Human Immunodeficiency Virus-1 Uses the Mannose-6-Phosphate Receptor to Cross the Blood-Brain Barrier

HIV-1 circulates both as free virus and within immune cells, with the level of free virus being predictive of clinical course. Both forms of HIV-1 cross the blood-brain barrier (BBB) and much progress has been made in understanding the mechanisms by which infected immune cells cross the blood-brain barrier BBB. How HIV-1 as free virus crosses the BBB is less clear as brain endothelial cells are CD4 and galactosylceramide negative. Here, we found that HIV-1 can use the mannose-6 phosphate receptor (M6PR) to cross the BBB. Brain perfusion studies showed that HIV-1 crossed the BBB of all brain regions consistent with the uniform distribution of M6PR. Ultrastructural studies showed HIV-1 crossed by a transcytotic pathway consistent with transport by M6PR. An in vitro model of the BBB was used to show that transport of HIV-1 was inhibited by mannose, mannan, and mannose-6 phosphate and that enzymatic removal of high mannose oligosaccharide residues from HIV-1 reduced transport. Wheatgerm agglutinin and protamine sulfate, substances known to greatly increase transcytosis of HIV-1 across the BBB in vivo, were shown to be active in the in vitro model and to act through a mannose-dependent mechanism. Transport was also cAMP and calcium-dependent, the latter suggesting that the cation-dependent member of the M6PR family mediates HIV-1 transport across the BBB. We conclude that M6PR is an important receptor used by HIV-1 to cross the BBB

The uptake of soluble and particulate antigens by epithelial cells in the mouse small intestine.

Intestinal epithelial cells (IECs) overlying the villi play a prominent role in absorption of digested nutrients and establish a barrier that separates the internal milieu from potentially harmful microbial antigens. Several mechanisms by which antigens of dietary and microbial origin enter the body have been identified; however whether IECs play a role in antigen uptake is not known. Using in vivo imaging of the mouse small intestine, we investigated whether epithelial cells (enterocytes) play an active role in the uptake (sampling) of lumen antigens. We found that small molecular weight antigens such as chicken ovalbumin, dextran, and bacterial LPS enter the lamina propria, the loose connective tissue which lies beneath the epithelium via goblet cell associated passageways. However, epithelial cells overlying the villi can internalize particulate antigens such as bacterial cell debris and inert nanoparticles (NPs), which are then found co-localizing with the CD11c+ dendritic cells in the lamina propria. The extent of NP uptake by IECs depends on their size: 20-40 nm NPs are taken up readily, while NPs larger than 100 nm are taken up mainly by the epithelial cells overlying Peyer's patches. Blocking NPs with small proteins or conjugating them with ovalbumin does not inhibit their uptake. However, the uptake of 40 nm NPs can be inhibited when they are administered with an endocytosis inhibitor (chlorpromazine). Delineating the mechanisms of antigen uptake in the gut is essential for understanding how tolerance and immunity to lumen antigens are generated, and for the development of mucosal vaccines and therapies

Defective Gap-Junctional Communication Associated with Imaginal Disc Overgrowth and Degeneration Caused by Mutations of the dco Gene in Drosophila

The lethal(3)discs overgrown (dco) locus of Drosophila melanogaster, located on the third chromosome at cytogenetic position 100A5,6–100B1,2, is necessary for normal development and growth control in the imaginal discs of the larva. Three recessive lethal alleles (dco^2, dco^3, and dco^(18)) in heteroallelic combinations and one allele (dco^3) when homozygous cause the imaginal discs to continue to grow beyond the normal disc-intrinsic limit during an extended larval period. Some degeneration also occurs in the overgrowing discs. The discs overgrow even when transplanted early in their development into wild-type hosts, whereas normal discs stop growth at about the normal final size under such conditions, indicating that the overgrowth is a disc-autonomous effect of the mutations. During overgrowth the imaginal discs retain their single-layered epithelial structure except near regions of degeneration, and they differentiate into disc-appropriate but abnormal adult structures when transplanted into wild-type larval hosts. When the mutant larvae are reared under certain conditions a small percentage develop to the pharate adult stage, and these animals show a characteristic syndrome of abnormalities including swollen leg segments with many extra bristles, small or missing eyes, duplicated antennae and palpi, and separated vesicles of cuticle. A fourth recessive lethal allele (dco^(le88)), when homozygous or in heteroallelic combination with the overgrowth alleles, causes the imaginal discs to degenerate, producing a “discless” phenotype. Gap junction-mediated communication was assayed by observing the intercellular transfer of injected fluorescein complexon (dye coupling). Dye coupling in the imaginal discs of the dco genotypes that cause overgrowth was dramatically reduced at 4 days after egg laying (AEL) compared with wild-type controls. Coupling was more normal although still significantly reduced at 7–8 and 12–14 days AEL. In c43^(hs1), another disc overgrowth mutant, the imaginal disc cells also showed very reduced dye coupling at 4 days and incomplete coupling at 9 days. In contrast, discs from wild-type larvae, two other imaginal disc overgrowth mutants, and a cell death mutant showed extensive dye coupling at all stages tested. Electron microscopic morphometry revealed a reduction in gap-junction length per unit lateral plasma membrane length in dco3dco18 and c43^(hs1) wing discs, although not in dco2dco3, compared with wild-type wing discs. The results suggest that gap-junctional cell communication may be involved in the cell interactions that limit cell proliferation in vivo

PKC1 Is Essential for Protection against both Oxidative and Nitrosative Stresses, Cell Integrity, and Normal Manifestation of Virulence Factors in the Pathogenic Fungus Cryptococcus neoformans▿ †

Cell wall integrity is crucial for fungal growth, survival, and pathogenesis. Responses to environmental stresses are mediated by the highly conserved Pkc1 protein and its downstream components. In this study, we demonstrate that both oxidative and nitrosative stresses activate the PKC1 cell integrity pathway in wild-type cells, as measured by phosphorylation of Mpk1, the terminal protein in the PKC1 phosphorylation cascade. Furthermore, deletion of PKC1 shows that this gene is essential for defense against both oxidative and nitrosative stresses; however, other genes involved directly in the PKC1 pathway are dispensable for protection against these stresses. This suggests that Pkc1 may have multiple and alternative functions other than activating the mitogen-activated protein kinase cascade from a “top-down” approach. Deletion of PKC1 also causes osmotic instability, temperature sensitivity, severe sensitivity to cell wall-inhibiting agents, and alterations in capsule and melanin. Furthermore, the vital cell wall components chitin and its deacetylated form chitosan appear to be mislocalized in a pkc1Δ strain, although this mutant contains wild-type levels of both of these polymers. These data indicate that loss of Pkc1 has pleiotropic effects because it is central to many functions either dependent on or independent of PKC1 pathway activation. Notably, this is the first time that Pkc1 has been implicated in protection against nitrosative stress in any organism

Inhibition of 40 nm NP uptake by CPZ leads to decreased concentration of NPs in the MLNs.

<p>NPs with or without CPZ were injected in the lumen of SI and 40 minutes later MLNs were snap-frozen. Tissue sections of MLNs from CPZ-treated and control mice were stained with phalloidin-Alexa 350 and imaged at 630×. The amount of NPs in MLN sections of CPZ-treated and control mice was quantified using Volocity software. Regions of MLNs with highest NP concentration from control and CPZ-treated mice (A–C) were analyzed separately from regions of MLN capsules from control and CPZ-treated mice (D–F). (A) The amount of NPs (red pixels per image) was significantly higher in control mice compared to CPZ-treated mice (p<0.05). (B, C) Stitched images of MLN regions with high NP concentration from tissues of control mice (B) and CPZ-treated mice (C). Eight images taken at 630× were stitched together to show large sections of MLNs. Insets: magnified representative images in which clumps of NPs and individual NPs can be visualized. (D) The amount of NPs (red pixels per image) was significantly higher in capsules of control mice compared to CPZ-treated mice (p<0.05). (E, F) Representative images of NP distribution in MLN capsules of control (E) and CPZ-treated mice (F). Data are representative of 3 experiments (6 mice). Group means were separated using Student's t-test and were considered significantly different at P<0.05. Data are expressed as mean ± SD of the mean.</p

Routes of uptake (entry) of soluble and particulate antigens in the small intestine (SI) of mice.

<p>Abbreviations: PO: Per-oral; IL: Intraluminal (injected in the lumen of the SI); N/E: Not evaluated. GAPs (Goblet Cell Associated Passageways); IECs (Intestinal Epithelial Cells).</p

Administration of CPZ inhibits the uptake of 40 nm NPs but does not affect the uptake of dextran via GAPs.

<p>(A) Green channel of a confocal image of SI villi taken in vivo showing the entry of dextran (green) into the LP via GAPs (arrows, inset) in CPZ-treated mouse SI. (B) The internalization of 40 nm NPs (red channel) is inhibited by CPZ, thus red fluorescence was detected only in the lumen of the SI (asterisk). (C–E) A representative IFM image of the villi from tissue sections of mice administered CPZ and lysine-fixable dextran (red). Goblet cells (GAPs) were stained with cytokeratin 18 (Cy-18) antibody (green). (C) Two color image showing actin staining (blue) and goblet cell staining (green). (D) A two color image showing the entry of dextran (red) via GAPs. (E) Overlap of image C and D showing co-localization of red dextran with Cy-18 positive GAPs (green). (F) Administration of CPZ does not alter the number of Cy-18+ cells in the villi. There were no differences in Cy-18+ cells present in the villi between CPZ-treated and control mice (p<0.05). (G) Administration of CPZ did not alter the entry of dextran into the LP via GAPs. There were no differences in the number of Cy-18+ GAPs co-localizing with dextran between CPZ-treated and control mice (p<0.05). Group means were separated using Student's t-test and were considered significantly different at P<0.05. Data (bars) are expressed as mean ± SD of the mean. In total over 200 villi and over 600 GAPs were counted per animal and per treatment group (+/− CPZ). For each treatment group 3 mice were used. Data are representative of 3 experiments.</p

The presence of NPs in the IECs isolated from the mouse SI.

<p>40(red) were injected into the lumen of the SI and 30 minutes later the SI was excised, Peyer's patches were removed (discarded), and IECs were isolated from the SI sections. (A–D) Isolated IECs from mice that were administered NPs (A, B) or PBS (C, D) were fixed then placed on a glass slide and imaged with a fluorescent microscope at 630× magnification. (A, B) A patch of IECs isolated from NP-treated mouse imaged in the green channel (autofluorescence) (A) and the red channel (red: NPs) (B). Characteristic GAPs that are not highlighted by NPs appear as black holes in isolated IEC patches (white arrows), while IECs exhibit strong red fluorescence due to the presence of NPs (similar to images taken in vivo). (C, D) No red fluorescence was detected in IEC patches isolated from a control mouse. (E) Expression of E-cadherin (green) in isolated IECs imaged with a fluorescence microscope. (F) A confocal image of IECs isolated from NP-treated mouse showing strong red fluorescence in IEC cytoplasm. (G) Distribution of E-cadherin (green) in a section of SI. Actin staining with phalloidin-Alexa 350 (blue) highlights the tissue architecture. (H) Western blot analysis of E-cadherin (120 kDa) expression in isolated IECs (Lane 1) or spleen lymphocytes (Control, Lane 3). Lane 2: Spectra™ multicolor protein ladder. Each image is a representative of at least 3 experiments.</p