Osteo-anabolic effects of human growth hormone with 22K- and 20K Daltons on human osteoblast-like cells.

Human growth hormone with 22,000 Dal (22K-hGH) stimulates proliferation and differentiation of osteoblasts as well as production of interleukin-6 in vitro and bone formation and remodeling in vivo. To investigate whether hGH isoform with 20Kd (20K-hGH), which accounts for 10% of circulating hGH, elicits similar metabolic effects on skeletal tissues, we studied the biological effects of 20K-hGH in cultured human osteoblast-like cells (HOB). HOB were obtained from trabecular bone explants and cultured in alpha-MEM supplemented with 10% FCS. In subconfluent cultures, 22K- and 20K-hGH stimulated [3H]thymidine incorporation by 62 +/- 27% and 63 +/- 23%, respectively (mean +/- SD, n=8, P>0.1). In confluent cultures, 22K- and 20K-hGH increased alkaline phosphatase activity by 38 +/- 23% and 41 +/- 23% (P>0.1), respectively, and increased the osteocalcin concentration in the presence of 10(-9) M 1,25-(OH)2D3 by 50% and 47% (P>0.1), respectively. Furthermore, both hGHs doubled the interleukin-6 (IL-6) concentration in the conditioned medium. RT-PCR analysis revealed that 22K- and 20K- hGH increased IL-6 gene expression 2.2 +/- 0.6 and 2.4 +/- 0.7 -fold, respectively. In summary, we have demonstrated that 20K-hGH elicits equipotent anabolic effects on HOB and stimulates to the same extent the production of IL-6, a cytokine which initiates osteoclastogenesis. These in vitro findings suggest that 22K- and 20K-hGH may equipotently stimulate bone remodeling and elicit anabolic effects on skeletal tissue when administered in vivo.

Expression of IL-6 receptor and GP130 in mouse bone marrow cells during osteoclast differentiation.

Interleukin-6 (IL-6) has been postulated as a possible mediator of bone loss after estrogen deficiency, and its signal is transduced via glycoprotein 130 (gp130) after binding IL-6 receptor (IL-6R) in the membrane of target cells. In this study, the expression of IL-6R and gp130 in bone marrow cells during osteoclast differentiation was investigated. Mouse bone marrow cells were isolated and cultured with or without 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]. During the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs), IL-6R and gp130 expression in the mononuclear cells, stromal cells, and TRAP-positive MNCs were quantitated, using a laser cytometer with a fluorescence confocal microscopy. With 1,25(OH)2D3 stimulus, the level of gp130 significantly increased, but that of IL-6R did not in the stromal cells. In contrast, the levels of both gp130 and IL-6R significantly increased in the mononuclear cells by the treatment with 1,25(OH)2D3. The high expression of both gp130 and IL-6R was observed in the TRAP-positive mononuclear cells. Moreover, both IL-6R and gp130 were expressed in the TRAP-positive MNCs and isolated murine osteoclasts. The treatment of TRAP-positive MNCs with IL-6 caused enhancement of the resorbing activity in a dose-dependent manner, and the effect was prevented by a neutralizing antibody against IL-6R. These data suggest that gp130 and IL-6R, as well as IL-6, are involved in the formation and activation of osteoclasts.

Comparison of the DNA content of megakaryocytes identified immunologically with that identified morphologically.

We devised a new microfluorometric method for determining the ploidy of megakaryocytes identified immunologically in bone marrow smears. The smears were immunostained by incubation with mouse monoclonal anti-glycoproteins (GP) IIb antibodies, followed by fluorescein isothiocyanate-conjugated goat antimouse IgG antibodies. They were then stained with 4',6-diamidino-2-phenylindole (DAPI). Megakaryocytes were identified by their GPIIb immunofluorescence using a microfluorometer and, after the filters were changed, their DNA content was assayed by measuring the intensity of DAPI fluorescence. This intensity was shown to be proportional to the DNA content when the aperture of the objective lens was reduced. We compared these results with those obtained when megakaryocytes were identified morphologically, using DAPI staining after Wright-Giemsa destaining. In all 12 normal controls, the ploidy peaks were shown to be 16N by both methods, and the mean ploidy detected by the immunological method was only reduced 0.961 times relative to the estimate from the morphological method. In contrast, in eight myelodysplastic syndrome (MDS) patients, the ploidy peaks were either 8N or 4N and the mean was reduced by 0.906 times (P = 0.018). Thus we could immunologically identify small megakaryocytes which we could not identify morphologically. Therefore, this method is useful for measuring megakaryocytic ploidy, especially in the pathological megakaryocytes of MDS patients.

Susceptibility to Infection Due to Diminished Interferon-α-Producing Capacity in Patients with Refractory Anemia with Excess of Blasts or Refractory Anemia with Excess of Blasts in Transformation.

Peripheral whole blood cells from normal subjects and from 8 patients with refractory anemia with excess of blasts (RAEB) and one patient with RAEB in transformation (RAEB-t) were studied to ascertain their interferon (IFN)-α-producing capacity. This capacity was determined by time-resolved immuno-fluoroassay 20 hrs after the blood cells had been incubated with 500 HA/ml of Sendai virus. The mean IFN-α-producing capacity in the peripheral whole blood cells of 35 normal males was 8718 ± 4683 IU/ml, while that of 49 normal females was 7549 ± 4731 IU/ml; not a significant difference. All of the RAEB and RAEB-t patients examined showed a significantly low (<2500 IU/ml) IFN-α-producing capacity in the peripheral whole blood cells. In addition, the levels of IFN-α-producing capacity in 1 ml cell suspension containing 1 x 10(6) peripheral mononuclear blood cells were markedly lower in all 5 RAEB patients and the RAEB-t patient examined than those in the normal subjects, when 5 HA of Sendai virus was used. The activities of natural killers were lower in the RAEB/RAEB-t patients than those in the normal subjects. These findings suggest that IFN-α-producing capacity is diminished in the peripheral whole blood cells of RAEB and RAEB-t patients, and great care is advised regarding infection by virus and bacteria in treatment of these patients.

Interleukin-6 (IL-6) inhibits thyroid function in the presence of soluble IL-6 receptor in cultured human thyroid follicles.

Interleukin-6 (IL-6), a pleiotropic cytokine, is postulated to be involved in the pathogenesis of sick euthyroid syndrome, although the direct in vitro effects of IL-6 on human thyroid function are controversial. Because IL-6 signal can be transduced when the complex of IL-6 and soluble IL-6 receptor (sIL-6R) binds to gp 130, an IL-6 signal transducer, we studied the effects of IL-6 and sIL-6R on thyroid function, using human thyroid follicles obtained from patients with Graves' disease. IL-6 alone had no inhibitory effect on TSH-induced thyroid function (125I incorporation and organic 125I release), even at supraphysiological concentrations. However, in the presence of physiological concentrations of sIL-6R (100 ng/ml), IL-6 inhibited thyroid function dose dependently and completely, accompanied with the decreased ratio of 125I-T3/125I-T4 not only in the thyroid follicles but also in the culture medium. Thyroid follicles did not secrete sIL-6R but produced IL-6 constitutively. Consistent with these findings, sIL-6R inhibited thyroid function slightly at high concentrations. Furthermore, RT-PCR analyses revealed that human thyroid follicles expressed the messenger RNAs for IL-6 and gp130 but scarcely messenger RNA for IL-6R. These in vitro findings suggest that IL-6 alone hardly affects thyroid function in thyroid follicles in which IL-6R gene is scarcely expressed. However, because sIL-6R is present abundantly in serum, IL-6 in vivo would be capable of inhibiting the synthesis and release of T4 and, to a greater extent, T3 from the thyroid gland. These in vitro findings are at least partly related to the development of sick euthyroid syndrome.

Determination of interferon-alpha-producing capacity in whole blood cultures from patients with various diseases and from healthy persons.

To assess the clinical value of determination of the interferon (IFN)-producing capacity of patients, IFN production induced by Sendai virus (HVJ) in vitro was measured in cell cultures of whole blood from patients with various diseases. IFN production in patients with lung cancer, myelodysplastic syndromes, noninsulin-dependent diabetes mellitus, pulmonary tuberculosis, and asymptomatic HIV-1 infection was lower than that in healthy persons. Furthermore, periodic measurements of IFN production revealed decreasing IFN producing capacities in patients with lung cancer with progression of the tumor stage. However, increased IFN-producing capacities were observed in patients with tuberculosis after standard therapy. Further experiments showed that the main type of IFN induced in whole blood cultures was IFN-alpha, and decreased IFN production in patients did not result from a decreased number of leukocytes but rather from an impairment of cellular IFN production. The evaluation of IFN production in whole blood cell cultures may be a feasible method of assessing the impaired immune status.

Relationship between morphological classification of the degree of maturation and the ploidy of micromegakaryocytes in myelodysplastic syndrome patients.

To clarify the biological behavior of micromegakaryocytes in myelodysplastic syndrome (MDS), the relationship between the morphological classification and the ploidy of megakaryocytes was studied in bone marrow aspirates obtained from patients with MDS and from normal controls. The morphology was determined according to Feinendegen's classification, which is considered to reflect megakaryocytic maturation, and the ploidy was determined by microcytofluorometry, using 4',6-diamidino-2-phenylindole (DAPI) staining after the removal of Wright-Giemsa stain. Most micromegakaryocytes (i.e., megakaryocytes < 20 microns in diameter) in MDS were morphologically mature, as were those in the normal controls. The peak micromegakaryocytic ploidy was 4N or 8N, whereas that of the megakaryocytes in normal controls was 16N. These findings indicated that the micromegakaryocytes in MDS were morphologically mature but had impaired polyploidization.

Prognostic significance of loss of a chromosome 17p and p53 gene mutations in blast crisis of chronic myelogenous leukaemia.

In 31 cases of chronic myelogenous leukaemia (CML) we examined the prognostic significance of chromosomal loss of a 17p and p53 mutations at the onset of blast crisis (BC). p53 mutations were closely related to a shortened survival in CML-BC (P < 0.005 by the logrank test), whereas loss of a 17p by itself was not a poor prognostic indicator. The prognostic significance of loss of a 17p, however, emerged when combined with its predominance in the metaphases analysed. This predominance might easily and rapidly be screened by polymerase chain reaction-based analysis in about half of the cases.

[[Aging of the population and social policy: lessons from Sweden]].

"In this paper I tried to show how the ageing of the population influences the change in the growth of employment, employment structure, the savings ratio, economic growth and the cost of social security [in Sweden]. In the latter part of the paper I suggested a close correlation between the average marriage age of women, the total fertility rate and the work participation ratio of women." (SUMMARY IN ENG)

Increased and highly stable levels of functional soluble interleukin-6 receptor in sera of patients with monoclonal gammopathy.

Soluble human interleukin-6 receptor (sIL-6R) was measured in the serum of 30 healthy individuals, 32 individuals with monoclonal gammopathy of undetermined significance (MGUS), 20 patients with early multiple myeloma (MM) and 54 patients with overt MM. The serum activity recognized by an immunoradiometric assay was determined to be sIL-6R, because of its binding capacity to IL-6 and its molecular mass of 55 kDa. All sera of healthy individuals contained sIL-6R (mean value: 89 ng/ml, range 17-300 ng/ml). Serum sIL-6R levels were increased by 51% in patients with MGUS (mean value: 135 ng/ml, p < 0.005), by 44% in patients with early myeloma (mean value: 128 ng/ml, p < 0.001) and by 116% in patients with overt MM (mean value: 193 ng/ml, p < 0.001). In patients with MM, a complete lack of correlation (p > 0.7) was found between serum sIL-6R levels and other previously recognized prognostic factors in this disease, particularly serum IL-6 levels and those factors related to tumor cell mass. The independence of serum sIL-6R levels on tumor cell mass was directly demonstrated by studying four patients with MM treated with autologous bone marrow transplantation for periods of between 320 and 760 days. These levels were found to be remarkably stable and constant, independent of whether patients relapsed or achieved complete remission. Finally, physiological concentrations of sIL-6R were found to increase by tenfold the sensitivity of human myeloma cell lines to IL-6. These observations suggest a high control of the sIL-6R level in vivo, and, possibly, an important functional role of this circulating protein in patients with monoclonal gammopathies.

Quantitative and qualitative characteristics of colony-forming unit-erythroid colonies in myelodysplastic syndrome patients.

The microcytofluorometrical method was applied to determine the relative hemoglobin (Hb) content in the bone marrow colony-forming unit-erythroid (CFU-E) colonies from 6 patients with myelodysplastic syndromes (MDS) and 10 healthy subjects. This method relies on a photochemical reaction, by which intracellular Hb is converted into fluorescent porphyrin using a 0.2 M mercaptoethylamine solution (an SH donor) and violet light (lambda = 405 nm). The relative Hb content was determined as a function of the intensity of emitted porphyrin fluorescence. The number of colonies identified by porphyrin fluorescence was smaller in MDS patients than in normal subjects. The relative Hb content was also lower in MDS patients than in normal subjects. In addition, the coefficient of variation of the relative Hb content in the CFU-E colonies was larger in MDS patients than in normal subjects. These findings suggest that colonies with low relative Hb content undergo impaired erythropoiesis and that the CFU-E colonies undergoing the impaired erythropoiesis are mixed with CFU-E colonies showing normal erythropoiesis in the bone marrow of MDS patients.

Megakaryocytic ploidy in myelodysplastic syndromes.

The megakaryocytic ploidy was microfluorometrically measured in 12 normal controls and 15 myelodysplastic syndrome (MDS) patients using DAPI (4',6-diamidino-2-phenylindole) staining after destaining of the Wright-Giemsa (WG) stain. MDS patients had slightly more immature megakaryocytes when compared with normal controls. The megakaryocytic ploidy distribution had a peak at 16N in normal controls, at 8N in the 11 of the 15 MDS patients, and at 4N in the remaining 4 patients, which is suggestive of impaired polyploidization in MDS patients. In MDS, micromegakaryocytes were shown not to be immature but much more impaired in polyploidization than non-micromegakaryocytes. However, there was no difference in the megakaryocytic ploidy pattern among the type of the modification of Feinendegen' classification in each case for both the normal controls and the MDS patients, suggesting that the megakaryocytic ploidy is probably determined at the maturation level of the megakaryoblasts or the precursor cells. The study of megakaryocytic ploidy before and after therapy in the case of refractory anemia with excess of blasts might suggest that the remission of MDS patients might be qualitatively different from that seen in acute leukemia patients. Furthermore, the DNA histogram of the megakaryocytes from one of the two MDS patients obtained by the new method, which is able to determine the amount of DNA in the immunologically identified megakaryocytes microfluorometrically, using the monoclonal anti-glycoproteins IIb/IIIa antibody on bone marrow smears, showed a shift towards small ploidy compared with those defined on the basis of WG staining. This finding indicates that the micromegakaryocytes or the megakaryoblasts which could not be identified morphologically can be identified immunologically.(ABSTRACT TRUNCATED AT 250 WORDS)

[Clinicopathological review of Japanese cases with neoplastic angioendotheliosis].

The patient was a 76-year-old female who had been referred to our hospital because of fever of unknown origin on October 15, 1987. On admission, the body temperature was 38.6 degrees C and atonic palsy of the left upper limb was noted. Abnormal laboratory findings included CRP5+, an increase in LDH, Hb 7.9 g/dl. The cause of the fever could not be identified. The fever did not respond to various treatment. The patient developed DIC in late October and died on November 5. In autopsy histological examination revealed tumor cells in the vessels of the generalized organs. A diagnosis of neoplastic angioendotheliosis (NAE) and immunohistologically B lymphoma was made. We reviewed the literature on 37 Japanese cases of NAE. The cases, consisting of 19 males and 18 females, were aged 37-87 years with a median value of 60 years. The symptoms observed during the course were most frequently mental or neurological symptoms and fever, and rash was uncommon. Laboratory findings were non-specific and biopsy was needed for definitive diagnosis. By autopsy, lesions were noted more frequently in the brain, kidneys, and lungs, and the findings in the skin were indeterminate. These observations suggest that when NAE should be considered, kidney, lung or skin biopsy should be performed for definitive diagnosis.

IL-6 increases endothelial permeability in vitro.

The effect of interleukin 6 (IL-6) on endothelial permeability was examined by measuring fluorescein isothiocyanate-labeled albumin flux across an endothelial cell monolayer. Bovine vascular endothelial cells (BVEC) were cultured up to confluency on collagen-coated polycarbonate micropore filters and then the filters were mounted on modified Boyden chambers. Treatment of the BVEC with IL-6 at 100 ng/ml for 21 h caused a remarkable increase in the permeability of fluorescein isothiocyanate-labeled albumin across the endothelial monolayer. This effect of IL-6 was concentration dependent, in the range from 10-200 ng/ml of IL-6. The effect of IL-6 was also time dependent, the maximal level being reached at 21 h from the beginning of the treatment. This stimulatory effect of IL-6 on albumin clearance was completely abolished by the addition of anti-IL-6 antibody. Light microscopic observation of a cross-section of a monolayer showed that the IL-6-induced increase in the permeability was correlated with changes in cell shape and rearrangement of intracellular actin fibers. IL-6 did not show any cytotoxicity toward or growth inhibition of endothelial cells, even at more than 200 ng/ml. The enhancing effect of IL-6 on the increase in the permeability was reversible; when IL-6 was removed by a medium change and the cells were incubated for a further 24 h without IL-6, the permeability was restored to the control level. These results suggest that IL-6 can induce an increase in endothelial permeability in vitro by rearranging actin filaments and by changing the shape of endothelial cells.

Inhibitory effects of interleukin 6 on prostaglandin I2 production in cultured bovine vascular endothelial cells.

The effect of interleukin 6 (IL-6) upon arachidonic acid (AA) metabolism was studied in cultured bovine vascular endothelial cells (BVECs). Although, in those BVECs pretreated with IL-6 for 48 h, there was no effect upon cell proliferation, it was discovered that IL-6 suppressed the conversion of AA to prostaglandin I2 (PGI2) in cellular homogenates. First signs of the suppression occurred 3 h after incubation, and thereafter the suppression increased with time until it leveled off at 12 h. This effect of IL-6 was concentration-dependent, ranging from 20 ng/ml to a maximum of 100 ng/ml with IL-6 at 47% of control. IL-6 had no effect upon AA conversion when exogenously added to the BVEC homogenates. To investigate the mechanism of the suppression induced by IL-6, we studied its effect upon PGI2 synthesizing enzymes. We found that IL-6 had no effect upon the release of AA from phospholipids, but inhibited cyclooxygenase, the key enzyme for PGI2 production from AA. To amplify PGI2 production, AA was added exogenously to both control and IL-6-treated BVECs, and in this case also, the conversion activity in IL-6-treated BVEC's was suppressed. Through immunoblot analysis with an antibody to cyclooxygenase, it was determined that the level of cyclooxygenase protein was lowered in IL-6-treated cells. This is the first report to confirm down expression of cyclooxygenase in addition to the first observation as to the effect of IL-6 on endothelial cells.

[The relationship between ploidy and morphological classification of human megakaryocytes].

The relation between polyploidy and morphological classification of human megakaryocytes was studied in bone marrow aspirates from five normal individuals. On a Wright-Giemsa stained smear, megakaryocytes were morphologically classified into four groups according to a modification of Feinendegen's classification which is considered to reflect megakaryocyte maturation. The DNA of the morphologically classified cell is measured by microcytofluorometry using DAPI (4',6-diamidino-2-phenylindole) staining after removing the Wright-Giemsa stain. Most of the normal megakaryocytes were classified into type III (mature megakaryocytes) and the maximum peak in population of the megakaryocyte ploidy was observed at 16N. In each individual, the ploidy showed a similar pattern regardless of the classification. These findings suggest that the development of ploidy depends on a factor different from the one that determined the megakaryocyte maturation of cytoplasm and the ploidy is determined at the level of a megakaryocyte precursor or the most juvenile megakaryocyte.

Histochemical study on the maturation of human megakaryocytes using microfluorometry.

A microcytofluorometrical DNA measurement was basically studied and was applied to single megakaryocytes previously identified on a Wright-Giemsa stained smear. The smear was first photographed and the location of each megakaryocyte was recorded on a cell map. The smear was then bleached with 50% acid ethanol and absolute methanol, and re-stained with 4',6-diamidino-2-phenylindole (DAPI) reagent (pH 7.4) at 4 degrees C. Nuclear blue fluorescence was observed and the intensity of this fluorescence was proportional to the amount of DNA with the coefficient of variation (CV) of 3.6% when stained for 30 min. After 30 min DAPI staining, the DNA measurement was microcytofluorometrically performed in single megakaryocytes which had been morphologically classified into 4 groups on the basis of cytoplasmic maturation, Bessis' classification, assessed on Wright-Giemsa-stained bone-marrow smears from normal human beings. The histograms of the cells did not show any difference in DNA ploidy distribution among the classes: that is, the DNA histograms disclosed ploidy distribution from 4 N to 64 N with the largest population of 16 N. These findings suggest that nuclear DNA synthesis is completed before platelet production starts. This method is useful for comparing the morphological features and DNA content of single megakaryocytes.

Shift in the megakaryocyte ploidy in MDS patients: microcytofluorometry with DAPI staining after destaining of Wright-Giemsa stain.

We applied DAPI (4',6-diamidino-2-phenylindole) staining to the determination of nuclear DNA content in single megakaryocytes in 12 normal subjects and 12 patients with myelodysplastic syndrome (MDS). After the megakaryocytes had been identified on Wright-Giemsa stained smear and classified according to modified Feinendegen's classification, they were photographed. Then Wright-Giemsa stain was removed by immersion in 50% ethanol at 37 degrees C for 1 h and 100% methanol at 37 degrees C for 1 h. The specimens were then stained with DAPI solution (DAPI 0.01 mg/ml, pH 7.4 Tris-EDTA-2Na buffer solution and 0.01 M 2-mercaptoethylamine hydrochloride mixed at the ratio of 0.5:98.5:1.0) for more than 30 min. The amount of nuclear DNA in the previously identified megakaryocytes was measured by microcytofluorometry. The maximum population of megakaryocytes ploidy was in 16N in normal subjects, 8N in 10/12 MDS patients, and 4N in the remaining two patients. These findings suggest impairment of the development of the megakaryocytes nucleus in the MDS patients.

[Qualitative and quantitative evaluation of CFU-E colonies in myelodysplastic syndromes].

Hemoglobin (Hb) synthesis in colony-forming-unit erythroid (CFU-E) colonies from the bone marrow of 10 normal subjects and 6 patients with myelodysplastic syndromes (MDS) was studied using microcytofluorometry. Hb content was determined utilizing a photochemical reaction in which the intracellular Hb is converted into fluorescent porphyrin. Briefly, the CFU-E in plasma clots was assayed according to Tepperman's method. Bone-marrow mononuclear cells were then dispensed onto petri dishes containing the plasma clots. The cells in plasma clots were cultured for seven days and then air-dried. The samples were then fixed with pure methanol. Thereafter, the dishes were exposed to ultraviolet light (lambda = 405 nm) in the presence of an SH-donor (0.2 M mercaptoethylamine hydrochloride). The intensity of porphyrin fluorescence was measured using a microcytofluorometer. When the photochemical reaction was carried out for 50 min, the intensity of fluorescence was found to be proportional to the mean corpuscular Hb (MCH) level, suggesting that the intracellular Hb level can be determined as a function of the intensity of this fluorescence. The number of CFU-E colonies in MDS patients was smaller than that in normal subjects. In addition, intracolony Hb level was markedly lower in the MDS cases than in normal subjects. Our findings suggest that the maturation process of CFU-E is impaired in MDS patients.

[Acute unclassified leukemia with bone marrow necrosis].

Massive bone marrow necrosis was seen in a 42-year-old male with acute leukemia. In December, 1988, on admission, laboratory data revealed pancytopenia and a high level of serum LDH and ALKP. Bone marrow aspiration resulted in dry-tap and showed bone marrow necrosis in the bone marrow biopsy specimen. A bone marrow scintigraphy with 111In faintly visualized the bone marrow but visualized area was expanded in the extremities compared with normal subjects. The second bone marrow biopsy showed proliferation of blasts. In the middle of March, blasts began to appear in peripheral blood. The blasts were cytochemically negative for POX, Es, PAS, AcP, TdT and had surface markers CD3-, CD19-, CD33-, CD13-, LCA-, HLA-DR-. Even by investigation on rearrangement of the immunoglobulin heavy chain region, an origin of the blasts could not be determined. In April, the number of blasts in peripheral blood increased and hepatosplenomegaly developed rapidly. Therefore, he was put on the chemotherapy with vincristine and prednisolone, but he died of cerebral hemorrhage. The autopsy revealed widespread bone marrow necrosis. It has rarely been reported that massive bone marrow necrosis is found prior to the occurrence of acute unclassified leukemia.