The immature platelet fraction is a useful marker for predicting the timing of platelet recovery in patients with cancer after chemotherapy and hematopoietic stem cell transplantation.

Prediction of the timing of platelet recovery after chemotherapy and hematopoietic stem cell transplantation (HSCT) allows for optimal platelet transfusion. We assessed the clinical utility of the percentage value of the immature platelet fraction (IPF%) monitored using an XE-2100 automated hematology analyzer to predict the timing of platelet recovery after chemotherapy and HSCT. The IPF% was serially monitored in 31 patients with cancer who received 66 courses of chemotherapy and HSCT. In patients with cancer undergoing chemotherapy and HSCT, a transient increase in IPF% was observed 1-11 days prior to platelet recovery (>30 × 109 /l). In patients undergoing chemotherapy with a peak IPF% >10%, platelet recovery occurred significantly earlier than in those with IPF% peak values ≤10% (median periods were 2 and 5 days; P < 0.05). Platelet recovery appears to occur earlier in patients undergoing HSCT with a peak IPF% >10% than in those with IPF% peak values ≤10% (median periods were 2 and 6 days). Thus, the IPF% peak value is a useful parameter for predicting the timing of platelet recovery after chemotherapy and HSCT and has the potential to facilitate optimal platelet transfusion.

Immature platelet fraction measurement in patients with chronic liver disease: a convenient marker for evaluating cirrhotic change.

Platelet number is often used as an indicator of the severity of liver disease. Although inadequate thrombopoietin production and decreased platelet production have been proposed as major causes of cirrhotic thrombocytopenia, the underlying mechanism has not yet been fully clarified. We examined whether the measurement of the immature platelet fraction (IPF) in thrombocytopenic patients with liver dysfunction is useful as a rapid and noninvasive method for the differential diagnosis of chronic liver diseases. We examined 20 liver cirrhosis patients, 56 patients with chronic hepatitis, 9 patients with fatty liver, and 86 patients without liver disease. The percentage value of IPF (IPF%) was measured using an XE-2100 multiparameter automatic hematology analyzer. Using a receiver operating characteristic curve, we found diagnostic significance of the absolute platelet count and the absolute number of the IPF between cirrhotic patients and noncirrhotic patients, and developed a powerful multivariate discriminant analysis (MDA) function based on the platelet count and the IPF%. The diagnostic accuracy obtained by the MDA function was superior to that obtained by the absolute number of platelets and the IPF. We therefore propose our IPF% measurement for the diagnosis of liver cirrhosis.

Growth hormone prevents Fas-induced apoptosis in lymphocytes through modulation of Bcl-2 and caspase-3.

OBJECTIVE: Growth hormone (GH) has been reported to have a potent effect on the immune system. However, the detailed mechanism of the effect of GH on the immune system has not yet been clarified. This study was designed to investigate the nature of this mechanism. METHODS: In the present study, we investigated the effects of GH on the susceptibility of both human CEM/C7 lymphocytes and human IM-9 lymphocytes to Fas-induced apoptosis. RESULTS: Both cell lines expressed GH receptor mRNA. GH rescued Fas-induced suppression of [(3)H]-thymidine incorporation into each cell line. GH prevented Fas-induced apoptosis in each cell line without changing Fas antigen expression. We next investigated the mechanisms of the prevention of Fas-induced apoptosis, by focusing on intracellular molecules related to the apoptotic signal. Bcl-2 expression was increased by GH treatment in both CEM/C7 and IM-9 lymphocytes. GH also downregulated caspase-3 expression and inhibited activation of caspase-3 in both cell lines. CONCLUSION: These findings suggest that GH regulates the human immune system through inhibition of Fas-induced apoptosis in activated T and B lymphocytes.

Secondary myeloid/natural killer cell precursor acute leukemia following essential thrombocythemia.

The de novo leukemic transformation of essential thrombocythemia is a rare event, and usually associated with previous treatments. We describe a patient who received treatments with nitrosourea for long-standing essential thrombocythemia and subsequently developed extramedullary tumors, tentatively diagnosed as lymphoblastic lymphoma. Combination chemotherapy was initially successful, but relapsed with marked bone marrow involvement. Surface marker analysis revealed that the tumor cells had CD5, CD7, CD33, CD34, and CD56 antigens but lacked other T-cell, and B-cell markers. Immunogenotypical studies revealed germline configurations for both T-cell receptors and immunoglobulin genes. These clinical and phenotypical features are consistent with a myeloid/natural killer cell precursor leukemia, a recently proposed distinct clinical entity. To our knowledge, this is the first report of secondary leukemia of myeloid/ natural killer cell precursor origin, and suggest that myeloid/natural killer cell precursor might be a potent target of therapy-related leukemia.

Specific skin manifestations in CD56 positive acute myeloid leukemia.

We found 16 CD56+ cases (29.6%) among 54 acute myeloid leukemia (AML) patients; they showed significantly frequent cutaneous involvement compared to CD56- cases (43.8% vs. 15.8%, p<0.05). Four of the CD56+ AML cases with specific skin manifestations were reviewed histologically. In all cases, cutaneous leukemic cells were seen in the dermis and subcutaneous tissue with accentuation around the adnexa/nerve, but sparing the epidermis. In addition, angiocentric/ angiodestructive and prominent cohesive tumor cell growth were seen in two cases, respectively. These findings suggest that the expression of CD56 may often be associated with the cutaneous involvement in AML, and that the above histological findings should remind us of the possibility of specific skin manifestations in CD56+ AML.

A novel mature B-cell line (DOBIL-6) producing both parathyroid hormone-related protein and interleukin-6 from a myeloma patient presenting with hypercalcaemia.

A novel human EBV-negative B-cell line, designated DOBIL-6, was established from a patient with non-secretary myeloma. The DOBIL-6 cell has cytoplasmic gamma protein and expresses CD19, 20, 38, 45RO, VLA-4 and PCA-1 antigens, but lacks CD10, 45RA and VLA5 antigens. Chromosome analysis showed that DOBIL-6 cells had many complex structural abnormalities, including t(11;4) (q13;q32), which were consistent with that of the fresh tumour cells. Interestingly, abundant interleukin-6 (IL-6) and parathyroid hormone-related protein (PTHrP) accumulated in the culture supernatant of DOBIL-6 cells. Hypercalcaemia and splenomegaly associated with plasma cell proliferations which resulted in the expansion of the light zones in the follicles were observed in DOBIL-6 transplanted nude mice. RT-PCR analysis detected mRNA for PTHrP, and IL-6 as well as its receptor (GP80) in DOBIL-6 cells. Treatment of the DOBIL-6 cells with neutralizing anti-IL-6 antibody inhibited their growth in a dose-dependent manner, whereas the addition of exogenous IL-6 stimulated it in serum-depleted conditions. These findings suggest that both IL-6 and PTHrP are produced in DOBIL-6 cells, and that IL-6 promotes its growth by an autocrine mechanism. Since IL-6 is known to stimulate not only the growth of B-cell neoplasms but also osteoclastic bone resorption by cooperating with PTHrP, this simultaneous production of IL-6 and PTHrP might be synergistically linked and play a role in the development of hypercalcaemia of the patient. The DOBIL-6 cell is a useful tool to clarify the mechanism of hypercalcaemia associated with mature B-cell neoplasms.

A new cytokine-dependent monoblastic cell line with t(9;11)(p22;q23) differentiates to macrophages with macrophage colony-stimulating factor (M-CSF) and to osteoclast-like cells with M-CSF and interleukin-4.

Monocytes/macrophages exert a series of important functions in vivo. To facilitate detailed investigation of their functional capacity and the mechanism leading to their differentiation, several cell lines have been established from primary material. We present here a new human monoblastic cell line, designated UG3. UG3 cells are characterized by the following features. (1) UG3 cells harbor the t(9;11)(p22;q23) translocation that results in fusion of the MLL and the AF9 genes and produce the corresponding AF9-MLL and MLL-AF9 fusion transcripts. (2) UG3 cells rely on the presence of exogenous growth factors for viability and proliferation, such as interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), or macrophage colony-stimulating factor (M-CSF). (3) When cultured in the presence of G-CSF, UG3 cells differentiate along the granulocytic lineage, as evidenced by segmentation of nuclei and positive staining for neutrophilic alkaline phosphatase and peroxidase. (4) When cultured in the presence of GM-CSF or M-CSF, UG3 cells differentiate into mature macrophages while preserving surface expression of CD14 and CD68 and also start to release cytokines into cell-culture supernatants. Under these culture conditions, UG3 cells also take up acetylated LDL. (5) When cultured in the presence of M-CSF and IL-4, UG3 cells differentiate into osteoclast-like multinucleated giant cells capable of bone resorption and display tartrate-resistant acid phosphatase (TRAP) activity. UG3 cells thus provide features to qualify them as a useful model to further investigate the mechanism underlying these processes and also to further elucidate the functional role of mature monocytes/macrophages or osteoclasts.

Alterations of p53 and Rb genes in a novel human GM-CSF-dependent myeloid cell line (OHN-GM) established from therapy-related leukaemia.

A novel GM-CSF-dependent myeloid cell line, OHN-GM, was established from a patient who developed acute myelogenous leukaemia (AML) as a consequence of myelodysplastic syndrome (MDS). As the patient had previously received cytotoxic chemotherapy for Hodgkin's disease, the MDS and AML were probably related to such therapy. Sequential karyotypic analysis established a del(5q) as the initial cytogenetic abnormality. Additional alterations, including t(10;13)(q24;q14), had developed subsequently during disease progression. Southern blot analysis of OHN-GM cells suggested deletion of one allele of the IRF-1 gene, although no aberrant transcripts were detected. Fluorescence in situ hybridization analysis revealed the deletion of the Rb gene due to the t(10;13)(q24;q14) translocation, and Western blot analysis demonstrated the absence of Rb protein in OHN-GM cells. Finally, the OHN-GM cells exhibited two missense point mutations in highly conserved regions of the p53 gene. These observations suggest that a multistep process, involving alterations of Rb and p53 genes, may have contributed to the patient's disease development and progression. To our knowledge, OHN-GM is the first cell line derived from a therapy-related AML. These cells may aid the investigation of leukaemogenesis as well as the biology of secondary leukaemia.

GM-CSF- and IL-3-dependent CD34 expressing myeloid cell line (SAS-1) established from CD7 and CD34 expressing acute myeloblastic leukemic cells.

A novel factor-dependent human myeloid leukemia cell line (SAS-1) was established from a 69-year-old Japanese male suffering from CD7 and CD34 expressing acute myeloblastic leukemia (AML M2 in FAB classification). Morphological and cytochemical staining showed that SAS-1 cells were round with basophilic cytoplasm which is positive for peroxidase. Analysis of surface markers revealed that SAS-1 cells were myeloblasts derived from an immature progenitor origin, which express CD34. The consensus karyotype of the cell line was 41 XY 5q-, -7, 11p-, 12p+, -13, -14, -16, -17, -19, -22, with two markers. The proliferation of SAS-1 cells was dependent on the presence of either granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3), and GM-CSF- and IL-3-induced proliferation was dose dependent. Neither, stem cell factor (SCF) nor granulocyte colony-stimulating factor (G-CSF) alone supported the growth of SAS-1 cells, but supported their viability for more than 4 days and arrested them in the G0/G1 phase. SCF also enhanced GM-CSF- or IL-3-induced growth, but other cytokines did not have this synergistic effect. Clonogenic assays revealed that SAS-1 cells formed 36.0 +/- 5.7 or 41.5 +/- 0.7 colonies/1000 cells in the presence of GM-CSF or IL-3, respectively. SCF also increased the number of colonies formed by GM-CSF or IL-3 treatment, while SAS-1 cells did not form colonies in the presence of SCF alone. SAS-1 cells may prove to be a useful tool for studying the regulation of the cell cycle, myeloid proliferation, and differentiation.

Thrombopoietin modulates platelet activation in vitro through protein-tyrosine phosphorylation.

To determine the roles of thrombopoietin (TPO) in platelet function in vitro, we examined the effects of TPO on platelet aggregation. Although several proteins in platelets were tyrosine-phosphorylated by TPO treatment, TPO alone was unable to induce platelet aggregation. However, the secondary wave of platelet aggregation induced by adenosine diphosphate (ADP) was enhanced by TPO in a dose-dependent manner. TPO in conjunction with ADP augmented tyrosine phosphorylation of platelet proteins, including tyrosine-phosphorylated proteins induced by TPO alone. Genistein inhibited protein-tyrosine phosphorylation in platelets induced by TPO with ADP and suppressed TPO-enhanced platelet aggregation. Moreover, tyrosine phosphorylation of MAP-kinases induced by TPO alone and TPO with ADP was consistent with TPO-enhanced platelet aggregation. These findings in the present study suggest that signal transduction involved in TPO-enhanced platelet aggregation is mediated in part by tyrosine-phosphorylated proteins, including MAP-kinases, in platelets through TPO-stimulated c-Mpl, TPO receptor.

Wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase, inhibits erythropoietin-induced erythroid differentiation of K562 cells.

To elucidate the role of phosphatidylinositol-3-kinase (Pl3K) in erythropoietin receptor (EPOR)-mediated signaling, we examined the effects of wortmannin (WT), a specific inhibitor of Pl3K on the proliferation of erythropoietin (EPO)-induced erythroid differentiation in K562 human erythroleukemia cells. Percentage of benzidine-positive cells synthesizing hemoglobin and level of glycophorin A expression in the cells were increased after EPO treatment. EPO-enhanced Pl3K activity was suppressed by WT treatment in a dose-dependent manner and constant inhibition of Pl3K by WT interfered with both hemoglobin synthesis and glycophorin A expression promoted by EPO. Wortmannin, however, did not inhibit hemin-induced erythroid differentiation. These findings in the present study suggest that Pl3K plays a crucial role in the transducing the erythroid differentiation signal through EPOR activated by EPO-binding ib K562 cells and that the signaling pathways involved in EPO-induced erythroid differentiation differ from those involved in hemin-induced differentiation.

Alkaline phosphatase activity in the human promyelocytic leukemia cell line, HL-60, induced by retinoic acid and recombinant human granulocyte colony-stimulating factor.

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) not only enhanced the growth of HL-60 cells, but also significantly increased NBT-reducing ability and alkaline phosphatase (ALP) activity of the cells, which were enhanced by the treatment with retinoic acid (RA). Protein kinase C inhibitors (H-7 and staurosporine) significantly suppressed this induction of ALP. The pretreatment with RA followed by rhG-CSF treatment showed almost the same degree of ALP activity as that induced by the simultaneous treatment with RA and rhG-CSF. This study suggests that RA and rhG-CSF are the potent inducers of ALP activity of HL-60 cells and protein kinase C is supposed to have a role in this induction of ALP.

[Hematological study in 27 patients with myelodysplastic syndrome (MDS)].

The clinical, histologic, and cytogenetic features of myelodysplastic syndrome (MDS) were analyzed in 27 patients consisting of 21 males and 6 females (M: F ratio = 3.5:1) whose median age was 60.9 years. Analysis of the peripheral blood of the patients revealed pancytopenia (8 patients), bicytopenia (12), and cytopenia in one cell lineage (5). The patients had high serum iron value and low UIBC value. More than 20% of all erythroid cells in the bone marrow consisted of ringed sideroblasts in not only RARS patients but also RAEB, CMMoL, and RAEB-T patients. Morphological changes of the bone marrow cells were observed in patients with all types of MDS. Eighty percent and 60% of RAEB and RAEB-T patients, respectively, showed trilineage myelodysplasia. Chromosomal abnormalities were observed in 52% of MDS patients. The rate of leukemic transformation was high in RAEB, CMMoL, and RAEB-T patients.

[The effects of retinoic acid and recombinant human granulocyte colony-stimulating factor on alkaline phosphatase activity of HL-60 cells].

We examined alkaline phosphatase (ALP) activity in the HL-60 cell induced by retinoic acid (RA) and recombinant human granulocyte colony-stimulating factor (rhG-CSF). rhG-CSF induced a small but significant increase of NBT-reducing ability and ALP activity of the HL-60 cells. Among various inducers of cell differentiation, 1,25(OH)2D3 and dimethylsulfoxide (DMSO) caused the increase of the NBT-reducing ability and the suppression of ALP activity induced by rhG-CSF, while RA enhanced both of them. Protein kinase C inhibitors (H-7 and staurosporine) but not a protein kinase A inhibitor (HA1004) significantly suppressed the ALP activity induced by the simultaneous treatment with RA and rhG-CSF.