Murine and human hematopoietic colony formation: a possible regulatory role for intracellular histamine.

Increasing number of data suggests that locally produced histamine is involved in regulation of hematopoiesis. In this study the granulocyte/macrophage (CFU-GM) colony formation by normal murine or human bone marrow cells, leukaemic colony formation (CFU-L) by a murine leukemia cell line (WEHI 3B), and colony formation by bone marrow cells from patients with chronic myeloid leukemia (CML) have been examined. We detected mRNA and protein expression of histidine decarboxylase (HDC), the only enzyme responsible for histamine synthesis both in normal bone marrow progenitor cells and in leukaemic progenitors. The significance of in situ generated histamine was shown on colony formation by inhibitory action of alphaFMH (blocking HDC activity, i.e. de novo histamine formation) and by N,N-diethyl-2-[4-(phenylmethyl)phenoxy]-ethanamine-HCl (DPPE) disturbing the interference of histamine with intracellular binding sites. These data provide further confirmation of the role of histamine in development and colony formation of bone marrow derived cells.

Blast colony-forming cell binding from CML bone marrow, or blood, on stromal layers pretreated with G-CSF or SCF.

Blast colony-forming cells (CFU-BL) represent a specific subpopulation of special primitive progenitors characterized by colony formation only in close contact with a preformed stromal layer. CFU-BL derived from bone marrow of chronic myeloid leukaemia (CML) patients have been proved to adhere poorly to bone marrow derived stromal layers suggesting that the appearance of progenitors and precursors in the circulation is due to a defective adhesion of these cells to the bone marrow microenvironment. In the present experiments the effect of short-term incubation of preformed normal bone marrow stroma on the adherence of CML derived CFU-BL was studied. For stroma cultures bone marrow cells were cultured in microplates in the presence of hydrocortisone. Cultures were used when stromal layers became confluent and no sign of haemopoiesis could be observed. CFU-BL were studied by panning plastic non-adherent mononuclear (PNAMNC) bone marrow or blood cells. 8.9 +/- 2.4 colonies/103 PNAMNC (six experiments) were formed from normal bone marrow on stromal layers and 4.8 +/- 2.1 colonies/103 PNAMNC (five experiments) from CML bone marrow. Colony formation from normal bone marrow was not increased if stromal layers were incubated with 100 ng/mL granulocyte colony-stimulating factor (G-CSF) or stem cell factor (SCF). Incubation of stroma with G-CSF or SCF, however, increased the colony formation of PNAMNC from CML bone marrow or blood significantly. These findings suggest that local concentration of haemopoietic growth factors at the time of panning may influence the attachment of CML progenitors to the stroma.

Leukaemogenic potency of WEHI-3B cells grown in vitro or in leukaemic mice.

In order to quantify contaminating leukaemia inducing cells in blood or bone marrow from WEHI-3B bearing BALB/c mice (injected with 10(5) WEHI-3B suspension culture cells), in vitro colony forming leukaemia cells (CFU-L) and survival rate and/or survival time of mice transplanted with cells from WEHI bearing mice or suspension culture were correlated. ED(50) (inoculum inducing leukaemia in 50% of the animals) was 109 CFU-L (33-361) from suspension culture cells. Three weeks after initiation of leukaemia 2 x 10(5) BM or 0.5-2.0 x 10(6) blood cells induced 100% mortality of recipients. After mobilisation with CY or CY and G-CSF, the same amount of blood or BM cells did not induce leukaemia in recipients. A significant negative correlation was found between the survival time of leukaemic mice and the log number of CFU-L inoculated from in vivo sources. In terms of CFU-L cells, leukaemia induction to BM or WBC obtained 3 weeks after leukaemia induction were more potent; those from BM or WBC also obtained at 3 weeks but after mobilisation were less potent inducers than those from suspension culture. These data suggest that CFU-L and leukaemogenic cells are associated, but not identical.

Autologous hematopoietic stem cell transplantation in chronic myeloid leukemia with different clinical stages.

Seven patients with Philadelphia (Ph) chromosome-positive chronic myeloid leukemia (CML) were treated with an ICE-based regimen plus G-CSF with the aim of mobilizing and collecting Ph-negative peripheral stem cells in the setting of an autologous transplant program. Five patients had CML in the first chronic phase and 2 in the accelerated phase. All patients had been previously treated with interferon-alpha. Median value and ranges for harvested mononuclear cells, CD34+ cells and CFU-GM, respectively: 5.65 x 10(8)/kg (2.61-11.38); 1.48 x 10(6)/kg (0.216-3.5), and 3.43 x 10(4)/kg (0.243-11.6). FISH was the only useful method for detection of minimal residual disease on apheresis product showing <5% t(9;22) positive cells in 2 cases and <10% positive cells in 4 other cases. Four of seven autologous grafts have been transplanted to date. Busulfan conditioning was used in 1 case and TBI/Cy conditioning in 3 other cases. All patients are alive and well following transplantation and are on interferon-alpha therapy.

[Autologous hematopoietic stem cell transplantation in chronic myeloid leukaemia with different clinical stages] / Autológ hemopoetikus ôssejt-transzplantáció eredményei chronicus myeloid leukaemiában.

For most chronic myeloid leukaemia patients the option of a potentially curative allogeneic stem cell transplantation is not available because of age or lack of donor. Alternative therapy with interferon-alpha appears to prolong survival but is probably not curative. The aim of the study is to analyse the clinical results of the first Hungarian autologous transplantations in CML. METHODS: Seven patients were treated with ICE-based regimen plus G-CSF with the aim of mobilising and collecting Ph-negative peripheral stem cells in the setting of autologous transplant program. Five patients had CML in first chronic phase and two in accelerated phase. All patients have been previously treated with interferon-alpha. RESULTS: Median value and ranges for harvested mononuclear cells, CD34(+) cells and CFU-GM were: 5.65x10(8)/kg (2.61-11.38), 1.48x10(6)/kg (0.216-3.5) and 3.43x10(4)/kg (0.243-11.6), respectively. Four out of seven autologous grafts have been transplanted. Busulfan conditioning was used in one case and TBI/Cy conditioning in three patients. All patients are alive and well post-transplant being on interferon-alpha therapy. CONCLUSIONS: Based on the clinical advantages of autologous transplantation including long-term chronic phase, achievement of second chronic phase and improved response to interferon-alpha therapy, the procedure can offer an alternative treatment in CML in lack of HLA-identical donor.

Lymphoma cell contamination of PBSC: a murine model.

In P-388 bearing BDF1 mice stem cell mobilisation was tested on the survival of lethally irradiated isologous recipients. Contamination of the graft with lymphoma cells was evaluated by the number of clonogenic lymphoma cells (CFU-L) and the ability of the graft to induce lymphoma in non-irradiated recipients. A mobilisation protocol (200 mg/kg cyclophosphamide (CY) i.p. or 200 mg/kg CY followed by 125 microg/kg G-CSF administered every 12 h for 3 consecutive days, starting 14 days after lymphoma initiation) that resulted in a substantial stem cell mobilisation in normal mice, mobilised too few CFU-L to induce lymphoma in the recipients: 50 microl of blood obtained after mobilisation protected lethally irradiated mice but did not induce lymphoma in normal recipients. A minimum graft of bone marrow (2 x 105 cells, with 5580 CFU-L) from untreated P-388 bearing donors killed irradiated recipients presumably by lymphoma and not bone marrow failure. The mobilisation protocol reduced CFU-L so much that no lymphoma-associated death occurred even after the transplantation of 10 x 105 bone marrow cells. These data suggest that, although the PBSC mobilisation protocol may also mobilise some clonogenic lymphoma cells, with the minimum graft size no lymphoma transfer can be observed.

[Diagnostic value of serum erythropoietin levels]. / A szérum eritropoetin szint vizsgálatának diagnosztikai jelentösége.

Erythropoietin (Epo) is a hormone-like glycoprotein regulating erythropoiesis. Under normal conditions Epo stimulates mitoses of the erythroid progenitors and precursors, decreases apoptosis, decreases "ineffective erythropoiesis" and stimulates the synthesis of the specific protein, haemoglobin. Epo producing cells in the kidney sense the O2 tension of kidney tissue and react to hypoxia with increased Epo production and to O2 saturation (polycythaemia) with decreased or completely abolished Epo production. Normal level of Epo in the serum is 3-20 mU/ml. If Epo production is functioning normally there exists a strict inverse correlation between serum Epo level and hematocrit: an exponential increase in Epo level can be observed if hematocrit decreases. Any damage in Epo production lead to inadequate production (e.g. renal anaemias). This paper analyses the Epo content of 278 serum samples assayed in the Laboratory of Experimental Bone Marrow Transplantation of the National Institute of Hematology and Immunology between August 1996 and December 1997 for their diagnostic value. Those samples are primarily discussed where Epo assay was meant to decide diagnosis of polycythaemia vera or those where decision of Epo treatment of anaemic patients depended on their serum Epo level.

Rapidly mobilizable stem cells. Do they belong to a special subpopulation?

Some characteristics of rapidly mobilized stem cells as a possible distinct subset of the murine bone marrow stem cell population are overviewed. Some of the agents that rapidly mobilize stem cells are toxic and possibly act through disrupting anchorage to the microenvironment. The mobilization occurring days after cytostatics and/or colony-stimulating factors (CSF), however, is a consequence of increased production or differentiation. While stem cells (colony-forming units-spleen; CFU-S) circulating normally in blood have low self-renewal capacity (SRC), SRC of rapidly mobilized CFU-S is closer to that of bone marrow stem cells and is similar to that of the late mobilized stem cells. The survival rate of mice after transplantation of rapidly mobilized stem cells did not differ from that of bone marrow stem cells. One year after transplantation of rapidly mobilized stem cells, the SRC value of bone marrow did not differ from those transplanted with bone marrow cells. Replacement of a rapidly mobilizable stem cell pool requires 48 h under physiological conditions and a longer time after damage to hemopoiesis (irradiation, hydroxyurea injection). Possible physiological mechanisms in the anchorage of stem cells are discussed.

[Diagnostic value of hematopoietic stem cells]. / A vérképzö elödsejtek vizsgálatának diagnosztikai jelentösége.

Proliferation and differentiation of the haemopoietic stem cell are critical for the maintenance of normal haemopoiesis, damage to haemopoiesis, onset of malignant haemopoiesis and repopulation of haemopoiesis after bone marrow transplantation. To evaluate the actual state of haemopoiesis a quantitative assay of haemopoietic stem cells would be needed. Morphology of haemopoietic stem cells cannot be detected by their microscopic morphology. In human practice no stem cell assay is available, therefore, the quantity of human pluripotent stem cells is characterised by the frequency of the progenitors differentiating into erythrocyte, granulocyte or macrophage cell lines (CFU-GEMM, BFU-E, CFU-GM, CFU-Meg). Progenitor cells, can be easily tested by their ability to form in vitro colonies in soft gel cultures in the constant presence of the specific regulator of the cell line ("colony stimulating factor" = CSF). Seven-14 days after explantation of haemopoietic cells colonies containing 40 to 1000 cells are formed. These colonies are proved to be of clonal origin. The quantitative assay of colony forming progenitors has a critical significance in haematological diseases due stem cell damage and bone marrow transplantation. The present paper deals with the diagnostic value of progenitor assays.

Ultrastructural study on the in vitro interaction between haemopoietic cells and stromal cells. A new method using gel technique.

The ultrastructural study of the interaction between stroma and haemopoiesis is not an easy task because the loose attachment may be damaged during manipulation. This paper describes a technique by which the loose connection between preestablished stromal layer and attached haemopoietic cells (derived from blast colony forming cells) can be preserved and studied ultrastructurally. Stromal cultures were obtained from human bone marrow cells. Blast colony forming cells were studied by co-incubating the stroma with fetal calf serum supplemented McCoy's medium containing bovine plasma, thrombin and calcium to form a gel ('plasma clot'). Colony formers attached to the stroma formed myeloid colonies within 6 days. The semisolid plasma clot which solidifiers rapidly on the addition of glutaraldehyde or formaldehyde entraps the blastic colonies and haemopoietic cells in their position. Even the non-attached or mobile cells can be entrapped by this technique. The immature cells were found to be attached to the stromal surface and/or to the extracellular matrix, while the more mature cells migrated either to the surface of the colony or attached to the non-covered areas of the plastic surface. This method may offer a special technique to study dynamic interactions in other situations (e.g. chemotaxis etc.), too.

Blast colony forming cell-binding capacity of bone marrow stroma from myelodysplastic patients.

A specific stroma function can be quantitatively assessed by counting the stroma-adherent blast cell colonies (CFU-BL) that are formed from normal plastic nonadherent mononuclear bone marrow cells (PNAMNC) after a short-term coincubation ("panning") with the preformed stromal layer. In order to obtain information of stroma function in myelodysplasia (MDS), the "CFU-BL-binding capacity" of stroma from normal bone marrow and from patients with MDS were compared. Stromal cell cultures were established from mononuclear bone marrow cells in microplate cultures cultured with or without 10(-6) M hydrocortisone. CFU-BL-binding capacity was studied by counting blast colonies seven days after panning, and the results were expressed as CFU-BL/10(3) PNAMNC. Normal marrow stromal layers bound CFU-BL only if they were cultured with hydrocortisone, while MDS stromal layers also bound CFU-BL in the absence of hydrocortisone. For further studies of the function of MDS stroma, the effect of growth factors (stem cell factor [SCF], G-CSF, interleukin 3 [IL-3] and their combinations) on CFU-BL binding by normal or MDS stroma has also been compared. Twenty-hour incubation of the stromal layers with a standard dose (100 ng/ml) of various hemopoietic growth factors (IL-3 alone or in combination with SCF, G-CSF alone or in combination with SCF) did not have any effect on CFU-BL binding by normal marrow stroma, but increased the CFU-BL binding by stromal layers from MDS bone marrow. These findings suggest that although stromal microenvironment in MDS is capable of supporting hemopoiesis, bone marrow stroma from MDS patients differs in some characteristics from the normal stroma.

Blast colony forming cells in umbilical cord blood: frequency and correlation to other haemopoietic progenitors.

Umbilical cord blood has been suggested as a potential source of haemopoietic stem cells for clinical transplantation. Assay of stroma adherent blast colony forming cells (CFU-BL), as a cell type that may predict marrow repopulation, has already been suggested to predict the outcome of bone marrow transplantation. In the present experiments the frequency of CFU-BL in plastic non adherent mononuclear cell fraction (PNAMNC) of umbilical cord blood was studied. If PNAMNC from cord blood was co-incubated with a pre-established stromal layer from normal bone marrow a strict linear correlation between panned PNAMNC and blast colonies were observed. Frequency of CFU-BL in cord blood was significantly lower than that in bone marrow. In cord blood, CFU-GEMM frequency was found to be comparable to that of bone marrow, CFU-GM and BFU-E frequency was reduced. A good correlation between CFU-BL and BFU-E frequency was found both in cord blood and bone marrow. On the other hand, irrespective of their source, correlation between CFU-BL and CFU-GEMM or CFU-GM was weak.

Comparative heat sensitivity of murine and human hemopoietic progenitors and clonogenic leukemia cells.

The aim of this study was to compare the thermal sensitivity of normal murine and human hemopoietic progenitors to that of leukemic murine and human clonogenic cells in order to assess the clinical relevance of experimental data. Colony forming units-granulocyte-macrophage (CFU-GM) from normal human bone marrow and from bone marrow of patients with acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), and Hodgkin's disease in complete remission proved to be less sensitive to 42.5 degrees C in vitro hyperthermia (D0: 93.9 min) than murine bone marrow CFU-GM (D0: 49.6 min). Leukemic colony forming cells (CFU-L) from HL-60 suspension culture--when compared to human CFU-GM--showed significantly increased thermal sensitivity (D0: 22.8 min). While the thermal sensitivity of CFU-L from a murine leukemia cell line (WEHI 3-B) was not statistically significant when compared to that of CFU-L from HL-60 (D0 values 17.0 versus 22.8 min), the vertical difference between the parallel regression lines suggested an approximately three-fold greater survival for human CFU-L. Although carefully controlled hyperthermia is an easy purging technique, the relevance of murine data to human clinical practice must be considered critically.

The effect of a combined purging with mafosfamide and hyperthermia on murine haemopoietic stem cells and leukaemogenic cells.

The in vitro purging effect of mafosfamide combined with hyperthermia was studied in a murine model. The survival of normal clonogenic progenitors (d-9 CFU-S and CFU-GM) and WEHI 3-B leukaemic clonogenic cells (CFU-L) were compared. At 37 degrees C, CFU-L proved to be significantly more sensitive to mafosfamide than either of the normal progenitors. When mafosfamide was combined with 42.5 degrees C hyperthermia for 1 h, an additive effect was observed: at a dose of 5 micrograms/ml mafosfamide, the survival of CFU-L was nearly two logs lower than that observed at 37 degrees C, while 37.7% of CFU-S survived the purging. The repopulating capacity of surviving bone marrow CFU-S was not altered: a similar 60 d survival of supralethally irradiated recipients transplanted with comparable graft sizes from purged or non-purged bone marrow was observed. When bone marrow suspensions containing WEHI 3-B cells were purged with 5 micrograms/ml mafosfamide at 42.5 degrees C and the minimal amount of bone marrow cells needed to protect supralethally irradiated mice were injected, leukaemia incidence was reduced to less than 10% as opposed to 100% of those injected with untreated bone marrow. Our results suggest that ex vivo hyperthermia may enhance the purging efficiency of mafosfamide.

Stromal cell growth and blast colony-forming cells in AML bone marrow.

Bone marrow cells (BMC) from normal donors and from patients with acute myeloid leukaemia (AML) were cultured. Growth kinetics and the efficiency of stromal layers in supporting the adhesion of normal blast-colony forming cells (BL-CFCs) were studied. BMC from treated AML patients formed confluent stromal layers faster than normal BMCs. BL-CFC binding capacity of normal and AML stromal layers did not differ: on normal stromal layers 67.3-147, on AML stromal layers 63-117 colonies per 5 x 10(5) plastic non-adherent BMC were formed. The amount and/or binding capacity of BL-CFCs was found to be normal in two AML patients in complete remission, while a significantly reduced number and/or binding capacity of BL-CFCs was found in AML non-treated patients and in patients within 4 weeks after the last cytostatic course.

Growth kinetics and blast-colony forming cell binding capacity of aplastic anaemic stromal cells.

The kinetics of bone marrow cell growth and a special function of stromal cells (the capability of binding blast colony forming cells) were studied in patients with aplastic anaemia (AA). All 10 patients studied showed faster growth of bone marrow stromal cells. The time for a confluent stromal layer formation was 24.5 days for AA bone marrow as opposed to 33.0 days for normal bone marrow. This faster growth rate could also be observed if normal bone marrow cells, depleted of plastic non-adherent fraction, were plated, suggesting that at least one of the reasons for altered stromal cell growth kinetics in AA is the changes in the ratio of plastic adherent/non-adherent cells. Functionally, i.e. in supporting the growth of normal bone marrow blast colonies, AA stromal layers did not differ from that of normal stromal layers, independently of the clinical state of the disease (AA or SAA; in one patient before or after ATG treatment; in two patients after successful allogenic bone marrow transplantation). Moreover, in some AA patients this blast colony forming cell binding function of AA stromal layers could also be detected in samples cultured without hydrocortisone (i.e. in the absence of fat cells), suggesting that AA stroma also differs qualitatively from normal stroma without inducing a defective microenvironment for stem cell homing.

Growth kinetics and blast-colony forming cell binding capacity of stromal cells in various haematological malignancies.

Growth kinetics of bone marrow stromal layers from normal, AML, ALL and CML patients was studied. Significantly reduced time for confluency was observed in AML patients in complete remission, in CML patients in chronic phase, or CML patients after allogenic bone marrow transplantation. The functional capacity of these stromal layers did not differ: they all bound similar amounts of blast colony forming cells (BL-CFC) from normal bone marrow. The stromal layers from bone marrow transplanted patients varied in their BL-CFC binding capacity: two CML patients (10.5 and 49 months after transplantation) showed normal values, while two ALL patients (1.5 and 3 months, respectively, after transplantation) as well as one patient transplanted for CML (19.5 months after transplantation) showed significantly reduced BL-CFC binding capacity.

Survival and characteristics of murine leukaemic and normal stem cells after hyperthermia: a murine model for human bone marrow purging.

The effect of hyperthermia in vitro on the survival and leukaemogenic effectiveness of WEHI 3-B cells and on the survival and transplantation efficiency of bone marrow cells was compared in a murine model system. Normal murine clonogenic haemopoietic cells (day 9 CFU-S and CFU-GM) proved to be significantly less sensitive to 42.5 degrees C hyperthermia (Do values: 54.3 and 41.1 min, respectively) than leukaemic clonogenic cells (CFU-L) derived from suspension culture or from bone marrow of leukaemic mice (Do: 17.8 min). Exposure for 120 min to 42.5 degrees C reduced the surviving fraction of CFU-L to 0.002 and that of CFU-S to 0.2. If comparable graft sizes were transplanted from normal or heat exposed bone marrow, 60-day survival of supralethally irradiated mice was similar. Surviving WEHI 3-B cells were capable of inducing leukaemia in vivo. The two log difference in the surviving fraction of CFU-L and CFU-S after 120 min exposure to 42.5 degrees C suggests that hyperthermia ex vivo may be a suitable purging method for autologous bone marrow transplantation.