Concanavalin A-induced Suppressor Cell Activity in Lipoid Nephrosis

Suppressor cell activity (SCA) was analysed in 28 patients with lipoid nephrosis (LN) and I patients with chronic proliferative glomerulonephritis (CGN). We have assessed the ability of peripheral blood lymphocytes (PBL) stimulated by concanvalin A(Con A) to inhibit the proliferative response of normal allogeneic lymphocytes by both Con A and phytohaemagglutinin (PHA). It was found that the LN patients in the earlier phase of relapse had significantly increased levels of suppression index (SI) when compared with the values obtained with normal controls. In contrast, the mean suppression values in the PBL from LN patients in remission and CGN patients with or without nephrotic syndrome, whether the mitogen used was Con A or PHA, were similar in those of the control subjects. Moreover, when individual patients were followed through their clinical illness. LN patients had high levels of SI, particularly in the beginning of acute exacerbations; the SI levels then decreased sharply in the latter phase of relapse and again increased to relatively normal levels with the onset of clinical remission. These in vitro findings suggest that there exists an alteration in Con A-induced SCA in a group of patients with LN.     

Effect of temperature and pH on release of enzymes from lysosomes of the bovine retinal pigment epithelium in vitro

The effects of buffer concentrations, pH, and temperatures on the release of enzymes from lysosomes of the bovine retinal pigment epithelium were studied in vitro. Cathepsin D, arylsulfatase, and acid phosphatase were used as lysosomal marker enzymes.Elevation of temperature caused a marked increase in the release of cathepsin D and arylsulfatase from lysosomes, but little changes in the release of acid phosphatase. Acidic conditions accelerated the release of arylsulfatase and acid phosphatase. Different buffer concentrations had little effect on the release of these enzymes from lysosomes.     

PEG-appended beta-(1-->3)-D-glucan schizophyllan to deliver antisense-oligonucleotides with avoiding lysosomal degradation

Schizophyllan is a natural beta-(1-->3)-d-glucan existing as a triple helix in water and as a single chain in dimethylsulfoxide (DMSO). As we already reported, when a homo-polynucleotide [e.g., poly(dA) or poly(C)] is added to the schizophyllan/DMSO solution and subsequently DMSO is exchanged for water, the single chain of schizophyllan forms a complex with the polynucleotide. One of the potential applications for this novel complex is an antisense-oligonucleotide (AS ODN) carrier. The present paper describes a modification technique that enabled us to introduce PEG only to the side chain of schizophyllan. This technique consisted of periodate oxidation of the glucose side chain and subsequent reaction between methoxypolyethylene glycol amine and the formyl terminate, followed by reduction with NaBH4. Subsequently, we made a complex from PEG-appended schizophyllan and an AS ODN sequence, and carried out an in vitro antisense assay, administrating the AS ODN complex to depress A375 c-myb mRNA of A375 melanoma cell lines. The PEG-SPG/AS ODN complex showed more enhanced antisnese effect than naked AS ODN dose, i.e., the same level as that of RGD-appended SPG. Here, the RGD system has been shown one on the most effective AS ODN carrier (Science 261 (1993) 1004-1012). When we added nigericin to the assay system, the antisense effect was not affected in the PEG-SPG system, on the other hand, it was almost eliminated in the RGD system. Nigericin is well known to interrupt transport from endosome to lysosome. Therefore, the difference between the PEG and RGD complexes indicates that, in the PEG system, AS ODN was able to escape from lysosomal degradation. The present work has thus proposed a new strategy to delivery AS ODN using schizophyllan as a new carrier.     

Src Tyrosine Kinase Inhibitor,M475271, Suppresses Subcutaneous Growth and Production of Lung Metastasis Via Inhibition of Proliferation,Invasion, and Vascularization of Human Lung Adenocarcinoma Cells

Src, a proto-oncogene, has been strongly implicated in the growth, progression and metastasis of a number of human cancers. Its role in lung cancer is, however, still unknown. In the present study, we assessed the expression of Src in three different human lung adenocarcinoma cell lines (PC-9, PC14PE6, A549), and explored the effect of a novel Src kinase inhibitor, M475271, on the behavior of the cell lines. The three cell lines expressed various levels of auto-phosphorylated Src. While M475271 reduced Src-phosphorylation and invasiveness of all three cell lines, it inhibited the proliferation of PC-9 and A549 cells with highly phosphorylated Src, but not PC14PE6 cells. We further examined the effect of M475271 on subcutaneous tumors and lung metastasis caused by PC-9 and/or A549 cells in NK-cell depleted SCID mice. Daily oral treatment with M475271 inhibited the growth of subcutaneous tumors with PC-9 and A549 cells via inhibition of tumor cells proliferation, VEGF production and/or vascularization in the mice in a dose-dependent manner. In the metastasis model with A549 cells, the lung weight in the M475271 (50 mg/kg)-treated group was less than that of the control group, despite no difference in the number of metastatic nodules. Our results suggest that inhibition of tyrosine kinase Src by M475271 could reduce the growth, invasion and VEGF-mediated neovascularization of lung adenocarcinoma cells, resulting in inhibition of growth of subcutaneous tumors and lung metastasis. Therefore, a novel Src tyrosine kinase inhibitor, M475271, might be helpful for controlling the progression of human lung adenocarcinoma.     

Mechanical properties and bond strength of silicone-based resilient denture liners

To evaluate the ease of manipulation and durability of 11 commercially available silicone-based resilient denture liners, extrusion force, hardness, weight change, and bond strength were determined. Extrusion force from the cartridge of each material ranged from 0.25 to 1.26 MPa at an extrusion rate of 1 cm/min. Durometer hardness, after set materials were stored in distilled water at 37 degrees C for one day, ranged from A5.9 to A47.7, and after four weeks their values increased by 4.0 to 275%. Bond strength ranged from 1.01 to 2.88 MPa after set materials were stored in distilled water at 37 degrees C for one day, but decreased to 0.59 to 1.99 MPa after 10,000 thermal cycles. These results suggested that except for one material, the rest of the evaluated materials exhibited good handling properties--for example, mixing and spreading of material can be done easily. However, some materials exhibited inadequate durability for clinical service, because hardness increased during storage and/or bond strength decreased after thermal cycling.     

Engraftment of NOD/SCID/gammac(null) mice with multilineage neoplastic cells from patients with juvenile myelomonocytic leukaemia

Several lines of evidence indicate the clonal nature of juvenile myelomonocytic leukaemia (JMML), involving myeloid, erythroid, megakaryocyte and B-lymphoid lineages. However, it is unclear whether the T-lymphocyte lineage is involved. We demonstrated that cells from six patients with JMML repopulated in non-obese diabetic/severe combined immunodeficient/gammac(null) mice and differentiated into granulocytes, monocytes, erythrocytes, B lymphocytes, T lymphocytes and natural killer cells. The percentage of human CD45 antigen-positive cells ranged from 41% to 73% in the murine bone marrow 12 weeks after transplantation. To examine the involvement of lymphocyte subpopulations, we purified human CD3(+), CD19(+) and CD56(+) cells from murine bone marrow cells transplanted from a patient with monosomy 7. Fluorescence in situ hybridization (FISH) showed the clonal marker in 96-100% of purified CD3(+), CD19(+) and CD56(+) subpopulations. These findings support the concept that JMML originates in transplantable multilineage haematopoietic stem cells. This novel murine xenotransplant model should be useful for investigating the nature of stem cells and testing new therapies for patients with JMML.     

Identification of DKC1 gene mutations in Japanese patients with X-linked dyskeratosis congenita

Dyskeratosis congenita (DC) is a rare inherited multisystem disorder characterized by the triad of abnormal skin pigmentation, nail dystrophy and mucosal leucoplakia. X-linked recessive inheritances are recognized in approximately 40% of the patients. DKC1 has been identified as the gene responsible for X-linked DC, and genetic analyses have been performed in a worldwide study. Here, we performed genetic analysis of five Japanese patients with presumed X-linked DC, and identified four mutations in the DKC1 gene, including two novel missense mutations (Q31K and T357A). Such genetic analysis is useful for the definite diagnosis and genetic counselling of patients.     

Usefulness of plasma brain-type natriuretic peptide level to differentiate left ventricular diastolic dysfunction from preserved diastolic function in patients with systolic dysfunction

The extent of left ventricular (LV) diastolic dysfunction is related to the finding that some patients with cardiomegaly due to LV systolic dysfunction have good exercise tolerance, although others have limited tolerance. A brain-type natriuretic peptide level of >/=104 pg/ml reliably enables the detection of relatively worse LV diastolic function in patients with systolic dysfunction, and this value may provide clinically useful information for the management of patients with cardiomegaly.     

Gefitinib ("Iressa", ZD1839), an epidermal growth factor receptor tyrosine kinase inhibitor, reverses breast cancer resistance protein/ABCG2-mediated drug resistance

Gefitinib ("Iressa", ZD1839) is an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor, and the single agent is clinically effective in non-small cell lung cancer. Although gefitinib combined with various cytotoxic agents has been reported to enhance cytotoxicity in vitro and in mouse models, the mechanism remains undetermined. Here, to explore the mechanism with topoisomerase I inhibitors, we focused on the efflux pump of the breast cancer resistance protein (BCRP/ABCG2), and then examined whether gefitinib restored drug sensitivity in multidrug-resistant cancer cells overexpressing BCRP. We used PC-6 human small cell lung cancer cells and multidrug-resistant PC-6/SN2-5H cells selected with SN-38 of the active metabolite of irinotecan, and BCRP-overexpressing MCF-7/MX cells selected with mitoxantrone and BCRP cDNA transfectant MCF-7/clone 8 cells. Drug sensitivity against anticancer drugs was determined by tetrazolium dye assay, and intracellular topotecan accumulation by FACScan. The topotecan transport study was done using the plasma membrane vesicles of PC-6/SN2-5H cells. The resistant PC-6/SN2-5H cells overexpressed BCRP but not epidermal growth factor receptor mRNA. Ten micromoles of gefitinib reversed topotecan, SN-38, and mitoxantrone resistance, and increased the intracellular topotecan accumulation in the resistant cells but not in the parental cells. Furthermore, gefitinib inhibited the topotecan transport into the vesicles, and the K(i) value was 1.01 +/- 0.09 micromol/L in the Dixon plot analysis, indicating direct inhibition of BCRP by gefitinib. However, gefitinib was not transported into the vesicles with the high-performance liquid chromatography method. These results indicate that gefitinib reverses BCRP-mediated drug resistance by direct inhibition other than competitive inhibition as a BCRP substrate. Combination of gefitinib and topoisomerase I inhibitors could be clinically effective in cancers expressing BCRP.