ssGPL does not contribute to the resistance of <i>M</i>. <i>avium</i> to LL-37.

<p>CFU determination of <i>M</i>. <i>avium</i>^ssGPL and <i>M</i>. <i>avium</i>^ΔssGPL serovar 8 after incubation with 0, 10, 25, and 100 μg/ml LL-37 and 20 μg/ml gentamicin. ***p<0.0001. Data are the mean ± SEM of 3 independent experiments.</p

<i>E</i>. <i>coli</i> is susceptible to LL-37.

<p>(A) Log₁₀ CFU of <i>E</i>. <i>coli</i> after 4 hours of incubation with 0, 10, or 25 μg/ml of LL-37. **p< 0.001; ***p<0.0001. Data are the mean ± SEM of 6 independent experiments. (B) Images were taken of each serial dilution on LB agar from <i>E</i>. <i>coli</i> cultures incubated for 4 hours in the absence or presence of 25 μg/ml of LL-37.</p

Loss of LL-37 activity after exposure to NTM or NTM-derived lipids.

<p><i>E</i>. <i>coli bioassays</i> were used to evaluate LL-37 activity. (A) LL-37 remaining in NTM (but not <i>Mtb</i>) culture supernatant no longer kills <i>E</i>. <i>coli</i>. (B) <i>E</i>. <i>coli</i> survives in untreated or boiled NTM culture supernatants to which fresh LL-37 was added. (C) <i>E</i>. <i>coli</i> survival following incubation with <i>M</i>. <i>abscessus</i> or <i>M</i>. <i>intracellulare</i> derived cell fractions. CM = cell membrane, CW = cell wall, ICW = insoluble cell wall fraction.</p

LL-37 demonstrates broad-spectrum antimicrobial activity.

<p>Log₁₀ CFU after 1–8 hours of incubation of a (A) laboratory isolate of <i>Salmonella enteriditis</i> or clinical isolates of (B) <i>Salmonella enteriditis</i> (Uganda) or (C) <i>Salmonella non-typhi</i> (Nairobi) with 0–50 μg/ml of LL-37. *p< 0.01; **p< 0.001; ***p<0.0001. Data are the mean ± SEM of 3–6 independent experiments. (D) <i>Mtb</i> H37Rv were incubated with 10 μg/ml LL-37 and the percent change in CFU calculated after 96 hours incubation. n = 3 independent experiments.</p

nsGPL do not mediate the resistance of <i>M</i>. <i>abscessus</i> to LL-37.

<p>(A-B) CFU determination of <i>M</i>. <i>abscessus</i>^nsGPL(+) in the presence of the indicated concentrations of native LL-37 (p = 0.69). Data are the mean ± SEM of 4 independent experiments. (B) CFU determination of <i>M</i>. <i>abscessus</i>^nsGPL(-) in the presence of the indicated concentrations of native LL-37. Data are the mean ± SEM of 4 independent experiments. *p<0.01; **p< 0.001; ***p<0.0001. (C) Thin-layer chromatography demonstrates the presence and absence of GPL in <i>M</i>. <i>abscessus</i>^nsGPL(+) and <i>M</i>. <i>abscessus</i>^nsGPL(-), respectively. (D-E) CFU determination of <i>M</i>. <i>abscessus</i>^nsGPL(+) and <i>M</i>. <i>abscessus</i>^nsGPL(-), respectively in the presence of the indicated concentrations of scrambled LL-37 peptide. Data are the mean ± SEM of 3 independent experiments. *p<0.01; **p< 0.001; ***p<0.0001.</p

Transfer of Intracellular HIV Nef to Endothelium Causes Endothelial Dysfunction

<div><p>With effective antiretroviral therapy (ART), cardiovascular diseases (CVD) are emerging as a major cause of morbidity and death in the aging HIV-infected population. To address whether HIV-Nef, a viral protein produced in infected cells even when virus production is halted by ART, can lead to endothelial activation and dysfunction, we tested Nef protein transfer to and activity in endothelial cells. We demonstrated that Nef is essential for major endothelial cell activating effects of HIV-infected Jurkat cells when in direct contact with the endothelium. In addition, we found that Nef protein in endothelial cells is sufficient to cause apoptosis, ROS generation and release of monocyte attractant protein-1 (MCP-1). The Nef protein-dependent endothelial activating effects can be best explained by our observation that Nef protein rapidly transfers from either HIV-infected or Nef-transfected Jurkat cells to endothelial cells between these two cell types. These results are of <i>in vivo</i> relevance as we demonstrated that Nef protein induces GFP transfer from T cells to endothelium in CD4.Nef.GFP transgenic mice and Nef is present in chimeric SIV-infected macaques. Analyzing the signal transduction effects of Nef in endothelial cells, we found that Nef-induced apoptosis is mediated through ROS-dependent mechanisms, while MCP-1 production is NF-kB dependent. Together, these data indicate that inhibition of Nef-associated pathways may be promising new therapeutic targets for reducing the risk for cardiovascular disease in the HIV-infected population.</p></div

Nef enhances live dye transfer between cells and the formation of nanotubes.

<p>A–B. Live dye(green) transfer from cDNA transfected Jurkat cells (A) or Nef-transfected Jurkat cells (B) to endothelial cells after 24 h coculture. Endothelial cells (red) were stained by phalloidin. C. Live dye stained Nef or control cDNA transfected Jurkat cells were cocultured with HCAEC either in direct contact, or separated by transwell membranes for varying time points. Percentage of live dye transfer was determined and quantified by confocal microscopy. The fold dye transfer was normalized to cDNA controls for each condition. B is the representative figure form direct control experiments, which were quantified in C. D. Nanotube-like conduit formation (white arrows) between Nef transfected T cells labeled with live dye (green) and phalloidin labeled endothelial cells (red). E. Nef-transfected Jurkat cells were cocultured with HCAEC for varying time points, and HCAEC were stained for Nef to determine the time course of Nef transfer from Jurkat cells to endothelial cells. Endothelial cells were washed with PBS to ensure no adhesion of Jurkat cells. Any remaining Jurkat cells were gated from endothelial cells by FACS based on forward scatter and side scatter profiles. F. Nef transfer to HCEAC after 24 hr co-culture. Endothelial cells were stained with phalloidin (red) and Nef (green). Right corner insert indicates Nef accumulation in HCAEC without an overlay. Original magnification, X 60. Scale bars represent 10 µm.</p

Nef is present in endothelial cells in vivo.

<p>A–C. Heart sections of single CD4-GFP (A) and double CD4-Nef-GFP (B, C) transgenic mice (N = 3; at least 4 pictures/slide) were double stained with GFP antibody (green, white arrows) and the endothelial marker vWF (red). Shown is GFP within the endothelial lining (arrows). Original magnification, X 60. Scale bars represent 100 µm. D–F. Macaque heart sections (N = 5; at least 4 pictures/slide) were double stained with IgG control (D) or Nef (E, F, red) and the endothelial marker vWF (green). Shown are cells double positive for Nef and vWF in coronary arteries (arrow). Original magnification, X 60. Scale bars represent 100 µm.</p

Nef is sufficient to induce endothelial activation and dysfunction.

<p>A–C. Endothelial MCP-1 release (A), apoptosis (B) and ROS formation (C) were determined in endothelial cells transfected with cDNA (mock), WT (WT Nef) or SH3 binding site mutated Nef (NefΔSH3) after 24 h. Data were expressed as fold MCP-1 production and apoptosis, normalized to the mean of control measurements (N = 6. *P<0.05, and **P<0.01).</p

Nef is necessary for endothelial cell activation.

<p>A–B. 80% confluent endothelial cells were co-cultured in direct or indirect contact with HIV-infected Jurkat cells with HCAEC alone (Mock), HCAEC in direct contact with uninfected Jurkat cells (EC+T cell), EC in indirect contact with infected Jurkat cells (EC+HIV) or HCAEC in direct contact with infected Jurkat cells (EC+T cells+HIV). MCP-1 release from HIV infected Jurkat cells was also included (HIV-Jurkat). MCP-1 release was analyzed by ELISA (A); endothelial apoptosis was determined by TUNEL assay (B). C–D. MCP-1 release (C) and apoptosis (D) in endothelial cells were determined after co-culture of endothelial cells with uninfected Jurkat cells (mock), HIV-infected Jurkat cells (WT HIV) or Nef deleted HIV-infected Jurkat cells (ΔNef HIV). E–F. Endothelial MCP-1 production (E) and apoptosis (F) were determined in HCAEC alone (EC) or after 24 h coculture with cDNA (THP-1+ EC) or Nef transfected THP-1 cells(THP-1+ Nef+EC). Data were expressed as fold MCP-1 production and apoptosis, normalized to the mean of control measurements (N = 6. *P<0.05, and **P<0.01).</p