D. Niethammer

Biochemical characterization of a factor released by macrophages

Abstract Peritoneal macrophages from the mouse are able to release a factor, which inhibits the incorporation of [ 3 H]thymidine into the DNA of lymphocytes. The biochemical characterization of this factor reveals that this factor is thymidine, a degradation product of cells dying in culture.

How and when should we monitor chimerism after allogeneic stem cell transplantation

Summary:Chimerism analysis has become an important tool for the peri-transplant surveillance of engraftment. It offers the possibility to realize impending graft rejection and can serve as an indicator for the recurrence of the underlying malignant or nonmalignant disease. Most recently, these investigations have become the basis for treatment intervention, for example, to avoid graft rejection, to maintain engraftment and to treat imminent relapse by pre-emptive immunotherapy. This invited review focuses on the clinical implications of characterization of hematopoietic chimerism in stem cell transplantation.

Inhibition of 3H-thymidine incorporation of lymphocytes by a soluble factor from macrophages

Abstract Supernatants of peritoneal macrophages cocultivated with spleen cells or thymocytes contain a factor suppressing the 3 H-thymidine ( 3 H-TdR) incorporation of PHA-stimulated lymphocytes. The suppressing activity is not due to cytotoxicity and does not affect the rate of cell transformation or cell proliferation. The factor reduces only the 3 H-TdR incorporation and not the DNA synthesis of lymphocytes. It does not show target cell specificity. The factor is dialysable, heat stable, and generated by macrophages.

Clinical scale isolation of T cell-depleted CD56+ donor lymphocytes in children.

We present a clinical scale method for immunomagnetic separation of CD56+ donor natural killer cells for adoptive immunotherapy of pediatric leukemias after allogeneic transplantation. This time-saving and partially automated procedure employed CD56+ selection followed by CD3+ depletion, resulting in a median purity of 98.6% NK cells and a four-log depletion of T cells. The enriched NK cells demonstrated high cytotoxic activity against K562 target cells and fresh leukemic blasts with low HLA class I expression, which could be further enhanced by IL-2 stimulation. Lysis of NK-insensitive leukemic cells with high HLA class I expression could also be demonstrated via ADCC. Due to the high degree of T cell depletion, alloreactive proliferation in mixed lymphocyte cultures and response to T cell-specific mitogen stimulation was profoundly decreased. Our results suggest that, even in the case of mismatched donors, infusions of donor NK cells with extremely low T cell content may be a promising treatment option for leukemic minimal residual disease after allogeneic transplantation without risk of inducing severe GVHD.Bone Marrow Transplantation (2002) 29, 497–502. doi:10.1038/sj.bmt.1703406

8-Methoxypsoralen and ultraviolet A therapy for cutaneous manifestations of graft-versus-host disease.

The potentially beneficial effect of 8-methoxypsoralen and ultraviolet A (PUVA) irradiation for treatment of drug-resistant cutaneous manifestations of graft-versus-host disease led us to investigate the effect of this therapy in a larger series of patients with GvHD. To date, 11 patients with histologically demonstrated cutaneous GvHD (acute GvHD grade III-IV in 4 patients, extensive lichenoid chronic GvHD in 6 patients, sclerodermatous chronic GvHD in 1 patient) have received PUVA treatment for 2-24 weeks. All patients have been on CsA for GvH prophylaxis; 5 with mismatched grafts had additionally received methotrexate or monoclonal antibody campath-1 after bone marrow transplantation. Seven patients were on CsA and prednisolone; 2 patients on CsA, prednisolone, and azathioprine; 1 patient on azathioprine and prednisolone; and 1 patient had no immunosuppressive treatment for the duration of PUVA treatment. The 8-methoxypsoralen (0.6 mg/kg bw) was given as photosensitizer before each ultraviolet A irradiation (0.3-8.5 joules/cm2). The only observed adverse reaction was mild nausea. In all patients improvement of cutaneous lesions could be observed with complete response in 5 patients and partial response in 6 patients. Immunosuppressive drugs could be withdrawn in 2 patients and reduced in 8 patients after initiation of PUVA treatment. These findings suggest that PUVA therapy may be a useful adjunct to conventional therapy for cutaneous GvHD.

Clinical scale isolation of highly purified peripheral CD34+progenitors for autologous and allogeneic transplantation in children.

We present our experience with three clinical scale isolation methods for positive selection of CD34+ progenitors from peripheral blood for autologous and allogeneic transplantation in children. A combination of the CellPro device and the Magnetic Activated Cell Sorting system (MACS), as well as two different combinations of MACS systems were used (VarioMACS-SuperMACS and SuperMACS-SuperMACS). With the CellPro-MACS combination (16 separations), a median purity of 96.2% and a median recovery of 42% CD34+ cells could be achieved, whereas the two step MACS systems (55 and 29 separations) showed a median purity of 97.6% and 98.0% and a median recovery of 96.5% and 97%, respectively. Depletion of T cells was profound (4–5 log). A total of 34 patients in the autologous and 18 patients in the allogeneic setting have been transplanted with highly enriched CD34+ cells, obtained by these methods. Only one patient failed to engraft, all other patients showed a rapid and sustained hematological engraftment with the longest follow-up of 3 years. In summary, especially the two step MACS systems have proven to be appropriate tools for enrichment of CD34+ cells, yielding both high purity and good recovery, and can thus be used for tumor cell purging in the autologous setting and for effective T cell depletion in the allogeneic setting.

Isolation and transplantation of highly purified autologous peripheral CD34(+) progenitor cells: purging efficacy, hematopoietic reconstitution and long-term outcome in children with high-risk neuroblastoma.

We have investigated the purging efficacy of positive selection of autologous mobilized CD34+ peripheral stem cells in 22 children with high-risk neuroblastoma. CD34+ cell selection was performed using the method of magnetic-activated cell sorting (MACS). The median purity of the CD34+ cells post selection was 97.6% (range 81.7–99.7). For detection of contaminating neuroblastoma cells before and after CD34+ selection, the chimeric anti-disialoganglioside GD2 antibody delta ch 14.18 was used. Prior to positive selection, various numbers of contaminating neuroblastoma cells were found in 17 patients. After positive CD34+ cell selection, low numbers of neuroblastoma cells were only detectable in four patients. In 18 patients, high-dose chemotherapy was performed and the isolated CD34+ cells were reinfused. In all patients, a rapid neutrophil recovery was seen with a median time to reach 0.5 × 109/l neutrophils of 12 days (range 8–24 days). Nine of the 18 patients are free of progression with a median follow-up of 55 months (range 45–70 months). Two patients are alive with relapse, six patients died due to progression or relapse and one patient died due to secondary AML 10 months after transplant while in remission from neuroblastoma. In summary, we show that, through a highly effective positive selection method, a high purging efficacy can be obtained without compromising the hematopoietic reconstitution capacity of the graft.

Transplantation of highly purified peripheral-blood CD34 + progenitor cells from related and unrelated donors in children with nonmalignant diseases

Summary:Transplantation of allogeneic stem cells is currently the only curative treatment for some nonmalignant pediatric diseases. We investigated whether transplantation of purified CD34+ stem cells prevents acute and chronic GvHD and reduces transplant-related mortality. A total of 25 pediatric patients with nonmalignant diseases underwent allogeneic transplantation from 26 donors (matched related n=4, matched or partially matched unrelated n=14, mismatched related n=8). All grafts were purified peripheral-blood CD34+ stem cells mobilized with G-CSF. Patients received a median of 12.9 × 106 CD34+ progenitor cells with a median of 6.1 × 103 contaminating T-lymphocytes per kilogram of body weight. No post transplant immunosuppressive drugs were given for prophylaxis of GvHD. Engraftment was seen in 21 patients. Three patients engrafted after a second transplant and one patient failed to engraft. Two patients had autologous reconstitution 1.5 years post transplant and one of them was successfully retransplanted. No acute GvHD >grade II was seen, and only two patients developed limited, chronic GvHD. In all, 22 patients (88%) are alive with a median follow-up of 3.7 years. In total, 19 patients (76%) are free of disease or of progression. Transplantation of highly purified peripheral-blood CD34+ stem cells is associated with low toxicity in patients with nonmalignant diseases.

Rituximab mediates in vitro antileukemic activity in pediatric patients after allogeneic transplantation

Summary:Relapse is a major problem after allogeneic transplantation in children with acute B-lineage lymphoblastic leukemias (ALL) and lymphomas and additional therapeutic strategies are needed to increase graft versus leukemia effects without inducing graft versus host disease (GvHD). Several studies have shown the efficacy of a humanized CD20 antibody (rituximab) for treatment of CD20+ malignancies together with conventional chemotherapy but less is known about its post transplant usefulness. We studied the ability of rituximab to mediate antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) with effector cells and complement from patients who were transplanted with T-cell-depleted grafts from unrelated or mismatched related donors. Highest lytic activity (ADCC) was observed against leukemia-derived MHH4 cells and Burkitts lymphoma-derived Raji cells in the first months after transplantation, corresponding to the high percentage of regenerating CD56+CD16+ cells. Moreover, primary cryopreserved ALL-blasts from a pediatric patient were also efficiently lysed. Increased lysis was obtained after stimulation with interleukin-2. Combination of ADCC and CDC had additive effects. These findings encourage clinical trials on the use of rituximab for improving minimal residual disease control and relapse prevention after allogeneic high-risk transplantation in the small group of pediatric patients with CD20+ leukemias/lymphomas.

IDENTIFICATION OF HIGH-RISK PATIENTS WITH APLASTIC ANÆMIA IN SELECTION FOR ALLOGENEIC BONE-MARROW TRANSPLANTATION

Abstract Of 75 patients with aplastic anaemia treated between 1968 and 1975, 33 were retrospectively considered as potential candidates for allogeneic bone-marrow transplantation on the basis of their age and severity of marrow failure. The prognosis of these patients with conservative treatment was assessed from parameters obtained at the time of the initial diagnosis. Initial peripheral-blood granulocyte or platelet concentrations were not of prognostic value. In contrast, initial reticulocyte concentrations, allowed separation of the patients into two groups with poor and good prognosis. Low initial reticulocyte concentrations (less than 10 000/μl) indicated those patients at extremely high risk of succumbing to their marrow aplasia (there were no survivors 36 months after diagnosis). In contrast, 75% of those patients with more than 10 000 reticulocytes per μl at diagnosis survived for 3 years. Initial peripheral-blood reticulocyte concentrations thus appear to indicate the extent of the marrow failure in aplastic anaemia more accurately than granulocytes or platelets. Low initial reticulocyte concentrations may indicate, among patients with severe aplastic anaemia, those for whom allogeneic bone-marrow transplanation should be seriously considered; patients with higher initial reticulocyte concentrations may benefit from conservative treatment.