Lubomir Arseniev

Allogeneic bone marrow transplantation in a patieni with Shwachman-Diamond syndrome

Shwachman-Diamond syndrome (SDS) is a rare inherited disorder involving concomitant neutropenia and exocrine pancreatic insufficiency. About 25% of patients develop hematopoiesic malignancies. We describe a 24-year-old male patient with SDS who underwent allogeneic bone marrow transplantation (BMT) because of progression into acute myeloid leukemia (AML) following myelodysplastic syndrome (MDS). The BMT preparative regimen consisted of busulfan (16 mg/kg body wt.), followed by cyclophosphamide (120 mg/kg). Cyclosporin A and short methotrexate were used for graft-versus-host disease (GvHD) prophylaxis. The post-transplant period was complicated by staphylococcal septicemia, CMV infection, renal insufficiency, and acute GvHD grade III. Hematological recovery was delayed (post-transplant day +55). The patient was discharged at day +68 in complete remission without any evidence of MDS. RFLP fingerprint analysis showed complete engraftment of the donors hematopoiesis. The patients leukemia relapsed 9 months post-transplant, and death followed due to CMV infection and multiorgan failure. Despite the fatal course in this patient, allogeneic BMT could be an option for curative treatment of the hematopoietic failure in SDS. The interaction of BMT with pancreatic insufficiency still has to be ascertained.

Technical and safety aspects of blood and marrow transplantation using G-CSF mobilized family donors.

An allogeneic transplantation programme using immunoselected blood progenitor and bone marrow CD34+ cells has been established. Thirteen healthy HLA-matched, MLC negative sibling donors received two doses of 5 micrograms kg-1 G-CSF (s.c. daily) for 5 days. On days 4 and 5, large-volume mononuclear cell aphereses were performed (COBE Spectra) and on day 5 one unit of autologous blood was obtained. Mononuclear cells were pooled and cryopreserved after CD34+ cell-immunoselection on day 5. Bone marrow (BM) of the same donors was procured under routine conditions 10-45 days later (median: 27 days). The final graft consisted of blood CD34+ cells with either complete BM (n = 5) or immunoselected BM CD34+ cells (n = 8). The present paper describes the progenitor cell mobilization and apheresis protocol and analyzes the cell loss by BM and peripheral blood progenitor cell (PBPC) donation. Considerably larger amounts of mononuclear cells (CD45+), T-lymphocytes (CD3+) and platelets were lost by the apheresis as compared to bone marrow without apparent immediate clinical consequences for the donors. Owing to cross-cellular contamination of the apheresis concentrate, blood platelet count (PC) significantly decreased (mean PC after the second apheresis 116 x 10 microL-1); furthermore on average 3.04 x 10(10) CD3+ cells were removed by two apheresis sessions. This loss did not lead to long-term total lymphocyte count changes (2370 microL-1 versus 1889 microL-1) as observed during the long-term follow-up of 7/13 donors (mean 290 days). Subjectively, the PBPC collections were better accepted than BM donations in all but one family donor.

CD34 positive blood cells for allogeneic progenitor and stem cell transplantation.

The transplantation of allogeneic peripheral blood progenitor cells (PBPC) provides complete and sustained hematopoietic and lymphopoietic engraftment. In healthy donors, large amounts of PBPC can be mobilized with hematopoietic growth factors. However, the high content of immunocompetent T-cells in apheresis products may expose recipients of allogeneic PBPC to an elevated risk of acute and chronic graft-versus-host disease. Thus, the use of appropriate T-cell reduction, but not depletion might reduce this risk. The hazards of graft rejection and a higher relapse rate can be avoided by maintaining a portion of the T-cells in the graft. The positive selection of CD34+ cells from peripheral blood preparations simultaneously provides an approximately 1000-fold reduction of T-cells. These purified CD34+ cells containing committed and pluripotent stem cells are suitable for allogeneic transplantation and can be used in the following instances: 1. As hematopoietic stem and progenitor cell transplantation instead of bone marrow cells, from HLA-identical family donors; 2. for increasing the stem cell numbers from HLA-mismatched or three HLA-loci different family donors in order to reduce the incidence of rejection but without increasing the T-cell number; 3. boosting of poor marrow graft function with stem cells from the same family donors; 4. transplantation from volunteer matched unrelated donors; 5. split transplantation of CD34+ and T-cells; 6. addition of ex vivo expanded CD34+ cells to blood cell or bone marrow transplantation; 7. generation of antigen specific immune effector cells and antigen presenting cells for cell therapy.

Outcome of peripheral blood stem cell mobilization in advanced phases of CML is dependent on the type of chemotherapy applied

Abstractu2002High-dose chemotherapy with autologous transplantation of in vivo purged PBSC is a novel investigational approach to treating chronic myelogenous leukemia (CML) patients not responsive to conventional therapy with interferon-α (IFN-α) and not eligible for allogeneic transplantation. PBSC mobilization using either 5+2/7+3-type chemotherapy or mini-ICE/ICE chemotherapy was investigated in 43 patients with advanced phases of Philadelphia (Ph)-positive CML. Thirty patients were in late chronic phase (>12u2009months post diagnosis) and 13 patients in accelerated phase (AP) or blast crisis (BC). Contamination with Ph-positive cells was evaluated in harvests from 37/43 patients. The outcome of PBSC mobilization was dependent on the type of chemotherapy administered: a complete or major cytogenetic response (<35% Ph-positive metaphases) in leukapheresis collections was obtained in ten of 15 patients treated with mini-ICE/ICE but in only three of 28 patients treated with 5+2/7+3 chemotherapy. One patient (1/43) in blast crisis died during mobilization therapy (2%). Twenty-five patients underwent PBSC transplantation and all of them engrafted successfully. Transplantation-related mortality was 0%. The data show that in advanced phases of CML the chance of harvesting Ph-negative peripheral blood stem cells depends on the type of chemotherapy used for mobilization.

Cyclic-adenosine 3', 5'-monophosphate-stimulated c-fos gene transcription involves distinct calcium pathways in single β-cells

Abstract In β-cells activation of the cyclic AMP (cAMP)-signaling cascade stimulates c-fos mRNA expression, which involves cAMP- and Ca 2+ -mediated mechanisms. To delineate potential crosstalk between both pathways at the transcriptional level we simultaneously measured c-fos promoter-driven enhanced green fluorescent protein (EGFP) expression and cytosolic free calcium ([Ca 2+ ] i ) in single β-cells (HIT-T15). Forskolin stimulated a rapid rise in cellular cAMP and in [Ca 2+ ] i through activation of voltage-sensitive Ca 2+ -influx and enhanced wild-type c-fos promoter-driven EGFP (pF711d2EGFP) expression about 4-fold after 6 h. The voltage-sensitive Ca 2+ channel (VSCC)-blocker nifedipine, which completely blocked the forskolin-induced rise in [Ca 2+ ] i , partially inhibited the forskolin-induced increase in pF711d2EGFP expression, while it was completely abolished in Ca 2+ -free medium. VSCC-dependent Ca 2+ -influx per se when stimulated by K + (45 mM) increased pF711d2EGFP expression only minimally. No correlations could be delineated between the forskolin-induced amplitude of the Ca 2+ signal and the expression of pF711d2EGFP at the single cell level, which may indicate that small rises in [Ca 2+ ] i are sufficient to fully activate the Ca 2+ -dependent pathways required for cAMP-dependent c-fos promoter regulation. In experiments with various deletion constructs of the c-fos promoter, it could be shown that cAMP-mediated activation of the c-fos promoter involves both the cAMP-responsive element (CRE) and the serum-responsive element (SRE). While nifedipine completely abrogated the cAMP-dependent activation of c-fos transcription via the SRE, the CRE-mediated effect of cAMP on the c-fos promoter remained unaffected by nifedipine. Thus, cAMP and Ca 2+ are required for full c-fos promoter activation by the cAMP-signaling pathway in β-cells. cAMP-dependent Ca 2+ -influx through VSCC is crucial for c-fos gene transcription via the SRE, whereas cAMP-mediated activation of the CRE demands Ca 2+ -influx, which is distinct from voltage-sensitive Ca 2+ -influx. This indicates a complex interplay between cAMP and Ca 2+ in controlling c-fos gene transcription and suggests that the mode of Ca 2+ entry may differentially act on signaling pathways leading to gene transcription in β-cells.

Mitoxantrone, Cytosine Arabinoside and VP-16 (MAV) for De Novo Acute Myeloid Leukemia: A Pilot Study

In several studies it could be shown that mitoxantrone has considerable antileukemic activity [1–3]. In a previous report we showed that the combination of mitoxantrone with cytosine arabinoside and VP-16 is effective in relapsed and refractory acute myeloblastic leukemia (AML) [4]: 21 (58.3%) of 36 patients achieved a complete hematological remission with a therapy consisting of mitoxantrone (M) 10 mg/m2 i. v., cytosine arabinoside (A) 100 mg/m2 continuous infusion and VP-16 (V) 100 mg/m2 i. v., each for 5 days (MAV protocol). Encouraged by the high antileukemic activity and moderate toxicity, we applied this triple combination in elderly patients with untreated AML. The question was whether the effectiveness of MAV therapy could lead to a high response rate in this group of patients with intermediate risk.