Mechanisms by which intravesical bacille Calmette-Guerin (BCG) treatment mediates antitumor activity are currently poorly understood. We have determined that both human bladder tumor (T-24) and mouse bladder tumor (MBT-2) cells are capable of internalizing the BCG and have characterized this process in vitro. The internalization of BCG by T-24 and MBT-2 cells was verified by histochemistry and electron microscopy. Time dependent internalization of BCG was observed with a maximum occurring at three hrs. Internalization was significantly inhibited by both incubation at 4C and cytochalasin B; conditions known to inhibit phagocytosis. Ultrastructural studies suggested that BCG were transported to membrane bound intracellular compartments and were degraded. The compartments containing the degraded mycobacteria labeled with the fluid phase marker HRP which is known to be transported to lysosomes in a variety of cell types. To determine if the internalization process occurred in vivo, we examined bladder washings of patients treated with intravesical BCG. The majority of the cells in the bladder washings were inflammatory cells which contained ingested BCG. In addition, internalized and degraded BCG were identified in urothelial cells. These data demonstrate that BCG are internalized by transitional epithelial cells both in vitro and in vivo. The internalization process is inhibitable by temperature and microfilament disruption. The potential therapeutic implications of this process and its relationship to antitumor activity of BCG therapy are currently being investigated.

Surface-active phospholipid-containing particles are traditionally considered to be the product of type II pneumocytes. We now demonstrate membrane-bound lamellar cytoplasmic organelles in adult and suckling rat enterocytes that are densely reactive with phospholipid-staining reagents. These structures were seen in the basolateral space, within the intercellular junctions, and unraveling on the lumenal surface, and were more abundant after fat feeding. Light scrapings of intestinal mucosa and lumenal washings that contained these bodies, as evidenced by morphology and biochemical analysis, lowered surface tension in a pulsating bubble assay. Production by normal enterocytes of material with surfactant-like appearance and properties demonstrates that these structures are present in extrapulmonary epithelia, and extends the possible range of their function beyond gaseous exchange, e.g., solute exchange or lubrication on membrane surfaces.

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We have initiated a characterization of the cis-acting regulatory elements of the murine low-affinity NGF receptor (p75NGFR) gene. Despite studies in cultured cells that suggest the p75NGFR promoter is constitutive, a detailed analysis of this promoter in five lines of transgenic mice demonstrated a high degree of cell-type specificity: 8.4 kb of 5′ flanking sequence directs expression of a lacZ reporter to retinal and CNS neurons normally expressing p75NGFR. A transgene with 470 bp of 5′ flanking sequence is also expressed in the CNS, but its regulation is aberrant, with a loss of basal forebrain expression. In non-neural tissues, both transgenes were expressed only in the testis, kidney, anterior pituitary, and pancreatic islets; with the exception of the renal pattern of expression, transgene activity was confined to appropriate cells within these tissues. In contrast, although expression of both transgenes was prominent in adrenal medulla and gastrointestinal myenteric neurons, neither construct was active in several sensory or sympathetic ganglia that strongly express the endogenous p75NGFR gene, indicating that genetic elements necessary for expression in these neurons are not present in these promoter sequences. In addition, neither transgene was activated in Schwann cells during Wallerian degeneration of sciatic nerve. We conclude that regulation of the p75NGFR gene is complex, with the first 470 bp of 5′ flanking sequence sufficient for expression in enteric and CNS neurons and additional elements within the first 8.4 kb of 5′ flanking sequence required for restriction to appropriate CNS neurons. Further regulatory elements are possibly required for expression in at least some sensory and sympathetic neurons in the PNS and in Schwann cells. To identify potential regulatory elements in the 470 bp of 5′ flanking sequence from the smaller transgene, we compared the sequences of equivalent regions from the mouse, rat, and human p75NGFR genes. This “phylogenetic footprint” identified conserved motifs potentially important for the regulation of this gene in the CNS.