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SUMMARYT cells expressing CD57 (a natural killer cell marker) with interferon-γ (IFN-γ) producing capacity increase under various conditions. CD57+ T cells are also present in the bronchoalveolar lavage fluid (BALF) of sarcoidosis, and several phenotypical and functional analyses of these cells have been reported. In the present study, BALF T cells obtained from 52 patients with sarcoidosis were classified further into CD4+CD57+ T cells, CD4+CD57– T cells, CD8+CD57+ T cells and CD8+CD57– T cells and their phenotypes and functional characteristics were assessed. Substantial proportions of these T cell subsets expressed natural killer cell markers CD161 and CD122. The biased expansion of Vβ2 T cells was observed in both CD4+CD57+ T cells and CD4+CD57– T cells in BALF from most patients, while the expansion of other Vβ T cells was also observed in some patients. Unexpectedly, the biased expansion of certain Vβ T cells was also seen in either CD8+CD57+ T cells or CD8+CD57– T cells, while the expanded Vβ T cells in CD8+ T cells differed substantially among individuals. BALF T cells showed a remarkably lower T cell receptor (TCR) intensity than that of peripheral blood T cells. Both CD8+ T cell subsets in BALF of sarcoidosis expressed the intracellular perforin/granzyme B, while all four subsets expressed intracellular IFN-γ after in vitro activation, and CD4+ T cells, especially CD4+CD57+ T cells, expressed tumour necrosis factor-α. These findings indicate that CD57+ T cells as well as CD57– T cells in the BALF are phenotypically and functionally different from peripheral blood T cells and may play an important role in the Th1 dominant state and inflammation in pulmonary sarcoidosis.

We propose a novel self-organizing waveguide formation method "Self-Organizing Lightwave Network" (SOLNET). SOLNET utilizes an interaction between light beams in photo-refractive materials. The interaction is an attractive force, so that the beams merge together into one optical path. This enables an automatic waveguide construction between optical devices, tolerating the mutual positional misalignment and mode size mismatching. SOLNET can also construct straight and downtapered waveguides in a free space. Applications of SOLNET are proposed, including self-aligned coupling between two optical devices and three-dimensional (3-D) optical wiring. Proof-of-concept of SOLNET is demonstrated by computer simulations and experiments. Optoelectronic scalable substrate (OE-SS) and film optical link module (FOLM) are described, showing their potentiality to reduce cost/space/noise and provide scalability/standardized-interface capability in optical interconnect hardware. The expected applications of SOLNET to OE-SS and FOLM are discussed.

A 40-dB dynamic-range variable-gain amplifier (VGA) designed for the code division multiple access (CDMA) cellular phone has been developed. A wide-dynamic-range VGA under a low control voltage of 2.0 V, compatible with high linearity and low distortion characteristics, essential for CDMA, is realized by the new gain control technique. It greatly contributes to the high performance and small size of RF circuits for CDMA cellular handsets.

In this work, the reliability of enhancement-mode GaN MIS-HEMTs with gate-recess structure was investigated. Stress test under the positive gate bias was mainly considered. By the positive gate bias stress, R on and V p was varied. This shift was reversible. The deep-level optical spectroscopy (DLOS) suggested deep levels at the gate recessed region. These results lead to the conclusion that R on and V p shift was attributed by the deep level at the gate recessed region. By controlling the trap density at recessed region, we can suppress the R on and V p shift.

To explore the biologic significance of the membrane-anchored form of macrophage colony-stimulating factor (M-CSF), we examined whether interaction between membrane-bound M-CSF and its receptor mediates intercellular adhesion as well as cell proliferation and differentiation. Human M-CSF receptors were expressed on a murine interleukin-2 (IL-3)-dependent cell line, FDC-P2, by DNA transfection with the c-fms gene (FDC-P2-MCSFR). A human bone marrow-derived stromal cell line, KM102, was used in the cell adhesion assay. The expression of membrane-bound M-CSF on KM102 cells was demonstrated by flow cytometry and immunoblot analysis. After the incubation of parent and transformed FDC-P2 cells on confluent KM102 cells, nonadherent cells were removed and the cells attached to KM102 cells were examined microscopically. Almost all parent FDC-P2 cells were nonadherent, whereas a significant number of FDC-P2-MCSFR cells bound to KM102 cells. The addition of anti-M-CSF or anti-M-CSF receptor neutralizing antibodies dose-dependently inhibited the binding of [3H]-thymidine- labeled FDC-P2-MCSFR cells to KM102 cells. An excess amount of M-CSF also inhibited the binding. On the other hand, the addition of antibodies against some representative adhesion molecules (vitronectin receptor, Pgp-1/CD44, and VLA-4) did not inhibit the adhesion between FDC-P2-MCSFR cells and KM102 cells. These results support the idea that membrane-anchored M-CSF and its receptor may function as mediators of cell-cell adhesion.

We have developed a highly reliable halogen free (hf) flame-resistant epoxy dielectric for build-up printed wiring boards (PWBs). This material would not generate environmentally hazardous products such as polybrominated dibenzodioxins/furans and hormone disruptive agents like Bisphenol-A and its derivatives. The dielectric is composed of a mixture of a reactive type oligomeric organic phosphate and Al(OH)/sub 3/ as the flame retardant, a thermally stable polyaromatic epoxy resin as the main component together with a rubber-modified epoxy, and a flexible phenolic epoxy hardener. High Cu conductor corrosion resistance under high-temperature-humidity-bias conditions has been obtained due to the cross-linkable organic phosphate that would reduce the free phosphate ion content. The polyaromatic epoxy resin, which would prevent over-etching of the resin surface during KMnO/sub 4/ etching process, significantly improved the dielectric adhesion to the Cu conductor. The newly developed dielectric exhibited good thermal properties and mechanical properties (Tg of 155/spl deg/C) such as high flexibility and shock resistance that are very essential in mobile electronic devices like hand phones and laptop-PCs. This material may also have potential usage in high-density packaging.

A 40 dB-dynamic-range variable gain amplifier designed for CDMA cellular phones has been developed. A wide dynamic range variable gain amplifier under low control voltage of 2.0 V compatible with high linearity and low distortion characteristics essential for CDMA is realized by the new gain control technique. It greatly contributes to the high performance and small size RF circuits of CDMA cellular handsets.

This report introduces a new approach on a recognition system of three dimensional, i.e. 3D, objects. The proposed system is based on a restricted multi-layered neural network. For the performance evaluation of this network, the experiment in which some similar objects are used for recognition is demonstrated.

This paper reports a new variable gain amplifier which has low NF of 1.7 dB, low distortion of IM/sub 3//spl les/50 dBc and low power consumption of less than 30 mA. These performances are realized by the new input-signal control circuit.

This report introduces a new approach on a recognition system of three dimensional, i.e., 3-D, objects. The proposed system is based on a restricted multi-layered neural network. In order to greatly reduce the total calculation cost of preprocessing for the recognition, directional coupled nodes in a network are introduced. This report also shows that the system can recognize some 3-D objects correctly in experiments.

We propose a novel self-organizing waveguide formation method "Self-Organizing Lightwave NETwork (SOLNET)". SOLNET utilizes an interaction between light beams in photorefractive materials. The interaction is an attractive force, so that the beams merge together into one optical path. This enables an automatic waveguide construction between optical devices, tolerating the mutual positional misalignment. SOLNET can also construct straight and downtapered waveguides in a free space. Applications of SOLNET are proposed, including self-aligned coupling between two optical devices and 3D optical wiring. Proof-of-concept of SOLNET is demonstrated by computer simulations and experiments. Optoelectronic (OE) interconnect hardware, OE Scalable Substrate (OE-SS) and Film Optical Link Module (FOLM), as well as a device integration process, Self-ORganizing Transfer (SORT), which have been proposed by FCPT, are presented. OE-SS and FOLM have potentiality to reduce cost and required space in OE systems. Expected applications of SOLNET to OE-SS and FOLM are shown.

This paper presents a two step power control driver MMIC for wideband-CDMA handsets. It has the self current control function coupled with the output power level. It realizes low current consumption of 12 mA at an average output power level (Pave) without any sacrifice of high performance at the high output power level. It greatly contributes to the realization of compact and low power consumption W-CDMA handsets.

We describe a patient with myelodysplastic syndrome (MDS) that transformed to Burkitt's acute lymphoblastic leukaemia (ALL). The leukaemic blasts were negative for peroxidase staining, and expressed CD10, CD19, CD22, CD38, human leucocyte antigen (HLA)-DR and surface immunoglobulin (sIg) M, but neither sIgD nor sIgG were expressed. Chromosomal study during the ALL phase showed t(8;22)(q24;q11) in addition to the karyotypes determined during the MDS phase. Furthermore, overexpression of c-myc mRNA was confirmed in ALL blasts. These findings indicate that MDS transformed to Burkitt's ALL through multiple cytogenetic evolutions, the final event of which seems to be overexpression of the c-myc gene.

More than 20 different partner genes with MLL have been cloned from leukemia cells with translocations involving chromosome 11 band q23 (11q23). All reported partner genes fused in-frame to MLL and the fusion cDNA encode chimeric MLL proteins with a significant portion derived from the partner genes. We analyzed one patient with de novo acute monoblastic leukemia with t(11;14)(q23;q24) and identified that a human homologue of gephyrin (human gephyrin) fused with MLL. Gephyrin is a rat glycine receptor-associated protein, which forms submembranous complexes and anchor glycine or gamma-aminobutyric acidA receptors to microtubules. Alternative splicing of human gephyrin gene created two different forms of fusion cDNA. In one form, human gephyrin gene fused in-frame to MLL exon 9, and the chimeric product had COOH terminus of human gephyrin protein, including the tubulin binding site. In the other, the reading frame terminated shortly after the fusion point. As a result, only seven amino acids with no known function were attached to the NH2 terminus of MLL protein. The functional significance of this de facto truncated MLL gene product is not clear.

Although studies using magnetic resonance imaging (MRI) in multiple sclerosis (MS) patients have focused on findings in the white matter because of its demyelination pathogenesis, Drayer et al. have reported a high incidence of low signal intensity on T2 weighted MR imaging (MRI) in gray matter such as the thalamus and putamen. In Japan there has been no investigation of MRI findings of the basal ganglia in MS patients. Therefore, we attempted to examine the incidence and clinical significance of the imaging phenomenon in 34 Japanese patients with MS (12 male, 22 female, ages 18-54 years). As it is well known that the spinal cord and optic nerves are more frequently involved in MS than the brain in Japanese patients, we divided the patients into two subgroups based on their clinical features and the major sites of demyelination on MRI. One group included the 17 patients whose demyelinations occurred in the brain (brain-type), and the other group included the 17 patients whose abnormalities were found in the spinal cord with or without optic nerve involvement (non-brain type). As a control, MRI studies were also performed in age-matched patients with headache without any neurological signs. On T2 weighted MRI, decreased signal intensity in the thalamus was found in only four patients with MS, 11.8% of the total number examined, and in the putamen in three patients with MS, 8.8% of the total examined. All of the patients who showed abnormal decreased signal intensity in the thalamus and/or putamen belonged to the brain-type group, and these incidences were 23.5% in the thalamus and 17.6% in the putamen among the brain-type patients. No patient belonging to the non-brain type showed this imaging sign. This imaging sign was well correlated with the degree of white matter abnormalities in the brain estimated as a score according to modified Callanan et al.'s method. In addition, this sign was also correlated with the expanded disability status scales (EDSS) in the brain-type patients. These observations suggest that the axonal damages due to severe demyelination may induce the impaired transport of iron resulting in an accumulation of ferritin in the thalamus and putamen, and would cause decreased signal intensity on T2 weighted MRI. The relatively low incidence of decreased signal intensity in the thalamus and putamen in this study may be associated with differences in the clinical phenotype of MS between Japan and the USA. In brain-type patients the evaluation of basal ganglia on T2 weighted MRI may be a useful tool for estimating patients' disabilities.

The formation of multinucleated giant cells (MGCs) from monocytes/macrophages is controlled by various cytokines, the roles of which are not fully understood. Both interleukin (IL)-4 and 1alpha,25(OH)(2) vitamin D(3) (D(3)) are known to induce MGC formation from monocytes/macrophages. D(3) is also known as a stimulator of osteoclast formation in the presence of stroma cells, and IL-4 as an inhibitor. Previously, we showed that IL-4-induced MGCs from monocytes/macrophages expressed tartrate resistant acid phosphatase (TRAP) activity and hydroxyapatite-resorptive activity in the presence of M-CSF without stroma cells. In this study, we examined the effects of D(3) and/or IL-4 on MGC formation and the characteristics of these MGCs using a monoblastic cell line (UG3), to elucidate the involvement of these factors in osteoclast development without stroma cells. D(3)-induced MGCs showed none of the markers of osteoclasts, such as TRAP activity, calcitonin receptor (cal-R) expression, hydroxyapatite-resorptive activity, and bone-resorptive activity. A low concentration of D(3) synergistically stimulated IL-4-induced TRAP-positive MGC formation, whereas a high concentration of D(3) inhibited it. When IL-4 was added on day 7 of the 2-week culture with D(3), TRAP positivity reached maximum. On the other hand, delayed addition of D(3) on day 7 of culture did not increase the TRAP positivity. Although the fusion rate increased during the first week of the 2-week culture in the presence of D(3), it increased further in the second week following the addition of IL-4 on day 7. Furthermore, IL-4-induced, or IL-4- and D(3)-induced MGCs differentiated into functional osteoclasts with bone-resorptive activity following coculture with osteoblastic cells, whereas D(3)-induced MGCs did not acquire bone-resorptive activity even after coculture with osteoblastic cells in the presence of D(3). These findings suggest that IL-4 initiates osteoclast development of UG3 cells, although stroma cells were necessary for development of functional osteoclasts. On the other hand, D(3) had only a "supportive" effect on this differentiation. IL-4 and direct contact with stroma cells may regulate different stages in the multistep process of osteoclastogenesis of UG3 cells.

Transforming growth factor beta (TGF-beta) enhanced the growth-inhibitory activities of dexamethasone (Dex) and 1alpha,25-dihydroxyvitamin D3 (VD3) on human monocytoid leukemia U937 cells. TGF-beta and VD3 synergistically increased the expression of differentiation-associated markers such as the CD11b and CD14 antigens, whereas TGF-beta and Dex did not. On the other hand, TGF-beta and Dex synergistically increased the number of Apo2.7-positive cells, which represents the early stage of apoptosis, whereas TGF-beta and VD3 did not, suggesting that TGF-beta enhanced apoptosis with Dex and enhanced monocytic differentiation with VD3. In the presence of TGF-beta, the retinoblastoma susceptibility gene product, pRb, was synergistically dephosphorylated by Dex as well as VD3. TGF similarly enhanced the expression of the p21Waf1 gene in U937 cells treated with Dex and VD3. TGF-beta dose-dependently increased the expression of Bcl-2 and Bad and decreased the expression of Bcl-X(L) in U937 cells. Dex enhanced the down-regulation of Bcl-X(L) expression in TGF-beta-treated cells, whereas VD3 blocked this down-regulation of Bcl-X(L). However, the down-regulation of Bcl-X(L) by treatment with the antisense oligomer did not affect the apoptosis or differentiation of U937 cells. The apoptosis of CD14-positive cells was suppressed in the VD3 plus TGF-beta-treated cultures. These results suggest that the expression of CD14 is involved in the survival of differentiated cells.

A 38-year-old woman showed symptoms of myasthenia gravis (MG) three months after receiving thymectomy for malignant thymoma. She was treated with anti-acetylcholine esterase drugs and azathioprine over 10 years with two exacerbations, which were controlled by plasmapheresis and large amounts of steroid. Nephrotic syndrome developed suddenly at the age of 48, was progressive even after azathioprine withdrawal, and resistant to several immunosuppressive therapies such as steroids and cyclosporine A, and plasmapheresis. She died of systemic infection one-and-a-half years after the onset of nephrotic syndrome. Immunological studies revealed several abnormalities of cellular immunity. The expansion of gamma-delta T cells and CD57+ lymphocytes in peripheral blood was characteristic findings. These cells are thought to originate from the extrathymic process. Nephrotic syndrome has been thought to be sometimes complicated with thymoma. Although some pathogenetic possibilities about combination of nephrotic syndrome and thymoma were supposed, none has yet been clarified. As we noticed the remarkable increase in the number of CD57+ cells, we examined its proportion in the peripheral blood of patients with MG and/or thymoma, as well as in individuals without sickness. The study revealed the expansion of CD57+ cells in MG thymoma patients (32.3 +/- 15.9%), compared with healthy controls (15.2 +/- 5.4%), MG non-thymoma patients (20.3 +/- 11.5%), and thymoma non-MG patients (15.2 +/- 12.0%) statistically (Mann-Whitney's U test). Therefore, we supposed that the peripheral CD57+ cell expansion was associated with parathymic immunological abnormalities, such as MG, thymoma, and nephrotic syndrome.

We explored the potential involvement of Sjögren's syndrome as a cause of diffuse lung disease. A prospective clinical study was performed with measurements of saliva production made using the Saxon test. Sixty-seven diffuse lung disease patients who did not exhibit xerosis were examined. The group included 43 patients with sarcoidosis, 11 with interstitial pneumonia, 3 with primary pulmonary lymphoma (PPL), 2 with idiopathic BOOP, 2 with chronic eosinophilic pneumonia, and 6 with other diseases. Decreased saliva production was detected in 11 (16.4%), and Sjögren's syndrome was diagnosed in 4 (6.0%). Lung lesions displayed by the group with Sjögren's syndrome included PPL, bronchiolitis, sarcoidosis, and interstitial pneumonia. We concluded that in patients with diffuse lung diseases, it is always important to discriminate between those with sicca syndrome and Sjögren's syndrome. In our study, the Saxon test proved highly effective as a screening procedure for this purpose.

A 66-year-old woman presented with left chest pain. Left pleural effusion was seen on a chest X-ray film and a large mass disclosed by chest computed tomography. However, the patient refused to undergo a recommended operation. Six months later, she was admitted without any symptoms. A huge (18 cm diameter) mass was detected by magnetic resonance imaging (MRI), and consisted of heterogeneous solid and cystic components. Angiography and endoscopic sonography disclosed a suspected abdominal tumor, which was resected by thoracolaparotomy. Gastric stromal tumor was diagnosed on the basis of histological findings. Chest pain and pleural effusion are rare as initial clinical symptoms of such tumors.

A 69-year-old man was admitted to our hospital due to pancytopenia and a marked bleeding tendency. On admission, he had a white cell count of 2.8 x 10(9)/l, hemoglobin level of 6.0 g/dl, and a platelet count of 3 x 10(9)/l. He was given a diagnosis of refractory anemia on the basis of bone marrow aspiration findings, which disclosed trilineage myelodysplasia. After discharge, the patient remained dependent on blood transfusions. The sole administration of an active form of vitamin D3 (calcitriol) was started in July 1997, and one and a half years later, the patient's transfusion dependency disappeared. However, bone marrow aspiration findings at this point disclosed marked cell dysplasia of erythroid lineage and a prognostically unfavorable chromosomal abnormality (monosomy 7) that had not been found during the initial examination. Nonetheless, the patient's hemoglobin level and platelet count increased to more than 9 g/dl and about 1.0 x 10(11)/l, respectively. The finding in this case suggested that vitamin D3 therapy is useful for refractory anemia even if aggravated marrow cell dysplasia and cytogenetic anomalies develop.

The clinical effects of concomitant use of granulocyte-colony stimulating factor (G-CSF) on empiric antibiotic therapy in febrile neutropenic patients were evaluated in a randomized fashion. Two hundred and fourteen neutropenic febrile episodes (neutrophil counts1.0 x 10(9)/l) were treated with flomoxef sodium and tobramycin with or without G-CSF. The resolution of fever at day 4 (excellent response) or at day 7 (good response) was deemed effective. Among 157 evaluable episodes, the observed excellent responses were 31 (38.8%) and the good responses were 20 (25.0%) in the G-CSF group; those in the control group were 26 (33.8%) and 25 (32.5%), respectively. The overall efficacy rate was 63.8% (51/80) in the G-CSF group and 66.2% (51/77) in the control group (not significant). The initial neutrophil count was 0.186 +/- 0.249 x 10(9)/l in the G-CSF group and 0.235 +/- 0.290 x 10(9)/l in the control group, and rose to 2.889 +/- 4.198 x 10(9)/l and 0.522 +/- 0.844 x 10(9)/l, respectively, at day 7. These results indicate that G-CSF does not affect the rate of response to empiric antibiotic therapy in febrile neutropenic patients, although a significant effect of G-CSF was observed on neutrophil recovery.

Macrophage colony-stimulating factor (M-CSF) is a potent stimulator of the effector cells such as monocytes and macrophages. To evaluate the effect of M-CSF on malignant gliomas, we transfected the rat gliosarcoma cell line (9L) with human M-CSF expression vector (pCEF-MCSF) by a liposome method. Transfectants were selected using G418-containing medium. As a control, 9L cells transfected with pRc/CMV and selected by G418 were used. The effects of M-CSF gene transfection on tumor cell proliferation in vitro and in vivo were examined. All growth rate did not change in vitro. While the control 9L cells formed progressively enlarging masses, 9L cells transfected with the M-CSF gene did not develop into tumors after the injection into rats. On the other hand, in rats receiving anti-asialo GM1 antibody, 9L cells transfected with M-CSF gene developed into tumors, though at a slower rate than control 9L cells. Histologic examination after transplantation of 9L cells transfected with M-CSF gene disclosed intense infiltration of macrophages in the tumor. Thus M-CSF gene transfection into glioma cells stimulates an antitumor effect.

Of various PGs, PGE1 and PGE2 are shown to be the most potent stimulators of osteoclastogenesis in vitro. PGE receptors have been classified into four subtypes, EP1-EP4. Little is known about PGE receptors functioning in bone cells. In this study, using mouse marrow culture, we investigated which PGE receptors are important in osteoclast-like cell (OCL) formation induced by PGE. 11-deoxy-PGE1 (EP2, EP3 and EP4 agonist) stimulated OCL formation potently. Butaprost (EP2 agonist) stimulated it slightly, while sulprostone (EP1 and EP3 agonist) and ONO-AP-324-01 (EP3 agonist) did not. AH23848B (EP4 antagonist) inhibited PGE2-induced OCL formation in a dose-dependent manner. The expression of EP4 mRNA in mouse bone marrow was confirmed by RT-PCR. The results indicate an important role of EP4 in PGE2-induced OCL formation in marrow cultures and suggest therapeutic potential of EP4 antagonists in some clinical conditions with accelerated bone resorption.

Differentiation inhibitory factor nm23 gene has been found to be expressed in high quantities in acute myelogenous leukemia (AML), especially in acute monocytic leukemia (AML-M5) and is suggested as a new prognostic factor in AML-M5. We report an example of elevated expression of nm23 mRNA in a patient with chronic myelogenous leukemia (CML) who developed monoblastic crisis. Relative levels of nm23-H1 and -H2 mRNA extracted from the patient's peripheral blood mononuclear cells and bone marrow mononuclear cells were measured by quantitative reverse transcriptase polymerase chain reaction. The level of nm23-H1 mRNA in CML cells at the chronic phase was as high as that in bone marrow cells from healthy volunteers. The mRNA level of nm23-H2 was slightly below the normal level. At blastic crisis, however, expression of both nm23-H1 and -H2 mRNA was elevated to about three to nine times of that at the chronic phase. Proliferated blastic cells were positive for non-specific esterase, and the serum lysozyme level was elevated and diagnosed as monoblastic crisis. The patient received combined chemotherapy but response was partial. These findings are compatible with our previous report that nm23 gene is overexpressed in monocytic leukemia.

Macrophage colony-stimulating factor (M-CSF) was found to be a glycoprotein with a molecular weight of 85 kDa which stimulated macrophage colony formation of mouse bone marrow cells in a semisolid agar culture system in 1978. M-CSF stimulates differentiation of progenitor cells to mature monocytes, and prolongs the survival of monocytes. It enhances expression of differentiation antigens and stimulates chemotactic, phagocytic and the killing activities of monocytes. Macrophage CSF also stimulates production of several cytokines such as granulocyte-macrophage CSF, granulocyte CSF and interleukin (IL)-6 by priming monocytes, and directly stimulates production and secretion of IL-8 and reactive nitrogen intermediates. In addition to the stimulation of hematopoiesis, M-CSF also stimulates differentiation and proliferation of osteoclast progenitor cells and cytotrophoblasts. Proteoglycan type M-CSF, which contains chondroitin sulfate chains, was found in 1992. In a large-scale double-blind controlled study on acute myeloid leukemia (AML), it has been shown that the administration of M-CSF to patients after consolidation chemotherapies shortens the periods of neutropenia and thrombopenia after chemotherapy and reduces the incidence and shortens the duration of febrile neutropenia, as well as shortening the period required to finish three courses of intensive consolidation therapy.

The novel uracil analog, 6-chloro-5-(2-propenyl)uracil (TI90), inhibited the growth of myeloid leukemia cells and induced morphologic and functional differentiation of the cells. Although TI90 was a weak inducer of differentiation, it greatly enhanced the growth inhibition and differentiation of the leukemia cells previously induced by 1alpha,25-dihydroxyvitamin D3 (VD3) or all-trans retinoic acid (ATRA). TI90 cooperated with VD3 much more effectively than with ATRA in inhibiting cell growth and inducing differentiation. It also decreased the effective concentration of VD3 to the 10(-10) M level. On the other hand, there was no significant synergy between VD3 and the other uracil analogs. TI90 did not affect VD3 metabolism or the number and affinity of VD3 receptors (VDR) in HL-60 cells. Signals from VD3 are predominantly mediated by VDR and the ligand-activated binding of VDR to vitamin D-responsive element (VDRE) as a heterodimer with the retinoid X receptor (RXR). According to the results of a gel shift assay, TI90 enhanced the intensity of the retarded band with synthetic VDRE oligomer in the presence of VD3, suggesting that TI90 increases the number of phosphorylated receptors by inhibiting phosphatase activity, and also stimulates the formation of a functional complex of VDR with RXR.

Estrogen deficiency puts individuals at risk of developing osteoporosis because it causes increased bone resorption. However, the mechanism by which this occurs is not known. We have shown, using a recently described two-phase human bone marrow culture system, that estradiol (17beta-E2) added to phase I results in inhibition of bone resorption by reducing the number of osteoclasts (identified as vitronectin receptor and/or calcitonin receptor-positive cells) formed in the cultures. The addition of 17beta-E2 in phase II was without effect, indicating that it does not interfere with the bone resorptive process. 17Beta-E2 down-regulated mRNA expression and protein synthesis of the membrane form of macrophage colony-stimulating factor (M-CSF). 17Beta-E2 did not the alter the expression of the 4.0 kb M-CSF transcript. However, it increased protein synthesis of the proteoglycan form of M-CSF, but not the 85 kDa soluble form in the same cultures. Finally, addition of M-CSF to the cultures reversed the 17beta-E2-induced inhibitory effect. These observations suggest that regulation of the synthesis of membrane-bound M-CSF plays a role in 17beta-E2-induced inhibition of bone resorption.

We compared the biologic activities of 85-kDa macrophage-CSF (85-kDa M-CSF), which is fully active, and a proteoglycan M-CSF (PG-M-CSF). Both originate from the same precursor, but the latter retains the carboxyl-terminal portion, which must be proteolytically removed from the precursor to generate 85-kDa M-CSF and which is uniquely modified by a chondroitin sulfate glycosaminoglycan chain. PG-M-CSF supported the formation of murine macrophage colonies such as 85-kDa M-CSF. Furthermore, PG-M-CSF stimulated the proliferation of murine bone marrow macrophages, an M-CSF-dependent murine cell line, and an M-CSF-responsive human cell line established by transfer of the human M-CSF receptor gene. PG-M-CSF and 85-kDa M-CSF had equivalent specific biologic activities on a molar basis in all bioassays. The activity of PG-M-CSF was not affected by enzymatically removing the glycosaminoglycan chain when assayed by the formation of macrophage colonies and proliferation of the bone marrow macrophages. We analyzed the phosphorylation on tyrosine residue(s) of the M-CSF receptor in response to these M-CSFs that trigger mitogenic responses. PG-M-CSF rapidly (within 10 min) induced receptor phosphorylation in human cells with the same potency as 85-kDa M-CSF. These results indicate that PG-M-CSF is not a latent form or precursor of 85-kDa M-CSF but a fully biologically active cytokine.

Macrophage colony-stimulating factor (M-CSF) enhances tumoricidal activities of macrophages. We transduced human M-CSF cDNA into the mouse lymphoid cell line, L1210, and examined the antitumor effect of the locally expressed M-CSF. Mice injected with the M-CSF-producing subline showed improved survival in comparison with the mock-transfected cell line or parental cell line plus M-CSF administration (20 microg/kg for 3 days) at inoculated cell numbers of 10(2) or 5 x 10(3). The survival rate at 50 days after injection of 10(6) high M-CSF-expressing cells was 80%, significantly higher than that after injection of the mock-transfected cells, which killed all the mice by day 23. The survival rate appeared to depend on the amount of M-CSF produced. Moreover, all surviving mice after intravenous injection of the M-CSF-expressing sublines were rechallenged with 10(6) parental L1210 cells at day 50, and all survived up to day 100, demonstrating that M-CSF-expressing cells induced immune protection against the parental cells. The same improvement of survival was observed in mouse M-CSF-expressing cell lines. These observations imply that M-CSF cDNA is a candidate gene for use in gene therapy in leukemia.

We have investigated the stimulatory effect of recombinant human macrophage colony-stimulating factor (rhM-CSF) on interleukin-8 (IL-8) production by human peripheral blood monocytes. When monocytes were prepared from peripheral blood and cultured for 24 hours, the average amount of IL-8 in the culture medium was about 8.9 +/- 3.2 ng/2 x 10(5) cells. In contrast, the production of IL-8 by monocytes increased to a level of 19.2 +/- 4.7 ng/2 x 10(5) cells in response to rhM-CSF. This induction by rhM-CSF was dose-dependent. Northern blot analysis showed that expression of IL-8 at the pretranslational level was enhanced after M-CSF treatment. Kinetic studies showed that secretion of IL-8 from monocytes was enhanced within 2 hours after exposure to rhM-CSF, and a saturation level, which was reached around 48 hours, was two-fold higher than that of cells without M-CSF treatment. In addition, conditioned medium of M-CSF-stimulated monocytes activated the chemotaxis of human neutrophils, and this activity was significantly inhibited by anti-IL-8 antibody. These results, taken together, suggest that M-CSF can affect many cellular functions through regulation of IL-8 expression in monocytes.

Although three molecular forms of macrophage colony-stimulating factor (M-CSF) have been reported, Western blot analysis of immunoaffinity-purified M-CSF from human blood has shown that the major species of M-CSF in the serum has a molecular weight (MW) of 85 kD. Superose-12 gel filtration chromatography of immunoaffinity-purified serum M-CSF showed the presence of M-CSF-positive fraction in a higher MW area compared with the elution profile of recombinant human (rh) 85-kD M-CSF. Western blot analysis of the higher MW fraction showed that the M-CSF was the same as rh 85-kD M-CSF (not proteoglycan form of M-CSF), indicating that, in the serum, M-CSF exists bound to some serum proteins. To detect the serum proteins, we performed M-CSF-bound column chromatography. The eluate contained at least three serum proteins including albumin and IgG. This result was supported by chromatography using biotinylated M-CSF and avidin-agarose. The binding between rhM-CSF and albumin or IgG was also demonstrated by high-performance liquid chromatography (HPLC) fractionation of the mixture and enzyme-linked immunosorbent assay (ELISA) of the fractions. In the serum, a fraction of M-CSF seems to be complexed with serum proteins such as albumin and IgG.

Glucocorticoids inhibit the proliferation of lymphoid leukemia cells, whereas most myeloid leukemia cells are resistant to glucocorticoids. However, this study showed that glucocorticoids significantly and preferentially inhibited growth of monocytoid leukemia cells in combination with a low concentration of transforming growth factor beta (TGF beta). Combined 1 alpha,25-dihydroxyvitamin D3 and TGF beta markedly induced monocytic differentiation of U937 cells, whereas dexamethasone (Dex) and TGF beta essentially did not, although both combinations similarly inhibited the growth of U937 cells. The growth inhibition was accompanied by a block in the cell cycle progression from G1 to S phase (G1 arrest). Expression of glucocorticoid receptors was not affected by TGF beta, although they are induced during the monocytic differentiation of myelogenous leukemia cells and have increased sensitivity to glucocorticoids. The expression of TGF beta receptors also was not enhanced by Dex. TGF beta significantly stimulated glucocorticoid responsive element-mediated transcription activity. Combined Dex and TGF beta stimulated the expression of c-jun and c-fos early responsive genes in U937 cells, although Dex or TGF beta alone did not. The combination synergistically induced expression of c-jun gene, reaching a maximum level at 24 h. On the other hand, expression of c-fos gene was induced by TGF beta alone and increased additively in combination with Dex. Treatment with antisense oligonucleotide complementary to the first exon of c-jun mRNA reduced the growth-inhibitory effect of Dex and TGF beta in a dose-dependent manner. However, exposure of U937 cells to the sense oligomer of c-jun mRNA or an antisense oligomer of c-fos mRNA did not affect the growth inhibition. These results suggested that the preferential expression of c-jun and stimulation of glucocorticoid responsive element-mediated transactivation are closely associated with the growth arrest of U937 cells incubated with Dex and TGF beta.

Immunoblot analysis of macrophage colony-stimulating factor (M-CSF) in KM 102 cell-conditioned medium showed the presence of two M-CSF molecular types, one being 85-kd M-CSF, the other a proteoglycan form (PG-M-CSF) carrying a chondroitin sulfate chain of variable length. When KM 102 cells were stimulated by TNF-alpha, they produced more M-CSF than that produced in unstimulated condition, in which PG-M-CSF had a shorter chondroitin sulfate chain. Although PG-M-CSF has binding affinity for type V collagen, the PG-M-CSF with the shorter chondroitin sulfate chain shows lower affinity. This spreads in type V collagen-containing agarose gel more easily than does PG-M-CSF with a longer chondroitin sulfate chain.

We studied 79 patients with adult T-cell leukaemia/lymphoma (ATL) and 11 human T-lymphotropic virus-type I (HTLV-I-carriers to investigate the clinical significance of absolute monocyte counts in peripheral blood. Monocytosis was observed in 20% of ATL patients, but in none of the HTLV-I carriers. ATL patients with absolute monocyte counts above 1.5 x 10(9)/l had a poorer prognosis than those with counts less than 1.5 x 10(9)/l. We also investigated serum levels of human monocytic colony-stimulating factor (hM-CSF) in 7 ATL patients and 11 HTLV-I carriers at the time of diagnosis before chemotherapy, and also in 5 normal healthy individuals. The differences among the 3 groups were not statistically significant. However, markedly increased serum hM-CSF levels were found in 3 ATL patients who were considered to be in the accelerated phase of the disease and 2 of whom were previously diagnosed and had received chemotherapy. There was no correlation between serum hM-CSF level and absolute monocyte count in ATL patients or HTLV-I carriers. The results of hM-CSF assay of supernatants of cultured ATL cells revealed that the ATL cells did not produce hM-CSF themselves. In an ATL patient with pleural involvement, the pleural hM-CSF level was lower than the serum level. These facts indicate that absolute monocyte count is one of the prognostic factors in ATL and the source of the elevated hM-CSF level in some patients with ATL is not ATL cells.(ABSTRACT TRUNCATED AT 250 WORDS)

In severe infection, the host responds to foreign agents and produces cytokines to activate lymphocytes and macrophages. Some of these cytokines can modulate growth and differentiation of myeloid leukemia cells. We examined differentiation-inducing activities in the sera from 9 patients with leukemia or lymphoma. These results indicate that some sera from infected patients, even with acute leukemia, have significant differentiation-inducing activities on both mouse and human leukemia cells, and that cytokines having differentiation-inducing activities varied for different specimens.

Protein kinase activities are involved in cellular proliferation and differentiation, and inhibitors of these activities are useful for studying the mechanisms of induction of differentiation. We found that staurosporine, an inhibitor of protein kinase activities, induced morphological differentiation of human myeloblastic leukemia ML-1 cells along myelomonocytic lineage and also induced functional differentiation (increase in nitroblue tetrazolium-reducing and lysozyme activities) in the cells. Several other protein kinase inhibitors such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), sphingosine, N-(6-aminoethyl)-5-chloro-1-naphthalenesulfonamide and 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-9) did not induce the differentiation of ML-1 cells. Treatment with staurosporine induced formation of granules in ML-1 cells, and the granules showed metachromasia by toluidine blue staining; however, histamine content did not increase. The "metachromatic" ML-1 cells were positive for CD14, indicating that staurosporine induced the differentiation of ML-1 cells into metachromatic monocytes/macrophages, 1 alpha,25-dihydroxyvitamin D3 (VD3) enhanced appearance of metachromatic granules in staurosporine-treated cells. These results suggest that modulation of protein phosphorylation by a staurosporine-sensitive protein kinase(s) may be associated with differentiation of ML-1 leukemia cells.

The localization of the human macrophage colony-stimulating factor (hM-CSF) in human trophoblast cells (syncytiotrophoblast and cytotrophoblast), decidual stromal cells, decidual lymphocytes, macrophages, and endometrial gland cells during the early stage of pregnancy was determined by the immunohistochemical examination and in situ hybridization. A large amount of hM-CSF was detected in the supernatant of the primary cultured cytotrophoblasts and decidual stromal cells. The decidual stromal cells secreted approximately 2 to 20 times as much hM-CSF as the trophoblast cells. Northern blot analysis showed that the placental and decidual tissues expressed the 4.0 kb hM-CSF-mRNA, however a larger amount of hM-CSF-related mRNA was found in the decidual than in the placental tissue. We also demonstrated the localization of hM-CSF mRNA expression in human decidual macrophages and CD16- CD56+ NK cells using the RT-PCR method. These findings indicate that hM-CSF is produced by decidual stromal cells, decidual macrophages, decidual CD16- CD56+ NK cells, and endometrial gland cells, as well as by trophoblasts. This increased production of hM-CSF may play a role in decidual function and placental function by the autocrine and paracrine system.

Serum levels of monokines, including macrophage colony-stimulating factor (M-CSF), tumor necrosis factor-alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha) and IL-1 beta (IL-1 beta), were measured in patients with chronic renal failure in an attempt to clarify the kinetics of these cytokines in the course of renal anemia. M-CSF was the only monokine detectable in the serum from all patients as well as healthy donors, making this cytokine feasible and reliable for serial evaluations. On all occasions, the level of M-CSF in uremic patients was significantly higher than that in healthy donors (29.4 +/- 12.3 vs. 5.5 +/- 1.1 ng/mL). In patients undergoing hemodialysis, the serum level of M-CSF was greater than that in patients undergoing continuous ambulatory peritoneal dialysis or in uremic patients without dialysis therapy. No difference was observed, however, in the levels of IL-1 alpha, IL-1 beta and TNF-alpha levels in these groups. Patients with severe anemia were subsequently treated with 60 to 80 U/kg per week of human recombinant erythropoietin (rhEpo) for 3 months. After this replacement therapy, hemoglobin levels increased with a variable change ranging from 0 to 3.5 g/dL. The pretherapy M-CSF level, however, was found to predict statistically the response to the therapy (p0.05). Patients with a lower pretherapy value responded better to rhEpo therapy; those with a higher level showed a minor degree of response. From these results, we postulate that the elevated M-CSF serum level in uremic patients is in part a consequence of the dialysis procedure and that rhEpo therapy is more effective in patients who are under sophisticated dialysis protocol and have a lower M-CSF level.