5A), suggested that these complexes may enter cells via an endocytic pathway. confirmed by experiments using inhibitors of endosomal acidification (bafilomycin A1, monensin) and by immunocytochemical colocalization of internalized phage particles with early endosome-associated protein-1 (EAA1) . In contrast, microtubule-targeting brokers (nocodazole, taxol) increased the efficiency of antibody-enhanced phage gene transfer. These results reveal an unexpected antibody-dependent, FcRI-mediated enhancement of phage transduction in mammalian cells, and suggest new approaches to improve bacteriophage-mediated gene transfer. (Eguchi et al., 2001; Zanghi et al., 2007) and (Clark and March, 2004; Lankes et al., 2007; March, Clark, and Jepson, 2004). The ability of lambda phage particles to transduce mammalian cells depends only partially on phagocytic uptake of phage, and is increased when mice are pre-immunized with bacteriophage lambda (Lankes et al., 2007). Antibody-dependent enhancement (ADE) of computer virus infection is usually a paradoxical phenomenon in which computer virus specific antibodies fail to completely neutralize computer virus infectivity, and in stead permit the more efficient contamination of susceptible host cells such as monocytes and macrophages (Takada and Kawaoka, 2003). This process can be mediated through cellular receptors specific for the Fc portion of IgG, and has been reported to occur in a wide range of mammalian viruses and computer virus infections, including dengue computer virus, HIV-1, influenza computer virus, measles computer virus, murine gamma herpesvirus 68, rabies computer virus and yellow fever computer virus (among others) (Gotoff et al., 1994; Guillon et al., 2002; Iankov et al., 2006; Littaua, Kurane, and Ennis, 1990; Peiris and Porterfield, 1979; Porterfield, 1981; Rosa et al., 2007; Schlesinger and Brandriss, 1981; Takeda, Nice, and Ennis, 1990; Takeda, Tuazon, and Ennis, 1988; Tamura, Webster, and Ennis, 1991; Tamura, Webster, and Ennis, 1994; Wallace et al., 2003). ADE has also been reported to occur with mammalian computer virus vectors, and can lead to enhanced transduction of antigen-presenting cells by neutralized adenovirus-immune complexes, in a Fc receptor-dependent fashion (Leopold et al., 2006; Mercier et al., 2004). In the present study, we sought to develop an model for antibody-dependent enhancement of mammalian cells by bacteriophage vectors, and to determine the underlying mechanism(s) involved in this process – including the role of cellular Fc gamma receptor (FcR) receptors. FcRs are expressed on a wide range of NVP-AEW541 hematopoieitic cells (including B cells, macrophages, monocytes, natural killer cells, and neutrophils), and play an essential role in the acknowledgement Itgb3 and removal of immune complexes and immunoglobulin G (IgG)-opsonized pathogens (Daeron, 1997; Ravetch and Bolland, 2001). The FcR family includes NVP-AEW541 both high affinity receptors (such NVP-AEW541 as FcRI or CD64) and low affinity receptors such as FcRIIA (CD32), and FcRIII (CD16) (Ravetch and Bolland, 2001). Binding of immune complexes to these receptors results in their ligation and in the internalization of the bound complexes, either via endocytosis (in the case of small/soluble complexes) or phagocytosis (in the case of IgG-opsonized microorganisms) (Daeron, 1997; Ravetch and Bolland, 2001). FcR crosslinking also results in the activation of cell signaling and kinase pathways that contribute to phagocytosis and endocytosis, and that mediate the downstream effects of receptor ligation (such as the degranulation, initiation of host inflammatory responses and cytokine production) (Huang et al., 2006; Ravetch and Bolland, 2001. A number of FcRs have been shown to contribute to antibody-dependent enhancement of computer virus contamination, including FcRIA (CD64) and FcRIIA (CD32) in the case of dengue computer virus (Rodrigo et al., 2006; Schlesinger and Chapman, 1999), and FcRIIA (CD32) in the case of adenovirus vectors (Leopold et al., 2006). Our experiments revealed that only FcRIA, but not other FcR family members (FcRIIA, FcRIIB and FcRIIIA), was capable of supporting antibody-dependent enhancement of bacteriophage-mediated gene transfer; coexpression of the FcR associated chain was not required for ADE. Finally, studies using pharmacologic inhibitors and immunocytochemical staining techniques revealed that clathrin-mediated endocytosis and actin filaments NVP-AEW541 played a critical role in antibody-enhancement of phage gene transfer, while the microtubule network was found to play an inhibitory role in this process. Results Bacteriophage lambda vectors are capable of transducing mammalian cells model for.
