g(m1)-ganglioside and thiazolyl-blue

The complement system is one potential cytotoxic effector mechanism that might be effective in immunotherapy of cancer using monoclonal antibodies (mAb) directed against tumor antigens. In order to evaluate the treatment outcome from trials using mAb in cancer patients, assessment of complement-dependent cytotoxicity (CDC) may therefore be of interest. Here we describe the elaboration of a CDC assay in vitro using a rat hepatoma cell line, H4-II-E, as target cells sensitised with mAb F12, directed against the tumor-associated ganglioside antigen fucosyl-GMI. Sensitised cells were incubated with various concentrations of fresh serum as complement source for 48 h and cytotoxicity was then assessed by the tetrazolium bromide (MTT) test. A large variation in CDC efficacy was observed between individual serum donors. No differences in CDC could be seen between healthy donors and cancer patients. The CDC showed a strong correlation to the serum concentrations of complement factor C4, supporting the validity of the assay. Our results suggest that there may be significant variations in complement function within and between individuals that might influence the outcome of clinical mAb therapy. The H4/F12 CDC assay described here, together with measurement of individual complement factors. such as C4, should be further validated in cancer patients at various disease stages and phases of treatment.

Topics: Animals; Antibodies, Monoclonal; Cell Death; Cell Division; Chemistry, Clinical; Complement C3c; Complement C4; Complement System Proteins; Dose-Response Relationship, Immunologic; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; G(M1) Ganglioside; Humans; Immunoglobulin G; Mice; Neoplasms; Rats; Tetrazolium Salts; Thiazoles; Time Factors; Tumor Cells, Cultured

The anti-proliferative activity of cocaine was determined in PC12 phenochromocytoma cells. The effects of nerve growth factor (NGF) and ganglioside GM1 (GM1) on the toxicity of this stimulant of abuse was examined over a period of 72 h. Cocaine (40 microM-320 microM) exhibited a dose-dependent inhibition of cellular proliferation as determined by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) reduction. While NGF (100 ng/ml) and GM1 (100 microM) alone partially reversed the cocaine-induced inhibition of proliferation, the combination of NGF and GM1 afforded additional protection that was greater than that of either agent individually.

Topics: Animals; Cell Division; Cocaine; G(M1) Ganglioside; Nerve Growth Factors; PC12 Cells; Rats; Tetrazolium Salts; Thiazoles

Excitatory dicarboxylic amino acid neurotransmitters, particularly glutamate, have been implicated in mediating neuronal cell injury in brain ischemia-anoxia, epilepsy, and stroke. Glutamate neurotoxicity has been demonstrated in several in vitro models, as well as its prevention by a variety of agents, including several sialic acid-containing glycosphingolipid species, gangliosides. We have now examined ganglioside effects in anoxic exposed cultures of granule cells from Postnatal Day 8 rat cerebellum. Cells between 10 and 12 days in vitro were placed into an anoxic atmosphere or subjected to a chemical model of anoxia by a pulse exposure to rotenone. Widespread neuronal degeneration of neuronal cell bodies and their associated neurite network was seen the following day. These effects on cell vitality at the morphological level were quantitatively confirmed by measuring the photometric reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide to a blue formazan product. This neuronal injury was abolished by the specific N-methyl-D-aspartate receptor noncompetitive antagonists Mg2+, phencyclidine and MK-801, suggesting that this subtype of glutamate receptor is involved in the pathogenesis of anoxic granule cell injury. Pretreatment for 30 to 60 min or more or concurrent treatment with ganglioside GM1 largely prevented the ensuing neuronal death (ED50 = 25 microM), even 4 days later. Degeneration induced by exogenous glutamate was equally reduced. Asialo GM1 (lacking sialic acid) was ineffective. These results are consistent with the observed beneficial effects of the gangliosides in ischemic brain injury models in vivo.

Topics: Animals; Cell Survival; Cells, Cultured; Coloring Agents; G(M1) Ganglioside; Gangliosides; Hypoxia; Microscopy, Phase-Contrast; Neurons; Nitrogen; Rotenone; Tetrazolium Salts; Thiazoles