Davies W A
J Reticuloendothel Soc. 1982 Dec;32(6):461-76.
Conventional methods of assessing antibacterial activities of macrophages by viable counting are limited by the precision of the statistics and are difficult to interpret quantitatively because of unrestrained extracellular growth of bacteria. An alternative technique based on the release of radioactive DNA from labeled bacteria has been offered as overcoming these drawbacks. To assess it for use with macrophages I have made a correlation with the conventional viable counting method using a mathematical model. Opsonized Listeria monocytogenes labeled with 3H-thymidine were exposed to rat macrophages for periods up to 4 hr. Numbers of viable bacteria determined after sonication increased exponentially in the absence of live cells and this growth rate was progressively inhibited by increasing numbers of macrophages. After a lag period of 30-60 min soluble 3H appeared in the supernatant, the amount increasing with time and numbers of macrophages. To correlate these data I developed a mathematical model that considered that changes in numbers of viable organisms were due to the difference between rates of 1) growth of extracellular bacteria and 2) killing within the macrophage. On the basis of this model curves of best fit to the viable counts data were used to predict the release of radioactivity, assuming that death of a bacterium led to the total release of its label. These predictions and the experimental data agreed well, the lag period of 30-60 min between death of the bacterium and release of radioactivity being consistent with intracellular digestion. Release of soluble radioactivity appears to be an accurate reflection of the number of bacteria killed within the macrophage.
通过活菌计数评估巨噬细胞抗菌活性的传统方法受统计精度限制，且由于细菌在细胞外不受限制地生长，难以进行定量解释。一种基于从标记细菌中释放放射性DNA的替代技术被认为可以克服这些缺点。为了评估其在巨噬细胞中的应用，我使用数学模型将其与传统的活菌计数方法进行了关联。用³H-胸腺嘧啶标记的经调理的单核细胞增生李斯特菌与大鼠巨噬细胞接触长达4小时。在无活细胞的情况下，超声处理后测定的活菌数量呈指数增长，且这种生长速率随着巨噬细胞数量的增加而逐渐受到抑制。在30 - 60分钟的滞后期后，上清液中出现可溶性³H，其含量随时间和巨噬细胞数量增加。为了关联这些数据，我开发了一个数学模型，该模型认为活菌数量的变化是由于1）细胞外细菌生长速率与2）巨噬细胞内杀伤速率之间的差异所致。基于该模型，假设细菌死亡导致其标记物完全释放，将拟合活菌计数数据的最佳曲线用于预测放射性释放。这些预测与实验数据吻合良好，细菌死亡与放射性释放之间30 - 60分钟的滞后期与细胞内消化一致。可溶性放射性的释放似乎准确反映了巨噬细胞内被杀死的细菌数量。
