This proposal is designed to examine the potential bioeffects of ultrasound on B cell development using both in vivo and in vitro methodologies. Specifically, the effects of ultrasound on B cell development, antibody class expression, and the diversification of B cell specificities in the developing fetus will be monitored. Pregnant Balb/c mice will be sham-treated or exposed to ultrasound radiation and sacrificed at several times during the gestation period. Fetal livers and spleens will then be dissected and analyzed directly or cultured first in an in vitro fetal organ culture system. Both uncultured and cultured fetal lymphoid cells will then be tested for the proportion of surface immunoglobulin cells (B cells), cytoplasmic Mu positive cells (pre B cells), and pre B cell marker positive cells by immunocytochemical staining. In addition, cultured and uncultured fetal lymphoid cells obtained from normal vs ultrasound exposed animals will be antigenically stimulated in the splenic focus assay and the resulting B cell clones analyzed for isotype production. Finally, cultured and uncultured fetal cells of various ages of gestation will be tested for the frequency and kinetics of appearance of B cells responsive to several different haptens. B cells specific for these haptens have been previously shown to arise in a predictable temporal order. Moreover, the appearance of individual clonotypes and the process of VH selection will be explored. If differences as a result of ultrasound are observed, B cell development will also be examined in neonatal mice. Finally, it will be determined if there are critical periods of sensitivity to ultrasound exposure. These studies should detect potential effects of ultrasound on B cell development, an important analysis given the widespread clinical use of ultrasound radiation.
