16 research outputs found
Rapid and Efficient Clearance of Blood-borne Virus by Liver Sinusoidal Endothelium
The liver removes quickly the great bulk of virus circulating in blood, leaving only a small fraction to infect the host, in a manner characteristic of each virus. The scavenger cells of the liver sinusoids are implicated, but the mechanism is entirely unknown. Here we show, borrowing a mouse model of adenovirus clearance, that nearly all infused adenovirus is cleared by the liver sinusoidal endothelial cell (LSEC). Using refined immunofluorescence microscopy techniques for distinguishing macrophages and endothelial cells in fixed liver, and identifying virus by two distinct physicochemical methods, we localized adenovirus 1 minute after infusion mainly to the LSEC (βΌ90%), finding βΌ10% with Kupffer cells (KC) and none with hepatocytes. Electron microscopy confirmed our results. In contrast with much prior work claiming the main scavenger to be the KC, our results locate the clearance mechanism to the LSEC and identify this cell as a key site of antiviral activity
Regulated protein degradation controls PKA function and cell-type differentiation in Dictyostelium
Cullins function as scaffolds that, along with F-box/WD40-repeat-containing proteins, mediate the ubiquitination of proteins to target them for degradation by the proteasome. We have identified a cullin CulA that is required at several stages during Dictyostelium development. culA null cells are defective in inducing cell-type-specific gene expression and exhibit defects during aggregation, including reduced chemotaxis. PKA is an important regulator of Dictyostelium development. The levels of intracellular cAMP and PKA activity are controlled by the rate of synthesis of cAMP and its degradation by the cAMP-specific phosphodiesterase RegA. We show that overexpression of the PKA catalytic subunit (PKAcat) rescues many of the culA null defects and those of cells lacking FbxA/ChtA, a previously described F-box/WD40-repeat-containing protein, suggesting CulA and FbxA proteins are involved in regulating PKA function. Whereas RegA protein levels drop as the multicellular organism forms in the wild-type strain, they remain high in culA null and fbxA null cells. Although PKA can suppress the culA and fbxA null developmental phenotypes, it does not suppress the altered RegA degradation, suggesting that PKA lies downstream of RegA, CulA, and FbxA. Finally, we show that CulA, FbxA, and RegA are found in a complex in vivo, and formation of this complex is dependent on the MAP kinase ERK2, which is also required for PKA function. We propose that CulA and FbxA regulate multicellular development by targeting RegA for degradation via a pathway that requires ERK2 function, leading to an increase in cAMP and PKA activity
Abundant intracellular IgG in enterocytes and endoderm lacking FcRn.
FcRn, a non-classical MHCI molecule, transports IgG from mother to young and regulates the rate of IgG degradation throughout life. Brambell proposed a mechanism that unified these two functions, saying that IgG was pinocytosed nonspecifically by the cell into an FcRn-expressing endosome, where, at low pH, it bound to FcRn and was exocytosed. This theory was immediately challenged by claims that FcRn specificity for ligand could be conferred at the cell surface in neonatal jejunum. Assessing Brambell's hypothesis we found abundant nonspecifically endocytosed IgG present in the cytoplasm of FcRn(-/-) enterocytes. Further, IgG was present in the intercellular clefts and the cores of FcRn(+/+) but not FcRn(-/-) jejunum. FcRn specificity for ligand could be determined within the cell
Comparison of distribution of IgG between FcRn<sup>+/+</sup> and FcRn<sup>β/β</sup> YS endoderm.
<p><b>A.</b> Photomicrographs illustrating YS sections from FcRn<sup>+/+</sup> (a, b, c) and FcRn<sup>β/β</sup> (d, e, f) YS labeled to visualize IgG (green) as was done in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070863#pone-0070863-g005" target="_blank">Fig. 5</a>. The phalloidin (gray) and DAPI (blue) labeling were used to mark cell boundaries and nucleus, respectively, shown in b and e. For orientation DIC images in c and f are shown. The solid arrows and arrow heads point to IgG in apical endoderm and mesenchyme, respectively. The barβ=β10 Β΅m. The inset of a and b, containing a higher magnified view of an outlined area of FcRn<sup>+/+</sup> and FcRn<sup>β/β</sup> yolk sac are shown. The solid arrows and arrow heads, respectively, point to IgG in apical and in mesenchyme areas in FcRn<sup>+/+</sup>. The lack of IgG in FcRn<sup>β/β</sup> mesenchyme and the presence of dense blobs in apical areas are shown by arrow heads and solid arrows respectively. <b>B.</b> Quantitative comparison of IgG (average intensity) between FcRn<sup>+/+</sup> and FcRn<sup>β/β</sup> in whole cell and apical areas of ED. Fluorescence images such as shown in panel A (a and c) were collected on a confocal microscope. The images were quantified, averaged for 300 cells, and plotted as average intensity for each strain (nβ=β3 FcRn<sup>+/+</sup> or 3 FcRn<sup>β/β</sup> mice). <b>a.</b> The image shows that total IgG in FcRn<sup>β/β</sup> is slightly higher but not statistically different than FcRn<sup>+/+</sup> (<i>P</i>β=β0.681). <b>b.</b> The image shows that IgG is more intense in FcRn<sup>β/β</sup>apical areas than in FcRn<sup>+/+</sup>. <b>C.</b> Number of pixels positive for IgG signal was quantified. Fluorescent images were converted to binary images and then used for quantifying the percent positive pixels. <b>a.</b> A difference could not be found in the percent of pixels positive for IgG in whole cell of FcRn<sup>+/+</sup> and FcRn<sup>β/β</sup> were almost equal (<i>P</i>β=β0.554). <b>b.</b> Percent positive pixels in FcRn<sup>β/β</sup> apical portion was significantly higher than in FcRn<sup>+/+</sup>.</p
Mean (Β±1 standard deviation) ratios illustrating differential expression of FcRn between YS endoderm and gut enterocyte cells.
<p>P, peripheral; I, internal; P<sub>A,</sub> apical periphery; P<sub>B,</sub> basal periphery; I<sub>A</sub>, apical internal and I<sub>B</sub>, basal internal.</p
Specificity of hamster anti-mFcRn antibody in IF assay.
<p>Photomicrographs illustrating gut sections from FcRn<sup>+/+</sup> (a, b, c) and FcRn<sup>β/β</sup> (d, e, f) strain neonates labeled to visualize FcRn (red) with Armenian hamster anti-mouse FcRn antibody. The phalloidin (gray) and DAPI (blue) labeling were used to mark cell boundaries and nucleus, respectively, shown in b and e. For orientation DIC images in c and f are shown. The barβ=β10 Β΅m. See the complete lack of FcRn labeling in FcRn<sup>β/β</sup> tissues (d) treated in parallel and in identical manner with FcRn<sup>+/+</sup> tissues.</p
A comparison of IgG distribution between FcRn<sup>+/+</sup> and FcRn<sup>β/β</sup> neonatal gut.
<p><b>A.</b> Photomicrographs illustrating gut sections from FcRn<sup>+/+</sup> (a, b, c) and FcRn<sup>β/β</sup> (d, e, f) neonates labeled to visualize IgG (green) with goat anti-mouse IgG heavy chain Fc antibody. The phalloidin (gray) and DAPI (blue) labeling were used to mark cell boundaries and nucleus, respectively, as shown in b and e. For orientation DIC images in c and f are shown. The barβ=β10 Β΅m. The insets of a and b contain a higher magnified view of an outlined area of FcRn<sup>+/+</sup> and FcRn<sup>β/β</sup> neonatal gut villi, respectively. The arrow heads point to the dense green labeling in intercellular areas of FcRn<sup>+/+</sup> enterocytes and lack of it in FcRn<sup>β/β</sup>. The presence or lack, respectively, of dense green blobs in FcRn<sup>β/β</sup> and FcRn<sup>+/+</sup> apical areas is marked by a solid arrow. <b>B.</b> Quantitative analyses of IgG in apical, intercellular and core villi areas are shown. Fluorescence images such as shown in panel A (a and c) were collected on a confocal microscope. The images were quantified, averaged for 300 cells, and the average intensity plus or minus standard deviations was plotted for each strain (nβ=β3 FcRn<sup>+/+</sup> and 3 FcRn<sup>β/β</sup> mice). <b>a.</b> The FcRn<sup>β/β</sup> apical area filled with green structures measured to be βΌ7 times more intense than FcRn<sup>+/+</sup> areas. <b>b.</b> The intercellular IgG in FcRn<sup>+/+</sup> is βΌ3 times more intense than FcRn<sup>β/β</sup> intercellular areas. <b>c.</b> IgG in the FcRn<sup>+/+</sup> jejunum villi core is βΌ4 times more intense than the FcRn<sup>β/β</sup> villi core areas.</p