By generating a mouse lacking expression of the WAS protein (WASP) selectively in B lymphocytes (B/WcKO), we and others have revealed a nonredundant B-cell-intrinsic role of WASP in immune homeostasis and prevention of autoimmunity, as well as in marginal zone (MZ) development and regulation of the germinal center (GC) reaction.3-5 Neural WASP (N-WASP, encoded by the Wasl gene) is another member of the WASP family of proteins; it is ubiquitously expressed and shares 50% homology with WASP.6 Similar to WASP, N-WASP undergoes a conformational change upon activation that enables initiation of actin polymerization, 7,8 thereby linking cellular activation to cytoskeletal modifications. 9 Selective deletion of N-WASP in B lymphocytes of Was knockout (WKO) mice resulted in the aggravation of B-cell abnormalities, including a strong decrease of intracellular calcium flux and Bruton's tyrosine kinase (Btk) and Src homology 2-containing inositol 59 phosphatase phosphorylation upon B-cell receptor (BCR) stimulation, 10 further worsening of MZ B-cell depletion, 11 and defective somatic hypermutation. 12 However, lack of WASP expression in multiple hematopoietic cells may have indirectly contributed to B-cell abnormalities in these models.To investigate the B-cell intrinsic role played by WASP and N-WASP in immune homeostasis and regulation more specifically, we have developed a double conditional mouse model (B/DcKO) in which deletion of both Was and Wasl floxed alleles in B lymphocytes is driven by the Cre recombinase expressed under the B-cell-specific promoter mb1. Here we show that deletion of N-WASP in B cells impairs B-cell activation and T-cell-dependent antibody responses and

Somatic cell hybrids secreting monoclonal antibodies against the core-glycolipid portion of enterobacterial endotoxin were derived from mice immunized with Escherichia coli J5 or Salmonella minnesota R595 heat-killed organisms or lipopolysaccharide (LPS). Eight antibodies were selected for their ability to cross-react with several members of a panel of gram-negative bacterial antigens in a radioimmunoassay. This panel represented five genera and two families of organisms: E. coli O111:B4, E. coli O55:B5, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella minnesota, and Serratia marcescens. The binding sites for six of the antibodies were unequivocally localized within the lipid A moiety of the endotoxin molecule by using the radioimmunoassay on LPS and free lipid A. The anti-lipid A antibodies were further characterized for their ability to interact with LPS variants by using a sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunostaining procedure. The monoclonal antibodies bound almost exclusively to the low-molecular-weight species of LPS on the polyacrylamide gel. These components corresponded to LPS isolated from rough strains of organisms (strains which lack O-specific carbohydrate). These results suggested that the cross-reactive component of antisera raised against rough mutants of gram-negative bacteria contain antibodies of lipid A specificity. Moreover, the determinant within the lipid A moiety of LPS may have been accessible to the monoclonal antibodies only in those endotoxin molecules on the outer membrane surface which lack the O-specific carbohydrate.