Fas ligand in human serum.

The Fas ligand (FasL), a member of the tumor necrosis factor family, induces apoptosis in Fas-bearing cells. The membrane-bound human FasL was found to be converted to a soluble form (sFasL) by the action of a matrix metalloproteinase-like enzyme. Two neutralizing monoclonal anti-human FasL antibodies were identified, and an enzyme-linked immunosorbent assay (ELISA) for sFasL in human sera was established. Sera from healthy persons did not contain a detectable level of sFasL, whereas those from patients with large granular lymphocytic (LGL) leukemia and natural killer (NK) cell lymphoma did. These malignant cells constitutively expressed FasL, whereas peripheral NK cells from healthy persons expressed FasL only on activation. These results suggested that the systemic tissue damage seen in most patients with LGL leukemia and NK-type lymphoma is due to sFasL produced by these malignant cells. Neutralizing anti-FasL antibodies or matrix metalloproteinase inhibitors may be of use in modulating such tissue damage.

Intrabullous blood injection for lung volume reduction.

Bronchoscopic treatment for emphysematous lung diseases has attracted clinical attention, and several different approaches are being investigated. We present a case of emphysematous bullae that was effectively treated with a newly developed bronchoscopic intervention, autologous blood injection. A 59-year-old man was referred to our institution with exertional dyspnoea. Chest CT showed emphysema and bullae with a diameter of 12 cm in the right upper lobe. Bronchoscopic treatment was introduced as an alternative to surgery. Autologous blood and fibrinogen solution were infused into bullae via the transbronchial catheter, under fluoroscopic guidance. Post-treatment CT showed marked contraction of bullae to a diameter of 3 cm, corresponding to a volume reduction of 800 ml on body plethysmography. A significant reduction in dyspnoea was also noted. This therapeutic approach is less invasive and may represent a good option for reducing lung volume.

Ganglioside complexes containing GQ1b as targets in Miller Fisher and Guillain-Barre syndromes.

BACKGROUND: Serum antibodies to GQ1b are associated with Miller Fisher syndrome (MFS) and Guillain-Barré syndrome (GBS) with ophthalmoplegia. Antibodies to ganglioside complexes (GSCs) have not yet been examined in a large population of patients with MFS or GBS. This study aimed to determine the clinical significance of antibodies to GSCs in MFS and GBS. METHODS: The study investigated serum anti-GSC antibodies and the clinical features in 64 MFS patients, 53 GBS patients with ophthalmoplegia (GBS-OP(+)) and 53 GBS patients without ophthalmoplegia (GBS-OP(-)). RESULTS: Thirty patients with MFS (47%), 25 with GBS-OP(+) (47%) and none with GBS-OP(-) had antibodies to GSCs containing GQ1b or GT1a. Patients with MFS and GBS-OP(+) were subdivided according to the antibody reactivities; patients with antibodies specific to GQ1b and/or GT1a (without anti-GSCs antibodies) were placed in Group 1, those with antibodies against GSCs with a total of two sialic acids in the terminal residues, such as GQ1b/GM1, were placed in Group 2, and those with antibodies against GSCs with a total of three sialic acids in the terminal residue, such as GQ1b/GD1a, were placed in Group 3. In MFS, sensory disturbances were infrequent in Group 2 compared with the other groups (p<0.0001). Antibodies specific to GQ1b were observed more often in MFS than in GBS-OP(+) (p = 0.0002). CONCLUSIONS: IgG antibodies to GSCs containing GQ1b or GT1a were closely associated with the development of ophthalmoplegia in GBS, as well as MFS. Both GQ1b and clustered epitopes of GSCs containing GQ1b or GT1a may be prime target antigens for MFS and GBS-OP(+).

Anti-ganglioside complex antibodies associated with severe disability in GBS.

Ganglioside complexes (GSCs) are known as target antigens in Guillain-Barré syndrome (GBS). To elucidate the clinical importance of the anti-GSC antibodies in GBS, we investigated serum antibodies to GSCs containing two of the gangliosides, GM1, GD1a, GD1b and GT1b, and analyzed clinical features of anti-GSC-positive GBS patients. Thirty-nine (17%) of 234 GBS patients had IgG anti-GSC antibodies. Anti-GSC-positive GBS had antecedent gastrointestinal infection and lower cranial nerve deficits more frequently than control GBS. The presence of antibody specificity to GD1a/GD1b and/or GD1b/GT1b was significantly associated with severe disability and a requirement for mechanical ventilation.

High-yield synthesis of conductive carbon nanotube tips for multiprobe scanning tunneling microscope.

We have established a fabrication process for conductive carbon nanotube (CNT) tips for multiprobe scanning tunneling microscope (STM) with high yield. This was achieved, first, by attaching a CNT at the apex of a supporting W tip by a dielectrophoresis method, second, by reinforcing the adhesion between the CNT and the W tip by electron beam deposition of hydrocarbon and subsequent heating, and finally by wholly coating it with a thin metal layer by pulsed laser deposition. More than 90% of the CNT tips survived after long-distance transportation in air, indicating the practical durability of the CNT tips. The shape of the CNT tip did not change even after making contact with another metal tip more than 100 times repeatedly, which evidenced its mechanical robustness. We exploited the CNT tips for the electronic transport measurement by a four-terminal method in a multiprobe STM, in which the PtIr-coated CNT portion of the tip exhibited diffusive transport with a low resistivity of 1.8 kOmega/microm. The contact resistance at the junction between the CNT and the supporting W tip was estimated to be less than 0.7 kOmega. We confirmed that the PtIr thin layer remained at the CNT-W junction portion after excess current passed through, although the PtIr layer was peeled off on the CNT to aggregate into particles, which was likely due to electromigration or a thermally activated diffusion process. These results indicate that the CNT tips fabricated by our recipe possess high reliability and reproducibility sufficient for multiprobe STM measurements.

Triterpenoid CDDO-Im downregulates PML/RARalpha expression in acute promyelocytic leukemia cells.

The triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) induces differentiation and apoptosis of diverse human tumor cells. In the present study, we examined the effects of the CDDO imidazolide imide (CDDO-Im) on the NB4 acute promyelocytic leukemia (APL) cell line and primary APL cells. The results show that CDDO-Im selectively downregulates expression of the PML/retinoic receptor alpha fusion protein by a caspase-dependent mechanism and sensitizes APL cells to the differentiating effects of all-trans retinoic acid (ATRA). CDDO-Im treatment of APL cells was also associated with disruption of redox balance and activation of the extrinsic apoptotic pathway. In concert with these results, CDDO-Im sensitizes APL cells to arsenic trioxide (ATO)-induced apoptosis. Our findings indicate that CDDO-Im may be effective in the treatment of APL by: (i) downregulation of PML/RARalpha; (ii) enhancement of ATRA-induced differentiation; and (iii) sensitization of ATO-induced APL cell death.

Anti-ganglioside complex antibodies in Miller Fisher syndrome.

BACKGROUND: Some ganglioside complexes (GSCs) are target antigens for serum antibodies in patients with Guillain-Barré syndrome (GBS). Anti-GSC antibodies may be associated with particular clinical features of GBS. OBJECTIVE: To investigate antibodies to GSCs in the sera of patients with Miller Fisher syndrome (MFS) characterised by elevation of the IgG anti-GQ1b antibody. RESULTS: In all, 7 of 12 (58%) consecutive patients with MFS were found to have IgG antibodies to GSCs containing GQ1b, of whom 5 had IgG antibodies to GQ1b-GM1 complex (GQ1b/GM1) and 2 had antibodies to GQ1b/GD1a; 4 of 5 patients without sensory symptoms had anti-GQ1b/GM1 antibodies. CONCLUSIONS: At least three different specificities in MFS-associated antibodies, GQ1b-specific, anti-GQ1b/GM1-positive and anti-GQ1b/GD1a-positive, were observed. In patients with MFS not only GQ1b itself but also clustered epitopes of GSCs, including GQ1b, may be considered to be prime target antigens for serum antibodies. A tendency to escape sensory disturbances is shown by anti-GQ1b/GM1-positive MFS.

Disinhibition of somatosensory evoked potential recovery in alcoholics.

The pathogenesis of cognitive impairment in alcoholics remains unclear. Previous studies suggested that diffuse white matter atrophy is associated with cognitive impairment in alcoholics. To elucidate this issue, the present study evaluated alcoholics with cognitive impairment using the somatosensory evoked potential (SEP) recovery method, which is suitable for detecting subtle dysfunction at the cortical level. Subjects comprised 12 alcoholics with mild cognitive impairment [Mild group: Mini Mental State Examination Score (MMSE), > or =24; mean, 27.9 +/- 1.6], 12 alcoholics with moderate to severe cognitive impairment (Moderate group: MMSE score, < 24; mean, 21.0 +/- 2.5) and 12 normal subjects (Control group). SEP was recorded from the hand sensory area contralateral to the median nerve stimulated at the wrist. Single-pulse or paired-pulse stimuli at various interstimulus intervals (10-300 ms) were administered. Recovery functions of N9 (a peripheral nerve component), N20, N20-P25 and P25-N33 (cortical components) were studied. N20 recovery curves of both alcoholic groups were less suppressive than those of Controls, and P25-N33 recovery curves of the Moderate group were more excitatory than those of the Mild or Control groups. A disinhibited recovery pattern of N20 indicates subcortical dysfunction, and a disinhibited pattern of P25-N33 would be induced by cortical dysfunction. Therefore, subcortical dysfunction indicated by an abnormal N20 recovery pattern may contribute to the early cognitive impairment of alcoholics, whilst the cortical dysfunction indicated by an abnormal P25-N33 recovery pattern may contribute to the later cognitive impairment of alcoholics.

In vitro differentiation of leukemic progenitor cells in various types of acute nonlymphocytic leukemia.

The effects of colony-stimulating factors on differentiation of leukemic progenitor cells were investigated in various types of acute nonlymphocytic leukemia. Two different sources of colony-stimulating factors were used in this study: human placental conditioned medium; and phytohemagglutin-stimulated leukocyte-conditioned medium. At the end of culture, colony types were determined by dual esterase staining in permanent preparations. The majority of the colonies formed in acute myeloblastic and acute promyelocytic leukemias were neutrophilic even when stimulated by phytohemagglutinin-stimulated leukocyte-conditioned medium, which contains a potent stimulator of macrophage colony growth from normal marrow cells. On the other hand, both neutrophilic and monocytic colonies were formed in acute myelomonocytic leukemia (AMMoL). The proportions of these two types of colonies were variable, depending on the nature of added colony-stimulating factor and its concentration. These findings suggest that the leukemic progenitors in acute myeloblastic and acute promyelocytic leukemias have a tendency to differentiate mainly into a neutrophilic lineage in vitro and that the leukemic progenitors in AMMoL differentiate into both neutrophilic and monocytic lineages in vitro, in two cases of esterase-negative AMMoL, both neutrophilic and monocytic colonies were detected as in the other well-defined cases of AMMoL. This study seems to be of value in understanding the nature of leukemic progenitor cells and also shows that morphological analysis of leukemic colonies may be helpful in the classification of acute nonlymphocytic leukemia.

Radiation sensitivity of leukemic progenitor cells in acute nonlymphocytic leukemia.

The radiation sensitivity of leukemic progenitor cells in 12 cases of acute nonlymphocytic leukemia was compared with that of normal myeloid progenitor cells (colony-forming units in culture), using in vitro cloning techniques. The D0 value for normal colony-forming units in culture was almost constant (130 +/- 14 rads). On the other hand, marked patient-to-patient variations were observed in the radiosensitivity of leukemic progenitor cells; namely, the D0 values in the present cases ranged from 30 to 210 rads. These variations seemed to be partly due to different cell cycle status and partly due to the intrinsic nature of leukemic progenitor cells. Moreover, in six of seven clinically drug-sensitive cases, the leukemic progenitor cells were proved to be relatively radiosensitive. Similar to in vitro drug sensitivity tests, this test may serve to predict the clinical response to chemoradiotherapy.

Inhibitory activity on murine granulocytic colony formation of bone marrow cell-conditioned medium obtained from colony-stimulating factor-producing tumor-bearing nude mice.

The effects of bone marrow-conditioned medium obtained from colony-stimulating factor-producing tumor-bearing nude mice (G-BM-CM) on mouse and human granulocyte-macrophage colony formation and mouse erythroid colony and burst formation were studied. Addition of G-BM-CM into the mouse granulocyte-macrophage colony-forming system containing colony-stimulating activity more strongly inhibited granulocyte colony formation than did mixed granulocyte-macrophage and macrophage colony formation, while it did not change the number of granulocyte colonies formed by human bone marrow cells stimulated by human granulocyte colony-stimulating activity. Addition of G-BM-CM slightly increased mouse erythroid colony and burst numbers when it was added into an erythroid colony-forming system stimulated by erythropoietin (1 unit/ml), and into the erythroid burst-forming system stimulated by erythropoietin (1 unit/ml) and 7% spleen cell-conditioned medium. These results might indicate that G-BM-CM mainly blocked commitment of mouse granulocyte-macrophage colony-forming cells to granulocytic progeny.

Clonal growth of human acute myeloid leukemia cells (ML-1 and HL-60) in serum-free agar medium.

Human acute myeloid leukemia (ML-1 and HL-60) cells grew continuously in the serum-free liquid medium supplemented with human transferrin and bovine insulin. Both ML-1 and HL-60 cells formed clusters and colonies in the serum-free agar medium supplemented with bovine serum albumin, human transferrin, cholesterol, and L-alpha-phosphatidylcholine. Medium conditioned by phytohemagglutinin-stimulated leukocytes prepared in the absence of serum had three types of colony-stimulating factors on normal human bone marrow cells. When fetal calf serum was present, medium conditioned by phytohemagglutinin-stimulated leukocytes stimulated the clonal growth of HL-60 cells at the lower concentration. However, it inhibited that of ML-1 cells. In contrast, under serum-free conditions, medium conditioned by phytohemagglutinin-stimulated leukocytes promoted the clonal growth of both ML-1 and HL-60 cells at the lower concentrations. The study using a Sephadex G-200 column revealed that, in the serum-supplemented cultures, HL-60 cells responded to one of the three colony-stimulating factors and an activity with molecular weight of around 12,000, while ML-1 cells responded only to an activity with molecular weight of around 12,000. In the serum-free cultures, both ML-1 and HL-60 cells were stimulated by activities with molecular weights of 62,000 and 54,000, respectively. These studies demonstrate that the determination of growth factors for cell lines is dependent on culture conditions, particularly on serum component; that there is a heterogeneity of ML-1 and HL-60 cells in response to the growth factors; and that there is potential importance of demonstration of heterogeneity among different cell lines in establishing requirements for different stages of differentiation.

Human colony-stimulating activity-producing tumor: production of very low mouse-active colony-stimulating activity and induction of marked granulocytosis in mice.

We reported previously a human lung cancer which induced marked granulocytosis both in the patient and in the tumor-transplanted nude mice (G-1 mice), and whose conditioned media (G-1-T-CM) contained both human-active colony-stimulating activity (h-CSA) (810 colonies/ml) and mouse-active colony-stimulating activity (m-CSA) (530 colonies/ml), suggesting that the CSA produced by the tumor caused granulocytosis both in the patient and in G-1-mice. We reported here another human lung cancer which induced marked granulocytosis both in the patient and in the tumor-transplanted nude mice (G-2-mice). However, in contrast to the tumor reported by us previously, the conditioned media of this tumor (G-2-T-CM) contained only very weak m-CSA (86 colonies/ml), although h-CSA was very strong (7332 colonies/ml). The ratio of m-CSA to h-CSA in G-1-T-CM (0.654) was 55-fold higher than that in G-2-T-CM (0.012). Gel filtration of G-2-T-CM on a Sephadex G-150 column denied the presence of colony-inhibiting activity which inhibited m-CSA in G-2-T-CM. Since the addition of serum obtained from G-2 mice did not enhance m-CSA in G-2-T-CM, it seems unlikely that the G-2 tumor produced an inactive form of m-CSA which was activated in G-2 mouse serum. G-2-T-CM tended to maintain the number of colony-forming units in spleen in mouse bone marrow during liquid cultures, while G-1-T-CM did not. These results may indicate that the G-2 tumor produced a humoral factor which has very weak CSA and acts on stem cells rather than on committed progenitors.

t(11;14)(q23;q24) generates an MLL-human gephyrin fusion gene along with a de facto truncated MLL in acute monoblastic leukemia.

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.

Enhancing effect of human monocytic colony-stimulating factor on monocyte tumoricidal activity.

Human monocytic colony-stimulating factor (hM-CSF) enhances several effector functions of human peripheral blood monocytes. In this study, we investigated the effect of the Mr 85,000 form of hM-CSF on the tumoricidal activity of human monocytes against several leukemic cell lines using a 12-h chromium release assay. Human peripheral blood monocytes preincubated with hM-CSF for 48 h showed more effective killing activity towards K562, U937, Daudi, and HL60 cells as compared with the cells preincubated with medium alone. Maximal enhancement of the tumoricidal activity was achieved by hM-CSF at concentrations of 50-100 ng/ml. A trace amount of lipopolysaccharide contained in the hM-CSF did not seem to contribute to the enhancing effect, as the addition of a lipopolysaccharide-neutralizing agent, polymyxin B, to the preincubation mixture did not reduce this effect. Anti-tumor necrosis factor antiserum partially blocked the tumoricidal activity mediated by hM-CSF, indicating that tumor necrosis factor may participate in the hM-CSF-mediated increase of monocyte tumor cell-killing activity. These results suggest that in addition to other monocyte-activating factors, hM-CSF augments monocyte tumoricidal activity against a wide spectrum of tumor targets.

A human osteoblastic cell line, MG-63, produces two molecular types of macrophage-colony-stimulating factor.

A human osteoblastic cell line, MG-63, mouse primary osteoblasts, and a mouse osteoblastic cell line, MC3T3-E1, were shown to produce macrophage-colony-stimulating factor (M-CSF) by bone-marrow-cell colony assay, using a specific neutralizing antibody for M-CSF. Immunoblot analysis of M-CSF, produced by MG-63 cells, revealed the presence of a higher-molecular-weight species of M-CSF, in addition to the 85-kDa M-CSF. The higher-molecular-weight species had a high affinity to the DEAE-Sephacel column and was sensitive to chondroitinase ABC and AC. These physico-chemical profiles were wholly compatible with those of the proteoglycan form of M-CSF (PG-M-CSF), which was recently identified by our group in the conditioned medium of Chinese hamster ovary cells transfected with the 4.0-kb cDNA of the M-CSF gene. Conditioned medium of MG-63 cells was fractionated by DEAE-Sephacel column chromatography, and the M-CSF of each fraction was measured by both enzyme-linked immunosorbent assay and bone-marrow-cell colony assay. The fractions eluted by 0.3-0.6 M NaCl, which were shown to contain only PG-M-CSF on immunoblot analysis, also have macrophage-colony-stimulating activity.

Differentiation of human monoblastic leukemia U937 cells induced by inhibitors of myosin light chain kinase and prevention of differentiation by granulocyte-macrophage colony-stimulating factor.

Inhibitors of protein kinase activities are useful for the study of intracellular signal transduction and some of these inhibitors are reported to induce differentiation of human leukemia cells. We examined effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) in combination with several kinase inhibitors on differentiation of human leukemia U937 cells. Nitroblue tetrazolium (NBT)-reducing activity, a typical marker of myelomonocytic differentiation, of U937 cells was induced by genistein and GM-CSF enhanced this activity. GM-CSF also induced the NBT-reducing activity of the cells in combination with 2,5-dihydroxycinnamic acid methyl ester, psi-tectorigenin and staurosporine, although each of them did not induce the activity. Inhibitors of myosin light chain kinase, 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-9) and 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7), induced in U937 cells NBT-reduction, and lysozyme and alpha-naphthyl acetate esterase activities. GM-CSF inhibited this differentiation and counteracted the anti-proliferation effect of the kinase inhibitors. These results suggest that some protein kinases are involved in differentiation of U937 cells and the kinases inhibited by ML-9 and ML-7 are associated with signal transduction of GM-CSF.

Restriction endonuclease sensitivity of DNA containing globin genes in different murine erythroleukemia cell lines.

The arrangement of the globin structural genes has been examined in murine erythroleukemia cells, strain DS19, and several related inducer-resistant variant cell lines. One fragment larger than 20 kilobases and six globin gene-containing fragments between 10 and 1.9 kilobases in size are detected in EcoRI-cleaved purified DNA prepared from strain DS19. By comparison, when isolated nuclei are digested with EcoRI, only two globin gene-containing fragments are detected, one greater than 20 kilobases and the other 1.9 kilobases. Of seven cell lines derived from DS19 and resistant to inducers, six had similar patterns to DS19 of globin gene-containing EcoRI-generated DNA fragments from nuclei and from purified DNA. One cell line, DR10, a DMSo-resistant cell line, lacks the 1.9 kilobase fragment after digestion of either nuclei or purified DNA. The 1.9 kilobase fragment hybridizes with alpha-globin cDNA but not with the beta-globin cDNA, suggesting either rearrangement or deletion of an alpha-globin gene-like fragment in DR10 DNA.

Differanisole A, a novel antitumor antibiotic, enhances growth inhibition and differentiation of human myeloid leukemia cells induced by 9-cis retinoic acid.

Differanisole A, 3,5-dichloro-2-hydroxy-4-methoxy-6-n-propylbenzoic acid, inhibited growth of human myeloid leukemia cells. The compound induced G1 arrest and granulocytic differentiation of HL-60 cells, although the differentiation-inducing effect was modest. Differanisole A and 9-cis retinoic acid (9cisRA) synergistically inhibited the growth and induced functional and morphologic differentiation of HL-60 and NB4 cells, whereas the combined treatment with differanisole A and all-trans retinoic acid or 1alpha,25-dihydroxyvitamin D3 was less effective. Similar results were obtained in primary culture of leukemia cells from a patient with acute promyelocytic leukemia. The synergistic effect on growth inhibition and induction of differentiation required simultaneous treatment with differanisole A and 9cisRA. Differanisole A and an RXR-specific ligand (Ro47-5944) cooperatively inhibited the cell growth, while the combined effect of differanisole A and an RAR-specific ligand Am80 was just additive. Differanisole A in combination with 9cisRA may have implications for therapy of acute promyelocytic leukemia patients.

Quantitative analysis of the two macrophage colony-stimulating factor mRNA expressed in a human stromal cell line by reverse transcription-polymerase chain reaction (RT-PCR).

We established a quantitative analysis system for 4.0 kb and 1.6 kb macrophage colony-stimulating factor (M-CSF) mRNA, using reverse transcription-polymerase chain reaction. Using this system, we performed quantitative analysis of the two mRNAs expressed in the human stromal cell line, KM102, in the resting condition and when stimulated by various concentrations of interferon-gamma (IFN-gamma). The expression of 1.6 kb M-CSF mRNA was more efficiently stimulated by IFN-gamma than that of 4.0 kb M-CSF mRNA. The alternative splicing of a single M-CSF gene has been shown to generate several M-CSF proteins with different localization; we believe that molecular analysis of the transcription products by this system is important to better understand the physiological significance of the different species of M-CSF derived from each mRNA.