Prophylactic use of clopidogrel in paediatric cardiac patients.

Thromboembolic complications in infants with congenital heart defects are common despite inhibition of platelet function with acetylsalicylic acid (ASS). Yet there is still insufficient pharmacologic data on the use of clopidogrel in infants. The adult dose of 75 mg/d is significantly higher than the dose lately recommended in infants (0.2 mg/kg/d). Moreover, we know of nonresponders to both acetylsalicylic acid and clopidogrel. Normal coagulation tests fail to identify those patients.Prospective monocentric study on 14 children (median age 5, range 0.7-84 months, 9 male, 5 female). Shunt thrombosis had occurred in 4 infants on ASS therapy. Seven days after starting clopidogrel (0.2 mg/kg/d), platelet function was tested by stimulation with ADP (4 and 10 µmol/l). We considered the range for the clopidogrel effect to be optimal if the maximum aggregation on ADP 4 µmol/l was between 30-50%.Clopidogrel 0.18-0.24 mg/kg/d in addition to ASS 2-4 mg/kg/d resulted in effective inhibition of platelet function in 93% (ADP 4 µmol/l: median 38%, range 30-63). All patients were responders. We observed neither any thromboembolic events nor severe bleeding episodes during the median 11-month follow-up period (range 1-30 mo).Testing platelet function makes clopidogrel dosing safer, and simplifies therapy adjustments in long-term treatment. A clopidogrel dose of 0.2 mg/kg/d was safe and effective in combination with ASS in this small patient cohort.

Donor natural killer (NK1.1+) cells do not play a role in the suppression of GVHD or in the mediation of GVL reactions after DLI.

Donor regulatory T cells (CD3+ alphabetaT-cell receptor [TCR]+) derived from the repopulating host thymus have been shown to be primarily responsible for suppression of GVHD following DLI therapy in murine BMT models. However, natural killer (NK) T cells also have regulatory properties, and a role for NK T cells in suppression of GVH reactivity has not been completely excluded. NK cells may also contribute to the graft-versus-leukemia (GVL) effect associated with DLI therapy. In this study, we used a murine BMT model (C57BL/6 into AKR) to study whether depletion of donor NK cells had any impact on the suppression of GVH reactivity after DLI or on the DLI-induced GVL effect against acute T-cell leukemia. Depletion of donor NK cells was accomplished in vivo by giving DLI-treated bone marrow chimeras multiple injections of anti-NK1.1 monoclonal antibody (MoAb). The chimeras treated with anti-NK1.1 MoAb had significantly fewer splenic NK1.1 cells than nontreated chimeras, and splenocytes from anti-NK1.1-treated mice were deficient in the ability to generate lymphokine-activated lytic activity. Results presented here showed that NK-cell depletion had no effect on the suppression of GVH reactivity after DLI. When DLI-treated chimeras were challenged with an acute T-cell leukemia, NK-cell depletion had no discernible effect on GVL reactivity. These preclinical data suggest that donor NK cells do not have a significant role in the suppression of GVHD after DLI or in the mediation of GVL reactivity induced by DLI.

Sca1(+)/Mac1(+) nitric oxide-producing cells in the spleens of recipients early following bone marrow transplant suppress T cell responses in vitro.

Spleen cells collected from allogeneic chimeras early after bone marrow transplantation (BMT) consistently showed suppressed proliferative responses to interleukin-2 in vitro and failed to proliferate in mixed lymphocyte reaction (MLR) assays. However, isolation of Thy 1.2(+) T cells from the heterogeneous spleen cell suspension prior to culture resulted in heightened proliferation, suggesting the presence of cells capable of suppressing T cell responses in vitro. When separated into subpopulations by negative and positive selection with specific monoclonal antibodies, a non-T, non-B population with immunosuppressive properties was identified. The suppressive cells were found in the spleens of both allogeneic and syngeneic chimeras, but not normal donor mice. Suppressor activity was transient and typically declined by 3 weeks post-BMT. The cells suppressed the response of alloactivated T cells isolated from BMT chimeras as well as naive donor T cells in MLR assays in a dose-dependent manner. To explore the mechanism(s) involved in the suppression, the effects of interferon-gamma (IFN-gamma)-specific mAb and the nitric oxide (NO) synthase inhibitor NG-methyl-l-arginine were examined. The results support a role for both IFN-gamma and NO in the suppressive activity. Separation of cells based on Mac-1 expression indicated that there were both Mac-1-enriched and Mac-1-depleted cells capable of producing NO, but that the Mac-1-depleted cells were the most potent suppressors in MLR assays. The Mac-1-depleted cells still contained a residual population of Mac-1(dim) cells which showed increased levels of Mac-1 expression after overnight culture. Intracellular staining with an inducible nitric oxide synthase (iNOS)-specific mAb indicated that the NO-producing cells expressed the cell surface markers Mac-1 and Sca-1. When iNOS knockout transgenic mice were used as transplant donors, in vitro suppression of T cell responses was reduced but not eliminated, suggesting that other mechanism(s) could contribute to the suppression. Collectively, these results demonstrate that Sca-1(+)/Mac-1(+) cells capable of producing NO are present in the spleens of recipients early after BMT and suggest that these cells may have immunoregulatory roles in vivo.

The graft-versus-leukemia effect of post-transplant donor leukocyte infusion.

Tumor relapse remains a major obstacle to the success of allogeneic bone marrow transplantation (BMT) as a treatment for leukemia. Due to limited treatment options, the outlook for most patients that relapse following allogeneic BMT has been poor. The infusion of normal immunocompetent leukocytes from the original marrow donor has become a promising new option for treating/preventing leukemia relapse in allogeneic BMT recipients. This form of treatment has often been referred to as donor leukocyte infusion (DLI) therapy. Our laboratory is using murine models of allogeneic BMT to address important unresolved issues regarding DLI therapy in an effort to make the treatment more effective. These include identification of the antileukemic effector cells, augmentation of the antileukemic effect, and understanding why graft-versus-host-disease (GVHD) is less severe than anticipated. This article reviews our work in murine models of DLI and introduces our current working hypotheses concerning DLI therapy.

Use of anti-CD3 epsilon F(ab')2 fragments in vivo to modulate graft-versus-host disease without loss of graft-versus-leukemia reactivity after MHC-matched bone marrow transplantation.

The use of T cell-specific mAb in vivo for prevention and treatment of graft-vs-host disease (GVHD) and its impact on graft-vs-leukemia (GVL) reactivity was examined in a murine model of MHC-matched bone marrow transplantation (BMT). F(ab')2 fragments of a CD3 epsilon-specific mAb were administered to irradiated AKR (H-2k) hosts after transplantation of BM plus spleen cells from B10.BR donors (BMS chimeras). The effects on GVH and GVL reactivity were Ab dose- and schedule-dependent. A short course of mAb (qe2d, days 0 to 8) prevented clinical evidence of GVHD and mortality. Anti-CD3 F(ab')2 mAb reversed clinical symptoms of acute GVHD when delayed up to 18 days post-transplant. Anti-host (Mls-1a)-specific V beta 6+ cells were absent from the spleens of GVH-negative control mice, but persisted in Ab-treated BMS chimeras despite the absence of GVHD. Leukemic mice given 16.7 micrograms of Ab on days 0, 2, and 4 survived leukemia-free without developing severe GVHD. A longer course of Ab completely prevented GVHD, but led to leukemia relapse in tumor-bearing hosts, despite engraftment of donor T cells. The GVL effect was quantitatively stronger when Ab was used for GVH therapy as compared with GVH prevention. Some Ab-treated, GVH-free chimeras relapsed with lymphomas in unusual sites, suggesting that occult tumor cells may persist in nonlymphoid tissues. These experiments demonstrate that T cell-specific mAb can be used successfully in vivo to avoid severe GVHD, but that excessive or ill-timed administration of Ab may eliminate GVL reactivity.