doi:10

doi:10.1099/vir.0.019505-0. We gathered normal intestinal samples from sites adjacent to excised colorectal carcinoma samples for mechanical fragmentation, enzyme digestion, and Percoll density gradient centrifugation (GE Healthcare). The granulocyte fraction was harvested, and CD117+ mast cells were positively selected using anti-CD117 or anti-FcR1 antibody-coated magnetic beads (Fig. 1A). In the anti-CD117 antibody-enriched cells, 97% of the cells presented a CD203c+ phenotype, and no or little expression of CD123 was observed (Fig. 1B). All cells showed a tryptase-positive reaction on intracellular staining, and the majority of purified cells expressed the high-affinity IgE receptor FcR1 and displayed binding with soluble IgE immunoglobulin (Fig. 1B). Tryptase is one of the granule components of mast cells and could be observed by confocal microscopy of intracellular staining (Fig. 1C), and ongoing degranulation of cells was also observed after toluidine blue staining (Fig. 1D). Under transmission electron microscopy, purified cells exhibited a characteristic phenotype, with the monolobed nuclei and numerous narrow, elongated folds around the cells (Fig. 1E) that are common of mast cells (31). Open in a separate window FIG 1 Characteristics of intestinal mucosal mast cells. (A) Enrichment and purification of mucosal mast cells from human healthy colorectal tissues. (B) Phenotype of LY2603618 (IC-83) purified mast cells as analyzed by immunostaining with specific antibodies and flow cytometry. (C) Intracellular immunostaining of tryptase (red) was confirmed by confocal microscopy; nuclei were stained with DAPI. DIC, differential interference contrast. (D) Positive staining of mast cells by toluidine blue. (E) Visualization of mast cells by transmission electron microscopy. LY2603618 (IC-83) Human mucosal mast cells express HIV-1 attachment factors for viral capture. To investigate LY2603618 (IC-83) the conversation of mast cells with HIV-1, we first explored the binding of viruses to cells. Freshly isolated mast cells were pulsed with HIV-1-gag-GFP/JRFL VLPs, and VLPs/Env, which do not incorporate HIV-1 envelope proteins, were used to monitor nonspecific binding. Viral association was quantified by flow cytometry to detect green fluorescent protein (GFP) levels. At 4C, about 22.3% of mast cells were found to capture JRFL VLPs, and no obvious binding LY2603618 (IC-83) was observed with VLPs/Env, indicating that the binding was envelope dependent and that the cell-associated HIV-1 particles LY2603618 (IC-83) could be removed by trypsin treatment (Fig. 2A). Confocal microscopy was also used to visualize and confirm viral surface binding (Fig. 2B), and replication-competent HIV-1 AD8 was used to visualize the binding of virus to mast cells by TEM (Fig. 2C). To confirm that HIV-1 binding is usually envelope dependent, we examined the binding of recombinant HIV-1 gp120 glycoprotein to mast cells. As shown in Fig. 2D, HIV-1 JRFL-derived gp120 glycoproteins were found to bind to mast cells. Open in a separate window FIG 2 Intestinal mucosal mast cell-mediated HIV-1 capture. (A) Detection of HIV-1 VLP binding on mast Rabbit Polyclonal to Tubulin beta cells by flow cytometry. VLPs made up of Gag-GFP were pulsed with mast cells at 4C, and VLPs/Env were used as the control to monitor nonspecific binding. Trypsin treatment was used to remove surface-bound viruses. (B) HIV-1 VLP association with cells was observed by confocal microscopy. (C) Binding of replication-competent HIV-1 AD8 on mast cells as visualized by TEM. Arrows indicate viruses. (D) Binding of gp120.