Susceptibility of primary cDCs to <i>ex-vivo</i> infection with HIV-1.

<p>(A): Flow cytometry gating strategy for defining cDCs in bulk PBMC cultures. (B): Representative flow cytometry dot plots indicating GFP expression in gated CD11c⁺ HLA-DR⁺ cDCs from Neg, CP, EC and HAART individuals after 24, 48 and 96 hours of <i>ex-vivo</i> infection with GFP-encoding HIV-1. Numbers in dot plots reflect the proportion of GFP-positive cells within gated cDCs. (C–D): Proportion (C) and GFP MFI (D) of GFP⁺ cDCs from Neg, CP, EC and HAART subjects at 96 hours after infection with HIV-1 (n = 20 tested subjects for each cohort). Horizontal lines represent the median for each specific cohort and experimental condition. Differences among cohorts were tested using a Kruskal-Wallis test with post-hoc Dunn’s test (* p<0.05; *** p<0.001) or using Mann Whitney U test (# p<0.05; ## p<0.01).</p

Type I IFN secretion and activation in cDCs after <i>ex-vivo</i> exposure to HIV-1.

<p>(A): IFNα and IFNβ mRNA levels in isolated BDCA1⁺ cDCs from HIV-negative persons (Neg), individuals with chronic progressive HIV-1 infection (CP), Elite controllers (EC) and HAART-treated HIV-1 patients (HAART) at 24 and 48 hours after exposure to HIV-1 (HIV) or to media only (Med) as negative control. Horizontal lines represent the median for each specific cohort and experimental condition. (B): Mean Fluorescence Intensity (MFI) reflecting surface expression of CD86, CD83 and CD40 in cDCs from the different study cohorts at 24 hours after infection with HIV-1 (HIV) or after exposure to poly(I:C) (PIC). MFI values are expressed as fold-changes in comparison to baseline levels. Intra-individual differences were tested for statistical significance using Wilcoxon matched-pairs signed rank tests (above each cohort), differences between cohorts were tested using a Kruskal-Wallis test with post-hoc Dunn’s test; * p<0.05; ** p<0.01; *** p < 0.001; **** p< 0.0001. Horizontal lines represent the median for each specific cohort and experimental condition. (C): Heatmaps reflecting gene expression patterns of 28 interferon-stimulated genes (ISG) in cDCs from Neg (n = 6), CP (n = 6) and EC (n = 6) at 48 hours after infection with HIV-1. (D): Heatmaps reflecting correlations among gene expression intensities of all 28 ISGs in indicated study cohorts. Color-coding reflects Pearson’s correlation coefficient indicating strengths of statistical association between expression intensities of given gene pairs.</p

Antigen-presenting properties of cDCs after exposure to HIV-1.

<p>(A): Representative flow cytometry plots reflecting proliferation of CD4⁺ T cells after stimulation with allogeneic uninfected (Med) or HIV-1 infected (HIV) cDCs from Neg, CP, EC and HAART patients. Numbers in flow cytometry plots represent the proportion of proliferating CFSE^low T cells. (B–C): Induction of allogeneic CD4 (B; n = 8) and CD8 T (C; n = 7) cell proliferation after exposure to HIV-1 infected cDC from indicated study cohorts. Horizontal lines represent the median for each specific cohort and experimental condition. (D): Representative flow cytometry dot plots reflecting IFN-γ secretion in an HLA-A2-SL9 (SLYNTVATL)-specific CTL cell line after 16 hours of co-culture with uninfected or HIV-1-infected cDCs from Neg, CP, EC and HAART individuals. (E): Proportion of IFNγ⁺ cells in the SL9 CTL cell line after exposure to HIV-1-infected cDCs from indicated study groups. Cumulative data from n = 8 experiments are shown. (B,C,E): Differences within and among study groups were tested for statistical significance using a Wilcoxon matched-pairs signed-rank test rank test or a Mann Whitney test corrected for multiple comparisons using Bonferroni method, respectively. * p<0.05; ** p<0.01. (F): Induction of allogeneic CD4 (left; n = 7) and CD8 (right; n = 7) T cell proliferation after exposure to cDC from EC cultured in the presence of media (Med) or infected with HIV-1 in the presence or absence of AZT or RAL. Statistical significance of differences was tested using a Wilcoxon matched-pairs signed rank test. Bonferroni correction was applied for multiple comparisons.* p<0.05. (E-F): Horizontal lines represent the median for each specific cohort and experimental condition.</p

Induction of cytosolic DNA sensors in cDCs from EC.

<p>(A): Fold change in cGAS, STING and IFI16 mRNA expression levels in indicated study cohorts at 24 (upper panels) and 48 (lower panels) hours after <i>ex-vivo</i> infection with HIV-1. Induction of mRNA expression in comparison to baseline levels was tested for statistical significance using Wilcoxon matched-pairs signed-rank test tests. Significant differences between distinct cohorts were calculated using a Mann Whitney test. No correction for multiple comparisons was applied (B): IFNα and IFNβ expression in primary cDCs nucleofected with scrambled (SC) or cGAS-specific siRNAs, followed by infection with HIV-1. Data from n = 4 experiments are shown. Data were normalized to results from experiments with scrambled siRNA sequences. Differences in type I IFN responses between untreated or SC- or cGAS-nucleofected cDCs were tested for statistical significance using a Kruskal-Wallis test with post-hoc Dunn’s test * p<0.05; ** p<0.01.</p

HIV-1 replication patterns in cDCs from EC.

<p>(A): Early and late HIV-1 reverse transcripts (RT) and 2-LTR circles in cDCs from indicated study subjects at 48 hours after <i>ex vivo</i> infection with HIV-1. (B): Analysis of integrated HIV-1 DNA in primary cDCs from the different study cohorts. Basal levels of integrated HIV-1 DNA are shown in the left panel, right panel shows <i>de novo</i> HIV-1 integration in cDCs after subtraction of baseline levels of integrated HIV-1 DNA. (C): Analysis of late HIV-1 RT products and 2-LTR circles normalized to levels of <i>de novo</i> integrated HIV-1 DNA in cDCs at 48 hours after infection. (A–C): Differences between different cohorts were tested for statistical significance using a Kruskal-Wallis test with post-hoc Dunn’s test or using Mann Whitney U test (# p<0.05; ## p<0.01). (A,B,C). Horizontal lines represent the median for each specific cohort and experimental condition. (D): Inhibition of IFNα and IFNβ mRNA expression in cDCs from EC cultured in media (Med) or infected with HIV-1 (HIV) in the presence or absence of AZT, Efavirenz (EFV) or Raltegavir (Ral). Data reflect mean and standard error from qPCR values of IFNα and IFNβ mRNA levels after normalization to β-actin endogenous expression from n = 5 experiments. Numbers above bars represent the mean percentage of inhibition induced by each drug. Differences were tested for statistical significance using a one-tailed Wilcoxon matched-pairs signed rank test, * p<0.05.</p

Additional file 4: Table S3. of A Reproducibility-Based Computational Framework Identifies an Inducible, Enhanced Antiviral State in Dendritic Cells from HIV-1 Elite Controllers

DE Analysis. DE analysis results as described in “Methods,” including contrasts for c1 vs c3–5, c2 vs c3–5, c1 vs c2, and intra-c1 exposure differences. Numbers of cells in each comparison are printed above log-fold-change columns. Gene sets from Fig. 2c are included. (XLSX 3450 kb

Empirical flashover model of EHV post insulators based on ISP parameter in cold environments

The main objective of this contribution is to present an empirical model of ice-covered insulator flashover in accordance with an important flashover index, called icing stress product (ISP). The ISP as the product of the ice mass per centimeter of insulator length and the electrical conductivity of the melted ice accretion is used to establish an empirical model to determine the flashover voltage under icing conditions. To achieve this model, several tests were carried out on post station insulators typically used in Hydro-Quebec 735-kV substations, under DC and AC voltage, to determine the relationship between flashover stress and the ISP. The ISP-based method offers a good tool not only to select insulators for locations exposed to freezing conditions, but also to compare flashover results obtained under different test conditions. Moreover, to study the optimizing method of insulator flashover, the influence of air gaps on the flashover stress of ice-covered post insulators is investigated. The results reveal that the number of air gaps significantly affects the flashover stress. The test results are highly meaningful as they can be used as a reference for ranking several other insulator types and configurations in order to select the appropriate one for cold environments. Moreover, several mitigation options to improve insulator reliability in cold environments are provided

Additional file 7: of A Reproducibility-Based Computational Framework Identifies an Inducible, Enhanced Antiviral State in Dendritic Cells from HIV-1 Elite Controllers

AOM. Additional online materials. (PDF 243 kb

Spearman correlation between LILRB2 binding strength and mVL in HIV-1-infected individuals.

<p>Spearman correlation between LILRB2 binding strength and mVL in HIV-1-infected individuals.</p

Effect of LILRB2-HLA binding strength and individual class I alleles on viral control (controllers vs. non-controllers) in white patients.

<p>Logistic regression model with stepwise selection included all <i>HLA</i> class I alleles with phenotypic frequencies of >2% and one of the A, B, C or ABC binding scores at a time. The results are shown for the p<0.05 cut-off. The C binding score did not stay in the model. ORs for binding scores reflect a change of 0.1 units.</p>1<p>stayed in the model with the p<0.01 cut-off but not with the p<0.001 cut-off.</p>2<p>stayed in the model with the p<0.01 and p<0.001 cut-offs.</p