Ultrasound-guided biopsy of the cricoarytenoideus lateralis muscle: technique and safety in horses.

REASONS FOR PERFORMING STUDY: Current diagnosis of recurrent laryngeal neuropathy (RLN) depends upon disease recognition in the clinically affected horse. Biopsy of the intrinsic laryngeal muscles may provide a method to identify the changes in fibre-type composition that occur in RLN before clinical signs become apparent. OBJECTIVE: To develop an ultrasound-guided biopsy technique of the left cricoarytenoideus lateralis muscle (CALM) and evaluate its efficacy and safety in vivo. STUDY DESIGN: A longitudinal descriptive study. METHODS: Six standing horses underwent ultrasound-guided biopsy of the left CALM. Frozen muscle cores were obtained with a breast biopsy tool. Serial endoscopic, ultrasonographic and physical examinations before and for 8 weeks after the biopsy were assessed for iatrogenic trauma. Histologies of representative muscle core cross-sections were analysed for the total number of muscle fibres obtained with each biopsy. RESULTS: There were no immediate complications of the procedure and the left CALM was harvested in all instances. Biopsy samples had an average weight of 0.043 g (range = 0.023-0.077 g) and contained 3418 fibres in cross-section (range = 711-7143). Laryngeal endoscopic grade did not change significantly between prebiopsy and the end of the 8 week follow-up. The left CALM had significantly greater echogenicity than the right throughout the study (P<0.001), but there was no difference between the prebiopsy CALM echogenicity and that at completion of the study. CONCLUSIONS: Ultrasound-guided biopsy of the left CALM is safe and well tolerated, providing a minimally invasive method to obtain muscle from healthy horses. This new technique may be applicable in research and clinical settings.

Restored gap junctional communication in non-tumorigenic HeLa-normal human fibroblast hybrids.

Gap junctional intercellular communication (GJIC) has been implicated in homeostasis, development, differentiation, wound healing or regeneration and adaptive responses of differentiated cells. The dysfunction of homologous or heterologous GJIC has been associated with the tumorigenic phenotype. Restoration of growth control and the suppression of the tumorigenic phenotype have been previously associated with the up-regulation of GJIC by various anti-tumorigenic chemicals or transfection of connexin genes into tumor cells. To test the hypothesis that 'tumor suppressor' genes may be associated with the up-regulation of GJIC, we tested clones of tumorigenic HeLa, several non-tumorigenic HeLa-normal human fibroblast somatic cell hybrids and a tumorigenic segregant of one of the non-tumorigenic hybrids for GJIC. The parental HeLa cells (D98 AH.2) had no detectable GJIC but expressed detectable connexin 43 transcripts, while the non-tumorigenic HeLa-human fibroblast hybrids, which contained the chromosome 11 from the normal human fibroblast (CGL-1, CGL-2, ESH15 and EHS15c1), expressed ample connexin 43 transcripts and showed proficient GJIC. The tumorigenic segregant (CGL-3) from the non-tumorigenic HeLa-human fibroblast hybrid showed no GJIC or connexin 43. These results show that the presence of GJIC is closely linked to the suppression of the tumorigenic phenotype in the HeLa-human fibroblast hybrid and further suggest that GJIC may be associated with the mechanisms of tumor suppression. The mechanism by which the tumor suppressor gene(s) on the normal chromosome in the HeLa-human fibroblasts induces the up-regulation of connexin 43 is not yet explained.

Pulse treatment with the tumor promoter TPA delays the onset of desensitization response and prolongs the inhibitory effect on gap junctional intercellular communication of a rat liver epithelial cell line WB F-344.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is an inhibitor of gap junctional intercellular communication (GJIC) of the rat liver epithelial cell line, WB F-344. We have previously reported that prolonged treatment of the WB cells with TPA (10 ng/ml) caused a reversal of the inhibition of GJIC that was initially induced (Oh, S.Y., et al. (1988) Carcinogenesis, 9, 135-139). Under this condition, addition of fresh TPA did not inhibit GJIC of these cells. In the present investigation we examined whether pulse exposure to TPA delays the onset of this desensitization response. Cultures were treated for 5 or 15 min with TPA and shifted to normal medium. Intercellular communication was measured at 15 min, 1 h and 6 h after the 5 or 15 min pulse treatments. Under these pulse treatment conditions, GJIC of the cells was markedly inhibited for up to 4 h and gradually reverted to near control levels by 6-8 h. At every sixth hour of pulse treatment the cells were given an additional pulse treatment (5 or 15 min) and the inhibitory effect of TPA on the GJIC of the cells was assayed 15 min after each such treatment. The results clearly showed that, when the cells were treated with 10 ng/ml TPA for 5 or 15 min every 6 h they maintained their sensitivity to the inhibitory effect of TPA on GJIC. This response to TPA was sustained for a considerably longer time when the duration of the pulse treatment was 5 min. Our data suggested that pulse exposure to TPA delays the desensitization response normally observed in prolonged treatment regimens and that this delay is possibly due to maintenance of the TPA activatable pool of protein kinase C under these conditions.

Effect of a tobacco-related nitrosamine on intercellular communication in human urothelial cells: a possible factor in smoking-related bladder carcinogenesis.

Bladder cancer is associated with smoking. Among the tobacco-derived carcinogens suspected of being involved in initiating the disease are nitrosamines found in urine. In this study a nitrosamine found in the urine of smokers was tested using a tissue culture model of normal human urothelium. Explant cultures were established from ureters and exposed to 5 ng/ml of the derivative. This level had been demonstrated previously to induce a variety of changes associated with initiation of carcinogenesis. Proliferation of the cultures was increased following exposure to the carcinogen, and the gap junction intercellular communication was reversibly inhibited. Examination of the connexin 43 protein and message status showed that the mRNA was unaffected, but the protein was not detectable using anti-connexin 43 antibody. The expression of the protein recovered within 24 h of removal of the carcinogen, indicating that the continued presence of the agent was necessary. Given the roles of cell proliferation and cell communication in carcinogenesis, the results may suggest a mechanism involving pre- or post-initiation deregulation of cell communication systems. Whether the enhanced growth is a separate effect or a consequence of reduced communication is an intriguing question.