Remission induction chemotherapy induces in vivo caspase-dependent apoptosis in bone marrow acute myeloid leukemia blast cells and spares lymphocytes.

BACKGROUND: The goal of new therapeutic strategies is to adapt the treatment of acute myeloid leukemia (AML) patients to the prognostic and/or to the hematological response. METHODS: We analyzed in vivo apoptosis induction in blast cells and in lymphocytes of AML patients receiving remission induction treatment. RESULTS: We show, on 12 peripheral blood samples, that the increase of peripheral apoptotic blast cells cannot be considered as the earliest marker of the treatment efficiency, because the significant increase of apoptosis followed the white blood cell and the peripheral blast cell count reductions, probably due to an efficient clearance of circulating apoptotic cells. Furthermore, the study of 65 bone marrow samples at d15 showed that the treatment induced apoptosis of blast cells while sparing the lymphocytes. This apoptosis was evidenced both at the caspase and at the membrane levels using respectively fmk-VAD-FITC and Annexin V binding assays. We found that less than 50% of apoptosis, measured with the fmk-VAD-FITC, in the d15 residual bone marrow blast cells, correlated with lower disease-free survival probability. CONCLUSION: More studies are needed in larger series and earlier during the remission induction treatment to confirm the possible prognostic significance of in vivo apoptosis induction.

Protein synthesis and storage in human platelets: a defective storage of fibrinogen in platelets in Glanzmann's thrombasthenia.

In vivo metabolic labelling experiments were performed to investigate the ability of human platelets to synthesize and store fibrinogen and thrombospondin. Newly synthesized proteins were analyzed by SDS-polyacrylamide gel electrophoresis. Results were compared with those obtained for the platelets of a patient with Glanzmann's thrombasthenia where endogenous fibrinogen levels were severely reduced. Normal human platelets were able to synthesize the different subunits of fibrinogen and thrombospondin and to assemble them into native fibrinogen and thrombospondin molecules. This synthesis was inhibited by cycloheximide. Synthesis of both fibrinogen and thrombospondin was observed in the platelets of the Glanzmann's thrombasthenia patient. However, radiolabelled fibrinogen was no longer detected after an 18-h non-radioactive chase, although it was retained in the control platelets. Neosynthesized thrombospondin of the patient was normally preserved during the same chase period. When the fate of the radioactive fibrinogen was studied, it was found to be degraded in Glanzmann's thrombasthenia platelets to the same extent as neosynthesized cytoplasmic proteins, whereas in control platelets less degradation had occurred. We conclude that human platelets maintain a residual capacity to synthesize fibrinogen and that its deficiency in Glanzmann's thrombasthenia results from a storage abnormality and not from a synthesis defect.

Bcr-abl translocation can occur during the induction of multidrug resistance and confers apoptosis resistance on myeloid leukemic cell lines.

Apoptosis was studied in parental and mdr-1 expressing U937, HL60 and K562 myeloid leukemic cell lines using mdr unrelated inducers of apoptosis such as Ara-C, cycloheximide, serum deprivation, ceramide, monensin and UV irradiation. Apoptosis was efficiently induced by all these treatments in U937 and HL60 cells while K562 cells exhibited an apoptosis-resistant phenotype except with UV and monensin. The pattern of apoptosis resistance in mdr-1 expressing U937 (U937-DR) and HL60 (HL60-DR100) was similar to that presented by K562. This apoptosis-resistant phenotype of mdr cells was not overcome by concentrations of verapamil inhibiting the P-gp 170 pump. The acquisition of this phenotype was posterior to the mdr-1 expressing phenotype since a HL60-DR5 variant, selected at the beginning of the induction of resistance, presented a low level of mdr-1 expression without resistance to apoptosis. The variations observed in the Fas (CD95) expression between sensitive and resistant cells were not sufficient to account for apoptosis resistance. However, a high expression in Abl antigen was found in all the apoptosis-resistant cells. RT-PCR and Western blot analysis showed that this increase in Abl antigen content was accompanied by the expression in U937-DR and HL60-DR100 cells of a hybrid bcr/abl mRNA and a 210 kD Bcr/Abl protein which was constitutive in K562. This expression was due to the translocation of abl and the amplification of the bcr-abl translocated gene. These results are in agreement with the role of Bcr/Abl tyrosine protein kinase as an inhibitor of apoptosis independently of the mdr-1 expression. They also suggest that translocation of the abl gene in the bcr region is a highly probable rearrangement in the mdr-1 expressing myeloid cells and that Bcr/Abl tyrosine kinase effect on apoptosis needs the regulation of intracellular pH and is inactive against UV-induced apoptosis.

Direct protein sequencing of wheat mitochondrial ATP synthase subunit 9 confirms RNA editing in plants.

RNA editing, a process that results in the production of RNA molecules having a nucleotide sequence different from that of the initial DNA template, has been demonstrated in several organisms using different biochemical pathways. Very recently RNA editing was described in plant mitochondria following the discovery that the sequence of certain wheat and Oenothera cDNAs is different from the nucleotide sequence of the corresponding genes. The main conversion observed was C to U, leading to amino acid changes in the deduced protein sequence when these modifications occurred in an open reading frame. In this communication we show the first attempt to isolate and sequence a protein encoded by a plant mitochondrial gene. Subunit 9 of the wheat mitochondrial ATP synthase complex was purified to apparent homogeneity and the sequence of the first 32 amino acid residues was determined. We have observed that at position 7 leucine was obtained by protein sequencing, instead of the serine predicted from the previously determined genomic sequence. Also we found phenylalanine at position 28 instead of a leucine residue. Both amino acid conversions, UCA (serine) to UUA (leucine) and CUC (leucine) to UUC (phenylalanine), imply a C to U change. Thus our results seem to confirm, at the protein level, the RNA editing process in plant mitochondria.

Study of apoptosis-related responses of leukemic blast cells to in vitro anthracycline treatment.

Anthracyclines trigger an apoptotic cell death but their molecular targets are not totally explored. We investigated the apoptotic response of blast cells and lymphocytes from medullary samples of 31 de novo acute leukemia. Mononuclear cells were treated in vitro by therapeutic concentrations of either daunorubicin (DNR) or idarubicin (IDA) for 1 h, washed and cultured for 18 h. A multivariate analysis using flow cytometry and a CD45 gating on lymphocytes and blast cells was performed. DNR and IDA induced a Fas enhancement on both leukemic and normal cells. In blast cells the DEVDases were activated and the caspase 3 was cleaved in relation to phosphatidyl serine exposure, showing a caspase-dependent pathway in anthracycline-induced apoptosis. Apoptotic percentages were always higher for blast cells than for lymphocytes, confirming that anthracycline toxicity mainly affected tumor cells. Moreover, drug-induced apoptosis was not related to spontaneous apoptosis, suggesting that variations in response intensities were due to individual variations of sensitivity rather than to programmed life span time. The apoptotic response of P-glycoprotein-expressing blast cells was not significant, giving biological argument for the poor prognosis of multidrug resistance leukemia. Finally, Fas induction and anthracycline-induced apoptosis on blast cells were significantly higher when a complete remission was achieved, thus shedding light on potential new prognostic factors in acute leukemia.

Nitric oxide induces the apoptosis of human BCR-ABL-positive myeloid leukemia cells: evidence for the chelation of intracellular iron.

Anti-leukemia activity of human macrophages involves the generation of nitric oxide (NO) derivatives. However, leukemic transformation may involve mechanisms that rescue cells from NO-mediated apoptosis. In the present work, we analyzed the effects of exogenous NO on the proliferation of BCR-ABL(+) chronic myelogenous leukemia (CML) cells. As normal leukocytes, the proliferation of leukemia cells was inhibited by SNAP (S-nitroso-N-acetyl-penicillamine), GEA (Oxatriazolium amino-chloride), and SIN-1 (Morpholino-sydnonimine), whereas SNP (sodium nitroprusside) had no effect on leukemia cell growth. SIN-1 induced higher anti-proliferation activity in BCR-ABL(+) cells, compared to normal hemopoietic cells. Inhibition of leukemia cell proliferation correlated with increased apoptosis and DEVDase activity. The simultaneous addition of exogenous iron reversed NO-mediated inhibition of cell growth, caspase activation and apoptosis in all BCR-ABL(+) cells tested. The quantification of intracellular iron levels in leukemia cells indicated that NO induced an early, dose-dependent decrease in ferric iron levels. Accordingly, elevation of intracellular iron protected leukemia cells from NO-mediated apoptosis. Together, the present work reveals the presence of an iron-dependant mechanism for leukemia cell rescue from NO-induced growth inhibition and apoptosis.

Resistance to daunorubicin-induced apoptosis is not completely reversed in CML blast cells by STI571.

The leukemogenic property of BCR-ABL in chronic myeloid leukemia (CML) is critically dependent on its protein tyrosine kinase activity. STI571 inhibits the BCR-ABL kinase activity, the growth and the viability of BCR-ABL expressing cells. In this study, we report the apoptotic effect of STI571 in combination with daunorubicin (DNR) on peripheral blood mononuclear cells from 11 CML patients and four BCR-ABL-positive cell lines: AR230, LAMA84, K562 and KCL22. Primary blast cells were identified by flow cytometry on the basis of their low CD45 expression. Nucleus fragmentation, exposure of phosphatidylserines and decrease in mitochondrial membrane potential were measured using acridine orange, FITC-annexin V and DiOC6(3), respectively, to evaluate apoptosis. On cell lines, the effect of DNR was negligible, whereas STI571 induced 10 to 35% of apoptosis in 18 h. STI571 sensitized AR230, LAMA84 and K562 cells to DNR when apoptosis was measured at the mitochondrial and membrane but not the nuclear levels. On CML blast cells, phosphatidyl serine exposure was significantly induced by both DNR and STI571 and was higher when these drugs were used in combination (P < 0.0003). However, the effects of this drug combination were only additive and no sensitization of blast cells to DNR by STI571 was observed. Interestingly, sensitization was evidenced in CML but not normal lymphocytes. These results suggest that other mechanisms additional to Bcr-Abl tyrosine kinase activity could be responsible for DNR resistance, and further investigations are needed to understand its origin.

Endothelial cell support of hematopoiesis is differentially altered by IL-1 and glucocorticoids.

We investigated the ability of endothelial cells (EC) to support hematopoiesis in contact and non-contact cocultures with isolated CD34+ or CD34/CD38low cells. In the absence of exogenous cytokines, umbilical vein EC (HUVEC) efficiently support proliferation of hematopoietic cells and generation of colony-forming cells (CFC). Cytokines (IL-6, LIF, G-CSF, GM-CSF, M-CSF, but not IL-1, IL-3, IL-7) were detected in HUVEC coculture supernatants. Neutralization of these cytokines profoundly inhibited the ability of EC supernatants to support the differentiation of hematopoietic progenitors and led to an accumulation of immature cells. Contact cocultures were significantly more efficient than non-contact cocultures. The expanded cell population essentially belonged to the myeloid and monocytic lineages. Contact cocultures generated cells expressing the CD61 or CD41 antigens. Interleukin-1alpha (IL-1alpha) augmented EC capacity to support hematopoiesis, this property resulting from the upregulation of cytokine expression. Glucocorticoids (GC) reduced this capacity by downregulating the biosynthesis of cytokines by EC and not by a direct effect on the progenitor cells. EC from the bone marrow microvasculature (BMEC) support the proliferation and the differentiation of hematopoietic progenitors. Synergistic increase in progenitor cells expansion and generation of CFC occurred when EC cocultures were added with exogenous cytokines. Supernatants of IL-1alpha-stimulated EC potentiated the effects of an association of IL-1, IL-3, IL-6, LIF, SCF, Flt3-ligand, TPO, G-CSF, GM-CSF, M-CSF and IL-11 on the proliferation of hematopoietic progenitors suggesting that EC may produce other soluble growth factors potentiating the action of the above set of cytokines.

Flow cytometry CD45 gating for immunophenotyping of acute myeloid leukemia.

A flow cytometry method has been introduced into the routine investigation of whole bone marrow samples following red blood cell lysis on the basis of a primary CD45/side scatter (SSC) gating procedure. Blast cells were first identified by CD45/SSC gating in 74 cases of acute myeloid leukemia (AML) and the results were compared to a conventional FSC/SSC gating procedure and to MGG-staining smears. The percentages of blast cells in these samples as defined by the morphological analysis of MGG smears correlated better with the values determined by CD45/SSC gating (r = 0.94) than with the blast cell counts recorded with FSC/SSC gating (r = 0.76). These findings were not surprising because while CD45 expression was regularly lower on leukemic blasts than on normal lymphoid and monocytic cells, the FCS/SSC characteristics of these populations were overlapping. In 53 samples, the blast cell populations were also analyzed with a panel of FITC-conjugated monoclonal antibodies that were utilized in double labeling with CD45-PE. We show that the CD45/SSC gating procedure improved phenotypic determination of the blast cells in three ways: (1) by discriminating between leukemic blast cells and residual normal cells; (2) by excluding normal cells from the phenotypic analysis of leukemic blast cells; and (3) by identifying blast cell heterogeneity in many cases of leukemia on the basis of different CD45 display. Moreover, this immunophenotyping procedure on whole bone marrow samples also allowed an efficient discrimination between the various cell lineages and facilitated the analysis of leukemic blasts present in low proportions.

Flow cytometric study of the activation of polymorphonuclear cells.

The activation of human polymorphonuclear cells (PMN) by the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), or the protein kinase C activator, phorbol myristate acetate (PMA), was studied using flow cytometry. Two probes were used to evaluate PMN activation: 1) a monoclonal antibody (MoF11) directed against an antigen (Ag) expressed on the membrane of monocytes and of activated PMN; 2) rhodamine phalloidin was used at the cytoplasmic level to measure the F-actin content. The expression of MoF11 antigen was found to be 3 to 5 times greater on the membrane of PMN activated by either FMLP or PMN as compared with membrane expression of the same Ag on resting PMN. This increase was found to be dose dependent for the two activators. Kinetic studies showed that a maximum response was observed in 1 to 2 min at 37 degrees C when FMLP was used, whilst a similar response required 10 min when PMA was used. The same discrepancy with activators was observed when actin polymerization was measured by labelling with rhodamine phalloidin. However, pretreatment of PMN with cytochalasin B inhibited actin polymerization whilst MoF11 antigen expression was increased, suggesting that the MoF11 antigen could be stored in granules of resting PMN. The study of actin polymerization and of MoF11 antigen expression, separately or in combination, could be a useful tool for the detection of activated PMN in biological samples.

Radiobiological features of acute myeloblastic leukemia: comparison of self-renewal versus terminally differentiated populations.

PURPOSE: To evaluate the radiosensitivity of self-renewing progenitor cells in acute myeloblastic leukemia (AML), we have compared the radiosensitivity of the cells grown either in methylcellulose alone for 7 days, or first in suspension culture for 7 days before being plated in methylcellulose. Methylcellulose selects for terminal-dividing cells and suspension cultures have been developed because they allow self-renewal to occur: The exponential growth of the progenitors of AML cultured in suspension is due to self-renewal. METHODS AND MATERIALS: Cells were harvested from previously untreated leukemic human bone marrows. The myeloblastic lineage of the colonies was assessed by morphological, cytochemical, and immunophenotypic analysis, and by the use of growth factors that did not stimulate the growth of T-lymphocytes. The cell-cycle distribution of the blasts was analyzed by flow cytometry and was comparable for all samples. The irradiation was performed with gamma-photons at a dose-rate of 0.05 Gy/min, similar to the clinical conditions used in our institution for total body irradiation (TBI). RESULTS: The culture methods selected aggressive leukemias. There were large variations of the individual radiosensitivity whatever culture method was used. The progenitor cells capable of self-renewal were more radiosensitive than terminal dividing cells. In two cases, a shoulder was found in the initial part of the cell-survival curves of cells capable of self-renewal. In these two cases, the best fit for the data was the linear quadratic model (survival = e-alpha D-beta D2) with alpha/beta values of 1.49 Gy and 3.12 Gy, respectively. CONCLUSION: The very low values of alpha/beta suggest a reduced antileukemic effect in case of fractionated TBI, and may lead to more reliable screening methods to determine the most appropriate technique for radiation ablation of bone marrow prior to bone marrow transplantation (BMT).

Influence of selected heparins on human neutrophil functions in vitro.

The effects of four heparin derivatives [unfractionated heparin (UFH) at 5-50 IU/ml, low molecular weight heparin (LMWH) at 2-20 IU/ml, pentasaccharide (Penta) at 5 IU/ml and a synthetic heparinoid (PPS) at 10(-6)-10(-5) M] on various polymorphonuclear (PMN) leukocyte end-functions (aggregation, chemotaxis, phagocytosis and burst) were examined. CR3 expression, actin polymerization and membrane surface charge were also studied to gain more insight on the mechanisms of the action of heparins on PMN. The different heparins were found to have rather different actions. PMN were found to be hyperreactive to PPS. Pentasaccharide hat no effect on PMN functions, while UFH and LMWH had intermediate reactivity, modulating responses in an adenosine-like manner. Interactions of heparins with PMN were attributed to biophysical properties of the molecules rather than to the presence of a specific sequence such as a pentasaccharide. Our results show that certain heparin derivatives, apart their well-known anticoagulant action, modulate polymorphonuclear leukocyte functions that may be involved in vascular injury.

Megakaryocytes from the marrow of a patient with Glanzmann's thrombasthenia lacked GP IIb-IIIa complexes.

Although it is recognized that glycoprotein (GP) IIb-IIIa complexes are deficient in platelets in Glanzmann's thrombasthenia, little is known of the origin of the defect. We have examined the megakaryocytes in a bone marrow aspirate obtained from a thrombasthenia patient during surgery. Analysis of platelet proteins by SDS-polyacrylamide gel electrophoresis confirmed the patient to be of the type I subgroup. The megakaryocytes were examined by immunofluorescence or by immunocytochemical procedures combined with electron microscopy. Antibodies used included the murine monoclonal antibody, AP-2 and the human allo-antibody, IgG L, both of which recognize determinants on GP IIb-IIIa complexes. Bound antibody was detected by anti-IgG antibodies coupled to fluorescein isothiocyanate or absorbed on gold particles. In the immunofluorescence studies, permeabilized megakaryocytes were identified by double staining using an antibody to von Willebrand factor (vWF). Whereas mature megakaryocytes and their small precursor cells from normal individuals were strongly fluorescent with AP-2 and IgG L, most vWF positive cells from the Glanzmann's thrombasthenia patient were negative and the remainder gave but a weak background fluorescence. Immunogold staining on the surface of marrow cells was severely reduced. Our results confirm a deficiency of GP IIb-IIIa complexes in megakaryocytes in thrombasthenia.

Mepacrine labelling test and uranaffin cytochemical reaction in human megakaryocytes.

5-HT storage organelles were observed by electron microscope analysis in human megakaryocytes. They were less numerous per unit of surface than in platelets. Their number depended on the visualization technique employed. Thus after fixation with calcium-enriched glutaraldehyde a higher number of very opaque organelles was observed than of uranaffin-positive organelles after the cytochemical uranaffin reaction. With conventional electron microscopy deep black granules characteristic of dense bodies were not observed. Fluorescent microscopy showed greenish-yellow granules distributed throughout the whole cytoplasm in 96 +/- 1.4% of normal megakaryocytes incubated with mepacrine. In 85.6 +/- 5%, 5.28 +/- 1.28 granules per 10 microns 2 were observed. With the mepacrine labelling test, 74% of the megakaryocytes of a patient with Hermansky-Pudlak syndrome contained no granules. A similar finding was made in the platelets of the same patient. This suggests that mepacrine also stains the dense bodies in the megakaryocytes and that in the Hermansky-Pudlak syndrome the platelet anomaly is secondary to a megakaryocyte anomaly.

Flow cytometric study of idarubicin and daunorubicin accumulation and the effect of verapamil in leukemic cell lines and fresh cells from patients with acute non-lymphoblastic leukemia.

By using flow cytometry, the intracellular accumulation (Acc) of idarubicin (IDA) and daunorubicin (DNR) and the effect of verapamil (VRP) on both anthracycline accumulation (VRP index) were studied in leukemic cell lines (K562 and HL60 and their two DNR-resistant subclones) and fresh leukemic cells. IDA accumulated more than DNR in both parental (K562: p < 0.03 and HL60: 0.09) and resistant cell lines (p < 0.01 for both cell lines) irrespective of whether or not they were treated with VRP. VRP index was higher for DNR than for IDA (p < 0.05). Similar results were observed in fresh leukemic blasts from 25 patients with ANLL (IDA Acc superior to DNR Acc: p < 0.0001; higher VRP index for DNR than for IDA: p < 0.01). The higher Acc of IDA than DNR seen in fresh leukemic cells could explain the better clinical efficacy of IDA reported in patients with ANLL.

Influence of rhGM-CSF on Ara-C sensitivity of patients with acute myeloid leukemia in relapse: a flow cytometry study.

Twenty-three patients with acute myelogenous leukemia (AML) in first relapse were treated with high-dose cytosine-arabinoside (Ara-C) and amsacrine or idarubicin. To prime the cells, the patients were given rhGM-CSF. We studied the influence of 48-h infusion of rhGM-CSF on proliferation and Ara-C sensitivity of leukemic cells both ex vivo and in vitro. We found that a 48-h infusion of rhGM-CSF increased both white blood cell counts and peripheral blood blast cell percentages. Using a Bromodeoxyuridine/DNA (BrdUrd/DNA) staining in flow cytometry, we found an non-constant increase in cells in the S-phase. Ex vivo 48-h culture of leukemic cells with or without rhGM-CSF, with or without other hematopoietic growth factors (HGFs), showed a greater increase of the cells in the S-phase with GF but no correlation with the ex vivo results. We used a method of quantitation of the DNA synthesis previously described (Lacombe F., et al. (1992) Cytometry 13, 730) to monitor the Ara-C sensitivity of the cells in S-phase before and after 48-h infusion with rhGM-CSF. We observed a great variation in the Ara-C sensitivity of the leukemic cells before and after infusion with rhGM-CSF from one patient to another. The BrdUrd/DNA method seems a convenient method to study the influence of HGFs on Ara-C sensitivity of the patients.

Study of the apoptosis induced in vitro by antitumoral drugs on leukaemic cells.

A flow cytometric method for simultaneous apoptotic cell detection and cell cycle analysis was applied on the U937 cell line. Four antitumoral drugs currently used in the treatment of acute myeloid leukaemia were studied in vitro: DNR, IDR, MITO and Ara-C. Our results show a dissociation between the cytostatic effect (the block in the cell cycle observed for low drug concentrations) and the cytotoxic effect (the induction of apoptosis induced by higher concentrations) for all the tested molecules. Low concentrations of Ara-C induced a block in the S phase while higher concentrations (>10(-7) M) induced apoptosis at the G1-S boundary. Low concentrations of anthracyclines (<40 nM DNR and <20nM IDR) induced a block in G2 without apoptosis. Apoptosis was induced in G1 and/or early S phases by higher concentrations of anthracyclines. The concentration inducing 50% apoptosis (IC50) was found to be, respectively, 200 and 40 nM for DNR and IDR. Analysis of MITO-treated cells showed a parallel increase in the percentages of S phase and apoptotic cells. However, the bivariate analysis showed that apoptosis did occur in a population with G1 DNA content. For two other drugs (CAM and COLC), apoptosis occurred for the same concentrations and in the same phase as the block (in S and G2M, respectively). The IC50 of MITO was found to be 100 nM. Cotreatment of the cells with colchicin and either Ara-C or IDR showed that the passage through mitosis was not necessary for the completion of apoptosis at the G1-S boundary. Short incubations of U937 cells with high concentrations of anthracyclines were found to be efficient in inducing further apoptosis. We conclude that, for all the assayed molecules, the cytotoxic and/or cytostatic effects of the antitumoral drugs tested greatly depend on the concentrations used and that, depending on their in vivo pharmacokinetics, the induction of apoptosis could be an important mechanism of action for some of these drugs.

Interferon gamma is effective for BM purging in a patient with CML.

We report a case of Ph-positive CML where the BM was incubated for 24 h with 10(3) IU/ml IFN gamma and then cultured in liquid media for 4 weeks. After 24 h incubation, there was no differential sensitivity of CML CFU-GM to IFN gamma compared with untreated BM. Subsequent long-term culture (LTC) of the IFN gamma treated CML BM, however, demonstrated a 75% inhibition of production of CFU-GM from the second week onwards. Using PCR, we were able to demonstrate two types of BCR-ABL transcript in the diagnostic BM. After 4 weeks of LTC, the J(bcr b3/ABL II) RNA transcript persisted in the untreated BM, whereas neither BCR/ABL RNA transcripts were detected in the culture established with IFN gamma-treated CML BM. This study has two points of interest with the demonstration of (1) a possible antileukaemic effect of IFN gamma on the progenitors generated in the LTC system, and (2) the use of highly sensitive PCR technology to evaluate the effectiveness of IFN gamma to purge CML BM of Ph-positive cells.

Mixed chimerism after sex-mismatched allogeneic BMT: evaluation of two molecular techniques.

Two different molecular techniques were used to monitor chimerism following 17 non-T cell-depleted BMTs from female donors to male recipients: pHY10, a Y chromosome-specific probe (Southern or slot blots), and a set of primers for Y chromosome sequence-specific amplification by the polymerase chain reaction (PCR). On Southern blots, male DNA was detectable at a level less than 1% of 10 micrograms DNA while cross-reactivity with autosomal sequences was avoided. On slot blots, male DNA was reliably detectable at levels less than 0.5%, even in small sample (0.5 microgram DNA). With the PCR technique, male DNA was detectable at levels of 1:10(6) to 1:10(7) of 0.5 microgram DNA. Slot blot and PCR results were concordant in 19 of 23 samples. Both techniques demonstrated a constant small mixed chimerism during the first year after BMT and in four of nine patients, this chimerism persisted even longer (up to 29 months after BMT).

Stable mixed chimerism without relapse after related allogeneic umbilical cord blood transplantation in a child with severe aplastic anemia.

Umbilical cord blood (UCB) cells from HLA-matched donors are used as an alternative to bone marrow for allogeneic transplantation and reports of successful UCB transplantation in patients with severe aplastic anemia (SAA) are scarce. SAA was discovered in a 4-year-old girl in February 1990. Transfusion support started in August 1990 and standard treatments were unsuccessful. The birth of an HLA-compatible brother in October 1993 permitted the cryopreservation of UCB. In December 1994 UCB transplantation was decided upon. No toxicity occurred. G-CSF was started at day 28. WBC and PMN reached 0.5 x 10(9)/l at days 33 and 37. RBC and platelet transfusion independence were reached at days 50 and 52. Mixed chimerism was demonstrated in blood cells at 1.5, 4 and 6 months after UCBT by molecular biology (VNTR). FISH studies yielded similar results at 15 and 18 months. Twenty months after UCBT, molecular biology showed full donor chimerism. Clinical follow-up (last follow-up: 32 months post transplant) is unremarkable. We suggest that CY and ATG may be a suitable regimen for related HLA-compatible UCBT in patients with SAA. Residual recipient cells can disappear even very late after UCBT, permitting the establishment of complete donor chimerism.