Effects of beta2-glycoprotein I and monoclonal anticardiolipin antibodies on extrinsic fibrinolysis.

Antiphospholipid antibodies (aPLs) are associated with an increased incidence of thrombosis, but the mechanisms responsible for thrombosis are unclear. The present study investigated the effect of both beta2-glycoprotein I (beta2-GPI) and aPLs on the activity of extrinsic fibrinolysis. The remaining tissue-plasminogen activator (t-PA) of the sample consisting of beta2-GPI, two-chain recombinant t-PA, plasminogen activator inhibitor (PAI) -1 was measured by a chromogenic assay using synthetic substrate S-2251, Glu-plasminogen, and soluble fibrin monomer. Without PAI-1, beta2-GPI did not affect t-PA activity. When 14.3 ng/ml PAI-1 was added to 3.6 U/ml t-PA, the remaining t-PA activity was increased from 48.9% to 60.4% by the addition of beta2-GPI (190 microg/ml). The effect of beta2-GPI did not require phospholipids. The beta2-GPI seems to protect t-PA activity from the inhibition by PAI-1. When monoclonal anticardiolipin antibodies (aCLs), EY1C8, and EY2C9, which were established from a patient with antiphospholipid syndrome, were further added to the mixture with a diluted phospholipid (Platelin) to investigate the influence of aPL, the remaining t-PA activity decreased to 50.1 and 80.7%. Monoclonal aCLs appeared to inhibit the effect of beta2-GPI, that is, these monoclonals inhibited the fibrinolytic activity by an elevation in PAI-1 activity. These results suggest the possibility that the impairment of fibrinolytic activity by aCLs is one of reasons for the increased incidence in thrombosis in patients with aCLs.

Role of physiologic concentrations of stem cell factor in leukemic type growth of myelodysplastic CD34+ cells.

The stem cell factor (SCF: a ligand for c-kit) plays a central role in the growth of myelodysplastic (MDS) progenitor cells with leukemic type growth. In this study, the role of physiologic concentrations of SCF on the proliferation and differentiation on MDS progenitor cells was further analyzed in the presence of combined cytokines. For this purpose, marrow CD34+ cells were purified up to 94% for 12 normal individuals and 90% for 18 MDS patients, using monoclonal antibodies and immunomagnetic microspheres. The purified CD34+ cells were cultured for 14 days with saturating doses of cytokines, including recombinant human macrophage colony stimulating factor (rM-CSF), granulocyte-CSF (rG-CSF), granulocyte/macrophage-CSF (rGM-CSF), interleukin-3 (rIL-3) and rSCF. The clonal growth of MDS CD34+ cells supported by a combination of all the above cytokines was then subdivided into the two patterns of leukemic or non-leukemic. The role of various concentrations of rSCF (0, 0.5, 5, 50 and 500 ng ml(-1)), with or without the above cytokines, in proliferation and differentiation of MDS CD34+ cells was analyzed in each group. The physiologic concentration of SCF at 5 ng ml(-1) significantly increased undifferentiated 'blast cell' colonies or clusters in leukemic type growth of MDS CD34+ cells over that seen in normal CD34+ cells. SCF is present in plasma at a level of ng ml(-1). This means that progenitor cells are continuously exposed to stimulation by SCF in vivo and that MDS leukemic cells have a growth advantage over normal blasts.

Large scale purification of human blood CD34(+) cells using a nylon-fiber syringe system and immunomagnetic microspheres.

BACKGROUND: To establish an available, economical technique for the large-scale purification of CD34(+) cells we used a nylon-fiber syringe (NF-S) for depletion of adherent cells and then selected CD 34(+) cells from peripheral blood mobilized by G-CSF, using MAb and magnetic heads. METHODS: PBSC were mobilized and collected from adult, healthy volunteers. With the effect of concentration of anti-CD34 MAb (9C5) on the purification of CD34(+) cells from 1 2 10(8) NF-S treated cells (NF cells), the recovery and the purity of CD34(+) cells was identical for 5, 10, 20, 40 microg of 9C5. In this study, therefore, the concentration of 9C5 was maintained at >5 microg/10(8) NF cells, with a cellhead ratio of 1:10. RESULTS: When half to one-third of leukapheresis product containing 121.5 + or - 26.0 2 10(8) mononuclear cells (mean + or - SD; n = 6) was processed for CD34(+) cell purification, 5.26 + or - 3.01 2 10(7) total purified cells were obtained with a purity of CD34(+) cells at 94.9 - 8.5%. The numbers of CD34(+) cells and total progenitor cells recovered in this process were 4.71 + or - 2.33 2 10(7) cells and 145.9 + or - 121.8 2 10(5) cells, respectively. The total recovery of CD34(+) cells was 37.0 + or - 21.0%. The depletion of monocyte by NF-S reduced the 9C5 anti-human CD34 MAb needed for purification of CD34(+) cells to one-third. Two patients were grafted with peripheral blood CD34(+) cells selected by this procedure and achieved a rapid and consistent hematopoiesis. DISCUSSION: Our clinical data show that these cells are capable of rapid reconstitution of hematopoiesis after high-dose chemotherapy. The procedure contains an additional step; however, consistent high purity of CD34(+) cells in the purified population and cost reduction were achieved, both critical issues in the better purging of malignant cells and for a wide clinical application.

In vitro expansion of CD34+/CD41+ cells from human peripheral blood CD34+/CD41- cells: role of cytokines for in vitro proliferation and differentiation of megakaryocytic progenitors.

The aim of this study is to clarify the transitional change of the proliferation and differentiation of human peripheral blood CD34+ cells to megakaryocytic lineage, focusing on its clinical application. We developed a rapid system to purify human peripheral blood CD34+ cells from healthy volunteers, which produced CD34+ cells with a 90% purity. The purified CD34+ cells predominantly consisted of CD41- cells, and the rate of coexpression of CD41 was 0.6% +/- 0.5%. When the purified cells were cultured in liquid phase for 10 days in the presence of recombinant human stem cell factor (rSCF: a ligand for c-kit), interleukin-3 (rIL-3), and thrombopoietin (rTPO: a ligand for Mpl), the number of CD34+/CD41+ cells increased to 19% +/- 7% of total expanded cells on day 4 (4 days of liquid culture) and then gradually decreased to 2.2% +/- 0.6% on day 10. The absolute number of CD34+/CD41+ cells increased and reached a plateau on day 6, and 1.7 +/- 0.6 x 10(5) CD34+/CD41+ cells were produced by 1 x 10(5) CD34+/CD41- day 0 cells. The CD34-/CD41+ cells appeared on day 6, continuously increased in number until day 10, and constituted the main population of expanded cells on day 10, with a value of 38% +/- 18%. On day 10, 19.5 +/- 10.6 x 10(5) of CD34-/CD41+ cells were produced by 1 x 10(5) CD34+/CD41- day 0 cells. The deletion of rTPO from this cytokine combination decreased the number of CD34+/CD41+ and CD34-/CD41+ cells, after days 6 and 8, respectively. Day 0 cells required rIL-3 for promoting colonies containing megakaryocytes, whereas rTPO alone promoted almost no megakaryocytic colonies from day 0 cells. Thus, a combination of IL-3 and SCF expands CD34+/CD41+ cells from CD34+/CD41- cells, and TPO mainly acts to increase CD34-/CD41+ cells. This study suggests that if the expansion of CD34+/CD41+ is performed in vitro, the 6 days' culture of peripheral blood CD34+/CD41- cells with a combination of IL-3 and SCF with TPO provides the most rapid and stable products of CD34+/CD41+ cells for the rapid recovery of platelets in patients with peripheral blood stem cell transplantation.

[Autoimmune thrombocytopenia following syngeneic peripheral blood stem cell transplantation].

A 35-year-old man with non-Hodgkin's lymphoma (NHL) (follicular small cleaved, B cell, stage IVB) received double myeloablative chemotherapy with syngeneic peripheral blood stem cell transplantation (PBSCT). Although platelet recovery was delayed until day 29 after the second transplantation, thereafter trilineage hematopoietic reconstitution was achieved. The evaluation after PBSCT did not detect any residual tumor. The patient was in good health until day 138, when his platelet count suddenly began falling; on day 150, it had fallen to 1.5 x 10(4)/microliter, and the patient was re-admitted for treatment. The bone marrow was normocellular with a normal count and megakaryocyte structure. Other examinations, including serological tests and computed tomography of the neck, chest, abdomen, and retroperitoneum, did not indicate a recurrence of NHL or reveal the cause of thrombocytopenia. The patient's platelet-associated IgG (PAIgG) level was at 70.9 ng/10(7) platelets (normal range: 9-25 ng/10(7) platelets); a diagnosis of thrombocytopenia due to an autoimmune mechanism such as idiopathic thrombocytopenic purpura (ITP) was made. Prednisolone therapy increased the platelet count and reduced the PAIgG level. Thrombocytopenia with an ITP-like mechanism rarely occurs more than 100 days after autologous or syngeneic stem cell transplantation, and should be taken into consideration as a late complication of PBSCT.

[Gamma-heavy chain disease associated with MALT lymphoma of the duodenum].

We report a case of a 63-year-old woman with gamma heavy chain disease (HCD) associated with mucosa-associated lymphoid tissue (MALT) lymphoma of the duodenum. She was suffering from drug-resistant tonsillitis with high fever. Examination on admission showed leukocytopenia and thrombocytopenia. Bone marrow aspirate revealed granulocytosis and a hypocellular marrow with no increase in plasma cells or atypical lymphocytes. Serum electrophoresis disclosed, in addition to hypogamma-globulinemia, an abnormal band due to the presence of gamma HCD protein. This abnormal protein was a molecular weight of approximately 40 kd as determined by Western blots technique, and belonged to the IgG1 subclass as determined by ELISA with monoclonal antibodies against IgG. An endoscopic examination of the patient's duodenum found a small tumorous lesion, which was confirmed pathologically to be MALT lymphoma. HCD is known to be associated with lymphoproliferative diseases. In this case, gamma HCD had developed as a secondary complication of MALT lymphoma. gamma HCD associated with MALT lymphoma of the duodenum is rare in the literature.

The influence of beta2-glycoprotein I on tissue factor activity.

The beta2-glycoprotein I (beta2-GPI) is known for its procoagulant as well as anticoagulant properties. The influence of beta2-GPI on tissue factor (TF) activity was investigated by chromogenic assays for both factor Xa generation and factor VIIa activity. When 1.36 mg/mL beta2-GPI was added to phospholipids prior to the addition of TF and factor VIIa, the TF activity was moderately inhibited by beta2-GPI in a concentration-dependent manner. Inhibition of TF activity by beta2-GPI required incubation for at least 20 min. When beta2-GPI was added to the mixture of phospholipids and TF followed by the addition of factor VIIa, the TF activity was little affected by the addition of beta2-GPI. The TF activity was inhibited when beta2-GPI was added after forming a complex with TF and factor VIIa. The results suggest that beta2-GPI inhibits both the reconstitution of TF with phospholipids and factor Xa generation by the TF factor-VIIa complex but does not affect the formation of a TF factor-VIIa complex.

Subclinical alterations in coagulation and fibrinolysis in patients undergoing autologous peripheral blood stem cell transplantation.

We monitored 30 laboratory hemostatic parameters in an attempt to better comprehend alterations in coagulation and fibrinolysis in 10 patients with hematological malignancies subjected to autologous peripheral blood stem cell transplantation (APBSCT). These parameters were assessed before and just after high-dose conditioning chemotherapy, on days 1, 7, 14 and 28. Although, clinical manifestations associated with fibrino-coagulation disorders never occurred, including veno-occlusive disease, a statistically significant increase was seen in 7 of 30 parameters, compared to values seen before conditioning chemotherapy. These were subdivided into early and late phase parameters. The early phase parameters, which increased during the first day after the conditioning chemotherapy was given, then returned to baseline values, included protein C, plasma tissue factor and tissue-plasminogen activator. The late phase parameters, which increased over baseline values during days 7 to 28, included free-protein S, fibrinogen, plasmin-alpha2-plasmin inhibitor complex and soluble-thrombomodulin. The increase of early phase parameters, as produced by the liver and by endothelial cells, may reflect tissue damage by conditioning chemotherapy. Late phase parameters increased in parallel with C-reactive protein, which suggests a correlation with the degree of inflammation, such as the presence of infective disease during neutropenia. These subclinical alterations in coagulation and fibrinolysis which take on a biphasic pattern during the course of APBSCT should be kept in mind by the attending physicians during therapy.

Protection by alpha2-macroglobulin of tissue plasminogen activator against inhibition by plasminogen activator inhibitor-1.

Tissue plasminogen activator (tPA) is widely used in the treatment of acute myocardial infarction (MI). However, its thrombolytic efficacy does not correlate with the dose administered. The interactions between tPA, alpha2-macroglobulin (alpha2-M), and plasminogen activator inhibitor-1 (PAI-1) were investigated both in vitro and in patients undergoing tPA therapy for MI in an attempt to identify variables that might affect the clinical efficacy of tPA. Purified alpha2-M (5.4 mg/ml) protected 16.0% or 22.4% of tPA (12.5 IU/ml) activity from inhibition by PAI-1 at 4 or 8 IU/ml in vitro. Of nine patients treated with 5-20 mega IU of tPA for MI, the plasma activity of tPA remained increased for 15-30 min after the cessation of infusion in eight; the patient who failed to exhibit a persistent increase in tPA activity had a low plasma concentration of alpha2-M. Total tPA activity, derived from the area under the activity-versus-time curve (AUC), showed a significant inverse correlation with the ratio of the plasma PAI-1 activity to the plasma alpha2-M concentration. Total tPA activity did not correlate with plasma PAI-1 activity or plasma alpha2-M concentration alone. Results suggest that alpha2-M, by binding to tPA, protects the latter against inhibition by PAI-1.

Role of cytokines in leukemic type growth of myelodysplastic CD34+ cells.

The clonal growth of progenitor cells from myelodysplastic syndromes (MDS) can be subdivided into four growth patterns: (1) normal, (2) no growth or low plating efficiency, (3) low colony and high cluster number, and (4) normal or high colony number with a large number of clusters. The former two (1 and 2) can be referred to as nonleukemic patterns and latter two (3 and 4) as leukemic. In a search for a role for cytokines in leukemic-type growth of MDS progenitor cells, marrow CD34+ cells were purified up to 94% for 8 normal individuals and 88% for 12 MDS patients, using monoclonal antibodies and immunomagnetic microspheres (MDS CD34+ cells). The purified CD34+ cells were cultured for 14 days with various combinations of cytokines, including recombinant human macrophage colony-stimulating factor (rM-CSF), granulocyte-CSF (rG-CSF), granulocyte-macrophage-CSF (rGM-CSF), interleukin-3 (rIL-3), and stem cell factor (SCF; a ligand for c-kit) in serum-free medium. The clonal growth of MDS CD34+ cells supported by a combination of all of the above cytokines was subdivided into the two patterns of leukemic or nonleukemic, and then the role of individual or combined cytokines in proliferation and differentiation of MDS CD34+ cells was analyzed in each group. Evidence we obtained showed that SCF plays a central role in the leukemic-type growth of MDS CD34+ cells and that G-CSF, GM-CSF; and/or IL-3 synergize with SCF to increase undifferentiated blast cell colonies and clusters over that seen in normal CD34+ cells. SCF is present in either normal or MDS plasma at a level of nanograms per milliliter, and this physiologic concentration of SCF can stimulate progenitor cells. This means that progenitor cells are continuously exposed to stimulation by SCF in vivo and that MDS leukemic cells have a growth advantage over normal blast cells. This depends, at least in part, on cytokines such as G-CSF, GM-CSF, IL-3, and SCF.

Synthesis of novel poly(ethylene glycol) derivatives having pendant amino groups and aggregating behavior of its mixture with fatty acid in water.

Novel poly(ethylene glycol) (PEG) derivatives having pendant amino groups were prepared by copolymerization of allyl glycidyl ether with ethylene oxide followed by chemical modification of the double bond side chains. Dropwise addition of the mixture of monomers to the anionic initiator gave an almost monodisperse (Mw/Mn = 1.05) random copolymer. 1H NMR spectra showed that addition of 2-aminoethanethiol to the pendant allyl groups of the copolymer was completely carried out in methanol without catalyst, and an aminated PEG derivative with a definite structure was obtained. Acetylation of the pendant amino groups was readily performed by acetic anhydride with triethylamine. A gel permeation chromatogram of the acetylated polymer showed a very narrow molecular weight distribution (Mw/Mn = 1.06) of the polyamine. These cationic PEG derivatives make amphiphilic polyion complexes with fatty acids, and then aggregate in water. A fluorescence study using pyrene as a microenvironment probe revealed that the aminated PEG-lauric acid ion complex could take up the hydrophobic fluorescence probe into the lipophilic field inside, and they also had a critical aggregation concentration at [lauric acid] = 0.7 mM. It is much lower than the critical micelle concentration of the corresponding fatty acid sodium salts, indicating high stability of the polymer-fatty acid aggregate.

[A case of virus-associated hemophagocytic syndrome (VAHS) complicated by rhabdomyolysis which were associated with herpes-simplex virus infection].

A 60-year-old male was admitted to our hospital complaining abdominal pain and fatigue. Complete blood count showed as follows; WBC 3,900/microliters (48% of monocytes), Hb 11.5 g/dl, Plt 0.9 x 10(4)/microliters. Marrow smears showed the presence of phagocytic histiocytes that consist 22.4% of total nuclear cells. Laboratory findings showed as follows; BUN 109.5 mg/dl, Creatinine 7.4 mg/dl, CPK 1,259 IU/l, Aldolase 195 IU/l, Myoglobin 4,200 mg/dl. Serological studies showed a 16-fold increase in herpes-simplex virus (HSV) antibody titers 4 weeks after admission. So we diagnosed his illness as virus-associated hemophagocytic syndrome (VAHS) and rhabdomyolysis that were associated with HSV. We performed three times of hemodialysis for acute renal failure and used prednisolone for VAHS. These treatments were successful, and he made a complete recovery from illness. VAHS complicated by rhabdomyolysis is very rare, and we think this case is full of suggestions.

Efficacy of delayed granulocyte colony-stimulating factor after full dose CHOP therapy in non-Hodgkin's lymphoma: a pilot study for a leukocyte count oriented regimen.

Bone marrow toxicity is a great challenge for physicians treating patients with non-Hodgkin's lymphoma (NHL) and prescribed chemotherapy. Granulocyte colony-stimulating factor (G-CSF) prevents myelotoxicity, but the optimal timing and scheduling of G-CSF administration has not been ascertained. We investigated leukocyte count oriented G-CSF administration schedules, as related to full dose administration of cyclophosphamide, adriamycin, vincristine, and prednisolone (CHOP) chemotherapy, with shortened intervals. Thirty-eight Japanese patients with NHL were included in this study. The standard CHOP combination was administered in six cycles. Patients were given G-CSF in a dose of 2 micrograms/kg/day, subcutaneously starting the day when total leukocytes were < 3,000/microliters. When leukocyte count remained at > 3,000/microliters, G-CSF was started 10 days following CHOP. Treatment with G-CSF was discontinued after the leucocyte count reached > 10,000/microliters, and CHOP was started the next day (CHOP-G treatment; CHOP-G). Doses were not modified in any patient. Patients over 70 years of age received 2/3 of the standard dosage. In the first cycle of CHOP, the day of initiation of G-CSF was 9.6 days following CHOP in the first cycle and 7.7 to 8.5 days during 2 to 6 cycles. The mean duration of G-CSF injection was 7.4 days with a range from 6.8 to 8.0 days, in each CHOP cycle. The mean intervals of CHOP-G was 14.7 days during six consecutive courses, and there was no prolongation of the intervals, even in later cycles. In 23 patients who received all six cycles of CHOP-G, the overall response rate was 91.3% (73.9% complete remission; CR and 17.4% partial remission; PR). In 32 patients with intermediate grade NHL, the overall response rate was 84.4% (65.5% CR and 18.8% PR). Thus, the full dose CHOP with G-CSF, based on the leukocyte count oriented schedule, can be achieved with shortened intervals, an approach which will increase the quality of life (QOL) for the patients by reducing the days of treatment as well as the cost of G-CSF.

Interferon-alpha-induced apoptosis in human erythroid progenitors.

Recombinant human interferon-alpha (rIFN-alpha) inhibits erythropoiesis, in vivo and in vitro. In an attempt to clarify mechanisms related to this inhibition, effects of rIFN-alpha on highly purified human peripheral blood burst-forming units-erythroid (BFU-E) (20-60% purity) were compared with effects on erythroid progenitors in various stages of development. Day-1 and -7 cultured cells were equivalent to primitive BFU-E and colony-forming units-erythroid (CFU-E), respectively. Day-1 BFU-E supported by recombinant human erythropoietin (rEpo) and interleukin-3 (rIL-3) was inhibited by rIFN-alpha in a dose-dependent manner, and a significant inhibition occurred at 2000 U/mL rIFN-alpha. Limiting dilution analysis demonstrated that rIFN-alpha directly inhibits BFU-E rIFN-alpha inhibited the proliferative capacity or the colony expression of erythroid progenitors, with no relation to the stage of development, but inhibition of differentiation was not apparent. This evidence suggested that apoptosis of erythroid progenitors was induced by IFN-alpha. When day-7 cells were incubated with IFN-alpha in the presence of rEpo, there was an increased breakdown of total cellular DNA into DNA fragments of less than 5 kb; hence, the inhibitory effect of IFN-alpha on erythroid progenitors may mediate apoptosis.

Proliferation and differentiation of myelodysplastic CD34+ cells in serum-free medium: II. Response to combined colony-stimulating factors.

To investigate the role of colony stimulating factors (CSFs) in the proliferation and differentiation of progenitor cells from myelodysplastic syndromes (MDS), marrow progenitor cells from 18 MDS patients were highly purified using CD34 monoclonal antibody and immunomagnetic microspheres (MDS CD34+ cells). These cells were cultured in serum-free medium with various combinations of five colony stimulating factors (CSFs): recombinant human interleukin-3 (rIL-3), granulocyte/macrophage-CSF (rGM-CSF), granulocyte-CSF (rG-CSF), macrophage-CSF (rM-CSF), and erythropoietin (rEP). Among the tested CSFs, such as rM-CSF, rG-CSF, rGM-CSF and rIL-3, a combination of the first three CSFs was the most effective stimulus for the proliferation of non-erythroid MDS progenitor cells. An increase of undifferentiated "blast" cell colonies in 5/18 MDS patients occurred and these 5 patients belonged to the high-risk group. In the presence of these three CSFs, rIL-3 had no effect on the proliferation and differentiation of MDS CD34+ cells; however, IL-3 was efficient for the proliferation of MDS CD34+ cells to the erythroid lineage. rGM-CSF or rIL-3 alone did not efficiently support proliferation and differentiation of CD34+ cells. M-CSF is present in normal human serum at a concentration of 550 +/- 110 U/ml, a concentration exceeding that used in this study (100 U/ml). Therefore, in vivo administration of G-CSF combined with GM-CSF to MDS patients may be one of the most effective CSF combinations for proliferation of MDS progenitor cells to the non-erythroid lineage. However, the effect on the capacity for differentiation was minimal, especially in patients belonging to the high-risk group.

Proliferation and differentiation of myelodysplastic CD34+ cells: phenotypic subpopulations of marrow CD34+ cells.

In a search for a mechanism to explain the impaired growth of progenitor cells in patients with myelodysplastic syndromes (MDS), marrow CD34+ cells were purified up to 94.9% +/- 4.2% for normal individuals and 88.1% +/- 17.6% for MDS patients, using monoclonal antibodies and immunomagnetic microspheres (MDS CD34+ cells). Phenotypic subpopulations of these CD34+ cells were analyzed for CD38, HLA-DR, CD33, CD13, CD14, CD41 and CD3 plus CD19, in association with proliferative and differentiative capacities. The 15 studies performed included 12 MDS patients. Coexpression rate of CD13 significantly increased in the MDS CD34+ cell population with a value of 91.4% +/- 11.6% and ranging from 60.3% to 100%, and exceeded 99% in four studies, whereas that of normal CD34+ cells was 49.9% +/- 15.8%, ranging from 28.2% to 70.1% (P < .001). Coexpression rate of CD38, HLA-DR, CD33, CD14, and CD3 plus CD19 in MDS CD34+ cells did not significantly differ from that of normal CD34+ cells. The total number of colonies and clusters grown from 100 normal marrow CD34+ cells was 40.4 +/- 8.6, the range being from 27.2 to 50.3; this varied in MDS marrow CD34+ cells with a value of 34.0 +/- 28.7, the range being 0 to 95.9. The lineage of colonies and clusters promoted by MDS marrow CD34+ cells was predominantly committed to nonerythroid with impaired differentiation in 13 of 15 studies (87%). CD13 is first expressed during hematopoiesis by colony-forming unit granulocyte-macrophage and is absent in erythroid progenitors. Therefore, this study provides direct evidence for the lineage commitment of MDS CD34+ cells to nonerythroid with impaired differentiation and explains the mechanism of nil or low colony expression of MDS progenitor cells to erythroid lineage.

Transitional changes in immunophenotypic subpopulations of human peripheral blood CD34+ cells expanded in vitro.

We determined the appropriate incubation period to expand human peripheral blood (PB) CD34+ cells for clinical application and the role of recombinant human (rh) interleukin-3 (rhIL-3) in the expansion and differentiation of these cells. The cells were purified up to 40 +/- 16% and transitional changes in immunophenotypic subpopulations in suspension culture were examined following stimulation with three different combinations of rh colony-stimulating factors (rhCSFs): 1) rhIL-3 alone, 2) rhIL-6, rhSCF, rhG-CSF, plus rhGM-CSF, and 3) the four CSFs plus rhIL-3. With all three CSF combinations, the total cells increased continuously after day 5 until day 14, and a combination of the five CSFs always gave rise to the highest number of total cells. Immunophenotypic analysis of the expanded cells showed that with all three CSF combinations CD34+ cells reached a maximal rate on day 5 and then decreased in an inverse correlation between the logarithm of CD34 positive rate and the duration of suspension culture. The maximum expansion of CD34+ cells and PB progenitor cells (PBPC) with rhIL-3 alone, the four CSFs, or the five CSFs was observed on day 5, 10, or 7, respectively. The combination of the five CSFs was identified as the most potent stimulus for expansion of PBPC and CD34+ cells, as it increased non-erythroid PBPC 89 +/- 69-fold, with a range of 24 to 204-fold on day 7. However, differences in the expansion rate of these cells on days 5, 7, and 10 were not statistically significant. The majority of purified CD34+ cells coexpressed CD38 (91 +/- 3%) but were negative for CD33 (85 +/- 4%), and one-half coexpressed CD13. With all three combinations of CSFs, the mature CD34+ cells that coexpressed CD38, CD33, or CD13 expanded in parallel with the total CD34+ cells, while an increase in relatively immature CD34+ cells, which do not express CD38, CD33, or CD13, was only statistically significant with the five CSFs. Thus, rhIL-3 played a critical role when combined with the four CSFs by increasing both mature and immature CD34+ cells.

[Chronic cold agglutinin disease accompanied with an increase of CD20+/CD5+ cells; a case report].

A 61-year-old male complained of acrocyanosis and dark urine when exposing to cold temperatures. This had continued for several years. His physical examinations showed neither lymphadenopathy nor hepatosplenomegaly. Laboratory findings were as follows; RBC 305 x 10(4)/microliters, Hb 10.3 g/dl, reticulocytes 4.32%, platelets 27.3 x 10(4)/microliters, WBC 7,400/microliters with 50% lymphocytes, and a high cold agglutinin titer (2,048-fold) with anti-I specificity. Bone marrow smear preparations showed erythroid hyperplasia and increase of lymphocytes (52%). Immunophenotypic analysis showed an increase of CD20+/B-lymphocytes in peripheral blood (32.6%) and in bone marrow, and 94% of these cells co-expressed CD5. Most B-lymphocytes expressed surface IgM-lambda, suggesting a monoclonal proliferation of B-lymphocytes. At this point we diagnosed cold agglutinin disease (CAD) because there was no evidence of lymphoma, and the absolute number of peripheral blood lymphocytes was lower than the criteria of chronic lymphocytic leukemia (CLL) proposed by the International Workshop (1989). However, there still remains the possibility of the transitional form between "idiopathic" CAD and B-CLL or lymphoma.

[A case with AML (M4EO) accompanied by recurrent reactive histiocytosis which showed spontaneous remission].

A 30-year-old female was admitted to our hospital complaining of high fever and fatigue. Laboratory findings showed as follows; WBC 41,500/microliter (40% of blasts), Hb 8.5g/dl, platelets 4.4 x 10(4)/microliter. Cytochemical staining of blasts was positive for peroxidase and non-specific esterase with NaF inhibition. Chromosome analysis showed 46, XX, inv (16p+,q-). AML with eosinophilia was diagnosed. During myelosuppression after remission induction therapy, she developed high fever, and did not respond to transfusions. Marrow smears showed the presence of phagocytic histiocytes consisting of 18% total nuclear cells. A diagnosis of reactive histiocytosis (RH) was made. She recovered spontaneously, but suffered two episode of recurrence during subsequent chemotherapy. Reactive histiocytosis is characterized by proliferation of histiocytes which phagocyte blood cells in immunodeficient cases, e.g. a myelosuppressive state after chemotherapy. RH causes high fever and prolonged myelosuppression. It is considered to be one of the poor prognostic factors in AML during chemotherapy, and spontaneous recovery is rare. In this report, the effect of hydrocortisone on histiocytes derived from patient marrow was also investigated in vitro.

Recombinant human interleukin-4 inhibits the production of granulocyte-macrophage colony stimulating factor by blood mononuclear cells.

The effect of recombinant human (rh) interleukin-4 (rIL-4) on human blood BFU-E was investigated using two populations of cells: platelet-depleted low-density mononuclear cells (FH,Pl- cells), as unpurified cells, and highly purified BFU-E. When FH,Pl- cells were cultured with rh-erythropoietin (rEp), rIL-4 inhibited BFU-E growth in a dose-dependent manner. However, the addition of rIL-4 did not affect rh-interleukin-3 (rIL-3) supported BFU-E growth. Limiting dilution analysis (LDA) of FH,Pl- cells showed that rIL-4 suppressed endogenous production of burst-promoting activity (BPA) by accessory cells. Highly purified BFU-E were used as target cells to measure BPA in the conditioned medium (CM) that was prepared by FH,Pl- cells. When 100 purified BFU-E were cultured in 0.5 ml clots with 20% (vol/vol) of the CM, the number of BFU-E colonies was increased by the CM. The increase was significantly reduced by the addition of the CM prepared in the presence of rIL-4, but anti-IL-4 blocked the effect of rIL-4. The concentration of IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) in CM was determined by an enzyme-linked immunoadsorbent assay (ELISA). The spontaneous production of GM-CSF but not IL-3 was detected, and this was significantly decreased in the presence of rIL-4. Anti-GM-CSF but not anti-IL-3 inhibited CM supported BFU-E growth, indicating that the main BPA in the CM is GM-CSF and that rIL-4 suppresses the spontaneous production of GM-CSF by accessory cells. From these studies, we conclude that rIL-4 has a unique mechanism as a negative regulator on erythropoiesis through the inhibition of BPA production by blood mononuclear accessory cells.