scholarly journals Inhibition of Stimulated Interleukin-2 Production in Whole Blood: A Practical Measure of Cyclosporine Effect

1999 ◽  
Vol 45 (9) ◽  
pp. 1477-1484 ◽  
Author(s):  
C Michael Stein ◽  
John J Murray ◽  
Alastair JJ Wood
Keyword(s):  
Whole Blood ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
Major Effect ◽  
Biologically Relevant ◽  
Maximum Inhibition ◽  
Before And After ◽  
Dose Dependent Fashion ◽  
The Relationship

Abstract Background: Prediction of cyclosporine (CSA) efficacy and toxicity in individual patients is difficult. There is no practical, biologically relevant, pharmacodynamic measure of CSA effect. A major effect of CSA is to decrease interleukin-2 (IL-2) production; however, measurement of this effect in isolated lymphocytes as a marker of response to CSA has been problematic. Methods: CSA inhibition of phytohemagglutinin-P (PHA)-stimulated IL-2 production, measured by ELISA, was studied ex vivo in whole blood drawn before, and after subjects received 4 mg/kg oral CSA. Results: Four hours after CSA was administered, the mean (± SD) CSA concentration was 702 ± 196 μg/L and PHA-stimulated IL-2 production decreased by 68.7% ± 17.2% (P <0.0001; n = 17). Twenty-four hours after CSA was administered, concentrations were low (64 ± 24 μg/L), with no inhibition of IL-2 production. A rapid, concentration-dependent response occurred. Maximum CSA concentrations (944 ± 187 μg/L) and maximum inhibition of IL-2 production (86.9% ± 13.7%) occurred 90 min after subjects received CSA. In vitro, 32.5–1200 μg/L CSA also inhibited PHA-stimulated IL-2 production in whole blood in a dose-dependent fashion with a similar IC50 (∼300–400 μg/L) ex vivo and in vitro. Conclusion: In the search for a pharmacodynamic marker to better guide immunosuppressive therapy, the relationship between this simple, biologically relevant measure of CSA effect and clinical outcome should be determined.

Monitoring Anticoagulant Therapy by Activated Partial Thromboplastin Time: Hirudin Assessment

1994 ◽  
Vol 72 (05) ◽  
pp. 685-692 ◽  
Author(s):  
Michael T Nurmohamed ◽  
René J Berckmans ◽  
Willy M Morriën-Salomons ◽  
Fenny Berends ◽  
Daan W Hommes ◽  
...  
Keyword(s):  
Whole Blood ◽  
Partial Thromboplastin Time ◽  
Ex Vivo ◽  
Fold Increase ◽  
Heparin Therapy ◽  
Activated Partial Thromboplastin Time ◽  
Recombinant Hirudin ◽  
Interindividual Differences ◽  
The Relationship

SummaryBackground. Recombinant hirudin (RH) is a new anticoagulant for prophylaxis and treatment of venous and arterial thrombosis. To which extent the activated partial thromboplastin time (APTT) is suitable for monitoring of RH has not been properly evaluated. Recently, a capillary whole blood device was developed for bed-side monitoring of the APTT and it was demonstrated that this device was suitable to monitor heparin therapy. However, monitoring of RH was not evaluated.Study Objectives. To evaluate in vitro and ex vivo the responsiveness and reproducibility for hirudin monitoring of the whole blood monitor and of plasma APTT assays, which were performed with several reagents and two conventional coagulometers.Results. Large interindividual differences in hirudin responsiveness were noted in both the in vitro and the ex vivo experiments. The relationship between the APTT, expressed as clotting time or ratio of initial and prolonged APTT, and the hirudin concentration was nonlinear. A 1.5-fold increase of the clotting times was obtained at 150-200 ng/ml plasma. However, only a 2-fold increase was obtained at hirudin levels varying from 300 ng to more than 750 ng RH/ml plasma regardless of the assays. The relationship linearized upon logarithmic conversion of the ratio and the hirudin concentration. Disregarding the interindividual differences, and presuming full linearity of the relationship, all combinations were equally responsive to hirudin.Conclusions. All assays were equally responsive to hirudin. Levels up to 300 ng/ml plasma can be reliably estimated with each assay. The manual device may be preferable in situations where rapid availability of test results is necessary.

Ex vivo blood stimulation assay as a translational research tool in the development of the ticilimumab (CP-675,206)

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 2542-2542 ◽  
Author(s):  
R. Millham ◽  
D. Pavlov ◽  
P. Canniff ◽  
D. Guyot ◽  
D. Hanson ◽  
...  
Keyword(s):  
Cancer Patients ◽  
T Lymphocyte ◽  
Whole Blood ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
Screening Experiments ◽  
Batch Release ◽  
Ctla4 Blockade

2542 Background: Cytotoxic T Lymphocyte-associated Antigen 4 (CTLA4) is an activation-induced T lymphocyte negative costimulatory receptor which down-regulates cellular immune responses. CTLA4 blockade may break peripheral immunological tolerance, leading to an effective immune response to cancer. Tools for assessing the effects of such a blockade are limited, as CTLA4 is not constitutively expressed on circulating T cells, and because activated lymphocytes are difficult to access in vivo. Therefore, we have employed ex vivo blood stimulation assays to define pharmacodynamic properties of the anti-CTLA4 antibody, ticilimumab. Methods: Ex vivo blood stimulation assays employed staphylococcal enterotoxin A (SEA) to stimulate isolated peripheral blood mononuclear cells (PBMC) or whole blood. Stimulation was monitored by production of interleukin 2 (IL-2). This assay was used preclinically to predict in vivo responses in animal models and in samples from cancer patients, as a batch release assay for production runs of ticilimumab, and clinically as a pharmacodynamic measurement in clinical trials of ticilimumab. Results: Screening experiments using the SEA assay allowed us to identify the lead candidate mAb with optimal CTLA4 blockade activity, ticilimumab. Dose-dependent increases in IL-2 production were observed in PBMC and whole blood samples up to an in vitro concentration of 100 ug/mL of ticilimumab, with 10 ug/mL identified as the minimum predicted efficacious concentration (Ceff). This functional potency assay was adapted for qualification of production lots of ticilimumab. Whole blood taken from cynomolgus monkeys dosed with ticilimumab demonstrated significant enhancement of IL-2 production at the same magnitude observed in in vitro experiments. Additionally, longitudinal samples taken from healthy volunteers and cancer patients suggested that an enhancement of 2.8 fold would be indicative of a pharmacodynamic effect of ticilimumab. Conclusions: The SEA assay provides a functional assessment of ticilimumab activity and can be used to guide the clinical development of this agent. Our data suggest that T cell reactivity is enhanced in the presence of ticilimumab in vitro, in primate models and in humans. [Table: see text]

Plasminogen Depletion during Streptokinase Treatment or Two-Chain Urokinase Incubation Correlates with Decreased Clot Lysability Ex Vivo and In Vitro

1993 ◽  
Vol 70 (06) ◽  
pp. 0998-1004 ◽  
Author(s):  
Páll T Önundarson ◽  
H Magnús Haraldsson ◽  
Lena Bergmann ◽  
Charles W Francis ◽  
Victor J Marder
Keyword(s):  
Myocardial Infarction ◽  
Ex Vivo ◽  
Time Dependent ◽  
State Variables ◽  
Thrombolytic Treatment ◽  
Direct Proportion ◽  
Plasmin Activity ◽  
Plasma Exposure ◽  
The Relationship

SummaryThe relationship between lytic state variables and ex vivo clot lysability was investigated in blood drawn from patients during streptokinase administration for acute myocardial infarction. A lytic state was already evident after 5 min of treatment and after 20 min the plasminogen concentration had decreased to 24%, antiplasmin to 7% and fibrinogen 0.2 g/1. Lysis of radiolabeled retracted clots in the patient plasmas decreased from 37 ± 8% after 5 min to 21 ± 8% at 10 min and was significantly lower (8 ± 9%, p <0.005) in samples drawn at 20, 40 and 80 min. Clot lysability correlated positively with the plasminogen concentration (r = 0.78, p = 0.003), but not with plasmin activity. Suspension of radiolabeled clots in normal plasma pre-exposed to 250 U/ml two-chain urokinase for varying time to induce an in vitro lytic state was also associated with decreasing clot lysability in direct proportion with the duration of prior plasma exposure to urokinase. The decreased lysability correlated with the time-dependent reduction in plasminogen concentration (r = 0.88, p <0.0005). Thus, clot lysability decreases in conjunction with the development of the lytic state and the associated plasminogen depletion. The lytic state may therefore limit reperfusion during thrombolytic treatment.

Monitoring of r-Hirudin Anticoagulation during Cardiopulmonary Bypass – Assessment of the Whole Blood Ecarin Clotting Time

1997 ◽  
Vol 77 (05) ◽  
pp. 0920-0925 ◽  
Author(s):  
Bernd Pötzsch ◽  
Katharina Madlener ◽  
Christoph Seelig ◽  
Christian F Riess ◽  
Andreas Greinacher ◽  
...  
Keyword(s):  
Cardiopulmonary Bypass ◽  
Whole Blood ◽  
Heart Surgery ◽  
Ex Vivo ◽  
Open Heart Surgery ◽  
Clotting Time ◽  
Blood Samples ◽  
Alternative Approach ◽  
Ecarin Clotting Time

SummaryThe use of recombinant ® hirudin as an anticoagulant in performing extracorporeal circulation systems including cardiopulmonary bypass (CPB) devices requires a specific and easy to handle monitoring system. The usefulness of the celite-induced activated clotting time (ACT) and the activated partial thromboplastin time (APTT) for r-hirudin monitoring has been tested on ex vivo blood samples obtained from eight patients treated with r-hirudin during open heart surgery. The very poor relationship between the prolongation of the ACT and APTT values and the concentration of r-hirudin as measured using a chromogenic factor Ila assay indicates that both assays are not suitable to monitor r-hirudin anticoagulation. As an alternative approach a whole blood clotting assay based on the prothrombin-activating snake venom ecarin has been tested. In vitro experiments using r-hirudin- spiked whole blood samples showed a linear relationship between the concentration of hirudin added and the prolongation of the clotting times up to a concentration of r-hirudin of 4.0 µg/ml. Interassay coefficients (CV) of variation between 2.1% and 5.4% demonstrate the accuracy of the ecarin clotting time (ECT) assay. Differences in the interindividual responsiveness to r-hirudin were analyzed on r-hirudin- spiked blood samples obtained from 50 healthy blood donors. CV- values between 1.8% and 6% measured at r-hirudin concentrations between 0.5 and 4 µg/ml indicate remarkably slight differences in r-hirudin responsiveness. ECT assay results of the ex vivo blood samples linearily correlate (r = 0.79) to the concentration of r-hirudin. Moreover, assay results were not influenced by treatment with aprotinin or heparin. These findings together with the short measuring time with less than 120 seconds warrant the whole blood ECT to be a suitable assay for monitoring of r-hirudin anticoagulation in cardiac surgery.

Early Platelet-Collagen Interactions in Whole Blood and Their Modifications by Aspirin and Dipyridamole Evaluated by a New Method (Basic Wave)

1985 ◽  
Vol 54 (04) ◽  
pp. 799-803 ◽  
Author(s):  
José Luís Pérez-Requejo ◽  
Justo Aznar ◽  
M Teresa Santos ◽  
Juana Vallés
Keyword(s):  
Whole Blood ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
White Blood Cells ◽  
Reversible Aggregation ◽  
Inhibitory Compounds ◽  
Rapid Generation ◽  
Stimulation Of ◽  
Collagen Stimulation

SummaryIt is shown that the supernatant of unstirred whole blood at 37° C, stimulated by 1 μg/ml of collagen for 10 sec, produces a rapid generation of pro and antiaggregatory compounds with a final proaggregatory activity which can be detected for more than 60 min on a platelet rich plasma (PRP) by turbidometric aggregometry. A reversible aggregation wave that we have called BASIC wave (for Blood Aggregation Stimulatory and Inhibitory Compounds) is recorded. The collagen stimulation of unstirred PRP produces a similar but smaller BASIC wave. BASIC’s intensity increases if erythrocytes are added to PRP but decreases if white blood cells are added instead. Aspirin abolishes “ex vivo” the ability of whole blood and PRP to generate BASIC waves and dipyridamole “in vitro” significantly reduces BASIC’s intensity in whole blood in every tested sample, but shows little effect in PRP.

Dipyridamole Inhibits Platelet Aggregation in Whole Blood

1983 ◽  
Vol 50 (04) ◽  
pp. 852-856 ◽  
Author(s):  
P Gresele ◽  
C Zoja ◽  
H Deckmyn ◽  
J Arnout ◽  
J Vermylen ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Significant Negative Correlation ◽  
Significant Inhibition ◽  
Oral Drug ◽  
Human Blood Samples ◽  
Induced Aggregation

SummaryDipyridamole possesses antithrombotic properties in the animal and in man but it does not inhibit platelet aggregation in plasma. We evaluated the effect of dipyridamole ex vivo and in vitro on platelet aggregation induced by collagen and adenosine- 5’-diphosphate (ADP) in human whole blood with an impedance aggregometer. Two hundred mg dipyridamole induced a significant inhibition of both ADP- and collagen-induced aggregation in human blood samples taken 2 hr after oral drug intake. Administration of the drug for four days, 400 mg/day, further increased the antiplatelet effect. A significant negative correlation was found between collagen-induced platelet aggregation in whole blood and dipyridamole levels in plasma (p <0.001). A statistically significant inhibition of both collagen (p <0.0025) and ADP-induced (p <0.005) platelet aggregation was also obtained by incubating whole blood in vitro for 2 min at 37° C with dipyridamole (3.9 μM). No such effects were seen in platelet-rich plasma, even after enrichment with leukocytes. Low-dose adenosine enhanced in vitro inhibition in whole blood.Our results demonstrate that dipyridamole impedes platelet aggregation in whole blood by an interaction with red blood cells, probably involving adenosine.

scholarly journals In Vivo Role of Dendritic Cells in a Murine Model of Pulmonary Cryptococcosis

2006 ◽  
Vol 74 (7) ◽  
pp. 3817-3824 ◽  
Author(s):  
Karen L. Wozniak ◽  
Jatin M. Vyas ◽  
Stuart M. Levitz
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
Mouse Lung

ABSTRACT Dendritic cells (DC) have been shown to phagocytose and kill Cryptococcus neoformans in vitro and are believed to be important for inducing protective immunity against this organism. Exposure to C. neoformans occurs mainly by inhalation, and in this study we examined the in vivo interactions of C. neoformans with DC in the lung. Fluorescently labeled live C. neoformans and heat-killed C. neoformans were administered intranasally to C57BL/6 mice. At specific times postinoculation, mice were sacrificed, and lungs were removed. Single-cell suspensions of lung cells were prepared, stained, and analyzed by microscopy and flow cytometry. Within 2 h postinoculation, fluorescently labeled C. neoformans had been internalized by DC, macrophages, and neutrophils in the mouse lung. Additionally, lung DC from mice infected for 7 days showed increased expression of the maturation markers CD80, CD86, and major histocompatibility complex class II. Finally, ex vivo incubation of lung DC from infected mice with Cryptococcus-specific T cells resulted in increased interleukin-2 production compared to the production by DC from naïve mice, suggesting that there was antigen-specific T-cell activation. This study demonstrated that DC in the lung are capable of phagocytosing Cryptococcus in vivo and presenting antigen to C. neoformans-specific T cells ex vivo, suggesting that these cells have roles in innate and adaptive pulmonary defenses against cryptococcosis.

HTLV-II down-regulates HIV-1 replication in IL-2–stimulated primary PBMC of coinfected individuals through expression of MIP-1α

Blood ◽  
2000 ◽  
Vol 95 (9) ◽  
pp. 2760-2769 ◽  
Author(s):  
Claudio Casoli ◽  
Elisa Vicenzi ◽  
Andrea Cimarelli ◽  
Giacomo Magnani ◽  
Paolo Ciancianaini ◽  
...  
Keyword(s):  
T Cells ◽  
Neutralizing Antibodies ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
Peripheral Blood Mononuclear ◽  
Human T Cell ◽  
Cc Chemokines ◽  
Kinetics Of ◽  
Hiv 1

The influence of human T-cell leukemia/lymphoma virus type II (HTLV-II) in individuals also infected with HIV-1 is poorly understood. To evaluate the reciprocal influence of HTLV-II and HIV-1 infection, primary peripheral blood mononuclear cell (PBMC) cultures from coinfected individuals were established in the presence of interleukin 2 (IL-2). In these cultures, the kinetics of HTLV-II replication always preceded those of HIV-1. Noteworthy, the kinetics of HIV-1 production were inversely correlated to the HTLV-II proviral load in vivo and its replication ex vivo. These observations suggested a potential interaction between the 2 retroviruses. In this regard, the levels of IL-2, IL-6, and tumor necrosis factor- (TNF-) were measured in the same coinfected PBMC cultures. Endogenous IL-2 was not produced, whereas IL-6 and TNF- were secreted at levels compatible with their known ability to up-regulate HIV-1 expression. The HIV-suppressive CC-chemokines RANTES, macrophage inflammatory protein-1 (MIP-1), and MIP-1β were also determined in IL-2–stimulated PBMC cultures. Of interest, their kinetics and concentrations were inversely related to those of HIV-1 replication. Experiments were performed in which CD8+ T cells or PBMCs from HTLV-II monoinfected individuals were cocultivated with CD4+ T cells from HIV-1 monoinfected individuals separated by a semipermeable membrane in the presence or absence of antichemokine neutralizing antibodies. The results indicate that HTLV-II can interfere with the replicative potential of HIV-1 by up-regulating viral suppressive CC-chemokines and, in particular, MIP-1. This study is the first report indicating that HTLV-II can influence HIV replication, at least in vitro, via up-regulation of HIV-suppressive chemokines.

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