) and IL-15 (MFI=4865, n=14) when compared to IL-2 CTL (MFI=3530, n=13, p=0.02 vs. IL-7, p=0.05 vs. IL-15), suggesting an increased in vivo survival ability. We hypothesize that IL-15 cultured CTL have greater GVL effects due to either higher in vivo survival, greater bone marrow homing efficiency, or both. Future experiments are planned to evaluate in vivo administration of IL-2 to enhance CTL survival in the host. In conclusion, IL-15 cultured CTL had significantly greater in vivo GVL effects compared to IL-2 and IL-7 CTL in the NOD/scid mouse model. This model can be utilized to evaluate the mechanism of T cell mediated GVL against ALL and potentially other human malignancies.

Transgenic Expression of Inducible Caspase9 Suicide Gene for In Vivo Elimination of Antigen Specific Cytotoxic T Cells (CTLs) Engineered To Produce Cytokines.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 137-137
Author(s):  
C. Quintarelli ◽  
V. Juan ◽  
B. Savoldo ◽  
G. Giordano ◽  
A. Foster ◽  
...  
Keyword(s):  
T Cells ◽  
Mouse Model ◽  
Scid Mouse ◽  
Cytotoxic T Cells ◽  
Safety Margin ◽  
Suicide Gene ◽  
Cytokine Release ◽  
Transgenic Expression ◽  
Scid Mouse Model

Abstract Transgenic expression of cytokines such as IL2 and IL15 by tumor specific cytotoxic T cells (CTLs) can improve the survival and expansion of these cells within the tumor microenvironment. Using our model of tumor antigen-Epstein-Barr virus-specific CTLs (EBV-CTLs), we found that transgenic production of either cytokine could sustain CTL expansion ex vivo, and improve their anti-tumor effect in vivo in a SCID mouse model. Although the proliferation of cytokine gene transduced CTLs remained antigen dependent, clinical application of this approach likely requires the inclusion of a suicide gene to deal with the potential development of T-cell mutants with autonomous, antigen-independent, growth. We constructed a novel suicide gene based on the inducible caspase9 gene [Straathof et al Blood 2005:105;4247] and showed that EBV-CTLs expressing this suicide gene can be eliminated after exposure to the small molecule chemical inducer of dimerization (CID) AP20187. However, it has been suggested that the anti-apoptotic effects of transgenic cytokine expression would counteract the pro-apoptotic action of icaspase9. To discover the general applicability of our inducible suicide gene approach, we generated tricistronic retroviral vectors encoding full length hIL2 or hIL15, a truncated human CD34 molecule (DCD34) as a marker, and the inducible Caspase9 suicide gene. The three genes were linked using 2A peptide cleavable sequences, which allows equal expression of the 3 transgenes. Established EBV-CTLs were transduced either with IL2.DCD34.iCasp9 or IL15.DCD34.iCasp9 or DCD34 empty vector. After stimulation with the antigen (LCLs) at a E:T ratio of 1:1, we observed significant expansion of CTLs/IL2.DCD34.iCasp9 (221 fold expansion, range 84–452) and IL15.DCD34.iCasp9 (182, range 48–355) after 28 days of culture, while CTLs/DCD34 maintained without cytokines had <2 fold expansion. Cytokine release after antigen stimulation was 6.4±3.4 pg/mL and 157±99pg/mL for IL15 and IL2, respectively. Transgenic CTLs maintained the same phenotype as control T cells (>90% CD3+/CD8+) and remained polyclonal as assessed by staining for the TCRVb repertoire. To evaluate the cytotoxic capacity of the suicide gene, control and transgenic CTLs were incubated with CID AP20187 at 20nM. After 24 hours the survival of transgenic CTLs was estimated from residual CD34+ expression. For both CTLs/IL2.DCD34.iCasp9 and CTLs/IL15.DCD34.iCasp9 the percentage of CD34+ cells was reduced by >1.5 logs. Moreover, the few residual cells were CD34dim, and produced no measurable cytokine release as measured by ELISA. To assess activity in vivo, we used an LCL-engrafted SCID mouse model, injected with CTLs transgenic for the firefly-luciferase gene. These cells were tracked in vivo using the Xenogen-IVIS bioluminescence system. We found that CTLs transgenic for either IL2 or IL15 both migrated to the tumor site, and had increased expansion compared to DCD34 control CTLs. Mice were then treated with 2 −3 doses of CID AP20187 (50 mg/mice i.p). The bioluminescence signal diminished to background levels by 48hr, suggesting efficient elimination of transgenic CTLs. In conclusion, these data indicate that induction of a transgenic caspase9 suicide gene can effectively destroy adoptively transferred T cells, even when these lymphocytes are expressing anti-apoptotic cytokines such as IL2 and IL15. This approach may increase the safety margin of gene modified CTL therapies.

Drug-induced thrombocytopenia: development of a novel NOD/SCID mouse model to evaluate clearance of circulating platelets by drug-dependent antibodies and the efficacy of IVIG

Blood ◽  
2010 ◽  
Vol 116 (11) ◽  
pp. 1958-1960 ◽  
Author(s):  
Simon X. Liang ◽  
Mykola Pinkevych ◽  
Levon M. Khachigian ◽  
Christopher R. Parish ◽  
Miles P. Davenport ◽  
...  
Keyword(s):  
Mouse Model ◽  
Scid Mouse ◽  
Immune Thrombocytopenia ◽  
Human Platelets ◽  
Ivig Treatment ◽  
Drug Induced ◽  
Beneficial Effects ◽  
Nonobese Diabetic ◽  
Scid Mouse Model ◽  
Drug Dependent

Abstract Drug-induced immune thrombocytopenia (DITP) is an adverse drug effect mediated by drug-dependent antibodies. Intravenous immunoglobulin (IVIG) is frequently used to treat DITP and primary immune thrombocytopenia (ITP). Despite IVIG's proven beneficial effects in ITP, its efficacy in DITP is unclear. We have established a nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model of DITP in which human platelets survive for more than 24 hours, allowing platelet clearance by DITP/ITP antibodies to be studied. Rapid human platelet clearance was uniformly observed with all quinine-induced thrombocytopenia (QITP) patient sera studied (mean platelet lifespans: QITP 1.5 ± 0.3 hours vs controls 16.5 ± 4.3 hours), consistent with the clinical presentation of DITP. In contrast, clearance rates with ITP antibodies were more variable. IVIG treatment partially prevented platelet clearance by DITP and ITP antibodies. Our results suggest that the NOD/SCID mouse model is useful for investigating the efficacy of current and future DITP therapies, an area in which there is little experimental evidence to guide treatment.

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