Effect of HIV-1 Nef on cytokine and chemokine secretion by untreated or IFN-γ treated intestinal epithelial cells.

<p>Caco-2 cells were grown and differentiated on inserts and then left untreated (medium) or treated with graded amounts of Nef (ranging from 0.01 to 1 µg/ml), boiled Nef (0.1 µg/ml, bNef) or Nef_F12 (0.1 µg/ml). After 48 h supernatants were harvested from the basolateral chambers and tested for cytokines (TNF-α, IL-6, IL-10) and chemokines (MIP-3α, IL-8) by ELISA. In some experiments Nef and IFN-γ were preincubated with an anti-Nef mAb (10 µg/ml) or anti-Tat mAb (10 µg/ml) for 1 h at 37°C and then added to Caco-2 cells for 48 h. Results are expressed as pg/ml and are means ± SEM from 4 independent experiments in duplicate. *p<0.05 vs medium; ^○p<0.05 vs IFN-γ; ^Δp<0.05 vs Nef.</p

Effect of HIV-1 Nef on tight junction protein expression and localization in untreated or IFN-γ treated intestinal epithelial cell monolayer.

<p>(A) Differentiated Caco-2 cells were left untreated (medium) or were treated for 48 h with Nef, IFN-γ or their combination. Cells were then lysed and proteins were probed on Western blots with anti-occludin or anti-ZO1 mAbs. (B) The expression of the proteins was estimated by densitometry after normalization with the β-actin signal. Data represent values from 1 experiment representative of 3 performed. (C) Immunofluorescence analysis of ZO1 distribution in untreated (medium) or 48 h Nef-, IFN-γ - or IFN-γ/Nef- treated Caco-2 monolayers. The experiment was repeated 3 times to ensure reproducibility.</p

HIV-1 Nef uptake in intestinal epithelial cells.

<p>Differentiated Caco-2 cells were left untreated (medium) or were treated for 1, 3, 6 and 24 h with biotynilated-Nef alone or in combination with IFN-γ. Nef uptake was analyzed by CLSM (central optical sections). Nuclei are reported in blue (DAPI). Nef was detected as a diffuse intracytoplasmatic positivity (green). Scale bar, 20 µm. Panels are representative of 3 independent experiments.</p

Effect of HIV-1 Nef on paracellular flux of FITC-dextran and transepithelial electrical resistance (TEER) in untreated or IFN-γ treated intestinal epithelial cell monolayer.

<p>(A) Caco-2 cells were grown and differentiated on inserts and then left untreated (medium) or treated with graded amounts of Nef (ranging from 0.01 to 1 µg/ml), boiled Nef (0.1 µg/ml, bNef) or Nef_F12 (0.1 µg/ml) for 48 h. FITC-dextran (FITC-DX) was added in the apical compartment and after 3 h, the fluorescence intensity was measured in the basolateral compartment. Data represent means ± SEM of 3 independent experiments in duplicate. F.I. fold increase. (B) Caco-2 cells were treated for 48 h with supernatants from 293T cells transfected with either a pcDNA3.1 vector expressing wt Nef HIV-1 (SN wt Nef, containing 0.01 µg/ml Nef), or with the vector alone (SN mock). The amount of Nef in supernatants was estimated by semi-quantitative anti-Nef Western blot assay against quantified amounts of recombinant Nef. (C) Caco-2 cells were left untreated or treated with Nef (0.1 µg/ml), IFN-γ (600 U/ml) or their combination for 48 h. Nef and IFN-γ were preincubated with an anti-Nef mAb (10 µg/ml) or anti-Tat mAb (10 µg/ml) for 1 h at 37°C and then added to Caco-2 cells for 48 h. Means ± SEM from 4 independent experiments in duplicate, are shown. (D) Caco-2 cells were cultured on filter inserts for 21 days and then left untreated (medium ○) or treated with Nef (0.1 µg/ml ▪), IFN-γ (600 U/ml ▴) or IFN-γ/Nef (Δ). The TEER values were measured at the indicated time points and were normalized with values obtained at the start point of the analysis. Data represent means ± SEM of 8 independent experiments in duplicate. *p<0.05 vs medium; ^○p<0.05 vs IFN-γ; ^Δp<0.05 vs Nef.</p

Drug classes used as comedication when beginning DAA therapy, by severity of liver disease, among HCV-infected patients.

<p>Drug classes used as comedication when beginning DAA therapy, by severity of liver disease, among HCV-infected patients.</p

Sociodemographic and virological characteristics and comedications used, by severity of liver disease, among HCV-infected patients undergoing DAA therapy.

<p>Sociodemographic and virological characteristics and comedications used, by severity of liver disease, among HCV-infected patients undergoing DAA therapy.</p

Number of co-medications used and percentage of patients, by DAA regimen, among HCV-infected patients.

<p>(A) Patients with mild liver disease. (B) Patients with moderate-to severe-liver disease. SOF/RBV: sofosbuvir plus ribavirin, SOF/SIM: sofosbuvir plus simeprevir, SOF/DCV: sofosbuvir plus daclatasvir, SOF/LDV: sofosbuvir plus ledipasvir, 3D: paritaprevir/ritonavir, ombitasvir, dasabuvir. The percentage of patients who took one drug (in blu), two drugs (in red), three drugs (in green) and more than 3 drugs (in violet) are reported considering the total number of patients reported for each regimen in both Fig 1A and Fig 1B at the same manner.</p

Category of potential DDIs, by DAA regimen and severity of liver disease, among HCV-infected patients.

<p>Comedication used in patients with mild liver disease (A) or in (B) patients with moderate-to severe-liver disease (B). DAA regiments and number of comedications used are shown. SOF/RBV: sofosbuvir plus ribavirin, SOF/SIM: sofosbuvir plus simeprevir, SOF/DCV: sofosbuvir plus daclatasvir, SOF/LDV: sofosbuvir plus ledipasvir, 3D: paritaprevir/ritonavir, ombitasvir, dasabuvir. Category 0: Classification not possible due to lack of information; Category 1: No clinical interaction possible; Category 2: May require dose adjustment/closer monitoring.</p

The most common drugs with a potential DDI among HCV-infected patients with moderate-to-severe liver disease.

<p>The most common drugs with a potential DDI among HCV-infected patients with moderate-to-severe liver disease.</p

Failure rates following the first DAA regimen, by HCV genotype and treatment regimen in patients who completed the 12 weeks post treatment evaluation (n = 3,830 patients).

<p>Failure rates following the first DAA regimen, by HCV genotype and treatment regimen in patients who completed the 12 weeks post treatment evaluation (n = 3,830 patients).</p