Assay Contact Line

Comparison of GT-3 and GT-4. Time dependent assays of the ether extracted GT-3 reported by Miller et al. (16) indicated that it had the same time course of action and irreversible effects as described for GT-4. When the ileal preparation was exposed to 10 ng/ml of GT-3 for 15 minutes a 50% inhibition from control response was observed. Upon contact with the preparation, the toxin caused a slow tonic contraction. Washing the segment with clean saline following the 15 minute exposure period caused a gradual relaxation of the preparation to near the base line levels observed in the control. However, when challenged by agonist at any time following the latency period, and irreversible loss of activity was evident. 

Key words Skin sensitization, Contact allergy, Allergic contact dermatitis, Local lymph node assay, In vitro alternatives, Direct peptide reactivity assay, KeratinoSens, Human cell line activation test... 

The cytotoxicity tests were performed with the use of the direct contact method on the extracts obtained by an 8-d incubation of the polymer PSU and PSU/Ag composite samples, placed at the bottom of the well of a 24-well culture plate, in 2 ml of culture mediiun. The incubation was conducted at 37°C in air atmosphere with a 5% content of C02and 100% of relative air humidity. Next, 0.2 ml of ihe obtained extract and its fourfold dilution in a proper culture medium was dosed for the cultures of human osteoblasts (HTB-85 cell line, ATCC, USA) and human fibroblasts (CRL-7422 cell line, ATCC, USA) adhered to the bottom of the well of a 96-well culture plate. In the case of the control test, the extract of the examined samples was replaced by the proper volume of culture medium. The plates were incubated at 37°C in air atmosphere with a 5% content of CO and 100% of relative air humidity. The incubation time for two parallelly conducted experiments was 24 h and 48 h. The cytotoxicity of PSU and the PSU/Ag composites was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium (MTT) assay [9, 10] and by lactate dehydrogenase (LDH) activity measured with the use of a commercial cytotoxicity assay kit (Roche Diagnostics GmbH, Mannheim, Germany), [11]. Each of the indications was repeated three times. 

To determine whether cell-cell contact is required for respecification of anterior ectoderm, we carried out two types of experiment. In the first, medium conditioned by a visceral endoderm-l e cell line was tested for its ability to substitute for VE tissue in the induction assay. Indeed, activation of an embryonic p-Iike globin gene was detected using RT-PCR (our unpublished results), indicating that at least some functions of the visceral endoderm can be mediated by soluble molecules. In the second type of experiment, transgenic anterior ectoderm and non-transgenic VE were cultured on opposite sides of a... 

In the heptatocyte-mediated mutagenesis studies, freshly isolated hepatocytes were mixed in a ratio of about 2 1 with liver epithelial cells before inoculation into culture flasks. After attachment, the two cell types were found to be well mixed and in close contact. Exposure of mixed cultures to several activation-dependent carcinogens that produce DNA repair in hepatocytes also produced mutagenesis in the cocultivated rat liver epithelial lines (Table 7) under conditions of exposure in which the carcinogens were not mutagenic to the lines in the absence of cocultivated hepatocytes. Thus, activated metabolites produced by the hepatocytes were transferred to the epithelial cells. Efforts to simplify this assay by the use of stored frozen hepatocytes are in progress. 

In contact-irritant assays, a test cylinder lined with a treated net is fixed to a darkened control cylinder. A valve between test and control unit is closed and 10 test mosquitoes are released into the treated cylinder. After an adaptation period of 30 seconds, the valve is opened and the distribution... 

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