Efficacy of prophylactic Immucothel in patients pretreated with conventional drugs to prevent recurrence of superficial bladder carcinoma.

OBJECTIVES: Fifty patients enrolled in clinical trials with Immucothel((R)) were reevaluated for their disease-free intervals. METHODS: Of the 37 evaluable patients, 16 had received mitomycin C, 3 bacillus Calmette-Guérin, 3 Adriamycin and 15 miscellaneous pretreatments prior to Immucothel. They thus served as their own controls. RESULTS: Although their prognosis was worse at the time when Immucothel therapy was started than at the time of initiation of pretreatment, the mean recurrence-free intervals of all patients increased from 17.0 months under pretreatment to 35.1 months under Immucothel. CONCLUSION: The difference was statistically significant (Wilcoxon matched-pairs signed-ranks test).

The antitumor activity of the platinum complex D-17872 is associated with tumor cell differentiation.

The novel cisplatin analogue D-17872 was studied for its anticancer activity using in vivo and in vitro preclinical models. The compound at the sublethal dose of 215 mg/kg (ca. 50% of the approximate LD50) induced no nephrotoxic effect strong enough to increase the blood urea level in rats. It had good in vivo antitumor efficacy against murine P388 (max. ILS: D-17872 132%, cisplatin 55%) and L1210 leukemia (max. ILS: D-17872 43%, cisplatin 38%), L5222 leukemia of the rat (max. ILS: D-17872 163%, cisplatin 163%) and murine B16 melanoma. Activity against P388 leukemia substantially exceeded that of cisplatin. Moreover, the M5076 reticulum cell sarcoma implanted into the subrenal capsule and the DMBA-induced mammary tumor of the rat were inhibited by D-17872 to a greater extent than by cisplatin (min. T/C: D-17872 -3%, cisplatin 11%). Using clonogenic microassays, D-17872 was active in vitro against a variety of human and rodent tumor cell lines, albeit at higher concentrations than cisplatin (IC50 values: D-17872 2.6-12.7 mumol/l, cisplatin 0.13-0.42 mumol/l). Apart from its cytotoxic action it was able to induce in vitro differentiation of the human HL-60 and K562 and of the murine M1-T22 cell lines, while cisplatin induced differentiation only in the HL-60 cell line. Thus D-17872 exhibited a pharmacological and toxicological profile different from that of the parent compound. The results suggest that induction of differentiation contributes to the antineoplastic efficacy of this novel cisplatin derivative.

Selective cytostatic activity of hexadecylphosphocholine against tumor-cells invitro leads to the establishment of an invivo screening system for phospholipid analogs.

Hexadecylphosphocholine (HPC, D-18506, INN: Miltefosine) was characterized in a clonogenic micro assay in vitro with respect to its cytotoxicity against a panel of human and murine tumor cell lines and murine bone marrow cultures (GM-CFC). The KB human epithelial carcinoma line was found to be more sensitive by at least one order of magnitude than the murine tumor cell lines which are in vivo insensitive to the drug. Xenotransplants of the KB cell line into nude mice were highly sensitive to HPC with optimal treatment resulting in total regression of the tumor. Twenty-seven structural analogues of HPC were tested in vivo in our normal screening system, the dimethylbenz(a)anthracene(DMBA)-induced mammary carcinoma of the rat, and against KB-xenotransplants. 88 % of the compounds rated identically in both models, no compound was classified falsely negative in the KB model. These results allow to adopt the KB-model, which is easier to perform, as a reliable primary screening system for phospholipid analogues without the risk of missing active compounds.

Lymphokine-activated killer cells: determination of their tumor cytolytic capacity by a clonogenic microassay using agar capillaries.

A lymphokine activated killer (LAK) cell assay has been developed using a clonogenic microassay in agar-containing capillaries with KB tumor target cells. The assay avoids the problems of the commonly used 51Cr release assay and mimics physiological conditions more closely. The assay procedure has been optimized, resulting in the following conditions: LAK cells are generated by incubating nonadherent peripheral blood mononuclear cells from normal donors with 20 U/ml interleukin-2 for 3 days and cocultivated with 10(4) KB human squamous carcinoma cells/ml at 5:1, 10:1, and 20:1 effector:target ratios for 24 h. The cocultivation mixture is then seeded into agar-containing glass capillaries, allowing undamaged tumor cells to form colonies. The colony number is proportional to the number of tumor cells seeded. The present microassay requires up to 90% less cells and agent quantities compared with other clonogenic assays. It thus provides a useful tool to quantitate LAK cell activity and its alteration by immunomodulatory agents.

Effects of low-dose mafosfamid on the lymphokine-activated killer cell activity of peripheral blood mononuclear cells determined by a clonogenic microassay.

The effects of mafosfamid on lymphokine-activated killer (LAK) cells were examined, using a newly developed clonogenic microassay with tumor target cells instead of the common 51Cr release. When nonadherent peripheral blood mononuclear effector cells were exposed to 1 x 10(-9) or 1 x 10(-8) M mafosfamid 1 h prior to activation with interleukin 2 (IL-2), LAK-cell activity increased, resulting in fewer colonies of human KB squamous carcinoma cells than without mafosfamid treatment. In contrast, when effector cells were exposed to mafosfamid after IL-2 activation LAK-cell activity was inhibited. Notably, the in vitro immunomodulatory effects of mafosfamid were detected at concentrations 100-1000 times below those shown to be cytotoxic. Although the mechanisms remain to be elucidated, our results might be an in vitro representation of the known in vivo immunostimulation of low doses of cyclophosphamide.

Clonogenic assays for hematopoietic and tumor cells using agar-containing capillaries.

The so-called hematopoietic stem cells from mouse and human bone marrow can be induced in vitro to form colonies, provided that the appropriate hormones (colony stimulating factors, CSFs) and culture conditions have been selected. Such hematopoietic colony cultures are of interest for in vitro cytotoxicity testing of a great number of drugs. In fact, it has been increasingly recognized that serious side effects of many drugs involve injury to hematopoietic organs. Moreover, several hormones and inhibitors (including chalones) that regulate hematopoiesis have attracted clinical interest and their identity can be determined by such in vitro cultures. Therefore hematopoietic stem cells seem to provide identified targets to assay for either stimulatory, inhibitory, or cytotoxic drug effects.

Defined, serum-free culture conditions for the GM-micro-clonogenic assay using agar-capillaries.

Culture conditions were determined and optimised for the serum-free growth of granulocyte-macrophage (GM) colonies, derived from mouse bone marrow cells, in agar-containing glass capillaries. The standard medium is DMEM/F-12 (1:1) mixture containing per ml: 10 mg BSA, 35 micrograms transferrin, 20 micrograms soybean lipids and 5 micrograms insulin. In contrast to previous attempts by others, GM-colony yield in serum-free medium was found to be equal to that in serum-containing medium (around 25 colonies/capillary) and to correlate satisfactorily with the cell density at seeding.--The role of polyamine oxidases in cell-proliferation experiments could be studied by this microclonogenic assay without interference from any type of serum.

T-lymphocyte colony formation of murine thymocytes in agar contained in glass capillaries.

Optimal growth conditions are presented for a new colony test with mouse thymocytes in agar contained in glass capillaries. The kinetics of colony growth and the dependence from the PHA-, I1-2-, agar- and 2-mercaptoethanol concentration are shown. The colony forming cells are identified as T-lymphocytes by usual morphology and by an indirect immunoperoxidase method using mouse anti-Thy 1.2 antibodies.

Cloning and analysis of the K1 capsule biosynthesis genes of Escherichia coli: lack of homology with Neisseria meningitidis group B DNA sequences.

Genes coding for production of the K1 polysaccharide capsule of Escherichia coli have been cloned. Complementation, insertion, and deletion analyses were used to localize the K1 genes and demonstrated that a minimum of 9 kilobases of DNA split into at least two gene blocks is involved in synthesis and assembly of the capsule. One of the gene blocks is responsible for biosynthesis of the polysaccharide, and the other is responsible for extracellular appearance of capsular material. Using cloned K1 genes as probes in Southern blot experiments, we detected homology to DNA from strains of E. coli capsular types K92, K7, and K100. In contrast, no homology was apparent between K1 genes and DNA from meningococcus group B, although the K1 and group B capsules are chemically and immunologically identical.

Is the IS1-flanked r-determinant of the R plasmid NR1 a transposon?

The 23 kilobase multiple drug resistance r-determinant (r-det) of the R plasmid NR1 is an IS1-mediated transposon, Tn2671. Drug-resistant Escherichia coli transductants isolated after infection with bacteriophage P1::Tn2671 derivatives carry the intact r-det in their chromosomes. Independently isolated transductants carry the r-det at different locations on the chromosome. From the E. coli chromosome, Tn2671 can transpose to various locations on the phage P7 genome. Throughout these processes, r-det is maintained as a stable unit. Various possible molecular mechanisms, which all might contribute with characteristic frequencies to the transposition of Tn2671, are discussed. The results presented are relevant to the understanding of mechanisms for a wide spreading of drug resistance genes.