High dose of antithrombin III induces indefinite survival of fully allogeneic cardiac grafts and generates regulatory cells.
ABSTRACT The authors investigated whether antithrombin III (AT-III) could induce unresponsiveness to alloantigens.
CBA mice were given intravenous injection of 50 or 500 U/kg AT-III or control plasma the same day as transplantation of a heart from a C57BL/6 mouse. An adoptive transfer study and mixed leukocyte culture analysis were also performed.
Naive CBA mice rejected C57BL/6 cardiac grafts acutely (median survival time [MST], 9 days). The 50-U/kg dose of AT-III induced a moderate increase in graft survival (MST, 25 days), whereas control mice rejected their graft acutely (MST, 7 days). With the 500-U/kg dose of AT-III, all grafts survived indefinitely (>100 days) and regulatory cells were generated. In vitro, AT-III suppressed proliferation of mixed leukocyte responses and generation of interleukin-2.
AT-III can be not only an antithrombotic agent but also a strong immunomodulating agent when used at high dose.
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ABSTRACT: The hypothesis that cyclooxygenase-2 (COX-2) is involved in the myocardial inflammatory response during cardiac allograft rejection was investigated using a rat heterotopic abdominal cardiac transplantation model. COX-2 mRNA and protein in the myocardium of rejecting cardiac allografts were significantly elevated 3 to 5 days after transplantation compared with syngeneic controls (n=3, P<0.05). COX-2 upregulation paralleled in time and extent the upregulation of iNOS mRNA, protein, and enzyme activity in this model. COX-2 immunostaining was prominent in macrophages infiltrating the rejecting allografts and in damaged cardiac myocytes. Prostaglandin (PG) levels in rejecting allografts were also higher than in native hearts. Because NO has been reported to modulate PG synthesis by COX-2, additional transplants were performed using animals treated with a selective COX-2 inhibitor (SC-58125) and a selective inhibitor of the inducible nitric oxide synthase (iNOS) N-aminomethyl-L-lysine. At posttransplant day 5, inhibitor administration resulted in a significant reduction of COX-2 mRNA expression (3764+/-337 versus 5110+/-141 arbitrary units, n=3, P<0.05) and iNOS enzymatic activity (1.7+/-0.4 versus 22.8+/-14. 4 nmol/mg protein, n=3, P<0.01) compared with vehicle-treated allogeneic transplants. Allograft survival in treated animals was increased modestly from 5.4 to 6.4 days (P<0.05). However, apoptosis of cardiac myocytes (TUNNEL method) was only marginally reduced relative to vehicle controls in treated graft recipients. The intensity of allograft rejection was also similar in the treated and untreated allografts. The data indicates that COX-2 expression is enhanced in parallel with iNOS in the myocardium during cardiac allograft rejection.Circulation 03/2000; 101(4):430-8. · 15.20 Impact Factor
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ABSTRACT: Antithrombin III (AT-III) is a physiological inhibitor of thrombin and other serine proteases, and has antiinflammatory properties. Thrombin is known to enhance T lymphocyte activation in vitro and serine proteases can act as costimulators for lymphocyte proliferation and cytokine production. We have previously shown that AT-III significantly inhibited allograft rejection in a highly histoincompatible model of rat lung transplantation and in vitro cell proliferation in ConA-stimulated rat spleen cells. In this study, we examined the involvement of cytokine gene expression in the above inhibitory effect of AT-III. We also examined the effect of AT-III on several in vitro immune reactions in human peripheral blood mononuclear cells (PBMCs). mRNA expression of cytokines/cytokine receptor important in lymphocyte activation was examined. Rat spleen cells were stimulated with Con-A with/without AT-III and submitted for reverse transcriptase-polymerase chain reaction (RT-PCR). To assess the effect of AT-III on human PBMCs, we examined the effects of AT-III on cell proliferation of human PBMCs stimulated in mixed lymphocyte reaction (MLR) (allogeneic stimulation), with OKT3 (T cell receptor activation) and with PHA (mitogenic stimulation). The effect of AT-III on PWM-stimulated immunoglobulin (Ig) production by human PBMCs was also examined. All experiments for cell proliferation were performed in 10% serum and in serum-free (SF) media to determine whether AT-III exerted its effects through its interaction with thrombin in serum. mRNA expression of IL-2, gamma-IFN and IL-4 in ConA-stimulated rat spleen cells was nearly completely inhibited by AT-III at 15 IU/ml. mRNA levels for IL-6, IL-2R and TGF-beta1 were not significantly affected by AT-III. AT-III showed a dose-dependent inhibition of cell proliferation in human PBMCs. At 15 IU/ml, cell proliferation was inhibited by approximately 86%, approximately 81% and approximately 56% in the MLR-, OKT3- and PHA-stimulated PBMCs, respectively in both serum and SF media. AT-III inhibited PWM-stimulated Ig production in a dose-dependent manner. IgG, IgM and IgA production was reduced by approximately 60%, 80% and 70%, respectively in cultures incubated with 15 IU/ml AT-III. (1) Inhibition of IL-2, gamma-IFN and IL-4 mRNA expression might be responsible for inhibition of cell proliferation by AT-III in ConA-stimulated rat spleen cells, (2) AT-III inhibits cell proliferation in the MLR-, OKT3- and PHA-stimulated human PBMCs, and Ig production in PWM-stimulated human PBMCs, (3) The immune regulatory effects of AT-III are independent of its interaction with thrombin since similar levels of suppression were seen in SF media, and (4) These results suggest that AT-III has potent inhibitory effects on lymphocyte activation and cytokine production and may have potential applications as an immunomodulatory agent.Transplant Immunology 11/2001; 9(1):1-6. · 1.52 Impact Factor
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ABSTRACT: To evaluate the effect of the AT III concentrates upon the clinical evolution and hemostatic parameters. Prospective, open, randomized trial. Septic and multiple trauma patients admitted to our Intensive Care Unit. Levels of AT III below 70% were used as criteria to choose 36 patients, 20 of whom received treatment with AT III and 16 did not. AT III concentrates were administered at an initial dose of 60 U/kg followed by 10 U/kg every six hours. The administration of AT III neither contributes to alterations in haemostasis, nor the clinical evolution (evaluated according to Apache II score). The results suggest that the administration of AT III concentrates to critical patients with acquired low levels, but without manifest DIC, may not be justified; although further studies on a larger population are required to establish definite conclusions.Intensive Care Medicine 12/1994; 20(8):577-80. · 5.26 Impact Factor