The muscle-tendon junctions of the extensor carpi radialis longus and brevis muscles from adult Balb C Bailey/J mice have been examined tensiometrically and ultrastructurally following removal of cellular membrane and soluble cytoplasm by exposure to nonionic detergent. As judged by the ability of the extracted muscle to generate tension upon exposure to ATP and to transmit the generated tension to the tendon, detergent extraction leaves the muscle-tendon junction functionally intact. Electron microscopic analysis of the extracted muscle-tendon junctions reveals that the relationship between the terminal myofilaments and the lamina densa of the basal lamina is retained, despite the extensive extraction of the plasma membrane. Fine filaments (2-7 nm) are seen to connect the lamina densa with an electron-dense intracellular layer into which terminal actin filaments appear to insert. These fine filaments are considered to represent an important component of the structural linkage between myofilaments and connective tissue and hence to be a significant component of the tension transmitting mechanism. Their precise nature is not known, but some part of the filaments must pass through the hydrophobic compartment of the plasma membrane and thus must be a transmembrane component of considerable tensile strength. These studies suggest that detergent-extractable membrane lipids play no significant role in the transmission of tension at the muscle-tendon junction, and that fine filaments, probably protein, are responsible for transmitting tension from myofilaments, through the plasma membrane, to the lamina densa of the basal lamina.
A method of culturing canine tracheal smooth muscle cells in vitro is described. The morphology of these cells is monitored up to 60 days in culture and selected stages are illustrated. The characteristics of these cells are numerous mechanical attachments, the presence of thick filaments in suitably processed cells, and their contractile response to in vitro administration of carbachol, a cholinomimetic drug. They also possess nexus formations and both thin (actin) filaments and 10-nm filaments. Mitosis is found in the nonconfluent preparations up to 16 days after culturing. Cultures of 2 to 8 days appear to be most useful as pharmacological test vehicles. This system will be used to explore the phenomenon of adrenergic beta-2 receptor desensitization in airway smooth muscle, to attempt to localize these receptor sites and to determine how receptor affinity and/or number may be regulating cell response to pharmacologic agents.
Chemosensitivity testing in vitro of breast cancer has been difficult because of small tumour volume, an even smaller yield of viable cells after disaggregation, and the low evaluability rate and sensitivity of current assays. We have employed an alternative approach that quantitates intracellular adenosine triphosphate (ATP) as a measure of cell viability. This ATP-cell viability assay (ATP-CVA) determines in vitro tumor cell viability after exposure to chemotherapeutic agents in comparison to untreated controls following 6 days of incubation. Sixty-one fresh breast cancer specimens upon testing yielded an evaluability rate of 95%. Forty-seven of the tumors were untreated primary breast cancers, the remaining 14 were from patients with metastatic disease. Correlations of in vitro drug sensitivity with in vivo response were obtained for 17 treatment regimens in 14 patients with metastatic breast cancer. The level of sensitivity was 90% and the specificity 86%. These preliminary data demonstrated the ATP-CVA to be a practical in vitro approach to breast cancer testing. It will require a larger clinical study for confirmation.
A panel of 14 monoclonal antibodies (MoAbs) (4 raised against breast cancer, 6 against colon cancer and 4 against melanoma) were used to phenotype frozen sections of tumor biopsies obtained from 110 patients, by avidin-biotin-peroxidase complex techniques. We observed heterogeneity of antigen expression among the multiple metastatic lesions of single patients, as well as among tumor lesions from different patients with similar tumor histotypes. A wide range of cross-reactivity of anti-(breast-carcinoma) and anti-(colon-carcinoma) MoAbs with other carcinoma histotypes and limited reactivity with melanoma and sarcoma was detected. Some of our anti-melanoma MoAbs were also found to cross-react with selected carcinomas. Nine of the 14 MoAbs most reactive with carcinomas of diverse histotypes have been identified. A mixture or 'cocktail' of different MoAbs could be selected for each individual patient in order to achieve binding of MoAbs with most, if not 100% of tumor cells. This study illustrates the approach that we have taken to individualize the cocktail of MoAbs for the development of patient-specific therapeutic immunoconjugates.
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