A NOVEL METHOD FOR MONITORING MYCOBACTERIUM-BOVIS BCG TRAFFICKING WITH RECOMBINANT BCG EXPRESSING GREEN FLUORESCENT PROTEIN

To better understand intracellular and extracellular trafficking of My cobacterium bovis bacillus Calmette-Guerin (BCG) when used as an intra vesical agent in the treatment of transitional cell carcinoma (TCC) of the bladder, recombinant BCG (rBCG) expressing the jellyfish green fl uorescent protein (GFP) was created. When the MB49.1 murine TCC cell l ine was incubated with GFP-expressing rBCG, internalization of the pat hogen could be directly visualized by UV microscopy and quantitated by flow cytometry. The in vitro internalization of the GFP rBCG by the b ladder tumor cells,vas temperature dependent, occurring most readily a t 37 degrees C and being severely inhibited at 4 degrees C. Optimum in ternalization was achieved in vitro at a 10:1 BCG-to-tumor cell ratio over 24 h during which approximately 16% of the tumor cells became inf ected. Cytochalasin B, a phagocytosis inhibitor, abrogated the ingesti on by almost 100% at a concentration of 200 mu g/ml, indicating that c ontractile microfilaments likely played an important role in this proc ess. By using mitomycin, a DNA cross-linking reagent, to inhibit proli feration of MB49.1 cells, clearance of about 40% of the green rBCG was achieved by 3 days postinfection, No significant difference between t he GFP rBCG and wild-type BCG was observed in the ability to induce th e expression of cell membrane proteins of major histocompatibility cla sses I and II, ICAM-I and -II, B7-1 and -2, or Fas from MB49.1 cells o r cytokine production from mouse spleen cells. These results indicate that GFP rBCG may serve as a useful substitute for wild-type BCG in fu ture studies of in vivo trafficking during experimental and clinical i mmunotherapy.