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A unique subclone of a bone marrow-derived stromal cell line, BMS2.4, produces soluble factors that inhibit proliferation of several types of hematopoietic cell lines. An understanding of these molecules may be informative about negative regulatory circuits that can potentially limit blood cell formation. We used expression cloning to identify interleukin-6 (IL-6) as one factor that suppressed growth of a pre-B–cell variant line, 1A9-M. Moreover, IL-6 induced macrophage-differentiation and apoptosis of 1A9-M cells. During this process, IL-6 downregulated expression of BCL2 in 1A9-M cells and stimulated BCL-XL expression, but had no effect on p53, Bax, or Bak gene expression. Mechanisms for transduction of IL-6–induced signals were then evaluated in IL-6–stimulated 1A9-M cells. Whereas the signal transducer and activator of transcription 3 (Stat3) was phosphorylated and activated, there was no effect on either Stat1 or Stat5. The importance of BCL2 and Stat3 on IL-6–induced macrophage-differentiation and apoptosis was studied with 1A9-M cells expressing human BCL2 or a dominant-negative form of Stat3, respectively. IL-6–induced apoptosis, but not macrophage-differentiation, was blocked by continuously expressed BCL2. A dominant-negative form of Stat3 inhibited both macrophage-differentiation and apoptosis induced by IL-6. However, diminished Stat3 activity did not prevent IL-6–induced downregulation of the BCL2 gene. Therefore, activation of Stat3 is essential for IL-6–induced macrophage-differentiation and programmed cell death in this model. Whereas overexpression of BCL2 abrogates the apoptotic response, Stat3-independent signals appear to downregulate expression of the BCL2 gene.

The c-kit receptor tyrosine kinase (KIT) is constitutively activated by naturally occurring mutations in either the juxtamembrane domain or the kinase domain. Although the juxtamembrane domain mutations led to ligand-independent KIT dimerization, the kinase domain mutations (Asp814 → Val or Tyr) did not. In an effort to determine if the kinase domain mutant could transfer oncogenic signaling without receptor dimerization, we have constructed the truncated types of c-kitWild and c-kitTyr814 cDNAs (c-kitDel-Wild and c-kitDel-Tyr814 cDNAs, respectively), in which ligand-binding and ligand-induced dimerization domains were deleted. When c-kitDel-Wild and c-kitDel-Tyr814 genes were introduced into a murine interleukin-3 (IL-3)–dependent cell line Ba/F3, KITDel-Tyr814 was constitutively phosphorylated on tyrosine and activated, whereas KITDel-Wild was not. In addition, Ba/F3 cells expressing KITDel-Tyr814(Ba/F3Del-Tyr814) grew in suspension culture without the addition of exogenous growth factor, whereas Ba/F3 cells expressing KITDel-Wild (Ba/F3Del-Wild) required IL-3 for growth. The factor-independent growth of Ba/F3Del-Tyr814 cells was virtually abrogated by coexpression of KITW42 that is a dominant-negative form of KIT, but not by that of KITWild, suggesting that KITDel-Tyr814 may not function as a monomer but may require receptor dimerization for inducing factor-independent growth. Furthermore, KITDel-Tyr814 was found to be coimmunoprecipitated with KITWild or KITW42 by an ACK2 monoclonal antibody directed against the extracellular domain of KIT. Moreover, KITW42 was constitutively associated with a chimeric FMS/KITTyr814 receptor containing the ligand-binding and receptor dimerization domain of c-fmsreceptor (FMS) fused to the transmembrane and cytoplasmic domain of KITTyr814, but not with a chimeric FMS/KITWildreceptor even after stimulation with FMS-ligand. These results suggest that constitutively activating mutation of c-kit at the Asp814 codon may cause a conformation change that leads to receptor self-association not in the extracellular domain and that the receptor self-association of the Asp814 mutant may be important for activation of downstream effectors that are required for factor-independent growth and tumorigenicity.

Recombinant human granulocyte-macrophage colony-stimulating factor (rHuGM-CSF) produces dose-related therapeutic and toxic effects; however, relationships between its pharmacokinetics and pharmacodynamics have not been extensively evaluated. The following studies were undertaken to investigate patterns in the disposition of rHuGM-CSF administered after high-dose chemotherapy (cyclophosphamide, cisplatin, carmustine) and autologous bone marrow support. Continuous 14 or 21 day intravenous infusions or daily 4-hour infusions were studied at doses of 1.2 to 19.2 micrograms/kg/d. GM-CSF was measured by an enzyme-linked immunosorbent assay from serum and urine samples collected throughout drug administration. Pharmacokinetic parameters were determined by compartmental (4-hour infusions) or noncompartmental methods (continuous infusions). GM-CSF was rapidly eliminated from the serum. Average systemic exposure increased with dose, although wide interpatient variability was evident. Approximately one half of the patients receiving continuous infusions demonstrated increasing GM-CSF clearance that corresponded to the appearance of white blood cells in the periphery. Conversely, clearance decreased in those experiencing renal dysfunction during the infusion. The percentage of a GM-CSF dose found in 24-hour urine collections was substantially reduced in the latter group. A subset of patients who developed renal dysfunction also experienced significant hypotension. Rapidly increasing serum GM-CSF concentrations corresponded to the hypotensive episodes. GM-CSF serum concentration monitoring may be useful for evaluation of therapeutic and toxic effects in patients receiving high-dose chemotherapy with autologous bone marrow support.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), Interleukin- 3 (IL-3), and Steel Factor (SF) induce proliferation of hematopoietic cells through binding to specific, high-affinity, cell surface receptors. However, little is known about postreceptor signal transduction pathways. In previous studies, we noted that each of these three factors could independently support proliferation of the human MO7 cell line, and also that each factor induced a rapid increase in protein-tyrosyl phosphorylation. Although the proteins phosphorylated on tyrosine by GM-CSF and IL-3 are similar or identical in MO7 cells, many of the proteins that are phosphorylated on tyrosine after SF are different. However, two proteins, p42 and p44, were prominently phosphorylated in response to all three of the factors. In MO7 cells, the tyrosyl phosphorylation of p42 and p44 was transient, peaking at 5 to 15 minutes. In contrast to many of the other proteins which are tyrosyl phosphorylated in response to these factors, phosphorylation of p42 and p44 was temperature-dependent, occurring at 37 degrees C, but not at 4 degrees C. We identified the p42 protein as p42 Mitogen- Activated Protein Kinase (p42mapk, ERK-2) and the p44 as a p42mapk- related protein using monospecific antisera to MAP kinase. GM-CSF, IL- 3, and SF were each found to induce MAP kinase activity when assayed in vitro using myelin basic protein (MBP) as a substrate. Remarkably, we found that GM-CSF-induced tyrosyl phosphorylation of p42 and p44 even in nonproliferative cells (neutrophils) that respond to this CSF, and that p42 and p44 were two of the most prominently tyrosyl phosphorylated proteins following GM-CSF stimulation of these cells. These results implicate p42mapk and p44 as important signal transducing molecules in myeloid cells, and it is likely that these kinases play a role as part of a sequential “kinase cascade” linking growth factor receptors to mitogenesis and other cellular responses.

We investigated the expression, degree of phosphorylation, and activation of the proto-oncogene c-kit product before and after stimulation with the c-kit ligand in a human factor-dependent myeloid leukemia cell line, MO7E. The culture supernatant of the BALB/3T3 fibroblast cell line, which contains the ligand for the murine c-kit product, was found to stimulate proliferation of the MO7E cell line in a dose-dependent manner. The proliferation was significantly inhibited by a tyrosine kinase inhibitor, genistein. An immunoblot technique with a monoclonal antibody specific for phosphotyrosine, showed that there was rapid, dose-dependent tyrosine-phosphorylation of the c-kit product in response to murine c-kit ligand. Furthermore, the murine c-kit ligand increased autokinase activity of the c-kit product in vitro. Similar results were obtained with human stem cell factor (SCF), a recombinant human ligand for the c-kit product. These results suggest that the phosphorylation and activation of the c-kit product are involved in proliferative signals of some human leukemia cells, as well as of normal hematopoietic cells.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein that is required for the survival, growth, and differentiation of hematopoietic progenitor cells. Although the primary structure of GM-CSF is known from cDNA cloning, the relationship between structure and function of GM-CSF is not fully understood. Fifteen different monoclonal antibodies (MoAbs) to human GM-CSF were generated to map immunologically distinct areas of the molecule. Each of the MoAbs was biotinylated and shown by enzyme-linked immunosorbent assay to bind to recombinant GM-CSF that had been affixed to a solid phase. Each of the 15 unconjugated MoAbs was then used to compete with each biotinylated MoAb for binding to GM-CSF. These cross-blocking studies identified eight distinct epitopes of native GM-CSF. Seven of these epitopes were also present in denatured GM-CSF by Western blotting, and four of the epitopes were at least partially conserved on GM-CSF that was reduced in beta-mercaptoethanol. MoAbs to four of eight epitopes neutralized both recombinant (glycosylated and nonglycosylated) and natural human GM-CSF in a GM colony-forming unit (CFU-GM) assay and blocked GM-CSF-induced activation of neutrophils. For most of the antibodies there was a good correlation between neutralizing activity and the capacity to block binding of 125I-GM-CSF to neutrophils or blasts. Non-neutralizing antibodies to one epitope partially blocked binding of 125I-GM-CSF to neutrophils. None of the MoAbs neutralized interleukin-3, G-CSF, or M-CSF. The locations of seven of the epitopes could be partially mapped with regard to the amino acid structure by determining reactivity to GM-CSF synthetic peptides or to human-mouse chimeric GM-CSFs. The neutralizing antibodies were found to map to amino acids 40–77, 78–94, or 110–127. Thus, these MoAbs are useful to identify functional domains of GM-CSF and in identifying regions that are likely to be involved in receptor interaction.

The product of the c-raf-1 proto-oncogene, Raf-1, is a 74,000-dalton cytoplasmic serine/threonine protein kinase that has been implicated as an intermediate in signal transduction mechanisms. In the human factor- dependent myeloid cell line MO7, both granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-3 (IL-3) were found to induce rapid, dose-dependent phosphorylation of Raf-1, which resulted in altered Raf-1 mobility in sodium dodecyl sulfate-polyacrylamide gels. The increase in phosphorylation was due primarily to an increase in phosphoserine, with only a minor component (less than 2%) of phosphotyrosine. PMA (12-phorbol 13-myristic acid) also induced Raf-1 phosphorylation in MO7 cells, but the resulting alteration in electrophoretic mobility was different than that observed after GM-CSF or IL-3. GM-CSF and IL-3 rapidly and transiently increased Raf-1 kinase activity using Histone H1 as a substrate in an immune complex kinase assay in vitro. These results suggest that phosphorylation of Raf-1 could play a role in some aspect of GM-CSF and IL-3 signal transduction.

The main objective of this study was to clarify the role of aPLs in the pathogenesis of arteriosclerosis obliterans (ASO), ischaemic heart disease (IHD) and cerebral vascular disorder (CVD) in patients with SLE.We evaluated 155 patients with SLE by using objective tests for diagnosing ASO, IHD and CVD and laboratory tests including ELISA for aCL/beta2-glycoprotein I antibodies (aCL/beta2-GPI) and anti-phosphatidylserine/prothrombin antibodies (anti-PS/PT).Twenty-five (16.1%) of the 155 SLE patients were diagnosed with ASO. Both aCL/beta2-GPI and anti-PS/PT levels were significantly higher in SLE patients with ASO (mean +/- S.E., 104.3 +/- 38.8 U/ml for aCL/beta2-GPI, P0.01; 72.6 +/- 48.9 U/ml for anti-PS/PT, P0.05) than in SLE patients without ASO (22.8 +/- 9.9 U/ml for aCL/beta2-GPI; 18.3 +/- 4.4 U/ml for anti-PS/PT). Multivariate logistic analysis including aCL/beta2-GPI, anti-PS/PT and traditional risk factors (hypercholesterolaemia, hypertension and diabetes mellitus) confirmed that the presence of aCL/beta2-GPI was the most significant risk factor for ASO in SLE patients [odds ratio (OR) 3.45; 95% CI 1.40, 8.56; P0.01]. Furthermore, the prevalence of ASO was associated strongly with IHD (OR 11.8; 95% CI 3.45, 40.1; P0.0001) but not CVD (OR 1.84; 95% CI 0.65, 5.21; P = 0.25).The presence of aCL/beta2-GPI contributes to the risk of development of ASO, which may represent an important mechanism for the pathogenesis of IHD in patients with SLE.

There is increasing evidence that cytokines such as granulocyte-macrophage (GM)-CSF can profoundly affect the adhesion, aggregation, and mobility of neutrophils both in vitro and in vivo. However, the mechanisms whereby these factors might alter the adhesive properties of neutrophils are incompletely understood. A new family of cellular adhesion molecules has recently been identified by cDNA cloning. The members of this family include human leukocyte adhesion molecule-1 (LAM-1), the human endothelial-leukocyte adhesion molecule, and the mouse leukocyte homing receptor for high endothelial venules, MEL-14. LAM-1 is the human homologue of murine MEL-14, and is believed to mediate binding of leukocytes to human high endothelial venules. LAM-1 can be identified by mAb TQ-1, Leu 8, or anti-LAM1.1. The expression and regulation of LAM-1 on granulocytes, monocytes, and their precursors was investigated using flow cytometry and the anti-LAM-1.1 mAb. Neutrophils, eosinophils, monocytes, marrow myeloid cells, granulocyte/macrophage colony-forming unit, and burst-forming unit for erythroid cells were LAM-1+ by flow microfluorimetry. The regulation of LAM-1 expression was tested by treating various cell populations with cytokines or other stimuli for 0-90 min. Exposure of neutrophils, monocytes, and marrow myeloid cells to GM-CSF induced rapid and complete loss of LAM-1 from the cell surface, but had no effect on LAM-1 expression by lymphocytes. The loss of LAM-1 was temporally correlated with up-regulation of CD11b (Mo1), an adhesion molecule involved in neutrophil aggregation. Several other factors known to activate neutrophils also caused down-regulation of LAM-1 and up-regulation of CD11b, including TNF, FMLP, and leukotriene B4. Interestingly, granulocyte-CSF and IFN-gamma had minimal effects on neutrophil LAM-1 expression. Similar results were observed on monocytes and myeloid precursor cells. Thus, exposure of neutrophils to GM-CSF results in a profound change in surface expression of adhesion molecules, with coordinated up-regulation of CD11b and down-regulation of LAM-1. These changes in adhesion proteins are likely to alter aggregation and mobility of both mature myeloid cells and their precursors in patients receiving certain types of cytokine therapy.

We describe the evaluation of the EMIT(®)2000 cyclosporine specific assay kit, an enzyme‐multiplied immunoassay for cyclosporine in whole blood, with a COBAS MIRA‐plus analyzer. The enzyme used for the assay was glucose‐6‐phosphate dehydrogenase (EC 1.1.1.49 G6PDH) from Leuconostoc mesenteroides; the monoclonal antibody is fairly specific for cyclosporine, and is not reactive with most metabolites. The assay principle is based on competitive immunoassay with G6PDH‐labeled cyclosporine and cyclosporine in sample to the anticyclosporine mouse monoclonal antibody binding site. The within‐assay coefficient of variation (CV) of this method was 2.7–4.2% (n = 10) at the levels of 56.2–339.7 μg/L. Day‐to‐day CVs ranged from 4.2–8.1% at the levels of 47.2–350.2 μg/L. The within‐day CVs ranged from 2.0–6.4% at the levels of 43.3–330.5 μg/L. The functional detection limit was 24.9 μg/L. Samples treated with pretreatment reagent were stable at least 5 hr. Calibration was stable at least 10 days. The analytical recovery was 81–109%. The correlation between values obtained with the EMIT(®)2000 cyclosporine specific assay kit (y) and fluorescence polarization immunoassay (FPIA) (TDxFLx) (x) was: y = 0.880x – 13.053 μg/L (r = 0.984, Sy/x = 15.968, n = 71) with a mean difference of 31.42 ± 19.89 μg/L ((TDxFLx – EMIT(®)2000) ± SD); for the FPIA (AxSYM) (x): y = 0.989 – 4.144 μg/L (r = 0.981, Sy/x = 17.478, n = 71) with a mean difference of 5.56 ± 17.38 μg/L ((AxSYM – EMIT(®)2000) ± SD); and for the radioimmunoassay (RIA, CYCLO‐Trac SP) (x): y = 0.893 – 6.764 μg/L (r = 0.993, Sy/x = 10.582, n = 71) with a mean difference of 22.18 ± 14.98 μg/L ((RIA – EMIT(®)2000) ± SD) using the Bland‐Altman technique. J. Clin. Lab. Anal. 15:319–323, 2001. © 2001 Wiley‐Liss, Inc.

Anti-prothrombin antibodies (anti-prothrombin) and anti-beta2-glycoprotein I antibodies (anti-beta2-GP I) are the most common and characterized anti-phospholipid antibodies (aPL) detected using specific enzyme-linked immunosorbent assay (ELISA) systems. Recently, lupus anti-coagulant (LA) activity detected by a phospholipid-dependent coagulation assay was reported to be associated with anti-prothrombin and/or anti-beta2-GP I. Here we show that the co-existence of IgG anti-prothrombin and LA activity might be an essential risk factor for venous thromboembolism (VTE) in patients with systemic lupus erythematosus (SLE). We examined not only the levels of antibodies to prothrombin and anti-beta2-GP I (both IgG and IgM isotypes) using an ELISA system, but also LA activity detected using both diluted Russell's viper venom time (dRVVT) and STACLOT LA test in 124 patients with SLE. The SLE patients were divided into four groups according to the results of ELISA and LA assay results for each aPL: group A, ELISA+ and LA+ group B, ELISA+ and LA-; group C, ELISA- and LA+ group D, ELISA- and LA-. Regarding IgG anti-prothrombin, the prevalence of VTE was significantly higher in group A (16/35 cases, 45.7%, P0.001, Fisher's exact probability test) than in the other groups (B, 2/30, 6.7%; C, 1/22, 4.5%; D, 1/37, 2.7%). With respect to IgM anti-prothrombin and IgG or IgM anti-beta2-GP I, the prevalence of VTE was higher in both groups A and C than in group D, but no statistical difference in prevalence was found between groups A and C. Multivariate logistic regression analysis of risk factors for VTE confirmed that the co-existence of IgG anti-prothrombin and LA activity was the only significant risk factor for VTE (odds ratio, 19.13; 95% confidence intervals, 4.74-77.18).

Anti-phospholipid (aPL) antibodies (Abs) frequently found in the plasma of patients with systemic lupus erythematosus (SLE) have been associated with thrombotic complications. Our aim was to clarify the roles in thrombosis of aPL Abs that react with complexes of phospholipids and plasma proteins such as beta(2)-glycoprotein I (beta(2)-GPI), prothrombin, protein C, protein S, and annexin V.We determined the prevalence of aPL Abs to various phospholipid-binding plasma proteins in SLE patients with arterial thrombosis (30 cases), venous thrombosis (19 cases), thrombocytopenia (14 cases), fetal loss (14 cases), and patients without complications (91 cases). The aPL Abs were measured by an ELISA system in which human plasma proteins (beta(2)-GPI, prothrombin, protein C, protein S, and annexin V) were immobilized on gamma-irradiated or plain polystyrene plates.All types of aPL Abs were frequently observed in the patients with SLE when gamma-irradiated polystyrene plates were used (51 of 168 cases positive for anti-beta(2)-GPI, 94 of 168 cases positive for anti-prothrombin, 36 of 168 cases positive for anti-protein C, 47 of 168 cases positive for anti-protein S, and 50 of 168 cases positive for anti-annexin V), whereas no Abs to these plasma proteins were detected when plain polystyrene plates were used. Multivariate analysis confirmed that both anti-beta(2)-GPI and anti-prothrombin Abs were significant risk factors for arterial thrombosis [odds ratios (ORs), 8.8 and 14.5, respectively; 95% confidence intervals (CIs), 3.2-25 and 1.8-116, respectively] but not for venous thrombosis. The presence of anti-protein S Abs was a significant risk factor for venous thrombosis (OR, 30.4; CI, 3.3-281) but not for arterial thrombosis. The only significant risk factor for fetal loss was the presence of anti-annexin V Abs (OR, 5.9; CI, 1.4-14.8).Patients with SLE frequently have some aPL Abs to beta(2)-GPI, prothrombin, protein C, protein S, and annexin V. Thrombotic complications in SLE may depend on the antigenic specificities of these Abs, alone or in combination.

PET with a double-head gamma camera (hybrid PET) is a new approach to tumor imaging with 18F-FDG. This study was conducted to clarify the feasibility of whole-body FDG hybrid PET in the staging of non-Hodgkin's lymphoma (NHL) in comparison with PET with a dedicated camera (dedicated PET) and to compare the results of both FDG studies with those of CT and 67Ga scanning as conventional imaging studies (CIS).Thirty patients with NHL were prospectively evaluated. The results of the imaging studies regarding detection of the sites involved and staging were compared with each other and with those of the reference standard based on the final overall clinical evaluation.Of the total of 206 sites, whole-body FDG hybrid PET and dedicated PET detected 159 sites (77.2%) and 179 sites (86.9%), respectively. Eighteen of the 20 sites missed by hybrid PET alone consisted of lesions1.5 cm. Both FDG studies provided concordant staging results in all but 2 patients. CIS, on the other hand, detected 164 (79.6%) of the 206 sites, 137 of which were also detected by hybrid PET. Hybrid PET detected an additional 22 sites not found by CIS, whereas CIS detected 27 additional sites. Hybrid PET and CIS provided concordant staging results in 19 patients. Hybrid PET correctly staged NHL in 5 additional patients, whereas CIS correctly staged NHL in only 1 additional patient.Whole-body FDG hybrid PET appeared to be an accurate method of staging NHL. Despite its poorer image quality compared with dedicated PET, hybrid PET provided NHL staging results comparable with those of dedicated PET. Hybrid PET also yielded results comparable with those of CIS. However, whole-body FDG hybrid PET is currently inadequate as a single modality for staging NHL and is complementary to CT.

The remaining portion of a laboratory specimen is usually used for education, research, and quality control of laboratory tests in hospitals, but informed consent has not been obtained because of the high volume of patients who undergo laboratory tests. However, patients must be informed in some manner. Therefore, we decided to inform patients that any remaining specimen would be used for various purposes by placing such a notice on walls in the central clinical laboratory and hospital lobby. We then obtained a signature on a dissent document, instead of a consent document, from any patient who dissented from such use. This indirect process for obtaining informed consent was approved by the ethics committee of Osaka University Medical School. The number of dissent documents sent in to the director was 54 of about 400,000 patients who underwent laboratory tests over the last 3 years, and there was no complaint against this "informed consent process".

Sinonasal lymphoma is one of the constituents of lethal midline granuloma, which is a clinical term for progressive, destructive lesions affecting the midline of the face. The majority of sinonasal lymphomas, especially those showing polymorphous patterns of proliferation and thus termed polymorphic reticulosis, recently were categorized as sinonasal natural killer/T-cell lymphomas. They are more prevalent in Asia than Europe or North America and are associated with EBV infection. Twenty-three cases with sinonasal natural killer/T-cell lymphomas were collected from two high-incidence regions: Beijing, China (14 cases) and Osaka, Japan (9 cases). c-kit mutations were analyzed on paraffin-embedded specimens by PCR-single-strand conformation polymorphism followed by direct sequencing; the c-kit proto-oncogene encodes a receptor of tyrosine kinase, which plays an important role in the regulation of normal and neoplastic hematopoiesis by the interaction with its specific ligand, termed stem cell factor. Twelve single nucleotide substitution mutations were seen in 23 cases. Ten of 14 Chinese cases (71.4%) had mutations at exon 11 or exon 17, whereas only two of nine Japanese cases (22.2%) had mutations, showing a significant difference in frequency between Chinese and Japanese cases. Furthermore, seven of eight mutations (92%) in exon 17 occurred at codon 825 and three of four mutations (75%) in exon 11 occurred at codon 561. Such a specificity has not been reported before, and these results, taken together, suggest that location-specific differences in etiological factors cause specific mutations in c-kit gene.

Cytokines exert pleiotropic effects on target cells in a manner dependent on the cell type or stage of differentiation. To determine how instinctive cell properties affect biological effects of cytokine, we introduced an erythroid/megakaryocyte lineage-specific transcription factor, GATA-1, into a murine myeloid cell line M1, which is known to undergo macrophage differentiation in response to interleukin 6 (IL-6). Overexpression of GATA-1 changed the phenotype of M1 cells from myeloid to megakaryocytic lineage. Furthermore, GATA-1 blocked both IL-6-induced macrophage differentiation and apoptosis of M1 cells. Although STAT3 is essential for IL-6-induced macrophage differentiation of M1 cells, GATA-1 had little or no effect on tyrosine phosphorylation, DNA binding, and transcriptional activities of STAT3 in Western blot analysis, electropholic mobility shift assay (EMSA), and luciferase assays. During IL-6-induced macrophage differentiation of M1 cells, IL-6 down-regulated cyclin D1 expression and induced p19(INK4D) expression, leading to reduction in cdk4 activities. In contrast, sustained expression of cyclin D1 and a significantly lesser amount of p19(INK4D) induction were observed in IL-6-treated M1 cells overexpressing GATA-1. Furthermore, although bcl-2 expression was severely reduced by IL-6 in M1 cells, it was sustained in GATA-1-introduced M1 cells during the culture with IL-6. Both IL-6-induced macrophage differentiation and apoptosis were significantly abrogated by coexpression of cyclin D1 and bcl-2, whereas overexpressions of cyclin D1 or bcl-2 inhibited only differentiation or apoptosis, respectively. These results suggested that GATA-1 may not only reprogram the lineage phenotype of M1 cells but also disrupt the biologic effects of IL-6 through the sustained expression of cyclin D1 and bcl-2. (Blood. 2000;95:1264-1273)

Antiphospholipid antibodies (aPL) are well known to be associated with arterial and venous thrombosis. In a series of 180 patients with systemic lupus erythematosus (SLE), the prevalence of arterial thrombosis was obviously higher in the patients who had both anticardiolipin antibodies (aCL) and lupus anticoagulant (LA) (17/35, 48.6%, p0.05) (Table 1) than in the other patients bearing aCL or LA alone or neither of them (2/145, 1.4%). Since a substantial fraction of the former group of patients with arterial thrombosis also had thrombocytopenia (12/17, 70.6%), there was a possibility that aCL and LA might have enhanced platelet activation and aggregation. To test this possibility, we studied the in vitro effects of aCL and LA on the enhancement of platelet activation by flow cytometric analysis using anti-CD62P and anti-CD41 monoclonal antibodies directed against platelet activation-dependent granule-external membrane (PADGEM) protein and platelet glycoprotein IIb (GPIIb), respectively. Platelet activation defined by the surface expression of CD62P was not induced by aCL+ x LA+ plasma only, but was significantly augmented by aCL+ x LA+ plasma in combination with adenosine diphosphate (ADP) at a low concentration that had only a modest effect on platelet activation. In contrast, aCL+ x LA-, aCL- x LA+ and aCL- x LA- plasma samples were incapable of enhancing platelet activation in the presence or absence of ADP stimulation. In addition to plasma samples, the purified IgG from aCL+ x LA+ plasma (aCL+ x LA+-IgG) also yielded apparent enhancement of platelet activation induced by ADP. Furthermore, platelet activation was generated by the mixture of aCL+ x LA--IgG and aCL- x LA+-IgG fractions prepared from individual patients, but not by each fraction alone. These results suggest that aCL and LA may cooperate to promote platelet activation, and may be involved, at least partially, in the pathogenesis of arterial thrombosis and thrombocytopenia in patients with SLE.

Very late antigen (VLA)-4 integrin has been suggested to play an important role in haemopoiesis. However, little is known concerning the roles of the fibronectin (FN)/VLA-4 interaction in the proliferation of human B cells. In this study we investigated the effect of immobilized FN on the proliferation of various B-cell lines, including a newly-established B-cell line, OPM-3, and human tonsillar B cells, that primarily express VLA-4 but not VLA-5. Immobilized FN significantly promoted the proliferation of OPM-3 cells and normal B cells via VLA-4. The cross-linking of beta1 integrins of OPM-3 cells resulted in the phosphorylation of the focal adhesion kinase (FAK) associated 90 kD protein, an increase in FAK-associated kinase activity, and the phosphorylation of Raf-1. Furthermore, the MEK1 inhibitor, PD98059, inhibited the FN-promoted proliferation of OPM-3 cells. These results demonstrate that the FN/VLA-4 interaction transmits the growth signal(s) which may be mediated by Ras pathway in OPM-3 cells, and suggest that OPM-3 cells may be of great value in studying the roles of the FN/VLA-4 interaction in human B-cell growth.

L-Arginine (L-Arg), one of the essential amino acids, has been reported to have an immunomodulatory effect. The precise mechanism of the L-Arg-induced natural killer (NK) cell activation remains unresolved,and the effect of L-Arg on NK cells in chronic fatigue syndrome (CFS) patients has not been estimated.NK cell function was evaluated in 20 subjects with CFS and compared with that in 21 healthy individuals.In healthy control subjects, NK activity was significantly increased after treatment with L-Arg, an NK function enhancer, for 24 h, whereas the same treatment failed to enhance NK activity in the CFS patients. We thus focused on L-Arg metabolism, which involves nitric oxide (NO) production through NO synthase (NOS). The expression of inducible NO synthase (iNOS) transcripts in peripheral blood mononuclear cells was not significantly different between healthy control subjects and CFS patients. The L-Arg-mediated NK cell activation was abolished by addition of NG-monomethyl-L-arginine, an inhibitor for iNOS. Furthermore, incubation with S-nitroso-N-acetyl-penicillamine, an NO donor, stimulated NK activity in healthy control subjects but not in CFS patients.These results demonstrate that the L-Arg-induced activation of NK activity is mediated by NO and that a possible dysfunction exists in the NO-mediated NK cell activation in CFS patients.

The mi locus encodes a member of the basic-helix-loop-helix-leucine zipper protein family of transcription factors (hereafter called MITF). We have reported that expression of several genes was impaired in cultured mast cells (CMCs) of mi/mi mice due to a defective transactivation ability of mutant MITF (mi-MITF). Because attachment of mi/mi CMCs to fibroblasts is impaired, we examined the expression of integrin genes in mi/mi CMCs in the present study. Among the integrin genes examined, the expression of integrin alpha4 subunit was barely detectable in mi/mi CMCs, and the alpha4 protein was not detected by flow cytometry either. The specific adhesion to vascular cell adhesion molecule-1 (VCAM-1), the ligand for alpha4 subunit, was observed in +/+ CMCs but not in mi/mi CMCs, indicating that the expression of integrin alpha4 subunit at a functional level did not occur in mi/mi CMCs. In the promoter region of the alpha4 subunit gene, there was a CACTTG motif to which normal MITF (+- MITF) bound. The coexpression of +-MITF but not of mi-MITF transactivated the promoter of the alpha4 subunit gene. The deletion or mutation of the CACTTG motif abolished the transactivation by +-MITF, suggesting that +-MITF directly transactivated the gene encoding alpha4 subunit of integrin.

To determine expression of c-kit and stem cell factor (SCF) mRNA in liver specimens from healthy adult dogs and to investigate whether differentiation of mast cells in the liver of dogs is supported by the c-kit receptor tyrosine kinase/SCF system.3 healthy adult Beagles.The nucleic acid sequence of the canine c-kit fragment of the intracellular tyrosine kinase domain was determined. Magnitudes of c-kit and SCF mRNA expression in liver samples was determined by means of northern blot analysis, using probes based on canine sequences. To determine which cells were expressing c-kit and SCF mRNA, in situ hybridization was performed.Expression of c-kit and SCF mRNA in liver samples was weak but appreciable. In situ hybridization revealed that c-kit and SCF mRNA expression was restricted to mast cells and plasma cells, respectively.Expression of SCF mRNA was detected in liver from healthy adult dogs. The c-kit receptor tyrosine kinase/SCF system may, possibly in combination with other growth factor and receptor systems, be involved in proliferation and differentiation of liver mast cells in dogs.

The expression of thrombopoietin (TPO) mRNA is observed in several tissues, including liver, kidney, brain, skeletal muscle, intestine, spleen, and bone marrow. Among these organs, the highest expression of TPO mRNA is detected in the liver. We identified cells producing TPO by means of in situ hybridization of adult rat liver using digoxigenin-11-UTP-labeled cRNA probes. We found that the cells expressing TPO mRNA also expressed serum albumin mRNA. TPO mRNA was detected in parenchymal cells (hepatocytes) but not in non-parenchymal cells (including endothelial cells, epithelial cells, and so forth). To determine the location of TPO expression in embryogenesis, sections of fetal mice were further analyzed by in situ hybridization. TPO mRNA was detected only in hepatocytes of fetal liver, which was also the major site of hematopoiesis. The expression of TPO mRNA in fetal liver was observed from 12.5 days postcoitus. Northern blot analysis showed that mouse liver transcribed the same size of TPO mRNA in the fetus and in the adult. These results clearly demonstrate that hepatocytes are the primary site of TPO production in the liver from fetus to adult.

We investigated the mechanism of constitutive activation of c-kit receptor tyrosine kinase (KIT) found in the FMA3 murine mastocytoma cell line, and compared it with the mechanisms observed in other tumor mast cell lines (the HMC-1 human mast cell leukemia cell line, the RBL-2H3 rat mast cell leukemia cell line, and the P-815 murine mastocytoma cell line). The c-kit gene obtained from FMA3 cells was found to have 21-base deletion at the juxtamembrane domain of KIT, thereby leading to the constitutive activation of KIT. The deletion at the juxtamembrane domain resulted in constitutive dimerization of c-kit proteins, whereas the point mutation that were detected at the kinase domain of KIT in HMC-1, RBL-2H3, and P-815 cells caused constitutive activation of KIT without dimerization. These constitutively activating mutations of c-kit may play a role in development of mast cell tumors.

beta 1-4 N-acetylglucosaminyltransferase (GnT-III) catalyzes the formation of bisecting N-acetylglucosamine (GlcNAc) in the biosynthesis of N-linked oligosaccharides. To examine the effect of bisecting GlcNAc on the natural killer (NK) cytotoxicity, the GnT-111 gene was introduced into NK-sensitive K562 cells that have no detectable GnT-III activity. We obtained three clones stably expressing high GnT-III (positive transfectants). Introduction of the GnT-III gene resulted in an increase of bisecting GlcNAc and a decrease of external sialic acid as well as tri- and tetraantennary sugars, as judged by flow cytometry. Compared to controls, the NK cytotoxicity was completely blocked against positive transfectants. The binding of effector cells to positive transfectants was also decreased. After s.c. injection into nude mice, positive transfectants produced spleen colonization, although no spleen lesions were formed by control cells. In nude mice depleted of NK cells by anti-asialo GM1 antibody, both positive transfectants and controls produced spleen colonization equally. These results indicate that K562 cells expressing GnT-III are resistant to NK cytotoxicity, resulting in spleen colonization in nude mice.

Thrombopoietin (TPO) is a recently identified hematopoietic growth factor that is essential for the growth and development of megakaryocytes. We have previously shown that TPO induces proliferation of acute myeloblastic leukemia (AML) cells in vitro. In this study, we have examined the expression of TPO and its receptor c-mpl in a series of AML cases and human leukemia cell lines. The mRNA transcripts of TPO were detectable in 18 of 50 AML cases and in some myeloid leukemia cell lines (HEL, M07E and CMK) by means of reverse transcriptase polymerase chain reaction (RT-PCR). In addition, TPO transcripts were coexpressed with c-mpl transcripts in 10 of 50 AML cases and in HEL, M07E and CMK cells. With regard to the French-American-British (FAB) classification, coexpression OF TPO and c-mpl was observed with high frequency in AML cases of M7-type. Despite the TPO expression in a substantial fraction of leukemia cells, biological activity of TPO was not found in the conditioned medium that was obtained from cultivation of TPO mRNA-positive leukemia cells. These results suggest that TPO may not commonly participate in the abnormal growth of AML cells as an extracellular autocrine growth factor.

Signaling through the c-kit receptor tyrosine kinase (Kit) is essential for development and survival of mast cells but not of basophils. Moreover, we recently found an activation mutation of Kit in several tumor mast cell lines.

The c-kit protooncogene encodes a receptor tyrosine kinase that mediates signals required for differentiation, proliferation and survival of mast cells. We have already shown the constitutive activation of c-kit receptor tyrosine kinase (KIT) in a human mast cell leukemia line (HMC-1) and a murine mastocytoma cell line (P-815). We here examined whether such constitutive activation of KIT occurred in the rat tumor mast cell line RBL-2H3 as well, which is frequently used as a tool for studying functions of mast cells. In RBL-2H3 cells, KIT was constitutively phosphorylated on tyrosine and activated in the absence of autocrine production of its ligand, stem cell factor (SCF). Sequencing analysis revealed that one of c-kit genes of RBL-2H3 cells had a point mutation, resulting in amino acid substitution of Tyr for Asp in codon 817. When rat wild-type c-kit cDNA and mutant-type c-kit cDNA encoding KITTyr817 were transfected into cells of a human embryonic kidney cell line (293T), only mutant form KITTyr817 was constitutively phosphorylated on tyrosine and activated in the absence of SCF. Since mutations at the same Asp codon constitutively activated KIT in all the human HMC-1, murine P-815, and rat RBL-2H3 cell lines, and since the incorporation of antisense oligonucleotides of c-kit messenger RNA significantly suppressed the proliferation of RBL-2H3 cells, the activating mutations in the Asp codon of the c-kit gene appeared to be involved in neoplastic growth of mast cells.

The proliferation of normal hematopoietic cells is strictly factor dependent, while leukemic cell lines and primary leukemic cells are frequently factor independent. Although autocrine growth stimulation of human leukemias is occasionally observed in vitro, it is possible that mutations of signal-transduction or cell-cycle control genes may also be important in the development of factor independence. We have previously shown that the proto-oncogene Raf-1, a 70-kd serine/threonine protein kinase, is rapidly phosphorylated and activated by hematopoietic growth factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and Steel factor and is likely to be an important intermediate in mitogenic signal transduction pathways in hematopoietic cells. In an effort to better understand the possible role of abnormal signal transduction in the development of factor independence, we compared the state of phosphorylation and associated kinase activity of Raf-1 between a series of factor-dependent human and murine-myeloid normal cells or cell lines and a series of factor-independent myeloid cell lines. In factor-dependent myeloid cells (normal neutrophils; monocytes; and the cell lines MO7, 32Dc13, and FDC-P1), Raf-1 phosphorylation and associated kinase activity was strictly regulated by the supply of growth factor. In contrast, each of eight factor-independent leukemic cell lines examined, HL-60, KG-1, K562, U937, JOSK-S, JOSK-M, JOSK-K, and JOSK-I, expressed hyperphosphorylated Raf-1 with increased Raf-1 associated kinase activity in the absence of growth factor addition. To further explore the relationship of Raf-1 to factor-independent growth, factor-independent sublines were derived from two factor-dependent cell lines, MO7 and FDC-P1, by culture in CSF-deprived medium. Also, several factor-independent sublines were derived by transfection of a cDNA encoding p210BCR/ABL into three different cell lines: MO7, 32Dc13, and FDC-P1. In each case, the new sublines expressed constitutively hyperphosphorylated and activated Raf-1. The correlation of hyperphosphorylation of Raf-1 with factor independence was also observed with primary acute myeloblastic leukemia cells. The rate of "spontaneous" proliferation of primary acute myeloblastic leukemia (AML) cells in vitro correlated with the extent of Raf-1 phosphorylation. These results suggest that the evolution of myeloid leukemic cells to factor independence is associated with phosphorylation and activation of Raf-1, implicating Raf-1 and signal transduction pathways which activate RAf-1 in this process.

The c-kit proto-oncogene encodes a receptor tyrosine kinase that is known to play a crucial role in mast cell growth and differentiation. In a human mast cell leukemia cell line (HMC-1), KitR was found to be constitutively phosphorylated on tyrosine, activated and associated with phosphatidylinositol 3-kinase (P13K) in the absence of autocrine production of SCF. Sequencing of c-kit cDNA revealed that c-kit genes of HMC-1 cells were composed of a normal, wild-type allele and a mutant allele with two point mutations in codon 560 and codon 816, resulting in intracellular amino acid substitutions of Gly-560 for Val and Val-816 for Asp, respectively. Murine c-kit mutants encoding Gly-559 and/or Val-814, corresponding to human Gly-560 and/or Val-816, were constructed by site-directed mutagenesis and expressed in cells of a human embryonic kidney cell line (293T). In the transfected cells, KitR (Gly-559 + Val-814) and KitR (Val-814) were strikingly phosphorylated on tyrosine and activated in the absence of SCF, whereas tyrosine phosphorylation and activation of KitR (Gly-559) or wild-type KitR was modest or little, respectively. These results suggest that constitutive activation of KitR in HMC-1 results from the activating mutations of c-kit gene, and raise the possibility that the activating mutations, particularly at codon 814 of murine c-kit or at codon 816 of human c-kit, may participate in oncogenesis of mast cells.

This study was undertaken to elucidate the effects of epidural analgesia on vibro-acoustic stimulation test (VAST) during normal labor and auditory brainstem response (ABR) in fifty full term parturients without any complication. The safety of VAST in relation to the ABR of neonates was also studied. The results were as follows, 1) There were some changes in CTG monitoring in five cases (10%) in the 10 minutes after epidural analgesia. However, these changes were not ominous signs but all related to the coiling of the cord, as previously reported. 2) The ABR latency-intensity curve (audio-function of the newborn infants) revealed no hazardous change in VAST-loaded infants or in the controls. The response threshold was 10dB in this study. The application of VAST to the parturients under epidural analgesia was therefore not only harmless to the fetuses but also a useful way to evaluate fetal well-being.

The W locus of mice encodes the c-kit receptor tyrosine kinase. Recently, we characterized a novel mutant allele, Wn, and demonstrated that the c-kit protein synthesized in Wn/Wn cultured mast cells (CMC) was reduced in size and not expressed on their surface (Tsujimura et al., 1993). In this study, we further examined biochemical nature of the mutant form of c-kit protein, by using Wn/Wn CMC and 293T cells transfected with Wn-type c-kit cDNA (c-kitWn). The c-kit product synthesized in Wn/Wn CMC was truncated almost all cytoplasmic domain and was less glycosylated. In c-kitWn-transected cells, both glycosylation and extracellular expression of c-kit protein was also impaired, however, no truncation was detected. These results indicate that Wn-mutant form of c-kit product is insufficient in maturation, which is associated with impairments in the transport to the plasma membrane, and retention of the mutant protein in endoplasmic reticulum is suggested. This is the first demonstration of the c-kit mutation affecting posttranslational processing its product.

Human interleukin 2 (IL-2) is a member of the class of crucial regulators of lymphocyte proliferation. The action of IL-2 is known to be mediated through binding to a specific IL-2 receptor (IL-2R) which comprises at least two distinct proteins: IL-2R alpha (p55) and IL-2R beta (p70-75). However, the expression and function of IL-2R are largely unknown in acute myeloblastic leukemia cells. In a human granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3, or stem cell factor-dependent myeloid leukemia cell line (M07E), IL-2 was found to stimulate proliferation in a dose-dependent manner and to augment GM-CSF- and stem cell factor-induced proliferation of M07E cells. The expression of IL-2R beta on M07E cells was detectable with 125I-IL-2 binding and affinity cross-linking analyses and with a monoclonal antibody against IL-2R beta, Mik-beta 1. Although the expression of IL-2R beta was not down-regulated but somewhat up-regulated by treatment with GM-CSF in both mRNA and protein levels, GM-CSF was found to compete (75%) with radiolabeled IL-2 for binding to IL-2R on M07E cells, whereas no competition of GM-CSF binding was observed with IL-2 even at a 400-fold molar excess. These results suggest that IL-2R may be functionally expressed in some cases of acute myeloblastic leukemia cells and raise the possibility that IL-2 may have some effects on human myelopoiesis.

The protooncogene product, Raf-1, is a serine/threonine kinase and has been implicated as an intermediate in signal transduction mechanisms. We examined neoplastic and normal B cells for phosphorylation and activation of Raf-1 protein in response to anti-immunoglobulin antibody (anti-Ig). Anti-Ig induced rapid phosphorylation of Raf-1 protein in both neoplastic B-cells of hairy cell leukemia and normal tonsillar B-cells which proliferated well in response to anti-Ig. The increase in phosphorylation was due primarily to an increase in phosphoserine. The immune complex kinase assay using Histone V-S as an exogenous substrate also showed an increase in Raf-1-associated kinase activity. An inhibitor of protein kinase C, H7, inhibited the proliferation as well as the Raf-1 phosphorylation in response to the proliferative signal of anti-Ig. Further, downregulation of protein kinase C by the treatment with 12-phorbol 13-myristic acid significantly abrogated the induction of Raf-1 phosphorylation. These results suggest that, in human B-cells, Raf-1 protein may be involved in the signal transduction pathway mediated by surface immunoglobulin, and that it may be, at least partially, phosphorylated by activated PKC.

Patients with refractory or relapsed acute myeloid leukemia (AML) are often treated with regimens containing high-dose cytosine arabinoside (ARA-C), resulting in complete response rates of approximately 30% to 50%, with a median remission duration of approximately 4 months [1]. Likewise, patients with the myeloid blast crisis of chronic myeloid leukemia (CML) are often treated with high dose ARA-C, with re-establishment of a short-lived stable phase in only about 20% of cases [2]. An important reason for treatment failure in these high-risk patients is likely to be pharmacologic resistance to ARA-C, which may be due to multiple mechanisms including decreased intracellular levels of deoxycytidine kinase and decreased intracellular uptake of ARA-C. Since ARA-C is a cell-cycle-specific drug, another potential mechanism of resistance may be related to the observation that a large fraction of leukemic clonogenic cells is not in S phase at the time of ARA-C exposure [3]. We have attempted to overcome kinetic resistance to ARA-C in vitro by recruiting leukemic cells into S phase through the use of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF), a humoral factor which supports the in vitro proliferation of clonogenic cells from the majority of patients with AML [4]. We have previously shown in tissue culture that this agent is able to effectively increase the fraction of leukemic clonogenic cells in S phase, and that this effect is associated with a significant increase in ARA-C-mediated cytotoxicity [5]. Since rhGM-CSF is biologically active and well-tolerated when administered to patients with normal bone marrow function [6–8], we chose to study the in vivo kinetic and therapeutic effects of combined rhGM-CSF and high dose ARA-C treatment in a population of high risk patients with myeloid leukemia. We now report the results of this treatment strategy in six patients with relapsed or refractory AML, and in two patients with the myeloid blast crisis of CML.

The treatment of patients with relapsed or refractory acute myeloid leukemia (AML) with high dose cytosine arabinoside (ara-C) results in short-lived complete response rates of 30-50%. We have previously shown that entry of myeloid leukemic cells into S phase can be accelerated in vitro through the use of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF), resulting in enhancement of ara-C-mediated cytotoxicity. In order to evaluate the in vivo biological and clinical effects of this strategy in patients with high risk AML, we treated three patients with either refractory or relapsed disease with a continuous infusion of rhGM-CSF (0.45 micrograms/kg/h aglycoprotein) for 18 h, followed by the institution of high dose ara-C and continuation of rhGM-CSF throughout the 4 day duration of ara-C treatment. Prior to therapy, no patient had detectable levels of circulating rhGM-CSF, and there was no evidence of GM-CSF receptor occupancy in leukemic myeloblasts. After 18 h of rhGM-CSF therapy, all patients had biologically active levels of circulating rhGM-CSF (7.9-12.0 ng/ml), and two patients showed a significant degree of leukemic GM-CSF receptor occupancy without evidence of GM-CSF receptor down-regulation. A significant rise in the S phase fraction of leukemic myeloblasts was observed at 18 h of rhGM-CSF treatment in all three patients (29-56% increment). The toxicity of combined rhGM-CSF/ara-C therapy included pericarditis and cerebellar degeneration in one patient, fever and mild renal dysfunction in two patients, and mild hepatic dysfunction in all three patients. Each patient showed a transient rise in the absolute neutrophil and blast count during rhGM-CSF/ara-C administration, followed by profound, but clinically tolerable, myelosuppression. No patient developed clinical evidence of leukostasis. There was one death related to pericardial tamponade, one death related to refractory disease, and one clinical and cytogenetic remission. These results suggest that exogenously administered rhGM-CSF is capable of rapidly mobilizing leukemic cells into S phase in vivo and theoretically should be useful in overcoming kinetic resistance to ara-C. Clinical trials of this regimen in patients with high risk AML who are not already pharmacologically resistant to ara-C are warranted.

A 48-year-old woman was admitted in September 1987, because of lumbago and galactorrhea. Peripheral blood analysis showed neutrophilia and eosinophilia without abnormal lymphocytes. The antibody to adult T-cell leukemia (ATL) virus-associated antigen was detected and a hyperprolactinemia was observed. The blastogenic responses to PHA, ConA and PWM were lowered. Brain CT and MRI scannings showed no abnormalities in the hypophysis and hypothalamus, but abdomen CT revealed markedly enlarged abdominal lymph nodes. Two months after the administration of OK432 and PSK, the lymph node swellings disappeared and the responses to PHA, ConA and PWM were normalized, but hyperprolactinemia and galactorrhea persisted. After four months of the remission period, the patient developed lymph node swellings again, and was diagnosed from the biopsy specimen of the retroperitoneal lymph node as having malignant lymphoma of diffuse mixed cell type. Southern blot analysis showed a monoclonal integration of HTLV-I proviral DNA. Despite repeated combination chemotherapies, she died of pneumonia in February 1989. Autopsy revealed marked infiltrations of lymphoma cells in the liver, spleen and lungs, but no abnormality accounting for hyperprolactinemia was detected in the suprasellar regions. This case was of interest in that immunotherapy was effective in achieving a remission and in normalizing immuno-parameters in ATLL.

The role of phorbol myristate acetate (PMA: a protein kinase-C (PKC) activator) and calcium ionophore A23187 in the induction mechanism of the interleukin 2 receptor (IL2R) on B-cell chronic lymphocytic leukemia (B-CLL) cells was studied. B-CLL cells from five patients were cultured with PMA or A23187 for 72 h and used for the following experiments. Interleukin 2 (IL2) cross-linking assays showed that PMA induced the expression of IL2R subunits (p55 and p70/75) in all cases examined, but that A23187 induced neither subunit. Radiolabeled IL2 binding assays also demonstrated that PMA induced both high-affinity IL2R (HA-IL2R) and low-affinity IL2R (LA-IL2R) on B-CLL cells, but that A23187 did not. After treatment with PMA, three of five cases did not respond to IL2 even though they expressed HA-IL2R, suggesting impaired signal transduction. No cases responded to IL2 after treatment with A23187. In conclusion, PMA but not A23187 stimulates B-CLL cells to induce the expression of p55 and p70/75, indicating that the PKC pathway plays a more important role than the calcium pathway in the induction of IL2R subunits in B-CLL cells.

Although precursors of mast cells are derived from the bone marrow, phenotypes of mast cells are influenced by the tissues in which final differentiation occurs. Connective tissue-type mast cells (CTMC) and mucosal mast cells (MMC) are different in morphological, biochemical, immunological, and functional criteria. The purpose of the present study was to obtain information about the differentiation process of MMC. First, we compared changes in irradiation susceptibility in mice during the differentiation process of CTMC and MMC. The decrease in irradiation susceptibility was remarkable in the CTMC differentiation process, but it was moderate in that of MMC. Some morphologically identifiable CTMC in the peritoneal cavity had proliferative potential and were highly radioresistant, whereas such a radioresistant population of MMC was not detectable in the gastric mucosa. Second, we estimated the turnover of CTMC and MMC by determining the proportion of mast cells that were labeled with continuously administered bromodeoxyuridine. The turnover of MMC was significantly faster than that of CTMC. The absence of the radioresistant mast cell population in the gastric mucosa appeared to be related to the short life span of MMC.