The toxicity of chronic immunosuppressive agents required for organ transplant maintenance has prompted investigators to pursue approaches to induce immune tolerance. We developed an approach using a bioengineered mobilized cellular product enriched for hematopoietic stem cells (HSC) and tolerogenic CD8+/TCR− graft facilitating cells (FC) combined with nonmyeloablative conditioning that allows engraftment, durable chimerism, and tolerance induction in highly mismatched related and unrelated donor-recipient pairs. Eight recipients of HLA-mismatched kidney and FC/HSC transplants underwent conditioning with fludarabine, 200 cGy total body irradiation, and cyclophosphamide followed by post-transplant immunosuppression with tacrolimus and mycophenolate mofetil. Subjects ranged in age from 29 to 56 years. HLA match ranged from 5 of 6 related to 1 of 6 unrelated. The absolute neutrophil counts nadired approximately one week after transplant, with recovery by two weeks. Multilineage chimerism at one month was 6% to 100%. The conditioning was well tolerated with outpatient management after postoperative day two. Two subjects exhibited transient chimerism and have been reduced to low-dose tacrolimus monotherapy. One subject developed viral sepsis two months after transplant and experienced renal artery thrombosis. Five subjects have durable chimerism, with immunocompetence and donor-specific tolerance by in vitro proliferative assays and were successfully weaned off all immunosuppression one year after transplant. None of the recipients produced anti-donor antibody or exhibited engraftment syndrome or graft-versus-host disease. These results suggest that manipulation of a mobilized stem cell graft and nonmyeloablative conditioning represents a safe, practical, and reproducible means of inducing durable chimerism and donor-specific tolerance in solid organ transplant recipients.
Achieving the capacity to detect minimal numbers of neoplastic cells is a major cancer diagnostic challenge. Chromosomal translocations such as the t(14;18)-(q32;q21) found in follicular and some nonfollicular lymphomas provide a tumor-specific molecular marker. The 14;18 breakpoints are focused at one of six immunoglobulin heavy chain joining (JH) regions on chromosome 14 and a small major breakpoint region (MBR) of the BCL2 gene on chromosome 18. We utilized universal oligonucleotide primers of a region 5' to the BCL2 MBR and at the 3' end of JH segments to initiate a DNA polymerase chain reaction that amplified these BCL2-JH junctures. Use of thermostable DNA polymerase enabled annealing and synthesis steps at temperatures approaching the melting point of the primers, providing a sensitive and specific assay capable of detecting 1 lymphoma cell in 106 normal cells. This technique identified the subclinical presence of leukemic cells in all seven patients examined, including two in clinical remission. It also assessed the effectiveness of protocols designed to purge malignant cells from marrow. Moreover, this approach enabled the rapid DNA sequencing of chromosomal breakpoints without their molecular cloning. This assay markedly refines the capacity to detect minimal residual disease and should improve the ability to determine the stage of disease, stratify treatment, and evaluate therapy.
We prospectively randomized 27 granulocytopenic patients who experienced a total of 30 episodes of gram-negative septicemia. The control group received an appropriate antibiotic regimen alone, whereas the "transfusion" group received infusions of granulocytes in addition to the antibiotics. Five of 14 controls survived, and 12 of 16 in the transfusion group survived, and 12 of 16 in the transfusion group survived (P less than 0.04). An important factor in the outcome of treatment was the recovery of bone-marrow function (return of peripheral granulocyte count greater than or equal to 1000 per microliter). Eighty-three per cent (five of six) of the control group and all (four of four) of the transfusion group with recovery of granulocyte levels survived the episode of sepsis. In contrast, none of the eight control patients, as compared to 67 per cent (eight of 12) of the transfusion group, survived persistent granulocytopenia (P less than 0.005). Granulocyte transfusions appear to complement appropriate antibiotic treatment of gram-negative-septicemia due to granulocytopenia.
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