A safe therapeutic apheresis protocol in paediatric patients weighing 11 to 25 kg.

BACKGROUND AND OBJECTIVES: Erythrocytapheresis and leukapheresis (LPE) of small children are logistically complex and many centres are reluctant to perform these procedures. In children, both sickle cell and leukaemic emergencies demand prompt action to prevent additional morbidity but detailed protocols for small children are lacking, and often are performed using guidelines shown to work in larger patients. We report a 3-year experience with children weighing 11-25 kg at a large academic medical centre. MATERIALS AND METHODS: All patients were treated with the COBE® Spectra apheresis system; circuit was primed with blood not adjusted for haematocrit and anticoagulant citrate dextrose A was used as anticoagulation. Procedures were performed in the paediatric intensive care unit by apheresis nursing staff. RESULTS: Twenty-five apheresis procedures in 19 patients were performed; 17 of 19 patients presented with sickle cell-related acute complications and two (2/19) with newly diagnosed acute leukaemia and hyperleucocytosis. None of the patients required medications during the procedures. Vital signs and clinical condition remained stable and did not worsen during or postapheresis. One patient had a delayed haemolytic transfusion reaction 1 week posterythrocytapheresis as he developed alloantibodies as a result of the procedure. All sickle cell patients achieved a target haematocrit of 21-30% and Haemoglobin A of ≥68%. Both leukaemia patients who underwent LPE had no further signs of leukostasis and achieved marked reductions in leucocyte counts. CONCLUSIONS: Apheresis of children weighing 11-25 kg can be safely performed without increased morbidity. We outline a protocol that can be used to perform apheresis with minimal complications.

Primary intestinal posttransplant T-cell lymphoma.

There have been only five reported cases of primary posttransplant T-cell lymphoma. We report the first case associated with the use of sirolimus (Rapamycin, Wyeth-Ayerst, Philadelphia, PA). The patient, receiving prednisone, cyclosporine, and sirolimus treatment, developed ascites, diarrhea, and weight loss 7 months after his second renal transplant. Tissue obtained at laparotomy established the diagnosis of primary T-cell lymphoma. Latent membrane protein-1 for Epstein-Barr virus was negative, but in-site hybridization test for Epstein-Barr-encoded RNA was positive. Despite aggressive chemotherapy, the patient died 8 months posttransplant. This is the sixth reported case of primary intestinal posttransplant T-cell lymphoma, but it is the first case associated with the use of sirolimus. The incidence of posttransplant lymphoproliferative disease in patients receiving sirolimus should be studied.

Premature TCR alpha beta expression and signaling in early thymocytes impair thymocyte expansion and partially block their development.

In thymocyte ontogeny, Tcr-a genes rearrange after Tcr-b genes. TCR alpha beta transgenic (Tg) mice have no such delay, consequently expressing rearranged TCR alpha beta proteins early in the ontogeny. Such mice exhibit reduced thymic cellularity and accumulate mature, nonprecursor TCR(+)CD8(-)4(-) thymocytes, believed to be caused by premature Tg TCR alpha beta expression via unknown mechanism(s). Here, we show that premature expression of TCR alpha beta on early thymocytes curtails thymocyte expansion and impairs the CD8(-)4(-) --> CD8(+)4(+) transition. This effect is accomplished by two distinct mechanisms. First, the early formation of TCR alpha beta appears to impair the formation and function of pre-TCR, consistent with recently published results. Second, the premature TCR alpha beta contact with intrathymic MHC molecules further pronounces the block in proliferation and differentiation. These results suggest that the benefit of asynchronous Tcr-a and Tcr-b rearrangement is not only to minimize waste during thymopoiesis, but also to simultaneously allow proper expression/function of the pre-TCR and to shield CD8(-)4(-) thymocytes from TCR alpha beta signals that impair thymocyte proliferation and CD8(-)4(-) --> CD8(+)4(+) transition.

Cellular requirements for the monoclonal antibody-mediated eradication of an established solid tumor.

Following subcutaneous implantation, the murine lymphoma E.G7 [a variant of EL-4, transfected with the chicken ovalbumin (OVA) gene] up-regulates the CD4 molecule. We previously showed that the administration of an anti-CD4 monoclonal antibody (mAb) to EG.7-bearing mice leads to a rapid and complete regression of large established tumors. This tumor regression was shown to require both CD8+ cells and functional Fcgamma receptors (FcgammaR), as it failed to occur in mice depleted of CD8 cells, or mice genetically deficient in FcgammaI/III (gamma-/-mice). Using adoptive transfer, we now show that the FcgammaR+ cells required for this regression are the CD11b+ (phagocytic) cells. Furthermore, experiments using peptide tolerization demonstrated that the critical CD8 CTL population in this model is tumor specific. Analysis of tumors at various stages of regression revealed a massive CD11b+FcgammaR+ and a marginal CD8 infiltration. In the presence of the CTL determinant OVA-8 on tumor cells and of the antitumor mAb, this CD8 infiltration became remarkable, and correlated with tumor regression. These results identify the specific cellular effectors essential for the mAb-mediated tumor regression, and suggest that FcgammaR-activated macrophages induced an expansion of tumor-eliminating CTL in situ.

Synergy between an antibody and CD8+ cells in eliminating an established tumor.

We investigated the effector mechanisms operating during the rejection of a transplantable solid lymphoma E.G7 (H-2b) which expresses the gene encoding chicken ovalbumin (OVA). Anti-OVA cytotoxic T lymphocytes (CTL) completely and specifically protected animals from the onset of, but could not eradicate established, E.G7 tumors. The growth of the same lymphoma was also effectively prevented by the antibody GK1.5, whose target molecule, CD4, was expressed on E.G7 cells in vivo. Furthermore, GK1.5 was able to eradicate established solid E.G7 tumors. GK1.5-mediated tumor elimination was due to its antitumor activity, and not to the elimination of regulatory CD4+ cells, based on unimpaired tumor growth in the absence of GK1.5 in animals that genetically lack CD4 T cells. In vitro, GK1.5 did not kill tumor cells: complement activation or apoptosis induction were not evident. In vivo, GK1.5-mediated tumor regression did not depend on natural killer cells, but it absolutely required CD8+ cells and intact Fcgamma receptor. We conclude that, in the E.G7 model, the collaboration of antibody and CTL immunity was crucial for the successful immunotherapy of established tumors. The mechanism of this collaboration is discussed.

CD4-independent in vivo priming of murine CTL by optimal MHC class I-restricted peptides derived from intracellular pathogens.

CTL combat intracellular pathogens by killing infected cells. The molecular targets of their attack are foreign peptides bound to self MHC encoded class I molecules. Immunization of mice with peptides containing CTL determinants was shown to elicit CD4-dependent CTL. Here, we have achieved in vivo CTL priming with naturally processed 8-10 amino acid long class I-restricted peptides emulsified in an adjuvant. A potent, reproducible and physiologically relevant response was obtained using peptides from an intracellular bacterium and five viruses (including HIV) in two murine MHC haplotypes. This method is suitable for multiple vaccination, since a 'cocktail' of peptides derived from three pathogens elicited effector CTL against each pathogen. Most importantly, peptide-induced CD8+CD4- CTL were CD4(+)-independent. These results have implications for CTL induction in situations where CD4 T cells are depleted or compromised, as is the case in HIV infection.