PRT060318, a Novel Syk Inhibitor, Prevents Heparin-Induced Thrombocytopenia in a Transgenic Mouse Model

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 269-269 ◽  
Author(s):  
Michael P. Reilly ◽  
Uma Sinha ◽  
Pierrette Andre ◽  
Scott M. Taylor ◽  
Yvonne Pak ◽  
...  
Keyword(s):  
Body Weight ◽  
Mouse Model ◽  
Transgenic Mouse ◽  
Platelet Activation ◽  
Transgenic Mouse Model ◽  
Heparin Induced Thrombocytopenia ◽  
Syk Inhibitor ◽  
Induced Aggregation

Abstract Heparin-induced thrombocytopenia (HIT), in which patients develop antibodies to complexes formed by heparin and platelet factor 4 (PF4), is the most frequent drug-induced immune thrombocytopenia. Extensive studies in vitro and our previous studies in vivo using a transgenic mouse model of HIT have shown that antibodies reactive with heparin-PF4 complexes lead to FcgRIIa receptor-mediated platelet activation. In this study we investigated whether PRT060318 (PRT318), a novel Syk inhibitor, prevents HIT antibody-mediated platelet activation both in vitro and in vivo. PRT318 at concentrations of 0.3 to 3 μM completely inhibited HIT immune complex (IC)-induced aggregation in both human and transgenic mouse platelets. In the absence of the inhibitor, HIT IC-induced final aggregation was 50–60%. At concentrations of PRT318 less than 0.1 μM, or in the presence of vehicle only, there was no inhibition of aggregation. Aggregation was not inhibited by PRT318 at any concentration when platelets were stimulated by ADP (5–20 μM final concentration). We also show that PRT318 prevents HIT IC-induced thrombocytopenia in vivo using a transgenic mouse HIT model. All mice were treated with KKO, a mouse monoclonal HIT antibody. On days 1 to 4 following antibody injection, the experimental group (n = 13) received orally dosed PRT318 (30 mg/kg body weight) twice a day by gavage while the control group (n = 11) was similarly treated with vehicle only (water). Both experimental and control mice were injected with heparin (1600 U/kg body weight, SQ, once daily). Nadir platelet counts of PRT318-treated mice were significantly higher than control mice (89.8 ± 1.1% of baseline vs. 48.8 ± 6.7%; p = 0.00003). The PRT318 concentration, 2 hrs post dose, in mouse plasma from treated mice was measured as 7.1 μM, consistent with the concentration which blocked FcgRIIa-mediated platelet activation in vitro. These studies demonstrate that Syk inhibitor PRT318 is an active agent in HIT. Figure Figure

scholarly journals PRT-060318, a novel Syk inhibitor, prevents heparin-induced thrombocytopenia and thrombosis in a transgenic mouse model

Blood ◽  
2011 ◽  
Vol 117 (7) ◽  
pp. 2241-2246 ◽  
Author(s):  
Michael P. Reilly ◽  
Uma Sinha ◽  
Pierrette André ◽  
Scott M. Taylor ◽  
Yvonne Pak ◽  
...  
Keyword(s):  
Mouse Model ◽  
Transgenic Mouse ◽  
Immune Complex ◽  
Transgenic Mouse Model ◽  
Control Group ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Syk Inhibitor ◽  
Induced Aggregation

AbstractHeparin-induced thrombocytopenia (HIT) is a major cause of morbidity and mortality resulting from the associated thrombosis. Extensive studies using our transgenic mouse model of HIT have shown that antibodies reactive with heparin-platelet factor 4 complexes lead to FcγRIIA-mediated platelet activation in vitro as well as thrombocytopenia and thrombosis in vivo. We tested PRT-060318 (PRT318), a novel selective inhibitor of the tyrosine kinase Syk, as an approach to HIT treatment. PRT318 completely inhibited HIT immune complex-induced aggregation of both human and transgenic HIT mouse platelets. Transgenic HIT model mice were treated with KKO, a mouse monoclonal HIT-like antibody, and heparin. The experimental group received orally dosed PRT318, whereas the control group received vehicle. Nadir platelet counts of PRT318-treated mice were significantly higher than those of control mice. When examined with a novel thrombosis visualization technique, mice treated with PRT318 had significantly reduced thrombosis. The Syk inhibitor PRT318 thus prevented both HIT immune complex-induced thrombocytopenia and thrombosis in vivo, demonstrating its activity in HIT.

scholarly journals Optogenetic Modulation of Cortical Neurons Using Organic Light Emitting Diodes (OLEDs)

2019 ◽  
Author(s):  
Arati Sridharan ◽  
Ankur Shah ◽  
Swathy Sampath Kumar ◽  
James Kyeh ◽  
Joseph Smith ◽  
...  
Keyword(s):  
Mouse Model ◽  
Transgenic Mouse ◽  
Cortical Neurons ◽  
Microelectrode Array ◽  
Light Emitting Diodes ◽  
Green Light ◽  
Transgenic Mouse Model ◽  
Light Emitting

ABSTRACTObjectiveThere is a need for low power, scalable photoelectronic devices and systems for emerging optogenetic needs in neuromodulation. Conventional light emitting diodes (LEDs) are constrained by power and lead-counts necessary for scalability. Organic LEDs (OLEDs) offer an exciting approach to decrease power and lead-counts while achieving high channel counts on thin, flexible substrates that conform to brain surfaces or peripheral neuronal fibers. In this study, we investigate the potential for using OLEDs to modulate neuronal networks cultured in vitro on a transparent microelectrode array (MEA) and subsequently validate neurostimulation in vivo in a transgenic mouse model.ApproachCultured mouse cortical neurons were transfected with light-sensitive opsins such as blue-light sensitive channel-rhodopsin (ChR2) and green-light sensitive chimeric channel-rhodopsin (C1V1tt) and stimulated using blue and green OLEDs (with 455 and 520 nm peak emission spectra respectively) at a power of 1 mW/mm2 under pulsed conditions.Main resultsWe demonstrate neuromodulation and optostimulus-locked, single unit-neuronal activity in neurons expressing stimulating and inhibiting opsins (n=4 MEAs, each with 16 recordable channels). We also validated the optostimulus-locked response in a channel-rhodopsin expressing transgenic mouse model, where at least three isolatable single neuronal cortical units respond to OLED stimulation.SignificanceThe above results indicate the feasibility of generating sufficient luminance from OLEDs to perform neuromodulation both in vitro and in vivo. This opens up the possibility of developing thin, flexible OLED films with multiple stimulation sites that can conform to the shape of the neuronal targets in the brain or the peripheral nervous system. However, stability of these OLEDs under chronic conditions still needs to be carefully assessed with appropriate packaging approaches.

The Syk inhibitor fostamatinib disodium (R788) inhibits tumor growth in the Eμ- TCL1 transgenic mouse model of CLL by blocking antigen-dependent B-cell receptor signaling

Blood ◽  
2010 ◽  
Vol 116 (23) ◽  
pp. 4894-4905 ◽  
Author(s):  
Mirza Suljagic ◽  
Pablo G. Longo ◽  
Sara Bennardo ◽  
Emerald Perlas ◽  
Giuseppe Leone ◽  
...  
Keyword(s):  
Mouse Model ◽  
B Cell ◽  
Transgenic Mouse ◽  
Cell Receptor ◽  
Transgenic Mouse Model ◽  
B Cell Receptor ◽  
Promising Therapeutic Approach ◽  
Bcr Signaling ◽  
Syk Inhibitor

Abstract Inhibition of antigen-dependent B-cell receptor (BCR) signaling is considered a promising therapeutic approach in chronic lymphocytic leukemia (CLL), but experimental in vivo evidence to support this view is still lacking. We have now investigated whether inhibition of BCR signaling with the selective Syk inhibitor fostamatinib disodium (R788) will affect the growth of the leukemias that develop in the Eμ-TCL1 transgenic mouse model of CLL. Similarly to human CLL, these leukemias express stereotyped BCRs that react with autoantigens exposed on the surface of senescent or apoptotic cells, suggesting that they are antigen driven. We show that R788 effectively inhibits BCR signaling in vivo, resulting in reduced proliferation and survival of the malignant B cells and significantly prolonged survival of the treated animals. The growth-inhibitory effect of R788 occurs despite the relatively modest cytotoxic effect in vitro and is independent of basal Syk activity, suggesting that R788 functions primarily by inhibiting antigen-dependent BCR signals. Importantly, the effect of R788 was found to be selective for the malignant clones, as no disturbance in the production of normal B lymphocytes was observed. Collectively, these data provide further rationale for clinical trials with R788 in CLL and establish the BCR-signaling pathway as an important therapeutic target in this disease.

scholarly journals Bystander Activation of Cytotoxic T Cells: Studies on the Mechanism and Evaluation of In Vivo Significance in a Transgenic Mouse Model

1997 ◽  
Vol 185 (7) ◽  
pp. 1241-1252 ◽  
Author(s):  
Stephan Ehl ◽  
Joachim Hombach ◽  
Peter Aichele ◽  
Hans Hengartner ◽  
Rolf M. Zinkernagel
Keyword(s):  
T Cells ◽  
Mouse Model ◽  
Transgenic Mice ◽  
Vaccinia Virus ◽  
Transgenic Mouse ◽  
Rare Event ◽  
Transgenic Mouse Model ◽  
Bystander Activation

Bystander activation, i.e., activation of T cells specific for an antigen X during an immune response against antigen Y may occur during viral infections. However, the low frequency of bystander-activated T cells has rendered it difficult to define the mechanisms and possible in vivo relevance of this nonspecific activation. This study uses transgenic mice expressing a major histocompatibility complex class I–restricted TCR specific for glycoprotein peptide 33-41 of lymphocytic choriomeningitis virus (LCMV) to overcome this limitation. CD8+ T cells from specific pathogen-free maintained, unimmunized “naive” TCR transgenic mice can differentiate into LCMV-specific cytolytic effector CTL during infections with vaccinia virus or Listeria monocytogenes in vivo or mixed lymphocyte culture in vitro. We show that in these model situations (a) nonspecifically activated CTL are able to confer antiviral protection in vivo, (b) bystander activation is largely independent of the expression of a second T cell receptor of different specificity, (c) bystander activation is not mediated by a broadly cross-reactive TCR, but rather by cytokines, (d) bystander activation can be mediated by cytokines such as IL-2, but not α/β-IFN in vitro; (e) bystander activation is, overall, a rare event, occuring in vivo in roughly 1 in 200 of the LCMV-specific CTL during infection of TCR transgenic mice with vaccinia virus; (f) bystander activation does not have a significant functional impact on nontransgenic CTL memory under the conditions tested; and (g) even in the TCR transgenic situation, where unphysiologically high numbers of T cells of a single specificity are present, bystander activation is not sufficient to cause clinically manifest autoimmune disease in a transgenic mouse model of diabetes. We conclude that although bystander activation via cytokines may generate cytolytically active CTL from naive precursors, quantitative considerations suggest that this is usually not of major biological consequence.

Hemopoietic lineage commitment decisions: in vivo evidence from a transgenic mouse model harboring μLCR-βpro-LacZ as a transgene

Blood ◽  
2000 ◽  
Vol 95 (4) ◽  
pp. 1274-1282 ◽  
Author(s):  
Thalia Papayannopoulou ◽  
Gregory V. Priestley ◽  
Alex Rohde ◽  
Kenneth R. Peterson ◽  
Betty Nakamoto
Keyword(s):  
Mouse Model ◽  
Transgenic Mouse ◽  
Fetal Liver ◽  
Transgenic Mouse Model ◽  
In Vitro Assays ◽  
Live Cells ◽  
Published Data ◽  
Lineage Specific Genes

A substantial body of published data suggests activation of lineage-specific genes in multipotential hemopoietic cells before their unilineage commitment. Because the behavior and plasticity of cells isolated in vitro away from microenvironmental constraints exercised in vivo may be altered, one wonders whether similar findings can be observed in a physiologic setting in vivo. We used a transgenic mouse model harboring human micro LCR together with β promoter sequences as a transgene to examine activation of lineage-specific programs in vivo. By using LacZ as a reporter, we had the ability to detect, quantitate, and select live cells with different levels of LacZ activation. We found strong expression of LacZ by X-gal staining in 2 lineages—erythroid and megakaryocytic. Activation in the latter was a novel finding not previously observed when similar transgenes were used. We also found activation of μLCR-βpro at low levels in progenitor cells of granulocytic-macrophagic, erythroid, or megakaryocytic lineage detected by in vitro assays, suggesting activation before commitment to a specific lineage pathway. In particular, the expression of LacZ was graded among progenitors, so that in a proportion of them activation occurred only after commitment to erythroid or megakaryocytic lineage. In addition, we found quantitative reduction in LacZ expression between fetal liver and bone marrow-derived cells, the basis of which is unclear. Collectively our data provide in vivo evidence supporting the view that lineage-specific genes are expressed in a graded fashion in pluripotential cells before their irreversible unilineage commitment.

scholarly journals Tau Secretion and Propagation Is Regulated by p300/CBP via Autophagy-Lysosomal Pathway in Tauopathy

2018 ◽  
Author(s):  
Xu Chen ◽  
Yaqiao Li ◽  
Chao Wang ◽  
Yinyan Tang ◽  
Sue-Ann Mok ◽  
...  
Keyword(s):  
Mouse Model ◽  
Transgenic Mouse ◽  
Transgenic Mouse Model ◽  
Cellular Mechanism ◽  
Autophagic Flux ◽  
Impaired Function ◽  
Unconventional Secretion ◽  
Lysine Acetyltransferase

SUMMARYThe trans-neuronal propagation of tau has been implicated in the progression of tau-mediated neurodegeneration. Tau secretion from neurons is the first step in tau transmission, but little is known about the cellular mechanism. Here, we report that p300/CBP, the lysine acetyltransferase that acetylates tau and regulates its homeostasis and toxicity, serves as a key regulator of tau secretion by inhibiting the autophagy-lysosomal pathway (ALP). Increased p300/CBP activity was associated with impaired function of this pathway in a tau transgenic mouse model. p300/CBP hyperactivation increased tau secretion by blocking autophagic flux. Conversely, inhibiting p300/CBP genetically or pharmacologically promoted autophagic flux, and reduced tau accumulation, tau secretion, and tau propagation in fibril-induced tau spreading models in vitro and in vivo. Our findings show that p300/CBP-induced impairment in the ALP underlies excessive unconventional secretion and pathogenic spread of tau.

The TCL-1 Transgenic Mouse Is an Effective Tool for Pre-Clinical Drug Development in Chronic Lymphocytic Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 51-51 ◽  
Author(s):  
Amy J. Johnson ◽  
Ryan Edwards ◽  
Lisa L. Smith ◽  
David M. Lucas ◽  
Carlo M. Croce ◽  
...  
Keyword(s):  
Mouse Model ◽  
Protein Binding ◽  
Transgenic Mouse ◽  
Plasma Levels ◽  
Transgenic Mouse Model ◽  
In Vitro Cytotoxicity ◽  
Leukemia Cells ◽  
Preclinical Studies

Abstract A TCL-1 transgenic mouse model of CLL was recently described that develops a slowly proliferating CD19+/CD5+/IgM+ clonal leukemia at 10–13 months of age, characterized by lymphocytosis, splenomegaly, and lymphadenopathy. To determine if this model has therapeutic properties similar to human CLL, we performed in vitro cytotoxicity assays using splenic B-cells isolated from leukemic TCL-1 mice. Results indicated that fludarabine, a common treatment for CLL, as well as experimental agents, 17-AAG, valproic acid, flavopiridol, and a novel kinase inhibitor OSU03012, each have activity against the mouse TCL-1-mediated leukemia at concentrations similar to that observed in human CLL cells in vitro. Subsequent in vivo studies in TCL-1 transgenic mice with evidence of leukemia were performed with low dose fludarabine (34 mg/kg IP, day 1–5, every month) versus saline control (n=10 per group) and were treated for three cycles. Mice receiving fludarabine had a notable decline in blood lymphocyte count. Furthermore, the median survival time for the fludarabine treated mice was 61 days, as compared to 31 days for the control mice (p=0.046). However, as seen in human CLL, treated mice became resistant to fludarabine over time as determined by lymphocyte counts. Thus, our preliminary data suggest the TCL-1 transgenic mouse model of CLL has a similar therapeutic response pattern to human CLL. We next assessed the ability of a novel PDK1/AKT inhibitor OSU03012 to mediate in vivo activity in this model. This agent previously has been shown to mediate activity against both CLL cells and TCL-1 leukemia cells in vitro through a caspase and bcl-2 independent pathway. Surprisingly, OSU03012 demonstrated no early or late in vivo activity at a dose (200 mg/kg) previously demonstrated to inhibit prostate xenograft tumor growth in vivo. The lack of efficacy with this compound in the TCL-1 transgenic mouse prompted us to examine in vivo properties such as protein binding, non-linear absorption of drug that could prevent attaining the concentration of free drug necessary to mediate apoptosis, or stromal cell contact that could promote cellular resistance to OSU03012. As measured using a liquid chromatography/tandem mass spectrometry assay, plasma levels of OSU03012 reached peak levels of 7.5–10μM in the treated mice. Interestingly, very high protein binding of OSU03012 to murine plasma (98–99%) was detected. This finding was further corroborated by parallel in vitro cytotoxicity studies in fetal bovine serum and mouse serum, in which the IC50 concentrations were 3.558μM and 14.15μM, respectively. These studies indicated that plasma levels of OSU03012 sufficient to mediate cytotoxicity against TCL-1 leukemia cells were not achieved in vivo. A second therapeutic study using higher doses of OSU03012 is underway to attain these higher concentrations to confirm in vivo activity of OSU03012. Preclinical studies of other highly effective agents (e.g., Flavopiridol) have recently demonstrated the importance of defining differences in plasma protein binding and schedule of administration of novel agents. It is important to consider the impact of plasma protein binding in designing early phase I studies in patients. Hopefully, this murine model will facilitate future preclinical studies to optimize the schedule and dose of drug administration to confirm the in vivo activity of promising new agents, including OSU03012.

The Syk Inhibitor R788 (FosD) Inhibits Tumor Growth in the TCL1 Transgenic Mouse Model of CLL by Blocking Antigen-Dependent BCR Signaling.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 887-887
Author(s):  
Mirza Suljagic ◽  
Pablo G Longo ◽  
Luca Laurenti ◽  
Dimitar G Efremov
Keyword(s):  
B Cells ◽  
Mouse Model ◽  
B Cell ◽  
Cytotoxic Effect ◽  
Transgenic Mouse Model ◽  
Control Group ◽  
Bcr Signaling ◽  
Syk Inhibitor

Abstract Abstract 887 CLL B-cells depend on various signals from the microenvironment for survival and proliferation. Among these, antigenic stimuli that are propagated through the B-cell receptor (BCR) are considered particularly important for the development and progression of CLL, suggesting that the BCR signaling pathway could be an important target for therapeutic intervention. We have previously characterized some of the critical components of the signaling pathway downstream of the BCR in CLL B cells and identified the protein tyrosine kinase Syk as a promising therapeutic target. In a recent study we showed that CLL B-cells frequently have increased basal/constitutive Syk activity and are moderately sensitive to the cytotoxic effect of the selective Syk inhibitor R406 [Gobessi et al, Leukemia 2009]. More importantly, the survival signal induced by sustained BCR engagement was completely abolished by R406, suggesting that this compound may exert an even greater effect in vivo by inhibiting antigen-dependent Syk activation. We have now tested this possibility in the Eμ-TCL1 transgenic mouse model of CLL. Aged Eμ-TCL1 mice develop CD5+ B-cell leukemias that, similar to aggressive human CLL, show features of an antigen-driven process, including expression of stereotyped BCRs and reactivity with common autoantigens and microbial agents [Yan et al, Proc Natl Acad Sci USA 2006]. For our experiments we used a TCL1 leukemia (TCL1-002) that does not grow in vitro, but can be propagated in syngeneic recipients in vivo. TCL1-002 cells express an unmutated stereotyped BCR encoded by the VH12/VK4 combination, which reacts with phosphatidylcholine, an autoantigen exposed on the surface of senescent erythrocytes. In vitro experiments showed that R406 is not cytotoxic for TCL1-002 cells, although it completely inhibited both the basal and BCR-induced activation of signaling pathways downstream of Syk. The absence of a direct cytotoxic effect provided a unique opportunity to investigate whether inhibition of BCR signaling will affect leukemia growth in vivo. For this purpose, 1×107 TCL1-002 cells were injected intraperitoneally in 18 syngeneic mouse recipients. Three days later treatment was started in 8 mice with R788, which is the water-soluble prodrug of R406, at a daily dose of 80mg/kg during 18 consecutive days. Because of the rapid clearance of the drug (serum half-life <2 hours) R788 was administered in 3 divided doses at 4 hour intervals. Two weeks after the end of treatment leukemia developed in all mice from the control group (median WBC counts 131×106/ml, range 12-300×106/ml), whereas all R788-treated mice showed normal WBC numbers (median 6×106/ml, range 3-8×106/ml, P<0.001). Three weeks later all mice in the control group had died (median survival 46 days), whereas all mice in the R788 group were still alive and only two of them had detectable leukemic cells. R788 also showed some efficacy in the treatment of mice with overt TCL1-002 leukemias (WBC >50×106/ml). Whereas all mice from the control group (n=9) died between 6 and 18 days from the beginning of therapy, 4 out of 9 mice from the R788 group survived for more than 33 days. The mechanism of R788 activity was primarily related to inhibition of leukemic cell proliferation, as evidenced by a substantial decrease in the percentage of Ki67-positive cells after 7 days of treatment (30% before, 5% after therapy, P<0.001). To investigate whether R788 will also be effective against other TCL1 tumors we treated five TCL1 mice with preleukemic mono- or oligoclonal B-cell expansions during a four week period. R788 reduced the percentage of CD5+/B220+ cells in 2 cases, whereas in 2 other cases the percentage increased. Interestingly, the pattern of clonal Ig gene rearrangements changed during therapy, suggesting that only certain TCL1 clones are sensitive to R788 treatment. In summary, this study shows that R788 can effectively inhibit the growth of certain TCL1 tumors and provides the first in vivo experimental evidence suggesting that inhibition of antigen-dependent BCR signaling could be an effective therapeutic approach in CLL. Disclosures: No relevant conflicts of interest to declare.

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