Resistance of infant leukemia with MLL rearrangement to tumor necrosis factor-related apoptosis-inducing ligand: a possible mechanism for poor sensitivity to antitumor immunity.

Malignant cells generally acquire some immune escape mechanisms for clonal expansion. Immune escape mechanisms also contribute to the failure of graft-versus-leukemia (GVL) effect after allogeneic hematopoietic stem cell transplantation (allo-SCT). Infant leukemias with mixed-lineage leukemia (MLL) rearrangement have a remarkably short latency, and GVL effect after allo-SCT has not been clearly evidenced in these leukemias. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)- and FasL-mediated cytotoxic pathways play important roles in cytotoxic T-lymphocyte- and natural killer cell-mediated antitumor immunity and optimal GVL activity. We investigated the in vitro sensitivity of MLL-rearranged acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML) cells to TRAIL- and FasL-mediated cytotoxicity. Most of cell lines and primary leukemia cells were highly resistant to TRAIL primarily owing to low cell-surface expression of death receptors in ALL and simultaneous expression of decoy receptors in AML. Nearly half of cell lines and majority of primary leukemia cells showed low sensitivity to FasL. These results suggest that resistance to death-inducing ligands, particularly to TRAIL, could be one of the mechanisms for a rapid clonal expansion and a poor sensitivity to the GVL effect in infant leukemias with MLL rearrangement.

Mechanical strain effect on bone-resorbing activity and messenger RNA expressions of marker enzymes in isolated osteoclast culture.

Adaptive modeling and remodeling are controlled by the activities of osteoblasts and osteoclasts, which are capable of sensing their mechanical environments and regulating deposition or resorption of bone matrix. The effects of mechanical stimuli on isolated osteoclasts have been scarcely examined because it has proven to be difficult to prepare a number of pure osteoclasts and to cultivate them on mineralized substratum during mechanical stimulation. Recently, we developed an apparatus for applying mechanical stretching to the ivory slice/plastic plate component on which cells could be cultured. The loading frequency, strain rate, and generated strain over an ivory surface could be controlled by a personal computer. Using this apparatus, we examined the role of mechanical stretching on the bone-resorbing activity of the osteoclasts. Mature and highly enriched osteoclasts were cultured for 2, 12, and 24 h on the ivory/plate component while being subjected to intermittent tensile strain. The stretched osteoclasts showed enhanced messenger RNA (mRNA) expression levels of osteoclast marker enzymes, tartrate-resistant acid phosphatase (TRAP), and cathepsin K and increases of resorbed-pit formation, suggesting that the mechanical stretching up-regulated the bone-resorbing activity of the osteoclasts. A stretch-activated cation (SA-cat) channel blocker significantly inhibited the increases of the mRNA level and pit formation after 24 h of stretching. This study suggested the possibility that the mature osteoclasts responded to mechanical stretching through a mechanism involving a SA-cat channel in the absence of mesenchymal cells and, as a result, up-regulated their bone-resorbing activity.

Osteopontin involvement in integrin-mediated cell signaling and regulation of expression of alkaline phosphatase during early differentiation of UMR cells.

To clarify the function of osteopontin in osteoblast differentiation, we have examined the signal transduction pathway in an osteoblastic cell line (UMR106-6) bound to osteopontin, fibronectin, vitronectin and collagen type I surfaces. This was done by investigating the production and autophosphorylation of focal adhesion kinase (FAK) and the expression of alkaline phosphatase (ALP) at the transcription level. Results suggest that osteopontin was not only responsible for the autophosphorylation of FAK but regulated the expression of ALP, which was strongly correlated with FAK activity. These results suggest that osteopontin might act as a trigger in the early differentiation of osteoblasts.

Zinostatin stimalamer-transcatheter arterial embolization for hepatocellular carcinoma: a comparison with lipiodol-transcatheter arterial embolization.

Twenty patients with unresectable hepatocellular carcinoma were treated by zinostatin stimalamer-transcatheter arterial embolization (SMANCS-TAE). After administration of SMANCS, a superselective catheterization technique was used to inject gelatin sponge particles into the artery or artery branch supplying the cancer-bearing segment. We compared the results of SMANCS-TAE with Lipiodol (Yamanouchi, Tokyo, Japan)-TAE performed during the same period. In 18 of 20 patients (90%), a tumor necrosis rate of 100% (grade 4) was obtained after one or two courses of SMANCS-TAE. The SMANCS group was superior to the Lipiodol-TAE group in terms of the tumor reduction rate, alpha-fetoprotein reduction rate, and cumulative 1.5-year survival rate, but not significantly. No severe side effects were noted after SMANCS-TAE. SMANCS-TAE appears to have potential as a new treatment for hepatocellularcarcinoma, and patients treated with this technique will be monitored to elucidate the long-term effects.

Comparison of various lazaroid compounds for protection against ischemic liver injury.

Lazaroids are a group of 21-aminosteroids that lack steroid action but have a potent cytoprotective effect by inhibiting iron-dependent lipid peroxidation. However, there have been conflicting reports on the effectiveness and potency of the various lazaroid compounds. In this study, we compared the effectiveness of three major lazaroids on warm liver ischemia in dogs using a 2-hr hepatic vascular exclusion model. The agents were given to the animals intravenously for 30 min before ischemia. The animals were divided into 5 groups: Control (n=10), no treatment; Group F (n=6), U-74006F (10 mg/kg); Group G (n=6), U-74389G (10 mg/kg); Group A1 (n=6), U-74500A (10 mg/kg); Group A2 (n=6), U-74500A (5 mg/kg). The effect of treatment was evaluated by two-week animal survival, hepatic tissue blood flow, liver function tests, blood and tissue biochemistry, and histological analyses. Animal survival in all treated groups was significantly improved compared with the control (83-100% versus 30%). Elevation of liver enzymes after reperfusion was markedly attenuated in treated groups, except for an early significant increase in Group G. Postreperfusion hepatic tissue blood flow was much higher in all treated animals (50% of the preischemic level vs. 25% in the control). Lazaroids, particularly U-74500A at 5 mg/kg (Group A2), suppressed adenine nucleotide degradation during ischemia and enhanced the resynthesis of high-energy phosphates after reperfusion. Although structural abnormalities in postreperfusion liver tissues were markedly ameliorated in all treated groups, Group A2 showed significantly less neutrophil infiltration. Liver injury from warm ischemia and reperfusion was attenuated with all lazaroid compounds, of which U-74500A at 5 mg/kg exhibited the most significant protective activity.

Mucosal damage and recovery of the intestine after prolonged preservation and transplantation in dogs.

BACKGROUND: Although much is known about the mucosal damage that occurs after intestinal warm ischemia and reperfusion and its recovery, little is known about the effect of cold preservation and transplantation on the mucosa. We studied the electrophysiological, biochemical, and histological changes of the intestinal mucosa after preservation for 24 hr and subsequent transplantation. METHODS: The small intestines from adult mongrel dogs were harvested. The intestines were orthotopically autotransplanted immediately (control group) or after preservation for 24 hr (preservation group). Jejunal and ileal tissues were taken before harvesting, at the end of preservation, 1 hr after reperfusion, and on postoperative days 3, 7, 14, and 28. The Ussing chamber method was used to study the electrophysiologic changes. Tissue maltase, diamine oxidase, and ornithine decarboxylase were measured. A histological analysis was also performed. RESULTS: Control group grafts showed no evident deterioration in electrophysiology, biochemistry, or morphology. In contrast, preservation group grafts exhibited electrophysiological and biochemical degradation, complete denudation of the villi, and crypt injury (especially in the ileum) after reperfusion. Electrophysiologic function and the mucosa biochemical marker recovered within 3 days in the jejunum and within 7-14 days in the ileum; however, histological recovery of mucosal injury required 28 days in the jejunum and more than 28 days in the ileum. CONCLUSIONS: Our study showed that despite severe destruction of mucosal integrity by prolonged preservation and transplantation, the intestinal mucosa has an enormous regenerative capacity. Our study also showed that regeneration was more pronounced in the jejunum than in the ileum.

Lazaroid U-74389G for 48-hour canine liver preservation.

Lazaroids have been reported to attenuate preservation and reperfusion injury. In this study, we examined whether lazaroids can improve the outcome after 48-hr canine liver preservation and transplantation. Adult female beagle dogs were randomized into 4 dosage groups (5 animals each). Lazaroid U-74389G was intravenously administered at a dose of 0 mg/kg, 6 mg/kg, 10 mg/kg, or 15 mg/kg to donors 30 min before harvesting and also to recipients 30 min before revascularization. Control animals (0 mg/kg) were given the lazaroid vehicle. The liver grafts were orthotopically transplanted after 48 hr of hypothermic preservation in UW solution. Lazaroid treatment significantly improved outcome after transplantation. Five-day animal survival increased from 0% in the control to 60% in the 6 mg/kg group, 100% in the 10 mg/kg group, and 80% in the 15 mg/kg group. Lazaroid protected the hepatocytes from damage during preservation, and enhanced energy charge and hepatic blood flow after reperfusion. Histological alterations were significantly less severe in the lazaroid-treated groups. The area of necrotic hepatocytes decreased from 43.7 +/- 17.7 in the control to 13.5 +/- 3.0 in the lazaroid 10 mg/kg group. These results indicate that lazaroid U-74389G has potential for improvement of clinical liver preservation.

The binding ability to matrix proteins and the inhibitory effects on cell adhesion of synthetic peptides derived from a conserved sequence of integrins.

The beta peptide (113-125), derived from a conserved sequence of the beta subunit of integrins, was synthesized to investigate its adhesive properties to matrix proteins and the effects on cell adhesion to immobilized fibronectin. In this study, we observed that the biotinylated beta peptide was able to bind efficiently to immobilized fibronectin, fibrinogen, collagen Type I and vitronectin with different degrees of affinity. It was also demonstrated that biotinylated fibronectin or fibrinogen could bind to the coated beta peptide. This kind of binding, which might be non-covalent linkage, was partially blocked by coincubation with the peptide GRGDS or EDTA, but not by SDGRG. Cell adhesion experiments were performed to study the effect of the beta peptide. The data showed that the beta peptide partially inhibited both fibroblast L929 and MC3T3-E1 osteoblastic cells from adhering to immobilized fibronectin in a dosage-dependent manner. In the presence of 100 microM concentration of the beta peptide, the inhibition rate of cell adhesion was 34% for fibroblast L929 cells and 54.1% for MC 3T3-E1 osteoblastic cells. This research suggests that the beta peptide might act independently as an adhesive region of the beta subunit of integrins and may occupy the cell-binding site within fibronectin.

Postoperative intravenous administration of lysine acetylsalicylate: effect on pain relief and platelet function.

The effects of lysine acetylsalicylate, a new injectable salicylate, on postoperative pain relief and platelet function were studied in ten men who had surgery for peptic ulcer. Four of five patients receiving lysine acetylsalicylate had satisfactory pain relief. One patient required an additional injection of 15 mg of pentazocine. Of the five patients in the control group, an average (+/- SD) dose of 90 +/- 23.7 mg of pentazocine was required to achieve adequate postoperative pain relief. Lysine acetylsalicylate decreased platelet aggregability but without resulting in hemorrhage. We concluded that this new salicylate administered intravenously to patients in the postoperative period provided adequate analgesia while allowing effective hemostasis despite its inhibitory effect on platelet aggregation.

Stimulation of rat hepatocyte proliferation in vitro and in vivo by factors derived from the bovine small intestinal mucosa.

A factor with a molecular weight of less than 1 kDa in the mucosa of the bovine small intestine (low molecular weight factor or LMW factor) stimulated DNA synthesis in rat hepatocytes in primary culture. This factor only showed its activity when it was added with a larger factor with a molecular weight of 30 kDa that was also found in the same tissue (high molecular weight factor or HMW factor). The LMW factor probably acts to enhance the action of a hepatotrophic growth factor, since EGF and HGF can substitute for the HMW factor. The action of the LMW factor was not due to the actions of low molecular weight substances such as norepinephrine, estradiol, triiodothyronine, and putrescine, which enhance the action of EGF or HGF, since substantial amounts of these substances were not found in the extract. When intraperitoneally administered into rats, after two-thirds hepatectomy, the LMW factor enhanced hepatocyte proliferation without the administration of the HMW factor. In the regenerating liver, a hepatotrophic growth factor(s), which acts synergistically with the LMW factor, might be properly provided, but the supply of the LMW factor might be below the level that maximally stimulates hepatocyte proliferation.

Characterization of two esteroproteases from the male mouse submandibular gland.

Three major esteroproteases, proteases A and D and P-esterase, obtained from the glands were studied kinetically and chemically; two (proteases A and D) were identified. Protease A is composed of a single subunit, molecular weight (27,600) similar to the native molecule (27,000); protease D consists of three subunits, approximate molecular weights of 9200, 7600 and 4600. P-esterase contains two subunits, approximate molecular weights of 7100 and 14,000. Protease A exhibits a strong kinin-releasing activity; the other two enzymes have low activity. Protease D binds to low molecular weight-epidermal growth factor, forming a complex which has an electrophoretic mobility similar to that of high molecular weight-epidermal growth factors. When beta-nerve growth factor was incubated with protease A, the amino-terminal amino acid, serine, was lost from the growth factor and a new amino-terminal amino acid, methionine, appeared. These data indicate that proteases D and A are the same proteins as epidermal growth factor-binding protein and beta-nerve growth factor endopeptidase, respectively. From a comparison of the peptide maps of trypsin-digests of the enzymes, the proteases A and D were inferred to have a similar primary structure.

Brasilinolide A, a new macrolide antibiotic produced by Nocardia brasiliensis: producing strain, isolation and biological activity.

A new 32-membered macrolide antibiotic, brasilinolide A was isolated from the fermentation broth of Nocardia sp. IFM 0406. The producer was identified as Nocardia brasiliensis. The antibiotic was only active against Aspergillus niger, but not active against other fungi including yeasts as well as other filamentous like fungi and bacteria. Brasilinolide A exerted an immunosuppressive activity in the assay system of a mixed lymphocyte reaction (MLR).

Brasiliquinones A, B and C, new benz[alpha]anthraquinone antibiotics from Nocardia brasiliensis. I. Producing strain, isolation and biological activities of the antibiotics.

New benz[alpha]anthraquinone antibiotics (brasiliquinones A, B and C) with an ethyl group at C-3 were isolated. The producer was identified as Nocardia brasiliensis. The antibiotics were active against Gram-positive bacteria including Mycobacterium sp., but not active against Gram-negative bacteria or fungi. They were also active against multiple drug-resistant P388/ADR tumor cells.

Brasilicardin A, a new terpenoid antibiotic from pathogenic Nocardia brasiliensis: fermentation, isolation and biological activity.

A novel tricyclic diterpenoid antibiotic, brasilicardin A, was isolated from the culture broth of Nocardia brasiliensis IFM 0406. The antibiotic exhibited immunosuppressive activity in a mouse mixed lymphocyte reaction (MLR) assay system and its IC50 value was 0.057 microg/ml. Although the inhibitory activity of cyclosporin A (CyA) against IL-2 production was confirmed in the MLR assay system, brasilicardin A did not have the activity. The results of in vitro toxicity testing of brasilicardin A against various human cell lines were compared with those of CyA.

Glutamine metabolism of intestine grafts: influence of mucosal injury by prolonged preservation and transplantation.

The demand for glutamine increased only in the preserved intestine in the early postoperative period (3 days after transplantation). Glutamine demand of the preserved grafts returned to control and immediate levels 7 and 14 days after transplantation. Three days after intestinal transplantation, when the intestinal mucosa was actively regenerating, the demand for glutamine was markedly enhanced. The enhanced demand for glutamine was met by increased output of glutamine by the liver and skeletal muscle. Glutamine uptake by the intestinal graft was enhanced by a brief infusion of glutamine. Thus, we believe exogenous glutamine supplementation may be beneficial for the recovery of intestinal grafts with severe mucosal injury.

A simple and rapid purification procedure for the preparation of tetramethylrhodamine-labeled antibodies.

A conjugate preparation of antibody (Fab) and tetramethylrhodamine (TMR) generally adsorbs free TMR which is very difficult to remove because of its strong hydrophobic binding. On the basis of criteria such as sodium dodecyl sulfate (SDS) gel electrophoresis and staining of the plasma membrane of live cells, we found that simple extraction with n-butyl alcohol or iso-amyl alcohol could remove the contaminating free dye. The procedure is especially useful when one needs to prepare conjugates with low nonspecific binding for the study of lateral diffusion of cell membrane-associated antigens.

Intrathecal baclofen therapy; patient selection & team approach.

Intrathecal baclofen therapy (ITB therapy) is useful for severe spasticity. However, the Ministry of health, labour and welfare has not allowed the therapy in Japan. A clinical trial of intrathecal baclofen therapy is currently under way. In this paper the situation in Japan with regard to ITB therapy is described and information for physicians in Japan given from our experience. Team approach is important for ITB therapy. Special attention should be paid to patient selection.

[Changes in metabolism and peritumoral circulation after radiosurgery of metastatic brain tumors: evaluation by three dimensional dual isotope SPECT].

Concurrent use of two different isotopes, 201T1C1 and 123I-IMP, in SPECT is useful in separative evaluation of tumor metabolism and peritumoral circulation. Three dimensional SPECT employed in our study has an obvious advantage over two dimensional SPECT for its accurate imaging of tumors and peritumoural areas. Changes of tumor metabolism and regional circulation in peritumoral edematous tissues were investigated by fused 3-D SPECT images using 201T1C1 and 123I-IMP. In this study, the volume of isotope accumulative and isotope defective regions were measured. Fusion of SPECT images was performed by the use of panning visualization software; Application Visualization System Medical View (K.G.T.). The threshold of 3-D rendering was determined by conforming the volume of the hemisphere and of the tumor estimated on CT to the volume of 123I-IMP and 201T1C1 accumulating area respectively. Accumulative volume of 201T1C1 in the tumor decreased remarkably at 7 days after radiosurgery (p < 0.01). Defective volume of peritumoral hypoperfusion was measured on 3-D SPECT. The average volume was 80.5 + 32.5cm3 before radiosurgery. It decreased by approximately 60% at 7 days after radiosurgery (p < 0.05). Analysis of 3-D SPECT images using two different isotope tracers is reliable and useful to evaluate early the changes of metabolism and peritumoral circulation in or around intracerebral tumors.

[Cord blood transplantation with two mismatched HLA loci in a child with acute lymphoblastic leukemia in second remission: follow-up of minimal residual disease using a clone-specific probe].

A 9-year-old girl with acute lymphoblastic leukemia in second remission underwent cord blood transplantation (CBT) from an HLA-mismatched (2 loci by serotype, 3 loci by genotype) unrelated donor. The infused nucleated cell count was 1.95 x 10(7)/kg. FK506 and mini-MTX were used to prevent graft-versus-host disease (GVHD), but grade II acute GVHD developed on the skin (stage III). The GVHD subsided after administration of corticosteroid, but marked hyperglycemia developed, which required transient insulin therapy for its control. Minimal residual disease (MRD) was assessed using a clone-specific probe for the JH region. MRD was positive before CBT, but became negative one month after CBT. Now, at 14 months after CBT, the patient is in a disease-free state without detectable MRD. These observations suggest that CBT with two mismatched HLA loci can be performed safely, and that sequential analysis of MRD is useful for evaluation of the disease status after CBT.

[Changes of regional perfusion in metastatic brain tumor and peritumoral area after radiosurgery: a study by 123I-IMP dynamic SPECT].

Changes of regional perfusion in the tumor, peritumoral edematous area and juxtatumor brain after radiosurgical treatment for metastatic brain tumor were investigated by dynamic SPECT using 123I-IMP. The SPECT was performed in 12 patients before and 1, 7 and 30 days after stereotactic irradiation. A region of interest (ROI) was selected each in the tumor, peritumoral edematous area, juxtatumor brain and ipsilateral cerebellum. Radioactivity in each ROIs was counted on early SPECT based on dynamic SPECT from 0 to 5 minutes. Mean count/pixel in each ROIs was divited by mean count/pixel in the ROI of the ipsilateral cerebellum and its value was designated as a count ratio (CR). Assuming the pre-treatment CRs are 1.0, relative changes of post-treatment CRs investigated. rCR in the tumor did not show any significant change after radiosurgical treatment. rCR in the edematous area and the juxtatumor brain increased at 7 days after irradiation [Mean +/- SD 1.43 +/- 0.409 (p < 0.05), 1.248 +/- 0.228 (p < 0.05) by Mann-Whitney test] and at 30 days [1.359 +/- 0.245 (p < 0.01), 1.301 +/- 0.287 (p < 0.01)] respectively. Computed tomography revealed no change in the maximum diameter of the tumor at 1 month after irradiation but a significant reduction in the diameter [0.744 +/- 0.227 (p < 0.02)] at 2 months. Early improvement of regional cerebral blood flow in the juxtatumor areas after radiosurgery suggested that radiosurgery could be effective treatment for metastatic brain tumor.