Fetal alcohol syndrome (FAS) children have defects in host defense and concomitant increase in the incidence of life threatening infections. Some children with FAS have long lasting deficiencies in both humoral and cell mediated immunity, suggesting that intrauterine exposure to ethanol may interfere with critical events in development of the immune system. Recent studies from this laboratory have shown that in utero exposure to ethanol caused a retarded development of B lymphocytes in neonatal mice. Phenotypic analysis of bone marrow and spleen showed that several intermediates in the B cell development pathway were affected by the fetal ethanol exposure. It addition, we noted that an unknown cell type, with an five weeks of age the splenic B cell population had returned to near normal levels, however, the B cell intermediates in the bone marrow remained below normal values at >5 weeks of age in the fetal ethanol animals. The overall goal of this proposal is to determine the mechanisms by which fetal ethanol exposure uterine exposure to ethanol on B cell developmental intermediates. We will determine the effects of intradiet containing ethanol, to (a) animals whose mothers will be pair fed a control liquid diet and (b) animals whose mothers will fed a stock diet. We will set up timed pregnancies and the dams will be fed the appropriate liquid diet for various periods (13-19 days), which will span the critical period of fetal B cell in these studies will be prepared from fetal liver taken at days 13-19 of gestation, and from bone marrow and spleens of mice 1 day old and then weekly through 6-8 weeks of age. We will determine the absolute of B cell progenitors at different stages of development using flow cytometry and fluorescence microscopy. B cell intermediates will be enriched in cell population by using panning on antibody coated plates and.or sorting by flow cytometry. The colony forming units of the purified progenitors will be determine by measuring the clonal proliferation of the B cell progenitors in soft agar in response to selected growth factors and culture conditions. The developmental potential of the B cell intermediates will be determined by measuring the frequency of cells that can give rise to a more differentiated cell. The long term effects on fetal ethanol will be assessed by testing the immune response of animals of different ages to Type-1 and -2 T independent antigens and to T dependent antigens. Completion of these experiments should provide a thorough understanding of the cellular basis for the effects of alcohol on the differentiation of B cells and suggest potential mechanisms for the clinical immune suppression associated with FAE.
