A Novel Animal Model for Detecting Damage to Human Platelet Transfusion Products: In Vivo Recovery and Survival in Severe Combined Immunodeficient Mice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1891-1891
Author(s):  
John T. Piper ◽  
Jaroslav G. Vostal
Keyword(s):  
Animal Model ◽  
Human Platelets ◽  
Human Cells ◽  
Scid Mice ◽  
Survival Times ◽  
Post Infusion ◽  
The Impact ◽  
Induced Aggregation

Abstract Clinical performance of platelet products processed or stored under novel conditions is difficult to predict based on in vitro studies alone. Evaluation of such products involves determination of recovery and survival of radiolabeled platelets in human volunteers as a surrogate endpoint for platelet efficacy. Such human studies pose some risk to volunteers, are a financial burden on the sponsor, and stifle innovation in the development of platelet products. The development of an animal model for evaluating human platelets has been limited by rapid, immunemediated clearance of human cells. In the current studies, severe combined immunodeficient (SCID) mice were used to circumvent the need to block the reticuloendothelial system and prolong circulation of human cells. Human platelets were infused via tail vein into normal and SCID mice, and the recoveries and survival times compared. Mouse whole blood was collected at various time points post-infusion, and human platelets were detected by flow cytometry using an anti-human CD41 monoclonal antibody. Recovery was defined as percent human platelets in circulation relative to time zero, and survival time in circulation as the t1/2 of the human platelets. Recoveries and survival times were different between normal and SCID mice, with a maximal difference in recovery of 60.3% at 4 hours post-infusion (normal recovery, 11.1 ± 9.1%; SCID recovery, 71.4 ± 8.8%), and survival times of 1.4 ± 0.4 hours and 10.7 ± 2.3 hours in normal and SCID mice, respectively (N=3). Chemically treated and aged platelets were used to evaluate the ability of the model to detect differences in control and damaged platelets. Chemical damage was induced by carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler which mimics the platelet storage lesion. Platelets were exposed to 10 μM CCCP in methanol, control platelets were exposed to an equal volume of methanol (N=3). CCCP treatment of platelets decreased agonist-induced aggregation (Control aggregation, 73.3 ± 6.8%; CCCP-treated platelet aggregation, 13.8 ± 5.3%). Recovery of control and CCCP-treated platelets were 31.5 ± 16.9% and 7.9 ± 5.1%, respectively, at 4-hours post-infusion. Survival times were 1.3 hours for control and 1.9 hours for CCCP-treated platelets. For storage studies, in vitro cell quality parameters were evaluated in three products, and each product was infused into 3 animals on Day 1 and 3 different animals on Day 7. In Day 7 platelets, in vitro platelet parameters were decreased compared to Day 1. Platelet counts decreased an average of 22.8% ± 2.2% between Day 1 and Day 7. pH decreased from 6.7 ± 0.1 at Day 1 to 5.8 ± 0.1 at Day 7. All platelet products had visible swirl on Day 1 and no swirl on Day 7. Platelets stored for 7 days showed decreased recovery over Day 1 platelets at 4 hours post-infusion (Day 1, 66.9 ± 12.8%; Day 7, 0.2 ± 0.08%). The SCID mouse may be a useful model for evaluating the impact of new technologies (apheresis devices, anticoagulants, storage containers, pathogen inactivation systems) on the in vivo efficacy of human platelets. In two different models of platelet damage (chemical and storage induced damage), this model can distinguish between normal and damaged platelets. Recovery of Infused Day 1 and Day 7 Human Platelets in SCID Mice Recovery of Infused Day 1 and Day 7 Human Platelets in SCID Mice

Comparison of UVA and UVB Induced Lesions in Human Platelets Evaluated in an Animal Model.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2883-2883 ◽  
Author(s):  
Monique P. Gelderman-Fuhrmann ◽  
Jessica L. Lewis ◽  
Elisabet K. Tassis ◽  
Alex C. Vostal ◽  
Jaroslav G. Vostal
Keyword(s):  
Animal Model ◽  
Scid Mouse ◽  
Human Platelet ◽  
Uv Light ◽  
Human Platelets ◽  
Scid Mice ◽  
Uv Exposure ◽  
Post Infusion ◽  
In Vivo Recovery

Abstract UV light can be used to irradiate platelets to reduce immune responses and activate chemically-mediated pathogen reduction agents. Platelets subjected to UV-based processing methods develop demonstrable losses of in vivo performance. We compared the “lesions” induced by the different wavelengths of UV light on human platelet performance in a SCID mouse animal model of recovery and survival. Previously we demonstrated that severe combined immunodeficient (SCID) mice could be used as an animal model to identify both severely damaged (Blood 106(11), p537a, 2005) and moderately damaged human platelets (Blood 108(11), p175–176a, 2006). Apheresis human platelets, stored for 1 or 7 days, were exposed to UVA(320–400 nm) or UVB(290–320 nm) light for 20 min (4.8 J/mL) or 40 min (9.6 J/mL). Control platelets were processed in the same manner without UV exposure. Effects on platelets were compared on platelet counts, activation measured by p-selectin (anti-human CD62P, clone AK-4) and in vivo recovery. Platelet counts and in vivo recovery are expressed as % of control platelets, p-selectin measurement represents % of cells expressing the antigen. For in vivo recovery, approximately 1×10 9 platelets (UV-treated or control) were injected into the tail vein of SCID mice (n=4 per each condition) and serial blood samples were collected. Human platelets were detected in mouse whole blood by flow cytometry using an anti-human GPIIbIIIa mAb (clone P2). Recovery was defined as percent of human platelets in mouse circulation 30 minutes post infusion. Comparison of recovery between control and UV treatment platelets was done at 2 hours post infusion as shown in Table 1. These results indicate that UVA produces less activation of platelets and less damage to human platelets recognized by the in vivo model than UVB. The UVA lesion detected by increased in vivo clearance is not cumulative in that longer exposures do not cause an increased loss of in vivo recovery. In comparison, UVB mediated damage is associated with lower in vivo recovery and the damage appears to be cumulative with longer exposure. These differences suggest that UVA and UVB exposure may produce platelet lesions through different mechanism(s). Further investigation into the molecular mechanisms of UVA and UVB lesions may lead to methods that could reduce the negative aspects of UV exposure. The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy. Table 1 Treatment Day 1 Day 7 1= percent of control platelets;2=human platelet recovery in SCID mouse model Platelet count1 P-selectin (%) In vivo recovery2 Platelet count1 P-selectin (%) In vivo recovery2 UVA 20 min 100 13.3±2.7 55.2±7.6 100 20.3±3.8 58.2±4.9 40 min 100 9.9±0.9 55.5±2.9 100 17.3±2.9 54.3±6.7 UVB 20 min 82±3.7 34.6±5.0 30.8±8.9 86±5.3 51.1±6.4 18.5±4.6 40 min 51±10.1 29.5±1.5 6.9±2.6 63±10.5 36.2±1.8 2.47±0.9

Detection of UV Induced Damage to Human Platelets by an In Vivo Animal Model.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 582-582
Author(s):  
Monique P. Gelderman-Fuhrmann ◽  
Sheena F. Siddiqui ◽  
Jaroslav G. Vostal
Keyword(s):  
Animal Model ◽  
Scid Mouse ◽  
Uv Light ◽  
Human Platelets ◽  
In Vitro Tests ◽  
Post Infusion ◽  
Scid Mouse Model ◽  
In Vivo Recovery

Abstract Evaluation of novel platelet products for transfusion currently involves determination of recovery and survival of radiolabelled platelets in human volunteers since in vitro studies are not predictive of platelet clinical performance and a practical animal model has not been universally accepted. We previously demonstrated that severe combined immunodeficient (SCID) mice could be used as a model to identify severely damaged human platelets (Blood 106 (11), p537a, 2005). To further characterize the sensitivity of this model we used exposure of human platelets to UV light to induce moderate damage, and quantitated this lesion by in vitro tests and in vivo recovery in the SCID mouse model. This type of damage is clinically relevant since UV radiation of blood-platelet concentrates is used to prevent the development of post-transfusion alloimmunization and has been used as part of pathogen reduction systems to inactivate bacteria and viruses. Apheresis platelet products, stored for 1 or 7 days, were exposed to UV light (280–315 nm) for either 20 or 40 min (intensity: 750 μW/cm2). In vitro testing included platelet count, pH, CD62P expression, mitochondrial membrane potential and collagen-induced (2 mg/ml) aggregation. The mitochondrial membrane potential was measured by flow cytometry (FL2-to-FL-1 ratio of platelets loaded with JC-1 (final: 2μM) showed a ratio 6.8 ± 1.5 for the 1 day old platelets and 3.25 ± 0.75 for the 7 day old platelets. Additional flow cytometric testing showed an increase of p-selectin (ant-human CD62P, clone AK-4) expression for 1 day old platelets (31.4 ± 3.5%) and 7 day old platelets (54.6 ± 6.6) after 20 min of UV light. Tables 1 and 2 show in vitro and in vivo results as percent of control (except for pH and JC-1). Table 1. 1 Day Old Platelets UV Exposure Plt Count pH JC-1 Ratio Collagen Aggregation in vivo Recovery 20 min 76.2 7.4 6.5 ± 1.15 101.6 83.8 ± 13.8 40 min 42.2 7.2 3.5 ± 0.4 66.1 56.8 ± 12 Table 2. 7 Day Old Platelets UV Exposure Plt count pH JC-1 Ratio Collagen Aggregation in vivo Recovery 20 min 78.1 7.2 2.85 ± 0.95 116.2 31.8 ± 6.2 40 min 66.1 7.1 nd 5.5 2.27 ± 0.31 For in vivo recovery, approximately 1x10 9 platelets (UV-treated or control) were injected into the tail vein of SCID mice (n=4 per each condition) and serial blood samples were collected. Human platelets were detected in mouse whole blood by flow cytometry using an anti-human GPIIbIIIa mAb (clone P2). Recovery was defined as percent of human platelets in mouse circulation five minutes post infusion. Comparison of recovery between control and UV treatments was done at 4 hours post infusion. UV light treatments, 20 and 40 minutes, reduced the in vivo recovery to 83.8 ± 13.8% and 56.8 ± 12% of control, respectively, for 1 day old platelets and to 31.8 ± 6.2% and 2.27 ± 0.31% of control, respectively, for 7 day old platelets. Our data suggest that the SCID mouse model can identify a relatively subtle UV-induced platelet lesion and that it may be more sensitive than in vitro tests. With further validation, this animal model could be useful for evaluation of the impact of new technologies on the in vivo efficacy of human platelets. The views of the authors represent a scientific opinion and should not be construed as FDA policy.

scholarly journals Impact of Bacterial Biofilm Formation on In Vitro and In Vivo Activities of Antibiotics

1998 ◽  
Vol 42 (4) ◽  
pp. 895-898 ◽  
Author(s):  
Silvia Schwank ◽  
Zarko Rajacic ◽  
Werner Zimmerli ◽  
Jürg Blaser
Keyword(s):  
Animal Model ◽  
Staphylococcus Epidermidis ◽  
Biofilm Formation ◽  
Bacterial Biofilm ◽  
Phenotypic Resistance ◽  
Vitro Model ◽  
The Impact ◽  
Isogenic Strains

ABSTRACT The impact of bacterial adherence on antibiotic activity was analyzed with two isogenic strains of Staphylococcus epidermidis that differ in the features of their in vitro biofilm formation. The eradication of bacteria adhering to glass beads by amikacin, levofloxacin, rifampin, or teicoplanin was studied in an animal model and in a pharmacokinetically matched in vitro model. The features of S. epidermidis RP62A that allowed it to grow on surfaces in multiple layers promoted phenotypic resistance to antibiotic treatment, whereas strain M7 failed to accumulate, despite initial adherence on surfaces and growth in suspension similar to those for RP62A. Biofilms of S. epidermidis M7 were better eradicated than those of strain RP62A in vitro (46 versus 31%;P < 0.05) as well as in the animal model (39 versus 9%; P < 0.01).

An Animal Model Suitable for Predicting In Vivo Behavior of Platelets in Humans: A Tool for New Product Development.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3628-3628
Author(s):  
Morris A. Blajchman ◽  
Junzhi Li ◽  
Myron Kulczycky ◽  
Zofia Woskowska ◽  
Henry Pieters ◽  
...  
Keyword(s):  
Animal Model ◽  
New Product Development ◽  
Isotope Labeling ◽  
Uv Light ◽  
Human Platelets ◽  
Light Dose ◽  
Blood Component ◽  
Survival Times ◽  
And Control

Abstract Testing platelet in vivo activity in an animal model remains an attractive approach for the pre-clinical evaluation of platelets that have been treated or manipulated by a new technology or product. Mirasol PRT™ is a novel pathogen reduction technology based on riboflavin photochemistry for the safety of blood component transfusion. In this study, rabbit platelets were tested for viability after exposure to 2, 3 and 5 J/cm2 of UV light in the presence of 50 μM riboflavin in a polypropylene bag (Sangewald). In order to minimize the potential variations caused by different isotope labeling and by individual rabbit differences, a pool of platelets was split into two for the treatment and control groups. Eight to ten rabbits were used in each group as recipients for each dose of light. Half of the rabbits in the group received concurrent injections of 111In-labeled PRT treated platelets and 51Cr-labeled control platelets. The other half received 51Cr-labeled PRT-platelets and 111In-labeled control platelets. The recovery and survival times were determined post-infusion for both treated and control platelets using the multiple hit model (γ-function) and data from the various groups were pooled and analyzed. The platelets treated with 2, 3 and 5 J/cm2 retained recoveries of 63.2% (SD=17, n=9), 55.3% (SD=10.8, n=10) and 44.0% (SD=12.8, n=8) of control platelets, respectively. The survival times of the platelets treated with 2, 3 and 5 J/cm2 were 59.5% (SD=15.7, n=9), 60.1% (SD=19.5, n=10) and 55.1% (SD=18.1, n=8) of control, respectively. Both values of recovery and survival times were correlated with UV light dose with correlation coefficients of 0.90 (r2) and 0.73 (r2) respectively. In a subsequent single site human clinical study, human platelets were treated with UV light at a dose of 3 J/cm2 in a Sangewald bag, radiolabelled with Indium at day 5 of storage and infused into autologous subjects. Recovery and survival values for these subjects were obtained. At a dose of 3 J/cm2, recovery and survival activities retained 54.9% (SD=3.1, n=4) and 71.7% (SD=21.5) of control platelets, respectively. Good agreement with observations made in the rabbit model was thus obtained. Based on this work, a light dose was selected for standard treatment of human platelets using the Mirasol PRT system. In conclusion, this kinetic study on UV light dose in an animal model was predictive of human platelet in vivo activity, making it a potentially valuable model for estimating human clinical trial outcomes for new platelet storage and treatment methodologies.

In Vivo Eradication of Human Clonigenic Acute Lymphoblastic Leukemic Cells Expressing the Wilms Tumor Protein, WT-1, by HLA Restricted WT-1 Specific T-Cells in NOD/SCID Mice Monitored by Bioluminescent Imaging.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 581-581
Author(s):  
Ekaterina Doubrovina ◽  
Mikhail Doubrovin ◽  
Elena Kanaeva ◽  
Richard J. O’Reilly
Keyword(s):  
T Cells ◽  
Wilms Tumor ◽  
Scid Mice ◽  
Leukemic Cells ◽  
Luciferase Reporter ◽  
Bioluminescent Imaging ◽  
Acute Leukemias ◽  
Post Infusion

Abstract WT-1 is expressed in 60–80% of acute leukemias, CML and high risk forms of MDS. Its expression has been hypothesized to be critical to the growth or survival of leukemic stem cells. Previously, alloreactive HLAA0201− T-cells recognizing a complex of WT-1 peptide and HLA A0201 were reported to prevent growth of leukemic HLA A0201+ CD34+ Ph+CML progenitor cells in NOD/ SCID mice (Transplantation, vol 75, No9, 2003). In this study, we have assessed the capacity of HLA-restricted, WT-1 peptide specific CTL (WT1-CTL) lacking alloreactivity to prevent the outgrowth of a human acute preB-lymphocytic leukemia (B-ALL)in NOD/SCID mice. This leukemia contained 65% of the blasts expressed WT-1 as determined by FACS analysis. For these studies the leukemic cells were transduced to express a luciferase reporter gene, permitting sequential monitoring of growth in vivo by bioluminescent imaging. WT-1 specific T-cells were generated from normal HLA A0201+ donor PBMC by in vitro sensitization with autologous dendritic cells loaded with the immunogenic HLA A0201 binding WT-1 peptide, RMFPNAPYL, and shown to be selectively cytotoxic against HLA A0201+WT-1+ leukemias and peptide loaded PHA blasts. T-cells from the same donor sensitized with autologous EBV BLCL and exhibiting HLA A0201 restricted EBV-specific cytotoxic activity served as controls. WT-1-CTL or EBV CTL were co-incubated in vitro with the WT-1+ HLA A0201+ BALL-LUC at a 4:1 effector target ratio for 7 hours at 37°C. Thereafter, separate groups of 5 NOD/SCID mice received intravenous infusions of cells from each of the co-cultures, at doses providing 12 × 106 WT1 CTL or EBVCTL and 3 × 106 BALL-LUC cells/mouse. A third group received 3×106 BALL-LUC alone. Leukemia growth was monitored at 2–3 day intervals from day 1–45 post infusion. In all 3 groups, BALL-LUC could be detected in the thorax by imaging at day 1. In mice treated with BALL-LUC alone or together with EBV-CTL, signal accumulation in the thorax increased steadily through 45 days of observation. By day 17, BALL-LUC were also detected throughout the head, abdomen and pelvis, and thereafter also increased until sacrifice at day 45. Autopsy confirmed presence of leukemic nodules in the lung and leukemic cells in blood, spleen and marrow as well as other organs. In contrast, in mice treated with WT1-CTL+ BALL LUC, signal intensity in lung decreased by day 4. In 4/5 of these mice, BALL-LUC could not be detected thereafter. In one mouse from this group, BALL-LUC were first detected in the head 31 days post infusion. At autopsy on day 45, this mouse had detectable BALL in the skull but in no other sites. WT-1 expression of residual leukemic cells is being analyzed. The other mice treated with WT-1 CTL had no detectable residual disease. These results suggest that clonogenic BALL cells express WT-1 and are susceptible to eradication in vivo by WT-1 peptide specific cytotoxic T-cells. The elimination of such clonogenic leukemic cells is sufficient to prevent subsequent development of leukemia.

Protein Kinase C Mediates UVB Light Induced Changes In Human Platelets that Lead to Inflammation In a Two-Event Animal Model of Transfusion Mediated Acute Lung Injury

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3358-3358
Author(s):  
Li Zhi ◽  
Xuan Chi ◽  
Monique Gelderman-Fuhrmann ◽  
Jaroslav Vostal
Keyword(s):  
Animal Model ◽  
Acute Lung Injury ◽  
Lung Injury ◽  
Human Platelets ◽  
Pkc Inhibitor ◽  
Kinase C ◽  
Uvb Exposure ◽  
Induced Changes ◽  
Induced Aggregation

Abstract Abstract 3358 Pathophysiology of acute lung injury (ALI) includes an inflammatory component with recruitment and activation of neutrophils to the lungs. One proposed mechanism of transfusion related acute lung injury (TRALI) involves two events; the first is a generalized inflammatory response, as would occur in sepsis, which leads to activation of endothelial cells and sequestration of neutrophils to the lungs. The second is an infusion of a transfusion product that contains HLA or HNA antibodies or biologic modifiers such as lipids from stored cells. The second event activates the neutrophils sequestered in the lungs which lead to neutrophil degranulation, superoxide release and localized tissue damage. Growing evidence suggests that platelets exert proinflammatory actions which include supporting tissue infiltration of neutrophils in septic lung injury. In a separate 2010 ASH abstract we show that ultraviolet B light (UVB, 2.4 J/cm2) exposed human platelets (HPs) mediate lung injury in a two-event animal model of ALI. UVB exposure has been reported to activate platelet protein kinase C (PKC). We compared the effects of UVB exposure to PKC activation by a PKC agonist, PMA (30 nM), in aggregation, activation and potential to cause lung injury in the two-event animal model. HPs were collected by apheresis and stored overnight with experiments performed on day 1 post collection. Platelet aggregation induced by increasing concentrations of ADP (5-20 mM) was potentiated by pretreatment with UVB or PMA. TRAP (20 mM) induced aggregation was inhibited by UVB, but unchanged by PMA pretreatment. Both UVB and PMA increased platelet PAC-1 binding and p-selectin expression. Pretreating HPs with a PKC inhibitor prevented all of PMA induced PAC-1 binding and inhibited UVB induced PAC-1 binding by 40%. Furthermore, the PKC inhibitor partially reduced p-selectin expression on PMA and UVB treated HPs, whereas p-selectin expression on control HPs remained unchanged. The UVB HPs or PMA HPs were evaluated in the two-event animal model of ALI. Immunodeficient (SCID) mice were used to minimize the species difference (Piper et al., Transfusion 47:1540-9, 2007). MIP-2 elevation in plasma is a marker of acute inflammation and was increased following LPS administration. Infusion of control HPs as the second event moderately increased MIP-2. When UVB HPs or PMA HPs were infused MIP-2 was significantly elevated compared to control HPs. Pretreatment of UVB HPs with the PKC inhibitor (RO31-8425) reduced MIP-2 elevation to the level of control platelets. In summary, UVB HPs can cause ALI in animals pretreated with LPS (separate 2010 ASH abstract as mentioned above). Changes to the platelets induced by UVB appear to be mediated by PKC since a PKC agonist (PMA) has similar effects on platelets in aggregation and activation as does UVB and PKC inhibitor partially inhibits UVB induced platelet activation. In vivo, both UVB and PMA treated HPs elevated MIP-2 plasma levels when injected after LPS and this response was prevented by treatment of platelets with a PKC inhibitor prior to UVB exposure. The UVB induced activation leads to a conformational change in GpIIb/IIIa which potentiates weak agonist induced aggregation and mediates an acute in vivo inflammatory response that may be responsible for the acute lung injury in the animal model. Understanding the underlying mechanisms of UVB exposure induced changes in platelets would be beneficial in designing methods to reduce the UVB associated ALI in an animal model and potentially in patients susceptible to TRALI by a primary sensitizing event and infused with high dose UVB exposed platelets. The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Dysfunctional platelet membrane receptors: from humans to mice

2004 ◽  
Vol 92 (09) ◽  
pp. 478-485 ◽  
Author(s):  
Jerry Ware
Keyword(s):  
Biochemical Characterization ◽  
Ex Vivo ◽  
Human Platelets ◽  
Membrane Receptors ◽  
Protease Activated Receptors ◽  
In Vivo Models ◽  
Depth Analysis ◽  
The Impact

SummaryInsights into hemostasis and thrombosis have historically benefited from the astute diagnosis of human bleeding and thrombotic disorders followed by decades of careful biochemical characterization. This work has set the stage for the development of a number of mouse models of hemostasis and thrombosis generated by gene targeting strategies in the mouse genome. The utility of these models is the in depth analysis that can be performed on the precise molecular interactions that support hemostasis and thrombosis along with efficacy testing of various therapeutic strategies. Already the mouse has proven to be an excellent model of the processes that support hemostasis and thrombosis in the human vasculature. A brief summary of the salient phenotypes from knockout mice missing key platelet receptors is presented, including the glycoprotein (GP) Ib-IX-V and GP IIb/IIIa (αIIb/β3) receptors; the collagen receptors, GP VI and α2/β1; the protease activated receptors (PARs); and the purinergic receptors, P2Y1 and P2Y12. A few differences exist between mouse and human platelets and where appropriate those will be highlighted in this review. Concluding remarks focus on the importance of understanding the power and limitations of various in vitro, ex vivo and in vivo models currently being used and the impact of the mouse strain on the described platelet phenotype.

The in Vitro Effects of Mithramycin on the Aggregation and the Calcium Uptake of Human Platelets

1973 ◽  
Vol 29 (03) ◽  
pp. 712-721 ◽  
Author(s):  
Francis C. Chao ◽  
James L. Tullis
Keyword(s):  
Calcium Uptake ◽  
Calcium Influx ◽  
Human Platelets ◽  
Biphasic Effect ◽  
Ionic Calcium ◽  
In Vitro Effects ◽  
High Doses ◽  
Induced Aggregation

SummaryThe in vitro incubation of platelets with mithramycin caused a biphasic effect on ADP-induced aggregation. At a low dose of 10 [xg/ml, mithramycin caused an enhancement of aggregation whereas at high doses above 50 [μg/ml, it produced variable effects on aggregation with inhibition predominating. Effects on ADP-induced aggregation similar to those caused by mithramycin were produced by the simple addition of CaCl2 solution to the plasma medium. Mithramycin in the range which increased aggregation altered neither the whole blood clotting times nor the ionic calcium concentrations, but did increase the uptake of radioactive calcium by platelets. The results confirmed the in vivo observations that mithramycin treatment during tumor chemotherapy may directly alter the aggregation response of circulating platelets to ADP. The effects of mithramycin on aggregation may be mediated by alteration of calcium influx in normal platelets.

Role of Thromboxane A2 as a Mediator of Platelet-Activating-Factor-Induced Aggregation of Human Platelets

1989 ◽  
Vol 77 (1) ◽  
pp. 99-103 ◽  
Author(s):  
R. K. McCulloch ◽  
J. Summers ◽  
R. Vandongen ◽  
I. L. Rouse
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activating Factor ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Minor Role ◽  
A Minor ◽  
Induced Aggregation

1. At present it is unclear whether platelet-activating-factor (PAF)-induced aggregation is mediated by thromboxane. To obtain further information about this event we have compared the affects of aspirin on platelet aggregation and secretion induced by PAF and collagen. 2. Collagen and PAF induced aggregation and secretion in human platelets in a dose-related manner. 3. Aspirin inhibited the magnitude of both platelet aggregation and secretion induced by PAF and collagen, but the degree of inhibition was much greater for collagen. 4. Aspirin strongly inhibited the aggregation rate of collagen-induced platelet aggregation, but had no measurable effect on the rate of PAF-induced aggregation. 5. Inconsistencies reported in previous studies of the effect of aspirin on PAF-induced platelet aggregation may be explained, in part, by the doses of PAF used and the method of inactivating cyclo-oxygenase (in vitro compared with in vivo). 6. Our results suggest that the initial events of PAF-induced aggregation are independent of thromboxane A2 formation and that thromboxane A2 plays only a minor role in the later phase of PAF-induced aggregation.

scholarly journals 1276. Evaluation of CD377, a Novel Antiviral Fc-Conjugate (AVC), In Vitro Activity and In Vivo Efficacy in Immune-Competent and -Deficient (SCID) Lethal Mouse Models

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S654-S654
Author(s):  
James Levin ◽  
Karin Amundson ◽  
Allen Borchardt ◽  
Thanh Lam ◽  
Tom Brady ◽  
...  
Keyword(s):  
Mouse Models ◽  
Survival Data ◽  
Scid Mice ◽  
Vitro Activity ◽  
In Vitro Activity ◽  
Prevention And Treatment ◽  
The Impact ◽  
Efficacy Studies

Abstract Background Cidara’s AVCs are novel, potent, antiviral agents conjugated with the Fc domain of human IgG1. CD377 is an AVC development candidate being evaluated for prevention and treatment of seasonal and pandemic influenza, including in immune-deficient populations unable to benefit from vaccination. We evaluated CD377 in vitro and in SCID mice to determine the impact of compromised immune status on efficacy. Methods CD377 and comparators (oseltamivir [OS], zanamivir [ZA], baloxavir marboxil [BM]) were evaluated in vitro by neuraminidase inhibition (NAI), and cytopathic effect (CPE) assays. The pharmacokinetics (PK) and efficacy of CD377 were determined in immune-competent (IC; BALB/c) and immune-deficient (ID; BALB/c SCID) mice. Efficacy was assessed by intranasal challenge at 3x the LD95 of influenza A/Puerto Rico/8/1934 (H1N1), followed by a single subcutaneous (SC) dose of CD377, 2 hours post-challenge. The SCID study also evaluated the efficacy of BM at 3 mg/kg (BID x 1 day). Body weights (BW) were monitored for 21 days, with 20% BW loss recorded as mortality. Results In vitro evaluation by NAI showed CD377, OS, and ZA to be approximately equipotent, with IC50 values between 0.5 and 1.7 nM. However, by CPE, CD377 was ~4.5-fold more active than BM and &gt;1,000-fold more active than OS and ZA. In vivo, the PK of CD377 was found to be comparable in IC and ID mice. In subsequent efficacy studies, CD377 was protective at 0.1 mg/kg in IC mice (P=0.0020 vs. vehicle), while control groups fully succumbed to infection by Day 7 (Fig. 1A). In a similar study with ID mice, CD377 dosed at 0.1 mg/kg was also fully protective (P=0.0020). In contrast, mice treated with 6 mg/kg (total dose) of BM were only partially protected until day 13 (40% mortality by study end) (Fig. 1B). The potency of CD377 was further supported by BW data, which mirrored the survival data in both studies. Conclusion CD377 exhibited potent in vitro activity and had similar PK in IC and ID mice. In efficacy studies, CD377 demonstrated robust potency in both IC and ID mouse models at equivalent doses (0.1 mg/kg, SC, single dose). These results support further development of CD377 as a novel antiviral for the prevention and treatment of influenza infection, including in people with immune deficiencies and higher risk of infection. Balb/c and Balb/c SCID Disclosures James Levin, PhD, Cidara Therapeutics (Shareholder) Karin Amundson, B.S., Cidara Therapeutics (Shareholder) Allen Borchardt, PhD, Cidara Therapeutics (Employee) Thanh Lam, PhD, Cidara Therapeutics (Shareholder) Tom Brady, PhD, Cidara Therapeutics (Shareholder) Elizabeth Abelovski, B.S., Cidara Therapeutics (Shareholder) Simon Döhrmann, PhD, Cidara Therapeutics (Shareholder) Jason Cole, PhD, Cidara Therapeutics (Shareholder) Voon Ong, PhD, Cidara Therapeutics, Inc. (Employee, Shareholder) Les Tari, PhD, Cidara Therapeutics (Shareholder)

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