The Wiskott-Aldrich syndrome protein (WASP) family of molecules integrates upstream signalling events with changes in the actin cytoskeleton. N-WASP has been implicated both in the formation of cell-surface projections (filopodia) required for cell movement and in the actin-based motility of intracellular pathogens. To examine N-WASP function we have used homologous recombination to inactivate the gene encoding murine N-WASP. Whereas N-WASP-deficient embryos survive beyond gastrulation and initiate organogenesis, they have marked developmental delay and die before embryonic day 12. N-WASP is not required for the actin-based movement of the intracellular pathogen Listeria but is absolutely required for the motility of Shigella and vaccinia virus. Despite these distinct defects in bacterial and viral motility, N-WASP-deficient fibroblasts spread by using lamellipodia and can protrude filopodia. These results imply a crucial and non-redundant role for N-WASP in murine embryogenesis and in the actin-based motility of certain pathogens but not in the general formation of actin-containing structures.
Cytotoxic T lymphocytes (CTL) are important to the control of viral replication and their presence may be important to disease outcome. An understanding of the spectrum of proteins recognized by hepatitis C virus (HCV)-specific CTL and the functional properties of these cells is an important step in understanding the disease process and the mechanisms of persistent infection, which occurs in the majority of HCV-infected individuals. In this report we identify HCV-specific CTL responses restricted by the HLA class I molecules A2, A3, All, A23, B7, B8, and B53. The epitopes recognized by these intrahepatic CTL conform to published motifs for binding to HLA class I molecules, although in some cases we have identified CTL epitopes for which no published motif exists. The use of vectors expressing two different strains of HCV, HCV-1 and HCV-H, revealed both strain-specific and cross-reactive CTL. These HCV-specific CTL were shown to produce cytokines including IFN-y, TNF-a, GM-CSF, IL-8, and IL-10 in an antigen-and HLA class I-specific manner. These studies indicate that the CTL response to HCV is broadly directed and that as many as five different epitopes may be targeted in a single individual. The identification of minimal epitopes may facilitate peptide-specific immunization strategies. In addition, the release of proinflammatory cytokines by these cells may contribute to the pathogenesis of HCV-induced liver damage. (J. Clin. Invest. 1995. 96:2311-2321
To assess the role of the DNA-PKcs nonhomologous DNA end-joining (NHEJ) protein in Ig heavy chain class switch recombination (CSR), we assayed CSR ability of DNA-PKcs-deficient (DP-T) B cells generated via complementation of DP-T mice with Ig heavy chain and light chain knock-in transgenes (DP-T/HC/LC mice). DP-T/HC/LC mice were severely deficient for all serum IgH isotypes except IgM and, unexpectedly, IgG1. Upon appropriate stimulation, DP-T/HC/LC B cells showed normal proliferation, germline C(H) gene transcription, and AID induction, indicating that DNA-PKcs deficiency did not affect cellular events upstream to CSR. Yet, in vitro activated DP-T/HC/LC B cells again underwent switching only to IgG1 and, like wild-type cells, frequently underwent CSR to gamma1 on both chromosomes. We conclude that DNA-PKcs is required for CSR to most C(H) genes but that CSR to gamma1 occurs via a DNA-PKcs-independent mechanism.
Hepatitis C virus (HCV) is a common infection worldwide, and in most persons, it leads to persistent viremia and liver damage. Efforts to identify the correlates of protective immunity are hampered by this high rate of persistent infection in both infected humans and the only animal model, the chimpanzee. Peripheral blood mononuclear cells from seronegative persons were stimulated with synthetic peptides that represent epitopes recognized by HCV-specific cytotoxic T lymphocytes (CTL) after natural infection. In addition, CD4+ proliferative responses to recombinant HCV proteins were examined in these same persons. CTL responses directed against a peptide epitope of HCV and proliferative responses in 2 HCV-seronegative persons with possible occupational exposure to HCV were found. These otherwise healthy persons were not viremic, suggesting that they may have recovered from acute HCV infection. Characterization of virus-specific immune responses in exposed but seronegative persons may provide important clues as to the nature of protective immunity in HCV.
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