Intramyocardial bone marrow cell injection does not lead to functional improvement in patients with chronic ischaemic heart failure without considerable ischaemia.

BACKGROUND: It has been suggested that bone marrow cell injection may have beneficial effects in patients with chronic ischaemic heart disease. However, previous trials have led to discrepant results of cell-based therapy in patients with chronic heart failure. The aim of this study was to evaluate the efficacy of intramyocardial injection of mononuclear bone marrow cells in patients with chronic ischaemic heart failure with limited stress-inducible myocardial ischaemia. METHODS AND RESULTS: This multicentre, randomised, placebo-controlled trial included 39 patients with no-option chronic ischaemic heart failure with a follow-up of 12 months. A total of 19 patients were randomised to autologous intramyocardial bone marrow cell injection (cell group) and 20 patients received a placebo injection (placebo group). The primary endpoint was the group difference in change of left ventricular ejection fraction, as determined by single-photon emission tomography. On follow-up at 3 and 12 months, change of left ventricular ejection fraction in the cell group was comparable with change in the placebo group (P = 0.47 and P = 0.08, respectively). Also secondary endpoints, including left ventricle volumes, myocardial perfusion, functional and clinical parameters did not significantly change in the cell group as compared to placebo. Neither improvement was demonstrated in a subgroup of patients with stress-inducible ischaemia (P = 0.54 at 3­month and P = 0.15 at 12-month follow-up). CONCLUSION: Intramyocardial bone marrow cell injection does not improve cardiac function, nor functional and clinical parameters in patients with severe chronic ischaemic heart failure with limited stress-inducible ischaemia. CLINICAL TRIAL REGISTRATION: NTR2516.

T-cell-pre-stimulated monocytes promote neovascularisation in a murine hind limb ischaemia model.

AIM: Monocytes play a significant role in neovascularisation. The stimuli that differentiate monocytes along a pro-angio-/arteriogenic-supporting pathway are currently unclear. We investigated whether pre-stimulation of human monocytes with soluble T-cell-derived factors improves revascularisation in murine hind limb ischaemia as a new option for therapeutic angio- and arteriogenesis. DESIGN: Human monocytes were cultured with or without soluble T-cell-derived factors. Unstimulated and pre-stimulated monocytes were transfused after induction of hind limb ischaemia in nude mice. METHODS: Blood flow was measured with laser Doppler perfusion imaging. Collaterals were visualised by immunohistochemistry and angiography. Monocytes were characterised by flowcytometry and Bio-Plex assays. RESULTS: Transfusion of T-cell-pre-stimulated monocytes significantly improved blood flow recovery after hind limb ischaemia and increased collateral size and collateral and capillary number in the post-ischaemic paw. Pre-stimulated monocytes produced a wide variety of factors that support neovascularisation such as platelet-derived growth factor-BB, vascular-endothelial growth factor, interleukin-4 and tumour necrosis factor-α. Few transfused human cells were detected in the muscle tissue, suggesting that paracrine rather than direct effects appear responsible for the enhanced recovery of blood flow observed. CONCLUSION: These results show a beneficial role for T-cell-pre-stimulated monocytes in neovascularisation, rendering the monocyte a potential candidate for regenerative cell therapy that promotes revascularisation in peripheral arterial disease patients.

Inhibition of human macrophage colony formation by interleukin 4.

We investigated the effects of human rIL-4 on in vitro hematopoiesis. A profound inhibition of macrophage colony formation by IL-4 was observed, whereas colony growth of other lineages was not affected. Inhibition of macrophage colony growth was not restricted to GM-CSF-induced colony growth but was also present in cultures stimulated with M-CSF. This inhibition was not only observed in cultures of light density bone marrow cells, but also in cultures of monocyte- and T lymphocyte-depleted bone marrow cells. Since a similar inhibition was observed in cultures of CD34+HLA-DR+-enriched bone marrow cells, a direct action of IL-4 on monocyte-committed progenitor cells is suggested.

Human hematopoietic progenitor cells in long-term cultures express HLA-DR antigens and lack HLA-DQ antigens.

The expression of HLA-DR antigenic determinants on human hematopoietic progenitor cells (HPC) capable of differentiating into mature blood cells, as determined in semisolid cultures, has been demonstrated previously (3-7). Here, we investigated the expression of class II determinants on HPC responsible for the sustained proliferation of colony-forming units of granulocyte/macrophage (CFU-GM), of multilineage HPC (CFU-GEMM, granulocyte/erythrocyte/macrophage/megakaryocyte), and burst-forming units of erythroid cells (BFU-E) in liquid long-term cultures. Using both fluorescence-activated cell sorting and complement-dependent cytotoxicity assays, HLA-DR determinants could be identified on virtually all these HPC capable of proliferating in long-term cultures. Experiments in which the stromal layer had been irradiated provided evidence that the HPC themselves were truly HLA-DR+, and that the sustained proliferation of HPC was not due to activation of HLA-DR- residual HPC in the stromal layer by reinoculated HLA-DR+ accessory cells. Furthermore, it was shown that all HPC recognized in semisolid and liquid long-term cultures were HLA-DQ-. These results suggest that the human true pluripotential stem cell is HLA-DR+. These results open the possibility of studying class II-dependent regulation of hematopoiesis in liquid long-term cultures.

Cellularly defined minor histocompatibility antigens are differentially expressed on human hematopoietic progenitor cells.

Previously, five CTL lines directed against minor histocompatibility (mH) antigens designated HA-1-5 have been established from peripheral blood of patients after allogeneic bone marrow transplantation (BMT), and have been characterized using population and family studies. All cell lines showed specific HLA class I-restricted lysis of PHA-stimulated peripheral blood target cells from donors positive for the particular mH antigens. After 4 h of incubation of the mH antigen HA-3-specific CTL line with bone marrow cells from HA-3+ donors, complete class I-restricted inhibition of colony growth of the hematopoietic progenitor cells was observed even at low E/T ratios, indicating that the HA-3 antigen is strongly expressed on hematopoietic stem cells. Therefore, this antigen may be a target structure in the immune-mediated rejection of the hematopoietic graft in case of incompatibility for this determinant between donor and recipient in allogeneic BMT. In contrast, incubation of bone marrow cells with the antigen-specific anti-HA-1, -2, -4, and -5 CTL lines did not result in growth inhibition of the hematopoietic progenitor cells tested. After a prolonged incubation time and using a very high E/T ratio, progenitor cells from HA-2+ or HA-5+ donors were killed to some extent by the anti-mH-specific CTL lines, although the growth inhibition observed was minor and variable. Our results show that mH antigens are differentially expressed on human hematopoietic progenitor cells. Therefore, only some of these antigens may be targets in immune-mediated rejection of the bone marrow graft.

Interleukin 6 is a permissive factor for monocytic colony formation by human hematopoietic progenitor cells.

Since monocytes and macrophages that arise during the culture of bone marrow progenitor cells are potential sources of interleukin 6 (IL-6), we investigated whether auto- or paracrine production of this factor is involved in colony formation by normal hematopoietic progenitor cells. We added a polyclonal anti-IL-6 antiserum and a monoclonal anti-IL-6 antibody to cultures of monocyte- and T cell-depleted bone marrow cells. Colony formation was stimulated with granulocyte/monocyte-colony-stimulating factor (GM-CSF), monocyte-CSF, or IL-3. Addition of anti-IL-6 antibody resulted in decreased numbers of monocytic colonies to 40-50% of control values, whereas the numbers of granulocytic colonies were not altered. The inhibitory effect was preserved in cultures of CD34(+)-enriched bone marrow cells. As a second approach, we added a monoclonal antibody directed against the IL-6 receptor to cultures of monocyte- and T cell-depleted bone marrow cells. This antibody almost completely inhibited the growth of monocytic colonies, again without decreasing the number of granulocytic colonies. Finally, the importance of IL-6 in monocytopoiesis was demonstrated in serum-deprived bone marrow cultures: addition of exogenous IL-6 to cultures stimulated with GM-CSF resulted in increased numbers of monocytic colonies. Our results indicate that the permissive presence of IL-6 is required for optimal monocytic colony formation by bone marrow progenitor cells.

Colony growth of human hematopoietic progenitor cells in the absence of serum is supported by a proteinase inhibitor identified as antileukoproteinase.

Serum contains many growth factors and nutrients that stimulate colony formation of hematopoietic progenitor cells (HPC) in semisolid cultures. In the absence of serum, no proliferation of HPCs could be obtained in semisolid medium cultures of partially purified bone marrow cells in the presence of multiple hematopoietic growth factors, insulin, cholesterol, and purified clinical-grade human albumin. This appeared to be due to a suppressive activity induced by monocyte- and T lymphocyte-depleted accessory cells on CD34+ HPCs. Serum-free conditioned medium from the bladder carcinoma cellline 5637 could replace serum to support the growth of HPCs in these cultures. After gel filtration and reverse-phase high-performance liquid chromatography of 5637 supernatants, this activity could be attributed to a 15-kD protein that was further identified by NH2-terminal sequence analysis as the serine proteinase inhibitor antileukoproteinase (ALP). The growth-supportive activity from the 5637 conditioned medium and the (partially) purified fractions could be completely neutralized by a polyclonal rabbit IgG antibody against human ALP (huALP). Similar supportive effects on the growth of HPC could be obtained in the presence of recombinant huALP. We demonstrated that the COOH-terminal domain of ALP containing the proteinase inhibitory activity was responsible for this effect. alpha-1 proteinase inhibitor was capable of similar support of in vitro HPC growth. These results illustrate that proteinase inhibitors play an important role in the in vitro growth of hematopoietic cells by the neutralization of proteinases produced by bone marrow accessory cells. This may be of particular relevance for in vitro expansion of human hematopoietic stem cells in serum-free media.

Minor histocompatibility antigen-specific cytotoxic T cell lines, capable of lysing human hematopoietic progenitor cells, can be generated in vitro by stimulation with HLA-identical bone marrow cells.

Recipient-antidonor alloreactivity before HLA genotypically identical bone marrow transplantation (BMT) between donor-recipient pairs that are negative in the mixed lymphocyte reaction (MLR), the cell-mediated lympholysis (CML) assay, and the lymphocyte crossmatch was not detectable in the majority of cases, using recipient peripheral blood lymphocytes (PBL) collected before BMT as responder cells and donor PBL as stimulator cells. However, when donor bone marrow mononuclear cells (BMMNC) instead of PBL were used as stimulator cells, we could detect donor-specific alloreactivity in 7 of 10 HLA genotypically identical donor-recipient pairs. To demonstrate that this alloreactivity was minor histocompatibility (mH) antigen specific and not directed against HLA class I splits or variants, two cytotoxic T lymphocyte (CTL) lines were tested in further detail against phytohemagglutinin (PHA) blasts from pairs of HLA genotypically identical siblings positive for the HLA class I restriction molecule. Both CTL lines recognized mH antigens, as illustrated by the differential recognition of PHA blasts of one of the two siblings from several pairs. The potential role of these mH antigen-specific CTLs in bone marrow graft rejection was demonstrated by the mH antigen-specific growth inhibition of hematopoietic progenitor cells from the original bone marrow donor and from HLA class I restriction molecule-positive individuals who expressed the mH antigens on their PBL and BMMNC. Our assay can be used in HLA genotypically identical BMT to detect a recipient-antidonor response, directed against cellularly defined mH antigens expressed on donor HPC, BMMNC, and PBL, before transplantation.

Growth inhibition of clonogenic leukemic precursor cells by minor histocompatibility antigen-specific cytotoxic T lymphocytes.

Minor histocompatibility (mH) antigens appear to play a major role in bone marrow transplantation (BMT) using HLA-identical donors. Previously, we reported the isolation of major histocompatibility complex (MHC)-restricted mH antigen-specific cytotoxic T lymphocytes (CTL) from patients with graft-vs.-host disease or rejection after HLA-identical BMT. We have demonstrated that mH antigens can be recognized on hematopoietic progenitor cells, and residual recipient CTL specific for mH antigens expressed on donor hematopoietic progenitor cells may be responsible for graft rejection in spite of intensive conditioning regimens in HLA-identical BMT. Here, we investigated whether mH antigen-specific CTL directed against the mH antigens HA-1 to HA-5 and the male-specific antigen H-Y were capable of antigen-specific inhibition of in vitro growth of clonogenic leukemic precursor cells. We demonstrate that mH antigen-specific CTL against all mH antigens tested can lyse freshly obtained myeloid leukemic cells, that these mH antigen-specific CTL can inhibit their clonogenic leukemic growth in vitro, and that this recognition is MHC restricted. We illustrate that leukemic (precursor) cells can escape elimination by mH antigen-specific CTL by impaired expression of the relevant MHC restriction molecule. We suggest that mH antigen-specific MHC-restricted CTL may be involved in vivo in the graft-vs.-leukemia reactivity after BMT.

Long-term follow-up results after autologous haematopoietic stem cell transplantation for severe systemic sclerosis.

OBJECTIVE: Systemic sclerosis (SSc) is a generalised autoimmune disease, causing morbidity and a reduced life expectancy, especially in patients with rapidly progressive diffuse cutaneous SSc. As no proven treatment exists, autologous haematopoietic stem cell transplantation (HSCT) is employed as a new therapeutic strategy in patients with a poor prognosis. This study reports the effects on survival, skin and major organ function of HSCT in patients with severe diffuse cutaneous SSc. PATIENTS AND METHODS: A total of 26 patients were evaluated. Peripheral blood stem cells were collected using cyclophosphamide (4 g/m2) and rHu G-CSF (5 to 10 microg/kg/day) and were reinfused after positive CD34+ selection. For conditioning, cyclophosphamide 200 mg/kg was used. RESULTS: After a median follow-up of 5.3 (1-7.5) years, 81% (n = 21/26) of the patients demonstrated a clinically beneficial response. The Kaplan-Meier estimated survival at 5 years was 96.2% (95% CI 89-100%) and at 7 years 84.8% (95% CI 70.2-100%) and event-free survival, defined as survival without mortality, relapse or progression of SSc, resulting in major organ dysfunction was 64.3% (95% CI 47.9-86%) at 5 years and 57.1% (95% CI 39.3-83%) at 7 years. CONCLUSION: This study confirms that autologous HSCT in selected patients with severe diffuse cutaneous SSc results in sustained improvement of skin thickening and stabilisation of organ function up to 7 years after transplantation.

Evaluation of 'out-of-specification' CliniMACS CD34-selection procedures of hematopoietic progenitor cell-apheresis products.

BACKGROUND: Immunomagnetic selection of CD34(+) hematopoietic progenitor cells (HPC) using CliniMACS CD34 selection technology is widely used to provide high-purity HPC grafts. However, the number of nucleated cells and CD34+ cells recommended by the manufacturer for processing in a single procedure or with 1 vial of CD34 reagent is limited. METHODS: In this retrospective evaluation of 643 CliniMACS CD34-selection procedures, we validated the capacity of CliniMACS tubing sets and CD34 reagent. Endpoints of this study were the recovery and purity of CD34+ cells, T-cell depletion efficiency and recovery of colony-forming units-granulocyte-macrophage (CFU-GM). RESULTS: Overloading normal or large-scale tubing sets with excess numbers of total nucleated cells, without exceeding the maximum number of CD34+ cells, had no significant effect on the recovery and purity of CD34+ cells. In contrast, overloading normal or large-scale tubing sets with excess numbers of CD34+ cells resulted in a significantly lower recovery of CD34+ cells. Furthermore, the separation capacity of 1 vial of CD34 reagent could be increased safely from 600 x 10(6) CD34+ cells to 1000 x 10(6) CD34+ cells with similar recovery of CD34(+) cells. Finally, T-cell depletion efficiency and the fraction of CD34+ cells that formed CFU-GM colonies were not affected by out-of-specification procedures. DISCUSSION: Our validated increase of the capacity of CliniMACS tubing sets and CD34 reagent will reduce the number of selection procedures and thereby processing time for large HPC products. In addition, it results in a significant cost reduction for these procedures.

Intramuscular or combined intramuscular/intra-arterial administration of bone marrow mononuclear cells: a clinical trial in patients with advanced limb ischemia.

AIM: Recent evidence indicates that bone marrow mononuclear cells (BMC) promote collateral vessel formation in patients with severe peripheral arterial disease (PAD). However, aspects concerning optimal administration mode, durability and long-term safety require consideration. Combined intra-arterial (IA) plus intramuscular (IM) BMC delivery may be more effective than exclusive intramuscular injections. The aim of this study was to evaluate feasibility, safety and effect of exclusive IM versus combined IM+IA delivery of autologous BMC in patients who were not candidates for surgical or endovascular treatment. METHODS: Twenty-seven patients were treated with either combined IA+IM (N=12) or sole IM (N=15) administration of autologous BMC. Efficacy was assessed after 1, 6 and 12 months. Limb salvage, pain-free walking distance, ankle-brachial pressure index (ABI) and pain scores were evaluated. RESULTS: There were no adverse reactions related to injection of the cells. Three patients died within the first year of follow-up due to non-procedure related causes. Two patients in the IA+IM group required limb amputation because of ongoing critical ischemia versus 7 patients in the IM group (P=0.17). BMC treatment in the remaining patients resulted in a significant and sustained (>12 months) improvement. Pain-free walking distance improved from 81+/-56 meters at baseline to 257+/-126 meters at t=6 months (P=0.0002). Mean ABI increased 23% after 6 months (P=0.01) and pain score reduced for up to 50% as shown by Brief Pain Inventory (P=0.001). CONCLUSION: Both IM and combined IM/IA delivery of autologous BMC are safe, and result in relevant and sustained improvement in a considerable proportion of patients with severe PAD who are not amenable for conventional treatment.

Minor histocompatibility antigen H-Y is expressed on human hematopoietic progenitor cells.

Polymorphic minor transplantation antigens probably play an important role in immune mediated graft rejections of bone marrow transplants. Mapping of these antigens on hematopoietic progenitor cells (HPC) is important since these antigenic determinants may serve as target structures in the rejection process, and it ultimately opens the possibility to match for these antigens. Using a cell-mediated cytotoxicity assay with H-Y-specific cytotoxic T lymphocytes as effector cells, a dose-dependent growth inhibition up to 100% of myeloid (CFU-GM), erythroid (BFU-E) and multipotential (CFU-GEMM) HPC of male donors was obtained, indicating expression of the H-Y antigen on these progenitor cells. In contrast, inhibition of relatively mature erythroid and myeloid progenitor cells was only 40-50%, indicating that the recognition of the H-Y antigen diminished during maturation of erythroid and myeloid HPC. Our results show that the H-Y antigen can be recognized on HPC as a target for cytotoxic T cell responses. This may be important in graft rejection of male donor bone marrow grafts by female recipients.

Stem cell transplantation in Europe: trends and prospects.

The aim of the present study was to identify trends in numbers of European patients treated with autologous and allogeneic haematopoietic stem cell transplantation (HSCT) as well as to provide anticipated transplant rates for the upcoming years. The following indications were considered: haematological malignancies (acute leukaemias, myeloproliferative disorders, lymphoproliferative disorders and multiple myeloma), solid tumours and non-malignant diseases. Numbers of patients treated from 1990 to 2004 were extracted from the European Group for Blood and Marrow Transplantation database, extrapolated to 2012 using mathematic models and adjusted to the literature study and expert opinion. In Europe, a 13% raise in HSCT utilisation is to be expected from 2005 to 2010, mostly due to the growing application of reduced-intensity conditioning regimens followed by allogeneic HSCT. Growing transplant rates are likely to exert health expenditure budgets and put pressure on health care providers and health insurers in Europe. Therefore, the rapid expansion would ideally imply a simultaneous increase in HSCT budgets.

Long-term follow-up of myeloablative allogeneic stem cell transplantation using Campath "in the bag" as T-cell depletion: the Leiden experience.

Graft-versus-host disease (GVHD) is a major cause of mortality and morbidity after allogeneic stem cell transplantation (alloSCT) but can be prevented by removing T-lymphocytes from the graft. Campath (anti-CD52) antibodies have been widely used in vivo for T-cell depletion following conventional and reduced intensity conditioning regimens. The use of Campath in vivo was associated with a significant reduction in GVHD but at the cost of impaired immune reconstitution. We evaluated the long-term outcome of 73 myeloablative allogeneic stem cell transplants with HLA-identical sibling donors using Campath "in the bag" as method of in vitro T-cell depletion. All patients engrafted and hematopoietic recovery was uneventful, resulting in a median of 99% donor chimerism at 3 months after alloSCT. Cytomegalovirus (CMV) reactivation occurred in 53% of the patients. No CMV disease was observed probably as a result of pre-emptive (val)ganciclovir treatment. The incidence of aGVHD was low (22% grade II). No grades III-IV aGVHD was observed and extensive chronic GVHD (cGVHD) occurred in 19% of the patients. The low incidence of GVHD and successful pre-emptive antiviral therapy resulted in low TRM of 8%. Sixteen patients died due to disease relapse after alloSCT, resulting in an overall survival of 48% at 5-years after alloSCT.

[Intramyocardial injection of autologous bone-marrow stem cells in a 74-year-old man with untreatable angina pectoris and demonstrated myocardial ischaemia]. / Intramyocardiale injectie van autologe stamcellen bij een 74-jarige man met onbehandelbare angina pectoris en bewezen myocardischemie.

For a 74-year-old man with angina pectoris and demonstrated myocardial ischaemia, optimal medical treatment was insufficient. He was ineligible for coronary revascularization. To improve myocardial perfusion, autologous bone-marrow stem cells were injected into the ischaemic myocardium. During the months following the injection, the frequency of angina attacks decreased and myocardial perfusion and cardiac function improved.

A phase I study for intravenous autologous mesenchymal stromal cell administration to patients with severe emphysema.

BACKGROUND: Mesenchymal stromal cells (MSCs) may reduce inflammation and promote tissue repair in pulmonary emphysema. AIM: To study the safety and feasibility of bone marrow-derived autologous (BM-) MSC intravenous administration to patients with severe emphysema. DESIGN: A phase I, prospective open-label study registered at ClinicalTrials.gov as NCT01306513 Eligible patients had lung volume reduction surgery (LVRS) on two separate occasions. During the first LVRS bone marrow was collected, from which MSCs were isolated and expanded ex vivo After 8 weeks, patients received two autologous MSC infusions 1 week apart, followed by the second LVRS procedure at 3 weeks after the second BM-MSC infusion. METHODS: Up to 3 weeks after the last MSC infusion adverse events were recorded. Using immunohistochemistry and qPCR for analysis of cell and proliferation markers, emphysematous lung tissue obtained during the first surgery was compared with lung tissue obtained after the second surgical session to assess BM-MSC effects. RESULTS: From 10 included patients three were excluded: two did not receive MSCs due to insufficient MSC culture expansion, and one had no second surgery. No adverse events related to MSC infusions occurred and lung tissue showed no fibrotic responses. After LVRS and MSC infusions alveolar septa showed a 3-fold increased expression of the endothelial marker CD31 (P = 0.016). CONCLUSIONS: Autologous MSC treatment in severe emphysema is feasible and safe. The increase in CD31 expression after LVRS and MSC treatment suggests responsiveness of microvascular endothelial cells in the most severely affected parts of the lung.

Treatment of T-cell prolymphocytic leukemia with human CD52 antibody.

PURPOSE: T-prolymphocytic leukemia (T-PLL) is an aggressive malignancy of mature T cells refractory to conventional chemotherapy, with a median survival duration of 7.5 months. We report here promising results with the use of a genetically reshaped human CD52 antibody, CAMPATH-1H. PATIENTS AND METHODS: Fifteen patients with T-PLL, most of whom had received the purine analog deoxycoformycin (DCF), were treated with CAMPATH-1H. Results were compared with those of 25 patients treated with DCF. RESULTS: Major responses occurred in 11 patients (73%) treated with CAMPATH-1H compared with 40% with DCF. Complete remissions (CRs) were documented in nine (60%) of the CAMPATH-1H cases and only three (12%) were obtained with DCF. CRs with CAMPATH-1H were durable, and re-treatment with the antibody resulted in second CRs in three relapsed patients. Two of them were successfully autografted with peripheral-blood and bone marrow stem cells collected during the first CR. Apart from first-dose reactions, infusions of CAMPATH-1H were well tolerated. However, two responding patients developed severe bone marrow aplasia that was fatal in one; the second remained moderately pancytopenic 21 weeks after stopping CAMPATH-1H therapy. The cause of this adverse effect is unknown. CONCLUSION: CAMPATH-1H is an effective agent in T-PLL and represents a significant improvement over other types of therapy. However, CAMPATH-1H alone is not sufficient for long-term remissions, and the role of autologous stem-cell transplantation needs further investigation.

Graft dysfunction and delayed immune reconstitution following haploidentical peripheral blood hematopoietic stem cell transplantation.

For many children with life-threatening hematological diseases, hematopoietic stem cell transplantation (HSCT) is the only curative option. In children lacking a matched related or unrelated donor and with the certainty that, left untreated, death will ensue alternative donors must be sought. Haplo-identical peripheral blood stem cell transplantation (PBSCT) from a healthy parent is a feasible alternative. To reduce the risk of fatal graft-versus-host disease (GvHD) as a complication of transplant across major histocompatibility antigens, intense T-cell depletion is required. Large numbers of purified, cytokine mobilized peripheral stem cells (the so-called mega-dose concept) are required to compensate for the significantly increased risk of either graft failure or early rejection. In our unit, despite this approach, graft dysfunction has, in a significant group of children, proved problematic and, despite salvage attempts at re-transplantation, usually fatal. In children with hematological malignant disease, our overall relapse-free survival is 41%. However, successful transplant outcome has been associated with considerable delays in immune reconstitution that can be implicated in subsequent viral reactivation. We are investigating new strategies to improve the outcome of haplo-identical PBSCT, which may allow us to offer this form of treatment to more children requiring urgent HSCT.

Inhibitory effect of interleukin-4 on the proliferation of acute myeloid leukemia cells with myelo-monocytic differentiation (AML-M4/M5); the role of interleukin-6.

Since interleukin-4 (IL-4) specifically inhibits monocytic colony formation in human bone marrow cultures, we investigated whether a similar inhibition could be observed in cultures of optimally stimulated acute myeloid leukemia cells with myelomonocytic differentiation (AML-M4/M5). Sensitivity to IL-4 was tested in 19 cases of AML-M4/M5, using both a 3H-thymidine incorporation assay and a clonogenic assay. Proliferation was stimulated with a combination of IL-3, granulocyte-monocyte colony-stimulating factor (GM-CSF), and conditioned medium from phytohemagglutinin-stimulated leukocytes (PHA-CM). In 13 out of 14 evaluable cases, IL-4 inhibited 3H-thymidine incorporation; stimulation was seen in one case. Using a clonogenic assay, IL-4 inhibited colony formation in all evaluable cases (n = 7). Addition of IL-6 did not alter the observed inhibition by IL-4 in 9 out of 10 cases tested. We conclude that IL-4 inhibits the proliferation of optimally stimulated AML-M4/M5 cells in most cases tested, and that this effect is not generally caused by inhibition of autocrine IL-6 production.